Advances in Applied Microbiology, Volume 9

ADVANCES IN Applied Micro biology VOLUME 9 CONTRIBUTORS TO THIS VOLUME R. F. Christman J. Ross Colvin C. H. Driver...

Author: Wayne W . Umbreit

29 downloads 1201 Views 16MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

ADVANCES IN

Applied Micro biology VOLUME 9

CONTRIBUTORS TO THIS VOLUME

R. F. Christman J. Ross Colvin

C. H. Driver G. F. Gause

I. H. Harrison Richard M. Hyde June Jenkins

L. Juragek Ralph E. Kunkee Birgitta Norkrans

R. T. Ogelsby Wesley 0. Pipes A. D. Russell

D. R. Whitaker

ADVANCES IN

Applied Microbiology Edited by WAYNE W. UMBREIT Department of Bacteriology Rutgerr, The State University N e w Brunswick, N e w Jersey

VOLUME 9

@

1967

ACADEMIC PRESS, New York and London

COPYRIGHT 6:

1967, BY ACADEMIC PRESS

INC.

ALL RIGHTS RESERVED. NO PART OF TIIIS BOOK MAY BE REPRODUCED IN ANY FORM,

BY PHOTOSTAT, MICROFILM,

on

ANY OTHER MEANS, WITHOUT

WRITTEN PERMISSION FROM THE PUBLISHERS.

ACADEMIC PRESS INC. 111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS INC. (LONDON) LTD. Berkeley Square House, London W . l

LIBRARYOF CONGRESS CATALOG CARDNUMBER: 59-13823

PRINTED IN T H E UNITED STATES O F AMERICA

CONTRlBUTORS Numbers in parentheses indicate the pages on which the authors' contxibutions begin. R.

F. CHRISTMAN, Departments of Civil Engineering and College of Forestry, University of Washington, Seattle, Washington (171)

J . ROSS COLVIN, Division of Biosciences, National Research Council, Ottawa, Canada (131) C.

Departments of Civil Engineering and College of Forestry, University ofwashington, Seattle, Washington (171)

H . DRIVER,

G. F .

CAUSE,

lnstitute of New Antibiotics, Academy of Medical Sciences, Moscow,

U.S.S.R. (69) I. H . HARRISON, Department of Pharmaceutics, Welsh School of Pharmacy, Welsh College of Advanced Technology, Cardiff, Britain ( 1 ) M. HYDE, Department of Microbiology, School of Medicine, University of Oklahoma, Oklahoma City, Oklahoma (39)

RICHARD

Department of Pharmaceutics, Welsh School of Pharmacy, Welsh College of Advanced Technology, Cardiff, Britain (1)

JUNE JENKINS,

L. JURASEK, Division of Biosciences, National Research Council, Ottawa, Canada (131)'

RALPH E. KUNKEE, Department of Viticulture and Enology, University of California, Davis, California (235) BIRGITTA NORKRANS, Department of Marine Botany, University of Goteborg, Goteborg, Sweden (91)

R. T. OGLESBY, Departments of Civil Engineering and College of Forestry, University of Washington, Seattle, Washington (171) WESLEY0. PIPES, Department of Civil Engineering, Northwestern University, Evans-

ton, Illinois (185) A. D. RUSSELL, Department of Pharmaceutics, Welsh School of Pharmacy, Welsh College of Advanced Technology, Cardiff, Britain (1) D. R. WHITAKER, Division of Biosciences, National Research Council, Ottawa, Canada (131)

'Present address: Stitny drevarsky vyskumny ustav, LamaGska 5, Bratislava IX, Czechoslovakia. V

This Page Intentionally Left Blank

PREFACE

The scope of this year’s volume is as broad as applied microbiology itself. Its problems range from the making of wine to the bulking of sludge, which may seem rather too wide to the uninitiated but which the professional will recognize as not entirely unrelated. Since cellulose comprises perhaps a third of the carbon in the world, we have devoted two separate papers to it. The knowledge of the transformation of lignin to humus is brought up-to-date - no one could say that the problem is solved. The preservation of pharmaceuticals, unfamiliar to many applied microbiologists, is brought to our attention. In addition, the present status of the changing relation between microbial metabolic research and the cancer problem is reviewed. This is the ninth volume of this serial publication, and for almost a decade I have watched and occasionally assisted its growth. The next volume will complete a decade and, as a matter of principle, one man ought not to edit a publication for a longer period of time particularly when he bears the sole responsibility for the work. Inevitably, the content will reflect his own interests and contacts, and these will not necessarily result in contributions containing the most important developments in the field. Therefore, for the next volume, Dr. David Perlman, of the University of Wisconsin will join me as coeditor, and in subsequent volumes he will be the sole editor. It has been evident that Advances in Applied Microbiology is a useful and valuable addition to the literature of the subject and, as editor, I am happy to have served this discipline.

W. W. UMBREIT

Rutgers University October, 1967

vii


CONTENTS CONTRIBUTORS .......................................................................................... PREFACE................................................................................................... CONTENTSOF PREVIOUSVOLUMES ..............................................................

V

vii xi

The Inclusion of Antimicrobial Agents in Pharmaceutical Products A. D. RUSSELL,JUNEJENKINS,AND I. H. HARRISON

I. Introduction ............................... ........... 11. Injections ....................._...........,............................ , ...... ..................... 111. Immunological Products ............., ....................................................... IV. Eye Drops ................................................................... istration ......, ....................... ........................ V. Medicines VI. Preparations for External Use ............................................................. References ................................... ..............

1 2 15 22 28 32 35

Antiserum Production in Experimental Animals RICHARDM. HYDE Introduction .... ................ .................................................................. Methods Employed in Antiserum Collection .......................... ............. . Animal Species Employed in Antiserum Production. Routes of Inoculation Employed in Antiserum Produ V. Factors Affecting Antibody Production ...................... ..... ..... ..... ............ VI. Specific Examples of Antiserum Production VII. Conclusion ............ ..................... ,.................... ....................... .... ...... graphy .................. ... ............

1. 11. 111. IV.

...........................................

39 40 41 43 47

58 58 59 63

Microbial Models of Tumor Metabolism C. F. CAUSE I. Introduction

......, ....

.................................................. .........

11. Disturbance of Control Mechanisms in the Metabolism of Tumors

References .......................................................................................

69 70 77 88 88

Cellulose a n d Cel Iu lolysis BIRGITTANORKRANS I. Introduction .......... ............................................................................ 11. Cell Wall Morphology and Chemistry ................................................. 111. Cellulose Chemistry and Supramolecular Morphology .................. ......... IV. Biosynthesis of Cell Wall Polysaccharides ....................... ... ..................

ix

91 92 95 98

X

CONTENTS

V. Degradation of Cellulose by Bacteria and Fungi ................................... VI . Applications for Cellulases ,. .................. ... ........................ .................. References ....... ,...................................... .....................................'. .

.

101 124 125

Microbiological Aspeck of the Formation and Degradation of Cellulosic Fibers L. J u R A ~ E K ,J. Ross COLVIN, AND D. R. WHITAKER 131 131 141

155 166

The Biotransformation of Lignin to HumusFacts and Postulates R. F . CHRISTMAN, AND DRIVER ................................................... 1. Introduction ............... .... 11. Mechanisms of Lignin Bio n ...................... ................. . ............. Humification .... IV. The White-Rot Fungi .................... ............................................. sis and Humification V. Other Organisms .................................. References ....... ........ ...................

171 173 175 178 182 183

Bulking of Activated Sludge 0 . PIPES

...... ..................... . ...... .................. 185 189 ................................................................. 11. Types of Settling P 111. Superficial Aspects of Bulking ..................... . ........................ 204 IV. Fundamental Aspects of Filmentous Bulking ........................ 217 229 V. Summary ........................ ............. ......................... References ............ ...................... 232 I. Introduction

.........

I

Malo-lactic Fermentation RALPH E. KUNKEE

..............

235 236 239 ................................ IV. Malo-lactic Bacteria ............ ............... ..... ... ............. .............. ... ......... 24 1 ......... ................. 259 V. Detection of Malo-lactic Fermentation 260 ........................ 270 VII. Control of Conclusions ...... ......................... 273 274 281 297 SUBJECT INDEX

.

CONTENTS OF PREVIOUS VOLUMES

A Commentary on Microbiological Assaying F . Kavanagh

Volume 1

Protected Fermentation

MiloH Herold and Jan NeEasek

Application of Membrane Filters

The Mechanism of Penicillin Biosynthesis Arnold L. Demain

Richard Ehrlich Microbiol Control Brewery

Preservation of Foods and Drugs by Ionizing Radiations

in

the

Gerhard J . Hass

W . Dexter Bellamy

Newer Development in Vinegar Manufactures

The State of Antibiotics in Plant Disease Control

Rudolph J . Allgeier and Frank M . Hildebrandt

David Pramer Microbial Synthesis of Cobamides

The Microbiological Transformation of Steroids

D. Perlman

T. H . Stoudt

Factors Affecting the Antimicrobial Activity of Phenols E. 0.Bennett

Biological Transformation of Solar Energy William J . Oswald and Clarence G .

Golueke

Germfree Animal Techniques and Their Applications Arthur W. Phillips andJames E . Smith

SYMPOSIUM ON ENGINEERING ADVANCES IN FERMENTATION PRACTICE

Insect Microbiology S . R. Dutky

Rheological Properties of Fermentation Broths

The Production of Amino Acids by Fermentation Processes

Fred H. Deindoerfer and John M . West

Shukuo Kinoshita

Fluid Mixing in Fermentation Processes J . Y. Oldshue

Continuous Industrial Fermentations Philip Gerhardt and M . C . Bartlett

Scale-up of Submerged Fermentations

W .H. Bartholemew

The Large-Scale Growth of Higher Fungi Radclife F. Robinson and R. S.

Air Sterilization Arthur E. Humphrey

Davidson AUTHOR INDEX-SUBJECT

Methods

INDEX

Sterilization of Media for Biochemical Processes

Volume 2

Lloyd L. Kempe

Newer Aspects of Waste Treatment

Nandor Porges

Fermentation Kinetics and Model Processes

Aerosol Samplers

Fred H. Deindoerfer

Harold W . Batchelor

xi

xii


Continuous Fermentation W. D. Muxon Control Applications in Fermentation George]. Fuld

AUTHOR INDEX- SUBJECT INDEX Volume 4

Induced Mutagenesis in the Selection of Microorganisms S. 1. Alikhanian

Volume 3

Preservation of Bacteria by Lyophilization RobertJ. Heckly

Sphuerotilus, Its Nature and Economic Significance Norman C . Doridero

The Importance of Bacterial Viruses in Industrial Processes, Especially in the Dairy Industry F . 1.Babel Applied Microbiology in Animal Nutrition Harlow H . Hall

Large-Scale Use of Animal Cell Cultures Donald 1. Merchant and C . Richard Eidam

Biological Aspects of Continuous Cultivation of Microorganisms T. Holme

Protection against Infection in the Microbiological Laboratory: Devices and Procedures Murk A . Chatigny

Maintenance and Loss in Tissue Culture of Specific Cell Characteristics Charles C . Morris

Oxidation of Aromatic Compounds by Bacteria Martin H . €logo8

Submerged Growth of Plant Cells L. G. Nickel1 AUTHOR INDEX- SUBJECT INDEX Volume 5

Screening for and Biological Characterizations of Antitumor Agents Using Microorganisms Frank M . Schabel, Jr., und Robert F . Pittillo

Correlations between Microbiological Morphology and the Chemistry of Biocides Adrien Albert

The Classification of Actinoinycetes in Relation to Their Antibiotic Activity Elio Baldacci

Generation of Electricity by Microbial Action 1.3. Davis

The Metabolism of Cardiac Lactones by Microorganisms Elwood Titus

Microorganisms and Biology of Cancer G . F . Gause

Intermediary Metabolism and Antibiotic Synthesis /. D. Bu’Lock

Rapid Microbiological with Radioisotopes Gilbert V. Levin

Methods for the Determination of Organic Acids A . C . Hzrlme

The Present Status of the 2,S-Butylene Glycol Fermentation Sterling X . Long and Roger Patrick

the

Molecular

Determinations

...

Xlll


Aeration in the Laboratory W . R. Lockhart and R. W. Squires Stability and Degeneration of Microbial Cultures on Repeated Transfer

Fritz Reusser Microbiology of Paint Films Richard T . Ross The Actinomycetes and Their Antibiotics

AUTHOR INDEX-SUBJECT

INDEX

Volume 7

Microbial Carotenogenesis

Alex Ciegler Biodegradation: Problems of Molecular Recalcitrance and Microbial Fallibility M . Alexander

Selman A. Waksman Fuse1 Oil

A. Dinsmoor Webb and John L. lngraham AUTHOR INDEX - SUBJECT INDEX Volume 6

Global Impacts of Applied Microbiology: An Appraisal Carl-Goran Heden and Mortimer P.

Starr Microbial Processes for Preparation of Radioactive Compounds D. Perlman, Aris P. Bayan, and Nancy

A. Giufre

Cold Sterlization Techniques John B . Opfell and Curtis E . Miller Microbial Production of Metal-Organic Compounds and Complexes

D. Perlman Development of Coding Schemes for Microbial Taxonomy S. T. Cowan Effects of Microbes on Germfree Animals

Thomas D. Luckey Uses and Products of Yeasts and Yeastlike Fungi Walter J . Nickerson and Robert G.

Brown Secondary Factors in Fermentation Processes

P. Margalith Nonmedical Uses of Antibiotics Herbert S. Goldberg Microbial Aspects of Water Pollution Control K . Wuhrmann Microbial Formation and Degradation of Minerals Melvin P. Silverman and Henry L.

Ehrlich Enzymes and Their Applications

Irwin W . Sizer A Discussion of the Training of Applied Microbiologists B . W . Koft and Wayne W . Umbreit

Microbial Amylases

Walter W . Windish and Nagesh S. M ha tre The Microbiology of Freeze-Dried Foods

Gerald J. Silverman and Samuel A. Goldblith Low-Temperature Microbiology ]udith Farrell and A. H . Rose AUTHOR INDEX-SUBJECT INDEX Volume 8

Industrial Fermentations and Their Relations to Regulatory Mechanisms Arnold L. Demain Genetics in Applied Microbiology S. G. Bradley

xiv


Microbial Ecology and Applied Microbiology Thomas D . Brock The Ecological Approach to the Study of Activated Sludge Wesley 0. Pipes Control of Bacteria in Nondomestic Water Supplies Cecil W . Chambers and Norman A . Clarke The Presence of Human Enteric Viruses in Sewage and Their Removal by Conventional Sewage Treatment Methods Stephen Alan Kollins

Oral Microbiology Heiner Hoffman Media and Methods for Isolation and Enumeration of the Enterococci Paul A . Hartmun, George W . Reinbold, und Deui S. Saruswut Crystal-Forming Bacteria Pathogens Martin H . Rogoff

as

Insect

Mycotoxins in Feeds and Foods Emanuel Borker, Nino F . Insalata, Colette P . Leui, and John S . Witzeman AUTHOR INDEX-SUBJECT

INDEX

ADVANCES IN

Applied Micro biology VOLUME 9


The Inclusion of Antimicrobial Agents in Pharmaceutical Products A. D. RUSSELL,JUNE

JENKINS, AND

I. H. HARRISON

Department of Pharmaceutics, Welsh School of Pharmacy, Welsh College of Advanced Technology, Cardiff, Britain I. Introduction 11. Injections ...................... A. Introduction ............................... B. Methods of Sterilization ......................................... C. Vehicles ............................................................... D. Containers and Closures E. Selection of Bactericides ...... 111. Immunological Products .............................................. A. Types of Immunity ................................................ B. Products Requiring a Preservative IV. Eye Drops .................................................................. A. Preparation and Sterilization .................................. B. Preservatives ........................................................ V. Medicines for Oral Administration A. Introduction ........................ B. Preservatives in Common Use ................................ VI. Preparations for External Use ....................................... A. Introduction ......................................... References .................

1 2 2 3 6 7 9 15 15 16 22 22 23 28 28 31 32 32 33 35

I. Introduction Certain pharmaceutical products require the inclusion of an antimicrobial substance in their formulation. This term antimicrobial substance may itself be interpreted as having a wide meaning, and may be thought to include such groups as the antibiotics and sulfonamides in addition to other antibacterial and antifungal substances. However, this paper deals only with those substances which are deliberately included in pharmaceutical formulations ( a ) for reasons of preservation, or ( b ) when combined with heat, as a method of sterilization. Such substances thus do not comprise the active medicament(s) of the products, and in fact, the concentration of the agents is usually much smaller. The choice of a preservative is governed by the type of product and its chemical and physical properties, because of the possibility of interaction occurring between the agent and the other constituents of the preparation. Incompatibility may result in the inactivation of the 1

2

A. D. RUSSELL, J. JENKINS, AND I. H. HARRISON

drug and/or the agent or in undesirable changes in the nature of the product, e.g., precipitation, coagulation, or separation. Although in many cases it is possible to predict that a given preservative will be unsuitable for use in a certain product, the converse is less easily predictable. Moreover, data on the antimicrobial activity of a preservative obtained from studies on nutrient media may be misleading. It is therefore important to test the final formulation thoroughly to ensure that it will produce the required results and that the preservative present will in fact protect the product against all the microorganisms likely to contaminate it in practice. To detect changes which occur slowly, prolonged storage tests under a variety of storage conditions are necessary. Some of the problems encountered in this field and methods for determining the efficacy of preservatives have been discussed by Rdzok et al. (1955). Products that require the inclusion of an antimicrobial compound include injections, immunological products, eye drops, creams, and other two-phase systems, tinctures, liquid extracts, and certain mixtures. Although normally administered parenterally, immunological products are considered separately from injections because of their special nature of preparation and composition. These groups of products, together with the antimicrobial substances which may be incorporated into their formulation, are discussed below. In each section, brief details of the types and preparation of the constituent products are given; it is hoped that such introductory data will be of some use to those microbiologists not engaged in this type of project. The abbreviations used throughout this paper are as follows: B.P. (British Pharmacopoeia), B.P.C. (British Pharmaceutical Codex), U.S.P. (United States Pharmacopoeia), U.S.N.F. (United States National Formulary) and A.N.F. (Australian National Formulary). II. Injections

A. INTRODUCTION

1 . Definition The International Pharmacopoeia (I.P.) defines injections as “a class of sterile pharmaceutical solutions, emulsions or suspensions, packaged in containers which will maintain sterility, and intended for parenteral administration, i.e., under or through one or more layers of skin or mucous membrane.” The parenteral route is used for several reasons: the drug may be destroyed when given orally, or it may be

ANTIMICROBIAL AGENTS IN PHARMACEUTICALS

3

inactive when given by that route. In addition, drugs given by injection often act more quickly: they may be used to produce a rapid localized effect, or in some cases to produce a prolonged action.

2. Routes Injections may be subdivided according to the route of administration. The British Pharmaceutical Codex (1963) lists seven, which Cooper and Gunn (1965) have defined. Intradermal or intracutaneous injections are made into the skin, between the dermis and the epidermis. Subcutaneous or hypodermic injections are made under the skin, into the subcutaneous tissue; the term hypodermic has also been used more generally, as in hypodermic syringe, but such syringes are not limited to subcutaneous injections. Intramuscular injections are made into the muscle tissue itself. Injections made into a vein, that is, intravenous injections, may include large volume injections, more specifically called infusion fluids. The three remaining, intrathecal, intracisternal, and peridural injections, are made into the cerebrospinal fluid.

3. Types Injections may be obtained in single dose or multidose containers, depending upon a number of factors, which include the nature of the active constituent and the requirements of both patient and doctor. However, the physical form of the injection varies more widely. The majority are aqueous solutions, but in other cases the medicament is dissolved in a suitable oil, ester, or alcohol. Some injections are formulated as suspensions or emulsions, while in the case of a watersoluble, but readily hydrolyzable drug, the formulation may be presented in two ampoules, a powder ampoule and an ampoule containing a solvent, the contents of the one to be dissolved in the other immediately before use. If the extended definition of the B.P. (1963) is considered, it will be noted that certain injections used in radiography, e.g., iodized oil fluid injection, may be introduced into the body cavities-the lungs, uterus, or urethra, as a contrast medium. For the purposes of this paper, these latter injections will not be considered.

B. METHODS OF

STERILIZATION

Alin and Diding (1949) demonstrated that the autobactericidal properties of a number of solutions for injection are insufficient to give a sterile product, and all injections must therefore be subjected

4


to a suitable, efficient sterilization procedure. Nonsterile injections are a potential danger to the patient, especially since he may already be suffering from a condition which has weakened the body's natural defenses. The introduction of such contaminated injections into blood, tissue, or spinal fluid provides an opportunity for the rapid growth of microorganisms, and must at all times be avoided. Furthermore, it has been reported (Kedzia et al., 1961) that the presence of bacteria in a solution, albeit not necessarily of a pathogenic species, may cause the breakdown of the drug, with resultant loss of activity. A number of different methods of sterilization are available, and the choice of method is determined by the nature of the active constituent of the injection, which must not be adversely affected b y the sterilization procedure selected. Methods may be physical, chemical, or physicochemical. Sykes (1963a) gives a useful commentary upon the methods of sterilization commonly used, but the following are the most important in the preparation of injections.

1 . Methods Involving Heat a . Dry Heat This method is suitable for oily solutions and suspensions, and for powders which are to be dissolved in sterile solvents before use. T h e choice of suitable temperature and times is influenced b y the stability of the product, and some variation is found in official recommendations (Table I).

OFFICIAL

TABLE I RECOMMENDATIONSFOR DRYHEAT STERILIZATION

Reference

Temperature ("C.)

B.P. (1963) I.P. U.S.P. (1965)

Duration

150 1 hour 150 2 hours General recommendations

b. Heating in an Autoclave. Moist heat is generally accepted as being a more efficient sterilizing agent than dry heat, and steam under pressure provides a convenient and effective method of sterilization. This method, when carried out correctly, is the most suitable for aqueous injections of thermostable substances. Suggested sterilization times and temperatures are given in the B.P. (1963), U.S.P. (1965), and I.P., but large volumes will


5

require increased periods of exposure to the sterilization temperature to ensure that the whole of the product has been subjected to the correct temperature for the required time. The B.P. (1963) recommends a sterilizing temperature of 115" to 116"C., while the U.S.P prefers the higher temperature of 121°C. with a correspondingly shorter exposure time. The B.P. (1963), U.S.P. (1965), and I.P. all state that when a container is sealed so as to permit the withdrawal of successive doses on different occasions, the solution or preparation should contain sufficient amounts of a suitable bactericide to prevent the growth of microorganisms (Table 11). The term bactericide, in this context, TABLE I1 BACTERIOSTATS RECOMMENDED IN DIFFERENTPHARMACOPOEIAS Reference B.P. (1963)

I.P.

U.S.P. (1965)

Recommended bacteriostat Phenol, 0.5% w./v. Cresol, 0.5% w./v. Chlorocresol, 0.1% w./v. Phenylmercuric nitrate, 0.001% w./v. Phenol, 0.5% w./v. Cresol, 0.3% w./v. Chlorbutanol or its hydrate, 0.5% w./v. Phenylmercuric nitrate, 0.001% w./v. Phenylmercuric borate, 0.001% w./v. General recommendations

indicates a substance in a bacteriostatic concentration, and will hereafter be referred to as a bacteriostat. Bacteriostats must not be included in any injection for intrathecal, intracisternal, or peridural use (these should be packed in single-dose containers), or in an intravenous injection of dose-volume greater than 15ml. Furthermore, a bacteriostat is unnecessary if the medicament itself has an antibacterial effect: leptazol is an example of this (Gilbert and Russell, 1963). Other commonly used injections are also claimed to exert some bacteriostatic effect (though not sufficient to preclude the addition of a bacteriostat) and Kohli et al. (1950) and Sen Gupta (1951) have studied these with a view to designing suitable sterility tests for them. It may be noted, in passing, that a number of injections containing ratioactive materials, although they may be packed in multidose containers, do not contain a bacteriostat. The reason for this omission has

6


not yet been satisfactorily established but research by the British Pharmacopoeia Commission (1966) in this field is continuing.

c . Heating w i t h a Bactericide.

The solution or suspension is prepared using a vehicle containing a given percentage of a suitable bactericide, and heated for a time which ensures that the whole of the product in each container is maintained at 98" to 100°C. for 30 minutes. The choice of bactericides is smaller than that of bacteriostats but neither bactericide nor bacteriostat should interfere with the therapeutic efficacy of the drug, nor cause a turbidity. In the B.P. (1963) the two recommendations are 0.2% w./v. chlorocresol and 0.002% w./v. phenylmercuric nitrate, in each case, twice the recommended bacteriostatic strength. The U.S.P. (1965) does not mention this process per se. The I.P. permits the two bactericides already mentioned, arid also 0.002% w./v. phenylmercuric borate.

2. Filtration Solutions which are sterilized by filtration may contain enough suitable bacteriostat to prevent the growth of microorganisms, although when such solutions are to be used for intrathecal, intracisternal, or peridural injection, or when they are to be used for intravenous injection in doses exceeding 15 ml., the bacteriostat is to be omitted. Solutions prepared by this method are packed in their final containers using aseptic technique, and after sealing, each batch must b e subjected to a test for sterility with which they must comply. The value of this method lies in the fact that it can be used for thermolabile medicaments, but it is a technique requiring skill and must be carried out by trained operators. Sterilization b y heating methods, or by filtration are the most commonly employed; however, other methods are available, including gaseous and radiation sterilization, but will not be considered. C. VEHICLES

1 . Water for Injection The majority of injections are aqueous solutions and the nature of the water used in their preparation must be considered. Potable water contains a wide range of contaminants, depending upon its source, subsequent treatment, and storage, (Taylor and Burman, 1956) and the degree of bacterial contamination, although not normally high,


7

may be sufficient to render it unsuitable for use in the preparation of injections. Dissolved chemicals may cause incompatibilities with the medicament, resulting in turbidity, discoloration, precipitation, or inactivation. Water for injection (B.P. 1963; sterile water for injection, U.S.P., 1965) is potable water distilled from a special still and sterilized in its final containers by heating in an autoclave, or by filtration, without the addition of a bacteriostat. Saunders and Shotton (1956) have examined the preparation of water for injection, and indicate that deionized water is not a suitable substitute for freshly distilled potable water, since the resins used in a deionizing column frequently become contaminated with bacterial colonies, and the water obtained from such columns, although free from chemical substances, may contain microorganisms. It is, however, conceivable that deionized water may, at some time in the future, be used as a vehicle in the preparation of injections.

2 . Nonaqueous Vehicles Aqueous solvents are the vehicles of choice in the preparation of injections, but certain formulations, for reasons of stability, solubility, or usage must be made in nonaqueous vehicles. Oils or fatty acid esters may be used to give a depot effect, while alcohols, propylene glycol, benzyl benzoate, and other substances are used to aid solution of a drug or to preserve its activity. The requirement to add a suitable bacteriostat to injections sealed so as to permit the withdrawal of successive doses also applies to oily injections, although the B.P. (1963) states that “bactericides are ineffective in oils.” Their inclusion would appear to be as a safeguard rather than as having a proved value.

D. CONTAINERS AND CLOSURES 1 . Suitable Materials

Injections may be packed in single-dose or multiple-dose containers, although the former are generally regarded as more desirable, since in the withdrawal of successive doses from a multidose container, there is the continued risk of the introduction of contaminants. Kohan et al. (1962) in a study of 490 multiple-dose vials, found that 13 had become contaminated during use. However, Ravnick and Yatoko (1962) working with 141 vials, found that none had been contaminated. The presence of a bacteriostat does deter the multiplication of microorganisms, but obviously a single-dose container is

8


more satisfactory in this respect, although it might not be as convenient in administration. Single dose containers may be ampoules or cartridges; large-volume infusion solutions may be packed in graduated bottles suitable for use in a transfusion apparatus. Multidose injections which have the advantage of flexibility of dose volume, are usually packed in small bottles of the Clinbritic type. The containers should be made of glass which has been tested for its suitability for packing injections: certain types of glass have a high extractive, and yield, among other substances, alkali, which may substantially alter the pH of the injection, with consequent deleterious effects upon the medicament and added substances. Tests for the limit of alkalinity of glass may be of the “whole-container” type (B.P., 1963, I.P.) or of the “crushed-glass” type (U.S.P., 1965). Materials other than glass are permitted for use as containers, provided that they do not react with the medicament, affect its therapeutic properties, or yield small solid particles. Amber glass may be used for injections which must be protected from light.

2. Absorption of Bactericides by Rubber

The ideal method of sealing an injection is by fusion of glass, as in ampoules, but since such a sea1 is broken to withdraw the dose, it can only be used for single-dose containers. Multidose containers are sealed by a rubber closure which will permit the withdrawal of successive doses. A plastic or metal oversea1 must be applied to prevent the removal of the closure before or during use. Ideally, the rubber used in the preparation of the closures should not contribute anything to the solution (Wing, 1958), should not absorb substances from it (Royce and Sykes, 1957) nor allow volatilization of contents through it, or absorption of air and moisture by the contents. Careful formulation of the rubber can reduce to a minimum the possibility of the closure contributing to a solution. Synthetic rubbers have been examined and some types are found to be comparatively inert, but because of expense, poor tensile strength, and other properties, are not always suitable for use in pharmaceutical closures. However, the problem of rubber absorbing bactericides from an injection solution is less easily overcome. Royce and Sykes (1957) have shown how common preservatives will be distributed between two immiscible solvents-that is, water and rubber- according to their partition coefficients. Their experiments showed that rubber can absorb from 15% (benzyl alcohol) to more than 95% (phenylmercuric nitrate) of an injection preservative when a solution of bacteriostatic strength is studied. The figures quoted are at equilibrium and may


9

not be reached for several weeks, but it is evident that, during storage, a high percentage of preservative may be removed from an injection, with subsequent risk of bacterial contamination. To minimize extraction from injections, rubber closures should be subjected to a treatment which will ensure that they are saturated with the preservatives to be used, before use. The B.P. (1963) recommends that the caps, which should be made of good quality compounded, natural or synthetic rubber, should, after thorough washing, be autoclaved at 10 p.s.i. for 30 minutes in a solution of the bactericide of concentration at least twice that to be used in the preparation. The closures are then stored in the autoclaved solution for at least 7 days. If sodium metabisulfite is to be used as an antioxidant in the injection formulation, 0.1% w./v. should be included in the solution in which the caps are stored. At the moment, no such treatment is indicated in the U.S.P. ( 1965). The volatilization of contents through rubber closures can be reduced by several methods (Royce and Sykes, 1957) but when possible, it it better to use one of the less volatile preservatives. In general, the B.P. (1963) recommends that a multidose container should not contain an excessive number of doses (U.S.P., 1965 limits the volume of a multidose container in most cases to 30 ml.), nor should the period of time between the withdrawal of the first and last doses be unduly prolonged. These precautions help to ensure that the period during which an injection is being used (and thus the period during which it is most liable to contamination) is not long. In certain cases, an injection may have a recommended shelf life, e.g., injection of heparin (B.P., 1963): if kept in a container sealed with a rubber closure, it may not maintain a satisfactory concentration of bactericide for more than 3 years.

E.

SELECTION OF

BACTERICIDES

1 . Desirable Properties The number of bactericides available is considerable, but those suitable for use in injectable products are limited by several factors, the most important being suitability for injection into the body, and good antibacterial activity. Guillot (1950) points out that the choice and concentration of bactericide is governed by the type and number of contaminants likely to occur, their resistance to the bactericide, the duration of contact, and the physical and chemical properties of the injection, e.g., pH, osmotic pressure. In addition, the official requirements indicate that the chosen bactericide should not interfere with

10


the therapeutic efficacy of the drug, nor cause a turbidity. Felix (1964) suggests that the ideal bactericide should be compatible with a wide range of medicaments, effective over a reasonable p H range, against a wide range of microorganisms. It should also be soluble over normal and refrigerated temperatures in the concentration used and should remain stable during the preparation and shelf life of the injection. Finally, it should not be adversely affected by the closure or container, should be nontoxic and nonirritant.

2 . Choice and Evaluation of Bactericides

The range of official bacteriostats and bactericides has already been indicated, but this does not cover all the substances used in this field. The wide range of substances used shows that, as yet, there is no substance which can be regarded as suitable for use in every injection. In the production of new formulations, it is necessary to confirm bactericidal effect after storage tests, for it cannot be assumed that a bactericide previously effective in one set of conditions will remain so in another. Moreover, studies carried out by Loosemore and Russell (1963), Russell and Loosemore (1964), Davison (1951), Klarmann (1959), and other workers on the recovery of spores after treatment by certain bactericides have shown that substances in concentrations originally thought to be bactericidal are, in actual fact, bacteriostatic, and in suitable recovery media, organisms can be recovered from supposedly “bactericidal” solutions. Thus it is apparent that tests carried out on a bactericide should ensure its effectiveness against both microorganisms and their spores. Sykes (1958) has pointed out the profound effect that concentration has upon the bactericidal properties of certain commonly used substances. Substances at even half their recommended strengths have considerably reduced or in some cases practically no lethal powers. The margin between the effective lethal concentration and the minimum inhibitory Concentration may be very narrow. Bearing in mind the readiness with which some substances are absorbed by rubber, it will be seen how quickly the originally bactericidal strength may be reduced below an effective level. Hess (1965), who emphasizes that the concentration exponent of the phenolic bactericides is very high, also draws attention to the fact that bacteriostats commonly used do not have the same degree of activity, e.g., 0.1% chlorocresol is significantly more active than 0.3%cresol. The ideal antimicrobial compound is effective against all microorganisms and should kill bacteria, molds, fungi, viruses, and also spores. However, it is manifestly impossible to test a bactericide


11

against the wide range of organisms which may contaminate an injection. The normal practice is to subject a representative sample of the batch to a test for sterility by subculturing a suitable sample into media which provide optimum growth conditions for as large a number of types of microorganism as possible. The design of sterility tests varies in different pharmacopoeias but it is interesting to note that the World Health Organization Report (1960) suggests the setting up of two tests, with the incubation of one at 35" to 37"C., and the other at 15" to 22°C. to facilitate the growth of bacteria and molds. Davies and Fishburn (1946) suggested an alternative type of sterility testthe filtration of the solution through a membrane filter, followed by subculture of the membrane. This method is especially valuable when the injection contains substances which may inhibit the growth of bacteria, as the membrane can be eashed free of any such substance. An alternative to the test for sterility used to assess the effectiveness of a bactericide is used by Rdzok et al. (1955) who described a standardized test whereby the preservative effectiveness of a formulation was challenged by the deliberate introduction of common contaminants. This method was developed by Kenny et al. (1964) who applied it to a wide range of products, including a multidose injection preserved with benzyl alcohol. It is noteworthy that while this product maintained a satisfactorily low leveI of contamination for 28 days, it was found that at the end of this period, the preservative had been completely lost, apparently via the rubber closure. It is evident that any contaminant introduced during the subsequent use of the injection could multiply without hindrance. It is difficult to determine the most likely cause of contamination of parenteral products. Contamination during preparation is always possible and the need for extreme care at this stage cannot be overemphasized. In certain cases, organisms may be introduced during storage, and the use of the oversea1 in multidose containers plays a large part in preventing this. However, hairline cracks in containers, which may arise during sterilization processes, can allow the ingress of organisms. The possibility of the introduction of Contaminants during the use of a multidose container has already been indicated and the inclusion of a bacteriostat reduces the risk of such organisms developing.

3. Bactericides and Bacteriostats in Common Use

a. Phenols and Cresols. Phenol itself has been used as a bacteriostat (strength 0.5%w./v.)

12


in injections for many years. It is accepted as a standard in the B.P. (1963) which states that, apart from the list of bacteriostats recommended, other substances may be used, provided that they have an activity not less than 0.5% w.lv. phenol in solution in water for injection. This strength solution also possesses considerable bactericidal properties, although Sykes (1958) points out that it is not rapid in action. This author also mentions that spores may survive treatment with phenol, a fact which has been confirmed by many subsequent workers. Phenol (and indeed, all phenolic bacteriostatics), has a high concentration exponent; Hess (1965) quotes in example the fact that a 1% w./v. solution of phenol kills a certain inoculum 50 times faster than a 0.5% w.lv. solution. It is fairly free from incompatibilities, although Cooper and Gunn (1965) point out that it reacts unfavorably after sterilization in injections of aneurine hydrochloride, hexamethonium, and quinine hydrochloride, and in injection of procaine penicillin. In the case of hexamethonium, the incompatibility is therapeutic in nature. Phenol also has the disadvantage that it is prone to oxidation and discoloration on storage, and that it is volatile. Royce and Sykes (1957) have demonstrated that its distribution between rubber and water is 25 : 75, a fact which makes its high concentration exponent the more significant. Acid conditions enhance its activity, as is shown by the fact that in injection of insulin, which is of p H 2.5 to 3.5, both the B.P. (1963) and the U.S.P. (1965) permit a lesser percentage of phenol as bacteriostat than is normally used. Conversely, its activity in alkaline solution is reduced or entirely lost. Finally, it has the advantage that its activity in the presence of serum is not markedly reduced. Cresol B.P., which has been deleted from the current U.S.P., is a mixture of cresols and other phenols. It is used as a bacteriostat in strengths up to 0.5% w./v. and has the virtue of ease of solubility and a wide range of activity. Cooper and Gunn (1965) mention only three cases of incompatibility (carbachol, ergometrine, and quinine hydrochloride). Cresol is of some value in immunological products, since its activity is not extensively reduced in the presence of organic matter. It is also used as a bacteriostat in certain insulin injections. In the case of isophane insulin injection, both the B.P. (1963) and the U.S.P. (1965) specify that meta-cresol should be used. Royce and Sykes (1957) state that the distribution between rubber and water is 33 : 67. It is generally found that the halogenated cresols are more active than their parent compounds and Hess (1965) states that a 0.1%w.lv. solution of chlorocresol (the recommended bacteriostatic strength in


13

the B.P. 1963) has significantly greater activity than 0.3%w./v. cresol. Chlorocresol, which is used both as bacteriostat and bactericide (0.2% w./v.) is frequently used in the sterilization of injections, despite the fact that it is incompatible with a number of medicaments. The solid form is subject to oxidation and discoloration and the resulting impurities may account for some of these incompatibilities. In addition, the bactericidal solution is virtually a semisaturated one (chlorocresol is soluble 1 in 260) and strong solutions of medicaments may cause salting out of the bactericide or even of the bacteriostat. McEwan and MacMorran (1947) have pointed out that strong solutions of sodium chloride cause the precipitation of chlorocresol, although it has also been suggested that low concentrations of sodium chloride will potentiate the action of chlorocresol. Chlorocresol is volatile, and is preferentially dissolved in rubber to a high degree, the distribution ratio between rubber and water being 85: 15 (Royce and Sykes, 1957). Davies and Davison (1947) used a filtration technique in the study of injection contamination and found spores of B . subtilis survived the B.P. process of heating with a bactericide using 0.2% w./v. chlorocresol. However, the degree of contamination used in the study (5000 spores/ml.) is not normally met with in injection, which should always be prepared in clean conditions. Another bacteriostat whose use in the preservation of injections is indicated in the U.S.P. (1965) and the B.P. (1958) is chlorbutol 0.5% w./v. Hopkins (1960) points out that chlorbutol is compatible with a wide range of drugs, and that it has some analgesic properties. Up to 90%may be absorbed by rubber vaccine caps (Royce and Sykes, 1957). It is volatile and difficult to dissolve. Its main disadvantage lies in the fact that it hydrolyzes rapidly above p H 3, the hydrochloric acid released in the reaction lowering the pH and causing loss of bactericidal activity. In neutral and alkaline solutions it is ineffective (Felix, 1964; Nair and Lach, 1959).

b. Mercurial s The most commonly used of the mercurial compounds are the phenyl mercuric salts, phenyl mercuric nitrate and phenyl mercuric acetate being the general choice, although the I.P. also permits the use of the borate. The main difference in these salts lies in their solubility, the nitrate being relatively insoluble (1 in 1500 at room temperature) while the acetate and borate are more soluble. Hess (1965) points out that the salts are more soluble in alkaline than in acid solution but Hopkins (1960) indicates that the changes in p H do

14


not significantly affect activity. Phenyl mercuric nitrate is official (B.P., 1963) as a bacteriostat (0.001%w./v.) and bactericide (0.002% w./v.). One of the virtues of these compounds is that they have a low concentration exponent and are not readily diluted out, but unfortunately, as demonstrated by Royce and Sykes (1957), up to 95% may be absorbed by rubber vaccine caps. For this reason, these authors do not recommend the use of phenylmercuric salts as bactericides or bacteriostats in multidose containers. Hess (1965) also states that the mercurials may react with certain types of rubber, and numerous instances of incompatibilities may be found in the literature (Hadgraft and Short, 1947); McEwan and MacMorran, 1947).Hopkins (1960) states that phenylmercuric acetate is a satisfactory preservative for antigens. Thiomerosal (thiomersal or Merthiolate) is also used in certain kinds of injections, mainly biological products, and further reference to it will be found under that section. It has both bacteriostatic and fungistatic properties and is used in strengths ranging from 0.01% w.lv. to 0.02%w.lv.

c . Other Substances Alchols also possess bactericidal properties, and of these, the one most commonly used in injections is benzyl alcohol. It has bacteriostatic rather than bactericidal properties and in addition, has some local anesthetic effect. It is included in compound injection of benzocaine, B.P.C. (1954) (5% w./v.), and injection of ethanolamine oleate, B.P. (1963) (2% w./v.). P-phenylethyl alcohol in strengths of 0.7 to 1.5%w.lv. is also bacteriostatic, but its greater use lies in the field of ophthalmic preparations. A 0.1% w./v. solution of benzoic acid has both bacteriostatic and bactericidal properties, but above pH 5, only the bacteriostatic property is retained. Due to this difficulty, the acid is not suitable for use in injections, but the esters of the hydroxylated acid (methyl p hydroxybenzoate and propyl p-hydroxybenzoate) have been used for their bacteriostatic properties in insulin zinc suspensions. In general, however, the esters are not used in injections. The quaternary ammonium compounds are useful in the formulation of injection suspensions, e.g., procaine penicillin injection. Being surface-active agents and having bactericidal properties, they serve a dual purpose, but care must be taken to avoid any incompatibility. From what has been written, it can be seen that, at present, there is no ideal bactericidal substance which can be used in all injections;

ANTIMICROBIAL AGENTS I N PHARMACEUTICALS

15

many of the bactericides in use, of which by no means all have been discussed, present problems of incompatibility, instability, absorption b y rubber, and other difficulties. It is difficult to foresee the development of the ideal bactericide and so pharmacists have to apply their knowledge to the improvement of substances at present in use. I I I. lmmu no1og ica I Products A. TYPESOF IMMUNITY Immunity may be defined as the resistance presented b y the host to an infecting organism. Without taking account of racial and species immunity, it is conveniently subdivided into:

1 . Passive Immunity a. Natural Immunity This is brought about by the passage of antibodies from mother to child.

b. Arti$cial Acquired Immunity This type of immunity involves no work on the part of the body defense mechanisms, since antibody is produced in another animal, usually the horse. Immunity to a specific organism is immediate, but is short-lasting. Products which confer this type of immunity are antibacterial, antitoxic, and antiviral sera, the last-named including sometimes the gamma-globulins.

2. Active Zmmunity a. Natural Zmmunity

This is exemplified by cases where a single attack of a disease confers increased resistance against a second attack.

b. Arti$cial Acquired Immunity An antigen is administered with the specific intention of inducing an antibody response in the recipient. Immunity is slow to develop, but is relatively long-lasting, and may easily be restored. The antigen may consist of living or dead bacteria, rarely of toxins, of toxoids, of living or inactivated virus particles, and of dead rickettsiae. A further group of products consists of diagnostic reagents. This group includes the tuberculins, and the Schick and Dick test toxins and their controls.

16

A. D.

RUSSELL, J. JENKINS,

AND 1. H. HARRISON

B. PRODUCTS REQUIRINGA PRESERVATIVE

The U.S.P. (1965) emphasizes that the choice of antibacterial agents for use in immunological products requires especial care. It is desirable that such an agent should suppress microbial growth, be compatible with the product, b e stable at normal temperatures, and b e nontoxic; in addition, there should be relative freedom from inactivation of the therapeutic principle, and no absorption into the rubber used in multiple-dose containers. A substance that is sometimes used for preservation of some vaccines and other biological preparations is thiomersal (thimerosal, sodium ethylmercurithiosalicylate, Merthiolate); however, it has been found that its concentration diminishes with storage, presumably due to its absorption by rubber (Birner and Garnet, 1964a) and not to the presence of zinc released from the rubber (Birner and Garnet, 1964b). Nevertheless, thiomersal occupies an important place in the preservation of immunological products. The products that require the addition of a preservative are: 1 . Antisera a . Antibacterial Sera These are prepared against certain bacteria which do not produce exotoxins, e.g., Leptospira antisera.

b . Antitoxic sera These sera are prepared against specific exotoxins, which are first converted into toxoids, by means of formaldehyde. The toxoids are injected into horses and the blood collected and processed. c. Antiviral Sera Apart from rabies antiserum, which is obtained from animals injected with the specific virus, antiviral sera are usually obtained from human convalescents, from adults who have had the disease in the past, or from persons who have been artificially immunized. Antisera exist in liquid form. A suitable antibacterial substance may be included; this is essential when antisera are present in multidose containers. No preservative is specifically named, but phenol is suitable.

2 . Vaccines a . Bacterial Vaccines Vaccines are defined (B.P., 1963) as being suspensions of living


17

organisms (bacteria, rickettsiae, or viruses), sterile suspensions of the organisms, or toxoids. Thus, toxoids are included in the official definition of vaccine. For convenience, however, toxoids are here considered separately. Two factors that need to be considered in preparing killed bacterial vaccines are the method employed in killing the bacteria, and the choice of a preservative. In neither case must damage occur to the antigenic potency (antigenic identity) of the organism. Cohen and Wheeler (1946), for example, described a method of preparing pertussis vaccine from Phase I cultures in which the organisms were killed with 0.01% w.lv. thiomersal, which was also present as a preservative in the final vaccine. This had no deleterious effect on the antigenic identity of Bordetella pertussis. More recently, Gardner and Pittman (1965) investigated the stability of pertussis vaccine in the presence of different preservatives, and showed that the potency of the vaccines preserved with 0.01%w./v. thiomersal was more stable than that of vaccines preserved with 0.0025% w.lv. benzethonium chloride (Phemerol) or a mixture of 0.15% w./v. and 0.02% w./v., respectively, of methyl and propyl p-hydroxybenzoates. The potency of vaccines containing no preservative was more stable than those containing benzethonium or parabens, but less stable than those containing thiomersal. Olson et aZ. (1964) had just previously shown that the saturation of negative sites on the pertussis cell with cations prior to the addition of benzethonium chloride prevented its uptake b y the cell and stabilized the antigenic potency of the vaccine. The histamine-sensitizing factor is also unstable in the presence of benzethonium chloride or the parabens. Formaldehyde, also, has an adverse effect on the potency of pertussis vaccine (Pittman and Cox, 1965). Further applications of these findings are considered in Section 111,2,d. At present, whole cells of B. pertussis are used in the preparation of vaccines. However, it has been found (see, e.g., Sutherland, 1963) that cell wall material from B. pertussis, prepared by Mickle disruption followed by purification by various chemical treatments, is a good protective agent. van Hemert et al. (1964) considered that chemical solubilization of the outer layers of the cell was a better approach in isolating the cell fraction responsible for immunizing man against whooping cough. This type of research opens up interesting possibilities for the future; at present, however, there is no information available as to a suitable preservative for this type of product, although it is not unreasonable to suggest that thiomersal would be of use.

18


The preparation of cholera vaccine has been described by Dick (1959) and by the World Health Organization (1959b), which points out that considerable differences occur in various countries in the choice of strains, their virulence, method of killing, and use of preservatives. It further stipulates that a suitable preservative be incorporated into cholera vaccines issued in multiple-dose containers and that such a preservative “shall have been demonstrated, to the satisfaction of the national control laboratory, not to affect the antigenicity and safety of the vaccine.” A suitable preservative appears to be 0.5%phenol (Dick, 1959). Typhoid-paratyphoid A and B vaccine (TAB vaccine) consists of Salmonella typhi and S . paratyphi A and B. In addition to the 0 and H antigens, it contains the Vi antigen in the case of S . typhi. Typhoidparatyphoid A, B, and C vaccine (TABC vaccine) consists of s. typhi and S . paratyphi A, B, and C. In addition to the 0 and H antigens, it contains the Vi antigen in the case of S . typhi and S . paratyphi C . The vaccines may be either alcohol-killed alcohol-preserved, or heatkilled phenol-preserved (phenolized). It had been found by Felix (1941) that the former type of vaccine conferred greater protection on mice, and that phenol tended to destroy the Vi antigen. However, alcoholized vaccines were later shown to be disappointing, since the protection obtained in the mouse was not reproduced in man (Parish and Cannon, 1963, 1964; Parish, 1965). Controlled clinical trials later confirmed that the phenolized vaccine gave better protection than the alcoholized one (Parish and Cannon, 1963), thus indicating that the importance of the Vi antigen was less than originally thought. In fact, Morgan (1958) has stressed that it had not been demonstrated that it played an essential role in protection. The U.S.P. (1960) lists 0.5% phenol or 0.4% cresol as a suitable preservative for TAB vaccine. An interesting development in the prevention of typhoid would appear to concern the finding that glycine-induced spheroplasts of S . typhi possessed a far higher immunogenic ability, and much lower toxicity, than intact cells (Diena et al., 1964). Difficulties of storing the spheroplasts for long periods could be discounted, since lysed spheroplast suspensions were as efficient as intact spheroplasts in conferring protection in mice. No indication of a suitable preservative for use in a multidose container is given, but 0.02% w./v. thiomersal was used to kill the spheroplasts, so that in the event of spheroplasts or their lysates becoming an accepted method of vaccine preparation, it is conceivable that this antibacterial agent would be investigated for its effect over longer periods.


19

b. Toxoids Gram-positive bacteria generally produce the so-called “soluble” toxins, or exotoxins, whereas most Gram-negative bacteria produce endotoxin (see Luderitz et al., 1966). Exotoxins, but not endotoxins, can be toxoided by means of formaldehyde to give the group of immunological products known as toxoids. An example of such products is diphtheria vaccine, which occurs in five different forms: formol toxoid, toxoid-antitoxin floccules, alumprecipitated toxoid, purified toxoid aluminum phosphate, and purified toxoid aluminum hydroxide. With the last three, phenols and cresols adversely affect the antigenic potency (B.P., 1963), and a suitable preservative is thiomersal. Phenol (0.5% w./v.) may be used as a preservative for the toxoid-antitoxin floccules.

c. Viral Vaccines These vaccines may be subdivided into living vaccines, e.g., poliomyelitis (Sabin) and smallpox vaccines, and inactivated vaccines, e.g., poliomyelitis (Salk) vaccine. Pivnick et al. (1963) and Tracy et al. (1964) have studied the use of differentpreservatives in polio (Salk) vaccine. Several antimicrobial agents may be present in this vaccine, e.g., streptomycin, neomycin, and polymycin may be employed to inhibit bacterial contamination of tissue culture (in Britain, penicillin and streptomycin are sometimes, but not necessarily, used); formaldehyde is added as a virucidal agent, although it is normally neutralized by sodium metabisulfite before the blending of individual components into the trivalent vaccine; and preservatives added to the finished vaccine. The World Health Organization (1959a) states that any preservative incorporated must have no deleterious effect on the product. Benzethonium chloride is a suitable agent (McLean, 1957; Schuchardt et al., 1960)and it has been recommended that it should preferably be used in vaccines to which sodium metabisulfite had not been added. Stable antibiotics contribute considerable antibacterial activity, and formaldehyde (if not neutralized) is active against fungi as well as bacteria (Pivnick et al., 1963). However, Pivnick et al. (1963) considered that benzethonium added little if any antibacterial activity, but did have some antifungal activity. The parabens are not harmful to poliomyelitis vaccine, and their addition to inactivated vaccine containing nonneutralized formaldehyde was found to give a mixture of preservatives inhibitory against high numbers of both bacterial or fungal contaminants.

20


Mercurials appear to be deleterious to the potency of inactivated polio vaccine (Gardner and Pittman, 1965) and thus cannot normally be employed as preservatives in this case. However, Davisson et al. (1956) have shown that thiomersal may be used as such a preservative when ethylenediaminetetraacetic acid (EDTA) is present. The chelating agent protects the thiomersal from breakdown to products which affect the antigenicity of the vaccine. Since polio viruses are labile agents, oral (living) vaccines must be handled carefully to prevent inactivation of virus, and a consequent loss of vaccine potency. Sabin vaccine stabilized with magnesium chloride was found by Melnick and Wallis (1964) to retain its potency for much greater periods than control vaccines (MgC12 absent). In addition to protecting the live virus, the concentration of magnesium salt used, 1to 2 M , was inhibitory to most bacteria and fungi. Smallpox vaccine is prepared by growing the virus (i) in the skin of living animals, (ii) in the chick embryo, or (iii) in tissue culture. It is a living vaccine; however, vaccine prepared by method (i) is likely to contain extraneous microorganisms, which must be reduced to < 10OO/ml. (also certain specific bacteria must be absent). If this vaccine is issued in liquid form, treatment consists of the addition of glycerol with or without another antibacterial substance, e.g., 1% w./v. phenol. In vaccines prepared by methods (ii) and (iii), extraneous organisms will be absent, but the World Health Organization (1959~) recommends the addition of glycerol with or without an antibacterial substance as a precaution against later contamination. Smallpox vaccine (liquid) is issued in single- or multidose containers. Each container of dry vaccine should be issued with an ampoule of sterile reconstituting fluid, which may contain glycerol with or without an antibacterial substance. A suitable antibacterial preservative for this (in addition to glycerol, which itself possesses bacteriostatic properties) is 0.5% w./v. phenol. Antibiotics may be used in the preparation of smallpox vaccine b y method (iii) but the World Health Organization ( 1 9 5 9 ~ )recommends that the use of antibiotics as additives to smallpox vaccine should be discouraged. The U.S.P. (1965) proposes that smallpox vaccine contains 40 to 60% glycerol (or sorbitol) with not more than 0.5% w./v. phenol as a preservative. Glycerol itself has been used as a preservative for over a century; its advantages, e.g., valuable bacteriostatic properties, good dispersing agent, and its disadvantages, e.g., fairly rapid rate of inactivation of virus particles, have been considered by Amies (1962), who has described an improved smallpox vaccine containing 0.4% w./v. phenol as preservative, except when the vaccine is lyophilized.


21

Other viral vaccines, e.g., influenza and rabies vaccines, must contain a suitable preservative. The U.S.P. (1960) stipulates in the case of influenza vaccine that if formaldehyde is used for inactivation, the finished vaccine contains > 0.02% w./v. free formaldehyde. Thiomersal, 0.013%, is used in Britain as a preservative in influenza (inactivated) vaccine.

d . Combined Prophylactics Diphtheria, tetanus, and pertussis vaccine (DTP/Vac) has, in recent years, been combined with polio vaccine (inactivated) to give a “quadruple antigen” vaccine, DTPP. Pittman (1962) and Edsall et al. (1962) have independently found that in this combination, the pertussis vaccine was unstable. In addition to containing the poliomyelitis component, DTPP differed from DTP in having a different preservative. Thiomersal has been used as a preservative in pertussis vaccine, and in DTP. However, mercurials are deleterious to the potency of polio vaccine (inactivated) and thus when the conditions in DTP were adjusted so as to be optimal for the less stable polio vaccine, the pertussis component became unstable. (Gardner and Pittman, 1965). McLean (1957) and Schuchardt et al. (1960) showed that benzethonium was not harmful to polio vaccine, and benzethonium thus replaced thiomersal as preservative when DTP was combined with polio vaccine to form DTPP. It has also been found, however, that benzethonium chloride causes a loss of potency of the pertussis vaccine component (Gardner and Pittman, 1965; Olson et al., 1964) and was thus responsible for the instability of pertussis in DTPP. Likewise, the parabens, which do not reduce the antigenic potency of polio vaccine, could not be used as a preservative in DTPP, as in their presence, the pertussis component was again unstable (Gardner and Pittman, 1965). Other combinations of pertussis, e.g., pertussis and tetanus vaccine, and pertussis and diphtheria vaccine, may all be preserved with thiomersal. Phenol (0.5%w./v.) is a suitable preservative for typhoidparatyphoid A and B and tetanus vaccine, and for typhoid-paratyphoid A and B and cholera vaccine.

3. Diagnostic Reagents These reagents include Dick and Schick test toxins (and their controls), old tuberculin and old tuberculin, purified protein derivative (PPD). PPD contains 0.5% phenol as a preservative (B.P., 1963; Landi, 1963). When used in the Mantoux test, this stock solution is diluted

22


to contain graded amounts. The potency of diluted PPD solutions is, however, reduced by phenol (see Pivnick et al., 1965), and a diluent for PPD that contained 8-hydroxyquinoline sulfate was devised by Magnusson et al. (1958).However, because of reports that 8-hydroxyquinoline was ineffective as a preservative in PPD, Pivnick et al. (1965) reinvestigated its antimicrobial activity, and found that it was an effective preservative against contamination of PPD with certain yeasts and molds, and that it had a bactericidal action against Pseudornonas aeruginosa provided that not more than 100 viable cellsiml. were present. Although results of its activity against Staphylococcus uureus are not clear-cut, other work has shown that low concentrations of 8-hydroxyquinoline inhibit the growth of strains of this organism. Old tuberculin contains, as a result of its preparation, a high concentration of glycerol, which itself exerts a bacteriostatic effect. In diluted preparations, which are stable for 3 months, phenol or thiomersal may be employed as a preservative. IV. Eye Drops

A. PREPARATION AND STERILIZATION It is widely agreed that, at the time of dispensing, eye drops should b e sterile. The B.P.C. (1963), U.S.P. (1965), and U.S.N.F. (1960) list methods for preparing and sterilizing eye drops. More recently, the B.P.C. Addendum (1966)has made important and far-reaching amendments to these methods as a result of further work on the part of research groups both in Britain and abroad. In addition, eye drops need no longer be made isotonic with the lachrymal secretions. Basically, the methods of preparation and sterilization listed by the B.P.C. Addendum (1966) are as follows: the medicament is dissolved in the aqueous vehicle containing one of the prescribed antimicrobial substances, and the solution is sterilized by heating in an autoclave at 115"C.,by heating at 98" to 10o"C.,or by filtration through a bacteriaproof filter, the actual method depending to a great extent on the stability to heat of the active constituent. In the case of oily e y e drops, an aseptic technique is used. As is the case with the rubber caps used in multidose injection containers (see Section 11, D2), the rubber teats used in eye-drop bottles may absorb antimicrobial substances from the solutions, and they must thus be impregnated with the selected agent, with which they must be compatible.


23

0. PRESERVATIVES

1. The Ideal Preservative The B.P.C. Addendum (1966) stipulates that aqueous eye drops must contain a vehicle that is bactericidal and fungicidal, and lists the following as suitable substances: 0.002% w./v. phenylmercuric nitrate (PMN), 0.002% w./v. phenylmercuric acetate (PMA), 0.01% w./v. benzalkonium chloride, and 0.01% w./v. chlorhexidine acetate. These replace the parabens which were used in many B.P.C. (1959) eye-drop formulations, then deleted from, but later brought back to, several B.P.C. (1963)formulations. The ideal antimicrobial compound for use in eye drops should have the following properties (based in part on Foster, 1965): (i) possess a wide antimicrobial spectrum, (ii) be compatible with the active medicament, (iii) be nontoxic and nonirritant, (iv) be stable to heat, moisture, and on storage, (v) show little or no absorption into rubber teats. To these may be added: (vi) a sterilizing time of < 1 hour, since Kohn et al. (1963b) propose that an antibacterial substance that has a sterilizing time of > 1 hour may be arbitrarily considered too slowacting for use as a preservative in multidose ophthalmic solutions; (vii) no reduction of activity due to composition of the container: as recently pointed out (Editorial, 1966), the use of plastic containers is still limited to some extent b y incomplete information on interactions between drugs, preservatives, and plastics; (viii) the antimicrobial compound should not significantly alter the pH and toxicity of ophthalmic solutions (Lawrence, 1955a). The ideal antimicrobial agent for use in eye drops has not yet been discovered, however (Hopkins, 1966),and the search for such a substance continues. The U.S.P. (1965)and the U.S.N.F. (1960)have stated that eye-drop preparations for use in surgical procedures on an injured eye should not contain an added antimicrobial agent, since this may be irritating to tissues lining the anterior chamber and should be in single-dose containers. Ophthalmic solutions may be packed in multiple-dose containers when intended for use on eyes with intact corneal membranes, and the most frequently used preservative is 0.01% w./v. benzalkonium chloride. Other preservatives that may be used are 0.5% w./v. chlorbutanol, 0.5% w./v. phenylethyl alcohol, 0.05%w./v. chlorocresol, and PMN at a concentration of 0.004%(U.S.N.F., 1960) or 0.001% (U.S.P., 1965). The Australian National Formulary (A.N.F.) requires all eye drops to be sterile, and states that 0.01%w./v. chlor-

24


hexidine is a suitable bactericide; in cases where difficulty of formulation is experienced, this may be replaced by 0.5% w./v. chlorbutanol. These and other substances which have been, or are, used as antimicrobial substances in e y e drops are considered in more detail below. A major difficulty in their use is presented by the abnormally high resistance of Pseudornonas aeruginosa (P. pyocyanea) to chemical substances; furthermore, some species of B . subtilis and C. welchii are implicated as eye pathogens (Brown and Norton, 1965; see also Beloian and Koski, 1964).

2 . Substances in Use a . Parebens Hugo and Foster (1964a) found that P . aeruginosa NCTC 7244, a strain originally isolated from an infected human eye, would grow readily without previous adaptation in solutions of esters of p-hydroxybenzoic acid, with or without mineral supplementation. The concentrations of esters conformed to those (methyl, 0.02%w./v. propyl, 0.01% w./v.) used in solution for eye drops (B.P.C., 1959). This paper was criticized by Montgomery and Halsall (1964a,b) who pointed out that no variation in ester concentration had been made, that the manufacturers of the esters recommended a total ester concentration of at least O.OS%, and that in their personal experience this concentration was at the least bacteriostatic and probably bactericidal against P . aeruginosa. These findings were confirmed by McIver (1964) who also found 0.08% w./v. Nipasept (a mixture of methyl, ethyl, and propyl p-hydroxybenzoates) to be effective. These criticisms were answered by Foster (1964a,b) who stated that a 0.08% (Me:Pr ratio of 2 : 1) total ester concentration was just bacteriostatic for P.aeruginosa, and that a total concentration of 0.2% w./v. was needed for a bactericidal action within 30 minutes at room temperature; at this concentration, the esters were too irritant. Confirmation of these results was later produced (Hugo and Foster, 1964b). Other workers have also produced evidence to show that the parahens are virtually inactive against P . aeruginosa (Lawrence, l955a; Kohn et at., 1963a; Brown et al., 1964).

+

b. Chlorocresol This replaced solution for eye drops in the B.P.C. (1963), but before this became official, chlorocresol was deleted as an eye drop preservative, and solution for eye drops reinstated. The reasons for this apparently hasty action have been described (Editorial, 1963): a


25

report was received to the effect that a solution of normal saline containing 0.1% w.lv. chlorocresol had caused damage to the corneal epithelium when used in a series of operations involving the anterior chamber of the eye, and the Faculty of Ophthalmologists requested that action be taken to find an alternative substance to maintain the sterility of eye drops. However, chlorocresol is not unduly irritant to intact noninflamed eyes, and is known to be an effective antibacterial agent. Furthermore, it has been used for some years in America as a preservative in e y e drops intended to be used on eyes with intact corneal membranes, and it is thus reasonable to propose that the possibility of using it for this purpose in Britain could well be reinvestigated,

c. Mercury Compounds The three mercury compounds that may be used as eye-drop preservatives are PMN, PMA, and thiomersal. There is conflicting information about the activity of PMN against P . aeruginosa, e.g., Lawrence (1955a) found that PMN was considerably more active than thiomersal in its effects against various strains of P . aeruginosa, but Kohn et al. (1963a) showed that thiomersal and PMN individually required 6 hours to exert a bactericidal effect. Anderson et al. (1964a) found that, in fluorescein eye-drop preparations, PMN failed to inhibit P . aeruginosa in one formulation, and both Pseudomonas and Proteus sp. in another, but a concentration of 0.004% w.lv. PMN was effective in a third formulation. Differences in technique could account for some of the discrepancies noted by various authors in the effectiveness of various antimicrobial agents (Brown and Norton, 1965), e.g., in the use of a suitable inactivator or neutralizing agent (Russell, 1964). It has been shown that organic mercurials were less active in the presence of active medicaments of eye drops than in their absence (Lawrence, 1955a) and Kohn et al. (1963a) did not recommend mercury compounds as being suitable preservatives. Also, although PMN is bactericidal in low concentrations against vegetative organisms, it is only slowly sporicidal (see review by Russell, 1965). However, the U.S.N.F. (1960) recommends its use at a concentration of 0.004%. Foster (1965) considers that thiomersal may prove to be a suitable eye-drop preservative; some of the above findings may suggest otherwise. With regard to the toxicity of organic mercury compounds, Abrams (1963) showed that mercurialentis (deposition of mercury on the lens) may result from exposure of the e y e to 0.004% w.lv. PMN over

26


a long period of time; this did not occur with thiomersal (Abrams et al., 1965). Foster (1965), however, has stated that mercurialentis is a rare event, may be innocuous, and should not be allowed to detract from the usefulness of PMN.

d . Benzalkonium Chloride Various authors have shown that benzalkonium chloride is an effective preservative in eye-drop formulations. Thus, Lawrence (1955a,b) found it to be the most effective of seven commonly used chemicals in destroying Pseudomonas and Proteus contaminants. Benzalkonium is a quaternary ammonium compound, and possesses lower activity against Gram-negative than against Gram-positive bacteria. Also, the activity of quaternary ammonium compounds may be reduced in the presence of certain metallic ions. The chelating agent, EDTA, has been found to reduce the resistance of P . ueruginosa to a quaternary ammonium compound (MacGregor and Elliker, 1958), and recently it has been shown that the activity of polymyxin B sulfate, chlorhexidine diacetate, and benzalkonium chloride against P . aeruginosa NCTC 8203 was substantially increased in the presence of EDTA (Brown and Richards, 1965). This potentiating action was blocked by Mg++ and Ca++,and it was postulated that EDTA was synergistic with these antibacterial agents by a mechanism involving removal of Ca++ or Mg++ions or both from the bacterial cytoplasmic membrane. This potentiation by EDTA finds application in eye drops of prednisolone sodium phosphate (B.P.C., 1963); also the U.S.N.F. (1960) states that resistant strains of P . aeruginosa are made more sensitive to benzalkonium in the presence of 0.01 to 0.1% EDTA. I n addition, a combination of 0.01% w./v. benzalkonium and 1000 USP units/ml. of polymyxin B sulfate may be effective against resistant strains of this organism and yet be sufficiently nonirritating to eye tissues (USP, 1965). A concentration of 0.02% w./v. is rapidly bactericidal against some strains of P . aeruginosa (Kohn et al., 1963a); Foster (1965) considers this concentration to be rather high, and the B.P.C. Addendum (1966) and the U.S.P. (1965), in fact, recommend a concentration of 0.01% w./v. Thus, the evidence in favor of benzalkonium chloride being the most reliable of the preservatives available (Brown et al., 1964) appears to be overwhelming. However, some dissension from this has been voiced by Anderson et al. (1964b), who find benzalkonium to be almost inactive against P . aeruginosa, and who quote the results obtained by Riegelman et al. (1956) in support of their claims; how-


27

ever, it would appear from the work of Kohn et al. (1963a) that Riegelman’s findings have now been disproved. The question of whether the quaternary ammonium compounds are toxic to the e y e has led to some confusion. Klein et al. (1954) in an otherwise interesting paper, misquoted the results of Ginsberg and Robson (1949) and in consequence reported that these substances could prove harmful by solubilizing the intercellular cement of the corneal epithelium. In fact, Ginsberg and Robson did not use a quaternary ammonium compound, and the quaternary ammonium compounds (including high concentrations of benzalkonium chloride) do not cause loss of intercellular cohesion (Buschke, 1949). Benzalkonium chloride suffers from the disadvantage that some rubber teats may be incompatible with it, and the B.P.C. Addendum (1966) thus recommends the use of silicone rubber teats for those eye drops in which this antimicrobial substance is included.

e. Chlorhexidine Anderson et al. (1964a) found that chlorhexidine (bis-p-chlorophenyldiguanohexane) was an efficient bacteriostatic agent in 69 out of 75 eye-drop formulations tested. Later (Anderson et al., 1964b), they again claimed its superiority as an eye-drop preservative over benzalkonium and cetrimide. The A.N.F. use chlorhexidine as such a preservative, and it has recently been advocated for this purpose in the B.P.C. Addendum (1966). Unfortunately, chlorhexidine is incompatible with some active medicaments of e y e drops, such as borates, phosphates, sulfates, fluorescein, and physostigmine. Chlorhexidine is not listed by the U.S.P. (1965) as being a suitable eyedrop preservative.

f. Chlorbutanol This is usually employed at a 0.5% w./v. concentration as a preservative in e y e drops. Provided that it is permitted to act upon the organisms for a sufficient length of time (several hours), it is effective in destroying Pseudomonas and Proteus cultures (Lawrence, 1955a,b; Kohn et al., 1963a). It is thus doubtful whether chlorbutanol has any significant value in eye-drop formulations. In addition, it hydrolyzes to hydrochloric acid, thus causing a decrease in pH, which occurs rapidly during heating and slowly at room temperature.

3. Conclusions At present, no one substance fills all the requirements of the ideal eye-drop preservative. As to the future, it might be more rewarding to

28


attempt to understand the nature of the resistance of P . aeruginosa to chemical inactivation rather than to investigate new agents (Brown and Norton, 1965).Certainly the work to date on the effects of EDTA and of Tween 80 (Richards and Brown, 1964) in reducing the resistance of P . aeruginosa to chemical substances is a start in this direction. V. Medicines for Oral Administration

A. INTRODUCTION Liquid medicines for oral use are dispersions of the therapeutically active drug(s), either solid or liquid, in a liquid which is called the vehicle. The vehicle is usually aqueous, either water itself or an aqueous solution of a flavoring agent, etc. Some vehicles possess therapeutic properties while others are pharmacologically inert. The dispersion may be a solution, a suspension, or an emulsion. The medicines are of several types and often have names indicative of their particular use or formulation, e.g., a “linctus” is a thick syrupy product intended to be sipped and swallowed slowly so as to exert a soothing effect on the lining of the throat; and “elixir” is a sweetened and flavored preparation containing a potent or nauseating drug and may contain a high proportion of ethanol, glycerol, or propylene glycol. The substances used in liquid medicines may be incorporated as such, e.g., sodium bicarbonate, morphine sulfate, or may be incorporated in the form of a “galenical” or other preparation. A galenical is a preparation made by macerating or percolating a crude drug with a solvent, e.g., ethanol, of an appropriate strength. The object is to extract the active principles of the drug as completely as possible while leaving behind the unwanted substances. Among the most common galenicals are tinctures (these are alcoholic preparations of vegetable or animal drugs containing rather low concentrations of active principles); and fluid extracts (these are similar to tinctures but contain high concentrations of active ingredients; usually 1 ml. of fluid extract contains the active principles from 1 gm. of drug). Other preparations of drugs used to make medicines include syrups (concentrated solutions of sucrose containing therapeutic or flavoring substances), spirits (alcoholic solutions of volatile substances), and solutions (aqueous solutions of solids, liquids, or gases, often containing sufficient ethanol to inhibit microbial growth). The galenicals and other preparations per se are usually resistant to microbial growth, either because of their ethanol content, or their high osmotic pres-


29

sure due to their sucrose concentrations (syrup). However, on dilution with water they frequently become ideal media for microbial growth. The addition of a relatively small amount of water may make all the difference; the B.P.C. 1963 states that solutions of sucrose containing less than 65% w.lw. will ferment, whereas those containing 66.7% w.lw. seldom permit microbial growth. The simplest mixture that could be made would consist of a solution or suspension of a solid drug in water. In many cases such a preparation would not support microbial growth and hence would not require the addition of a preservative. Certain drugs in aqueous solution, however, will support microbial growth; cocaine hydrochloride is an example (Extra Pharmacopoeia, 1958). Few mixtures consist solely of a drug and water since it is frequently necessary to include other substances to make a stable and palatable product. The pH of a mixture may have to be adjusted so as to achieve optimal stability of the drug or to obtain its optimal therapeutic effect; the resulting product may encourage microbial growth. If the drug has an unpleasant taste, it is usual to disguise this by the addition of suitable flavoring and/or sweetening agents. Probably the most commonly used sweetening agents used in pharmaceutical products are sucrose, sorbitol (especially suitable for preparations for diabetics), glycerol, and propylene glycol. Barr and Tice (1957a) studied the inhibition of growth of several bacteria ( S . aureus, B . subtilis, P . aeruginosa) and molds (Aspergillus niger, P . notatum, and Monilia (Candida) albicans) in aqueous solutions of either glycerol or propylene glycol containing various concentrations of sorbitol. Of the microorganisms studied, M . albicans was the most resistant but its growth was inhibited by 50% w.lv. glycerol and by 30% w.lv. propylene glycol. When more than 10% w./v. sorbitol was present, the inhibitory concentrations of glycerol and propylene glycol were decreased. Barr and Tice attributed the inhibition to the osmotic pressure of the solutions. In a subsequent paper (Barr and Tice, 1957b) they reported on the inhibitory effects of other sugars, including dextrose, levulose, and invert sugar. They found that 60% w./w. invert sugar solution inhibited Aspergillus niger, but solutions containing 50% w./w. of either levulose or dextrose did not. Stable solutions of these last two substances containing more than 55% w.lw. could not be made. It is evident, therefore, that unless mixtures are made in almost pure syrup, or contain very high concentrations of glycerol or propylene glycol, the presence of a preservative in sweetened aqueous preparations is essential.

30


The physical stability of many suspensions and emulsions is enhanced by the presence of suspending and/or emu1sifying agents. Some of these agents, e.g., acacia, tragacanth, are carbohydrates and are liable to microbial attack. Various cellulose derivatives, e.g., methyl cellulose, are also used as suspending or emulsifying agents. Although these were formerly believed to be resistant to microbial growth (Mueller and Deardorff, 1956), there is now evidence that under certain conditions suitable microorganisms do attack them and destroy their pharmaceutical usefulness (Brown, 1961). Moreover, methyl cellulose has been shown to form complexes with a number of common preservatives, notably the parabens (Tillman and Kuramoto, 1957). The synthetic nonionic surface-active agents of the fatty acid ester type (e.g., sorbitan esters and their polyoxyethylene derivatives) are also widely used in pharmaceutical products as emulsifying or solubilizing agents. Barr and Tice ( 1 9 5 7 ~showed ) that various microorganisms, e.g., P. aeroginosa, A. niger, P. notatum, and M . albicans, could grow in solutions and dispersions of such substances and produce esterases which split the ester linkages. The adverse effects of this splitting of the surface-active molecules on the physical properties of the product are obvious as is the need for the inclusion of an effective preservative. In another paper Barr and Tice (1957d) described the results obtained when they screened over 50 antimicrobial substances and combinations for effectiveness in preserving both solutions of and preparations containing these nonionic agents. Among the effective preservatives were sorbic acid (0.1 to 0.2%), the phenylinercuric salts (about 0.01%), hexylene glycol (3.0%),and benzalkonium chloride (0.1%).The phenols were ineffective, probably due to complexation with the surface-active agent. As to whether a particular formulation will, or will not, support microbial growth, only thorough testing in a laboratory will show. However, by a suitable choice of ingredients, galenicals, etc., it may be possible to achieve the desired result without adding an antibacterial agent as such. This is the preferred procedure because it avoids the possibility of interaction between the antibacterial and the drugs, etc., present in the mixture. The use of a pleasantly flavored tincture in sufficient quantity to provide a final ethanol content of about 15% should prevent microbial attack especially if the pH is low (Gabel, 1921). Another important factor is the length of time the preparation is required to “keep.” Medicines made extemporaneously for a given patient are usually consumed within a few days, and preservation of


31

these is relatively simple. The problem becomes more difficult when a manufacturer wishes to prepare a product having a shelf life of perhaps 2 years. The storage conditions required by the drug are also important. Some drugs are only stable in mixtures when refrigerated; such mixtures would not require additional preservatives. The desirable properties of an antimicrobial for use in oral preparations include freedom from an unpleasant taste and smell, in addition to the usual pharmaceutical requirements of compatibility, absence of toxicity, etc.

B.

PRESERVATIVES IN COMMON U S E

1 . Benzoic Acid Benzoic acid is a useful preservative for mixtures having a p H of 5 or less; about 0.1%w.lv. of the acid is sufficient to inhibit microbial growth under these conditions. In the United Kingdom the acid is normally used in the form of benzoic acid solution B.P.C. (which contains 5.5% w.lv. benzoic acid and 75% propylene glycol in water) because the acid itself is difficult to dissolve in water. This solution is used in a number of pediatric mixtures included in the B.P.C.

(1963). 2. Esters of p-Hydroxybenzoic Acid (Parabens) These esters are tasteless, stable, and nontoxic, but are incompatible with certain substances used in medicines, e.g., nonionic surfaceactive agents. The parabens have been widely studied over many years. Neidig and Burrell (1944) comprehensively reviewed the extensive literature appertaining to their use as preservatives up to the early 1940’s. This work covered bacterial studies, toxicity studies, and the preservation of cosmetics, food, and pharmaceutical products (170 references to the latter). Subsequent work by Aalto e t al. (1953) and by Husa and co-workers (Littlejohn and Husa, 1955; Schimmel and Husa, 1956) and others, have confirmed the original findings that these esters are useful preservatives for many pharmaceutical products, especially syrups and medicines containing syrups or gums. Prickett e t al. (1961) demonstrated the potentiation of the preservative action of the parabens on the addition of small amounts (2%or 5%)of propylene glycol. None of the elixirs, linctuses, or mixtures mentioned in the current B.P.C. contain these esters as preservatives though some manufacturers use them in their proprietary products, e.g., Lederle Laboratories use several combinations in their antibiotic mixtures.

32


3. Chloroform This has been widely used for many years as a preservative for oral medicines because of its sweet and pleasant taste. In addition, its carminative action is useful in certain preparations. The chief disadvantage of chloroform is its volatility which leads to its loss from a medicine unless the container is well closed. Sykes (1963b) stated that 0.15 to 0.25% chloroform is sufficient to inhibit or even kill bacteria and molds, but below 0.1%it is ineffective. Chloroform per se is seldom used in preparing medicines because of the small amount required and the difficulty of dissolving it in water. The B.P. contains several preparations which overcome these difficulties. Chloroform water contains 0.25%v./v. chloroform and is used as the vehicle for a number of mixtures, e.g., chalk and opium mixture B.P.C. Strong solutions of certain salts sometimes throw the chloroform out of solution, but, apart from this, chloroform water is compatible with most ingredients of medicines. Sometimes for purely pharmaceutical reasons, more concentrated chloroform preparations are preferred, e.g., chloroform spirit B.P. (15% chloroform in 90% v./v. ethanol) or chloroform emulsion B.P. (5%chloroform emulsified in water with quillaia tincture).

4 . Sorbic Acid (2,4-hexadienoic acid) This has been reported by Puls et al. (1955) to be an effective fungistat for use in mucilages of vegetable gums and in diluted syrups. About 0.2% w./v. will prevent the growth of most molds. It does not appear to be used much in the preservation of oral pharmaceutical products, though it is used in various foodstuffs.

5. Sulfur Dioxide and Sulfites Sulfur dioxide, either as such, or present in the form of sulfites, can

be used to preserve fruit syrups and other preparations. Recently, the 1966 Supplement to the B.P.C. 1963 increased the recommended sulfur dioxide content of raspberry syrup from 350 to 420 1J.p.m. because the former has been found to be ineffective in preventing mold growth. VI. Preparations for External Use

A. INTRODUCTION Included in this section are lotions, liniments, creams, ointments, and pastes for application to the skin, and various “drops,” douches, and other preparations intended for insertion into body cavities, e.g.,


33

ear, nose. Ophthalmic preparations have been dealt with separately (Section IV). The various preparations may be liquid or semisolid, and may be made with either aqueous or oily vehicles. Those made with oily bases, e.g., ointments, pastes, and some liniments, seldom permit microbial contaminants to flourish and hence do not usually require the inclusion of a preservative. Many of the products are designed to treat local infections and consequently contain antimicrobial agents as their chief active ingredients. However, it is occasionally necessary to add another agent to extend the antimicrobial spectrum of the product to inhibit possible contaminants. Sometimes, an antimicrobial substance is included in a preparation primarily to exert some other effect. An example of this is phenol, which is included in some lotions to exert a mild local anesthetic effect and alleviate itching; the concentration required for this purpose (about 1%)is generally sufficient to prevent microbial growth in the lotion. SimiIarly, alcohol is often included to provide a cooling effect on the skin and again the concentration used is normally sufficient to inhibit the growth of microorganisms. €3. TYPESOF PRODUCT

In addition to the usual pharmaceutical requirements of compatibility, etc., the preservatives used in preparations for external use should be nonirritant and nonsensitizing. Hjorth and Trolle Larsen (1962) have reported on the latter aspect with regard to the parabens and sorbic acid. The antimicrobial agents commonly used in preparations for external use include most of those used in parenteral, ophthalmic, or oral products, viz., parabens, phenols, chlorinated phenols, organic mercurials, quaternary ammonium compounds, etc. The aqueous preparations can be divided into four types: solutions, suspensions, emulsions, and jellies. It will be convenient to discuss the preservation of each type of product separately.

I. Solutions T h e preservation of these is normally a relatively simple matter. A wide variety of suitable agents is available and the choice is usually governed by questions of compatibility and cost. The most commonly used agents include chlorocresol, alcohol, phenylmercuric salts, and quaternary ammonium compounds.

2. Suspensions The preservation of suspensions is complicated by two possible factors, the adsorption of the antimicrobial agent onto the suspended

34


material and/or inactivation of the agent by the suspending agent. Harris (1961) showed that cationic antiseptics including cetrimide and chlorhexidine, were inactivated by suspensions containing 5% bentonite.

3. Emulsions The literature pertaining to the preservation of emulsions has been thoroughly reviewed and discussed by Wedderburn (1964). Persons interested in this topic are strongly recommended to study this article which covers in detail the many factors involved, and lists the preservatives most commonly used in cosmetic and pharmaceutical emulsions.

4 . Jellies Jellies are used in medicine for a variety of purposes. Some are formulated for application to the skin or to a body cavity, e.g., nose or vagina, while others are formulated for lubricating surgical instruments prior to insertion into a body cavity. Basically, a jelly consists of approximately 1 to 5% of a gel-forming substance, e.g., tragacanth, methyl cellulose, gelatin, sodium alginate, up to about 10% of a dispersant (e.g., glycero1 or propylene glycol), the active drug, and sufficient water to make 100%. Most gel-forming substances are prone to microbial attack especially when a lot of water is available, so a preservative is almost invariably essential. Jellies used as lubricants for surgical instruments must, of course, be sterile, but sterility is usually less important in the jellies for application to the skin. Interactions between various preservatives and gel-forming substances have been reported. Tillman and Kuramoto (1957), using a spectrophotometric analysis technique, found evidence for complex formation between methyl cellulose 400 and methyl-, propyl-, and butylparabens. Miyawaki et al. (1959), also using physical methods, found that methyl- and propylparabens interacted to some extent with methyl cellulose, polyvinylpyrrolidone, and gelatin. They found no evidence for a significant degree of interaction between methylparaben and carboxymethyl cellulose or tragacanth. They concluded that in the concentrations of polymers generally used, binding is insufficient to prohibit the effective application of the parabens as preservatives. Eisman et al. (1957) found that in jellies containing 3% tragacanth and buffered at pH 7 the bactericidal activity of a number of preservatives, e.g., chlorbntanol, benzalkonium chloride, methyl- and


35

propylparabens, was markedly reduced. The properties of phenol, Merthiolate, and PMA were less affected. The test organism employed was Staphylococcus uureus. Taub et ul. (1958) studied jellies containing 2% tragacanth and 5% propylene glycol. Jellies were prepared having specific p H values in the range pH 3 to 7 , and one of the following preservatives added: 0.2% benzoic acid, 0.5% chlorbutanol, and a combination of 0.2% methyl- and 0.05% propylparabens. The jellies were sterilized and then inoculated with the following microorganisms: S . aweus, B . subtilis, Escherichia coli, and Candida albicans. Samples of the jellies were removed at various time intervals and inoculated into culture media, care being taken to ensure that bacteriostatic concentrations of the preservatives were not carried over into the plating medium. The results obtained showed that the combination of parabens sterilized the jellies over the pH range 3 to 7 except when the contaminant was B . subtilis, though even with this organism the bacterial population was much reduced. Almost identical results were obtained with jellies which had been stored for 28 days prior to inoculation and testing, showing that no inactivation of the preservative occurred during storage. As might be expected, the results obtained with benzoic acid as preservative varied with the pH of the jelly, being satisfactory below pH 5. Chlorbutanol exerted some preservative action but was inferior to the parabens. Fiedler and Lee (1955)found a heavy black mold growth on samples of ephedrine sulfate jelly N.F. (containing 1%tragacanth). The jelly also suffered from a number of other imperfections, and thus a better formulation was sought. It was shown that when the tragacanth was replaced by 4% sodium alginate, a clear jelly was formed which could be preserved using 0.2% sodium benzoate. A mixture of methyl- and propylparabens would also preserve this new formulation but the resulting jelly was cloudy. More recently, Swafford (1960) suggested the use of 2% Carbopol934 (B. F. Goodrich Chemical Co., Cleveland, Ohio, U.S.A.) as the gel-forming agent in ephedrine sulfate jelly. Carbopol 934 is a synthetic hydrophilic gum and appears to have a number of advantages over the natural products, not least of these being its ability to resist bacterial and fungal attack. REFERENCES Aalto, T. R., Firman, M . C., and Rigler, N. E. (1953).J . Am. Pharm. Assoc. Sci. Ed. 42, 449-457. Abrams, J. D. (1963). Trans. Ophthalmol. Soc. U . K . 83, 263.

36


Abrams, J. D., Davies, T. G., and Klein, M. (1965). Brit. J. Ophthalmol. 49, 146-147. Alin, K., and Diding, N. (1949). F a m . Reuy 48, 545. Amies, C. R. (1962). J . H y g . 60,473-481. Anderson, K. F., Lillie, S., and Crompton, D. 0.(1964a).Pharm.J.192,593-594. Anderson, K. F., Lillie, S., and Crompton, D. 0.(1964b). Pharm.J.193,165. Barr, M., and Tice, L. F. (1957a).J. Am. Pharm. Assoc. Sci. E d . 46,217-218. Barr, M., and Tice, L. F. (1957b).J. Am. Pharm. Assoc. Sci. E d . 46,219-221. J . Am. Pharn~Assoc. Sci. E d . 46, 442-445. Barr, M., and Tice, L. F. (1957~). Barr, M., and Tice, L. F. (1957d).J. Am. Pharm. Assoc. Sci. E d . 46, 445-451. Beloian, A., and Koski, T. (1964).J. Assoc. Ofic. Agr. Chemists 47,804-807. Birner, J., and Garnet, J. R. (1964a).J. Pharm. Sci. 53,1264-1265. Birner, J., and Garnet, J. R. (1964b).]. Pharm. Sci.53,1266-1267. British Pharmaceutical Codex (1954). British Pharmaceutical Codex (1959). British Pharmaceutical Codex (1963) British Pharmaceutical Codex Addendum (1966). British Pharmacopoeia (1958). British Pharmacopoeia (1963). British Pharmacopoeia Commission (1966). Personal communication. Brown, M. R. W., and Norton, D. A. (1965). J. SOC. Cosmetic Chemists 16, 369-387. Brown, M. R. W., and Richards, R. M. E. (1965). Nature 207, 1391-1393. Brown, M. R. W., Foster, J. H. S., Norton, D. A., and Richards, R. M. E. (1964). Pharm. J. 192, 8. Brown, W. R. L. (1961). Pharm. J. 187,221-222. Buschke, W. (1949). J . Cellular Comp. Physiol. 33, 145-149. Cohen, S. M., and Wheeler, M. W. (1946). Am . ]. Public Health 36,371-376. Cooper, J. W., and Gunn, C. (1965). “Dispensing for Pharmaceutical Students,” 11th Ed. Pitman, New York. Davies, G. E., and Davison, J. E. (1947). Quart J . Pharm. Pharmacol. 30, 212-218. Davies, G. E., and Fishburn, A. G. (1946). Quart. J . Pharm. Pharmacol. 29, 365-372. Davison, J. E. (1951). J. Pharm. Phamacol. 3,734-740. Davisson, E. O., Powell, H. M., MacFarlane, J. O., Hodgson, R., Stone, R. L., and Culbertson, C. G. (1956). j . Lab. Clin. Med. 47, 8-19. Dick, G. W. A. (1959). Practitioner 183,305-312. Diena, B. B., Wallace, R., and Greenberg, L. (1964). Can. J . Microbiol. 10, 555-560. Editorial (1963). Pharm. J. 192, 587-588. Editorial (1966). Pharm. J . 196, 159-160. Edsall, G., McComb, J. A,, Wetterlow, L. H., and Ipsan, J. (1962). New Engl. J. Med. 267,687-689. Eisman, P. C., Cooper, J., and Jaconia, D. (1957). J . Am. Pham. Assoc. Sci. E d . 46, 144-147. Extra Pharmacopoeia (1958). Pharmaceutical Press, London. Felix, A. (1941). Brit. Med. J. i, 391. Felix, R. I. (1964).J. SOC. Cosmetic Chemists 16, 1-12. Fiedler, W., and Lee, C. 0. (1965). J . Am. Pham. Assoc. Pract. Pharm. E d . 16, 101. J. 192,429. Foster, J. H. S. (1964~).Pha~m. Foster, J. H. S. (1964b). Pharm. J. 192,461. Foster, J. H. S. (1965). Mfg. Chemist 36, Pt. 5 , 45-50; Pt. 6, 43-46. Gabel, L. F. (1921). I . Am. Pharm. Assoc. 10,767-768. Gardner, R. A., and Pittnr,iii. \ I . (1965). Appl. Microbiol. 13, 564-569.


37

Gilbert, R. J., and Russell, A. D. (1963). Pharm. J. 190, 111-112. Ginsberg, M., and Robson, J. M. (1949). Brit. J . Ophthalmol. 33,574-579. Guillot, M. (1950).J . Pharm. Pharmacol. 2, 345-360. Hadgraft, J. W., and Short, P. (1947). Pharm. J. 158,202. Harris, W. (1961). Australasian J. Pharm. 42,583-584, 587-588. Hess, H. (1965). Pharm. Weekblad 100, 764-774. Hjorth, N., and Trolle Larsen, C. (1962). Am. Pe$umer 77, 43-45. Hopkins, S. J. (1960). M . B . Pharm. Bull. Hopkins, S . J. (1966). Pharm. J. 196, 141-142. Hugo, W. B., and Foster, J . H. S. (1964a).J. Pharm. Pharmacol. 16,209. Hugo, W. B., and Foster, J. H. S. (1964b). J . Pharm. Pharmacol. 16, 124T-126T. Kedzia, W., Lewon, J., and Wisniewski, T. (1961). J. Pharm. PharmacoZ. 13,614-616. Kenney, D. S., Grundy, W. E., and Otto, R. H. (1964). Bull. Parenteral Drug. Assoc. 18,lO-19. Klarmann, E. G. (1959).Am.J . Pharm. 131,86-91. Klein, M., Millwood, E. G., and Walther, W. W. (1954).J. Pharm. Pharmacol. 6,725-732. Kohan, S . , Carlin, H., and Whitehead, R. (1962).Am . ]. Hosp. Pharm. 19,83. Kohli, J. D., Chopra, I. C., and Chander, K. (1950). ZndianJ. Med. Res. 33, 413-416. Kohn, S . R., Gershenfeld, L., and Barr, M. (1963a).J. Pharm. Sci. 52,967-974. Kohn, S . R., Gershenfeld, L., and Barr, M . (1963b).J. Pharm. Sci. 52,1126-1129. Landi, S. (1963). Appl. Microbiol. 11, 408-412. Lawrence, C. A. (1955a).J. Am. Pharm. Assoc. Sci. Ed. 44,457-464. Lawrence, C . A. (1955b). Am. J . Ophthalmol. 39,385-394. Littlejohn, 0. M., and Husa, W. J. (1955). J . Am. Pharm. Assoc. Sci. Ed. 44, 305-308. Loosemore, M., and Russell, A. D. (1963).J . Pharm. Pharmacol. 15, 558. Luderitz, O., Staub, A. M., andwestphal, 0. (1966). Bacteriol. Rev. 30,192-255. McEwan, J. S., and MacMorran, G. H. (1947). Pharm.]. 158,260-262. MacGregor, D. R., and Elliker, P. R. (1958). Can.J.Microbiol. 4,499-503. MvIver, A. K. (1964). Pharm. J. 192,429. McLean, I. W. (1957). US.Patent No. 2,763,160. Magnusson, M., Guld, J., Magnus, K., and Waaler, H. (1958). Bull. WorldHealth Organ. 19, 799, 828. Melnick, J. L., and Wallis, C. (1964). Clin. Med. 71, 2053-2067. Miyawaki, G. M., Patel, N. K., and Kostenbauder, H. B. (1959). J. Am. Pharm. Assoc. Sci. Ed. 48, 315-318. Montgomery, W. F., and Halsall, K. G. (1964a). Pharm. J . 192,407. Montgomery, W. F., and Halsall, K. G. (196413). P h a m . J. 192,461. Morgan, H. R. (1958). In “Bacterial and Mycotic Infections of Man” (R. J. Dubos, ed.), 3rd Ed. Pitman, New York Mueller, W. H., and Deardo& D. L. (1956).J. Am. Pharm. Assoc. Sci. Ed. 45,334-341. Nair, A. D., and Lach, J. L. (1959).J. Am. P h a m . Assoc. Sci. Ed. 48,390-395. Neidig, C. P., and Burrell, H. (1944). Drug. Cosmetic Znd. 54,408-410,481-489. Olson, B. H., Eldering, G., and Graham, B. ( 1 9 6 4 ) ~Bacteriol. . 87,543-546. Parish, H. J. (1965). “A History of Immunisation.” Livingstone, Edinburgh and London. Parish, H. J . , and Cannon, D. A. (1963). Practitioner 190, 75-80. Parish, H. J., and Cannon, D. A. (1964). “Antisera, Toxoids, Vaccines and Tuberculins,” 6th Ed. Livingstone, Edinburgh and London. Pittman, M. (1962).J . Am. Med. Assoc. 181,25-30 Pittman, M., and Cox, C . B. (1965). AppE. Microbiol. 13, 447-456. Pivnick, H., Tracy, J. M., and Glass, D. G. (1963). J . Pharm. Sci. 52, 883-888.

38

A. D. RUSSELL, J. JENKINS, AND 1. H. HARRISON

Pivnick, H., Siebenmann, C. D., Landi, S., and Ashford, W. R. (1965).J . Pharm. Sci. 54, 640-642. Prickett, P. S., Murray, H. L., and Mercer, N. H. (1961).J . Pharm. Sci. 50, 316-320. Puls, D. D., Lindgren, L. B., and Cosgrove, F. P. (1955).J.Am. Pharm. Assoc. Sci. Ed. 44,85-87. Ravnick, A,, and Yatoko, J . (1962).Am. J . Hosp. Pharm. 19,469-471. Rdzok, E. J., Gmndy, W. E., Kirchmeyer, F. J . , and Sylvester, J. C. (1955).J . Am. Pharm. Assoc. 44,613-616. Richards, R. M. E., and Brown, M. R. W. (1964).J . Pharm. Pharmacol. 16, 360-361. Riegelman, S., Vaughan, D. G., and Okumoto, M. (1956).J . Am. Pharm. Assoc. Sci. Ed. 45, 93-98. Royce, A,, and Sykes, G. (1957).J . Pharm. Phavmacol. 9, 814-822. Russell, A. D. (1964). Lab. Pruct. 13, 114-122. Russell, A. D. (1965).Mfg. Chemist 36, 38-45. Russell, A. D., and Loosemore, M. (1964).App2. Microbiol. 12,403-407. Saunders, L., and Shotton, E. (1956).J. Pharm. Pharmacol. 8,832-847. Schimmel, J., and Husa, W. J . (1956).J.Am. Pharm. Assoc. Sci. Ed. 45,204-208. Schuchardt, L. F., Munoz, J., and Verwey, W. F. (1960). Am. J . Public Health, 50, 321-328. Sen Gupta, P. N. (1951). IndianJ. Med. Res. 40, 115-119. Sutherland, I. W. (1963). Immunology 6, 246-254. Swafford, W. B. (1960).Am. J . Pharm. 132,383-384. Sykes, G. (1958).J . Pharm. Pharmacol. 10,40T-46T. Sykes, G. (1963a).Practitioner 190,52-57. Sykes, G . (196313).“Dissinfection and Sterilisation,” p. 366. Spon, London. Taub, A., Meer, W. A., and Clausen, L. W. (1958).J. Am. Pharm. Assoc. Sci. Ed. 47, 235-239. Taylor, E. W., and Burman, N. P. (1956).J . Pharm. Pharmacol 8,817-831. Tillman, W. J., and Kuramoto, R. (1957).J . Am. Phurm. Assoc. Sci. Ed. 46, 211-214. Tracy, J . M., Glass, D. G., Nicholson, M. J., and Pivnick, H. (1964).J . Pharm. Sci. 53, 659-663. U S . Natl. Formulary (1960). U.S. Pharmacopoeia (1960)XVIth Revision. U.S. Pharmacopoeia (1965).XVIth Revision. van Hemert, P., van Wezel, A. L., and Cohen, H. H. (1964). Nature 203, 774-775. Wedderburn, D. (1964).Aduan.Pharm. Sci. 1,195-268. Wing, W. T. (1958).Proc. Inst. Rubber Ind. (Trans. Inst. Rubber Ind.) 5, 67-72. World Health Organization (1959a).Tech. Rept. Ser. 178. World Health Organization (195913).Tech. Rept. Ser. 179. World Health Organization (1959~).Tech. Rept. Ser. 180. World Health Organization (1960). Tech. Rept. Ser. 200.

Antiserum Production in Experimental Animals

RICHARD M. HYDE Department of Microbiology, School of Medicine University of Oklahoma, Oklahoma City, Oklahoma

I. Introduction ............................................................. 11. Methods Employed in Antiserum Collection ................ 111. Animal Species Employed in Antiserum Production ....... IV. Routes of Inoculation Employed in Antiserum Production ................

39 40 41

43 47 47 50 C. Presence of Specific Antibody ............................... 52 D. Age of Animal ..................................................... 54 ................................... ......... 56 F. Other ................................... ................. 57 VI. Specific Examples of Antisemm Production ................. 58 VII. Conclusion ............................................................... 58 VIII. Selected Bibliography.. ....................... 59 References ................. ............................. 63 V.

I. Introduction The production of specific antibodies in experimental animals has become a problem of practical importance to investigators in many areas of biology. Thus, the endocrinologist may wish to obtain an antibody to a specific hormone in order to study the role of the hormone in the physiology of the animal, or perhaps to ascertain the cellular site of production of the hormone. The need for such an antibody requires that the investigator understand the basic concepts of immunology. It is the purpose of this review to discuss certain aspects of immunization practices which will guide scientists wishing to produce specific antiserum to substances of biological interest. Numerous reviews have been written concerning immunization methods and vaccines available for the prevention of human disease (Menzin, 1961; Edsall, 1963, 1965; Hilleman, 1964; Riley, 1966), hence this .subject will not be dealt with here. No attempt will be made to review the methods employed for the induction of delayed hypersensitivity in experimental animals. Individuals interested in this particular facet of the immune response are referred to the excellent articles by Arnason and Waksman (1964) and Chase (1965). Certain aspects covered in this review have been considered recently by White (1963) and Edsall (1966). The reader is referred to these sources for supplementation of information presented herein. 39

40

RICHARD M. HYDE

II. Methods Employed in Antiserum Collection Detailed procedures for bleeding rabbits, chickens, guinea pigs, rats, and mice b y both cardiac and venous puncture are described by Campbell et at. (1964).An even more extensive discussion of the techniques employed for collection of blood from experimental animals is presented by Moreland (1965).The latter included in his discussion methods for the collection of lymph and other body fluids from experimental animals. In addition, the author presents methods employed for the collection of blood, etc., from dogs, cats, monkeys, various birds, and ruminants, as well as the animal species considered by Campbell et al.. In general, it is desirable to fast the animal for approximately 18 hours prior to bleeding, thus reducing the amount of lipid in the serum. Occasionally, excessive hemolysis presents a problem. This may be minimized by collecting the blood specimen in an anticoagulant solution, centrifuging to remove the erythrocytes, and recalcifying the plasma supernatant. Alternately, the blood may be drawn into a chilled vacutainer tube (Becton, Dickinson, and Co.) and kept at a low temperature (to prevent clotting) until the erythrocytes have been spun down. After centrifugation of the chilled blood, the fibrin can be broken away from the glass and, after recentrifugation, the clear serum can be aspirated off. The latter method has the further advantage that the serum can be harvested from the blood less than 1 hour after the specimen has been removed from the animal. The success of either of these methods of avoiding hemolysis during the collection of blood will depend upon knowledge of the appropriate amount of anticoagulant to employ or, alternately, the clotting time of the blood in question. Information of this nature can be readily obtained from the Biology Data Book (Altman and Dittmer, 1964). A discussion of some of the steps involved in processing of blood and preparation of glassware and reagents can be found in Tocantins and Kazal (1964). The volume of blood removed from an animal is determined solely by the blood volume of that animal and consideration for the amount that can safely be withdrawn without jeopardizing the life of the donor. Bleeding has apparently no adverse effect on the antibody content of the serum. Smolens et al. (1957) subjected a group of human volunteers to plasmaphoresis (the removal of plasma while returning the cellular portion of the blood to the donor) for a period of 1 year, during which time 5200 ml. of plasma was removed from each individual. They reported that the bacterial and viral antibody content

ANTISERUM PRODUCTION IN EXPERIMENTAL ANIMALS

41

of the serums persisted at peak levels throughout the period of the experiment and for 1 year after the cessation of plasmaphoresis. In fact, several observers have recorded a stimulation of antibody production as a result of excessive or repeated bleedings. This observation is discussed by Wilson and Miles (1964). 111. Animal Species Employed in Antiserum Production Many practical considerations must be evaluated in the selection of the animal to be used as the source of antiserum, e.g., the amount of antiserum desired, the ease of bleeding the animal, the cost of purchase and maintenance of the animal, etc. No attempt will be made here to recommend guidelines in this regard, as each investigation will have its own requirements and limitations. However, a few comments of a general nature may be appropriate. The ability to synthesize antibodies in response to antigen exposure appears to be restricted to vertebrate animals. Attempts to induce antibody formation in various invertebrates such as sea anemones (Phillips and Yardley, 1960), earthworms (Triplett et al., 1958), caterpillars (Bernheimer et al., 1952), and insects (Stephens, 1959) have met with failure. The body fluids of the invertebrate animals under study failed to react in serological tests with the antigens employed in immunization. In lower vertebrates the immune response appears to increase in intensity as one ascends the phylogenetic scale. Papermaster et al. (1964) reported that the hagfish appeared to be completely devoid of any inducible immune responses. The lamprey possessed a low level of immunologic competence, while the capacity to form circulating antibody and demonstrate immunologic memory was found to be reasonably well developed in the elasmobranchs and lower bony fishes. The phylogeny as well as the ontogeny of the immune responce has been reviewed recently by Good and Papermaster (1964). Antigens must be foreign to the circulation of the experimental animal. In general, the greater the phylogenetic disparity of the antigen and the experimental animal, the greater will be the immune response the antigen induces. Thus, human serum albumin is a stronger antigen for the chicken than is duck serum albumin (Ivanyi and Valentova, 1966). Tempelis (1965) studied an even closer phylogenetic relationship when h e examined the antibody response of chickens to gamma-globulin fractions of turkey and goose serum. Ninety-seven percent of the birds produced antibody to the later antigen, whereas only 82% had a demonstrable immune response when injected with

42

RICHARD M. HYDE

the turkey gamma-globulin. Furthermore, the goose gamma-globulin induced the production of approximately twice the amount of antibody in the chickens as did a comparable quantity of the phylogenetically more closely related turkey gamma-globulin. The ability of an animal to produce a specific antibody may be influenced by subtle genetic factors. It has been known for many years that mice (Avery and Goebel, 1933) and humans (Francis and Tillett, 1930; Felton et al., 1935) respond to the injection of pneumococcal polysaccharide with the production of circulating antibodies, while rabbits (Avery and Goebel, 1933; Downie, 1937a) are apparently unable to do so. Fink and Quinn (1953) presented evidence suggesting that there is a genetic variation in the ability of inbred mice to produce demonstrable antibody to either egg albumin or pneumococcal polysaccharide. Ipsen (1954) studied the ease of immunization of 10 inbred mouse strains with tetanus toxoid. Three of the strains examined were relatively easy to immunize against the lethal effects of the toxin, whereas the remaining seven strains required from 10 to 30 times more toxoid to achieve the same degree of protection. In a later study, the same investigator found that the strength of the anamnestic response to a second injection of antigen correlated with the ease of primary immunization. Strains of mice that were more efficiently immunized b y a primary injection of the toxoid also demonstrated a greater anamnestic response upon booster injection (Ipsen, 1959). When random-bred animals are employed in antiserum production it is common to obtain a wide range of antibody titers. Ipsen (1961), studying the primary immune response of rabbits to tetanus toxoid, observed that antitoxin was demonstrable in 73 of 79 immunized animals, with a geometric mean titer of 0.06 unitslml. However, the titers ranged from less than 0.005 antitoxin unitslml to 1.6 unitslml. Burnet (1964) states that even when immunizing genetically uniform inbred mice it is common to obtain a wide range of antibody titers. Hyde et al. (1965) employed immunoelectrophoresis to evaluate the antibody response of rabbits to human serum antigens. Of 14 animals injected with human serum, four failed to produce detectable antialbumin antibodies, whereas only one animal did not respond to transferrin, thus demonstrating that the percentage of animals producing antibody to a particular antigen will vary with the antigen employed. Better antigens apparently induce a higher percentage of reactive sera. Experiments conducted recently in several laboratories have shown that some strains of animals are genetically unable to respond to certain antigens. Pinchuck and Maurer (1965a) examined the anti-


43

genicity of synthetic polypeptides in inbred strains of mice and found that three inbred strains produced antibodies to the antigen under study, while four strains were unable to do so. All seven strains tested were able to respond to two other synthetic polypeptides differing from the first terpolymer only in the percentage of the three amino acids present in each. They also tested the antigenicity of the first polypeptide in random-bred Swiss mice and observed that 47%of the animals were able to respond to the antigen. Through mating studies in this random-bred line they were able to obtain evidence which suggested that the antibody response to the polymer was controlled by a codominant Mendelian factor. Similar evidence for a genetic control of antibody response was obtained by McDevitt and Sela (1965) who examined the sera of two strains of inbred mice immunized with branched, multichain synthetic polypeptides. Arquilla and Finn (1965) have observed a similar genetic control of antibody production in inbred guinea pigs. They found that although both strain 2 and strain 13 animals produced antibodies to alum-precipitated insulin, they did not produce antibodies of identical specificity, i.e., strain 2 guinea pigs produced antibodies to one determinant group on the insulin molecule while strain 13 animals produced antibodies to a second (different) determinant group. Thus, both strains were able to recognize insulin as an antigen, but they did not recognize the same region of the molecule. Pinchuck and Maurer (1965a) discussed a similar observation in strain 2 and strain 13 guinea pigs. They found that strain 13 animals were completely unable to produce antibodies to a synthetic polypeptide copolymer, while all of the strain 2 animals immunized with the same material responded with antibody production. Further, they state that only 35% of the Hartley strain guinea pigs (a random-bred animal) recognized the polypeptide as antigenic. IV. Routes of Inoculation Employed in Antiserum Production The most common routes of parenteral inoculation of antigenic materials are intravenous (IV), intradermal (ID), subcutaneous (SC), intramuscular (IM), and intraperitoneal (IP). Descriptions of the techniques involved in these methods can be found in Campbell et al. (1964) and in Moreland (1965). In general, antigens in suspension are inoculated via the IV or IP routes, while antigens in solution are given by one of the other routes. This is an oversimplification and, as will be developed later in this section, the route selected will often affect the level of antibody attained in the animal. Better immuniza-

44

RICHARD M. HYDE

tion usually results if the antigen is given in a manner that stimulates the entire antibody-synthesizing apparatus of the host and not just the lymphatics draining the site of injection. For example, when employing the SC route of immunization, it is desirable to place aliquots of the inoculum at several different sites on the body, and thus involve a larger portion of the lymphatic system in the ensuing antibody response. Special methods of immunization may be required to solve particular problems. For example, if the investigator is interested in studying the antibodies that might be concerned with immunity to a particular infectious agent, it may be desirable to expose the animal to the antigen by use of the natural route of infection for the agent under study. Similarly, if the study is designed to establish a method for immunization of large population groups, the investigator may wish to follow a procedure such as that used for jet injections. An excellent review on the latter method of immunization has been written recently by Hingson et al. (1963).A brief discussion of some of the routes of inoculation for specialized studies follows:

1 . Oral

The Sabin polio vaccine is an excellent example of the efficiency of this route of immunization. However, the oral administration of antigen is not always effective. Mascoli et al. (1966)attempted immunization of humans by oral feeding of live rhinoviruses in enteric-coated capsules. None of the individuals involved in the study developed neutralizing antibodies against the viruses ingested. Attempts to induce protective immunity to helminths by parenteral injection of worm extracts or dead worms have not been successful, with the exception of certain cestodes (Soulsby, 1962). Numerous workers in the past 20 to 40 years have shown that, in most cases, infection via the natural route does provide protection against a subsequent challenge with an animal parasite. In many instances, however, the immunizing infection debilitates the host or causes significant weight loss. In the case of Dictyocaulus vipiparous (lung worm of cattle) and Ancylostoma caninum (hookworm of dogs) the debilitating effects can be avoided by immunizing with irradiated, but still viable worms (Jarrett and Sharp, 1963; T. A. Miller, 1966). A rise in antibody titer after natural infection can be detected by most conventional serologic techniques. However, with the exception of the cestodes, attempts to demonstrate protective antibody via passive transfer experiments have shown either no protection or only


45

slight protection. Such findings suggest that cellular elements must play an important role in this immunity.

2. Ocular The results of numerous studies indicate that the introduction of soluble antigens into the ocular tissues of various laboratory animals induces the production of circulating antibodies (Thompson et al., 1957; Breebaart and James-Witte, 1959; Fernando, 1960). Parks et al. (1961) inoculated rabbits with identical doses of bovine gammaglobulin via either the intracorneal, intravitreous, intravenous, or intramuscular route and measured the effect that the route of inoculation had upon the development of antibodies. They reported that antibody was produced more consistently and in higher titer following primary inoculation of antigen into ocular tissues than via the conventional parenteral routes. The highest titers were obtained after intracorneal injection, followed by intramuscular injection of the antigen incorporated in incomplete Freund’s adjuvant. Intravitreous injection resulted in the production of demonstrable antibody in every animal tested. Only two of six animals receiving intramuscular injections of the antigen in the absence of adjuvant produced detectable antibody, while none of the six animals receiving antigen via the intravenous route produced enough antibody to be detected by the passive cutaneous anaphylaxis assay. Experimental herpetic keratoconjunctivitis has been induced in rabbits to study the production of precipitating antibodies against the virus. Mantyjarvi (1965) reported the production of seven to eight precipitin lines in his diffusion-in-gel detection system when he analyzed the serum from the infected animals. Pinkerton and Webber (1964) devised a method of injecting small laboratory animals by the ophthalmic plexus route. When done with care, this method facilitates the introduction of materials into the circulation of the animal, and offers at least an alternate route when the tails of the animal being immunized have become so scarred as to make further injections impossible. Further, they report that leakage of the inoculum is not a problem, nor does the procedure impair the eyesight of the animal.

3. Respirutory The efficacy of active immunization against airborne infections by inhalation of attenuated microorganisms has been demonstrated in experimental animals and man. Middlebrook (1961) has reviewed

46

RICHARD M. HYDE

some of the immunologic aspects of the host’s reactions to inhaled antigens. Vaccination by the aerogenic route has also been discussed by Jemski and Phillips (1965) and was considered in the Second International Conference on Aerobiology (Lepper and Wolfe, 1966), hence this subject will not be dealt with in any detail here. Yamashiroya et al. (1966) compared the aerosol method of immunization with the more conventional subcutaneous route. They immunized guinea pigs with fluid tetanus toxoid b y one or the other of these two routes and concluded that, although the serum antibody titers were lower after aerosol vaccination, definite immunity was conferred after inhalation of the toxoid. Interestingly, the anamnestic response in the animals surviving toxin challenge was greater in the aerosol-immunized group than in the animals that received a subcutaneous injection of the toxoid.

4 . Other Essentially all areas of the body can be, and have been, employed as routes of immunization. Perhaps one of the most inclusive studies was that of Draper and Sussdorf (1957) who studied the nature of the hemolysin response following the injection of heated sheep erythrocyte stromata by various routes. Compared to the mean log peak titers in intravenously injected rabbits, the titers were: (a)higher following injection into the liver or femoral bone marrow; ( b )lower after injection into the appendix, kidney fat, or subcutaneous tissues of the lumbar region; and ( c ) not different after injection into the peritoneal cavity, thigh muscle, mesenteric lymph nodes, spleen, or hind footpads. The intravaginal route of immunization has been employed by Menzoian and Ketchel (1966) to test the antigenicity of homologous and heterologous seminal fluid. Antigen was placed deep into the vagina of the test rabbit by means of a glass tube with a polished end, care being taken to avoid trauma to the vaginal lining. Tuba1 ligations were performed on some of the rabbits to prevent absorption of antigen through the peritoneum. The rabbits produced antibody to heterologous bovine seminal plasma but not to homologous seminal plasma. Further, these workers found that it took approximately 10 times more antigen to induce detectable antibody by the intravaginal route than by the subcutaneous or intravenous routes.

5. Effects of Route of Immunization on Antibody Production The effect of the route of immunization on both the quantity and the quality of the antibody response has been well established. Webster


47

(1965) studied the influence of route and schedule of vaccination on the time course of the immune response in a group of rabbits injected with influenza virus antigens. H e found, as expected, that these two variables markedly influenced the maximal level of serum antibody attained. The routes tested, ranked in order of decreasing efficiency, were; intravenous, intraperitoneal, and subcutaneous. Secondary stimulation with antigen (first booster injection) caused an 8- to 10fold rise in maximal antibody titers by all routes of inoculation. Tertiary stimulation (second booster injection) of all treatment groups served only to return the maximal titers of antibody to those obtained in the secondary response, but did not stimulate higher levels of antibody. The use of an improper route of immunization may even result in the complete absence of antibody formation. Sulzberger (1929) found that he was able to induce skin hypersensitivity to arsphenamine in guinea pigs if he injected the animals intradermally. However, if he administered a second aliquot of the antigen intracardially within 24 hours of the primary injection, the animals failed to develop any allergic manifestations upon subsequent challenge with arsphenamine. The significance of this antigen-induced refractory state remained obscure until Chase and others (reviewed by Chase, 1959) reinvestigated the phenomenon. This type of antigenic inertness is now considered to be an example of route-dependent immunologic tolerance.

V. Factors Affecting Antibody Production A. QUALITYOF THE ANTIGEN Antigenicity (immunogenicity) can be defined as the ability of a molecule to induce the production of homologous antibodies when introduced into an animal. There are certain qualities, or characteristics, intrinsic to a molecule which will determine its ability to initiate antibody synthesis. These characteristics are: (a) foreignness (F); (b) size (S); and (c) chemistry (C). Thus, the antigenicity (A) of a molecule can be expressed as an algebraic equation, A=F+S+C 1 . Foreignness

In general, the greater the phylogenetic distance between the source of the antigen and the animal that will be used to produce the antiserum, the greater will be the antigenicity of the material. For example, human serum albumin is a better antigen in chickens than is duck serum albumin (Ivanyi and Valentova, 1966).

48

RICHARD M. HYDE

2. Size

The significance of particle size in determining the antigenicity of a molecule is indicated by the observation that nonantigenic or weakly antigenic substances can sometimes be made antigenic by absorbing them onto particulate carriers such as colloidion particles or charcoal (Landsteiner and Jacobs, 1932). Further, high molecular weight dextrans are antigenic in man while dextrans below 50,000 molecular weight appear to be nonantigenic (Maurer, 1965). The minimum molecular weight of protein antigens is usually accepted to be approximately 10,000. Generally, proteins of high molecular weight (above 40,000) are considered to be good antigens, while molecules below 10,000 are relatively ineffective in inducing antibody production. However, some low molecular weight polypeptide materials such as the pancreatic hormone glucagon (MW 3485) and synthetic polymers (MW 5000) can function as effective antigens (Unger et al., 1959; Maurer, 1963). The state of aggregation of the antigen also influences its ability to induce antibody formation. Nossal et al. (1963) studied the immune response of rats to various flagellar preparations from Salmonella adelaide and observed that the antigenicity of the materials increased with their complexity. Thus, the intact, isolated flagella induced the best antibody response; the low molecular weight protein, flagellin, prepared by acid treatment of the flagella, was the poorest antigen; and a polymer prepared from flagellin proved to be of intermediate antigenicity. A similar observation was made by Biro and Garcia (1965) who injected rabbits with either aggregated or aggregate-free human gamma-globulin (HGG). They found that the animals injected with aggregated HGG produced large amounts of precipitating antiHGG antibodies, whereas those injected with aggregate-free HGG produced no detectable antibodies and, in fact, became inimunologically unresponsive to HGG. A procedure has been developed recently whereby bovine serum albumin (BSA) can be rendered insoluble without concomitant alterations in its characteristic determinant groups (Hirata and Campbell, 1965). This finding led Hirata and Sussdorf (1966) to a comparison of the immunogenicity of insolubilized BSA and its native counterpart. They found that the use of the insoluble protein as immunizing antigen in rabbits resulted in both a larger number of responding animals and in markedly higher serum antibody levels than those obtained with soluble BSA. All of these observations may be related to the suggestion made by


49

Frei et al. (1965) and Nossal (1965) that phagocytosis of the antigen is a crucial step in the induction of an immune response. In fact, Medawar (1963) has proposed that the outcome of a direct encounter between immunologically competent lymphoid cells and antigen may be the specific loss of antibody-synthesizing ability, whereas antigen presented “uia the proper diplomatic channels” (i.e., after processing by phagocytic cells) results in the production of antibodies. It is known that serum proteins which have been aggregated are very actively cleared from the circulation b y phagocytic cells of the reticuloendothelial system (Thorbecke et al., 1960). If, indeed, phagocytosis is an essential prerequisite of antibody synthesis, then the influence that particle size and physical state have upon antibody formation would be relatively easily explained.

3. Chemistry The chemical nature of a molecule markedly influences its antigenicity. In general, proteins are better antigens than carbohydrates, while nucleic acids and lipids are not antigenic in themselves but can function as haptens when attached to appropriate carrier molecules. For example, it has been found that denatured deoxyribonucleic acid becomes immunogenic after it has formed a complex with methylated bovine serum albumin (Plescia et al., 1964). These same investigators have recently summarized some pertinent data dealing with the antigenicity of nucleic acids (Plescia et al., 1965). Unfortunately, the question that remains is why it is necessary to couple such complex materials to carrier molecules in order to induce the synthesis of antibodies specific for determinants of the nucleic acids. Another interesting, but as yet poorly understood observation on the effect of the carrier molecule on antihapten antibodies was made recently by Siskind et al. (1966).These investigators were studying the antibody response of guinea pigs to dinitrophenol hapten conjugated to various protein carriers and observed that the carrier affected not only the amount of antibody synthesized by the animal but also affected the avidity of the resulting antibody. For example, when bovine gamma-globulin was compared with bovine serum albumin as a hapten carrier, it was found that the animals produced more than twice as much antibody if the dinitrophenol were conjugated with the former carrier. There appears to be a minimum degree of complexity which a molecule must possess in order to be able to function as a complete antigen. This was demonstrated by the work of Pinchuck and Maurer

50

RICHARD M. HYDE

(1965b) who observed an apparently consistent nonimmunogenicity in mice of synthetic polymers of two amino acids, and the appearance of immunogenicity upon the introduction of a few mole percent of a third amino acid, without regard to the nature of the third amino acid. There are many other facets of the chemistry of a molecule which influence its antigenicity, e.g., the presence of charged determinant groupings, the shape of the molecule, susceptibility to the hydrolytic enzymes of the host, etc. Recent investigations on the structural requirements for antigenicity have been reviewed by Eisen and Pearce (1962), Maurer (1964, 1965), and by Sela (1966) and will not be discussed further here. In addition to these intrinsic characteristics of the molecule which determine its antigenicity, there are extrinsic factors which influence the quantity of antibody produced in response to a given antigen. In the following sections a brief discussion of some of these quantitative determinants of antigenicity will be presented. B. QUANTITYO F ANTIGEN Over 50 years ago, Smith and St. John-Brooks (1912) suggested that a direct mathematical relationship exists between the amount of antigen injected into an experimental animal and the peak antibody response obtained. Stevens (1956) reviewed several sets of published data on the relationship of dose to response and concluded that the relationship is a logarithmic one with a straight-line function. H e also stated that protein antigens appeared to be approximately 50 times as effective as polysaccharide antigens in inducing a comparable increase in antibody levels. That is to say, to obtain a 10-fold increase in homologous antibody titer, it would require 50 times more polysaccharide antigen than protein antigen. However, this straight-line relationship between dose and response may not always occur. Stille et al. (1959) have reported that the relationship of antibody response to increasing amounts of influenza vaccine will vary with the route of injection employed. They obtained a straight-line relationship by using the intracutaneous route of injection and a sigmoid curve when the subcutaneous route was used. This difference in the antibody response may have been due to differences in the rate of antigen absorption from the site of inoculation, but it in no way alters the fact that the amount of antigen employed plays a major role in determining the extent of the immune response. Normally, two effects follow the first injection of an antigen: (a) the production of specific antibodies; and ( b ) the development of in-


51

creased responsiveness to a second injection of the same antigen (the anamnestic response). However, under appropriate conditions, e.g., animal immaturity or excessive amounts of the antigen, the first injection may reduce or eliminate both of these responses with the resultant development of immune tolerance. This state, also referred to as immunological unresponsiveness, is specific for the antigen which induced it and is not comparable to the general immunologic anery which follows administration of immunosuppressive drugs (cf. Section V, F.). The phenomena of immunologic paralysis and antigen overload seen with carbohydrate and protein antigens, respectively, are examples of immune tolerance induced in adult animals. They will not be discussed here as this subject has been amply reviewed elsewhere (Smith, 1961; Hasek et al., 1962; Makinodan et al., 1965; Crowle, 1966). The minimum amount of antigen necessary to induce a demonstrable immune response will vary with the quality of the antigen under study, the method of immunization employed, as well as other factors discussed in this review. Some representative studies of the minimum amounts of antigen needed to induce an immune response are presented in Table I. Another variabIe which will influence the TABLE I MINIMUMAMOUNTSOF ANTIGEN NEEDEDTO INDUCE

Antigen Bovine serum albumin Bovine gammaglobulin Poliovirus type I Poliovirus type I1 Pneumococcal pol ysaccharide type I Flagellin

Micrograms injected

AN

IMMUNE RESPONSE

Route

Animal

Reference

60.0

IV

Rabbit

Farr and Dixon (1960)

6.0

IV

Rabbit

Farr and Dixon (1960)

4.0 0.8 0.05

IM IM IP

Man Man Mouse

Charney et al. (1961) Charney et al. (1961) Neeper and Seastone (1964)

0.001

SC

Rat

Nossal et al. (1964)

amount of antigen necessary to induce an immune response is the sensitivity of the assay method employed to detect the antibody produced. Obviously, if a very sensitive method of antibody detection is used, the quantity of antigen needed to induce a detectable immune response may appear lower, as it will take less antibody to reach the

52

RICHARD M. HYDE

threshold of the assay system. This fact makes it difficult to compare results such as those presented in the table, since different methods of assay were employed. In general, repeated injections of small amounts of antigen are employed in the production of antiserums. The range of amounts over which effective antigenicity can be demonstrated is extremely wide, Thus, Downie (1937b) found that he could effectively induce immunity to pneumococcal infection with injections of as little as 0.01 to 1 micrograms of polysaccharide, while he did not reach inhibitory levels until he employed 0.1 to 1 milligram amounts. Repeated injections of antigen, however, may not always be successful in inducing an anamnestic response. Heidelberger et al. (1946) failed to obtain a rise in antibody titer when they administered a booster injection of pneumococcal polysaccharide to human volunteers 2 years after their primary immunization. A similar absence of anamnesis was observed by Maurer (1957) in his studies on the antibody response of human volunteers to polyvinylpyrrolidone. In both of these studies the investigators were unable to elicit a rise in the titer of an antibody which was already present in the serum of the experimental subjects. A different type of refractoriness was observed by Cryan et al. (1966) in their attempts to produce antiserums specific for tumor antigens. They found that the appearance of antibody was transient and was unresponsive to booster inoculation. Antibody was detectable in one or two bleedings, but it subsequently disappeared and did not reappear following booster injection.

c. PRESENCE O F

SPECIFIC ANTIBODY

The three possible effects that specific antibody may have on immunization are: ( a ) suppression of the antibody response; ( b ) enhancement of antibody production; and ( c )no apparent effect. The influence that antibody exerts will depend upon the ratio of antigen to antibody as well as upon the immunologic status of the animal being used for antiserum production. Most of the recent investigations on the effect of antibody on immune responses have dealt with the primary immune response (antibody production following the first injection of antigen), since secondary antigenic stimulation is reasonably refractory to the presence of specific antibody, particularly when the amounts of antigen are increased, as they commonly are in booster injections. In general, large amounts of antibody inhibit a primary immune response while small amounts may enhance it. More specifically, if the ratio of antigen to antibody molecules in the immune precipitate


53

favors the antigen, antibody synthesis will ensue; whereas, if the ratio favors antibody then de novo production of antibodies may be inhibited (Leskowitz, 1960).The enhancing effect of low levels of specific antibody has been studied by Segre and Kaeberle (1962). They found that 3-week-old piglets deprived of colostrum (and hence possessing very little gamma-globulin) gave a markedly suppressed response to diphtheria and tetanus toxoid as compared to conventionally reared pigs. Further, they obtained successful antitoxin responses in the colostrum-deprived animals by mixing the toxoid with a minute amount of specific antitoxin, suggesting that the active principle in the colostrum was specific antibody which had been passively acquired from the mother by the colostrum-fed animals. This conclusion was substantiated by the observation that the enhancing effect of the antiserum could b e removed by specific absorption with toxoid. A similar enhancement of the antibody response by small amounts of specific antibody was observed in mice by Terres and Wolins (1961). In a subsequent publication, Terres and Stoner (1962) reported the results of their studies on the specificity of the antibody-mediated enhancement and showed that the augmentation of antibody production was demonstrable only to the antigens found in the specific precipitate. The mice demonstrated enhanced antibody production to both the antigen present in the precipitate as well as to the rabbit gamma-globulin that was employed as a heterologous antiserum source. However, the animals responded poorly to extraneous antigens incorporated in the injection materials. The suppressive effect of large amounts of passively acquired antibody on the primary immune response is well known (Osborn et al., 1952; Talmage et al., 1956; Perkins et al., 1958). However, the mechanism of this inhibition remains obscure. It is not known whether the effect is due to diversion of the antigen from its normal pathway to the induction of antibody synthesis or to a more direct effect that preformed antibody may have on the antibody-producing cells themselves. Uhr and Baumann (1961) found that antibody from heterologous species was more efficient in the inhibition of de novo antibody synthesis than was homologous antiserum. Moeller (1964) noted that antibody synthesis was not inhibited b y incubation of normal lymphoid cells prior to their transfer to, and stimulation in, X-irradiated recipients. Inhibition occurred if the mice were passively immunized prior to injection of the antigen or if the antiserum was added to the antigen prior to its injection into the animals. Tao and Uhr (1966) examined the capacity of enzyme-digested antibody preparations to

54

RICHARD M. HYDE

inhibit active antibody formation and concluded that inhibition was due to the fragments of the antibody molecule which contain the combining sites for antigen. All of the above observations support the concept that inhibition is brought about through interaction with the antigen, rather than through direct action on the antibody-forming cells themselves. Specific antibody appears to be without effect in an animal that has already received one injection of the antigen. Rowley and Fitch (1964) studied the effect of passive antibody on the hemolysin response of rats immunized with sheep erythrocytes. Although they noted inhibition of the primary response to the antigen, the secondary response (antibody production following booster injection) was unaffected by the presence of passively administered antibody. Wigzell (1966) employed the Jerne plaque technique to study antibody-induced suppression of hemolysin response at the level of the lymphoid cells actively synthesizing the antibody. He concluded that the inhibitory action of antibody was not due to direct suppression of cells already producing antibody, but was an effect mediated by removal from the system of some stimulus (possibly antigen) required for the maintenance of antibody production. The use of antigen-antibody precipitates in immunization procedures may offer certain advantages. For example, it is possible to employ such precipitates to produce antisera to the antigen portion of the complex when this portion is too toxic for injection in the uncombined form. Copeman et al. (1922) used underneutralized mixtures of diphtheria toxin and antitoxin for human immunization before toxoid had been developed. Specific precipitates have also been employed to obtain antiserums of high specificity. Thus, Treffers and Heidelberger (1941) obtained antibody to horse gamma-globulin b y immunizing rabbits with a precipitate of pneumococcal polysaccharide (nonantigenic in rabbits) and its corresponding horse antibody. Similarly, Pace and Pappenheimer (1959) obtained a highly specific antistreptococcal diphosphopyridine nucleotidase rabbit serum by using precipitates of the enzyme and its homologous rabbit antibody as the immunizing agent.

D. ACE

OF

ANIMAL

The immune response of most animals is poorly developed at birth but rapidly matures during infancy and reaches maximum efficiency during adulthood. Usually, a decrease in the efficiency of antibody production is seen during senescence. The absence of antibody forma-


55

tion in the fetus is probably due to the placental barrier which shields the offspring from exogenous antigenic stimuli. The ability of various animal species to produce antibodies in fetal life, or as neonates, has been reviewed recently by J. F. A. P. Miller (1966)and will not be discussed further here. The process of aging is commonly accompanied by a decrease in immune responsiveness. Goullet and Kaufmann (1965) examined the ability of young (3 months of age) and old (22 months of age) rats to produce antibodies to bovine serum albumin. They concluded that antibody production in the “aged” animals was definitely inferior to that obtained in the younger animals. A similar conclusion was reached b y Aoki and Teller (1966) who compared the ability of young and old mice to reject histoincompatible tumor homogenates. They also observed a decreased ability of the older mice to form cytotoxic and opsonic antibodies. However, not all animal species may show this age-associated decrease in immunologic competence. Wolfe et al. (1957) were unable to demonstrate a significant decrease in precipitin production by chickens up to 8 years of age. Comprehensive studies of the age-associated change in the immune system of mice have been conducted to ascertain the physiologic basis of this observation. Makinodan and Peterson (1964) studied the primary antibody-forming potential of spleen cells from mice ranging in age from 1 to 126 weeks. They concluded that the growth and aging of the immune system of this animal are due to an increase and decrease, respectively, in the number of potential antibody-forming cells rather than to a change in the efficiency of the cells. The relative number of potential antibody-forming cells increased approximately 600-fold by the fortieth week of life. After 40 weeks of age the number decreased gradually. At 120 weeks it was only 25% of that of the 40-week-old mice, thus suggesting that the immune potential of an individual does not remain at a plateau level after young adulthood but actually decreases. If this is the case, then the common practice of adjusting the dose of antigen in terms of body weight is of questionable validity. Wigzell and Stjernsward (1966) examined the immune responsiveness of mice ranging from neonates to animals 36 months of age. They observed a rapid exponential increase in immunologic reactivity immediately after birth which was followed by a slower rise. The peak of reactivity was reached when the animals were about 6 months of age. Although they noted that immunologic reactivity declined after this age, no evidence was obtained to indicate that individual anti-

56

RICHARD M. HYDE

body-forming cells from senile mice differed from those of younger animals. They concluded, as did Makinodan and Peterson, that the decline in immunologic responsiveness seen with increasing age is due to a decrease in the relative and absolute numbers of potential antibody-forming cells.

E. ADJUVANTS Adjuvants may be defined as substances which, when mixed with the antigen to be injected, enhance antigenicity and increase the amount of antibody produced over that obtained by injection of the antigen alone. To site a specific example, Dixon et al. (1966) reported that the incorporation of hernocyanin in incomplete Freund’s adjuvant enhanced its immunogenicity approximately 1000-fold. Many materials have been reported to have adjuvant activity, e.g., zymosan (Cutler, 1960), crystalline silica particles (Pernis and Paronetto, 1962), branched-chain, saturated, noncyclic hydrocarbons (Wilner et al., 1963),aliphatic nitrogenous bases (Gall, 1966),lipid (Youngnerand Axelrod, 1964), acrylamide gel (Weintraub and Raymond, 1963), cellulose (Olovnikov and Gurvich, 1966), saponin (Gill, 1965), endotoxin (Munoz, 1964), and calcium alginate (Amies, 1959). Infection of mice with the lactic dehydrogenase virus has even been found to greatly enhance their immune response to human gamma-globulin (Notkins et ul., 1966).Aluminum salts and water-in-oil emulsions are the most widely used adjuvants and they have been discussed extensively in other reviews (White, 1963; Edsall, 1966; Freund, 1947, 1951, 1956; Berlin, 1963), hence only brief consideration of them will be given here. The effect of the concentration of antigen in water-in-oil adjuvants on the initiation of antibody synthesis was studied by Farr and Dixon (1960). They noted that the critical factor in eliciting an antibody response in rabbits appeared to be the concentration of antigen in the aqueous phase. By maintaining the concentration of antigen constant, it was possible to reduce the total amount of antigen injected by a factor of 10 without significantly reducing the proportion of animals producing antibody. The use of adjuvants may make it possible to convert an apparently nonantigenic substance into an effective antigen. Maurer and Lebovitz (1956) demonstrated that modified fluid gelatin was antigenic in rabbits only when it was injected in water-in-oil emulsions. No antibody production was noted when the antigen was injected as a saline solution or as an alum precipitate.


57

A substance may demonstrate adjuvant activity with one antigen and yet be completely devoid of any ability to enhance the immune response to a second antigen. Gamble (1966) studied the effect of phytohemmagglutinin on the primary antibody response of mice and noted that, while a significant enhancement of antibody production occurred to rat erythrocytes, there was an unexpected suppression of antibody formation to human gamma-globulin.

F. OTHER Any factor which upsets the physiologic balance of the host can potentially interfere with the immune response; although, in general, a severe imbalance must occur before marked inhibition of antibody synthesis is noted. For example, Northey (1965)reported that shaved rabbits, maintained at 4" C. prior to and during the period of immunization with various protein antigens, failed to demonstrate any appreciable impairment of antibody production. In an earlier study, Trapani (1960)had observed a slight decrease in the immune response of rabbits similarly shaved and housed at -15°C. The hormonal balance of the host may play a significant role in determining the outcome of immunization. Sang and Sobey (1954) reported that both pregnant and lactating rabbits have a significantly decreased response to diphtheria or tetanus toxoid. Likewise, the nutritional status of the animal may influence the immune response. Panda and Combs (1963) studied the agglutinin response of 4-weekold chicks fed diets deficient in vitamin A, pantothenic acid, or riboflavin and noted an impairment of antibody production which, when compared with that obtained with control animals fed a complete diet, was significant at the 0.01 level. The inhibition of antibody production by immunosuppressive drugs has been amply reviewed elsewhere (Berenbaum, 1965; Schwartz, 1965; Makinodan et al., 1965), as has the competition of antigens occasionally observed when animals are injected with a mixture of several antigenic materials (Adler, 1959, 1964), consequently these subjects will not be considered here. The effect of the time interval between primary and secondary injections of antigen on the immune response has also been investigated. Brown et al. (1964) immunized 373 human infants with diphtheria, tetanus, pertussis, and polio vaccines and found that the second injection was more efficient if the interval between primary and secondary injection was increased from 1 month to 2 months. The efficiency was evidenced by higher antibody titers after secondary

58

RICHARD M. HYDE

injection, as well as by a greater response to booster injections administered 1 year later. The variation in secondary antibody response as a function of the interval between injections has also been studied by Fecsik et al. (1964).These investigators employed a subcutaneous injection of diphtheria toxoid in mice as their test system and found that, as the interval between two antigen injections lengthens, the magnitude of the secondary response increases. The optimal time for secondary stimulation of mice with the dose of antigen employed was found to be between 20 and 40 days. The duration of the capacity to respond to a booster injection has also been investigated. An apparently unimpaired capacity to respond to booster injections of tetanus toxoid has been found as long as 9 to 11 years after primary immunization (Turner et al., 1954; Peterson et at., 1955). The anamnestic reactivity of rabbits to sheep erythrocyte hemolysin has been reported to persist for at least 2.5 years (Taliaferro and Taliaferro, 1966). VI. Specific Examples of Antiserum Production

The selected bibliography (Section VIII) has been included in this review to serve as a guide to immunization procedures currently in use. Appropriate review articles have been cited whenever possible. The review by Ouchterlony (1962) is also recommended as a source of references for the production of specific antiserums. It includes an antigen-index bibliography which allows the investigator to obtain a reference to any particular antigen employed as an immunizing agent. VII. Conclusion Because of the variation in antibody response seen in experimental animals, at least two precautions should be taken when establishing an immunization schedule. First, sufficient numbers of animals should be used to assure that, even if the proportion of animals producing antibody is low, some of them will produce the antibody desired. Second, periodic bleedings should be done during the immunization period and the serum should be examined for the presence of antibody. When the antibody level has reached a plateau, the animal can be exsanguinated or bled successively over a period of days. From the preceding brief discussion of some of the variables that influence the outcome of an immunization procedure, it is obvious that there is no universal vaccination procedure that will guarantee maximum antibody production. How, then, does the investigator who


59

wishes to produce an antiserum proceed? One possibility is to search the literature and obtain a reference article that is appropriate to the needs of the experiment. Alternately, an immunization procedure can be determined empiricaIIy. It is hoped that some of the information contained in this review will be helpful, regardless of the approach taken. ACKNOWLEDGMENTS The preparation of this article has been assisted by U.S.P.H.S. Training Grant 5TlAI162. The author is grateful to Mrs. Kay Christiansen for excellent clerical assistance and to Dr. Robert A. Patnode for his critical reading of the manuscript.

VI II. Selected Bib1iogra phy

A. BLOOD

1 . Cellular Elements Dietrich, F. M. (1966). The immune response to heterologous red cells in mice. Immunology 10,365-376. Gray, J. G., Monaco, A. P., Wood, M. L., and Russell, P. S. (1966). Studies on heterologous anti-lymphocyte serum in mice. I. In vitro and in uiuo properties. I. Immunol. 96,217-228. Harris, S., and Harris, T. N. (1966). Suppression of rabbit lymph node cells by rabbit anti-leucocyte serum demonstrated in vitro by the antibody plaque test.]. Immunol. 96,478-487. Kochan, I., Christopher, J. A., and Kupchyk, L. (1966). Study on the cellular factor of delayed hypersensitivity.]. Allergy 38,280-289. Ovary, Z. (1964). Antigenicity of hemoglobin and its constituents. I. Antigenicity of human adult hemoglobin and its structural units (alpha and beta chains). fmmunochemistry 1,241-248. Reichlin, M., Hay, M., and Levine, L. (1965). Immunochemical studies on inter-species molecular hybrids of hemoglobin. lmmunochemistry 2,337-350. Stone, W. H., and Irwin, M. R. (1963). Blood groups in animaIs other than man. Aduan. Immunol. 3,315-350.

2 . Plasma (Serum) Berglund, G. (1965). Preparation of antiserum to an antigen of low molecular weight (fibrinopeptide).Nature 206,523-524. Bergmann, F. H., Levine, L., and Spiro, R. G . (1962). Fetuin: Immunochemistry and quantitative estimation in serum. Biochim. Biophys. Acta 58,41-51. Bergquist, L. M., Carroll, V. P., Jr., and Searcy, R. L. (1961). Evaluation of a specific antiserum for serum-P-lipoprotein estimations. Lancet 1,537-538. Fudenberg, H. H. (1965).The immune globulins. Ann. Rev. Microbiol. 19,301-338. Gitlin, D. (1966). Current aspects of the struchire, function and genetics of the immunoglobulins. Ann. Reu. Med. 17,l-22. Matheson, A., Jensen, R. S., and Donaldson, D. M. (1966). Serologic relationships between P-lysins of different species.]. Immunol. 96,885-891.

60

RICHARD M. HYDE

Robbins, K. C., and Summaria, L. (1966). An immunochemical study of human plasminogen and plasmin. lmmunochemistry 3,29-40. West, C. D., Davis, N. C., Forristal, J., Herbst, J., and Spitzer, R. (1966). Antigenic determinants of human PlC- and P l G . globulins.]. Immunol. 96,650-658. Williams, R. C., Jr. (1964). Anti-albumin antibodies after immunization with whole and and enzyme-digested autologous rabbit albumins.]. Immunol. 93,850-859.

B. TISSUESA N D

ORGANS

Beernink, K. D., Courcon, J., and Grabar, P. (1965). Immunochemical studies on bone marrow of the rat. Immunology 9,377-390. Clarke, W. M., and Fowler, I. (1960). The inhibition of lens-inducing capacity of the optic vesicle with adult lens antisera. Deuelop. Biol.2,155-172. Dumonde, D. C. (1966).Tissue-specific antigens. Aduun. Immunol. 5,245-412. Flickinger, R. A. (1962).Embryological development of antigens. Aduun. Immunol. 2, 310-366. Harvie, N. R., and Elberg, S. S. (1961). Observations on cytotoxic effect of antihistiocyte serum. PTOC.Sac. Exptl. Biol.Med. 107,8-11. Hook, W. A., Muschel, L. H., and Faber, J. E. (1966).Antibodies to X-irradiated rabbit testes. Immunology 10,245-248. Inoue, K. (1961).Precipitin reactions and developmental arrest by antisera in amphibian embryos. Develop. Biol. 3,657-683. Kurata, Y., and Okada, S. (1966). Immunological studies of insoluble lipoproteins. I. Antigenic analysis of thyroidal lipoproteins. Intern. A d . Allergy Appl. lmmunol. 29,495-509. LeVeen, H. H., Falk, G., and Schatman, B. (1961). Experimental ulcerative colitis produced by anticolon sera. Ann. Surg. 154,275-280. Loewi, C., and Muir, H. (1965).The antigenicity of chondromucoprotein. Immunology 9,119-128. Milgrom, F., Kasukawa, R., and Calkins, E. (1966). Studies on antigenic composition ofamyloid.]. Immunol. 96,245-252. Myers, J., Frei, J. V., Cohen, J . J., Rose, B., and Richter, M. (1866).Basement membrane specific antisera produced to solubilized tissue fractions. Immunology 11,155-162. Rajam, P. C., Bogoch, S., Rushworth, M. A., and Forrester, P. C . (1966). Antigenic constituents of basic proteins from human brain. Immunology 11,217-222. Rothbard, S., and Watson, R. F. (1965). Immuiiologic relations among various animal collagens.]. Exptl. Med. 122,441-454. Smith, D. E., and Lewis, Y. S. (1961). Preparation and effects ofan anti-mast cell serum. ]. Exptl. Med. 113,683-692. Spragg, J., Austen, K. F., and Haber, E. (1966). Production of antibody against bradykinin: demonstration of specificity by complement fixation and radio-immunoassay. ]. Zmmunol. 96,865-871. Tamanoi, I., Yagi, Y., and Pressman, D. (1961). Rate of localization of anti-rat lung antibody. Proc. Sac. Exptl. Biol. Med. 106,769-772. Warren, B., Johnson A. G . , and Hoobler, S. W. (1966). Characterization of the reninantirenin system.]. Exptl. Med. 123,1109-1128. Yakulis, V. J., and Heller, P. (1962). The detection of myoglobin by means of imniunologic technics. Am.]. Clin. Puthol. 37,253-256.

ANTISERUM

PRODUCTION IN EXPERIMENTAL

ANIMALS

61

C. BIOLOGICAL FLUIDS 1 . Hormones Bourdel, G. (1961). Effect of rabbit antiserum to sheep pituitary interstitial-cell stimulating hormone in immature female rats. Gen. Comp. Endocrinol. 1,375-380. Geschwind, I. I. (1963). The chemistry and immunology of gonadotropins. In “Gonadotropins” (H. H. Cole, ed.). Freeman, San Francisco, California. Goodfriend, T., Fasman, G., Kemp, D., and Levine, L. (1966). Immunochemical studies of angiotensin. lmmunochemistry 3,223-232. Levi-Montalcini, R., and Angeletti, P. U. (1966). Immunosympathectomy. Pharmacol. Rev. 18,619-629. Levy, R. P., and Sampliner, J. (1962). Prolactin, immunologic evidence of species specificity. Proc. Soc. Exptl. Biol. Med. 109,672-673. McGarry, E. E., and Beck, J. C. (1963). Some studies with antisera to human FSH. Fertility Sterility 14,558-564. Pope, C. G. (1966).The immunology of insulin. Aduan. Immunol. 5,209-244. van Hell, H., Goverde, B. C., Schuurs, A. H. W. M., D e Jager, E., Matthijsen, R., and Homan, J. D. H. (1966). Purification, characterization, and immunochemical properties of human chorionic gonadotropin. Nature 212,261-262. Wallace, A. L. C., and Sobey, W. R. (1965). Immunological studies of a bovine growth hormone preparation. J . Endocrinol. 32,321-327. Weigle, W. 0. (1965). The induction of autoimmunity in rabbits following injection of heterologous or altered homologous thyroglobulin. J . Exptl. Med. 121,289-308. Wolstenholme, G. E. W., and Cameron, M. P., eds. (1962). “A Ciba Symposium: Immunoassay of hormones.” Churchill, London. Zimmering, P. E., Beiser, S. M., and Erlanger, B. F. (1965). Purification and some properties of anti-testosteroneantibodies. J . Immunol. 95,262-272.

2. Enzymes Arnon, R., and Schechter, B. (1966). Immunological studies on specific antibodies against trypsin. Immunochemistry 3,451-462. Barrett, J. T. (1965). An immunochemical study of procarboxypeptidase A and its enzyme. Immunology 8,129-135. Barrett, J. T., and Thompson, L. D. (1965). Immunochemical studies with chymotrypsinogen A. Immunology 8,136-143. Cinader, B. (1957).Antibodies against enzymes. Ann. Reu. Microbiol. 11,371-390. Cinader, B. (1963). Antibodies to enzymes-a three-component system. Ann. N . Y. Acad. Sci. 103,493-1154. Glimp, H. A., and Tillman, A. D. (1965). Effect of jackbean urease injections on performance, anti-urease production and plasma ammonia and urea levels in sheep. J . Animal Sci. 24,105-112. Lehrer, H. I., and van Vunakis, H. (1965). Iminunochemical studies on carboxypeptidase A. Immunochemistry 2,255-262.

3. Other Abelli, G. (1962). Immunoelectrophoretic studies on colostrum and milk using specific immune sera. Panminerua Med. (EnglishEd.)4,181-184.

62

RICHARD M. HYDE

Abuelo, J. G., and Ovary, Z. (1965). Dinitrophenylated bacitracin A as an antigen in the guineapig.J. Zmmunol. 95,113-117. Beard, R. L. (1963). Insect toxins and venoms. Ann. Reu. Entomol. 8,1-18. Halloran, M. J., and Parker, C. W. (1966). The production of antibodies to mononucleotides, oligonucleotides and DNA.]. Zmmunol. 96,379-385. Hochwald, G. M., and Thorbecke, G. J, (1962).Use ofanantiserumagainstcerebrospinal fluids in demonstration of trace proteins in biological fluids. PTOC.Soc. E x p t l . Biol. Med. 109,91-95. J O I I S S O I ~ ,J., and Paleus, S. (1966). Studies on the antigenic capacity of chicken atrtl bovine cytochrome c. Intern. Arch. Allergy A p p l . Zmmunol. 29,272-277. Kaminski, M. (1965). The analysis of the antigenic structure of protein molecules. P‘TOgT. Allergy 9,79-157. Katsh, S., and Katsh, G . F. (1965). Perspectives in immunological control of reproduction. Paci$c Med. Surg. 73, Suppl. l A , 28-43. Keegan, H. L., Whittemore, F. W., Jr., and Flanigan, J. F. (1961). Heterologous antivenin in neutralization of North American coral snake venom. Public Health Rept. (U.S.)76,540-542. Mange, A. P., and Stone, W. H. (1962). Tests on immune sera produced against calf thymus chromosome. Proc. Soc. E x p t l . Biol. Med. 109,42-44. Rohbins, K. C., Wu, H., and Hsieh, B. (1966). Physical, chemical, and immunochernical studies on a low ragweed pollen antigen. Zmmunochemistry 3 , 7 1 4 0 . Rumke, P., and Sluyser, M. (1966).Antigenicity of histones. Bi0chem.J. 101,1C-2C. Stevens, K. M., and Fost, C. A. (1964). Sperm and antibody production in rabbits following immunization with sperm and semen. PTOC. Soc. E x p t l . B i d . Med. 117, 125-127.

D.

MALIGNANCIES

Boyle, W., and Davies, D. A. L. (1966). Antigens of the surface of mouse ascites tumor cells. I. Studies with rabbit anti-mouse cell sera. Immunology 11,353-360. Caso, L. V. (1965). The relation of the immune reaction to cancer. Aduan. Cancer Res. 9,47-141. Day, E. D. (1965). “The Immunochemistry of Cancer.” Thomas, Springfield, Illinois. Garb, S., Stein, A. A., and Sims, G. (1962). The production of anti-human leukemic serum in rabbits. J. Zmmunol. 88,142-152. Klein, G. (1966).Tumor antigens. Ann. Reu. Microbiol. 20,223-252. Old, L. J., and Boyse, E. A. (1964).Immunology of experimental tumors. Ann. Reu. Med. 15,167-186. Pilch, Y. H. (1964). Immunogenicity of cell free extracts of neoplastic tissue. S U T ~ . F O ~ U V15,348-350. ~

E. MICROORGANISMS Barber, C., Vladoianu, I. R., and Dimache, G. (1966). Contributions to the study of Salmonella immunological specificity of proteins separated from Salmonella typhi. Immunology 11,287-296. Basaca-Sevilla, V., Pesigan, T. P., and Finkelstein, R. A. (1964). Observations on serological responses to cholera immunization. Am. /. Trop. Med. H y g . 13, 100107.


63

Blyth, W. A., Reeve, P., Graham, D. M., and Taverne, J. (1962). The production of antisera that neutralize inclusion blennorrhoea virus. Brit. ]. Exptl. Pathol. 43, 340343. Cuadrado, R. R. (1966). Agar gel diffusion method for the study of antigenic components of mumps virus. J. Immunol. 96,892-897. Daniel, T. M. (1965). Observations on the antibody response of rabbits to mycobacterial antigens. J. Immunol. 95,100-108. Eaton, M . D. (1965). Pleuropneumonia-like organisms and related forms. Ann. Reu. Microbial. 19,379-406. Estrup, F., and Santer, M. (1966).Immunological analysis of the proteins of Escherichia coli ribosomes.]. Mol. Biol. 20,447-452. Houba, V., and Hana, I. (1966).The difference in immunological characteristics of two streptokinases. Immunology 11,387-398. Hsiung, C. D., Chang, P. W., Cuadrado, R. R., and Isacson, P. (1965). Studies of parainfluenza viruses. 111. Antibody responses of different animal species after immunization. J. Immunol. 94,67-73. Kwapinski, J. B. (1966). Serological and chromatographic characterization of exoantigens of the Dermatophilus. Australian]. E x p t l . Biol.Med. Sci. 44,87-92. Levine, L., Wasserman, E., and Murakami, W. T. (1966). Immunochemical studies on bacteriophage DNA. VI. Renaturation of T, DNA. Immunochemistry 3,41-50. McCarty, M., and Morse, S. I. (1964). Cell wall antigens of Cram-positive bacteria. Advan. Immunol. 4,249-286. Matthews, R. E. F. (1957). “Plant Virus Serology.” Cambridge Univ. Press, London and New York. Nell, E. E., and Hardy, P. H. (1966). Studies on the chemical composition and immunologic properties of a polysaccharide from the Reiter treponeme. lmmunochemistry 3,233-246. Norris, J. R. (1962). Bacterial spore antigens: A review. J . Gen. Microbiol. 28, 393408. Salvin, S. B. (1963).Immunologic aspects of the mycoses. Progr. Allergy 7,213-331. van Heyningen, W. E., and Arseculeratne, S. N. (1964). Exotoxins.Ann. Reu. Microbiol. 18,195-216. Weidanz, W. P., Jackson, A. L., and Landy, M. (1964). Some aspects of the antibody response of rabbits to immunization with enterobacterial somatic antigens. Proc. Soc. E x p t l . Biol. Med. 116,832-837. REFERENCES Adler, F. L. (1959).In “Mechanisms of Hypersensitivity” (J. H. Shaffer, G. A. LoGrippo, and M. W. Chase, eds.), p. 539. Little, Brown, Boston, Massachusetts. Adler, F. L. (1964).Progr. Allergy 8,41-57. Altman, P. L., and Dittmer, D. S. (1964). “Biology Data Book.” Federation Am. SOC. Exptl. Biol., Washington, D.C. Amies, C. R. (1959).]. Pathol. Bacterial. 77,435-442. Aoki, T., and Teller, M. N. (1966).Cancer Res. 26,1648-1652. Arnason, B. G., and Waksman, B. H. (1964).Aduan. Tuberc. Res. 13,l-97. Arquilla, E. R., and Finn, J. (1965).]. E x p t l . Med. 122,771-784. Avery, 0.T., and Goebel, W. F. (1933).]. Exptl. Med. 58,731-755. Berenbaum, M. C. (1965).Brit. Med. Bull. 21,140-146.

64

RICHARD M. HYDE

Berlin, B. S. (1963).Ann. Allergy 21,82-90. Bernheimer, A. W., Caspari, E., and Kaiser, A. D. (1952).J.E x p t l . Zool.119,23-35. Biro, C. E., and Garcia, G. (1965). Immunology 8,411-419. Breebaart, A. C., and James-Witte, J. (1959).A m . ] . Ophthalmol. 48,37-47. Brown, G . C., Volk, V. K., Gottshall, R. Y., Kendrick. P. L., and Anderson, H. D. (1964). Public Health Rept. ( U S . )79,585-602. Burnet, M. (1964).Nature 203,451-454. Campbell, D. H., Carvey, J. S., Cremer, N. E., and Sussdorf, D. H. (1964). “Methods in Immunology.” Benjamin, New York. Charney, J., Tytell, A. A., Machlowitz, R. A,, arid Hilleman, M. R. (1961).J. Am. Med. A S ~ O177,591-595. C. Chase, M . W. (1H50).Attii. RUG.Microbiol. 13, 349-376. Chase, M. W. (1965). Med. Clin. N . Am. 49,1613-1646. Copeman, S. M., O’Brien, R. A., Eagleton, A. J., and Glenny, A. T. (1922). B r i t . ] . E x p t l . Puthol. 3,42-50. Crowle, A. J. (1966).Ann. Allergy 24, 195-214. Cryan, W. S., Hyde, R. M., and Garb, S. (1966).Cancer Res. 26,1458-1465. Cutler, J. L. (1960).J.Immunol. 84,416-419. Dixon, F. J., Jacot-Guillarmod, H., and McConahey, P. J. (1966).J . Immunol. 97, 350355. Downie, A. W. (1937a).J.Pathol. Bacteriol. 45,149-155. Downie, A. W. (1937b).J.Pathol. Bacteriol. 45,131-147. Draper, L. R., and Sussdorf, D. H. (1957).]. Infect. Diseases 100,147-161. Edsall, G. (1963). In “Modern Trends in Immunology” (R. Cruickshank, ed.). Butterworth, London and Washington, D.C. Edsall, G . (1965).Med. Clin. N.Am. 49,1729-1743. Edsall, G. (1966).Ann. Reu. Med. 17,39-48. Eisen, H. N., and Pearce, J. H. (1962).Ann. Reu. Microbiol. 16,101-126. Farr, R. S., and Dixon, F. J., Jr. (1960).J.Immunol. 85,250-257. Fecsik, A. I., Butler, W. T., and Coons, A. H. (1964).J.E x p t l . Med. 120,1041-1049. Felton, L. D., Sutliff, W. D., and Steele, B. F. (1935).J.Infect. Diseases 56,101-110. Fernando, A. N. (1960).A.M.A. Arch. Ophthalmol. 63,515-539. Fink, M. A., and Quinn, V. A. (1953).]. Immunol. 70,61-67. Francis, T., Jr., and Tillett, W. S. (l930).J.E x p t l . Med. 52,573-585. Frei, P. C., Benacerraf, B., and Thorbecke, G. J. (1965). Proc. Natl. Acad. Sci. U . S. 53, 20-23. Freund, J. (1947).Ann.Reu. Microbiol. 1,291-308. Freund, J. (1951).A m . ] . Clin. Pathol. 21,645-656. Freund, J . (1956).Advan. Tuberc. Res. 7,130-148. Gall, D. (1966).Immunology 11,369-386. Gamble, C. N. (1966). Intern. Arch. Allergy Appl. Immunol. 29,470-477. Gill, B. S. (1965).]. Comp. Pathol. Therap. 75,233-240. Good, R. A., and Papermaster, B. W. (1964).Aduan. Zmmunol. 4,l-115. Goullet, P., and Kaufmann, H. (1965).Experientia 21,46-47. Hasek, M., Lengerova, A,, and Vojtiskova, M. (1962). “Mechanisms of Immunological Tolerance.” Academic Press, New York. Heidelberger, M., Macleod, C. M., Kaiser, S. J., and Robinson, B. (1946).]. E x p t l . Med. 83,303-320. Hilleman, M. R. (1964).An,. Rev. Respirat. Diseases 90,683-706.


65

Hingson, R. A,, Davis, H. S., and Rosen, M. (1963).Military Med. 128,516-524. Hirata, A. A., and Campbell, D. H. (1965).lmmunochemistry 2,195-205. Hirata, A. A., and Sussdorf, D. H. (1966).J.Zmmunol. 96,611-613. Hyde, R. M., Bennett, A. J., and Garb, S. (1965). Intern. Arch. Allergy Appl. Immunol. 28,271-279. Ipsen, J., Jr. (1954).J . Zmmunol. 72,243-247. Ipsen, J., Jr. (1959).J . Immunol. 83,448-457. Ipsen, .I Jr. ., (1961).J . Zmmunol. 86,50-55. Ivanyi, J., and Valentova, V. (1966).Folia Biol. (Prague)12,36-48. Jarrett, W. F. H., and Sharp, N. C. C. (1963).]. Perasitol. 49,177-189. Jemski, J . V., and Phillips, G. B. (1965).In “Methods of Animal Experimentation” (W. I. Gay, ed.). Academic Press, New York. Landsteiner, K., and Jacobs, J. (1932).Proc. Soc. Exptl. B i d . Med. 29,570-571. Lepper, M. H., and Wolfe, E. K. (1966).Bacteriol. Reu. 30,485-698. Leskowitz, S . (1960).J . Immunol. 85,56-66. McDevitt, H. O., and Sela, M. (1965).J.Exptl. Med. 122,517-531. Makinodan, T., and Peterson, W. J. (1964).J . Zmmunol. 93,886-896. Makinodan, T., Albright, J. F., Perkins, E. H., and Nettesheim, P. (1965). Med. Clin. N . Am. 49,1569-1596. Mantyjarvi, R. (1965).Acta Pathol. Microbiol. Scand. 65,581-586. Mascoli, C . C., Leagus, M. B., Weibel, R. E., Stokes, J., Jr., Reinhart, H., and Hilleman, M. R. (1966).Proc. Soc. Exptl. Biol. Med. 121,1264-1268. Maurer, P. H. (1957).J.Immunol. 79,84-88. Maurer, P. H. (1963).J.Zmmunol. 90,493-504. Maurer, P. H. (1964).Progr. Allergy 8,l-40. Maurer, P. H. (1965).Med. Clin. N.Am. 49,1505-1516. Maurer, P. H., and Lebovitz, H. (1956).J.Zmmunol. 76,335-341. Medawar, P. B. (1963). In “Acquired or Natural Immune Tolerance Toward Defined Protein Antigens” (A. Bussard, ed.). C. N. R. S. Symposium, Academic Press, New York. Menzin, A. W. (1961).Arch. Internal Med. 107,409-429. Menzoian, J. O., and Ketchel, M. M. (1966).Nature 211,133-135. Middlebrook, G . (1961). Bacteriol. Reu. 25,331-346. Miller, J. F. A. P. (1966).Brit. Med. Bull. 22,21-26. Miller, T. A. (1966).J.Parasitol. 52,512-519. Moeller, G. (1964). Transplantation 2,405-415. Moreland, A. F. (1965). In “Methods of Animal Experimentation” (W. I. Gay, ed.). Academic Press, New York. Munoz, J. (1964).Aduan. Zmmunol. 4,397-440. Neeper, C. A,, and Seastone, C. V. (1964).J . Zmmunol. 93,867-871. Northey, W. T. (1965).J . Zmmunol. 94,649-657. Nossal, G . J . V. (1965).Australasian Ann. Med. 14,321-328. Nossal, G . J. V., Ada, G. L., and Austin, C. M. (1963).Nature 199,1257-1262. Nossal, G. J. V., Ada, G. L., and Austin, C. M. (1964). AustralianJ. Exptl. Biol. Med. Sci. 42,283-294, Notkins, A. L., Meryenhagen, S. E., Rizzo, A. A., Scheele, C., and Waldmann, T. A. (1966).J.E x p t l . Med. 123,347-362. Olovnikov, A. M., and Gurvich, A. E. (1966).Nature 209,417-419. Osborn, J. J., Dancis, J., and Julia, J. F. (1952).Pediatrics 10,328-334.

66

RICHARD M. HYDE

Ouchterlony, 0. (1962).Progr. Allergy 6,30-154. Pace, M. G., and Pappenheimer, A. M. (1959).J.Immunol. 83,83-86. Panda, B., and Combs, G. F. (1963).Proc. SOC. Exptl. Biol. Med. 113,530-534. Papermaster, B. W., Condie, R. M., Finstad, J., and Good, R. A. (1964).J . Exptl. Med. 119,105-130. Parks, J. J,, Leihowitz, H. M. I., and Maumenee, A. E. (1961).J.Immunol. 87, 199-204. Perkins, F. T., Yetts, R., and Gaisford, W. (1958).Brit. Med. J. 2,68-71. Pernis, B., and Paronetto, F. (1962).Proc. Soc. E x p t l . Biol. Med. 110,390-392. Peterson, J. C., Christie, A., and Williams, W. C:. (1955). Am. J. Diseases Children 89, 295-303. Phillips, J. H., and Yardley, B. J. (1960). Nature 188,728-730. Pinchuck, P., and Maurer, P. H. (1965a).J.Exptl. Med. 122,673-679. Pinchuck, P., and Maurer, P. H. (1965b).J.Exptl. Med. 122,665-671. Pinkerton, W., and Webber, M. (1964).Proc. S O C . Exptl. Biol. Med. 116,959-961. Plescia, 0 .J., Braun, W., and Palczuk, N. C. (1964). Proc. Natl. Acad. Sci. U . S. 52,279285. Plescia, 0. J., Braun, W., and Palczuk, N . C. (1965). In “Molecular and Cellular Basis of Antibody Formation” (J. Sterzl, ed.). Academic Press, New York. Riley, H. D., Jr. (1966).Pediat. Clin. N . Am. 13,75-104. Rowley, D. A., and Fitch, F. W. (1964),J.Exptl. Med. 120,987-1005. Sang, J . H., and Sobey, W. R. ( 1 9 5 4 ) ~Immunol. . 72,52-65. Schwartz, R. S. (1965).Progr. Allergy 9,246-303. Segre, D., and Kaeberle, M. L. (1962).J.Immunol. 89,782-789. Sela, M . (1966).Advan. Immunol. 5,29-129. Siskind, G . W., Paul, W. E., and Benacerraf, B. (1966).]. E x p t l . Med. 123,673-688. Smith, J. H., and St. John-Brooks, R. (1912).J.H y g . 12,77-107. Smith, R. T. (1961).Aduan. Immunol. 1,67-129. Smolens, J., Stokes, J., Jr., and Vogt, A. B. (1957).]. Immunol. 79,434-439. Soulsby, E. J. L. (1962).Aduan. Immunol. 2,265-309. Stephens, J. M. (1959).Can.J . Microbiol. 5,203-228. Stevens, K. M . (1956).J.Imrnunol. 76,187-191. Stille, W. T., Woolridge, R. L., and Gundelfinger, B. F. (1959).J . Lab. Clin. Med. 53, 751-754. Sulzberger, M. B. (1929).Arch. Dermatol. Syphilol. 20,669-697. Taliaferro. W. H., and Taliaferro, L. G . (1966).Proc.Natl. Acad. Sci. U. S. 56,1151-1154. Talmage, D. W., Freter, G . G . , and Thomson, A. (1956).J.Infect. Diseases 99,246-252. Tao, T., and Uhr, J. W. (1966).Nature 212,208-209. Tempelis, C. H. (1965).J.Immunol. 94,705-709. Terres, G . ,and Stoner, R. D. (1962).PTOC. Soc. Exptl. Biol. Med. 109,88-91. Terres, G., and Wolins, W. (1961).]. lmmunol. 86,361-368. Thompson, R., Olson, V. H., and Sibal, L. R. (1957).J.Immunol. 79,508-515. Thorbecke, G . J., Maurer, P. H., and Benacerraf, B. (1960). Brit. J . Exptl. Pathol. 41, 190- 197. Tocantins, L. M., and Kazal, L. A. (1964). “Blood Coagulation, Hemorrhage, and Thrombosis.’’ Grune & Stratton, New York. Trapani, I. L. (1960).Federation PTOC.19, Suppl. 5,109-114. Treffers, H. P., and Heidelberger, M. (1941).J.E x p t l . Med. 73,125-140. Triplett, E. L., Cushing, J. E., and Durall, G. L. (1958).Am. Naturalist 92,287-293. Turner, T. B., Stafford, E. S., and Goldman, L. (1954). Bull.]ohns Hopkins Hosp. 94, 204-2 17.


67

Uhr, J. W.,and Baumann, J. B. (1961)./. Exptl. Med. 113,935-957. Unger, R. H., Eisentraut, A. M., McCall, M. S., Keller, S., Lanz, H. C., and Madison, L. L. (1959).Proc. S O C . Exptl. Biol. Med. 102,621-623. Webster, R. G. (1965).Immunology 9,501-519. Weintraub, M., and Raymond, S. (1963). Science 142,1677-1678. White, R. G. (1963).Brit.Med. Bull. 19,207-213. Wigzell, H. (1966)./.E x p t l . Med. 124,953-969. Wigzell, H., and Stjernsward, J. (1966).J.Natl. Cancer Inst. 37,513-517. Wilner, B. I., Evers, M. A. Troutman, H. D., Trader, F. W., and McLean, I. W. (1963). J . Immunol. 91,210-229. Wilson, G. S., and Miles, A. A. (1964). “Principles of Bacteriology and Immunity,” p. 1372. Williams & Wilkins, Baltimore, Maryland. Wolfe, H. R., Mueller, A,, Neess, J., and Tempelis, C. (1957).]. Zmmunol. 79, 142-146. Yamashiroya, H. M., Ehrlich, R., and Magis, J. M. (1966).]. Bacteriol. 91,903-904. Youngner, J. S., and Axelrod, V. (1964)./. Immunol. 92,879-884.


Microbial Models of Tumor Metabolism

G. F. GAUSE lnstitute of New Antibiotics, Academy of Medical Sciences, Moscow, U.S.S.R.

.................................

I. Introduction

69

anisms in the Metabolism

of Tumors ....................................... A. Tumor Metabolism in the Evolutionar

70

.................

70 72 73

111. Disturbance of Control Mechanisms in the

Respiratory-Deficient Yeast ....................................... A. Metabolism of the Respiratory-Deficient Yeast B. Disturbance of Control Mechanisms in the Respiratory-Deficient Yea ......................... C. Distorted Organization of Respiratory-Deficient Yeast.. ...... IV. Summary ................... References ..............................................................

77 77 83 85 88 88

I. Introduction Theories are like living organisms that can survive only b y evolving in order to adapt themselves to new demands. If the concept of microbial models of cancer cells were restricted to its initial formulation, it would experience the fate of other theories and wither away, or at least become mummified. Fortunately, the concept is acquiring new life and becoming more fruitful of understanding, because its scope is being widened. At first focused on some resemblances between respiratory-deficient mutants of microorganisms and cancer cells, it is now taking cognizance of the multiplicity of similarities in the molecular organization of cancer cells and of their microbial equivalents. Seen from this broader point of view, microbial models of cancer cells may stimulate investigation of the nature of malignancy, and act as a creative force in setting goals for research (Gause, 1966). In recent times considerable attention has been paid in microbiology to biochemical correlates of respiratory deficiency in yeast and to cytoplasmic transformation, when mitochondria from normal yeast cells restore the respiratory capacity of the respiratory-deficient mutants. This research in microbiology now closely approaches one of the most fundamental problems of cancer research, namely, the search 69

70

G. F. GAUSE

for an experimentally controlled reversion of the malignant cell to its “normal” counterpart (Pitot and Cho, 1965). There is a growing feeling among investigators concerned with analysis of control mechanisms in the normal and neoplastic cell that microbiology and cancer research have much to learn from each other. The purpose of this review is to summarize the available literature on disturbance of control mechanisms in the respiratory-deficient mutants of yeast, and to compare it with more recent data on disturbance of control mechanisms in the metabolism of tumors. II. Disturbance of Control Mechanisms in the Metabolism of Tumors

A. TUMORMETABOLISM IN

THE

EVOLUTIONARY PERSPECTIVE

It is reasonable to begin a discussion of tumor metabolism with a consideration of the contributions of Warburg, extended over almost half a century. His first paper on this subject was published in 1923, a book entitled “The Metabolism of Tumors” in 1926, and the most recent contribution to this series of investigations appeared in 1965. Two features are characteristic of Warburg’s approach: the first is his consideration of tumor metabolism in the evolutionary perspective, and the second is his continued reference to microbial models, and particularly to yeast cells, for better understanding of the essence of tumor metabolism. The transformation of the normal cell into a malignant one, or the process of malignization, can be considered as a transition to an evolutionarily more primitive type of metabolism, d u e to impaired respiration and greater dependence upon “life without oxygen,” observed b y Pasteur in yeast cells almost a century ago. Respiration is a much more complicated and subtle process than fermentation, and it is more vulnerable to the effect of injurious carcinogenic agents, responsible for the malignization of cells. The discovery by Warburg in 1923 of the impaired respiration of tumor cells and of their greater dependence upon fermentation was an important turning point in the study of tumor metabolism. This phenomenon has various aspects. One of the consequences of the metabolic shift in tumors is the capacity of tumor cells to live and multiply under anaerobic conditions for a long period of time, which differentiates them from normal cells. This characteristic feature of tumor cells can be demonstrated with the aid of different techniques, including a very elegant microbiological experiment of Malmgren and Flanigan (1955). It is well known that tetanus bacteria belong to

MICROBIAL MODELS OF TUMOR METABOLISM

71

anaerobic microorganisms; tetanus spores germinate and grow only in the absence of oxygen. When injected intravenously into the body of normal or of pregnant mice, tetanus spores do not germinate anywhere in the organism, as far as complete anaerobiosis is practically excluded in the body. However, Malmgren and Flanigan (1955) observed that the injection of washed tetanus spores, which was without untoward effect in normal animals, uniformly resulted in tetanic death in the tumor-bearing host in approximately 48 hours, regardless of the tumor size. The production of a lethal toxin in the vegetative state makes determination of germination of these spores both simple and conclusive. On the basis of examination of comparable tumors in untreated animals prior to spore injection, it appears likely that necrotic areas were absent. It is therefore possible to conclude that tetanus bacilli can grow and multiply only among cancer cells in the tumors. This experiment clearly demonstrates that cancer cells, and cancer cells alone, grow and multiply in the organism under anaerobic conditions. However, growth under anaerobic conditions may occur only temporarily in the life of tumor cells. Warburg et al. (1965) compared it with an anaerobically growing yeast, which afterward can be brought into contact with oxygen and continue to grow aerobically. In the same way, metastasizing tumor cells migrate into the new areas of the body provided with oxygen. The observations of Warburg were for several decades the subject of controversy, which to a very considerable extent depended upon insufficient development of theoretical oncology and the absence of proper “control” cells for the evaluation of metabolic shifts in tumors. This situation has improved considerably only recently, with the wide availability of a new class of transplantable and primary tumors of animals that were named “minimal deviation” tumors by Potter and his colleagues (Potter et al., 1960). They were discovered in the course of a systematic search for liver tumors, induced by feeding animals with various carcinogens, and differing as little as possible from normal liver. The first example of this type of tumor was described by Morris et al. (1960), and designated the Morris hepatoma 5123. Following the discovery of this hepatoma, a systematic search for other representatives of the class was made, and a growing list of minimal deviation hepatomas is now available. Different strains of minimal deviation hapatomas in the rat grow with different speed: some of them double their weight in 2 weeks, while others do it in 10 months. The rate of growth may be taken as an index of malignancy: the better tumor cells grow, the more malignant

72

6. F. GAUSE

they are. It is very remarkable therefore that Burk and his colleagues (Burk et al., 1965, 1967) observed in this series of tumors a significant correlation between malignancy and glycolysis, which is shown in Table I. The initial rates of anaerobic and aerobic glycolysis of TABLE I CONNECTION BETWEENGLUCOSE FERMENTATION AND GROWTH W T E IN THE SPECTRUMOF MORRISRAT HEPATOMAS" ~~

Anaerobic glycolysis* Tissue Normal liver Host liver Slowest-growing hepatomas (subminimal deviation) Slow-growing hepatomas (minimal deviation) Intermediate-deviation hepatomas Fast-growing (advanced) hepatomas

Minimum

Maximum

Average

No. of specimens studied

0.18 0

0.50 0.68

0.32 0.33

6 26

0.40

1.00

0.69

9

0.58

1.80

1.03

13

2.00

6.40

4.10

15

4.5 11.8

8.0 17.5

6.7 13.7

3 6

From Burk et ul. (1967).

'Qco2 N20.1to 0.5% glucose

in medium.

minimal deviation hepatoma cells are up to several-fold higher than those of liver tissue from the host animal or from hepatoma-free animals. These metabolic differences between normal and tumor cells gradually increase with the increase of malignancy and rate of growth of tumors, and they clearly confirm observations made by Warburg on other strains of tumor tissue some years ago.

B. DISTURBANCE O F CONTROL MECHANISMS IN METABOLISM OF TUMORS

THE

A comparison of cancer cells with anaerobic yeasts is illuminating, but it is only partially valid. What is characteristic for a cancer cell is the disturbance of cellular control mechanisms on various levels of its organization (Pitot and Cho, 1965). Chance and Hess (1959)in the rapid measurements of intracellular events with the aid of spectroscopic methods observed that the respiratory system of tumor cells is not used to the extent of its capabilities. They suggested that there is a control mechanism imposed upon the respiratory activity of the


73

intact tumor cell. Another manifestation of the work of this control mechanism can be seen in the fact that the oxidative rate of tumors is fixed, and quite refractory to the stimulation seen when excess carbohydrate or fatty acid is added to normal tissues. It might be said, therefore, that the respiratory activity of neoplastic material is a maximal one, while normal cells have ample reserves. This aspect of tumor metabolism represents only a special case of deficiency in biochemical control mechanisms, which has been described by Potter (1964) in a wider biological perspective as the biochemical inflexibility of tumors. H e believes that cancer cells probably exhibit irreversible changes in regulatory genes. It is this disturbance of control mechanisms which makes the cancer cell different from anaerobic yeast. In the latter case the respiration is decreased and glycolysis increased due to low oxygen tension, but regulatory genes are not lost, and in response to a change of environment, metabolic regulations are observed. It is only in the respiratorydeficient mutants of yeast that metabolic regulations are also irreversibly lost, and these mutants can therefore be approached with some justification as potential microbial models for the study of some aspects of metabolism resembling the metabolism of tumors.

c. MOLECULARORGANIZATION OF TUMORMITOCHONDRIA Impaired respiration and increased glycolysis of tumors were discovered by Warburg in the early 1920’s, in the beginning of an era of luxurious flowering of research on biological oxidations, which occurred with relatively little attention to mitochondria. Analyzing the major events of this period Lehninger (1964) wrote: “Actually, very few biochemists concerned themselves with the possible importance of the fact that respiratory enzymes were found to be associated with particulate matter of cells and tissues. It was a part of the biochemical Zeitgeist that particles were a nuisance and stood in the way of purification of the respiratory enzymes. Yet it almost seems paradoxical that it was two biochemists who had many years earlier made important discoveries on the occurrence of biological oxidationreduction mechanisms in granular elements of the cell. Michaelis had shown in 1898 that mitochondria of unfixed cells reduced oxidation-reduction indicators such as Janus green. , . . It is interesting that Warburg in 1913 found respiration to be associated with granular, insoluble elements of cell structure, which he recovered b y filtration of tissues dispersions, but the significance of his observation was not developed further by cytologists of the day.”

74

C . F. CAUSE

Almost 40 years later, in the early 1950’s, the mitochondria were first recognized to be the “power plants” of cells. At that time the isolated mitochondria were found to carry out the oxidation, and, most importantly, oxidative phosphorylation, at a rate compatible with that of the intact cell. On the other hand, the isolated mitochondria failed to catalyze the reactions of glycolysis, indicating the specificity of respiratory enzyme localization in mitochondria. Since the early 1950’s, biochemical studies of the enzymic mechanisms of the oxidative cycles, electron transport, and oxidative phosphorylation have proceeded in confluence with studies of the structure and cell biology of the mitochondrion. The molecular structure and the dynamic function of the mitochondrion are now beginning to illuminate and define each other. In the mechanism of energy-coupled respiration may lie the secret of the ultrastructure of the mitochondrion and, conversely, in the molecular organization of the mitochondrion may lie the secret of electron transport. The central objectives in attaining a molecular description of the mitochondrion are the isolation of the enzymic components of the oxidative cycles, definition of their molecular structure and action, analysis of their participation in the multi-enzyme systems in the mitochondrion, and mapping of their location in the mitochondrial structure. Another characteristic of mitochondrial enzymology is the phenomenon of compartmentation of enzymes and substrates in the mitochondrial structure. These features are intricately involved in the control and channeling of enzymic pathways within the mitochondrion and in its metabolic interactions with the surrounding cytoplasmic matrix (Lehninger, 1964). If at the time of Warburg’s early work on the impaired respiration of tumors the concept of oxidative metabolism was entirely “scalar,” it is now much more “vectorial,” and therefore an analysis of a possible reflection of the malignant state in the distorted structure of mitochondria is of great interest. The molecular organization of tumor mitochondria is at present but little investigated, and in his excellent monograph entitled “The Mitochondrion,” Lehninger (1964)has only a few words to say on this subject. He notes that integration of mitochondrial respiration with extramitochondrial glycolysis is distorted in tumor cells, presumably in view of the genetic deletion of some enzymes. However, there is now significant evidence that mechanochemical activities of the mitochondrial membrane are different in normal and in tumor cells. Changes of mitochondrial shape and volume have recently been found to be coupled to the energy-conserving mech-


75

anisms of the respiratory chain. When isolated mitochondria respire in the absence of phosphate acceptor, they undergo swelling with uptake of water (and solutes) from the medium. On the other hand, when respiration is poisoned or the mitochondria are kept anaerobic, they do not swell. The reverse process, the shrinkage or contraction of mitochondria, with extrusion of water, may be brought about by instituting phosphorylating respiration. It was found that mitochondria contain the contractile protein, which is a contraction factor. Ohnishi and Ohnishi (1962), as well as Neifakh and Kazakova (1963), by extraction of mitochondria of normal liver cells with KC1 solutions of high ionic strength, were able to separate a protein that was similar in many respects to the actomyosin of skeletal muscle. It is of considerable interest therefore that Neifakh et al. (1964) reported the failure of all attempts to extract the contractile protein from mitochondria of tumor cells. This observation does not mean that the contractile protein is absent from tumor mitochondria, particularly in view of the fact that the latter display marked contractility. Nevertheless, this observation clearly indicates certain anomalies in the structure and properties of contractile protein in tumor mitochondria which make impossible extraction by a procedure appropriate for normal cells. It is clear that the nature of mitochondrial contractile protein and of its distortion in the tumor cell deserve further study. Neifakh et al. (1965) suggested that mitochondria may play an important part in the control of glycolysis, and that distorted structure of contractile protein in tumor mitochondria may be related to impaired control of glycolysis in tumor cells. It was in fact shown that liver, heart, brain and tumor cell mitochondria release into incubation medium glycolysis-stimulating factors (Neifakh et al., 1964). One of these factors is nicotinamide-adenine dinucleotide (NAD), and in normal cells its excretion depends upon reversible changes in the structure of mitochondrial membranes. Preincubation of mitochondria with adenosine triphosphate (ATP), magnesium, or ethylenediaminetetracetate (EDTA), i.e., with substances that cause membranes to contract, results in a strong decrease in the release of NAD, as shown in Fig. 1. Preincubation with phosphate or potassium chloride, causing the relaxation and swelling of mitochondrial membranes, leads to intensified release of NAD. In the case of tumor mitochondria the results are quite different (Fig. 1). The liberation of NAD from tumor mitochondria does not depend upon their swelling or contraction, and is out of any control. It seems highly probable therefore that in the tumor cell, due to a genetic abnormality in the structure of contractile protein, mitochondrial membranes are exempt from control, so

76

G . F. GAUSE

mATP EDTA KCI

Mouse liver

Ehrlich ascites carcinoma

Fig. 1. T h e effect of ATP, EDTA, KCI, and phosphate upon the release of NAD from normal and tumor mitochondria (from Neifakh et al., 1964).

that they release the stimulating factors incessantly. Tumor mitochondira are “leaky” mitochondria. This may be the condition responsible for monotonous continuation of aerobic glycolysis, occurring at a high level in the tumor cell. Lehninger (1964, p. 201) concludes that the swelling-contraction cycle of mitochondria may be an important cybernetic mechanism permitting self-adjustment of respiration and phosphorylation in the intact cell. Because these mechanochemical changes are brought about by intermediate reactions of oxidative phosphorylation, they may give some important clues to molecular relationships between the mechanical changes and the enzymes of the respiratory chain. It is of considerable interest that certain defects in the structure and properties of the contractile protein in tumor mitochondria may be correlated with the impaired control of respiration and glycolysis in the tumor cell. One possible approach in the study of molecular organization of tumor mitochondria may be concerned with the study of their cytochromes. In fact, cytochromes are important members of the respiratory assembly of the highly integrated mitochondrion, that is, of an organized arrangement of macromolecules located in a geometrical pattern favorable for their interaction. It is only reasonable to expect that any distortion in the molecular organization of tumor mitochondria may be reflected by alterations of cytochrome pattern. Therefore it is of considerable interest that in the Zajdela ascites hepatoma of the rat, as well as in the Ehrlich ascites tumor of the


77

mouse, the complete or nearly complete absence of cytochrome b has been recorded in a low-temperature spectrographic study (Monier et al., 1959). Although there is now considerable evidence for the role of mitochondria in metabolic regulation of both respiration and glycolysis, the concept of mutant mitochondria in tumors is supported not only by the data of bioenergetics. Graffi (1940) had shown many years ago that carcinogenic hydrocarbons are preferentially accumulated in mitochondria, and it appeared probable that the malignization might be based upon a mutation of mitochondria. The most recent data from the laboratory of the same author (Graffi et al., 1965) demonstrate significant differences of mitochondrial protein synthesis in vitro in tumor and normal cells. It was observed that the rate of incorporation of amino acids into the mitochondrial protein of malignant tumors is very low. These differences may finally depend upon differences in nucleic acids governing this process. This possibility is being investigated now in a number of laboratories. Fiala and Fiala (1966a) concluded that the tumor mitochondria differ from normal liver mitochondria by the depletion of the structural protein. These authors also conclude that the scarcity of mitochondria, impaired respiration, and increased anaerobic glycolysis are fundamental features of the “minimal deviation” hepatoma (Fiala and Fiala, 1966b). These observations are particularly interesting in the light of recent work on biogenesis of mitochondria, indicating that one group of proteins is synthesized inside mitochondria and the other group is synthesized on non-mitochondria1 cytoplasmic ribosomes and later transferred to the mitochondria (Haldar et al., 1966). 111. Disturbance of Control Mechanisms in the Respiratory-Deficient Yeast

A. METABOLISMO F T H E RESPIRATORY-DEFICIENT YEAST One advantage of using microorganisms in the study of biochemical systems is the possibility of isolating mutants and of employing these for better understanding of metabolic organization. Since yeast can acquire necessary energy for growth through fermentation, it can survive with lesions in the respiratory chain, making it possible to study mutants having various blocks in the cytochrome system. Mutants of this type were first observed by stier and Castor (1941), Whelton and Phaff (1947), and much studied by Ephrussi, Slonimski, and other investigators since 1949 (see Slonimski and Ephrussi, (1949). I n fact, more than one hundred papers were published be-

78

G . F. GAUSE

tween 1949 and 1965 dealing with this type of metabolic mutant (Sherman, 1965). Respiratory-deficient mutants of yeast in many cases exceed the parent culture in their glycolytic power, and in view of this combination of impaired respiration with enhanced glycolysis they have been repeatedly discussed as possible micorbial models for metabolic organization of the cancer cell. The literature in this field has been reviewed recently (Gause, 1966), and there is no need to repeat it here. Slonimski and Hirsch (1952) have measured the activities of some enzymes of the glycolytic, tricarboxylic acid cycle and electron transport mechanisms in normally respiring Saccharomyces cerevisiae and in a respiration-deficient mutant. Their findings indicated the mutant yeast lacked cytochrome oxidase, succinic dehydrogenasecytochrome b complex, and NADHz cytochrome c reductase. Only the cytochrome c component was present in normal concentrations in both yeasts. Kovachevich (1964) compared oxidation rates of reduced pyridine nucleotides (NADHZ and nicotinamide-adenine dinucleotide phosphate, NADPHZ) in both normal and respiratory-deficient mutant yeasts in the presence and absence of antimycin A. In normal yeast the bulk of NADHZ and NADPHz oxidations with concomitant cytochrome c reduction occurs via an antimycin A sensitive pathway. In contrast, mutant yeast cell-free extracts demonstrated very low oxidation rates of both NADHZand NADPHZ, as measured by cytochrome c reduction, and both systems were only slightly affected by antimycin A. The very low activity of NADHz cytochrome c reductase in the mutant yeast shown in Table I1 is consistent with observations made earlier by Slonimski and Hirsch (1952). A number of metabolic impairments is associated with respiration deficiency in yeast. Lomander and Gundersen (1963),as well as Avers et al. (1965a) noted, for example, that none of the respiratory-deficient mutants sporulated, while respiratory-competent cells sporulated readily. It is of considerable interest that respiratory-deficient mutants in Saccharomyces cerevisiae are not identical, but belong to a number of different types. The apparent uniformity of mutants, which indeed are much unlike their parents, for some time precluded the recognition of their variety. Avers et al. (1965b) supposed that the variety of mutants has been overlooked by earlier workers because of the lack of suitable methods for identification, and they recognized among the respiratory-deficient mutants of Saccharomyces cerevisiae four

79

MICROBIAL MODELS O F TUMOR METABOLISM TABLE I1 COMPARISON OF OXIDATION RATESOF REDUCEDPYRIDINE NUCLEOTIDES BY CELL-FREEEXTRACTSOF NORMALAND RESPIRATORY-DEFICIENT MUTANTYEAST Saccharomyces cerevisiaeU NADH, as substrate

NADPH2 as substrate

Without antimycin A With antimycin A (1.2 pg./ml.)

Normal yeast

Mutant yeast

Normal yeast

Mutant yeast

74.8*

1.9

30.0

7.4

10.5

1.6

18.6

7.0

From Kovachevich (1964). bActivities are expressed as mpM of ferrocytochrome c formed/minute/0.05 ml. of cell-free extract as measured at 549 mp. a

types differing in the character of alteration of their mitochondria1 enzymes: Mitochondrial cytochrome oxidase Mitochondria1 succinic dehydrogenase

Type 1

Type 2

Type3

Type4

Reduced

Reduced

Absent

Absent

Normal

High

High

Normal

The variety of types of mutants in the respiratory-deficient yeast can be recognized also b y the study of their aerobic glycolysis. Cause et al. (1957) observed a number of mutants in Saccharomyces cere-

visiae with respiration impaired quantitatively in the same degree, which differed in the intensity of their aerobic glycolysis: in some glycolysis was strongly increased, while in others it did not differ from that of their parents. Similar relations were also observed by Trenina et al. (1965) in another yeast, Torulopsis holmii, and these are shown in Table 111. Respiratory-deficient mutants in yeast can be induced by different mutagens, notably by acriflavine (Ephrussi et al., 1949), 5-fluorouracil (Moustacchi and Marcovich, 1963), carcinogenic agent 4-nitroquinoline N-oxide (Mifuchi et al., 1963), and a water-soluble carcinogen, Styryl 430 (Constantin, 1964). Biochemical mechanisms of action of these mutagens are different, and it has been shown, for example, that methylene blue, which by itself is ineffective in the induction of

80

G . F. GAUSE

respiratory-deficient mutants of Succhuromyces cereuisiue, strongly suppresses the induction of these mutants by acriflavine but does not TABLE 111 RESPIRATION AND AEROBIC GLYCOLYSIS IN THE PARENTCULTUREAND RESPIRATORY-DEFICIENT MUTANTSOF Torulopsis holmif'

Torulopsis holmii, strain 424 Mutant 8 Mutant 30

57.8 1.4 1.7

IN THE

125.1 216.6 125.2

From Trenina et al. (1965). TABLE IV AND AEROBIC GLYCOLYSIS IN THE PARENTCULTUREOF RESPIRATION Saccharomyces chevalieri 406 AND IN THE RESPIRATORY-DEFICIENT MUTANTS INDUCED BY VARIOUS MUTAGENS. AVERAGE DATAOF Foun SERIESOF EXPERIMENTS" Culture Parent Mutant A-1 Mutant A-2 Mutant A-3 Mutant A-4 Mutant A-4-2 Mutant A-5 Mutant A-7 Mutant A-8 Mutant A-10 Mutant A-13 Mutant F-1 Mutant F-3 Mutant F-5 Mutant F-5-1 Mutant F-7 Mutant F-8 Mutant F-11 Mutant F-12 Mutant F-19 Mutant F-20 Mutant N-3-3 Mutant N-3-5 Mutant N-4

Mutagen Acriflavine, 50 pg.flml. Acriflavine, 50 pg./ml. Acriflavine, 50 pg./ml. Acriflavine, 50 pg./ml. Acriflavine, 50 pg./ml. Acriflavine, 25 pg./ml. Acriflavine, 25 pg./ml. Acriflavine, 12 pg./ml. Acriflavine, 12 pg./mI. Acriflavine, 6 pg./ml. 5-Fluorouracil, 500 pg./ml. 5-Fluorouracil, 500 pg./ml. 5-Fluorouracil, 500 pg./ml. 5-Fluorouracil, 250 pg./ml. 5-Fluorouracil, 250 pg./ml. 5-Fluorouracil, 250 pg./ml. 5-Fluorouracil, 250 pg./ml. 5-Fluorouracil, 250 pg./niI. 5-Fluorouracil, 125 pg./ml. 5-Fluorouracil, 125 pg./ml. 4-Nitroquinoline N-oxide, 1.25 pg./ml. 4-Nitroquinoline N-oxide, 1.25 pg./ml. 4-Nitroquinoline N-oxide, 1.25 pg./ml.

From Gause et al. (1967).

Qo

Qco

50.4 1.8 3.5 4.2 1.2 1.2 1.5 4.9 1.3 3.0 8.5 4.9 5.2 2.5 4.9 2.2 3.6 2.6 2.1 3.6 7.5 3.2 3.2 4.1

261.1 345.2 317.2 333.2 292.9 331.2 265.2 295.2 309.6 338.6 347.9 352.5 371.1 308.1 333.4 447.6 354.3 325.2 299.6 311.6 312.9 343.0 360.5 301.5

81

MICROBIAL MODELS O F TUMOR METABOLISM TABLE V RESPIRATIONAND AEROBICGLYCOLYSIS IN THE PARENTCULTUREOF Saccharomyces ouiformis 493 AND IN THE RESPIRATORY-DEFICIENT MUTANTSINDUCED BY VARIOUS MUTAGENS.AVERAGE DATAOF FOUR SERIES OF EXPERIMENTS" Culture

Mutagen

Qo 2

QcoB

50.8 5.2 3.4 2.7 2.6 2.9 2.5 2.8 1.2 6.3 2.6 3.7 3.6 3.0 1.8 3.3 1.9 1.3 1.5 2.3 1.2 2.0 2.7 3.7 3.0 4.8 3.4 4.3 5.6

72.7 246.6 248.1 260.7 246.4 282.9 217.9 201.4 210.6 237.8 268.0 255.4 205.3 235.0 201.6 262.3 225.9 233.0 229.1 250.1 252.2 260.3 330.9 270.1 241.2 248.4 238.1 232.9 220.6

~

Parent Mutant A-2 Mutant A-3 Mutant A-4 Mutant A-5 Mutant A-6 Mutant A-8 Mutant A-12 Mutant A-13 Mutant A-14 Mutant A-22 Mutant F-1 Mutant F-3 Mutant F-3-2 Mutant F-7 Mutant F-7-2 Mutant F-10 Mutant F-12 Mutant F-13 Mutant F-15 Mutant F-16 Mutant F-18 Mutant N-2 Mutant N-4 Mutant N-5-2 Mutant N-5-3 Mutant N-5-4 Mutant N-10 Mutant N-12

Acriflavine, 50 pg./ml. Acriflavine, 50 pg./ml. Acriflavine, 25 pg./ml. Acriflavine, 25 pg./ml. Acriflavine, 25 pg./ml. Acriflavine, 25 pg./ml. Acriflavine, 6 pg./ml. Acriflavine, 6 pg./ml. Acriflavine, 6 pg./ml. Acriflavine, 12 pg./ml. 5-Fluorouracil, 500 pg./ml. 5-Fluorouracil, 500 pg./ml. 5-Fluorouracil, 500 pg,/ml. 5-Fluorouracil, 250 pg./ml. 5-Fluorouracil, 250 pg./ml. 5-Fluorouracil, 125 pg./ml. 5-Fluorouracil, 125 pg./ml. 5-Fluorouracil, 62 pg,/ml. 5-Fluorouracil, 62 pg./ml. 5-Fluorouracil, 62 pg./ml. 5-Fluorouracil, 31 pg./ml. .~ 4-Nitroquinoline N-oxide, 0.25 pg./ml. 4-Nitroquinoline N-oxide, 1.25 pg./ml. 4-Nitroquinoline N-oxide, 0.62 pg./ml. 4-Nitroquinoline N-oxide, 0.62 pg./ml. 4-Nitroquinoline N-oxide, 0.62 pg./ml. 4-Nitroquinoline N-oxide, 1.25 pg./ml. 4-Nitroquinoline N-oxide, 0.62 pg./ml.

From Cause eta.?.(1967).

show any effect on the frequency of mutations induced by 4-nitroquinoline N-oxide (Morita and Mifuchi, 1965). Gause et aZ. (1967) investigated alterations of aerobic glycolysis in respiratory-deficient mutants of yeast induced by mutagens with different mechanisms of action. Experiments were made with three mutagens (acriflavine, 5-fluorouracil, 4-nitroquinoline N-oxide) employing two species of yeasts, Saccharomyces chevalieri and Saccharomyces oviformis. Tables IV and V show metabolic alterations in respiratory-deficient

82

G . F. CAUSE

mutants of yeast induced by different mutagenic agents. If the rate of aerobic glycolysis of the parent culture is taken for a unit, the degree of increase of glycolysis in mutants can be expressed in the relative form. The results of these calculations are shown on Fig. 2. It can be S. chevolieri

S. owformis

Increase of glycolysis-

FIG.2. The relative increase of aerobic glycolysis in respiratory-deficientmutants of Saccharomyces cheualied and Saccharomyces ouiformis induced by different mutagenic agents (from Gause et al., 1967).

seen that in each species of yeast mutagens of different nature produce approximately the same effect, as far as values of enhanced aerobic glycolysis in these mutants are of the same order of magnitude. At the same time there are very strong differences between different species. In mutants of Saccharomyces chevalieri the aerobic glycolysis is increased on the average by 30%, and in Saccharomyces ouiformis by 230%. This observation is confirmed by the work with another species of yeast, Saccharomyces formosensis, where aerobic glycolysis in mutants is increased by 450%, as can be seen from the data given in Table VI. The increase of aerobic glycolysis evidently depends upon some distortions in mitochondria of mutant yeasts, and it seems that in different species of yeast, mitochondria are differently susceptible to such distortions. These results of the work with microorganisms have much in common with the experience acquired in the study of the action of chemical carcinogens upon mammalian cells (Kaiser and Barton, 1966).

83

MICROBIAL MOLELS O F TUMOR METABOLISM

TABLE VI RESPIRATIONAND AEROBICCLYCOLYSIS IN THE PARENT CULTURE O F Saccharomyces fomosensis 428 AND IN THE RESPIRATORY-DEFICIENT MUTANTS INDUCED BY ACRIFLAVINE. AVERAGEDATAOF FOURSERIES OF EXPERIMENTS* Culture

Qo I

Qco

Parent Mutant A-9 Mutant A-10 Mutant A-11 Mutant A-16 Mutant A-17

42.9 0.8 1.0 1.9 1.5 1.9

47.0 285.8 266.2 232.5 244.7 266.9

a

From Cause et al. (1967).

B. DISTURBANCE OF CONTROL MECHANISMSIN RESPIRATORY-DEFICIENT YEAST

THE

It has been mentioned earlier in this review that a fundamental difference between parent cells and their mutants with the impaired respiration is related to the loss of regulatory mechanisms in mutant cells. This can be clearly seen in the work with respiratory-deficient mutants of Saccharomyces cerevisiae induced by acriflavine, which have been investigated by Gause et al. (1967). Table VII indicates TABLE VII RESPIRATIONAND AEROBIC GLYCOLYSIS IN THE PARENT CULTURE OF Saccharomyces cereuisiae AND IN THE RESPIRATORY-DEFICIENT MUTANTS PREVIOUSLY CULTURED UNDER AEROBIC AND ANAEROBIC CONDITIONS. AVERAGEDATAOF FOURSERIESOF EXPERIMENTS~ Qo

QW

Strain

Aerobic culture

Anaerobic culture

Aerobic culture

Anaerobic culture

Parent Mutant ST-4 Mutant ST-I0

37.5 2.6 3.1

26.1 3.7 3.2

194.9 349.8 313.0

270.8 327.7 304.3

From Cause et al. (1967).

that in the study of metabolism of parent cells previously grown under aerobic or anaerobic conditions, one can see that the respiration is

84

G . F. CAUSE

decreased and glycolysis increased due to low oxygen tension at the time of cultivation. These regulatory mechanisms are entirely lost in mutant cells. Their aerobic glycolysis is not only quantitatively increased but it is no longer regulated, and its intensity does not depend upon oxygen tension at the time of cultivation. Another manifestation of biochemical inflexibility of the respiratorydeficient yeast was revealed in the study of the adaptive formation of ubiquinone or coenzyme Q. As is well known, ubiquinone serves as a carrier in the electron transport system in mitochondria, although there is still considerable uncertainty about its exact role in this process (Lehninger, 1964).The ubiquinone content in the hepatoma is one-quarter to one-tenth of that of the normal liver (Sugimura et al., 1962),and this observation evidently reflects the impaired respiration in tumor cells. It was also recorded that in a respiration-deficient mutant of Saccharomyces cerevisiae induced by acriflavine, the ubiquinone content of aerobically cultured cells ranged from 1/5 to 1/15 of that of the normal parent cells (Sugimura et al., 1964). What is essential, however, is not only the low content of ubiquinone in the cells of the respiratory-deficient mutant yeast, but also a strongly impaired adaptive response of mutants in the synthesis of ubiquinone after aeration. Table VIII shows that after aeration the content of TABLE VIII INCREASE OF THE UBIQUINONE ( U Q ) CONTENT IN ANAEROBICALLY CULTURED CELLS OF Saccharomyces cereuisiae AFTER AERATION"

UQs Strain Parent Respiratory-deficient mutant T-5

a From

Treatment

(pg.1100 mg. N)

Increase

Before aeration After aeration

13 274

x 21.0

Before aeration After aeration

42

9 X

4.6

Sugimura et al. (1964).

ubiquinone in the cells of parent yeasts increases by 21 times, while under similar conditions the content of ubiquinone in the cells of mutants increases only by 4.6 times. The nature of the defect in respiratory-deficient yeast mutants, as it pertains to adaptive ubiquinone synthesis, appears to be complex and its elucidation mdst await further knowledge of the pathways and intermediates involved in the normal synthesis of ubiquinone.

MICROBIAL MODELS O F TUMOR METABOLISM

85

The delay of synthesis of various inducible enzymes has been repeatedly recorded in the work with respiratory-deficient mutants of yeast. Lindegren et al. (1957) noticed the slow adaptation to galactose or maltose fermentation in these mutants. Reilly and Sherman (1965) observed that if parent and mutant cells were first grown for 1 day in glucose medium, and then inoculated into melibiose or raffinose medium, prolonged delays in growth of mutant cultures could be seen. When compared with parents, delays of about 1 day before growth is initiated in melibiose medium, and of over 3 days in raffinose medium have been recorded for respiratory-deficient mutants of the yeast Saccharomyces cerevisiae. This situation is shown in Fig. 3. It is of considerable interest that many defects in synthesis of various inducible enzymes have been recorded also in neoplastic cells (Pitot and Cho, 1965).

[

Melibiose

z f i n o s e

Hours

FIG.3. Adaptation to growth on melibiose and raffinose in normal cells of Saccharomyces cereuisiae (A) and in the respiratory-deficient mutant (B) (from Reilly and Sherman 1965).

c. DISTORTED ORGANIZATION O F MITOCHONDRIAIN T H E RESPIRATORY-DEFICIENT YEAST Respiratory-deficient mutants of yeast fail to form a substantial portion of the electron transport chain and are deficient in cytochromes a and b, ubiquinone, and associated enzymes. T h e simultaneous disappearance of these compounds suggests that the respiratory apparatus of the cell develops, at least in part, as a unit (Lascelles,

86

G . F. CAUSE

1965). It is also clear that the respiratory-deficient yeast represents a unique model for the study of organization of the highly integrated mitochondrion. Sherman and Slonimski (1964) pointed out that respiratory-deficient mutants of yeast lack cytochromes which are strongly particle-bound (cytochromes a and b) and retain cytochromes which are loosely bound (cytochrome c). This could be due to either a modification of a structural component common to several cytochromes, or to a common controlling mechanism. As suggested by Ycas (1956), the formation of one cytochrome could depend on the formation of another, e.g., the absence of cytochrome a may be only a secondary effect of the cytochrome b deficiency. It can be easily envisaged that the structural relationships between different bound cytochromes can take place at different levels of organization of the mitochondrion. For instance, cytochromes a and b could contain an identical polypeptide chain controlled by a single determinant; the formation or attachment of cytochromes a and b to the mitochondrial membrane may be conditioned by another protein. There is some evidence that a specific structural protein, which has precisely the property to form polymers with cytochromes a and b, and not with cytochrome c, does exist in mammalian mitochondria (Criddle ut al., 1962). If the structural protein is under genetic control, then it would be expected that the loss or alteration of this protein could result in the loss or diminution of several cytochromes. At a higher level of organization, multienzyme deficiencies could be due to the alterations in the lipoprotein matrix of the mitochondrial membrane. Whatever the exact nature of the lesions in these respiratory-deficient mutants, it is clear that there should be different ways of producing multienzyme deficiencies in such a highly organized organelle as a mitochondrion. Yotsuyanagi (1962) observed with the electron microscope that respiratory-deficient strains of yeast differed from normal strains by having a modified structure of the mitochondria. He suggests that as far as respiratory enzymes are arranged in the rigid and precise pattern on the mitochondrial membrane, the distorted architecture of mitochondria may represent the cause of the distorted electron transport. It is of great interest that alterations of mitochondrial structural protein in respiratory-deficient mutants of Neurospora have been reported (Woodward and Munkres, 1966). Schatz et al. (1963) studied the structure of mitochondria in the respiratory-deficient mutants of the yeast Saccharomyces cerevisiae and came to the following conclusions: (a) The number of mitochondria per cell in mutants is strongly reduced. ( b )Mitochondria of


87

mutants sediment more slowly in the sucrose density gradient; this observation suggests an altered structure of mitochondria in mutants correlating with their loss of cytochromes a and b. ( c )Mitochondria of mutants are labile and more easily breakable in the preparation of a cell-free system. Mahler et al. (1964a) reported that respiratory particles prepared from respiratory-deficient mutants of the yeast Saccharomyces cerevisiae are devoid of cytochrome a and cytochrome b, are incapable of catalyzing the reduction of cytochrome c by succinate and the oxidation of reduced cytochrome c by oxygen, but exhibit a pattern of primary dehydrogenases similar to those of the wild type. The study of antigenic properties of respiratory particles (Mahler et al., 1964b) has shown that respiratory particles from respiratory-deficient mutants of yeast contain a cross-reacting material capable of binding (or reacting with) some components of sera prepared against particles from wildtype cells. An attempt has been made recently to investigate the molecular properties of mitochondrial DNA in the respiratory-deficient yeast (Moustacchi and Williamson, 1966). Density-gradient centrifugation of protoplast lysates of yeast cells indicates that a satellite component corresponding to low-density mitochondrial DNA is absent in the respiratory-deficient mutants of yeast. This problem was investigated in greater detail by Mounolou et al. (1966). They observed in the respiratory-deficient mutants of yeast a specific change in the buoyant density of mitochondria1 DNA. Whether this alteration in density corresponds to changes in base composition of mitochondrial DNA in mutants or to the presence of unknown components remains to be seen. A very important step in the study of respiratory-deficient mutants of yeast was reached with the demonstration of the possibility of cytoplasmic transformation (Tuppy and Wildner, 1965). In these experiments the mitochondria from a wild-type parent yeast Saccharomyces cerevisiae restored the respiratory capacity of the respiratory-deficient mutant yeast. The cells of mutants were converted into spheroplasts and the latter were treated with mitochondria isolated from normally respiring parent cells. A sizable portion of the treated cells acquired the ability to respire and to form normal colonies, indicating that normal mitochondria had been incorporated into the cells of mutants and in this way transformed mutants into parent cells. Very similar results were independently reached by Diacumakos et al. (1965) in the work with the fungus Neurospora crassa. In this case a mitochondrial fraction isolated from an abnormal strain of

88

G. F. GAUSE

Neurosporu produced drastic changes in the rate of growth, morphology, reproductive characteristics, and cytochrome spectra of normal strains when single hyphal compartments were microinjected and isolated, whereas the mitochondria1 fractions of the wild type produced no effect. These results provide evidence for the transmission of biochemical and biological characters when mitochondria are transferred to new nucleocytoplasmic environments. This transformation of mutants into normal cells, which has been already achieved on microbial models, evidently opens the way for new attempts to investigate whether or not a reversion of the malignant process in mammalian cells is experimentally possible.

IV. Summary

It can be concluded from the discussion and examples presented on the preceding pages that the disturbance of control mechanisms in the respiratory-deficient yeast has much in common with the disturbance of control mechanisms in the metabolism of tumors. In particular, distorted organization of mitochondria in the respiratorydeficient yeast may be instructive as a model for understanding some aspects of molecular organization of tumor mitochondria. A number of similarities between respiratory-deficient mutants of yeast and cells of malignant tumors in the increased but uncontrolled glycolysis, in the defective synthesis of various inducible enzymes, and in some other respects indicate that microbiology and cancer research have much to learn from each other. REFERENCES Avers, C. J., Rancourt, M. W., and Lin, F. H. (1965a). Proc. Natl. Acud. Sci. U S . 54, 527-535. Avers, C. J., Pfeffer, C. R., and Rancourt, M. W. (1965b).J. Bucteriol. 90, 481-494. Burk, D., Woods, M., and Hunter, J. (1965). Proc. Am. Assoc. Cancer Res. 6,9. Burk, D., Woods, M., and Hunter, J. (1967).J. Natl. Cancer Inst. (in press). Chance, B., and Hess, B. (1959). Science 129,700-708. Constantin, T. (1964). Compt. Rend. Soc. B i d . 158,2263-2268. Criddle, R. S . , Bock, R. M., Green, D. E., and Tisdale, H. (1962). Biochemistry 1, 827-842. Diacumakos, E. G., Garnjobst, L., and Tatum, E. L. (1965). J. Cell B i d . 26, 427-443. Ephrussi, B., Hottinguer, H., and Chimenes, A. M. (1949). Ann. Inst. Pasteur 76, 351-362. Fiala, S., and Fiala, A. (1966a). Proc. Am. Assoc. Cancer Res.7,20. Fiala, S., and Fiala, A. (1966b).Naturwissenschaften 53,228. Cause, G . F. (1966). “Microbial Models of Cancer Cells.” Saunders, Philadelphia, Pennsylvania.


89

Cause, G. F., Kochetkova, G. V., and Vladimirova, G. B. (1957). Dokl. Akad. Nauk SSSR 117,138-141. Cause, G. F., Kochetkova, G. V., Sarukhanova, L. E., and Vladimirova, G. B. (1967). Mikrobiologiya 36,340-352. Graffi, A. (1940). Z. Krebsforsch. 50,196-219. Graffi, A., Butschak, C., and Schneider, E. S. (1965). Biochem. Biophys. Res. Commun. 21,418-423. Haldar, D., Freeman, K., andWork,T. S. (1966).Nature211,9-11. Kaiser, H. E., and Barton, J. C. (1966). Federation Proc. 25 (2), 661. Kovachevich, R. (1964). Biochem. Biophys. Res. Commun. 14,48-53. Lascelles, J. (1965). In “Function and Structure in Micro-Organisms,” Proc. 15th Symp. Soc. Gen. Microbiol., London (M. Pollock and M. Richmond, eds.), pp. 32-56. Cambridge University Przss. Lehninger, A. L. (1964). “The Mitochondrion. Molecular Basis of Structure and Function.” Benjamin, New York Lindegren, C., Ogur, M., Pittman, D., and Lindegren, G. (1957). Science 126,398-399. Lomander, L., and Gundersen, K. (1963).J. Bacteriol. 86,956-965. Mahler, H. R., Mackler, B., Grandchamp, S., and Slonimski, P. P. (1964a).Biochemistry 3,668-677. Mahler, H. R., Mackler, B., Slonimski, P. P., and Grandchamp, S. (196413).Biochemistry 3,677-682. Malmgren, R. M., and Flanigan, C. C. (1955). Cancer Res. 15,473-478. Mifuchi, I., Morita, T., Yanagihara, Y., Hosoi, M., and Nishida, M. (1963). Japan J . Microbial. 7, 69-77. Monier, R., Zaidela, F., Chaix, P., and Petit, J . F. (1959). Cancer Res. 19, 927-934. Morita, T., and Mifuchi, I. (l965).JapanJ. Microbiol. 9,123-129. Morris, H. P., Sidransky, H., Wagner, B. P., and Dyer, H. M. (1960). Cancer Res. 20, 1252-1254. Mounolou, J. C., Jakob, H., and Slonimski, P. P. (1966). Biochem. Biophys. Res. Commun. 24,218-223. Moustacchi, E., and Marcovich, H. (1963). Compt. Rend. 256,5646-5649. Moustacchi, E., and Williamson, D. H. (1966). Biochem. Biophys. Res. Commun. 23, 56-61. Neifakh, S. A., and Kazakova, T. B. (1963). Nature 197,1106. Neifakh, S. A,, Gaitskhoki, V. S., and Kazakova, T. B. (1964).Acta, Unio-Intern. Contra Cancrum 20,1285-1287. Neifakh, S. A., Avramov, J. A., Gaitskhoki, V. S., Kazakova, T. B., Monakhov, N. K., Repin, V. S., Turovski, V. S., and Vassiletz, I. M. (1965). Biochim. Biophys. Acta 100,329-343. Ohnishi, Ts., and Ohnishi, To. (1962). J . Biochem. (Tokyo) 51,380-384. Pitot, H. C.,andCho, Y. S . (1965). Progr. Exptl. TumorRes. 7,158-223. Potter, V. R. (1964). In “Cellular Control Mechanisms and Cancer” (P. Emmelot and 0.Muhlbock, eds.), pp. 190-210. Elsevier, Amsterdam. Potter, V. R., Pitot, H. C., Ono, T., and Morris, H. P. (1960). Cancer Res. 20,1255-1261. Reilly, C., and Sherman, F. (1965). Biochim. Biophys. Acta 95,640-651. Schatz, G., Tuppy, H., and Klima, J. (1963). Z. Naturj‘orsch. 18b, 145-153. Sherman, F. (1965). Colloq. Intern. Centre Natl. Rech. Sci. (Paris) 124,465-479. Sherman, F., and Slonimski, P. P. (1964).Biochim. Biophys. Acta 90,l-15. Slonimski, P. P., and Ephrussi, B. (1949).Ann. Znst. Pasteur 77,47-53.

90

G . F. CAUSE

Slonimski, P. P., and Hirsch, H.M . (1952).Compt. Rend. 235,741-743. Stier, T.,and Castor, J. (1941)~. Gen.Physiol. 25,299-233. Sugimura, T.,Okabe, K., and Baba, T.(1962).Gunn 53,171-181. Sugimura, T.,Okabe, K., and Rudney, H. (1964).Biochim. Biophys. Actu 82,350-354. Trenina, G . A.,Bibikova, M. V., and Samkhanova, L. E. (1965).Mikrobiologiya 34,300-

304. Tuppy, H., and Wildner, G. (1965).Biochem. Biophys. Res. Commun. 20, 733-738. Warburg, 0.(1923).Biochem. 2. 142,317-351. Warburg, 0.(1926).The Metabolism of Tumors. Constable Press, London. Warburg, O., Gawehn, K., Geissler, A., Kayser, D., and Lorenz, S. (1965).Klin. Wochschr. 43,289-293. Whelton, R., and Phaff, H. (1947).Science 105,44-45. Woodward, D.O.,and Munkres, K. D.(1966).PTOC.Nutl. Acud. Sci. U.S. 55,872-880. Ycas, M.(1956).E x p t l . Cell Res. 11, 1-6. Yotsuyanagi, Y. (1962).J . Ultrustruct. Res. 7 , 141-158.

Cel I ulose

CI

nd Cel lulolysis '

BIRGITTA NORKRANS Department of Marine Botany, University of Goteborg, Goteborg, Sweden

I. Introduction .............................................................. 11. Cell Wall Morphology and Chemistry

.........................

111. Cellulose Chemistry and Supramolecular Morphology ... IV. Biosynthesis of Cell Wall Polysaccharides .................... A. Cellulose Synthesis ............................................. B. Resynthesis of Cellulose ............................ ... V. Degradation of Cellulose by Bacteria and Fungi ........... A. Degradation of Cellulose by Bacteria ..................... B. Degradation of Cellulose by Fungi ........................ C. Cellulolytic Enzymes ........... D. Inhibition of Cellulases ........ VI. Applications for Cellulases ......................................... References ......

91 92 95 98 99 101 101 102 103 109 122 124 125

I . Introduction Cellulolytic microorganisms are involved in the deterioration of all types of cellulosic material, contributing to man's material needs. They are destructive to the raw materials and end products and detrimental in the manufacturing processes of wood, cotton, and other natural textile fibers. They cause damage to fruits and vegetables, and many species are phytopathogenic. The annual damage by woodrotting microorganisms in the United States amounts to 3 billion dollars. Furthermore, it has been reported that attacks solely on spruce by just one organism, the root-rotting Polyporous annosus, in Sweden cause an annual loss of 100-200 million Crowns. Even if similar economic losses caused by cellulolytic organisms are calculated for all parts of the world, it must be realized that this negative role is very small in comparison with their vital role as regulators of the dynamic equilibrium in nature. Calculations for the organic net production by the photosynthetic process indicate that about 3 x 1O'O tons of carbon in the form of COz are transformed yearly into plant materials over the earth as a whole. Approximately one third of the organic material produced is cellulose, which occurs in most plants as the skeletal 'The survey of literature pertaining to this review was concluded in November 1966. The references cited have partly been selected for the scope of information provided by summarizing reviews, lectures at Symposia, etc. than in recognition of original sources.

91

92

BIRCITTA NORKRANS

substance of the cell. If microbial degradation of cellulose did not occur, the atmospheric carbon dioxide pool, which is generally the limiting factor for photosynthesis in nature, would -because of the equilibrium between carbon dioxide in the air, that dissolved in the sea, and carbonates in marine sediments (Sillen, 1963)-presumably not be consumed within decades as has been theorized, but dead vegetation would pile up and suppress new production. The consequences for a world population growing in a logarithmic scale can easily be imagined. In working toward the prevention of the disintegration of useful cellulosic material and toward facilitating the breakdown of dead plant debris, the enzymic process of the biological degradation of cellulose has to be clarified. Native cellulose is not simply a glucose anhydride chain, showing a characteristic insolubility in water at even very short chain lengths, but a high polymer arranged in supramolecular structures, laid down in cells and tissues and embedded in a noncellulosic matrix, often surrounded by lignin. These structural features hardly facilitate the understanding of the degradation processes. Several investigators have attacked the problem, and their work has been reviewed in extensive monographs and reviews (e.g., Cowling, 1958; Fghraeus, 1958; Gascoigne and Gascoigne, 1960; King, 1961; Seifert, 1962; Siu, 1951; Wood, 1960). Three years ago the present author compiled a review on the degradation of cellulose (Norkrans, 1963a). Since then, research work within this field has enlarged our knowledge about the micromorphology of wood decay and of the initial phase of degradation of native crystalline cellulose. It has brought new ideas to our concept of the structure of cellulose and its biosynthesis, which is of importance for our understanding of cellulose degradation. In the following discussion these topics will be dealt with in greater detail.

II. Cell Wall Morphology and Chemistry Microscopically, three fundamental parts associated with the plant cell wall can b e recognized: primary wall, defined as the part of the wall produced during surface growth; secondary wall, produced after the surface growth has ceased; and between adjacent cells in the tissue there exists the middle lamella. In the secondary wall of normal xylem fiber or tracheid, formed when successive polysaccharide layers are deposited next to each other, three distinct layers can be demonstrated. The outer layer, S,, is a thin transition layer, the middle S2 layer forms the bulk of the secondary wall, whereas S3, when

CELLULOSE AND CELLULOLYSIS

93

present, is a thin layer adjacent to the cell lumen. In wood fiber, S z is 1-5 p thick, S1 and Ss 0.1-0.2 p. Chemical fractionation of the cell wall by means of dilute alkali (usually 17.5% NaOH) divides the polysaccharide compounds into an alkali-insoluble component, a-cellulose, and an alkali-soluble part comprising noncellulosic polysaccharides, i.e., “hemicelluloses” and pectic substances. Most chemical analyses deal with the overall composition of whole plant tissues, and deductions about the cell wall composition are made on the assumption that the polysaccharides and lignin are derived solely from these structures. With cambial and other soft tissues having no secondary thickening, the composition of the primary cell wall may be studied directly. Setterfield and Bayley (1961) and later Roelofsen (1965) summarize quantitative data from about thirty investigations dealing with primary wall material from different species and parts of plants. Generally, the a-cellulose constitutes only 20-35% of the dry wall materials. They also include investigations of cotton, the unicellular seed hair, which has about 50% cellulose in the primary wall (see Tripp et al., 1954), and a study of separate cambial cells of Acer pseudoplatanus (Northcote, 1963), which has been subjected to enzymic fractionation, instead of being analyzed by the usual chemical method (Lamport, 1965). With regard to the noncellulosic polysaccharides, it is more difficult to give precise figures for their percentage composition, mainly because of the difficulties involved in subgrouping. If, however, galactans, arabans, and galacturonides are grouped as a “pectic triad,” 10-20% are pectic substances, 3550% may be considered to consist of hemicelluloses. Proteins comprise 3-lo%, and lipids 2-7%. By far the most common analyses, however, deal with woody tissues from conifers (softwoods) or deciduous trees (hardwoods) in which xylematic cells with secondary thickening dominate. Thus the analytical values mainly reflect the composition of secondary walls. An average cellulose content of 43+2% for both hardwood and softwood, and 1-4% of pectic substances has been reported (Timell, 1965b). Hemicelluloses amount to about 40% in hardwood and 30%in softwood (Meier, 1964). The chemistry of wood hemicelluloses has recently been discussed in detail (Timell, 1964,1965a). By analyzing fibers from the outermost part of the xylem of birch, pine, and spruce at various stages of maturation, Meier and co-workers (Meier, 1964) have been able to deduce the proportions of the indi-

94

BIRGITTA NORKRANS

vidual polysaccharides in the different layers of the fiber wall. For all these three woods, the cellulose and hemicellulose content is lowest in the middle lamella and primary wall layer (taken together at the analysis), increasing successively in the inner part of the cell to a maximum in the Sz-& layers. Pectic substances are mainly present in the middle lamella-primary wall layer. Figure l a illustrates this general trend. Microfibril (crystalline phase)

-

-

Continuous matrix (amorphous)

r

M I

1

I

I

I I

I

I I ~

Cellulose 50 I 55

I

YO

1

“Hemicellulose“

%

I

I

I

I I I

GDP-glucose

I

48 I 64

1 I

I 48 tI

24 I

P e ic substances -

B

I

Soluble pool of hexose phosphate

Glucose

2

60

-;j 35 24

UDP-glucose UDP-glucuronic acid UDP-xylose GOLGI BODY UDP-galactose UDP-galacturonic acid

P 46

FIG. 1. a. The cell wall, its different layers and their relative amounts (percentage) of chemical constituents in xylem fibers of pine (p)and birch (b). M, middle lamella; P, primary wall; S, secondary wall; S,, Sz, and S,, its different layers; S:% adjacent to cell lumen. The analytical data derive from Meier (1964), where values from pine and spruce were very much the same. Values for glucuronoxylan, by far the most important “hemicellulose” in hardwoods, have been given here as percentages of “hemicellulose” in birch. (A small percentage of glucomannan was also found in each layer.) For the same reason, the percentage glucomannan has been given as pine “hemicellulose.” In this case about 12% glucurono-arabino-xylan was also present in each layer.) Values for arabinan and galactan have been added and given as “pectic substances” (to which the nonanalyzed amounts of pectic acids should be added to get a complete account of pectic substances). In the Si pine value of 9% given, 8% derive from galactan, which might be due to the presence of the “hemicellulosic” galacto-glucomannan). b. Differences in (1) the nucleotide component of the precursors and ( 2 )possible sites of synthesis for the polvsaccharides formed by a I l l a i i t c,ell. nccordinq to tlw l r \ . l ~ o t l i c ~ s iII!,s Northcote and Pickett-Heaps (1966), when glucose was offered to the cell. Otherwise the plastid sugar will serve as donator to the hexose-phosphate pool.


95

The final phase in the differentiation of fibers and tracheids is that of lignification. Basically, lignin consists of a three-dimensional polymer of phenylpropane units, present as guaiacylpropane in softwoods and guaiacyl- and syringylpropane in hardwoods. Lignin synthesis in vitro and in vivo has been treated by, e.g., Freudenberg (1964) and Neish (1964), and in a very readable article Stewart (1966) discusses the course of lignification during fiber senescence and the postmortem enzymic reactions. In softwoods, about 60% of the total lignin (amounting to 30%) is localized in the region of the lamella and primary wall, the corresponding figure for hardwoods, which contain 20% lignin, being about 90% (Meier, 1964). For some fiber strands in phloem of flax, hemp, jute, and ramie, the a-cellulose amounts to about 70% (Treiber, 1957), for cotton to about 90%. Cotton has about 5% noncellulosic polysaccharides, and no lignin. 111. Cellulose Chemistry a n d Supramolecular Morphology

Cellulose is a high polymer of p-1,4-linked D-glucose residues. All methods for the isolation, purification, and solubilization of cellulose seem to depolymerize the molecule more or less. However, by improved methods giving a minimum of depolymerization, higher values for the degree of polymerization (DP)are obtained. Goring and Time11 (1962) found DP values of 8000 to 10,000 for wood cellulose. Similar values were found for all the angiosperms, gymnosperms, and ferns tested. Species of Equisetum, Lycopodiurn, and Psiloturn, descendants of plants preceding the ferns in the course of evolution, had DP values of 2000-4000. A value as high as 15,000 was found for cellulose from unopened cotton balls, corresponding to a molecular weight of 2.4 x lo6and a chain length of about 7 p (unfolded chain). Marx-Figini and Schulz (1966b)obtained the same values for cotton, and are of the opinion that cellulose derived from the secondary wall of all higher plants will have a DP of about that magnitude. Native cellulose is an aggregate of well-defined partly crystalline microfibrils of indefinite length. Three different morphological models have been proposed. 1. From early X-ray diffraction studies on cellulose, its partially crystalline structure was proposed to fit into a model called the fringe micelle. When the concept of this morphological model was more developed, cellulose could be described as an aggregate of individual glucose anhydride chains arranged more or less parallel to

96

BIAGITTA NORKRANS

each other in the microfibril. This parallel arrangement occurs to a much lesser degree in amorphous and paracrystalline regions, whereas in crystallinic micelles (or crystallites) the chains are oriented strictly parallel by means of hydrogen bonds. Micelles measure at least 600 A along the fiber axes and about 50 to 60 A perpendicular to the axis, corresponding to a maximum of 100 in electron microscopical studies (RBnby, 1958);the cellulose molecules are believed to be long enough to pass through several micelles (Fig. 2a). The microfibril has been described as a flat ribbon, according to most authors 100 to 300 A wide, and 40 to 100 thick. Frey Wyssling (1959) and Muhlethaler (1960) have a somewhat different concept. They envisage “elementary fibrils” of 35 x 35 A, each having paracrystalline sheaths, which would occur either freely or aggregated. All experimental values for microfibrils found by them or others should thus be considered as multiples of 35

A

A

A.

a

b

C

-Y

FIG.2. Morphological models of native cellulose. a. The fringe micelle. Models with folded cellulose chains: b. Folded along the fiber axis according to Marx-Figini and Schulz (1966a,b). c. Molecules forming a flat ribbon by folding back and forth perpendicular to the ribbon axis, the ribbon being wound into a helix according to Manley (1964) (after Marx-Figini and Schulz, 1966b).

Since the concept of a folding process generally associated with the crystallization of macromolecules is gaining ground (cf. RBnby and Noe, 1961; Davidovits, 1966), this idea has been adopted even for the molecular morphology of cellulose (models 2 and 3).


97

2. Manley (1964) made electron microscopical studies of aqueous suspensions of microfibrils from ramie, obtained by ultrasonic disintegration, by using the negative staining technique. The individual filament had a diameter of 35 and appeared to have a periodic variation along its length, giving it a beaded appearance. The same was observed in preparations from cotton, wood, and bacterial cellulose. According to Manley, the “beads” may possibly appear if the microfibril is composed of a flat ribbon wound into a tight helix. From X-ray diffraction studies it is known that the molecular chain axis is arranged parallel to the length of the microfibrils. In Manley’s folded model, this is achieved in the following way: the chain molecules form a flat ribbon by folding back and forth in a “concertinafashion,” nearly perpendicular to the ribbon axis, the ribbon then being wound into a tight helix (see Fig. 2c). Manley (1965a) suggested 4 to 5 glucose units in the fold and hypothesized that hydrolysis of cellulose involves preferential attack at the chain folds. When studying a number of regenerated celluloses, Manley (1965b) found that they were apparently also composed of filamentary structure elements, remarkably similar to those in native celluloses. The average length of the filaments reconstituted in vitro was found to increase with the molecular weight of the dissolved cellulose. It means that the elementary microfibril, characteristic of natural as well as of regenerated celluloses, is in fact a cellulose molecule. 3. Marx-Figini and Schulz (1966a) suggested a flat ribbon formed by the molecule chain folded along the ribbon axis (see Fig. 2b). Heyn (1966) made observations on cell walls of cotton, ramie, and jute fibers by high-resolution electron microscopy on negatively stained ultrathin sections. This new technique has advantages over the study of isolated fibers used hitherto in that the original arrangement of microfibrils is better preserved and their collapse into larger fibrillar units is prevented. Again, a certain periodicity could be observed, giving a beaded appearance to the microfibril, measuring about 35 A in diameter as determined by X-ray diffraction. Furthermore, it was found that the average distance between neighboring fibrils is equal to, or less than, the diameter of the microfibril, constituting the maximum space which can be occupied by amorphous material. These values, however, will naturally vary with the degree of swelling. Judging from the photomicrographs, Heyn’s material gives always a beaded appearance. When discussing the probability for a folded cellulose molecule, Muhlethaler (1965)points out that a few molecules might be folded, but it is unlikely that all cellulose

A

98

BIRGITTA NORKRANS

molecules are folded in the native material, because many of the physical properties, e.g., tensile strength, can only be explained if straight molecules are present. None of the fibril structures, however, has been definitely proved, and more work has to be done in order to prove or disprove them. The microfibrils are embedded in a continuous matrix of noncellulosic polysaccharides. Such an organization into microfibrils and matrix can be distinguished during the whole wall development. The microfibrils are oriented a t random in the primary wall, and in a more or less steep helix in the different layers of the secondary wall. The orientation of microfibrils has been extensively discussed (see Roelofsen, 1965). Whether or not any chemical linkage exists between cellulose and hemicelluloses, or between cellulose and incrusting material such as lignin, has been discussed several times. According to Lindberg (1960) and Meier (1964), linkages between cellulose and lignin seem improbable, although there are indications for linkages between hemicellulose and lignin. The same authors also point to the lack of evidence for chemical linkages between cellulose and hemicelluloses, or for the intermixing of, e.g., xylose residues with the glucose residues into the cellulose chain. The question of such chemical associations has arisen because of the difficulty in removing from a cellulose sample all of the hemicellulose components, chiefly xylose residues from hardwood, and mannose residues from softwood, and the difficulty in obtaining glucose as the sole sugar upon cellulose hydrolysis. Increasing knowledge of the biosynthesis of cellulose and other complex polysaccharides in the plant cell wall, however, should eventually yield definite information about the composition and structure of cellulose. IV. Biosynthesis of Cell Wall Polysaccharides Recently, Northcote and Pickett-Heaps (1966) presented a hypothesis for the localization and course of formation of cell wall polysaccharides. Their hypothesis is based on radioautographic studies of root tips of wheat, incubated with D- (1- or 6-) g l ~ c o s e - ~ H for short periods, followed by incubation with unlabeled glucose. The incorporation of labeled material was followed by means of electron microscopy on thin tissue sections and by chemical analysis. Within the Golgi apparatus (a cytoplasmic organelle consisting of a stack of about six cisternae, each 0.6 to 1p across, arranged on top of one another like a pile of plates and bound by a unit membrane) a pool of precursors for the synthesis of polysaccharides, containing galactose,


99

galacturonic acid, and arabinose occurs. After the synthesis of these polysaccharides, which are probably identical with pectic substances, they are passed to the vesicles associated with the Golgi bodies, and transferred within these across the cytoplasmic membrane to be incorporated into the cell wall. Later on, a vesicle transport of polysaccharides from the hemicellulose fraction containing glucose, xylose, glucuronic acid, and mannose residues, may take place. The labeled glucose derivatives, which probably do not emerge from the Golgi body, are not immediately available for cellulose synthesis. From work by Hassid and co-workers on the synthesis of pectins and hemicelluloses by cell-free enzyme preparations (Villemez et al., 1965; Pridham and Hassid, 1966), it seems reasonable to assume that uridine-diphosphate sugars and sugar derivatives serve as precursors in these syntheses. In addition, Barber and Hassid (1965) demonstrated cellulose synthesis from guanosine-diphosphate-D-glucose, (GDP-glucose), with particulate enzyme preparations from cotton balls. Four to eight-day-old balls were the most active ones. Since GDP-glucose could not be replaced by other glucose nucleotides such as UDP-, ADP-, or CDP-glucose, both the nucleotide precursors and the site of the synthesis within the cell must be different for cellulosic and noncellulosic polysaccharides, which is illustrated in Fig. lb, modified from Northcote and Pickett-Heaps (1966). Ledbetter and Porter (1963) first demonstrated microtubules in plant cells. They suggested that these structures, situated just beneath the surface of the protoplast, and reflecting the orientation of the microfibrils in the adjacent cell wall, may be involved in fibril formation. Pickett-Heaps and Northcote (1966) and Pickett-Heaps (1966) further develop this idea and also propose the concept of the endoplasmic reticulum functioning in the transport and synthesis of cell wall material.

A. CELLULOSE SYNTHESIS Our knowledge of cellulose synthesis is very incomplete and the mechanisms for the formation of the supramolecular structure will not be understood until we know: (1)the biochemical pathway for the formation of the precursor of the glucose monomer and its polymerization into a p-1,4 glucose anhydride chain, (2) its transformation into a microfibril, and (3) the incorporation and orientation of the microfibrils into the material comprising the plant cell wall. Only a few years ago, almost all available information dealt with cellulose formed as a “by-product” by some bacteria, notably Acetabacter xylinum, studied mainly by Colvin and co-workers. Their concept of this

100

BIRGTTTA NORKRANS

synthesis (Colvin, 1964; Dennis and Colvin, 1965) is summarized by the following formulation: glucose, or a glucose precursor such as dihydroxyacetone, gluconate, or related compound, enters the cytoplasmic membrane, where it is activated. (The route by means of glucose 1-phosphate and UTP to UDPG could not be found, and neither guanosine, GDP, or GTP seemed to have any effect on the synthesis.) The glucose residue is then transferred to a lipid carrier, presumably a lipophilic 30-carbon long polyhydroxy alcohol, which diffuses across the cell wall and attaches the glucose residue presumably directly to the tip of the elongating microfibril, occurring free in the medium. The microfibril thus grows by end synthesis and is not produced by aggregation of preformed chains. Formation of the microfibril is, however, still a matter of dispute (cf. Ben-Hayyim and Ohad, 1965; for further references, see Horecker, 1966). It is probably synthesized directly in crystalline form (Sumi et al., 1963). Marx-Figini and Schulz (1966a,b) investigated cellulose synthesis by a series of mainly kinetic studies of cellulose formation in cotton balls, during a period of 24 to 72 days after pollination. According to these authors, cellulose synthesis takes place in two distinct phases, the first one proceeding slowly and yielding only a small amount of cellulose of nonuniform molecular size, D P 2000 to 6000. The second phase is rapid, resulting in the bulk of the cellulose being formed. Independent of time elapsed and amount of glucose available for synthesis, cellulose of a high and uniform degree of polymerization, D P about 14,000, is formed in this phase. The first phase, lasting up to 35 days, corresponds to the formation of primary wall, the second to that of the secondary wall. Low D P values of cellulose from the primary wall have been reported previously (Wardrop, 1962). In order to explain the formation of the uniformly large macromolecules, a template mechanism was suggested; the template could be situated inside the microtubules. The enzymic combination of activated glucose molecules with the cellulose chain along the template is associated with the folding of the chain. The synthesized, folded chain may be fixed at the growing end of the fibril by hydrogen bridges. If the synthesis of “secondary wall cellulose” is dependent on structures like endoplasmic reticulum, microtubules, and cytoplasmic membrane, many of the unsuccessful efforts to synthesize cellulose in uitro might be attributable to the lack of undamaged structures in the enzyme preparations. In the aforementioned in uitm synthesis by an enzyme system from young cotton balls (Barber and Hassid, 1965), not only GDP-glucose


101

but also GDP-mannose was incorporated in the polysaccharide formed. According to Marx-Figini and Schulz this polysaccharide corresponds to “primary wall cellulose.” T h e difference between primary wall cellulose’’ and glucomannan, characterized as a hemicellulose, may perhaps be quantitative rather than qualitative and it brings to mind Preston’s (1961) statement from algal studies: “Cellulose is a mixed polysaccharide.” ‘I

B. RESYNTHESIS OF CELLULOSE Much of the aforementioned data is based on the assumption that cellulose is irreversibly deposited. This may be true for mature plant tissues; in young cells, however, breakdown of cellulose b y enzymes of endogenous origin has been discussed as a factor affecting the plasticity of the primary wall (Matchett and Nance, 1962; Maclachlan and Perrault, 1964) and involved in cell fusion processes (Sassen, 1965).Cellulases have been found in these cases, as well as in some plants studied by Reese and Mandels (1963). Evidence supporting the idea of reversible cellulose synthesis has been obtained by 14C studies on isolated wheat roots b y Margerie and Lenoel(l961). It has been shown later (Peaid-Lenoel and Axelos, 1965) that wheat roots previously depleted of sugars had a reduced capacity for synthesizing cellulose, a P-l,S-glucan, sophoran, not previously known from higher plants, being formed instead. Since Mandels and Reese (1959) have found sophorose to be a very active cellulase (C,) induced for Trichoderma virada, one might feel tempted to suggest a reuse of cellulose in starved cells by means of sophoraninduced cellulases. Besides the breakdown of cell wall material during seed germination (for refs. see Norkrans, 1963a), a resynthesis of cellulose may take place, which surely is not the case during the ripening process, where cellulases of endogenous origin are supposed to be involved (Hall, 1964; Dickinson and McCollum, 1964). V. Degradation of Cellulose by Bacteria and Fungi

Cellulase production is the common denominator for cellulolytic organisms. The cellulosic material occurring in nature, however, varies greatly. It ranges from heartwood and sapwood in living, more or less lignified trees, through nonliving trunks, stumps, felled timber, forest litter, nonwoody plants, soft tissues of fruits, to transformed debris of all this matter mixed in soil or water, material offering conditions for growth selective for microorganisms. Thus, even in cases of

102

BLRGITTA NORKRANS

about equal cellulolytic potential per se for the organisms concerned, we will find them divided in specific groups specially adapted to compete on one or another of the natural substrata.

A. DEGRADATION OF CELLULOSE BY BACTERIA Most cellulolytic organisms are found among bacteria and fungi. In more or less anaerobic, closed environments, as in guts of herbivores, in the digestive juices of invertebrates, and in the rumen of cattle, bacteria are the outstanding cellulose decomposers. Our knowledge of this latter system, with a mixture of cellulolytic bacteria and protozoan is fairly good, as is evident from Hungate’s recent extensive monograph “Rumen and its Microbes” (1966). Anaerobic eubacterial cellulose decomposers are also to be found in soil (Enebo, 1954; Skinner, 1960), in addition to aerobic species belonging mainly to the genera Cellvihrio and Cellulomonas. Furthermore, some actinomycetes and myxobacteria, including the imperfect genera Sporocytophaga and Cytophaga, are known as strong cellulose decomposers in soil. Their cellulolytic activity and mucopolysaccharide formation have been studied thoroughly since the days of van Iterson and Winogradsky (Stanier, 1942; Fihraeus, 1947; Charpentier, 1965). Cytophaga species also play an important role in the unique cellulolytic marine bacterial flora treated by Kadota (1959). Cellulolytic activity ought to contribute to the ability of parasitic microogranisms to spread through the host plant tissues. Correlation between cellulolytic ability and pathogenicity, however, is presumed for only very few bacteria, and has been convincingly demonstrated for only one, namely the wilt bacterium Pseudomonas solanacearum, studied b y Kelman and co-workers (Kelman and Cowling, 1965; Husain and Kelman, 1959). By means of quantitative chemical analysis of experimentally diseased tomato stem tissue, in a way which has been frequently used in case of wood decay studies but seldom applied to phytopathological studies of nonwoody plants, it could be shown that the a-cellulose content, the ratio of a-cellulose to heniicellulose, and the DP of holocellulose decreased during pathogenesis. The cellulolytic activity tested in vitro, however, was weak in comparison with that of Trichoderma viride. Bacteria have always been considered to play an insignificant role, if any, in cellulolytic attack on wood. There have been recent findings of cellulose-decomposing hyphae-forming actinomycetes from the genus Micromonospora, but also of eubacteria, in softened wood from old foundation poles and logs (Harmsen and Nissen, 1965a,b).


103

Anaerobic conditions may have existed. Attack on wood by Bacillus polymyxa has been experimentally shown (Greaves and Levy, 1965). The bacteria degraded rayparenkymatic tissues, and the permeability of the wood to liquids was greatly increased. Courtois (196613) has studied the bacterial attack on coniferous wood. The micromorphological cell wall deterioration is different from that caused by fungi. Irregular, ruffled corrosion starts from cell lumen and (or) the middle lamella. Close contact between bacteria and cell wall is necessary for decomposition, and the cell wall layers are destroyed successively one after another. In all cases, however, the bacteria show slow action within a restricted area.

B. DEGRADATION OF CELLULOSE BY FUNGI The strong cellulolytic attack of fungi might partly depend on their hyphal organization, which gives these organisms a penetrating capacity and presumably a continuous cellulase-releasing phase. No solely budding yeast of cellulolytic importance is known to the author. Ascomycetes and fungi imperfecti play an important role as decomposers of plant residues in soil, and may form a considerable part of the zymogenous microflora in soil sensu Winogradsky (1949). A closely allied flora is involved in the deterioration of cellulosic textile fibers, for which extensive lists of the genera and species involved have been presented by Reese and co-workers. Besides giving an idea about the succession of invaders in wood, a recent investigation on colonization of wood by soil fungi (Merrill and French, 1966) is quite revealing with respect to the soil flora population. Fusarium sotani was the dominating pioneer invader, followed by Trichoderma viride, Aspergillus ustus, and some species of Penicillium, all together accounting for more than 90% of the isolates from invaded wood. Furthermore, Rhizoctonia solani, previously known to competitively colonize and decompose cellulose in soil (Garrett, 1962), was found among the wood invaders. Even phycomycetes, otherwise known to produce cellulolytic enzymes only in their role as pathogens (e.g., Winstead and McCombs, 1961; Unestam, 1966) were found to be wood invaders. Their growth, however, may have been supported b y some easily available cytoplasmic nutrients. Correlation between cellulase activity and pathogenicity has been discussed for varieties of wilt-inducing Fusarium oxysporum (Husain and Dimond, 1960; Deese and Stahmann, 1962). Cellulolytic activity has been found in Rhizina undulata, a root rotter on young pine plants (Norkrans and Hammarstrom, 1963; Norkrans, 1967).

104

BIRGITTA NORKRANS

1 . Wood Decay Ever since the time of Robert Hartig (1878), it has been known that fungi are responsible for decay of wood. Different fungi give rise to different types of wood rot, brown-, white- and soft-rot having been distinguished. The blue-staining fungi, also first recognized by Hartig, are associated with wood, although not causing decay. The composition of decaying wood has been studied in numerous investigations, early as well as recent ones (Seifert, 19661, and in some studies the wood analysis has been extended even to determinations of degree of polymerization, particle-size distribution, and X-ray diffraction (Cowling, 1961, 1963; Levi and Preston, 1965). Biochemical studies have dealt with the character of the enzyme systems involved (Lyr, 1959a,b; Lyr and Novak, 1962; Levi, 1964), and microstructural changes caused by all types of wood-inhabiting fungi have received much attention in recent microscopical and electron microscopical studies (Corbett, 1965; Courtois, 1963a,b, 1966a; Greaves and Levy, 1965; Levy and Stevens, 1966; Liese, 1966; Schmid and Liese, 1965; Wilcox, 1965). Some information obtained from these studies is briefly sunimarized below. In wood, the rays generally containing easily available nutrients for fungal growth afford initial penetration paths for fungi. This initial penetration is followed by a passive penetration via the pits until all the easily available nutrients have been utilized for the mycelial build-up. From this stage onward, active penetration with subsequent degradation of the cell wall material begins. a. Blue-Staining Fungi

The blue-staining fungi seem not to cause any enzymic breakdown of wall substances. Their ability to penetrate lignified cell walls has been attributed to their characteristic club-shaped hyphae, the heads of which are provided with a spearlike projection. T h e finding that these “transpressoria” are able to pass through metal foils suggests that mechanical forces are involved, although the possible presence of some lytic enzymes, localized to the spear, has not been excluded. The blue-staining fungi all belong to the Ascomycetes and Fungi Imperfecti. The above-mentioned results were obtained in studies of Aureobasidium pullulans (=Pullularia pullulans), Ceratocystis pilifera (=Ophiostoma coemleum), Ceratocystis piceae, and Phialocephala phycomyces (=Scopularia phycomyces). The flora of bluestaining fungi is known from extensive studies on wood pulp and timber by Meliri arttl co-workers (Lagerberg et at., 1927; Melin and


105

Nannfeldt, 1934). Several of these fungi are dark-coloured per se, in other cases iron reactions between fungus and substrate give the discolored sap (Gadd, 1965).

b. Soft-Rot

The term soft-rot was coined (Savory, 1954) for the softening produced in the surface layers of wood by the action of Chaetomium globosum, isolated from the wood of water-cooling towers, when cylindrical cavities with conical ends appeared with certain constancy in the secondary cell wall of the decayed wood. The term soft-rot is now used whenever the characteristic cavity pattern is present, even if no softening of the wood surface has occurred. These cavities, first studied by Bailey and Vestal (1937), may be formed by Chaetomium globosum in the following way: Hyphae invade the tracheid lumen and align themselves parallel to the long axis. Hyphae within the lumen form some thin hyphae, which penetrate the wall of the tracheid laterally at right angles to its long axis. When reaching the S z layer, these penetration hyphae reassume their original thickness and branches out at right angles to its penetration path in a direction parallel to the tracheids’ long axis, thus assuming the shape of a T. The cellular substances around this hypha become decomposed, giving rise to an elongated cavity. Generally, a fine hypha grows out from the apex of this cavity, thus giving rise to another cavity. A chain of cavities, separated from the next by a short distance, is formed in this way and may be seen aligned in the same spiral arrangement as the cellulose microfibril in the S z layer. Where enzymic action has been considerable, the cavities have coalesced to form a large cavity. Courtois (1963a) has described in detail a series of somewhat similar decay patterns. Chemical analysis of beech wood undergoing attack b y Chaetomium globosum has shown that the marked cellulose depletion taking place in the early stages of the attack is accompanied by an initial increase in the high polymer fraction (Levi and Preston, 1965). This increase reaches a maximum at about 20% weight loss, whereafter it decreases slowly. Alkali solubility is regarded as a measure of the amount of degradation products present in excess of those not immediately being utilized by the attacking fungus. When red beechwood was decayed to 5396, both alkali solubility and soluble sugars amounted to zero (Seifert, 1966). In this respect soft-rot resembles white-rot. When the wood had decayed to about 80% weight loss, three-quarters of the residue was lignin. I n this respect soft-rot resembles brown-rot.

106

BIRGITTA NORKRANS

Soft-rot is more common in deciduous wood than in coniferous; Courtois (196313) suggested that the reason for this is attributable rather to qualitative differences in the lignin than to quantitative differences, as has been previously supposed (Meier, 1955). The methoxyl content in hardwood lignin is generally higher (>21%) than that in conifers (ca. 14%). Like all other wood-destroying fungi, those responsible for soft-rot also remove methoxyl from lignin; this seems to be the main effect on lignin of soft-rotters as well as of brown-rotters. Accordirg to Levi and Preston, the longitudinal path for diffusion of the “lignin-modifying” or “cellulose-freeing” enzyme will lie between the microfibrils while the transverse path will lie through lignin deposited in the “amorphous” regions of the microfibrils. Assuming then that longitudinal diffusion is more rapid than transverse, a pathway having a conical pattern will be opened for the real cellulolytic enzymes producing the typical, conically ended cavities. This is a modification of Roelofsen’s mesh hypothesis (1956). Soft-rot is produced by ascomycetes and fungi imperfecti. Their importance is based not so much on their deterioration capacity as on their ability to grow under conditions too moist to favor growth of basidiomycetes and on their ability to survive during dry periods (Liese and Ammer, 1964). In a review dealing with soft-rot fungi, Levy (1965) makes mention of a list, compiled by Corbett (1963), containing all the fungi recorded as inducing soft-rot both in hardwoods and in softwoods in controlled cultures. Though the list records some forty organisms, it omits marine fungi, which have been given by Jones (1963).To a list of fungi isolated from wood in watercooling towers and later tested for soft-rot activity (Courtois, 1963b), some other rot-inducing organisms can be added: species of Paecilomyces, Chaetomium, Ceratocystis, Rhizoctonia, and Stysanus, these being the most active ones with respect to weight loss, formation of hyphae, and structural changes caused; they are much more aggressive than the strains of Trichoderma viride used. None of the phycomycetes isolated had any activity. Several species, listed above as bluestaining fungi, can also be found here. It has also been pointed out that the action of soft-rot fungi appears to be intermediate between that of blue-staining fungi and wood-rotting basidiomycetes, showing properties of both under certain conditions.

c . Brown-Rot and White-Rot Brown- and white-rot, first described by Falck (1926) as destruction and corrosion rot, respectively, are caused by basidiomycetes. The


107

fungal hyphae are located within the cell lumina; they pass from one

cell to another through the pits or by producing bore holes. The brown-rotting fungi decompose cellulosic and noncellulosic polysaccharides. They depolymerize the cellulose more rapidly than the depolymerization products are utilized by the organism in the early stages of decay. Since they start their decaying process from hyphae within the cell lumen and break down the S2 and later on the S1 layer while the S3 layer, closest to the lumen, still is intact, d i h s i bility of the enzymes produced has been postulated. From electron microscopical studies of the attack of six different species on samples of sapwood from beech, spruce, or pine, however, Courtois (1966a) concluded that S3 was attacked before the S z layer. Certain difficulties may be associated with the interpretation of the different layers in electron microphotographs. Liese (1966) pointed to the observations of slime substances around the fungal hyphae which might previously have been attributed to the S3 layer. The white-rotting group is a more heterogenous collection of organisms having in common a capacity for decomposing lignin, and for producing enzymes oxidizing phenolic compounds -probably related to lignin-a fact which has been utilized in diagnostic color tests for identifying white-rotting fungi ever since it was first described (Bavendamm, 1928). The relative amounts of lignin and cellulose destroyed and utilized vary. A gradual thinning of the cell wall takes place, characterized by essentially complete removal of one wall layer at a time, beginning from lumen with Ss and progressing toward the middle lamella. Lignin solubilization is supposed to precede cellulose decomposition. The depolymerization of wood polysaccharides is slight, each constituent being depolymerized only as rapidly as the degradation products are metabolized. Among the investigations mentioned above, however, only that by Lyr and Ziegler (1959) relates the study of enzymes formed with decay produced. Two white-rotters, Phellinus igniarius Quel. and Collybia velutipes Curt. were studied by these authors. The attack on wood was polyenzymic, production of the different enzymes being simultaneous. The enzymes active against insoluble wood components were extracellular. The use of such heterogeneous a substrate as wood, however, presents a lot of difficulties and uncertainties. Information about the inducibility was later obtained from growth experiments on several wood-rotting and wood-inhabiting fungi, including the brown-rotting Coniophora cerebella, and subsequent examination of the extracellular polysaccharide-degrading enzymes

108

BIRGITTA NORKRANS

produced. C . cerebella has recently been studied in a similar way b y King (1966) who used a highly active strain which produced rapid and extensive weight losses in the standard decay tests. Enzymes that degraded carboxymethyl-cellulose and precipitated cellulose (a-cellulose to only very limited extent), spruce xylan, and glucomannan, were found in the culture filtrates, besides starch-degrading glucoamylase and laminarinase [p-(1,3)-glucanase]. All were adaptive except the laminarinase, which however, may be a self-induced rather than a constitutive enzyme, associated with the lysis of older areas of mycelium (p-1,3-glucans have been found in the hyphal walls of fungi, see Clarke and Stone, 1963; Mitchell and Sabar, 1966). Approximately 2000 species of basidiomycetes have been classified as wood-decaying fungi. Approximately one-tenth, mostly polyporoid fungi, can attack the heartwood of living trees and a few of them are active exclusively at this site (Wagener and Davidson, 1954). Another group can attack living sapwood, whereas most of these basidiomycetes decompose dead trunks, stumps, and felled timber (Cartwright and Findlay, 1958). Some are also specially adapted to specific species of tree. The factors governing this specialization among the decay organisms are not well understood. It may be due, in part at least, to varying abilities of different organisms (1)to efficiently utilize bound nitrogen, present only in very small amounts in wood (the C : N ratio of wood varies from about 350: 1 to 1250: 1) and to reuse mycelial nitrogen (Merrill and Cowling, 1966) or, possibly, to fix atmospheric nitrogen, (2)to grow at low oxygen pressure (Gundersen, 1961) or at high wood moisture as well as at lack of moisture (Ammer, 1964), (3) to decompose adherent and incrusting material besides lignin resins, waxes, etc. (Hata et al., 1966), (4) to tolerate or eliminate substances in the wood which are toxic to the organism itself, as in the case of thujaplicine, pinosylvine, and other heartwood extractives (Lyr, 1962; Rudman, 1962) or which are inhibiting to its enzymes (cf. Reese, 1965). The ability to initiate attack on litter and other lignified plant material under aerobic conditions seems mainly to b e restricted to hymenomycetes, which exhibit differences among themselves with respect to the relative amounts of cellulose and lignin they decompose (Lindeberg, 1946; Haider et al., 1964).

2. Fiber Decay

The attack on a lignified fiber, i.e., jute fiber, has been studied by


109

Basu and Ghose (1962a,b) who give details for the pattern of attack. Growth inside the lumen is not a universal feature although exhibited by nearly all the dark-colored ascomycetes and fungi imperfecti tested, e.g., Alternaria sp., Memnoniella echinata, Stachybotrys atra, and Myrothecium verrucaria. Internal damage along the lumen was noted in these cases. Photographs given for Chaetomium indicum (Basu and Ghose, 1962a) bring to mind those of soft-rot in wood, with thin penetration hyphae and attack in the secondary wall parallel to the fiber axis. Cracks across the fiber walls are a common feature. Complete breaks were also found as well as V-shaped corrosions and notches in the surface, and helical cracks. Fungal attack on cotton fiber usually involves growth of hyphae within the lumen, accompanied by complete or partial dissolution of wall material from within with transverse cracking and spiral fissures visible at the early stages. Since cotton is frequently used as substrate in the cellulase assays, the correlation between structural changes and enzymic attack will be discussed below. The following section will deal with more or less purified fungal and bacterial cellulase systems, their formation and mode of action. C. CELLULOLYTIC ENZYMES

1 . Extracellular Production As cellulose under physiological conditions is insoluble and cannot permeate cell membranes, the microbial cellulase has been supposed to be extracellular. In culture filtrates of cellulolytic fungi, cultivated under suitable conditions, extracellular cellulases can generally be demonstrated. According to Lyr and Sch6ni.l (1964), an active secretion into the culture medium is to be assumed; autolytic liberation can be excluded, since the activity of preparations from corresponding amounts of mycelia always were low. In replacement media, the cellulase synthesis could be shown to change with age of mycelium. For most fungi tested, the highest production was obtained from young mycelia and hyphal tips, which is surely of ecological importance for these wood rotters. Older mycelia, however, were more productive in, e.g., Fomes marginatus. Bacterial cellulases are more firmly bound to the cells, or to the cellulose, since culture filtrates of cellulolytic bacteria do not always

110

BIRGITTA NORKRANS

contain cellulolytic enzymes. They can generally be obtained b y treatment with autolytic agents, though even such treatment did not release cellulases from C ytophaga cultures (FHhraeus, 1947). Hungate (1966) states that “the cellulase is quite firmly attached to the cellulose and unable to diffuse until the substrate has been digested.” King (1961) reports that there are indications that cellulases occur on the cell surface of rumen bacteria. The importance of culture conditions for cellulase production has often been pointed out (e.g., Whitaker and Thomas, 1963).This has to be stressed again, since apparently insignificant differences in the conditions may influence not only the quantity of enzymes produced but even the quality. Among the factors influencing cellulase production may be mentioned: composition of culture medium (quantity and quality of cellulose used, the amount of metal salts present), pH, temperature, the adequacy of the oxygen supply, and the way in which it is obtained (Norkrans, 1963b; Lyr, 1964).

2. Induced Synthesis Cellulolytic enzymes are generally considered to be formed only in the presence of cellulose. According to Mandels and Reese (196O), however, not cellulose itself but the soluble cellobiose, an hydrolysis product, is the true inducer of cellulase in cellulose cultures. Other compounds having P-glycosidic linkages, such as lactose and salicin, can serve a s inducers; sophorose, mentioned above, is 200 times more active than cellobiose. However, not all soluble compounds with P-glycosidic linkages are inducers. Glucose and cellobiose offered in amounts as large as those used in standard growth experiments generally depress the yield of cellulolytic enzymes (e.g., Norkrans, 1957a).

a. Inducer-Repressor Mechanism in the Relationship of Higher Plant and Fungus Recently, Horton and Keen (1966) studied the sugar repression on cellulase synthesis in the case of Pyrenochaeta terrestris, a fungus involved in the formation of onion pink root. P. terrestris has been observed to produce carboxymethyl cellulase in infected onion roots. The enzyme is also produced in quantities in cultures of the fungus containing cellulose. Studies on replacement media showed that cellulase synthesis was repressed to the basal level by glucose concentrations of ij x 10V’M or above. Krebs cycle intermediates had the same effect. By dilution of the medium, re-formation of a maximum cellulase level followed, identical with that obtained before sugar


111

depression had occurred. This has been taken as evidence for the regulation of the cellulase synthesis by cellulolytic products through a repressor-inducer mechanism. From studies with toluene-treated cultures on various types of celluloses, differing in their susceptibility to enzymic hydrolysis, it appeared that hydrolytic liberation of more than 0.01 mg./ml. glucose per 48-hour periods invoked the repressorinducer regulation of cellulase synthesis (for the repressor-inducer system, see Jacob and Monod, 1963; Pollock, 1959). Thus for phytopathogenic microorganisms in general, it may be said that high sugar levels in the plants would delay development of disease through repression of cellulase synthesis, whereas low sugar levels would promote pathogenic invasion as a result of decreased repression of enzyme synthesis. Since the repression to basal level occurs at such a low concentration as 5 x 1 W M in the case of P. terrestris, cellulolytic enzymes may not be important in its pathogenesis, as sugar concentrations of that low level will exist only in very localized areas within the plants. Endopolygalacturanase repression in P. terrestris occurs first at hundred times higher sugar concentrations (Keen and Horton, 1966), hence this enzyme may be of more importance in connection with pathogenic invasion in the plant. In general, cellulolytic enzymes have been found less important than pectinolytic ones for phytopathogens, and the obligate parasites seem to be found among “high sugar” organisms, according to Horsfall and Dimond (1957). A similar sugar depression system has been suggested to work in a highly balanced symbiontic system, namely, in the formation of ectendotrophic mycorrhiza. Studies of different Tricholoma species (Norkrans, 1950b) revealed that, contrary to what had previously been supposed and later confirmed (Ritter, 1964), some mycorrhiza-forming species produced cellulolytic enzymes. Tricholoma f u m o s u m Fr. (non Pers.), a facultative mycorrhiza-former on pine, produced (when cultured on cellulose as the sole carbon source) cellulolytic enzymes in amounts as high as those produced by any of the litter decomposers. Other symbiontic species of TTichoZoma showed no cellulase production, except T. imbricatum (Fr.) Quel. and T. vaccinum (Pers. ex Fr.) Quel., in which very weak formation of cellulase was attained by adding some “start-glucose,” and indeed only after the glucose had been consumed. Studies by Melin (1923) and later by Melin and Nilsson (1957) and Bjorkman (1942, 1944) had shown that ectotrophic mycorrhizal fungi require soluble carbohydrates from trees. From these different facts, the following assumption was made: “The mycorrhizal mycelium obtains glucose (or soluble carbohydrates) from the plant. CelluIase production would be depressed as long as

112

BIRGITTA NORKRANS

glucose or other soluble carbohydrates attacked by ‘constitutive’ enzymes are present. The cell wall, however, constitutes a cellulose substrate which will induce an increase in cellulase production when the plant, for some reason, no longer forms a surplus of soluble carbohydrates. This may furnish a possible explanation of the ectendotrophic mycorrhiza.” (Norkrans, 1950b).

3. Assay of Cellulolytic Enzymes and Purijicution Native cellulose is crystalline and possesses the complex molecular structure mentioned above. It is combined with noncellulosic material even in cotton fiber. These factors reduce its accessibility and susceptibility to cellulolytic degradation. Cellulase investigators have tried to obviate some of these difficulties by using well-purified, more or less modified celluloses. Some of the common substrates used, including the water-soluble substrates, namely, oligoglucosides, and some cellulose derivatives are presented in Table I, which also gives the methods of measuring the activity. Solubility greatly increases the accessibility of the linkages-providing that the degree of substitution (DS) of the derivatives is not too high-resulting in high TABLE I ASSAY CELLULASE

SUBSTRATES TO

Substrate Insoluble: Native cellulose: Cotton, undried Cotton, dried

“Native cellulose” Cotton, dewaxed

Physically modified cellulose (11): a-Cellulose from wheat straw Hydrocellulose

Determination

References

Morphological changes by: Microscopic observations Marsh (1957) Electron microscopic observations Porter et ul. (1960) Marsh et al. (1953) Tensile strength and alkaliswelling (weight increase) Reese and Gilligan (1854)

Microscopic observations Cellulose residue (gravimetrically or colorimetricall y)

Blum and Stahl(N52) Halliwell(lSl63) Selby (1961)

Formation of reducing groups

Grimes et ul. (1957)

Decrease in turbidity and Formation of reducing groups,

Norkrans (l950a) Li et ul. (1963)

c ~ ( ~ 1 I i i l oresidue w

__

.

113

CELLULOSE AND CELLULOLYSIS TABLE I (Continued) TO ASSAY CELLULASE SUBSTRATES Substrate

Determination ~

References ~

-

Cotton, swollen (HsP04)

Decrease in D P Formation of reducing groups

Walseth (1952) Gilligan and Reese (1951 Myers and Northcote (1959) Whitaker (1953)

Colloidal cellulose sol (alkali) DP 200-300

Decrease in turbidity and DP Formation of reducing groups

Norkrans (1950a,b) Norkrans and RQnby ( 1956)

Cellodextrin (acetolysis) (DP 24)

Formation of reducing groups; ratio moles oligosaccharides

Whitaker (1956)

Soluble: Chemically modified cellulose: Cellulose derivatives DS

Advances in Applied Microbiology, Volume 9

Advances in applied microbiology

Advances in Applied Microbiology, Volume 26

Advances in Applied Microbiology, Volume 42

Advances in Applied Microbiology, Volume 37

Advances in Applied Microbiology, Volume 49

Advances in Applied Microbiology, Volume 73

Advances in Applied Microbiology, Volume 14

Advances in Applied Microbiology, Volume 60

Advances in Applied Microbiology, Volume 45

Advances in Applied Microbiology, Volume 10

Advances in Applied Microbiology, Volume 17

Advances in Applied Microbiology, Volume 18

Advances in Applied Microbiology, Volume 60

Advances in Applied Microbiology, Volume 39

Advances in Applied Microbiology, Volume 11

Advances in Applied Microbiology, Volume 49

Advances in Applied Microbiology, Volume 68

Advances in Applied Microbiology, Volume 56

Advances in Applied Microbiology, Volume 6

Advances in Applied Microbiology, Volume 36

Advances in Applied Microbiology, Volume 72

Advances in Applied Microbiology, Volume 12

Advances in Applied Microbiology, Volume 63

Advances in Applied Microbiology, Volume 47

Advances in Applied Microbiology, Volume 75

Advances in Applied Microbiology, Volume 8

Advances in Applied Microbiology, Volume 65

Advances in Applied Microbiology, Volume 48

Advances in Applied Microbiology, Volume 43

Advances in Applied Microbiology, Volume 3

Advances in Applied Microbiology, Volume 9