In Vitro Toxicity Testing Protocols

CHAPTER1

Alternative

Method

of Assessing

Toxicity

Chris K Atterwill 1. Introduction The safety assessment of new chemicals and pharmaceuticals and the incorporation of these data into a human risk assessmentpackage requires a large number of expensive, regulated tests in animal species including, in some cases, nonhuman primates (I-3). There are currently a wide range of animal replacement alternative opportunities in industrial chemical and drug development (Table 1) (4,.5). Although in vitro methodology has long been used as a basic laboratory tool for defining biological and toxicological processes in different cellular systems, application and use alternatives in industrial compound discovery (i.e., research and development) is slow. Coupled with a relatively low innovation rate in the design of new in vivo tests for the toxicological and safety evaluation of new compounds, this has both ethical and resource implications. From a basic scientific viewpoint in vitro toxicological models have made important contributions in elucidating, e.g., the cellular and molecular mechanisms involved in apoptotic and necrotic cell death and in carcinogenesis and the role of mediators, such as free radicals and oncogenes, in these processes (6). The “take-up” of in vitro systems in toxicity testing is, however, now gradually occurring in industry and resources are being invested slowly into the area for both ethical and scientific reasons. A lot of emphasis currently lies on the ethical question as public sensitivity to animal use in safety testing increases. This has occurred most significantly in the case From Methods m Molecular Wology, Edited by S. O’Hare and C. K Atterwlll

Vol 43 In Wtro Tox/c/ty Testmg Protocols Copyright Humana Press Inc , Totowa, NJ

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Atterwill Table 1 Ammal Replacement Alternatives Improved storage, exchange, and use of information, so that unnecessary repetition of experiments on animals could be avoided. Maximum use of predictions based on physical and chemical properties of molecules Mathematical modeling of quantitative structure-activity relationships. Molecular modeling and the use of computer graphics. Mathematical modeling of biochemical, physiological, pharmacological, and toxlcological systems and processes. The use of lower organisms not protected by legislation, including invertebrates, plants, and microbes.a The use of embryonic and larval vertebrates before they reach the developmental stage at which time they become protected by law a The use of in vitro methods including subcellular fractions, perfused organs, tissue shces, and cell suspensions; and cell organotypic cultures * Human studies, including epidemiology, postmarketing surveillance, and the properly regulated use of volunteers.a % vitro areas

of cosmetic and toiletry safety assessmentand the safety testing of chemical intermediates used in drug synthesis, which have classically used the controversial Draize, guinea pig, and rabbit tests, and for which valid alternatives now exist. Furthermore, in drug development the classic LD,, tests for acute toxicity have not only been partially replaced by the “fixed dose” procedure, but much work is being carried out by Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) on new and rapid in vitro predictors for acute cytotoxicity using human cell lines. 2. Compound Development Utilizing Alternative Test Models The development and registration of both new drugs and chemicals currently requires the submission of a large battery of in vivo toxicity data derived from a number of species for the “risk-assessment” process (1-3). The compilation and validation of the animal batteries has been largely empirical over the years and, although being fairly well-proven for detecting toxic phenomena in animal species, can have limited predictive value for human safety assessment for some of the reasons listed in Table 2 (a,b). There are large lists of chemicals with good animalhuman toxic correlations, but equally, lists of compounds exist that have been withdrawn from the market because of the increasing number of clinically reported adverse reactions (ADRs).

Alternative

Method of Assessing Toxicity

3

Table 2a Reasons for Incorrect Predictions from Animal Toxicity Studies False negative responses Effect not looked for Use of inappropriate assay methoda Improper timing of assay Insufficient target organ exposure0 Incorrect evaluation of an experimental finding0 Failure to consider absence of preexisting pathological condition Inability to identify and measure adverse effect0 False negative and false positive responses Failure to consider differences in metabolic activation and detoxrfrcationa Disregard of anatomrcal and physiological differences between species“ Inability of animals to express human-specific reaction pattern9 Table 2b Conversion Factors for Predictmg from Animal Studies to Individuals Toxic effects Molecular Subcellular Cellular Tissue Organ Whole animal

From experiment to humans! Bioavailability Pharmacokinetics Protein bindinga Metabolism0 Dose and time Concentrationa Receptor sensitivitya Anatomical characteristics Physiological characteristicsa Repau mechanismsa Species-specific responsea Mechanisms of actiona

at Risk

From humans to individuals at risk Healthy men and women Babies and children Pregnant women Elderly People at genetic risk Diabetics People with infections Immunosuppressed individuals Alcohol or drug abusers Smokers Patients with organic disease Occupationally exposed indivrduals

OAreas in which alternatlves tests can have an impact. (Data taken from table compiled by G Zbinden, personal communication.)

It is also well accepted that during the development of a “safe” and effective pharmaceutical (or agrochemical agent) there is a large attrition rate throughout the safety assessment process with massive financial implications. When one superimposes on this the varying worldwide regulatory requirements for administration of a new compound to humans, one can see a number of important reasons for developing in vitro toxicity testing systems either as prescreens or as adjuncts to current in vivo test packages. This, together with harmonization of the cur-

4

Atterwill

rent regulatory requirements, will hopefully improve the sensitivity and specificity of animal tests (3).

3. Summary

of Gains

1. Financial gains: Reduce attrition rate by use of prescreening strategies prior to full regulatory animal study packages and develop better predictors of human toxic phenomena. 2. Scientific gains: Describe more effectively the lesions seen m vivo from regulatory studies and give better definition of safe concentration and clinical dose. Define “direct toxicant effects” on target organs as opposed to indirect effects, and give human reaction mdication using primate cells. “Adjunct” studies will improve sensitivity and specificity of animal studies. 3. Ethical gains: Implementation of 3R strategy (reduce, refine, replace amma1testing). Supplement and reduce current m vivo toxicity tests, particularly those involving distressing procedures and the use of large mammals and primates.

4. Principles, and Types of In Vitro

Aims, Toxicity

Testing

The general advantages and disadvantages of in vitro testing for toxicity are described in Table 3. 5. Validation The successful use and industrial and regulatory acceptance of a new in vitro test model depends on a certain degree of validation (7,s). Detailed validation is generally required if the in vitro test is to replace an in vivo test or is to be used as a prescreen where financial factors are critical. However, when the result from an in vitro procedure is submitted to a regulatory authority along with that from an in vivo test package in order to explain a lesion, then full validation is not formally necessary as long as good laboratory practice (GLP) procedures have been adopted in the execution of that test and the test and endpoints have acceptable relevance. For example, the gradual replacement of the Draize procedure by tests such as the EYETEX or SKIN2 tests has required extensive validation of that test. So, for example, would the use of a prescreen for a new immunosuppressant with adrenal toxicity where there were a limited number of available backup compounds or they were very expensive to synthesize. On the other hand, if a drug company were trying to confirm to the Food and Drug Administration (FDA) that a particular drug had no

Alternative

Method of Assessing Toxicity

5

Table 3 Advantages and Disadvantages of In Vitro Systems for Detection of Xenobiotic-Mediated Toxicity Advantages (general) Detect direct (vs Indirect) toxrc/cytotoxic effects on target organ. Use controlled conditions of exposure-concentration of toxrcant known. Study parent compound vs metabolite (rt liver S9 metabolizing fractions from different species) Study effects on cells vs subcellular organelles. Has resource implications (time, animals, number of compounds tested). Disadvantages (general) Systems not always representative of mature, differentiated target organs (cells dedifferentiated in cell lines?). Xenobiotic concentrations not representative of those in vivo (e.g., plasma protein bindmg factors). Biological barriers absent (e.g., blood-brain barrier in neural cultures of CNS). Metabolite profiles differ Difficulty of culturing/maintaining certain target organs in vitro.

direct neurotoxic effect in humans despite some minor behavioral changes detected in the rat, then submission of data from cultured human exposed neurons would be acceptable, probably without full validation of that particular culture model. Accepted validation criteria for an in vitro system are described in Table 4 and include definition of the specificity, sensitivity, and predictive value of such a test. The validation parameters are obtained by conducting blind validation trials. It is my belief that the requirements for in vitro test validation can be summarized as follows: 1. Full validation involving multicenter coordination: replacement.

To support in vrvo test

2. In-house validation: In vivo test reduction, to support,e.g., development

of a prescreen, 3. Limited inter- or intralaboratory: To supportrefinement or supplementa-

of in vivo toxicity test data,to develop adjunct tests;use of the model to define basic scientific toxic phenomena.

tion

It is believed that validation should not be used as an excuse for nonadoption, nondevelopment, or nonacceptance of in vitro methodology. Sadly, and largely for political reasons, this scenario still exists in many companies and countries.

Atterwill Table 4 Validation Criteria for In Vitro Test Models A formal validation study will require: Careful selection of chemicals (mimmum 20-40?) Use of chemical pairs Toxicological classification from m vivo data “Blind” testing to be performed Method for evaluation of test outcome (absolute values) Method for evaluation of test performance Methods of expressing test performance Other points Are there good in vivo comparative data for compounds chosen? Which kind of in vivo assay trying to emulate/evaluate

in vitro?

Agree with collaborating centers in validation trial at beginning who will be organizing and collatmg data.

6. Spectrum of Available In Vitro Toxicity Tests The currently available models in In Vitro Toxicology (Table 5) (5-8) span six main areas: reproductive toxicity, mutagenicity, irritancy testing, immunotoxicity, target organ toxicity (including endocrine and neurotoxicity), and ecotoxicity involving the use of fish, invertebrates, and so on. Within these main areas there are also important subareas. As alluded to above there are various modes in which to operate these tests in an industrial setting and generally the mode predominance varies significantly according to both scientific area and whether or not one is operating in the drug, chemical, or cosmetic industry. For example, a test system might progress from unvalidated use in the fine description of a pathologically identified lesion for a lead development compound, to the subsequent, semivalidated use of this system in a prescreening mode for second-generation drug candidate compounds. Alternatively, the agrochemical industry has developed a tiered in viva/in vitro hierarchical model for the labeling of industrial chemicals as skin irritants. This latter development was performed under the auspices of the British Toxicological Society, showing how the scientific and industrial communities can interact so well on such issues. Here, a chemical for irritancy classification would proceed from tests on isolated skin or cells in vitro to tests in a limited number of animals in vivo depending on negative or positive outcomes in the initial in vitro tests.

Alternative

Method of Assessing Toxicity

7

Table 5 In Vttro Models Currently Available in Toxicology Mutagenicity testing Irritancy testing Reproductive toxicity testing Quality-Structure Activity Relationship (QSAR) Target organ toxicity Immunotoxicity Hemtc system Endocrine toxicity Neurotoxtctty Acutelcytotoxicity testing

7. Recent Successes and Developments in In Vitro Toxicity

Testing

It is refreshing to observe the momentum that is now gathering in this field (see Table 6) and the way in which “in the face of adversity” some tests are being accepted as full replacement alternatives. It is noteworthy to say that a lot of this energy has been provided by the public and by academic research centers. Apart from the mutagenicity test area where many innovations continue to occur, some of the following recent developments in other areas warrant attention. 1. Eyetex, Skmtex, and Corrosrtex tests for eye and skin irritancy (Ropak Corporation Ltd) and the SKIN2 Model (Advanced Tissue Sciences). More recently, vitro.

the Ropak

Solatex

system for predicting

photoirritation

in

2. The use of hepatocyte “couplets” for in vitro investigation of xenobiotic effects on bile flow. Together with measurements of hepatotoxicity and fatty acid accumulation by these cells, rt may now be possible to obtain a

complete hepatoxicological profile in one in vitro model. 3. Luminescent bacteria (Microtox test) for measuring the ecotoxic potential of industrial effluent. 4. More sensitive in vitro toxicity measurementsusing, for example, the mitochondrial MTT test for succurate dehydrogenase activity. This test gives a more sensitive and earlier prediction of toxicity than classical LDH or neutral red measurements.

Atterwill Table 6 Orgamzatlons Involved m the Development of Alternative Testing Bodies for promotion of alternatlve nonanimal testing FRAME-Fund for Replacement of Animals in Medical Experiments ERGAT-European Research Group for Alternative Testing EURONICHE-European group for alternatlve methods for biology teaching CAAT-Center for Alternatives to Animal Testings (Johns Hopkins Medical School, Baltimore, MD) Dr. Hadwen Trust-Nonammal research and testing strategies (UK-based) Societies, conferences, and journals advancing alternative testing PIVT-Practical In Vitro Toxicology conference IVTS-In Vitro Toxicology Society (UK) Scandinavian

Cellular

Toxicology

Society

FRAME Toxicity Committee and Conference TIV-Toxicology In Vitro, Journal ATLA-Alternatwes to Laboratory Animals, Journal Hildegard Doerenkamp and Gerhard Zbinden Foundation for Reahstlc Animal Protection and Scientific Research, Switzerland

5. Measurements of calcium accumulation in single cultured neurons for the measurement of neurotoxicity. 6. Tlered tests involving both simple and organotyplc organ systems; hierarchical proceeds involving a battery of m vitro and in vivo models.

8. Conclusions Industry and academia have come far in developing in vitro alternatives, and bodies such as Fund for Replacement of Animals in Medical Experiments (FRAME), European Centre for Validation in Alternative Methods (ECVAM), and Center for Alternatives to Animal Testing (CAAT) (USA), have simultaneously enhanced public and regulatory awareness (Table 6). The regulatory and industrial acceptance of new alternative tests depends on proper, well-coordinated validation trials at a level befitting the intended use of the alternative test. This has started through FRAME and European Community (EC) initiatives and good examples have been set by the cosmetics and toiletry industries. More commercial “takeup” is still required for these new tests at the toxicological prescreening and in vivo adjunct testing level. Regulatory harmonization of in vivo animal testing is occurring for both ethical and resource reasons. The gradual replacement of the LD,, test by the “Fixed

Alternative

Method of Assessing Toxicity

9

Dose” procedure for acute toxicity testing and the realization that 6 mo chronic testing (I) is sufficient to identify the most important pathology (excluding carcinogenicity) are most welcome changes. The potential risks for humans in adopting alternative toxicity tests are few and benefits great if the data generated is used correctly. The imminent replacement of all in vivo tests is unlikely but in the future may gradually occur. In the meantime in vitro tests will continue to supplement the somewhat “impirical” animal tests for human toxicity. References 1. Volans, G. N., Sims, J., Sullivan, F. M., and Turner, P. (eds.) (1989) Basic Science in Toxzcology, V International Congress of Toxicology (ICTV), Taylor & Francis. 2 Poole, A. and Leslie, G. B , eds. (1989) A Practical Approach to Toxological Investigations, Cambridge University Press. 3. Lumley, C., Parkmson, C, and Walker, S. R. (1992) An international appraisal of the minimum duration of chronic animal toxicity studies. Hum. Exp. Toxicol. 11, 155-162 4. Parish, W. E. and Hard, G. C. (eds.), Toxicology In Vitro. Proceedings of Second International Conference on Practical In Vitro Toxicology 4/5. 5. Atterwill, C. K. and Steele, C. E., eds. (1987) In-Vitro Methods in Toxicology, Cambridge University Press, Cambridge. 6. Walum, E., Stenberg, K., and Jenssen, D. (1990) Understanding Cell ToxicologyPrinciples and Practice, Ellis Horwood, New York, London, Sydney. 7. OECD Environment Monograph No. 36. Scientific Criteria for Validation of In Vitro Toxicity Tests, Sept. 1990. 8. FRAME 21st Anniversary Issue (1990) ATLA, 18.

CHAPTER 2

LLC-RKl

Cell Screening for Nephrotoxicity

David

X&t

J. White and Chris Seaman

1. Introduction In this test, kidney-derived cells are cultured in the presence of test compounds whose cytotoxicity is then determined by the Neutral Red method, and serves as an indicator of potential nephrotoxicity (1). Healthy LLC-RKl cells (an established cell line, ATCC CCL) maintained in culture continuously divide and multiply over time but still retain certain characteristics of kidney cells in vivo (2). Compounds that have a deleterious effect on these cells may, therefore, be considered as potential in vivo nephrotoxins. In this test system, harmful effects on cell viability are determined by monitoring the uptake of the vital dye Neutral Red into the lysosomes of healthy cells. LLC-RKl cells are maintained in culture and exposed to varying concentrations of test compounds, The cultures are incubated for 48 h. The cultures are then rinsed and incubated for 3 h in medium containing Neutral Red that is taken up by viable cells. After rinsing, the dye present in the cell population is liberated and the amount is quantified using a spectrophotometer, in order to obtain an indication of cell number. Comparison of the number of cells in control and test cultures provides an index of cytotoxicity and an indication of potential nephrotoxicity in vivo. The maintenance and culture of a cell line such as LLC-RKl cells is a relatively simple and inexpensive technique. Additionally, LLC-RKl cells exhibit many features in common with kidney cells in vivo. Among these is the unidirectional transport of solutes via the Na+K+ ATPase From. Methods m Molecular Biology, Ed&d by. S. O’Hare and C. K Atterwtll

Vol. 43 In Wtro Tox/c/ty Testing Protocols Copyright Humana Press Inc , Totowa, NJ

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White and Seaman

system. As a result of this, one-way transport “blisters” are formed in the monolayer, a feature in common with primary kidney cells and other cell lines in culture (2). The application of such cultures to determine nephrotoxicity may potentially allow the rapid, highly reproducible testing of many chemicals on a routine basis. There are, however, disadvantages associated with using a cell line in culture. The cells grow rapidly and are nondifferentiating. Additionally, the cells in culture inevitably lose many characteristics of those in vivo. In particular, the loss of xenobiotic metabolizing activity may affect the sensitivity of the cells to certain chemicals and may raise concern when trying to directly extrapolate results to the in vivo situation. 1.1. Neutral Red Uptake Assay Neutral Red is preferentially taken up into the lysosomes/endosomes of the cell. Absorbances obtained using the Neutral Red assayshave been shown to correlate linearly with cell number over the specific optical density range obtained using this method. Any chemical having a localized effect on the lysosomes/endosomes will, therefore, result in an artificially low (or possibly high) reflection of cell viability and cell number. This factor does, however, make the system useful to detect other chemicals that selectively affect the lysosomes, especially when it is used in conjunction with other tests capable of determining cell number (3).

1. 2. 3. 4. 5. 6. 1. 2. 3. 4.

2. Materials 2.1. Equipment 37°C incubator,hurmdified, 5% CO,/95% air. 75 cm2tissue culture flasks. 24-well tissueculture plates. Inverted phasecontrastmicroscope. Spectrophotometer. Hemocytometer. 2.2. Reagents Cell line rabbit kidney LLC-RKl cells. Dulbecco’s formulation tablets,without magnesiumand calcium. PBS Trypsin/EDTA, 1X cone (dispenseas 10 rnL aliquots into universals and storeat -2O’C), Glbco Europe Ltd. (Paisley, Scotland). Eagle’s minimum essentialmedium (MEM) supplementedwith 1% penicillin/streptomycin, 5% fetal calf serumN.B. Omit penicillin and streptomycin if the test compoundis an antibiotic.

LLC-RKl

Cell Screening

Test

5. 10,000 U/mL penicillm/l0,000 pg/mL streptomycin solution in saline Neutral Red stock solution; 100 mL 0.4% w/v Neutral Red in distilled water, filter sterilized. Store at 4°C until required. 6. Neutral Red medium: dilute the stock dye solutron (1 in SO)just prior to use with culture medium to give a final concentration of 50 pg/rnL. 7. Formaldehyde. 8. Glacial acetic acid. 9. Ethanol. 10. Calcium chloride. 11. Trypsin/EDTA solution: 0.5 g trypsin (1:250) and 0.2 g EDTAIL of Puck’s salme A. 12. Ca2+- and Mg2+-free PBS: Make up Dulbecco’s tablets as indicated by supplier. 13. Neutral Red wash solution: 10% CaC12in formaldehyde. 14. Neutral Red resorb solutron: 1% glacial acetic acid, 50% ethanol, 49% distilled water. 15. Test compounds: These should be drssolved in sterile water, ethanol, methanol, or drmethylsulfoxrde (DMSO), as approprrate at loo-fold the required final concentratron. The final solvent concentration should be kept at a constant level of 1% in the culture medium.

3. Methods 3.1. Cell Maintenance 3.1.1. Preparation of Cells for Freezing 1. Count the cells and dilute to l/O.9 of the intended final concentration of l-2 x 106/mL in complete culture medium. 2. Add DMSO to a final concentration of 10% to the cell suspensron immediately prior to adding to the vials. 3. Aliquot 1.8 mL of cell suspension per vial and freeze at a rate of l”C/min in liquid nitrogen, 1. 2. 3. 4. 5.

3.1.2. Thawing and Culture of Cells When required, thaw the cells rapidly in a 37°C water bath to avoid damage owing to the high DMSO concentration. Transfer immediately to a 75 cm2 tissue culture flask containing -30 mL medium (i.e., l-2 x lo6 cells/flask). After 24 h, rinse the culture with 5-10 mL of PBS at 37OC. Add -30 mL fresh medium. Subculture the cells 2-3 times following thawing before using for test purposes.

14

1. 2. 3. 4. 5. 6. 7.

White and Seaman 3.1.3. Subculture of Cells When the cultures approach confluence remove the cells from the dish by trypsinization. (N.B. If the cells are not subcultured or used for test purposes, the medium should be changed every 3-4 d.) Decant the medium and rinse the cultures with 5-10 mL of PBS at 37OC. Add 10 mL trypsin/EDTA (37°C) and incubate at 37°C. Remove the flasks after 20-30 s and examine visually to ensure the cells have begun to detach (i.e., round up). Discard the trypsin/EDTA solution and return the flask to 37°C. After a further 1 mm, examine the cells and if necessary tap the side of the flask to aid detachment. Add 10 mL of complete medium to neutrahze the trypsin activity and spht or use for test purposes.

3.2. Test Procedure 1. After growing up the cells and preparing a cell suspension as described above, remove an aliquot of suspension and count the number of cells using a hemocytometer and dilute to a concentration of lo5 cells/ml medium. 2. Add 1 mL of the diluted suspension to all the wells of a 24-well plate. Incubate overnight to allow adherence and recovery from the trypsm exposure. 1.

2. 3. 4. 5.

3.2.1. Range Finder Prepare the following test chemical concentrations (diluting the stock solutions 1: 100 in medium) immediately before use: a. 0.5,5,50,500, and 5000 pg/rnL. b. 1% solvent control. Remove growth medium and replace with 1 mL of each chemical dilution in the appropriate wells in 24-well plates. Shake the plates gently to ensure an even distribution. Incubate for 48 h at 37°C. Remove the medium and determine the cell number by the Neutral Red assay (see Note 3). From the preliminary results select six concentrations, spanning the range of O-100% cell death, for an accurate determination of cytotoxicity.

3.2.2. Determination of IDzO, ID,, and IDgO 1, Test each chemical concentration m triplicate on three separate occasions. 2. Prepare: a. The appropriate solvent controls. b. Six concentrations of the test chemical.

LLC-RKl

Cell Screening

Test

15

3. Prepare the 24-well plates as before. 4. After overmght incubation, remove growth medium and replace with 1 mL of the test chemical or the control to random wells (thus minimizing bias), but ensuring that a careful note is made of the treatment received by the cells in each well. Shake the plates gently to ensure even distribution. Incubate for 48 h. 5. Estimate the cell number using the Neutral Red assay. 3.2.3. Neutral Red Assay (1) 1. After 48 h, remove the medium from all the wells. Wash gently with PBS. Add 1 mL of Neutral Red medium per well. Incubate for 3 h at 37OC,5% CO2 in a humidified atmosphere. 2. After 3 h, remove the Neutral Red medium. Wash quickly with the Neutral Red wash solution. Add 1 mL of resorb solution to each well. Agitate the plates intermittently for a period of 15-20 min. Transfer the solutions to cuvets and measure their absorbance at 540 nm using the resorb solution as a blank.

3.2.4. Results 1. Determine the mean value for the absorbance of the control cultures and adjust all indivtdual absorbances accordingly. Mean the values for each treatment group and plot graphically. Determine the ID,,, ID,,, and ID,, values from the curve. 2. Mean the ID values from three separate experiments and give the final concentrations expressed as pg/mL or mmol/L. Rank the chemicals for toxicity using the ID,, value (the section of the curve most likely to be linear and subject to least variation).

4. Notes 1. Volatile chemicals tend to evaporate under the conditions of the test, thus the ID,, value may be variable, especially when the toxicity of the compound is fairly low. Chemicals that are unstable or explosive in water are also difficult to test (4J). Neutral Red is preferentially taken up into the lysosomes/endosomesof the cell. Absorbances obtained using the Neutral Red assayshave been shown to correlate linearly with cell number over the specific optical density range obtained using this method. Any chemical having a localized effect on the lysosomes/endosomeswill, therefore, result in an artificially low (or possibly high) reflection of cell viability and cell number. This factor does, however, make the system useful to detect other chemicals that selectively affect the lysosomes, especially when it is used in conjunction with other tests capable of determining cell number.

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White and Seaman

2. One major drawback of the assay is the precipitation of the Neutral Red dye into visible, fine, needle-like crystals. When this occurs it is almost impossible to reverse, thus producing inaccurate readings. Some chemicals induce this precipitation therefore a visual inspection stage in the procedure 1svery important. 3. If the intensity of the color is too great, it may be necessary to add a further 2 n-L of resorb solution to some wells. If this is required, carry out the procedure for all the wells. References 1, Borenfreund, E. and Puerner, J. A. (1985) Toxicity determined in vitro by morphological alterations and Neutral Red absorption. Toxicol. Lett. 24, 118. 2 Williams, P. D., Laska, D A., Tag, L K., and Hottendorf, G. H. (1988) Comparative toxicities of cephalosporin antibiotics in a rabbit kidney cell line (UC-RKl) Antimicrob. Agents Chemother. 32(3), 314. 3. Riddell, R. J., Clothier, R. H , and Balls, M. (1986) An evaluation of three in vitro cytotoxicity assays. Fd. Chem. Toxic01 24,469-47 1 4 Riddell, R. J., Panacer, D. S , Wilde, S M., Clothier, R. H., and Balls, M. (1986) The importance of exposure period and cell type in in vitro cytotoxictty tests. ATM 14,86-92.

5 Knox, P., Uphill, P. F., Fry, J. R., Benford, D. J., and Balls, M. (1986) The FRAME multicentre project on in vitro cytotoxicology. Fd Chem. Tox. 24,457-463.

CHAPTER3

Preparation and Use of Cultured Astrocytes for Assay of Gliotoxicity Mark R. Cookson, R. McClean, and Vie tor W. Pentreatk 1. Introduction Cultured astrocytes provide a valuable and important system for predictive testing and mechanistic analysis of neurotoxic compounds. The culture procedures allow relatively rapid assessment of different chemicals or their metabolites over a range of concentrations, using cells derived from a restricted source. The use of multiwell plates for the cultural astrocytes means that multiple samples can be analyzed with a high degree of statistical accuracy and the cell environment can be carefully monitored or manipulated for content of nutrients, ions, agonists, antagonists, or modulators. On the other hand, cultured astrocytes are devoid of their normal integrative functions, and the lack of a blood-brain barrier, the absence of neuron-glial metabolic interactions, and metabolism of substances outside the CNS, together with local regional astrocyte heterogeneity and limited survival time (about 3 mo) are potential important shortcomings that require correlative reference to other neuronal, coculture, and in vivo studies. A recent review on astrocyte culture for evaluation of neurotoxic-induced injury can be found in ref. 1. The preparation and use of astrocytes is a well established and documented procedure for which there is general consensus regarding the principal steps (see refs. 2,3). There are, however, many variations in the From- Methods m Molecular B/ology, Edited by: S. O’Hare and C K Atterwill

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Cookson, M&lean,

and Pentreath

detailed methodology of, for example, cell separation and media composition, and our descriptions are appropriate for several tests for gliotoxicity. Cell purity of the primary cultures are checked by immunostaining for glial fibrillary acidic protein (GFAP), with typical values in the 9095% positive range. For toxicological studies, subcultured astrocytes are advantageousbecausehigher cell densities and purities may be obtained. Keys to the assay and understanding of gliotoxic mechanisms will lie in the accurate measurement of cell membrane perturbations together with the subsequent intracellular biochemical and metabolic effects. Because a large number of physiological and biochemical properties of astrocytes have been measured in culture systems across different disciplines in neurobiology, a considerable range of potential targets or endpoints are available for neurotoxicological evaluation. Some examples are described in ref. 1. The choice of procedures that may be useful as a preliminary predictive screen is therefore critical and is currently the subject of extensive studies by us. To date we have analyzed cell viability, total cell protein, energy utilization, and membrane integrity as likely key indicators. These assays can be completed relatively quickly, with cell viability and total protein combined in the same experimental procedure and with the energy utilization and membrane integrity also evaluated together by uptake and backflux of radiolabeled 2-deoxyglucose (2-DC). The findings show that these have a high degree of validity with, for example, a variety of gliotoxic substances causing increases in total protein (measured in pg/104 cells) at certain critical concentrations. However, additional valuable procedures will undoubtedly be described in the future. Below we describe convenient methodologies for the preparation of astrocyte cultures and the application of procedures. 2. Materials 1. Poly-L-lysine (mol wt 75,000-150,000): 0.1 mg/mL in sterile deionized water. 2. BME: 0.05% TrypsmIEDTA (5.3 mm EDTA) solution in with 25 mm HEPES and Hank’s Salts without L-glutamine (BME).

3. 0.1% Trypsin mhibrtor in BME (asabove)plus 200 uL deoxyribonuclease (Type 1, from bovine pancreas)per 5 mL of solution, 4. Serum Supplemented Growth Medium: Dulbecco’s Modified Eagle’s

Medium (DMEM) containing 4500 mg glucose,110 mg sodium pyruvate, and 110 mg sodium bicarbonatewithout L-glutamine plus 10% fetal calf serum and 1% antibiotic solution (see Note 1).

Astrocytes

for Gliotoxicity

Assay

19

5. Hank’s Balanced Salt Solution with sodium bicarbonate, without calcium chloride or magnesium sulfate (HBSS). 6. TrypsmiEDTA solution: 0.1% made up in HBSS as above. 7. Sodium hydroxide: 0.2M. 8. Trypan Blue dye solution: 0.4% in 0.8% NaCl and 0.06% K2HP04. 9. Coomassie blue based protem assayreagent kit plus bovine serum albumin (BSA) standards (1 mg/mL). 10. 2-deoxy-D-[13H] glucose ([3H]2-DG). 11. Ultima Gold High Flashpoint Liquid Scintillation Cocktail. 12. Pony vial H/L. Miniature polyethylene “Rangin” vials. 13. Phosphate buffered saline (PBS), pH 7.4, consisting of in wt/vol; 0.8% NaCl, 0.02% KCl, 0.02% KHZ P04, and 0.115% Na2HP04 made up both with and without 5.6 mM n-glucose (see ref. 6). 14. 0.2M HCI.

3. Methods of Astrocyte Cultures 1. Poly-L-lysine (PLL) coating: Cells can be grown either on glass coverslips (13-mm diameter; No. I thickness) in 24 well multidishes or in 25 cm2 flasks (see Note 2). Coverslips are flamed after dipping in 95% ethanol to sterilize, then placed in the wells of the culture dish. To each well, 100 pL of poly+lysine solution is added and allowed to dry for 5 min. The coverslips are then rinsed with sterile water and allowed to dry thoroughly before use. If coverslips are not required, the PLL can be added directly to the cells of the plate. 2. All instruments are sterilized by being flamed in ethanol and are supported on a piece of aluminum foil in a sterile laminar flow hood. Neonatal rat pups are obtained and the heads are quickly wiped in alcohol to sterilize them. Pups are sacrificed by decapitation and the heads are placed in a sterile Petri dish with a few milliliters of BME. 3. A small cut is made m the skin at the posterior of the skull and the skin removed. The skull is very fragile and can be easily removed using two pairs of forceps. The whole brain is then removed from the skull cavity by carefully scooping it out. 4. The neocortical tissue is isolated by pinching off the olfactory lobes at the anterior and the superior colliculi and the developing cerebellum at the posterior of the brain. The meninges are teased off and are separated carefully from the cortical hemispheres using fine forceps. 5. The cortical tissue is chopped into about eight pieces. These are placed in a sterile tube containing 10 mL of Trypsin/EDTA solution in BME, then are covered and incubated in a water bath at 37°C for 25 min.

3.1.

Production

and

Maintenance

20

Cookson, MeClean, and Pentreath

6. The cells are then triturated using a sterile Pasteur pipet and centrifuged at 1000 r-pm in a benchtop centrifuge. 7. The supernatant is removed and 2.5 mL of trypsin inhibitor plus DNase added. The tissue is triturated using a fresh Pasteur pipet. This suspension is then recentrifuged at 1000 rpm for 5 mm. 8. Step 7 is repeated. 9. After discarding the supernatant, 2 mL of supplemented DMEM are added to resuspend the cells. This suspension can be plated out at 50 pL/well or 1 n&/25 cm2 flask. 10. After incubating the cells for l-2 h at 37°C in a 5% CO2 humidified atmosphere, the cells are fed with 500 pWwel1 or 5 ml/flask of serum supplemented DMEM. These are then incubated for 1 wk and thereafter are fed twice weekly (see Note 3). 11. Staining for Glial Fibrillary Acidic Protein (GFAP) after 1 wk culture (4) should demonstrate that over 90% of such cultures are astrocytes. Both anti-GFAP and secondary, fluorescein-conjugated antibodies are commercially available. However, for various reasons it is often preferable to subculture at least once before usmg the cells in toxicological evaluations, mainly to give a greater yield of cultures per rat and to ensure even plating densities across the wells of the multiwell plates. 3.2. Astrocyte Subculture 1. Coverslips with attached cells are rinsed three times in HBSS using sterile Pasteur pipets. 2. Trypsin/EDTA solution in HBSS is added to cover the cells: 200 pL/ multiwell is sufficient. The covered plates are incubated at 37°C until the cells round up and float off the coverslips. The amount of time required to do this depends largely on the density of the cultures and is generally around 5-10 min (see Note 4). 3. Samples from the wells are removed and pooled in a sterile tube, centrrfuged for 5 min at 1000 rpm, and the supernatant replaced with serum supplemented DMEM as above and plated out at a density of about 2 x lo4 cells/well, as assessedby a hemocytometer count. 3.3. Measurement of Cell Viability and Total Protein 1. Cells on coverslips are washed in HBSS three times and are trypsinized wrth 200 /.tL of Trypsm/EDTA m HBSS per well until the cells detach from the coverslips. 2. Trypsin inhibitor (150 pLWwel1)IS added and the contents of each removed and placed in labeled microcentrifuge tubes,

Astrocytes for Gliotoxicity

Assay

21

3. Of this sample, 50 pL is removed and mixed with 50 l.tL of Trypan blue dye solution, and this sample is counted in a hemocytometer. Viability is expressed as percent dye excluding cells divided by the total number of cells. 4. The remainder of the cell suspension is centrifuged at 2000 rpm and the supernatant is removed. The small pellet of cells is resuspended in 500 pL of 0.2M NaOH, vortexed to disperse the pellet, and left overnight at 4OC. 5. BSA standards are made up in the following series; 1, 2.5, 5, 10, 15, 25, and 50 l.tg/mL. These are pipeted in duplicate into wells of a microtiter plate (150 pL/well). Likewise, 150 ltL of each sample is added in duplicate to a series of wells in the plate, followed by 150 pL of protein assay reagent to each well of the plate (5). The color develops within 5 min at room temperature and lasts for several hours. The plates are read at 570 nm using a plate reader (see Note 5).

3.4. Measurement 1. 2. 3. 4. 5. 6.

7.

of Cell Membrane Integrity with PHl2-DG Following the specified incubation with toxicant (see Notes 6 and 7), the medium is aspirated and each well washed twice with 1 mL PBS (37OC). PBS with 5.6 mM o-glucose (450 u,L/well) (37OC) is added followed by 50 PL 0.5 l.tCi/rnL [3H]2-DG. Incubation is carried out at 37OCin a 5% CO,/ 95% air humidified incubator for 15-45 min (7). Incubation is terminated by aspiration (with legitimate disposal) of the medium (see Note 8) followed by three washes with 1 mL ice-cold PBS. Cells are digested using 300 l.tL 0.2it4 NaOH and left overnight. The solution is neutralized with 0.2M HCI. Two 150 pL samples are taken directly from the well to the microtiter plate for protein determination and treated as in Section 3.3.5. BSA standards are made up in 0.2M NaCl. The remaining solution is transferred to a miniature scintillation vial containing 3 mL scintillation cocktail. A serial dilution of [3H]2-DG (0.50.0005 l&i) is made up in parallel to act as a standard. Samples are shaken and left overnight in the dark at 4OC. Samples are counted on a liquid scintillation counter (see Note 9). Backflux is determined by incubating cells in PBS with 5 rnM o-glucose containing 0.5 l&i [3H]2-DG as described above. At the end of the incubation time the medium is aspirated and each well washed three times with 1 mL PBS (37°C). A further incubation is carried out (15-45 min) in 500 FL PBS without [3H]2-DG. This [3H]2-DG solution is treated as in Section 3.4.6. (see Note 10).

22

Cookson, l&Clean,

and Pentreath

8. The remaining cells are treated for protein determination as in Section 3.4., steps 4 and 5 and scmtillation counting as in Section 3.4., step 6.

4. Notes 1. Suitable antibiotics are either penicillin/streptomycin (stock 10,000 p,Penicillin G, 10 mg streptomycm/mL) or gentamycin (10 mg/mL stock solution). We commonly use the latter. 2. Multiwell dishes are convenient for toxicological evaluations since one can perform experiments at five concentrations, plus a control, m quadruplicate from a 24-well plate. 3. It takes around 2 wk for these cells to reach confluency, depending on the age of the rat. The fastest growing cultures are prepared from neonates of ~24 h old. It is possrble to grow cells from older rats (we have used up to 5 d), but in older rats the more developed meninges are difficult to remove and may contaminate the culture with fibroblast-like cells. 4. Time of trypsinization should be kept to a minimum to prevent excess cellular damage. The progress of the reaction can be monitored using an inverted microscope. 5. Control values for total protein are typically around l-2 l.tg/104 cells. 6. We have used 6, 12, and 25 h incubation periods with toxicants. Cells are fed within 48 h preceding dosing with toxicant. 7. The uptake of [3H]2-DG in older cultures may become reduced. We recommend that primary cultures be used soon after confluence (2-3 wk) or 4-5 d after the first subculture. 8. Uptake and backflux can be measured together rf Section 3.4., step 7 is proceeded to. The total uptake of [3H]2-DG is equal to that contained in the cells plus that in the medium. 9. Typical control values for uptake are in the range of l-10 pmol/mg protein/min. 10. Typical control values for backflux are between 5 and 10% of uptake.

References 1. Aschner, M. and Kimelberg, H. K. (1991) The use of astrocytes in culture as model systems for evaluating neurotoxic-induced-injury. Neurotoxicology 12,505-518. 2 Shahar, A., de Vellis, J., Vernadakis, A., and Haber, B (eds.) (1989) A Dissection and Tissue Culture Manual of the Nervous System Lrss, New York. 3. Hertz, L., Juurlink, B. H. J., Szuchet, S., and Walz, W. (1986) In Neuromethods, Vol. I, GeneralNeurochemical Techniques. (Boulton, A. A and Baker, G. B., eds.), Humana, Clifton, NJ, pp. 117-167. 4. Raff, M., Fields, K., Mirsky, R., Press, R., and Winter, J. (1979) Cell type specific markers for distinguishing and studying neurons and the maJor classes of glial cells in culture. Brain Res. 174,283-308

Astrocytes for Gliotoxicity

Assay

23

5 Redinbaugh, M. G. and Campbell, W. H. (1985) Adaptation of the dye binding protein assay to microtitre plates. Anal. Biochem. 147,144-147 6. Brookes N. and Yarowsky P. J. (1985) Determinants of deoxyglucose uptake in cultured astrocytes. the role of the sodium pump. J. Neurochem. 44,473-479. 7. Yarowsky P. J., Boyne A. F., Weirwille R., and Brookes N. (1986) Effect of monensin on deoxyglucose uptake in cultured astrocytes: energy metabolism is coupled to sodium entry J Neurosci. 6,859-866.

CHAPTER4

Human

Thyroid

Carmel

Culture

Mothersill

1. Introduction The technique described in this chapter enables the culturing of thyroid cells without loss of differentiation and medium change. It is potentially useful for the long-term study of drug effects on the thyroid gland. Human thyroid cells obtained during surgery can be maintained in culture for periods of up to 2 mo without losing morphological or functional differentiation (1). In the clinical situation the thyroid may be exposed to long-term drug or radiation treatment that may have adverse effects on the functioning of the thyroid gland. These adverse effects can be assessed in culture by studying morphological and biochemical changes after exposure to test chemicals, cytotoxics, or radiation and extrapolated to the likely toxicity in humans. Sections of human thyroid are incubated in a trypsin/collagenase solution. The resulting supernatant is filtered and centrifuged twice. The cells are collected and resuspended in growth medium and any undigested thyroid tissue is reincubated in the trypsin/collagenase solution on two further occasions, Each supematant is filtered and centrifuged. The digests are pooled and plated out in flasks containing Eagle’s Medium. The cultures are incubated for 48 h and are then exposed to test chemicals and morphology, epithelial cell growth, and biochemical parameters. A long-term culture system for sheepthyroid has been established that retains many of the characteristic functional and morphological features of the gland. The human thyroid culture has been adapted from this sheep culture with only minor modifications. Some morphological differentiaFrom Methods m Molecular Slotogy, Edlted by. S O’Hare and C. K. Atterw~ll

Vol 43. In Vltfo Toxmty Testing Protocols Copyright

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Mothersill tion time discrepancies occur. Follicles develop in sheep cultures in 5-8 d and in human cultures after 15-20 d. Undifferentiated areas are more common in human cultures and are visualized as patches of epithelial cells devoid of follicles. The unusual glucose and lactate metabolism of the sheep system permits a prolonged culture period. Glucose is rapidly metabolized to lactate, and then the lactate is utilized by the cultures over their remaining life-span. Exhaustion of lactate in the culture medium coincides with cell death, but the latter can be delayed by adding concentrated glucose to the medium just before this occurs. The metabolism of glucose to lactate and subsequentlactate utilization, follows the same pattern in human cultures, but at a much slower rate because of the lesser degree of differentiation (lactate use correlates strongly with morphological differentiation). The major factor in establishing a human thyroid culture is the amount and character of healthy tissue obtained. The best cultures are from 5-10 g samples of thyroid tissue, although even samples of 0.05 g have been cultured. The slower rate of differentiation of the human culture system is advantageous when long-term studies of drugs or radiation effects on the human thyroid are required. The test chemical can be added directly above the differentiated monolayer without disturbing the media or the degree of differentiation that occurs. Although human thyroid cultures have been established and utilized by other scientists, they have been short-term systems or subcultures maintained by the use of hormones or growth enhancers. In general, these have been used to study the biochemical behavior of the cultures in the short-term or in the characteristics of the subculture. In these cases the primary culture was not maintained for more than 7 d. This culture system correlates well with the in vivo situation. Thyroid cultures have a limited life-span in humans (15-20 doublings), which equates to the deterioration of cells in culture after the third or fourth subculture. The best endpoints for determining that the cells are functioning correctly are the T4 assay or 1251trapping ability. The thyroid culture shows a progressive loss of differentiation after repeated subculture. It is postulated that this may be a result of the effects of the trypsination, which causes the release of a receptor component into the medium that binds thyrotrophin. This receptor is regenerated when the cells are replated, but it is thought that the regeneration declines after repeated subcultures.

Human

Thyroid Culture

27

A large number of scientists use whole animals or animal cell culture systems (mostly rodent) that have limited use in relation to the study of human disease and toxicity. The heterogeneity of the source material in terms of genetic makeup and previous history of cytotoxic insult is a disadvantage in relation to the development of a standardized routine test system. 2. Materials 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Normal and diseased human thyroid tissue excised during surgery. Blood administrator set. 100 j.t.mfine surgical gauze. Centrifuge. 40 mL sealed flasks. Sterile scalpel. Radioimmunoassay kit for T4. Liquid scintillation counter. Perchloric actd. 2 #Zi/mL Na ‘?odtde. Scintillation fluid. PAS stain. Collagenase (Type IV from Clostridium vibrio) solution:Make up a 1 mg/mL solution in BSS. Trypsin/collagenase solution: Prepare a 0.25% w/v trypsin in 1 mg/mL collagenase solution. Glucose solution: Prepare a 10 mg/mL solution in Eagle’s balanced salt solution. Methimazole solution: Prepare a 2 mM solution in Eagle’s balanced salt solution. Balanced salt solution: Ca2+ and Mg2+ free balanced salt solution (BSS) containing 200 IU/mL penicillin, 200 pg/rnL streptomycin, and 40 IU/mL gentamicin. Growth medium: 500 mL Eagle’s medium, 2 mM L-glutamine, 20% v/v lamb serum, 0.1 ug/mL hydrocortisone, 10 mL U/n-& insulin, 1 @4 potassium iodide, 20 IU/mL penicillin, 20 pg/mL streptomycin, 4 IU/mL gentamicin, 1 pg/rnL fungizone, 40 mL U/r& thyrotrophin, 12.5 mL 1M HEPES buffer.

3. Methods 3.1. Culture Procedure 1. Add pieces of human thyroid to ice-cold BSS continumg antibiotrcs. Chop the thyroid into small pieces, preferably of 5-10 g, using a sterile scalpel.

28

Mothersill

2. 3. 4. 5. 6.

Add the chopped tissue to 12 mL trypsin/collagenase solution at 37°C. Incubate for 30 min at 37OC. Filter the supernatant (containing freed cells) through a blood admmistration set. Filter again through a fine surgical gauze, thus removing any fat and fibrous debris. Centrifuge the filtrate at 4OOg. Resuspend the tissue digest in an equal volume of growth medium containing 40% serum, neutralizing trypsin activity. Incubate any undigested thyroid pieces m the trypsin/collagenase solution and incubate for two further periods of incubation (depending on the size of the tissue) followed by filtration, Centrifuge. Pool the cells from all three digests. Count the cells and adjust a 0.5 mL cell suspension to -1 x lo6 cells. Add the adjusted cell suspension to 5 mL of growth medium in a 40 mL flask. Plate directly into a flask containing the minimum amount of medium necessary to wet the surface of the plastic. Leave for 24 h (the explant should have adhered). Add further medium up to a total volume of 5 mL.

The following

3.2. Testing assays can be performed to evaluate any thyrotoxicity.

1. After 48 h expose the cultures to test chemicals (0.05-0.1 mL in culture medium or DMSO/ethanol), cytotoxics, or irradiation as follows: a. 5 replicates for each chemical. b. 5 or 10 controls. c. 3 replicate experiments. 2. Assessthe toxic effect using the following parameters: a. Lactate/glucose levels (2,3). b. Monitor the perchloric acid extracts of media samples throughout the culture period and compare to the control. c. T4 measurement. d. Use a T4 radioimmunoassay kit to monitor T4 release into the medium. e. Iodine trapping ability: Add 0.1 mL of 2 mM methimazole and 0.1 mL of 2 pCi/mL sodium 125I to the medium of the cell culture at room temperature, Incubate for 90 min. Wash the culture thoroughly with 4 aliquots of 5 mL Earle’s BSS to remove all traces of free radioactivity. Trypsinize the cells with 5 mL trypsin/EDTA solution and resuspend in -10 mL fresh growth medium. Count the cells and determine the 1251levels by liquid scintillation. Calculate the 1251counts/106 cells. Plot a graph of 1251counts/lO”cells as a function of time in culture for the differentiated cultures.

Human

Thyroid Culture

1

0

6

29

18

12

DAYS

IN

2~

30

36

12

CULTURE

Fig. 1. The levels of T, (ng/106 cells) detected in medium from differentiated huan thyroid cultures (dertved from multinodular goiter) over a 40-d life span. (n = 6). 3. Epithelial cell outgrowth: Monitor the tissue outgrowth by measuring its area and by performing autoradiography. Study the effects of the toxic agents on the different cell types present using immunocytochemical analysis for intermediate filaments or surface antigens.

3.3. Results 3.3.1. T4 Plot a standard curve for T4. Read off the values for T4 found in the medium samples at regular time intervals. Plot a graph of T4 levels over 40 d (Fig. 1).

30

Mothersill

z r \

2x10L

1= r= 0

lxlOL

"-

5 x103

\ ul ; 2x103 2 0 1x103 DAYS

IN

CULTURE

Fig. 2. The iodine trapping (count&O6 cells) of human thyroid cells over a 40-d life span (n = 6). Solid circle-multinodular goiter; open circle-adenoma; triangle-fibroblast culture. 3.3.2. Iodine Trapping Ability Calculate the 125Icounts/106 cells. Plot a graph of 1251counts/106 cells as a function of time in culture for the differentiated culture (Fig. 2).

4. Notes 1. If the tissue sample is small, incubate with the trypsin/collagenase solution and then plate directly mto a flask. After 48 h, expose the cultures to test chemicals as above. 2. Fibroblast contamination can occur in cultures derived from low initial cell numbers. Seeding high numbers of cells probably inhibits the proliferation of any fibroblasts present. 3. If the amount of tissue is very small (co.5 cm3), incubate the tissue sample with trypsin/collagenase solutron for 30 mm at 37°C. 4. Cultures rapidly metabolize the available glucose after which cell death occurs, therefore, it may be necessary to prolong the culture life-span by adding 0.1 mL of a concentrated glucose solution. This can be assessed initially by using a glucose assay,or once experienced with the technique, by judging the color of the medium.

Human

31

Thyroid Culture References

1 Mothersill, C., Seymour, C., Moriarty, M. .I., and Cullen, M. J. (1985) Long-term culture of differentiated human thyroid tissue. Acta Endocrinologica 108, 192-199. 2. Schmidt, F. H (1961) Enzymatic determination of glucose in biological samples. Klin. Wochenschr.

39,1244

3. Gutmann, I. and Wahlefel, A. W. (1974) Enzymatic measurement of lactic acid in samples of serum and plasma, in Methods of Enzymatic Analysis (Bergmeyer, H U , ed ), Academic, New York, pp. 1464-1467

CHAPTER5

Thyroid Follicular Cells in Monolayer Culture In Vitro Models for Thyroid

Stephen

Toxicity

Testing

I? Bidey

1. Introduction The development of in vitro models that preserve the functional characteristics of the normal thyroid gland has been a challenging objective of recent experimental thyroidology. A major aim of such endeavors has been to facilitate the development of simple, reliable, reproducible testing strategies for compounds interacting with, and perturbing the function of, the thyroid follicular cell (TFC). The earliest experimental thyroid models were based on organ culture or tissue slice preparations or, alternatively, short-term cell suspensions. Subsequently, however, it has become possible to maintain TFCs as monolayer cultures in which a high level of thyroid-specific differentiation and responsiveness may be preserved over a prolonged period. Such characteristics, which allow experimental intervention and the subsequent study of cell function and morphology, have facilitated the development of biological assays for thyrotrophin (TSH) (I) and thyroid autoantibodies (2) in serum, and have recently begun to encompass applications in the field of cellular toxicology, where the application of these new investigative tools has enabled the identification of the sites and mechanisms of action of agents demonstrating direct thyroid toxicity in vivo (3). The preservation of TSH-dependent responses in cultured TFCs, together with the relative ease with which large numbers of identical, From Methods in Molecular Biology, Edited by S O’Hare and C K Atterwill

Vol. 43: In Vitro Toxicity Testing Protocols Copynght

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Bidey

34

replicate cultures may be maintained, has made the TFC monolayer the model of choice for the quantitative assessment of the agents interfering with or modifying TSH-receptor interaction, transmembrane iodide movement, or cell proliferation. The most widely adopted in vitro functional markers of TFC stimulation have included intracellular CAMP accumulation and thyroid-radioiodide uptake, the latter being a response unique to the thyroid cell. Although cell proliferation within the normal adult thyroid gland is minimal, growth is enhanced by dietary goitrogens, such as the cyonogenie glucosides and thioglucosides, which inhibit iodiode uptake, leading to an impaired synthesis of thyroid hormones and a compensatory rise in pituitary TSH secretion. In vitro strategies for estimating the rate of cell proliferation involve the determination of the incorporation of C3H] thymidine into subconfluent TFC monolayers or, alternatively, assessment of the metaphase index of the culture (i.e., determination of the percentage of cells with chromosomes visibly in the “S” phase) (4). Despite the widespread use of primary thyroid cultures derived directly from thyroid tissue as a fundamental tool in thyroid cell biology, the inherent viability in agonist and antagonist responsiveness between preparations of cells derived from different individual thyroid tissues, coupled with the progressive dedifferentiation of cultures with increasing duration of in vitro maintenance, has limited the use of this system to essentially short-term, qualitative investigations of thyroid function and proliferation, Recently, however, a number of stable cell lines have been isolated that retain major features of the differentiated follicular cell. Foremost among these is FRTL-5, a cloned Fischer rat thyroid cell (5-7). 2. Materials 2.1. Basic

Maintenance

Medium

FRTL-5 cell monolayers are maintained in Ham’s F- 12 medium (Coon’s modification) containing various supplements,asdescribedbelow. Each liter of working medium is prepared by adding 2.5 g NaHC03 to 12.08 g of powdered medium and making this to 1 L with triple glass-distilled water. The medium is then filtered (0.22 pm) into presterilized 100 mL bottles and may be stored at 4°C for up to 3 mo. Immediately prior to use, a supplement of 4 hormones (as detailed below), MEM nonessential amino acids, penicillin and streptomycin are filtered into the medium.

Thyroid Toxicity Testing In Vitro

35

2.2. Hormone Supplements The hormones comprising this supplement are somatostatin (1 mg/L), hydrocortisone (0.33 mg/L), transferrin (OS g/L), and glycyl-histidyllysine acetate (2 mg/L). The stock supplement is prepared as follows: 1. Somatostatin:50 ug is dissolvedin 500 uL of Ca2+h4g2+free Hank’s balancedsalt solution (HBSS), and then madeto 5 mL with Ca2+/Mg2+free HBSS. 2. Hydrocortisone: 1 mg is dissolved into 1.5 rnL absoluteethanol, and 100 uL then is addedto 10.9mL Ca2+/Mg2+free HBSS. 3. Glycyl-histidyl lycine acetate:1 mg is dissolved in 1 mL Ca2+/Mg2+free HBSS. 4. Transferrin: 25 mg is dissolved into 5 mL Ca2+/Mg2+free HBSS. To prepare stock aliquots of the combined hormone supplement, 5 rnL of each of the solutions detailed above is added to 25 rnL Ca2+h4g2+free HBSS, mixed and stored as 1 mL aliquots at -70°C until required. After thawing, each is diluted in 100 mL of basic maintenance medium. The preparation of complete maintenance medium also requires addition of 200 mmol glutamine/L, nonessential amino acids (stored as 1 mL aliquots at 4”C), penicillin (100 U/mL) and streptomycin (100 pg/niL) (stored as 1 mL aliquots at -2OOC). Immediately before use, supplements are filtered (0.22 pm pore size) into 90 rnL of base medium, and sterile newborn calf serum (NCS) added. 2.3. TSH Preparations A number of biologically active preparations of TSH are available from commercial sources, which may be used as reference thyroid cell stimulators. Bovine TSH (First International Standard of Thyrotrophin) (pituitary TSH, bovine, for bioassay; coded 53/11) and human TSH (Second International Standard, coded 80/558) are specifically recommended for this purpose, and may be obtained from the National Institute for Biological Standards and Control (South Mimms, Potters Bar, Herts, UK). Both are supplied as ampuled, lyophilized preparations having a uniform, stated activity, and are stable at -20°C over prolonged periods (i.e., years). After reconstitution, preparations are stored as aliquots at -7O”C, and used within 6 mo. It is particularly important that partially used aliquots are not refrozen, since this will diminish their biological activity.

36

Bidey

Fig. 1. Phase-contrast photomicrograph of a monolayer colony of the rat thyroid follicular cell strain FRTL-5,48 h after passaging (200x magnification). 2.4. FRTL-5 Cells FRTL-5 cells are available from the American Type Culture Collection (Rockville, MD). They are routinely passaged in Coon’s modified Ham’s F12 medium supplemented with the “4H” hormone mixture described above, together with 10 pg/mL insulin, 100 pU/mL TSH, and 5% (v/v) NCS. Cells are grown in lo-cm diameter Petri dishes in 5% COz in air at 37°C. In the presence of TSH, the cells proliferate as uniform, round colonies (Fig. 1). 1. 2. 3. 4.

2.5. Giemsa 1 mL Giemsa stock reagent. 40 mL distilled water. 1.25 mL methanol. 2 drops 1M NaHC03.

Stain

3. Methods 3.1. Preparation of Cell Suspension from Stock FRTL-5 Cultures 1. Aseptically aspirate the growth medium from stock cultures of FRTL-5 cells. Rinse the cultures with prewarmed Ca2+/Mg2+-free HBSS.

Thyroid

37

Toxicity Testing In Vitro

2. To each culture, add a sterile solution of trypsm (1 mg/mL) and collagenase (20 U/n& in Ca*+/Mg*+-free HBSS). Ensure that the liquid covers the monolayer, Return the cultures to the incubator for 2-5 min. 3. Use a sterile Pasteur pipet to transfer the suspension of detached cells into a sterile 25 mL universal tube. Clumped cells should be dispersed by repeatedly pipetmg the suspension. 4. Add calf serum (0.5% v/v) to inactivate the trypsin. 5. Close the tube and shake gently to obtain a uniform cell suspension. 6. Centrifuge (lOOg, 5 min at room temperature) to obtain a cell pellet. 7. Remove the supernatant solution with a sterile Pasteur pipet, and resuspend the cells to an appropriate density in a small volume (e.g., 5 mL) of the plating medium.

3.2. Preparation

of Replicate

FRTL-5

Cell Monolayers

1, Having prepared a suspensionof single, viable FWI’L-5 cells from stock cultures, initiate the replicate monolayers that will form the bioassay “target” tissueby adding aliquots of the cell suspensionto 24-well tissueculture dishes. 2. After initiating the test cultures maintain the bioassay plates at 37°C under 5% CO2 in air, in a water-saturated atmosphere to prevent evaporation of the culture medium.

3.3. Prebioassay

Treatment

of Cells

1. A change of culture medium will be necessary 3-4 d after initiation of cultures. Remove the exhausted medium from the monolayers using a sterile Pasteur pipet attached to a vacuum suction pump and collection jar. 2. Add fresh medium (500 pLWwel1)to each monolayer with a minimum of delay, so that the cultures do not become dry. 3. Imtiate the bioassay by the addition of test solutions at the time of, or shortly after the first medium change.

3.4. Procedure for Extracting and Determining Intracellular CAMP Assessment of the effect of a compound on TSH-dependent adenylate cyclase activity is made by determining the final intracellular CAMP levels attained in the presence of a serial dilution of that compound, in cells simultaneously exposed to a standard dose of TSH, compared with the CAMP level attained in cells exposed to TSH alone. 1. Remove the maintenance medium from subconfluent monolayers by aseptic aspiration, and replace with 500 pL Leibovitz (L-15) medium. 2. Add 3-isobutyl-1-methylxanthine (IBMX) to a final level of 0.4 mM m all wells, to inhibit CAMP-dependent phosphodiesterase activity.

3. Add the test compound in multiple dilution, and, if appropriate, control diluent to triplicate sets of incubation wells. 4. Add a standard dose of TSH (e.g., 100 pU/mL) to each well. 5. After incubation for 15 min, remove the medium, and add 500 ltL of icecold absolute ethanol to each culture. This treatment both arrests the incubation reaction and releases intracellular CAMP from the lysed cells. 6. Seal the culture plates in wrapping film to prevent evaporation of ethanol, and transfer them to a -20°C freezer for 24 h. 7. Remove 200 p,L aliquots of the ethanolic fractions and transfer these to small glass test tubes. 8. Evaporate the tube contents to dryness under a stream of nitrogen. 9. Redissolve the dried residues in 25 mM Tris, 50 mM NaCl, 8 n&f or other appropriate assaybuffer. 10 Determine the CAMP content by conventional radioimmunoassay (e.g., usmg a commercial kit). 11, Express the final CAMP level attained within each set of triplicate cultures as mean + SD level/culture. 3.5. Procedure for Determining Cellular Iodide Uptake The accumulation of inorganic iodide against a concentration gradient is a thyroid specific CAMP-dependent process that is maintained by the FRTL-5 line. 1, Remove the maintenance medium from subconfluent monolayers by aseptic aspiration, and replace with a fresh 500 l.tL ahquot of the same medium in each well. 2. Add 3-isobutyl-1-methylxanthine (IBMX) to a final level of 0.1 mM m all wells, to inhibit CAMP-dependent phosphodiesterase activity. 3. Add the test substance or control diluent, in multiple dilution, to triplicate sets of incubation wells, leaving a set of wells as controls. 4. Add a standard dose of TSH (e.g., 100 pU/mL) to both test and control incubation wells. 5. After incubation for 24 h, remove the medium, and add a fresh aliquot of maintenance medium containing 1 ltCr Na1251.Continue to incubate for a further 24 h at 37°C. 6. After incubation, remove the radioactive media and carefully discard. Briefly rinse (X2) each culture with 50 pL ice-cold HBSS. 7. After washing, add 500 l.tL 100 pJ4 sodium perchlorate to each well. This inhibits iodide pump activity, allowing intracellular iodide to discharge into the medium. 8. After 20 min, remove duplicate 100 l.tL portions of the sodium perchlorate solution, and determine 1251content using a y-scintillation counter. Express

Thyroid

Toxicity

Testing

In Vitro

results as a percentage of the mean 1251uptake value obtained in replicate cultures exposed to the standard TSH dose alone. 3.6. PH] Thymidine Incorporation as a Marker of Cell Proliferation 1. Remove the maintenance medium from subconfluent monolayers by aseptic aspiration, and replace with a fresh 500 pL aliquot of the same medium in each well. 2. Add the test substance,in multiple dilution, to triplicate sets of incubation wells, leaving a set of wells as controls. 3. Followmg a 24 h incubation period in the presence of appropriate combinations of test substances,dilute an aliquot of [methyl 3H] thymidine stock solution to 5 mL with serum-free “4H” medium, and add 50 pL to each well (1 pWwel1). Continue to incubate the cultures at 37OC,under 5%C02/ 95% air for a further 8 h. 4. Terminate the incubation by removal of the medium. 5. Briefly rinse each culture (X2) with ice-cold 10% (w/v) trichloroacetic acid (TCA), followed by a further addition of 500 pL 10% TCA/well. Leave the cultures for 3-4 h at 4°C to allow protein precipitation. 6. Remove the acid supernatants using a fresh Pasteur pipet for each well. Add 250 pL of 1N NaOH/L to each well. Seal the plates, wrap in aluminum foil, and leave overnight at room temperature to allow cellular digestion. 7. Remove duplicate 100 pL aliquots, and transfer these to scintillation vials. Add liquid scintillant (e.g., “Hisafe 2” scintillant, Pharmacia, Uppsala, Sweden) (4 mL) to each vial and determine [3H] thymidine activity using a p-scintillation counter. 3.7. Metaphase Index Determination 1. Follow the procedure shown in 3.5 for iodide uptake to include step 4. 2. After incubation for 44 h, remove the medium and add 200 PL fresh, serum-free maintenance medium together with 30 pL colcemid, a mitoticspindle arresting agent. 3. Incubate cultures for a further 3 h at 37°C. 4. Remove the incubation medium, and add 200 pL of freshly-prepared 30% (w/v) glacial acetic acid/70% ethanol to fix the cells. 5. After 15 min, remove the fixative with a Pasteur pipet, and rinse the monolayers twice in 70% ethanol. Leave to dry overnight. 6. After drying, add 100 pL diluted Giemsa stain to each well. Leave for 150 mm, replenishing the stain after 1 h. 7. Rmse the cell layers twice in 70% ethanol, then once with 95% ethanol. 8. Dry the cell layers, and observe for metaphase figures under a 40x objective.

40

Bidey

9. Calculate the Metaphase Index (X/100) x lOO%, where X= no. of cells/100 displaying figures.

4. Notes 1. Since the uniformity of the monolayer test cultures is crucial in obtaming a high level of precision and sensitivity of the assay system, cells must be dispensed into the culture wells with the aid of a fixed-volume repeating pipet, using disposable micropipet tips previously sterilized by autoclaving or y-irradiation. Assuming an even distrtbution of smgle cells within the starting suspension, tt 1spossible to obtain a between-culture variation in cell plating density approaching & l-2%. In the case of standard 24-well plates, a suitable starting inoculum may consist of lo5 cells/well. 2. L-15 medium does not require an equilibrating CO2 gas phase, so that incubations may be performed in room air at 37°C. 3. It is important to recogmze that the calculation in Section 3.4., step 11, assumes that the population densities of cells within replicate cultures are closely identical (i.e., within Z!Zl-2% limit). The between-culture variation in density may be estimated on the basis of cell protem or DNA estimations in a separate series of cultures wtthin each batch of assay plates. 4. One of the major advantages of the FRTL-5 is that unlike primary cultures, these cells may be maintained in long-term culture, having a reproducible, fully characterized behavioral pattern and population-doubling time. Given stable culture conditions, therefore, the responses of sequential passages of FRTL-5 cells should be entirely predictable. This has the important advantage of enabling large numbers of replicate and uniformly responsive test monolayers to be established, while also generating the inocula of FRTL-5 cells required to initiate subsequent stock cultures. 5. It may be desirable to investigate, m parallel cultures, for actions of TFC function on both CAMP accumulatton and transmembrane iodide uptake. Thus, although the latter is dependent on the functtonal and structural integrity of the cell membrane, specific inhibition of iodide pump or Na+/ K+-dependent ATPase activity may not necessarily, at least in the short term, adversely affect adenylate cyclase activity in a cell membrane that is otherwise structurally intact. However, if both CAMP accumulation and iodide uptake are diminished after incubation of cells with the test material, an effect on cell viability should be suspected. 6. In order to investigate for thyroid-directed toxic actions of nonwater soluble molecules, after solubilizing these in a nonpolar solvent, it is important that equivalent levels of the solvent are also included within TSHcontaining control cultures.

Thyroid Toxicity Testing In Vitro 7. The potential toxicity of any given compound toward TFCs may result from either a change in the normal functioning of that cell, through direct and specific interference (e.g., with TSH receptors or iodide pump activity) or, alternatively, through a more fundamental perturbation of basal cell activity owing to generalized cytotoxic actions. In the latter case, thyroid-specific function will also be impaired as a consequence of the interaction of the compound with the basal cell membrane, although the fundamental toxicity of the compound may, of course, not be restricted to TFCs. Thus, for example, since accumulation of iodide is dependent on the correct functionmg of a Na+/K+ ATPase and an iodide pump m the basolateral TFC membrane, in addition to the TSH receptor itself, nonspecific damage to the basolateral membrane may also impatr TFC function. The converse is also true, and if iodide accumulation remains unimpaired in the presence of a given compound, then the basolateral membrane is unlikely to have suffered any fundamental damage. The question as to whether a drug-induced decrease in iodide uptake is secondary to a nonthyroid specific impairment in cell viability, can, in extreme cases,be ascertained by morphological observation. However, more subtle changes may be uncovered by investigating for the release into the medium of membrane-associated enzymes, such as lactate dehydrogenase. References 1. Robertson, W. R. and Bidey, S. P. (1991) The in vctro bioassayof peptide hormones, in Peptide Hormone Secretion, A Practical Approach (Hutton, J. C and Siddle, K., eds ), IRL, Oxford, UK, pp 121-157. 2 Vitti, P., Rotella, C. M., Valente, W. A , Cohen, J., Aloj, S. M., Laccetti, P., Ambesi-Impiombato, F. S , Grollman, E. F., Pinchera, A., Toccafondi, R., and Kohn, L. D. (1983) Characterisation of the optimal stimulatory effects of Graves’ monoclonal and serum IgGs on cyclic AMP production m FRTL-5 thyroid cells: a potential climcal assay J. Clin. Endocrinol. Metabol. 57,782-79 1. 3. Fowler, K. L. and Atterwill, C. K. (1989) Potential use of thyroid FRTL-5 cells for predicting the toxic effects of xenobiotics, in FRTL-5 Today: Proceedings of the First International

Workshop

on the Characterization

and Standardization

of an

In Vitro Thyrozd Cell System (Ambesi-Impiombato, F. S and Perrild, H., eds.), Exerpta Medica, Amsterdam, pp. 3 l-35. 4. Ealey, P. A , Emmerson, J , Bidey, S P., and Marshall, N. J. (1985) Thyrotrophin stimulation of mitogenesis of the rat thyroid cell strain FRTL-5: a metaphase index assay for the detection of thyroid growth stimulators. J. Endocrinol. 106,203-210 5. Ambesi-Impiombato, F. S., Picone, R., and Tramontano, D. (1982) Influence of hormones and serum on growth and differentiation of the thyroid cell strain FRTL, in Growth of Ceils in Hormonally-Defined Media, vol. 9. (Sato, G. H., Pardee, A., and Sirbasku, D. A., eds ), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 483-492.

Bidey 6. Ambesi-Impiombato, F. S. (1989) The FRTL-5 cells, an in vitro thyroid system: introduction and development, in FRTL5 Today: Proceedings of the First International Workshop on the Characterization and Standardization of an In Vitro Thyroid Cell System, (Ambesi-Impiombato, F. S. and Perrild, H., eds.), Exerpta Medica, Amsterdam, pp. 3-7 7. Bidey, S. P., Lambert, J., and Robertson, W. R. (1988) Thyroid cell growth, differentiation and function in the FRTL-5 cell line: a survey J. Endocrinol. 119, 365-376.

CHAPTER6

Dust Toxicity in Rat Alveolar Macrophage Cultures Yrjii

Collan

and

Veli-Matti

Kosma

1. Introduction Macrophage cells in culture may be exposed to particulate matter, and resultant effects on cell viability are determined by vital dye exclusion and enzyme leakage assays. Many compounds, when inhaled as dust particles, have been found to be toxic to the respiratory system, with long-term exposure resulting in the development of pneumoconiotic fibrotizing lung disease. One of the initiating factors in fibrogenic lung disease is believed to be the direct damage inflicted on the alveolar macrophages, hence, these cells may represent a suitable in vitro screening system to investigate whether particulate matter is likely to be harmful when inhaled over an extended period (l-4). Macrophages can be isolated, cultured, and exposed to suspensions of particulate matter. Damage may then be assessedin two ways. The fast is simply by estimating cell death. The second parameter, i.e., lactate dehydrogenase (LDH) activity in the supernatant, provides a means of determining cell membrane damage as indicated by leakage of the enzyme, LDH, out of the cells into the medium (5). The damageto the cells as a result of exposure can then be usedto assesswhether inhalation of the dust may be harmful in vivo. This test system provides a rapid, sensitive, and relatively inexpensive means of assessing the harmful effects of dust particles, In vitro hemolysis, an acute toxicity test, has been used in the past to detect the potential toxicity of particulate matter. However, since alveoFrom- Methods m Molecular Brology, Vol. 43 In V&o Toxicity Testmg Protocols Edlted by- S. O’Hare and C K Atterw~ll Copynght Humana Press Inc , Totowa, NJ

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Collan and Kosma

44

lar macrophages appear to be one of the primary sites of damage in the initial stages of fibrogenic lung disease, they are probably a more suitable cell to be used in an in vitro screening system and are likely to reflect the in vivo situation more closely. A number of enzyme activities have been examined by the authors including aspartate amino transferase, acid phosphatase, and alanine amino transferase activity. Although a certain degree of reproducibility and correlation to cell damage were found, LDH activity was the most sensitive and readily detectable of the enzyme activities examined (1,2,6). Determination of this enzyme activity is, therefore, the preferred indicator of cell damage. There are three possible methods of examining the cells after exposure to potential toxins (6): 1. The medium of the cultures with the naturally detached floating macrophages is collected and analyzed. 2. The medium of the cultures IS removed, fresh medmm is added, macrophages are detached from the bottom of the well, then new medium wrth the harvested cells IS collected and analyzed. 3. The macrophages are detached from the bottom of the well without adding fresh medium; these are collected and analyzed. The third option has proved to be the most effective and consistent in identifying cell damage and best correlates to the occurrence of cell death. Modifications of this test system that use shorter incubation times are currently being developed by colleagues of the authors.

2. Materials 1. Animal-rat, e.g., BN-Kuo, Osborne-Mendel, Wistar: A number of different strains of rat have been used to provide cells for this procedure. Although the response of the cells to test compounds does not appear to be dependent on strain, it should be noted that the yield of cells may vary greatly. 2. Sterile dissection equipment. 3. Petri dishes. 4. Lammar flow cabinet. 5. Venflon-catheter. 6. 10 mL syringe. 7. Sterile centrifuge tubes. 8. Centrifuge, capable of 1000-3000 rpm. 9. Burker’s chamber. 10. 6 well plates with 1” diameter wells.

Dust Toxicity 11, 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23, 24. 25. 26. 27. 28. 29. 30.

31.

1. 2. 3. 4.

in Cultures

45

Incubator, 37”C, 5%/95% COZ in au, 95% humidity. Rubber policeman. Filters-1.2,0.8,0.45, and 0.22 pm pore. Automatic Multistat III analyzer-Intr. Lab. Microc. Analyzer, USA (for LDH determination). Sodium thiopental. Medium 199. 2.9% L-glutamine. Hanks’ balanced salt solution, pH 7.4. Hanks’ balanced salt solution, Ca2+- and Mg2+-free, pH 7.1-7.4. Phosphate buffered saline, Ca2+-and Mg2*-free, pH 7.2. Trypan blue. 2M hydrochloric acid. 2M NaOH. UV-LDH working solution. Na-pyruvate starter for LDH assay. Sodium chloride solutton: 0.9% NaCl, containing 100 U/mL penicillin, 100 I.t.g/mL streptomycin. Physiological saline: 0.9% NaCl, 10 U/mL heparin, 100 U/mL penicillin, 100 I.tg/mL streptomycin. 0.5% solution of Trypan blue dye in 0.9% NaCl solution. Culture medium: 1X medium 199 supplemented with 100 mL/L FCS, 100 U/mL penicillin, 100 p,g/mL streptomycin, and 10 mL/L 2.9% L-glutamine. Add 100 U/mL penicillin and 100 pg/mL streptomycin to the following: a. Ca2+- and Mg2+-free Hanks’ balanced salt solution. b. Hanks’ solution containing Ca2+and Mg2+. c. Ca2+- and Mg2+-free phosphate buffered saline. d. Heat treatment of fetal calf serum (see item 31). To free the FCS of LDH activity: a. Heat the serum at 56°C for 30 min. b. Keep at pH 3.5 (adjust with 2M hydrochloric acid) for 2 h. c. Adjust the pH to 7.4 (add 2M NaOH). d. Filter through 1.2,0.8,0.45, and 0.22 i.trn Millex nitrocellulose filters. 3. Methods 3.1. Cell Preparation Remove the lungs from anesthetized animals and lavage. Centrifuge the alveolar macrophages thus obtained and suspend m culture medium. Estimate cell number, dilute to 1 x lo6 cells/n& and plate out. Once the cells have adhered rinse the cultures and add fresh medium.

46

Collan and Kosma

5. After 24 h incubation the cells should be exposed to medium containing particulate matter for a further 24 h period. After this time the medium and harvested cells should be collected and the percent cell death established (using a Trypan blue staining technique). 6. The cells should then be centrifuged and an aliquot of the supernatant (culture medium) be removed for assessmentof LDH activity. 1. 2. 3. 4.

3.2. Macrophage Collection Anesthetize the rats with a subcutaneous injection of sodium thiopental (1.2 g/kg). Remove the lungs, ensuring the trachea and bronchi remain intact. Wash the outside of the lungs with sterile ice-cold sodmm chloride solution (containing antibiotics). Keep in covered Petri dishes on crushed ice for 10-20 min if not processed immediately.

Carry out the following procedures in the laminar flow cabinet. 1. 2. 3. 4. 5. 6. 7. 8. 9.

Cannulate the trachea with the catheter. Draw out the needle from within the catheter. Push the catheter deeper into the lumen of the trachea. Slowly introduce 10 mL of the physiological saline solution (4°C) into the lungs through the catheter using a 10 mL syringe. When filled, gently compress the lungs with the fingertips to encourage flow between lung compartments. Withdraw the fluid back into the syringe. Transfer to a sterile centrifuge tube. Keep tubes on ice for up to a maximum of 30 mm. Repeat the lavage 5-7 times for each animal.

3.3. Macrophage Culture 1. Centrifuge the cells at 4°C for 3 min at 2000 rpm, followed immediately by 7 min at 1000 rpm. Suspend in Ca2+- and Mg2+-free Hanks’ balanced salt solution (containing antibiotics) (4°C). Centrifuge the cells (3 min at 2000 rpm, 7 min at 1000 rpm). 2. The cells obtained from several animals may be pooled and suspended in a volume of culture medium (4°C) equivalent to 2.5 mL/ammal. 3. Transfer to plastic tubes kept on ice. Estimate cell number using a Burker’s Chamber and adjust to 1 x lo6 cells/ml. Perform a Trypan dye exclusion test at this point (200 p,L cell suspension, 200 FL 0.5% Trypan blue). 4. Add 2 mL of the cell suspension to each well of a multidish. Incubate for l-2 h at 37°C (5% CO2 m air, 95% humidity). Once the cells have adhered carefully remove the supernatant. Wash the cells for 5 min with Hanks’

Dust Toxicity

47

in Cultures

solution (containmg antibiotics) (room temperature). Aspirate off. Add 2 mL of culture medium/well (room temperature). Incubate for 24 h.

3.4. Preparation

of Dust Samples

1, Add 2 mg of the dust samples to be tested to 1 mL of Ca2+-and Mg2+-free PBS (containing antibiotics). Mix thoroughly. 2. Add 100 pL or 200 pL dust concentrate per 2 mL tissue culture fluid to give a final concentration of 100-200 pg/mL. 3. Incubate for 30 min at 37°C. 4. Place in an ultrasound bath for 3 min.

3.5. Testing Remove the culture medium from the wells. Wash each well carefully with Hanks’ solution containing antibiotics (room temperature). Set up

the plate as follows: 1. 2. 3. 4.

5. 6. 7. 8. 9. 10.

Zero control: culture medium only, no cells. Cell-free control: culture medium containing dust particles, no cells. Dust-free control: dust-free culture medium. Dust control: a. Titanium dioxide dust in culture medium. b. Chrysotile dust in culture medium. c. Additional control dusts in culture medium. d. Test condition-dust sample to be tested in culture medium. Add 2 mL of relevant medium (room temperature) to each well. Incubate for 24 h at 37°C 5% COz.in air. At the appropriate time detach the cells from the dish with a rubber policeman. Collect the cell-containing medium for analysis. Cell death: Remove a 200 pL aliquot of the cell suspension and add 200 pL of 0.5% Trypan blue. Count the number of viable and nonviable cells using a Burker’s chamber. LDH activity: Spin down the detached cells at 3000 rpm for 15 min. Remove 9 pL of supernatantfor assessmentof lactic dehydrogenase activity. Add 91 pL UV-LDH working solution and 5.2 pL Na-pyruvate starter.

The assay proceeds automatically to determine absorption at 340 nm and 37°C and express the result as IU/L LDH. 3.6. Results 3.6.1, Cell Death

Calculate the number of dead cells present in each situation as a percentage of the total number of cells, The dust-free situation represents the control, i.e., cell wastage, which occurs naturally. The positive con-

48

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and Kosma

trols and test situations may be compared to assess whether a significant increase in cell death has occurred. 3.6.2. LDH Activity Zero control provides an indication of LDH activity present in medium. Cell-free control indicates whether the dust particles are affecting the LDH assay. Dust-free control indicates the level of cell death and LDH activity present in control, nonexposed cultures. Dust control provides a positive control, i.e., a standard against which the harmful effect of test compounds may be judged (I--3,6).

4. Notes 1. Care should be taken to treat the serum used to supplement culture medium so that any lactate dehydrogenase activity can be removed. It should also be noted that the serum may, to some extent, exert an mhibitory effect on the enzyme assay,although the incorporation of appropriate controls in the experimental protocol will allow for this eventuality. 2. Under ideal conditions, the zero control and the cell-free control will be the same. 3. Each situation should be corrected with respect to any activity found in the cell-free control. An increase in LDH activity over that observed in the dust-free cultures indicates that damage has occurred to the cells. The degree of damage may be estimated by comparing to standard positive controls. 4. Cells can be left for up to 3 h without markedly influencing the results but the larger the number of cells present the higher the enzyme value; therefore, ensure careful standardization of cell number and technique. 5. There should be a good correlation between the fraction of dead cells and the results of the enzyme assays.

References 1. Holopainen, M., Collan, Y , Kosma V.-M , Kalliokoski, P , Kulju, T., Anttonen, H , Tossavainen,A , and Kauppinen, H. (1986) Evidence for toxicity of phlogopite in hemolysis and macrophagetests,in Proceedings of the 2nd International Symposium on Occupattonal Health and Safety in Mining and Tunneling. Prague. pp. 65-72. 2. Committee on Enzymesof the ScandinavianSociety for Clinical Chemistry and Clinical Physiology (1974) Recommendedmethodsfor the determination of four enzymesin blood. Stand. J. Clin. Lab. Invest. 33,290-306 3 Pasanen,J. T. (1982) Alveolaarlset ja peritoneaalisetmakrofagit pdlyJen sytotoksisuudentestauksessa in vitro. Pro gradu-tutkielma.Jyvaskyllin yliopisto. Mom&e, Tybterveyslaitos, Helsinki.

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Toxicity

in Cultures

49

4. Collan, Y., Kosma, V -M , Kalhokoski, P., Seppa, A., Kulju, T., Vaantinen, I., Remola-Pgirssmen, E., Mlettmen, R., Pretila, L., Gidlund-Marjanen, A.-L., Manninen, R., Anttonen, H , Tossavainen, A., Husman, K., Huuskonen, M. S., Rytkiinen, E., Lehtinen, A., Kauppinen, H., Mikkonen, A., Koistinen, S., Karjalainen, T., and Harm&i, 0. (1985) Siilinjiirven Apatiittiesiintymiin Richteriitti: Biologinen Vaikutus ja Tybhygieeninen Merkitys. Kuopio 5. Collan, Y., Kosma V.-M , Anttonen, H., and Kulju, T (1986) Toxicity of richterite in hemolysls test and macrophage cultures. Toxic interfaces of neurones, smoke and genes. Arch. Toxic01 Suppl. 9,292-293. 6 Collan, Y., Kosma, V.-M., Kulju, T., Vaananen, I., Remola-Parssmen, E., Pesonen, E., Puhakainen, R., Rytoluoto-Kfirkkainen, R., Manninen, R., and Pasanen, J. (1988) Estimation of dust toxicity in rat alveolar macrophage cultures, in Safety Evaluation of Chemicals on Laboratory Animals, Proceedmgs of the Finnish-Soviet Symposium, Kuoplo, 20-22 May 1986 (Nevalainen, T., Voipio, H -M., and Haataja, H., eds.), Kuopio, Finland, pp. 105-123

CHAPTER7

Hepatoma Cell Cultures as In Vitro Models for Hepatotoxicity Margherita

Ferro

1. Introduction This test is designed to detect irreversible toxic effects on both cell growth and survival, by theevaluation of colony-forming (CF) efficiency, in hepatoma cell lines derived from humans, rat, and mouse. The liver is a major target organ for the cytotoxicity of many xenobiotics. It has been suggested that hepatoma cell lines may provide an appropriate in vitro model for the assessment of likely hepatotoxicity in vivo. It should be noted, however, that the usefulness of such systems largely depends on the ability of the cells to maintain differentiated functions. The procedure presented here suggests a simple means of assessing the cytotoxicity of compounds to hepatoma cell lines. The method involves exposing the cells to xenobiotics, after which colony formation is monitored and compared to that of nonexposed control cultures. The method can easily be adapted for many hepatoma cell lines. In this particular procedure, six hepatoma cell lines have so far been used: 1. HepG2, a human hepatoblastoma cell line that shows basal and inducible levels of monooxygenases; 2. MH&, a rat hepatoma cell line, that shows basal and inducible levels of monooxygenases; 3. 7777, a rat hepatoma cell line that shows basal and inducible levels of monooxygenases; 4. HTC, a rat hepatoma cell line that does not show monooxygenase activities; 5. JM2, a rat hepatoma cell line that does not show monooxygenase activities; and From’ Methods m Molecular Biology, Edited by S O’Hare and C K Atterwlll

Vol. 43’ In Vitro Toxmty Tesbng Protocols Copyright

51

Humana

Press

Inc , Totowa,

NJ

Ferro 6. Hepa 1~1~7,a murine hepatomacell line that shows basal and inducible levels of monooxygenases. A small number of cells (250-500) are plated into complete medium in 6 well (35~mm diameter) culture plates. After 24 h, the medium is removed and the cells are exposed to different concentrations of the test compounds in serum-free medium. After 1,6, or 24 h, the experimental medium is removed and the cells are grown in standard conditions for 8-15 d. The colonies are then stained and those at least 0.3 mm in diameter are scored with the naked eye and the percentage survival is calculated with reference to control cultures. A decrease in the number of colonies formed is an indication of test compound toxicity. Colony formation may provide a much more sensitive measure of toxicity than certain other commonly employed methods. For example, it was found to be more sensitive than the LDH leakage assay (I), because it depends on cell growth-related mechanisms rather than cell membrane damage. Colony formation was also found to be a more sensitive parameter of toxicity than cell viability, assessed by total macromolecular content of the attached monolayer (2,3). This increased sensitivity could be because colony formation is assessedwhile the cells are in a state of proliferation, and thus more susceptible to toxic effects. Moreover, the measurement of total macromolecular content is carried out in a larger number of cells, which may mask some dose-dependent effects since the test compound is distributed over a large population of target cells. The sensitivity of the colony-formation assay, and the fact that dose and time-dependent effects are detectable, enables acute and chronic exposure periods to be investigated as well as permitting recovery studies. It has been found that exposure of the cells to test compounds before seeding rather than 24 h post attachment is not an effective way of assessing toxicity. Colony formation may be employed as an endpoint in many different hepatoma cell lines. The method must be standardized, however, for each cell type. There are cell lines, for example H4IIEC3, whose cells spread out from the colony, thus impairing distinction of separate colonies. Ideally the cells should be plated out at a suitable level so that over a period of approx 7-15 d they form between 50 and 100 colonies. The level of plating and period of incubation for any particular cell line can be established this way.

Hepa toma Cell Cd t ures Basal Colony-Forming Cell line JM2 7777

HTC Hep G2 MI-WI Hepa lclc7

53

Table 1 Abilities (CFA) of the Various Hepatoma Cell Lines No. cells/well 250 250 250 500 300 200

CFA (%) 37f 30*

6 2

39 f 10 19 * 12 16f 4 37 Ik 15

The different hepatoma cell lines vary in their sensitivity to different chemicals. A comparison of HTC (rat hepatoma) and Hep G2 (human hepatoma) cells (3) showed differences that arise from the fact that Hep G2 cells retain some cytochrome P450-dependentfunctions, whereas HTC cells are practically devoid of bioactivating enzymes. Thus, the IC,a values in mM for paracetamol, amitryptiline and nicotine (see Table 1) are approximately ten times greaterfor HTC cells than for Hep G2 cells, showing that metabolism by the latter contributes to the toxicity. However, Hep G2 cells are also more sensitive than HTC to iron sulfate, digoxin, and potassium cyanide, substances that are assumed to have a direct toxic effect. This could be caused by less specific mechanisms operating in the human cell line. Similarly, a difference in sensitivity to benzaldehyde was found between the two rat cell lines, MHiCr and HTC (I), because aldehyde dehydrogenase is present in much greater quantities in HTC cells. Thus, although the effects of a chemical on colony formation in a hepatoma cell line will provide an indication of cytotoxicity, interpretation of the results depends on many factors, including the metabolic competence of the cells with respect to the test compound. The degree of expression of the various monooxygenase isozymes by different hepatoma cell lines must therefore be taken into consideration, The relevance of results obtained in various hepatoma cell lines to human toxicity in vivo, and therefore the definition of the most appropriate cell lines to use for specific testing purposes has yet to be established. The inclusion of this system into the Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) program may begin to answer these questions. The cells are exposed to the test compounds in serum-freemedium so that the direct toxic effect can be estimated in the absenceof any confounding effects arising from the interaction of the compounds with serum proteins.

Ferro

54

2. Materials 2.1. Cell Lines 1. Human hepatoma cell line: Hep G2 (#), ECACC: 85011430, ATCC: HB8065. Obtained from B. Knowles, Wistar Institute of Anatomy and Biology, Philadelphia, PA. 2. Rat hepatoma cell lines. a. MHiCt (5), ECACC: 85112702, ATCC: CCL 144. Obtained from the American Type Culture Collection, Rockville, MD. b. McA-RH 7777 (6), ECACC: 90021504, ATCC: CRL 1601. Obtained from R. Lindahl, Dept. Biochemistry and Molecular Biology, Umversity of South Dakota, Vermillion, SD. c. JM2 (7). Obtamed from R. Lmdahl, Dept. Brochemrstry and Molecular Biology, University of South Dakota. d. HTC (8). Obtained from Flow Laboratories (Irvine, Scotland). 3. Murine hepatoma cell line: Hepa lclc7 (9). Obtained from G. Bellomo, Dept. of Experimental Medicine and Oncology, Universrty of Turm, Italy. 2.2. Equipment 1. Incubator: 37”C, humidrfied atmosphere, 5% CO,/95% air. 2. COSTAR Nucleopore 6 well (35-mm diameter) cluster dishes. 3. Corning tissue culture flasks, area 25 or 75 cm2. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12.

2.3. Materials D-MEM/F12 (1: 1) (Grbco, Paisley, UK). MEM (Boehringer Mannheim GmbH, Germany). Nonessential ammo acids supplement for culture medium (Gibco). Vitamin supplement for culture medmm (Gibco). Newborn calf serum (Sera-Lab Ltd., Crawley Down, Sussex, UK). Fetal bovine serum (Sera-Lab Ltd.). Gentamycin (Gibco). Trypsm-EDTA (O.OS-0.02%) solution (Seromed, Berlin, Germany). HBSS or PBS. Stammg solution: 1% crystal violet m a 3: 1 v/v mixture of acetic acid and 97% ethanol. Complete medium for MHtC,, HTC, JM2, McA-RH 7777, and Hepa lclc7 cells: D-MEM/F12 (1: 1) supplemented with 0.1% NEA (by volume from a 100X solution), 0.1% vrtamms (by volume from a 100X solution), 10% newborn calf serum, 50 pg/mL gentamycin. Complete medmm for HepG2 cells: MEM supplemented with 0.1% NEA (by volume from a 100X solution), 10% fetal bovine serum, 50 ,uglrnL gentamycin.

Hepatoma

55

Cell Cultures

2.4, Test Compounds All test compounds/drug solutions should be prepared in serum-free medium on the day of use. Any test compounds/drug solutions that are insoluble in water should be dissolved in dimethylsulfoxide (DMSO), methanol, or ethanol. The final maximum solvent concentration should not exceed 0.25% v/v. 3. Methods 3.1. General Maintenance of the Hepatoma Cell Lines 1. Thaw cells rapidly (i.e., in a few minutes) from frozen stock. Suspend the cells in the appropriate medium and seed in tissue culture flasks (37”C, 5% CO2 in air) as follows: in 25 cm* flasks: a. 3 x lo5 MH,CI cells in 5 mL medium. b. 1 x lo5 HTC cells in 5 mL medium. c. 1 x lo5 7777 cells in 5 mL medium. d. 1 x lo5 JM2 cells in 5 mL medium. e. 1 x lo5 Hepa lclc7 cells in 5 mL medium. f. 3 x lo5 Hep G2 cells in 5 mL medium. in 75 cm* flasks: g. 1 x lo6 MH,H, cells in 15 mL medium. h. 3 x lo5 HTC cells in 15 mL medium. i. 3 x lo5 7777 cells in 15 mL medium. J. 3 x lo5 JM2 cells in 15 mL medium. k. 3 x lo5 Hepa lclc7 cells in 15 mL medium. 1. 1 x lo6 Hep G2 cells m 15 mL medium. 2. Change the medium every 2 d. Add 5 or 15 mL fresh medium to 25 and 75 cm* flasks, respectively. Grow as monolayer cultures. 3. To subculture the cells: Aspirate off medium and rinse cells with either HBSS or PBS. Add 1 (or 2) mL trypsin-EDTA to detach cells. Incubate flasks at 37°C for l-5 min. Monolayers should detach after 2-5 mm. Resuspend the cells m the appropriate complete medium and seed as above unless the cells are to be used for cytotoxiclty assessment.

3.2. Cytotoxicity

Test by Colony-Forming

Efjkiency

1. Cells should be harvested at the late-log phase of growth (i.e., when the cells are Just approaching confluency) in the manner described m Section 3.1., item 3. This ensures maximum colony-forming efficiency. 2. Count the cell suspensions and aliquot correct volumes (1.5 mL medium/ well) into 6-well cluster plates to seed the following numbers of cells/well:

56

Ferro 200 Hepa lclc7 cells, 250 HTC, 7777, and JM2 cells, 300 MH,C1 cells, 500 Hep G2 cells.

3.3. Exposure

to Test Compound

1. Ikventy four hours after the mmal seedmg,replace growth medium with complete (or serum-free) medium containmg the xenobiotic under investigation. 2. Acute exposure: After 1, 6, or 24 h, remove the xenobiotic-containing medium as described below and replace with fresh growth medium. Do not disturb the cultures until the end of the incubation period. 3. Chronic exposure: Leave cells exposed to the xenobiottc-containing medium. Do not disturb the cultures until the end of the incubation period. 4. Colony formation: At the end of the exposure period, aspirate off the experimental medium. Wash cells briefly with fresh medium. Grow cells in complete medium in standard conditions for 8-15 d. At the end of the culture period, rinse dishes with cold PBS. Stain with crystal violet staining solution. Score stained colonies over 0.3 mm in diameter (20-50 cells/ colony).

5. Calculate the number of colonies formed m the presence of test compounds as a percentage of those occurring m control cultures. Use these results to produce a dose-response curve from which the IC5a value (i.e., the concentration of test compound

that reduces colony formatton

to 50% of that in

controls) can be obtained.

4. Notes 1. Some serum-free batches of these media will also be required for dilution of test compounds. 2. It is important to handle colony-forming cultures with great care. All mediakolutrons

should be prewarmed before use.

3. The system should be standardized so that 70-100 colonies are obtained after 7 (HTC, 7777, JM2, and Hepa lclc7

cells) or 15 (MHIC1,

Hep G2

cells) d of incubation using control cultures. 4. After certain treatments,

the colonies produced are very small but visible

to the naked eye, thus, only colonies that can be seen without magnification are considered.

References 1. Ferro, M., Bassi, A. M., and Nanni, G. (1988) Hepatoma cell cultures as in vitro models for the hepatotoxlcity of xenobtottcs. ATLA 16(l), 32-37. 2. Bassi, A. M., Piana, S., Pence, S., Bosco, O., Brenci, S., and Ferro, M. (1991) Use of an established cell line in the evaluation of the cytotoxlc effects of various chemicals. Boll. Sot. It. Biol. Sper. 8, 809-816.

Hepatoma

Cell Cultures

57

3. Bassi,A. M., Bosco, 0 , Brenci, S , Adamo, D., Pence, S , Piana, S., Ferro, M., and Nanni, G. (1993) Evaluation of the cytotoxicity of the first 20 MEIC chemicals m two hepatoma cell lines with different xenobiotic metabolism capacities. ATLA 21, 65-72. 4. Knowles, B., et al (1980) SCWW 209,497-499. 5. Richardson, U. I , et al. (1969) J. Blol. Chem. 40,236-247. 6. Becker, J. E., et al (1976) in Oncodevelopmental Gene Expression. Academrc, New York, pp. 259-270 7 Novicki, D., et al (1983) In Vitro 19, 191-202. 8 Thompson, E. B., et al. (1966) Proc. Nat1 Acad Scl USA 56,296-303 9 Hankinson, 0. (1979) Proc. Natl. Acad. Sci. USA 76,373-376

CHAPTER8

Cytotoxicity, DNA Fragmentation, and DNA Repair Synthesis in Primary Human Hepatocytes Giovanni

Brambilla

and Antonietta

Martelli

1. Introduction The well-documented existence of interspecies differences in the responses elicited by exposure to chemical agents (1-3) represents the main difficulty in the extrapolation to humans of the results of toxicity studies carried out in laboratory animals or in in vitro systems employing as targets prokaryotes, lower eukaryotes, or mammalian cells. Growing evidence indicates that the most important cause of these species-related differences is the different handling of xenobiotics by metabolic activation/detoxification processes.Primary cultures of human hepatocytes that can be prepared from material discarded during the course of prescribed surgery offer the unique advantage of directly assessing the cytotoxic and genotoxic effects of chemicals in cells of our species possessing a comprehensive metabolic capability. Isolation and culture of human hepatocytes were first described by Strom and others in 1982 (4,5). In this chapter, we describe our procedure for hepatocyte isolation and culture, as well as the techniques for the evaluation of cytotoxicity, DNA fragmentation, and DNA repair induced by the exposure to chemical agents. Examples of the use of human hepatocyte primary cultures in cytotoxicity and genotoxicity studies are offered by several papers (6-l 1). Up to now chemicals tested for their cyto- and genotoxic effects in human hepaFrom Methods m Molecular Biology, Vol 43 ln V/fro Toxmty Teshng Protocols Edlted by S O’Hare and C K Atterw~ll Copynght Humana Press Inc , Totowa, NJ

59

60

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and Martelli

tocytes are only a minimal fraction of those potentially hazardous for our species, but some provisional considerations can already be drawn. Human hepatocytes from different donors display a great variability in response. This can be ascribed to the fact that human donors differ in age, sex, diet, drug intake, lifestyle, and pathological conditions of the liver. For the large majority of compounds tested on human hepatocytes, the great interindividual variability of the response results in an overlapping of the genotoxic potencies with those observed in rat hepatocytes, but some chemicals, such as cimetidine, tripelennamine, P-naphtylamine, and unleaded gasoline are negative in humans and positive in rats; the converse occurs for 2,4-diaminotoluene and 5methylchrysene. Our survey of the literature data suggests that a standardization of the methods employed is necessary in order to avoid differences of results attributable rather to different experimental conditions than to true interspecies differences. Human liver may be obtained from discarded surgical material. Hepatocytes are isolated by collagenase perfusion. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

2. Materials Heating water bath, 42OC. Peristaltic pump, Mastefflex model 7014, tubing set. Plastic polythene catheters, 18-20 gage. Large glass Petri dish. Stainless steel dog comb and scissors. Inox or nylon mesh (74 pm pore size). Refrigerated centrifuge, swinging-bucket type. Hemocytometer and microscope. Hanks’ balanced salt solution (HBSS). Disodmm EDTA. Ca2+- and Mg2+-free HBSS, Collagenase (type I or IV), Sigma (St. Louis, MO). Williams’ E medium (WE). Fetal bovine serum (FS). Gentamicin, 50 pg/mL m WE. Trypan blue, 0.4% in saline. Plastic culture dishes, 60- and 35-mm diameter. Slide flasks. Collagen, 25 pg/mL. Inverted microscope. Plate shaker. Spectrophotometer (or microplate reader photometer),

Cytotoxicity

in Human Hepatocytes

61

23. 24. 25. 26. 27.

Trypan blue, 0.4% in saline. Neutral red dye, 50 pg/mL WE. Fixative: 1% formaldehyde, 1% CaC& in distilled water. Destain solution: 1% acetic acid, 50% ethanol, 49% distilled water. Stainless steel filter holder wrth an upper cylindrical funnel section and a lower collecting section. 28. Tubing set. 29. Peristaltic pump Gilson (Paris, France) Miniplus 2. 30. Spectrophotofluorometer.

2.1. Solutions

for DNA El&ion

1, Merchant’s solutron: 0.14M NaCl, 2.7 mIt4 KCI, 1.47 mZt4KH,PO,, 8.1 nuI4 Na2HP04, 0.53 miI4 disodium EDTA, pH 7.5. 2. Lysing solution: 0.2% sodium lauroyl sarcosinate, 2A4NaCl, 20 mZt4disodium EDTA, pH 10.0. 3. Eluting solution: 0.06M tetraethylammonium hydroxide, 20 mM disodium EDTA, pH 12.3. 4. Washing solution: 20 mIt4 disodium EDTA, pH 10.0. 5. Millipore mixed estersof cellulose filters, 25 mm diameter, 5 pm pore size.

2.2. Solutions

for DNA Determination

1. 33258 Hoechst dye stock solution (0.15 miI4 m distilled water); prepare and store in the dark; stable for at least 1 wk at 4°C. 2. Citrate buffer: 0.154MNaCl,O.O15M sodium citrate, pH 7.0. 3. 0.2M KH2P04 solution. 4. Dark room. 5. Heating water bath at 38°C for meltmg of photographic emulsion. 6. Black hermetic box. 7. Microscope (x 1250 magnification). 8. [Methyl-3H] thymidine (specific activity 20-40 Ci/mmol). 9. Sodium citrate, 1% solution. 10. 1:3 Acetic acid: ethanol solution. 11. Glycerol jelly. 12. Kodak NTB-2 emulsion. 13. Dl 1 Kodak developer. 14. Unifix Kodak fixer. 15. May Grtinwald-Giemsa stain.

3. Methods 1. After surgical removal the tissue is immediately placed in me-cold saline. Perfusion should start within 1 h from excision; a longer time interval reduces the hepatocyte viability.

Brambilla

and Martelli

2. Remove any damaged areas of the liver sample. The fragment for use should be at least 10 g m weight, and cut 2 cm apart from the damaged areas. It is better if the fragment is enclosed m the hepatic capsula on all sides, except for the one cut surface. 3. Place this fragment m a large sterile glass Petri dish, and insert plastic polythene catheters mto vascular orifices on the cut surface. Usually a mmimum of 2 or a maximum of 4 catheters are placed; the canulae may be moved from one cut vessel to another in order to obtain a more homogeneous blanching of the liver. 4. The perfusion starts with Ca2+- and Mg2+-free HBSS supplemented with 0.5 mM disodmm EDTA. Set up the peristalttc pump so as to perfuse at a rate of 14 mL/min for about 20 min. Dissolve collagenase m complete HBSS to a concentratton of 0.4-0.5 mg/mL, and continue the perfusion with this solutton, at the same rate, for a further 20-40 mm. The length of this step is dependent on the hver conditions. Since the buffers should be delivered to the hepatic site at a temperature of 37”C, the buffers should be maintained durmg perfusion m a heating water bath at 42°C; a loss of temperature occurs over the tubing set. 5. A few minutes after startmg the perfusion, wedge-shaped areas of blanched tissue should become evident on the surface of the liver. By the end of about 40 min perfusion approx 70% of the surface should have this appearance. 6. The following steps are carried out at 4°C in order to stop the collagenase digest and endonuclease activity. The Glisson membrane is cut with scissors, and the perfused hepatic parenchyma is gently combed in HBSS with the dog comb to obtain a liver cell suspension. Filter this suspension through a 74 pm pore mesh. The volume of HBSS used m this step should be sufficiently large to dilute bile that will prevent the subsequent cell sedimentation performed in a refrigerated centrifuge at 5Og for 4 mm. The pellet is resuspended m serum-free WE medium and centrifuged again Repeat this procedure twice, and finally resuspend the hepatocytes in complete WE medium, i.e., WE supplemented with 10% FS, and 50 yglmL gentamicm. Add 0.1 mL sample of cell suspension to 0.4 mL of 0.4% trypan blue solution; count the number of viable and nonviable (blue-stained) cells in a hemocytometer after 3 min resting. The fraction of viable cells usually varies from 70-90%, but since human liver fragments are not so easily obtamed, even hepatocyte suspensions 60% viable may be used. 7. The hepatocyte suspension is diluted m complete WE medium to the required concentration of 5 x lo5 cells/ml. The cell yield obtained with this procedure usually ranges from 5 x lo6 to 10 x 107/g of liver.

Cytotoxicity

in Human Hepatocytes 3.1. Cell Culture

and

63 Treatment

The method of seeding the cells depends on the assayto be carried out. 1. For cytotoxicity evaluation, 2 mL cell suspension (-1 X lo6 cells) are seeded in 35 mm dishes precoated with collagen; the total number of dishes depends on the test performed; for the trypan blue exclusion test only 1 dish/dose may be sufficient; for the neutral red test 3 dishes/dose are required. For the DNA damage/alkaline elution assay, 4 mL cell suspension (-2 x lo6 cells) are seeded in 60 mm dishes not coated with collagen, in order to allow an easy cell detachment; 2-3 dishes/dose are needed. For unscheduled DNA synthesis (UDS), 2 mL cell suspension are seeded in 35 mm plastic dishes or in slide flasks; the collagen coating of the flasks may be difficult and is usually avoided; 2 dishes or flasks/dose are needed. 2. The cultures are incubated in a 95% sir/5% CO2 humidified atmosphere for about 3 h, in order to allow cell attachment. Then remove the medium, rinse the culture with WE serum-free medium, and start the exposure to the test compound. 3. Appropriate concentrations of the compound to be tested are prepared m the medium used to set up the cultures. If the test compound is not directly soluble m medmm, use a solvent vehicle, such as ethanol or dimethyl sulfoxide, at maximum concentrations of 3 and 0.5%, respectively. Solvent control cultures receive equal concentrations of the solvent alone. The length of treatment is usually 20 h. 3.2. Cytotoxicity

Assays

Since not all laboratories are equipped with a microplate reader photometer suitable to allow the use of multiwell plates, the procedure for the neutral red assay will be described for cultures in 35 mm plastic Petri dishes. Multiwell plates are more convenient where lower numbers of cells are required. 1. Trypan blue assay: At the end of the treatment the cultures are washed with two changes of salme. Then 0.8 mL 0.4% trypan blue solution are added. After 2-3 min incubation, determine the fraction of nonviable (bluestained) cells in 10 randomly chosen fields with a graduated inverted microscope. 2. Neutral red (NR) assay:At the end of the treatment the medium is removed and replacedwith 2 mL serumfree medrumcontaining 50 ug NR/mL. The cultures are returned to the Incubator for further 2 h to allow for the uptake of the vital dye into the lysosomes of viable cells. Thereafter the medium is removed, and the cells are rapidly washed with 3 changes of fixative, fol-

64

Brambilla

and Martelli

lowed by addition of 2 ml/culture of destain solution. Plates are shaken for 10 min, and the extracted dye is read at 540 nm. Results are expressed as a percentage of the optical density determined with extract from control cultures at 540 nm. 3.3. DNA FragmentationlAlkaline El&ion Assay 1. At the end of the treatment wash cultures with cold Merchant’s solution and incubate dishes for 4-5 min at 4°C in 2 mL of the same solution. Merchant’s solution, containing EDTA that binds Ca2+ ions, allows the detachment of hepatocytes without scraping, which could cause mechanical DNA damage. Cells will be gently harvested in the same solution with a Pasteur pipet, and about 1 x lo6 cells of this suspension are loaded onto a 25 mm diameter, 5 urn pore size Millipore filter supported by a stainless steel holder with an upper cylinder funnel section and a lower collecting section terminatmg with an outflow to which a flexible tube can be attached. After an additional wash with 5 mL Merchant’s solution, pass 4.5 mL of lysing solution through the filter to lyse the cells. Filters are then washed with 3 mL 20 mM disodium EDTA at 22OC.Single-strand DNA is eluted from the lysate m the dark with a controlled flow (0.13 mL/min) of 13 mL eluting solution, The eluate is collected at lo-min intervals (10 samples). Once the elution is complete, break up the filter in 3 mL of eluting solution. 2. The DNA content of each eluate and that remammg on the filter is determined by the microfluorometric procedure of Cesarone et al. (12). Bring to pH 7.2 with 0.2M KH2P04 1 mL aliquots of DNA samples and blank (eluting solution), and dilute to a final volume of 2 mL with citrate buffer. Add to each sample 2 mL of a 1:50 dilution m citrate buffer of the 33258 Hoechst stock solution, Shake and mcubate for 10 min in the dark. The fluorescence spectra of the dye-DNA complex and of the free dye solution are recorded in a spectrophotofluorometer with an excitation wavelength at 360 nm and emission at 450 nm. 3. Calf thymus DNA diluted to an mitial concentration of 1 mg/mL m citrate buffer is used as standard solution, A DNA standard is run through the system after every 20 elution assays. 3.4. Unscheduled DNA Synthesis (UDS) 1. In the case of the UDS assay, treatments are carried out in serum-free medium supplemented with [methyl-3H] thymidine (10 mCi/mL). The length of exposure is 18-20 h. At the end of incubation, wash the cultures with cold saline; then add 2 ml/dish of 1% sodium citrate solution. After 5-10 min incubation, remove the citrate solution, add 2 ml/dish 1:3 acetic acid:ethanol, and incubate for 10 min. Repeat the exposure to the acetic acid:ethanol a further two times in order to fix the cells.

Cytotoxicity

in Human Hepatocytes

65

2. Cultures are air-dried; then a section from the bottom of the dish is cut out and glued to a microscope slide using glycerol jelly. Alternatively, plastic disposable slrde flasks may be used. 3. Dip the slides in Kodak NTB-2 emulsion for autoradiography, and expose for 7 d at 4OC in a black hermetic box. Develop autoradiographs in Dll Kodak developer at room temperature for 2-5 min in the dark. Wash the slides for 30 s in 1% acetic acid, and fix for 4 min m Umfix Kodak fixer. Stain the slides with May Grtmwald-Giemsa. 4. Count autoradiographic grains in the nucleus and m an equal-sized randomly chosen area of the cytoplasm of 50 consecutive cells of each slide using a microscope at 1250x magnification. The net number of nuclear grains owing to DNA repair synthesis is calculated by subtracting the cytoplasmic grain count from each nuclear count.

4. Notes 1. A variety of standard media such as Leibowitz-15, Waymonth MB 752/l, or Dulbecco’s modification of MEM have been successfully used for these cultures, but WE medium is the most convenient and the most often employed. Human hepatocyte primary cultures need a CO, incubator. 2. The NR contammg medium must be preincubated overnight at 37°C and centrifuged prior to use to remove precipitates of dye crystals. 3. In Section 3.4., step 1 acetic acid is essential to remove the excessof radioactive thymidme. 4. Results should report the net nuclear count but also nuclear and cytoplasmic counts separately. As a matter of fact, some chemicals could influence cytoplasmic labeling and interfere in the evaluation of DNA repair.

References 1.

Langenbach,R., Nesnow, S.,andRice, J. M. (1983) Organ and Species Specificuy

in Chemical Carcinogenesis Plenum, New York. 2 Hsu, L. C , Harris, C C , Lipsky, M. M , Snyder, S , and Trump, B F. (1987) Cell and species differences in metabolic activation of chemical carcinogens. Mutat. Res. 177, l-7. 3. Philhpson, C. E. and Ioannides, C. (1984) A comparative study of the broactivatlon of nitrosamines to mutagens by various animal species includmg man Carcinogenesis 5,1091-1094 4. Strom, S., Jirtle, R. L , Jones, R. S., Novicki, D L , Rosenberg, M. R , Novotny, A., Irons, G., MC Lain, J. R., and Michalopoulos, G. (1982) Isolation, culture, and transplantation of human hepatocytes. J. Natl. Cancer Inst. 68,771-778 5 Maekubo, H., Ozaki, S., Mitmaker, B., and Kalant, N (1982) Preparation of human hepatocytes from primary culture In Vitro 18,483-49 1. 6 Butterworth, B E., Bermudez, E , Smith-Oliver, T , Earle, L , Cattley, R , Martin, J., Popp, J. A , Strom, S , Jirtle, R., and Mrchalopoulos, G. (1984) Lack of genotoxic

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activity of di(2-ethylhexyl) phthalate (DEHP) in rat and human hepatocytes. Curcmogenesis 5, 1329-1335 7 Robbiano, L., Gazzamga, G. M., Martelh, A., Pino, A , and Brambilla, G. (1986) DNA-damagmg activity of tripelennamine m primary cultures of human hepatocytes. Mutat. Res. 173,229-232. 8. Martelli, A., Robbiano, L., Ghia, M., Giuliano, L., Angelim, G., and Brambilla, G. (1986) A study of the potential genotoxicity of cimetidme using human hepatocyte primary cultures; discrepancy from results obtained in rat hepatocytes. Cancer Lett

30,11-16. 9. Martelli, A., Robbiano, L., Gazzaniga, G M., and Brambilla, G (1988) Comparative study of DNA damage and repair induced by ten N-mtroso compounds in primary cultures of human and rat hepatocytes. Cancer Res. 48,4144-4152. 10 Butterworth, B E , Smith-Oliver, T , Earle, L , Loury, D J , White, R D , Doolittle, D. J., Working, P K., Cattley, R C , Jlrtle, R., Michalopoulos, G., and Strom, S. (1989) Use of primary cultures of human hepatocytes in toxicology studies. Cancer Res 49,1075-1084 11 Robbiano, L., Martelh, A., Allavena, A., Mazzei, M., Gazzaniga, G. M., and Brambilla, G. (1991) Formation of the N-nitroso derivatives of SIX P-adrenergicblocking agents and their genotoxic effects m rat and human hepatocytes Cancel Res. S&2273-2279. 12. Cesarone, C. F., Bolognesi, C., and Santa, L. (1979) Improved microfluorometric DNA determmation in biological material using 33258 Hoechst. Anal. Biochem

100,188-197.

CHAPTER9

Model

Cavity

Method

INVITTOX 1. Introduction The model cavity method enables the in vitro cytotoxicity testing of dental restorative materials that may then be related to dental toxicity likely to occur in vivo. The test system has been designed to mimic, as closely as possible, the contact that occurs between dental restorative and tooth pulp when cavities are filled in the clinical situation, A monolayer of cells is maintained in culture, the test material being in contact with the medium. A reduction in cell number, compared to control (nonexposed) cultures, indicates that the compound is cytotoxic and, hence, may cause damage to a tooth in the in vivo situation. A hole is bored through the center of a Petri dish lid and a borosilicate glass cylinder; the lower aperture, sealed with a microfilter, is attached to the underside. The resultant chamber, which represents the cavity, is packed with test material. The lid is placed over a culture dish containing a monolayer of either fibroblasts or macrophages (which represents the pulp), in such a way that the filter is just in contact with the medium. After a set exposure period the number of cells in the culture is determined and compared to that of control cultures. A reduction in cell number provides an indication of the cytotoxicity of the compound. If the reduction is 10% the compound is tested again in the presence of dentine. Artificial cavities are prepared as above with the addition of powdered dentine (obtained from noncarious human teeth) compacted into a 0.5 mm layer at the base of the cylinder. (Or, alternatively, the filter can From: Methods m Molecular Biology, Vol 43. In V&o Toxfaty Tesbng Protocols Edlted by S O’Hare and C K Atterwlll Copynght Humana Press Inc , Totowa, NJ

67

INVITTOX be replaced by an intact dentine slice.) The cytotoxicity of test compounds is then reassessedas before. This system provides a very reproducible, simple technique for the screening of large numbers of compounds. It is less time-consuming and relatively inexpensive, especially in terms of animal usage, compared to in vivo tests. 1.1. Comparison with the In Vivo Situation In vivo testing of dental restoratives is performed on the teeth of primates, dogs, or ferrets. The wide variation in results, together with species differences, complicates any extrapolation to the situation in humans. It has been suggested that this method, which attempts to mimic cavities in human teeth, may provide a more appropriate test system for comparing the relative toxicities of compounds, especially in view of its reproducibility. At the present time, however, the test cannot be categorically stated to be superior to in vivo tests. 1.2. Comparison with Other In Vitro Test Systems This test has several advantagesover other in vitro test systems because it conforms more accurately to the conditions that exist in the clinical situation. Certain in vitro systems produce inaccuracies, often because of the unrealistically large volume of material being testedproducing toxic effects that would not be seen in vivo. In this test the ratio of volume and surface area of the test material, to the volume of the culture medium, is similar to that found in the clinical situation, thus producing more applicable results. Contact between the test material and underlying cells is through a permeable filter enabling the materials to be introduced into the system in their freshly mixed state when they are most irritant, as occurs in use. In conventional in vitro test systems the test chemical is often in direct contact with the target cells (representing the pulp cells of the tooth). In humans, however, when a tooth is prepared for a filling it is usual for a certain amount of dentine to remain intact. This layer of dentine would, therefore, separatethe material used to fill the cavity from the pulp of the tooth. This test system has been adapted so that, should a compound appear toxic, it can be retested in the same system, but one that incorporates a dentine component. The test compound must, therefore, penetrate the dentine before coming into contact with the medium bathing the pulp (i.e., macrophages or fibroblasts).

Model

Cavity

Method

The dentine component is prepared from noncarious human teeth (usually extracted wisdom teeth), which are readily available and again increase the applicability of the system for direct comparison to the clinical situation in humans. Dentine can be prepared as a powder or in slices. In its powdered form the dentine provides an alternative to intact dentine as a chemically reactive substrate,moderating toxicity and binding potentially toxic elements released from dental material. It is unsatisfactory, however, when the permeability characteristics of the dentine are important. In this instance, a dental slice provides a better simulation of the clinical situation. The slices also possessan intact smear layer (a layer of cutting debris that serves as a natural cavity liner) that limits the access of certain toxic agents to the pulp. The choice between using slices or powdered dentine ultimately depends on the importance of standardization against structural normality. However, if a material is to be tested according to the British Standard, dentine powder must be used. of Cell Type The dental pulp is a specialized area of connective tissue composed of a peripheral layer of odontoblasts and a central mass of fibroblastic tissue. Although odontoblasts are the most likely to be initially affected by a toxic material, they are highly specialized and difficult to grow in culture. Therefore, the cells of choice are either fibroblasts or macrophages. Mouse macrophages can be used as the target cells. The pulp beneath a carious dentine lesion may contain macrophages. Although much of this lesion is removed prior to placing a restoration, the inflammation would still be present. In addition, trauma induced during cavity preparation prior to placing a restoration can result in the presence of inflammatory cells. Macrophages from two strains of mice have been compared (7) and no difference in responsiveness was observed. The authors stress, however, that this does not guarantee that differences would not occur if other strains were used. On a routine basis, fibroblasts rather than macrophages are used for several reasons, e.g., ease of handling and maintaining a cell line rather than having to obtain fresh cells, lack of animal involvement, and so on. Pulp fibroblasts can be cultured as a primary diploid culture but this takes time, and the characteristics of the cells may vary from culture to culture. Fibroblasts are probably the most important cell type. It should be stressed that, since the two types of cell respond differently to certain 1.3. Choice

INVITTOX toxic constituents of dental restoratives, results obtained using fibroblasts are not necessarily comparable to those obtained with macrophages. The model cavity was recently adopted as a British Standard. The model cavity system is now under consideration as an international testing method. Dental materials have been tested from many different manufacturers.

2. Materials 2.1. Tissue Culture 1. Incubator, temperature 37 f l”C, humidified, 5% COJ95% air. 2. 50-mm Diameter sterile disposable vented Petrt dishes (tissue culture grade) 3. 35-mm Diameter sterile disposable vented Petn dishes (tissue culture grade). 1. 2. 3. 4. 5.

2.2. Model Cavity System 35 mm sterile plastic disposable vented Petri dishes (tissue culture grade). Dental wax. Borosilicate glass cylinder, BS 2598 (7 mm outside diameter x 3 mm inside diameter x 4 mm hrgh). Microfilter cement. Cellulose acetate filter (retainmg particles 2 0.45 ~JV!in size). 2.3. Preparation

1. 2. 3. 4. 5.

of Dentine

Phers. Steel bur. Ball mill. Liquid N2. Packing tool (2.5-mm diameter). 2.4. Solutions

1. 2. 3. 4. 5. 6. 7.

95% ethanol solution. Filter sterilized salme solution, 9 g/L NaCI. Penicillin-streptomycin solution (100 U/mL and 0.1 mg/mL, respectively). Trypsm-EDTA solution (0.5 g/L and 0.2 g/L, respectively). Dulbecco’s phosphate-buffered saline solution (D-PBS). Conditioning liquid, e.g., 50% citric acrd, acids or EDTA. Fibroblast growth medium Composition, mL/lOO mL Eagle’s minimal essential medium Tryptose phosphate broth Glutamine (29.24 g/L) Penicillin-streptomycin Newborn calf serum

76 10 2 2 10

Model

Cavity

Method

71

8. Macrophage culture medium Composition, mL/lOO mL RPM1 1640 medium (HEPES buffered) 87 1 Glutamine 2 Penicillin-streptomycin Newborn calf serum 10 Composition, mWlO0 mL 9. Macrophage lavage medium D-PBS 90 Heparin BP solution (500 U/mL) 0.4 10 Newborn calf serum 10. Test materials: Prepare any test material immediately before use according to the manufacturer’s instructions. The material should be processed under consistent conditions of temperature and humidity. 11. Teeth: Noncarious freshly-extracted human wisdom teeth. Store in saline solutron containing 1 g/L thymol at 0-4”C, until required. Although it has been suggested that teeth can be stored for several months, the author prefers to use them within 1 mo. 12. Animal: BKW mice, 6-8 wk old. 3. Methods

3.1. Cell Culture 3.1.1. Preparation

of Mouse Macrophages

Use aseptic techniques throughout. Kill mice by asphyxiation with carbon dioxide, Subject each animal to peritoneal lavage using 2.5 mL of macrophage lavage medium. Pool the aspirated material from each mouse. Distribute -1.5 mL aliquots into 35mm diameter Petri dishes to give 3.5 x lo6 cells (including erythrocytes and small lymphocytes) per dish. Incubate (37”C, humidified, 5% CO,/95% air) for 2 h to allow the cells to attach. Remove nonadherentcells by washing twice with D-PBS solution (37°C). Add 2 mL vol of macrophage culture medium to each dish and incubate. 3.1.2. Routine Fibroblast Cell Maintenance 1. BHK-21 (C13) fibroblasts: Passagerecently thawed BHK-21 (C13) cells twice per week for 6-8 wk. Culture the cells m sterile disposable vented Petri dishes (50 mm diameter) containing 5 mL of fibroblast growth medium (37OC, humidified, 5% COJ95% air). 2. Subculture: For routine subculture BHK-21 (C13) fibroblasts are passaged at 1:5 ratio. 3. Wash the cell monolayer with D-PBS (37”C), to remove cell debris. Repeat. Add 0.3 mL of the trypsm/EDTA solution per Petri dish. Incubate

72

IIVVITTOX the dishes at 37°C for 10 min, to detach all the cells. Add 5 mL of fibroblast growth medium to each dish and disaggregate the cells by vigorous pipetmg to produce a fibroblast cell suspensron. Add 1 mL of the cell suspension to 4 mL of fresh medium m 50 mm culture dishes.

3.2. Model

Cavity

System

Drill a 5 mm diameter hole in the center of a vented 35 mm sterile tissue culture grade plastic Petri dish lid. Fix a borosilicate glass cylinder directly beneath the hole, using dental wax. Stick the cellulose acetate filter to the underside of the cylinder using microfilter cement.

3.3. Preparation

of Test Cultures

1. Prepare a fibroblast suspensionculture as described previously. Dilute with fibroblast growth medium until rt contams 1 x lo5 cells/ml. Plate out 4 mL of the suspensron m the base of the Petri dish; or 2. Prepare a monolayer of macrophage cells as described previously (3.5 x lo6 macrophages/dtsh in 4 mL of macrophage culture medium). Incubate for 24 h, remove the supernatant medium, and replace it with 4 mL of fresh macrophage culture medium. Then incubate monolayers for 24 h at 37”C, humidified 5% CO,/95% air.

3.4. Exposure

to Dental

Restoratives

Pack the cylinder (i.e., model cavity) with freshly mixed test material until it is flush with the top of the lid. Sterilize the lid assembly by swabbing the lid and filter with 95% ethanol. Allow to evaporate. Place the lid

assembly over the Petri dish. Agitate the dish so that the fluid contacts the filter. Expose the fibroblast or macrophage cultures for 24 h (incubate at 37”C, humidified 5% CO,/95% air) to the test material. 3.5. Control

Cultures

1. Use lid assemblies that do not contain test materials. 2. Test each material in quadruplicate. 3. Repeat each test on three separate occasrons.

3.6. Assessment

of Cell Damage

1. Frbroblast culture: After incubation for 24 h, remove the supernatant medmm. Wash the cells twice with prewarmed (37°C) D-PBS medium. Add 100 pL of trypsin/EDTA solutron. Incubate at 37°C for 30 min. Occasionally agitate the dishes. Add 2 mL of saline solutron that contams 10% v/v newborn calf serum, to each dish. Suspend and disaggregate the cells

Model

Cavity

Method

73

by vigorous pipeting. Dilute cell suspension with 9.6 mL sterilized saline solution. Determme the number of cells present, 2. Macrophage culture: After incubation for 24 h, remove the supernatant medium. Wash the cells twice with prewarmed (37°C) D-PBS solution, Add 2 mL of a lignocaine hydrochloride in RPM1 1640 medium to each dish and incubate for 12-15 min at 23 Z!Z2°C (this has been shown to aid the detachment of macrophage cells). Scrape the cells off the bottom of the dish and suspendthem by pipetmg. Add 1.5 mL of the suspendedcells to 8.5 mL of filter sterilized saline solution. Determine the number of cells present. Determine cell number of control cultures. Determine cell number of test cultures and present as a percentage of that present in controls. Statistically compare control results to test results. Test any material that gives a statistically significant reduction (p < 0.05) in cell number, or a reduction of 10% compared with controls, in the presence of dentine.

3.7. Test Procedure

in the Presence

of Dentine

1. Preparation of dentine powder: Take noncarious freshly-extracted human permanent wisdom teeth from storage. Wash the teeth in water and air-dry. Place m absolute ethanol and redry. Break off the crowns with pliers and remove the cementum and root canal contents with a steel bur. Grind the remaining dentine m a ball mill under liquid nitrogen to a particle size of ~0.2 mm. (Ground dentine can be stored at -2OOC;wash before use). Using a packing tool, apply a pressure of 4.2Nlm2 to compact 4.7 X 10m3-1.2 X lo-4 g of dentine powder to a depth of 0.5 f 0.1 mm in the bottom of a borosilicate glass cylinder. Repeat the test procedure as before. 2. Preparation of dentine slice: Cut 100, 500, or 1000 pm sections transversely through the crowns of freshly extracted teeth (down to the level of the pulp horns) using a ground section machine. Float the sections on conditioning liquid for 30 s to remove the lower pulpal smear layer. (N.B. The upper smear layer may be removed if it is applicable to the clinical use of the test material.) Attach the slices to the base of the cavity m place of the Millipore filter. Repeat the test procedure as before. 4. Notes 1, If using an electronic cell counter determine values for amplification, aperture diameter, current, and lower threshold value. 2. A reduction in fibroblasts indicates direct toxicity and/or effects on cell growth. 3. A reduction m the number of macrophages is indicatrve of irreversible damage.

74

IN??ITTOX References

1. Rabinovitch, M. and Destefano, M J. (1975) In Vitro 11,379-381. 2. Tyas, M. J. (1977) A method for the in vitro toxicity testmg of dental restorative materials. J. Dent Res. 56, 1285-1290 3. Meryon, S. D. and Browne, R. M. (1983) Evaluation of the cytotoxicity of four dental materials in vitro assessed by cell viability and enzyme cytochemistry. J. Oral Rehab. 10,363-372 4. Meryon, S. D. and Browne, R. M. (1983) Test methods for assessing the cytotoxrcity of dental restorative materials using an in vitro model cavity system, in Ceramics in Surgery (Vincenzini, P., ed.), Elsevier, Amsterdam, pp 127-135. 5. Meryon, S D., Stephens, P. G., and Browne, R. M. (1983) A comparison of in vitro cytotoxiclty of two glass monomer cements J. Dent. Res. 6769-773. 6. Meryon, S. D. and Browne, R. M (1984) in vitro cytotoxicity of a glass ionomer cement of a new generation. Cell Biochem. Funct. 2,43-48 7. Meryon, S. D., Uphill, P F., Cordery, A. D., and Browne, R. M (1985) A reproducibility study of the model cavity method for the in vitro toxicity testmg of dental restorative materials. ATLA 12,215-223 8. Meryon, S. D. (1988) Model cavity method incorporating dentine. Znt. Endo. .Z 21, 79-84.

CHAPTER10 Human

Esophageal Cannel

Culture

Mothersill

1. Introduction This chapter describes a method for establishing short-term explant cultures of esophageal mucosa. Adverse effects produced by exposure to radiation or test compounds can be detected as an inhibition of cell outgrowth. When mucosa explants are plated into culture there is an initial migration of cells outward, followed by a period of mitotic activity resulting in a pronounced outgrowth. Such explant cultures can be exposed to test compounds and radiation, after the initial period of migration has occurred, and the effects of these can then be quantified as an inhibition on the rate of cell growth. As the cells grow outward they form a definite area surrounding the tissue initially plated that, after fixing and staining, can readily be quantified by visual examination. Mucosa samples are dissected from the esophagus; the pieces of tissue are placed in growth medium and cut into small sections. They are then incubated in a trypsinkollagenase mixture for a short period, then suitable tissue samples are selected and cultured in growth medium until required for testing. After -2 d, test chemicals are added to the cultures and, after the required exposure period, toxicity is assessedby fixing, staining, and measuring the area of outgrowth of the epithelial cells. Primary cultures of epithelial cells are very difficult to achieve, require a high degree of technical expertise, and can be very time-consuming to maintain. Although a number of primary “normal” cell lines have been establishedfrom adenomatoustissue,it is quite common for theseto become From Methods m Molecular Brology, Edlted by S O’Hare and C K Atterwill

Vol 43: In Vitm Toxmty Testmg Protocols Copynght Humana Press Inc , Totowa, NJ

75

Mothersill contaminated with fibroblasts and, thus, they can no longer be considered “normal.” A further complication with regard to maintaining esophageal mucosa cells in culture is their lack of clonogenicity. The epithelial nature of the outgrowth was confirmed in representative samples of cultures using a low mol-wt general cytokeratin antibody with indirect peroxidase development. The presence of stromal or endothelial elements can be monitored using antivimentin and antihuman endothelium. This method, however, provides a relatively simple, fast, and easy technique for establishing short-term cultures of relatively pure populations of mucosal epithelial cells. Should contamination with fibroblasts occur, these cells are readily discernable after staining and can be allowed for in the calculation of cell growth. Unfortunately, the major drawback of this technique is that the cultures are only viable for a limited period of -2 wk (but up to 4 wk is possible). The viability of the culture can be extended by changing half the medium weekly. In addition, each new culture is dependent on a fresh supply of human tissue, which may not always be readily available. It should also be noted, however, that the technique is readily adapted to healthy and diseased tissue, thus enabling the differential response to compounds to be examined in both tissue types. Cell outgrowth is measured directly by quantifying the area of cells around the explant. This may provide a more reliable measure of cell growth and survival than other methods currently used, e.g., growth curve extrapolation in monolayer cell culture systems. The possibility that the outgrowth of cells from the original explant is simply a result of a process of cell migration has been considered. This is largely true in the initial stages of culture, however, the contribution of migrating cells to overall outgrowth at later stages is insignificant. This has been confirmed through experiments designed to measure the incorporation of tritiated thymidine (coupled with autoradiographic analysis) and examination of cultures by electron microscopy, which have illustrated that the cells present in the area of outgrowth have a high rate of mitotic

activity and are actively dividing (1). Cell proliferation in cultures canbe studied using the monoclonal antibody, Ki 67 (Dako, Santa Barbara, CA) and an indirect peroxidase immunocytochemical development technique. The effects of irradiation can also be studied using this cell system either in the presence or absence of the test compounds. Irradiation is carried out 2 d after the explant is established and/or 12 h after exposure

Human

Esophageal

77

Culture

to the test compound. Preliminary results using this culture system have shown that a reduction in the outgrowth of cells from the explant occurs after radiation treatment, The dose-response relationship that is obtained is within normal mammalian limits and tissue specific differences can be detected (2).

2. Materials 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11,

12. 13.

14. 15. 16.

Tissue: Normal or cancerous human esophageal mucosa explants. Sterile scissors. Sterile forceps. 90 mm Sterile Petri dish. 25 cm2 Tissue culture flasks. Incubator, 37”C, humidified atmosphere, 5%/95% CO2 in air. Macroscopic or microscopic grid: Either a grid is drawn on transparent paper or the flasks/Petri dishes are bought with grids on their surfaces. Eagle’s Balanced Salt Solution (EBSS). Eagle’s Basal Medium containing L-glutamme (EBM). Trypsin/collagenase solution: 0.25% trypsin and 10 mg/mL collagenase type IV in EBSS. Complete growth medium: EBM supplemented to give final concentration of: 20% fetal calf serum (FCS) 0.1 pg/mL hydrocortisone, 10 U/mL insulin, 20 U/r& penicillin, 20 pg/mL streptomycin, 1 ug/mL fungizone, 4 U/mL gentamicin sulfate, 50 U/n& mycostatin. Fixative: Make up a solution of formalm m 10% formaldehyde solution Test compounds: Test compounds should be dissolved in culture medium if water soluble. If insoluble in water, use DMSO or ethanol. Prepare a concentrated stock solution and dilute by at least 1: 100 in culture medium prior to addition to cells. Hematoxylm. Eosin. Periodic Acid Schiff Reagent (PAS).

3. Methods 3.1. Tissue

Preparation

The most common source of tissue is that available when patients are undergoing surgery for esophageal tumor removal. 1. Fix 2 mm3 samples of tissues for histological examination using\ 10 mL formalin at 20°C. Place the main sections (not fixed) into complete growth medium at 20°C (room temperature).

Mothersill

78 3.2. Culture

Technique

1. Rapidly chop the tissue into -2-3 mm2 pieces using sterile scrssors and forceps. 2. Place all the pieces m 10 mL of the trypsin/collagenase mixture at room temperature and incubate for 30 mm at 37°C. 3. Place the tissue segments m a 90 mm sterrle Petrt dish and select appropriate pieces for culture (nonfibrous, nonfatty, and of a uniform size). 4. Place each piece of explant into a separate 25 cm2 tissue culture flask containing 2 mL growth medium at room temperature. Maneuver the explant to the middle of the flask and carefully transfer to an incubator (37OC, 5%/ 95% CO, m au) without dislodging it. 5. Leave for 2 d after plating before commencmg with testmg, to ensure that the explant is attached and the mmal mrgratlon of cells has started.

3.3. Exposure

to Test Compounds

1, Discard any flasks that are heavily contaminated with frbroblasts or show no discernible migration of cells. 2. Establish the base line counts of cell numbers at rusk (usmg the method in Section 3.4.5., but without fixation and staining of the culture). 3. Set up 5 flasks/test concentration and controls. 4. Add 0.05-o. 1 mL of test compound, or vehicle, to each flask and return to the mcubator. 5. After the required period of exposure, remove the cultures and assess cell growth. 6 Remove the compound after the exposure period and replace wrth fresh medium. 7. Incubate for 2 wk.

3.4. Area

Measurement

1. Drain the flask of medium and rinse the culture wtth BSS at 20°C. 2 Add 5 mL of acetic acid, methanol, or formalin to fix the cultures at 20°C. 3. Leave the cultures for 15 mm m the case of acetic acid/methanol or 24 h for formalin. 4. Add 5 mL of either hematoxylin and eosm or Periodic Acid Schtff Reagent (H and E standard concentrations for hrstologlcal sections). 5. Measure the area of the flask covered by cells using a macroscopic or mrcroscoprc grid, depending on the size of the explant culture. Exclude the size of the initial explant and any frbroblastic areas from the calculation (fibroblastrc areas are vlsuahzed as fibrous, densely stained areas). 6. Express the results as a percentage of the control area The results can be converted to cell numbers by countmg the number of cells m a representative number of grid squares (normally 25% of total outgrowth).

79

Human Esophageal Culture 4. Notes

1. No more than 1 h should elapse between removal of the sample and plating out in culture flasks. 2. The medmm should not be changed at any stage, since thus leads to the death of eprthehal cells and prohferation of frbroblasts. 3. The exposure period will depend on the half life of the compound/drug but should be in the range of l-24 h. 4. PAS ISused to illustrate secretory activity, for example, in adenocarcmomas.

References 1. Mothersill, C., Cusack, A., MacDonnell, M., Hennessy, T. P., and Seymour, C. B. (1988) Differential response of normal and tumour oesophageal explant cultures to radiation. Acta Oncologica 27,275-280 2. Mothersill, C., Cusack, A., and Seymour, C. B. (1989) Enhanced proliferation of cells from human tissue explants following Irradiation m the presence of envrronmental carcinogens Radiat. Enwon Biophys. 28,203-212.

CHAPTER11 The Application of In Vitro Models of Anterior Pituitary Function in Toxicity Testing Glenda E. Gillies and Julia

C. Buckingkam

1. Introduction Together, the hypothalamus and the pituitary gland form the functional unit called the neuroendocrine system. For the purposes of introducing this system, therefore, the two will be considered together, although the methods for assessing their function will be considered separately (the pituitary gland in this chapter and the hypothalamus in Chapter 12). The hormones of the anterior pituitary gland, or adenohypophysis, namely growth hormone (GH), thyroid stimulating hormone (TSH), the gonadotrophins (luteinizing hormone, LH; follicle stimulating hormone, FSH), prolactin (Prl), and the pro-opiomelanocortin (POMC) family of peptides (adrenocorticotrophic hormone, ACTH; P-lipotrophin, P-LPH; P-endorphin; and N-terminal POMC peptides) play a key role in the maintenance of homeostasis. Their secretion is controlled by neurohormones (neuropeptides and dopamine), which are secreted by hypothalamic neurons mto the hypophyseal portal vessels and thereby conveyed directly to the anterior pituitary gland. The activity of the hypothalamopituitary axis is tightly regulated by a variety of mechanisms, including ascending and descending neural inputs to the hypothalamus, local regulatory mechanisms operating within the hypothalamus and pituitary gland, and blood-borne factors acting at the level of both the hypothalamus and pituitary gland (for review, see ref. I). The major secretory prodFrom. Methods m Molecular B/ology, Edited by S O’Hare and C K Atterw~ll

Vol. 43. In V/fro Toxmfy Testrng Protocols Copyright

81

Humana

Press

Inc , Totowa,

NJ

82

Gillies

and Buckingham

ucts of the posterior pituitary gland, or neurohypophysis, vasopressin, and oxytocin, will not be addressed in this chapter. Disturbances in hypothalamo-pituitary function result invariably in a spectrum of disorders of growth, physical and mental development, reproductive function, metabolism, and osmotic balance, many of which may not become apparent until some time after the original insult, by which time irreversible damage may have already occurred. In addition, the ability of the individual to respond appropriately to and/or cope with acute or long-term stress may be impaired with consequent insidious effects on both physical and mental health. Clinically, such conditions may be precipitated by pathological lesions (2) or by drugs and other xenobiotic agents, including environmental pollutants (I). Thus, there is growing awareness within the discipline of toxicology of the importance of experimental models for assessing hypothalamic and pituitary functional responsesover extended periods of time, and that simple measurements of endocrine organ weight or histological examination, which were once thought to be sufficient, may no longer be an adequate reflection of these parameters. Investigations of drug action on the hypothalamo-pituitary system in vivo are complicated by the inaccessibility and sensitivity of the tissues to experimental manipulation. Thus, although gross changes in neuroendocrine function induced by acute or repeated exposure to drugs may be readily assessedindirectly in vivo, by measuring changes in blood concentrations of the hormones produced by the pituitary gland or their peripheral target organs (e.g., the steroids of the adrenal cortex or gonads; thyroid hormones), such studies provide only limited insight as to the mode of action of the active agents or the point in the axis (hypothalamus, pituitary, or peripheral endocrine organ) at which they may act (1). These difficulties have prompted the development of a variety of in vitro models, which unlike their in vivo counterparts, permit direct examination of drug action within the anterior pituitary gland and hypothalamus at the cellular and molecular level. This and Chapter 12 describe the methodology of a number of in vitro preparations that may be used to mvestigate drug action on the anterior pituitary gland and the hypothalamus, drawing attention to their various advantages and limitations. Models of posterior pituitary function in vitro are less widely used and will not be covered here.

In Vitro Models

in Toxicity

Testing

83

It should be noted that all the methods described here involving the use of animal tissue may be subject to governmental approval and that appropriate permission should be obtained before the work is undertaken. 1.1. Basic Principles

of In Vitro Models

The techniques used to maintain adenohypophysial and hypothalamic tissue in vitro fall into two broad categories, short- and long-term. With respect to the anterior pituitary gland, short-term preparations (minuteshours) utilize tissue segments or enzymatically dispersed cells maintained in static or dynamic conditions, whereas long-term preparations (days-weeks) include primary cultures of mixed or “purified” cells and cell lines (e.g., ACTH producing AtT20 cells or growth hormone/prolactin, producing GH, cells). Short-term hypothalamic preparations include isolated nerve endings (synaptosomes) and whole hypothalami as well as tissue slices and fragments, whereas the most important long-term preparations are organotypic cultures derived from hypothalamic fragments and dissociated cell cultures. In all cases, the function of the tissue is assessedby measuring the output of the hormone under investigation, using, in most instances, biological or immunological assays. In designing studies and interpreting data several important points must be taken into account. For short-term studies, it is critical that the tissue should be derived from animals of the same strain, age, weight, sex, and, if female, stage of the estrous/menstrual cycle (which in the rat normally spans a 4-d period). In addition, the diet, housing, lighting, and handling regimes prior to autopsy should be tightly controlled, since these too may influence the subsequent activity of hypothalamic and pituitary tissue in vitro. Ideally, such stringent criteria should also be applied to tissue collection for long-term study. The viability of the tissue in vitro is obviously of paramount importance and, since small changes in the physicochemical environment (e.g., pH, ionic balance, O2 tension, and endotoxin content of commercially available media) may influence the level of cellular activity, the incubation conditions should be rigorously monitored. Finally, medium containing the pituitary or hypothalamic secretions and tissue samples should be stored in appropriate conditions (snap frozen and kept at -20°C for peptides or -70°C for dopamine) prior to stringent analysis by well validated, specific, precise methods. Individual drug treatments may be tailored as required and will

84

Gillies and Buckingham

depend on whether acute or long-term effects are to be investigated. In principle, the animals may be exposed to the drug under consideration in vivo and the respective glands removed and their functional activity assessedin vitro; alternatively, the tissues may be removed from naive animals and exposed to the drug in vitro. Ideally, responses to a range of doses/concentrations of drugs should be examined in two or more models. Particular care should be taken to include suitable vehicle controls, because in many instances, solvents (e.g., ethanol, polyethylene glycol, dimethyl sulfoxide) have been shown to exert marked effects on the basal and neurochemically evoked secretory activity of hypothalamic and pituitary tissue in vitro. 1.2. Advantages and Limitations of In Vitro Models of Anterior Pituitary

Function

The static incubation system utilizing pituitary segments has several advantages. First, the tissue retains its three dimensional structure and thus the cell-cell interactions are representative of those in vivo. Second, the system is highly precise and therefore lends itself to quantitative analysis of drug action. Third, it is simple and inexpensive to perform, requiring little specialized equipment. Its main disadvantage is that tissue viability may be limited by poor diffusion of nutrients into and of metabolites out of the tissue, and experiments should therefore not be continued for longer than 3 h. Tissue viability may be improved by the use of a perifusion system in which pituitary segments from 3-4 rats are incubated in a single chamber (3). Such preparations may provide valuable information about the dynamics of pituitary hormone release. They also permit detailed examination of the rate of onset, intensity, and duration of acute responsesto drugs and are particularly valuable in dynamic studies on drugs, such as steroids, that may exert biphasic actions. However, the potential of perifused tissue to exhibit pronounced variations in the magnitude of the secretory responses to repeated stimulation (3-5) limits their use in quantitive pharmacological studiesinvolving, for example, assessment of agonist/antagonist potency. Models utilizing cells dispersed enzymatically prior to incubation do not suffer from diffusion problems and cell viability is generally good. However, production of a single cell suspension inevitably destroys cellcell contacts that may be important physiologically, and reducesthe opportunity for transmission of autocrine and paracrine influences, which

In Vitro Models

in Toxicity

Testing

85

growing evidence suggests are present within the intact gland. Furthermore, when maintained in static incubation conditions, dispersed cells are often relatively insensitive to physiological secretagogs (6). Nevertheless, the static system described above, in which aliquots of cells derived from a single pool are used, readily lends itself to quantitative pharmacology, since inherent variation is minimal and the responses to a wide range of drug treatments (duration of contact/concentration) and/or secretagogs may be examined in parallel. In contrast to the static incubations, responsesfrom the perifused anterior pituitary cell column are virtually immediate, and this system provides a means whereby nutrient supply is continuously replenished and has rapid accessto all cells while metabolites are readily removed. These features undoubtedly contribute to the fact that this preparation is robust and the most sensitive of all the in vitro preparations described here (7). It is also particularly well-suited to the study of the dynamics of the secretory response. Because the pituitary gland contains large stores of hormones (unlike the hypothalamus), the perifused cells respond to repeated stimulation for periods of up to 8 h (in the region of 50 stimuli), so that several variables may be tested in replicate in one experiment. Since all the responses are produced by a single pool of cells, biological variation is minimized and standard errors are relatively small. However, because one stimulus could possibly influence the magnitude of a subsequent response, it is crucial to randomize doses with at least four replicates per dose. It is also important to realize that, although a single experiment may yield many data, the results are representative of only a single pool of cells, and that repetition of the experiment is essential for comprehensive statistical analysis. This point is also relevant to dispersed cells in static incubation. A criticism often leveled at the use of single cell suspensions is the possibility that the enzymes used for cell dispersal may perturb receptor structure. However, our studies, which substituted finely chopped anterior pituitary gland tissue for the enzyme dispersed cells in a perifusion system, show that responses to a variety of stimuli and combinations of stimuli are unaffected, except for a significant time lag in responsiveness, which we attributed to the altered diffusion characteristics. Cultured pituitary cells (primary culture or cell lines) permit examination of not only the acute but also the more long-term effects of drugs on pituitary hormone secretion in tightly controlled conditions. In many

Gillies

86 I Anterior segment

pttuitary (I/, or’/,

and Buckingham

gland cl ) per well

Lid of multiwell mul~iwell dish with holes bored in it Plastic tubing sealed glue Into lhe holes 1OOpm pore nylon

with mesh

>

FlnOnQ

nunYnG

f

Base portion of multiwell dash

I

/ lml of incubation medium

Incubate

at 37 ‘C asdescribed

in the text

Fig. 1. Adaptation of multiwell tissue culture plates, as described in the text, permits easy, rapid transfer of the pituitary gland segments to fresh medium without the need to handle the segmentsthemselves.

respects the advantages and limitations of cultured cells resemble those of static incubates of acutely dispersed cells. In addition, the cells may undergo some degree of (de)differentiation, and thus their functional activity may not be directly comparable with that of either freshly removed tissue or of the cells in vivo. Before utilizing cultured cells in toxicological studies it is therefore advisable to verify the integrity of the secretory system to be investigated, for example, by comparison with a well established acute in vitro model. 1. 2. 3. 4. 5. 6. 7. 8. 9.

2. Materials For dissection: guillotine, forceps (fine, straight, and bent watchmakers forceps are ideal). Multiwell tissue culture plates adapted as in Fig. 1. Sealed perspex box. 95% 02/5% cop 37°C incubator. Cell dispersal apparatus as described in Frg. 2. Collagenase or trypsin. 37OCwater bath. 37°C shaking water bath.

In Vitro Models in Toxicity Testing Teflon pulley and stirring paddle

Teflon bearing Polythene vial __)

b Tissue fragments

+c&

0 *

--J

d 4 d

f-------

D

I

h b

b

b . .

c

Trypsin solution

3.2 cm-

Fig. 2. Cell dispersal apparatus, 10. 11. 12. 13.

Bio-Gel P2,200-400 mesh. Perifuston apparatus as described in Frg. 3. Media and buffers: Earle’s balanced salt solution (EBSS) (see Table 1). Incubation medium for anterior pituitary segments dispersed cells and perifused cell column. Bovine serum albumin (0.25% w/v) Benzyl penicillin (100 IU/mL,) Streptomycin ( 100 pg/mL) Aprotonin (100 Kalhkrein inactivator U/mL) Earle’s balanced salt solution (100 mL) Note: Artificial cerebrospinal fluid (CSF) may also be used for incubation of pituitary tissue (8). Antimicrobtal drugs are not necessary for mcubation of the segments. 14. “Growth” medium for cultured anterior pituitary cells: Charcoal stripped fetal bovine serum (20%) Nonessential amino acids (1%) L-Glutamme (4 mM) Penicillin (100 U/nL)

Gentamycin (10 pg/mL) Dulbecco’s Modified Eagle’s Medium (100 mL)

Gillies

88 WASTE

-

and Buckingham

0 15ml/mr -+-y-l

TEST SUBSTANCES

I

FRACTION -COLLECTOR

Fig. 3. Perifusedisolated rat anterior pituitary cell bioassay. 3. Methods 3.1. Anterior Pituitary Segments For Materials see Section 2., numbers l-5.

(8,9)

1. Remove the anterior pituitary glands postmortem from 20-30 rats (agematched and of the same strain and sex) that have been handled regularly and housed for at least 1 wk before the experiment in a temperature- and lightcontrolled room (21-23”C, lights on 7 a.m.-7 p.m.) with food and water available ad libitum. Decapitatethe rats, remove the dorsal surface of the skull, lift the frontal lobes, cut the optic nerves, and reflect the whole brain to expose the prtuitary gland. Dissect away the overlymg diaphragma sellae, remove the gland, and discard the centrally positioned neurointermediate lobe. 2. Divide the anterior lobe into 4-6 pieces (dependent on the age and weight of the rat) of approx 1 mm3 size (0.8-1.0 mg wet wt). 3. Pipet an equal volume (1 .O n-L) of the (prewarmed) incubatron medium (see Section 2., number 12b), pH 7.4, into each well of a multiwell tissue culture plate (Fig. 1). Place one segment in each well and then transfer the plates into a sealed perspex box connected to a 95% O&5% CO, gas sup-

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Table 1 Earle’s Balanced Salt Solution (EBSS) Concentration,

mg/lOO mL Elevated K+

Basal K+ Constituent NaCl KC1 CaCl, . 2Hz0 MgS04. 7Hz0 NaH2P04 - 2Hz0 NaHC03 D-Glucose

56&K+ 680 40 36 20 15.8 220 100

14mA4K+ 615 104 36 20 15.8 220 100

28 mMK+ 534 209 36 20 15.8 220 100

56mMK+ 370 417 36 20 15.8 220 100

aNaCl is reduced as KC1 increases m order to maintain isotomcity. Ca2+/Mg2+free 56 mM K” as above, excluding Ca2+Cl, s 2H,O and Mg2+S0, s 7H,O Ca2+ chelatmg agent (e g , EGTA, 2 mM) may also be added to “Ca2+ free” medium

ply. Saturate atmosphere with 95% 0,/5% CO2 gas (5 min). Incubate for 2.5 h at 37”C, replacing the medium and stopper gas mletjoutlet and replenishmg 95% 02/5% CO2 atmosphere after 2 h and 2 h 15 min. Note: These incubations may also be performed m an incubator at 37’C in 5% CO,/ 95% air and 100% humidity. 4. Transfer the segments to fresh medium containing the test substancesand/ or appropriate secretagogs (e.g., releasing hormones) or, in the case of controls, an equal volume (1 .OmL) of medium alone or appropriately diluted vehicle, and then incubate in 95% 02/5% CO* saturated conditions as described above for a further 30 min. Note: If required, test substances/ vehicle may also be included m the premcubatlon period. 5. Collect aliquots (0.2 mL) of medium m plastic tubes, snap freeze, and store at -2OOC for hormone assay, or assay immediately. Weigh the pituitary tissue, chill rapidly (-7O”C), and store for subsequent biochemical or histological analysis. Results are normally expressed per mg tissue. 3.2. Dispersed Anterior Pituitary Cells in Static Incubation 3.2.1. General Method for Dispersal The method for cell dispersal is essentially common to all models, although many modifications may be found in the literature. In some cases these variations may depend on personal preference rather than scientific arguments.

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1. Collect anterior pituitary glands postmortem as described above, cut each lobe into approx 10 pieces with a fresh scalpel blade, and transfer the combined fragments of 5-8 anterior pituitary glands to a dispersal apparatus (Fig. 2) containing 10 mL of Earle’s balanced salt solution supplemented with collagenase (0.1% w/v) or trypsin (0.25% w/v) over which watersaturated 95% 02/5% CO2 gas is bubbled continuously. 2. Place the vial in a water bath maintained at 37°C and incubate for 30 min, agitating constantly by rotating the paddle at approx 200 rpm; aid dispersal with periodic gentle repetitive pipetmg using a clean 1 mL plastic pipet tip or a fire polished, siliconized glass Pasteur pipet. 3. Allow the prtmtary pieces to settle and pour the supernatant fluid mto a plastic tube and store at room temperature. Add a further 10 mL aliquot of enzyme containing medium to the pituitary fragments and repeat the dispersal procedure two more times until all the tissue is dispersed. 4. Centrifuge at 1OOgfor 10 min to recover the cells. Discard the supernatant fluids and resuspend each cell pellet in the medium (10 mL) employed for the subsequent incubation (see Section 2., number 12). 5. Repeat the centrifugation procedure and resuspend the cells from each harvest in medium (1 mL), pool, and filter through 100 pm pore nylon gauze (previously washed in sterile saline) to remove any remaining connective tissue or clumps of cells. 6. Take an aliquot (10 pL) of the cell suspension to determine the cell count. Verify cell viability at this stage using the trypan blue exclusion test (make a 1: 1 mix of cell suspension with 0.4% trypan blue solution m 0.9% NaCl, wait 5 min, and calculate the percentage of cells that do not take up the dye using a hemocytometer). Viability is usually >90% and should not be ~85%. 3.2.2. Static Incubation of Dispersed Anterior Pituitary Cells (6) 1. Dilute the cells to a known concentration, normally 3.3 x 105-5.0 x lo5 cells/ml in incubation medium (see Section 2., number 12), and transfer aliquots of the cell suspension (0.6 mL) to plastic tubes. 2. Incubate for 2 h at 37OCin a shaking water bath, gassing the medium continuously with water saturated 95% 02/5% C02. 3. Centrifuge the cell suspensions (lOOg, 10 min, 4”C), discard the supernatant fluids, and resuspend the cell pellets either in medium (0.6 mL) contaming appropriate drugs and/or secretagogs or, m the case of controls, an equal volume of medium alone or appropriately diluted vehicle. Incubate the cell suspensions for a further 60 min under the conditions described above. 4. Centrifuge the cells (lOOg, 10 min, 4”C), collect the supernatant fluid, snap freeze, and store at -20°C for hormone assay.

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3.

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3.2.3. For Preparation of the Perifused Anterior Pituitary Cell Column (7) Mix the filtered cell suspension (approx 1 mL) with 0.5 g Bio-Gel P2, 200-400 mesh, which had been previously swollen overnight in 0.9% NaCl, containing antibiotics, and then washed twice with the medium (see Section 2., number 12b). Draw the slurry into a column constructed from a plastic disposable syrmge (Fig. 3) (7), connect to a variable speed peristaltic pump, clamp vertically, and perifuse at a rate of 1 mL/min with medium containing trypsin lima bean inhibitor (0.1% w/v) if trypsin was used for dispersal. When the cells and Bio-Gel mixture have packed down to a volume of 1.5 mL (2-3 min), disconnect the column from the pump, and, taking care that the gel does not dry out, reduce the dead volume to 0.1-O-2 mL and recommence perifuslon with medium at a rate of 0.5 mL/min. Clamp the column vertically in a water bath at 37°C and perifuse for 1.5-2 h to allow hormone secretion to reach a steady baseline level. Connect the column outlet to a fraction collector and collect the eluate as 2-min fractions (Fig. 3). When 2-4 min pulses of hypothalamic regulatory factors, diluted in the perifusion buffer, are passed through the column a response occurs almost immediately and secretion rapidly returns to baseline such that a subsequent stimulus may be given 10 min later (7). Fractions containing the column effluent may either be assayed immediately or snap frozen and stored at -20°C.

3.3. Cultured

Anterior

Pituitary

Cells (10)

Use materials listed in Section 2., numbers 1,4, and 6-9. In addition, at all stages use sterile buffers, media, and plastics and perform the relevant manipulations in a sterile atmosphere provided by an appropriate microbiological safety cabinet. 3.3.1. Culture Preparation 1. Collect anterior pituitary glands postmortem from 9-15 rats, as described earlier, but under aseptic conditions. 2. Pool the tissue and wash several times in sterile Ca2+/Mg2+ free Earle’s balanced salt solution until all the blood has been removed. Place in a Petri dish and chop into cubes (1 mm3) using a sterile scalpel blade. 3. Transfer the pituitary fragments to a plastic vial containing 5 mL Ca2+/ Mg2+ free Earle’s balanced salt solution containing 25% (v/v) pancreatin 4X and 10% (v/v) collagenase (stored as 0.1% [w/v] in Tris buffered CaCl,), and incubate in a water bath at 37°C for 20-30 mm. At the end of

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Gillies and Buckingham this period, aid dispersion by gentle repetitive pipetmg. Allow the pituitary fragments to settle and transfer the supernatant fluid to a plastic tube containing 5 mL unstripped fetal bovine serum, stopper, and store at room temperature. Add a further 5 mL Ca2+/Mg2+free Earle’s balanced salt solution containing pancreatin 4X and collagenase to the remammg tissue and repeat the procedure. Collect four such harvests and centrifuge (lOOg, 5-10 min). Discard the supernatant fluids and resuspend each cell pellet in 5 mL “growth medium” (see Section 2., number 12~). Pool the cell suspensions and centrifuge (lOOg, 5-10 min). Discard the supernatant fluid and resuspend the cell pellet in 10 mL growth medium. Repeat the centrifugation, again suspending the resulting cell pellet in 10 mL growth medium. Take an ahquot (10 p,L) to determine the cell count and to verify cell viability using the trypan blue exclusion test as described earlier. Dilute the cells in the growth medium to a concentration of lo5 cells/ml. Add 200 PL aliquots of this cell suspension to each well of a 96-well culture plate and incubate m sterile conditions at 37°C in a 5% C02/95% air atmosphere for 72 h or until the cells grow to confluence.

3.3.2. Assessment of Function 1. Decant the growth medium and wash the cultured cells twice with incubation medium (see Section 2., number 12~). 2. Add a further 200 p.L medium to each well and incubate at 37°C for 1 h in an atmosphere saturated with 5% CO,/95% air. Decant the medium and replace with an equal volume (200 pL) containing appropriate drugs and/ or secretagogs, or m the case of controls, an equal volume of medium alone or appropriate diluted vehicle. 3. Incubate at 37°C in 5% CO,/95% an for 2 h. 4. Remove the supernatant fluid from each well and store at -20°C for assay. 4. Notes 1. All buffers, incubation media, and culture media are to be used prewarmed to 37°C. Media and additives are readily obtained from major suppliers. Unless indicated, additives are most conveniently stored at -20°C for up to 3 mo in 0. l-l .OmL vol aliquots at concentrations that, when added to 100 mL of medium, produce the desired concentration. For cell culture, solutions containing additives that are not originally sterile (e.g., powdered additives) need to be filter sterilized through 0.2 km pore disks that fit on to sterile disposable plastic syringes (commercially available). The osmolality of all media should be within the range 290-300 mosM/L.

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2. In order to obtain a high index of precision m the anterior pituitary seg3. 4. 5. 6.

ment model we recommend that n = 5 for each treatment and thus rt is feasible for a single operator to assess approx 16 variables/experiment. Owing to diffusion problems, static pituitary segment incubations should not be continued for more than 3 h. For static incubations of dispersed cells, relatively long periods of contact with secretagog (>l h) may be required before a response is seen. When using perrfused isolated cells, a response may be seen within mmutes. When cutting anterior pituitary glands into fragments use a clean, sterile scalpel blade wrth a smooth, not a dragging, motion to make the cut.

References 1. Buckingham, J C and Gillies, G. E. (1992) Hypothalamus and pituitary gland. xenobiotic induced toxicity and models for its investrgatton, in Endocrine Toxicology (Flack, J. D and Atterwrll, C., eds.), Cambridge University Press, pp 83-114 2. Besser, G M and Cudworth, A G. (eds.) (1987) Clinical Endocrinology Chapman and Hall, London. 3. Busbridge, N. J , Chamberlain, G. V. P , Grrffiths, A., and Whitehead, S. A. (1990) Non-steroidal follicular factors attenuate the self-prrmmg action of gonadotrophin releasing hormone on the pmutary gonadotroph. Neuroendocrinology 51,493-499. 4. Buckingham, J C and Cover, P. 0. (1986) Changes in the responsiveness of perifused rat adenohypophyseal cells to luteinizing hormone releasing hormone. Acta Endocrinol.

(Copenhag.)

113,479-486.

5. Cover, P 0 and Buckingham, J. C. (1989) Effects of estradiol and tamoxifen on GnRH self priming m perifused rat adenohypophysial cells. Acta Endocnnol. 121, 365-373 6. Cowell, A.-M., Flower, R. J., and Buckmgham, J. C. (1991) Studies on the roles of phospholipase A2 and elcosonotds in the regulation of corticotrophin secretion by rat pituitary cells m vitro. J. Endocr. 130,21-32.

7. Gilhes, G. E. and Lowry, P. J. (1978) Perfused rat isolated anterior pituitary cell column as bioassay for factor(s) controllmg release of adrenocortlcotropin:

valida-

tion of a technique. Endocrinology 103,521-527. 8. Buckingham, J. C. and Hodges, J. R. (1977) The use of corticotrophin production by adenohypophysial tissue in vitro for the detection and estimation of potential corticotrophin releasing factors. J. Endocr. 72, 187-193.

9. Hadley, A. D., Flack, J. D., and Buckingham,

J. C. (1992) Effects of selective

phosphodlesterase mhlbttors on the release of ACTH and LH from rat anterior pituitary segments in vitro. Pharmacol. Commun. 3,283-295. 10. Stone, M A., Carey, F , Cowell, A -M , and Buckingham, J C (1989) Etcosanoids

and piturtary function: a role for phospholipase AZ in the stimulation from cultured pituitary cells. Br. J. Pharmacol. 96, 162P.

of ACTH

CHAPTER12

The Application of In Vitro Models of Hypothalamic Function in Toxicity Testing GZenda E. GiZZies and Julia

C. Buckingham

1. Introduction The reader is referred to the previous chapter in which the background and rationale for monitoring hypothalamic function in vitro is discussed. 1.1. Advantages and Limitations of In Vitro Models of Hypothalamic Function Like other in vitro systems, the isolated whole hypothalamus permits the examination of hormone release in a precisely controlled physical and chemical environment. It also has the advantage that it largely retains the three-dimensional relationships between neurons and non-neuronal supporting cells and, thus, it is approximately representative of the prior state in vivo. Several workers have expressed concern about the viability of a tissue block of the size employed. This is an important consideration since, according to Lumsden (I), 1 mm3 is the maximum volume of brain tissue that will permit adequate diffusion of nutrients and metabolites to and from the center of the tissue and thereby maintain cellular integrity. Cells located near the edge of the larger explants retain their morphological and biosynthetic characteristics in vitro, but those at the center may undergo necrosis. The viability studies of Bradbury et al. (2) showed that the O2 consumption of the isolated hypothalamus is linear over a 3 h period (range 68.9-120 w/g/h, n = 5). Similar data have been reported by Berelowitz et al. (3), who also observed raised O2 consumption in the From Methods m Molecular B/ology, Edlted by: S O’Hare and C. K. Atterwlll

Vol. 43: In V/fro Toxmty Testmg Protocols Copyrtght Humana Press Inc , Totowa, NJ

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presence of an elevated K+ concentration (178 * 45 p,M/glh, mean + SE, n = 3). Examination histologically of the tissue after a 3 h incubation revealed signs of deterioration but, despite a variable degree of perineuronal edema, there was no indication of neuronal death. It thus appears that the tissue viability is limited. If one allows a 60 mm pre-incubation period to minimize leakage from severedneuronal surfaces and to enable the tissue to recover from the trauma of excision, the preparation is suitable only for short-term experimental studies involving at the most two successive stimulations. Nevertheless, there are reports of more prolonged incubations and one group (4) has successfully maintained the explants for more than 24 h. Thus, the isolated whole hypothalmus provides an invaluable tool with which to examine the physiology, pharmacology, and biochemistry of the hypophysiotrophic neurons. It is currently employed in this and other laboratories in studies on the maturation and agmg of hypothalamic neurons, the second messenger systems effecting peptide/transmitter release, the modes of action of steroids and other neuroactive drugs, and the interactions between the immune and neuroendocrine systems. As mentioned earlier, the problems of diffusion and viability may be reduced by using small tissue pieces and/or perifusion systems. Inevitably, the improvements obtained with tissue fragments are at the expense of maintaining cellular integrity and the three-dimensional structure, which limits the usefulness of the models. Perhaps the most effective compromise 1sthe bissected organ (5) in which the improvements in diffusion are coupled with preservation of a high degree of cellular integrity together with the normal intrinsic network and supporting glia, and so forth. This technique has been successfully exploited in several laboratories. Superfusion procedures appear to improve viability, and hrstological studies indicate that there is little deterioration for periods of up to 4-5 h. Theoretically, therefore, such preparations may be expected to lend themselves to repetitive stimulation. However, in practice they frequently exhibit marked tachyphylaxis and, since very small quantities of neuropeptides are released, it is often necessary to concentrate/lyophylize the eluate fractions in order to make reliable measurements, even though as many as 12 hypothalami may be used in each perifusion chamber. In our hands, therefore, we feel that the disadvantages of the perifusion technique outweigh its advantages.

In Vitro Models The advantages of using hypothalamic cells in culture include the virtual removal of diffusion problems and the provision of an opportunity to study cellular responses directly under controlled conditions (especially when using defined medium) without interference from the many homeostatic mechanisms that operate through the hypothalamus. This model, therefore, allows one to investigate repeated responses over many weeks in vitro and also the effects of prior treatments (acute vs chronic) on subsequent responses in a manner that cannot be achieved in vivo or in other in vitro experiments. Because fetal or early neonatal tissue has to be used, hypothalamic cultures are particularly useful for the study of factors that influence the development of the neuroendocrine system. It should be noted, however, that the development of the cells may proceed in a distinctly different manner, depending on the culture medium used. In particular, many cultures are grown in the presence of serum that, although promoting growth and survival, is a variable, undefined, and unphysiological cocktail of bioactive substances that do not normally come into contact with brain cells. Thus, we have shown that the responsiveness of hypothalamic peptidergic neurons becomes distinctly suppressed when grown in a serum-supplemented medium compared with those grown in a defined, serum-free medium and that the cell types that survive in each of these media are distinctly different (6-8). In fetal/early neonatal tissue the levels of certain neuropeptides may be low (e.g., corticotrophin-releasing hormone and vasopressin) and this could therefore, present a problem if detection systems are not sufficiently sensitive. Another potential disadvantage of using isolated hypothalamic cells is the loss of the normal cytoarchitecture found in VWO, but studies using cortical cultures suggest that a certain degree of histotypical reorgamzation occurs in vitro. Generally, however, cultured hypothalamic cells behave in many ways, as would be expected in vivo. They exhibit both a morphological and functional maturation (6-12) and they also exhibit electrical activity and synaptogenesis as well as Ca2+ dependence of their secretory activity. In context, therefore, they offer certain advantages over other systems. However, like all experimental models, this preparation should not be used in isolation, but results should be compared with other in vitro models as well as ultimately being verified in vivo.

Gillies

1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12. 13.

14.

15.

and Buckingham

2. Materials Guillotme, scissors, and forceps for dissection, Shaking water bath. 95% o,/co,. Anesthetizing box. Sterile culture facilities (aseptic working area, autoclave, sterilizing oven, humidified CO, incubator, Class II microbiologtcal safety cabinet, sterile plastics). Inverted microscope. Buffers and media. Incubation medium for hypothalamic tissue (artificial cerebrospinal fluid [CSFJ): 126 mM NaCl, 6 mM KCl, 1mM NaJ-IPQ, 0.877 mM MgSO,7H,O, 22 mMNaHCOs, 1.45 n&f CaC12,and 200 mg/% glucose. When madeup in distilled and deionized water, osmolarity should be 298 mosM/L. Sterile saline: 100 IU/mL pencillm, 100 ug/mL streptomycin, and 100 mL sterile saline (0.9% NaCl). Collection buffer: 0.01% bovine serum albumin (BSA Fraction V), 100 IU/mL pencillin, 100 pg/mL streptomycm, 20 mJ4 HEPES, and 100 mL Hanks’ Balanced Salt solution (HBSS, Ca2+/Mg2+free). Enzyme solution: 0.04% BSA, 100 IU/mL pencillin, 100 pg/mL streptomycin, 0.2 mg/mL Dispase I, 0.5 mg/mL DNase I, 20 rnil4 HEPES, and 40 mL HBSS (Ca2+/Mg2+free). DNase solution: 0.01% BSA, 100 IU/mL pencillin, 100 pg/rnL streptomycin, 20 mA4HEPES, 1 mg/mL DNase, and 20 mL HBSS. Serum supplemented culture medium: 10 mL heat inactivated fetal calf serum (FCS), 10 mL heat inactivated donor horse serum (HS), 100 IU/mL penicillin, 100 pg/mL streptomycin, 0.5 pg/mL fungizone, and 100 mL Dulbecco’s Modified Eagle’s Medium (DMEM). Defined medium: 50 mL DMEM, 50 mL Ham’s F12 nutrient medium, 100 IU/mL penicillin, 100 pg/mL streptomycin, 0.5 pg/mL fungizone, 10 n&f HEPES, 5 pg/mL insulin, 100 pg/mL transferrin, 2 x lo-*M progesterone, 3 x lo-*M selenmm, lOAM putrescme, 10-12M P-estradiol, and 10e9Mtriodothyronme. Release medium: 30 pg/mL ascorbic acid, 30 pg/mL bacitracin, 0.01% w/v bovme serum albumin, 0.1% w/v glucose, 10 n&f HEPES buffer, 100 IU/mL penicillin, 100 ug/mL streptomycin, 80 KIU/mL trasylol, and 100 mL EBSS.

2.9. Siliconization of Pasteur Pipets 1. Wash 230 mm unplugged glass Pasteur pipets m distilled water for 1 h in a gas chromatography Jar, rinse in methanol, and dry at 100°C in an oven.

In Vitro Models 2. When cool, rinse the pipets individually in the repel coat, dimethyldichlorosilane solution, in a 100 mL measuring cyclinder for approx 10 s and dry at 100°C before the final distilled water wash (1 h), followed by a brief rinse in methanol and final drying in the oven. 3. When cooled again, fire polish the Pasteur tips in a Bunsen flame to give tips of varying diameters with no rough edges. With non-absorbent cotton wool, pack into a metal box and heat sterilize at 180°C for 2 h. Once opened, sterile Pasteur storage boxes are kept in the microbio-

logical safety cabinet. 3. Methods 3.1. Short Term Incubation of Hypothalamic Fragments (13,14) Use materials in Section 2., items l-3. 1. Collect hypothalami post mortem from rats (age-matched and of the same strain and sex) that have been handled regularly and housed for at least a week before the experiment in a temperature- and light-controlled room (21-23”C, lights on 7 AM-~ PM)with food and water available ad libitum. Remove the dorsal area of the skull immediately after decapitation and lift the frontal lobes gently. Cut the exposed optic nerves and reflect the whole brain. Dissect out the hypothalamus using fine scissors;the tissue block taken is bordered rostrally by the optic chiasma, laterally by the hypothalamic fissures, caudally by the mammillary bodies, and dorsally by the ventral surface of the thalamus. 2. Lift the tissue carefully handling only by the cut ends of the optic nerves and transfer to a plastic incubation vial containing 1.0 mL of medium (Section 2.1.), pH 7.4., pre-warmed to 37OC and pregassed with watersaturated 95% 02/5% C02. 3. Incubate for 1 h at 37°C in a shaking water bath in an atmosphere saturated with 95% 0,/5% COZ. Replace the medium after 30 min. 4. Transfer the hypothalami either to medium (1 .OmL) containing the test substance and/or appropriate secretagogs or to a corresponding volume of medium alone or appropriate diluted vehicle (controls). Incubate for a further 30 min in the conditions described above, Collect medium, freeze, and store at -70°C for assay of peptides and/or neurotransmitter substances.Freeze the tissue in liquid Nz or on solid CO2 and store (-70°C) for subsequent biochemical studies or histological examination.

If the actions of a drug with a slow onset is to be investigated (e.g., steroids), the compound may also be included during earlier incubation periods.

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3.2. Modifications Utilizing Hypothalamic Fragments andfor Perifision Systems The viability of hypothalamic tissue in vitro may be prolonged for more long-term studies (up to 12 h) by utilizing smaller tissue pieces and/or perfusion systems. Thus, the hypothalamic blocks (removed as described above) may be bisected by a mid-sagittal incision, quartered by perpendicular mid-sagittal and coronal cuts, or sliced (200-250 pm) in the sagittal plane. Alternatively, a smaller total hypothalamic mass, for example the medial basal hypothalamus or median eminence, may be employed, the dissections of which are described in detail elsewhere (15,16). In addition, perifusion techniques may be applied to whole hypothalami (17,18) and to hypothalamic fragments (15,19-22) or slices (23,24) in a manner essentially similar to that described above for pituitary tissue. 3.3. Primary Cultures of Dissociated Hypothalamic Cells (6,7) Use materials in Section 2., items 1-6. All procedures described below involve aseptic techniques. All dissection instruments are either heat sterilized (large scissors, forceps, and so forth) or autoclaved (fine forceps, scissors, and so forth that have autoclavable protective plastic tips, and so on) before use. The polythene cell dispersal pot (as described in Fig, 2 of Chapter 11) is sterilized by soaking in 70% ethanol for 24 h with thorough rinsing with sterile saline before use. The Teflon head of the dispersal apparatus is autoclaved before use. Cells and sterile medium are handled only in the microbiological safety cabinet with sterile pipets. 3.3.1. Tissue Dissection 1. Removal of fetal rats from the pregnant mothers IS carried out m a nonsterile, clean area outside the trssueculture laboratory. Place the timed 18 d pregnant rats, one at a time, in an anesthetizing box. After inhalation of ether vapor, dams rapidly become anesthetized and removal of fetuses is performed under contmued anesthesia (after whrch the mothers are killed by methods as approved by the Home Office). 2. Soak the abdomen with 70% ethanol and make a large rmd-lme incision. Hold back the skm and abdommal walls wrth Spencer Wells clasps, ensuring that the fur does not touch the uterus.

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3. Remove the uterus containing the fetuses and place it in a 70-mm bacteriological sterile plastic Petri dish containing sterile saline supplemented with antibiotics (Section 2.2.). 4. Transfer the uterus to the tissue culture laboratory and remove the fetuses one by one from the uterus. 5, Decapitate the fetuses and place the heads in a sterile Petri dish containing a piece of filter paper, moistened with sterile saline supplemented with antibiotics to provide a non-slip surface for the subsequent dissection. 6. While securing the head with forceps in the eye sockets, make one midline cut through the dorsal fetal skull, ease out the whole brain, and invert it through 180°C so that the hypothalmus is visible. 7. Remove the hypothalami using fine watch-makers’ forceps to take a “pinch” of tissue that includes the “hypophysiotrophic area” bordered by the hypothalamic sulci laterally, the mamillary bodies caudally, and the optic chiasm rostrally. The area removed, therefore, includes the secretory nuclei known to contain the hypothalamic factors that control anterior pituitary hormone secretion and weighs approx 1 mg (wet weight). 8. Place the dissected hypothalami into a sterile Petri dish containing the collection buffer (Section 2.3.) that has been warmed previously to 37°C in an incubator. If more than five pregnant rats are used, a second Petri dish containing warmed and gassed collection buffer is used for subsequent collection of hypothalami while the first dish is kept in a humidified CO2 incubator at 37°C. With practice, up to 100 hypothalami (approx 10 mothers) may be collected by two workers in 80 min. 3.3.2. Cell Dispersal From this stage onward all procedures take place in the sterile environment provided by a microbiological safety cabinet. 9. Transfer all hypothalami to a 30 mL plastic sterile Universal vessel using an automatic pipet fitted with a sterile disposable pipet and add fresh collection buffer. 10. Gently pellet the tissue at 800 rpm for 3 min and then resuspend in 15 mL of previously warmed (37°C) enzyme solution (Section 3.4.) and transfer to the sterile dispersal pot (Fig. 2). 11. Place the dispersal apparatus into a clean water bath at 37OCcontaining fresh tap water and Roccal disinfectant. The contents of the dispersal apparatus are gently gassedwith 5% COZ: 95% O2 and mechanically agitated by the paddle of the apparatus, driven by a motor, rotating at 100 rpm for 30 min at 37°C.

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12. Remove the supernatant containing the dtspersed cells mto a Universal container and store at 37OC. 13. Add 2 mL. of fresh enzyme solution to the tissue fragments and gently triturate 10 times with a fire polished siliconized sterile Pasteur pipet with a tip bore diameter of approx 1 mm. Add a further 13 mL of enzyme solution and reconnect the dispersal apparatus to the motor for a further 15-20 min at 37°C. 14. Collect the supernatant as above. Any tissue fragments remaining after this second dispersal procedure can be gently trtturated with siliconized Pasteurs of decreasing tip bore diameters until all tissue appears to be dispersed. 15. Pool andcentrifuge dispersed cells at 1100 rpm for 8 min in a 30 mL Universal container; resuspendthe pellet in 2 mL of DNase solution (Section 2.5.). 16. Gently disperse the cell pellet using a wide diameter Pasteur pipet and then make up the volume of the DNase solution to 20 mL. This suspension is then allowed to stand at 37°C in the CO2 Incubator for 15 min. The aim of this step is to remove nucleic acid released from damaged cells because it would make the subsequent cell pellets very “sticky” and diffrcult to redisperse. 17. Centrifuge at 1100 rpm for 8 min and resuspend the pelleted cells in 2 mL of collection buffer previously kept at 37°C. 18. Gently layer the cell suspension onto 20 mL of collection buffer (Section 2.3.) containing an extra 4% w/v BSA and centrifuge at 1100 rpm for 8 min. 19. Resuspend the pelleted cells in normal collection buffer (Section 2.3.) and estimate the total number of cells and cell viabihty (trypan blue exclusion test as described m the prevrous chapter) using a hemocytometer. 20. Centrifuge at 1100 rpm for 8 min and resuspend in an appropriate volume of either serum supplemented or defined culture medium (Sections 2.6. and 2.7.) to yield 2.5 x lo6 cells/mL. 21. While continually gently swirling the cell suspension transfer 1 mL to 35-mm diameter sterile, plastic tissueculture dishes,swirling each dish gently to ensure even distribution of cells. All 35 mm dishes have been previously coated with filter sterilized poly-L-lysine (10 pg/mL) for 30 min at room temperature, followed by two rinses with HBSS before incubating at 37°C for 2-3 h with serum supplemented culture medium until ready for use. 22. Maintain the cells in an incubator at 37°C in 5% CO*, 95% air, and 100% hurmdity. 23. Change the medium after 4 d, decreasing the sera concentrations, if used, to 5%. Thereafter the medium ISchanged every 4 d or after experimentation.

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3.3.3. Functional Assessment Studies on the release of peptides and neurotransmitters (GABA and dopamine) that influence anterior pituitary hormone secretion will be described here, Release under basal or control conditions is compared to the release in the presence of the substance(s) under investigation. 24. Remove the growth medium and rinse the cells (on/off) with 1 ml/dish of basal release medium (Section 2.8.) prewarmed to 37OC.The cells are then equilibrated for 15 mm at 37°C again with release medium before the final timed mcubation. 25. For basal release, 500 PL of release medium is added to each culture dish for the required period of time. A further control should be included if the drug to be studied requires dilution m a vehicle other than the release medium. For stimulated peptide release, release medium containing 56 mM K+ (with Na+ concentration reduced to maintain isotonicity, as described m the previous chapter) or various concentrations of the substance(s) under investigation are added m 500 lrL vol to the dishes and incubated at 37OCfor the required period of time. 26a. If peptides are to be measured, proceed as follows: At the end of the experimental epoch, collect the medium from each dish into plastic tubes containing 50 pg Polypep and HCl(50 mL of l.OM). The samples are then heated to 70°C for 5 min to destroy peptidase activity and centrifuged at 11OOgfor 5 min to remove any precipitate or cell debris. Providing sensitive immunoassays are available, the neuropeptides may be measured directly in the release medium, which does not interfere significantly in the majority of assays(although this should always be checked). However, it is also possible to concentrate samplesfor pepttde detection (6,7) (see Note 1). b. If GABA is to be measured, proceed as follows: Using HPLC with electrochemical detection, GABA can be monitored after derivatization with o-phthaldehyde 2 min prior to sample injection in order to produce electrochemically detectable reactants (9). Incubation medium may be stored at -20°C so that measurements can be done at a later date. c. If dopamine is to be measured, proceed as follows: Collect the incubation medium onto a small volume of perchloric acid (PCA) to give a final concentration of 0.M. Flash freeze and maintain at -70°C for storage. Before measurement by HPLC with electrochemical detection, return the sample to pH 8.5 with Tris-EDTA buffer (pH 8.6, 1.5it4) and load onto alumina cartrrdges. Elute the catecholamines with 200 mL PCA (0.M) and inject onto the HPLC column (9). (N. B. This procedure is not as easy as it sounds, but further discussion is beyond the scope of thts chapter.)

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and Buckingham

Notes 1. Make up fresh each month. 2. Store as lyophyhzed powder. 3. Ethanol is required for the initial dissolution of a few milligrams. Subsequent dilutions to prepare stock solutions may be done in an aqueous vehicle (0.9% NaCl or EBSS) and hrgh drlution ensures that the vehicle has no effect on the cultures. 4. Concentration of samples for peptrde detection (6). To detect all three peptides (CRF-41, AVP, and SRIF), a concentration of medium 1s required using silica columns (C, and Cs) prepared in 1 mL disposable plastic syringes. The bottom of each column 1s sealed with porous Teflon disks cut from a sheet using a No. 2 cork borer. Silica is poured into the syringe to a depth of approx 6 mm and sealed on top with another Teflon disk. Newly prepared columns are washed with 5 x 1 mL methanol, pushed through the column usmg a 1 mL plasttc syringe plunger with a tip resistant to solvent corrosion. Columns are then washed with 5 x 1 mL 10 mM HsPO,, followed by 2 x 1 mL 80% acetomtrile/20% 10 mM H3P04 before presaturation of nonspecific bmding sites of the silica with polypep (1 mg/mL in 10 mM HsPO,). Samples are loaded first onto the C4 column and eluted directly onto the Cs column since no one resin gives optimal recovery for all 3 peptides. Peptides are eluted with 80% CH$ = N/20% H3P04 (10 mill), which is then evaporated to dryness under vacuum. Samples may be stored at -20°C prior to reconstitution m radtoimmunoassay buffer and measurement. 5. In general, hypothalamic trssue is more delicate and requrres more careful handling than pituitary tissue. 6. Although an “arttfictal CSF” has been recommended as the mcubation medium for hypothalamtc tissue (2), Earle’s balanced salt solution may also be used (26). These media may either be purchased or made up m deionized water immediately prior to collection of the hypothalamic tissue. The media may be supplemented with protease mhrbitors (e.g., aprotinin, 0.5%), reducing agents (e.g., ascorbic acid 10M3m, and albumm (e.g., bovine serum albumin, 0.25%) to minimize degradation or adsorptton of the released hormones. However, the necessity for such additives is disputed because, although the hypothalamus is rich in proteolytic enzymes (27), Berelowitz et al. (3) reported good recovery of somatostatin from supplement-free medium, possibly because the enzymes released mto the medium are inactive or effectively diluted out by the relatively large incubation volume. Drugs may be Included in the medium during the initial and/or final mcubation period. They are normally diluted in medium immediately prior to use and pH adjusted to 7.4

105

In Vitro Models

7. When making up media, dissolve all salts except CaC12in deiomzed water at room temperature: bubble water saturated 95% 0,/S% CO* gas through the solution for 5-10 min before adding CaCl, and, subsequently, glucose (this procedure prevents the CaCIZ from precipitating out). Check pH (7.4) and warm to 37°C while continuing to pass the 95% 02/5% CO2 gas through the solution. (N. B. The K+ concentration of this solution IS approximately twice that of mammalian CSF; this improves the viability of the isolated hypothalamus preparation 121). 8. When using acute hypothalamic incubations, we recommend that n = 5 per treatment group for reliable statistics. This enables a single operator to analyze up to 5 variables per experiment. 9. When usmg hypothalamic cultures, for reliable statistics we recommend that it = 4 per treatment and, since 28 wells may be plated for every 10 mothers used, 7 variables may be tested on any given day and retested at 48 h intervals over 4-6 wk in vitro. However great care must be taken with randomization of the cultures between treatments and each result should be repeated several times on different batches of cells. 10. As well as measurmg neurotransmitter/neuromodulator release or content, hypothalamic cultures may also be used to investigate the cellular localization of the substances under mvestlgation using immunocytochemistry (8,9) and we are currently investigating the use of in situ hybridization methods to identify their cells of synthesis. References 1. Lumsden, C L. (1986) Nervous tissue in culture, in Structure and Function of Nervous Tissue (Bourne, G H , ed.), Academic, New York, pp 67-140 2. Bradbury, M W. B., Burden, J , Hillouse, E W., and Jones, M. T (1974) Stimulation electrically and by acetylchohne of the rat hypothalamus in vitro. J. Physiol. 239,269-283. 3. Berlowitz, M., Kronheim, S., Pimstone, B , and Sheppard, M. (1978) Potassmmstimulated calcium-dependent release of lmmunoreactlve somatostatin from incubated rat hypothalamus. J. Neurochem. 3, 1537-1539 4 Calogero, A E , Gallucci, W. T , Bernardini, R., Saoutis, C , Gold, P W., and Chrousos, G. P (1988) Effect of cholinergic agonists and antagonists on rat-hypothalamic corticoctrophm-releasing hormone secretion in vitro. Neuroendocrinology47,303-308. 5 Maeda, K and Frohman, L A. (1980) Release of somatostatin and thyrotropmreleasing hormone from rat hypothalamic fragments in vitro. Endocrinology 106, 1837-1842. 6 Clarke, M J O., Lowry, P J , and Gdlies, G. E. (1987) Assessment of corticotropinreleasing factor, vasopressin and somatostatin secretion by fetal hypothalamic neurons in culture Neuroendocrinology 46,147-154.

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7 Clarke, M. .I. 0. and Gilhes, G. E. (1988) Comparrson of peptide release from fetal rat hypothalamic neurones cultured in defined media and serum-containmg media. J. Endocrinol.

116,349-3X

8. Davidson, K. and Gillies, G E (1993) Neuronal vs ghal somatostatin in the hypothalamus: a cell culture study of the ontogenesis of cellular locatron, content and release. Brain Res. 624,15-84 9 Murray, H. E. and Gilles, G. E. (1993) Investigation of the ontogenetic patterns of rat hypothalamic dopammergic neurone morphology and function in vitro J Endocnnol.

139,4031114.

10. Murray, H. E. and Gillies, G. E. (1992) Maturation of morphologrcally distinct subsets of dopaminergic neurons in primary cultures of dissociated hypothalamic cells. J. Endocrinol 132, Abstract 198 11. Davidson, K. and Gillies, G. E. (1992) GABAergic influences on somatostatm secretion from hypothalamic neurons cultured m defined medium. Neuroendocrznology 55,248-256

12. Gillies, G. E , Cover, P O., Loxley, H. D , and Buckmgham, J C. (1992) Evidence for a hypothalamic involvement durmg the stress hyporesponsiveness period in the rat using in vivo and in vitro models. (manuscript submitted). 13. Buckingham, J. C. and Hodges, J. R. (1977) Production of corticotrophin releasing factor by the isolated hypothalamus of the rat. J. Physiol. (Lond.) 272,469-479. 14 Loxley, H. D., Cowell, A.-M., Flower, R. J , and Buckingham, J C (1992) Modulation of the hypothalamic-pituitary-adrenocortical response to cytokmes m the rat by Lipocortin-1 and glucocorticoids: a role for lipocortin-1 in the feedback inhibiton of CRF41 release Neuroendocrinology 57,801-814. 15 Terry, L. C., Rorstad, 0. P., and Martin, J. B. (1980) The release of biologically and rmmunologically reactive somatostatin from perifused hypothalamus fragments. Endocrinology 107,794-800. 16. Negro-Vilar, A., Ojeda, S. R., Arimura, A., and McCann, S. M. (1978) Dopamme and norepine-phrine stimulate somatostatm release by median emmence fragments in vitro. Life Sci. 23, 1493-1498. 17. Kim, K. and Ramirez, V. D. (1982) In vitro progesterone stimulates the release of luteinizmg hormone-releasing hormone from superfused hypothalamic tissue from ovariectomized estradiol-primed prepubertal rats. Endocrinology 111,750-756 18. Redei, E., Branch, B. J., Gholami, S., Lin, E. Y. R., and Taylor, A. N. (1988) Effects of ethanol on CRF release in vitro. Endocrinology 123,2736-2743. 19. Gallardo, E. and Ramirez, V D. (1977) A method for superfusion of rat hypothalami: secretion of lutemizing hormone releasing hormone (LH-RH) (39749). Proc. Sot. Exp. Biol. Med.

155,79-84.

20. Drouva, S. V., Epelbaum, I., Heri, M , Tapia-Arancibia, L., Laplante, E , and Kordon, C. (1981) Ionic channels involved m the LHRH and SRIF release from rat medrobasal hypothalamus. Neuroendocinology 32, 155-162. 21. Shimatsu, A., Kato, Y., Matsushita, N , Katakami, H , Yanaihara, N , and Imura, H. (1982) Effects of glucagon, neurotensisn and vasoactive intestmal polypeptrde on somatostatin release from perrfused rat hypothalamus Endocrmology 110, 2113-2117.

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22. Gillies, G. E., Puri, A., Hodgkison, S., and Lowry, P. J. (1984) Involvement of rat corticotrophin-releasing factor-41-released peptide and vasopressin in adrenocorticotrophin-releasing activity from super-fused rat hypothalamic in vitro. J. Endocrinol.

103,25-29

23. Kelly, M. J., Condon, T. P , Levine, J. E., and Ronnekleiv, 0. K. (1985) Combined electro-physiological, immunocytochemical and peptide release measurements in the hypothalamic slice. Brain Res. 345,2&I-270 24. Nikolarakis, K. E., Almeida, 0. F. X., and Herz, A. (1986) Corticotropm-releasing factor (CRF) ‘inhibits gonadotropin releasing hormone (GnRH) release from superfused rat hypothalami in vitro. Brain Res. 377,388-390. 25. Gillies, G. E., Anderson, R., Davidson, K., and Cross, A. (1992) Release of endogenous GABA from primary cultures of drssociated hypothalamic neurons. J Endoc 132, Abstract 220. 26. Leposavic, G., Dashwood, M. R., Ginsberg, J., and Buckingham, J. C. (1990) Peripubertal changes in the nature of the GnRH response to alpha-adrenoceptor stimulation in vitro and their modulation by testosterone Neuroendocrinology 52, 82-89.

27. Griffiths, E C., Jeffcoate, S. L., and Holland, D. T (1977) Inactivation of somatostatin by peptidases in different areas of the rat brain. Acta Endocrinol. (Copenhagen) 85, l-10.

CHAPTER13 The FRAME Cytotoxicity (Kenacid Blue) Richard

Test

H. Clothier

1. Introduction The cytotoxic effect of chemicals on cells in culture is measured by the change in total cell protein (Kenacid Blue R dye binding method). Healthy 3T3-Ll cells (an established cell-line, ATCC CCL92. l), when maintained in culture continuously divide and multiply over time, The

basis of this test is that a cytotoxic chemical (regardless of site or mechanism of action) will interfere with this process and, thus, result in a reduction of the growth rate as reflected by cell number. The degree of inhibition of growth, related to the concentration of the test compound, provides an indication of toxicity. 3T3-Ll Cells are maintained in culture and exposed to test compounds over a range of concentrations. The cultures are visually examined after 24, 48, and 72 h, and the number of viable cells and/or the total cell protein content are determined, after either 24 or 72 h exposure, by the

Kenacid Blue method. This assay may be performed on cells previously used for the Neutral Red uptake assay. The number of cells in the presenceof test chemicals is compared with that observed in control cultures and the percent inhibition of growth calculated. The IDSa, ID,,, and IDso concentrations (i.e., the concentrations producing 20, 50, and 80% inhibition of growth) are determined and expressed as jtg/mL or n-H. These values enable a comparison of the relative cytotoxicity of the test compounds. From Methods m Molecular Biology, Vol. 43 In V/fro Toxioty Testrng Protocols Edlted by* S O’Hare and C K Atterw~ll CopyrIght Humana Press Inc , Totowa, NJ

109

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Clothier

The maintenance and culture of a cell line, such as 3T3-Ll cells, is a relatively simple and inexpensive technique. The application of such cultures to determine cytotoxicity enables the rapid, highly reproducible testing of many chemicals on a routine basis. There are certain limitations of the technique, some of which concern the character of the compounds to be tested: Volatile chemicals tend to evaporate under the conditions of the test, thus the ID,, value may be variable, especially when the toxicity of the compound is fairly low. This has been overcome to some extent by adapting the procedure for use in 96- rather than 24-well plates (1,2) since the smaller surface area of the well in these dishes reduces the extent of evaporation. Mineral oil can also be used on the medium to reduce evaporation (3). Other chemicals that are difficult to test include those that are unstable or explosive in water. Insoluble substances are also unsuitable for testing, although the author has adapted the method for use with some compounds, using mineral oil as the solvent. Other difficulties are related to the nature of the cell line, i.e., rapidly growing, nondifferentiating cells of very low metabolic activity, hence raising problems of direct extrapolation of results to the in vivo situation. The system is likely to underestimate the toxicity of chemicals that require metabolic activation to a toxic intermediary or product. Substances that specifically attack dividing cells may appear to be of a much higher order of toxicity than they would be in vivo. The toxicity of substances that bind to serum proteins (i.e., such as those found in newborn calf serum) may be also underestimated. 1.1. 24 us 72 h Exposure

Period

The procedure may be adapted to enable determination of cytotoxicity of chemicals after an exposure period of either 24 or 72 h. The authors would stress, however, that they believe the longer exposure period should be used routinely (4). 1.2. Kenacid

Blue

R Dye Binding

Assay

One of the drawbacks of this assay is that the Kenacid Blue dye may, on occasion, precipitate out. The likelihood of this occurring increases as the length of handling time increases, therefore 96-well plates should be agitated regularly and inspected visually for uneven blue color. The process is, however, readily reversed by agitation, so any odd reading should be retested after trituration to obviate the possibility of precipitation.

The FRAME

Cytotoxicity

Test

111

Another problem that may occur is the deposition of a ring of dried protein around the walls of the well, at the air/medium interface. This arises through excessive evaporation or if the culture medium is not properly removed. Such precipitated protein will give an inaccurate assessment of total cellular protein. Total protein measurement does not make allowances for necrotic cells that may still be attached to the culture dish and, therefore, may underestimate the toxicity of a compound. It should be noted, however, that the occurrence of adhering dead or dying cells is very rare. Advantages of this system include: 1, It can be repeatedmore than once on the samecells. 2. Cells can be fixed and the staining performed later. 3. The cell distribution can easily be seenwith the naked eye when stained with the Kenacid Blue before desorbing,thus giving a rapid indication of the successof the assay. Despite the limitations of the system, it provides a simple screen for the rapid assessment of the toxicity of compounds. The current validation of the system is producing results for a wide range of compounds, 150 of which are published (5). It would appear that the correlation to the in vivo situation (especially when problems concerning metabolic activation, and so on, are taken into account) is very good. When the in vitro cytotoxicities, i.e., IDS0 values, of 59 chemicals were compared with rat oral and mouse intraperitoneal LD,, values, correlation coefficients of 0.76 and 0.80, respectively, were obtained (6). The advantages and disadvantages of the Kenacid Blue protein assay are presented above. Under certain conditions a direct comparison of the Kenacid Blue and the Neutral Red methods may be of value. For example, certain chemicals, such as Chloroquine sulfate, and other antimalarial agents that target lysosomes may give different results (4). In such cases, performing both methods will yield extra information. The cytotoxicity test system has undergone a period of in-house development and an investigation into its interlaboratory validation has been performed in a collaborative study involving four different centers. The results of this study are presented in the paper by Knox et al. (I). The authors note that the initial cytotoxicity test employed a human embryonic cell line, BCL-Dl, but now uses a mouse embryonic cell line, 3T3-Ll . At present a number of research groups are evaluating the cur-

112

Clothier

rent procedure and comparing its performance to a variety of other test systems. The test is being used in cooperative schemes to compare different results, including those run by the European Commission, and the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) scheme organized by the Scandinavian Society of Cell Toxicology. These authors have now tested 225 pure chemicals and 100 formula-

tions in the system. The experimental data for 150, together with in vivo comparisons for 59 of these chemicals, is published (3-5). The cytotoxicity test system will be presented in two sections. The first outlines the maintenance of the cell-line and culture procedures, and describes the exposure of the cells to test chemicals. The second section presents the methodologies for the Kenacid Blue assay.

2. Materials of Cell Cultures to Test Compounds Cell line: 3T3-Ll cells, obtainable from the American Tissue Culture Collection (ATCC), code CCL92.1 or the European Collection of Animal Cell Cultures (ECACC) (Porton Down, UK). Incubator: 37”C, humidified, 5% C02/95% an. Tissue culture flasks, 80 cm. 96-Well tissue culture plates. Phosphate buffered saline (PBS), calcium and magnesium free. Prepared from Dulbecco’s formulation. PBS tablets, Trypsin-EDTA: 0.05% w/v trypsin, 0.02% w/v EDTA. Dulbecco’s supplemented medium; composition per liter: 730 mL stenle double distilled water, 100 mL Dulbecco’s minimum essential medmm (x10 concentrate), 100 mL newborn calf serum, heat inactivated. 50 mL Sodium bicarbonate (7.5%) 10 mL L-glutamme (200 mM), 100 mg. Streptomycin sulfate, 100,000 IU benzylpenicillin, UK, 2 mg fungizone. Solvents: Ethanol, methanol, dimethyl sulfoxide (DMSO), and, if appropriate, mmeral oil. All solutions, glassware, and so on, are sterile and all procedures are carried out under aseptic conditions and in the sterile environment of a laminar flow cabinet (biological hazard standard). 2.1.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Exposure

2.2.

Determination

of Cell

Growth

Inhibition

1. Rotatest shaker. 2. Micro ELISA Kontron SLT 210 plate reader: readings taken at 577 nm, reference blank at 404 nm, or Anthos 2001 plate reader: readings taken at 570 nm, reference blank at 404 nm. Note: Filters of 577 and 600 nm may also be used.

The FRAME

Cytotoxicity

Test

113

3. Phosphate buffered saline (PBS), pH 7.4. 4. Fixative: 1% glacial acetic acid, 50% ethanol, 49% distilled water. 5. Kenacid Blue stock solution: 0.4 g Coomassie Brilliant Blue R-250 stain, 250 mL ethanol, 630 mL distilled water. 6. Kenacid Blue stain: 12 mL glacial acetic actd, 88 mL Kenactd Blue stock solution. Prepared immediately prior to use. 7. Washing solution: 10% ethanol, 5% glacial acetic acid, 85% distilled water. 8. Desorbing solution: 98.15 g potassium acetate (lM), 300 mL distilled water, 700 mL ethanol.

3. Methods 3.1. Exposure of Cell Culture to Test Compound 3.1.1. Cell Maintenance and Culture Procedures 1. Stocks of 3T3-Ll cells can be stored in sterile, heat sealable ampules in liquid nitrogen or -8OOC freezer, following suspension in complete medmm containing 5% DMSO as a cryoprotective agent. The cell concentration used is approx 1 x lo6 cells/ml. Cells can be centrifuged for short periods, i.e., 5 min at low speeds (approx 1OOg). 2. When required, the cells should be thawed rapidly in a water bath at 37°C and suspended in sufficient medium in a tissue culture flask to ensure that the DMSO is diluted to yield a final concentration of ~0.1%. Once the cells have attached overnight, the medium should be replaced, i.e., approx 30 mL in a 80 cm tissue culture flask. 3. When the cells approach confluence they should be removed from the flask by trypsinization: Decant the medium and rinse the cultures with PBS at 37OC. Add 1 mL of Trypsin-EDTA. After 2-3 min give the flask 2 or 3 sharp taps to detach the cells into a single suspension (rocking the flask causes the cells to clump). When this occurs (judge by visual examination) add -10 mL of medium (37OC) to prevent enzymatic damage. Gentle trituration may be employed to ensure a single cell suspension is obtained. 4. Count a sample of the cell suspension (using a hemocytometer). Dilute the cell suspension with medium (37OC) to give a final concentration of 1.5-2 x lo4 cells/n& for the 72 h exposure period assay or 4-5 x lo4 cells/ml for the 24 h exposure period assay. 5. Dispense 150 pL of the diluted cell suspension into 95 of the 96 wells of each multiwell plate. Add 150 l.tL of cell-free medium only to the remaining well. Incubate the cells overnight to allow adherence and recovery from the exposure to trypsin.

114

Clothier 3.1.2. Range Finding

Experiment

1. Assess the solubility of each test compound in the following order: Highest possible in medium; 100 + mg/mL m methanol; 100 + mg/mL ethanol; 100 + mg/mL DMSO. 2. A preliminary mvestigatron should be performed for each chemical. Test eight different conditions: a. Control, medium only. b. Solvent control, medium containing 1% solvent (if necessary). c. Stx test chemical concentrations, e.g., 0.01, 0.1, 1.0, 10, 100, 1000 l.tg/ mL or up to the limit of solubility in medium (keeping solvent at 1%). Solutions >lOO mg/mL, or if a substance takes up 10% of the final volume, should be made up volumetrically. Results from wells containing any chemical precipitate should be disregarded. Two chemicals are tested per plate. 3. Prepare 96-well plates as outlmed above. (One row of 3 wells on each plate contains cell-free control medium only.) The outer 36 wells are not used for test chemrcals. 4. After the overnight mcubation aspirate the medium from the cells. Add 150 pL of medium containing the appropriate concentration of test chemical. Incubate for either 24 or 72 h. 5. Visual examination should be performed after 24, 48, and 72 h and an value noted. This enables detection of time-related approximate IDsa changes in cytotoxicity. 6. After the fmal visual examination remove the medium and determine protein content by the Kenacid Blue method, see Section 3.1. The results of this preliminary study enable the concentration range over which O-100% inhibition occurs to be identified. 7. If no toxicrty is found at 1000 pg/mL, another range finding expertment is carried out with a top concentration of 100,000 pg/mL or maximal solubility is obtained, i.e., the concentration at which the chemical begins to precipitate out of solution. 8. At least six concentrations spanmng this range can then be selected for an accurate determination of cytotoxicity.

3.1.3. Accurate Determination

of IDzO, IDsO, and IDS0 Values

1. Each chemical should be tested, which may be performed blind, on three separate occasions (allows for variation in weighing of chemical and plating of cells) and each of the six concentrations of a chemical and controls should be tested in triplicate. It is important that the same range of concentrations be tested on three separate occasions.

The FRAME

Cytotoxicity

115

Test

2. The following conditions should be set up: a. Nonsolvent control, medium only. b. Solvent control, medium + 1% solvent (where necessary). c. At least six compound concentrations. d. Positive controls should also be set up to ensure that the cells behave as expected. It is suggested that 70 p,g/mL dinitrophenol or Tween 20 at 250 pg/mL be employed in this respect. 3. Prepare 96-well plates as outlined above. (All corner wells on each plate contam cell-free control medium only.) After the overnight mcubation aspirate the medium from the cells. Add 150 j-tL of appropriate drug-containing or control medium. It is recommended that drug treatment of the outer wells be avoided. Incubate for either 24 or 72 h. An indrcation of the number of cells present at the initiation of the experiment should be obtained by removing, but not replacing, the medium from three of the wells at the treatment stage. They will then air dry and the protein content can be determined at the end of the incubation period. After 24 or 72 h exposure the Kenacid Blue method may be employed to give an mdication of cell number. 3.2. Determination of Cell Growth Inhibition 3.2.1. The Frame Kenacid Blue R Dye Binding Method (Nottingham Modification)

This method is basedon that of ref. I. The measurementof total cell protein provides a quantitative indication of cell number present in a culture. 3.2.1.1, MODIFICATION

OF ORIGINAL

METHOD

This method of protein determination has been adapted for use in 96-, rather than 24-multiwell plates, hence, certain parameters are different from those given in previously published papers (e.g., 8). Other modifications of the original Kenacid Blue method include: 1. Glutaraldehyde is no longer used as the frxattve because it has a heavy vapor, is not a rapid fixative, and is a penetrative, accumulative toxin. 2. A Kontron or Anthos 2007 plate reader 1snow used, which has a 577 or 570 nm filter read agamst a 404 nm filter (reference readmg). 3.2.2. Method 1. After 24 or 72 h exposure to the chemical, discard medium from cells, Rinse cultures twice wrth warm PBS (-37”C), to remove any remaining medium. Aliquot approx 150 pL of fixative to each well. Shake multiwell plates gently for 20 mm on a Rotatest Shaker. Remove fixative. If the

116

2.

3. 4.

5.

6. 7.

Clothier Kenacid Blue assay is to be performed on cells that have been tested with the Neutral Red method, the destain is removed and the Kenacld Blue assay 1scontinued as below. Prepare Kenacld Blue stain immediately prior to use: Add 12 mL glacial acetic acid to every 88 mL of stock Kenacid Blue R solution. Add 150 p.L of Kenacid Blue stain per well. Shake multiwell plates for a further 2-3 h on the Rotatest shaker. Remove stain. Fill each well with washing solution to remove excess stain. Repeat. Leave for 1 min. Replace this washing solution with fresh washing solution. Shake plates for a further 20 min. Remove washing solution and replace with precisely 150 PL desorbing solution. Rapidly agitate the plates for 20 min until the dye has completely gone mto solution and gives a homogeneous colored solution. Read the absorbance of each well directly at 577 nm using the plate reader with 404 nm reference blank, against the reference well contaming no cells. Set this well at zero. (Note: 570 or 600 nm measurement filters can be used.) The absorbance correlates linearly with cell number over specific optical density ranges 0.6-l .8 at 570 nm without subtraction of a reference filter using 24-well plates. When 96-well plates are employed and the optical density is 570 nm minus the 404 nm reference, the readings should be m the range of 0.4-1.2. The increase in optical density value between the original protein level and the value 72 h later should be 1.8x or more. If either of these conditions are not satisfied, the results should be discarded. The positive control should give a total protein value of 50% of the medium control. (Thirty to seventy percent is acceptable. Outside this range the results should be discarded.) The standard error of the mean for triplicate wells should not be more than 20% of the mean for results to be valid.

3.2.3. Calculation of Results 1. An estimation of the total cell protein (measured using the Kenacid Blue method) is made on each culture dish as outlined above. The results obtained under test conditions are compared to the appropriate control and converted to a percentage value. The six concentrations of each compound tested should span the range of no effect up to 95-100% inhibition of cell growth. 2. The mean result of trlphcates (i.e., 3 wells exposed to the same concentration) are plotted on a graph (usually sigmoidal or exponential in shape) as concentration vs percent inhibition. Precise ID,,, ID,,,, and ID,, values can be calculated from the curve. The concentration of test compound producmg just less than, and that producing Just over, the required level of inhlbi-

The FRAME

Cytotoxicity

Test

tion are joined on the graph by a straight line. The appropriate ID value is then calculated or read directly from the graph at the point the line intersectsthe 20, 50, or 80% inhibition mark. 3. The ID values from three separate runs are averaged to give final concentrations, usually expressed as pg/mL. Chemicals are normally ranked for toxicity using the ID,, value, since this is the section of the curve most likely to be linear. Ranking in terms of IDso mM values is preferred, since this reflects relative toxicity in terms of the same number of molecules to which the cells are exposed.

When linear regression analysis was used to compare in vitro (log ID,,) values and rat and mouse in vivo (log LDSO)acute toxicities, the following correlation coefficients (r values) were obtained: Rat oral/in vitro Mouse ip/in vitro Most toxic (rat or mouse)/in vitro Least toxic (rat or mouse)/in vitro

r r r r

= 0.76 = 0.80 = 0.81 = 0.78

Data on the comparison of the in vitro cytotoxicities and acute in vivo toxicities of 59 chemicals were taken from ref. 6. 4. Notes 1. The concentration m solvent should always be loo-fold greater than that required in the medium. This enables the concentration of solvent present in the medium to be maintained at a constant level, i.e., 1%. It should be noted that 1% ethanol or methanol exert little effect on the cells in culture but that 1% DMSO may cause a reduction in total protein to between 515% compared with solvent-free cultures. (If a larger decrease occurs in response to the solvent alone, the results of that plate should be discarded.) 2. All test chemical solutions should be made up immediately prior to addition to avoid problems of stability and to avoid problems of precipitation of medium proteins. 3. The authors note that they have detected a decrease in cell growth in the wells on the outer perimeter of the plate, therefore they recommend the use of the inner wells only. (That is only two chemicals per plate.) 4. Cells are plated out m 95 of the wells, whereas 1 well is left without cells, but with medium, to act as a reference. This well is set at zero when reading the absorbance. 5. The cells do not stain well if the acetic acid is omitted from the Kenacid blue stain.

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6. If any dark blue rings of Kenacid Blue stain are left on the sides of the wells at the orrgmal atr/medmm interface, carefully wipe these away between washes. This can be drffrcult rf not impossible in 96-well plates so if it occurs the results should be discarded.

Acknowledgments This is based on the INVITTOX protocol 3B, ISSN 0960-2194, INVITTOX, 34 Stoney St., Nottingham, NGl lNB, UK. References 1 Knox, P., Uphill, P. F , Fry, J. R , Benford, D. J., and Balls, M. (1986) The FRAME Multicentre ProJect on in vitro Cytotoxicology. Fed. Chem. Tox. 24,457463. 2 Riddell, R. J., Clothier, R. H , and Balls, M. (1986) An evaluation of three m vitro cytotoxicity assays. Fed. Chem. Tox~ol. 24,469-471 3. Smith, L. M., Clothier, R. H., Hilligde, S., and Balls, M. (1992) Modification of the FRAME Kenacid Blue method for cytotoxmity tests on volatile materials, ATLA 20,230-234. 4 Riddell, R J , Panacer, D S , Wilde, S M., Clothier, R H , and Balls, M (1986) The importance of exposure period and cell type in in vitro cytotoxicity tests ATLA 14,86-92. 5 Clothier, R. H , Hulme, L , Ahmed, A. B., Reeves, H L , Smith, M., and Balls, M. (1988) In vitro cytotoxicity of 150 chemicals to 3T3-Ll cells, assessed by the FRAME Kenacid Blue Method. ATLA 16,84-95. 6. Clothier, R. H., Hulme, L., Smith, M., and Balls, M. (1987) A comparison of the m vitro cytotoxicities and acute in vivo toxicities of 59 chemicals. Mol. Toxic01 1, 571-577. 7. Hulme, L., Reeves, H. L., Clothier, R. H., Smith, M., and Balls, M (1987) An assessment of two alternative methods for predicting the in vivo toxicities of Metallic compounds. Mol. Toxicol. 1,589-596. 8 Balls, M., Riddell, R. J , Horner, S. A, and Clothter, R H (1987) The FRAME approach to the development, validation, and evaluation of in vitro alternative methods, in In Vitro Methods tn Tox~ology - Approaches to Validation (Goldberg, A. M., ed.), Mary Ann Liebert, New York, pp 45-58

CHAPTER14 Allium

Test

Geirid Fiskev% 1. Introduction The Allium test provides a rapid screening procedure for chemicals, pollutants, contaminants, and so on that may represent environmental hazards. Root growth inhibition and adverse effects on chromosomes provide an indication of likely toxicity. The root tip is often the first part of any plant that is likely to come into contact with chemicals and pollutants found in soil and water supplies. Observation of the root tip system of the onion, AZ&m cepa, has shown that this plant is particularly sensitive to the harmful effects of such environmental contaminants. Gross effects can be quantified by measurement of inhibition of growth of the newly developing root system, whereas examination of the chromosomes of the individual cells of the root tip can indicate likely mutagenic effects. Twelve onions (A&m cepa) are prepared by removal of the outer scales and brownish bottom plate and put into test tubes filled with test liquids for 4 d, the liquid being changed every day. A further series of twelve onions are similarly prepared and maintained in pure water to provide a control population. The ten onions that appear to be developing the best in each series are selected for examination. On d 2 one or two root tips from each of 5 onions is prepared for microscopic examination, One hundred mitoses are scored from each of the 5 slides, as is the mitotic index (MI) for 400 cells. On d 4 the root length of each bulb is measured and the series photographed. (A recovery experiment can be performed by changing the medium for 5 of the 10 onions of each test From Methods m Molecular B/ology, EcMed by S O’Hare and C K Atterwlll

Vol 43 In Vitro Toxmty Testmg Protocols Copyright Humana Press Inc , Totowa, NJ

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series to control water after measurement on d 4, replenishing the liquid on d 5 and, finally, measuring the root length and photographing the series on d 6). Toxicity is measured by both macroscopic parameters (e.g., growth inhibition), where the degree of damage is used to assess the toxic status of the chemical tested and microscopic parameters, where the rate of chromosome breakage and damage may be used to predict mutagenesis. Plants are easy to store and handle and are plentiful and inexpensive. In general, the chromosome condition of plant cells is good, thus providing a high standard in control conditions. The Allium test is a relatively rapid, easy test. It is also both highly sensitive and reproducible. It also provides comparable results to a number of other test systems. Both macroscopic and microscopic effects may be observed, and there appearsto be a good correlation between the two. The macroscopic effect (inhibition of root growth) appears to be the most sensitive parameter. This is to be expected since any deleteriouseffect, direct or indirect, is likely to result in inhibition of growth. Microscopic examination allows assessment of chromosome damage and cell division disturbances, thus providing additional information as to the severity or mechanism of the toxic effect, or potential mutagenicity. The root cells possesscertain enzymes, the mixed function oxidases, that are instrumental in the activation of many promutagens to mutagens. This activating system will improve the detection of those chemicals that exert their toxic effect via a reactive metabolite. The system has a wide range of applications, e.g., for testing pure chemicals, drinking water, natural water, industrial waste, and so on, and is useful for evaluating and ranking environmental chemicals with reference to toxicity. The test can also be used to measure the relative toxicities of nonwater soluble compounds, provided they can be dissolved in a suitable solvent and then diluted in water so that the final concentration does not exceed certain limits. In cases such as these, solvent controls must also be incorporated into the test regime. The system operates over a wide pH range (3.5-11.0) without any obvious effects on the growth of the root systems. Thus, moderately acidic/alkali water samples, chemical solutions, and so on can be tested readily without pH correction being necessary. Although the pH itself may not affect the growth of the roots, it should be taken into consideration when assessing the toxicity of compounds

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since, in many cases, the pH can dramatically alter the toxic potential of these, e.g., by changing the state of ionization. Disadvantages of the system concern problems associated with the state of compounds being tested. The influence of pH on compounds present in solution and the resultant change in toxic profiles has already been mentioned. Another problem concerns the presence of insoluble compounds in waterways, industrial effluent, and so on. It is very difficult to look at the biological effect of such complex mixtures in the Allium system since particulate matter may exert indirect harmful effects, such as the prevention of uptake of nutrients. It is therefore recommended that samples such as these are also chemically analyzed. The Allium test is highly sensitive and, as such, positive toxic effects may result for a number of compounds that would not necessarily be deemed harmful when tested in other systems (especially higher organisms, such as fish). Although this may occasionally result in false positives, it also ensures that contaminations will not be overlooked. This is especially important when complex mixtures are to be tested. A positive result in this test system should, therefore, be taken to indicate a potential biological hazard. False negatives, on the other hand, have been shown to rarely occur in either the Allium test or other similar plant tests (I), therefore, any compound tested giving a negative result can be reliably considered nonmutagenic. An extrapolation of results from one test system to another (and eventually to humans) should, however, be based on the results of a battery of tests and with due consideration to the metabolic pathways of the compound tested. The Allium test system was first used in 1938 (2) and 1944 (3) to examine the effect on chromosomes of colchicine. It has received much attention since that time (for review see ref. 4). Certain modifications of the basic test system have been introduced to enable environmental monitoring of various chemicals, and also of complex mixtures such as those present in river water, industrial waste, and so on (5). The major modifications include the use of a series of bulbs (i.e., 10) for each condition tested (thus permitting an EC5, determination), and the immediate exposure of bulbs to test solutions. The old test allowed an initial growth period in pure water until roots reached an appropriate length, (l-2 cm), after which they were exposed to test compounds,

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1.1. Comparison to Other Short-Term Alternative Toxicity Test Systems This test has shown good agreement with results from other test systems, using many different organisms, eukaryotic as well as prokaryotic. Results of such comparisons are summarized below (5-8). 1.1.1. Chinese Hamster

Cell Line V79

In the absence of a metabolic activating system, V79 cells appear less sensitive than the Allium test in response to benzo(a)pyrene. The relative sensitivity is reversed when the cells are incubated in the presence of mixed function oxidases. Despite changes in sensitivities, the overall results between the two systems are comparable. 1.1.2. Human

Lymphocytes

Human lymphocytes seem slightly more sensitive than Allium root tip cells to the effects of organic mercury compound, although the overall ranking of chemicals by toxicity is similar. It should also be noted that when studied microscopically both cell types respond in a similar way (c-mitosis). 1.1.3. Autotrophic

Algae, Heterotrophic Microorganisms, and Activated Sludge

A number of chemicals have been tested in the Allium test and the results compared with those found using 16 different plankton algae (green algae and silicious algae), yeast (Succharomyces cerevisiae), protozoa (Tetruhymena pyriformis), and activated sludge (composed of bacteria, yeast, and protozoa). The tests were all comparable when the rank ordering of chemicals according to toxicity was examined, although differences in sensitivities were apparent. The majority of the algae were more sensitive than the Allium test, whereas the yeast, protozoa, and activated sludge were less sensitive. 1.1.4. Aquatic Animals and Plants A number of aquatic animals (Duphnia magna, Brachydario

rerio-

eggs or spawn, and Microtox test bacteria) and plants (lens and unicellular algae) appear to be less sensitive to certain classes of compounds compared with the Allium test, e.g., fish (Gusterosteus aculeatus). The Allium test, in this case, is probably a better test for environmental screening because of its higher sensitivity. Other animals (e.g., the crus-

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tacean Nitocru spinz’pes)and plants (e.g., lens and unicellular algae) give comparable results to the Allium test. A wide range of metals, industrial pollutants, compounds, and so on have been tested. Water from a variety of sources has also been examined. For examples, see refs. 9-15. 2. Materials 1, Onions: Equal size bulbs (l-5-2.0 cm diameter) of the speciesAZZiumcepa. After harvest, the bulbs should be stored in dry and well aerated conditions for a few months at lo-20°C before use (winter rest). Discard any onions that are dry, moldy, or have started shooting green leaves. 2. Test tubes: 1.5 cm diameter, 10 cm length. 3. Test tube racks. 4. Microscope, camera, slides, coverslips, and coverslip cement (to preserve the slides). 5. Materials: Fixation and maceration/hydrolysis: 9 parts 45% HAc and 1 part 1N HCl; 2% orcein in 45% HAc. a. Nutrient solutron for plant growth: Nutrient Stock Final concentratron Ca(N0,)2 . 4H20 KNOs MgS04. 7H20 KH2P04 Fe-EDTA e3H20 b. Trace elements MnS04 CuCl, NazMo04 ZnSO, H3BO3

l.Omh4

0.1 mM

2.0 mM

0.2 n-&l

l.Omh4 1.0 mlI4

0.1 mM 0.1 r&i

0.2 mM

0.02 mM

3.64pM 0.48 /.tM 0.0078 pA4 0.0042 piI4 3.7 ~.LM

0.364 PM 0.048 yM 0.00078pM 0.00042pM 0.37 pM

A stock solution (as above) should be prepared at tenfold the required concentration. Before use the solution should be diluted with distilled water. (Colchicine may be used for a specific purpose; see Section 4.2.) 6. Test samples: Control-Normal fresh tap water or prepare the nutrient solution for plant growth (see above). The water should be relatively hard,

e.g., Ca + Mg = 50-70 mg/L, with relatively neutral pH. No toxic ions should be present, e.g., copper ions from copper pipes (Cu2+ should not exceed 0.05 mg/L). Do not use distilled or deionized water alone. Insoluble compounds may be dissolved in certain solvents (i.e., ethanol, methanol, or acetone) and diluted to the required concentration in pure water. Final

Fiskesjti solvent concentration must not exceed 1% v/v. Chemicals should be tested initially over a concentration range of 1P2-lOAM, although in certain casesit may also be necessary to test higher or lower concentrations. Test materials may be stored under dry and well aerated conditions at lO-20°C until bulbs from the next harvest become available. All test/control solutions should be brought to room temperature before commencement of the experiment.

3. Methods 3.1. Preparation 0fAllium cepa Remove the outer scales and brownish bottom plate of the bulbs, leaving the root primordia

intact. If a large number of bulbs are being pre-

pared, place the peeled bulbs into fresh pure water to protect the primordia from drying. A number of bulbs in any population will be natu-

rally slow or poor growing (should not exceed 20%). To allow for this, a series of 12 bulbs for each control and test condition should be prepared, the 10 best bulbs being chosen at the appropriate time (d 1 or 2) for examination and continued observation. 3.2. Exposure

to Test Solutions

Remove bulbs from water and place on a soft layer of paper to remove place in test tubes filled with test liquids or control solutions and incubate for 2-4 (or up to 6) d, changing the test

excess water. Immediately

liquid every day. All tests should be performed at a relatively

constant

room temperature of about +2O”C and protected against direct sunlight. Run one control per each set of experiments and, where the chemical

is dissolved in a solvent, a second control series where pure water is supplemented with the relevant concentration of solvent. 3.3. Day 2

On d 2, prepare slides for microscopic examination. One or two root tips is removed from 5 bulbs out of each series of ten. These are then

used to prepare 5 slides in accordance with the standard procedure for orcein staining of squashed material (the method of choice because of its proven high and rapid performance).

Fixation and maceration/hydroly-

sis is a mixture of 9 parts 45% HAc and 1 part 1NHCl at 50°C for 5 min. Squash the material in 2% orcein in 45% HAc on slide and place coverslip over. Place a piece of blotting paper over the coverslip and press down to fix into position. With the other hand, press two fingers from

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Test

left to right over the coverslip to remove superfluous stain. This also helps to arrange the chromosomes in one plane. If coverslip cement is used, the slides may be stored in a refrigerator for 2 mo or more. Examine under a microscope. Score 100 mitoses for each slide; 5 slides for each series. Score the mitotic index for 400 cells on each of 5 slides. 3.4. Microscopic Parameters 1. Mitotic index: the number of dividing cells per 1000 observed cells. 2. Characterization of mitosis: 100 mitoses/slide. Normal metaphase and anaphase. it: Early normal anaphase: A higher number compared with the control indicates lower rate of cell division. Normal metaphase and anaphase. :: Observation of stickiness: This occurrence indicates a highly toxic, usually irreversible effect, probably leading to cell death. e and f. Clastogenic effects (chromosome fragments or bridges): This occurs as a result of chromosome- and chromatid-breaks, and is an indication of mutagenicity. g and h. Vagrant chromosomes (weak c-mitotic effect): This indicates a risk of aneuploidy. i and j. C-mitosis: A relatively weak toxic effect that may be reversible if the occurrence rate is low. It indicates a risk of aneuploidy. k. Other lessfrequently seen aberrations: Multipolar anaphases(weak c-mitotic effect); C-mitosis “with spindle;” “Banded” chromosome (6). 3.5. Day 3 or 4 1. On d 3 or 4, the root length and root appearanceshould be noted. Control roots will usually have reached a length of -5 cm. Root length can be measured in two ways: a. The most accurate method is to measure every root from each bulb requiring the removal of the roots and the termination of the experiment. Those roots that are mechanically bent or damaged are discarded. b. The length of the whole root bundle is measured outside the test tube by a ruler giving one value for each bulb. The second method is preferred because it allows the continuation of the experiment and does not appear to cause any significant reduction in

sensitivity or accuracyof the test (5). 2. Photograph the test series. 3.6. Macroscopic

Parameters

The most important parameter to measure is root length.

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Fiskesjii 3.7. Other

Parameters

Change of color: The root tip as well as the whole plant may change color either with a certain salt (e.g., blue-green from copper sulfate), or the root tips may turn brownish because of toxic effects causing cell death. 3.8. Root Form

1. Normal roots. 2. “Crochet hooks”: Bending of the roots or root tips that may occur especially after treatment with certain metal salts. 3. c-Tumor formation: This may be apparentafter 3-5 d of cultivation after various types of treatments,but is more obvious after a longer period of time and is observedas a swelling of the root tips 4. Broken root tips. 4. Modifications of Basic Procedure 4.1. Recovery Study

On d 4 (after measurement of root length) change the test liquid to control water in 5 of the 10 test tubes. Change the liquid on d 5. Measure root lengths and/or photograph the series on d 6. If the first 4-d treatment was not irreversible, the five onions in pure water will have new roots or roots longer than those in test liquids. 4.2. Colchicine

Treatment

This is performed specifically to study chromosome breaks in more detail; these are sometimes more evident in c-mitotic chromosomes. Add 0.1% colchicine (to one or two extra onions in those test series that are to be studied) l-2 h prior to the preparation of slides. In those cases where environmental monitoring is being performed, colchicine should not be used, because it may mask possible c-mitotic effects induced by the test chemicals themselves. 5. Results

Calculate the mean root length for each onion as a percentage of the control. Plot treatment concentration against root length as a percentage of the control. Calculate the EClO, ECSO,and ECgOfrom the curve (representing the Effect Concentration) causing 10,50, and 90% growth restriction in relation to the control.

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Some sample ECsO values are presented below: Methyl mercury chloride-g.0 x 1c7M. Mercury chloride-3.3 x 1O”M. Copper sulfate-2.7 x lOA. Nickel chloride-l .7 x 10m5A4. Cadmium chloride-3.1 x 10e5M. Beryllium sulfate4.8 x ltiiI4. Aluminum chloride-8 .Ox 1Od4M. Manganese chloride-5.2 x 10m3M. Lithium chloride-2.0 x 10M2M. References 1. Ennever, F K , Andreano, G , and Rosenkranz, H S (1988) The ability of plant genotoxrcity assays to predict carcinogenicity Mutat Res 205,99-105 2. Levan, A. (1938) The effect of colchicine on root mitoses m Alhum. Heredztas 24, 471-486. 3. Ostergren, G (1944) Colchrcme mitosis, chromosome contraction, narcosis and protein cham folding Heredztas 30,429-467. 4. Grant, W. F. (1982) Chromosome aberration assays m Allium. Mutat Res. 99, 273-29 1. 5. Fiskesjo, G (1985) The Allium as a standard m environmental monitoring. Heredztas 102,99-102. 6. FiskesJG, G. (1981) Benzo(a)pyrene and N-methyl-N-nitro-N-nitrosguanidine in the Allium test. Heredztas 95, 155-162. 7. Fiskesjo, G. (1988) The Allium test-an alternative m environmental studies, the relative toxicity of metal ions Mutat. Res 197(2), 243-260 8. Fiskesjo, G. (1993) The Allium test-a potential standard of assessment of environmental toxrcity Environmental Toxicology and Risk Assessment: 2nd Vol., ASTM STP 1216. (Gorsuch, J. W., Dwyer, F. J., Ingersoll, C G., and La Pomt, T W , eds.), American Society for Testing and Materials, Philadelphia, PA. 9 Berggren, D. and Fiskesjo, G (1987) Aluminum toxicity and speciation m soil liquids-experiments with Allzum cepa L. Environ. Tox. Chem. 6,771-779. 10 Fiskesjo, G. (198 1) Allium test on copper in drmkmg water. Vatten 17(3), 232-240 11. Fiskesjo, G., Lassen, C , and Renberg, L. (1981) Chlorophenoxyacetic acids and chlorophenols m the modified Allium test. Chem.-Biol. Znterac. 34,333-344. 12. Fiskesjd, G. (1982) Evaluation of short-term tests for toxicity and mutagenicity with special reference to mercury and selenium. PhD. thesis, Institute of Genetics, University of Lund, Sweden 13 Fiskesjo, G. (1983) Nucleolar dissolution induced by aluminium in root cells of Allium. Physiologzca plantarum 59, 508-5 11 14. Fiskesjo, G. (1985) Allmm test on river water from BraUn and SaxUn before and after closure of a chemical factory Ambio 14(2), 99-103 15 Fiske@, G. (1987) The Alhum test-an alternative ATLA 15,33-35

CHAPTER 15

Measurement of Cell Membrane Toxicity by Means of Z-Deoxy-D-Glucose Erik

Walum and Anna For&y

1. Introduction The membrane theory of toxicity (I) emanates from the fact that the plasma membrane is the first barrier met by a toxic agent on reaching the cell, and was first advanced to explain the toxic effects of heavy metals. The vital regulatory mechanisms inherent in the plasma membrane and its chemical composition make it susceptible to many toxic compounds (2). A toxic substance may cause structural alterations in the membrane by binding to or crosslinking proteins or by dissolving in the lipid matrix and thereby disturbing its order. The formation of functional holes in the membrane through the action of chemicals with detergent properties is common. Structural alterations often lead to specific functional changes, since the function of both integral and peripheral proteins are greatly influenced by their lipid environment. Furthermore, chemicals may induce toxic effects by direct interaction with functional proteins. Membrane leakage tests are frequently used to screen for cytotoxic effects in cultured cells. In one way or another these tests are designed to measure an increased permeability of the plasma membrane, and they are based on the assumption that leakage of intra- or extracellular substances indicates damage to the membrane. Furthermore, the molecular size of the leaked material is often regarded as indicative of the character of membrane damage, in terms of size of the lesions induced by the test compound (3). From. Methods m Molecular B/ology, Edited by- S. O’Hare and C K Attenwll

Vol 43’ In Vitro Toxicity Testing Protocols Copyright Humana Press Inc , Totowa, NJ

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Walum and Forsby

Tritiated 2-deoxy-o-glucose has proved suitable as a tool for determining general cell membrane permeability changes in monolayer cultures (4). As an analog of o-glucose, 2-deoxy-n-glucose is readily taken up into cells via the glucose transport system and it is then phosphorylated by hexokinase. The product, 2-deoxy-D-glucose-&phosphate, accumulates in the cell for several reasons: Hexokinase is not feedbackinhibited, the product is metabolically inert, and the cell membrane permeability for this compound is very low. By terminating the uptake and then measuring the efflux of radioactivity in the presence of a test compound, it is possible to quantify membrane impairment. In a study of membrane lesions in cultured neuroblastoma cells exposed to metal compounds, a membrane toxic concentration (MTC) was defined as the concentration of the tested chemical, giving rise to an increase in the relative efflux from 1.O to 1.2 in 60 min (5). Changes in the kinetics of efflux of tritiated 2-deoxy-o-glucose are well correlated with alterations in the structure of the cell surface as observed in the scanning electron microscope (6). Furthermore, it is possible to detect more subtle changes in the physical properties of the cell membrane by the use of 2-deoxy-o-glucase. This was shown in an investigation, where the influence of benzene and phenol on the activation energies for the efflux were studied in a number of cell lines (7). In a more recent paper, the effect of six sesquiterpenes on neuroblastoma cell membrane permeability is described. Using a quantitative structure-activity relationship analysis, it was possible to show a very close correlation between the chemical characteristics of the terpenesand their ability to alter 2-deoxy-o-glucose-efflux (8). Below, two different procedures are given for the application of 2deoxy-o-glucose in membrane toxicity studies. The first procedure is essentially identical to that recorded by the in vitro toxicology data bank INVITTOX (9) and involves the continuous perfusion of monolayer cultures during determinations (perfusion procedure). The second procedure has recently been published (8) and requires no perfusion (static procedure). 2. Materials

1. 2. 3. 4. 5.

CO2incubator, 37°C. Incubator, 37*C. Water bath, 37OC. Electronic cell counter. Scintillation counter.

Measurement

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Toxicity

131

Fig. 1. Perfusion chamber consisting of a standard culture dish (A) and a perfusion block (B) with inlet (C) and outlet (D) channels. 6. Tissue culture dishes, diameter 60 mm, culture surface 22 cm2. 7. Scintillation vials. 8. Perfusion chamber consisting of the tissue culture dish and a cylindershaped polycarbonate perfusion block (Sweden pat. 8101564-6, Eur pat. 0073773, US pat. 4,530,907; Peterson and Walum, 1983; Walum et al., 1990, see Fig. 1). Viton rubber O-rings. 9. Luer connections. 10. Silicone rubber tubings. 11. Multichannel peristaltic pump. 12. Perfusion buffer reservoirs. 13. Fraction collector. 14. 2-deoxy-o-[l-3H]glucose, aqueous solution, 15a. Phosphate buffered saline (PBS 1): 8.00 g NaCl, 0.20 g KCI, 0.10 g CaC12 * 2H20, 0.10 g MgCl2 * 6H2O, 1.15 g Na2HP0, * 12H20, 0.20 g/L KH2P04, pH 7.0, 1 mL of water. b. PBS 1 containing 0.5 pC!i [3H]2-deoxy-n-glucose/mL. c. PBS 1 supplemented with n-glucose (1 mg/mL) and containing appropriate concentrations of test compound. 16. 1M NaOH. 17. 1MHCI. 18. Scintillation cocktail.

Walum and Forsby

232 3. Methods 3.1. Perfusion

Procedure

1. Four days before an experiment detach cells in stock cultures, pool, and determme cell number using an electronic cell counter. Dilute and plate out cells in 22 cm* tissue culture dishes at a density of 1 x lo6 cells/dish. Maintain cells in a growth-promoting medium at 37OC, in a humidified atmosphere with approprrate amounts of CO,. 2. Remove growth medium from cell cultures and wash twice with 5 mL PBS 1 (37°C). Add immediately thereafter 4 mL of PBS 1 containmg tritiated deoxyglucose to each culture dish. Incubate dishes at 37OC, in a hunndtfied atmosphere without extra COZ, for 2 h. 3. Remove the incubation solution and wash cultures 3 times with 5 mL aliquots of ice-cold PBS 1. Mount culture dishes on perfnsron block (see Fig. 1). 4. The perfusion chamber consists of two parts; the standard tissue culture dish to which the cells are attached, and the perfusion block. The latter part is a cylinder-shaped polycarbonate block that has an outer diameter equal to the inner diameter of the culture dish. The top of the perfusion block has a turned counter-sink determining the volume of the perfusion chamber, Channels, which serve as in- and outlets for the perfusion solution, are placed at the periphery of the counter-smk (see Fig. 1). 5. Connect silicone tubmgs to the m- and outlets of the perfusion blocks, via simple luer connections. Connect inlet tubings to reservoirs containing perfusion solutions (PBS 1 with glucose and toxic substance). Adapt inlet tubings to peristaltic pump and outlet tubings to fraction collector. 6. Insert an O-ring around the top of the perfusion blocks. Press the culture dishes, containing adherent preloaded cells, onto the top of the perfusion blocks. Submerge the perfusion chambers and perfusion solution reservoirs in a water bath at 37°C (see Fig. 2). Start perfusion and continue for 1 h, at a rate of 1 r&/mm. Collect fractions of the perfusate mto scintillation vials at 5-min intervals (vials no. 1-12). 7. After 1 h stop the perfusion and remove buffer from each culture dish. Place that portion of buffer in a scintillation vial (no. 13). Add 1 mL of 1M NaOH solution to each dish and leave at room temperature for 30 min. Transfer the solubilized cell suspension into scmtillation vial no. 13. Wash each dish with 1 mL 1M HCl and transfer the acid into vial no. 13. 8. Add scintillation cocktail to vials containing perfusate fractions or solubilized cells and determine radioactivity as disintegrations per minute (DPM) by liquid scintillation counting. 9. Efflux kinetic calculations (perfusion procedure):

Measurement

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Toxicity

133

Fig. 2. Perfusion apparatus consisting of buffer reservoir (A), peristaltic pump (B), perfusion chamber (C; cf., Fig. l), scintillation vial (D), and fraction collector (E). a. Time dependence: The radioactivity remaining in the cells (calculated by subtracting the cumulative radioactivity released by the cells from the total radioactivity incorporated) is divided by the radioactivity in the cells at the beginning of the experiment (the total radioactivity incorporated) and plotted logarithmically vs time according to Kotyk and Jancek (10). The graph obtained (4) is resolved in its components, and the least-square fit of the lines calculated. Pool sizes, halflives, and rate constants are calculated from the equations of the lines. b. Temperature dependence: Rate constants are plotted vs the reciprocal of the absolute temperature (7) and activation energies determined from the straight lines obtained according to Arrhenius (11). c. Concentration dependence: For determinations of concentrationeffect curves the relative efflux is calculated according to: p, = a-/b, where u is the efflux, a the amount of radioactivity released in 60 mm, and b the total amount of radioactivity incorporated in the cells. The ratios of lt in the presence and absence of the test substance are then plotted vs the concentration of substance. In this graph (5) the membrane toxic concentration (MTC) may be determined as the concentration of the tested chemical giving rise to an increase in p,-ratio from 1.0 to 1.2.

3.2. Static Procedure Materials are the same as those outlined in Section 3.1. However, no perifusion apparatus is involved. 1. The cultures are prepared and the preloadmg of cells 1scarried out as in the perfusion procedure.

Walum

and Forsby

2. When the incubation with trrtiated deoxyglucose IS terminated, 2 mL of 37°C PBS 1 contanung glucose and test substance are added to each culture dish and the cultures incubated at 37°C m a humidified atmosphere without extra COz, for 1 h. 3. One minute after addition of the test substance, 50 pL IS taken from each dish and put into scintillation vials (no. 1). This procedure is then repeated 5, 10, 20, and 30 mm after the start of the incubatron (vials no. 2-5). At 30 min the remammg PBS 1 (1.75 mL) IS collected from the dishes and put into vials (no. 6). 4. The cells are then dissolved in 1 mL 1M NaOH, at room temperature for 30 min. The cell fraction IS transferred to vial no. 7, and the dish rinsed with 1 mL 1M HCl, which is also transferred to vial no. 7. 5. Scmtrllatron cocktail IS added to each 7-set of vials and the radioactivity determined in a liquid scintillation counter as DPM. 6. Efflux kinetics calculations (static procedure): The relative remaining radioactivity m the cells and concentratron dependence in each dish after each time interval (y,) is calculated as: y, = (DPMtot - [(DPM, . v,j50) + (DPMI to DPM, _ r)])/DPMt,r DPMtot is the total radioactivity taken up by the cells, DPM, is the amount of radioactrvrty in the 50 pL fraction taken from the incubation solution at time X, and DPM,- 1is the radioactivity in the fraction taken before that at time x. The expression cell membrane impermeability denotes the resistance of the membrane to radroactrvrty penetration and is calculated as yXin the presence of test substance divided by yX m the absence of test substance and multiplied by 100 to obtain a value as percent of control. This value is then plotted vs concentration of test compound for each time interval used (8). From these graphs values for the concentratrons that give a 5 or 20% increase in permeability (EC5 and EC& can be obtained. References 1. Rothstein,A. (1959) Cell membraneas a site of action of heavy metals. Fed. Proc l&1026-1035.

2. Kinter, B and Pritchard,J. B. (1977) Altered permeabrhtyof cell membranes,rn Handbook ofPhysiology (Lee, D H K , ed ), 9,563-576. 3 Thelestam, M. and Mollby, R. (1976) Cytotoxic effects on the plasma membrane of human diploid fibroblasts-a comparative study of leakage tests Med. Biol. 54, 39-49. 4 Walum, E. and Peterson, A (1982) Tritlated 2-deoxy-o-glucose as a probe for cell membrane permeablhty studies. Anal Bzochem. 120,8-l 1. 5. Walum, E (1982) Membrane lesions in cultured mouse neuroblastoma cells exposed to metal compounds. Toxicology 2567-74

Measurement

of Cell Membrane

Toxicity

135

6. Walum, E. and Marchner, H. (1983) Effects of mercuric chloride on the membrane integrity of cultured cell hnes. Toxic01 Lett. l&89-95. 7. Walum, E (1982) Temperature dependence of membrane permeability in cultured cells exposed to benzene and phenol. Biochem. Biophys. Rex Comm. 108,948-952. 8. Forsby, A., Walum, E., and Sterner, 0. (1992) The effect of six pungent sesquiterpenes with anttfeedant activity on cell membrane permeability in human neuroblastoma SH-SYSY cells. Chem. Biol. Interact. 84,85-95. 9. Warren, M., Atkinson, K., and Steer, S. (1989) Introducing INVITTOX: the ERGATT/FRAME in vitro toxicology data bank. ATLA 16,332-343. 10. Kotyk, A. and JanaZcek, K. (1970) Cell Membrane Transport: Principles and Techniques, Plenum, New York, pp. 240-244. 11 Arrhenms, S. (1915) Quantitative Laws in Biological Chemistry, Bell, London. 12. Peterson, A. and Walum, E. (1987) Growth and morphology of neuronal cell lines cultured in perfusion. In Vitro 19, 875-880. 13 Walum, E. and Jenssen, D. (1990) Understanding Cell Toxicology: Principles and Practice, Ellis Horwood, Chichester, p. 203.

CHAPTER16

MTT Assays Rosa Supino 1. Introduction The MTT calorimetric assay determines the ability of viable cells to convert a soluble tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] (MTT) into an insoluble formazan precipitate. Tetrazolium salts accept electrons from oxidized substrates or appropriate enzymes, such as NADH and NADPH. In particular, MTT is reduced at the ubiquinone and cytochrome b and c sites of the mitochondrial electron transport system and is the result of succinate dehydrogenase activity. This reaction converts the yellow salts to blue-colored formazan crystals that can be dissolved in an organic solvent whose concentration can be spectrophotometrically determined (Fig. 1). Owing to the many advantagesof the assay,it is today considereda significant advance over traditional techniques. In fact, it is rapid, versatile, quantitative, and highly reproducible with a low intratest variation between datapoints (+15% SD); it is useful in a large-scale, antitumor drug-screening program (1-3). Moreover, the test can also be used for floating cells, such as leukemias and small cell lung carcinoma, and always allows sufficient time for cell replication, drug-induced cell death, and loss of enzymatic activity, which generatesthe formazan product from the M’IT substrate(4). The MTT assay has to be set up for each cell line. The optimal cell number seeding (since cells during the assay have to be actively metabolizing), the duration of the experiment, and the time of MTT incubation necessary for an evaluable final optical density need to be selected in preliminary experiments. From: Methods m Molecular Biology, Edlted by: S. O’Hare and C K At&twill

Vol 43: In V/fro ToxrCrty Testing Protocols Copynght Humana Press Inc , Totowa, NJ

137

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Fig. 1. Absorption spectraof MTT formazanreagent(25 ug/mL) in DMSO (A) and 0.04N HCVisopropanol (B). A number of key points are relevant for the choice of the MTT assay. It is valid for a number of cell lines derived from a broad spectrum of solid tumors by a variety of isolation techniques. Most cell lines tested (106/l 11) exhibited acceptable calorimetric profiles (control growth absorbance > 0.500 U). Measurements of cell growth by MTT reduction correlated well with indices of cellular protein and viable cell number. At specific culture conditions and appropriate assay parameters, it provides reproducible indices of drug sensitivity. It is very sensitive since 32 human cells/well give an optical density of 0.05 U (optical density developed by murine cells is lower than that of human cells) (Fig. 2). It is comparable with other in vitro methods of drug cytotoxicity evaluation (Fig. 3).

1. 2. 3. 4. 5.

2. Materials Cells: exponentially growing cell line with recognizable levels of mitochondrial activity. 96-well tissue culture plates,U-bottomed or flat bottomed. Scanning multiwell spectrophotometer. Automatic plate shaker. Incubater, 37OC,humidified, 5% CO2atmosphere.

139

MTT Assays

cell number

(~10~)

Fig. 2. Relationship between cell number and optical density in B16V melanoma cells (0) and N592 small cell lung carcinoma (A). 6. Centrifuge. 7. 50 mL tubes. Make up the following: 8. Drugs made up to solutions 10x more concentrated than required. 9. Hank’s salt solution, composition (in g/L): KC1 (0.4), KH2P04 (O-06), NaCl (8), NaHCO, (0.35), Na2HP04. 7H,O (0.09). 10. MTI’ solution: 5 mg/mL in Hank’s salt solution. Note: MIT solution may be stored at 4°C for about 1 wk. 11. Culture medium, dependent on the cell line. 12. Dimethylsulfoxide (DMSO). 13. RPMI-1640. 14. Fetal calf serum. 15. 0.45 p filters. 16. Agarose. 17. HCl. 18. Isopropanol. 19. Collagenase. 20. Hyaluronidase. 21. Ficoll-Hypaque. 22. Percoll.

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Fig. 3. Clonogenic assay and MTT assay curves: effect of adriamycin, cisplatin, and vinblastine on V79 cells. 0 MTT assay;A Clonogenic assay. 3. Methods 3.1. MTT Method For the chemosensitivity test, exponentially growing cells were harvested, counted, and inoculated (at the appropriate concentrations in a vol of 100 pL) into 96-well microtiter plates; 8 replicates were prepared for each dose. U-bottom microplates were used for suspension-growing cells, whereas flat-bottom microplates were used for plastic-adherent cell cultures. Immediately or 24 h after cell seeding, 10 PL of different dilutions of drugs, prepared 10x more concentrated than requested, was added to each well. After different incubation times at 37°C in a humidified 5% CO, atmosphere, the MTT assay was performed. MTT (Sigma,

MTT Assays St. Louis, MO) was dissolved at a concentration of 5 mg/mL in Hank’s salt solution and filtered with a 0.45 l.~filter (in order to avoid MTT aggregates). Ten microliters of MTT solution was added to each well and also to the control wells without cells. In fact, additional controls consisted of media alone with no cells, with or without the various drugs. After 4-6 h of incubation, microtiter plates were centrifuged at 2000 rpm for 10 min; medium was then removed, and 100 PL of DMSO was added to each well. After thorough mixing with a mechanical plate mixer, absorbance of the wells was read in a scanning well microculture plate reader at test and reference wavelengths of 550 and 620 nm, respectively, that are approximately the peak and the lowest MTT wavelengths of absorption required to avoid quenching from growth medium, in particular phenol red. Absorbance values from all wells were corrected against these control absorbance levels, and the IDS0 was defined as the concentration of drug that produced 50% reduction of absorbance compared with untreated control cells. 1. Harvest, count, and inoculate, in 100 uL of complete medium, the appropriate number of cells. 2. Add 10 pL of drug solution 10x more concentratedthan requested. 3. Incubatefor different times in a humidified atmospherein 5% CO2at 37°C. 4. Add 10 FL of MTT solution (5 mg/mL). 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Incubate 4-6 h in 5% CO2 at 37°C. Centrifuge the plates at 2000 rpm for 10 min. Remove the medium. Add 100 pL of DMSO. Mechanically mix the plate until formazan crystals are dissolved. Read in a microculture plate reader at test and reference wavelengths of 550 and 660 nm, respectively. Calculate the mean of the optical density of different replicates of the same sample. Evaluate the percentage of each value vs the control. Plot in a semilog chart the percentage of optical density against drug concentration. Determine the ID5e from the dose-response curve. 3.2. HTCA-MTT

Method

Different authors have reported that the assay yields results very similar to the clonogenic tumor cell assay in Chinese Hamster Ovary, lung

142

Supino

cancer cells, and mouse melanoma cell lines and their derived pleiotropic drug-resistant counterparts (5). However, data obtained with one technique should be compared only with data obtained with the same technique and the same treatment schedule. The MTT assay is successfully used, since other valid tests such as HTCA (human tumor colony assay) often have several technical problems, such as low plating efficiency and longer assay time. However, the use of the MTT assay for drug sensitivity testing of tumor samples may result in a higher background, because contamination by normal cells may reduce tetrazolium dye as well. To reduce such contamination, an HTCA-MTT mixed test was thus set up. After treatment for 1 h with the drugs, samples were seeded (in 96well microtiter plates prefilled with a 50 PL underlayer of RPMI-1640 and 15% fetal calf serum [FBS] in 0.4% agarose) in 50 PL of RPMI1640 and 15% FBS in 0.25% agarose.Followmg incubation for l-7 d at 37°C in 5% COZ, 10 ltL of MTT (5 mg/mL) was added to each well, and the plates were incubated at 37°C for 4 h. Then, 100 ~JLof 0.04N HCI in isopropanol was added to each well, and the solution was mixed vigorously to solubilize the formazan product. Air bubbles were sucked out, and after 1 h at room temperature, the absorbance of the wells was measured in a 2-wavelength microplate photometer at test and reference wavelengths of 550 and 660 nm, respectively. 1. Fill 96-well microplates with 50 l&L of complete medium in 0.4% agarose. 2. Allow agarose to solidify. 3. Add 50 ~.LLof complete medium contammg the appropriate number of drug-treated cells in 0.25% agarose. 4. Incubate for l-7 d in a humidified atmosphere of 5% CO2 at 37°C. 5. Add 10 FL of MIT (5 mg/mL). 6. Incubate for 4-6 h at 37°C in 5% CO*. 7. Add 100 l.tL of 0.04/V HCI m isopropanol. 8. Mix vigorously. 9. Leave for 1 h at room temperature. 10. Read m a microplate photometer at test and reference wavelengths 550 and 660 nm, respectively. 11. Calculate the mean of the optical density of different replicates of the same sample. 12. Evaluate the percentage of each value vs the control.

MTT Assays

143

13. Plot in a semilog chart the percentageof optical density againstdrug concentration. 14. Determine the IDS0from the dose-responsecurve. The HTCA-MTT hybrid assay is successful, since the HTCA assay is time-consuming, complicated, costly, and applicable for a limited number of human solid tumors. However, the MTT assay results in a high background owing to contamination by normal cells (especially in solid tumors), which may reduce tetrazolium dye as well. Moreover, it has been reported that tumor tissues are more sensitive to various antitumor drugs than are adjacent normal mucosal tissues. In contrast, the HTCAMTT hybrid assay is much shorter than the HTCA assay (4 d vs 2-3 wk) and is also effective to measure cells with low colony-forming efficiency. Furthermore, normal cells (fibroblasts, lymphocytes, and endothelial cells) do not grow in the double layer of agarose. 3.3. Purification

of Tumor

Cells

Other authors have reported a technique for purification from normal cells of tumor cells using discontinuous Ficoll-Hypaque and Percoll gradients. Using this method they obtained a higher correlation between in vitro results and clinical response (6) (Fig. 4). Tumor cells were dispersed in complete medium containing collagenase (2 mg/mL, type V-S; Sigma, hyaluronidase (10 U/r& type IV-S; Sigma), and DNase-1 (0.4 mg/rnL; Sigma). After a 40-min incubation at 37OC, the cells were harvested, washed, and suspended in complete medium. In the case of ascites, cells after centrifugation at 400g for 5 min were suspended in complete medium. Cells were then centrifuged on Ficoll-Hypaque (specific gravity 1.077, Pharmacia, Uppsala, Sweden) gradients at 400g for 30 min in 50-mL tubes (400g at the bottom of tubes). Mononuclear and tumor cells at the interface were collected, washed, and suspended at 106/mL in complete medium. The cells were then layered on discontinuous gradients consisting of 10 mL of 100% and 15 mL of 75% Ficoll-Hypaque in 50-mL plastic tubes. After centrifugation at 400g for 30 min (400g at the bottom of tubes), a tumor cell-rich fraction was collected from the 75% interface. The tumor-cell-enriched suspension was then layered onto discontinuous gradients containing 4 mL each of 25, 15, and 10% Percoll (Pharmacia) in complete medium in 15-n& plastic tubes. Centrifugation was performed at 25g for 7 min (25g at the

144

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Fig. 4. Technique for Ficoll-Hypaque tumor cell purification from normal cells. bottom of tubes), and tumor cells depleted of lymphoid cells were col-

lected from the bottom and the 25% interface, washed, and suspended in complete medium at a concentration of 1 x 106/nL. The cells thus prepared were mainly tumor cells, with ~10% contamination by nonmalignant cells, as judged by morphological examination using Papanicolaou staining or carcinoembryonic antigen staining for CEA-positive tumor cells. The cells were found to be more than 90-95% viable by the Trypan blue dye exclusion test. The mean yield of purified tumor cells was 2.3 x 106, and the tumor cell count at the beginning of preparation was 13 x lo6 (rate of yield = 17.7).

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4. Notes 1. Considerable changes in optical density are brought about by the presence of different volumes of reaction medium. Low and constant evaporation microwell plates therefore have to be used (7), and variations over a medium volume htgher than 20 pL are unacceptable. 2. Phenol red at 10 mg/mL does not change the optical density of formazan in DMSO. 3. The MTT reductron is dependent on the o-glucose concentration in the culture medium and is independent of pH (8). 4. MTT formazan production is dependent on the MTT concentration in the culture medium (9) (Fig. 5). 5. The kinetics of MTT formazan production and the degree of saturation vary in a cell-line-specific manner. 6. Drug cytotoxrcrty evaluation may be influenced by the length of exposure to MTT (9) (Fig. 6). 7. Since MTT is cleaved by active mitochondria, the assayis effective, but to a lesser extent, also in the absence of cell proliferation. 8. For any given tumor cell line, the optical density of the solubilized formazan product is directly proportional, over a wide range, to the number of cells per well.

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Fig. 7. Dose-effect curves of adriamycin on N592 (0) and N592DX (0) cell lines. 9. The MTT assay is effective on parental cell lines and their pleiotropic drug-resistant counterpart, thus leading to a correlation between comparable cell lines (Fig. 7). 10. The assay is effective on many different clmmal anticancer drugs (I) (Table 1).

MTT Assays

147

Table 1 Chemosensitivity of Cells Based on ICscs @tg/mL) of Some Clinical Anticancer Drugs in the MTT Reduction Assay Cells B16FlO B16 B16Fl L929 Mouse spleen HPBMC LOVO K562 RPM17272 COL0320HSR L1210 Clone A WiDr COLO205 MCF7 COLO201 Cells

Dactinomycin

Doxorubicin

Ara-C

l >1 >lO >lO >lO 9.50 >lO >lO >lO >lO >lO >lO

Methotrexate

Vincristine