Acetaldehyde-Related Pathology: Bridging the Trans-Disciplinary Divide (Novartis Foundation Symposium 285)

Novartis Foundation Symposium 285

ACETALDEHYDERELATED PATHOLOGY: BRIDGING THE TRANSDISCIPLINARY DIVIDE

ACETALDEHYDE-RELATED PATHOLOGY: BRIDGING THE TRANS-DISCIPLINARY DIVIDE

The Novartis Foundation is an international scientific and educational charity (UK Registered Charity No. 313574). Known until September 1997 as the Ciba Foundation, it was established in 1947 by the CIBA company of Basle, which merged with Sandoz in 1996, to form Novartis. The Foundation operates independently in London under English trust law. It was formally opened on 22 June 1949. The Foundation promotes the study and general knowledge of science and in particular encourages international co-operation in scientific research. To this end, it organizes internationally acclaimed meetings (typically eight symposia and allied open meetings and 15–20 discussion meetings each year) and publishes eight books per year featuring the presented papers and discussions from the symposia. Although primarily an operational rather than a grantmaking foundation, it awards bursaries to young scientists to attend the symposia and afterwards work with one of the other participants. The Foundation’s headquarters at 41 Portland Place, London W1B 1BN, provide library facilities, open to graduates in science and allied disciplines. Media relations are fostered by regular press conferences and by articles prepared by the Foundation’s Science Writer in Residence. The Foundation offers accommodation and meeting facilities to visiting scientists and their societies. Information on all Foundation activities can be found at http://www.novartisfound.org.uk

Novartis Foundation Symposium 285

ACETALDEHYDERELATED PATHOLOGY: BRIDGING THE TRANSDISCIPLINARY DIVIDE

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Contents

Symposium on Acetaldehyde-related pathology: bridging the trans-disciplinary divide, held at the Novartis Foundation, London 5–7th September 2006 Editors: Derek J. Chadwick (Organizer) and Jamie Goode This symposium is based on a proposal made by Victor Preedy, Peter Emery and Mikko Salaspuro Peter Emery Chair’s introduction 1 David W. Crabb and Suthat Liangpunsakul Acetaldehyde generating enzyme systems: roles of alcohol dehydrogenase, CYP2E1 and catalase, and speculations on the role of other enzymes and processes 4 Discussion 16 Richard A. Deitrich, Dennis Petersen and Vasilis Vasiliou Removal of acetaldehyde from the body 23 Discussion 40 Shih-Jiun Yin and Giia-Sheun Peng Acetaldehyde, polymorphisms and the cardiovascular system 52 Discussion 63 Jun Ren Acetaldehyde and alcoholic cardiomyopathy: lessons from the ADH and ALDH2 transgenic models 69 Discussion 76 Mikko Salaspuro Interrelationship between alcohol, smoking, acetaldehyde and cancer 80 Discussion 89 v

vi

CONTENTS

Hiroto Matsuse, Chizu Fukushima, Terufumi Shimoda, Sadahiro Asai and Shigeru Kohno Effects of acetaldehyde on human airway constriction and inflammation 97 Discussion 106 Helmut K. Seitz The role of acetaldehyde in alcohol-associated cancer of the gastrointestinal tract 110 Discussion 119 Robert Tardif The determination of acetaldehyde in exhaled breath 125 Discussion 133 Mostofa Jamal, Kiyoshi Ameno, Mitsuru Kumihashi, Weihuan Wang, Ikuo Uekita and Iwao Ijiri Ethanol and acetaldehyde: in vivo quantitation and effects on cholinergic function in rat brain 137 Discussion 141 Ville Salaspuro Pharmacological treatments and strategies for reducing oral and intestinal acetaldehyde 145 Discussion 153 Victor R. Preedy, David W. Crabb, Jaume Farrés and Peter W. Emery Alcoholic myopathy and acetaldehyde 158 Discussion 177 Onni Niemelä Acetaldehyde adducts in circulation 183 Discussion 193 General discussion 198 M. Apte, J. McCarroll, R. Pirola and J. Wilson pathways and acetaldehyde 200 Discussion 211

Pancreatic MAP kinase

Shivendra D. Shukla, Youn Ju Lee, Pil-hoon Park and Annayya R. Aroor Acetaldehyde alters MAP kinase signalling and epigenetic histone modifications in hepatocytes 217 Discussion 224 Paul J. Thornalley Endogenous α-oxoaldehydes and formation of protein and nucleotide advanced glycation endproducts in tissue damage 229 Discussion 243

CONTENTS

C. J. Peter Eriksson Measurement of acetaldehyde: what levels occur naturally and in response to alcohol? 247 Discussion 256 Final discussion 261 Contributors Index 265 Subject index 267

vii

Participants

Emanuele Albano Dipartimento di Scienze Mediche, Università Amedeo Avogadro del Piemonte Orientale, Via Solaroli 17, 28100 Novara, Italy Minoti V. Apte Pancreatic Research Group, South Western Sydney Clinical School, The University of New South Wales, Room 517, Level 5, Wallace Wurth Building, UNSW, Sydney, NSW 2052, Australia Agustin Aranda Departamento de Bioquimica y Biologia Molecular, Universidad de Valencia y Departamento de Biotecnologia, Instituto de Agroquimica y Tecnologia de Alimentos, Apdo 73, Burjassot, 46100, Valencia, Spain David W. Crabb Indiana University School of Medicine, 545 Barnhill Drive, Emerson Hall, Room 317, Indianapolis, IN 46202-5124, USA Richard A. Deitrich University of Colorado Health Sciences Center at Fitsimons, Department of Pharmacology, Alcohol Research Center, 12800 E. 19th Ave, P O Box 6211, Mail stop 8303, Aurora, CO 80045-0508, USA Peter Emery (Chair) Department of Nutrition and Dietetics, King’s College London, The Franklin Wilkins Building, 150 Stamford Street, London SE1 9NH, UK C. J. Peter Eriksson Department of Mental Health and Alcohol Research, National Public Health Institute, KTL/ATY, POB 33, Helsinki, FIN-00251, Finland Mostofa Jamal Department of Forensic Medicine, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki, Kita, Kagawa, 761-0793, Japan Hiroto Matsuse Second Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan viii

PARTICIPANTS

ix

John B. Morris Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, 69 N Eagleville Road, Box U-3092, Storrs, CT 062693092, USA Onni Niemelä Department of Laboratory Medicine and Medical Research Unit, Seinäjoki Central Hospital and University of Tampere, FIN-60220 Seinäjoki, Finland Tomonori Okamura Department of Health Science, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu City, Shiga, 520-2192, Japan Victor R. Preedy Department of Nutrition and Dietetics, School of Life Sciences, King’s College London, The Franklin Wilkins Building, 150 Stamford Street, London SE1 9NH, UK Etienne Quertemont Psychologie Quantitative, Department of Cognitive Sciences, Boulevard du Rectorat 5/B32, University of Liège, Liège 4000, Belgium Radhakrishna K. Rao Department of Physiology, University of Tennessee, 894 Union Ave, Nash 426, Memphis, TN 38163, USA Jun Ren Center for Cardiovascular Research and Alternative Medicine, Division of Pharmaceutical Sciences, University of Wyoming, 1000 E. University Avenue, Department 3375, Laramie, WY 82071, USA Mikko Salaspuro Research Unit of Substance Abuse Medicine, Biomedicum Helsinki, Haartmaninkatu 8, Helsinki, 00029 HUS, Finland Ville Salaspuro Research Unit of Substance Abuse Medicine, Biomedicum Helsinki, Haartmaninkatu 8, Helsinki, 00029 HUS, Finland Helmut K. Seitz Laboratory of Alcohol Research, Liver Disease and Nutrition, Department of Medicine, Salem Medical Centre Heidelberg and University of Heidelberg, Heidelberg, Germany Shivendra D. Shukla Department of Medical Pharmacology & Physiology, School of Medicine, University of Missouri, Columbia, MO 65212, USA Sophie Tambour (Novartis Foundation Bursar) University of Liege, Département des Sciences Cognitives, Laboratoire de Neuroscience Comportementale

x

PARTICIPANTS

et de Psychopharmacologie, Boulevard du Rectorat 5/B32, B-4000 Liege, Belgium Robert Tardif Department of Occupational and Environmental Health, Faculty of Medicine, University of Montreal, 2375 Cote St-Catherine, Montreal, Québec, Canada H3T 1A8 Paul J. Thornalley Protein Damage and Systems Biology Research Group, Warwick Medical School & Systems Biology Centre, Clinical Sciences Research Institute, University of Warwick, University Hospital, Coventry CV2 2DX, UK Simon Worrall Alcohol Research Unit, Biochemistry and Molecular Biology, School of Molecular and Microbial Sciences, University of Queensland, Brisbane, QLD 4072, Australia Shih-Jiun Yin Department of Biochemistry, National Defense Medical Center, 161 Min-Chuan East Road, Section 6, Taipei 114, Taiwan

Chair’s introduction Peter Emery Department of Nutrition and Dietetics, King’s College London, The Franklin Wilkins Building, 150 Stamford Street, London SE1 9NH, UK

Although there are three proposers listed for this symposium, the idea for this meeting came largely from Victor Preedy, and his enthusiasm caught up Mikko Salaspuro and myself. I have been working with Victor for a few years on a number of projects, one of which has involved looking at aspects of acetaldehyde protein adducts. As we were working on this project, the idea came to us that although many people are interested in acetaldehyde because of its role as a metabolite of alcohol, there are in fact many other aspects of acetaldehyde that are equally important. The idea here was to bring together people who are approaching acetaldehyde from a number of different perspectives to try to understand more about what these different approaches can bring. Acetaldehyde is an appealingly simple small molecule that is pretty reactive in vitro, and has some fairly serious effects in vivo in a variety of physiological systems. I would like to outline some of the questions that we might want to explore during this meeting. As a nutritionist I tend to start from a simple point of view, asking questions such as ‘where does it come from?’ Many people will think of it mainly as the first metabolite of alcohol. Perhaps the key to understanding many of the damaging effects of acetaldehyde is the distribution of the alcohol dehydrogenase (ADH) enzymes in tissues. We know that there is a fair amount of acetaldehyde produced quite separately from the ingestion of ethanol. In particular, the bacteria in the oral cavity and throughout the gut can produce it. They may be contributing significant amounts of acetaldehyde: does this have systemic effects or is it a local phenomenon? We also know that acetaldehyde is a product of cigarette smoke. We will hear about the effects of acetaldehyde from this source and its interaction with alcohol intake. There are also increasing amounts of acetaldehyde in the air: it is a volatile molecule produced by combustion of hydrocarbons. As we live in an increasingly polluted world, more of our exposure to acetaldehyde may be coming from the air. There is also acetaldehyde in some foodstuffs, particularly fermented foods. We don’t know whether this is a significant source of acetaldehyde intake as well. 1

2

EMERY

Once it is in the body, how is it metabolized? It is metabolized by various dehydrogenase and oxidase enzymes. There are background levels of these activities, which may increase considerably in response to exposure. We will hear a lot about polymorphisms of these different enzymes, which give us useful biological models for studying exposure to acetaldehyde. People produce very different amounts of acetaldehyde in response to the same amount of alcohol intake. There are other aldehydes present, and some of the systems we have for metabolizing acetaldehyde will also metabolize other aldehydes. The interaction with other aldehydes could be key to understanding some of the physiological actions of acetaldehyde. This leads us to consider the whole question of the variety of antioxidant defences that may be induced and up-regulated when we are exposed to acetaldehyde. It comes in, it is metabolized, but what we really need to measure is how much acetaldehyde is present in various tissues and for how long. This is difficult with a molecule like this that is short-lived and moves around. Instead of tissue concentrations we may have to look at proxies such as blood levels. Saliva may be useful: of course, this will reflect what is produced in the mouth, but it could also reflect systemic production and exposure to an extent. Acetaldehyde is a volatile molecule so we may be able to measure it in the breath. We may be able to measure products of acetaldehyde metabolism in the urine; this could be a long-term integrative measure of exposure, rather than reflecting acute changes. A key question from a biochemical viewpoint is the mechanism by which damage is caused. I’m sure we’ll hear a lot about this fairly vague term ‘oxidative stress’. Hopefully, we can be more precise in our discussion to clarify what is meant by this term. As an electrophilic molecule, acetaldehyde will attack many nucleophilic centres in a variety of important molecules, particularly forming adducts with DNA and protein, and indeed the lipid components. The question then becomes, what is the subsequent damage caused by production of these adducts? Which sorts of proteins may be affected? Proteins in signalling pathways may be amplifying the signal and the damage that is caused. There could be effects within the nucleus through transcription factors or DNA repair enzymes, or epigenetic effects on histone decoration. We will consider the tissues that are affected. Victor Preedy always teaches his students about the effects of alcohol on different tissues. When we look at where acetaldehyde might be having its damaging effects, it concerns a great variety of tissues, and not just the liver. There is the gut, and many cancers through the gastrointestinal tract may relate to acetaldehyde damage. There could be effects on the brain, which could lead not only to behavioural effects but also degenerative diseases. There are effects on the heart and cardiovascular system, and effects on skeletal muscle. The lung will be exposed to acetaldehyde from the atmosphere, and asthma, bronchitis and emphysema have all been linked with acetaldehyde.

CHAIR’S INTRODUCTION

3

Breast cancer development seems to be sensitive to differences in acetaldehyde production. Finally, the question we would like to move towards: what can we do about it? We are developing the idea that acetaldehyde is a damaging molecule, so are there ways of avoiding or minimizing this damage? Are there agents that could be used to bind acetaldehyde and reduce the amount that is present? Can we affect the amounts that are produced by reducing bacterial populations or manipulating them to change their characteristics? What can we do with the enzymes that are involved in producing or removing acetaldehyde to minimize the amount of acetaldehyde we are exposed to? The following list highlights some of the questions about acetaldehyde that we might want to explore over the next few days: • • • • • •

How does it get into the body? How is it metabolized? How much is there? How does it cause damage? Which tissues are affected? What can we do about it?

So let’s start the story where it needs to begin, by looking at alcohol dehydrogenase and other enzymes involved in acetaldehyde production.

Acetaldehyde generating enzyme systems: roles of alcohol dehydrogenase, CYP2E1 and catalase, and speculations on the role of other enzymes and processes David W. Crabb and Suthat Liangpunsakul Indiana University School of Medicine and Roudebush VA Medical Center, Emerson Hall Room 317, 545 Barnhill Drive, Indianapolis, IN 46202, USA

Abstract. Most acetaldehyde is generated in the liver by alcohol dehydrogenase (ADH) during ethanol metabolism. Polymorphic variants of these genes encode enzymes with altered kinetic properties, and pathophysiological effects of these variants may be mediated by accumulation of acetaldehyde. Two additional pathways of acetaldehyde generation are by the cytochrome P450 2E1 (CYP2E1) and catalase. While the amount of ethanol oxidized by these enzymes comprises a small fraction of total body ethanol clearance, the local formation of acetaldehyde by these enzymes may have important effects. Additional sources of acetaldehyde include other minor enzymes (nitric oxide synthase, other cytochrome P450s, P450 reductase, xanthine oxidoreductase) as well as non-enzymatic pathways (formation of hydroxyethyl radicals from the reaction of ethanol with hydroxyl radical, and its subsequent decomposition to acetaldehyde). Acetaldehyde may have effects locally (in the cells generating it), or when delivered to other cells by the blood stream or saliva, or by diffusion from the lumen of the gastrointestinal tract. The ultimate determinants of acetaldehyde toxicity include rates of its formation, rates of oxidation, and the capacity of cellular systems to prevent or repair chemical effects of acetaldehyde (e.g. formation of protein adducts or modification of nucleic acid bases). 2007 Acetaldehyde-related pathology: bridging the trans-disciplinary divide. Wiley, Chichester (Novartis Foundation Symposium 285) p 4 –22

Chronic consumption of large amounts of ethanol has well known effects on the heart, liver, brain, muscles, fetus and pancreas, and is involved in the pathogenesis of a number of neoplasms. The susceptibility of individuals to the ill effects of alcohol consumption is due to complex interactions of genes and the environment. Many of the effects of ethanol are mediated by acetaldehyde, which is mainly 4

ACETALDEHYDE GENERATING ENZYME SYSTEMS NAD+

5

NADH

Ethanol

Acetaldehyde Alcohol dehydrogenase NADP+

NADPH, O2

Acetaldehyde

Ethanol

CYP2E1 H2O2

H2O

Acetaldehyde

Ethanol

Catalase

FIG. 1. Major enzymatic pathways for acetaldehyde formation. The major pathways of acetaldehyde formation, alcohol dehydrogenase, cytochrome P450 2E1 (CYP2E1), and catalase are shown with their cofactors, substrates and products.

generated by alcohol dehydrogenase (ADH), cytochrome P450 2E1 (CYP2E1) and catalase (Fig. 1). Some of the enzymes involved in acetaldehyde formation are genetically polymorphic; when the polymorphism alters the enzymatic properties of the enzyme or the level of its expression, different individuals may generate varying amounts of acetaldehyde in a given tissue. Furthermore, the enzyme activities are in some cases regulated by transcriptional or translational mechanisms, controls, concentrations of substrates and products, and by post-translational modification (Table 1). These enzymes, the regulation of their activity, and tissue distribution, as well as some minor enzymatic processes that form acetaldehyde, are the subject of this overview. Enzymology of acetaldehyde formation Alcohol dehydrogenases General description. The enzymes responsible for the bulk of alcohol oxidation are the ADHs. All are dimeric enzymes with subunit molecular weight of about 40 kDa; subunits are identified by Greek letters. These enzymes are grouped into classes based upon enzymatic properties and the degree of sequence similarities. Enzyme subunits belonging to the same class can heterodimerize. The general properties of these enzymes are summarized in Table 2. Class I contains α, β, and γ isozymes. These enzymes have a low Km for ethanol and are highly sensitive to inhibition by pyrazole derivatives. They are very abundant in liver, and play a major role in alcohol metabolism. Class II ADH (πADH) is also abundant in liver, has a higher Km for ethanol, and is less sensitive to pyrazole inhibition than class I enzymes (Ehrig

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CRABB & LIANGPUNSAKUL

TABLE 1 Properties of alcohol dehydrogenases (ADHs) in humans Gene locus Class I ADH1 ADH2 ADH3 Class II ADH4 Class III ADH5 Class IV* ADH7 Class V* ADH6

New nomenclature

Subunit type

Km (ethanol)

Vmax

4 0.05–34** 0.6–1**

54 — —

Tissue distribution

ADH1A ADH1B ADH1C

α β γ

ADH4

π

34

ADH5

χ

1000

ADH7

σ, µ

20

1510

Stomach, oesophagus, other mucosae

ADH6

−

30

?

Liver, stomach

40 —

Liver Liver, lung Liver, stomach Liver, cornea Most tissues

* Tentative assignments based upon sequence homologies. Km values are given in mM and Vmax values are given in terms of turnover number (min−1). Tissue distributions indicate tissues with relatively high expression; see Fig. 3 for more detailed distribution information. ** Kinetic constants vary with isozyme, see Table 2.

TABLE 2 Properties of polymorphic forms of human alcohol dehydrogenase (ADH) Gene locus ADH2 *1 (ADH1B *1) ADH2 *2 (ADH1B *2) ADH2 *3 (ADH1B *3) ADH3 *1 (ADH1C *1) ADH3 *2 (ADH1C *2)

Subunit type

Km (ethanol)

Vmax

Population

β1 β2 β3 γ1 γ2

0.05 0.9 34 1.0 0.63

9 400 300 87 35

Caucasians, African-Americans Asians African-Americans All groups Caucasians

The kinetic constants are noted for the homodimers of the subunits listed (Ehrig et al 1990). Heterodimers behave as if the active sites were independent. The Km values are in mM and the Vmax values are given in terms of turnover numbers (min−1), as in Table 1. The column labelled population indicates which populations have high allele frequencies for these variants. The alleles are not limited to those populations.

et al 1990). Class III ADH (χADH) is present in nearly all tissues, is virtually inactive with ethanol, but can metabolize longer chain alcohols, ω-hydroxy-fatty acids, and formaldehyde. A recent paper suggested that class III ADH might be more active towards ethanol in a hydrophobic environment, and argues that liver cytosol may be such an environment (Haseba et al 2006). The class IV enzyme was purified from stomach and oesophagus (Pares et al 1994). σADH has the highest Vmax of the known ADHs and is very active towards

ACETALDEHYDE GENERATING ENZYME SYSTEMS Genes

Expression variants: promoter, mRNA stability, translation efficiency

Protein

7

Post-translational modification Substrate/product

Altered enzyme mass

Enzyme activity

Altered kinetic properties

Protein stability

Saturation effects (high Km variants) Product inhibition

Coding region variants

Protein stabilization

FIG. 2. Factors which control the rate of enzymatic generation of acetaldehyde. Genetic variation can influence the expression of the gene (transcriptional effects) or the stability or translational efficiency of the mRNA, as well as alter the coding sequence. Transcriptional or mRNA effects will result in varying amounts of active enzyme, and thus determine the maximum flux through the pathway. Coding region variants for ADH have widely varying kinetic properties. Post-translational modifications can influence the activity of an enzyme or its susceptibility to degradation, as can the degree of substrate binding to the enzyme, as in the case of CYP2E1 stabilization by substrate. The enzyme activity is ultimately determined by the concentrations of substrate and product, the kinetic constants for each isozyme, and the total activity of the enzyme, as defined by the kinetic rate equation for the enzyme (Crabb et al 1983).

retinol. This may be relevant to its expression in numerous epithelia which are dependent on retinol for their integrity. Class V ADH, encoded by the ADH6 gene, is expressed in liver and in stomach, but the enzyme itself has not been purified. In vitro expressed enzyme had a high Km for ethanol (about 30 mM), and moderate sensitivity to pyrazole inhibition (Cheng & Yoshida 1991). Class VI ADH was reported in deer, mouse and rat liver; class VII ADH was cloned from chicken, but the human homologues have not been found. Genetic variants. The nomenclature for ADH genes was recently revised. The ADH1, 2 and 3 genes are now designated ADH1A, ADH1B, and ADH1C genes, respectively. Two of the seven human ADH gene loci are polymorphic, and the prevalence of the alleles depends on continental origin. The kinetic properties and population distributions of these allelic enzymes are shown in Fig. 3. The isozymes encoded by the three ADH1B alleles, differing at single amino acids, vary markedly in Km for ethanol and Vmax. β1 is most common in Caucasians, has a low Vmax and a very low Km for ethanol. β2 is found in Asians and Ashkenazi Jews. It has a substantially higher Vmax and somewhat higher Km compared with β1. The β3 isozyme was first detected in samples from African-Americans, and has also been found in Southwest Native Americans. It has a high Km for ethanol and high Vmax. Smaller differences in enzymatic properties are observed between the products of the ADH1C alleles. The γ1 isozyme has about twice the Vmax of the γ2 isozyme,

8

CRABB & LIANGPUNSAKUL ADH1C

Tissue

ADH4

ADH6

ADH7

CYP2E1

CAT

blood

0

17

0

0

53

367

bone

13

0

0

0

13

55

bone marrow

0

0

0

0

0

634

brain

27

0

1

0

19

47

connective tissue

74

0

0

0

0

65

adipose tissue

4251

0

0

0

0

144

liver

1930

729

252

0

843

319

pancreas

36

4

4

0

0

95

adrenal gland

611

0

0

0

0

32

thyroid

0

0

0

0

18

163

placenta

16

0

0

0

0

121

eye

9

0

0

19

0

67

cervix

62

0

20

0

0

41

ovary

0

0

9

0

28

0

uterus

217

0

8

0

4

62

prostate

32

0

0

0

6

51

testis

28

0

11

0

8

48

bladder

132

0

0

33

0

99

kidney

56

0

84

0

0

79

tongue

30

0

15

90

0

30

larynx

32

0

0

32

0

98

pharynx

0

0

0

0

0

0

salivary gland

0

0

48

0

0

146

heart

602

0

55

0

0

100

lymph node

10

0

0

0

0

146

spleen

416

0

0

0

0

37

thymus

135

0

0

0

13

0

mammary gland

450

29

23

0

29

58

muscle

122

0

8

17

8

69

lung

169

0

0

40

28

69

trachea

1444

0

0

288

0

20

skin

21

0

0

0

0

85

vascular

118

0

0

0

0

157

small intestine

1558

22

90

0

0

22

colon

153

0

14

0

0

84

stomach

254

0

48

9

0

19

esophagus

472

0

52

996

0

0

nerve tissue

550

0

0

0

39

118

FIG. 3. Tissue distribution of ADH, CYP2E1, and catalase transcripts reflected by the abundance of expressed sequence tags (ESTs). Tissue distribution of ESTs for the noted genes were obtained from the NCBI Unigene Database using the EST Profile Viewer (e.g. http://www.ncbi. nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Hs.78989 for ADH5). The number in each cell is the number of transcripts per million, a measure of the abundance of the transcripts. The spot intensity is based on the transcripts per million.

ACETALDEHYDE GENERATING ENZYME SYSTEMS

9

while the Kms for ethanol are similar. γ1 ADH is found at high frequency in Asians and African-Americans; Caucasians have about equal frequency of γ1 and γ2 ADH alleles (Burnell & Bosron 1989). The other ADH loci have not been found to be polymorphic to date. Individuals expressing ADH1B *2 and ADH1B *3 would be predicted to metabolize ethanol more rapidly and generate more acetaldehyde; however, effects of the polymorphism on ethanol elimination rates are small. Different ADH1B *2 genotypes are correlated with only a small fraction of the between-individual differences in alcohol elimination rates. The ADH1B *3 polymorphism confers a 10% increase in the rate of ethanol metabolism; both it and ADH1B *2 are protective against alcoholism (Edenberg et al 2006). The ADH1C polymorphism did not affect alcohol elimination rate, but recent data link the ADH1C *1 allele with head and neck, oesophageal, breast and hepatocellular carcinomas (Homann et al 2006), which could reflect increased rates of acetaldehyde formation. An additional ADH genetic variant is a Pvu II restriction fragment length polymorphism (RFLP) in an intron of the ADH1B gene. It is not known if the variant alters expression of the gene or is linked to another susceptibility locus; the B allele was found at higher frequency in alcoholics and in patients with alcoholic cirrhosis (Sherman et al 1993). Single nucleotide polymorphisms (SNPs) presumed to influence expression of the ADH4 gene have been linked to risk of alcoholism (Edenberg et al 2006); one polymorphism in the promoter affects gene expression (Edenberg et al 1999). Similarly, sequence variants in the promoter of ADH1C may affect its expression (Chen et al 2005). Control of expression of ADHs. The ADH1 promoters are all active in liver. They interact with ubiquitous transcription factors (e.g. TATAA binding factors, upstream stimulatory factor [USF], CTF/NF-I and Sp1-like factors), as well as tissue-specific factors (e.g. hepatocyte nuclear factor 1 [HNF-1], D-box binding protein [DBP] and CCAAT-enhancer binding proteins [C/EBPα and β]). An HNF-1 site was recently reported to serve as a master control for all three of the class I genes (Su et al 2006). The ADH5 and ADH7 promoters lack TATAA boxes. The ADH5 promoter is G+C rich, a characteristic of housekeeping genes and consistent with its ubiquitous expression. Binding sites for thyroid hormone, retinoic acid and glucocorticoid receptors have been identified in the upstream regions of ADH1 genes. In in vitro experiments, retinoic acid and glucocorticoids activated the promoters and thyroid hormone antagonized the effect of retinoic acid; these hormones had less dramatic effects in vivo. Growth hormone increased ADH activity in rats and cultured hepatocytes, while androgens and thyroid hormones decreased it. Chronic ethanol consumption can affect the expression of ADH. Ethanol increased hepatic ADH activity in male rats by reducing testosterone levels. The amount of ethanol consumed from conventional liquid diets did not alter liver ADH activity, whereas higher doses achieved by intragastric ethanol infusion induced liver

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ADH activity. This resulted from induction of the transcription factor C/EBPβ and suppression of C/EBPγ and a truncated, inhibitory form of C/EBPβ called LIP (He et al 2002). In addition, chronic intragastric infusion of ethanol increases portal vein endotoxin, which can induce ADH mRNA via increased binding of USF (Potter et al 2003). In humans, the amount of ADH in the liver was not induced by chronic drinking; however, with fasting, protein malnutrition and liver disease, ADH activity and the ethanol elimination rate were decreased. Orchiectomy increased alcohol elimination rates in humans. Little is known about expression of extrahepatic ADH, with the exception of gastric ADH, which is reduced with age, in women, and with heavy drinking (Seitz et al 1993). Post-translational modifications. No post-translational modifications of the ADH enzyme are recognized. However, peroxynitrite can oxidize the active site, causing disulfide formation and release of zinc, inactivating the enzyme (Daiber et al 2002); whether this is physiologically relevant remains to be seen. Role of substrate and product concentrations. The ADH isozymes with high Km for ethanol, e.g. β3, π, and σ will be more active when blood ethanol concentrations are high or in tissues of the upper gastrointestinal (GI) tract that are directly exposed to beverage ethanol. Modelling of alcohol oxidation in rat liver indicated that ADH activity was controlled by the total activity of the enzyme as well as product inhibition by NADH and acetaldehyde (Crabb et al 1983); thus ADH operates below its Vmax at steady state. Our laboratory determined the rate of ethanol oxidation by cells expressing ADH1B *1, ADH1B *2 and ADH1B *3. The inhibition constants for β1, β2 and β3 ADH were 1.5 ± 0.1, 22 ± 14 and 210 ± 5 µM, respectively (Matsumoto et al, unpublished data), indicating that activity of β1 and β2 ADH could be limited by the accumulation of acetaldehyde. Tissue distribution. ADHs are expressed in a variety of tissues. High levels of class I ADH mRNA were found in kidney, stomach, duodenum, colon and uterus of rats, with lower levels in many organs including the lung, small intestine and hepatic Ito cells, and much lower levels were found in brain, thymus, muscle or heart (Estonius et al 1996). Cytosolic ADH has been found in parotid gland, and chronic alcohol use was associated with parotid steatosis (Maier et al 1986). Class I ADH is found in blood vessels, which may be relevant to alcohol-induced flushing and cardiovascular effects of ethanol consumption. Class II ADH was detected in liver and duodenum (Estonius et al 1996). Gastric mucosa contains several ADHs (γ-, χ-, and σADH). σADH is absent in the stomach biopsies of about 30% of Asians, and those lacking this enzyme had lower first pass metabolism of ethanol (Dohmen et al 1996), suggesting that σADH is important in gastric oxidation of ethanol.

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Relative expression of various mRNAs can be estimated from the frequency of expressed sequence tags (ESTs) detected in cDNA libraries. Figure 3 shows the relative expression of ADH1C, ADH4, ADH6 and ADH7 transcripts in a number of tissues. Microorganisms express numerous forms of alcohol dehydrogenase, which can contribute to the formation of acetaldehyde in the lower GI tract, or wherever microbial overgrowth occurs. Cytochrome P450 2E1 General description. Ethanol can be metabolized by microsomal ethanol oxidizing systems, predominantly via cytochrome P450 2E1 (CYP2E1). Other cytochromes, CYP1A2 and CYP3A4, also contribute to a lesser extent (Lieber 2004). CYP2E1 is associated with NADPH-cytochrome P450 reductase in the endoplasmic reticulum, and reduces molecular oxygen to water as ethanol is oxidized to acetaldehyde. It is responsible for perhaps 10% of ethanol elimination. CYP2E1 is inducible by chronic drinking especially in the perivenular zone, and it may contribute to the increased rates of ethanol elimination in heavy drinkers. CYP2E1 is induced in fasting, diabetes and by a diet high in fat, which may relate to its ability to oxidize the ketone body acetone. Its Km for ethanol is about 10 mM; thus CYP2E1 may assume a greater role in ethanol metabolism at high blood alcohol levels. CYP2E1 is unusually ‘leaky’ and generates reactive oxygen species (ROS) including hydroxyl radical (OH• ), superoxide anion (O2 −), hydrogen peroxide (H2O2), and hydroxyethyl radical (HER• ). Thus, CYP2E1 is a major source of oxidative stress. CYP2E1 knockout animals had longer sleep times than normal counterparts, suggesting a role for CYP2E1 in brain sensitivity to ethanol (Vasiliou et al 2006). Genetic variants. An Rsa I (−1053C > T) polymorphism (the Rsa I+ allele is also named the c1 allele) is located in the 5′-flanking region of the CYP2E1 gene (Hayashi et al 1991) in a region interacting with HNF-1. The Rsa I− allele (c2) was more active in in vitro transcriptional assays, although a corresponding increase in CYP2E1 activity in vivo has not been unequivocally confirmed using the clearance of chlorzoxazone as a probe. The frequency of this polymorphism depends on continental origin: the c2 variant is found in 2–8% of Caucasians and in 25–36% of East Asians. Another polymorphism, detectable with the Dra I restriction enzyme, is located in intron 6. The distribution of the variant genotype (lacking the Dra I site) also depends on continental origin: 40–50% of East Asians carry this genotype, while only 10% of Caucasians lack the Dra I site. A more recently described polymorphism is the −71G > T polymorphism in exon 1, which has been associated with enhanced transcriptional activity of promoter constructs in HepG2 cells. Heterozygosity for this allele occurs in about 10% of Caucasians. The effects of the various genotypes on alcohol pharmacokinetics or risk of alcoholic

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complications have been inconsistent, and there is no direct evidence of differences in rates of acetaldehyde formation. Control of expression of CYP2E1. The human CYP2E1 gene spans 11 kb, contains 9 exons, and contains a typical TATAA box. HNF-1 is critical for its expression. Expression is also controlled both at the level of mRNA (high concentrations of ethanol can induce transcription of the CYP2E1 gene [Takahashi et al 1993]) and by the stabilization of the protein, as observed for ethanol, acetone and pyrazole derivatives, which reduce the rate of proteasomal degradation (Lieber 2004). Recent data suggest that additional signals may affect its expression. For instance, CYP2E1 can be induced by interleukin (IL) 4 in liver (Lagadic-Gossmann et al 2000) and by phorbol ester and other cellular stresses in astrocytes (Tindberg 2003). Insulin post-transcriptionally reduced the expression of CYP2E1 by destabilizing its mRNA. Role of substrate and product concentrations. Since CYP2E1 has a high Km for ethanol, it will generate more acetaldehyde when ethanol concentrations are elevated. There is no evidence that acetaldehyde is a product inhibitor of CYP2E1; in fact, CYP2E1 can oxidize acetaldehyde to acetate, although probably not in the presence of ethanol. Post-translational modification. CYP2E1 is reported to be a substrate for cAMPdependent protein kinase A (PKA). Phosphorylation of a serine residue inactivates the enzyme (Oesch-Bartlomowicz et al 1998). Whether this plays a physiological role in controlling activity of this enzyme is not clear, although in several conditions in which CYP2E1 activity is low (fasting, diabetes), hepatic PKA activity is high. Tissue distribution. CYP2E1 is expressed at highest levels in the liver, as well as numerous other tissues, as demonstrated by western blotting, mRNA, or EST analyses (Fig. 3). These include kidney, lung, oesophagus, biliary epithelium, pancreas, uterus, leukocytes, breast, brain, colon, urinary bladder, nasal mucosa and pancreatic beta cells. Western blots and activity assays have confirmed expression of CYP2E1 in oesophagus, pancreas and lung, among others. In brain, CYP2E1 was reported to be expressed in neurons and inducible by ethanol administration (Tindberg & Ingelman-Sundberg 1996). Catalase General description. The peroxisomal catalase is a tetrameric, haem-containing enzyme. In addition to converting hydrogen peroxide (H2O2) to water and oxygen, it can oxidize ethanol to acetaldehyde in an H2O2-dependent fashion. This pathway is not

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thought to be a major elimination pathway under most physiological conditions, but it may be important in certain tissues such as brain; in fact, acatalasaemic mice had longer sleep times than their normal counterparts (Vasiliou et al 2006). Genetic variation. The absence of active catalase (acatalasaemia) is encountered in Asian populations. A number of SNPs in the 5′ untranslated region and introns are reported, but there are no known effects of these variants on expression or activity of the enzyme, nor on responses to ethanol. Control of expression. Little is known regarding transcriptional control of catalase expression in mammalian cells. The rat catalase gene is a single-copy gene spanning 33 kb. The promoter region lacks a TATAA box and an initiator consensus sequence, contains multiple CCAAT boxes and GC boxes, and contains multiple transcription initiation sites, consistent with its housekeeping function. Chronic ethanol feeding was reported to increase catalase activity (Orellano et al 1998). The rat catalase promoter contains a peroxisome proliferator responsive element (PPRE [Girnun et al 2002]) and can be induced by peroxisome proliferators. Post-translational modification. In cells exposed to H2O2, Abl and Arg (non-receptor protein tyrosine kinases) associate with catalase and can activate it by phosphorylating two tyrosine residues. However, at higher concentrations of H2O2, phosphorylation of these residues can stimulate ubiquitination and proteasomal degradation of the enzyme (Cao et al 2003). Control by substrate and product levels. The activity of catalase depends upon the availability of H2O2. This was observed with perfused rat liver: when fatty acids were added to the perfusate, peroxisomal β oxidation generated H2O2 and stimulated ethanol oxidation. This raises the possibility that under conditions of oxidant stress (and H2O2 production) catalase-mediated ethanol oxidation may be increased. Tissue distribution. Catalase is expressed in nearly all tissues (Fig. 3). Catalase is also expressed by colonic micro-organisms and contributes to the formation of acetaldehyde from ethanol in the lower GI tract (Tillonen et al 1998). Other pathways of acetaldehyde generation A number of minor pathways of acetaldehyde generation have been suggested. Nitric oxide synthases 1 and 2 were reported to generate 1-hydroxyethyl radical from ethanol in the presence of NADPH and arginine. This is perhaps not

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surprising given the presence of a CYP motif within the structure of the enzymes. 1-Hydroxyethyl radical can break down to form acetaldehyde (Porasuphatana et al 2006). Castro et al (2001) reported that cytosolic xanthine oxidoreductase is capable of oxidizing ethanol to acetaldehyde. CYP reductase (in the absence of CYP proteins such as CYP2E1) was reported to oxidize ethanol to 1-hydroxyethyl radical and acetaldehyde, possibly via the semiquinone form of FAD (Diaz-Gomez et al 2000). Other investigators report formation of acetaldehyde from ethanol in tissue extracts for which the responsible enzymes have not been identified in studies using different cofactors and inhibitors. It is possible that other oxidant species (hydroxyl radical) formed non-enzymatically might be able to oxidize ethanol to acetaldehyde. In addition, acetaldehyde can be formed during the degradation of threonine, putatively by threonine aldolase.

Summary Three major enzymes and several minor enzymes can generate acetaldehyde when ethanol is present. These enzymes are present in virtually all cells; thus, the ability of acetaldehyde to alter cellular function or to modify DNA or proteins, will depend on the rate of acetaldehyde formation (related to ethanol concentration, activity of the enzyme, and the presence or absence of inhibitors of the enzymes), and of its further oxidation by aldehyde dehydrogenases.

Acknowledgements This work was supported in part by P60 A07611 to DWC and a Young Investigator Award from the Richard Roudebush VA Medical Center to SL.

References Burnell JC, Bosron WF 1989 Genetic polymorphism of human liver alcohol dehydrogenase and kinetic properties of the isoenzymes. In: Crow KE, Batt RD (eds) Human metabolism of alcohol. 11th Edn. CRC Press, Boca Raton, FL, p 65–75 Cao C, Leng Y, Liu X, Yi Y, Li P, Kufe D 2003 Catalase is regulated by ubiquitination and proteosomal degradation. Role of the c-Abl and Arg tyrosine kinases. Biochemistry 42: 10348–10353 Castro GD, Delgado de Layno AM, Costantini MH, Castro JA 2001 Cytosolic xanthine oxidoreductase mediated bioactivation of ethanol to acetaldehyde and free radicals in rat breast tissue. Its potential role in alcohol-promoted mammary cancer. Toxicology 160: 11–18 Chen HJ, Tian H, Edenberg HJ 2005 Natural haplotypes in the regulatory sequences affect human alcohol dehydrogenase 1C (ADH1C) gene expression. Hum Mutat 25:150–155 Cheng C-S, Yoshida A 1991 Enzymatic properties of the protein encoded by newly cloned human alcohol dehydrogenase ADH6 gene. Biochem Biophys Res Comm 181:743–747

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Crabb DW, Bosron WF, Li T-K 1983 Steady-state kinetic properties of purified rat liver alcohol dehydrogenase: application to predicting alcohol elimination rates in vivo. Arch Biochem Biophys 224:299–309 Daiber A, Frein D, Namgaladze D, Ullrich V 2002 Oxidation and nitrosation in the nitrogen monoxide/superoxide system. J Biol Chem 277:11882–11888 Diaz Gomez MI, Castro GD, de Layno AM, Costantini MH, Castro JA 2000 Cytochrome P450 reductase-mediated anaerobic biotransformation of ethanol to 1-hydroxyethyl-free radicals and acetaldehyde. Toxicology 154:113–122 Dohmen K, Baraona E, Ishibashi H et al 1996 Ethnic differences in gastric sigma-alcohol dehydrogenase activity and ethanol first-pass metabolism. Alcohol Clin Exp Res 20: 1569–1576 Edenberg HJ, Jerome RE, Li M et al 1999 Polymorphism of the human alcohol dehydrogenase 4 (ADH4) promoter affects gene expression. Pharmacogenetics 9:25–30 Edenberg HJ, Xuei X, Chen HJ et al 2006 Association of alcohol dehydrogenase genes with alcohol dependence: a comprehensive analysis. Hum Mol Genet 15:1539–1549 Ehrig T, Bosron WF, Li T-K 1990 Alcohol and aldehyde dehydrogenase. Alcohol Alcohol 25:105–116 Estonius M, Svensson S, Hoog JO 1996 Alcohol dehydrogenase in human tissues: localisation of transcripts coding for five classes of the enzyme. FEBS Lett 397:338–342 Girnun GD, Domann FE, Moore SA, Robbins ME 2002 Identification of a functional peroxisome proliferator-activated receptor response element in the rat catalase promoter. Mol Endocrinol 16:2793–2801 Haseba T, Duester G, Shimizu A, Yamamoto I, Kameyama K, Ohno Y 2006 In vivo contribution of Class III alcohol dehydrogenase (ADH3) to alcohol metabolism through activation by cytoplasmic solution hydrophobicity. Biochim Biophys Acta 762:276–283 Hayashi S, Watanabe J, Kawajiri K 1991 Genetic polymorphisms in the 5′-flanking region change transcriptional regulation of the human cytochrome P450IIE1 gene. J Biochem (Tokyo) 110:559–565 He L, Ronis MJ, Badger TM 2002 Ethanol induction of class I alcohol dehydrogenase expression in the rat occurs through alterations in CCAAT/enhancer binding proteins beta and gamma. J Biol Chem 277:43572–43577 Homann N, Stickel F, Konig IR et al 2006 Alcohol dehydrogenase 1C*1 allele is a genetic marker for alcohol-associated cancer in heavy drinkers. Int J Cancer 118:1998–2002 Lieber CS 2004 The discovery of the microsomal ethanol oxidizing system and its physiologic and pathologic role. Drug Metab Rev 36:511–529 Lagadic-Gossmann D, Lerche C, Rissel M et al 2000 The induction of the human hepatic CYP2E1 gene by interleukin 4 is transcriptional and regulated by protein kinase C. Cell Biol Toxicol 16:221–233 Maier H, Born IA, Veith S, Adler D, Seitz HK 1986 The effect of chronic ethanol consumption on salivary gland morphology and function in the rat. Alcohol Clin Exp Res 10: 425–427 Oesch-Bartlomowicz PR, Padma R, Becker B et al 1998 Differential modulation of CYP2E1 activity by cAMP-dependent protein kinase upon Ser129 replacement. Exp Cell Res 242:294–302 Orellana M, Rodrigo R, Valdes E 1998 Peroxisomal and microsomal fatty acid oxidation in liver of rats after chronic ethanol consumption. Gen Pharmacol 31:817–820 Pares X, Cederlund E, Moreno A, Hjelmqvist L, Jornvall H 1994 Mammalian class IV alcohol dehydrogenase (stomach alcohol dehydrogenase): structure, origin, and correlation with enzymology. Proc Natl Acad Sci USA 91:1893–1897 Porasuphatana S, Weaver J, Rosen GM 2006 Inducible nitric oxide synthase catalyzes ethanol oxidation to alpha-hydroxyethyl radical and acetaldehyde. Toxicology 223:167–174

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Potter JJ, Rennie-Tankersley L, Mezey E 2003 Endotoxin enhances liver alcohol dehydrogenase by action through upstream stimulatory factor but not by nuclear factor-kappa B. J Biol Chem 278:4353–4357 Seitz HK, Egerer G, Simanowski UA et al 1993 Human gastric alcohol dehydrogenase activity: effect of age, sex, and alcoholism. Gut 34:1433–1437 Sherman DIN, Ward RJ, Warren-Perry M, Williams R, Peters TJ 1993 Association of restriction fragment length polymorphism in alcohol dehydrogenase 2 gene with alcohol-induced liver damage. Br Med J 307:1388–1390 Su JS, Tsai TF, Chang HM, Chao KM, Su TS, Tsai SF 2006 Distant HNF1 site as a master control for the human class I alcohol dehydrogenase gene expression. J Biol Chem 281:19809– 19821 Takahashi T, Lasker JM, Rosman AS, Lieber CS 1993 Induction of cytochrome P-4502E1 in the human liver by ethanol is caused by a corresponding increase in encoding messenger RNA. Hepatology 17:236–245 Tillonen J, Kaihovaara P, Jousimies-Somer H, Heine R, Salaspuro M 1998 Role of catalase in in vitro acetaldehyde formation by human colonic contents. Alcohol Clin Exp Res 22: 1113–1119 Tindberg N 2003 Phorbol ester induces CYP2E1 in astrocytes, through a protein kinase C- and tyrosine kinase-dependent mechanism. J Neurochem 86:888–895 Tindberg N, Ingelman-Sundberg M 1996 Expression, catalytic activity, and inducibility of cytochrome P450 2E1 (CYP2E1) in the rat central nervous system. J Neurochem 67: 2066–2073 Vasiliou V, Ziegler TL, Bludeau P, Petersen DR, Gonzalez FJ, Deitrich RA 2006 CYP2E1 and catalase influence ethanol sensitivity in the central nervous system. Pharmacogenet Genomics 16:51–58

DISCUSSION Deitrich: I was wondering about the protection against alcoholism given by the ADH variants. Yedi Israel has pointed out that we have been measuring steady state acetaldehyde an hour or two after alcohol has been given, but in UChA rats which have a defective ALDH2 enzyme, the major effect on acetaldehyde levels is seen in 30 min or less (Quintanilla et al 2005, Israel et al 2006). He proposes that it is the burst of acetaldehyde rather than the steady-state level which is preventing people from going ahead and drinking, and not the steady state levels. When were these acetaldehyde levels measured? It could be the burst rather than the steady state level that is important. Crabb: This thought occurred to us as we were looking at the data from the cells that have ALDH2 and ADH. It seems possible that during the first pass of alcohol through the liver, there would not be that restraining effect of acetaldehyde or alcohol oxidation, and a pre-steady-state burst of acetaldehyde might come out in the hepatic veins. I think we need someone to do the hepatic vein catheterizations as were done in Finland many years ago, to catch that early time point. M Salaspuro: The question as to why some ADH isoforms may protect from alcoholism is very interesting. We are used to working with hepatocytes, and know

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very well how acetaldehyde formation and ethanol oxidation is regulated in the liver. But we don’t know much about how they are regulated in the mouth, especially in different populations. And we don’t know how either ADH and ALDH are expressed in various cell lines of the mouth mucosa. If acetaldehyde is released in the mouth some of its effects may be much more potent in the brain. Acetaldehyde for example may release histamine from the mast cells or mucosal cells and in this case it escapes the liver. Acetaldehyde may get to the CNS via the arterial tree. Crabb: I think you can say the same about the oesophagus. Acetaldehyde made there won’t pass through the liver, either. If the EST data are correct, acetaldehyde might even be formed beyond where we have been measuring it (i.e. in the hepatic veins) and closer to the brain—even in the arterial tree. Eriksson: On the other hand, there is work showing that when 4-methylpyrazole is used in normal conditions, there isn’t any effect on salivary acetaldehyde. This suggests that no measurable ADH-dependent levels are formed during normal conditions. If acetaldehyde is elevated, e.g. by deficient ALDH activity, then there is an effect of 4-methylpyrazole. I will speak more about this aspect in my paper. Apte: With regard to the local production of acetaldehyde, the pancreas is a bit of a forgotten organ in terms of its ability to produce acetaldehyde locally. It has been shown that the pancreas can metabolize alcohol. It has ADH. Interestingly, the kinetics of ADH in the acinar cells of the pancreas seems to match most closely to ADH5. It has a very high Km. In the cells I am interested in, the stellate cells which produce fibrosis, we think we have found ADH1. The problem I have with alcohol and acetaldehyde experiments is that when I read the literature I can’t work out whether people are using the concentrations of either ethanol or acetaldehyde that the cells may actually be exposed to in vivo. People use concentrations big enough to get an effect. This has always been a worry of mine: I’m concerned that in our own work we are using concentrations as high as 200 µM acetaldehyde, and we justify this by saying that local production during a burst of acetaldehyde might reach as high as that, and in the 30 min it persists for it has enough time to produce these toxic effects. Should we be looking more at steady-state levels? I also have a point regarding your table about ESTs. We have found CYP2E1 protein expression in the pancreas. Not only is it present, but it is also inducible in alcohol-fed rats. Crabb: I work with neurochemists, and they do interesting things such as in vitro microdialysis to get a sense of concentrations present at the pericellular level. I don’t know whether this has been done with the liver or other solid organs. If it has been, I don’t know whether our analytical methods are sensitive enough to detect acetaldehyde in those dialysates. We could do all sorts of things if we could get real time acetaldehyde concentrations. We need engineers and physicists to give us this kind of instrumentation.

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Emery: Are there any people here who have experience of microdialysis in tissues? Apte: Lieber’s lab used to use GC-MS to measure acetaldehyde in hepatocytes in culture. Emery: The real question is what is happening in vivo. Eriksson: This is a relevant aspect. Acetaldehyde is formed at a specific site in the cell. At that site the concentration is extremely high. In the cytosol, nature has created aldehyde dehydrogenases with high Km. Thus, 200 µM is quite appropriate for studying the effects of acetaldehyde. Also, Asian flushers may have average hepatic concentrations this high during alcohol drinking. Apte: I’m pleased about this because I have always worried about the concentrations we’ve used. If we talk about alcohol in a general meeting, people might think about their own drinking and then be surprised about the concentrations that we use in vitro. Eriksson: I’d like to add a point. There’s a source of acetaldehyde that is forgotten, which comes with drinking the alcoholic beverage. Some beverages contain millimolar concentrations of acetaldehyde. We are talking here about micromolar concentrations, so it means that this is a significant source. Systemically, the body has a fantastic capacity to remove acetaldehyde. Also, in liver the efficacy of removal is striking: more than 99% is removed directly. Emery: I think we can be comfortable with these sorts of concentrations extracellularly. What is the concentration going to reach within the cell, and within particular compartments where the effects occur? This comes back to the question of the local production within the cell and organelle. Niemelä: With regard to the possible associations between the expression of alcohol-metabolizing enzymes and organ damage in alcoholics, recent studies have shown that overexpression of CYP2E1 creates damage in the liver (Caro & Cederbaum 2004). Is there any evidence linking ADH expression with end-organ damage or certain ethanol-induced disease states? Crabb: Even more broadly than that, many years ago Christopher Day at the University of Newcastle upon Tyne and I were looking at these gene variants as risk factors for alcohol pathology. The literature contains a number of papers reporting such associations. Dr Day has continued to work in this area and he concludes that so far the only genetic risk factors for alcoholic liver disease are the ALDH2 *2 allele in Asians, and a polymorphism in the TNFα promoter, even though we have numerous publications from various groups (Stewart et al 2001). There are no other findings that have been reproduced in other laboratories and with other populations. He kids that I did well to stop working in this area because he has been frustrated by the inability to identify stronger correlations. He has one of the best databases of patients who have been well-characterized for alcoholic liver disease without hepatitis C. I believe one comes close to having a

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genetic risk factor if you combine several studies from Japan. It seems that ALDH2deficient individuals or β2 expressing individuals had somewhat higher risk of liver injury if they persisted in drinking heavily. This fits with our preconceptions of the role of acetaldehyde in liver injury. If you look at this from the other direction, the Consortium on Genetics of Alcoholism (COGA) have been working for 20 years, and only in the last couple of years a number of candidate genes came out of this study, which contribute to the risk of alcoholism. A couple of them are neurotransmitter receptors (which is kind of satisfying), one is a taste receptor for bitter substances, and the final one is ADH4, which is the gene for πADH. Because of its high Km and predominant expression in the liver, this was not thought to be a candidate gene before COGA, and it didn’t have coding region variants that we thought were important. Coming at it from this non-biased genome-wide screen, an enzyme of alcohol metabolism popped up that we hadn’t expected. Our next step in the field is to genotype people for ADH4 with these risk haplotypes, and then see what the difference is in their alcohol and acetaldehyde metabolism. Worrall: In your table of ESTs you had ‘brain’. Of course, the various regions of the brain vary in their metabolic capacity. Conventional wisdom is that brain has two to three orders less ethanol metabolic capacity than the liver. We have been measuring some adducts in alcoholic cerebellar degeneration. We are finding a lot more than one would expect. Is there another metabolizing system lurking in the brain that we are not seeing? Crabb: The compiled data in those Unigene sets will just say ‘brain’ without any further anatomical division. I don’t know whether it was libraries made from different regions all combined together, or whether it was whole brain. The first thing would be to go back and find existing data on an area of interest, or test for it in a specific area by measuring protein or RNA abundance. I think that Dr Deitrich is more able to answer the question about whether there are non-ADH generators of acetaldehyde than I am, because of what he published on catalase. Deitrich: We found that brain tissue will oxidise alcohol to acetaldehyde primarily through catalase, with some contribution from CYP2E1, but there’s still something that we can’t account for (Zimatkin et al 1999, 2001a,b, 2006, Zimatkin & Deitrich 1997, Gill et al 1992). I don’t know whether it is our deficiency in being able to measure those enzymes, or whether there is some other oxidative enzyme present. Person from the University of Washington thinks that there is another mechanism that is oxygen dependent which catalyses the formation of acetaldehyde from ethanol (Person et al 2000). It’s true that it’s a tiny amount compared with the liver, but it may be critical to the brain. Thornalley: I want to comment on the in situ reactivity of acetaldehyde in in vivo and in vitro experiments. In vitro experiment design can actually lead to studying acetaldehyde in a more activated state because a factor influential on aldehyde

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reactivity is binding to thiols in albumin. In most cell culture systems there is 10% serum, not 100% plasma as in vivo. The aldehyde has a lower binding to thiols extracellularly and is thereby activated. This is very important in the case of dicarbonyls. The in situ reactivity of dicarbonyls is increased 10-fold in medium with 10% serum in vitro compared to 100% plasma in vivo. In vivo these aldehydes are mostly reversibly bound to cysteine 34 in albumin. Albano: Returning to the issue concerning the genetic factors that might influence the susceptibility to alcoholic liver diseases, Zintzaras et al (2006) have recently published a meta-analysis showing that none of the polymorphisms of alcohol or acetaldehyde dehydrogenases-coding genes increase the risk of developing alcoholic cirrhosis. Also, in the case of CYP2E1, none of the polymorphisms so far characterized has been shown to have any influence on the risk of progression of alcoholic liver disease or an appreciable influence on ethanol metabolism (Zintzaras et al 2006, Hu et al 1997). Nonetheless, we have to consider that in humans there are phenotypic variances in the expression of CYP2E1. In a study performed together with the late Francois Mènez in a group of heavy drinkers from Brest (France) the functional evaluation of CYP2E1 activity by the chloroxazone oxidation test has shown that in about 20% of alcohol consumers CYP2E1 activity was not induced, despite high alcohol intake. In these subjects the formation of the hydroxyethyl radical was also significantly lower than in the patients with induced CYP2E1, and comparable to non-drinking controls (Dupont et al 1998). The presence of inter-individual differences in the inducibility of CYP2E1 has been subsequently confirmed by a study performed in Professor Seitz’ laboratory (Oneta et al 2002). The genetic basis of such phenotypic variability has not yet been established, but it is not dependent upon any of the SNPs so far detected in the CYP3E1 gene (Hu et al 1997). Its characterization might be important to provide more insight on genetic factors influencing alcohol toxicity because the low-inducible CYP2E1 phenotype is prevalent among the subjects with less severe liver disease. Seitz: We don’t see differences in genetics for liver disease and ADHs, but we may see differences with respect to other diseases such as cancer. Quite a number of studies did not show cancer in ADH1C homozygotes, but there are other studies including our own which showed an increased risk of certain cancers in ADH1C homozygotes, but not of liver disease. Shukla: You raised an interesting point about the differences between human liver cell lines versus the rat in terms of ADH activity. Some of the human cell lines have very poor ADH activity. One of the issues that comes to my mind is whether G2 cells also have similarly low ADH activity. Is this due to the nature of the transformed cell line? If one considers the normal human liver cells, is the metabolic capacity the same as the transformed human liver cell line? There may be an important difference here.

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Crabb: I think that is the case. The class I ADH expression is just as much a differentiated phenotype of a liver cell as making albumin or having these other functions. In cultured primary rat hepatocytes, ADH is lost fairly soon. If you want to use liver cell lines you need to test them to make sure they have the alcoholmetabolizing capacity you hope they do, or at least acknowledge the difference. Until we had measured alcohol metabolism and acetaldehyde generation in the experiments that I showed, we didn’t really know that much about the cells we had been working on for quite a long time. By learning that HeLa cells seem to be making acetaldehyde, we now have to double check what we are thinking when we say that if a certain phenomenon occurs in hepatoma cells but doesn’t occur in HeLa cells, that the explanation is that the phenomenon must be due to alcohol metabolism. We may have been misled. Eriksson: It is difficult to extrapolate to in vivo when we go to purer in vitro systems. You can see the difficulty. Based on enzyme kinetics in isolated pure systems the hepatic ratio between acetaldehyde and alcohol is surprisingly high to enable efficient alcohol oxidation. If you were to have a pure isolated system with only the ADH present there would be a shift in the oxidation/reduction equilibrium reaction towards alcohol at current in vivo conditions. Yet in vivo there is an alcohol oxidation taking place. This means that acetaldehyde is not in the same form in vivo and in vitro. The in vivo system is far more complicated.

References Caro AA, Cederbaum AI 2004 Oxidative stress, toxicology, and pharmacology of CYP2E1. Annu Rev Pharmacol Toxicol 44:27–42 Dupont I, Lucas D, Clot P, Ménez C, Albano E 1998 Cytochrome P4502E1 inducibility and hydroxyethyl radical formation among alcoholics. J Hepatol 28:564–571 Gill K, Menez JF, Lucas D, Deitrich RA 1992 Enzymatic production of acetaldehyde from ethanol in rat brain tissue. Alc Clin Exp Res 16:910–915 Hu Y, Oscarson M, Johansson I et al 1997 Genetic polymorphism of human CYP2E1: characterization of two variant alleles. Mol Pharmacol 51:370–376 Israel Y, Quintanilla ME, Sapag A, Tampier L 2006 Autosomal and maternal genes influence alcohol intake in alcohol drinker and nondrinker rat lines: role of the ‘acetaldehyde burst’. Alcohol Clin Exp Res 30:276A Oneta CM, Lieber CS, Li J et al 2002 Dynamics of cytochrome P4502E1 activity in man: induction by ethanol and disappearance during withdrawal phase. J Hepatology 36:47–52 Person RE, Chen H, Fantel AG, Juchau MR 2000 Enzymic catalysis of the accumulation of acetaldehyde from ethanol in human prenatal cephalic tissues: Evaluation of the relative contributions of CYP2E1, alcohol dehydrogenase, and catalase/peroxidases. Alcohol Clin Exp Res 24:1433–1442 Quintanilla ME, Tampier L, Sapag A, Israel Y 2005 Polymorphisms in the mitochondrial aldehyde dehydrogenase gene (Aldh2) determine peak blood acetaldehyde levels and voluntary ethanol consumption in rats. Pharmacogenet Genomics 15:427–431 Stewart S, Jones D, Day CP 2001 Alcoholic liver disease: new insights into mechanisms and preventative strategies. Trends Mol Med 7:408–413

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Zimatkin SM, Deitrich RA 1997 Ethanol metabolism in the brain. Addict Biol 2:387–392 Zimatkin SM, Liopo AV, Deitrich RA 1999 Oxidation of ethanol to acetaldehyde in brain and the possible behavioral consequences. Adv Exp Med Biol 463:231–236 Zimatkin SM, Liopo AV, Satanovskaya VI, Bardina LR, Deitrich RA 2001a Relationship of brain ethanol metabolism to the hypnotic effect of ethanol. II: Studies in selectively bred rats and mice. Alcohol Clin Exp Res 25:982–988 Zimatkin SM, Liopo AV, Slychenkov VS, Deitrich RA 2001b Relationship of brain ethanol metabolism to the hypnotic effect of ethanol. I: Studies in outbred animals. Alcohol Clin Exp Res 25:976–981 Zimatkin S, Pronko SP, Vasiliou V, Gonzalez FJ, Deitrich RA 2006 Enzymatic mechanisms of ethanol oxidation in the brain. Alcohol Clin Exp Res 30:1500–1505 Zintzaras E, Stefanidis I, Santos M, Vidal F 2006 Do alcohol-metabolizing enzyme gene polymorphisms increase the risk of alcoholism and alcoholic liver disease? Hepatology 43:352–361

Removal of acetaldehyde from the body Richard A. Deitrich, Dennis Petersen* and Vasilis Vasiliou* Department of Pharmacology, University of Colorado School of Medicine, University of Colorado, HSC, PO Box 6511, Mail Stop 8303, Aurora, CO 80045 and *Department of Pharmaceutical Sciences, University of Colorado School of Pharmacy, 4200 E. 9th Ave, C238 Denver, CO 80262, USA Abstract. The reduction of acetaldehyde back to ethanol via NAD-linked alcohol dehydrogenase is an important mechanism for keeping acetaldehyde levels low following ethanol ingestion. However, this does not remove acetaldehyde from the body, but just delays its eventual removal. Acetaldehyde is removed from the body primarily by oxidation to acetate via a number of NAD-linked aldehyde dehydrogenase (ALDH) enzymes. There are nineteen known ALDHs in humans, but only a few of them appear to be involved in acetaldehyde oxidation. There are many analogous enzymes in other organisms. Genetic polymorphisms of several ALDHs have been identified in humans that are responsible for several hereditary defects in the metabolism of normal endogenous substrates. The best known ALDH genetic polymorphism is in ALDH2 gene, which encodes a mitochondrial enzyme primarily responsible for the oxidation of the ethanol-derived acetaldehyde. This common polymorphism is known to dominantly inhibit its enzymatic activity resulting in reduced ability to clear acetaldehyde in both homozygote and heterozygote individuals. These individuals are generally protected against alcohol abuse but are susceptible to oesophageal cancer. For those enzymes that are capable of reacting with acetaldehyde, they may do so at the expense of their normal substrates, resulting in abnormal accumulation of these substrates. Examples of this are the aldehydes of the biogenic amines, dopamine, noradrenaline, adrenaline, serotonin and long chain lipid aldehydes such as nonenal. Not all of these enzymes are capable of efficient oxidation of acetaldehyde; however, it is possible that acetaldehyde may function as an inhibitor of these enzymes as well. The aldehydes whose metabolism is interfered with may also serve as inhibitors of ALDHs as well. However, this aspect of aldehyde function has not been extensively studied. A number of other mechanisms for the removal of acetaldehyde exist. For example, reaction of acetaldehyde with protein or nucleic acids is responsible for the disappearance of a small amount of acetaldehyde, but may be responsible for some pathological effects of acetaldehyde. There are a few other enzymes such as aldehyde oxidase, xanthine oxidase, cytochrome P450 oxidase and glyceraldehyde-3-phosphate dehydrogenase that are capable of oxidizing acetaldehyde. However, these enzymes account for only a small fraction of the total activity. 2007 Acetaldehyde-related pathology: bridging the trans-disciplinary divide. Wiley, Chichester (Novartis Foundation Symposium 285) p 23–51

Removal of acetaldehyde is essential to the metabolism of ethanol and critical to the survival of the animal. Ethanol could not be metabolized unless acetaldehyde 23

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was removed since the equilibrium constant is far in the direction of ethanol. Survival of the organism following ethanol intake depends on the removal of highly toxic acetaldehyde, either by reduction back to ethanol, oxidation to acetate, or removal via respiration, kidney excretion or protein binding. Acetaldehyde removal by reduction to ethanol: importance to toxicity of acetaldehyde While it is normally considered as the major producer of acetaldehyde from ethanol, the enzyme alcohol dehydrogenase can also serve an important function in limiting the accumulation of acetaldehyde since the equilibrium constant for ethanol to acetaldehyde is far in the direction of ethanol (Backlin 1958). In fact the ethanol concentration will be 5000 times greater than that of acetaldehyde in tissues that contain alcohol dehydrogenase, even if no acetaldehyde is removed by other means. This limits the amount of acetaldehyde present in those individuals with a deficiency of aldehyde dehydrogenase or in those who have ingested aldehyde dehydrogenase inhibitors. Acetaldehyde oxidation Aldehyde dehydrogenases (ALDHs) Mitochondrial ALDH (ALDH2). Early papers dealing with ALDH used various preparations of whole tissues such as acetone powders (Racker 1949), or whole tissue homogenates as well as a variety of substrates. These studies most likely dealt primarily with the mitochondrial enzyme since that enzyme is very active and has a low Km value. Later studies separated the enzymes on the basis of their tissue subcellular localization and their kinetic properties. It was not until the advent of molecular genetics that it became clear just how many ALDH enzymes there are (Sophos & Vasiliou 2003, Vasiliou & Nebert 2005, Vasiliou et al 1995). Early studies surveyed various tissues for NAD-dependent aldehyde oxidizing capacity utilizing indole-3-aldehyde as substrate because a sensitive assay could be developed by measuring the fluorescence of indole-3-acetic acid (Deitrich 1966). ALDH activity was observed in rat, rabbit, monkey and dog liver, lung and kidneys. ALDH activity was also found in the liver of other species such as cow, sheep and pig. The major work was in the rat where ALDH activity was observed in whole homogenates of liver, kidney, gonads, adrenal, brain, small intestine, heart, uterus and adipose tissue. Liver mitochondrial, cytosolic and microsomal fractions were found to have substantial ALDH activity; however, no enzymatic activity was observed in blood, perhaps because of a large blank value. An older review of the role of ALDHs in the removal of acetaldehyde from the body is available (Weiner 1979). The conclusion from this and other studies

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•LIVER ETHANOL 30,000 nmoles/gm

• ACETALDEHYDE • 200 nmoles/gm

(Eriksson, 1977)

FIG. 1. Liver metabolism of ethanol.

(Eriksson 1977, Eriksson & Sippel 1977) as well as a more recent review (Crabb et al 2004) is that the liver is primarily responsible for both generation and elimination of acetaldehyde. Only a small percentage of the acetaldehyde that is generated in the liver escapes into the blood. The mitochondrial ALDH enzyme has a very low Km value, of the order of 1 µM and, given the large amount of the enzyme, removes most of the acetaldehyde before it reaches the blood. However, this also demonstrates that the rate of production of acetaldehyde by the liver exceeds the ability of that organ to completely remove it (Fig. 1). The presence of the enzyme in liver, the site of the major production of acetaldehyde from ethanol, is protective of this and other organs of the body since relatively little acetaldehyde escapes. The presence of the enzyme in other tissues serves not only to protect these tissues from acetaldehyde borne by the blood or produced locally, but also to protect the tissues from other biogenic or exogenous aldehydes as well since the enzyme has broad substrate specificity (Allali-Hassani & Weiner 2001, Deitrich et al 1962, Petersen et al 1991, Wroczynski & Wierzchowski 2000, Yin et al 1995). On the other hand, acetaldehyde will compete with these other aldehydes (such as those from the biogenic amines, catechols amines and serotonin) for ALDH2 (Hellstrom & Tottmar 1982, MacKerell et al 1986, Nilsson 1988, Von Wartburg et al 1975). This inhibition may have behavioural consequences related to the actions of ethanol (Deitrich & Erwin 1980, 1975). Other endogenous substrates such as 4-hydroxynonenal also function as inhibitors of the enzyme (Hartley et al 1995a,b, Hartley & Petersen 1997, Honzatko et al 2005, Luckey et al 1999, Mitchell & Petersen 1991, Nguyen & Picklo 2003). Later studies showed that ALDH2 is located in the mitochondrial matrix (Tottmar et al 1973, Deitrich & Siew 1974, Siew & Deitrich 1976). Thus acetaldehyde, produced outside the mitochondria by alcohol dehydrogenase, cytochrome P450 or catalase must diffuse into the mitochondria in order to be oxidized by this enzyme. While the liver has the highest concentration of this enzyme in most animals, it exists in other tissues as well (reviewed in Agarwal et al 1989, Berkovitz et al 2001,

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Braun et al 1987, Harada et al 1980, Sladek 2002), including brain (Erwin & Deitrich 1966). A deficiency of ALDH2 in Asian populations is protective against development of alcoholism (Goedde et al 1992, Harada et al 1999, Xiao et al 1996, Yamamoto et al 2000) and perhaps carotid atherosclerosis (Narita et al 2003). However, this deficiency also has potentially severe consequences for those individuals who continue to consume ethanol and possibly in those that do not consume ethanol at all. There is a comprehensive analysis of the relationship between ADH1 and ALDH2 genotypes and head and neck cancer (Brennan et al 2004). The conclusion is that there is an increased risk factor for ALDH2-deficient individuals for head and neck cancer. There are also reports of increased incidence of cancer of the gastrointestinal tract (reviewed in Salaspuro 2003) in hepatocellular carcinoma (Kato et al 2003, Sakamoto et al 2005), oesophageal cancer (Morita et al 2005, Watanabe et al 2002) but not in colorectal adenomas (Hirose et al 2005). There is enhanced oxidative stress (Ohsawa et al 2003) perhaps related to late onset Alzheimer’s disease (Ohta et al 2004) although this is disputed (Kim et al 2004, Shin et al 2005). There has been a meta analysis of the relationship of inactive ALDH2 genotype with maternal inheritance of diabetes (Murata et al 2004). The conclusion was that there is an association between ALDH2 inactivity and maternal inheritance. The authors proposed an interesting hypothesis to explain this finding. ‘ALDH2 is the key enzyme for detoxifying the various aldehydes such as, not only acetaldehyde, but also 4-hydroxy-2-nonenal (4-HNE). 4-HNE is the by-product of lipid peroxidation. Therefore, in a chronic situation of diabetes (in the mother), increased tissue levels of 4-HNE could result from inactive ALDH2. Because 4-HNE reacts preferentially with sulfhydryl (SH)-containing molecules, the mitochondrial inner membrane is easily adducted with 4-HNE. The mitochondrial inner membrane adducted by 4-HNE alters the electron transfer chain system, which may increase free radicals production, leading to the production of mitochondrial DNA damage. The acquired mitochondrial DNA damages can be inherited exclusively from mother to offspring, and in the offspring, the genetic handicap may lead to the maternal inheritance of diabetes.’ This study points out that ALDH2 is normally responsible for the oxidation of many endogenous aldehydes. If the enzyme activity is compromised either by the presence of inhibitors or for genetic reasons, these aldehydes must be dealt with in other ways, such as reduction to the alcohol, oxidation by other ALDH enzymes or by reaction with proteins and nucleic acids. It is the latter possibility that may be responsible for damage even in the absence of exogenous acetaldehyde. The availability of Aldh2 knockout mice (Isse et al 2002) should help us to work out the mechanisms of these effects. It is known that mitochondria are damaged by chronic ethanol ingestion (Lieber 1997). Inhibition of ALDH in mitochondria could be accomplished by ‘mutually

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27 ACETATE

ACETALDEHYDE ALDH NONENAL

NONEONIC ACID

FIG. 2. Inhibition of ALDH in mitochondria can be accomplished by ‘mutually assured destruction’ by 4-HNE and acetaldehyde since they are competitive substrates and inhibit each other’s oxidation by ALDH.

assured destruction’ by 4-HNE and acetaldehyde (Fig. 2) since they are competitive substrates and inhibit each other’s oxidation by ALDH. In addition 4-HNE is a potent inhibitor as well as a substrate for ALDH2 (Doorn et al 2006). In spite of the studies on ALDH2 and the relationship between acetaldehyde and other endogenous aldehydes, studies have been largely confined to those enzymes where acetaldehyde is known to be a substrate. While a great deal is now known about the myriad of ALDH enzymes, relatively little is known about the effect of acetaldehyde on those ALDH enzymes for which acetaldehyde is not a substrate or a very poor substrate because of very high Km values. For many of these enzymes we do not know if acetaldehyde functions as an inhibitor since it is almost never studied in this light. Of course, it is necessary to measure the acid product concentration of the aldehyde substrate, not just the NAD(P)H produced when both aldehydes are added to the reaction mixture. Acetaldehyde certainly has the structure to function as a competitive inhibitor at the active site of ALDHs, even if it, for some reason, is not a substrate. While one would expect that the Km of acetaldehyde as a substrate would be the same as the Ki for acetaldehyde when it functions as a competitive substrate, this also has not been studied carefully. This consideration may be important in our understanding not only of the pathways of acetaldehyde removal, but also of the many effects that acetaldehyde has via the surrogate aldehydes allowed to escape metabolism by their preferred enzyme. This would result in a ‘multiplying’ or ‘amplification’ effect of the relatively small amount of acetaldehyde present. Other ALDHs There are several sources of information concerning the numerous ALDH enzymes in humans and other organisms (Vasiliou et al 1995, 1999, Vasiliou & Nebert 2005). In the tables that follow we have largely restricted the material to human ALDH enzymes with some reference to rat and mouse enzymes. It can be seen in Tables 1 and 2 that the myriad of aldehyde dehydrogenase enzymes outside of ALDH2 are unlikely to play a significant role in the direct removal of acetaldehyde from the body. However, they may play a role in the toxic effects

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TABLE 1 ALDH enzymes for which acetaldehyde (or propionaldehyde) has been reported to be a substrate Approved gene symbol

Trivial name

Km/Ki (mM) acetaldehyde

Endogenous substrate(s) Km (mM)

Exogenous substrates Km (mM)

Notes. Consequences of inhibition

ALDH1A1

ALDH1

50, 118

Retinal, 4-HNE

Aldophosphamide 2; 7-methoxy-1napthaldehyde; 0.85; 6-methoxy2-napthaldehyde; 100 mg/kg intraperitoneally) into mice for 10 days and they seemed fine. But we noticed that these mice showed a severe anterograde amnesia. The mice were unable to learn new tasks, especially operant tasks. Perhaps there is a toxic effect of acetaldehyde on the brain that has yet to be investigated. Is there a reason to believe that damage to the brain can be caused by acetaldehyde? Or should we be looking at an effect on the liver with a consequence for brain functioning. Deitrich: Ostrowska et al (2004) have shown that the 4-HNE levels in the brain are much higher than in the liver. Emery: Did you measure the levels of any of the other aldehydes in your animals? Quertemont: No. Thornalley: In physiological situations in which there is oxidative stress, the physiological situation does not stay unresponsive. It responds by inducing genes with antioxidant response elements in their promoters. One of the activators of this transcription factor-mediated event is 4-HNE. If this hypothesis is correct, we would expect to see alcoholism associated with a burst of induction of antioxidant response element-linked gene expression. Is there any evidence for this? Crabb: There is in the cell model that Art Cederbaum has used. These cells have CYP2E1 expressed, and most of the expected antioxidant gene battery was turned on. He was following levels of glutathione and worked backwards to this. Thornalley: You mentioned that acetaldehyde can be reversed to ethanol. Which enzymes are involved? Are aldoketo reductases involved?

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Deitrich: No, just plain old ADH. I have a question for David Crabb on the product inhibition of ADH by acetaldehyde. Is this a real dead-end product inhibition, or is it reversal of the reaction? Crabb: Kinetically speaking, it is not the equilibrium term; rather it is due to competitive product inhibition. This is why you get different answers for the different enzymes. If it was just a matter of reversing the equilibrium, it should apply to all of the enzymes, since the equilibrium is independent of the kinetics of the enzymes. Apte: Is the protective role of decreased ALDH2 due to the fact that these people drink less since they avoid alcohol because of its unpleasant side-effects? Eriksson: The heterozygotes do drink. They can become alcoholics. They are much more likely to develop cancers because of increased acetaldehyde exposure: the odds ratio is very much increased. With regard to liver damage the situation is less clear. Someone mentioned earlier this new meta analysis that shows that in Asians there isn’t a relationship. I did a similar meta analysis some years ago and found an association. Thinking about the telescoping effect, acetaldehyde is euphoric as well as being toxic. This can be seen in any restaurant in Japan or China. The flushers become very happy, just as non-flushers become with much higher doses of alcohol. There is no other reason than the acetaldehyde. Crabb: The group in San Diego has changed my way of looking at acetaldehyde. The story used to be that acetaldehyde makes you feel sick so you don’t drink. Now it seems that you are at most risk of becoming alcoholic when you don’t respond much to drinking, whether this is because of something you inherited, or some other reason (Luczak et al 2002). Eriksson: This is a different aspect. It is well known that people who are tolerant of alcohol have a higher risk of becoming alcoholics, but this could be simply explained by the fact that those who are tolerant drink more. The acetaldehyde connection is a different issue. Apte: You mentioned that there is acetaldehyde in different alcoholic beverages. Do different beverages affect people differently? Eriksson: This has been studied, but people doing epidemiological investigations find that people who drink wine or spirits tend to have different lifestyles and personalities, especially in northern countries. The studies done with different beverages are quite confounded. A lot of these complicating factors haven’t been acknowledged. Deitrich: David Crabb raised the issue of disulfiram (Antabuse). At least in the USA judges hand out disulfiram like candy. Alcoholics who offend often have the choice of going to jail or taking Antabuse. The medical profession should look into this because the people taking Antabuse will be in essence ALDH negative. The assumption is that they are not going to be drinking alcohol, but the drug also inhibits the ALDH for all these other substrates.

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Preedy: As I understand it, the ALDH2 inhibitors can have quite severe long-term side effects. This could be due to the drug, or because this knocks out the other enzymes that the tissue needs for its normal metabolism. Deitrich: It is not an innocuous drug. Eriksson: If we want to know about the consequences of knocking out ALDH2, 7% of the Asian population have this genotype. We have recently published a study about the Chinese population (Luo et al 2005). There wasn’t really any difference in factors such as mortality. It would be interesting to look at some of the rarer syndromes. They could be enriched to a degree that they could be picked up in a population study. Preedy: A study published some years ago (Ohta et al 2004) looked at the ALDH2deficient subjects, and hypothesized that because of the deficiency in this enzyme they had a higher risk of other diseases, such as Alzheimer’s. They related this to the role of ALDH2 in the oxidation of other aldehydes. Eriksson: On the general mortality level, we don’t find a significant effect of ALDH2. On the other hand, there may be negative effects which are balanced out by positive effects. If you are born with a deficient ALDH you don’t become an alcoholic, for example. There could be a combination of plus or minus effects. Deitrich: The difference between ALDH-negative subjects and people treated with Antabuse is that Antabuse has many other effects. It hits many of the other ALDHs. Crabb: And CYP2E1. All our inhibitors are turning out to be non-specific. Rao: If I remember correctly, did you say that alcoholic beverages have millimolar concentrations of acetaldehyde? Eriksson: There is a huge variation. The maximum level is 5 mM, which is unbelievably high. Rao: In tissues we are talking about levels one thousandth of this. Is the acetaldehyde level in some of these alcoholic drinks a matter of real concern? Eriksson: It is an interesting question. Where is the cancer formed? It is formed in the mouth and upper digestive tract—places that come into contact with the beverages. Alcohol doesn’t so much increase the other forms of cancer that come from deeper inside. It has never been surveyed well epidemiologically because of the confounding factors. The myth is that the worst alcoholic drinks are vodka because of its strength, but perhaps the truth is that those alcoholic beverages with high acetaldehyde levels could be worse for you. M Salaspuro: There is some epidemiological evidence coming from France. In Normandy, it has been common for people to drink a lot of Calvados together with hot coffee. After adjustment for other alcoholic beverages this habit appeared to explain almost half of the peak incidence of oesophageal cancer in the Northwest of France compared to the regions with lower oesophageal cancer risk (Launoy et al 1997). We have recently measured the acetaldehyde concentration of Calvados

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produced by factories and also made in small farms. The acetaldehyde level of these beverages was in the millimolar range. This is the only epidemiological evidence we have so far. There are many other food products that also have considerable levels of acetaldehyde. Companies making dairy products sometimes try to increase the acetaldehyde level in order to give a special aromatic taste. One example is yoghurt, where some have acetaldehyde levels as high as 800 µmoles/l. Apte: Did the Calvados study tease out smoking levels? M Salaspuro: Yes, it was one of the possible confounding factors that was excluded. Aranda: Acetaldehyde is an off flavour in wine caused by ethanol oxidation, and producers aim to keep it as low as possible. In sherry wines, however, the amount is pretty high at around 300–400 mg per litre (7–9 mM), because these wines are made in an oxidative style. The process of making these wines results in high levels of acetaldehyde that are good for the organoleptic properties of these wines. Seitz: Dr Salaspuro pointed out that acetaldehyde is present in certain beverages. However, the concentration of alcohol may also be important. 40% alcohol may have a local toxic effect, leading to mucosal hyper-regeneration. In this situation biogenic amines such as spermidine and spermine are produced. In the presence of these amino acids there is increased formation of acetaldehyde DNA adducts with mutagenic and carcinogenic properties. Thus, with high concentrated alcohols, hyper-regeneration and the production of certain acetaldehyde DNA adducts are favoured. Emery: Yes, we can’t just focus on alcohol. Perhaps we need to think of acetaldehyde coming from sources other than alcohol, too. Okamura: I’d like to add some information from a Japanese study concerning beverage types (Okamura et al 2004). The effect on serum lipids and blood pressure level is the same across beverages for the same level of ethanol intake. In Japanese populations, wine drinkers show the highest serum level of cholesterol because their eating pattern is quite different. We have some cohort study data concerning the relationship between ALDH2 genotypes and total mortality (personal communication). We don’t see a significant difference among ALDH2 genotypes at the same alcohol drinking level. In non-drinkers the mortalities are quite similar. Shukla: How do some of the contaminants that are found in alcoholic drinks, such as methanol, affect the ALDH levels? Would the metabolism of surrogate alcohols increase the toxicity? Deitrich: They certainly would, but I have no idea what the Km values would be for methanol to formaldehyde. Formaldehyde is a poor substrate for ALDH2. The other longer chain aldehydes are reasonably good substrates until you get to very long chain; then the fatty aldehyde dehydrogenase takes over and metabolizes those.

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Shukla: I remember that isopentanol, for example, has been a problem as a contaminant for some alcoholic drinks. Deitrich: I have a question about the acatalasaemic individuals that David Crabb mentioned. There is an acatalasaemic mouse, but it is missing catalase only in the blood. The levels in the brain are about 50% and the kidney and liver are normal. What is the situation with humans? What are the catalase levels in other tissues in acatalasaemic humans? Okamura: I major in epidemiology, so I can’t answer some of these questions about the more basic science. At least in humans aged 40 and over, we don’t observe significant differences in the relationship of some genotypes such as ALDH2 and mortality or other risk factors (Amamoto et al 2002). Eriksson: If you look at people dying aged 40 years, the cause of death at such an early age will bear a high relationship with alcohol consumption. If you don’t see such a relationship in Japan, then there is something strange. The ALDH2 homozygous deficient people are non-drinkers, and if they still die at the same frequency aged 40, we should investigate this. Okamura: We need more information about early death due to alcohol drinking in Asia. It is difficult to perform this sort of study in some social groups, but it is a good suggestion. I will try to analyse some data sets of cohort study with information of ALDH2 genotypes. However, the life expectancy in Japan at age 20 is longer than almost all other developed or developing countries. This suggests that there is no burden on life expectancy in Japan by ALDH2 deficit. Niemelä: Earlier on we discussed acetaldehyde toxicity and its association with oxidative stress. Studies both in experimental models and in humans have indicated that high fat diets or excess iron modulate the generation of acetaldehyde and associated tissue damage. It is quite possible that in humans, conditions such as obesity or diabetes also create the status of enhanced oxidative stress thereby adding to acetaldehyde toxicity in alcohol consumers. Apte: I’d like to be a bit provocative about the catalase issue. It’s my understanding that catalase plays a minor role in alcohol oxidation. Would it matter if people are catalase deficient, and is there any point in doing a large study on this? Deitrich: Catalase is the major alcohol oxidizing enzyme in the brain. Catalasedeficient individuals might have interesting things happening in the brain. Brain doesn’t have very much ALDH, either. The issue here is mainly one of CNS effects of alcohol as related to acetaldehyde production in the brain via catalase and CYP2E1. Eriksson: So the point is that catalase may matter, because if the underlying causation is the alcohol metabolism in the brain, then those systems that are specifically working in the brain are of great interest. Worrall: Catalase is 90–95% of the metabolic capacity in certain areas of the brain. CYP2E1 is vanishingly small.

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Emery: Presumably, in catalase-deficient animals other enzymes would be up-regulated. This would be different from acute pharmacological effects. Jamal: I have done these experiments looking at the effects of catalase, ADH and ALDH inhibitors on brain acetaldehyde concentrations after ethanol dosing, which we measured with in vivo microdialysis in the striatum of free-moving rats. Catalase has an effect, but it doesn’t make a major contribution. High peripheral acetaldehyde levels may be able to cross the blood–brain barrier and may produce a greater contribution to brain acetaldehyde accumulation. When there is more than 70 µM acetaldehyde in the blood it does cross the blood–brain barrier. Other enzymes such as CYP2E1 and ADH would be up-regulated in catalase-deficient animals, but it is not clear yet. It is well-established that >90% of ethanol metabolism occurs via ADH in the liver. CYP2E1, a microsomal oxidative enzyme, has a small contribution to the total metabolism (about 2 or 3%) in acute ethanol ingestion in the brain. Eriksson: Many years ago we investigated the break point, when acetaldehyde is also found in the brain. You mentioned 70 µM. This may be quite close to the cut-off point after which you will have increasing concentrations in the brain. However, under this level, the capacity for removing acetaldehyde at the blood– brain barrier is very efficient. However, there are parts of the brain where there isn’t such an efficient barrier, such as the hypothalamic area. In those individuals who are heterozygotes acetaldehyde can get up to levels which get into the brain. Under normal conditions we don’t find so much except maybe in the hypothalamic area. Worrall: Isn’t there some evidence that high blood alcohol levels make this barrier leaky? High acetaldehyde plus high alcohol might lead to more acetaldehyde getting in. Eriksson: That’s an interesting possibility. To my knowledge we don’t have any human data on this. Worrall: I think there’s some literature on this (see for example Haorah et al 2005). References Albano E 2002 Free radicals and alcohol-induced liver injury. In: Sherman CDIN, Preedy VR, Watson RR (eds) Ethanol and the liver. Taylor and Francis, London, p 153–190 Albano E, Clot P, Comoglio A, Dianzani MU, Tomasi A 1994 Free radical activation of acetaldehyde and its role in protein alkylation. FEBS Lett 348:65–69 Alderman J, Kato S, Lieber CS 1989 The microsomal ethanol oxidizing system mediates metabolic tolerance to ethanol in deermice lacking alcohol dehydrogenase. Arch Biochem Biophys 271:33–39 Amamoto K, Okamura T, Tamaki S et al 2002 Epidemiologic study of the association of low-Km mitochondrial acetaldehyde dehydrogenase genotypes with blood pressure level and the prevalence of hypertension in a general population. Hypertens Res 25:857–864

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Arteel GE 2003 Oxidant and antioxidant in alcohol-induced liver disease. Gastroenterology 124:778–790 Bailey SM, Cunningham CC 2002 Contribution of mitochondria to oxidative stress associated with alcohol liver disease. Free Radic Biol Med 32:11–16 Burnett KG, Felder MR 1978 Genetic regulation of liver alcohol dehydrogenase in peromyscus. Biochem Genet 16:443–454 Caro AA, Cederbaum AI 2004 Oxidative stress, toxicology and pharmacology of CYP2E1 Ann Rev Pharmacol Toxicol 44:27–42 Doorn JA, Hurley TD, Petersen DR 2006 Inhibition of human mitochondrial aldehyde dehydrogenase by 4-hydroxynon-2-enal and 4-oxonon-2-enal. Chem Res Toxicol 19:102–110 Haorah J, Knipe B, Leibhart J, Ghorpade A, Persidsky Y 2005 Alcohol-induced oxidative stress in brain endothelial cells causes blood-brain barrier dysfunction. J Leukocyte Biol 78:1223–1232 Hoek JB, Cahill A, Pastorino JG 2002 Alcohol and mitochondria: a dysfunctional relationship. Gastroenterology 122:2049–2063 Koll M, Beeso JA, Kelly FJ et al 2003 Chronic alpha-tocopherol supplementation in rats does not ameliorate either chronic or acute alcohol-induced changes in muscle protein metabolism. Clin Sci (Lond) 104:287–294 Launoy G, Milan C, Day NE, Faivre J, Pienkowski P, Gignoux M 1997 Oesophageal cancer in France: potential importance of hot alcoholic drinks. Int J Cancer 71:917–923 Luczak SE, Elvine-Kreis B, Shea SH, Carr LG, Wall TL 2002 Genetic risk for alcoholism relates to level of response to alcohol in Asian-American men and women. J Stud Alcohol 63:74–82 Luo HR, Israel Y, Tu GC, Eriksson CJ, Zhang YP 2005 Genetic polymorphism of aldehyde dehydrogenase 2 (ALDH2) in a Chinese population: gender, age, culture, and genotypes of ALDH2. Biochem Genet 43:223–227 Morris JB 1997 Dosimetry, toxicity and carcinogenicity of inspired acetaldehyde in the rat. Mutat Res 380:113–124 Nordmann R, Ribière C, Rouach H 1992 Implication of free radical mechanisms in ethanol induced cellular injury. Free Radic Biol Med 12:219–240 Ohta S, Ohsawa I, Kamino K, Ando F, Shimokata H 2004 Mitochondrial ALDH2 deficiency as an oxidative stress. Ann NY Acad Sci 1011:36–44 Okamura T, Tanaka T, Yoshita K et al 2004 Specific alcoholic beverage and blood pressure in a middle-aged Japanese population: the High-risk and Population Strategy for Occupational Health Promotion (HIPOP-OHP) Study. J Hum Hypertens 18:9–16 Ostrowska J, Luczaj W, Kasacka I, Rózanski A, Skrzydlewska E 2004 Green tea protects against ethanol-induced lipid peroxidation in rat organs. Alcohol 32:25–32 Reilly ME, Patel VB, Peters TJ, Preedy VR 2000 In vivo rates of skeletal muscle protein synthesis in rats are decreased by acute ethanol treatment but are not ameliorated by supplemental alphatocopherol. J Nutr 130:3045–3049 Shaw S, Jayatilleke E 1990 The role of aldehyde oxidase in ethanol-induced hepatic lipid peroxidation in the rat. Biochem J 268:579–583

Acetaldehyde, polymorphisms and the cardiovascular system Shih-Jiun Yin and Giia-Sheun Peng* Departments of Biochemistry and *Neurology, National Defense Medical Center, 161 Min-Chuan East Road Section 6, Taipei 114, Taiwan

Abstract. To date, the only genes that have been consistently replicated across racial and ethnic groups to influence alcoholism vulnerability are polymorphisms in the alcoholmetabolizing enzymes, i.e. cytosolic alcohol dehydrogenase 1B (ADH1B) and mitochondrial aldehyde dehydrogenase 2 (ALDH2). Both the variant ADH1B *2 and ALDH2 *2 alleles significantly protect against developing alcoholism. The protection has been thought to result from accumulation of acetaldehyde after drinking. Unlike ALDH2 *2, direct correlation between ADH1B *2 and blood acetaldehyde has not been verified. ALDH2 *2/ *2 homozygosity appeared to almost completely protect against alcoholism, whereas ALDH2 *1/ *2 heterozygosity appeared to reduce risk of the disease only about threefold. Direct correlations of blood ethanol and acetaldehyde concentrations, cardiovascular haemodynamic responses, and the subjective perceptions after challenge with low (0.2 g/kg) to moderate (0.5 g/kg) alcohol in individuals with different ALDH2 genotypes support the notion that full protection against alcoholism by ALDH2 *2/ *2 may derive from either abstinence or deliberate moderation in alcohol consumption due to strong discomfort from physiological and psychological responses caused by persistently elevated blood acetaldehyde after ingestion of a small amount of alcohol, and that the partial protection by ALDH2 *1/ *2 can be ascribed to significantly lower acetaldehyde build-up in blood and the according adverse reactions. 2007 Acetaldehyde-related pathology: bridging the trans-disciplinary divide. Wiley, Chichester (Novartis Foundation Symposium 285) p 52–68

Alcoholism is a multifactorial, polygenic behavioural disorder involving complex gene–gene and gene–environment interactions (Li 2000). The pharmacological and toxicological effects of ethanol depend on the duration of exposure and the concentrations of ethanol and its metabolite acetaldehyde in body fluids and tissue. Alcohol dehydrogenase (ADH; EC 1.1.1.1, alcohol:NAD+ oxidoreductase) and aldehyde dehydrogenase (ALDH; EC 1.2.1.3, aldehyde:NAD+ oxidoreductase) are the principal enzymes responsible for ethanol metabolism in humans, catalysing the conversion of ethanol to acetaldehyde, then acetaldehyde to acetate, respectively (see this volume: Crabb & Liangpunsakul 2007, Deitrich et al 2007). Interestingly, both ADH and ALDH exhibit genetic polymorphisms, which may 52

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influence drinking behaviour as well as vulnerability to development of alcohol dependence (Yin & Peng 2005, Yin et al 2006). In this chapter, we present the distribution of functional allelic variations of the alcohol-metabolism genes among racial populations, the protection against developing alcoholism by variant alleles ADH1B *2 and ALDH2 *2 in East Asians, and the pharmacokinetic and pharmacodynamic consequences in individuals with different ALDH2 genotypes following a low to moderate dose of ethanol, with emphasis on cardiovascular haemodynamic effects and the subjective perceptions. This work provides a physiological perspective for the roles of acetaldehyde in full protection by ALDH2 *2/ *2 homozygosity versus partial protection by ALDH2 *1/ *2 heterozygosity against development of alcoholism. Experimental procedures Recruitment of subjects and diagnosis of alcohol dependence were as described previously (Chen et al 1999a,b, Peng et al 1999, 2002). Determination of functional single-nucleotide polymorphic sites at the ADH1B, ADH1C and ALDH2 genes was carried out as described previously (Chen et al 1999a). Blood ethanol was determined by gas chromatography and blood acetaldehyde was determined as a fluorescent adduct by high-performance liquid chromatography (Peng et al 1999). Parameters for cardiac function before and after ingestion of alcohol were measured in the supine position by M-mode, two-dimensional Doppler echocardiography (Peng et al 1999). Systolic and diastolic pressure of the left brachial artery was measured using a sphygmomanometer. Extracranial and intracranial arterial blood flow were measured by Doppler ultrasonography (Peng et al 1999). Assessment of subjective responses to alcohol was performed by slight modification of the Subjective High Assessment Scale (SHAS) by Schuckit (1984) as described previously (Peng et al 1999). The chi-square test and logistic-regression analysis for genotype data were performed using the SPSS statistics program (SPSS, Chicago, USA). The pharmacokinetic and pharmacodynamic data were analysed by SPSS one-way ANOVA with the Scheffe’s test or with the Tamhane’s T2 test and the subjective self-ratings were evaluated by SPSS nonparametric Mann– Whitney test (Peng et al 1999). Values for the data are expressed as mean ± SEM. Results and discussion Functional polymorphism and alcoholism Human ADH constitutes a complex enzyme family (Duester et al 1999, Yin et al 2006). Class I ADH1A, ADH1B, and ADH1C, and class II ADH contribute to

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hepatic metabolism of ethanol, whereas Class I ADH1C and Class IV ADH participate in the gastric metabolism (Lee et al 2006). Class III ADH may be involved in gastrointestinal first-pass metabolism at high ethanol concentrations (Lee et al 2003, 2006). Allozymes ADH1B2 and ADH1B3, encoded by variant alleles ADH1B *2 and ADH1B *3 respectively, exhibit much higher activity than that of ADH1B1. ADH1C1, encoded by ADH1C *1, exhibits a higher activity than ADH1C2. Table 1 shows the distribution of ADH1B and ADH1C gene alleles in racial populations. ADH1B *1 is a predominant allele among the Caucasians, African Americans and American Indians, whereas ADH1B *2 is predominant among the East Asians including Han Chinese, Japanese, Koreans, and Taiwanese aborigines. ADH1B *3 occurs uniquely in the African populations (Osier el al 2002). It is interesting to note that among Caucasians, the Jews and Russians appear to have considerably higher frequencies of ADH1B *2, ∼20% (Hasin et al 2002) and 41% (Ogurtsov et al 2001), respectively. ADH1C *1 is predominant among the East Asians, African Americans and Taiwanese aborigines, but ADH1C *1 and ADH1C *2 are about equally distributed among the Caucasians and American Indians. Cytosolic ALDH1 and mitochondrial ALDH2 are the principal members of ALDH superfamily responsible for oxidation of acetaldehyde in mammals (this volume: Deitrich et al 2007). There is a common point mutation of the ALDH2 gene, denoted ALDH2 *2, which results in a glutamic acid to lysine substitution (Agarwal & Goedde 1992). Allele frequencies of ALDH2 *2 are relatively high in the Vietnamese (35%), Han Chinese (24%), Japanese (24%), and Koreans (16%) (Chen et al 1994, Higuchi et al 1995, Lee et al 1997, Chen et al 1999a), but rare in other ethnic groups including Caucasians, black Americans, American Indians, and TABLE 1 Allele frequencies of ADH1B and ADH1C in racial populations Allele frequency Population East Asians Caucasians African Americans American Indians Taiwanese Aborigines

ADH1B *1

ADH1B *2

∼0.30 ∼0.90

∼0.70 ∼0.10

0.84 0.93

0 0.01

0.09

0.91

ADH1B *3

ADH1C *1

ADH1C *2

∼0.90 ∼0.60

∼0.10 ∼0.40

0.16 0.06

0.85 0.56

0.15 0.44

0

0.99

0.01

0 0

Data are taken from Agarwal & Goedde (1992), Borràs et al (2000), Burnell & Bosron (1989), Chen et al (1999a), Higuchi et al (1995), Thomasson et al (1994) and Wall et al (1997). The ADH1B and ADH1C genes were previously denoted ADH2 and ADH3, respectively. For new nomenclature of human ADH gene family, see Duester et al (1999).

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Polynesians (Yin & Peng, 2005). Interestingly, autopsy livers from individuals homozygous for ALDH2 *2 or from those heterozygous for ALDH2 *2 and ALDH2 *1 lacked ALDH2 activity on starch gel electrophoresis, suggesting a dominance on loss of the enzyme activity by the variant allele (Crabb et al 1989). Kinetic and x-ray crytallographic studies support a partial dominance model of a dimer-ofdimers molecule (Larson et al 2005). The model predicts that residual activities of ALDH2 in mutant homozygotes and the heterozygotes would account for 0% and 25%, respectively, of the total activity in normal homozygotes. A recent study with surgical liver specimens reported that a considerably lower ALDH2 activity was found in the heterozygotes as compared to that in normal homozygotes, and that no measurable activity of the enzyme was found in mutant homozygotes (Wang et al 2002). We have genotyped allelic variations of ADH1B, ADH1C and ALDH2 from leukocyte DNA samples of 340 Han Chinese alcohol-dependent subjects and 545 non-alcoholic controls in Taiwan (Chen et al 1999a). The frequency of ADH1B *2 allele was significantly lower in alcoholics than that in controls, suggesting a protection against alcoholism by the variant allele. The ADH1C *1 allele frequency was also found significantly decreased in the alcoholic group but further haplotype analysis indicated that this is virtually due to linkage disequilibrium between the ADH1C *1 and ADH1B *2 alleles in ADH gene cluster on chromosome 4. The frequency of ALDH2 *2 variant allele decreased threefold in alcoholics compared to that in controls (Chen et al 1999a). Strikingly, no single alcoholic patient with homozygous ALDH2 *2/ *2 was found in this study. The reduction of risk for alcoholism by ADH1B *2 has also been found in the Japanese (Higuchi et al 1995), Atayal Natives of Taiwan (Thomasson et al 1994) and Caucasians (Borràs et al 2000, Ogurtsov et al 2001, Hasin et al 2002). Recent haplotype/diplotype studies examining a battery of single-nucleotide polymorphisms across the seven ADH genes in Caucasian alcoholics and controls indicate that class II, III and IV ADH genes may also independently influence susceptibility to alcohol dependence (Edenberg et al 2006, Luo et at 2006). The underlying functional causality for these candidate genes, however, remains unclear. It is noteworthy that Higuchi et al (1994) surveyed 1300 Japanese alcoholdependent subjects and found no single patient identified with ALDH2 *2/ *2. On the basis of genotype frequencies in the Japanese general population, it is expected 118 of the above alcoholic individuals would be ALDH2 *2 homozygotes. Thus, genetic epidemiological studies strongly suggest that homozygosity of variant ALDH2 *2 may fully protect against alcoholism both in Japanese and Han Chinese (Table 2). Intriguingly, the frequency of heterozygous ALDH2 *1/ *2 genotype varied from 2.5 to 13% for the Japanese alcoholics and from 10 to 18% for the Han Chinese alcoholics surveyed from different registry periods with a trend of higher frequency in more recent years (Table 2). This finding suggests that the heterozygos-

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TABLE 2 Protection against alcoholism by heterozygosity of ALDH2*1/*2 in East Asians Genotype frequency Alcoholics Japanese Han Chinese

Patients (n)

Registry period

ALDH2*1/*1

ALDH2*1/*2

ALDH2*2/*2

400 400 500 49 291

1979 1986 1992 1989–90 1994–7

0.975 0.92 0.87 0.90 0.82

0.025 0.08a 0.13b 0.10 0.18

0 0 0 0 0

Data for the Japanese and Han Chinese alcohol-dependent patients are taken from Higuchi et al (1994) and Chen et al (1999a), respectively. a P < 0.001 (1986 versus 1979); b P < 0.005 (1992 versus 1986).

ity can only afford partial protection against alcoholism, permitting other biological and environmental factors to have an influence. Unexpectedly, one case of a homozygous ALDH2 *2 alcoholic patient was discovered in a survey study in Taiwan (Chen et al 1999b). This patient displayed a unique drinking pattern to accommodate his inborn error of acetaldehyde metabolism, i.e. (a) drinking beer, instead of wine or spirit liquors, as his favourite alcoholic beverage; (b) sipping alcoholic beverages almost continuously throughout the day rather than fast, binge drinking; (c) consuming relatively low amounts of alcohol with three to five bottles (i.e. 350 ml of 4.5% by volume of ethanol or 12.4 g of ethanol per bottle) of beer per day. To address the question of interaction between functional polymorphisms of ADH1B and ALDH2 in protecting against alcoholism, we performed logistic regression analysis of the combinational genotypes for Han Chinese alcohol-dependent and control subjects (Chen et al 1999b). The risks for alcoholism are in the following order:ADH1B*2/*2–ALDH2*2/*2 0.05 vs. medium stimulation. (B) Acetaldehyde-induced NF-κB nuclear translocation. Representative micrographs from eight independent experiments are shown.

airway inflammation (Fig. 5). Although the underlying mechanism is yet to be determined, it is possible that acetaldehyde-induced GM-CSF could activate inflammatory cells including eosinophils, macrophages and dendritic cells. Concluding remarks Collectively, our experimental results indicate that acetaldehyde has potential effects on human airway by two distinct mechanisms. As a metabolite of alcohol, its elevation following alcohol consumption induces airway mast cells to release histamine, which results in exacerbation of asthma in susceptible populations. As an air pollutant contained in cigarette smoke, for example, its inhalation potentially increases airway inflammation. Acknowledgements This study is supported by a grant-in-aid for scientific research (No. 12670563) by the Japanese Society for the Promotion of Science, and a grant from the Kanae Foundation for Life & Socio-Medical Science.

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FIG. 5. Pulmonary pathology of (A) control, (B) acetaldehyde inoculated, (C) mite allergen sensitized, (D) mite allergen sensitized and acetaldehyde inoculated BALB/c mice. Representative photomicrographs of each group (n = 4) are shown.

References Dahl R, Henriksen JM, Henning H 1986 Red wine asthma, a controlled challenge study. J Allergy Clin Immunol 78:1126–1129 Enomoto N, Takada A 1990 Acetaldehyde metabolism and aldehyde dehydrogenase 2 gene. J Exp Med 154:823–828 Harada S 1990 Racial and genetic factors in ethanol and aldehyde metabolism. J Exp Med 154:817–822 Kawano T, Matsuse H, Kondo Y et al 2004 Acetaldehyde induces histamine release from human airway mast cells to cause bronchoconstriction. Int Arch Allergy Immunol 134:233–239 Koivisto T, Kaihovaara P, Salaspuro M 1999 Acetaldehyde induces histamine release from purified rat peritoneal mast cells. Life Sci 64:183–190 Machida I, Matsuse H, Kondo Y et al 2003 Acetaldehyde induces granulocyte macrophage colony-stimulating factor production in human bronchi through activation of nuclear factor-κB Allergy Asthma Proc 24:367–371 Matsuse H, Shimoda T, Fukushima C et al 2001 Screening for acetaldehyde dehydrogenase 2 genotype in alcohol-induced asthma by using the ethanol patch test. J Allergy Clin Immunol 108:715–719

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Ruiz CM, Gomes JC 2000 Effects of ethanol, acetaldehyde, and acetic acid on histamine secretion in guinea pig lung mast cells. Alcohol 20:133–138 Shimoda T, Kohno S, Takao A et al 1996 Investigation of the mechanism of alcohol-induced bronchial asthma. J Allergy Clin Immunol 97:74–84 Takao A, Shimoda T, Kohno S, Asai S, Harada S 1998 Correlation between alcohol-induced asthma and acetaldehyde dehydrogenase-2 genotype. J Allergy Clin Immunol 101:576–580 Takao A, Shimoda T, Matsuse H et al 1999 Inhibitory effects of azelastine hydrochloride in alcohol-induced asthma. Ann Allergy Asthma Immunol 82:390–394 Wyatt TA, Heires AJ, Sanderson SD, Floreanni AA 1999 Protein kinase C activation is required for cigarette smoke-enhanced C5a-mediated release of interleukin-8 in human bronchial epithelial cells. Am J Respir Cell Mol Biol 21:283–288 Wolff PH 1972 Ethnic differences in alcohol sensitivity. Science 175:449–450

DISCUSSION M Salaspuro: The blood ethanol levels in your experiments were rather low, at around 5–10 mM, even though you gave subjects a rather high dose of ethanol. The blood acetaldehyde levels ranged from 10–30 µM, which is similar to the range we see in our experiments. How did you measure your blood acetaldehyde? Matsuse: The concentration of blood acetaldehyde was measured by the outside lab. They used GC. M Salaspuro: In your study the flushing and other effects occurred immediately after the end of the experiment, and was associated with a rapid histamine release. Could you repeat your study but ask subjects to keep alcohol only in their mouth and not swallow it. In this case microbial acetaldehyde production starts immediately and might result in the local release of histamine from the mucosal mast cells without any systemic effects. Matsuse: We had never done such a study. Thank you for your good suggestion. Morris: Your results are intriguing because we don’t think of acetaldehyde as being that potent an irritant by itself. You might want to be careful in your animal models. I have shown that if you inhibit ALDH, this inhibits sensory nerve stimulation by acetaldehyde in the rat (Stanek et al 2001). Rodents may not be a good model for the human. In the rodent it seems that acetic acid is the key player for the respiratory tract effects. Matsuse: We agree with you. Actually, I wanted to develop an animal model of alcohol-induced asthma in mice, but mice aren’t good for histamine research. Morris: Was your effect specific for GM-CSF? Matsuse: That is all I have measured. Apte: As John mentioned it was surprising to see that there is an induction of NF-κB, but then no induction of TNF or interleukins. In terms of your results in the human studies you saw blood acetaldehyde levels going up and coming down around the 120 min mark. But the histamine levels keep rising, so could there be something other than acetaldehyde which keeps the mast cells in a state of degranulation.

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Matsuse: We have measured other mediators such as leukotrienes, but only histamine was elevated. I have no idea why this happens. Apte: Do mast cells themselves have ADH, or is acetaldehyde positively chemotactic for mast cells? As far as I could tell your patients didn’t have active asthma at the time. The asthma was under control, so there was presumably less inflammation and fewer mast cells, yet there is a huge histamine release. M Salaspuro: We have used isolated human mast cells and were able to show histamine release caused by acetaldehyde of about 100 µM. Matsuse: I have no idea whether mast cells themselves have ADH, or whether acetaldehyde is positively chemotactic for mast cells. Morris: The time course is interesting. Was bronchoconstriction immediate? Was there enough time to get mast cells coming in? Apte: It happened within 30 min. Is this enough time for mast cell migration? Morris: It is for neutrophils; I don’t know about mast cells. Matsuse: We don’t think histamine is released from migrating mast cells but it is released from mucosal mast cells. Apte: There are tissue mast cells that are already present. But even if it is all tissue mast cells, there needs to be some chemotaxis. Eriksson: The first point made by Mikko Salaspuro is important. This finding has been documented before in Japanese populations. It is well established that acetaldehyde is responsible. But which mast cells are involved, and where are they located? The first flushing is in the face and neck. It would be interesting to see where it starts and to uncover the mechanism. Rao: It is interesting that a small dose of acetaldehyde by itself doesn’t do anything, but in allergen-mediated inflammation it can have an effect. This touches on the discussion we had yesterday about the carcinogenic concentration of acetaldehyde. A low dose of acetaldehyde can, in concert with other factors, be injurious. Deitrich: You mentioned that there is acetaldehyde in plastic and rubber. There are a lot of other things that leach out of plastic bottles into alcohol, such as phthalates and triphenol phosphate (Goldstein et al 1987). Niemelä: I am curious about the possible immunological basis of this phenomenon. If you repeat the experiments several times on the same subjects, would the symptoms remain similar or would they get worse? In mast cell reactions, there may be other immunological players involved which we don’t yet know about. Matsuse: We repeated the experiment at least twice in the same subjects and found similar symptoms. Morris: Mast cells put out a variety of pro-inflammatory mediators, not just histamine. Continuous stimulation of mast cells would exacerbate the inflammatory aspects of asthma. Apte: Could you desensitize the mast cells by repeated challenge?

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Morris: In the process of desensitizing inflammation might get worse, but it might get better in the long term. Matsuse: Another group have used inhaled acetaldehyde to cause bronchoconstriction. They showed desensitisation with repeated challenge. Thornalley: In cigarette smoke there are many other aldehydes, some of which are far more potent than acetaldehyde and which have been shown to induce histamine secretion. These are also substrates for ALDH. Morris: In that context, asthmatic subjects are hyper-responsive to a wide variety of irritants. In the mouse models this is also the case. It may not be specific to acetaldehyde. Emery: This also brings us back to the comment made about pollution. Acetaldehyde may be one of the irritant molecules, but do we have any feel for how this relates to other air pollutants? Morris: In terms of respiratory tract irritation acetaldehyde is not considered to be a key player. There are other more potent irritants present in potentially much more hazardous concentrations. Matsuse: The number one irritant in human environments is formaldehyde. The next one is acetaldehyde. Does anyone know whether ALDH2 can metabolise formaldehyde? Morris: No, it requires formaldehyde dehydrogenase. Deitrich: Formaldehyde is a poor substrate for ALDH2. Morris: As an irritant formaldehyde is a 1000 times more potent than acetaldehyde. Deitrich: Formaldehyde is almost completely hydrated, and acetaldehyde is only about 50% hydrated. This may be why formaldehyde is not a good substrate for ALDHs. Apte: Just a point of clarification. Were your human subjects non-smokers? Or had they just stopped smoking? This could be a confounding factor. Matsuse: Some are current smokers and some are ex-smokers. Apte: So you had a mixture of people, some of whom were still smoking when they entered the study. That’s something to be careful of. Crabb: A prolonged increase in histamine would turn on gastric acid secretion if the concentration is high enough. Is there a connection with alcohol flushing and duodenal ulcers? Matsuse: I have no idea about duodenal ulcers. Emery: Are there any genetic data on ulcers? Could this be related to the polymorphisms? Yin: We published a paper in 1992 about human lung ADH and ALDH (Yin et al 2002). ADH1B is the only form present in human lung for ethanol oxidation. ADH1B2 is high activity. It is possible that high activity of ADH1B2 produces acetaldehyde that can be accumulated in the lung cells in individuals with deficient

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ALDH2 activity. The locally produced acetaldehyde may also contribute to bronchoconstriction and alcohol-related cytotoxicity. References Goldstein DB, Feistner GJ, Faull KF, Tomer KB 1987 Plasticizers as contaminants in commercial ethanol. Alcohol Clin Exp Res 11:521–524 Stanek J, Symanowicz PT, Olsen JE, Gianutsos G, Morris JB 2001 Sensory-nerve-mediated nasal vasodilatory response to inspired acetaldehyde and acetic acid vapors. Inhal Toxicol 13:807–822 Yin SJ, Liao CS, Chen CM, Fan FT, Lee SC 1992 Genetic polymorphism and activities of human lung alcohol and aldehyde dehydrogenases: implications for ethanol metabolism and cytotoxicity. Biochem Genet 30:203–215

The role of acetaldehyde in alcohol-associated cancer of the gastrointestinal tract Helmut K. Seitz and Nils Homann* Laboratory of Alcohol Research, Liver Disease and Nutrition, Department of Medicine, Salem Medical Center Heidelberg and University of Heidelberg, Heidelberg, Germany and *Medical University of SchleswigHolstein, Department of Gastroenterology, Luebeck, Germany

Abstract. Acetaldehyde has been classified as a carcinogen in experimental animal research. Acetaldehyde is highly toxic, mutagenic and carcinogenic. Acetaldehyde causes point mutations, sister chromatid exchanges and gross chromosomal aberrations. In the liver, acetaldehyde binds to DNA and the formation of stable adducts represents one mechanism by which acetaldehyde could trigger the occurrence of replication errors and/or mutations in oncogenes or tumour suppressor genes. In experimental colorectal carcinogenesis the inhibition of acetaldehyde dehydrogenase with elevated acetaldehyde levels results in an acceleration of cancer development. The production of acetaldehyde is reduced when germ-free animals are studied, emphasizing the role of bacteria in the generation of colorectal acetaldehyde. Acetaldehyde levels in the colorectum correlate with crypt cell production rate and result in hyper-regeneration, a precancerous condition. Genetic linkage studies give further evidence for acetaldehyde as a carcinogen. Individuals who accumulate acetaldehyde due to polymorphism and/or mutations in the genes coding for enzymes responsible for acetaldehyde generation and detoxification have an increased cancer risk. This is true for Asians with low acetaldehyde dehydrogenase 2 and for Caucasians with alcohol dehydrogenase 1C*1/1. In conclusion, there is an enormous body of evidence from in vitro studies, animal experiments and genetic linkage studies, that acetaldehyde is the major factor responsible for tumour development in alcohol-associated carcinogenesis of the gastrointestinal tract. 2007 Acetaldehyde-related pathology: bridging the trans-disciplinary divide. Wiley, Chichester (Novartis Foundation Symposium 285) p 110–124

Alcohol per se is not a carcinogen, while acetaldehyde has been identified as a mutagen and carcinogen. In this overview the mechanisms by which acetaldehyde acts in alcohol-associated carcinogenesis and the evidence for acetaldehyde as being the major carcinogenic principle in alcohol-associated cancer development will be discussed. Carcinogenesis of the upper alimentary tract and the liver will be considered only. It is beyond the scope of this review to discuss acetaldehyde effects 110

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on other tissues such as the breast as well as effects of acetaldehyde on metabolic and signal transduction pathways involved in carcinogenesis in detail. Acetaldehyde, a carcinogen The first and major metabolite of ethanol oxidation is acetaldehyde (AA). There is increasing evidence that AA rather than alcohol itself is responsible for the cocarcinogenic effect of alcohol (Pöschl & Seitz 2004). Numerous in vitro and in vivo experiments in prokaryotic and eukaryotic cell cultures as well as in animal models have identified AA as highly toxic, mutagenic and carcinogenic. Inhalation of AA in rats and hamsters resulted in the occurrence of carcinomas in the nasal mucosa and in the larynx (Woutersen et al 1986, Feron et al 1982). AA interferes at many sites with DNA synthesis and repair and may, consequently, result in tumour development (IARC 1999). AA causes point mutations in the hypoxanthine-guanine-phosphorybosyl transferase locus in human lymphocytes, induces sister chromatid exchanges and gross chromosomal aberration (for review see Pöschl & Seitz 2004). It induces inflammation and metaplasia of tracheal epithelium, delays cell cycle progression and enhances cell injury associated with hyperregeneration (Simanowski et al 1994). AA binds to proteins resulting in structural and functional alterations. AA weakens the antioxidative defence systems by binding to glutathione and thus increases oxidative stress indirectly. AA also injures microtubules and mitochondria. Decreased mitochondrial function results in an inhibition of fatty acid oxidation and ATP formation. Both factors favour the occurrence of fatty liver. In addition, mitochondrial damage induces apoptosis but also survival factors such as NF-κB. It has also been shown that AA interferes with the DNA repair machinery. AA directly inhibits O6-methylguanosyl transferase, an enzyme important for the repair of DNA adducts (Espina et al 1988). In the liver, AA forms adducts with intracellular proteins and DNA resulting in morphological and functional impairment of the cell and a humeral immune reaction towards de novo generated antigens. The binding to DNA and the formation of stable adducts represents one of the mechanisms by which AA could trigger the occurrence of replication errors and/or mutations in oncogenes or tumour suppressor genes. The occurrence of stable DNA adducts has been shown in different organs of alcohol-fed rodents and in leukocytes of alcoholics (Fang & Vaca 1997). It has been shown that the major stable DNA adduct N2-ethyl desoxyguanosine (N2-Et-dG) serves as a substrate of eukaryotic DNA polymerase (Matsuda 1999). Although N2-Et-dG has been shown to be present in DNA samples from white blood cells of human alcoholics and in the liver of rats (Wang et al 2006) that were administered ethanol in the drinking water, there is relatively little evidence that this lesion is mutagenic and its biological significance is unclear. However, this lesion can be detected in human urine samples,

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SEITZ ADH1C*1/1

ETHANOL

ALDH2*1/2

ACETALDEHYDE

ACETATE Antioxidant defence

Methyl transfer

DNA adducts

DNA repair

IL6

NF-κB

Apoptosis FIG. 1. Effect of acetaldehyde on important factors in carcinogenesis. Acetaldehyde accumulates due to either increased production or decreased detoxification. Acetaldehyde inhibits methyl transfer at various stages (e.g. folate deficiency and reduced activation of methionine to S-adenosyl-methionine) and injures the antioxidative defence system. Acetaldehyde forms DNAadducts and inhibits DNA-repair at the same time. Subsequently acetaldehyde results in an increased NF-κB, which is associated with inhibition of apoptosis and hyper-regeneration (Seitz & Stickel 2006).

suggesting that it may be useful as a biomarker of AA-related DNA damage. More recent data have shown that in the presence of basic amino acids or histones, AA reacts with deoxyguanosine in DNA to form a different DNA adduct, 1,N 2-propano-dG (PdG) (Brooks & Theruvathu 2005). In contrast to N2-Et-dG, PdG has been shown to be a mutagenic DNA lesion in vivo in mammalian cells. These AA-associated effects occurred at AA concentrations from 40–1000 µM ( Theruvathu et al 2005), which are similar to concentrations observed in human saliva following alcohol ingestion. It has been recently shown that the polyamines spermine and spermidine can also facilitate the formation of PdG from AA and dG at AA concentrations as low as 80 µM. The action of AA on DNA metabolism is summarized in Fig. 1. According to the International Agency for Research on Cancer there is sufficient evidence to classify AA as a carcinogen in experimental animals (IARC 1999). Acetaldehyde production in the gastrointestinal tract AA in the gastrointestinal tract can be produced either by mucosal enzymes or by gastrointestinal bacteria. Mucosal alcohol dehydrogenase (ADH) isozymes vary within the gastrointestinal tract (GIT) (Seitz & Oneta 1998). While ADH4 is the major ADH present in the mucosa of the upper GIT (oropharynx, oesophagus and the stomach), AHD1C is predominantly present in the stomach and in the small and large intestine. ADH3 is present in the entire GIT and ADH1B can be found in the muscular layer of the mucosa. The contribution of the various ADH

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isozymes to ethanol metabolism depends on their kinetic properties and on their distribution within the GIT. Gastrointestinal ethanol metabolism is dependent on the ethanol concentration in the alcoholic beverage consumed. Metabolism increases with increasing concentrations of ethanol. ADH1C with a Km of approximately 1 mM is usually saturated and contributes fully to ethanol metabolism. ADH4 with its intermediate Km value of 41 mM at ph 7.4 contributes only partly. ADH3 is insaturable for alcohol and contributes only at rather high alcohol concentrations. Moreover, genetic polymorphism in ADH1C and ethnic differences in the expression of ADH4 have been described and may further modify gastrointestinal ethanol metabolism. It is interesting that a high percentage of Asians lack class IV ADH in the stomach. All these ADHs are influenced by various factors in their activity including genetics, ethnicity, gender, age, concentration of alcohol, drugs and mucosal injury (Seitz & Oneta 1998). The net amount of AA accumulating in mucosal cells depends on the production of AA from ethanol and on its detoxification by acetaldehyde dehydrogenase (ALDH). Thus, the ratio of ADH to ALDH activity is of pathogenetic importance and an imbalance of this ratio has been reported for the distal colon and the rectum. Beside the fact that alcohol is metabolized to AA in the mucosal cells AA is also produced by bacteria (Homann 2001, Salaspuro et al 2006). This bacterial metabolism takes place in the upper GIT (Homann et al 1997), and in the large intestine (Jokelainen et al 1996a). Many microbes representing normal oral flora possess ADH activity with individual kinetic characteristics. Under aerobic or microaerobic conditions in the mouth the ADH reaction is reversed with AA being the end product (Salaspuro et al 1999). After drinking of alcohol, salivary ethanol is metabolised to AA via this reversed reaction and AA concentrations up to 100–150 µM can be detected in the saliva of healthy volunteers (Homann et al 1997). This AA can be reduced significantly by an antiseptic mouthwash underlining the essential role of bacteria in the production of salivary AA. The kinetic characteristics of microbial ADHs may vary to a great extent. Some microbial ADHs are not saturated with ethanol; therefore an increasing salivary ethanol concentration or production of AA is enhanced. Accordingly, at higher blood and salivary ethanol concentrations salivary AA levels are also higher. This may explain the well established epidemiological finding of increased cancer risk associated with heavier and more intoxicating drinking. In addition to that, smoke contains high amounts of AA and smoking also changes the bacterial oral flora towards a flora with a higher capacity to produce AA. Thus, smoking, poor oral hygiene and drinking alcoholic beverages with high concentrations of AA, such as Calvados, are associated with an increased risk for cancer (Salaspuro et al 2006). It has been shown that patients with an oropharyngeal cancer had indeed a higher concentration of AA in their saliva as compared to healthy controls (Jokelainen et al 1996b).

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AA can also be produced by colonic bacteria (Jokelainen et al 1996a). The amount of AA per gram of mucosal tissue in the colon is significantly higher than in other tissues following ethanol ingestion (up to 200 µM). This is primarily due to the production of AA from ethanol by faecal bacteria, as shown in experiments using germ-free rats (Seitz et al 1990). The toxic mucosal effects of AA result in decreasing cell numbers in the functional compartment of the colonic crypt, being answered secondarily by compensatory hyper-regeneration with increased crypt cell production rates and an extension of the proliferative compartment towards the lumen of the crypt (Simanowski et al 1986, 1994, 2001). This observation was paralleled by a significant increase in rectal mucosal ornithine decarboxylase activity (Seitz et al 1990). The alterations of crypt cell dynamics caused by AA favour the development of colorectal cancer (Seitz et al 1990). As the alcohol-associated hyper-regeneration of the colonic mucosa is especially pronounced with increasing age, chronic alcohol consumption during the lifetime may additionally result in an elevated risk of developing colorectal cancer. In contrast, cell differentiation with regard to the cytokeratin expression pattern was not influenced by chronic alcohol consumption as well as regulatory factors involved in carcinogenesis and/or apoptosis (Simanowski et al 2001). Changes in colonic cell regeneration caused by ethanol and AA have been observed in animal studies and in humans (Simanowski et al 1986, 1994, 2001). Evidence for the carcinogenic role of AA in alcohol-associated cancer Animal experiments It has been shown in rats that the administration of cyanamide, a potent ALDH inhibitor, increases AA concentrations in the blood but also in the colon accelerates chemically induced colorectal carcinogenesis. Under these conditions an earlier occurrence of tumours is observed (Seitz et al 1990). This is associated with an increased proliferation status of the colorectal mucosa which correlates with AA levels (Simanowski et al 1994). Such a hyper-regeneration of the gastrointestinal mucosa is also observed following chronic AA feeding resembling that following chronic ethanol feeding (Homann et al 1997, Simanowski et al 1993). Furthermore, colonic acetaldehyde concentrations correlate with mucosal folate levels in the colon. This is of major importance since folate deficiency is associated with an increased risk for distal colorectal cancer when it coincides with alcohol drinking (Giovannucci et al 1995). Genetic linkage studies Recent and striking evidence for the causal role of AA in ethanol-associated GIT carcinogenesis derives from genetic linkage studies in alcoholics. Individuals who

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accumulate AA because of polymorphism and/or mutation in the genes coding for enzymes responsible for AA generation or detoxification have an increased cancer risk. In Japan as well as in other Asian countries, a high percentage of individuals carry a mutation of ALDH2. Mitochondrial ALDH2 is primarily responsible for AA oxidation. Human ALDH2 is polymorphic, with two distinct alleles: ALDH2 *1 and ALDH2 *2. ALDH2 *2 results from a single-point mutation in chromosome 6. Blood AA levels of ALDH2 *2 homozygous individuals are 6–20 times higher compared to ALDH2 *1 individuals, in whom AA is hardly detectable after alcohol consumption. The elevated AA concentrations cause unpleasant side effects (flush syndrome) that protect these individuals from alcoholism. However, heterozygous individuals may become heavy drinkers or even alcoholics. Yokoyama et al (1998) were the first to report that the heterozygous mutation of the ALDH2 gene (ALDH2 *1,2) is a strong risk factor for oesophageal cancer in everyday drinkers and alcoholics. A comprehensive study of the ALDH2 genotype and cancer prevalence in Japanese alcoholics showed that the frequency of inactive ALDH2 increased remarkably among alcoholics with cancer of the oral cavity, oropharynx, hypopharynx, larynx, oesophagus and colorectum (Yokoyama et al 1998). Many epidemiological studies have uniformly shown that the risk of alcohol related GIT cancers is markedly increased in Asians with the low-activity ALDH2 enzyme. After adjustment for confounders the relative risks to those with the normal enzyme were 11.1 for oropharyngolaryngeal, 12.5 oesophageal, 3.5 stomach, 3.4 colon and 8.2 for lung cancer (Yokoyama et al 1998). In a recent meta-analysis including seven studies and 905 cases carried out in Japan, Taiwan and Thailand, these findings with regard to the risk of oesophageal cancer were confirmed (Lewis & Smith 2005). The review provided additional evidence for the important role of alcohol intake in the risk of oesophageal cancer. Individuals whose genotype results in markedly lower alcohol intake (homozygotic flushers) appear to be protected. However, the most important message of this meta-analysis was that AA may play an important carcinogenic role in the pathogenesis of oesophageal cancer. It is important to note that these individuals also have high AA levels in their saliva, and thus AA is delivered directly to the surface mucosa of the upper GIT in such individuals (Väkeväinen et al 2000). In addition to the mutation of the ALDH2 gene, polymorphisms of ADH1B and ADH1C may also modulate AA levels. Whereas the ADH1B *2 allele encodes for an enzyme that is approximately 40 times more active than the enzyme encoded by the ADH1B *1 allele, ADH1C *1 transcription leads to an ADH isoenzyme 2.5 times more active than that from ADH1C *2. However, the ADH1B *2 allele frequency is high in Asians but low in Caucasians. It protects from alcoholism because of the high amount of AA produced and its toxic side effects. Because of the low ADH1B *2 allele frequency and the lack of ALDH2 mutations in Caucasians, ADH1C polymorphism and its role in alcohol-associated carcinogenesis is ideally investigated in Caucasian populations.

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Studies on ADH1C polymorphism in Caucasians and GIT cancer have shown contradictory results. Whereas an increased risk of oropharyngeal and laryngeal cancer in individuals with the ADH1C*1 allele has been reported (Harty et al 1997, Coutelle et al 1997), others could not confirm such an association in case-control studies (Olshan et al 2001, Sturgis et al 2001, Schwarz et al 2001, Zavras et al 2002). One reason for this discrepancy is the fact that in all these studies, the percentage of cancer patients with high alcohol intake was rather low, sometimes extremely low. In the study by Sturgis et al (2001) the amount of alcohol ingested was not even reported. Thus, it is not surprising that a pooled analysis of all the studies published so far led to the conclusion that the ALDH1C allele is not a risk factor for alcohol-associated carcinogenesis (Brennan et al 2004). Visapää et al (2004) studied 107 alcoholic patients with high alcohol ingestion and oropharyngeal, laryngeal, hypopharyngeal and oesophageal cancer to compare their ADH1C genotype with 103 age-matched alcoholics with a similar alcohol consumption but without cancer, and he observed a significantly increased cancer risk in individuals with the ADH1C*1 allele. This was found to be associated with significantly elevated AA levels in the saliva of individuals homozygous for ADH1C*1 Increased salivary AA levels in these individuals as in individuals with ineffective ALDH activity may explain their increased cancer risk, because AA comes into direct contact with the mucosa. In this context, it is interesting to note that AA-fed rats showed a severe hyper-regeneration of the upper GIT mucosa (Homann et al 1997); this is very similar to the morphological changes observed after chronic consumption. These changes were only observed when the animals had functionally intact salivary glands. After sialoadenectomy, this proliferation disappeared, which supports the hypothesis that salivary AA is involved in carcinogenesis (Simanowski et al 1993). In this context, it has to be pointed out that chronic alcohol consumption alters salivary morphology and function. In addition, a more recent study in 818 patients with alcohol-associated cancers gives further strong evidence for AA being an important factor in the development of upper GIT and liver cancer (Homann et al 2006). Summary and Conclusion There is overwhelming evidence for the involvement of AA in alcohol-associated carcinogenesis which may be summarized as follows: • High AA levels occur in the saliva and in the colon following alcohol consumption. • Oral bacteria are capable of oxidizing AA from ethanol, and salivary AA levels are elevated in patients with upper GIT cancer as well as in individuals with a poor dental state and cigarette smokers, both conditions also favouring cancer risk.

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• AA leads to mucosal hyper-regeneration in the upper and lower GIT, and the colonic crypt cell production rate significantly correlates with AA levels in the colonic mucosa. • Animal experiments have shown an acceleration of colorectal carcinogenesis induced by the specific locally acting carcinogen acetoxymethyl-methylnitrosamine (AMMN), when cyanamide, an ALDH inhibitor, was applied and AA levels were increased. • Colonic AA levels show a significant inverse correlation with mucosal folate concentrations supporting in vitro data showing a destruction of folate by AA. • Individuals with the inactive form of ALDH2 resulting in elevated AA concentrations exhibit an increased risk for upper and lower GIT cancer due to chronic alcohol consumption. • In individuals homozygous for the ADH1C *1 allele, salivary AA levels are elevated thus increasing the risk of developing upper GIT cancer.

References Brennan P, Lewis S, Hashibe M et al 2004 Pooled analysis of alcohol dehydrogenase genotypes and head and neck cancer. A HuGE review. Am J Epidemiol 15:1–16 Brooks PJ, Theruvathu JA 2005 DNA adducts from acetaldehyde: implications for alcohol-related carcinogenesis. Alcohol 35:187–193 Coutelle C, Ward PJ, Fleury B et al 1997 Laryngeal and oropharyngeal cancer, and alcohol dehydrogenase 3 and glutathione S-transferase M1 polymorphism. Hum Genet 99:319–325 Espina N, Lima V, Lieber CS, Garro AJ 1988 In vitro and in vivo inhibitory effect of ethanol and acetaldehyde on O6-methylguanine transferase. Carcinogenesis 9:761–766 Fang JL, Vaca CE 1997 Detection of DNA adducts of acetaldehyde in peripheral white blood cells of alcohol abusers. Carcinogenesis 18:627–632 Feron VJ, Kruysse A, Woutersen RA 1982 Respiratory tract tumours in hamsters exposed to acetaldehyde vapour alone or simultaneously to benzo(a)pyrene or diethylnitrosamine. Eur J Cancer Clin Oncol 18:13–31 Giovannucci E, Rimm EB, Ascherio A, Stampfer MJ, Colditz GA, Willett WC 1995 Alcohol, low-methionine-low-folate diets and risk of colon cancer in men. J Natl Cancer Inst 87: 265–273 Harty LC, Caporaso NE, Hayes RB et al 1997 Alcohol dehydrogenase 3 genotype and risk of oral cavity and pharyngeal cancers. J Natl Cancer Inst 89:1698–1705 Homann N 2001 Alcohol and upper gastrointestinal tract cancer: the role of local acetaldehyde production. Addict Biol 6:309–323 Homann N, Jousimies-Somer H, Jokelainen K, Heine R, Salaspuro M 1997 High acetaldehyde levels in saliva after ethanol consumption: methodological aspects and pathogenetic implications. Carcinogenesis 18:1739–1743 Homann N, Kärkkäinen P, Koivisto T, Nosova T, Jokelainen K, Salaspuro M 1997 Effects of acetaldehyde on cell regeneration and differentiation of the upper gastrointestinal tract mucosa. J Natl Cancer Inst 89:1692–1697 Homann N, Stickel F, Konig IR et al 2006 Alcohol dehydrogenase 1C*1 allele is a genetic marker for alcohol-associated cancer in heavy drinkers. Int J Cancer 118:1998–2002

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IARC 1999 Re-evaluation of some organic chemicals, hydrazine and hydrogen peroxide. In: Monographs on the evaluation of the carcinogenic risk of chemicals to humans. Acetaldehyde. vol. 77, p 319–335, International Agency for Research on Cancer, Lyon Jokelainen K, Matysiak-Budnik T, Mäkisalo H, Höckerstedt K, Salaspuro M 1996a High intracolonic acetaldehyde values produced by a bacteriocolonic pathway for ethanol oxidation in piglets. Gut 39:100–104 Jokelainen K, Heikkonen E, Roine R, Lehtonen H, Salaspuro M 1996b Increased acetaldehyde production by mouthwashings from patients with oral cavity, laryngeal or pharyngeal cancer. Alcohol Clin Exp Res 20:1206–1210 Lewis SJ, Smith GD 2005 Alcohol, ALDH2 and esophageal cancer: a meta-analysis which illustrates the potentials and limitations of a Mendelian randomization approach. Cancer Epidemiol Biomarkers Prev 14:1967–1971 Matsuda T, Terashima I, Matsumoto Y, Yabushita H, Matsui S, Shibutani S 1999 Effective utilization of N2-ethyl-2′-deoxyguanosine triphosphate during DNA synthesis catalyzed by mammalian replicative DNA polymerases. Biochemistry 38:929–935 Olshan AF, Weissler MC, Watson MA, Bel DA 2001 Risk of head and neck cancer and the alcohol dehydrogenase 3 genotype. Carcinogenesis 22:57–61 Pöschl G, Seitz HK 2004 Alcohol and cancer. Alcohol Alcohol 39:155–165 Salaspuro M, Salaspuro V, Seitz HK 2006 Interaction of alcohol and tobacco in upper aerodigestive tract and stomach cancer. In: Cho CH, Purohit V (eds) Alcohol, tobacco and cancer. Karger Basel, p 48–62 Salaspuro V, Nyfors S, Heine R, Siitonen A, Salaspuro M, Jousimies-Somer H 1999 Ethanol oxidation and acetaldehyde production in vitro by human intestinal strains of Escherichia coli under aerobic, microaerobic, and anaerobic conditions. Scand J Gastroenterol 34:967–973 Schwartz SM, Doody DR, Fitzgibbons ED, Rick S, Porter PL, Chen C 2001 Oral squamous cell cancer risk in relation to alcohol consumption and alcohol dehydrogenase-3 genotypes. Cancer Epidemiol Biomarkers Prev 10:1137–1144 Seitz HK, Oneta CM 1998 Gastrointestinal alcohol dehydrogenase. Nutr Rev 56:52–60 Seitz HK, Simanowski UA, Garzon FT et al 1990 Possible role of acetaldehyde in ethanol related rectal carcinogenesis in the rat. Gastroenterology 98:1–8 Simanowski UA, Seitz HK, Baier B, Kommerell B, Schmidt-Gayk H, Wright NA 1986 Chronic ethanol consumption selectively stimulates rectal cell proliferation in the rat. Gut 127:278–282 Simanowski UA, Suter P, Stickel F et al 1993 Oesophageal epithelial hyperregeneration following chronic ethanol ingestion: effect of age and salivary gland function. J Natl Cancer Inst 85: 2030–2033 Simanowski UA, Suter P, Russell RM et al 1994 Enhancement of ethanol induced rectal mucosal hyperregeneration with age in F244 rats. Gut 35:1102–1106 Simanowski UA, Homann N, Knuhl M et al 2001 Increased rectal cell proliferation following alcohol abuse. Gut 49:418–422 Sturgis EM, Dahlstrom KR, Guan Y et al 2001 Alcohol dehydrogenase genotype is not associated with risk of squamous cell carcinoma of the oral cavity and pharynx. Cancer Epidemiol Biomarkers Prev 10:273–275 Theravathu JA, Jaruga P, Nath RG, Dizdaroglu M, Brooks PJ 2005 Polyamines stimulate the formation of mutagenic 1,N2-propanodeoxyguanosine adducts from acetaldehyde. Nucleic Acids Res 33:3513–3520 Väkeväinen S, Tillonen J, Agarwal D, Srivastava N, Salaspuro M 2000 High salivary acetaldehyde after a moderate dose of alcohol in ALDH2-deficient subjects: strong evidence for the local carcinogenetic action of acetaldehyde. Alcohol Clin Exp Res 24:873–877 Visapää JP Gotte K, Benesova M et al 2004 Increased cancer risk in heavy drinkers with the alcohol dehydrogenase 3*1-allele possibly due to salivary acetaldehyde. Gut 53:871–876

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Wang M, Yu N, Chen L, Villalta PW, Hochalter JB, Hecht SS 2006 Identification of an acetaldehyde adduct in human liver DNA and quantitation as N2-ethyldeoxyguanosine. Chem Res Toxicol 19:319–324 Woutersen RA, Appelman LM, van Garderen-Hoetmer A, Feron JV 1986 Inhalation toxicity of acetaldehyde in rats. III. Carcinogenicity study. Toxicology 41:213–231 Yokoyama A, Muramatsu T, Ohmori T et al 1998 Alcohol-related cancers and aldehyde dehydrogenase-2 in Japanese alcoholics. Carcinogenesis 19:1383–1387 Zavras AI, Wu T, Laskaris G et al 2002 Interaction between a single nucleotide polymorphism in the alcohol dehydrogenase 3 gene, alcohol consumption and oral cancer risk. Int J Cancer 97:526–530

DISCUSSION Crabb: The mutagen you showed (AMN) looked like an adduct of acetaldehyde with nitrosamine. Does this actually form from acetaldehyde and other nitrosamines? Seitz: This is a nitrosamine which does not need activation, as the normal nitrosamines do. It is a compound that is very stable and acts directly. We don’t need any metabolic activation. Crabb: Could it be created in, say, the upper GIT with acetaldehyde and other nitrosamines? Seitz: Possibly. Emery: What did you mean by saying that acetaldehyde destroys folate? Seitz: The study was done in vitro by Spencer Shaw (Shaw et al 1989). The authors applied different amounts of acetaldehyde to folate, and couldn’t recover the folate as a folate molecule any more. I’m unsure of the chemical mechanisms. They found no effect with 50–100 µM acetaldehyde, but did when they went to 200 µM. Worrall: There are a couple of mechanisms that have been proposed. One is that acetaldehyde opens one of the rings. Another is that there are large modifications on the rings that alter the properties of the folate. Thornalley: If this is of physiological importance you might expect to see increased expression of the reduced folate carrier in these colonic epithelial cells. The reduced folate carrier is also the carrier by which thiamine monophosphate is exported from cells. This could be one of the mechanisms by which thiamine levels become depleted in alcoholism. Seitz: That could well be, but there could be other mechanisms of thiamine depletion. Thornalley: There are many adducts of dicarbonyls, such as glyoxal and methylglyoxal. These increase in oxidative stress. Conceivably in these sorts of situations there are several types of mutagenic adducts present. Seitz: I agree. I didn’t mention adducts created by oxidative stress. We are working on exocyclic etheno –DNA adducts coming from 4-HNE, for example. We measure

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them. I am not sure which of these adducts are more important. We should measure both types in a certain tissue. Thornalley: There are imidazopurinone adducts which are tricyclic adducts produced from glyoxal and methylglyoxal, which normally exist at levels similar to those of 8-hydroxydeoxyguanosine. These are possibly increased as well as adducts of acetaldehyde. If you do DNA digests with nuclease at high pH you destroy these. You need to do an acidic digest to see them. Rao: The evidence you have provided for the role of acetaldehyde in carcinogenesis is compelling. Is there a way to examine whether the acetaldehyde effect is more a promotion of carcinogenesis, or whether by itself it can induce carcinogenesis? Seitz: That is an important question. Although we see that there are adducts, I am not sure whether these adducts are more important than those coming from oxidative stress. The data we have indicate that it is more of a promoting effect of acetaldehyde than an initiating effect. However, we can’t exclude the latter possibility completely. Rao: Can you also analyse other types of mutations in these patients? Are there oncogenic mutations? Seitz: We haven’t specified certain genes at that time point. Shukla: The information you provided on hypermethylation is very relevant to carcinogenesis. Has anyone looked at the CpG island methylations and p53? Seitz: We have looked at p53 in the colon and haven’t found an effect. Apte: But there are hypermethylations of CpG islands in colon cancer. Seitz: Yes, but that isn’t related to p53. In p53 there is nothing. Shukla: There is some evidence that in relation to alcohol, hypomethylation of DNA can occur. Seitz: I agree. Emery: Are specific genes being hypomethylated? Shukla: It is more global. Apte: In addition to the proliferative effect you see, is there an effect on apoptosis? Is it a double effect with increased proliferation and decreased apoptosis? Seitz: We see an acetaldehyde effect on apoptosis in the liver. We also see an effect on NF-κB in the liver. We haven’t shown this clearly in the GI tract. We have looked at some antiapoptotic proteins in the colon of individuals, such as Bcl-2. There was no effect. We have also looked at cytokeratins and haven’t seen an effect. Apte: Have there been any in vitro studies with colonocytes? Seiz: No. Rao: One of the mechanisms involved in carcinogenesis and tumour invasion is the loss of cadherin based cell–cell contact. Studies in our laboratory have shown that acetaldehyde even at low concentrations (100 µM) can disrupt the cadherin–

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catenin complex by inducing phosphorylation of E-cadherin and β-catenin on tyrosine residues. Deitrich: In relation to the question about the nitrosamines, I’d like to point out that ethanol will react with NO to give ethyl nitrite (Deng et al 2004). I don’t know whether or not this is carcinogenic. It was marketed at the turn of the century as sweet spirits of nitre. It fell out of favour. The concentration in vivo is very low, but it might not need to be high to be carcinogenic. M Salaspuro: Yesterday we were discussing that we should be able to measure acetaldehyde in situ in different tissues. What kind of method did you use for this? Do you think your findings reflect something that is happening in the colonic mucosa, or do you think your results reflect the acetaldehyde concentration inside the colon, and then part of that is transported to the mucosal cells? We tried to infuse high acetaldehyde concentrations intracolonically. We could not detect anything in the portal blood. This means that the colon mucosa must be able to metabolize acetaldehyde efficiently. Seitz: I don’t think the enzymic machinery in the colon mucosa is sufficient to produce that high amount of acetaldehyde. We have done bacterial studies and believe that colonic bacteria are largely responsible for the acetaldehyde production. If we look at ADHs in individuals, these are usually the class I. We studied polyps in patients, and found sigma ADH (ADH7) in the polyps. We never found this in the normal mucosa of the colon, but we found it in the polyps. The idea was that polyps as a precancerous lesion probably need retinoic acid more for differentiation and to reduce proliferation than anywhere else. The idea is that there may have been a re-expression of ADH7 to produce retinoid acid which is lacking. This is another observation that needs following up. So to come back to your question, I think it is mostly bacteria producing acetaldehyde, which is then bound to the mucosa when we measured it in the mucosa. The method we are using is the one developed by Timothy Peters’ group, which is an HPLC method measuring adduct formation of acetaldehyde by fluorescence detection. We have compared this method with gas chromatography and they both produce equivalent results. Emery: So there is evidence that acetaldehyde can be transported into the colonic mucosa, but probably only if it is produced by the bacteria that are intimately associated with the mucosa. Yin: In your genetic study, you show that ADH1C polymorphism is associated with colon, liver and oesophageal cancer. Have you looked at the phenotype? We need to consider the enzyme kinetic parameters, the enzyme amount and the overall ADH family expression pattern in target tissue. In colon mucosa ADH1C is the major form but both the Vmax difference between ADH1C1 and ADH1C2 and the total enzyme amount don’t seem that high. In the upper digestive tract ADH1C seems very minor. In the liver it is only one of the ADH isozymes involved and its total contribution is not that high.

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Seitz: For the colon it isn’t clear. We did some phenotyping studies and found ADH1C present, probably not as high as in other tissues, but it is still there. These results are preliminary, and I’m still not sure of the mechanism. For the liver the only explanation I have is that in the Japanese studies there is a mixture of other risk factors such as hepatitis C in the control patients. Ren: Is there any evidence linking ALDH2 polymorphisms to homocysteine? Seitz: There is evidence that alcohol leads to homocysteinaemia. This is well known. But I am not aware that ALDH2 heterozygotes have a change in their homocysteine levels. Okamura: Two years ago, to my knowledge, there was no study looking at the relationship between ALDH2 genotypes and homocysteine. The relationship between alcohol drinking per se and homocysteine is controversial. Related to this, I have a question. As you mentioned, folate is a very important protective factor for acetaldehyde-related carcinogenesis. Vitamin B12 is also an important determinant for plasma homocysteine levels. How do we know about the relationship between vitamin B12 or folate and carcinogenesis related to alcohol drinking or acetaldehyde? In a study published a few years ago on a Japanese population (Moriyama et al 2002), we have examined the determinants of serum plasma homocysteine. Vitamin B12 is a strong determinant of plasma homocysteine in Japanese populations, especially when there is a genetic deficiency of MTHFR. Seitz: To my knowledge there are data on B12 effects on the relationship between alcohol and carcinogenesis. There are data from Dr Mason in Boston showing that B12 may modulate colon carcinogenesis. But I am not aware of any such data with alcohol. There are data on an increased risk of colon cancer in vitamin B6 deficiency (Larsson et al 2005). We know that acetaldehyde can lead to B6 destruction (Lumeng & Li 1974). Apte: I’d like to return to the colonic bacteria story. Earlier we heard how lactobacilli produce acetaldehyde and they are part of the normal colonic flora. There is a background level of acetaldehyde production all the time in the colon. Do you think the increase in acetaldehyde from this baseline is enough to cause the toxicity that we are talking about? Seitz: I am not sure. Yesterday we heard about the level of toxicity or carcinogenicity, which is between 50 and 100 µM. The level we find is far above this, while the background level is below this. If you drink regularly for a long time this could play an important role. Emery: Are there other factors in food that would stimulate bacterial production of acetaldehyde in the colon? Apte: People talk about not eating enough vegetables and fresh fruit as opposed to eating a lot of red meat. Is this connected to fermentation? Seitz: This is an important question. This is what we are thinking: the diet may modulate bacterial flora, and in turn this may modulate acetaldehyde production. If

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bacteria are modulated by an antibiotic such as metronidazole, this can kill anaerobics and increase aerobics in the colon. This would result in increased acetaldehyde production. This is probably one of the reasons that metronidazole plus alcohol increases acetaldehyde levels. Probiotics such as lactulose change the pH of the colon. If pH is decreased then acetaldehyde production is reduced. Eriksson: It seems that since the odds ratio of colon cancer is not so dramatic with alcohol, there is an effect but it doesn’t compare with the oesophageal cancer rates in Japanese populations with ALDH mutations. There may be a risk factor to start with so the increase is not that big regarding colon cancer in Europeans. If you differentiate different control populations you might be able to find this out. It means that acetaldehyde is still one of the main players, but its role isn’t so obvious because of the existing endogenous baseline production. This would also be interesting to study in the Japanese population. Controls there should have endogenously quite high acetaldehyde levels. On the other hand, their diet is a little different from European diets. Rao: The GI tract has evolved mucosal defence mechanisms to protect the mucosal tissue from a variety of injurious insults. There needs to be a balance between the protective factors and the injurious factors. Weak mucosal protection may increase the risk for acetaldehyde-mediated tissue injury. Worrall: Is there an increase of gut motility in these people? Seitz: We have not measured it. But alcohol per se is changing motility significantly in the whole GI tract. There is hypermotility in the small intestine and also quite a disturbed motility in the colon. Motility is affected by alcohol. Emery: Is that independent of other aspects of their diet? Seitz: We know that a lot of dietary factors influence motility, so probably not. Worrall: Presumably that low motility in the large intestine is giving the bacteria more chance to metabolize things? Seitz: Alcohol is actually having the opposite effect. One cause of alcoholassociated diarrhoea is hypermotility. Preedy: Although we have not measured motility disturbances in the rat, rats fed on an alcohol-containing diet using the Lieber-DeCarli regimen show reduced smooth muscle loss from the small intestine, but not the rectum or colon. This may contribute to your overall motility disturbances. The smooth muscle part of the gut is very sensitive to alcohol in terms of protein synthesis. Albano: You showed nice data on the effects of ADH polymorphisms on the risk of upper digestive tract cancer. However, I am a little confused. If a considerable amount of acetaldehyde is coming from the bacterial fermentation of alcohol, how can we see this strong influence of genetic background? This suggests that the ADH polymorphism is probably influencing the metabolism of other aldehydes. Seitz: That goes in the same direction as Dr Yin proposed. The genetic modification causes acetaldehyde to be produced within the cell (the colonocyte). The rest

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happens in the colonic lumen outside the colonocyte by bacteria. It may take several steps for the acetaldehyde to enter the cell and then the nucleus. Even if a low amount of acetaldehyde is produced in the colonocyte by genetic modification, this may be relevant because it is closer to the target. Apte: The retinol story is interesting. In the liver we know there is a small window for vitamin A reinforcement in alcoholics because it can cause toxicity. Since retinol is a precursor for retinoic acid, is there any place for retinol replacement? Seitz: The story has now been completed. If retinol or retinoic acid is given to alcohol-fed animals, both compounds are metabolized by CYP2E1, which is induced by alcohol. As a result of this increased metabolism apoptotic metabolites of retinol and retinoic acid appear in the liver. This is the major mechanism by which the combined application of alcohol, β-carotene, retinol or retinoic acid create toxicity. The therapeutic window for β-carotene is very small and replacement therapy of β-carotene, retinol or retinoic acid would be the same. We don’t recommend it. References Deng XS, Bludeau P, Deitrich RA 2004 Formation of ethyl nitrite in vivo after ethanol administration. Alcohol 34:217–223 Larsson SC, Giovannucci E, Wolk A 2005 Vitamin B6 intake, alcohol consumption, and colorectal cancer: a longitudinal population-based cohort of women. Gastroenterology 128:1830– 1837 Lumeng L, Li TK 1974 Vitamin B6 metabolism in chronic alcohol abuse. Pyridoxal phosphate levels in plasma and the effects of acetaldehyde on pyridoxal phosphate synthesis and degradation in human erythrocytes. J Clin Invest 53:693–704 Moriyama Y, Okamura T, Kajinami K et al 2002 Effects of serum B vitamins on elevated plasma homocysteine levels associated with the mutation of methylenetetrahydrofolate reductase gene in Japanese. Atherosclerosis 164:321–328 Shaw S, Jayatilleke E, Herbert V, Colman N 1989 Cleavage of folates during ethanol metabolism. Role of acetaldehyde/xanthine oxidase-generated superoxide. Biochem J 257:277–280

The determination of acetaldehyde in exhaled breath Robert Tardif Department of Occupational & Environmental Health, Faculty of Medicine, University of Montreal, 2375 Cote St-Catherine, Montreal, Québec, Canada H3T 1A8

Abstract. Breath acetaldehyde has been used to investigate the production of acetaldehyde after ethanol ingestion in ALDH2-deficient individuals, to compare ethanol and acetaldehyde metabolism, to study the toxicological outcome of metabolic inhibitors of ALDH2, and as a biomarker of exposure to ethanol vapours. A number of approaches have been developed to collect representative breath samples (mixed air or alveolar air) and to measure breath acetaldehyde. For instance, the highest breath concentration of acetaldehyde (∼50 nmoles/l) measured during pulmonary ethanol exposure (1000 ppm, 6 hours) is of the same magnitude as those measured after ingestion of 0.4–0.8 g/kg (∼60–80 nmoles/ l), whereas endogenous levels rarely exceed 1 nmole/l. The interpretation of breath acetaldehyde is compounded by several factors; smoking, ALDH2 polymorphism and alcohol drinking habits are associated with higher breath/blood levels. Some authors have considered that breath acetaldehyde, particularly low levels, cannot be used to estimate blood acetaldehyde. Despite the problems associated with its determination, breath acetaldehyde could be an interesting tool for estimating ethanol or acetaldehyde exposure. However, some additional research efforts will be necessary in order to standardize the technique used for breath sampling and to control the influence of the factors that are known to affect breath acetaldehyde determination. 2007 Acetaldehyde-related pathology: bridging the trans-disciplinary divide. Wiley, Chichester (Novartis Foundation Symposium 285) p 125–136

The determination of volatile organic chemicals (VOCs), such as acetaldehyde, in exhaled breath is useful for several applications in toxicology, epidemiology and pathology. Among other things, breath testing, which involves a safe and noninvasive approach, is being used to assess environmental or occupational exposure to chemicals, in several metabolic studies, and in the diagnosis of several pathologies or diseases. Indeed, the concentration of a given chemical in a breath sample, such as alveolar air (end-expired air), reflects its blood concentration and, as such, the internal dose. Hence, the determination of VOCs in breath samples represents a useful approach for estimating the internal exposure to toxic chemicals and the health risk as well. 125

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Acetaldehyde, the first metabolite of ethanol, is a very reactive chemical and has been shown to be mutagenic and carcinogenic in animals. It has been reported that acetaldehyde is produced by the lung cancer cell lines SK-MES and CALU-1 under in vitro conditions (Smith et al 2003). Acute toxic effects (e.g. irritation, bronchoconstriction, flushing syndrome) have been associated with the ingestion of ethanol or with the inhalation of ethanol or acetaldehyde vapours. Hald and Jacobsen were the first, in 1948, to isolate acetaldehyde chemically from the expired air of individuals previously treated with disulfiram (Antabuse)—a known metabolic inhibitor of acetaldehyde—and ethanol (Hald & Jacobsen 1948). A number of epidemiological and experimental studies have provided convincing evidence of the role of acetaldehyde as a significant risk factor for various diseases. These observations resulted in an increasing interest, particularly during the 1980s, in the determination of acetaldehyde in biological fluids (e.g. blood and plasma) for the assessment of acetaldehyde exposure resulting from local or systemic ethanol metabolism. However, several reports pointed out the difficulties affecting acetaldehyde measurement in blood/plasma, particularly due to the low levels usually encountered. Acetaldehyde, like other volatiles, crosses the alveolar–capillary membrane of the lungs and is in equilibrium with pulmonary blood. Therefore, in order to overcome the problems associated with blood/plasma analysis, some researchers considered the possibility of measuring acetaldehyde in breath samples as a surrogate to blood/ plasma acetaldehyde (Freund & O’Halloren 1965, Stowell et al 1980). Several aspects of acetaldehyde determination in breath have been previously reviewed by Jones (1995). The objective of this paper is to provide a historical perspective of the developments regarding acetaldehyde analysis in exhaled breath, with special emphasis on: (1) the various approaches that have been used to collect breath samples, (2) the analytical techniques applied for the detection/quantification of acetaldehyde in breath samples, and (3) the main factors that are known to influence the production/elimination kinetics of acetaldehyde and the levels of acetaldehyde in exhaled breath. Utility of breath acetaldehyde determination Breath acetaldehyde has been primarily used in studies aimed at characterizing the production of acetaldehyde after ethanol ingestion in normal individuals or in individuals with atypical forms of alcohol dehydrogenase (ADH2) and/or aldehyde dehydrogenase (ALDH2) (Freund & O’Hollaren 1965, Stowell et al 1980, Couchman & Crow 1980, Jones et al 1984, Stowell et al 1984, Sarkola et al 2002). Similarly, other researchers have used breath acetaldehyde to compare ethanol and acetaldehyde metabolism or to investigate the toxicological/toxicokinetic

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outcome resulting from the administration of metabolic inhibitors of ALDH2 (e.g. disulfiram, calcium carbamide) (Miozi et al 1980, Hald & Jacobsen 1948, Stowell et al 1984, Sarkola et al 2002). In some of these studies, breath acetaldehyde or ethanol was used to indirectly estimate their equivalent blood concentration (Stowell et al 1980, Jones et al 1984). Recently, acetaldehyde has been measured, and compared to ethanol levels, in breath samples obtained from volunteers exposed by inhalation to low levels (25–1000 ppm) of ethanol vapours (Tardif et al 2004). Analytical approaches Breath air sample The type of breath sample, either mixed (dead space) or end-exhaled air (alveolar), used for acetaldehyde determination differs from one study to another (see Tables 1–3). Moreover, from the authors’ description, it was sometimes impossible to clearly identify which type was used. Although it still remains difficult to assess the impact of breath sample type, it is reasonable to consider that it could influence the levels of acetaldehyde measured, depending on the dose of ethanol and the time of breath sampling. For instance, Pikkarainen et al (1981) measured acetaldehyde levels in dead space and alveolar air in volunteers who were administered ethanol (0.8 g/kg); compared to dead space levels, the levels measured in alveolar air (60–120 min post administration) showed a downward trend (−15% to −42%), although the differences were not statistically significant. Analysis of acetaldehyde Since the first study that reported acetaldehyde in breath, the analytical methodology used to collect breath samples and to measure breath acetaldehyde has considerably improved and has contributed to an increase in the sensitivity/accuracy of this analysis. In 1948, Hald and Jacobson collected breath samples through a valve connected to a U-shaped glass tube filled with an absorber, and measured acetaldehyde by spectrophotometry after a colorimetric reaction with 2,4dinitrophenylhydrazine (Hald & Jacobsen 1948). Later, acetaldehyde was analysed by injecting breath air directly into a gas chromatograph (Freund & O’Hollaren 1965). Other authors included a prior concentrating step, using either a cold trap (Dannecker et al 1981, Shaskan & Dolinsky 1985) or drawing a known volume of breath air through a glass tube containing a derivatizing agent such as 2hydroxymethylpiperidine (Tardif et al 2004). Similarly, Lin et al (1995) measured acetaldehyde by high performance liquid chromatography after reaction with 2,4-dinitrophenylhydrazine. More recently, some researchers took advantage of the high specificity and sensitivity provided by selected-ion flow-tube mass

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TABLE 1 Highest breath concentrations of acetaldehyde following ethanol administration, from studies directly reporting acetaldehyde concentrations in breath Ethanol dose

Subjects

n

Breath samples

Acetaldehyde (nmoles/l)

0.87 g/kg

Healthy men

6

Alveolar

147–227

38–70 g

Healthy men

6

Alveolar

6–27

0.4 g/kg 0.4 g/kg

Healthy men Healthy men (smokers) Healthy men (non-smokers) Healthy men Healthy men Healthy women (−OCc) Healthy women (+OC) Healthy men (n = 3) and women (n = 2)

29 11

Mixed air Alveolar

0.3 g/kg 0.5 g/kg

1000 ppm × 6h (∼0.2 g/ kg)d

Alveolar Alveolar

14 –36a 28 ± 18b 22 ± 15

Wong et al (1992) Sarkola et al (2002)

23 ± 12

12 5

Freund & O’Hallaren (1965) Couchman & Crow (1980) Mizoi et al (1980) Jauhonen et al (1982)

23 ± 3

12 5 13 10

58 ± 35 (SD) 52 ± 8 (SEM)

Reference

Alveolar

48 ± 10.8 (SD)e Tardif et al (2004)

a Values reported in Wong et al’s papers are in µmoles/l, which is impossible considering the dose administered.

b

Measured 75 minutes after ethanol administration. Women using (+OC) or not using (−OC) oral contraceptives. d Estimated considering 100% pulmonary absorption. e Concentration measured at 4 hours after the start of exposure. c

spectrometry (SIFT-MS) (Diskin et al 2003, Turner et al 2006). Lately, Mitsubayashi et al (2005) developed bioelectronic sniffers (gas sensors) incorporating enzymes such as ALDH or alcohol oxidase for the measurement of acetaldehyde or ethanol, respectively, in expired air. Levels of acetaldehyde in breath Almost all data on breath acetaldehyde are from studies where volunteers ingested various doses of ethanol. The concentrations obtained in some studies are reported in Tables 1 and 2. Some of them are directly reporting values of acetaldehyde (Table 1). Other values, however, have been indirectly estimated from the blood levels provided in the papers, since these blood levels were calculated from breath level values measured in volunteers; these blood levels have been divided by the

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TABLE 2 Highest breath concentrations of acetaldehyde following ethanol administration, from studies reporting blood acetaldehyde estimated from breath concentrationsa Ethanol dose 1.2 g/kg 0.4 g/kg 0.8 g/kg

0.25 g/kg 0.25 g/kg

Subjects

n

Healthy men Healthy men Healthy men (smokers) Healthy men (non-smokers) Healthy men Healthy men

5 5 3

Breath samples

Acetaldehyde (nmoles/l)

Mixed Mixed Not specified

32–84 32–159 70 ± 11 (SEM)

Alveolar Alveolar

18 ± 3 (SEM) 9–34

3 10 10

31 ± 5

Reference Stowell et al (1980) Stowell et al (1984) Pikkarainen et al (1981)b Jones et al (1987) Jones et al (1988)

a

Breath concentrations were estimated indirectly from reported blood levels as follows: BreathACETALDEHYDE = BloodACETALDEHYDE ∏ Acetaldehyde blood:air partition coefficient (Pb : air = 189) (Stowell et al 1980; Jones 1995). b Values reported in Jones 1995.

TABLE 3 Endogenous breath concentrations of acetaldehyde reported in humans Subjects

n

Breath samples

Acetaldehyde (nmoles/l)

Reference

Healthy men (n = 9) and women (n = 5) NA-NS NA-S A-NS A-S Healthy men (n = 3) and women (n = 2) Healthy men (n = 3) and women (n = 2) Healthy men (n = 19) and women (n = 11)

14

Mixed air

0.016–0.25

Dannecker et al (1981)

14 12 16 15 5

Not specified

0.10 ± 0.02 (SEM) 0.37 ± 0.06 0.33 ± 0.06 0.57 ± 0.05 0.045–0.11

Shaskan & Dolinsky (1985)

Not specified

5

Alveolar

30

Mixed air

0.57 ± 0.16 (SD) (0.48–0.81) 0.55 ± 0.39 (SD)

Diskin et al (2003) Tardif et al (2004) Turner et al (2006)

A, alcoholic; NA, non-alcoholic; S, smoker; NS, non-smoker.

value of acetaldehyde blood:air partition coefficient (Table 2) ( Jones 1995). Several of these studies reported breath values measured in normal individuals who received acetaldehyde metabolic inhibitors of ALDH2 prior to ethanol administration or in individuals carrying a deficient form of this enzyme (flushing individuals); both situations result in particularly high breath acetaldehyde levels as a result of reduced acetaldehyde metabolism. These values, however, are not presented in this paper.

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There is only one study that reported breath acetaldehyde concentrations after inhalation of ethanol vapours (Tardif et al 2004). Interestingly, this study showed that the highest concentration of acetaldehyde (∼50 nmoles/l) measured during an exposure to 1000 ppm of ethanol for 6 hours is of the same magnitude as those measured after ingestion of 0.4–0.8 g/kg of ethanol (∼60–80 nmoles/l) (Table 1). Nonetheless, few papers report endogenous levels of breath acetaldehyde measured in humans. In general, the levels reported are below 1 nmole/l and are strongly influenced by the individuals’ smoking habits and alcohol use (Shaskan & Dolinsky 1985) (Table 3). Factors modifying acetaldehyde in breath air Despite numerous studies on ethanol metabolism, we still do not have a clear idea of the extent to which breath acetaldehyde is representative of blood acetaldehyde and about how to reduce/avoid the influence of some factors that are known to affect acetaldehyde analysis in breath. Indeed, different sources besides acetaldehyde in blood contribute to acetaldehyde in breath. Pïkkarainen et al (1980, 1981) showed that acetaldehyde originates, in part, from lung microsomal enzymes (likely CYP2E1 known to be enhanced by chronic alcohol consumption) and from microbial ethanol oxidation in the oropharynx. This was confirmed by Homann et al (1997) who showed that the local production of acetaldehyde by oral microflora can be reduced by the use of antiseptic mouthwash. Lately, Turner et al (2006) reported that they have occasionally observed large concentrations of ethanol in breath that they attributed to the consumption of sweet drinks prior to breath sampling. They showed that whereas breath ethanol was enhanced by washing the mouth with sugary drinks, there was no apparent enhancement in the production of acetaldehyde within the mouth. However, this raises the question as to what extent the consumption of sugary food or drinks by individuals with poor oral hygiene might enhance the production of acetaldehyde in the mouth. Jauhonen et al (1982), in a study aimed at evaluating the suitability of breath acetaldehyde for estimating blood acetaldehyde, found that most of the acetaldehyde in the end-expired air of non-Oriental subjects (e.g. Caucasian), who produced low concentrations of acetaldehyde in blood after alcohol ingestion, was produced in the respiratory tract and did not correlate with blood levels. In such situations, calculations of blood acetaldehyde based on breath analysis result in overestimation of the actual concentration. They concluded that breath acetaldehyde is not a good indicator of blood levels when these levels are below 30–50 µM, which are very high concentrations compared to those generally reached after drinking alcohol (Tables 1–2). This was confirmed in a subsequent report by Stowell et al (1984),

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who concluded that blood acetaldehyde ‘. . . cannot under all circumstances be predicted from breath analysis with the same accuracy that blood ethanol concentration can.’ However, Jauhonen et al (1982) reported that the production of acetaldehyde in the respiratory tract, following alcohol ingestion, was clearly exaggerated in Japanese flushers (ALDH2 deficient), and by long-term cigarette smoking, which is also known to induce CYP2E1. Interestingly, Shaskan & Dolinsky (1985) reported that the endogenous levels of acetaldehyde in breath is higher in smokers, and that being an alcoholic smoker results in even higher levels (Table 3). McLaughlin et al (1990) also reported this positive contribution of cigarette smoking. Unfortunately, however, there are no data available for evaluating the impact of ALDH2 deficiency on endogenous levels of acetaldehyde in breath.

Potential impact of ethanol-blended gasoline on breath acetaldehyde The anticipated escalating use of ethanol-blend gasoline in certain countries will result in increasing the ethanol and acetaldehyde exposure of the general population. As a result, there is a demand by regulatory agencies for health-based evidence to assess the potential health risk associated with inhalation of ethanol vapours. Exposure to 25 ppm of ethanol vapours (for 6 hours), which is lower than the maximum ambient air level (46 ppm) reported to occur upon refuelling a vehicle, produced measurable levels of acetaldehyde in breath air (Tardif et al 2004). Accordingly, there is a need to understand better the potential impact of such long-term exposure on endogenous breath acetaldehyde levels and to assess the health risks associated with such exposure, particularly in individuals with ALDH2 deficiency or pulmonary diseases (e.g. asthma).

Conclusion The assessment of the health risk associated with exposure to ethanol or acetaldehyde in the general population or in more susceptible individuals requires a proper estimate of the exposure. Despite the problems associated with its determination, breath acetaldehyde remains an interesting tool for estimating ethanol or acetaldehyde exposure following ingestion or inhalation, with good toxicological relevance, since acetaldehyde is more directly related to toxic effects. In this case, however, some additional research efforts will be necessary in order, among other things, to standardize the technique used for breath sampling and to control/assess the influence of the several factors that are known to affect breath acetaldehyde determination and interpretation.

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References Couchman KG, Crow KE 1980 Breath acetaldehyde levels after ethanol consumption. Adv Exp Med Biol 132:451–457 Dannecker JR Jr, Shaskan EG, Phillips M 1981 A new highly sensitive assay for breath acetaldehyde: detection of endogenous levels in humans. Anal Biochem 114:1–7 Diskin AM, Spanel P, Smith D 2003 Time variation of ammonia, acetone, isoprene and ethanol in breath: a quantitative SIFT-MS study over 30 days. Physiol Meas 24:107–119 Freund G, O’Hollaren P 1965 Acetaldehyde concentrations in alveolar air following a standard dose of ethanol in man. J Lipid Res 6:471–477 Hald J, Jacobsen E 1948 The formation of acetaldehyde in the organism after ingestion of Antabuse (Tetraethylthiuramdisulphide) and alcohol. Acta Pharmacol Toxicol 4:305–310 Homann N, Jousimies-Somer H, Jokelainen K, Heine R, Salaspuro M 1997 High acetaldehyde levels in saliva after ethanol consumption: methodological aspects and pathogenetic implications. Carcinogenesis 18:1739–1743 Jauhonen P, Baraona E, Miyakawa H, Lieber CS 1982 Origin of breath acetaldehyde during ethanol oxidation. Effect of long-term cigarette smoking. J Lab Clin Med 100:908–916 Jones AW, Skagerberg S, Borg S, Anggard E 1984 Time course of breath acetaldehyde concentrations during intravenous infusions of ethanol in healthy men. Drug Alcohol Depend 14:113–119 Jones AW, Neiman J, Hillbom M 1987 Elimination kinetics of ethanol and acetaldehyde in healthy men during the calcium carbimide-alcohol flush reaction. Alcohol Alcohol Suppl 1:213–217 Jones AW, Neiman J, Hillbom M 1988 Concentration-time profiles of ethanol and acetaldehyde in human volunteers treated with the alcohol-sensitizing drug, calcium carbimide. Br J Clin Pharmacol 25:213–221 Jones AW 1995 Measuring and reporting the concentration of acetaldehyde in human breath. Alcohol Alcohol 30:271–285 Lin Y, Dueker SR, Jones AD, Ebeler SE, Clifford AJ 1995 Protocol for collection and HPLC analysis of volatile carbonyl compounds in breath. Clin Chem 41:1028–1032 McLaughlin SD, Scott BK, Peterson CM 1990 The effect of cigarette smoking on breath and whole blood-associated acetaldehyde. Alcohol 7:285–287 Mitsubayashi K, Matsunaga H, Nishio G, Toda S, Nakanishi Y 2005 Bioelectronic sniffers for ethanol and acetaldehyde in breath air after drinking. Biosens Bioelectron. 20:1573–1579 Mizoi Y, Hishida S, Ijiri I et al 1980 Individual differences in blood and breath acetaldehyde levels and urinary excretion of catecholamines after alcohol intake. Alcohol Clin Exp Res 4:354–360 Pikkarainen P, Baraona E, Seitz H, Lieber CS 1980 Breath acetaldehyde: evidence of acetaldehyde production by oropharynx microflora and by lung microsomes. Adv Exp Med Biol 132:469–474 Pikkarainen PH, Baraona E, Jauhonen P, Seitz HK, Lieber CS 1981 Contribution of oropharynx microflora and of lung microsomes to acetaldehyde in expired air after alcohol ingestion. J Lab Clin Med 97:631–636 Sarkola T, Iles MR, Kohlenberg-Mueller K, Eriksson CJ 2002 Ethanol, acetaldehyde, acetate, and lactate levels after alcohol intake in white men and women: effect of 4-methylpyrazole. Alcohol Clin Exp Res 26:239–245 Shaskan EG, Dolinsky ZS 1985 Elevated endogenous breath acetaldehyde levels among abusers of alcohol and cigarettes. Prog Neuropsychopharmacol Biol Psychiatry 9:267–272 Smith D, Wang T, Sule-Suso J, Spanel P, Haj AE 2003 Quantification of acetaldehyde released by lung cancer cells in vitro using selected ion flow tube mass spectrometry. Rapid Commun Mass Spectrom 17:845–850

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Stowell AR, Lindros KO, Salaspuro MP 1980 Breath and blood acetaldehyde concentrations and their correlation during normal and calcium carbamide-modified ethanol oxidation in man. Biochem Pharmacol 29:783–787 Stowell A, Johnsen J, Aune H, Vatne K, Ripel A, Morland J 1984 A reinvestigation of the usefulness of breath analysis in the determination of blood acetaldehyde concentrations. Alcohol Clin Exp Res 8:442–447 Tardif R, Liu L, Raizenne M 2004 Exhaled ethanol and acetaldehyde in human subjects exposed to low levels of ethanol. Inhal Toxicol 16:203–207 Turner C, Spanel P, Smith D 2006 A longitudinal study of ethanol and acetaldehyde in the exhaled breath of healthy volunteers using selected-ion flow-tube mass spectrometry. Rapid Commun Mass Spectrom 20:61–68. Wong MK, Scott BK, Peterson CM 1992 Breath acetaldehyde following ethanol consumption. Alcohol 9:189–192

DISCUSSION1 Worrall: When you talk about blood acetaldehyde levels, what are you referring to? There must be a complex set of equilibria between the plasma and red cells, and protein bound and non-bound acetaldehyde. Eriksson: Yes. I’ll say more about this in my paper. Preedy: This difference between bound and non-bound acetaldehyde is quite important. This ‘storage’ that you alluded to is interesting. I am aware that some studies have examined acetaldehyde dosing and noticed that long after the dosing there is acetaldehyde within the tissue (Heap et al 1995). This tissue was the brain. After the dosing period, the acetaldehyde was measurable in the brain but not in the liver or blood. This could be due to the fact that it was being released from another tissue. Eriksson: This is an important issue. I am speaking about the ‘free’ or ‘loosely bound’ acetaldehyde. Because acetaldehyde is so reactive, there is an enormous amount of different kinds of binding. Some are in equilibrium with the partition and some binding is released with a little bit of heating. It is certainly bound in such a way that it can accumulate. It could be interesting to take different types of tissue to see with different methods how the acetaldehyde could be released. You have gone another way, trying to measure the adducts themselves directly. One could also try to release the acetaldehyde itself. Preedy: In the measurement of breath acetaldehyde, do you have a similar pre-oral hygiene regime? Do subjects have to wash out their mouths? Eriksson: No, but if you look at Mikko Salaspuro’s data the washout affects the saliva acetaldehyde. We have done studies where we take breath measurements and saliva measurements at the same time. The early part of the breath sample could 1

Dr Tardif was unable to attend the meeting, and in his absence his paper was presented by Dr Eriksson, who also fielded the questions in this discussion session.

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have more resemblance to the salivary acetaldehyde and the last part of the alveolar air could match more closely the systemic acetaldehyde. No one has done this very systematically. Seitz: Has anyone taken the saliva and incubated it with headspace chromatography at 37 °C, then heated it up and measured again? In other words, how much acetaldehyde is released from the saliva at body temperature? Eriksson: Not exactly, but there is a lot of acetaldehyde formation at 37 °C which complicates the exact measurement of in vivo saliva acetaldehyde levels. Since the saliva sampling takes some time the detected acetaldehyde levels may overestimate the in vivo levels. Seitz: What percentage of saliva acetaldehyde could be released into the air? Eriksson: It seems that all free or quite loosely bound acetaldehyde can be released from saliva. V Salaspuro: There are salivary proteins that acetaldehyde might bind to. It isn’t clear. M Salaspuro: The biggest problem is the continuous microbial production of acetaldehyde. Evaporation occurs at about 18 degrees, which is a severe confounding factor. Has anyone measured breath acetaldehyde? We have many patients who are intubated, and in these we could easily measure breath acetaldehyde without bacterial contamination. Eriksson: I don’t think this has been reported. The breath acetaldehyde itself is partly of microbiological origin. After it is taken, nothing further happens. The evaporation problem isn’t as big as you propose. The boiling point is very low and initially the saliva is warmer than this. But the acetaldehyde doesn’t evaporate so dramatically. In five minutes you can have about 10% loss. This implies that in the saliva part of the acetaldehyde is in the form of Schiff bases, and in such a form that real partition is not so freely available. Worrall: You suggested trying to drive off acetaldehyde from reversible adducts. We are working on some mass spectrometry, and it doesn’t seem to work: what seems to happen is that some of the reversible adducts are converted into irreversible adducts—we actually change the adduct chemistry. Eriksson: How have you done this? Worrall: So far we have just been trying a modest temperature rise, going from say 30 to 50 °C. Eriksson: Try going a little bit higher. You get as much acetaldehyde as you want! The problem is you get it from other sources, through other chemical reactions. Worrall: That’s why we weren’t trying to go so high. Eriksson: What acetaldehyde you get depends on the binding and the chemistry. A few years ago we published this heating process and what it could achieve (Fukunaga et al 1993). The result depends very much on the temperature level.

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Apte: Once you have the sample what is the stability of acetaldehyde? Eriksson: That’s a good question. We chill everything, and we always try to measure acetaldehyde the same day we do the study. Acetaldehyde can disappear very easily. But it can be preserved at cold temperatures and in the right sort of container. Apte: Do you see any clinical role for this, given all the confounding factors and difficulties with measurement? Eriksson: Yes. First of all it is non-invasive. You can blow it with a tube directly into the machine. It simplifies measurements. It also measures something occurring inside the body. Rao: There is increasing concern over passive smoking. Does this increase acetaldehyde levels? Eriksson: The acetaldehyde content in the air will not be all that high. This needs to be investigated. Morris: In a smoky car, the airborne acetaldehyde concentration gets to be about 40 nM/litre (1 part per million). This is as high as it can get. In a smoky bar it is usually 100-fold lower. Eriksson: That’s about 8 µM in the blood if it is all absorbed. The lung is quite good at absorbing things. People are now using alcohol vapour to study chronic effects of alcohol. Morris: The partition coefficient of alcohol is about 2000 versus 200 for acetaldehyde. This favours the absorption of alcohol. Thornalley: About 10 years ago we addressed the issue of reversible aldehyde binding to plasma proteins with methylglyoxal. We distinguished between free, reversibly bound and irreversibly bound methylglyoxal by using ultrafiltration. If albumin is incubated with methylglyoxal and this is followed by limited ultrafiltration where there is less than 10% change of volume, the methylglyoxal in the first ultrafiltrate is the free form. If you then wash exhaustively three or four times by ultrafiltration then the reversibly bound methylglyoxal comes off the protein into the combined ultrafiltrate and can be quantified. Then there are the irreversible adducts. The main one is an arginine adduct which is quasi-reversible but with a half-life of about two weeks under physiological conditions. So for all intents and purposes this is irreversibly bound. Eriksson: If you have a biological sample you need to do things fast. So while this distinction between reversible and irreversible binding is a useful one, it has its practical limits. Thornalley: In measuring volatiles in breath, can you reference the acetaldehyde in breath to any other volatile that would be useful? Can you measure acetone, for example? Eriksson: Yes. We have an internal standard, but it is the alcohol. If you take saliva or blood at the same time you can overcome these difficulties quite elegantly.

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Ren: I have a point for clarification. Dr Tardif used the term ‘background’ levels of acetaldehyde. What was he referring to? Eriksson: Acetaldehyde is sometimes found as a pollutant in the air. Controls are absolutely essential, therefore. We used to work in the Finnish alcohol monopoly, and in that whole building there was an endogenous ethanol and acetaldehyde level in the air! Ren: How high are background levels? Morris: Background acetaldehyde concentrations in the USA are about 10 parts per billion, which is 0.4 nM/litre. Eriksson: It must depend very much on the environmental conditions. This summer we were faced with a problem because of the forest fires in Russia. Apte: You said earlier that venous blood acetaldehyde levels are almost non-detectable. Eriksson: They are almost undetectable in most conditions. The problem is not the detection limit but more the fact that any treatment of blood, even control blood, involves some artefactual acetaldehyde formation from internal or external alcohol which has to be controlled for. References Heap L, Ward RJ, Abiaka C et al 1995 The influence of brain acetaldehyde on oxidative status, dopamine metabolism and visual discrimination task. Biochem Pharmacol 50:263–270 Fukunaga T, Sillanaukee P, Eriksson CJP 1993 Problems involved in the determination of endogenous acetaldehyde in human blood. Alcohol Alcohol 28:535–541

Ethanol and acetaldehyde: in vivo quantitation and effects on cholinergic function in rat brain Mostofa Jamal, Kiyoshi Ameno, Uekita Ikuo, Mitsuru Kumihashi, Weihuan Wang and Iwao Ijiri Department of Forensic Medicine, Faculty of Medicine, Kagawa University, Ikenobe, Miki, Kita, Kagawa 761-0793, Japan

Abstract. First, ethanol (EtOH) and acetaldehyde levels were determined simultaneously in the striatum of free-moving rats after administration of their major oxidative enzyme inhibitors followed by EtOH. The results showed that acetaldehyde was present in the cyanamide (CY) + EtOH, CY + 4-methylpyrazole (4-MP) + EtOH and CY + sodium azide + EtOH groups. The CY + EtOH-induced peak acetaldehyde level was 195.2 ± 19.4 µM, and this value was significantly higher than those in the other groups. The peak EtOH level was 25.9 ± 2.3 mM in the CY + 4-MP + EtOH group, and this level was considerably higher than the value in EtOH. No significant difference in brain EtOH levels was found in any of the other groups studied. Second, the effects of EtOH and acetaldehyde on choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were investigated in the frontal cortex and hippocampus of high acetaldehyde-producing rats using RT-PCR and Western blot. The results showed that EtOH and acetaldehyde decreased ChAT expression at 40 and 240 min after EtOH dosing in the brain. The acetaldehyde-induced reduction in ChAT expression was significantly higher than that induced by EtOH. No remarkable alteration of AChE expression was observed. The study suggested that catalase made a significant contribution to acetaldehyde formation in the rat brain, and that EtOH and acetaldehyde decreased ChAT expression at 40 and 240 min after EtOH dosing. 2007 Acetaldehyde-related pathology: bridging the trans-disciplinary divide. Wiley, Chichester (Novartis Foundation Symposium 285) p 137–144

Brain acetaldehyde accumulation after ethanol (EtOH) intake has been a topic of debate. Two observations have been made with respect to acetaldehyde formation as well as acetaldehyde transport to the brain: (1) peripherally formed high acetaldehyde may be able to cross the blood–brain barrier (BBB) and enter the brain (Hoover et al 1981), and (2) the enzyme catalase is the chief mediator of acetaldehyde formation in the brain (Aragon et al 1992). It is, therefore, essential to measure the brain acetaldehyde level after inhibition of the major oxidative pathways of EtOH and acetaldehyde metabolism. The oxidation of EtOH occurs 137

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via several enzymatic pathways that include alcohol dehydrogenase (ADH), catalase and CYP2E1 (Ramchandani et al 2001). Another enzyme, aldehyde dehydrogenase (ALDH), breaks the acetaldehyde down into acetate. In this study, a very simple brain microdialysis method was employed for simultaneous quantitation of in vivo EtOH and acetaldehyde in the brain of free-moving rats after administration of their oxidative enzyme inhibitors. The neurons of the brain are adversely affected by the ingestion of alcohol. It is, therefore, of considerable interest to know whether these central effects are due, in part, to acetaldehyde, a metabolite of EtOH, in the brain. EtOH has a substantial effect on the cholinergic system, and this effect may share some features with Alzheimer’s disease (Arendt et al 1988). Therefore, the cholinergic deficits after EtOH consumption have been well documented, but acetaldehyde’s effects on the CNS have remained a matter of debate. Thus, we focused on choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) expression at 40 and 240 min after EtOH dosing in the brain of high acetaldehyde-producing rats in order to clarify the effects of EtOH and acetaldehyde on these enzymes. Materials and methods Animal Male Wistar rats (9–10 weeks-old, 250–300 g) were used throughout the study. For the quantitation of EtOH and acetaldehyde, the rats were divided into the following 7 experimental groups: (a) EtOH (1 g/kg), (b) cyanamide (CY, 50 mg/kg, an ALDH inhibitor) + EtOH, (c) CY + 4-methylpyrazole (4MP, 82 mg/kg, an ADH inhibitor) + EtOH, (d) CY + sodium azide (10 mg/kg, a catalase inhibitor) + EtOH, (e) 4MP + EtOH, (f) sodium azide + EtOH, and (g) CY alone. For the ChAT and AChE analyses, the rats were divided into saline (0.9%), EtOH, and CY + EtOH groups. In both trials, rats received an i.p. injection of EtOH (20% v/v) 30 and 60 min after a dose of sodium azide and CY or 4MP, respectively. Microdialysis The microdialysis procedure and chromatographic conditions were as previously described ( Jamal et al 2003). Using this method, no artefactual acetaldehyde was detectable in the dialysate. In vitro probe recovery of EtOH and acetaldehyde were 72.2 ± 3.6 and 52.6 ± 5.9%, respectively, and the values were corrected. RT-PCR and Western blot The procedures used for the ChAT and AChE analyses have been described in detail previously ( Jamal et al 2006).

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Results and discussion Table 1 shows the EtOH and acetaldehyde levels 30 min after EtOH dosing in the striatum. Acetaldehyde was detected in the CY + EtOH, CY + 4MP + EtOH, and CY + sodium azide + EtOH groups. The CY + EtOH-induced peak acetaldehyde level (195.2 ± 19.4 µM) was significantly greater than the values in the other groups. Treatment with inhibitors did not modify the levels of EtOH that reached the brain from the bloodstream, with the exception that treatment with CY + 4MP + EtOH showing the peak EtOH level (25.9 ± 2.3 mM) was significantly higher than the value for EtOH. Therefore, the results of this study support the hypothesis that catalase and the high level of peripherally-formed acetaldehyde contributed to brain acetaldehyde accumulation in the CY + EtOH group. Peripherally-formed high acetaldehyde played a role in brain acetaldehyde accumulation in the CY + sodium azide + EtOH and catalase played a role in brain acetaldehyde accumulation in the CY + 4MP + EtOH group. Thus, the data obtained from this study demonstrated that catalase made a remarkable contribution to brain acetaldehyde formation. Table 2 shows the percentage of ChAT and AChE expression at 40 and 240 min after EtOH injection in the brain. RT-PCR analysis revealed a significant decrease in ChAT levels at 40 min in the EtOH and CY + EtOH groups, respectively, in the frontal cortex and hippocampus. The hippocampal reduction in ChAT was significantly higher in the CY + EtOH group than in the EtOH group. These findings were consistent with the results of the Western blot analysis, but one discrepancy was found. ChAT mRNA expression was reduced significantly at 240 min in the frontal cortex and hippocampus, respectively, in the CY + EtOH group as revealed by RT-PCR. The reduction in ChAT levels was markedly higher in the CY + EtOH

TABLE 1 The Peak EtOH and acetaldehyde (ACe) levels in the brain Groups CY + EtOH CY + 4MP + EtOH CY + sodium azide + EtOH Sodium azide + EtOH 4MP + EtOH EtOH CY

EtOH level (mM)

ACe level (mM)

17.8 ± 1.1 25.9 ± 2.3 17.9 ± 1.9 16.3 ± 1.7 19.7 ± 1.9 20.1 ± 1.9§ ND

195.2 ± 19.4 129.3 ± 12.1* 76.9 ± 7.3†‡ ND ND ND ND

Data represent mean ± SD (n = 5), analysed by student’s t-test. § P < 0.05, for the difference between EtOH and CY + 4MP + EtOH. * P < 0.05, for the difference between CY + EtOH and CY + 4MP + EtOH, † P < 0.05, for the difference between CY + EtOH and CY + sodium azide + EtOH, and ‡ P < 0.05, for the difference between CY + 4MP + EtOH and CY + sodium azide + EtOH. ND, not determined.

140

TABLE 2 ChAT and AChE mRNA, and ChAT protein expression in the rat frontal cortex (A) and hippocampus (B) mRNAs (% of control) 40 min Groups (A) (B)

Saline EtOH CY + EtOH Saline EtOH CY + EtOH

ChAT 100 ± 10.1 72.8 ± 8.2* 71.6 ± 4.1† 100 ± 7.1 76.5 ± 6.3* 53.0 ± 4.2†‡

Protein (% of control) 240 min

AChE 100 ± 15.0 87.5 ± 19.0 82.8 ± 17.2 100 ± 19.0 115.0 ± 18.3 95.0 ± 15.0

ChAT 100 ± 15.3 92.2 ± 17.1 62.0 ± 9.0†‡ 100 ± 18.2 97.6 ± 17.5 65.5 ± 12.2†‡

AChE 100 ± 18.0 85.6 ± 10.0 99.8 ± 15.0 100 ± 9.1 102.9 ± 18.0 88.2 ± 15.3

40 min

240 min

ChAT

ChAT

100 ± 9.1 49.0 ± 8.2* 34.5 ± 5.0†‡ 100 ± 9.0 54.9 ± 6.1* 32.0 ± 8.3†‡

100 ± 9.1 86.5 ± 15.4 34.1 ± 9.2†‡ 100 ± 11.0 87.8 ± 13.0 49.6 ± 8.1†‡

Data represent mean ± SD (n = 5) as percentage of control, analysed by two-way ANOVA followed by post hoc Tukey-Kramer test. * P < 0.005, for the difference between saline and EtOH, † P < 0.005, for the difference between saline and CY + EtOH, and ‡ P < 0.005, for the difference between EtOH and CY + EtOH.

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group than in the EtOH group. The EtOH group did not show any significant change in the ChAT mRNA levels at 240 min. These findings were also consistent with the results of the Western blot analysis. No significant difference of AChE mRNA was found in either group. The brain EtOH levels were 20.1 ± 1.9 and 0.65 ± 0.1 mM at 30 and 240 min, respectively, and the acetaldehyde levels were 195.2 ± 19.4 and 51.7 ± 11.6 µM at 30 and 240 min, respectively. The data obtained from this study demonstrated that EtOH and acetaldehyde reduced ChAT expression at 40 and 240 min after EtOH injection in the rat brain, and the acetaldehydeinduced decrease in ChAT expression was significantly higher than the decrease by EtOH. Summary We conclude that the enzyme catalase contributed to the accumulation of acetaldehyde in the rat brain. EtOH and acetaldehyde both decreased ChAT expression in the rat brain, and the acetaldehyde-induced decrease in ChAT expression was considerably higher than EtOH-induced. Acknowledgement This study was funded by a Grant-in-Aid for Scientific Research (c) (No-18590637) from the Ministry of Education, Science and Culture, Japan.

References Aragon CM, Rogan F, Amit Z 1992 Ethanol metabolism in rat brain homogenates by a catalase H2O2 system. Biochem Pharmacol 44:93–98 Arendt T, Henning D, Gray JA, Marchbanks R 1988 Loss of neurons in the rat basal forebrain cholinergic projection system after prolonged intake of ethanol. Brain Res Bull 21:563–569 Hoover DJ, Brien JF 1981 Acetaldehyde concentration in rat blood and brain during the calcium carbimide–ethanol interaction. Can J Physiol Pharmacol 59:65–70 Jamal M, Ameno K, Kumihashi M et al 2003 Microdialysis for the determination of acetaldehyde and ethanol concentration in the striatum of freely moving rats. J Chromatogr B 798: 155–158 Jamal M, Ameno K, Ameno S et al 2007 Changes in cholinergic function in the frontal cortex and hippocampus of rat exposed to ethanol and acetaldehyde. Neuroscience 144: 232–238 Ramchandani VA, Bosron WF, Li TK 2001 Research advances in ethanol metabolism. Pathol Biol 49:676–682

DISCUSSION Quertemont: In your experiments, when you tried to measure acetaldehyde in the brain after an injection of ethanol alone, i.e. without any other drug that modulates

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ethanol metabolism, you found non-detectable levels of acetaldehyde. Many papers have been published showing that catalase inhibitors reduced many different alcohol-induced behaviours. In the brain, the major part of acetaldehyde is produced from ethanol metabolism by the enzyme catalase. Therefore, catalase inhibitors are believed to exert their behavioural effects through a decrease in brain acetaldehyde concentrations after alcohol administration, and these studies often interpret their results in terms of the role of acetaldehyde in alcohol’s effects (Quertemont et al 2005). One of the problems with this explanation is that so far it hasn’t been possible to show in vivo that catalase inhibitors reduce the concentrations of acetaldehyde within the brain. As you have an undetectable level of acetaldehyde after ethanol administration alone, would it be possible with your methodology to show a significant reduction of brain acetaldehyde concentrations with catalase inhibitors without the concomitant administration of cyanamide? Such a result would greatly improve our understanding of the role of acetaldehyde in the behavioural effects of alcohol. Jamal: I used a different group, cyanamide plus ethanol, which results in a remarkable increase in acetaldehyde in the blood and brain. In the ethanol only group, there is no detection of acetaldehyde in the brain. The ethanol in the brain is highly metabolized to acetaldehyde and then metabolized to acetic acid. This is the reason I couldn’t detect any acetaldehyde in the ethanol group. Catalase does important things. It produces acetaldehyde in the brain, but we didn’t know how much. So I used the catalase inhibitor powered by ethanol. Catalase inhibitor in combination with cyanamide produced acetaldehyde in the brain, but the cyanamide plus ethanolinduced acetaldehyde production was significantly greater. Eriksson: This was an old argument about the acetaldehyde levels in brain. It has been clearly shown that there is a cut-off point in brain (Eriksson 1977) after which it starts to begin to be detected. This is close to 200 µM. This doesn’t mean that acetaldehyde is not formed in the brain. Indeed, Dr Jamal’s data show that there is active catalase present. It works also during the normal metabolism, but the acetaldehyde is rapidly metabolised to acetate and is also bound or condensated in brain, which may be important. It may be the immediate reactions which are important in the brain and these are hard to study. Preedy: I think that’s an important point. It would be interesting to look at the level of adduct formation. You could correlate this with the actual levels of acetaldehyde. Eriksson: That would be perfect if it was possible, but so far it has not been possible to replicate adduct formation that can be correlated with normal concentrations of acetaldehyde. Worrall: I have measured adduct levels in brain from ethanol-fed rats and from human cerebellar degeneration. I’ll mention this in my paper.

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Deitrich: There is another complication with cyanamide. If you inhibit catalase, then cyanamide will not be effective since it is required to convert cyanamide to an active inhibitor of ALDH. There is a cross-talk with cyanamide which is an inhibitor of ALDH after reaction with catalase, and catalase inhibition, which renders cyanamide ineffective. There is therefore a complication in using the catalase inhibitor with cyanamide as an ALDH inhibitor (Deitrich 2004). Yin: Also, azide can inhibit cytochrome oxidase. There are a lot of complications with azide use. Quertemont: One of the problems with the studies that have been published on catalase inhibitors is that many of them have a lot of non-specific effects. Some of them are also toxic. Eriksson: Did I understand correctly that alcohol had dual effects, first increasing the acetylcholine, and then decreasing it after some time? Jamal: Yes. Our studies showed this. I would like to find out the reason for the short-term increase in acetylcholine. The results of the present findings have confirmed that the brief elevation in acetylcholine release is presumably due to the arousal from the injection of EtOH (Jamal et al 2005). Eriksson: This came to my mind: that there could be an initial reaction, which in itself would be an interesting finding. Jamal: The other thing I need to investigate is if the choline acetyltransferase and acetylcholinesterase transport proteins are responsible for the mechanism of acetylcholine reduction in the rat brain after ethanol and acetaldehyde exposure. Choline acetyltransferase catalyses the biosynthesis of acetylcholine in the cytoplasm of presynaptic terminals, whereas acetylcholinesterase is responsible for degradation of acetylcholine to acetate and choline in the synaptic cleft. Apte: I was interested in the ethanol effect on choline acetyltransferase at 40 min, which seemed to be lost at 240 min. What is the explanation? The acetaldehyde persisted. Jamal: Ethanol produces a short-term elevation in acetylcholine at 40 min followed by a decrease ( Jamal et al 2005). The present results indicated an opposite decrease in choline acetyltransferase at 40 min while ethanol reached a peak and acetaldehyde was not detected. This ethanol-induced elevation did not correspond with the result of choline acetyltransferase. There needs to be further studies to confirm this. Eriksson: There is acute tolerance to alcohol effects. 240 min is enough to get some tolerance. References Deitrich R 2004 Acetaldehyde: Deja Vu du Jour. J Stud Alcohol 65:557–572 Eriksson CJP 1977 Acetaldehyde metabolism in vivo during ethanol oxidation. Adv Exp Med Biol 85a:319–341

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Quertemont E, Tambour S, Tirelli E 2005 The role of acetaldehyde in the neurobehavioral effects of ethanol: a comprehensive review of animal studies. Prog Neurobiol 75:247–274 Jamal M, Ameno K, Wang W et al 2005 Inhibition of acetaldehyde metabolism decreases acetylcholine release in medial frontal cortex of freely moving rats. Brain Res 1039: 90–96

Pharmacological treatments and strategies for reducing oral and intestinal acetaldehyde Ville Salaspuro Research Unit of Substance Abuse Medicine, University of Helsinki, Biomedicum Helsinki, Finland

Abstract. Strong epidemiological, genetic and biochemical evidence indicates that local acetaldehyde exposure is a major factor behind gastrointestinal cancers especially associated with alcohol drinking and smoking. Thus, reducing the exposure to carcinogenic acetaldehyde either by decreasing the production or by eliminating acetaldehyde locally might offer a preventive strategy against acetaldehyde-induced gastrointestinal cancers. Thiol products, such as the amino acid cysteine, are known to be able to protect against acetaldehyde toxicity. Cysteine is able to bind acetaldehyde efficiently by forming a stable thiazolidine–carboxylic acid compound. Special cysteine preparations (such as lozenge and chewing gum) have already been developed to bind smoking and alcohol drinking derived acetaldehyde from the oral cavity. Most importantly, these type of drug formulations offer a novel method for intervention studies aimed to resolve the eventual role of acetaldehyde in the pathogenesis of upper digestive tract cancers. Acetaldehyde exposure could also be influenced by modifying the acetaldehyde producing microbiota. With regard to the upper digestive tract, acetaldehyde production from ingested ethanol could be significantly reduced by using an antiseptic mouthwash, chlorhexidine. In the large intestine acetaldehyde production could be markedly decreased either by reducing the Gram-negative microbes by ciprofloxacin antibiotic or by lowering the intraluminal pH by lactulose. 2007 Acetaldehyde-related pathology: bridging the trans-disciplinary divide. Wiley, Chichester (Novartis Foundation Symposium 285) p 145–157

Alcohol and tobacco are the main risk factors for upper digestive tract cancers ( Bagnardi et al 2001, Castellsagué et al 1999, Zeka et al 2003). A plausible explanation for the high cancer risk in alcoholics and smokers is the increased exposure to high local acetaldehyde concentrations in the upper digestive tract (Salaspuro & Salaspuro 2004, Salaspuro 2003). Carcinogenic acetaldehyde is produced from ingested alcohol by oral microbes during ethanol metabolism. On the other hand, during tobacco smoking acetaldehyde from the smoke becomes easily dissolved into the saliva. Both of these result in significant acetaldehyde concentration in the saliva which, via swallowing, is distributed to all parts of the upper digestive 145

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tract. Thus, combined exposure to acetaldehyde derived from tobacco smoke and microbial alcohol metabolism might explain the multiplicative and synergistic risk effect. There is also a causal link between alcohol consumption and cancers of the colon and rectum (Kune & Vitetta 1992, Scheppach et al 1999). In the large intestine there are many aerobic and/or facultative anaerobic bacteria capable of ethanol oxidation with subsequent production of acetaldehyde (Salaspuro 1996). Therefore ethanol consumption leads to marked intracolonic acetaldehyde concentration ( Jokelainen et al 1996, Seitz et al 1990). During ethanol metabolism acetaldehyde is produced by normal digestive tract microbiota. This microbially mediated pathway for acetaldehyde production can be modulated by treatment with antiseptics, antibiotics or prebiotics, which affect the microbial counts or the metabolic activity of the microbes. On the other hand, acetaldehyde can be inactivated by locally administered cysteine. Cysteine is a sulfur-containing amino acid present in dietary proteins. Decades ago it was found in experimental studies that free cysteine is able to protect against the lethal effects of acetaldehyde (Sprince et al 1974). Cysteine is able to ameliorate the toxicity of acetaldehyde by forming a stable 2-methyl-thiazolidine-4-carboxylic acid (MTCA) adduct (Cederbaum & Rubin 1976). This review presents known strategies and a novel experimental approach to decrease the local acetaldehyde exposure of the upper and lower digestive tracts, with an eventual goal of reducing the prevalence of digestive tract cancers.

Reducing the acetaldehyde exposure in the upper digestive tract Chlorhexidine After ingestion, ethanol is present in saliva in concentrations comparable to blood ethanol levels. Human oral flora is known to contain over 350 cultivatable species of bacteria and many of them are capable of producing mutagenic (>50 µM) amounts of acetaldehyde from ethanol. For instance, certain Streptococcus and Neisseria species present in the oral flora possess high alcohol dehydrogenase (ADH) activity and are able to produce high levels of acetaldehyde from ethanol in vitro (Kurkivuori et al 2006, Muto et al 2000). Poor oral hygiene is an accepted risk factor for oral cancer, which is associated with bacterial overgrowth and an increased in vitro salivary acetaldehyde production from ethanol (Homann et al 2000). Acetaldehyde produced in the saliva by oral microbes can be significantly reduced by using an antiseptic mouthwash, chlorhexidine. In a study of 10 volunteers salivary acetaldehyde levels after ethanol drinking decreased by about 40% after three-day treatment with chlorhexidine (Homann et al 1997). The acetaldehyde peak levels decreased from 143.3 µM to 49 µM. There is recent evidence suggesting that

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acetaldehyde levels over 50 µM are mutagenic ( Theruvathu et al 2005). In addition to chlorhexidine mouthwash, an invention involving chlorhexidine, formulated as a controlled-release chip fixed with a dental device has been hypothesized to be a rational strategy for reducing acetaldehyde production by oral microbiota (Rota & Poggi 2003). Cysteine Cysteine is a sulfur-containing amino acid. With respect to human physiology, it is considered as semiessential, since it can be synthesized from methionine and serine. Cysteine is normally consumed as a component of dietary proteins. The estimated average intake of cysteine is about 1 g/day. Cysteine, cystine (two cysteine molecules attached by disulfide linkage) and cysteine-containing peptides are absorbed after digestion from the small intestine. The importance of cysteine is related to the presence of a sulfur-containing functional thiol group in its side chain. Cysteine plays a key role in the regulation of cellular redox state and is also a rate-limiting amino acid in the synthesis of glutathione (Shoveller et al 2005). Earlier data strongly suggested that cysteine should be administered locally in order to be effective in the elimination of acetaldehyde. Due to its reactivity, it is unlikely that free, effective cysteine will reach the desired site of action through systemic delivery. Thus, the local liberation of cysteine to bind acetaldehyde should be preferred. This type of in situ method is associated with several benefits including (i) cysteine would not have to be absorbed from the gut, (ii) formed acetaldehyde is directly and immediately bound at the site of its formation and (iii) the total amount of delivered cysteine can be kept low. The condensation product, MTCA, is formed from the reaction of acetaldehyde with cysteine according to the following equation:

COOH CH3CHO +

H2N

CH

COOH

CH2 CH 3

SH Acetaldehyde

+

Cysteine

↔

HN

CH

CH

CH2

+

H 2O

S

2-methyl-thiazolidine-

+

water

4-carboxylic acid (MTCA)

There is some evidence that this thiazolidine derivative may undergo nitrosation in vivo, forming nitroso-thiazolidine-4-carboxylic acid. This substance is detected

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in vivo from human urine especially from smokers without cysteine supplementation, indicating endogenous deactivation of acetaldehyde by cysteine (Ohshima & Bartsch 1984). There exits a strong and linear correlation between salivary acetaldehyde and ethanol levels (Homann et al 1997). Therefore, acetaldehyde is continuously formed in the saliva during ethanol challenge. In order to bind the acetaldehyde during ethanol oxidation in the oral cavity, a special buccal drug formulation with Lcysteine is required. A tablet which slowly releases intact L-cysteine enables continuous and direct local binding of reactive acetaldehyde. In a human in vivo study, up to two-thirds of acetaldehyde could be removed from saliva after ethanol intake with this type of buccal tablet (Fig. 1). Tablets containing 100 mg of L-cysteine reduced the acetaldehyde exposure (expressed as area under the curve, AUC) of the volunteers from 162.3 ± 34.2 µM × h to 54.3 ± 11 µM × h (P = 0.003) as compared to placebo tablet, after ingestion of ethanol (0.8 g/kg of body weight) (Salaspuro et al 2002). This finding implies that this drug formulation could potentially be used for the prevention of the local toxic effects of acetaldehyde during alcohol consumption in the oral cavity and upper digestive tract. In addition to ethanol, another major source of acetaldehyde is tobacco, since acetaldehyde is one of the major toxic components of tobacco smoke (Fowles &

50 Placebo Cysteine

Acetaldehyde (µM)

40

30

20

10

0 0

50

100

150

200

250

300

min

FIG. 1. In vivo acetaldehyde levels (mean ± SEM) in saliva of volunteers with placebo or Lcysteine-containing buccal drug formulation after a dose of alcohol. Differences between concentrations are significant at all time points from 20 min to 320 min (P ≤ 0.001). Adapted from Salaspuro & Salaspuro (2004).

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Dybing 2003, Hoffmann & Hoffmann 1997). Acetaldehyde is easily dissolved from the mainstream tobacco smoke into saliva and is further distributed to the entire upper digestive tract of smokers via swallowing. During a smoking period, in vivo salivary acetaldehyde level increases markedly ranging from 100 µM to 400 µM. In a placebo controlled in vivo study with smokers it was shown that carcinogenic acetaldehyde can be totally inactivated by a lozenge containing 5 mg of L-cysteine (Salaspuro et al 2006). In addition, there was an inverse dose–response relationship between the cysteine content of the lozenge and salivary acetaldehyde concentration (Fig. 2). As acetaldehyde is totally eliminated from saliva the carcinogenic potential of acetaldehyde is probably eliminated also in the oesophagus and stomach. Most importantly, these type of drug formulations offer a novel method for intervention studies aimed to resolve the real role of acetaldehyde in the pathogenesis of upper digestive tract cancers. The idea of the chemopreventive mechanism of this tablet is that it is sucked during every smoking period. Because of the resolving time of about 6 minutes, the tablet designed for tobacco is not ideal for longer smoking periods as in the case of cigar smoking. After the tablet has dissolved, the salivary acetaldehyde increases after three minutes to the placebo level (Fig. 3). The use of the L-cysteine tablet is safe because L-cysteine, as a non-essential amino acid, has no known adverse effects in the concentrations described. Furthermore, the daily dose of L-cysteine would be very small even if one is a heavy smoker because an efficient tablet contains only 5 mg of L-cysteine.

Salivary acetaldehyde (µM)

400

300

200

* 100

**

**

**

5mg

10mg

0 0mg

1,25mg

2.5mg

Cysteine concentration in the tablet

FIG. 2. Salivary acetaldehyde levels immediately after tobacco smoking with placebo- or Lcysteine-containing tablet (*P = 0.007, **P < 0.001). Adapted from Salaspuro et al (2006).

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Acetaldehyde (µM)

300

250

200

150

100

50

0 0

3

6

9

12

15

18

21

min Smoking started with L-cysteine (5mg) tablet

Tablet totally dissolved

FIG. 3. Salivary acetaldehyde concentration in vivo during cigar smoking with a lozenge containing 5 mg of L-cysteine. Adapted from Salaspuro et al (2006).

Reducing the acetaldehyde exposure in the large intestine Intracolonic acetaldehyde emerges from microbial metabolism due to anaerobic fermentation or due to the oxidation of ingested ethanol. There is substantial evidence for microbial production of acetaldehyde from ingested ethanol under aerobic or microaerobic conditions prevailing near the mucosal surface of the large intestine ( Jokelainen et al 1996, Seitz et al 1990). After alcohol consumption ethanol is absorbed from the intestine and is thereafter distributed by blood circulation into the whole water compartment of the human body. Therefore, ethanol concentrations in the colon are comparable with that found in the blood. In the large intestine ethanol can be metabolized by intracolonic microbes to acetaldehyde. Consequently, high acetaldehyde levels have been found after alcohol administration in the colon of experimental animals. These acetaldehyde concentrations are significantly lower in germ-free animals than in conventional rats, highlighting the essential role of microbes in intracolonic acetaldehyde production (Seitz et al 1990). On the other hand, there are measurable levels of acetaldehyde in the large intestine even without ingested ethanol. This acetaldehyde is probably produced

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endogenously during alcoholic fermentation or from fermented ethanol (Visapää et al 1998). Thus, by modifying the microbiota or the environment with antibiotics or prebiotics it is possible to enhance or reduce the local acetaldehyde production in the large intestine. Ciprofloxacin Ciprofloxacin, an antibiotic, possesses excellent antibacterial activities against most aerobic and facultative anaerobic bacteria. It has been shown that ciprofloxacin treatment reduces the total ethanol elimination rate by 9–10% in both rats and humans, which is associated with a reduction in faecal aerobic flora and faecal ADH activity ( Jokelainen et al 1996, Tillonen et al 1999). In this way the formation of endogenous acetaldehyde in the large intestine could be abolished almost totally. Furthermore, the intracolonic production of acetaldehyde from ingested ethanol could also be almost totally inhibited by ciprofloxacin treatment. The mean intracolonic acetaldehyde concentration in the rats after a dose of ethanol (1.5 g/kg of body weight i.p.) was reduced by ciprofloxacin treatment from 483 ± 169 µM to 23 ± 15 µM (Visapää et al 1998). Lactulose Lactulose is a non-absorbable disaccharide, which is rapidly metabolized by bacteria representing normal large bowel microbiota to lactic acid. This results in a significant decrease in the pH of the contents of the large intestine. By acidifying the intracolonic environment with lactulose, the pH-dependent ADH-mediated microbial ethanol oxidation and acetaldehyde production is proportionally inhibited. It has been shown in rats that lactulose feeding effectively inhibits ethanol oxidation by colonic microbes and subsequently reduces intracolonic acetaldehyde levels (Zidi et al 2003). Interestingly, lactulose has also been shown to significantly decrease the recurrence rate of colorectal adenomas (Roncucci et al 1993), which thus could be related to the ability of lactulose to decrease the concentration of carcinogenic acetaldehyde in the large intestine. In conclusion, numerous studies emphasize the potential role of acetaldehyde in the carcinogenesis of the digestive tract in humans. L-cysteine effectively binds reactive acetaldehyde when used as different drug formulations designed to be used during alcohol drinking and/or tobacco smoking. As a safe amino acid cysteine could be used to reduce the local acetaldehyde exposure, especially among certain high risk groups, e.g. ALDH2-deficient Asians, smokers and heavy drinkers. Thus, these cysteine preparations warrant further clinical intervention studies to find out the eventual role of acetaldehyde in the pathogenesis of upper digestive tract cancers.

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In order to reduce the acetaldehyde exposure derived from microbial metabolism, good oral health is recommended. Also the use of antiseptic mouthwash (without alcohol) efficiently reduces the number of oral microbes which results in a decreased acetaldehyde exposure after alcohol consumption. With regard to the large intestine, experimental results show that ciprofloxacin and lactulose are efficient in reducing intracolonic acetaldehyde levels. In particular, lactulose, as a harmless prebiotic, should be considered as a tentative agent for further clinical trials.

References Bagnardi V, Blangiardo M, La Vecchia C, Corrao G 2001 A meta-analysis of alcohol drinking and cancer risk. Br J Cancer 85:1700–1705 Castellsagué X, Muñoz N, De Stefani E et al 1999 Independent and joint effects of tobacco smoking and alcohol drinking on the risk of esophageal cancer in men and women. Int J Cancer 82:657–664 Cederbaum AI, Rubin E 1976 Protective effect of cysteine on the inhibition of mitochondrial functions by acetaldehyde. Biochem Pharmacol 25:963–973 Fowles J, Dybing E 2003 Application of toxicological risk assessment principles to the chemical constituents of cigarette smoke. Tob Control 12:424–430 Hoffmann D, Hoffmann I 1997 The changing cigarette, 1950–1995. J Toxicol Environ Health 50:307–364 Homann N, Jousimies-Somer H, Jokelainen K, Heine R, Salaspuro M 1997 High acetaldehyde levels in saliva after ethanol consumption: methodological aspects and pathogenetic implications. Carcinogenesis 18:1739–1743 Homann N, Tillonen J, Meurman JH et al 2000 Increased salivary acetaldehyde levels in heavy drinkers and smokers: a microbiological approach to oral cavity cancer. Carcinogenesis 21:663–668 Jokelainen K, Matysiak-Budnik T, Mäkisalo H, Höckerstedt K, Salaspuro M 1996 High intracolonic acetaldehyde values produced by a bacteriocolonic pathway for ethanol oxidation in piglets. Gut 39:100–104 Kune GA, Vitetta L 1992 Alcohol consumption and the etiology of colorectal cancer: a review of the scientific evidence from 1957 to 1991. Nutr Cancer 18:97–111 Kurkivuori J, Salaspuro V, Kaihovaara P et al 2007 Acetaldehyde production from ethanol by oral streptococci. Oral Oncol 43:181–186 Muto M, Hitomi Y, Ohtsu A et al 2000 Acetaldehyde production by non-pathogenic Neisseria in human oral microflora: implications for carcinogenesis in upper aerodigestive tract. Int J Cancer 88:342–350 Ohshima H, Bartsch H 1984 Monitoring endogenous nitrosamine formation in man. In: Berlin A, Draper M, Hemminki K, Vainio H (eds) Monitoring human exposure to carcinogenic and mutagenic agents. International Agency for Research on Cancer, Oxford University Press, p 233–246 Roncucci L, Di Donato P, Carati L et al 1993 Antioxidant vitamins or lactulose for prevention of the recurrence of colorectal adenomas. Dis Colon Rectum 36:227–234 Rota MT, Poggi P 2003 Reduction of oral acetaldehyde levels using a controlled-release chlorhexidine chip as a prevention strategy against upper digestive tract cancer. Med Hypotheses 60:856–858 Salaspuro M 1996 Bacteriocolonic pathway for ethanol oxidation: characteristics and implications. Ann Med 3928:195–200

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Salaspuro M 2003 Alcohol consumption and cancer of the gastrointestinal tract. Best Pract Res Clin Gastroenterol 17:679–694 Salaspuro V, Salaspuro M 2004 Synergistic effect of alcohol drinking and smoking on in vivo acetaldehyde concentration in saliva. Int J Cancer 111:480–483 Salaspuro V, Hietala J, Kaihovaara P, Pihlajarinne L, Marvola M, Salaspuro M 2002 Removal of acetaldehyde from saliva by a slow-release buccal tablet of L-cysteine. Int J Cancer 97: 361–364 Salaspuro VJ, Hietala JM, Marvola ML, Salaspuro MP 2006 Eliminating carcinogenic acetaldehyde by cysteine from saliva during smoking. Cancer Epidemiol Biomarkers Prev 15:146–149 Scheppach W, Bingham S, Boutron-Ruault MC et al 1999 WHO consensus statement on the role of nutrition in colorectal cancer. Eur J Cancer Prev 8:57–62 Seitz HK, Simanowski UA, Garzon FT et al 1990 Possible role of acetaldehyde in ethanol-related rectal cocarcinogenesis in the rat. Gastroenterology 98:406–413 Shoveller AK, Stoll B, Ball RO, Burrin DG 2005 Nutritional and functional importance of intestinal sulfur amino acid metabolism. J Nutr 135:1609–1612 Sprince H, Parker C, Smith G, Gonzales L 1974 Protection against acetaldehyde toxicity in the rat by L-cysteine, thiamin and L-2-methylthiazolidine-4-carboxylic acid. Agents Actions 4:125–130 Theruvathu JA, Jaruga P, Nath RG, Dizdaroglu M, Brooks PJ 2005 Polyamines stimulate the formation of mutagenic 1,N2-propanodeoxyguanosine adducts from acetaldehyde. Nucleic Acids Res 33:3513–3520 Tillonen J, Homann N, Rautio M, Jousimies-Somer H, Salaspuro M 1999 Ciprofloxacin decreases the rate of ethanol elimination in humans. Gut 44:347–352 Visapää JP, Jokelainen K, Nosova T, Salaspuro M 1998 Inhibition of intracolonic acetaldehyde production and alcoholic fermentation in rats by ciprofloxacin. Alcohol Clin Exp Res 22:1161–1164 Zeka A, Gore R, Kriebel D 2003 Effects of alcohol and tobacco on aerodigestive cancer risks: a meta-regression analysis. Cancer Causes Control 14:897–906 Zidi SH, Linderborg K, Väkeväinen S, Salaspuro M, Jokelainen K 2003 Lactulose reduces intracolonic acetaldehyde concentration and ethanol elimination rate in rats. Alcohol Clin Exp Res 27:1459–1462

DISCUSSION Preedy: I have read that acetaldehyde or some protein adducts are actually toxic to some cells. Is it possible that the adduct that you are forming in very high concentrations could be toxic? V Salaspuro: Not that I am aware of. I have examined the rather sparse literature on this topic and there is no evidence of toxicity. Preedy: It would be interesting to test it in vitro in cell lines. Emery: Is the effect specific to cysteine or can you do a similar job with other amino acids? V Salaspuro: We have tried this with several amino acids and substances, such as N-acetylcysteine. This also binds to acetaldehyde but not so effectively.

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Worrall: We have done some kinetic studies on thiazolidine adduct formation and breakdown. The amino group is needed near the thiol to get the ring structure, otherwise it just makes a thio-hemi-acetal, which is not stable. I have a question. We use acid to break down the thiazolidine, so are you shifting the acetaldehyde from the mouth further down the gut into the stomach? V Salaspuro: This has occurred to me. If in vivo it is nitrosated, this is a stable compound. But I don’t know the in vivo role of this. In our in vitro studies we have not seen any release of acetaldehyde even in acidic conditions. That might be due to the surplus of cysteine as compared to acetaldehyde. Eriksson: Penicillamine is doing the same job as for making the ring structure. How much would then be hydrolysed in the stomach is an interesting question. Albano: What about the effect of peptides containing cysteine such as glutathione? V Salaspuro: We have tested glutathione and it is not as effective. Reactions would be quite rapid in the mouth and gluthatione seems to act quite slowly in the binding process. Worrall: Again, the cysteine is needed right on the end of the molecule for it to work. Internal cysteines don’t work. As you say, glutathione doesn’t seem to form adducts very well. Eriksson: The rat haemoglobin has these reactive SH groups with NH groups in proximity. They bind acetaldehyde very nicely. Rao: Is this effect stereospecific? Worrall: Both L and D work in vitro. Rao: You could use D-cysteine in addition to the alcoholic beverages. It won’t be absorbed. Worrall: You still have the potential problem of shifting the acetaldehyde further down. Crabb: Are there any bacteria that could live on acetaldehyde? Eriksson: Yes, they make alcohol out of it. V Salaspuro: You can modify ethanol and acetaldehyde production and metabolism by bacteria with different ADHs. Crabb: You could take acetaldehyde to acetate by a bacterium with high expression of an aldehyde oxidase, but it would be a genetically modified food. Deitrich: Making alcohol from acetaldehyde is an energy requiring system since a molecule of NADH is oxidized to NAD. Seitz: What was the background of the study with the lactulose and the polyps? How long was the lactulose given to these individuals? This is an interesting aspect. Given long-term, lactulose results in a softer stool. V Salaspuro: In the Roncucci study (Roncucci et al 1993) they gave lactulose to polypectomized patients 20 mg/day for 18 months and the treatment lowered the recurrence rate of the adenomas. Emery: Do other prebiotics have a similar effect on acetaldehyde?

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V Salaspuro: We haven’t tested this. Crabb: Do you know why pH has that effect on acetaldehyde formation? I take it the inside of a bacterium is somehow pH regulated. What is it about the pH that is changing what the bacteria do? Apte: As a corollary, are there any microbes that have inducible ADH or CYP2E1 activity? V Salaspuro: Yes. These changes have been discovered in smokers, for example. They have increased acetaldehyde production in smoking and ethanol consumption. There must be selection for bacteria with enhanced acetaldehyde production. We don’t know whether it is a selection of bacteria with higher ADH or induced ADH activity. Apte: The idea of having a cysteine lozenge during smoking seems attractive, but tobacco smoke has many other carcinogens and it would be dangerous to lull people into a false sense of security about their smoking. V Salaspuro: I agree. This helps us to find out the role of acetaldehyde, though. Eriksson: This statement is relevant, because many arguments are more derived by emotion rather than thinking. These are legal drugs, although they are causing harm. The idea is that if you reduce the harmful effects it is worthwhile. Rao: We showed that epidermal growth factor (EGF) can prevent acetaldehydemediated toxicity in intestinal epithelia (Seth et al 2004, Sheth et al 2004). Saliva secretes EGF at a very high level, nearly 1000-fold its concentration required for biological activity. Unfortunately, cigarette smoking also reduces this level of EGF secretion. Crabb: I was interested to come across some work from the 1990s which showed that alcohol feeding of animals caused some important changes in the salivary glands (Maier et al 1990). They get fatty parotid glands, and we know that human drinkers get big mumps-like glands. We have heard that it does not lead to acetaldehyde being formed in the saliva, but does it affect the EGF or other factors in the saliva? Seitz: We did a study in which we removed the salivary glands. We thought that this had some effect on growth factors. We couldn’t come up with a clear answer at that time. This was the idea we had. M Salaspuro: I have a comment on this. We cannulated the parotid gland duct on those with either normal or deficient ALDH2. Only those individuals with the deficient enzyme secreted acetaldehyde in the parotid gland saliva. This proves that salivary glands are metabolizing alcohol to acetaldehyde. When they aren’t capable of metabolizing acetaldehyde further, there is a spill-over to saliva. Crabb: Is the level in saliva higher than in the blood? M Salaspuro: Yes, about six times higher. Eriksson: I am not sure this has been proven. Acetaldehyde is penetrating the membranes very easily, so it can come from elsewhere. Why shouldn’t they find any acetaldehyde in the parotid gland without a local alcohol metabolizing system?

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And if there is a local system making acetaldehyde, one would expect to find it there. M Salaspuro: Penetrating from where? Eriksson: The saliva, the blood and the vicinity. M Salaspuro: If we put the cannula into the duct, we get just the pure saliva. Eriksson: This pure saliva has still been in the gland for some time and the acetaldehyde could have penetrated into this region. M Salaspuro: Saliva coming from the parotid gland is sterile and reflects the acetaldehyde concentration of the parotid gland cells. There is spill-over of acetaldehyde to saliva only in ALDH2-deficient individuals. In those who have the normal ALDH2 enzyme all acetaldehyde formed in the gland is metabolized further to acetate. Because blood acetaldehyde levels are much lower this is the only way to explain our findings. Eriksson: Then why should it only be formed when there is deficient ALDH? M Salaspuro: Because salivary gland cells can’t remove acetaldehyde efficiently in this case. Aranda: Do the cysteine tablets taste nice? V Salaspuro: No, because of the sulfur. Aranda: I am worried that if you put cysteine in the mouth that bacteria present can use cysteine as a nitrogen source. This will cause the production of all sorts of smelly sulfur products. V Salaspuro: Because of the sulfur cysteine doesn’t taste nice, so we have to add blackcurrant flavour. Smokers, with their reduced taste ability, can’t tell the difference between placebo and one with 5 mg of L-cysteine. Aranda: If you were to add a nitrogen source that is preferred by bacteria you could avoid the bacteria using cysteine as a nitrogen source. Preedy: In terms of taste, have you looked at the effects of eating food on oral acetaldehyde production? Does the change in flora that results change the acetaldehyde production? V Salaspuro: Our experiments are done after fasting. Eating causes a wash-out of bacteria, but this is hard to measure. Crabb: Is acetaldehyde only formed when tobacco is burned, or is there acetaldehyde present with snuff or smokeless tobacco? V Salaspuro: In Sweden a moist snuff is used, and there isn’t any acetaldehyde in this. Eriksson: Yes, acetaldehyde is produced by the burning process. Emery: The antibiotic experiments you reported were interesting. What effect does ciprofloxacin have on other bacteria within the gut? V Salaspuro: In humans a 7 day ciprofloxacin treatment reduced the number of faecal aerobic bacteria from 1.1 × 108 cfu/g to 6.5 × 106 cfu/g. Before the drug administration Enterobacteriaceae was the predominant aerobic flora, and

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Enterococcus sp. were also found. After the ciprofloxacin however, these species totally disappeared from the stool samples. In addition to this, species-specific changes were also detected but those were the major changes. References Maier H, Seitz HK, Mayer B, Adler D, Mall G, Born IA 1990 Lipomatous atrophy of the parotid gland in chronic alcohol consumption. Laryngorhinootologie 69:600–604 Roncucci L, Di Donato P, Carati L et al 1993 Antioxidant vitamins or lactulose for prevention of the recurrence of colorectal adenomas. Dis Colon Rectum 36:227–234 Seth A, Sheth P, Basuroy S, Rao RK 2004 L-glutamine ameliorates acetaldehyde-induced paracellular permeability in Caco-2 cell monolayer. Am J Physiol 287:G510–G517 Sheth P, Seth A, Thangavel M, Basuroy S, Rao RK 2004 Epidermal growth factor prevents acetaldehyde-induced disruption of tight junctions in Caco-2 cell monolayer. Alcohol Clin Exp Res 28:797–804

Alcoholic myopathy and acetaldehyde Victor R. Preedy*, David W. Crabb†, Jaume Farrés‡ and Peter W. Emery* * Nutritional Sciences Research Division, Department of Nutrition and Dietetics, The Franklin Wilkins Building, 150 Stamford Street, London SE1 9NH, UK, † Indiana University School of Medicine, Roudebush VA Medical Center, Emerson Hall, 545 Barnhill Drive, Indianapolis, IN 46202, USA and ‡ Department of Biochemistry and Molecular Biology, Faculty of Sciences, Universitat Autonoma de Barcelona, E-08193 Barcelona, Spain

Abstract. Alcoholic myopathy is characterized by biochemical and morphological lesions within muscle, ranging from impairment of muscle strength and loss of lean tissue to cellular disturbances and altered gene expression. The chronic form of the disease is five times more common than cirrhosis and is characterized by selective atrophy of type II (anaerobic) fibres: type I (aerobic) fibres are relatively protected. Although the causative agent is known (i.e. ethanol), the intervening steps between alcohol ingestion and the development of symptoms and lesions are poorly understood. However, acetaldehyde appears to have an important role in the aetiology of the disease. For example, alcohol is a potent perturbant of muscle protein synthesis in vivo, and this effect is exacerbated by cyanamide pre-dosage, which raises acetaldehyde concentrations. Acetaldehyde alone also reduces muscle protein synthesis in vivo and proteolytic activity in vitro. The formation of acetaldehyde protein adducts is another mechanism of putative importance in alcoholic myopathy. These adducts are formed within muscle in response to either acute or chronic alcohol exposure and the adducts are located preferentially within the sarcolemmal and sub-sarcolemmal regions. However, the significance of protein adduct formation is unclear since we do not currently know the identity of the adducted muscle proteins nor whether adduction alters the biochemical or functional properties of skeletal muscle proteins. 2007 Acetaldehyde-related pathology: bridging the trans-disciplinary divide. Wiley, Chichester (Novartis Foundation Symposium 285) p 158–182

In this paper we first define then review the nature of the disease entity ‘alcoholic myopathy’ and speculate on the role of acetaldehyde. We focus our attention on (i) the enzymes responsible for acetaldehyde formation and oxidation; (ii) protein adducts, (iii) protein degradation and (iv) protein synthesis. This does not mean to say that there are only four processes affected in alcoholic myopathy. Indeed, we have always argued that in alcohol toxicity, all pathways have the potential to be affected at the cellular and molecular levels and there may be hitherto unexplored inter-tissue mechanisms (Preedy & Watson 2005). For example, we have recently shown molecular involvement in that acute alcohol moderates the expression of at least 400 genes (Arno et al 2006). Furthermore, when endogenous 158

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acetaldehyde levels are raised with cyanamide + ethanol, there are significant increases in muscle mRNA encoding c-myc, which may represent a pre-apoptotic effect, or even a non-specific cellular stress response (Nakahara et al 2003). Studies by other groups in vitro have shown that acetaldehyde impairs biomechanical factors of muscle, such as irreversibly reducing the twitch and the tetanic tension in isolated muscle (Khan 1981). Acetaldehyde also interferes in the interaction of actin and myosin in muscle contraction and the dissociation of the actomyosin complex in a reversible manner in vitro (Puszkin & Rubin 1975). Change in muscle biomechanical features may be due to perturbations in calcium channel proteins and/or calcium regulation (Ohlendieck et al 2003, Oba & Maeno 2004). However, at present, the role of acetaldehyde in modulating muscle contraction and force generation in vivo has not been proven. Limitations in space preclude us from describing all the facets of alcoholic myopathy, and in this article we focus on the role of acetaldehyde in our studies. There are, however, some generalized reviews available elsewhere (Lang et al 2001, 2005, Preedy et al 2001b, 2001a, 2002, 2003, Adachi et al 2003, Urbano-Marquez & Fernandez-Sola 2004, Fernandez-Sola et al 2005).

Effect of alcohol on skeletal muscle Alcohol-induced muscle disease (AIMD; Table 1) is arguably the most prevalent skeletal muscle disorder in the Western Hemisphere but, paradoxically, one of the

TABLE 1 Definitions and features of alcoholic-induced muscle disease Alcohol induced muscle disease (AIMD) Acute alcoholic myopathy

Chronic alcoholic myopathy

A composite term to describe any pathology (molecular, biochemical, structural or physiological) affecting muscle as a consequence of either acute or chronic alcohol ingestion. A rare condition affecting approximately 1% of alcoholics, characterized by swollen painful muscles, usually with myoglobinuria, strikingly raised serum creatine kinase activities and often renal impairment. It occurs after severe alcoholic binges in malnourished alcoholics and affects both red and white muscle fibres (rhabdomyolysis). A common complication of alcoholism affecting approximately 50% of patients. It primarily affects the white muscle fibres of the pectoral and pelvic girdles i.e. a proximal metabolic myopathy though other muscle groups may be involved. Approximately 50% of affected patients show proximal muscle weakness. It is not associated with nutritional, vitamin or mineral deficiencies. The atrophy of the muscle fibres does not lead to an increase in serum creatine kinase and is reversible with 6–12 month abstinence.

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least studied. It occurs in up to two-thirds of all chronic alcohol misusers and is thus about four or fives times more common than alcoholic cirrhosis and ranks with the most common alcohol-induced pathologies (Fernandez-Sola et al 2005, Patussi et al 2005). In simple terms, AIMD can be divided into chronic and acute alcoholic myopathy. Working definitions of these are provided in Table 1. In chronic alcoholic myopathy, the diameters of fast-twitch white fibres are markedly reduced: in contrast the slow twitch red fibres are more resilient (Martin et al 1985) (Fig. 1). Patients may lose up to 30% of their muscle mass (Duane & Peters 1988). There may be enhancement of lipid deposition within the muscle, but no overt fibrosis, inflammation or necrosis (Martin et al 1985). In most patients with alcoholic myopathy mitochondrial changes are absent (Martin et al 1985), though there is reduced skeletal muscle strength (Martin et al 1985, Urbano-Marquez et al 1989, Aagaard et al 2003). Muscle strength assessment can form the basis of diagnostic tests, in the absence of facilities for biopsy and histology. Patients with overt liver disease such as cirrhosis also have reduced muscle strength. However, it is important to emphasise that the myopathy can arise independently of liver disease (Martin et al 1985, Aagaard et al 2003), neuropathy (Mills et al 1986) or malnutrition

Control subject Type I Type II

Alcoholic patient Type I Type II

FIG. 1. Type I and II fibres in a control and alcoholic patient. Micrographs show a specimen from a normal control and an alcoholic patient. Muscles have been stained for myosin-ATPase. Thus the lighter fibres are type I whereas the darker fibres are type II.

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(Duane & Peters 1988). The metabolic superimposition of concomitant disease or malnutrition will exacerbate the myopathy (Nicolas et al 2003). Acute myopathy is relatively rare, occurring in less that 1% of alcohol misusers (Martin et al 1985) (Table 1). Despite this, many of the older textbooks on skeletal muscle concentrate on the acute form of the disease. The full spectrum of rhabdomyolysis leading to renal failure can be fatal (Louboutin et al 1995, Hewitt & Winter 1995, Riggs 1998, De Francesco et al 2005). Animal models of alcohol-induced muscle disease In carrying out patient-based studies there are numerous constraints of a practical (such as limitations in obtaining sufficient material for biochemical analysis) or ethical (such as dosing of alcohol misusers with ethanol or drugs which will potentiate the effects of alcohol) nature. Moreover, the coexistence of many pathologies or malnutrition complicates the interpretation of clinical data. The use of animal models with pair-feeding regimens resolves many of these problems, as alcohol can be administered in defined amounts without concomitant nutrient deficiencies: controls are given identical amounts of the same diet in which ethanol is replaced with isoenergetic carbohydrate (for example Ohlendieck et al 2003) or fat (for example Hunter et al 2003). In such models, anatomically distinct skeletal muscles are taken to represent the different muscle fibre types. To investigate the type I fibres, the soleus is examined, whereas the plantaris or gastrocnemius are used to represent the type II fibres (Preedy & Peters 1988a, Preedy et al 1990) (see also Table 2). The suitability of this has been affirmed by studies showing that feeding rats a nutritionally complete liquid diet, containing ethanol as 35% of total dietary energy, causes a marked reduction in the weight of the plantaris compared to those muscles taken from control rats fed identical amounts of the same diet in which ethanol is replaced by isocaloric glucose, whereas the weight of the soleus is relatively unaffected by alcohol feeding (Preedy & Peters 1988b) (Fig. 2). It could be argued that these changes in muscle weight could be due to some hitherto unrecognized phenomena related to the anatomical location of the plantaris muscles. However, we examined the type II fibres within the soleus muscle and showed that the relative abundance of fibres with a small diameter increased, with a corresponding decrease in the proportion of fibres with a larger diameter (Fig. 3). This strongly suggests that the myopathy is related to fibre characteristics rather than the location of the muscle (Preedy et al 1989). The biochemical and physiological differences in the two fibre types are displayed in Table 2. Of potential interest in understanding the aetiology of AIMD disease is the higher activities of antioxidant enzyme systems such as catalase, superoxide dismutase and glutathione peroxidase in the type I fibres when compared to the more susceptible type II fibres.

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TABLE 2 Features of type I and II fibres or muscle types

Myoglobin Twitch/Myosin heavy chains Metabolism Glycogen Mitochondria Capillary density Muscle fibre diameter Antioxidant enzymes Superoxide dismutase Glutathione peroxidase Catalase Alpha-tocopherol concentration Imidazole dipeptides Myosin ATPase ALDH2

Red (type I)

White (type II)

High Slow Oxidative-aerobic Low High High Smaller

Low Fast Glycolytic-anaerobic High Low or absent Low Larger

High High High High Low Low High

Low Low Low Low High High Low

Muscle weight (mg)

In this table, the differences between the fibre and muscle types are compared. We have defined ‘muscle types’ as muscle that contains a particular prominence of fibre types. It is very rare for muscles to contain exclusively a single fibre type, and most anatomically distinct muscles contain a mixture of fibre types. Much of the information contained in this table is derived from animals where a muscle with a predominance of a particular fibre type has been dissected and analysed. For example the plantaris, EDL (extensor digitorum longus) or white portions of the gastrocnemius muscle can be taken to represent the white muscle. For red muscle, the soleus or red portions of the gastrocnemius are analysed. Compiled from various sources.

500 400 300 NS 200 100 0 Soleus (Type I)

P