The cytochrome P-450 isoenzyme CYP2E1 in the biological processing of industrial chemicals: consequences for occupational and environmental medicine

REVIEW

The cytochrome P-450 isoenzyme CYP2E1 in the biologicalprocessing of industrial chemicals: consequencesfor occupational and environmental medicine

Received: 29 August 2002 / Accepted: 26 October 2002 / Published online: 1 March 2003� Springer-Verlag 2003

Abstract The importance of the isoform CYP2E1 of thehuman cytochrome P-450 superfamily of enzymes foroccupational and environmental medicine is derivedfrom its unique substrate spectrum that includes anumber of highly important high-production chemicals,such as aliphatic and aromatic hydrocarbons, solventsand industrial monomers (i.a. alkanes, alkenes, aromaticand halogenated hydrocarbons). Many polymorphicgenes, such as CYP2E1, show considerable differences inallelic distribution between different human populations.The polymorphic nature of the human CYP2E1 gene issignificant for inter-individual differences in toxicity ofits substrates. Since the substrate spectrum of CYP2E1includes many compounds of basic relevance toindustrial toxicology, a rationale for metabolic interac-tions of different CYP2E1 substrates is provided.In-depth research into the inter-individual phenotypicdifferences of human CYP2E1 enzyme activities wasenabled by the recognition that the 6-hydroxylation ofthe drug chlorzoxazone is mediated by CYP2E1. Studieson CYP2E1 phenotyping have pointed to inter-individual variations in enzyme activities. There areconsistent ethnic differences in CYP2E1 enzymeexpression, mostly demonstrated between European andJapanese populations, which point to a major impact ofgenetic factors. The most frequently studied geneticpolymorphisms are the restriction fragment lengthpolymorphisms PstI/RsaI (mutant allele: CYP2E1*5B)located in the 5’-flanking region of the gene, as wellas the DraI polymorphism (mutant allele: CYP2E1*6)

located in intron 6. These polymorphisms are partlyrelated, as they form the common allele designatedCYP2E1*5A. Striking inter-ethnic differences betweenEuropeans and Asians appear with respect to the fre-quencies of the CYP2E1*5A allele (only approximately5% of Europeans are heterozygous, but 37% of Asiansare, whilst 6% of Asians are homozygous). Availablestudies indicate a wide variation in human CYP2E1expression, which are very likely based on complexgene–environment interactions. Major inter-ethnicdifferences are apparent on the genotyping and thephenotyping levels. Selected cases are presented whereinter-ethnic variations of CYP2E1 may provide likelyexplanations for unexplained findings concerning in-dustrial chemicals that are CYP2E1 substrates. Possibleconsequences of differential inter-individual and inter-ethnic susceptibilities are related to individual expres-sions of clinical symptoms of chemical toxicity, to resultsof biological monitoring of exposed workers, and to theinterpretation of results of epidemiological or molecular-epidemiological studies.

Keywords Cytochrome P-450 Æ CYP2E1 Æ Industrialchemicals Æ Inter-individual variability Æ Inter-ethnicvariability Æ n-Hexane, 1,3-butadiene Æ Acrylonitrile ÆAcrylamide

Introduction

Metabolism and toxicokinetics of chemicals foreign tothe organism (xenobiotics) play a key role in tox-icological processes. ‘Phase-l’ enzymes, e.g. cytochromeP-450, are mostly involved in the biological activation ofchemical toxicants, whilst a major role of ‘phase-II’ en-zymes (e.g. glutathione-S-transferases, sulfo-trans-ferases, glucuronyl-transferases) is detoxification, inorder to produce hydrophilic terminal metabolites thatcan be excreted in the urine. An overall balance betweenactivation and detoxification processes determines ulti-mately active target doses of many toxicants.

Int Arch Occup Environ Health (2003) 76: 174–185DOI 10.1007/s00420-002-0407-4

Hermann M. Bolt Æ Peter H. Roos Æ Ricarda Thier

H.M. Bolt (&) Æ P.H. RoosInstitut fur Arbeitsphysiologie,University of Dortmund,Ardeystrasse 67, 44139,Dortmund, GermanyE-mail: [email protected]: +49-231-1084403

R. ThierDepartment of Physiology and Pharmacology,University of Queensland, St. Lucia,Queensland, Australia

The ‘superfamily’ of cytochrome P450 (CYP)enzymes (Nebert et al. 1987) is mainly involved in thebiological oxidation of chemicals. During the past dec-ade a large genetic variability of human CYP enzymeshas been recognised, as many of these are geneticallypolymorphic in humans. A genetic polymorphism iscommonly defined as a gene variant that is prevalent atleast at a frequency of 1% in a defined population.

Owing to its particular substrate spectrum, theisozyme CYP2E1 is of great interest to industrial andenvironmental medicine. With respect to its tissuedistribution, the highest constitutive CYP2E1 enzymelevels appear in the liver, but lower levels are alsoexpressed in extrahepatic tissues (Botto et al. 1994;Subramanian and Ahmed 1995; Vieira et al. 1998;Mostafa et al. 1999) and can be of toxicological sig-nificance. For example, CYP2E1 is expressed in dopa-mine-containing cells of the substantia nigra and isbelieved to be connected to xenobiotic-induced devel-opment of M. Parkinson (Jenner 1998). Quantitatively,CYP2E1 is one of the major cytochrome P-450 iso-enzymes, as it comprises approximately 7% of all CYPisoforms that are constitutively expressed in human li-ver. Importantly, there are no major gender-relateddifferences in distribution and activity of CYP2E1 inhumans (Shimada et al. 1994). CYP2E1 is polymorphicin humans, and resulting genotypes could be char-acterised and have, in part, been connected with differ-ent levels of individual expression of enzyme activity(Bogaards et al. 1993; Raucy et al. 1993).

The importance of CYP2E1 for occupational andenvironmental medicine is derived from its substratespectrum that includes a number of highly importantbulk chemicals. Substrates of CYP2E1 include aliphaticand aromatic hydrocarbons, solvents and industrialmonomers. The polymorphic nature of the humanCYP2E1 gene is probably connected with human inter-individual differences in toxicity of its substrates(Deutsche Forschungsgemeinschaft (DFG) 2001).Moreover, many polymorphic genes, such as CYP2E1,show considerable differences in allelic distributionsbetween different human populations. Such variation ingenotypes and associated phenotypes of metabolicenzymes among different ethnic and/or geographicallylocalised populations is a very recent and expanding fieldof research (Garte et al. 2001; Thier and Bolt 2001).

The classical aspect: CYP2E1 as ‘microsomal ethanoloxidising system’

Lieber and de Carli (1968) found that hepatic microsomesof different species contained a cytochrome P-450 basedactivity for oxidising ethanol to acetaldehyde. In rats, thiscould be stimulated by ethanol pretreatment, very similarto the situation in human alcoholics. They called thisactivity the ‘microsomal ethanol oxidising system’(MEOS). The enzyme was later isolated, purified andsequenced (Song et al. 1986), and with the establishment

of the unified nomenclature of cytochrome P-450enzymes, it was designated ‘CYP2E1’ (Nebert et al. 1987;Nelson et al. 1996). Compared with alcohol dehy-drogenase (Km for ethanol below 0.5 mM) CYP2E1 hasa much lower affinity for ethanol as substrate (Km =10 mM). This explains why the microsomal pathway(MEOS) is normally of only minor importance for etha-nol oxidation. This, however, contrasts with the situationin human alcoholics, where CYP2E1may be induced by afactor of 3 to 5 (Lieber 1987).

A comprehensive review of the functionality ofCYP2E1 as a MEOS has recently been provided (Lieber1999).

Industrial chemicals as substrates of CYP2E1

Besides ethanol, CYP2E1 oxidises a significant number ofimportant industrial chemicals. Among these are alkanes,alkenes, aromatic and halogenated hydrocarbons, andrelated compounds, as well as therapeutic drugs. Table 1gives a compilation of established CYP2E1 substrates,inducers and inhibitors, mainly based on Meskar et al.(2001). Since the substrate spectrum of CYP2E1 includesmany compounds of basic relevance to industrial tox-icology, a rationale for metabolic interactions (enzymeinduction or inhibition) of different CYP2E1 substratescan be provided (e.g. disulfiram and chlorzoxazone,Kharasch and Thummel 1993; Kharasch et al. 1993; 1,3-butadiene and styrene, Laib et al. 1992).

In essence, it can be stated that CYP2E1 occupies akey position in the formation of toxic metabolites fromindustrial chemicals (Thier et al. 2002c).

Genetic variability and phenotyping studies

In discussing matters of inter-individual variability ofCYP2E1, one must distinguish between genotypic var-iations at the DNA level and phenotypic differences atthe protein or enzyme activity levels1. So far, the co-herence between these levels is not fully understood, asthis is a major focus of current research activity (Garteet al. 2001). Again, a classical field has been the inter-individual variations in the toxicokinetics of ethanol inrelation to genotypes of CYP2E1, alcohol dehy-drogenase (ADH) and aldehyde dehydrogenase (ALDH;Iwahashi et al. 1995).

In contrast to experimental animal models, humansshow large inter-individual variations in CYP-catalysedoxidation reactions of drugs and chemicals, and suchvariations have been connected with differential sus-ceptibilities of humans to pharmacological and tox-

1The current international nomenclature distinguishes the gene le-vel from the protein/enzyme level by typing genes and gene variantsin italics (CYP2E1 and its variant alleles, as opposed to CYP2E1for the protein and associated enzyme activity). This is usedthroughout this review.

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icological effectors (Guengerich 1989). As far as ex-pressions of CYP2E1 activities are concerned, such in-ter-individual variations were first established by thestudy of human liver microsomes (Yoo et al. 1988; Huntet al. 1990; Tassaneeyakul et al. 1993).

Against this background, Shimada et al. (1994)compared human liver microsomes from 30 Japaneseand 30 European (Caucasian) subjects, with regard toexpressed protein quantities of seven CYP isoenzymes,including CYP2E1, and the associated enzymic activitiestowards 13 test substrates. A high correlation appearedbetween the immunoreactive CYP2E1 protein contentsand enzyme activities of aniline-4-hydroxylase. There-fore, aniline appeared as a relatively selective substrateof CYP2E1. Most interestingly, the total cytochromeP-450 content of microsomes from Japanese subjects(mean: 0.26 nmol/mg protein) was consistently lowerthan those of European (Caucasian) origin (mean:0.43 nmol/mg protein). This may be related to majorethnic differences in CYP expression and dependentmetabolism of chemicals.

In-depth research into the inter-individual phenotypicdifferences of human CYP2E1 enzyme activities wasenabled by the recognition that the 6-hydroxylation ofthe drug chlorzoxazone is mediated by CYP2E1.Chlorzoxazone was therefore used as a phenotypicprobe of CYP2E1 enzyme activity in studies of humancollectives (Girre et al. 1994; Kim et al. 1995). Thereby,obesity, fasting (O’Shea et al. 1994), alcoholism (Girre

et al. 1994), and enzyme induction and inhibition byconcomitantly administered drugs (Kharasch et al. 1993;Zand et al. 1993), were shown to be modulators of hu-man CYP2E1 activity.

A further investigation into ethnic differences be-tween European and Japanese populations was con-ducted by Kim et al. (1996). The authors comparativelyinvestigated the pharmacokinetics of chlorzoxazone,orally administered to groups of 20 male volunteers ofEuropean (Caucasian) and Japanese origin. Plasmaconcentrations were significantly higher and rates ofelimination slower in the Japanese subjects than in theEuropeans. With regard to toxicokinetics, the clearancevalues were smaller in the Japanese, and the respectivedifferences (of approximately 30%) were apparent afternormalisation for body weight (b.w.) as well (3.74±1.23vs 5.05±1.41 ml per min and kg b.w.). These resultswere taken as an indication of consistent ethnic differ-ences, and were confirmed by further studies in vitrousing hepatic microsomes from individuals re-presentative of the two ethnic groups. In these micro-somal preparations, CYP2E1 protein levels were lower(by 61%) and chlorzoxazone and aniline hydroxylationswere reduced (by 22% and 35%, respectively) in pre-parations from the Japanese subjects, compared withthose from the Europeans. A connection of these phe-notypic differences with genomic restriction fragmentlength polymorphisms (RFLPs; here indicative of theallele CYP2E1*5A) could not be found in this in-

Table 1 Principle substrates,inhibitors and inducers ofCYP2E1 (based onMeskar et al.2001, modified andsupplemented)

Substrates Inducers Inhibitors

Endogenous compounds and food ingredientsAcetaldehyde DiallylsulphideAcetone DihydrocapsaicinFatty acids PhenylethylisothiocyanateEthanolGlycerol

Therapeutic drugs/anaesthetic gasesAcetylsalicylic acid Isoniazid (INH) ChlormethiazoleCaffeine Pyrazole DisulfiramChlorzoxazone MalatilateDapsoneParacetamolEnflurane, sevofluraneMethoxyflurane, halothane

Solvents, chemical productsAcrylonitrile, methacrylonitrile Ethanol 3-Amino-1,2,4-triazoleAlcohols, ethers, alkanes DiethyldithiocarbamateAcetone Acetone 4-MethylpyrazoleAnilineBenzene (and derivatives) BenzeneStyreneChloroform, CCl4 IsopropanolTrichloroethylene Dimethylsulphoxide

(DMSO)Pyrazole PyrazolePhenolPyridine, anilineAcrylamideNitrosamines (e.g., N-nitrosodimethylamine, NNK)Ethylcarbamate (urethane)Vinyl chloride, vinyl bromide

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vestigation. Other polymorphisms of CYP2E1, however,were not studied.

In essence, the studies on CYP2E1 phenotyping haveconsistently pointed to inter-individual variations inenzyme activities which might, in part, be ascribed tomodulating individual factors (physical/hormonal sta-tus, alcohol consumption, drug interferences; see above).However, there are obvious and consistent ethnic dif-ferences in CYP2E1 enzyme expression, demonstratedbetween European and Japanese populations, whichalso point to an impact of genetic factors.

Genotypic variations

A number of genetic polymorphisms have been de-scribed for the human CYP2E1 gene. The Human Cy-tochrome P450 Allele Nomenclature Committee notifiesestablished genetic variants of CYP isoenzymes underhttp://www.imm.ki.se/CYPalleles. Due to differentnumberings of specific nucleotide changes, assignmentsof RFLPs to the specific restriction enzymes used, anddifferent nomenclatures of alleles (Garte and Crosti1999), there are inconsistencies in the literature in as-signment of genetic variants. Table 2 shows the allelicmodifications recognised so far, based on the assign-ments of the Human Cytochrome P450 Allele Nomen-clature Committee.

CYP2E1*5A, CYP2E1*5B, CYP2E1*6

The most frequently studied genetic polymorphisms arethe RFLPs PstI/RsaI (mutant allele: CYP2E1*5B) lo-cated in the 5’-flanking region of the gene, which havebeen related to altered enzyme expression in vitro(Hayashi et al. 1991), as well as the DraI polymorphism

(mutant allele: CYP2E1*6) located in intron 6 (Uematsuet al. 1991). These polymorphisms are partly related, asthey form the common allele designated CYP2E1*5A(see Table 2).

Using the database of the International Project onGenetic Susceptibility to Environmental Carcinogens(GSEC), containing information from over 15,000control (non-cancer) subjects, Garte et al. (2001) havecompiled allele and genotype frequencies of commonlystudied metabolic genes, among them CYP2E1, in thehuman population. Table 3 summarises the frequenciesof the PstI/RsaI and DraI polymorphisms, as wellas those of the associated allele combinationsCYP2E1*5A, CYP2E1*5B and CYP2E1*6, accordingto this evaluation. For the European population, theCYP2E1*6 allele variant appears of interest, as itoccurs in approximately 10% of the population (beingheterozygous for this allele). Very recently, Haufroid etal. (2002) described a trend to lower ‘chlorzoxazonemetabolic ratios’ for individuals possessing at least onemutant CYP2E1*6 allele, compared to the homozygouswild type.

Striking inter-ethnic differences between Europeansand Asians appear with respect to the frequencies ofCYP2E1*5A allele (only approximately 5% of Eur-opeans are heterozygous, but 37% of Asians are, whilst6% of Asians are homozygous). The relationship of thisparticular type of genetic polymorphism with CYP2E1enzyme expression and the associated consequences inthe metabolism of chemicals remain to be definitely es-tablished, as available studies are conflicting in this re-spect (Hayashi et al. 1991; Kim et al. 1996). Because ofthe very clear-cut ethnic differences in prevalence of theCYP2E1*5A allele, further investigations to answerthese open questions should necessarily be focussed onpopulations from the Far East. For instance, LeMarchand et al. (1999) have studied this question in a

Table 2 Current nomenclature of CYP2E1 alleles, according to the Human Cytochrome P450 Allele Nomenclature Committee (http://www.imm.ki.se/CYPalleles), key information supplemented

Allele Protein SNP/nucleotide(s) RFLP Reference

CYP2E1*1A CYP2E1.1 None Umeno et al. 1988CYP2E1*1B CYP2E1.1 9893C>G TaqI- Brockmoller et al. 1996CYP2E1*1C CYP2E1.1 Six repeats (in flanking region) Hu et al. 1999

(Common state) Fritsche et al. 2000CYP2E1*1D CYP2E1.1 Eight repeats DraI, XbaI McCarver et al. 1998

Hu et al. 1999(From )2770 to )1672) Fritsche et al. 2000

CYP2E1*2 CYP2E1.2 1132G>A (Protein: R76H) Hu et al. 1997CYP2E1*3 CYP2E1.3 10023G>A (Protein: V389I) Hu et al. 1997CYP2E1*4 CYP2E1.4 4768G>A (Protein: V179I) Fairbrother et al. 1998CYP2E1*5A CYP2E1.1 )1293G>C; PstI+ Watanabe et al. 1994

)1053C>T; RsaI- Hayashi et al. 19917632T>A DraI- Persson et al. 1993

CYP2E1*5B CYP2E1.1 )1293G>C PstI+ Watanabe et al. 1994)1053C>T RsaI+ Hayashi et al. 1991

CYP2E1*6 7632T>A DraI- Persson et al. 1993CYP2E1*7A CYP2E1.1 )333T>A Fairbrother et al. 1998CYP2E1*7B CYP2E1.1 )71G>T; )333T>A Fairbrother et al. 1998CYP2E1*7C CYP2E1.1 )333T>A; )352A>G Fairbrother et al. 1998

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Japanese population in Hawaii and found the CY-P2E1*5A allele related to a low chlorzoxazone oxidisingactivity. A lower CYP2E1 expression, in associationwith CYP2E1*5A or CYP2E1*6 alleles is, to differentextents, also supported by Lucas et al. (1995), Kim et al.(1995), Carriere et al. (1996), Powell et al. (1998) andRaucy et al. (1999).

CYP2E1*2, CYP2E1*3, CYP2E1*4

Hu et al. (1997) have described two single nucleotidepolymorphisms (SNP) that lead to associated aminoacid changes in the CYP2E1 protein (CYP2E1*2,CYP2E1*3; see Table 2). However, based on the lowfrequencies of these CYP2E1 polymorphisms in Eur-opeans, they claimed that, in general, no quantitativelyimportant polymorphisms would occur in the openreading frame of CYP2E1. In view of the establishedphenotypic variability in CYP2E1 enzyme expression,this has prompted an intensified research into thevariability of regulatory sequences of the humanCYP2E1 gene.

In this context, it is worth mentioning that also theCYP2E1*4 mutation first described by Fairbrother et al.(1998) is located in the open reading frame of the gene andtherefore leads to an amino acid change (see Table 2).

CYP2E1*1C, CYP2E1*1D

McCarver et al. (1998) described an insertion (estimatedto be approximately 100 base -pairs) in the promoterregion of CYP2E1, found by Southern blot analysis, andtentatively localised it to a region between the nucleotide

positions )2270 and )1672. In order for a functionalsignificance of this mutation to be determined, subjectswere checked for this genomic insertion, and the meta-bolic activity of CYP2E1 was assessed, with chlorzox-azone being used as a metabolic probe. The presence ofthe insertion appeared to be associated with greatermetabolic activity, but only among individuals who ei-ther were obese or had recently consumed alcohol(P<0.01, both). These data were found to be consistentwith the idea that the DNA insertion is associated withaltered induction behaviour of CYP2E1, generallypointing to more complex gene–environment interac-tions. The incidence of the mutation was found to beconsistently higher in the black (African–American)than in the white (Caucasian) US population.

Hu et al. (1999) localised the polymorphic repeat se-quence between the base-pair positions 2178 and 1945 ofthe CYP2E1 gene. They determined two major alleles,carrying either six or eight sequence repeats, which theydesignated CYP*1C (common allele) and CYP2E1*1D(rare allele; see Table 2). The wild type, CYP2E1*1A,contains only five of these repeats (Hu et al. 1999). Infact, the CYP2E1*1D allele is very rare in the Europeanpopulation (Table 4). Therefore, it is not surprising thatPlee-Gautier et al. (2001) could not identify this pro-moter sequence repeat polymorphism as a major factorfor inter-individual differences in CYP2E1 expressionand susceptibility to alcohol-related disorders in aFrench population, where the CYP2E1*1D allele fre-quency was only 1.58% among 349 persons.

In order to characterise the repeat insertion fully,Fritsche et al. (2000) developed a polymerase chain re-action (PCR) method for easy detection of the variants,performed sequence analysis of the wild-type and mu-

Table 3 Frequencies of polymorphisms (RLFP: RsaI, DraI) and related frequencies ofCYP2E1*5A, CYP2E1*5B and CYP2E1*6, ac-cording to the database of the International Project on Genetic Susceptibility to Environmental Carcinogens (GSEC; Garte et al. 2001)

Frequencies of polymorphisms

RFLP Related alleles Caucasians n (%) Asians n (%)

RsaI CYP2E1*5A or CYP2E1*5BTotals 1,454 719Homozygous (wild type) 1,344 (92.4) 428 (59.5)Heterozygous 109 (7.5) 258 (35.9)Homozygous (variant) 1 (0.1) 33 (4.6)DraI CYP2E1*5A or CYP2E1*6Totals 1,360 286Homozygous (wild type) 1,162 (85.4) 138 (48.3)Heterozygous 187 (13.8) 121 (42.3)Homozygous (variant) 11 (0.8) 27 (9.4)

Gene allele frequencies

Gene Ethnicity n Heterozygous Homozygous Allele

CYP2E1*5A Caucasian 854 0.048 0.0012 0.0252Asian 286 0.367 0.0594 0.243

CYP2E1*5B Caucasian 854 0.0105 0 0.00525Asian 286 0.021 0 0.0105

CYP2E1*6 Caucasian 854 0.102 0.0023 0.0533Asian 286 0.126 0 0.0630

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tant alleles, and determined the allele frequencies inseveral ethnic groups (Table 4). They found that thesequence of the CYP2E1 promoter consisted of eightalmost identical repeats (designated I–VIII). Repeat Iwas 54 base pairs, repeats II to V and VIII were 48 basepairs each, and repeats VI and VII were 42 base pairseach. Comparison of the wild-type with the mutant allelerevealed an additional 96 base-pair insertion in themutant (’Ins96+’, identical to the CYP2E1*1D of Huet al. 1999). The Ins96+ consisted of a 90 base-pairinsertion at position 2029, containing exactly tworepeats (V and VI), and located on the same allele asanother six-base-pair insertion at position 2071 withinrepeat IV. In every Ins96+ allele sequenced, there wasalso a base change, )2245C>T, occurring along withthe insertion. Again, it is very striking that the mutantallele CYP2E1*1D appears much more frequently inAsians than in Europeans (Table 4).

CYP2E1*7B

Fairbrother et al. (1998) have identified a polymorphism(allocated as 297T>A, now being addressed as333T>A) that (in combination with the 71G>T de-tected by Hu et al., 1997) exhibited a higher in vitroactivity in a promoter construct than the wild-type. Thisled to increased interest in possible metabolic con-sequences of the CYP2E1*7 group of polymorphisms.

CYP2E1*7C

Case reports of industrial acrylonitrile poisonings havepointed to significant individual differences in humanacrylonitrile metabolism and toxicity (Thier et al. 2000).The reasons for these individual differences seem to becomplex, as exemplified later in this article. For instance,a cohort of 59 persons involved in the industrial hand-ling of low levels of acrylonitrile has repeatedly beenstudied from 1994 to 1999 as part of a medical surveil-lance programme. The analyses included adduct de-terminations of N-terminal N-(cyanoethyl)valine inhaemoglobin and genotypings of the following CYP2E1polymorphisms: )1293G>C and )1053C>T (twosubjects heterozygous), )352A>G (three subjects het-erozygous), )333T>A (15 subjects heterozygous),)71G>T (eight subjects heterozygous), 4768G>A (twosubjects heterozygous), 7632T>A (six subjects hetero-zygous). N-(cyanoethyl)valine adduct levels were, ifanything, only slightly influenced by smoking and mainly

determined by the external acrylonitrile exposures. Noinfluences of the investigated CYP2E1 polymorphismson the N-(cyanoethyl)valine levels appeared at the 5%level. However, there was a trend, at a level of P�0.1,pointing to higher acrylonitrile-specific adduct levels inpersons with the )352A>G mutation which is in-dicative of the CYP2E1*7C allele (see Table 2). Higheradduct levels would be compatible with a slowerCYP2E1-mediated metabolism of acrylonitrile and withlower extents of toxification to cyanide. Hence, furtherresearch appears to be warranted into the biologicalconsequences of the CYP2E1*7C allele (Thier et al.2002b).

Peculiarities in the regulation of CYP2E1 expression

It appears that CYP2E1-dependent metabolic capacityvaries individually due to polymorphisms of theCYP2E1gene. Furthermore, it is modulated by xenobiotics or bypathological processes at a post-genomic level. In con-trast to other CYP enzymes such as CYP1A1, CYP2B6and CYP3A4, which are induced by receptor-mediatedtranscriptional activation, CYP2E1 is primarily regu-lated on the post-transcriptional and post-translationallevels (Koop and Tierney 1990). Thus, the mode of actionof xenobiotics is the stabilisation of the CYP2E1 mRNAor protein. Increased half-lives of the enzyme or itsmRNA, in consequence, result in higher steady-stateenzyme levels. With respect to the protein, protease-mediated and hsp90-assisted degradation of CYP2E1is retarded (Goasduff and Cederbaum, 2000).

Generally, CYP2E1 expression appears to be undertight homeostatic control (Novak and Woodcroft 2000).This enzyme is usually not expressed in foetal tissues.However, increased levels are found within a few hoursafter birth. Levels of CYP2E1-mRNA rise during thefirst year of post-natal development. In the foetal stage,CYP2E1 expression is obviously prevented by hyper-methylated sites at the 5’-end of the gene, which pro-gressively disappear postnatally (Vieira et al. 1996).

Several hormones such as insulin, glucagon, leptinand epidermal growth factor influence the expression ofCYP2E1 (Novak and Woodcroft 2000) and, thus, themetabolic capacity of this enzyme for xenobiotics. It hasbeen shown that hepatic CYP2E1 levels are elevated indiabetic rats (Dong et al. 1988) and that the enzyme levelis increased, at least, in lymphocytes of humans withinsulin-dependent diabetes (Hannon-Fletcher et al.2001).

Table 4 Frequencies of genotypes and allele frequencies of CYP2E1*1D in different ethnicities, according to Fritsche et al. (2000)

Ethnicity (n) Genotype frequencies Allele frequencies

CYP2E1 *1C/*1C *1C/*1D *1D/*1D *1C *1DAsian (117) 86 (74%) 28 (24%) 3 (3%) 0.85 0.15African-American (107) 88 (82%) 17 (16%) 2 (2%) 0.9 0.1Caucasian (172) 165 (96%) 7 (4%) 0 (0%) 0.98 0.02

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Possible consequences of inter-individual variations ofCYP2E1 in occupational and environmental medicine

In essence, all available studies have indicated a widevariation in human CYP2E1 expression (Lieber 1999),which is very likely based on complex gene–environmentinteractions (v.s.). In addition, major inter-ethnic dif-ferences have become apparent, both on the genotypingand the phenotyping levels. The most thoroughlystudied populations are Asian (mostly Japanese) andEuropean, supplemented by limited data on African–Americans.

Aspects of inter-individual and inter-ethnic variationof CYP2E1 are of relevance for occupational medicine.Such variations ought also to be seen in combinationwith variations of glutathione-S-transferases (GSTs).Members of the GST superfamily of enzymes frequentlyinactivate products of the oxidative metabolism ofCYP2E1 (e.g. epoxides, quinones or semiquinones).Alternatively, they provide a competing reductivepathway leading to a differential toxicological profile (asfor a number of halogenated compounds). As importantGST isoenzymes are also polymorphic in humans(GSTM1, GSTT1, GSTP1), combinations of CYP andGST variabilities must essentially be considered (Thieret al. 2002a, 2002c). Such aspects have considerableweight in the assessment of inter-individual and inter-ethnic differences in risks due to chemical compounds(El Masri et al. 1999).

As an example, Lipscomp et al. (1997) have studiedthe variability of human oxidative metabolism of tri-chloroethylene in hepatic microsomal samples in vitro.As CYP2E1 was responsible for more than 60% of thetotal microsomal trichloroethylene metabolism ob-served, the variability in CYP2E1 expression appearedas a major factor to explain the total inter-individualmetabolic variability. As the reductive, GSH-dependentpathway of trichloroethylene is also influenced by

genetic polymorphisms, particularly of GSTT1 (Brun-ing et al. 1997), a range of genetic susceptibilities mustbe considered, with respect to human ne-phrocarcinogenesis following high occupational tri-chloroethylene exposures (Bruning and Bolt 2000).Similar factors have been discussed with respect to theacute and chronic toxicity of acrylonitrile (Thier et al.1999, 2000, 2001, 2002a).

In general, the consequences of inter-individual var-iations of CYP2E1, on the levels of phenotypes andgenotypes, should more extensively be examined in thefield of industrial medicine, e.g. in the chemical industrywhere substantial exposure to CYP2E1 substratesoccurs. Again, an important field of research is thecoherence of CYP2E1 genotypes and phenotypes, whichappears embedded in a complex network of gene–environment interactions and reflects the peculiarities inthe regulation of CYP2E1 expression (Peng and Coon2000; Wan et al. 2001; Woodcroft et al. 2002).

Aspects of inter-ethnic variations of CYP2E1for occupational medicine

Considering specific genetic and inter-ethnic variationsin occupational medicine opens new scientific andpractical fields. One finds that classical examples forthe necessity of such considerations have been derivedfrom the pharmacology of therapeutic drugs; there arealso well-studied variations in metabolism of and tol-erance to ethanol (Iwahashi et al. 1995; Thier et al.2002c).

In the following section, some selected cases arepresented where inter-ethnic variations of CYP2E1 mayprovide likely explanations for so-far unexplained find-ings concerning industrial chemicals which are CYP2E1substrates. The main metabolic pathways of these sub-strates are given in Fig. 1.

Fig. 1 Main metabolicpathways of industrialchemicals, discussed asexamples. A n-hexane, B 1,3-butadiene, C acrylonitrile

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Case studies

Case 1: biological monitoring of n-hexane

In 1994, the Senate Commission of the Deutsche For-schungsgemeinschaft for the Heath Evaluation of Working Mate-rials re-assessed the biological tolerance value (BAT) for n-hexane.In persons occupationally exposed to that neurotoxic solvent,biological monitoring is via the analysis of the urinary metabolite2,5-hexanedione (after acid treatment of the urine). The Commis-sion assessed five field studies focussed on this matter that had usedcomparable or even identical analytical procedures. Of these, threestudies were from Italy and two studies from Japan, all providingwell-founded correlation data between human airborne n-hexaneexposure and urinary excretion of 2,5-hexanedione. Recent studiesbased on ambient monitoring and determination of excreted hex-anedione confirm these early findings (Mayan et al. 2001, 2002).n-Hexane is metabolised to 2,5-hexanedione via CYP2E1 andADH (see Fig. 1), whereby CYP2E1 appears to be essential (Ibaet al. 2000). The first step in the reaction sequence, the 2-hydro-xylation, can be alternatively catalysed by CYP2B enzymes(Crosbie et al. 1997).

The urinary metabolite excretion equivalents to a daily (8-h)exposure to 50 ppm n-hexane (occupational exposure limit, MAK)were derived from the data sets of these five individual studies.There was clear coherence among the three studies from Italy(equivalent excretion means of 4.2, 4.5, and 4.9 mg/l 2,5-hex-andione in urine) on one the hand, but marked contrast betweenthese and the two studies from Japan (1.48 and 2.8 mg/l 2,5-hex-anedione in urine) on the other hand. The striking differencesbetween the results of Japanese vs Italian studies were tentativelyascribed to toxicogenetic differences by the Commission (DFG1994).

In view of the current knowledge of inter-ethnic differences ingenotypic and phenotypic expressions of CYP2E1 between Japa-nese and European populations, this explanation appears now verylikely. Thus, one must be quite careful in extrapolating quantitativebiological monitoring data between ethnic groups, when metabolicenzymes are involved that display high inter-ethnic variability(Thier et al. 2002c).

Case 2: 1,3-butadiene cancer epidemiology

1,3-Butadiene is an established carcinogen in animal experimentsand its carcinogenicity in humans appears very likely (category 1,according to DFG 1998). In the organism, the epoxidation of 1,3-butadiene (to the mono- and di-epoxide) is effected by CYP2E1 (seeFig. 1), while conjugation of the di-epoxide proceeds via thepolymorphic glutathione-S-transferase GSTT1 (Thier et al. 1996).Hence, a genetic variability in the balance of activation and in-activation of 1,3-butadiene is founded in an interplay of humanCYP2E1 and GSTT1 polymorphisms. Presumably, there are otherCYP enzymes besides CYP2E1 that contribute to 1,3-butadienegenotoxicity by metabolic activation, as evidenced from inhibitionstudies in animal experiments (Jackson et al. 2000).

When the International Agency for Research on Cancer(IARC) assessed the available epidemiological studies (IARC1992), an interesting aspect appeared from the epidemiologicalresults reported by Matanoski et al. (1989).

A cohort of 12,110 workers in eight styrene-butadiene-rubber(SBR) production plants in the US and Canada was followed,contributing a total of approximately a quarter of a million person-years of professional experience. When compared with the re-ference population of the US, the cohort showed no significantexcess in standard mortality ratio (SMR) for cancers. The popu-lation was divided into four work areas on the basis of the joblongest held. The three major groups were production (highestexposures), maintenance, and other work sites including labora-tories and administrative services. Only among black workers inthe production area, deaths from lymphopoietic cancers occurred

5.1-times more frequently and deaths 6.6-times more frequentlythan expected, based on population mortality rates, both ratiosbeing significantly high (Matanoski et al. 1989).

This result was discussed in the framework of epidemiologicalstudies on 1,3-butadiene, with the result, at that time, of ‘limitedevidence for the carcinogenicity of 1,3-butadiene in humans’(IARC 1992). The elevated cancer risk in a specific ethnic subgroupwas not considered plausible at that time. However, in the light ofthe present knowledge on differences in the occurrence of geneticCYP2E1 polymorphisms between the white and black populationswithin the US (see Table 4), this question seems worthy of being re-assessed. The resolution of such a problem would be possible by adiligent interplay between epidemiological and toxicological re-search strategies (Thier et al. 2002b).

Case 3: acute toxicity of acrylonitrile

Acrylonitrile and the structurally related acrylamide are provenmulti-site carcinogens in animal experiments (Leonard et al. 1999,IARC 1994, 1999). Both compounds are, on one hand, metabolisedby CYP2E1 to epoxide intermediates (oxidative pathway; Gha-nayemet al. 1999; Sumner et al. 1999); on the other hand, both acryliccompounds are conjugated with their activated double bond toglutathione. This reaction is catalysed byGST(s) (reductive pathway;Fennell et al. 1991). Owing to their chemical reactivities, the epoxideintermediates of the oxidative pathway are viewed in conjunctionwith genotoxic and carcinogenic activities (Kirkovski et al. 1998). Asecondary metabolite in consequence of the oxidative metabolism ofacrylonitrile (and the related methacrylonitrile) is cyanide, whichplays a key role in the acute toxicity of these nitriles (Mostafa et al.1999; Thier et al. 2000). The main pathways are given in Fig. 1.

The human and experimental toxicology of acrylonitrile wasrecently reviewed by the Scientific Committee on OccupationalExposure Limits (SCOEL) of the European Union. In this context,the contents of a recent ‘Toxic Substances Control Act (TSCA) 8(e)submission’ to the US Environmental Protection Agency (EPA),compiled by a group representing producers and users of acrylo-nitrile (Murray 2000), were discussed. This documentation (letterand attachments) provided heretofore unpublished data on healtheffects in Chinese workers occupationally exposed to acrylonitrile.These data from China suggested interesting dissimilarities, re-garding the clinical picture of acrylonitrile intoxication, againstpublished data from Europe. As this is in line with the known inter-ethnic differences of CYP2E1 genotypes and phenotypes, this newexample merits being highlighted in more detail.

Of the two competing metabolic pathways of acrylonitrile, theoxidative one is clearly mediated by CYP2E1. Of the reductive,GSH-dependent pathway the responsible GST isoenzyme(s) are notas well characterised so far; possibly the genetically polymorphicisoenzyme GSTP1 is involved (Thier et al. 2001). Marked differ-ences in the relative importance of the competing oxidative andreductive pathways occur between species (Thier et al. 2000).Clinical effects of incidental industrial acrylonitrile intoxication aremostly ascribed to the metabolic formation of cyanide, althoughthere is an array of inter-individual differences in the responsibleCYP2E1-mediated transformation of acrylonitrile (Thier et al.2000). The toxic effects occur after some latency period (in whichformation of cyanide occurs) and involve the respiratory, cardio-vascular, and, in particular, the central nervous system (commonlyreported are dizziness, headache, nausea, feebleness, dyspnoea,tachycardia). These symptoms are reported to be independent ofethnicity (e.g. Germany: Steffens et al. 1998, 2001; China: Chenet al. 1999; Philippino case report: Vogel and Kirkendall 1984).

These symptoms of acrylonitrile intoxication, which are at-tributed to cyanide, contrast, at least in part, to the results of ex-perimental studies in rats. In rats acutely intoxicated withacrylonitrile by inhalation, the cyanide antidotes 4-dimethylami-nophenol and sodium thiosulphate were found to be only mar-ginally effective, much in contrast to N-acetylcysteine, whichappeared as a very effective antidote (Appel et al. 1981). Thisspecies difference is concurrent with reports that the quantitatively

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major urinary metabolites of acrylonitrile in rats result from thereductive pathway of direct conjugation with glutathione (Burkaet al. 1994).

Additional information points to differential modes of actionbetween the species. When rats were acutely intoxicated with ac-rylonitrile, symptoms were observed that were indicative of in-creased parasympathomimetic activity (flow of tears, salivation,miosis), and atropine proved to be an effective experimental anti-dote (Appel et al. 1981). Another observation was an increasedrelease of acetylcholine from electrically stimulated chicken hearts,which occurred in the presence of acrylonitrile (Peter and Bolt1984). A dose-dependent acetylcholine-mimetic effect in the earlyneurotoxicity of acrylonitrile in rats was confirmed by Ghanayemet al. (1991), and Wei (1999a) found a dose-dependent decrease ofblood acetylcholine-esterase activity in rats subchronically treatedwith acrylonitrile by gavage (0, 20, 40 or 60 mg/kg daily for 8weeks). A likely explanation for these findings was that acryloni-trile, owing to its chemical reactivity, interacts with the activecentre of acetylcholine-esterase (Peter and Bolt 1984).

Parasympathomimetic symptoms, comparable to those ob-served in rat experiments, have not been reported in occupationallyexposed humans in Germany, where the clinical status of quite anumber of industrial acrylonitrile intoxications has been carefullyrecorded (case reports by Steffens et al. 1998, 2001). This was in-terpreted as being entirely consistent with the concept that theoxidative, CYP2E1-mediated metabolism of acrylonitrile, asso-ciated with the formation of cyanide, is much more prevalent inhumans than in the experimental situation in rats (Thier et al.2000). Hence, the faster oxidative, CYP2E1-mediated metabolismin humans vs rats leads to reduced effectiveness of the parentcompound, acrylonitrile (acetylcholine-esterase inhibition), in hu-mans, and to more pronounced effects of the oxidative metabolite,cyanide (Thier et al. 2000).

Against this background of studies in a European population,the observations from China that appear of greatest interest werecontained in the before-mentioned TSCA 8(e) submission to theUS EPA (Murray 2000).

Fong et al. (1999) provided Chinese clinical reports on acuteindustrial acrylonitrile intoxication. They pointed out that the cy-anide metabolite alone would not explain all observed manifesta-tions of acute acrylonitrile toxicity. Upon summarising 334 casesthat had occurred over 10 years they concluded that acrylonitrile,in the early intoxication phase involving toxic CNS effects, pro-duces clear manifestations of cholinergic nerve excitation. This wassupported by observations by Ding (1978) of clear symptoms ofmiosis in acutely acrylonitrile-intoxicated patients. This observa-tion was reported together with other ocular symptoms (ocularmuscle tremor and ocular muscle paralysis).

The occurrence of cholinomimetic effects of acrylonitrile inexposed Chinese individuals is corroborated by the determinationof blood acetylcholine-esterase levels in Chinese workers exposedto acrylonitrile. Wei (1999a, 1999b) conducted a field study of 273Chinese workers who used acrylonitrile in an industrial setting. Thetotal study group comprised subgroups (A, B, C) with differentexposure levels and from different workshops; comparison wasmade with a group of 184 controls. The average exposure level ofthe highest exposed group (A) was nearly 7-times over the existingMaximal Allowed Concentration of 2 mg/m3. Table 5 summarisesthese results of ambient air monitoring and total blood acet-ylcholine-esterase activities, and shows a clearly dose-dependentdecrease in the activities of this enzyme (Wei 1999a). Wei (1999b)also compared the data of the entire exposed group (A, B, C) with aset of 284 controls. The exposed persons showed a mean reductionin blood cholinesterase by approximately 50%, diminishment ofreduced glutathione (GSH) in blood (controls: 35.3±5.0 mg/100 ml; exposed: 27.2±4.9 mg/100 ml) and an elevation in bloodthiocyanate indicative of the acrylonitrile exposure (controls:1.32±0.52 mg/dl; exposed 1.63±0.54 mg/dl; mean ± SD).

These data, although fragmentary, suggest significant differ-ences in the clinical picture of acrylonitrile intoxication betweenEuropean and Chinese populations. Cholinomimetic symptoms,experimentally well established in rats intoxicated with acryloni-

trile, are seemingly not prevalent in Europe, but have been reportedin China. Whilst the oxidative metabolism of acrylonitrile, viaCYP2E1, appears as the preferential one in Europeans (as opposedto the experimental situation in rats), in populations of the FarEast with consistently lower CYP2E1 expression levels, differentrelative proportions of the two major metabolic pathways ofacrylonitrile (Fig. 1) must be expected. Thus, the established inter-ethnic variations in CYP2E1 genetics and enzyme expressionappear to be connected with clinically different pictures in acuteindustrial acrylonitrile intoxication.

Conclusion

The cytochrome P-450 isoenzyme CYP2E1 is of keyrelevance for the oxidative metabolism of importantindustrial chemicals. This plays a decisive role for thetoxicity of CYP2E1 substrates. Owing to its inter-individual and inter-ethnic variability in genotypes andphenotypic expression of the protein and enzyme activ-ities, CYP2E1 is of great interest to industrial andenvironmental medicine. Possible consequences of dif-ferential inter-individual and inter-ethnic susceptibilitiesare related to individual expressions of clinical symp-toms of chemical toxicity, to results of biologicalmonitoring of exposed workers, and to the interpreta-tion of results of epidemiological or molecular-epide-miological studies.

Urgent research needs appear when the scientific si-tuation is reviewed. More understanding is needed of thegenetic and phenotypic variabilities of CYP2E1 andtheir coherence. Epidemiological and field studies shouldbe related to genetically different types (and subtypes) ofpopulations, and more awareness by occupational andenvironmental physicians of the practical consequencesis necessary. General questions are related to assess-ments of individually different chemical risks and con-sequences for regulation and standard setting.

In the face of increasing migration world-wide,questions of inter-ethnic variability in susceptibility totoxicants will certainly gain further practical relevance inthe future.

Acknowledgement The authors thank the European ScienceFoundation (ESF) for sponsoring the international Genetic Sus-ceptibility to Environmental Toxicants (GENSUT) network during1998 to 2001.

Table 5 Acetylcholine-esterase (AcChE) activities in blood ofChinese workers employed with acrylonitrile (AN), related to ex-posure levels (subgroups A, B, C) and compared with non-exposedcontrols, according to report of Wei (1999a)

Exposuregroup

Mean AN exposure (n)(mg/m3 in

Mean bloodAcChE ± SD (n)

ambient air) (lKat/l whole blood)

Control group Not exposed 82.53±4.57 (184)Subgroup C 6.62 (25) 53.67±10.97 (28)*Subgroup B 7.10 (35) 43.14±11.30 (107)*Subgroup A 15.27 (38) 38.44±5.21 (138)*

*P<0.01 vs controls

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