Document zdZLRbwkXMkaE9z6K30xz1zD3

**i*^4*sis f Critical Reviews in. wpi kTT >iw Halogenated hydrocarbon . ,, $W" ' - Solvents 1 `ifrn Chloroform .f /^i, Carbon tetrachloride f v.-; .r/:* v:!.; : ` V-^* : \-:T 1 "r;'(* T-.j'* v"^' - 4v ; * -ri, ' .r*.\S/'Ltf"S^tfv-T. r rSfeH ')f-aMfffoil: T' 1,1,1,2-Tetrafluoroethane f~ 1,2^Dibromomethane JzMZl Ethylene dibromide ^^iVinyl chloride hwVinyl bromide (^Trichloroethylene uu ii. Anesthetics -; . .,, . , -;1,-.''v r&%L, -^ 14m- ^lHaiothane : ^ ..- rrv'V'r'.'f ' -> ...OR-OlgjrlM'lr: Sevoflurane 30^50-: Methoxyflurane^y .! .' ,- . ' '; . ' >`'ry -! V. .mV.'.A. . . ', -- H&ES/Tox. Library ' ' ` APR 1 2 1993 v Dow Chemical-1803 Roger O. McClellan  'flltfitl Ac w. .1 > m '/.m, 23( I 1.1 -20 (I<WS) Bioactivation of Halogenated Hydrocarbons by Cytochrome P4502E1 Judy L, Raucy* Toxicology Program, College o( Pharmacy, University of New Mexico, Albuquerque. NM 87131 James C. Kraner Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ 85721 Jerome M. Lasker Alcohol Research and Treatment Center, Bronx Veterans Administration Medical Center and Mt. Sinai School of Medicine. Bronx, NY 10468 * To whom >11 corTdpotkJencc should be addressed ' , , ,, ^ ABSTRACT: Numerous halogenated hydrocarbons of the alkane, alkcnc. and alkync classes arc metabolized by P450 enzymes to products that elicit cytotoxic and/or carcinogenic effects. Such halogenated hydrocarbons, include anesthetics (e.g., halothanc and cnfluranc) and industrial solvents (c.g., carbon tetrachloride, chloroform, and vinylidinc chloride). Formation of reaction intermediates from these compounds occurs via P450-promotcd deliaIntonation, reduction, or reductive oxygenation, with certain hydrocarbons undergoing all three reaction types. Of the multiple forms of P450 present in liver microsomcs, P4502EI has been identified as the primary catalyst of hydrocarbon bioactivalion in animals and, most likely, in humans as well. As hepatic concentrations of this P450 enzyme arc highly inducible by ethanol and simitar agents, prior exposure to 2E1 -inducing com pounds can play a pivotal role in halogenated hydrocarbon toxicity. Considering that metabolism governs the cytotoxicity and carcinogenicity of halogenated hydrocarbons, an understanding of the nicchanism(s) underlying 2EI induction in man becomes all the more important. KEY WORDS: P4502E1, P450, bioactivalion, halogenated hydrocarbons. , * ; , -r.) .S'. 'e-.` t I. INTRODUCTION - ' Halogenated hydrocarbons are a structurally diverse class of chemicals that have been widely used as solvents, pesticides, plasticizers, fuel ad ditives, flame retardants, anesthetics, and as pre cursors, intermediates, or byproducts of indus trial organic syntheses.1 Several of these halogenated compounds have been identified as environmental pollutants, and have been detected in air, food, water, soil, and in tissues of live stock. The general utility of halogenated hydro carbons leads to the manufacture and use of bil lions of pounds of these compounds'each year, resulting in environmental and occupational ex posure. Because of this exposure, it is important to understand their potential human toxicity. ., The toxic effects of halocarbons can be both general and specific. The effects observed in ex perimental animals or in occupational settings depend on many factors, including solvent struc ture, level and frequency of exposure, edneom- 1040-8444/93/S.50 1993 by CRC Picas, Inc. 1 4 I 4 30 SCO CJl o CD il:ml exposure lo oiliei agents. ami individual sensilivily, Cjener:il effects include lliose on ilie central nvtvotis 01 cardiovascular systems. Midi ;is depression. |i;iulysis. convulsion. ;ind death from respiralory or cardiovascular collapse. Spe cific effects include scleciivo organ toxicides, such us nepliro- und hepulotoxieity. Specific organ loxicitics caused by halogensued hydrocarbons are related directly to metab olism of these agents. The phenomenon of toxic metabolite generation is termed hioaetivation, and it is largely mediated by 1*450 enzymes.' How ever, not all metabolism of a given compound results in hioaetivation. Usually, multiple P450s convert the compound to one of several metab olites, with only one enzyme promoting forma tion of a reactive intermediate. The other P450s involved convert a large percentage of the dose to nontoxic metabolites, a process termed detox ication. In the absence of metabolism by P450 cn/.ymcs, halogcnatcd hydrocarbons generally do not exhibit specific organ toxicity. Many halogcnatcd hydrocarbons arc low mo lecular weight compounds and, as such, fit the substrate model for metabolism by one particular P450 enzyme, 2E1/ This P45Q is present both in animals and man, and is found not only in . liver, but also in several cxtrahepatic tissues. Regulation of 2E1 enzyme levels involves both endogenous and exogenous factors, and the ex tent to which the enzyme is expressed may play an important role in the outcome of human ex posure to halogcnatcd hydrocarbons. Endoge nous regulation of 2E1 expression entails phys iological conditions such as fasting, diabetes, and obesity/ Exogenous factors include exposure to xcnobiotics such as alcohol, which can result in the marked induction'of 2E1 enzyme levels/ This review focuses on three different areas ", of investigation. The first area discussed is the research on the metabolism of halogenated hy drocarbons, including those that require biotrans formation to exhibit toxicity. The emphasis is on haloalkancs and haloalkenes; aromatic halogenated compounds are not considered here. Evi dence also is provided for the involvement of specific P450 enzymes in the hioaetivation pro cess. Another area focused on is the functional characteristics of 2E1, the P450 enzyme that pro duces reactive intermediates from many halo- gcnalcd hydriv.uhons. I inal!>. ctmcnl theories on the iiiecliaiiiMli'. llitough u Inch cellular levels ol 2HI are rceulalcd are dc-wi ihed II. METABOLISM Hiotraitsformaiion of halogenated hydrocar bons has been known to occur for quite some time. Metabolism is promoted primarily by P450 enzymes, which often catalyze the initial steps in both activation and detoxification of xenohiotics and comprise a superfamily of multifunc tional forms encoded by distinct genes that arc independently influenced by factors such as he redity, sex, tissue, and age/ factors that affect the relative balance between activation and de toxification as catalyzed by the P450 enzymes can affect the outcome of exposure to toxins. Among these factors is the alteration in enzyme expression that occurs as a result of cither genetic polymorphisms or exposure to compounds that induce or repress individual enzymes. The fact that several P450 enzymes with differing sub strate.selectivity and modes of regulatory control existed led to the concept of variability in relative rates of substrate metabolism to both toxic and nomoxic products. Therefore, from a quantitative standpoint, the formation of toxic metabolites de pends on the concentrations of specific P450s present at the time of exposure to halogcnatcd hydrocarbons. Several human P450 enzymes have been identified whose expression can be influenced by cither polymorphism or induction/ These include members of the 1A, 2A, 2C, 2D, 2E, and 3A subfamilies. With regard to toxicity, 2E gene subfamily proteins arc among the most important due to their extensive capacity to metabolize drugs, solvents, and environmental procarcino gens to cytotoxic and/or carcinogenic metabo lites/-7 Studies to date indicate that structural features of the substrate play a critical role in determining whether catalysis by 2E1 will result in toxication or detoxication/ The initial step in the bioactivation of halocarbons occurs with the breakage of the C-H bond, which is usually the rate-limiting step "in metabolism/ Hepatotoxicity of these agents has been associated with the ease with which a hal- 2 ogcn'can be removed to produce ;i reactive metuholite. The toxicity of halogcuatcd hydroear- bons is known to increase as the numbers of halogens, si/e, and ease of hemolytic cleavage of the molecule increase.'1 Toxicity front Italogenuted hydrocarbons may arise as either cyto toxicity or carcinogenesis, and generally occurs in the liver and/or kidney. Of the many halogcnalcd hydrocarbons, evidence for carcinogenicity lias been found in the case of chloroform, carbon tetrachloride, vinyl Chloride, vinyl bromide, vinylidene chloride, and Tris (2,3-dibromopropyOphosphatc.' Methylene chloride, 1,1,2,2tctrachloroethanc, tciraclilorocthylene, and trichloroethylene arc strongly suspected to be carcinogenic.' The known hepatotoxins among these agents include carbon tetrachloride, chlo roform, 1,2-dibronioclhanc, trichloroethylene, vinylidene chloride, letrachlorocihanc, and 1,2dichloroclhane. These same compounds also arc nephrotoxic, as is ethylene dibromide,2 For the many halogcnated hydrocarbons that undergo bioactivation, our discussions arc di vided into two areas, solvents'and anesthetics, I.Trihatomethanes - ' The trihalomethancs cause damage to both the liver and kidney,, and have, been found to, promote cancer in those organs.10 The most widely studied of this class of compounds is chloroform.11 Chloroform-promoted liver damage has , been observed in humans as well as. in expert-, vi Jmental animals..12 The mechanism by which chlo- toxicity with chrome tissue regeneration rather than from the formation of chlorolorm-iferived DMA adducts.1 Evidence for the involvement of a specific I'd.xO in the bioactivation of chloroform was pro vided by Brady ct a!.," who demonstrated that purified 2EI from rat liver microsomcs metab olized chloroform to trichloromcthanol more efficiently than other purified P450 enzymes (Ta ble 1). Chloroform metabolism by liver micro somcs also was potentiated (threefold) when an imals were prcircatcd with 2E1 inducers, such as the secondary ketones acetone, 2-butunonc, and 2-hcxanonc.IJ Moreover, antibodies against this P-450 inhibited 81% of the chloroform metabo lism in microsomcs obtained from acctonc-trcatcd rats'1 and also were effective inhibitors of trich loromcthanol formation by human liver nticrosomes.* It has been proposed that the hepatotoxic and nephrotoxic effects of chloroform occur inde pendently as a result of the differential metabo lism of chloroform in the two tissues.14 Further more, gcndcr-spccific chloroform metabolism to promote toxicity in micc. Malc mice, both liver"; former i mechanism'probably involves version of chlorofom to a reactive'nwtatralite by^el^-v. ^ renal 2EI, which is under regulation by testos terone. Hong et a1.,s reported that 2E1 was found in kidney from male mice but not front female mice; however, treatment of females with tes tosterone resulted in renal expression of the cn- < zyme. In contrast, the corresponding liver en zyme docs not appear to be regulated in a sexspccific manner. Strain differences in the sus, ceptibiiity to chloroform kidney toxicity also have , ,, been shown to exist.16 DBA/2J mice are more - Z reactive intermediate that acylates proteins, giv- , 2. Tetrahalogenated Methanes ; zi ing rise to the hepatic centrilobular necrosis and. 'renal proximal, tubular necrosis ^observed' with - S.- Carbon tetrachloride (CC14), an exemplary * -`chloroform poisoning. Carcinogenesis occurring tetrahalogenated methane, is metabolized by re in these tissues may stem from recurrent cyto- / ductive pathways to reactive intermediates that TABLE 1 P450 Enzymes Involved in the Bioactivation of Halogenatcd Hydrocarbons Halogenatcd hydrocarbon Major P4S0 involved in bioactivatlon Ref. Solvents Chloroform Carbon tetrachloride 2E1 2E1 1,1,1,2-Tetrafluoroethane 1,2-Dibromomethane Ethylene dibromide Vinyl chloride Vinyl bromide Trichloroethylene 2B4 2E1. 1A1 2E1 2E1 2E1, 2B1/2 2E1, 2B1/2 2E1, 2B1/2, 2C11/6 8. 13 8. 25-28. 30, 31, 105 23 38 8 8 8, 41, 42 8. 41. 42 46-51. 85 Anesthetics Halothane - Enllurane - -;V Sevotlurane v. Methoxyflurane " ,1 .-o; . , <' :r ' > 2E1,2B4, 2B1/2, 2A2 2E1 2E1 ' 2E1, 2B1/2, 2B4 67, 68 71. 72 71 71, 73 .,r; can caus^hepaticgs, welhis renal damagc.^Ccn--^-^^or trichlorqrnethylpcroxyl) free radical, whereas. ^triIobul|mnecrosis^n^ in ^j y cartenc^meia may play an important role ^-thQTliliyi^^<yi^tl^w^and;^t^u^tl>e^^:in'CClftoxicity when oxygen tension is low,*1,^ ^ `^nKtpl^i^^f toxi^y^Myci to bed^ncd^both^X^^^ExiKnmental evidence suggests that 2E1 and,' lipid peroxidation and covalent binding of CC14- possibly; phcnobarbital-induciblc P450 enzymes derived reactive metabolites have been impli arc involved in the metabolism of CCI4 (Table cated in the process.17 The reductive pathway 1). In early studies, it was shown that animals initially involves a P450-mcdiated one electron treated with phenobarbital exhibited greater hep- reduction leading to the cleavage of a C-Cl bond atotoxicity upon CC14 exposure than untreated to yield a tnchloromethyl radical (CCly) and a animals,13 an effect most likely due to the gen chloride radical (Cl-).1? The tnchloromethyl rad eralized inductive properties of phenobarbital on ical may dismutatc to give chloroform, bind co drug-metabolizing enzymes. A majority of recent valently^ to critical cellular macromolecules, or studies, however, implicate 2E1 as the major ca ; [} attack' enoic fatty acids within the membranes of talyst of both reductive and oxidative CC14 bioac - ,'thp'>.ndnnlasmic!'reticulum = leadi tivation/ Pretreatment of rats with acetone or iso- 1 results in enhanced lipid peroxidation14 ^diuoi^thc. trichJoromethylAracIical can reaclwith ^ and covalent binding oFCCl* metabolites to pro^moieciil^m^g^ttf^om aneven more reactive ; tein.2^5.Tmhi,i:s.. enhanced bioactivation occurred ,w,,;it.hi species^the^tnchloromethylperoxyl.free radical, . a concomitant increase in metabolism of two spe ^'whicHfalso?exhibitscovalent bindingand/orelic--' cific'2E1 substrates, N-nitrosodimethylamine "i'-.'i v 4 * ** A -cs. v i" ^Vfjits lipid^peroxidatipn.j^fAltematively;'under, an-.. l (NDMA)23 andp-nitrophenol.14 Ethanol pretreat .. (,:; aerobicfconditions,'another reductive pathway of . ment also results in an increase in the rate of CC14 - CCl4"metabolism^canresultincarbene-typeprod-^ metabolism by rat liver microsomcs. This in ' ucts that-bind tq'.proteins and nucleic acids,11. crease in CC14 metabolism was accompanied by Thus,- it appears; that aerobiosis promotes cova- . a parallel increase in immunoreactive 2E1 con2 lent binding and. lipid peroxidation by CC14 via P450-catalyzed'formation of the tnchloromethyl < tent in liver microsomes.16 Administration of pyr. azole, acetone, or other aliphatic alcohols to rats . `Ci'rfjVi ij,' -v, . /- v,%. *!,i- , , i I . -:' S-& k*m V* ',;A*; WW- tetr" causes a 15- to 30-fold potentiation of CX3,-de pendent hcpatoio.vicity.'7-''' All of these audits itie known to he potent 2EI inducers.'1 A tole for this cn/ymc in die reduction of CCL also was provided by Johansson and Ingelman-Sundbcrg,;j who showed that purified rabbit 21* I, upon reconstitution, was 100-fold more active in me tabolizing CCIj to chloroform and in initiating lipid peroxidation compared to P450IA2 or P4502B4. Inhibition studies with selective inhib itors or antibodies also have demonstrated the involvement of 2EI in the bioactivation of CCI4. Disulfiram, a chemical inhibitor of the enzyme, was shown to block CC14 hcpatoxicicity in rats.2'1 methane, also are hepatoxic.-' Tetrachloroethtine is oxidatively dechlorinaled by. P450 to 2.2-dichloroacetyl chloride, which is then hvdroly/ed to dichloroacetic acid and aeylates proteins.*'' *' Administration of the 2EI-inducing agent pyri dine to rats results in an eightfold elevation in the rate of tetrafluorocthanc metabolism by liver microsomcs. Moreover, in reconstituted systems, purified 2EI catalyzes reductive defluorination of 1, 1, 1,2-tctrafluorocthanc at high rates com pared to other P450s including P4501A1. which is somewhat active toward the substrate, and P4502BI and P4501A2, which display negligible rates of defluorination (Table 1).'" Furthermore, anti-2El lgG produced marked in hibition (80%)'of CCIj reduction mediated by human liver microsomcs,*-1" indicating that 2E1 4. Halogenated Alkenes participates in CCI4 metabolism in man as well. . i- CC14 metabolism has been shown to be closely linked with the concomitant destruction of P450. Indeed, metabolic reduction of CCI4 was accom panied by'a'rapid loss in immunorcactivc 2E1 Mbnosubstitutcd halocthylcncs. such as vi nyl chloride and vinylidcnc chloride, arc metab olized to compounds that arc mutagenic and car cinogenic.1 A common mechanism for haloalkcnc oxidation may be through formation of an oxy- ^geriated intermediate' that,directly, forms r dchydc and haloacylhalidc'derivativcs. Thc na---:; ^^ ^turc of the intermediate has, been postulated a an 'oxo-iron complex that can cither collapse to - decrease in both low K,, NDMA demcthylasc attd the conversion of CC14 to chloroform has been reported in rats treated with CCIj,'- again suggosling that 2E1 was the target of' dc estruction. - v_' * a haloaldehydc (in the case of vinylidcnc chlo ride) or an epoxide (in the case of vinyl chloride), or can result in heme alkylation and P450 de struction.1 M Evidence demonstrates that of the halogenated cthylcncs, vinyl chloride, vinyl bro mide, and 1,1,2-trichlorocthylcnc (trichloroethy 3. Vicinal Halogenated Alkanes lene) arc oxidized primarily by 2E1 to cytotoxins 4- * i j^XCertain vicinal dihalogenated ethanes arc both i. 1 _4 - - j .1.1,1 n- 33 'T'L*. A (Table 1). ` The human carcinogenicity of vinyl chloride was established in 1974, when 50 cases of hepatic 'Ccliac necrosis have been observed after exposure . '. i Cr0-* -J 1 . .. ^-of humans to fatal doses of EDB.35 Recently, it ,: has' been shown' that 2E1 mediates'the hepatic ,'r .3j***rnetabo1ism' of.' both 1,2-dibromomcthanc and -:-EDB in man.* ^V^v'^ft5-Vicinal'ipolyltalogenated -ethanes;? such as i ;.i?!Si;f,2,2-tetrachloroethane and 1,1,12-tetrafluo- v-X:; ;x. tivity of vinyl chloride as well as that of vinyl bromide. Oxidative metabolism of vinyl c- hloride by P450 was found to result in the production of 'a reactive epoxide, 2-chIoroethyIene oxide, and " vicinal haloaldehydes,40 with evidence favoring the epoxide as the ultimate carcinogenic metabolite.*1 The haloaldehyde, 2-chloroacetald^hyde, also is an important alkylating agent derived from vinyl chloride.41 At least two P450 enzymes may be involved in the activation of vinyl chloride ' ' A,'s-V;U-;T'v'e4V:' -*v";'Vl? VI*i $ ft - ,^'5' X-. 4 ' V=' ' ! " 'y-"a*u1 - lIivf I : 'j ,S<** '*V ' . Ty R&S 150820 r .f 'e.--' !i>.- . I - I b- and, vinyl bromide. Micro.-iomex from mix pietreated with plicnobarbital exhibited high eonversion titles of these compounds to their cor responding epoxides compared to microsomes pregnancy, which are lactors that intluence 21:1 expression, both alter the extent ol 1 CE oxidation in rats.'1 Three-week-old animals converted I CE to Cl I at taster rates than did 18-week-old rats from naive animals. Purified rut P4502UI, the major P450 cn/.yme induced by phenobarbital treatment, was highly active in the conversion of vinyl chloride to its corresponding epoxide.'*1-'13 More recently, inhibitors or 2E1, c.g,, distill iram, were shown to prevent epoxidation of vinyl chloride and vinyl bromide by human liver mi crosomes,* implying that 2E1 also is involved in the activation of halogcnated alkcnes in man. (an approximate twofold difference). With regard to pregnancy. TCE metabolism in animals at day 21 of gestation was decreased 50% compared to nonpregnant rats or to those at day 10 ol gesta tion.51 These changes in microsomal TCE me tabolism paralleled those of microsomal 2EI con tent, suggesting that TCE metabolism is influenced by the same physiological factors that regulate 2EI levels. . Trichloroethylene (TCE) has been widely used as an organic'solvent and general anesthetic. It is weakly hepatocarcinogcnic in B6C3F mice and hcpatotoxic in rats and humans.2-43 TCE oxide has been detected as an oxidative metabolite, and B. Anesthetics 1, Halogenated Anesthetics may. be responsible for the covalent binding to liver,'proteins!and nucleic acids noted with .TCE.44-45 The only other TCE metabolite formed is by P450 in dctectablc'amounts is chloral hydrate The most widely used inhalation anesthetics arc polyhaloalkancs or alkyl ethers containing fluorine. Most of these anesthetics have been shown to be oxidized and/or reduced by P450 enzymes to reactive metabolites that can bind to .of which' P450;s proteins and lipids.53-53 This biotransformation rasted^ts'^and'tho'se%!<ivolatile anesthetics is of concern because ^treated'withphehobarbitalor cthanolexhibit cn- been linked in a number of instances to toxic hanccd rates of TCE metabolism;46-** the latter' effects, including hepatotoxicity and nephrotox- . treatments also potentiate TCE hcpatoxicity. icily.54 The best studied of the inhalation anes However, this enhancement of TCE toxicity by thetics is halothanc (l-bremo-l-chloro-2,2,2-tri- ethanol is noted when the animals arc exposed fluorocthane), an agent that causes hepatotoxicity to low TCE concentrations. In contrast, exposure . in man and animals.5-5'5! Evidence supports P450- > \ to markedly "higher TCE concentrations arc re dependent formation of the 2-chloro-l,I,l-tri-' quired to observe potentiation of TCE toxicity fluorocthyl radical as the major reactive and toxic by phenobarbital.4'''*7 Nakajima ct al.4*-50 showed metabolite produced under conditions of low ox . -. that 2E1 antibodies were potent inhibitors ofTCE ygen tension.5*-61 This reductive metabolite also acts as a suicide inhibitor of P450 and, under ; conditions, there is a significant of P450 and a parallel loss of the torv^witit1'phenobarbital-induced .microsomes at -$$$!thietic heme - 4 *3 higher substrate'concentrations.' Such results in- , -/.--ie-dicia:t.eothant 2iEiiEVLi ....I,.- -ts -^^enzyme , thane was found !' ? - :' tions of . x,' inducible . "! .?tabolism onlyat'much higher substrate' levels. 2El-catalyzed metabolism of TCE also has been , . shown to occur in human liver microsomes.* - Factors other than exposure to inducing agents , (appear to'affect metabolism of TCE. Age and - ' Oxidative biotransformation of halothanc i predominant in humans and results in lesions of a far more extensive nature than those produced by reductive metabolism.54 Oxidation of halo-? thane proceeds through a halohydrin to produce i',4 I . . . ........... 1 ? r . -- " . . '! :- j6 ; -tl k'M r- either (rifluoroaociyi chloride or bromide, which subsequently hydrolyzes lo mfluoioueeiic acid and acylaics proloins.''' A iri(luon>acctyl-!'45() adduct has been detected on the outer cell surface of rat hcpatocytc membranes after treatment with , halothane, This acylatcd P450 (Mr = 54,000) may be immunogenic and may promote the de velopment of halothane hepatitis, . The metabolism of halothane involves at least two different P450 enzymes (Table I). In rabbits, both phenobarbita! and imidazole induce metab olism of halothane, although the extent of in duction is much greater with imidazole/'7 More over, purified rabbit P4502B4 and 2EI catalyze the oxidation of halothane to trifiuoracctic acid. Immunoinhibilion studies have revealed that 2E1 is the major catalyst of halothane oxidation from imidazole-treated rabbits; whereas the reductive metabolism of halothane was shown to be cata lyzed mainly by phenobarbital-inducible en zymes.". Utilizing purified rat liver P450 en zymes, VanDyke crial.61< demonstrated that ... . P4502A2, P4502B1, and P4502B2 catalyzed the PA/41 ,Zlt AAH J-* f LnlAlUnnA t L *.11' __L. . valently to liver proteins in a manner analogous to that of halothane.'4 Several studies have revealed that 2EI is the predominant catalyst of cnfluranc metabolism (Table I ).71-72 Hoffman et cl." demonstrated that treatment of rabbits with imidazole resulted in a 250% increase in the microsomal oxidation of cnfluranc, mcthoxyfluranc, and scvoflurane, the latter being a structurally related halogcnalcd an esthetic. Antibodies to 2EI were found to inhibit microsomal cnfluranc defluorination by over 90% but inhibited mcthoxyfluranc dehulogcnation to a much lesser extent (40%). In the case of mcthoxyfluranc, such a finding was not unexpected inasmuch as other P450 enzymes (c.g., P4502B1) arc known to be involved in metabolism of this compound (Table l).73 However, reconstitution studies showed that 2E1 was capable of oxidizing not only cnfluranc, but also mcthoxyfluranc and scvoflurane.71 A role for 2EI as the major cnfluranc-oxidizing enzyme in liver microsomcs also was provided by Tsulsumi et al.,72 who found that liver microsomcs from ethanol-treated rats '. <: .;>;: t vl- - - ' " halothane, iii fact at rates comparable to niicrosomes from imidazole-treated rabbits.67 Further more,' metabolism of halothane was found to be elevated in obese individuals, as serum levels of inorganic fluoride, trifluoroacetic acid, and bro- mide ion were higher in these subjects than in non-obese subjects following halothane administration,6',, Because obesity is among those ;,;pathophysiological:conditions that results in 2E1 ' / induction.70 the elevated metahnlismnf halnrhnne were used by Tsutsumi and co-workers72 to dem onstrate involvement of the' ethanol-inducible P450 in dchalogcnation of the anesthetic. In clin ical studies, elevated serum fluoride ion levels were found in subjects who received isoniazid prior to enflurane administration.71 Obese pa tients also were found to exhibit enhanced rates of enflurane metabolism.7* These alterations in , enflurane disposition observed in both types of ' f.v^... ;r^ R&S 150822 4>, :y,- 7 X*-.- %$&&&*&* &&: :"V\,"}, . ji*:' ...: specific metabolic properties of the enzyme itself acetone levels arc elevated (e.g., starvation, di ;ts well as on mechanisms involved in its cellular abetic ketoacidosis, and obesity), hepatic 2EI regulation. levels are usually induced and, hence, increased The initial observation in humans that chronic conversion of acetone It' acelol and methoxygly- ethanol consumption resulted in proliferation of col. proposed gluconeogenic intermediates, may liver smooth endoplasmic reticulum1' ''' led to the occur. This pathway could constitute an impor discovery of a unique pathway of ethanol oxi tant source of glucose during starvation or dia dation. which was termed the microsomal ethanol betic ketoacidosis. oxidizing system or MEOS. MEOS was depen Multiple P450s also arc present in human dent on P450 and NADPH, and displayed in liver, and 2EI constitutes one of the enzymes creased activity following chronic ethanol ex found in most, if not all, individuals. Immuno posure in rats." The P450 enzyme responsible chemical studies using antibodies to rat or rabbit for this ethanol-oxidizing activity was initially 2EI first revealed the presence in human liver of characterized upon purification from ethanol- an enzyme (Mr = 54,000) that promoted aniline treated rabbits and was termed P450LM-3a.TM The hydroxylation and low K,,, NDMA dcmethyla- purified enzyme displayed an enhanced ability to iion.`*,-`'1 2E1 was subsequently purified in a cat- oxidize ethanol and other primary aliphatic al cohols to their corresponding aldehydes when alytically active state from human liver microsomes and, upon its reconstitution, was shown r compared to other'purified rabbit P450s.'* An to effectively oxidize ethanol, aniline, and homologous P450 enzyme, initially termed P450j, NDMA, the latter at concentrations similar to was then purified from isoniazid-treated rats."0 "1 those found in vivo.91 Other metabolic properties Rabbit LM-3a and rat P450j, now collectively later attributed to human 2E1 include activation designated 2E1," not only metabolized alcohols of acetaminophen to the reactive metabolite N- ebut also converted environmental procarcinogens , *> - acctyl-w-bcnzoquinone imtne," chlorzoxazone to ultimatecarcinogenicmetabolites yin fact; 2E1 hydroxylation,*4 benzene hydroxylation, and sty-tx-sf**'-^ - *5is MisJno^klmwh^^illTe^soIetmicrosomalMow K.Siii1 renc epoxidation;* the role of this hunian;P45O0f sNDMATdemcthyIasesin;most-animal species.I3;': enzyme in halocarbon bioactivation was already.;^; "-This P450 enzyme*also plays an important role * ** discussed. Furthermore, the amino acid sequence " in the generation of cytotoxic metabolites from of human 2EI, which was derived from the com acetaminophen, a widely used therapeutic agent.*4 plementary DNA, was found to exhibit an ex Other'compounds whose metabolism is catalyzed , V,;.- primarily by'2EKinclude aniline, pyridine, 4-. ,s ' t methylpyrazole;benzene,chlorzoxazone,carbon M _ disulfide; p^nitrophcnol; acetone, glycerol, di- cthyl^ether, and the'halogenated hydrocarbons . 'f.- >1 ^--* - ,'P - 'dVHisOcVuUsMsCeUtl eC-a4Xr1li1e1Vr1-1inU tlihuis rIVeYvIWieVwY.3- ' ....^.. '* tensive degree of homology with both the rabbit and rat enzymes.'"'-*7 Southern blot analysis revealed that the human CYP2E genesubfamily consists of only a single gene without closely related members.*4 In contrast, rabbits possess tw.Vo\J VCYitP2E ggveunve osubvifuaummiljyf,, umivetmuwbevrios,, 2EI maniud --i-" , ^ . . K_ - - --'v-.'i.vV*'ji ::r* Experimental'^studies have revealed thar xe- ' 2E2, which exhibit different modes of regula- ; ,'g' ^i^tS^'.'fel^nobiotic^otheritharivethanol'also incfease. ^El^ tion.**.The human CKP2E1 gene was mapped to; I r'S||J^.,;1|:'v;;''.I,''':J'v,thatTinduce42ElJals6;;are"substrate?^for'thVreh^^T microsomesfrom these subjects -were, found,to I `^:1lzyme^Ttie.'ability'of 2E1 to' metabolize acetoncT'!^.*; exhibit'enhanced rates of, NDMA metaboiism.^f^il^^-i-^^pl UnA^lA^^ *A7 rU A IU A4 K A* A M A ' ' #1 AMAJi 4 AM r V T. E A.U A,-,%4A AM ^ Irf 1 I In11/Xrt IH4C ff~\ 1 in/1 lA AA/tl V l S.*l tf^ 1 -I,:.: 1 ' I1 rrryr^i - f : %x:'~ , v /_- | exposure lo ctlmnol. liulngcnutcd hydrocarbons concluded that 'his transient increase was rail suf and other 2El-activaicd hepatutoxins.7 ficient to account lor the marked induction of the en/.yme by imidazole.'17 Wrighton el al.`n could A. Xenobiotic Regulation find no correlation between 2EI mRNA and 2E1 protein levels in human liver, and concluded that ethanol-mediated 2E1 in man. as is the case in Because 2EI-mediated metabolism is be rodents, docs not require the accumulation of 2E1 lieved to play such nil important role in the cy tianscripls. Such observations led to the consen totoxicity and carcinogenesis of halocarbons. the sus that the induction of 2E1 caused by xeno- mechanisms involved in regulation of this P450 biotics occurs primarily via post-translational also arc of importance. Indeed, the enhanced bioactivation of halocarbons noted after exposure mechanisms. A decrease in 2E1 protein degradation as a to certain xenobiolics stems from the elevation in 2E1 levels elicited by such agents. However, result of inducer-mediated enzyme stabilization has been proposed by a number of investiga the mechanisms underlying induction of this en tors. lu'`10i The rapid decrease in 2E1 protein lev zyme remain somewhat controversial. In this fi nal section of the review, we consider the various modes of regulation proposed for 2E1. The tox els noted in primary cultures of rat hcpatocytcs was shown by Eliasson ct al.101 to be prevented by including isopropanol, dimctbyisulfoxidc, or icological significance of 2E1 regulation as it relates to halocarbon bioactivation also is dis imidazole in the culture media. Song ct al.1'* measured the turnover of 2E1 in vivo in untreated cussed. Y;; The modulation of cellular 2E1 levels by xc- and acctonc-trcatcd rats and found that the former animals exhibited biphasic 2E1 degradation ki nobiotics has been demonstrated in several mam- netics with respective estimated half-lives of 7 ^ and 37 h. In animals treated with acetone, how-,.., c component (37 h) wass?s^ > byiwhichunduccrcM-1** include increases; in 2E1 mRNA sYcmming from/?' block' 2E1 enzyme degradation was ciramincd < transcriptional activation or rnRN[AA stabilization','' V Tierney et al.,'17 who'found'that*Yhice'' trcatcd^^-r^'^':?Si : enhanced, protein synthesis, and decreased en with 4-mcthylpyrazoIc possessed much lower zyme degradation. The three, mechanisms may levels of ubiquitin-conjugatcd microsomal pro-: -act individually or in concert with each other to tcin than did mice treated with CC14. These work .increase 2E1 content in the liver. Species dif-rYfefencesYthe''particular inducing agent,'and the ers proposed that ubiquitination is an intracellular signal for2El proteolysis, and that the inhibition ^ '-duration of inducer treatment (acute or chronic) of2El degradation by 4-methylpyrazolc resulted from inhibition of the formation of microsomal ubiquitin conjugates by this compound.17 How-.; . >, *;-r , Of .thejyarious' treatmentsythabincrease' he- 'Yv ever, because specific ubiquination of 2E1 was AtnatiC'2EUin rodents.' ethanol and similar avents if. not measured in this study,,the proposal..that in-... j _p t '_____i'll______ * ;*L' ..u; i ^ _ ! ' !' ;of.2El .induction in-' transenpts' with acetone for IsSTpi t; it ^ ]*/ . .c^vV; short periods of time (6 to 24 h), which paralleled ' the increase in newly synthesized 2EI protein. The concomitant exposure of rabbit hepatoeytes to acetone and a-amaniiin, an inhibitor of gene transcription, blocked the increase of both 2E1 mRNA and protein produced by the former com pound,11" suggesting that the enhanced rate of 2EI protein synthesis found in acetone-treated hepatoeytes may stem from increased transcrip tion of the CYP2El gene, at least in rabbits. Treatment of acetone appears to stimulate rates of 2E1 protein synthesis in rats as wcll.B7,1<w A single injection of acetone was found to increase aniline hydroxylation by liver microsomcs both 30 min and 48 h after administration: The more , rapid increase imthis drug-metabolizing activity (69%), which was maximal at 0.5 h, returned to control levels after 4 h while the slower increase. in activity (168%) peaked after 48 h. Bccausc occurring within the hepatic pcrivcnular zone is accompanied by an increase in 2EI transcripts within the same acinar zone, furthermore, it was found that levels of 2EI protein and mRNA in pcrivcnular, pericentral, and periportal hepato- - cytes were significantly correlated among all pa-' . lients studied.117 , Translational control of protein synthesis also may play a role in the 2E1 induction process. For instance, Kim and Novak11* found that pyr idine (and acetone) increased the hepatic 2E1 content, not by enhancing levels of 2E1 tran scripts but by increasing the efficiency with which preexisting transcripts were translated. The two- to fourfold increase in 2E1 content noted 6 to 24 , h following a single pyridine injection could be completely blocked by prior administration of cyclohcximidc, whereas actinomycin D, an in-' hibilor of transcription, had no effect. The stirn- w*.' :-&& 'M. ssinductiontKfEleyations of-SElfmRNAfand pro-jSz-; Iteii^^v^TCe^^ort^/fnlra^f^etKandiiin8 iirmrf iitifcM9?ac!w/*ii * nus recciving'contin- n interesting phenomjz enon' Was noted^wtth the"-continuous-infusion 1. Fasting/Diet/Obesity .(' model of ethanol treatment. Despite the fact that ' ; , :.- , ethanol was infused intragastrically at a constant Fasting markedly enhances microsomal drug- , \l: r: rate, blood ethanol levels were cyclical in nature, metabolizing activities, including those mediated, :f^^^^^^^^^an)d^^ge,dtfioi^TpT<^500'fm^dl':rovwfa<'7-day%';' :':' by 2E1. Twenty-four- and 48-h fasts were'found_^'^fM|^^^l ! " ; ' period.!11 At'blood ethanol levels"<250 mg/dl, ' to cause 59 and 116% increases, respectively, in a 6-Hd increase in hepatic 2ET content was microsomal NDMA demcthylation in rats. These i- ' j . - increases in enzyme activity were accompanied ; by parallel increases in 2E1 protein and its cor- responding mRNA;1!'induction of 2E1;protein) and mRNA was'noted mainly in the hepatic peri| tivenular zone. 12" Elevated 2Elprbtein1and>mRN/^,^^j_,^.^f,^ T liver and kidney also have been observed! fasted rabbits, although renal 2E1 protein' levels <'&$'**$/* s - X . j-,:,Y1'<' ,<u gene transcription or sta- ...'r filiation- of,2El- mRNJAA.*9*8-.,n19#.-i,3Mo IInnitliarlilay;l'-livn-A! 'tny.^.^^.^ 'r,'r v * ' -\SViW;4h? r'VZ. creased circulating ketone levels were proposed as the cause for induction inasmuch as the admin istration of kctogcnic compounds, such as n-hexanc, 2-hcxanone. and acetonyl acetone, in creased 2E1 concentrations in the liver.121 However, even prolonged fasting docs not pro duce blood ketone levels high enough to account for the extent of 2E1 induction observed. There fore, physiological factors other than increased circulating ketones arc apparently tesponsiblc for the elevation of 2E1 that occurs when animals are starved. Dietary' alterations also may result in the in duction of 2E1 protein. Ketogenie diets, includ ing those deficient in carbohydrate or high in fat, arc known to enhance metabolism of chloroform, CCI.,, 1,2-dichlorocthane, 1,1,-dichlorocthyIcnc, trichloroethylene, and cnfluranc in rats.122-12'The increased rates of metabolism observed with these halogcnatcd hydrocarbons most likely stem front the increase in 2E1 elicited by the high levels of circulating ketones attendant with the diets. Com pared to animals :fcd fat-free diets; animals fed ". ;; ," *4r,| ~| and it^coraspSnding^ ' activity,' NDMA dcmcthybtion, in liver micro- somes.12'1 Both hepatic and renal 2E1 were induccd by high fat diets, and the increase in cnzyme was accompanied by a threefold increase .................'. .in 2E1 mRNA,';25. Such results indicate that high fat diets can influence expression of 2E1, pos sibly in a manner similar to that observed with fasting,-' In addition, the long-term (6 months) feeding of high fat diets to render rats obese also .resulted in induction of 2E1 protein and its cor- , ' 05 I' - .. - ;'X the cause of the increase in 2E1 enzyme conceni; trations/The ability of high fat diets, as well as' die.^obesity produced by these'diets, to'elevate hepatic 2ET'content may have significant impli cations for man .' considering that these diets re flect; to a certain" degree, the human diet. This could place the obese individual at greater risk t0 halogenated hydrocarbon-promoted toxicity. , , Moreover, it has been shown that high fat diets potentiate the 2E1 induction mediated by xeno- hioiics. again demonslr.uuig the mleraetion beiween xemihiniic' and pliyvinlogieal factors in terms of 21:1 regulation. Administration of eth anol to rats in liquid diets containing 359r lal resulted in a greater induction of 2E1 compared to animals fed ethanol in liquid diets containing 5% fat.'2'1 Finally, feeding rats a diet deficient in thiamine resulted in an increase in 2E1 content and ND.VJA dcmethylasc activity in liver microsomcs.,2, As thiamine deficiency docs not affect ketone levels, the mechanism of 2EI induction remains obscure. 2. Diabetes Chemically induced and spontaneous dia betes are conditions that may influence the out come of exposure to halogcnatcd hydrocarbons bioactivated by 2E1. Uncontrolled diabetes re sults in a dramatic elevation of serum ketone levels. In fact, it has been shown in sponta neously diabetic (BB) rats that plasma (i-hydroxybutyrate levels (but not those of glucose or ^ insulin) correlated with microsomal 2E1 content Tand "associated activities, i.c., aniline hydroxylationand NDMA dcmethylation.12* Moreover, in rats made diabetic by the administration of sircpiozotocin or alloxan, both the 2E1 protein and 2E1 mRNA were elevated to levels found in fasted rats.,2, lw Interestingly, insulin treatment was found to reverse the induction of 2E1 caused by diabetes''1'132 via a mechanism that most likely involves reversal of the ketosis rather than through a direct effect on the CYP2E\ gene. Indeed, in sulin has no effect on 2E1 expression when added to cultured hcpatocytcs.l;,;, With regard to dia betes itself, the elevations noted in 2E1 protein and 2E1 transcripts are reportedly due to mRNA V stabilization.,M However, as ketones have been shown to increase 2E1 mRNA levels by activat ing CYP2EI gene transcription in fasted animals, it seems likely that a similar mechanism is at work in the diabetic animal. 3. Hormonal Regulation t At least two hormones, growth hormone and testosterone, have been implicated in the regu- 11 -NH laiion ol 2Iil expression, 'icstostemne appears genateil hydrocarbons. Because it is now realized to lx* important in regulating 2EI levels in the that 2EI is not only expressed but also is induced kidney ;ind is discussed in the next section. In in extrahepatie tissues, the halocarbon-promoied vestigations into the effects of growth hormone toxicity observed in these tissues also may he due on 2EI have been pet formed mainly with hy- to 2EI-mediated hioactivaiion. Most often, im pophyseciomizcd rats, which exhibit multiple munochemical procedures have been used to lo hormonal deficiencies. Hvpophysectomy results calize the enzyme in extrahepatie tissues. For in an increase in 2EI and. in a parallel fashion, instance, Shimizu ct al.l4J used protein blotting its corresponding niRNA.,,'`1'' Replacement to show that 2E1 was present and inducible by therapy with growth hormone reverses the liy- ethanol treatment in rat lung, kidney, and nasal pophyscctomy-mediuicd increase in enzyme con epithelial microsomcs. A more sensitive tech tent. These results imply that of the various hor nique, immunohistochcmistry, was used by these mones altered by removal of the pituitary, growth investigators to reveal expression and ethanol- hormone is the most important in regulating hc- mediated induction of the enzyme in rat duodenal oattc 2E1 expression. That 2EI protein and and jejunal villous cells, and in epithelial ceils mRNA levels arc both coordinately induced by derived from the check mucosa, tongue, csoph- hypophysectomy indicates that growth hormone agus, forestomach, and proximal colon.112 In rab acts directly on the CY/'iEl gene and suppresses bits, 2EI has been found in kidney, nasal mu its expression by inhibiting gene transcription.I V' cosa, and, interestingly, in bone marrow. 14,~14' It also has been suggested that growth hormone Treatment with ethanol or acetone induced the suppresses 2E1 concentrations through a soma enzyme nearly 5-fold in kidney and over 12-fold togenic receptor-mediated process.Although in bone marrow but was without effect in nasal 2EI protein content is markedly increased by hy- mucosa. The elevation of enzyme content in kid pophyscctomy, catalytic activities associated with ney and bone marrow was accompanied by a jz-.lheenzyme(jxnitrophcnol hydroxylation, NDMA ''similar enhancement of 2EI-catalyzed'activitiM!^?-Sj;,^f xdcmcthylatiom'rand aniline'hydroxylation) were . " (c.'g., aniline and,benzene hydroxylases) in these,; \clcvatcd only'slightlyaThis dissociation of 2E1 v- - tissues.'"-14-' 2E1 is present mainly within^hcT^f.ffi:;-1 ; content and activity is most likely the'result of a". -vd proximal tubules of mouse kidney. In contrast to decrease in P450 reductase concentrations caused the rat and rabbit homologs, however, mouse by hypophysectomy; P450 reductase content fails renal 2E1 is regulated in a sex-dependent manner. to return to control levels after growth hormone Kidney microsomcs obtained from adult male replacement therapy.,M-'-w Thus, the depression mice exhibit substantial levels of immunorcactivc . of P450 reductase, which is the rate-limiting en 2E1 and also dcmcthylatc NDMA, whereas those . zyme in many P450-dcpcndcnt reactions, may be obtained from female and immature male mice responsible for the lower 2E1-mediated drug me do not.'5-144 The role of testosterone in regulation tabolizing activities noted in hypophyscctomizcd of mouse renal 2E1 was shown by the appearance rats. , of a new microsomal low K,,, NDMA demeth- . ylasc as well as 2EI mRNA in kidneys from . t : _ , female animals treated with the hormone.^With. vlwjV _ C. Tissue Distribution/Localization : / regard to halocarbon toxicity, this gender-spe-, cific expression of renal 2E1 in mice most likely ^ V f ' Similar to most other P450 enzymes, the explains the kidney damage observed in male but highest concentrations of 2E1 are found in the not in female mice after exposure to chloro- . liver. Both prior to and after induction, 2E1 form.16 V. expression predominates among hcpatocytcs Besides the endoplasmic reticulum (i:e.,mi-; comprising the perivenular zone of the liver aci crosomcs), 2E1 also is found in the plasma mem- 1 ' nus.V3-IJ0-140-IJI -phis regiospecific pattern of 2E1 brane of rat hepatocytcs.147 In fact, purified prep expression in liver has been invoked to explain arations of plasma membranes demetjiylatc the enhanced susceptibility of perivenular cells NDMA at rates nearly one third of those obtained to damage from hepatotoxins, including halo- with microsomes, reflecting the difference in 2E1 <,,, /t a-.. i >L content Ix-twecn the hid organelles, 21:1 levels in tile Itcpatocyte plasma inembr.nie lime proved inducible,,J' an observation that may h:ive toxi cological significance with regard to the produc tion of acylated protein adducts on the cell surl;icc. The lornunion of acylated 21:1 adducts in plasma membranes as a result of lialocarbon biouclivatiun may, in turn, elicit the immuno genic (and toxic) response observed in certain types of drug-promoted hepatitis, such as that described for halothane.'''','''w, D. Polymorphism in Human 2E1 Expression Various genetically linked polymorphisms in oxidative drug metabolism have been described in man. Such polymorphisms play an important role in the differences observed among individ uals with regard to drug response and drug tox icity. One of these polymorphisms, namely that involving the CYP2D6 debrisoquinc hydroxylase locus/sicms'frpmV single base change in a CYP2D6 inironic splice site. This point mutation causcs'abcrrant splicing of 2D6 pre-mRNA and, hcncc/ thc production of a very unstable'2D6 protein.Inasmuch as the mechanisms un derlying other inheritable defects in human drug metabolism arc far less understood, these poly morphisms also may involve base changes as well as deletions in the corresponding P4S0 gene. Such mutations may result not only in the absence of P4S0 proteins, but also in the production of func tionally deficient enzymes. For instance, the S- mephenytoin 4-hydroxylasc polymorphism ap pears to stem from a mutation in a CYP2C gene subfamily member (e.g., 2C9, 2C10,2C18) with altered catalytic properties.130-111 ; ' One approach for identifying'structural changes in P450 genes is restriction fragment length polymorphism (RFLP) analysis. Muta tions of functional significance in the CYP2D6 gene have been detected using this type of anal ysis.132 With regard to the human CYP2EI gene, Taq\ and Dra\ RFLPs have been discovered,"3-134 the former within intron 7 and the latter within intron 2. Both are two allele polymorphisms that occur with a frequency of 0.1 (Taq\) and 0.24 (/>/-,/It. Whether either Rl l.I' ha-, ramifications in tcmis ol 21:1 protein expression or function is not known, although L'emalsu ct a!."4 reported that host susceptibility to lung cancer is associ ated with the C T/'2EI Pr,i\ Kl LP. More recently, two other RFLPs in the hu man 0'/'21:1 gene have been detected. /\wl and A'.vrd polymorphisms were both found in the 5'flanking (i.e.. regulatory) region of the gene, were in complete linkage disequilibrium w ith each other, and occurred with frequencies of O.til tel = Psi\ ', A'.vol ') and 0.19 (e2 = P.uI . A'.vnl ) among 202 unrelated Japanese subjects. The pre dominant homozygous allele, heterozygous al lele, and the homozygous rare allele were des ignated as type z\ (cl/cl), type B (cl/c2), and type C (c2/c2). respectively.1'-' Comparison of type A and type C gene structure revealed several point mutations within the CYP2E\ distal pro moter region (bases -- 1259 through -771) but no changes in the proximal promoter region of the gene.136 Nevertheless, these base substitu tions were shoxvn to have a marked effect on gene transcription. Hayashi ct al.134 subcloncd " the 5* regions from types A and C CYP2EI DNA j into pSV40CAT (chloramphenicol acctyltransferasc) plasmids, and then transfected the dif ferent plasmid constructs into the HcpG2 human hepatoma cell line. Whereas cells transfected with either the type A or type C DNA pCAT plasmid constructs expressed CAT activity, the level of CAT expression was tenfold higher in cells xvith the type C construct, indicating not only that CYP2El bases -- 1259 through --771 contain a as-acting clement capable of activating CAT gene transcription in HcpG2 cells, but also that the point mutations found in this CYP2EI DNA re gion have a profound influence on the extent of CAT gene transcription,134 If these polymorphisms are found to actually influence expression ` of the CYP.2EI structural gene itself, then indi viduals of the type C genotype will express more hepatic 2E1 protein than individuals of the type A genotype. Moreover, the type C genotype could give rise to a phenotype that, in all likelihood, exhibits more extensive metabolism of 2E1 sub strates, including halogenatcd hydrocarbons. The identification of such genotypes would be ofclin ical importance as it could aid in predicting those subjects at risk to exposure to halocarbons. 13 =' P.i; > - . T.i ' IV. CONCLUDING REMARKS agents (e.g.. benzene) requiring activation by 2EI, toxicity would lie reduced, whereas drugs ' In this review, we emphasized the importance (e.g.. eltlor/.oxa/one) normally converted by the of one particular 1*45(1 enzyme, ethanol-inducible enzyme to an inactive product would exhibit an 2E1. in the bioactivation of halogenatcd hydro exaggerated therapeutic response. Inasmuch as carbons. The presence of the enzyme in man can 2EI also may play a role in ketone body ho predict a variety of pathological sequelae that meostasis, suicide inactivation stemming from -' '.. Stem from 2EI-mediated metabolism of these halocarbon metabolism could have serious con ubiquitous environmental pollutants. Of the many sequences in terms of decreased acetone convcr- r , factors that affect halocarbon bioaclivation and sion to acciol and methoxyglycol, the latter being its resultant toxicity, one of the most prominent a putative glucose' precursor.' Halogenatcd hy concerns variations in 201 enzyme concentra drocarbons also could influence the metabolism tions. If conversion of a particular halocarbon to of other 2EI substrates in a different manner, its toxic metabolite is limited by the amount of i.c., through competitive inhibition at the sub- i enzyme present, then enzyme induction may stratc binding sitc(s) of the enzyme. <\. fl markedly, enhance toxicity of the compound. Genetic variability in 2EI expression and/or v."- Thus, the induction of 2E1 that results upon kc- its induction is another important factor that could tosis or upon exposure to any of several xeno. biotics, including alcoholic beverages, may be influcncc the outcome of halocarbon exposure. As discussed herein, inheritable polymorphisms ' ?V: '"'^'T . `especially detrimental to individuals simultane. ously exposed to halogenatcd hydrocarbons. From within the human CYP2E\ structural gene and upstream promoter regions have been discov- . v'.xh,. ; the cxpcrimcntaland the clinical studies, it has cred. However, it is not known yet whether such , become apparent that the regulation of 2EI ^`expression by both endogenous and exogenous polymorphisms ultimately affect the expression or the function of the encoded protein, as has 3Xv,> '-''4* ; ,compounds\isi,rather complex,: and involves at,. .. been found to be the ease for othet; human P450 ^|r^^. least three^distinct mechanisms operating cither ' ' .enzymes (e.g., P4502D6). If indeed this is the ^fe^a^^^Sjngularlv'ortjn conceit at tlie-level'of transcrip-0! ease,'then individuals'expressing high levels j^v^^X.yi^MSjtion^traroiatioitr qr^subsequent to translation. In ' 2E1 would exhibit enhanced metabolism of hal-^-Tr^r k^f fact,' because of the opposing effects of the many factors that influence 2E1 expression, it is some\ what difficult to predict the outcome of halogcn,. ated hydrocarbon exposure. Age, diet, hormonal ogenated hydrocarbons to reactive intermediates and. as a result, enhanced susceptibility to their cytotoxic effects. ; status,, and physiological status arc included : vf;' among "the endogenous factors affecting 2EI en zyme levels. The ketosis that results from star- ACKNOWLEDGMENT , vation, uncontrolled diabetes, or high fat intake can, by increasing 2E1 levels, potentiate liver 1 1___-L1_______.. _____________i /s/si w n sj s The authors wish to acknowledge support .,' from DHHS research grams AA08139 (JLR) and AA07842 (JML), .- 'V hepato-- ^toxicity,of-numerous halocarbonslTn addition,' : `` ^: halcnated hydrocarbons can induce their biqactivation`' again fresuliingr in increased Because'several.halogenatcd hydrocarbons, ' '*:i including CC1* and halothane, can act as suicide y inhibitors of 2El, halocarbon-mediated destruction of the enzyme could cause a marked decrease ; v,/r in the metabolism of other substrates. This could , result in either the reduction or potentiation of .' _ I? \ xenobiotic toxicity. In the case of environmental ; REFERENCES ,. , ... 1. Kadlubar, F. F. and Hammons, G. 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