Cytochrome P450, oxidative stress, and alcoholic liverinjury
The cytochrome P450 enzymes are a superfamily of heme-proteins that serve as terminal oxidases in the mixed-functionoxidase system for metabolizing various endogenous substrates,
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such as steroids and fatty acids, and xenobiotics, includingdrugs, toxins, and carcinogens [1]. Many different enzymesbelong to this P450 family; P450s are present in virtually allliving organisms. A systematic nomenclature system wasdeveloped for the P450 family which is based on the sequenceidentities of the various P450 enzymes [2,3]. The enzymes arenamed CYP for cytochrome P450, followed by an Arabicnumber denoting the family (more than 40% identity on theamino acid sequence level), a letter designating the subfamily(more than 55% identity), and finally an Arabic numeralrepresenting the individual gene in the subfamily. The P450scatalyze many different chemical reactions, including mono-oxygenation (insertion of an atom of oxygen into the substrate),peroxidation, reduction, dealkylation, epoxidation, and dehalo-genation [4–6]. Many different compounds of diverse structurescan be metabolized by P450 enzymes. A major function ofP450-catalyzed reactions is to convert a compound into a morepolar metabolite that can be easily excreted directly by theorganism or conjugated by phase II enzymes into more polarexcretable metabolites. With some compounds, e.g., carbontetrachloride or acetaminophen, metabolism by P450 can giverise to toxic metabolites which damage cells. For P450s tofunction catalytically, flavoprotein reductases such as NADPH-cytochrome P450 reductase, adrenodoxin, and adrenodoxinreductase, are necessary to transfer electrons from NADPH orNADH to reduce the heme from the ferric redox state to theferrous state. The latter is necessary to bind molecular oxygen toform the oxygenated P450 complex that catalyzes the diversechemical reactions mentioned above [7]. Cytochrome b5 mayalso play an important role in electron transfer to certain P450s.
It is important to recognize that oxygen activation by P450,necessary for the enzyme's catalytic function, can also result inthe production of reactive oxygen species (ROS). Smallamounts of the superoxide anion radical (O2
U−) can be producedfrom decay of the oxygenated P450 complex, whereashydrogen peroxide (H2O2) can form from either dismutationof O2
U− or decay of the peroxy P450 complex [8–10]. ROS havebeen implicated in many of the major diseases that plaguehumankind, including the toxicity of O2 itself; hyperbaric O2;ischemia–reperfusion injury; cardiovascular diseases; athero-sclerosis; carcinogenesis; diabetes; neurodegenerative diseases,including Parkinson disease and Alzheimer disease; toxicity ofheavy metals, e.g., iron; asbestos injury; radiation injury;vitamin deficiency; drug (e.g., redox cycling agents) toxicity;aging; inflammation; smoking toxicity; emphysema; andtoxicity of acute and chronic ethanol treatment [11–15]. ROScan be produced from many systems in cells, including themitochondrial respiratory chain [16]; the cytochrome P450s[10,17]; oxidative enzymes such as xanthine oxidase, aldehydeoxidase, cyclo-oxygenase, monoamine oxidase, and theNADPH oxidase complex [18,19]; and autoxidation of hemeproteins such as ferrohemoglobin or myoglobin or biochemicalssuch as catecholamines, quinones, or tetrahydrobiopterins. Inaddition to these cellular sources of ROS, environmentalsources of ROS include radiation, UV light, smoke, and certaindrugs which can redox cycle. ROS are toxic to cells becausethey can react with most cellular macromolecules, inactivating
enzymes or denaturing proteins and causing DNA damage suchas strand breaks, base removal, or base modifications, whichcan result in mutation, or peroxidation of lipids, which canresult in destruction of biological membranes and producereactive aldehydic products such as malondialdehyde or 4-hydroxynonenal [20,21]. A variety of enzymatic and non-enzymatic mechanisms have evolved to protect cells againstROS, including the superoxide dismutases, which remove O2
U−;catalase and the glutathione (GSH) peroxidase system, whichremove H2O2; glutathione transferases, which can removereactive intermediates and lipid aldehydes; metallothioneins,heme oxygenase, and thioredoxin, which remove various ROS;ceruloplasmin and ferritin, which help remove metals such asiron, which promote oxidative stress reactions; and non-enzymatic, low-molecular-weight antioxidants such as GSHitself, vitamin E, ascorbate (vitamin C), vitamin A, ubiquinone,uric acid, and bilirubin [22,23]. Oxidative stress or toxicity byROS reflects a balance between the rates of production of ROSand the rates of removal of ROS plus repair of damaged cellularmacromolecules. Whereas excess ROS can cause toxicity,macrophages and neutrophils contain an NADPH oxidasewhich produces ROS to destroy foreign organisms [24] and theenzyme myeloperoxidase catalyzes a reaction between H2O2
and chloride to produce the powerful oxidant hypochlorite(bleach) to help destroy foreign invaders. In addition, ROS atlow concentrations, especially H2O2, may be important in signaltransduction mechanisms in cells and thus be involved incellular physiology and metabolism [25].
The ability of acute and chronic ethanol treatment to increaseproduction of reactive oxygen species and enhance peroxidationof lipids, protein, and DNA has been demonstrated in a varietyof systems, cells, and species, including humans. Much hasbeen learned about alcohol metabolism, the various enzymesand pathways involved, and how alcohol, directly via itsmetabolism or indirectly via its solvent-like action affectingcellular membranes, impacts on cell function. Yet, despite thistremendous growth in understanding alcohol metabolism andactions, the mechanisms by which alcohol causes cell injury arestill not clear. A variety of leading mechanisms have beenbriefly summarized [13–15], and it is likely that many of themultimately converge as they reflect a spectrum of the organism'sresponse to the myriad of direct and indirect actions of alcohol.A major mechanism that is a focus of considerable research isthe role of lipid peroxidation and oxidative stress in alcoholtoxicity. Many pathways have been suggested to play a key rolein how ethanol induces “oxidative stress” (reviewed in [13–15]Again, many of these pathways are not exclusive of one anotherand it is likely that several, indeed many, systems contribute tothe ability of alcohol to induce a state of oxidative stress.
What is the evidence that ethanol-induced oxidative stressplays a role in cell injury? There are many studies which showthat administration of antioxidants or iron chelators or GSH-replenishing agents can prevent or ameliorate the toxic action ofethanol. The most convincing data that oxidative stresscontributes to alcohol-induced liver injury comes from thestudies using the intragastric infusion model of alcoholadministration. In these studies, alcohol-induced liver injury
was associated with enhanced lipid peroxidation, proteincarbonyl formation, formation of the 1-hydroxyethyl radical,formation of lipid radicals, and decreases in hepatic antioxidantdefense, especially GSH [26–30]. Replacement of polyunsatu-rated fat (required for lipid peroxidation to occur) with saturatedfat or medium-chain triglycerides in the diets fed to ratsintragastrically lowered or prevented the lipid peroxidation andthe alcohol-induced liver injury [29,30]. Thus, alcohol pluspolyunsaturated fat was required for the injury to occur.Addition of iron, known to generate ·OH and promote oxidativestress, to these diets exacerbated the liver injury [31].Importantly, addition of antioxidants such as vitamin E, ebselen,superoxide dismutase, and GSH precursors prevented thealcohol-induced liver injury [28] Because alcohol-inducedliver injury has been linked to oxidative stress, we investigatedthe effect of a compromised antioxidant defense system,copper–zinc superoxide dismutase (SOD1) deficiency, onalcohol-induced liver injury [32,33]. A rather moderate ethanolconsumption promoted oxidative stress and liver injury inSOD1-knockout mice, indicating that compromised antioxidantdefense promotes alcohol liver injury.
In addition to these in vivo studies, in vitro studies withhepatocytes also showed that ethanol can produce oxidativestress and hepatocyte toxicity. Studies with isolated hepatocytesfrom control rats or chronically ethanol-fed rats indicated thatethanol metabolism via alcohol dehydrogenase results in anincrease in ROS production, hepatocyte injury, and apoptosis,reactions blocked by antioxidants [34,35]. Studies in ourlaboratory with HepG2 cell lines expressing CYP2E1 showedthat addition of ethanol or polyunsaturated fatty acids or iron, ordepletion of GSH, resulted in cell toxicity, increased oxidativestress, and mitochondrial damage, reactions prevented byantioxidants [36] Recent reviews on the roles of oxidativestress in alcoholic liver disease can be found in [37,38]. BecauseCYP2E1 plays a role in ethanol-induced oxidant stress and is aminor pathway of ethanol oxidation, the biochemical andtoxicological properties of CYP2E1 will form the basis formuch of the remainder of this review.
CYP2E1 and the microsomal ethanol-oxidizing system
Alcohol dehydrogenase is the major enzyme pathway foroxidizing ethanol to acetaldehyde. The morphological observa-tions that chronic ethanol treatment causes proliferation of theliver smooth endoplasmic reticulum suggested that ethanol,similar to certain xenobiotics which are metabolized bycytochrome P450, may also be metabolized by P450 [39]. Amicrosomal ethanol-oxidizing system (MEOS) was character-ized by Lieber and associates and shown to be dependent onP450 [40]. The Km for ethanol oxidation by MEOS (about10 mM) was about an order of magnitude greater than the Km
for ethanol by alcohol dehydrogenase. Acetaldehyde is theproduct resulting from ethanol oxidation by MEOS, and it isclear that MEOS represents a minor pathway of ethanoloxidation, probably accounting for less than 10% of the livercapacity to oxidize ethanol [41]. Importantly, the activity ofMEOS is enhanced after chronic ethanol treatment, partly due
to an increased total content of P450 and partly due toinduction of CYP2E1, a member of the P450 family with highcatalytic activity with ethanol [40]. Induction of MEOS mayplay an important role in the metabolic tolerance found afterchronic ethanol treatment, i.e., the increased capacity to oxidizeethanol. Although there was early controversy over the natureof MEOS, the purification of an ethanol-inducible form ofP450 from rabbit liver microsomes firmly established the roleof P450 in MEOS [42]. Ethanol-inducible P450s have beenisolated from many species and although several P450s may beinduced by ethanol, the major inducible P450 is now referredto as CYP2E1.
CYP2E1 substrates
CYP2E1 metabolizes a variety of small, hydrophobicsubstrates and drugs (reviewed in [40,43–46]. Possiblephysiological substrates are acetone and fatty acids such aslinoleic and arachidonic acid [47]. From a toxicological point ofview, interest in CYP2E1 revolves around the ability of thisenzyme to metabolize and activate many toxicologicallyimportant compounds such as ethanol, carbon tetrachloride,acetaminophen, benzene, halothane, and many other haloge-nated substrates. Procarcinogens including nitrosamines andazo compounds are effective substrates for CYP2E1, e.g.,CYP2E1 is a low Km dimethylnitrosamine demethylase [48].Toxicity of the above compounds is enhanced after induction ofCYP2E1, e.g., by ethanol treatment, and toxicity is reduced byinhibitors of CYP2E1 or in CYP2E1-knockout mice [49]. Ofthe substrates, chlorzoxazone is of special value, as itshydroxylated product can readily be assayed in the blood andthe ratio of 6-hydroxychlorzoxazone/chlorzoxazone is widelyused to assess the approximate levels of CYP2E1 in humans,including alcoholics [50].
Molecular oxygen itself is likely to be a most importantsubstrate for CYP2E1. CYP2E1, relative to several other P450enzymes, displays high NADPH oxidase activity, as it seems tobe poorly coupled with NADPH-cytochrome P450 reductase[51,52]. CYP2E1 was the most efficient P450 enzyme in theinitiation of NADPH-dependent lipid peroxidation in recon-stituted membranes among five different P450 forms investi-gated. Furthermore, anti-CYP2E1 IgG inhibited microsomalNADPH oxidase activity and microsomal lipid peroxidationdependent on P450, but not lipid peroxidation initiated by theaction of NADPH-cytochrome P450 reductase [52]. In ourlaboratory, we found that microsomes isolated from rats fedethanol chronically were about two- to threefold more reactivein generating superoxide radical and H2O2 and, in the presenceof ferric complexes, in generating hydroxyl radical andundergoing lipid peroxidation compared to microsomes frompair-fed controls [53–56]. CYP2E1 levels were elevated aboutthree- to fivefold in the liver microsomes after rats were fed theLieber–DeCarli diet for 4 weeks. The enhanced effectiveness ofmicrosomes isolated from the ethanol-fed rats was prevented byaddition of chemical inhibitors of CYP2E1 and by polyclonalantibody raised against CYP2E1, confirming that the increasedactivity in these microsomes was due to CYP2E1.
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CYP2E1 is a minor pathway of ethanol oxidation as itcatalyzes the two-electron oxidation of ethanol to acetaldehyde.Interestingly, acetaldehyde is also a substrate for CYP2E1 and isoxidized to acetate; thus CYP2E1 can, at least theoretically,catalyze the oxidation of ethanol to acetate [57]. However, thisoxidation is likely to be negligible in the presence of ethanol,the substrate which generates acetaldehyde [58]. CYP2E1 canalso promote the one-electron oxidation of ethanol to the 1-hydroxyethyl radical. Detection of the 1-hydroxyethyl radical inthe bile after administration of ethanol to rodents has been amost valuable assay for determining ethanol-induced radicalformation and oxidant stress in vivo [26,59].
Mitochondrial CYP2E1
CYP2E1 is mainly found in the liver but significant amountsare also found in most organs, including the brain [60]. CYP2E1is expressed mainly in the hepatocytes of the liver; however,significant amounts are also found in the Kupffer cells [61] andhepatocyte and Kupffer cell CYP2E1 is inducible, e.g., byethanol. CYP2E1, like other xenobiotic-metabolizing P450s, ismainly located in the membrane of the endoplasmic reticulum(ER). CYP2E1 has also been detected in other cellularcompartments such as the plasma membrane [62–64].CYP2E1 located at the plasma membrane has been suggestedto play a role in the immune-mediated hepatotoxicity observedin patients suffering from drug toxicity and alcoholic liverdisease [65–67]. CYP2E1 was shown to be transported out ofthe ER to the Golgi apparatus, with subsequent transfer to theplasma membrane [68,69].
Ingelman-Sundberg and co-workers, and Avadhani and co-workers, have shown that CYP2E1 is also present in themitochondria [70–75]. Essentially two forms of CYP2E1 arepresent in the mitochondria, a highly phosphorylated formmediated via cAMP-dependent protein kinase A and ashortened 40-kDa amino-terminal-truncated form, which lacksthe N-terminal amino acids and which can be further NH2-terminally truncated to produce a mature mitochondrial form ofCYP2E1 lacking about 100 amino acids. The phosphorylationand amino-terminal truncation are hypothesized to causeconformational changes and altered interactions with molecularchaperones and signal recognition particles and direct theCYP2E1 to the mitochondria. The mitochondrial CYP2E1 iscatalytically active with typical substrates but requires, as do theother mitochondrial P450s, adrenodoxin and adrenodoxinreductase (and NADPH) as electron donors [70,73]. It is notclear what regulates either the phosphorylation or the amino-terminal truncation which directs CYP2E1 to the mitochondria.Importantly, the mitochondria isolated from rat liver and highlypurified, and essentially devoid of endoplasmic reticulumcontamination, contained CYP2E1, indicating the in vivopresence of mitochondrial CYP2E1 [70,71,73]. Robin et al.[73] showed that pyrazole treatment elevated not onlymicrosomal CYP2E1, but also mitochondrial CYP2E1. Themitochondrial CYP2E1 was present at about 30% of the level ofthe microsomal CYP2E1 under basal conditions and at 40% ofthe level of the microsomal CYP2E1 after pyrazole treatment
[73]. In a similar manner, streptozotocin-induced diabeteselevated microsomal CYP2E1 two- to threefold and mitochon-drial CYP2E1 five- to sixfold [75]; mitochondrial CYP2E1protein and catalytic activity was 25 to 35% that of microsomalCYP2E1 after treatment with streptozotocin. Raza and John[76] recently reported that 4-hydroxynonenal increasedCYP2E1 activity in the mitochondria and postmitochondrialsupernatant of PC12 cells, in association with elevatedmitochondrial oxidative stress.
To evaluate the functional consequences associated withexpression of mitochondrial CYP2E1, we established a HepG2cell line which expresses CYP2E1 in the mitochondria [77]. ACYP2E1 expression vector lacking the coding sequences foramino acids 2–34 was generated, cloned into a pCI-neoexpression vector, and transfected into HepG2 cells, and stablecell lines were established by selection for G418 resistance.Western blot analysis of whole-cell extracts and of isolatedmitochondria, and immunofluorescence of permeabilized cells,showed the presence of CYP2E1 in the mitochondrial fractionof mE10 and mE27 cells (HepG2 cells transfected with theamino-terminal-depleted CYP2E1), but not in pCI vector-transfected HepG2 cells or E47 HepG2 cells which expressCYP2E1 in the endoplasmic reticulum. Treatment with 0.1 mMBSO for 48 h to lower GSH levels caused a striking loss of cellviability in mE10 and mE27 cells, which contain mtCYP2E1,but not in the pCI-neo cells. Toxicity could be prevented byantioxidants such as glutathione ethyl ester and Trolox,suggesting that enhanced oxidant stress plays a role in thetoxicity. Indeed, ROS production (DCF fluorescence) waselevated after BSO addition to the mE10 and mE27 cells. Therewas an increase in 3-nitrotyrosine protein adducts and 4-hydroxynonenal protein adducts in the mE10 and mE27 cellstreated with BSO compared to plasmid vector controls. Themitochondrial membrane potential slightly declined in BSO-treated pCI-neo cells but dramatically declined in the mE10 andmE27 cells. This decline in MMP was prevented by cyclosporinA, and the BSO-induced loss of cell viability in the mE10 andmE27 cells was prevented by cyclosporin A. Importantly,ethanol was shown to elevate the levels of mitochondrialCYP2E1 in addition to the well-known increase in microsomalCYP2E1 [78]. It is interesting to speculate that damage tomitochondrial function and membrane potential produced bymitochondrial CYP2E1 may be an early event in liver cell injuryand that mitochondrial CYP2E1 may contribute to thebiochemical and toxicological effects which were previouslyascribed to CYP2E1 in the endoplasmic reticulum. However, atpresent it is not obvious how effects contributed by mitochon-drial CYP2E1 versus microsomal CYP2E1 in vivo or in primaryhepatocytes can be distinguished from each other, e.g., the lackof specific inhibitors.
Induction and regulation of CYP2E1
Many of the substrates for CYP2E1 can induce their ownmetabolism. This was initially observed with ethanol, which is asubstrate for CYP2E1 and elevates CYP2E1 levels [39,40]. Infact, these two properties explain the ability of ethanol to inhibit
the metabolism of certain substrates when it is present, i.e.,ethanol and the substrate compete for oxidation by CYP2E1,and to increase the metabolism of substrates when it is no longerpresent to compete, but had already elevated the levels of theCYP2E1 catalyst. Ethanol can be oxidized by other P450s inaddition to CYP2E1, notably CYPs 3A and 1A, and ethanoltreatment can elevate the levels of these CYPs [79,80]. Avarietyof heterocyclic compounds such as imidazole, pyrazole, 4-methylpyrazole, thiazole, and isoniazid have been shown toelevate CYP2E1 levels, as do solvents such as dimethylsulfoxide, various alcohols, benzene, and acetone [81–83].These low-molecular-weight compounds have been used invivo or in vitro to elevate or help prevent the loss of CYP2E1under tissue culture conditions and their mode of mechanismwill be discussed below.
CYP2E1 can also be induced under a variety of metabolic ornutritional conditions. For example, CYP2E1 levels wereelevated in chronically obese, overfed rats and in rats fed ahigh-fat diet [84]. Somewhat paradoxically, in rats levels ofCYP2E1 were also increased by fasting and by prolongedstarvation [85,86]. Diabetes has been reported to increase theexpression of CYP2E1 mRNA and protein levels several fold[87]. This may be related to actions of insulin whichdownregulated CYP2E1 expression at the posttranscriptionallevel in a rat hepatoma cell line [88,89] and in rat hepatocyteculture [90]. CYP2E1 levels were elevated in liver and kidneymicrosomes of rats treated with streptozotocin. CYP2E1induction in diabetes may be associated with the elevatedproduction of ketone bodies [91]. The carbohydrate content ofthe diet influences CYP2E1 levels, as a low-carbohydrate dietincreased the extent of induction of MEOS by ethanol [92] andhigh-fat/low-carbohydrate diets resulted in the highest levels ofCYP2E1 induced by ethanol [93]. In this respect, it isinteresting that alcohol-induced liver damage is magnified indiets with very low levels of carbohydrate and high levels of fat[94].
In addition to insulin, other hormones can affect CYP2E1levels. Hypophysectomy and triiodothyronine increaseCYP2E1 protein and mRNA levels in contrast to insulin,which lowers them [89,95]. In primary rat hepatocyte cultures,glucagon lowered CYP2E1 levels by accelerating the turnoverof the CYP2E1 protein by a cyclic AMP-dependent process[96]. Testosterone increased renal but not hepatic CYP2E1levels [97].
Considerable data have been reported elucidating themolecular mechanism of CYP2E1 regulation by exogenouscompounds as well as during pathophysiological conditions.CYP2E1 is regulated by multiple, distinct regulatory mechan-isms [83,98,99]. The CYP2E1 gene is under transcriptionalcontrol during development. In rats, immediately after birth, it isactivated and is maximally transcribed within the first week.Upon fasting or induced diabetes, the mRNA for CYP2E1 isincreased several fold due to posttranscriptional mRNAstabilization [100]. After administration of ethanol, acetone,or pyrazole to rats, Song et al. found that CYP2E1 mRNA levelsdid not increase [81]. The mechanism of induction was,therefore, suggested to be at the level of protein degradation.
CYP2E1 is not transcriptionally activated by an acute bolusdose or chronic administration of ethanol, acetone, or otherexogenous inducing agents. Although elevation of CYP2E1mRNA levels has been reported [101], most investigators havefound little induction or a slight reduction of CYP2E1 mRNAlevels after ethanol administration [81,83,102]. From in vivodata of CYP2E1 turnover in rats chronically treated withacetone [103] and in vitro hepatocyte culture systems [104,105],exogenous CYP2E1 inducers such as acetone, ethanol,imidazole, and 4-methylpyrazole (4-MP) were shown toincrease CYP2E1 by protein stabilization. Roberts et al.[106,107] reported that ethanol increases CYP2E1 by proteinstabilization. This phenomenon was observed not only in theliver but also in extrahepatic tissues such as kidney, brain, andintestine. In addition, CYP2E1 protein stabilization seemeddependent on blood ethanol or acetone concentration. Further-more, a turnover study, using in vivo radiolabeling of CYP2E1with [14C]NaHCO3 and immunopurification, demonstrated thatethanol treatment abolished the rapid phase of CYP2E1degradation, whereas biphasic degradation of CYP2E1 wasobserved in the control animals [108]. Pyrazole and 4-MPelevated liver and kidney CYP2E1 immunoreactive protein andcatalytic activity in the absence of an increase in CYP2E1mRNA levels [109–111]. In isolated rat hepatocyte cultures,CYP2E1 mRNA and protein levels and CYP2E1 catalyticactivity rapidly declined with time in culture. Addition ofpyrazole or 4-MP slowed the decline in CYP2E1 protein andactivity, without any effect on CYP2E1 mRNA levels [105].Similarly, McGhee et al. [112] reported the half-life of CYP2E1in a hepatoma cell line to be 1.8 h in the absence of ethanol and45 h in the presence of ethanol. It is clear that a major level ofregulation of CYP2E1 formation seems to be posttranscriptionalas various substrates and ligands increase the content ofCYP2E1 by protection against rapid degradation by intracel-lular proteolytic pathways.
What are the proteolytic systems responsible for CYP2E1turnover and prevented from their action on CYP2E1 byethanol? Roberts [113] provided evidence for a role of theproteasome in the degradation of several cytochrome P450s,including CYP2E1. Huan et al. [114] showed that in a HeLa cellline, inhibitors of the proteasome decreased the degradation ofCYP2E1 and CYP2B1. They found that ubiquitination ofCYP2E1 was not required for its degradation by the protea-some. However, Banerjee et al. [115], using molecular models,predicted a cytosolic domain of CYP2E1 which would functionas a putative ubiquitination–target/substrate interaction struc-ture. An antibody recognizing this domain (amino acids 317–340) quenched CYP2E1 ubiquitination and inhibited CYP2E1catalytic activity. They suggested that substrate binding shieldsthe CYP2E1 protein from turnover by blocking the ubiquitina-tion domain. Morishima et al. [116] reported that a HSP90inhibitor promoted CYP2E1 degradation by the proteasome.They found that purified bacterially expressed truncatedCYP2E1 (Δ3–29) is ubiquitylated by the E3 ubiquitin ligaseCHIP and concluded that CYP2E1 is a HSP90 “client” protein.In contrast, Huan et al. [114] found that three HSP90 inhibitorshad no effect on CYP2E1 turnover. We found that in an in vitro
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reconstituted system containing microsomes or cytosol orpurified 20S proteasome, geldanamycin, an inhibitor ofHSP90, decreased CYP2E1 degradation and this suggestedthat HSP90 helps present oxidized CYP2E1 to the proteasomefor degradation [117]. The proteasome complex was importantin the degradation of CYP2E1 in HepG2 cells, as proteasomeinhibitors proved to be effective in preventing CYP2E1degradation. Importantly, Bardag-Gorce et al. [118] showedthat the rapid loss of CYP2E1, which occurs in vivo after theethanol inducer is withdrawn, could be blocked by theproteasome inhibitor PS-341, thus establishing the critical roleof the proteasome in regulating CYP2E1 turnover in vivo.
CYP2E1 and alcohol-induced liver injury
Because CYP2E1 can generate ROS during its catalyticcircle, and its levels are elevated by chronic treatment withethanol, CYP2E1 has been suggested to be a major contributorto ethanol-induced oxidant stress and to ethanol-induced liverinjury. Initial suggestions of a role for CYP2E1 in alcoholicliver injury arose from studies with the intragastric model ofethanol feeding in which prominent induction of CYP2E1occurs and in which significant liver injury occurs [29–31]. Inthese models, large increases in microsomal lipid peroxidationhave been observed and the ethanol-induced liver pathology hasbeen shown to correlate with CYP2E1 levels and elevated lipidperoxidation [29,30,119,120]. Experimentally, a decrease inCYP2E1 induction was found to be associated with a reductionin alcohol-induced liver injury [121,122]. CYP2E1 inhibitorssuch as diallyl sulfide [123], phenethyl isothiocyanate[124,125], and chlormethiazole [126] blocked the lipidperoxidation and ameliorated the pathologic changes inethanol-fed rats. Polyenylphosphatidylcholine, another com-pound exerting anti-CYP2E1 properties [127], was effective inopposing alcohol-induced oxidative stress [40]. A strongassociation between dietary carbohydrate, enhanced CYP2E1induction, and hepatic necrosis was observed. No liver injurywas found if carbohydrate levels were elevated [128]. It wasconcluded that diet is an important factor in toxicity mediatedby ethanol because of modulation of the levels of CYP2E1[128]. Ethanol consumption in oral liquid diets does not causesignificant liver injury. However, micro- and macrovesicularsteatosis, occasional inflammatory foci, and a threefold increasein transaminase levels were observed in a nutritionally adequateethanol-containing liquid diet with a carbohydrate content of5.5%; no changes were found if the level of carbohydrate waselevated to 11% [94,129]. Thus dietary and nutritional factorsplay a key role in the toxic actions of ethanol on the liver, in partdue to modulation of the levels of CYP2E1. Recently, aCYP2E1 transgenic mouse model was developed that over-expresses CYP2E1. When treated with ethanol, the CYP2E1-overexpressing mice displayed higher transaminase levels andhistological features of liver injury compared with the controlmice [130]. We developed an adenoviral vector which expresseshuman CYP2E1 and showed that infection of HepG2 cells withthis adenovirus potentiated acetaminophen toxicity compared toHepG2 cells infected with a LacZ-expressing adenovirus [131].
Administration of CYP2E1 adenovirus in vivo to miceproduced significant liver injury compared to the LacZ-infectedmice as reflected by histopathology, markers of oxidative stress,and elevated transaminase levels [132].
On the other hand, studies by Thurman and colleagues havepresented powerful support for a role for endotoxin, activationof Kupffer cells, and cytokines such as TNFα in the alcohol-induced liver injury found in the intragastric infusion model[133,134]. They suggested that CYP2E1 may not play a role inalcohol liver injury based upon studies with gadoliniumchloride or CYP2E1-knockout mice [135,136]. FemaleCYP2E1 wild-type mice or knockout mice were given a high-fat liquid diet intragastrically with either ethanol or isocaloricmaltose–dextrin for 4 weeks. Mice given ethanol had elevatedtransaminases, mild steatosis, and slight inflammation andnecrosis with no differences in pathology between the wild-typeand the knockouts [135]. However, Bardag-Gorce et al. [137],using the same model, reported that ethanol-induced oxidativestress and inactivation of the proteasome complex werecompletely prevented in these mice. They concluded thatCYP2E1 induction by chronic ethanol treatment was respon-sible for the decrease in proteasome activity and accumulationof oxidized proteins in the liver. They speculated the pathologyfound in the CYP2E1 knockouts by Kono et al. [135] may bedue to upregulation of NADPH-cytochrome P450 reductase andother CYPs such as CYP3A and 4A [137] (see below). As totheir observations with gadolinium chloride, others havereported that gadolinium chloride does indeed decrease levelsof several P450 enzymes, including CYP2E1, and lowered theinduction of CYP2E1 by ethanol. Moreover, Leclercq et al.[138], using the same knockout mice, observed that other CYPs,notably CYP4A10 and CYP4A14, were upregulated in theCYP2E1-knockout but not the wild-type mice; these CYPswere, like CYP2E1, active generators of ROS and catalysts oflipid peroxidation and, in the absence of CYP2E1, served asalternative initiators of oxidative stress. Bradford et al. [139],using CYP2E1 and NADPH oxidase-knockout mice, concludedthat CYP2E1 was required for ethanol induction of oxidativestress to DNA, whereas NADPH oxidase was required forethanol-induced liver injury. Clearly, further studies arenecessary to resolve the above discrepancies. As mentionedearlier, it is likely that several mechanisms contribute to alcohol-induced liver injury and that ethanol-induced oxidant stress islikely to arise from several sources, including CYP2E1,mitochondria, and activated Kupffer cells.
Cell lines expressing CYP2E1
To characterize the biochemical and toxicological propertiesof CYP2E1, several investigators have developed cell lines toexpress CYP2E1. The first cell line to be developed was tointroduce human CYP2E1 into NIH 3T3 mouse fibroblasts viaretroviral infection followed by selection via G418 resistance[140]. Southern blot analysis showed the viral DNA wasintegrated into the cellular DNA. The transduced CYP2E1 wascatalytically active, oxidizing ethoxycoumarin to 7-hydroxy-coumarin and forming labeled covalent DNA adducts after
incubation with [14C]nitrosodimethylamine [140]. The cyto-toxic effect of N-nitrosodimethylamine was studied in theP450-expressing human fibroblast cell line GM2E1, whichexpresses the rat CYP2E1 [141]. N-Nitrosomethylamine wastoxic to the cells expressing CYP2E1; toxicity was decreased byCYP2E1 inhibitors and was partially prevented by antioxidants[141]. Toxicity was apoptotic in nature and could be preventedby caspase inhibitors [142]. A V79 Chinese hamster cell lineexpressing human CYP2E1 was used in toxicological studiesinvolving CYP2E1-mediated activation of N-nitrosodimethyla-mine and p-nitrophenol and for mutagenicity studies with theformer substrate [143]. Human CYP2E1 was also expressed inthe rat adrenal pheochromocytoma cell line PC12 [144].CYP2E1 metabolism of several different substrates wascharacterized in these cells; levels of enzyme activities wereabout 10% that of human liver microsomes, similar to what ourlab found with HepG2 cells expressing CYP2E1 [145]. ThePC12 cells were shown to metabolize acetaminophen, andactivation of this protoxin caused a loss of viability to the cells[144]. Acetaminophen toxicity was also characterized in ahuman hepatoma cell line, HLE, expressing human CYP2E1[146]. Treating these cells with buthionine sulfoximine to lowerGSH levels also produced a decrease in cell viability, whichcould be inhibited by ethanol or vitamin E. This cell line hasbeen used to examine changes of heme metabolism via assaysof δ-aminoleulinic acid synthase and heme oxygenase-1 (HO-1), the rate-limiting enzymes in heme synthesis and hemebreakdown [147]. Both enzymes were upregulated in the HLEcells expressing CYP2E1, perhaps due to the demand forincreased heme synthesis for holoCYP2E1 formation andperhaps increased availability of heme to induce hemeoxygenase. Our lab has also observed an increase in hemeoxygenase-1 mRNA, protein, and activity in HepG2 cellsexpressing CYP2E1, perhaps an increase in response toCYP2E1-generated oxidant stress [148]. Huan and Koop[149] established a tetracycline-controlled rabbit CYP2E1-expressing system in HeLa cells in culture. This system wasused to evaluate turnover of the rabbit CYP2E1, which wasrapid, with a half-life of 3.9 h in the absence of a stabilizingsubstrate or ligand. Addition of the latter, 4-methylpyrazole,decreased the degradation of CYP2E1. We observed similarresults in HepG2 cells expressing CYP2E1, as the half-life ofhuman CYP2E1 was about 3–6 h in the absence of substrate orligand and was elevated in the presence of various substratesand ligands [150]. The CYP2E1 half-life was also elevated byan inhibitor of the proteasome complex. Recently, a comparisonof mouse, rat, and human CYP2E1 activities in V79 Chinesehamster cell lines was made to study possible speciesdifferences in toxicity and metabolism of CYP2E1 substrates[151].
A HepG2 cell model expressing human CYP2E1 wasestablished by Patten et al. [152] using the vaccinia virusexpression system. The oxidation of several typical substrates ofCYP2E1 was evaluated in these cells and the ability ofcytochrome b5 to elevate CYP2E1 activity was shown. Asbriefly mentioned above, an approach that our laboratory hasutilized to try to understand the basic effects and actions of
CYP2E1 was to establish cell lines that constitutively expresshuman CYP2E1. HepG2 cell lines, which overexpressCYP2E1, were established either by retroviral infectionmethods (MV2E1-9 cells or E9 cells) or by plasmid transfectionmethods (E47 cells) [145,153]. Results utilizing E9 or E47 cellsto study CYP2E1-generated oxidant stress have been summar-ized in recent reviews [154–156]. We have characterized thetoxicity of ethanol, polyunsaturated fatty acids (PUFA) such asarachidonic acid (AA), and iron in E9 and E47 cells.Concentrations of ethanol or AA or iron which were toxic tothe CYP2E1-expressing cells had no effect on control HepG2cells not expressing CYP2E1 or on HepG2 cells expressing adifferent P450, CYP3A4 (3A4 cells). Toxicity to CYP2E1-expressing cells was found when GSH was depleted bytreatment with l-buthionine sulfoximine (BSO) [157]. Inhibitorsof CYP2E1 prevented the toxicity of the above treatments.Antioxidants such as vitamin E, Trolox, and ascorbate alsoprevented toxicity found when the CYP2E1-expressing E9HepG2 cells were treated with either ethanol or AA. The abovetreatment of CYP2E1-expressing cells with ethanol, AA, iron,or BSO resulted in an increase in oxidative stress to the cells asreflected by increased lipid peroxidation and enhanceddichlorofluorescein fluorescence. Low concentrations of ironand AA that are not cytotoxic by themselves can act as primingor sensitizing factors for CYP2E1-dependent loss of viability inHepG2 cells or rat hepatocytes. This synergistic toxicity wasassociated with elevated lipid peroxidation and could beprevented by antioxidants which prevent lipid peroxidation.Damage to mitochondria by CYP2E1-derived oxidants seems tobe an early event in the overall pathway of cellular injury.
Adaptation to oxidant stimuli is critical for short- and long-term survival of cells exposed to oxidative stress. We found thatthe levels of GSH and several antioxidant enzymes, such asglutathione S-transferase (GST), catalase, and heme oxygenase-1, were upregulated in the CYP2E1-expressing cells. Thisupregulation was prevented by antioxidants, suggesting thatROS generated by CYP2E1 were responsible for the transcrip-tional activation of these antioxidant genes. Because of thisactivation of antioxidant genes, the CYP2E1-expressing cellswere less sensitive to toxicity of H2O2, menadione, or 4-hydroxynonenal (HNE) than control cells. We believe that theupregulation of these antioxidant genes reflects an adaptivemechanism to remove CYP2E1-derived oxidants. Recentexperiments suggested that Nrf-2 plays a key role in theadaptive response against the increased oxidative stress causedby CYP2E1 ([158], reviewed in [159]).
Aworking model of CYP2E1-dependent oxidative stress andtoxicity is shown in Fig. 1. Ethanol increases levels of CYP2E1,largely by a posttranscriptional mechanism involving enzymestabilization against degradation. CYP2E1, a loosely coupledenzyme, generates ROS such as O2
U− and H2O2 during itscatalytic cycle. In the presence of iron, which is increased afterethanol treatment, more powerful oxidants including
UOH, ferryl
species, and 1-hydroxyethyl radical are produced. Initially, theliver cells respond to the CYP2E1-related oxidative stress bytranscriptionally inducing various antioxidant enzymes via theirantioxidant response elements. Ultimately, these protective
Fig. 1. Working model of CYP2E1-dependent oxidative stress and cytotoxicity.Please see the text for discussion.
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mechanisms are overwhelmed and the cells become sensitive tothe CYP2E1-generated oxidants. These various oxidants canpromote toxicity by protein oxidation and enzyme inactivation,oxidative damage to the DNA, and disturbing cell membranesvia lipid peroxidation and production of reactive lipid aldehydes,such as malondialdehyde (MDA) and 4-hydroxynonenal.Mitochondria seem to be among the critical cellular organellesdamaged by CYP2E1-derived oxidants. A decrease in ΔΨm,likely due to the mitochondrial membrane permeability transi-tion, causes the release of proapoptotic factors, resulting inapoptosis. A decrease in ATP levels will cause necrosis. SomeCYP2E1-derived ROS, such as H2O2, LOOH, and HNE, arediffusible and may exit hepatocytes and enter other liver celltypes such as stellate cells and stimulate these cells to producecollagen and elicit a fibrotic response [160,161]. We believe thatthe linkage between CYP2E1-derived oxidative stress, mito-chondrial injury, and GSH homeostasis contributes to the toxicactions of ethanol on the liver.
Other investigators have utilized E9 and E47 HepG2 cellsexpressing CYP2E1 in a variety of studies, including evaluatingthe effects of ethanol and acetaldehyde on activation of thetranscriptional factors AP-1 and NF-κB [162], proteomicstudies on ethanol-induced oxidation of mitochondrial andcytosolic proteins [163,164], and studies on ethanol-inducedinhibition of the proteasome and cytokeratin aggresomeformation [165], comparison of gene expression patternsinduced by alcohol in vivo and in vitro [166], CYP2E1-hepatitis C virus [167] or hepatitis B virus [168] interactions,acetaminophen alterations of the microsomal ryanodine calciumchannel [169], fatty acid ethyl ester toxicity [170], and ethanolpotentiation of TNFα cytotoxicity [171] and the role of p38MAPK pathways in ethanol plus TNFα toxicity [172]. A ratherinteresting HepG2 cell culture model in which CYP2E1 andalcohol dehydrogenase are both expressed has been effectivelyutilized to study ethanol–CYP2E1–proteasome interactions,interferon-γ induction of the proteasome, and interferon-γsignaling and antigen processing [173–175]. The use of thesecombined cell lines as a model of ethanol-elicited cytotoxicityhas recently been reported [176]. Thus, CYP2E1 biochemistry,
oxidant stress, and toxicology have been extensively studied ina variety of stable cell lines.
The CYP2E1-knockout mouse
CYP2E1-knockout mice were developed by Gonzalez andcolleagues to determine the role of CYP2E1 in xenobioticmetabolism and toxicity [49,177,178]. The development of theCYP2E1-knockout mouse has been of great value in establish-ing the role of CYP2E1 in the metabolism and toxicity ofvarious hepatotoxins. For example, there was no liver pathologyor elevation of transaminases induced by CCl4 in CYP2E1-knockout mice compared to wild-type mice, leading to theconclusion that CYP2E1 is the major factor in CCl4hepatotoxicity [179]. Blood acetone levels were elevated 2.5–4 times in wild-type mice after 48 h fasting but elevated 28-foldin the CYP2E1 knockout mice, leading to the conclusion thatCYP2E1 plays a critical role in catabolism of acetone afterfasting [180]. Formation of benzene metabolites such ashydroquinone, catechol, and phenol were lowered more than90% with microsomes from CYP2E1-knockout mice comparedto microsomes from controls [181]. The CYP2E1-knockoutmice have been used to validate the important role of CYP2E1in the metabolism of thioacetamide, trichloroethylene, acrylo-nitrile, and urethane [182–186] The half-life for urethane was0.8 h in wild-type mice expressing CYP2E1 and 22 h inCYP2E1-knockout mice [186]. Interestingly, no difference inthe oxidation of styrene to styrene oxide was observed betweenmicrosomes from wild-type mice and from knockout mice[187]. Because the styrene metabolite styrene oxide wascomparably toxic in wild-type and knockout mice, thedecreased sensitivity of the CYP2E1-knockout mice to styrenelikely should be due to decreased bioactivation of styrene tostyrene oxide. Yet no differences in styrene metabolism werefound, disconnecting the metabolism from the toxicity forunknown reasons [187]. The CYP2E1-knockout mice havebeen used to validate that hydroxylation of p-nitrophenol maybe used as a specific probe for CYP2E1 [188].
The metabolism of acetaminophen has been widely studied.CYP2E1-knockout mice were highly resistant to liver toxicitycompared to wild-type mice treated with acetaminophen [49].Mice lacking both CYP2E1 and 1A2 were almost completelyresistant to acetaminophen toxicity [189]. The combination ofethanol plus isopentanol caused an increase in acetaminophenhepatotoxicity in CYP2E1-knockout mice that was sensitive tothe CYP3A inhibitor triacetyloleandomycin, leading to thesuggestion that both CYP2E1 and CYP3A contribute toacetaminophen toxicity in ethanol plus isopentanol-treatedmice [190]. Recently, a CYP2E1-humanized transgenic mousemodel that expresses functional and inducible human CYP2E1was described [191]. Comparisons between CYP2E1-huma-nized mice, CYP2E1-knockout mice, and wild-type mice willallow a determination of whether the actions of humanCYP2E1 are similar to those of mouse CYP2E1 in vivo.Indeed, the CYP2E1-humanized mouse model was success-fully used to characterize acetaminophen toxicity by humanCYP2E1 [191].
With respect to the role of CYP2E1 in alcohol-induced liverinjury, as discussed above, Bardag-Gorce et al. [137] reportedthat ethanol-induced oxidative stress and inactivation of theproteasome complex were completely prevented in CYP2E1-knockout mice. They concluded that CYP2E1 induction bychronic ethanol treatment was responsible for the decrease inproteasome activity and accumulation of oxidized proteins inthe liver. In a very interesting study, Bradford et al. [139] foundthat ethanol treatment for 4 weeks led to an increase in oxidativeDNA damage and induction of expression of basic excisionDNA repair genes in wild-type mice but not in CYP2E1-knockout mice. The increase in DNA repair gene expression inwild-type mice was abolished by treatment with a P450inhibitor. The induction and the DNA damage induced byethanol were the same in wild-type mice and NADPH oxidase-deficient mice. The authors concluded that CYP2E1 but notNADPH oxidase is required for the ethanol induction ofoxidative stress to DNA and thus CYP2E1 may play a key rolein ethanol-associated hepatocarcinogenesis [139]. On the otherhand, as mentioned above, studies by Thurman and colleaguessuggest that CYP2E1 may not play a role in alcohol-inducedliver injury [135]. Instead, their studies have presented powerfulsupport for a role for endotoxin (lipopolysaccharide, or LPS),activation of Kupffer cells, and cytokines such as TNFα in thealcohol-induced liver injury found with the intragastric infusionmodel.
LPS/TNFα–CYP2E1 interactions
Abnormal cytokine metabolism is a major feature ofalcoholic liver disease as described in many review articles[192–195]. Rats chronically fed ethanol were more sensitive tothe hepatotoxic effects of administration of LPS and had higherplasma levels of TNFα than control rats [196–198]. In theintragastric model of chronic ethanol administration, thedevelopment of liver injury coincided with an increase inTNFα, associated with an increase in serum LPS [195,197–199]. The pioneering studies of Thurman and collaboratorsshowed that anti-TNFα antibody prevented alcohol liver injuryin rats [200], and mice lacking the TNFR1 receptor did notdevelop alcohol liver injury [133]. Taken as a whole, these andother studies clearly implicate TNFα as a major risk factor forthe development of alcoholic liver injury. One complication inthis central role for TNFα is that hepatocytes are normallyresistant to TNFα-induced toxicity. This led to the hypothesisthat in addition to elevating TNFα, alcohol somehow sensitizesor primes the liver to become susceptible to TNFα [201,202].Known factors which sensitize the liver to TNFα are inhibitorsof mRNA or protein synthesis, which likely prevent thesynthesis of protective factors; inhibition of NF-κB activationto lower synthesis of such protective factors; depletion of GSH,especially mitochondrial GSH; lowering of S-adenosylmethio-nine (SAM) coupled to elevation of S-adenosylhomocysteine(SAH), i.e., a decline in the SAM/SAH ratio; or inhibition of theproteasome [203–210]. Of major relevance to this review is thework by Hoek and collaborators showing that combinedtreatment with ethanol and TNFα is more toxic to hepatocytes
and HepG2 E47 cells, which express high levels of CYP2E1,than control hepatocytes with lower levels of CYP2E1 orHepG2 C34 cells, which do not express CYP2E1 [171]. Theethanol sensitization of TNFα toxicity in E47 cells andhepatocytes from chronically ethanol-fed rats also dependedon p38 MAPK signaling because SB203580, a p38 MAPKinhibitor, prevented this enhanced toxicity [172] In a RALAhepatocyte cell line model, Czaja and collaborators showed thathepatocytes with increased expression of CYP2E1 weresensitized to TNFα-mediated cell death [211]. Toxicity was amixture of necrosis and apoptosis, was associated withprolonged activation of JNK and phosphorylation of c-Jun,and could be prevented by a dominant negative c-Jun construct[211]. These results suggest that increased oxidant stress fromCYP2E1 may sensitize isolated hepatocytes to TNFα-inducedtoxicity.
Because CYP2E1 and LPS/TNFα are believed to be key riskfactors in the development of alcoholic liver injury, weevaluated possible interactions in promoting liver injurybetween them in vivo [212,213]. Sprague–Dawley rats weretreated with 200 mg/kg body wt pyrazole in the absence orpresence of LPS (10 mg/kg) and killed at 8 h after LPS [212].C57BL/6 mice were treated with 150 mg/kg body wt pyrazolein the absence or presence of LPS (4 mg/kg) and killed at 24 hafter LPS [213] The combination of LPS plus pyrazoletreatment resulted in elevated ALT and AST levels in rats andmice. Liver injury was confirmed by H&E staining. LPS aloneor pyrazole alone did not elevate transaminase levels and did notproduce liver injury under these conditions. Increased 3-nitrotyrosine protein adducts were observed at 8 (in rats) and24 h (in mice) after LPS plus pyrazole treatment [212,213].Positive staining for 4-hydroxynonenal adducts was found at24 h in the LPS plus pyrazole mice [213]. The CYP2E1inhibitor chlormethiazole (CMZ) protected against the elevationin ALT and AST in mice and the histopathology changes.CYP2E1 catalytic activity was decreased about 50% by theCMZ treatment [213]. We obtained CYP2E1-knockout micefrom Dr. Frank Gonzalez (National Cancer Institute, Bethesda,MD, USA). These mice were treated with pyrazole plus LPS.Compared to SV/129 wild-type mice, ALT and AST levels werelower in the CYP2E1-null mice, histopathology was normal,and TUNEL staining was much less [213]. Western blotanalysis confirmed the absence of CYP2E1 in the CYP2E1-knockout mice [213]. Based on such studies, we hypothesize[212,213] that increased production of ROS by CYP2E1 mayprime or sensitize the liver to LPS/TNFα, and such interactionsmay be important in alcohol-induced liver injury.
Nonalcoholic steatohepatitis (NASH) is a progressive liverdisorder that occurs in patients without significant alcoholconsumption. The pathogenesis of NASH is not well under-stood, although it has been suggested that oxidative stress andlipid peroxidation may play key roles in the pathogenesis ofNASH [214–217] Elevated CYP2E1 was observed under
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conditions such as obesity and high-fat/low-carbohydrate diets[84]. Weltman et al. [218] reported increased liver expression ofCYP2E1 in the methionine–choline-deficient model of NASH.CYP2E1 activity, protein levels, and mRNA levels were allelevated in this experimental model of NASH, although totalP450 content was decreased. Inhibitor studies further suggestedthat CYP2E1 was the major catalyst of lipid peroxidation inmice fed the methionine–choline-deficient diet [138]. However,CYP2E1-knockout mice fed with this diet still displayedelevated lipid peroxidation and NASH [138]. Under theseconditions, CYP4A10 and CYP4A14 but not CYP1A orCYP3A were upregulated and could replace the deficientCYP2E1 as catalysts for microsomal lipid peroxidation. Thus,although CYP2E1 contributes to the pathogenesis of NASH, itis not unique among P450 enzymes in promoting oxidant stress,as some CYP4A enzymes can serve as alternative initiators ofoxidant stress in the liver. Interestingly, antibody againstCYP2E1 strongly inhibited lipid peroxidation by microsomesfrom wild-type mice but antibody against CYP4A had littleeffect [138]. The opposite was found with microsomes fromCYP2E1-knockout mice as antibody against CYP4A blockedlipid peroxidation, whereas antibody against CYP2E1 had noeffect. Thus CYP4A can mediate lipid peroxidation as analternative pathway when CYP2E1 is absent [219].This canpartially explain the observations by Kono et al. [135] that in theintragastric infusion model of alcohol-induced liver injury,injury persisted in CYP2E1-knockout mice, i.e., possibleupregulation of CYP4A or other enzymes could replaceCYP2E1 as initiators or catalysts of oxidative stress.
Hepatic CYP2E1 levels were increased in patients withNASH [220]. Chalasani et al. [221] measured liver CYP2E1activity in a cohort of nondiabetic patients with NASH andcontrols. They found that chlorzoxazone clearance was greaterin the NASH patients compared with controls and lymphocyteCYP2E1 mRNA levels were also higher in the NASH patients.Increases in CYP2E1 correlated with increases in the ketonebody β-hydroxybutyrate. They suggested that although morestudies were necessary, CYP2E1 is a reasonable candidate in thepathogenesis of human NASH [221]. In a recent studyinvolving obese patients with nonalcoholic liver disease,increased CYP2E1 protein content and activity correlatedwith the development of liver injury [222].
Because CYP2E1 is elevated in pathophysiological condi-tions such as obesity and diabetes, we recently evaluated theeffects of CYP2E1 induction on promoting oxidative andnitrosative stress and liver injury in ob/ob mice, an experimentalmodel of obesity [223]. Ob/ob mice and lean controls weretreated with pyrazole or acetone to induce CYP2E1. CYP2E1protein and activity were elevated in acetone- or pyrazole-treated obese and lean mice. Acetone or pyrazole induceddistinct histological changes in liver and significantly higheraminotransferase enzymes in obese mice compared to obesecontrols or acetone- or pyrazole-treated lean mice [223].Increased malondialdehyde, protein carbonyls, 4-hydroxyno-nenal–protein adducts, levels of inducible nitric oxide synthase,and 3-nitrotyrosine protein adducts were found in livers ofpyrazole-treated obese animals, suggesting elevated oxidative
and nitrosative stress [223] Liver TNFα levels were higherin pyrazole-treated animals. The CYP2E1 inhibitor CMZand iNOS inhibitor N-(3-(aminomethyl)benzyl) acetamidine(1400W) abrogated the elevated toxicity (transaminases,caspase 3, triglyceride) and the oxidative stress (proteincarbonyl, HNE adduct formation, malondialdehyde) elicitedby the induction of CYP2E1 [223]. Peroxynitrite (ONOO−),formed by the rapid reaction between NO and O2
U−, has beenshown to nitrate free and protein-associated tyrosine residuesand produce nitrotyrosine; therefore, either decreased NOproduction by 1400W or a decline in O2
U− production by CMZprevented 3-nitrotyrosine formation [223]. These results showthat obesity contributes to oxidative/nitrosative stress and liverinjury and that induction of CYP2E1 may synergize with highfat in obesity to promote liver cell injury.
Future perspectives
Alcohol-induced liver injury is probably a multifactorialprocess involving several mechanisms. Future studies arerequired to further clarify how alcohol produces oxidativestress in various tissues. Some of the major proposed systemsrequire more detail about mechanism, e.g., how ethanol-derivedNADH, by itself or when reoxidized in the mitochondrialrespiratory chain, produces ROS. What is the role of ethanolmetabolism or ethanol metabolites like acetaldehyde in theproduction of ROS, and how is oxidative stress produced byethanol in tissues with limited ethanol metabolism? What arethe priming or sensitizing factors for ethanol-induced oxidantstress and cell injury? Can markers predictive of individualsparticularly sensitive to ethanol-induced oxidant stress and liverinjury be developed?
The role of CYP2E1 in the toxic effects of ethanol requiresfurther study as this remains a controversial issue. This issignificant not only from a mechanistic point of view butperhaps from a therapeutic treatment approach. If indeedCYP2E1-induced oxidative stress plays a central role inalcohol-induced liver damage, possible strategies for preventingthis stress may be effective in attempts to minimize thehepatotoxicity of ethanol in humans. The CYP2E1 inhibitorswhich were partially effective in preventing ethanol-inducedliver injury are not entirely selective and may be toxic, althoughCMZ [126] or polyenylphosphatidylcholine [127] may meritfurther consideration. YH439 is a novel synthetic compoundinhibiting CYP2E1 (but also other P450s) that is beingevaluated as a hepatoprotective agent [224]. Actually, naturalagents inhibiting CYP2E1, including diallyl sulfide (fromgarlic) mentioned above, phenylethyl isothiocyanate andsulforaphane (present in cruciferous vegetables), and berga-mottin (found in the essential oils of grapefruit and certainoranges) have been proposed as possible candidates forminimizing the ethanol-induced hepatotoxicity [225]. Inaddition, trans-1,2-dichloroethylene was reported to be aselective inhibitor of CYP2E1 [226].
Regulation of CYP2E1 protein levels is complex, withtranscriptional, translational, and posttranscriptional effectsobserved; more mechanistic details as to how ethanol modulates
CYP2E1 levels are required to define, e.g., effects on activity ofthe proteasome, ubiquitination, and how ethanol stabilizesCYP2E1. What are the factors which trigger the rapid turnoverof CYP2E1? Most studies on the biochemical and pharmaco-logical actions of CYP2E1 are derived from studies withrodents and rabbits and cultured hepatocytes: extrapolation tohuman studies is obviously necessary. The role of polymorphicforms of CYP2E1 on CYP2E1 expression, activity, and actionrequires further understanding, as current literature suggestssome possible relationships with certain types of cancers but notwith alcohol toxicity. Are there endogenous substrates forCYP2E1? At present, acetone and some fatty acids (ω-1hydroxylase activity) seem to be physiological substrates forCYP2E1, but further studies should be carried out becausealtered metabolism of such putative endogenous substrates, ifany, could contribute to the cellular actions associated withCYP2E1. CYP2E1 is present, although at relatively low levels,in other tissues, e.g., kidney, lung, brain, and gastrointestinaltract. Much less is known about the actions of CYP2E1 invarious pathophysiological conditions or after chronic ethanolexposure in these tissues. CYP2E1-nutritional interactionsrequire further study, especially interactions with pro-oxidants,such as iron; polyunsaturated fatty acids; or reagents that loweroxidant defenses, e.g., GSH levels. There is much currentinterest in synergistic interactions between alcohol and hepatitisB or hepatitis C virus, especially with respect to generatingoxidative stress. The role of CYP2E1 in such synergisticinteractions, if any, would be important to explore in view of themany chemicals and conditions that are known to elevateCYP2E1.
The ability of alcohol to promote oxidative stress and the roleof free radicals in alcohol-induced tissue injury clearly areimportant areas of research, particularly because such informa-tion may be of major therapeutic significance in attempts toprevent or ameliorate alcohol's toxic effects, e.g., by anti-oxidants, iron chelators, inhibitors of CYP2E1 or of cytokineproduction/actions, and GSH replenishment. As basic informa-tion continues to emerge regarding the role of oxidative stress indisease development and the mechanisms underlying ROS-related cellular toxicity, these findings will lead to more rationalantioxidant therapeutic approaches. Moreover, these findingscould result in the development of more effective and selectivenew medications capable of blocking the actions of CYP2E1and ROS and, consequently, the toxic effects of alcohol.
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