Oxidative and reductive metabolism by cytochrome P450 2E1.

Oxidative

and reductive

metabolism

by cytochrome

P450 2E1 DENNIS Department

R. KOOP of Pharmacology,

Oregon

Health Sciences

We are constantly exposed to many potentially toxic chemicals. Most require metabolic activation to species responsible for cell injury. Although cytochrome P450 2E1 is only one of many different forms of cytochrome P450 that catalyze these reactions, it has an important role in human health as a result of being readily induced by acute and chronic alcohol ingestion. The enzyme efficiently catalyzes the low Km metabolism of compounds commonly used as solvents in industry and at home as well as components found in cigarette smoke, many of which are established carcinogens and hepatotoxins. As a result, there is the potential for increased risk to low level exposure to such chemicals while cytochrome P450 2E1 is induced. Many substrates have been identified for cytochrome P450 2E1. Of the 52 substrates for the enzyme identified in this review, the demethylation of N,N-dimethylnitrosamine and the hydroxylation of p-nitrophenol and chlorzoxazone are the most effective for monitoring the level of this enzyme. In addition to oxidative reactions, cytochrome P450 2E1 is also an efficient catalyst of reductive reactions. CC14-induced hepatotoxicity is one of the best-documented cases for the participation of cytochrome P450 2E1 in a toxicologically important reductive reaction. The reduction of oxygen to superoxide and peroxide are also important reductive reactions of the enzyme and could be important in lipid peroxidation. However, the role of this reaction in vivo remains controversial. Koop, D. R. Oxidative and reductive metabolism by cytochrome P450 2E1. FASEBJ. 6: 724-730; 1992. ABSTRACT

Key lion

-

Words: P450 hepatotoxicity

2E1

ethanol-inducible

. lipid

peroxida-

DOCUMENTED THAT chronic ethanol ingestion can cause hepatotoxicity that predominates in the centrilobular region of the liver (1-3). In addition to the toxic effects of ethanol itself, ethanol also potentiates the toxicity of other chemicals including benzene, alkylnitrosamines, halogenated alkanes, and acetaminophen (3-5). Acute ethanol intake can inhibit metabolism and thus actually protect against toxicity. However, even low doses of ethanol will induce cytochrome P450 2EP (6) and when ethanol is cleared after acute treatment, potentiation of toxicity can be observed. Most chemicals that cause hepatotoxicity must be bioactivated to reactive species that initiate cell damage (5). This requirement for bioactivation, which occurs predominately via the liver microsomal cytochrome P450-dependent mixedfunction oxidase system, led to speculation that the induction of an ethanol-inducible form of P450 (P450 2El) was involved in the bioactivation reactions. An ethanol-inducible form of P450 was purified from rabbits (7) and has been characterized from many species, including rats, mice, hamsters, and humans, and it was demonstrated that the purified

IT IS WELL

724

University,

Portland,

Oregon

97201, USA

enzyme will catalyze bioactivation reactions (1, 3, 4). Additional observations support the involvement of P450 2E1 in ethanol-potentiated hepatotoxicity: other compounds that induce P450 2E1, such as acetone and other short-chain alcohols, also potentiate the same hepatotoxicity (4, 5). P450 2E1 is located in the cell layers near the terminal hepatic vein and is induced in the same region by all inducers examined (2). The centrilobular region of the liver is most susceptible to chemical toxins that are substrates for P450 2E1 (1-5). The number of compounds that have been identified as substrates for P450 2E1 has increased significantly since a similar compilation was done in 1986 (4). The list includes chemicals that are potential mechanism-based inhibitors for the enzyme as well as those compounds that provide a selective measure of P450 2E1 in hepatic microsomes, cell suspensions, and, potentially, in vivo. In addition to compounds that are substrates for oxidative metabolism by P450 2E1, substrates for reductive reactions will be reviewed and the potential role of these pathways in toxicity will be discussed.

CRITERIA

FOR

THE

PARTICIPATION

OF

P450

2E1

Multiple criteria are required to demonstrate a role for P450 2E1 in the metabolism of a xenobiotic. The experimental approach uses both direct and indirect experiments. Indirect experiments involve demonstrating that the activity is inducible by compounds known to induce P450 2E1 such as acetone, ethanol, and isoniazid (4, 6). All these compounds are easily administered in the drinking water and levels of induction from two- to eightfold are often observed after treatment from 1 to 7 days. As shown by intraperitoneal administration, pyridine and pyrazole are also potent inducers of the enzyme (4, 8)’. A correlation between an increase in the activity being measured and treatment with acetone is not definitive as acetone also induces P450 2B1 in rats (9). The activity measured should correlate with the level of P450 2E1 present

in various

microsomal

preparations.

The

concentra-

tion of microsomal P450 2E1 should vary over as wide a range as possible and not cluster around a single value. This is readily accomplished in animal models, as the dose and time of treatment can be manipulated, but it is more difficult with samples of human liver. The absolute concentration of P450 2E1 need not be established for these types of experiments. The relative concentration can be easily obtained by immunoblot analysis of microsomal samples. With the increased sensitivity of staining procedures currently available, it is possible to obtain excellent immunoblot data on as little as 0.05 tg of microsomal protein or 2 eg of total cell homogenates (10). It is useful to also quantify other forms of P450 in the same microsomal preparations to ensure that the activity does not correlate with other P450 forms present.

‘Abbreviation:

P450

2E1,

cytochrome

P450

2E1.

0892-6638192/0006-724/$01

.50.

©

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The activity under investigation can be correlated with the metabolism of other substrates for which P450 2E1 has been shown to be the principal catalyst. These include N,Ndimethylnitrosamine demethylation at substrate concentrations equal to or below 1 mM (11), chlorzoxazone 6-hydroxylation (12), and p-nitrophenol 2-hydroxylation (13). The activity used for comparison depends on a number of factors and will be discussed in more detail below. Selective inhibitors are an effective means to establish the participation of P450 2E1 in substrate metabolism. This approach is currently difficult to apply in that a specific inhibitor of P450 2E1 has not been described. As described below, some compounds appear to be mechanism-based inhibitors of the enzyme but their specificity has not been completely established. The use of reversible inhibitors is less effective, and in most cases the chemicals represent alternative substrates and/or ligands for other P450s as well. As the inhibition depends on the binding constant to P450 2E1 and to other forms of P450, some selectivity may be observed, but is often difficult to interpret. Specific inhibitory antibody to P450 2E1 is an effective and direct approach to identify a role of the enzyme in catalysis. This approach has been used extensively with both polyclonal and monoclonal antibodies. The most direct experimental approach to identify catalysis by P450 2E1 involves measuring the activity with the purified enzyme in a reconstituted system containing NADPH cytochrome P450 oxidoreductase and cytochrome b5. The inclusion of cytochrome b5 is important as this hemoprotein can significantly alter the activity toward many substrates by affecting both Vm, and Km (11, 13, 14). Ths approach can be misleading with some forms of P450 because the purified enzyme is not always catalytically competent. There are no reports of purified P450 2E1 from rats, rabbits, hamsters, or humans being inactive. Contamination of a P450 2El preparation with a very small amount of another form of P450 with high activity could lead to the false identification of an activity being associated with P450 2E1. This can be avoided by the expression of the enzyme in either bacterial or mammalian cells where P450 2E1 is the only P450 expressed (15). Under these conditions, if metabolism is observed, it can be attributed to the expression of P450 2El. In these types of experiments there must be ample P450 oxidoreductase expressed. As a result of the important role that cytochrome b5 can have in P450 2E1 function, the coexpression of this hemoprotein should also be considered. This is especially important as cytochrome b5 can decrease the apparent Km, and thus specificity for metabolism can be established at low substrate concentrations.

OXIDATIVE

SUBSTRATES

FOR

P450

2E1

Many compounds have been identified as substrates for P450 2E1, as summarized in Table 1. The compounds are, in general, all small, relatively polar compounds. The list includes many solvents used extensively in industry such as benzene, chloroform, and trichloroethylene. As a result, the bioactivation of these types of protoxicants by P450 2E1 places particular emphasis on this form of P450 in human health. The enzyme is readily inducible in humans (15, 20, 22) and there can be significant exposure to such substrates in the workplace. Chronic ethanol ingestion is not required for induction of P450 2E1; significant increases in the enzyme can be observed after a single dose of ethanol.

P450 2E1-DEPENDENT

METABOLISM

It is important to determine the catalytic activity of the human ortholog of P450 2E1, as in some cases significant differences in the activity of a structural ortholog have been reported. It should be emphasized that with respect to P450 2E1, all compounds that have been shown to be substrates in animal models are also substrates for the human ortholog. Human P450 2E1 catalyzes the demethylation of ANdimethylnitrosamine (15) and the activation of acetaminophen (20). In a more recent study, Guengerich et al. (22) described the metabolism of many low molecular weight cancer suspects by human P450 2E1. In this study, antibody to human P450 2El was not as effective an inhibitor of the oxidation of most of the halogenated alkanes when compared with substrates such as chlorzoxazone (22). The metabolism of CC14 was inhibited but the results presented were from a single experiment, and potential variation in the analysis cannot be dismissed. Whether the difference in inhibition by the antibody suggests a different mode of metabolism by the enzyme remains to be determined. If the antibody inhibits the transfer of electrons from P450 oxidoreductase to the P450 or access to the active site, the antibody should be equally effective with all substrates for the enzyme. The putative mechanism-based inhibitor diethyldithiocarbamate (22, 38) was not a very effective inhibitor of P450 2E1-dependent CC14 metabolism (which is reductive), but was very effective against the other halogenated alkanes tested (22). The results suggest that the inactivated enzyme may not catalyze oxygen activation but can still effectively transfer a single electron to Cd4. The potential for common organic solvents to act as substrates for P450 2El has important implications when other potential substrates are tested in metabolic assays. Solvent effects can be quite dramatic as relatively low K1 values were reported (i.e., DMSO, 390 tiM; 2-mercaptoethanol, 20 fsM; dimethylformamide, 90 sM; ethyl acetate, 220 tM) (39). Glycerol has a much higher K1 (53 mM) and Km (18 mM) (28), but this level of glycerol can be reached with the reconstituted enzyme when 20% (v/v) glycerol (2.74 M) is used in storage buffers. The inhibition observed will depend greatly on the stock concentration of the purified P450 and P450 oxidoreductase. For example, if both enzymes are stored as 30 iM stocks in 20% glycerol and are used at final concentrations of 0.1 and 0.3 aiM, the final glycerol concentration will be 36 mM in a 1.0 ml reaction mixture. The P450-dependent oxidation of glycerol to formaldehyde (28) suggests other vicinal diols as potential substrates for P450 2E1. These compounds include propanediol and butanediol, the blood levels of which are increased by alcohol treatment. Benzene is oxidized to muconic acid in vivo and traits, trnns-munconaldehyde is thought to be an intermediate in this biotransformation (40). The pathway for muconaldehyde formation has not been established. Latriano et al. (40) reported that this reactive dialdehyde was produced in vitro with microsomes from benzene-pretreated mice. Benzene treatment induces P450 2E1 (4), which is the principal catalyst of the oxidation of benzene to phenol presumably via the intermediate benzeneoxide (18, 19). As a result, the trans-dihydrodiol may be increased under these conditions depending on the relative concentration of epoxide hydrolase. By a reaction analogous to the oxidation of glycerol, the formation of muconaldehyde from the dihydrodiol may be possible (Fig. 1). In addition, a radical-mediated pathway for the formation of muconaldehyde was suggested (40). As discussed below, P450 2E1 very efficiently supports hydroxyl radical-mediated types of reactions (41, 42) and P450 2E1

725


TABLE

1. Substrates rnetabolized by cytoc/trorne P450 2E1 Product Measured

Substrate

Aromatic

Reference

compounds

Pyridine 3-Hydroxypyridine p-Nitrophenol Benzene

Pyridine N-oxide 2 ,5-Dihydroxypyridine 4-Nitrocatechol Phenol

16 17 13 18

Phenol Acetaminophen Pyrazole Chlorzoxazone

Hydroquinone; catechol Glutathione conjugates 4-Hydroxypyrazole 6-Hydroxychlorzoxazone

19 20 21 12

Styrene

Glutathione

22

Aniline

p-Aminophenol

Halogenated Chloroform

alkanes

conjugate

4

and alkenes/alkanes Glutathione

conjugate

22, 23

Pentane

product not measured

24

Chloromethane Dibromoethane Dichloromethane 1,2-Dichloropropane Ethyl carbamate 1,1,1 -Trichloroethane Trichloroethylene

Formaldehyde Glutathione conjugate Glutathione conjugates Glutathione conjugate 1,M-Ethenoadenosine 1,1,1 -Trichloro-2-hydroxyethane Chloral

22 22 22 22 22 22 22

Ethylene dibromide Ethylene dichloride Vinyl chloride Vinyl bromide Vinyl carbamate

1,M-Ethenoadenosine 1 ,N6-Ethenoadenosine 1 ,M-Ethenoadenosine 1 ,M-Ethenoadenosine 1,N6-Ethenoadenosine

22 22 22 22 22

Enflurane Halothane

Fluoride Trifluoroacetic

25 26

1,1,1 ,2-Tetrafluoroethane

Fluoride

27

Acetaldehyde Propionaldehyde

4 4

Isopropanol

Acetone

Butanol Pentanol Glycerol

Butyraldehyde Valeraldehyde Formaldehyde

4 4

4 28

Acetol

Methylglyoxal

4

Acetone Acetonitrile

Acetol Cyanide 1,M-Ethenoadenosine

Alcohols/ketones/nitriles Ethanol Propanol

(+

catalase)

Acrylonitrile

acid

4 29 22

Nitrosamines/azocompounds

N N-Dimethylnitrosamine Azoxymethane Methylazoxymethanol

Formaldehyde/nitrite

11

Azoxymethanol Methanol/formic

N N-Diethylnitrosamine N-Nitrosopyrrolidine N-Nitroso-2,6-dimethylmorpholine

Acetaldehyde 4-Hydroxybutyraldehyde N-Nitroso-(2-hydroxypropyl)-(2-oxopropyl)amine

30 30 11 31 4

Acetaldehyde Formaldehyde/t-butanol

32 33

Ethers Diethyl Methyl

ether i-butyl

ether

Reductive substrates Carbon tetrachloride Chromium (Cr”t) 13-Hydroperoxy-9,ll-octadecadienoic 1 5-Hydroperoxy-5 ,8, 11,1 3-eicosatetraenic Cumyl hydroperoxide t-Butylhydroperoxide

Oxygen

726

Vol. 6

January 1992

acid

Lipid peroxidation/chloroform Product not measured Pentane

acid acid

22, 34 35 36

Pentane Methane/acetophenone Methane/acetone

36

Superoxide/peroxide/water

37

The FASEB Journal

36

36

KOOP


0 H-1.._H

OH P450

2E1?

NADPH,

02

H

H71C-H

II 0

‘OH

trans-i

2-Dihydrobenzene- 1 2-dial

trans.trans-Uuconaldehyde

Figure 1. Proposed formation of muconaldehyde. way for the formation of trans,trans-muconaldehyde

dihydrobenzene-1,2-diol shown.

via

a P450

A potential pathfrom irans-1,2-

2E1-mediated

reaction

is

may participate in this manner in muconaldehyde formation in lieu of a direct oxidative mechanism. Of the many oxidatively metabolized substrates shown in Table 1, the most extensively investigated has been the P450 2E1-dependent demethylation of N,N-dimethylnitrosamine (reviewed by Yang et al. in ref 11). This nitrosamine was one of the first to be shown to be selectively metabolized by P450 2E1 at low concentrations of substrate (11). As with ethanol oxidation (43), there does not appear to be any stereoselectivity (i.e., for the (Z)- vs. (E)-isomers) in the initial oxidation reaction, which suggests very little restriction on the substrate in the active site of the enzyme (44). Yang et al. (11) proposed an active site model based on the structure of that permits the oxidation of small molecules such as those shown in Table 1. When P450 2E1 is presented with a nitrosamine such as N-methyl-N-butylnitrosamine, the methyl group is preferentially oxidized (as determined by the release of formaldehyde instead of butyraldehyde) whereas other isozymes, such as P450 2B1, preferentially oxidize the butyl group (11). P450 2E1 will oxidize the methyl group of all nitrosamines that contain one methyl group and another substituent such as propyl or benzyl, but the selectivity for catalysis by P450 2E1 is decreased significantly (4, 11). ‘450cam

METABOLIC

MARKERS

FOR

P450

2E1

It would be extremely desirable to have a specific assay for P450 2E1 that could be used to identify the presence of the functional enzyme as immunoblot analysis detects both apoand holoenzyme. The demethylation of N,N-dimethylnitrosamine at a concentration of less than 1 mM and the hydroxylation of p-nitrophenol and chlorzoxazone are the best metabolic markers for the presence of P450 2E1. The structure of each of these substrates and the position of hydroxylation are shown in Fig. 2. The choice of which marker to use depends on several factors. The measurement of pnitrophenol hydroxylation is simple and rapid, and the only instrumentation required is a spectrophotometer. The demethylation of N-methylnitrosamines is also relatively simple and the formaldehyde produced is measured colorimetrically. Both of these assays are limited by the sensitivity of the methods and can detect around 400 pmol of product. The nitrosamines are established carcinogens. As a result, special care must be used when handling these compounds, and routine use is more difficult and waste disposal is problematic. The sensitivity of these assays can be greatly enhanced with radioactive substrates, but again, this makes routine use more difficult and disposal is a problem. In contrast, the 6-hydroxylation of chlorzoxazone is about 10-fold more sensitive than the colorimetric assays. The substrate is an approved drug and not toxic at low doses. The 6-hydroxylase assay requires high-pressure liquid chromatography although a simple isocratic solvent system was

P4fl

7F1.DFPFNDENT

METABOLISM

described (12). A major advantage of chlorzoxazone metabolism is that the hydroxylation has the potential to be used as a noninvasive probe for the presence of P450 2El in animal models and humans, provided that the in vivo rate of metabolism is dependent on the concentration of P450 2El (i.e., the drug has low intrinsic clearance) under all conditions. Lucas et al. (45) recently concluded that of 12 different activities, the demethylation of N,N-dimethylnitrosamine was one of the best indicators of P450 2El induction. The hydroxylation of benzene and p-nitrophenol were also good indicators of P450 2E1 but were not as specific (45).

MECHANISM-BASED

INHIBITORS

OF

P450

2E1

Although microsomal studies can implicate P450 2El in catalysis in vitro, the availability of a specific inhibitor for the enzyme for the assessment of in vivo function would be extremely valuable. Mechanism-based inhibitors offer the greatest potential for specificity as catalysis is required for inhibition (46). There has been some progress toward this end in the past few years. Four compounds shown in Fig. S have been suggested to be mechanism-based inhibitors for P450 2E1. 3-Amino-1,2,4-triazole exhibited a number of kinetic criteria consistent with mechanism-based inhibition of P450 2E1 (47). The inhibition was time- and NADPH-dependent. The first order loss of p-nitrophenol hydroxylase activity was saturable, irreversible, and insensitive to exogenous nucleophiles, and had a pH dependence similar to other reactions catalyzed by P450 2E1. The mechanism of the inactivation was not established. Heme was not destroyed as determined by the reduced pyridine hemochrome but carbon monoxide binding was inhibited. Radiolabel from the C-S position of 3-amino-1,2,4-triazole was not incorporated into the protein. The compound is a well established mechanism-based inhibitor of catalase and the specificity for various P450 forms needs to be established (47). Gannett Ct al. (48) described the irreversible inactivation of rat liver P450 2E1 by the natural product from red peppers, dihydrocapsaicin. NADPH-dependent covalent binding of dihydrocapsaicin to P450 2E1 was demonstrated by immunoaffinity purification of the inactivated enzyme, but the stoichiometry was not established. Based on electrochemical studies, the authors suggested that P450 2E1 catalyzed the 1-electron oxidation of dihydrocapsaicin to the phenoxy radical that reacted with P450 2E1 (48). Because P450 2E1 is responsible for the bioactivation of many carcinogens, the inhibition of P450 2El by compounds such as dihydrocapsaicin may partially explain inhibition of tumorigenesis by some foods.

0

0H CI

CH3-CH3 NO2

N

Chlorzoxazone

N-Nitrosodimethylamine

p-Nitroplienol

Figure 2. Substrate probes for P450 2El. The structures of substrates useful to monitor the presence of P450 2El are shown. The position of hydroxylation is indicated by the arrow for each structure.

727


NH2

(-CH2CH2N=C=S

N’ 3-Amino-

Phenethyl

isothiocyanate

1,2,4-triazole

CH3CH2M

0

CH3CH2-

ii

Diethyldithiocarbamate

Figure 3. Mechanism-based of putative mechanism-based the text are shown.

Dihydrocapsaicm

inhibitors of P450 2E1. The structures inhibitors of P450 2E1 discussed in

Ishizaki et al. (49) recently reported that phenethyl isothiocyanate, another dietary constituent found in cruciferous vegetables such as cabbage and brussel sprouts, was an effective inhibitor of the microsomal demethylation of !‘1Ndimethylnitrosamine. A K1 of 1 eM for the reversible inhibition was reported. There was also a time- and NADPHdependent inactivation of the demethylation reaction which suggests mechanism-based inhibition. There was a significant time-dependent loss of activity in the absence of NADPH, which is not surprising due to the reactivity of the isothiocyanate group. The specificity and mechanism of the mechanism-based inactivation require further investigation. Disulfiram inhibited the carcinogenicity of 1,2-dimethylhydrazine (50) and the hepatotoxicity of CHC13, CC!4, acetaminophen, and Ps N-dimethylnitrosamine (38). Disulfiram is reduced to diethyldithiocarbamate, which was an effective inhibitor of P450 2E1 in rat (38) and human (22) microsomes. Brady et al. (38) reported a time- and NADPHdependent loss of rat liver microsomal N,N-dimethylnitrosamine demethylation. However, the specificity and mechanism were not addressed although the authors suggested the potential for an initial oxidation of diethyldithiocarbamate by the NADPH-dependent flavin containing monooxygenase in microsomes. Guengerich et al. (22) reported the preliminary characterization of diethyldithiocarbamate as a mechanism-based inhibitor of human P450 2E1. Diethyldithiocarbamate caused a timeand NADPH-dependent loss of both N,N-dimethylnitrosamine demethylation and chlorzoxazone hydroxylation. In competition assays using 300 eM diethyldithiocarbamate, which inhibited about 80-85% of the N,N-dimethylnitrosamine demethylation and chlorzoxazone hydroxylation, (± )-mephenytoin 4’-hydroxylation was inhibited about 40% whereas tolbutamide methyl hydroxylation and (±)-bufuralol 1-hydroxylation were inhibited only about 10% (22). These results suggest some specificity for the diethyldithiocarbamate inhibition. If the compound were used in preincubation reactions at low concentrations it might irreversibly inactivate all of the P450 2E1 with little effect on other isoforms.

REDUCTIVE

REACTIONS

The oxidative metabolism of well recognized. In addition, reactions. In most cases these oxygen, and so are generally

OF

P450

2E1

organic substrates by P450 is P450 also catalyzes reductive reactions are competitive with thought to occur only under

very low oxygen tensions. P450 2E1 is an effective catalyst of reductive reactions, and those that have been clearly established for this enzyme are also shown in Table 1. One of the best-documented cases of ethanol-potentiated toxicity after both acute and chronic ethanol ingestion is CC14-induced liver damage (1, 2, 5). Although many investigators have demonstrated that compounds that induce P450 2E1 also potentiate CCI4-induced hepatic injury, Johansson and Ingelman-Sundberg (34) directly demonstrated that the purified rabbit ortholog of P450 2E1 was 100-fold more active than rabbit P450 1A2 and P450 2B4 in initiating CC!4dependent lipid peroxidation. They also demonstrated that under anaerobic conditions, microsomes from imidazoletreated rabbits were about threefold more effective than microsomes from untreated rabbits in the production of CHC13 from CCI4 (34). Antibody to P450 2E1 inhibited the NADPH-dependent lipid peroxidation of hepatic microsomes from acetone-treated rats (51) and from humans (52). Although inhibition of the NADPH-dependent lipid peroxidation could be attributed to a decrease in the level of reactive oxygen produced and not to an inhibition of CCI4 reduction to the trichloromethyl radical, the formation of chloroform from CC14 suggests P450 2E1-dependent metabolism of CCJ4. The mechanism of CCI4 reduction is not clear. Isoniazid (1 mM) inhibited 70% of the NADPH oxidation and peroxide production by purified rat P450 2E1 (53). However, the same concentration of isoniazid inhibited CC!4 metabolism to chloroform by only about 22% (53). In contrast, Lindros et al. (54) reported that 2 mM isoniazid inhibited CC14 induced hepatocyte damage in perivenular cells by about 80%, a value consistent with the inhibition of microsomal lipid peroxidation by isoniazid (53). The formation of trichloromethyl radicals from CC!4 enhanced by ethanol treatment was reported by Reinke et al. (55). The effect of cytochrome b5 on the reductive reactions has not been carefully examined. It is important to understand the role of cytochrome b5 in the reductive reactions because it has been proposed that this cytochrome can function as a source of the second electron in the catalytic cycle of P450 (56). If cytochrome b5 efficiently donates an electron to the ferrousoxygen enzyme, then one might predict that the release of superoxide by autooxidation would decrease. The ferric peroxide intermediate formed would rapidly cleave to give water and an active oxygen intermediate that would be reduced by two electrons to give a second molecule of water (37, 56). If the peroxide is produced by dismutation of superoxide, then cytochrome b5 would decrease peroxide production. Gorsky et a!. (37) reported that P450 2E1 exhibited the greatest 4-electron oxidase activity of six purified rabbit forms of P450. Vaz et a!. (36) described the efficient reduction of hydroperoxides by P450 2E1. These reactions were conducted under anaerobic conditions, and produced hydrocarbons and either aldehydes or ketones from the original hydroperoxide. Lipid hydroperoxides were proposed as the physiological substrates, and the nearly stoichiometric formation of pentane and NADPH oxidation for the reaction with 13-hydroperoxy-9,11-octadecadienoic acid was reported (36). Other organic hydroperoxides such as cumyl hydroperoxide and tertiary butyl hydroperoxide were also effective substrates. The physiological significance of this reaction has not been demonstrated, but P450 2E1 appears quite capable of initiating lipid peroxidation (51, 57). Thus, although initially involved in the formation of lipid hydroperoxides, P450 2E1 may continue to function in the reduction of hydroperoxides as the oxygen concentration is

728 Vol. 6 january 1992 The FASEBjournal KOOP m www.fasebj.org by Kaohsiung Medical University Library (163.15.154.53) on August 27, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber(

depleted in the centrilobular region of the liver. This could contribute to the degeneration of the lipid bilayer. Terelius and Ingelman-Sundberg (24) demonstrated that pentane is an effective substrate for P450 2E1 with an apparent Km of about 9 tIM. Thus, as pentane is formed, P450 2E1 will be able to oxidize the compound if the oxygen tension is increased. Whereas the oxidation of other alkanes has not been reported, it is likely they would also be substrates for P450 2El. Also included in the list of substrates for reductive reactions is molecular oxygen. The NADPH-dependent reduction of oxygen by cytochrome P4SO in the presence and absence of substrates to peroxide is well documented for all forms of the enzyme. This reduction reaction - referred to as the oxidase activity of P450-is probably as important, if not more so, than the other reductive reactions listed in Table 1. Peroxide formation is thought to occur by the release and subsequent dismutation of superoxide, but the direct release of peroxide from the 2-electron reduced enzyme is possible (37, 56). The reduction of oxygen may be especially important for P4SO 2E1. This form of P450 is isolated in the high spin state (7) and reticulum,

if the

enzyme

is also high be readily

spin in the endoplasmic reduced by NADPH

it may cytochrome P450 oxidoreductase in the absence of substrate. Compared with other forms of P450, P450 2E1 exhibits a higher rate of oxidase activity when purified (37), and microsomes from animals pretreated with inducers of P450 2E1 also exhibit much greater rates of NADPH oxidation than microsomes from untreated animals (41, 42, 51, 57). Enhanced oxidase activity would result in the increased production of both superoxide and hydrogen peroxide, which in the presence of chelated iron can produce reactive hydroxyl radicals (41, 42). P450 2E1 exhibits a unique ability to potentiate iron-catalyzed Fenton chemistry in a reconstituted system, and increased rates of hydroxyl-radical mediated metabolism of substrates such as ethanol and dimethylsulfoxide were reported for microsomes isolated from animals pretreated to induce P450 2E1 (41, 42, 51, 53, 57). The increased capacity to produce active oxygen species is manifested by an increased rate of microsomal lipid peroxidation by microsomes or liposomes enriched in P450 2E1 (51, 53, 57). Antibody to P450 2E1 inhibited about 65% of microsomal peroxide production while almost completely inhibiting NADPHdependent

lipid

peroxidation

(51).

These

results

suggest

that

although there are other microsomal sources of peroxide, the participation of P450 2El is necessary for lipid peroxidation to be observed. However, the role of the oxidase reaction in vivo remains controversial. There is little experimental data to directly test the hypothesis that the peroxide produced in the cytosol is generated from the reduction of oxygen to superoxide and/or peroxide by cytochrome P450. Krieter et al. (58) monitored the biliary efflux of oxidized glutathione (OSSO) as a measure of peroxide formation in the presence and absence of aminopyrine, a substrate for some forms of P450 but not for P450 2E1. The efflux of GSSG was very low in the absence of substrate. In the presence of aminopyrine, the OSSG released was not dependent on glutathione peroxidase and peroxide as a selenium-deficient diet had no effect on GSSG release (58). In these experiments, the results suggested that the P450 system produced no peroxide in the absence of a hydroxylatable substrate, and the peroxide that was produced in the presence of aminopyrine was also not attributable to peroxide release. Studies in vitro indicate that the extent of uncoupling is dependent on the P450 form (37). Although the results of Krieter et a!. (58) suggest that the

PAcn

)t1flCPCNifl1N.JT

tAITARflI

cM

P450

population

treated

rat

present

liver

the case when

does

not

P450

2El

in untreated produce

and

peroxide,

phenobarbitalthis may not be

is induced.

the induction of P450 2E1 in the centrilobular region of the liver may participate in the accentuation of the oxygen gradient between the perivenous and centrilobular regions of the liver after ethanol treatment (1, 2). If the presence of P450 2E1 is the only reason for this lower oxygen tension, then induction of P450 2E1 by other treatments and also with single nontoxic doses of ethanol It has

should

been

also

suggested

increase

that

hypoxia

in the

centrilobular

region.

It

would be interesting to determine whether there is enhanced OSSO efflux into the bile under conditions of optimal P450 2El induction. The hypothesis that increased oxidase activity of P450 2El in the centrilobular region results in increased oxygen consumption while also increasing oxygen radical production that participates in lipid peroxidation is attractive. This would lead to the regioselective hepatotoxicity observed after ethanol treatment. However, it remains to be demonstrated that the enzyme is uncoupled in vivo. Work in the author’s laboratory was supported by USPHS grant AA-08608 from the National Institute on Alcohol Abuse and Alcoholism.

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(n,,rr,I


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Prediction of cytochrome P450 mediated metabolism.

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Reductive metabolism and protein binding of chromium(VI) by P450 protein enzymes.

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Multifactorial comparative proteomic study of cytochrome P450 2E1 function in chronic alcohol administration.

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Cytochrome P450 expression and metabolism in isolated rabbit renal epithelium.

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