Role of Cytochrome P-450 2E1 in Ethanol-, Carbon Tetrachloride- and Iron-dependent Microsomal Lipid Peroxidation TIRSOCASTILLO,~ DENNISR. KOOP,~ SEIICHIRO KAMIMURA,~ GEORGETRIADAFILOPOULOS~ AND HIDEKAZU TSUKAMOT03 'Division of Gastroenterology, Veterans Affairs Medical Center, Martinez, California 94553; 2Department of Pharmacology, Oregon Health Sciences University, Portland, Oregon 97005;and 3Division of Gastroenterology and Hepatology, MetroHealth Medical Center and Veterans Affairs Medical Center, Case Western Reserve University, Cleveland, Ohio 44109-1998

This study investigated the role of cytochrome P-450 2E1 in enhanced microsomal lipid peroxidation in experimentalalcoholic liver disease. We also examined the contribution of this isoform to the increased microsomal injury in alcoholic liver disease caused by carbon tetrachlorideinduced or iron-inducedoxidant stress. Adult male Wistar rats were intragastrically infusedwith a high-fat diet and ethanol or glucose for 16 wk; this resulted in hepatic lipid peroxidation and fibrogenesis in the ethanol-fed animals. Microsomes were isolated by differential centrifugation in the presence of 100 pmoUL deferoxamine,washed twice in buffer without deferoxamine and incubated in the absence or presence of ethanol (50 mmoUL), carbon tetrachloride (150 pmoUL),ferric citrate (60 Fmol/L)or ferric citrate plus ethanol at 37" C for 30 min in an NADPH-generating system. The basal rate of lipid peroddation in microsomes isolated from ethanol-fed rats was increased by 52% compared with that in microsomes from controls. Carbon tetrachlorideinduced and ferric citrateinduced lipid peroxidation were also accentuated in microsomes from ethanol-fed rats, by 76% and 108%,respectively. Ethanol added in uitro significantly reduced basal (-58%) and ferric citrateinduced ( - 48%) lipid peroxidation in microsomes from ethanol-fed rats, whereas it had an insignificant effect on that in control microsomes. In fact, this protective effect of ethanol on microsomes from ethanol-fed rats resulted in attenuation of the differencein the level of microsomal lipid peroxidation between the two groups. P-4502E1 IgG antibody added to the incubation completely blocked the enhanced lipid peroxidation observed in microsomes from ethanol-fed rats under the basal, ferric citrateinduced and carbon tetrachlorideinduced conditions. Immunoblot analpis of microsomal proteins with

Received January 21, 1992;accepted March 16,1992. This study was supported by the Department of Veterans Af€airs and by United StatesPublic Health SeMce grants AA06603 (HT) and AAOS608 (DRK). Address reprint requests to: Hidekazu Tsukamoto, D.V.M., Ph.D., Department of Medicine, MetroHealth Medical Center, 2500 MetroHealth Drive, Cleveland, OH 44109.

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P-4502E1 IgG antibody demonstrated a large increase in the level of this cytochrome in microsomes from ethanol-fed rats. The catalytic activity of P-460 2E1, measured by antibody-inhibitable p-nitrophenol hydroxylation, was increased 20-fold over control levels by the ethanol feeding. These results demonstrate (a) induction of P-460 2E1 at an advanced stage of experimentalALD, (b) a maljor role of this cytochrome in the enhancedbasal lipid peroxidation of microsomes obtained from livers with ALD; (c) a maljor contribution of P-4602E1 to the increased vulnerability of these microsomes to ferric citrate and carbon tetrachlorideinduced peroxidation and (d) the possible implication of ethanol withdrawal in M h e r peroxidative injury caused by the absence of the protective effect of ethanol. (HEPATOLOGY 1992;16992996.)

The microsomal ethanol-inducible cytochrome P-450 (P-4502E1) is incriminated in the pathogenesis of largely by its potential for alcoholic liver disease (ALD), free radical generation and subsequent induction of microsomal lipid peroxidation (1-3). Induction of this P-450isoform is evident in animals given ethanol (4-6) and may underlie the increased vulnerability of these animals to hepatic peroxidative injury by ethanol itself and by other xenobiotic substances. One such xenobiotic substance is CCl, (7-91, which is believed to exert its toxicity as a result of reductive metabolism by P-4502E1 (10). However, the contribution of P-4502E1 to microsomal lipid peroxidation in ALD and to the increased susceptibility of microsomes from animals fed ethanol over long periods to CC1, toxicity is unknown. It is well known that ionic iron and low-molecularweight iron chelates catalyze the initiation of lipid peroxidation through reactions yielding hydroxyl radicals. Of equal importance is reductive and oxidative decomposition of lipid hydroperoxidesby Fe2 and Fe3 to yield a new generation of lipid alkoxyl and peroxyl radicals (11).This transition-state metal is responsible for hepatocellular injury and cirrhosis in hereditary and secondary hemachromatosis (12),and the pathogenetic

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CYTOCHROME P-450 2E1 IN MICROSOMAL LIPID PEROXIDATION

importance of peroxidative injury has been strongly supported by studies employing experimental models of iron overload (13). Iron added in uitro to microsomes catalyzes NADPH-dependent generation of hydroxyl radical (14) and lipid peroxidation (15). Long-term alcohol consumption enhances these iron-mediated responses (16). Although P-450 2E1 has been shown to play a major role in microsomal lipid peroxidation (2),it remains to be determined to what extent P-450 2E1 contributes to ethanol-induced enhancement of ironcatalyzed oxidant stress. Our recent longitudinal study employing a rat model of ALD showed that the enhanced lipid peroxidation of microsomes and mitochondria occurs at the stage of alcoholic liver fibrogenesis ( 17).The study reported here used microsomes isolated from this model at this stage of ALD and an antibody against P-450 2E1 to examine in uitro the role of P-450 2E1 in enhanced lipid peroxidation of these organelles in ALD and their increased susceptibility to CC1, and iron-induced peroxidation.

TABLE 1. Lipid peroxidation in liver microsomes from ethanol-fed and control animals TBA-reactive substances (nmoU2 md30 min) ~

Additive

None CCl, (150 pmoUL) Fe3+ (50-100WmoW)

~~

Control

Ethanol-fed

1.65 ? 0.71” 2.51 t 0.91 25.64 t 5.24 45.06 2 4.91 38.83 t 21.66 80.60 t 17.89

p Value

0.06 < 0.05

or and diluting it with sodium citrate to achieve the stock Fe3+ (50 p,moVL:100 p,moVL) expectedly caused a concentrations of 5 mmol/L iron and 10 mmol/L citrate. Rabbit marked increase in lipid peroxidation of control miP-450 2E1 IgG antibody (anti-P-450 2E1) or preimmune IgG crosomes. However, these treatments induced lipid +

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f.

cc14

6dSdl

Control IgG Anti-P4502El

EthanolisiimMi

-

+

-

Frc. 1.In vitro effects of ethanol on (A) basal and (B)iron-induced microsomal lipid peroxidation.Note that ethanol (50 mmoVL)added in vitro to the reaction mixture (shown at the bottom of the figure as Ethanol. 50 mM +), significantly reduced (A) basal and (B)ironcatalyzed (Fes’: 50 pmoVL) lipid peroxidation in microsomes from ethanol-fed animals (Etoh-micro), but had an insignificant and minimal effect on those from controls (Cont-micro). * p = 0.06 and **p c 0.05 compared with control microsomes without ethanol incubation; t p < 0.05 compared with microsomes from ethanol-fed rats without ethanol incubation.

peroxidation in microsomes from ethanol-fed rats 70% to 100%greater than that in control microsomes. These results demonstrate that microsomes isolated from rats with advanced ALD indeed have a higher rate of peroxidative activity and that these microsomes are susceptible to more severe lipid peroxidation induced by CCl, and iron. In uitro effects of ethanol (50 mmoVL) on basal and iron-stimulated microsomal lipid peroxidation are depicted in Figure 1 A and B. Addition of ethanol significantly reduced the basal (Fig. 1A) and ironcatalyzed (Fig. 1B) lipid peroxidation in microsomes from ethanol-fed rats, whereas no significant effect was observed in control microsomes. Consequently, no significant differences were observed in basal and ironcatalyzed lipid peroxidation in these two groups of microsomes when they were incubated with ethanol. In the next experiment, anti-P-450 2E1 IgG was used to evaluate the involvement of this isoform in the enhanced basal lipid peroxidation and the potentiated responses to CCl, and Fe3+ observed with microsomes from ethanol-fed rats. As shown in Figure 2, addition of anti-P-450 2E1 IgG but not preimmune control IgG completely blocked enhanced microsomal lipid peroxidation in microsomes from ethanol-fed rats under all three sets of conditions. The inhibition by anti-P-450 2E1 IgG clearly indicates a role for P-450 2E1 in the enhanced microsomal lipid peroxidation in microsomes from ethanol-fed rats.

-

-

t

-

-

-

-

-

+

- - _

t

Fe It -

-

- +

-

- _ _

FIG.2. Effects of anti-P-450 2E1 IgG on basal and CC1,- or iron-induced lipid peroxidation of microsomes from ethanol-fed rats. Microsomes were incubated in the absence or presence of CCl, (150 FmoVL) or Fe3+ (50 pmoVL to 100 FmoVL) with or without anti-P-450 2E1 IgG or nonimmune IgG as described in “Materials and Methods.” Lipid peroxidation was determined by the production rate of thiobarbituric acid-reactive substances and expressed as the percentage of those measured in control microsomes under each of the three conditions (basal, added CCI, and added iron). Note that the enhanced lipid peroxidation seen in microsomes from ethanol-fed rats under all three conditions was completely blocked by addition of anti-P-450 2E1 IgG but not by control IgG. *p = 0.06 and **p < 0.05 compared with control microsomes; t p < 0.05 compared with lipid peroxidation of microsomes from ethanol-fed rats with addition of nonimmune control IgG.

To assess the induction of P-450 2E1 in microsomes from ethanol-fed rats, P-450 2E1 protein was immunoblotted and compared between the two groups. As clearly shown in Figure 3, a marked increase in the level of P-450 2E1 is evident in microsomes from ethanol-fed rats compared with that in control microsomes. The increase in functional enzyme was best assessed by gauging PNP hydroxylase activity, for which P-450 2E 1 was shown to be a principal catalyst (19). Microsomal PNP hydroxylation was increased about 12-fold by ethanol feeding. When the activity that was inhibitable by anti-P-450 2E1 IgG was determined, the apparent induction of P-450 2E1 was about 20-fold (Table 2). DISCUSSION

Several lines of experimental evidence suggest a role for microsomal enzymes in hepatic lipid peroxidation and pathological evolution of ALD. First, carboncentered radicals were detected in uiuo in the hepatic endoplasmic reticulums of ethanol-fed but not control rats (23). Second, the formation of radicals was potentiated by a high-fat diet (23), which has been shown to induce P-450 2E1 (24) and to exacerbate experimental ALD (25). Third, the centrilobular distribution of P-450 2E1 (26, 27) fits the pattern of regional predisposition for alcoholic liver injury. Last, the amount of P-450 2E1 correlates with the rate of microsomal lipid peroxi-

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RG.3. Representative immunoblot of microsomal P-450 2E1 from two pairs of control rats (Cont-micro 1 and 2 ) and two pairs of ethanol-fed (Etoh-micro 1 and 2 ) rats. Microsomal protein in amounts of la) 0.5 Fg, (b) 1 pg, (c) 2 bg. or (d) 4 pg wm analyzed by immunoblotting with anti-P-450 2E1 IgG as described in “Materials and Methods.” Note the marked induction of P-450 2E1 in microsomes from ethanol-fed rats (Etoh-micro 1 and Etoh-micro 2 ) compared with those from controls (Cont-micro 1 and Cont-micro 2 ) .

dation, which can be completely blocked by anti-P-450 2E1 IgG (2). Our results extend these observations and demonstrate in uitro that the higher rate of microsomal lipid peroxidation at the advanced stage of experimental ALD is caused by P-450 2E1. Ekstrom and IngelmanSnndberg (4) previously demonstrated a similar result in ethanol-fed rats with minimal liver damage. Our study represents the first attempt to relate this observation to the evolution of advanced liver damage induced by ethanol. Hepatic microsomes isolated from our model at the advanced stage of experimental ALD have a higher basal rate of lipid peroxidation, which is shown to be completely blocked by immunological inhibition of P-450 2E1. P-450 2E1 is also suggested to have a critical role in the increased susceptibility of liver microsomes in ALD to CC1, and iron-induced peroxidative injury. Cytochrome P-450 2E1 is one P-450 isoform involved in the metabolic reduction of CC1, to the toxic free radical CCl; (28). Thus induction of this cytochrome by long-term alcohol consumption is presumed to underlie the enhanced CC1, hepatotoxicity seen in alcoholic patients and in animal models (8, 28-30). Our results demonstrate for the first time that inhibition of P-450 2E1 with a specific antibody completely abolishes the enhanced CC1,-induced lipid peroxidation of microsomes from the ethanol-fed animals, establishing the unquestionable role of this cytochrome in potentiation of CC1,-induced lipid peroxidation. As a corollary, we have also observed similar inhibition by anti-P-450 2E1 IgG of the accentuated iron-induced lipid peroxidation in liver microsomes from animals with ALD. The synergism between the hepatotoxicity induced by excessive iron and alcohol has long been suggested (31-33), and clinical evidence also exists to implicate long-term alcohol intake as a permissive factor in the development of liver fibrosis in young patients with hereditary hemachromatosis (34).These observations may be mechanistically explained with the induction of P-450 2E1 by ethanol as the primary event leading to the potentiated peroxidative injury by iron of the hepatocyte organelle from livers with ALD.

TABLE2. Micrommal P-4502E1 activity m with hydroxylation ~

e

d

~

PNP hydroxykm with PNP hydroqration Microwmee

Control Ethan0l-expos-d

(nmoUminimg protein)

0.38 4.67

2 f

0.15“ 1.53’

anti-P-460 ZEl IgG (nmoUmidmg protein)

0.17

2

0.07

0.44 2 0 . 2 9

Hydroxylation of PNP by microsomes was determined with or without anti-P-450 2E1 I& a8 described in “Materials and Methods.” “Data expressed as mean IT S.D. ‘p < 0.05 compared with control microsomes.

Ethanol offered an in uztro protective effect on microsomes from animals with advanced ALD. Even though our study did not address mechanisms underlying this effect, it is possible that ethanol acted as a free radical scavenger (35) to provide the protective effect against lipid peroxidation. Alternatively, ethanol might have caused direct inhibition of P-450 2El-dependent free radical formation. The extrapolation of this in U ~ ~ F result to the possible implication in uivo is difficult. However, this may suggest that sudden withdrawal of ethanol from alcoholic patients with induced cytochrome P-4502E1 would create a situation in which hepatocytes become vulnerable to further oxidant stress. This may underlie our previous observation in our ALD mode1 that the degree of alcohoIic liver injury correlates with the magnitude of the oscillation for daily blood alcohol levels in these animals (36).Obviously, further in uivo experiments are needed to explore this possibility. REFERENCES I. Ingelman-Sundberg M,Johansson I. Mechanisms of hydroxyi radical formation and ethanol oxidation by ethanol-inducible and other forms of rabbit liver microsomal cytochrome P-450. J Biol Chem 1984;259:6447-6458. 2. Ekstrijm G, Ingelman-Sundberg M. Rat liver microsomal NADPHsupported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-45OIIEl). Biochem PharUCOI 1989;38:1313-1319. 3. Lieber CS. Mechanisms of ethanol induced hepatic iqjury, Pharm a d Ther 1990;46:1-41.

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a role of cytochrome P-450 in liver microsomal ethanol oxidation. 4. Koop DR, Crump BL, Nordblom GD, Coon MJ. Immunochemical Arch Biochem Biophys 1984;235:228-238. evidence for induction of the alcohol-oxidizingcytochrome P-450 of rabbit liver microsomes by diverse agents: ethanol, imidazole, 20. Schnier GG, Laethem CL, Koop DR. Identification and induction of cytochromes P450, P450IIEl and P450IA1 in rabbit bone trichloroethylene, acetone, pyrazole, and isoniazid. Proc Natl Acad Sci USA 1985;82:4065-4069. marrow. J Pharmacol Exp Ther 1989;251:790-796. 5. Ryan DE, Koop DR, Thomas PE, Coon MJ, Levin W. Evidencethat 21. Koop DR. Hydroxylation of p-nitrophenol by rabbit ethanolinducible cytochrome P-450 isozyme 3a. Mol Pharmacol 29: isoniazid and ethanol induce the same microsomal cytochrome 399-404. P-450 in rat liver, an isozyme homologous to rabbit liver cytochrome P-450 isozyme 3a. Arch Biochem Biophys 1986;246: 22. Koop DR. Inhibition of ethanol-inducible cytochrome P450 IIEl by 3-amino-1,2,4-triazole.Chem Res Toxicol 1990;3:377-383. 633-644. 6. Ardies CM, Lasker JM, Lieber CS. Characterization of the 23. Reinke LA, Lai EK, DuBose CM, McCay PB. Reactive free radical generation in uiuo in heart and liver of ethanol-fed rats: correcytochrome P-450 monooxygenase system of hamster liver milation with radical formation in vitro. Proc Natl Acad Sci USA crosomes: effects of prior treatment with ethanol and other 1987;84:9223-9227. xenobiotics. Biochem Pharmacol 1987;36:3613-3619. 7. Lindros KO, Cai Y, Penttila KE. Role of ethanol-inducible 24. YOO J-S H, Ning SM, Pantuck CB, Pantuck EJ, Yang CS. Regulation of hepatic microsomal cytochrome P450IIE1 level by cytochromeP-450 IIEl in carbon tetrachloride-induceddamage to centrilobular hepatocytes from ethanol-treated rats. HEPATOLOGY dietary lipids and carbohydrates in rats. J Nutr 1991;121:959-965. 25. 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FEBS Lett 28. Williams AT, Burk RF. Carbon tetrachloride hepatotoxicity: an 1985;183:265-269. example of free radical-mediated injury. Semin Liver Dis 1990; 11. Recknagel RO, Glende EA Jr, Britton RS. Free radical damage 10279-284. and lipid peroxidation. In: Meeks RG, Harrison SD, Bull RJ eds. Hepatotoxicology. Boca Raton, FL: CRC Press, 1991:401- 29. Reinke LA, Lai EK, McCay PB. Ethanol feeding stimulates trichloromethyl radical formation from carbon tetrachloride in 436. liver. Xenobiotica 1988;18:1311-1318. 12. Powell LW, Bassett ML, Halliday JW.Hemochromatosis: 1980 30. Hasumura Y, Teschke R, Lieber CS. Increased carbon tetraupdate. Gastroenterology 1980;78:374-381. chloride hepatotoxicity and its metabolism after chronic ethanol 13. Bacon BR, Tavill AS, Brittenham GM, Park CH, Recknagel RO. consumption. Gastroenterology 1974;66:415-424. Hepatic lipid peroxidation in vivo in rats with chronic iron 31. Powell LW. The role of alcoholism in hepatic iron storage disease. overload. J Clin Invest 1983;71:429-439. Ann NY Acad Sci 1975;252:124-134. 14. CederbaumAI,Dicker E, Cohen G. Role of hydroxyl radicals in the iron-ethylenediaminetetraaceticacid medicated stimulation of 32. Valenzuela A, Fernandez V, Videla LA. Hepatic and biliary levels of glutathione and lipid peroxides following iron overload in the microsomal oxidation of ethanol. Biochemistry 1980;19:3698rat: effect of simultaneous ethanol administration. Toxicol Appl 3704. Pharmacol 1983;70:87-95. 15. Puntarulo S, Cederbaum AI. Comparison of the ability of ferric complexes to catalyze microsomal chemiluminescence, lipid per- 33. Videla LA. Hepatic antioxidant-sensitive respiration: effect of ethanol, iron and mitochondrial uncoupling. Biochem J 1984;223: oxidation,and hydroxyl radical generation. Arch Biochem Biophys 885-891. 1988;264:482-491. 16. Dicker E, Cederbaum AI. Hydroxyl radical generation by mi- 34. Bassett ML, Halliday JW,Powell LW. Value of hepatic iron measurements in early hemochromatosis and determination of crosomes after chronic ethanol consumption. Alcohol Clin Exp Res the critical iron level associated with fibrosis. HEPATOLOGY 1986; 1987;11~309-314. 624-29. 17. Kamimura S, Gaal K, Britton RS,Bacon BR, TriadatilopoulosG, Tsukamoto H. Increased 4-hydroxynonenallevels in experimental 35. Klein SM, Cohen G, Lieber CS, Cederbaum AI. Increased microsomal oxidation of hydroxyl radical scavenging agents and alcoholic liver disease: association of lipid peroxidation with liver ethanol after chronic consumption of ethanol. Arch Biochem 1992;16:448-453. fibrogenesis. HEPATOLOGY Biophys 1983;223:425-432. 18. Tsukarnoto H, Gaal K, French SW. Insights into the pathogenesis 36. Tsukamoto H, French SW, Largman C. Correlation of cyclical of alcoholic liver necrosis and fibrosis: status report. HEPATOLOGY blood alcohol levels with progression of alcoholic liver injury. 1990;12:599-608. Biochem Arch 1985;1:215-220. 19. Koop DR, Nordlom GD, Coon MJ. Immunochemicalevidence for

Role of cytochrome P-450 2E1 in ethanol-, carbon tetrachloride- and iron-dependent microsomal lipid peroxidation.

This study investigated the role of cytochrome P-450 2E1 in enhanced microsomal lipid peroxidation in experimental alcoholic liver disease. We also ex...
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