Glutathione, lipid peroxidation and regulation of cytochrome P-450 activity

Life Sciences, Vol. 31, pp. 779-784 Printed in the U.S.A.

Pergamon Press

GLUTATHIONE, LIPID PEROXIDATION AND REGULATION OF CYTOCHROME P-450 ACTIVITY Walter G. Levine Department of Molecular Pharmacology and Liver Research Center Albert Einstein College of Medicine Bronx, New York 10461 (Received in final form June ii, 1982) Summa ry Depletion of hepatic glutathione leads to an increase in lipid peroxidation and depression of cytochrome P-450-catalyzed metabolism of the azo dye carcinogen, N,N-dimethyl-4-aminoazobenzene. This contributes to the marked decrease in biliary excretion of N-demethylated metabolites of the dye. Parallel time courses are seen for decreased hepatic glutathione, enhanced lipid peroxidation and depressed excretion of dye metabolites. In vitro metabolism of DAB by hepatic i0,000 g supernatant fractions is depressed by iron only after glutathione depletion. In view of the iron requirement for microsomal lipid peroxidation, it is proposed that glutathione depletion leads to an increase in the intracellular iron available for activation of lipid peroxidation. In this way, glutathione may contribute to the regulation of cytochrome P-450 activity. Many xenobiotics form conjugates with hepatic glutathione (GSH) (1,2) catalyzed by several cytosolic GSH S-transferases (2). Frequently the substrate is a highly electrophilic metabolite of a potentially toxic or carcinogenic xenobiotic (3), which implies a significant role for hepatic GSH in detoxication of these chemicals. The hepatocarcinogen, N,N-dimethyl-4-aminoazobe~zene (DAB) is metabolized primarily by N-demethylation and ring-hydroxylation and conjugated products are excreted in bile (4-6). The two demethylation steps and ring-hydroxylation are preferentially catalyzed by different isozymes of cytochrome P-450 (7). Depletion of hepatic GSH leads to depressed formation of demethylated products while ring-hydroxylation is relatively unaffected (8). The present study suggests that depression of DAB metabolism may be mediated in part by lipid peroxidation which is induced after GSH depletion. Materials and Methods Male Wistar rats (180-250 g) were housed under controlled lighting conditions (12 hr light, 12 hr dark) and given free access to food and water. Where indicated, they were injected with diethylmaleate (i.0 ml/kg) or phorone (150 mg/kg) i.p. in corn oil. For GSH determination, the rats were decapitated and livers rapidly removed, homogenized in 4 volumes of 5% trichloroacetic acid in 5 mM EDTA and centrifuged 20 min at 12,000 g. Two-tenths ml of supernatant material was added to 4 ml Ellman's reagent (9) and absorbance determined at 412 nm. 0024-3205/82/080779-06503.00/0 Copyright (c) 1982 Pergamon Press Ltd.

780

GSH, Lipid Peroxidation

and P-450

Vol. 31, No. 8, 1982

Lipid peroxidation was determined by the rate of malondialdehyde formation using thiobarbituric acid (i0). Incubation conditions are described below. Biliary metabolites of [14C]DAB were determined as previously decribed (8). Briefly, this involved collection of bile from anesthetized (urethane 1 g/kg) animals injected i.v. with 1 mg [14C]DAB, hydrolysis of conjugates with Glusulase, ether extraction of primary metabolites and separation by thin layer chromatography. Spots which c0-chromatographed with reference standards were counted by liquid scintillation spectrometry. DAB metabolism was carried out in vitro in the presence of hepatic i0,000 g supernatant fraction or microsomes as previously described (8). [14C]DAB was obtained from New England Nuclear (Boston, MA) and shown to be 96% radiochemically pure by thin layer chromatography (8). Other sources of chemicals were: thiobarbituric acid, NADP and isocitrate, Sigma Chemical (St. Louis, MO); 5,5'-dithiobis-(2-nitrobenzoic acid) (Ellman's reagent), Aldrich Chemical (Milwaukee, WI); DAB, Pfaltz and Bauer (Stamford, CT); Glusulase, Endo Labs. (Garden City, NY). Results Rats were injected with phorone, ~ 0 mg/kg and hepatic GSH and lipid peroxidation determined at various times thereafter. GSH concentrations were maximally depressed at 2 hr, in agreement with others (ii), and gradually returned to control levels by 24 hr (Table I). Similarly, lipid peroxidation was maximally induced at 2 hr and returned to control levels at 24 hr (Table I). In a parallel experiment, metabolism of DAB was measured in vivo at various times after phorone injection by quantitation of biliary metabolites. Production of aminoazobenzene (AB), the di-demethylation product of DAB, was markedly depressed 2 hr after phorone, but returned to normal at 24 hr (Table II). Production of ring-hydroxylated metabolites, 4'-OH-dimethylaminoazobenzene (4'-OH-DAB), 4'-OH-methylaminoazobenzene (4'-OH-MAB) and 4'-OH-aminoazobenzene (4'-OH-AB) was unaffected throughout the experimental period. The results confirm our previous observations that depletion of hepatic GSH leads to selective suppression of biliary N-demethylated metabolities (8). Microsomal lipid peroxidation is highly sensitive to, and apparently dependent on, the presence of iron (12-14). Addition of low concentrations of ferrous sulfate to a i0,000 g supernatant preparation resulted in marked stimulation of lipid peroxidation with GSH-depleted preparations while little effect was seen with control preparations (Table III). Even in the absence of added iron, lipid peroxidation proceeded far more rapidly with GSH-depleted preparations compared to controls. Activity was almost totally inhibited by the addition of EDTA. The greater rate of lipid peroxidation in GSH-depleted preparations was reversed by the addition of cysteine or GSH but not methionine, which actually stimulated activity (Table IV). In vitro metabolism of DAB, measured by disappearance of substrate (DAB) or formation of N-demethylated product (AB), was inhibited by added iron only when using GSH-depleted liver preparations (Table V). In a separate experiment, N-demethylation of DAB by washed microsomea was shown to be considerably more sensitive to added iron than was ring hydroxylation. The values for N-demethylation and ring hydroxylation were 0.233 and 0.090 nmol/min/mg protein, respectively, in the absence of added iron,and 0.035 and 0.078 after preincubation with 0.01 mM iron. Thus, N-demethylation was inhibited 85% and ring hydroxylation 13%.

Vol. 31, No. 8, 1982

GSH, Lipid Peroxidatlon and P-450

781

TABLE I Effect of Phorone on Hepatic GSH and Lipid Peroxidation

Hr after phorone administration 0 2 5 i0 15 24

GSH concentration a'b ~moles/g liver 8.40 1.25 1.90 3.80 5.60 7.85

± ± ± ± ± ±

0.62 0.12 O.ii 0.29 0.50 0.80

Lipid peroxidation a,c AB3 ? 0.040 0.625 0.470 0.317 0.195 0.075

± ± ± ± ± ±

0.003 0.026 0.030 0.026 0.005 0.009

aEach value is the mean ± S.E. of 3-4 rats; assays were carried out in triplicate. bGSH concentrations in controls at O, I0, and 24 hr were 8.4, 7.6 and 8.2 ~moles/g liver, respectively. CFor determination of lipid peroxidation, 0.2 ml of i0,000 g liver supernatant fraction was incubated for 30 min at 37 ° with i0 m M M g C I 2, 250 ~M NADP and 5 mM isocitrate in 0.i M phosphate buffer, pH 7.4, total volume = 1.0 ml. Malondialdehyde was determined by the addition of 2 ml thiobarbituric acid, 0.375% in 15% trichloroacetic acid, and heating at 90 ° for 15 min. After centrifugation, the absorbance at 532 nm was measured. Lipid peroxidation (A532) in controls at O, I0 and 24 hr was 0.040, 0.090 and 0.035, respectivel~ TABLE II Effect of Phorone on Biliary Metabolites of DAB

Hr after phorone administration 0 2 5 i0 24

Percent extractable biliary metabolites a AB 4'-OH-DAB 4'-OH-MAB 4'-OH-AB 24 ± 2 29 ± 4 8.2 ± 0.i 8.0 ± 1.0 3.9 ± 0.7 27 ± 2 ii ± 1 7.1 ± 0.1 17 ± i 29 ± 4 7.8 ± O.5 7.O ± 0.8 16 ± 2 28 ± 2 7.6 ± 0.3 6.6 ± 0.6 26 ± 5 22 ± 3 8.6 ± 0.8 7.1 ± 0.4

avalues are the mean ± S.E. of bile samples from 4 rats. They are calculated from thin-layer chromatograms of ether extracts of Glusulase treated samples.

782

GSH, Lipid Peroxidation and P-450

Vol. 31, No. 8, 1982

TABLE III Iron Stimulation of Lipid Peroxidation

Fe 2+ concentration 0 IO-7M 5 x IO-7M 10-6M 5 x 10-6M IO-5M 5 x 10-5M IO-4M

Controls 0.068 0.048 0.060 0.067 0.088 0.109 0.104 0.138

10-4EDTA no added Fe 2÷

± ± ± ± ± ± ± ±

0.002 0.001 0.002 0.006 0.007 0.003 0.005 0.018

0.010 ! 0.001

Lipid peroxidation (A532) a DiethylmaleatePhoronetreated treated 0.772 ± 0.061 0.972 ± 0.033 0.877 ± 0.067 0.936 ± 0.053 0.937 ± 0.053 1.068 ± 0.079 1.069 ± 0.079 i.ii0 ± 0.063 1.355 ± 0.118 1.189 ± 0.054 1.490 ± 0.055 1.559 ± 0.151 1.315 ± 0.020 0.996 ± 0.018 1.253 ± 0.020 0.974 ± 0.088

0.028 ! 0.003

aEach value is the mean ± S.E. of triplicate determination. TABLE I.

0.003 ~ 0.001 Methods as in

TABLE IV Reversal of Lipid Peroxidation after GSH Depletion

Conditions Controls GSH depleted (DEM) + cysteine + GSH + methionine

Lipid Peroxidation (A~q2) a 0.041 ± 0.006 0.486 ± 0.041 0.044 ± 0.005 0.046 ± 0.003 0.722 ± 0.043

aMethods as in TABLE I. TABLE V Effect of Iron on the in vitro Metabolism of DAB by Control and GSH-Depleted Rats

Fe 2+ concentration

0 IO-7M IO-6M lO-5M IO-4M aEach value is the mean

DAB metabolism a nmoles/min/g liver Substrate (DAB) Product (AB) disappearance formation GSHGSHcontrol depleted control depleted 73 ± 6 59 ± 7 14.5 ± 1.0 14.5 ± 1.2 74 ± 5 65 ± 6 16.1 ± 1.7 15.0 ± 1.6 77 ± 7 62 ± 4 14.2 ± 1.2 16.3 ± 0.9 77 ± 8 48 ± 4 17.8 ± 1.9 8.3 + i.i 71 ± 8 39 ± 4 13.0 ± I.U 6.0 + 0.4 S.E. of 3-4 rats.

Vol. 31, No. 8, 1982

GSH, Lipid Peroxidation and P-450

783

Discussion Increased lipid peroxidation measured in vitro has been seen after administration of GSH depleting agents in vivo and in isolated hepatocytes (15,16). The present investigation reveals a similar relationship using i0,000 g supernatant fractions. Younes and Siegers (ll) reported an inverse relationship between hepatic GSH concentration and lipid peroxidatlon but found that the latter activity did not increase appreciably until hepatic GSH concentrations were below i mM. On the other hand in our experiments, lipid peroxidation was well above control levels when GSH concentrations were as high as 67% of control values of 5.6 mM, 16 hr after phorone administration (Table I). It is felt therefore, that i mM GSH may not necessarily be the critical value for lipid peroxidation as proposed by Younes and Siegers. In this investigation, lipid peroxidation was assayed with hepatic i0,000 g supernatant fractions. Preparations from GSH-depleted animals carried out lipid peroxidation far more rapidly than did control preparations and were more sensitive to added iron (Table III). We find that washed microsomes do not carry out lipid peroxidation to an appreciable extent in the absence of added iron (W.G. Levine and J. Yee, unpublished observations) in confirmation of others (12,14). It is therefore tempting to speculate that iron normally present in the i0,000 g supernatant fraction, and ostensibly within the cell, is in some manner sequestered or otherwise protected by GSH. Iron complexes with GSH have been demonstrated (17) and may exist within the cell. Alternatively GSH may block iron-dependent microsomal lipid peroxidation directly through interaction with the free radicals generated or by way of a GSH-dependent cytosolic factor which may (18) or may not (19) be peroxidase. At least one of the several GSH S-transferases may contribute to this effect. Lipid peroxidatlon destroys cytochrome P-450 activity under various circumstances (20-22). The parallel time courses in vivo of depressed GSH, induced lipid peroxidation and inhibited N-demethylation of DAB after phorone administration (Tables I and II) are consistent with the hypothesis that depressed formation of demethylated metabolite (AB) after GSH depletion is in part mediated by iron-induced lipid peroxidation. This is further supported by the finding that both lipid peroxidation and DAB metabolism in vitro are more sensitive to added iron when carried out with 10,O00 g supernatant fractions from GSH-depleted livers compared to controls. Furthermore, Ndemethylation of DAB catalyzed in vitro by 10,O00 g supernatant fractions from GSH-depleted rat livers is depressed relative to that of controls, while ring hydroxylation is relatively unaffected (8). Recently, a GSH conjugate of demethylated product of DAB was identified in rat bile (23). Depressed formation of this conjugate may contribute materially to the decreased excretion of DAB metabolites after GSH depletion. However, even after 90% depletion, hepatic GSH concentration is still well above the Km for GSH transferase activity (24) and exceeds by an order of magnitude the amount of DAB processed by the liver under conditions of suppressed biliary excretion of metabolites. The recent finding (25) of a single pool for hepatic GSH negates the argument that the 10% residual GSH may represent a pool unavailable for conjugation. We considered the possibility that GSH depleting agents directly supress DAB metabolism. However, two facts cast doubt on this explanation. One, cysteine readily reverses GSH depletion and suppression of demethylation i_~n vivo (8). Two, the concentrations of each of the depleting agents required to inhibit DAB metabolism in vitro are 10-30 times those which are maximally possible within the liver in vivo after injection of doses sufficient to depress DAB demethylation (26).

784

GSH, Lipid Peroxidation

and P-450

Vol. 31, No. 8, 1982

It is concluded that the relatively selective depression of N-demethylation of DAB after depletion of hepatic GSH is largely mediated by an increase in iron-dependent lipid peroxidation. Therefore, the influence of GSH on xenobiotic metabolism is associated not only with conjugate formation but with regulation of cytochrome P-450 activity as well. Acknowledgements Excellent technical contributions were made by Ira Kalfus, Charles Walton, Geraldine Cousins and Judy Yee. This study was supported in part by grants CA 14231, National Cancer Institute, and AM 17702, National Institute of Arthritis, Metabolism and Digestive Diseases. References i. 2. 3.

4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15.

L. F. CHASSAUD, Adv. Cancer Res. 2 9 175-274 (1979). W.B. JAKOBY, Adv. Enzymol. 48 383-4]4 (1978). D.M. JERINA and J.R. BEND, in Biological Reactive Intermediates, Formation, Toxicity and Inactivation (D.J. JOLLOW, J.C. KOCSIS, R. SNYDER and H. VAINEO, eds.), pp. 207-236 Plenum Press, New York (1976). M. ISHIDATA, Z. TAMURA and K. SAMEJIMA, Chem. Pharm. Bull. ii, 1014-1021 (1976). W.G. LEVINE and T.T. FINKELSTEIN,Drug Metab. Dispos. 6, 265-272 (1978). W.G. LEVINE, Drug Metab. Dispos. 8, 212-217 (1980). W.G. LEVINE and A.Y.H. LU, Drug Metab. Dispos. i0, 102-109 (1982). W.G. LEVlNE and T.T. FINKELSTEIN, J. Pharmacol. Exp. Ther. 208 399-405 (1979). G.L. ELLMAN, Arch. Biochem. Biophys. 82, 70-77 (1959). J.A. BUEGE and S.D. AUST, Methods in Enzymol. 5 2 302-310 (1978). M. YOUNES and C.P. SIEGERS, Chem.-Biol. Interact. 34 257-266 (1981). E.D. WILLS, Biochem. J. 113 325-332 (1969). B.A. SVINGEN, J.A. Buege, F.O. O'NEAL and S.D. AUST, J. Biol. Chem. 254 5892-5899 (1979). D.J. KORNBRUST and R.D. MAVIS, Mol. Pharmacol. 1 7 400-407 (1980). M. YOUNES and C.P. SIEGERS, Res. Commun. Chem. Path. Pharmacol. 27 119-

128 (1980). 16. 17.

18. 19.

I. ANU~)I, J. HOGBERG and A.H. STEAD, Acta Pharmacol. Toxicol. 45 45-53 (1979). D.L. RABENSTEIN, R. GRUEVREMONT and C.A. EVANS, in Metal Ions in Biological Systems (H. SIGEL, ed.), pp. 103-141, Marcel Dekker, Inc., New York (1979). D.D. GIBSON, K.R. HORNBROOK and P.B. McCAY, Biochim. Biophys. Acta 620, 572-582 (1980). R. BURK, M.J. TRUMBLE and R.A. LAWRENCE, Biochim. Biophys. Acta 618 35-41

(1980). 20. 21. 22. 23. 24. 25. 26.

W. LEVIN, A.Y.H. LU, M. JACOBSON and R. KUNTZMAN, Arch. Biochem. Biophys. 158 842-852 (1973). T. KAMATAKI and H. KITAGAWA, Biochem. Pharmacol. 22 3199-3204 (1973). R.R. MILES, J.R. WRIGHT, L. BOWMAN and H.D. COLBY, Biochem. Pharmacol. 29 565-570 (1980). B. KETTERER, S.K.S. SRAI, B. WAYNFORTH, D.L. TULLIS, F.E. EVANS and F.F. KADLUBAR, Chem.-Biol. Interact. 38 287-302 (1982). W.H. Habig, M.J. PABST and W.B. JAKOBY, J. Biol. Chem. 249 7130-7139 (1974). B.H. LAUTERBERG and J.R. MITCHELL, J. Clin. Invest. 67 1415-1424 (1981). W.G. LEVINE, Drug Metab. Rev. (1982) In press.