- Email: [email protected]
Predominant role of alcohol-inducible P-450s in oxidative damage
Cytochrome P450 and Oxidative Stress
17.1 CYTOCHROMEP-450 AND OXIDATIVE STRESS
Ronald W. Estabrook and Julian A. Peterson, Department of Biochemistry, University of Texas Southwestern Med. Ctr, Dallas, Texas 75235, USA The superfamily of hemoproteins, known as cytochrome P-450, contains over 100 different proteins that "activate" oxygen for the oxidative transformation of a great diversity of endogenous and xenobiotic chemicals. The P-45Os catalyze oxygenase, peroxygenase and oxidase type reactions as well as epoxide and quinone reduction and peroxide rearrangements. One can readily demonstrate in v i t r o the formation of hydrogen peroxide (via superoxide generation) during NADPHoxidation reactions involving P-450s derived from mammalian tissues, in contrast, studies with purified bacterial P-450s show a very tight coupling of substrate metabolism to oxygen u t i l i z a t i o n with l i t t l e or no formation of hydrogen peroxide. However, the addition of "uncoupling chemicals" can f a c i l i t a t e the formation of hydrogen peroxide by both mammalian and bacterial P-450s. The role of the P-450s i n vivo for the formation of peroxides remains unclear. Recent mutagenesis studies suggest the role of a specific amino acid (threonine) in the protein sequence of the bacterial P-450,~, for the coupling of oxygen reduction to subs~P~te hydroxylation. In situ mammalian P-450s have access to r e l a t i v e l y low oxygen concentrations. Studies measuring the influence of oxygen tension on the f a t t y acid hydroxylating a c t i v i t y of a purified bacterial P-450 suggests the operation of a second order reaction mechanism for oxygen u t i l i z a t i o n . Clearly, P-450s play a significant role in oxygen metabolism.
17.3
A N T I O X I D A N T I N T E R V E N T I O N STUDIES IN C Y T O C H R O M E P 4 5 0 - D E P E N D E N T A N D P450I N D E P E N D E N T M O U S E LIVER INJURY M O D E L S Albrecht Wendel, Biochemical Pharmacology, Faculty of Biology, University of Konstanz, D - 7 7 5 0 Konstanz, F.R.G. Four different types of experimental liver injury models in mice were compared with respect to the influence of the modulation of the antioxidant status on the extent of liver injury. I. P450-dependent, acetamlnophen-lnduced liver injury is characterized by massive llpid peroxidatlon (LPO). GSH depletion potentiated liver injury, liposomal vitamin E protected, while desferrioxamine or allopurinol pretreatment had no effect. 2. Allylalcohol-induced liver injury, led to P450-independent extensive LPO. Total protection was achieved by desferrioxamine. Vitamin E or allopurinol did not protect. 3. Hepatitis induced by endotoxin in galactosamine-sensltized mice occurred without any detectable lipid peroxidation. GSH depletion protected, as did dexamethasone, vitamin E, or allopurinol. 4. Galactosamine-sensitized mice treated with rTNFa developed a fulminant hepatitis. GSH depletion protected, while none of the antioxidants tested was protective. These findings demonstrate under in vJvo conditions: a) a P450-mediated LPO-induced hepatotoxiclty. b) a reductive stress by metabolism of allylalcohol via iron redox cycling, c) an involvement of ROS in the production of cytokines, d) a GSHdependent step in the cytotoxic action of TNFa.
PREDOMINANT ROLE OF ALCOHOL-INDUCIBLE P-450s IN OXIDATIVEDAMAGE Minor J. Coon, Elizabeth S. Roberts, and Alfin D. N. Vaz Department of Biological Chemistry, Medical School, The University of Michigan, Ann Arbor, M148109, U.S.A.
161
17,2
As shown previouslyin thislaboratory,purifiedrabbitliver microsomal cytochrome P-450 form 2 (P-450IIIg)catalyzes the reductlve cleavage of hydroperoxides to yield hydrocarbons and eitheraldehydes or ketones. W e have found that lipid hydroperoxides are the physiological substratesfor thisreactionand have recentlyshown thatthe alcohol-inducibleform of liver rnicrosomalcytochrome P450 (form 3a; UEI) is the most activeof the variousisozymes [Proc. Natl. Acad. Sci. U S A (1990), in press]. Epidemiologicaievidence in severallaboratorieshas revealeda link between chronic alcohol intake and cancer and other
pathological states, and increasing attention has been paid in recent years to the role of P-450 in alcohol-related chemical toxicities, mutagenesis, and carcinogenesis. In many instances,as in the oxidative activationof procareinogens and prohepatoxins, alcohol-inducibleP-450 is primarily involved. Furthermore, the "leakiness,"or poor coupling between 0 2 reductionand substrateoxygenation,with this particularP-450 contributesto the increased formation of
potentially damaging species such as hydrogen peroxide, superoxide, and hydroxyl radicals. Our results indicating the relatively high activity of P-450 HE1 in the reductive cleavage of lipid hydroperoxides raise the interesting question of whether the enhancement of this reaction, with a consequent loss of membrane integrity, may be yet another harmful effect of increased levels of the alcohol-inducible cytochrome after chronic ethanol intake. The role of P-450 form 3d (IIE2), which differs in only 16 amino acid residues from IIE1, is presently under study in all of these respects. (Supported by NIH Grant DK-10339.) POTENTIATION BY 1,2-DIBROMOETHANE OF LIPID PEROXIDATION AND IRREVERSIBLE HEPATOCYTE DAMAGE DUE TO CARBON TETRACHLORIDE Gluseppe Poll*, Elena Chlarpotto*, Fiorella Blasi*, Manuela Aragno§, Ollvlero Dannl§ and Adrlana Comogllo* Department of Experimental Medicine and Oncology, General Pathology Section*, Environmental Pathology Sectlon§, University of Torlno, Italy Together with the interaction among different antloxldants during cellular oxidative damage, it is essential to consider the possible interactlon among pro-oxldant compounds. Xenobiotlcs are more often present in the environment as mixtures and a variety of factors Is in principle able to modulate their toxicity. The association of 1,2-dlbromoethane (DBE) to carbon tetrachlorlde (CC14) poisoning in the isolated rat hepatocyte model produces a significant potentiation on both lipid peroxldatlon and cell enzyme leakage CCI~induced. The increase in malonaldehyde production chronologically precedes hepatocyte damage. When hepatocytes are isolated from alpha-tocopherol preloaded rats, the synergism between CCI. and DBE on both parameters 4 disappears almost completely, l.e. the stimulation of lipid peroxldatlon by the mixture is totally Inhlblted and cell damage significantly reduced. Defined changes in glutathlone metabolism and alpha-tocopherol content have been observed in the treated hepatocytes and their contribution to the reported potentiation discussed.
17.4
17.1 CYTOCHROMEP-450 AND OXIDATIVE STRESS
Ronald W. Estabrook and Julian A. Peterson, Department of Biochemistry, University of Texas Southwestern Med. Ctr, Dallas, Texas 75235, USA The superfamily of hemoproteins, known as cytochrome P-450, contains over 100 different proteins that "activate" oxygen for the oxidative transformation of a great diversity of endogenous and xenobiotic chemicals. The P-45Os catalyze oxygenase, peroxygenase and oxidase type reactions as well as epoxide and quinone reduction and peroxide rearrangements. One can readily demonstrate in v i t r o the formation of hydrogen peroxide (via superoxide generation) during NADPHoxidation reactions involving P-450s derived from mammalian tissues, in contrast, studies with purified bacterial P-450s show a very tight coupling of substrate metabolism to oxygen u t i l i z a t i o n with l i t t l e or no formation of hydrogen peroxide. However, the addition of "uncoupling chemicals" can f a c i l i t a t e the formation of hydrogen peroxide by both mammalian and bacterial P-450s. The role of the P-450s i n vivo for the formation of peroxides remains unclear. Recent mutagenesis studies suggest the role of a specific amino acid (threonine) in the protein sequence of the bacterial P-450,~, for the coupling of oxygen reduction to subs~P~te hydroxylation. In situ mammalian P-450s have access to r e l a t i v e l y low oxygen concentrations. Studies measuring the influence of oxygen tension on the f a t t y acid hydroxylating a c t i v i t y of a purified bacterial P-450 suggests the operation of a second order reaction mechanism for oxygen u t i l i z a t i o n . Clearly, P-450s play a significant role in oxygen metabolism.
17.3
A N T I O X I D A N T I N T E R V E N T I O N STUDIES IN C Y T O C H R O M E P 4 5 0 - D E P E N D E N T A N D P450I N D E P E N D E N T M O U S E LIVER INJURY M O D E L S Albrecht Wendel, Biochemical Pharmacology, Faculty of Biology, University of Konstanz, D - 7 7 5 0 Konstanz, F.R.G. Four different types of experimental liver injury models in mice were compared with respect to the influence of the modulation of the antioxidant status on the extent of liver injury. I. P450-dependent, acetamlnophen-lnduced liver injury is characterized by massive llpid peroxidatlon (LPO). GSH depletion potentiated liver injury, liposomal vitamin E protected, while desferrioxamine or allopurinol pretreatment had no effect. 2. Allylalcohol-induced liver injury, led to P450-independent extensive LPO. Total protection was achieved by desferrioxamine. Vitamin E or allopurinol did not protect. 3. Hepatitis induced by endotoxin in galactosamine-sensltized mice occurred without any detectable lipid peroxidation. GSH depletion protected, as did dexamethasone, vitamin E, or allopurinol. 4. Galactosamine-sensitized mice treated with rTNFa developed a fulminant hepatitis. GSH depletion protected, while none of the antioxidants tested was protective. These findings demonstrate under in vJvo conditions: a) a P450-mediated LPO-induced hepatotoxiclty. b) a reductive stress by metabolism of allylalcohol via iron redox cycling, c) an involvement of ROS in the production of cytokines, d) a GSHdependent step in the cytotoxic action of TNFa.
PREDOMINANT ROLE OF ALCOHOL-INDUCIBLE P-450s IN OXIDATIVEDAMAGE Minor J. Coon, Elizabeth S. Roberts, and Alfin D. N. Vaz Department of Biological Chemistry, Medical School, The University of Michigan, Ann Arbor, M148109, U.S.A.
161
17,2
As shown previouslyin thislaboratory,purifiedrabbitliver microsomal cytochrome P-450 form 2 (P-450IIIg)catalyzes the reductlve cleavage of hydroperoxides to yield hydrocarbons and eitheraldehydes or ketones. W e have found that lipid hydroperoxides are the physiological substratesfor thisreactionand have recentlyshown thatthe alcohol-inducibleform of liver rnicrosomalcytochrome P450 (form 3a; UEI) is the most activeof the variousisozymes [Proc. Natl. Acad. Sci. U S A (1990), in press]. Epidemiologicaievidence in severallaboratorieshas revealeda link between chronic alcohol intake and cancer and other
pathological states, and increasing attention has been paid in recent years to the role of P-450 in alcohol-related chemical toxicities, mutagenesis, and carcinogenesis. In many instances,as in the oxidative activationof procareinogens and prohepatoxins, alcohol-inducibleP-450 is primarily involved. Furthermore, the "leakiness,"or poor coupling between 0 2 reductionand substrateoxygenation,with this particularP-450 contributesto the increased formation of
potentially damaging species such as hydrogen peroxide, superoxide, and hydroxyl radicals. Our results indicating the relatively high activity of P-450 HE1 in the reductive cleavage of lipid hydroperoxides raise the interesting question of whether the enhancement of this reaction, with a consequent loss of membrane integrity, may be yet another harmful effect of increased levels of the alcohol-inducible cytochrome after chronic ethanol intake. The role of P-450 form 3d (IIE2), which differs in only 16 amino acid residues from IIE1, is presently under study in all of these respects. (Supported by NIH Grant DK-10339.) POTENTIATION BY 1,2-DIBROMOETHANE OF LIPID PEROXIDATION AND IRREVERSIBLE HEPATOCYTE DAMAGE DUE TO CARBON TETRACHLORIDE Gluseppe Poll*, Elena Chlarpotto*, Fiorella Blasi*, Manuela Aragno§, Ollvlero Dannl§ and Adrlana Comogllo* Department of Experimental Medicine and Oncology, General Pathology Section*, Environmental Pathology Sectlon§, University of Torlno, Italy Together with the interaction among different antloxldants during cellular oxidative damage, it is essential to consider the possible interactlon among pro-oxldant compounds. Xenobiotlcs are more often present in the environment as mixtures and a variety of factors Is in principle able to modulate their toxicity. The association of 1,2-dlbromoethane (DBE) to carbon tetrachlorlde (CC14) poisoning in the isolated rat hepatocyte model produces a significant potentiation on both lipid peroxldatlon and cell enzyme leakage CCI~induced. The increase in malonaldehyde production chronologically precedes hepatocyte damage. When hepatocytes are isolated from alpha-tocopherol preloaded rats, the synergism between CCI. and DBE on both parameters 4 disappears almost completely, l.e. the stimulation of lipid peroxldatlon by the mixture is totally Inhlblted and cell damage significantly reduced. Defined changes in glutathlone metabolism and alpha-tocopherol content have been observed in the treated hepatocytes and their contribution to the reported potentiation discussed.
17.4