Abstract
The mechanism of benzene oxygenation in liver microsomes and in reconstituted enzyme systems from rabbit liver was investigated. It was found that the NADPH-dependent transformation of benzene to water-soluble metabolites and to phenol catalyzed by cytochrome P-450 LM2 in membrane vesicles was inhibited by catalase, horseradish peroxidase, superoxide dismutase, and hydroxyl radical scavengers such as mannitol, dimethyl sulfoxide, and catechol, indicating the participation of hydrogen peroxide, superoxide anions, and hydroxyl radicals in the process. The cytochrome P-450 LM2-dependent, hydroxyl radical-mediated destruction of deoxyribose was inhibited concomitantly to the benzene oxidation. Also the microsomal benzene metabolism, which did not exhibit Michaelis-Menten kinetics, was effectively inhibited by six different hydroxyl radical scavengers. Biphenyl was formed in the reconstituted system, indicating the cytochrome P-450-dependent production of a hydroxycyclohexadienyl radical as a consequence of interactions between hydroxyl radicals and benzene. The formation of benzene metabolites covalently bound to protein was efficiently inhibited by radical scavengers but not by epoxide hydrolase. The results indicate that the microsomal cytochrome P-450-dependent oxidation of benzene is mediated by hydroxyl radicals formed in a modified Haber-Weiss reaction between hydrogen peroxide and superoxide anions and suggest that any cellular superoxide-generating system may be sufficient for the metabolic activation of benzene and structurally related compounds.
Highlights
NADPH-dependent transformation of benzene to mediated benzene oxygenation mechanism in microsomes was water-soluble metabolites and to phenol catalyzed by cytochrome P-450 LM2 in membrane vesicles was inhibited by catalase, horseradish peroxidase, superoxide dismutase, and hydroxyl radical scavengers such as mannitol, dimethyl sulfoxide, and catechol, indicating the participatioonf hydrogen peroxide, superoxide anions, and hydroxyl radicals in the process
Microsomal phospholipids were extracted from liver microsomes obtained from phenobarbitalmation of benzene metabolites covalently bound to pro- treated rabbits according to Bligh and Dyer [18] and stored under tein was efficiently inhibited by radical scavengersbut nitrogen in sealed tubes at -20 “C. Cytochrome P-450LM2I and not byepoxide hydrolase
The results indicatethat the NADPH-cytochrome P-450 reductase was purified to apparent hommicrosomal cytochrome P-450-dependent oxidation of ogeneity from liver microsomes of phenobarbital-treated rabbits esbenzene is mediated by hydroxyl radicals formed in a modified Haber-Weissreaction between hydrogen peroxide and superoxide anions and suggest that any cellular superoxide-generating system may be sufficient for the metabolic activation of benzene and structurally related compounds
Summary
Incubations of benzene (17 p ~in)the presence of NADPH with reconstituted membrane vesicles containing NADPHcytochrome P-450 reductase and cytochrome P-450 LM2 resulted in the production of several lipid- and water-soluble metabolites, phenol constituting the major product (Fig. 1). The formation of both phenol and the other metabolites was strongly inhibited by the addition of the hydroxyl radical scavenger Me2S0 orof catalase to the incubation system This indicates that hydrogen peroxide and hydroxyl radicals participate in the cytochrome P-450-dependent metabolic transformation of benzene. The cytochrome P-450 LM2-dependent conversion of benzene to water-soluble products, including the formation of covalently protein-bound benzene metabolites, was compared with respect to the inhibition profile obtained by addition of scavengers of hydrogen peroxide, superoxide anions, and hydroxyl radicals with the hydroxyl radical-mediated destruction of the deoxyribose. Nmol of cytochrome P-450 as described under “Experimental Proce- In ~iposomescontaining NADPH-c+,tochrome p-450 reducdures.”
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