BIOACTIVATION OF DIETARY PHENOLS BY HEMOPROTEINS

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There is a great deal of interest in naturally occurring phenols because of their enormous potential as agents for antioxidant therapy and cancer prevention. Unfortunately, however, these substances also lead to the formation of free radicals, mutations, tumors, and cytotoxicity. Mechanisms underlying the various biological effects of phenols must be elucidated before those factors which influence the blend of beneficial and determinant properties can be more fully understood. The metabolic activation of these compounds by cytochrome P450 involves well known reactive metabolites such as epoxides and quinones, but phenoxy radicals produced by P450-catalyzed one-electron oxidation can add molecular oxygen para to the hydroxy group to form hydroperoxy-cyclohexadienones, referred to in the chemical literature as 'peroxyquinols.' This novel pathway has not been widely recognized because peroxyquinols do not accumulate, but are rapidly degraded in biological systems. During the current funding period, it has been demonstrated, using a series of alkyl-substituted phenols, that peroxyquinol formation occurs to a significant extent both in liver microsomes and in isolated hepatocytes. The relative contribution of this pathway is influenced by the substrate structure and P450 isozyme. Peroxyquinols are rapidly degraded by various hemeproteins along complex pathways, form radicals and electrophilic products, selectively oxidize methionine residues of intact proteins, and severely damage cells. The data strongly support continued studies on the roles of peroxyquinols in mediating the biological properties of phenolic compounds. We now propose to extend the work from relatively simple alkylphenols to more complex compounds that contain structural features common to naturally occurring phenols. The specific aims are as follows: (1) Investigate the contribution of peroxyquinol formation to the microsomal and cellular metabolism of mononuclear phenols containing ortho-methoxy or meta-hydroxy substituents, and polynuclear phenols with methyl substituents. Determine if estrogens are oxidized via the peroxyquinol pathway. (2) Identify products formed and pathways involved in the degradation of peroxyquinols by hemeproteins. Data obtained with hepatic cytochromes P450, and site-specific mutants of P450cam and myoglobin will elucidate hemeprotein mechanisms, and provide information on biologically active products of peroxyquinols. (3) Study the effects of peroxyquinols on rat hepatocyte and murine keratinocytes. Determine the consequences of peroxyquinol-mediated oxidations of methionine residues in non-heme proteins, and determine if DNA-bound metal ions reduce peroxyquinols to radical species that damage DNA. Accomplishing these specific aims will significantly enhance our knowledge of this novel oxidative pathway and its potential to mediate the biological effects of certain phenolic compounds.