Relationship between covalent binding to microsomal protein and the destruction of adrenal cytochrome P-450 by spironolactone.

Previous investigations have demonstrated that guinea pig adrenal microsomes catalyze an NADPH-dependent activation of spironolactone (SL) resulting in the degradation of cytochrome(s) P-450 and decreases in steroidogenic enzyme activities. Studies were done to evaluate the relationship between the destruction of cytochrome P-450 and the covalent binding to microsomal protein by SL and by 7 alpha-thiospironolactone (7 alpha-thio-SL), an obligatory intermediate in the activation pathway. NADPH-dependent irreversible binding to guinea pig adrenal microsomal protein was demonstrable with 22-14C- and with 35S-labelled SL or 7 alpha-thio-SL as substrates. In the absence of NADPH, there was relatively little binding. NADPH-dependent covalent binding was not demonstrable with hepatic microsomal preparations. The amount of covalent binding to adrenal microsomes was far greater with 7 alpha-thio-SL than with SL and also greater with 35S-labelled than with 14C-labelled substrates. The latter results suggest the possibility of more than one reactive metabolite. Time-course experiments revealed a good correlation between covalent binding and P-450 destruction by SL and by 7 alpha-thio-SL. In addition, the 17 alpha-hydroxylase inhibitor, SU-10'603, and the 17 alpha-hydroxylase substrate, progesterone, prevented both the degradation of cytochrome P-450 and the NADPH-dependent covalent binding by 7 alpha-thio-SL. Reduced glutathione also decreased covalent binding but did not diminish P-450 destruction. The latter results indicate that some of the covalent binding is unrelated to the degradation of cytochrome P-450. However, all of the data are consistent with the hypothesis that 7 alpha-thio-SL is a suicide inhibitor of adrenal cytochrome P-450 and that covalent binding to protein is involved in the degradation of cytochrome P-450.

Alterations of pulmonary benzo[a]pyrene metabolism by reactive oxygen metabolites.

Superoxide anion radical and hydrogen peroxide (H2O2) are reactive oxygen metabolites which are thought to be involved in oxidant-induced lung injuries. Therefore, we studied their effects on the pulmonary metabolism of benzo[a]pyrene (BP) in rat lung microsomes. The microsomes were incubated with xanthine and xanthine oxidase to generate superoxide anion (effects verified with superoxide dismutase) or H2O2 and then the products formed during the metabolism of BP were measured. Both oxygen metabolites inhibit BP hydroxylase activity, i.e., the production of 3- and 9-hydroxybenzo[a]pyrene (phenols) in a concentration-dependent manner. The phenols account for approximately 75% of metabolite formation and are the major products of BP metabolism. Two components of the monooxygenase system responsible for BP metabolism, cytochrome P-450 and NADPH-cytochrome P-450 reductase, are also inhibited by the two oxygen metabolites in a similar manner. Superoxide anion is more effective than H2O2 in the inhibition of both BP hydroxylase and the monooxygenase components. Neither oxygen metabolite has any effect on the formation of minor metabolites of benzo[a]pyrene, i.e., BP-quinones and BP-dihydrodiols. These are the BP metabolites thought to produce toxic effects and which may lead to the formation of carcinogens and/or mutagens. The results of all these experiments suggest that exposure of lung microsomes to oxygen metabolites can lead to a slowing of overall BP metabolism and the increased accumulation of potentially toxic BP metabolites.

Spironolactone metabolism in target tissues. Characteristics of deacetylation in kidney, liver, adrenal cortex, and testes.

Prior investigations demonstrated that many of the actions of spironolactone (SL) required deacetylation of the parent compound as the first step in the formation of biologically active metabolites. Studies were done to characterize the process of deacetylation in several target tissues. The reaction was catalyzed by microsomal and cytosolic fractions of livers, kidneys, adrenal glands, and testes. Microsomal activity was greatest in liver and kidney and far exceeded cytosolic metabolism in those tissues. In adrenal glands and testes, by contrast, deacetylation was greater in cytosolic than microsomal fractions. The metabolism-mediated destruction of adrenal microsomal cytochromes P-450 by SL was enhanced by coincubation of microsomes with cytosol, illustrating the potential importance of combined microsomal and cytosolic metabolism in the actions of SL. The deacetylation of SL was decreased by various esterase inhibitors; the organophosphate compounds were the most potent inhibitors. The effectiveness of the esterase inhibitors varied from tissue to tissue, as well as from microsomes to cytosol within each tissue. The results indicate that SL deacetylation is catalyzed by microsomal and cytosolic esterases in various target tissues; several isozymes appear to be involved. These and prior observations suggest that tissue metabolism of SL is of major importance in the actions of the drug.

Adrenal mitochondrial metabolism of spironolactone. Absence of metabolic activation.

Previous investigations have established that spironolactone (SL) administration to guinea pigs decreases adrenal mitochondrial and microsomal cytochrome P-450 content, and that the latter requires microsomal activation of the drug. Studies were carried out to determine if adrenal mitochondrial metabolism (activation) of SL was similarly involved in the effects of the drug on mitochondrial cytochrome P-450 destruction. Incubation of guinea pig adrenal mitochondria with SL in the absence of NADPH resulted in the formation of 7 alpha-thio-SL as the only metabolite. In the presence of an NADPH-generating system, an unknown polar metabolite was also produced. The mass spectrum of the unknown compound suggested that it was a hydroxylated derivative of SL. Incubation of mitochondrial preparations with 7 alpha-thio-SL also resulted in the formation of a polar metabolite, but the latter had a different HPLC retention time than that of the SL metabolite. Formation of the polar SL metabolite was prevented by metyrapone, an 11 beta-hydroxylase inhibitor, and was greatest in mitochondria from the adrenal zone having the highest 11 beta-hydroxylase activity. Steroid substrates for 11 beta-hydroxylation inhibited the production of the SL metabolite. Mitochondrial incubations with SL or with 7 alpha-thio-SL in the presence or absence of an NADPH-generating system did not affect cytochrome P-450 concentrations. The results indicate that, unlike the microsomal effects of SL, local activation of SL is not responsible for the destruction of adrenal mitochondrial cytochromes P-450. The major adrenal mitochondrial metabolites of SL appear to be 11 beta-hydroxy-SL and 7 alpha-thio-SL.