Jan Noordhoek

The development of sex differences in the demethylation of ethylmorphine and in its interaction with components of the hepatic microsomal cytochrome P450 system in mice

Abstract The development of sex differences in ethylmorphine N -demethylation and several components of the reaction chain were studied in hepatic microsomes from mice of the CPB-SE strain between 3–11 weeks of age. Sex-specific changes were observed in demethylation rate, type 1 spectral interaction, cytochrome P450 content, and ethylmorphine-induced stimulation of NADPH-cytochrome P450 reductase activity. These changes occurred mainly between weeks 3–7 and were confined to females. It is concluded that the development of the cytochrome P450 system is repressed by androgen during sexual maturation. The kinetic constants of demethylation developed differently from ethylmorphine binding constants. Changes in demethylase were mainly restricted to K m , whereas the changes in type 1 binding only involved the maximum spectral change. In combination with differences observed between the developmental patterns of demethylation rate and cytochrome P450 reductase activities, this demonstrated that the reduction of cytochrome P450-substrate complex is not rate-limiting in ethylmorphine demethylation. The type 1 spectral change was correlated with the amount of cytochrome P450 only when a large portion of the cytochrome was considered inactive in ethylmorphine binding. It is suggested that immature animals possess a low basal level of ethylmorphine binding type 1 sites, which is elevated selectively in females during sexual maturation.

Calculation of competitive inhibition of substrate binding to cytochrome P-450 illustrated by the interaction of d,l-propranolol with d,l-hexobarbital

Various forms of cytochrome P-450 exist. A substrate may bind to these forms completely or partially. In addition, during the interaction of two substrates, several cytochrome P-450 forms may also be involved, either partially or completely. Now a method is described which allows the estimation of the spectral dissociation constants for the binding of two compounds to cytochrome P-450 subforms common to both. The method is illustrated by the competitive interaction of d,l-hexobarbital and d,l-propranolol with cytochrome P-450 which show only a partial overlap for type I binding sites using rat hepatic microsomes. Different subforms of cytochrome P-450 may be characterized by means of crossover studies with several type I compounds using this method.

Sex Differences in the Kinetic Constants of Ethylmorphine Demethylation and Type I Binding to Hepatic Microsomal Cytochrome P-450 in Mice. The Influence of Castration and Testosterone

1. In the CPB-SE mouse strain sex differences were observed in the Km and Vmax of ethylmorphine demethylation and in the deltaAmax of its type I binding to cytochrome P-450. In the CPB-V strain a small sex difference in the Vmax of the demethylation was found, whereas Ks and deltaAmax of type I binding differed considerably. 2. Testosterone pre-treatment of female CPB-SE mice abolished all sex differences, as did castration of males, except in Vmax, which was partially decreased. In the CPB-V strain testosterone pre-treatment of females abolished sex differences in type I binding, but had no effect on ethylmorphine demethylation. 3. Km values exceeded the corresponding Ks in all cases and sex differences in deltaAmax far exceeded those in Vmax. It is concluded that the Km is determined not only by the Ks of type I binding and the reduction rate of the type I complex between ethylmorphine and cytochrome P-450. The larger sex differences in deltaAmax as compared with Vmax may be attributable to type I binding of ethylmorphine to cytochrome P-450 subspecies not involved in its demethylation.

METABOLISM OF FORMALDEHYDE DURING IN VITRO DRUG DEMETHYLATION

Abstract Formaldehyde formation is usually determined as a measure of the metabolism of drug substrates in vitro. We have studied the fate of formaldehyde added to incubates of rat liver microsomes or 9000 g supernatant fraction as well as the effectiveness of semicarbazide in protecting formaldehyde from metabolic degradation. Formaldehyde is known to be oxidized by a cytosolic NAD- and GSH-dependent (form)aldehyde dehydrogenase. We found that, in addition, some other NAD-independent reactions take place in the cytosol. We observed, moreover, that formaldehyde is also metabolized by the 9000 g supernatant fraction fortified with cofactors for hepatic monooxygenase in the absence of NAD. This finding could be attributed to a Hitherto unknown, cytosolic NADP-dependent, GSH-requiring dehydrogenase. The microsomal fraction metabolized formaldehyde only to a small extent. Therefore, in order to use formaldehyde formation as a parameter of drug metabolism, semicarbazide is necessary to protect formaldehyde from further metabolism in the 9000 g supernatant fraction and microsomes. By determining amounts of both formaldehyde and p-chlor-aniline duringp-clor-N-methylanilinedemethylation, it was shown that semicarbazide (4 mM) only partially protected for-maldehyde from further metabolism in the 9000 g supernatant, although semicarbazone formation from the added formaldehyde and semicarbazone progressed more rapidly than formaldehyde metabolism. As higher semicarbazide concentrations inhibit microsomal demethylations, it is concluded that determination of formaldehyde is not a suitable method for determining drug demethylation by the 9000 g liver supernatant. In microsomal incubates, only a low semicarbazide concentration (1.0 mM) was necessary to protect formaldehyde from further metabolism.

The use of competitive inhibition of substrate-binding to cytochrome P450 in the determination of spectral dissociation constants for substrates with multiple types of binding, as illustrated with 1-butanol

Abstract A method is described, which allows (a) the detection of competitive inhibition of binding to cytochrome P450 between two substrates which elicit the same type of spectral change, and (b) the estimation of dissociation constants for one type of spectral binding of a substrate which exhibits multiple interaction with cytochrome P450. This method was used to investigate the interaction of 1-butanol with the type I binding site of cytochrome P450 in liver microsomes from female mice. 1-Butanol was found to competitively inhibit the binding of ethylmorphine, and has an apparent dissociation constant for type I binding of 30 mM.

Sex- and strain-dependent hepatic microsomal ethylmorphine N-demethylation in mice: the roles of type I binding and NADPH-cytochrome P-450 reductase.

The roles of type I binding and NADPH-cytochrome P-450 reductase in ethylmorphine demethylation were investigated in two strains of mice, using sex differences in these activities as a tool. In the CPB-SE strain, females metabolize ethylmorphine faster than males. Sex differences in cytochrome P-450 content and endogenous NADPH-cytochrome P-450 reductase activity were too small to account for this. On the other hand, the differences in the magnitudes of type I spectra and ethylmorphine-induced enhancement of cytochrome P-450 reduction were considerable larger than those in the rates of demethylation. All parameters, except endogenous cytochrome P-450 reduction, were modified in a similar way by testosterone pretreatment: in females they were depressed to the male level, whereas in males they remained unchanged. Castration had no effect in females and enhanced the activities in males. The CPB-V strain exhibited little or no sex differences in ethylmorphine demethylation, cytochrome P-450 content and endogenous cytochrome P-450 reduction. Testosterone pretreatment had little or no influence on these activities. Type I binding and reductase stimulation, however, showed sex differences, comparable to those observed in the CPB-SE strain, which were also abolished by testosterone. A relationship between reductase stimulation and type I binding was observed, which was, apparently, independent of sex or strain. It is concluded that androgen primarily influences the amount of cytochrome P-450-substrate complex formed, but that the reduction of this complex is not rate-limiting in the demethylation of ethylmorphine.

METABOLISM OF HEXOBARBITAL ENANTIOMERS AND INTERACTION WITH CYTOCHROME P-450 IN MALE AND FEMALE MICE AND RATS

ABSTRACT The metabolism of hexobarbital enantiomers by 9000g liver supernatant was determined in mice and rats. Both male and female mice and rats metabolize hexobarbital stereospecifically. In mice the 1-enantiomer is metabolized faster, whereas the opposite was observed in rats. The kinetics of metabolism and type I binding to cytochrome P-450 of the enantiomers was compared in female mice and male rats. In mice K m equaled K S for type I binding, but in rats K m exceeded K S . This suggests that in mice the type I binding site is the active site of the enzyme, whereas in rats this cannot be proved. In female rats the A max for type I binding of the enantiomers differs, indicating a binding to different cytochrome P-450 subspecies. Comparison of the enhancement of NADPH-cyt. P-450 reductase activity by the enantiomers with their rate of metabolism at the same concentration revealed that in rats the reduction of the cytochrome P-450-substrate complex cannot be rate-limiting for the total reaction, whereas in mice this cannot be disproved. This would explain the apparent discrepancy between the kinetics of type I binding and metabolism in rats, which was not found in mice.

The relation between the sex-dependency of type I binding of ethylmorphine and the 1-butanol-induced spectral change in mouse liver microsomes

Abstract 1. A sex difference in the spectral interaction of 1-butanol with liver microsomes from adult mice was observed. In males a profound reverse type I spectrum was elicited, whereas only a small spectral change of irregular shape was apparent in females. This sex difference is the opposite of that observed in the type I binding of ethylmorphine. In immature animals no sex difference was found. Testosterone pretreatment of female mice increased the size of the 1-butanol spectrum concomitantly with a decrease in ethylmorphine binding. 2. Microsomes from males and females did not contain different levels of endogenous substrates. Thus, the presence or displacement of such substrates does not explain the sex differences in type I and reverse type I binding respectively. 3. 1-Butanol was found to interfere with both type II and type I binding. It is concluded that the 1-butanol-induced spectral change consists of at least two components and that the sex difference is due to a sex-dependent type I component.