Enzymology and regulation of xenobiotic and steroid metabolism in placenta.

(Petters et al., 1983) permits the determination of small amounts of 7-aminoclonazepam (about 20 pmol) and is thus useful for measuring low activities in various tissues. All investigated foetal livers catalysed the nitroreduction reaction at different rates. The mean activity was in the same range as that in Rhesus monkey liver, but only about 15% of the activity in human adult liver. Such discrepancies between foetal and adult livers are usually encountered for many drug-oxidation reactions. The enzyme reaction obeyed Michaelis-Menten kinetics and the apparent K , values are in reasonable agreement. The levels that are normally encountered in treated patients (Sjo et al., 1975; Stah1 et al., 1983) are far below these K , values, indicating that the enzymes are operating in uiuo under first-order conditions. Our results are in agreement with the data of Juchau (1970, 1971). In the presence of CO the reduction of clonazepam was almost completely inhibited. Omission of NADP yielded very low metabolic rates, indicating that this cofactor is required for the reaction. We also demonstrated an extreme sensitivity to O2 in that the presence of air in the incubation tubes inhibited the nitroreduction more than 95%. As far as the dependence on NADP and sensitivity to CO and O2 is concerned, the reaction conforms with the concepts of the microsomal nitroreductase (Gillette et al., 1968) and may thus be catalysed by cytochrome P-450. Reduction reactions are catalysed not only in microsomes but also in the soluble fraction of the liver (Fouts & Brodie, 1957). We did not investigate the reduction in the soluble fraction of the human foetal liver, but assume that is has less quantitative significance than the microsomal metabolism. Nitroreductase activity in the homogenates of foetal hepatocyte cultures may thus be derived both from soluble and microsomal enzyme. The specific activity when based on total protein in the homogenate was only about 3% of microsomal activity, which is consistent with the dilution effect by non-microsomal protein. Our study thus has shown that the human foetal liver contains a microsomal nitroreductase enzyme system which catalyses the reduction of clonazepam, an anti-epileptic drug. Although the metabolic rates are low in human foetuses and hence the quantitative importance of this enzyme for the foetal clearance of clonazepam is doubtful, its presence may have toxicological implications in the metabolism of other nitro compounds, since many sideeffects of drugs are thought to be mediated via the formation of N-oxygenated metabolites from amines (Uehleke, 1973). Some of the toxic effects of chloramphenicol and 5nitrofurans (Zenser et al., 1981) have been ascribed to their nitroreduced metabolites, although different opinions on this issue have been expressed. Whether the reduced clonazepam metabolite may account for some of the sideeffects of this drug is not yet known.