Comparative studies on hepatic dimethylnitrosamine demethylase and some xenobiotic-metabolizing enzymes in the rat.

Dimethylnitrosamine is a potent hepatocarcinogen in a number of animal species including the rat (Magee & Barnes, 1956). Previous investigations into the hepatic metabolism of dimethylnitrosamine in vitro have shown that this compound is metabolized to formaldehyde, a process requiring NADPH and molecular oxygen (Magee & Vandekar, 1958). The requirement for these two cofactors has led to the suggestion (Brouwers & Emmelot, 1960; Mizrahi & Emmelot, 1963) that dimethylnitrosamine is N-demethylated by enzymes of the hepatic mixed-function oxidase system dependent on cytochrome P-450, the terminal oxygenase (Mason et al., 1965). However, in a previous communication (Lake et ul., 1974) we reported several important anomalies between the enzymic degradation of dimethylnitrosamine and of some typical substrates that are metabolized by microsomal mixed-function oxidases. In the present communication we present results extending our previous findings and, in addition, report the detection of methanol formed as a consequence of the metabolism of dimethylnitrosamine by rat liver preparations. Male Wistar albino rats (80-12Og) were used in these studies; they were allowed free access to laboratory diet and water. Assays of ethylmorphine N-demethylase (Holtzman et al., 1968), aniline Chydroxylase (Nakanishi et al., 1971) and dimethylnitrosamine demethylase (Lake et al., 1974) were performed on hepatic lOOOOg supernatant fractions. Dimethylnitrosamine was determined in deproteinized tissue incubates in a Pye series 104 gaschromatographequippedwithaflame-ionizationdetector andwitha 10%Carbowax 20M column at 120°C. Methanol was similarly measured in a Chromosorb 102 column at 89°C. Compound SKF 525A and metyrapone [2-methyl-l ,2-bis(3-pyridyl)-l-propanone] are known inhibitors of hepatic microsomal xenobiotic metabolism (Anders & Mannering, 1966; Leibman, 1969; Anders, 1971). In the first series of experiments the effects of these two compounds on the activities of dimethylnitrosamine demethylase, ethylmorphine N-demethylase and aniline Chydroxylase in vitro were investigated. Compound SKF 525A, at concentrations of 0.01 and 1 mM, significantly inhibited the activities of ethylmorphine N-demethylase and aniline Chydroxylase (Table 1) examples of a type I and a type I1 substrate of the mixed-function oxidase system (Schenkman et al., 1967). In contrast, the percentage inhibition of dimethylnitrosamine demethylase was only significant at the higher inhibitor concentration. Metyrapone inhibited the activity of all three enzymes at a concentration of lOmM but only ethylmorphine N-demethylase was significantly inhibited in the presence of 1 mwmetyrapone. However, dimethylnitrosamine demethylase activity was stimulated in the presence of 1 m-metyrapone. Our previous work has shown that pyrazole and 3-amino-l,2,4-triazole, potent inhibitors of methanol and ethanol metabolism (Feytmans & Leighton, 1973), profoundly inhibit the metabolism of dimethylnitrosamine in the intact animal (Phillips et al., 1974). The addition of 1 mwpyrazole produced a marked inhibition of dimethylnitrosamine demethylase activity in vitro, however, the activities of the two mixed-function oxidase enzymes remained unaffected (Table 1). 3-Amino-l,2,4-triazole at a concentration of 1 0 m produced a 75 % inhibition of dimethylnitrosamine demethylase but only 25 and