The Absorption, Distribution and Excretion of Einalool in the Rat

5 % respectively of the total (whole homogenate) activity; the postmitochondrial (1OOOOg) supernatant contained the whole of the activity. Further, on resuspending the microsomal fraction in the cell sap, all the dimethylnitrosamine demethylase activity was restored. The kinetics of dimethylnitrosamine demethylase were investigated in hepatic 1OOOOg supernatants over a substrate concentration range of 0.05-100mM. The data obtained were plotted according to Hofstee (1959) and were found to contain three distinct lines (Fig. 1). Apparent Michaelis constants were 0.32, 1.5 and 3 5 m ~ , with corresponding maximal rates of 0.88, 1.8 and 4.8pmol of formaldehyde formed/h per g of liver. The stability of dimethylnitrosamine demethylase was investigated by storing 10 OOOg supernatant preparations under N2 at 1-2"C, and determining dimethylnitrosamine demethylase activity after 0,1,3,6,9,13 and 17 days of storage. In addition, microsomal cytochrome P-450 and the activities of two known cytochrome P-450-dependent Ndemethylases, namely ethylmorphine N-demethylase and 4-chloro-N-methylaniline Ndemethylase were also determined. Activities of all four parameters were expressed as percentages of the activities on the day of preparation of the tissue fractions (day zero). The microsomal content of cytochrome P-450 decayed with storage (Fig. 2), being about 40% of the day zero activity after 17 days. The decay curve of the haemoprotein was closely paralleled by the activities of ethylmorphine and 4-chloro-N-methylaniline Ndemethylases. In contrast, dimethylnitrosamine demethylase activity declined only very slowly with time (Fig. 2) and was about 85 % of the day zero activity after 17 days. These results may suggest that cytochrome P-450 and perhaps other components of the hepatic microsomal mixed-function-oxidase complex are not wholly involved in the demethylation in vitro of dimethylnitrosamine. It would appear that cytochrome P-450 is not a rate-limiting step in dimethylnitrosamine demethylation. Two further observations also serve to confirm that hepatic dimethylnitrosamine demethylation is essentially not a cytochrome P-450-dependent process. First, spectral interaction studies showed that dimethylnitrosamine at concentrations of 0.1-7.0m~ did not exhibit either a Type I or a Type I1 spectrum with cytochrome P-450 in microsoma1 preparations obtained from both normal and sodium phenobarbitone treated animals. Secondly, enzyme inhibition studies revealed that dimethylnitrosamine at concentrations of or 10-2~ did not significantly inhibit the microsomal activities of aniline 4-hydroxylase, biphenyl 4-hydroxylase, ethylmorphine N-demethylase, 4-chloroN-methylaniline N-demethylase and 4-nitroanisole 0-demethylase. Our results indicate that the demethylation of dimethylnitrosamine is likely to be a multi-component process and not a single-step enzymic oxidative demethylation.