Quantification of cytochrome P-450-dependent cyclohexane hydroxylase activity in normal and neoplastic reproductive...

It is well established that liver microsomal cytochrome P-450 participates in steroid metabolism and probably also in the metabolism of anti-oestrogens such as tamoxifen (Nolvadex). Thus it is possible that variations in cytochrome P-450 levels may influence the responsiveness of human breast and endometrial carcinomas to endocrine therapy. Therefore a simple sensitive spectrophotometric assay for determining levels of cytochrome P-450-dependent cyclohexane hydroxylation activity in breast and uterine microsomes (microsomal fractions) has been developed. Cyclohexane was chosen as a substrate because of the relatively high levels of cyclohexane hydroxylase activity in tumour microsomes and because cyclohexane serves as a substrate for several forms of cytochrome P-450. As previously described [Senler, Dean, Pierce & Wittliff (1985) Anal. Biochem. 144, 152-158], a direct method utilizing isotope-dilution/gas chromatography-mass spectrometry was also developed in order to confirm the results of the spectrophotometric assay. The average activity (cyclohexane-dependent NADPH oxidation) for 139 human breast-tumour microsome preparations was 1.34 nmol/min per mg, which is in the range of that found in untreated mammalian liver (1-3 nmol/min per mg). Also, high enzyme activity was demonstrated in human ovary, normal uterus as well as uterine leiomyomas. Endocrine status appeared to influence enzyme levels, in that mammary tissue from virgin rats contained significantly (P less than 0.025) higher amounts of activity than did tissues from either pregnant or lactating rats. Furthermore, carbon monoxide, as well as an antibody against rat liver cytochrome P-450, completely inhibited NADPH oxidation by breast-carcinoma microsomes. These results strengthen our hypothesis that tumours with high levels of cytochrome P-450 may have a reduced response to additive endocrine therapy.