Cytochrome P-450 activity in human leiomyoma and normal myometrium.

Variations in cytochrome P-450 levels may influence the responsiveness of uterine and breast tissue as well as carcinomas to endocrine therapy and may be of particular importance with agents such as tamoxifen (Nolvadex) where hydroxylation is known to alter therapeutic activities. Therefore, a sensitive spectrophotometric assay of cytochrome P-450 levels in reproductive tissue microsomes was developed to measure cyclohexane hydroxylase activity. Cyclohexane served as a substrate for several forms of cytochrome P-450. Human uterine leiomyomas (uterine fibroid tumor) contained significantly higher (p less than 0.01) cytochrome P-450 activity than adjacent normal myometrium. Specific activities for both leiomyomas (2.87 +/- 0.26 nmol/min/mg) and normal myometrium (1.60 +/- 0.11 nmol/min/mg) were in the range of those observed for untreated rabbit liver microsomes (1 to 3 nmol/min/mg). The contribution of smooth muscle in the specimen, the phase of the menstrual cycle, and the clinical diagnosis did not influence the level of cytochrome P-450 activity.

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

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.

Procedures for measuring cytochrome P-450-dependent hydroxylation activity in reproductive tissues.

Cytochrome P-450 is present in the endoplasmic reticulum at varying concentrations in almost all tissues. However, the existence and role of cytochrome P-450 in normal and neoplastic reproductive tissues has not been clearly demonstrated. Our interest lies in the possibility that variations in cytochrome P-450 levels may influence the responsiveness of breast and endometrial carcinomas to endocrine therapy. This may be of particular importance with agents such as tamoxifen where hydroxylation reactions are known to alter therapeutic activities. Therefore, a simple, sensitive spectrophotometric assay for determining levels of cytochrome P-450-dependent cyclohexane hydroxylase activity in breast and uterine microsomes has been developed. Cyclohexane was chosen as a substrate because of the relatively high levels of cyclohexane hydroxylase activity in tumor microsomes and because cyclohexane serves as a substrate for several forms of cytochrome P-450. In order to confirm the results of the spectrophotometric assay, a direct method utilizing isotope dilution gas chromatography/mass spectrometry (GC/MS) has been developed for detecting low levels of the hydroxylated product, cyclohexanol. By employing a stable isotopically labeled analog of cyclohexanol (cyclohexanol-d12), good agreement was demonstrated between the simple, indirect method (measuring NADPH oxidation at 340 nm) and the more complex, direct method (measuring cyclohexanol formation) utilizing GC/MS. The agreement of results obtained using these two techniques indicates that they are equally valid measures of NADPH-dependent cyclohexane hydroxylase activity. The use of the spectrophotometric method is proposed for rapid, multiple assays such as in the clinical setting, reserving GC/MS analysis for use as a research tool.