Precision cut liver slices from humans and rats were used to investigate the covalent binding of xenobiotics to the DNA by means of the (32)P-postlabeling technique. Human liver slices were incubated with the structurally related steroid hormones chlormadinone acetate (5 mu g/ml), cyproterone acetate (0.01-5 mu g/ml), megestrol acetate (5 mu g/ml), and the positive control 2-aminofluorene (0.01-5 mu g/ml), which is known for its marked ability to form DNA-adducts in vivo. Rat liver slices were incubated with cyproterone acetate in concentrations of 0.1, 1, and 5 mu g/ml. The functional viability and metabolic activity of the slices were shown to be sufficiently maintained during the incubation time by measurement of the intracellular K(+)-content and the metabolic turnover of the model substrate 7-ethoxycoumarin, respectively. All three test substances and the control induced DNA-adducts in human liver slices, however, with a different adduct pattern. While the total DNA-adduct levels obtained with cyproterone acetate and megestrol acetate were in the same order of magnitude (on average 1000 DNA-adducts/10(9) nucleotides after incubation with 5 mu g /ml), the relative adduct labeling calculated for chlormadinone acetate was about 400. Following in vitro incubation of rat liver slices with cyproterone acetate, the relative adduct labeling values increased proportionally with increasing concentrations and added linearily to in vivo generated DNA-adducts. At the level of liver slices, different DNA-adduct patterns were induced by cyproterone acetate in rat and man. In contrast to the finding of others, using rat hepatocytes, the relative adduct labeling values of cyproterone acetate and megestrol acetate were in the same order of magnitude after incubation with human liver slices. The present study indicates that liver slices are a useful tool to investigate the in vitro DNA-adduct inducing potential of xenobiotics.
1 We are reporting investigations into the potential of the steroid hormones chlormadinone acetate (CMA), cyproterone acetate (CPA), ethinylestradiol (EE2) gestodene (GEST), megestrol acetate (MGA), norethis terone acetate (NET-Ac), estradiol (E 2), and progester one (P) to form DNA-adducts in rat liver in vivo. 2 Compound-related DNA-adduct spots were detected in male and female rat liver following CMA, CPA, and MGA using the 32P-postlabeling-technique. Substance- specific DNA-adducts were also observed in male rats after administration of E2. The other compounds showed no DNA-adduct formation. After treatment with CMA, CPA or MGA, the relative adduct labeling (RAL) differed sex- and substance-specifically.
Cyproterone acetate (CPA) is a synthetic steroid hormone used in the therapy of prostate cancer in men and different forms of acne and hirsutism in women. CPA has been shown by 32P-postlabeling analysis to bind covalently to hepatic DNA of rats in vivo and in vitro. A prerequisite for DNA adduct formation of CPA is metabolic activation of the drug to a reactive intermediate. In the present study bile was collected from [3H]CPA-treated female rats and, following chromatographic separation of bile extracts, fractions of the eluate were examined for the presence of reactive metabolites which were able to form adducts with calf thymus DNA in vitro. The formation of adducts was detected by 32P-postlabeling analysis. One major metabolite of CPA present in the bile extracts was isolated and, following a thorough structural elucidation by mass spectrometry and 1H-NMR, this metabolite was identified as 3 alpha-hydroxy-cyproterone acetate (3 alpha-OH-CPA). This metabolite was able to form the same major adduct in vitro which has been observed before in CPA-treated rats in vivo and in rat hepatocytes in vitro. A number of already known or putative metabolites of CPA were available as authentic standards and these were also examined for their propensity to form adducts in vitro. A positive result was obtained for 3-O-acetyl-cyproterone acetate, which formed the same major adduct as 3 alpha-OH-CPA. However, the presence of this putative metabolite in rat bile could not be demonstrated. Besides 3 alpha-OH-CPA, additional reactive metabolites of CPA were present in the bile extracts, however, since these were only minor components, their chemical structures could not be elucidated.
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