Use of precision cut human liver slices for studying the metabolism and genotoxic potential of xenobiotics by means of the (32)P-postlabelling technique: steps towards method validation using testosterone and 2-aminofluorene.

In the present study, a new in vitro model combining the short-term incubation of precision-cut human liver slices with DNA-adduct analysis by the (32)P-postlabelling technique is proposed for investigation of the genotoxic potential of xenobiotics. For method validation, the metabolic turnover of testosterone (TES) and the DNA-adduct inducing potential of 2-aminofluorene (2-AF) were used. Precision-cut human liver slices were prepared from a total of 12 human liver samples which were freshly obtained as parts of resectates from liver surgery. The slices were incubated as submersion cultures with TES and 2-AF for up to 6 h in 12-well tissue culture plates at concentrations of 10-50 and 0.06-28 µM, respectively. Slices recovered from the slicing procedure in the 4 °C cold Krebs-Henseleit buffer as indicated by intracellular potassium concentrations which increased for 2 h and then remained stable until the end of the incubation. TES was extensively metabolized by human liver slices with a similar metabolite pattern as observed in vivo. Almost 90% of the metabolites were conjugates. Major phase-I metabolites were androstendione, 6ß-OH-androstendione, 6ß-OH-TES, and 15ß-OHTES. After incubation with 2-AF, substance related DNA-adducts were detected which increased dose-dependently from 12 to 1146 adducts per 10(9) nucleotides. The adduct pattern consisted of one major adduct spot, A, representing 80-90% of the total adduct level and up to four minor adduct spots, B-E. In summary, the present data demonstrate that precision-cut liver slices are a valuable alternative in vitro system for DNA-adduct determination to screen chemicals for potential genotoxicity in humans.

DNA adduct formation of selected sex steroids in human liver slices in vitro.

We report investigations into the potential of the steroid hormones chlormadinone acetate (CMA), cyproterone acetate (CPA), dexamethasone (DEX), estradiol (E(2)), ethinylestradiol (EE(2)), gestodene (GEST), levonorgestrel (LNG), megestrol acetate (MGA), medroxy progesterone acetate (MPA), mifepristone (MIFE), norethisterone (NET), prednisolone (PRED), progesterone (P) and testosterone (T) to form DNA adducts in precision-cut human liver slices in vitro from 14 male and female donors using the (32)P-postlabelling technique. The synthetic steroid hormones CPA, CMA and MGA generated DNA adducts in human liver slices obtained from all donors. MPA-related adduct spots were only observed in some of the livers tested. No DNA adduct formation was detectable with DEX, EE(2), E(2), GEST, LNG, MIFE, NET, PRED, P and T. The total DNA adduct levels and adduct patterns were different for each compound. On average, total DNA adduct levels decreased in the following order: CPA>MGA>CMAMPA. The DNA adduct levels varied inter-individually. At a treatment concentration of 1mug/ml, the coefficient of variation of the total adduct levels ranged from 38% to 101%. A sex-specific distribution of the DNA adduct formation was only detected after incubation with MPA. MPA-related adduct spots were observed predominantly in the livers of female donors.

Formation of DNA-adducts by selected sex steroids in rat liver.

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), norethisterone acetate (NET-Ac), estradiol (E2), and progesterone (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.

Use of rat and human liver slices for the detection of steroid hormone-induced DNA-adducts in vitro by means of the (32)P-postlabeling technique.

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.

Identification of 3 alpha-hydroxy-cyproterone acetate as a metabolite of cyproterone acetate in the bile of female rats and the potential of this and other already known or putative metabolites to form DNA adducts in vitro.

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.

Reactivity and genotoxicity of arylnitrenium ions in bacterial and mammalian cells.

Electrophilic arylnitrenium ions are considered to be the ultimate reactive intermediates formed by metabolism of mutagenic and carcinogenic arylamines and nitroarenes; they can produce DNA damage by reaction with specific sites on DNA bases. We studied their formation, reactivity and the genotoxic sequelae of their reactions with cellular DNA to understand the mutagenic and carcinogenic activities of arylamines and nitroarenes as a function of their chemical structure. Arylnitrenium ions were generated by the convenient non-metabolic procedure, photolysis of arylazides, to study the reactivity of these ultimate intermediates with DNA, by means of 32P-postlabelling, and the induction of histidine reversions in Salmonella, HPRT mutations and sister chromatid exchange in mammalian (Chinese hamster V79) cells. Good correlations were observed between the DNA-binding potencies and the mutagenic and SCE-inducing potencies of the arylnitrenium ions, among these the nitrenium ions derived from the heterocyclic food mutagens/carcinogens MeIQ, IQ, and MeIQx. This suggests that the reactivity of the arylnitrenium ions and the quantity of adducts formed with DNA are the principal determinants of the final quantity of genetic alterations in Salmonella and in V79 cells. Conversely, the quality of the adducts, that is, the structure of the arylamine residue bound, appears to be of less significance.

32P-postlabelling studies on the DNA adducts of the food mutagens/carcinogens IQ and PhIP--adduct formation in a chemical system, and by rat and human metabolism.

The DNA adducts of the food mutagens/carcinogens IQ and PhIP were studied by means of 32P-postlabelling techniques. Adducts were generated in vitro and in vivo by three techniques: by photolysis of azido-IQ and azido-PhIP in the presence of dGp, calf-thymus DNA or Salmonella; by administration of IQ and PhIP to rats; and by incubation of cultured COS-1 cells with IQ. These cells expressed the human cytochromes P450 1A1 or P450 1A2 and/or the human N-acetyltransferases NAT1 or NAT2. The data demonstrate that, in the photolytic, rat and human systems, a common IQ metabolite and a common PhIP metabolite are formed together with common sets of IQ adducts and PhIP adducts. The data obtained in the human system show that N-hydroxy-IQ, formed by cytochrome P450, binds poorly to DNA, whereas more efficient binding occurs in the presence of NAT1 and most efficient binding in presence of NAT2. This indicates an O-acetyltransferase activity of human NAT1 and NAT2 and formation of N-acetoxy-IQ as an intermediate and immediate precursor of the ultimate arylnitrenium ion. The effect of the polymorphic NAT2 suggests a critical role for the human acetylation polymorphism in the DNA-binding of IQ in humans and in its genotoxic implications.