Dose dependent induction of DNA adducts, gene mutations, and cell proliferation by the antiandrogenic drug cyproterone acetate in rat liver.

1. CPA does not only induce the formation of DNA adducts but also of mutations in female rat liver. 2. The mutation frequency exhibited a characteristic time course. Within a period of 3 days post administration, a tremendous increase was noted, which remained at a high level until 2 weeks post exposure. Thereafter, most mutation-carrying cells were eliminated within a period of 2 weeks leaving a cell population remaining at a constant level for another 4 weeks. Thus, the length of the observation period post exposure, i. e. the manifestation time, seems to be a critical factor for the strength of the mutagenic response. The highest as observed between 1 and 2 weeks post exposure. Correspondingly, the dose response curve recorded 2 weeks post exposure showed a higher mutagenic response than the curve after 6 weeks of exposure recorded previously. 3. When CPA-induced mutations were recorded as a function of the dose, mutation frequencies at the lower dose range were found that did not differ from those of controls. The non-effective dose recorded 2 weeks post exposure was much lower than that recorded after 6 weeks of exposure indicating that it is a function of the manifestation time. Since DNA adducts were formed in high amounts at the non-effective doses, we assume that the mitogenic activity required for the conversion of DNA adducts into mutations was not sufficiently strong. The liver of adult animals exhibits a very low endogeneous proliferation rate, which is not likely to contribute significantly to the expression of mutations. We conclude that it is the mitogenic activity of CPA itself, which stimulates the expression of mutations.

Establishment of a novel in vitro system for studying the interaction of xenobiotic metabolism of liver and intestinal microflora.

We developed a new two-chamber system for the coculture of hepatocytes and fecal microflora under aerobic and anaerobic conditions, respectively, to investigate the sequential metabolism of chemicals by the liver and microflora in vitro. The culture device consisted of two chambers separated by a permeable polycarbonate membrane. In the aerobic compartment, hepatocytes were cultivated as a monolayer on the membrane and in the anaerobic compartment fecal microflora as a suspension. To characterize the metabolic capacity of the microflora and hepatocytes, various marker enzymes were studied. Azoreductase, nitroductase, beta-glucuronidase, beta-glucosidase and sulphatase were tested in the microflora of the feces from three volunteers who had had significantly different eating habits for years (daily meat, mixed diet, vegetarian). The microflora exhibited significant activities and the various enzymes differed only moderately in the samples from the three volunteers. For rat hepatocytes the activities of various cytochrome P450 forms and conjugating enzymes served as markers. The enzyme activities were tested in the coculture system during a 4-h culture period intended for the test protocol. Deethylation of ethoxycoumarin and 2alpha-, 6beta- and 16alpha-hydroxylation of testosterone decreased by about 30%, 25%, 40% and 20%, respectively, while there was no loss of glucuronidation and sulphonation of 3-OH-benzo(a)pyrene nor of glutathione conjugation of 1-chloro-2,4-dinitrobenzene during the 4-h culture period. The activities of the tested hepatic phase I and II enzymes were not changed after coculture of the hepatocytes with the microflora for 4 h. The applicability of the in vitro system for studying the metabolic interaction of liver and microflora was demonstrated using 7-ethoxycoumarin and the developmental drug EMD 57033, a thiadiazinon derivative from Merck KGaA, as model compounds. Both compounds were oxidized and conjugated by liver cells. In the coculture of hepatocytes and fecal microflora the resulting glucuronides and sulphoconjugates were split by hydrolytic enzymes of the intestinal microflora.

Genotoxicity of urban air pollutants in the Czech Republic. Part II. DNA adduct formation in mammalian cells by extractable organic matter.

The study was aimed at determining the genotoxic potential of extractable organic matter (EOM) from ambient air particles PM10 (<10 micrometer) using mammalian cells in culture as test system. Air samples were collected in the course of summer and winter periods in two regions of the Czech Republic representing low and high levels of air pollution, the districts of industrial Teplice and rural Prachatice, respectively. EOM was fractionated by acid-base partitioning and silica gel column chromatography. Aliquots of fractions were incubated with cultured hepatocytes derived from male rats or Chinese hamster lung V79NH cells expressing nitroreductase activity but virtually no cytochrome P450 activity. DNA adduct levels were analyzed by 32P-postlabeling using butanol extraction for adduct enrichment. In hepatocytes, crude extracts caused the formation of substantial amounts of DNA reactive material being detectable in a broad diagonal radioactive zone (DRZ) in the chromatograms. Highest DNA adduct levels were found in the aromatic fractions and slightly polar fractions which contain most of the polycyclic aromatic hydrocarbons (PAH) and nitro-substituted PAH (nitro-PAH), respectively, comprising 75-90% of total adducts. This partitioning was independent of the sampling period and locality. In agreement with the higher average ambient air concentrations of PAH in the winter than the summer, 3-4-fold higher DNA adduct levels were detected in extracts sampled in the winter. Calculated on the basis of EOM/m(3), DNA adduct levels of samples collected in winter period were 10-fold higher than those collected in the summer period and 2-fold higher in Teplice than in Prachatice. Pretreatment of hepatocytes with 2,3,7,8-tetrachlorodibenzo-p-dioxin decreased DNA binding by 50-75%. In contrast to the findings in hepatocytes, in V79NH cells about 80% of the DNA adducts were caused by material in the slightly polar fractions appearing as distinct spots in the radiochromatograms. Seasonal variation of DNA adducts in V79NH cells was greater than variation between localities. Our results suggest that PAH as well as nitro-PAH are the main contributors to the genotoxicity of EOM derived from both industrial and rural areas. The results, furthermore, indicate that analysis of DNA adducts in mammalian cells in culture offers a suitable method for monitoring the genotoxicity of complex mixtures of environmental chemicals.

Decreased apoptosis as a mechanism for hepatomegaly in streptozotocin-induced diabetic rats.

Insulin-dependent diabetes mellitus in both humans and animals leads to structural and functional changes including hepatomegaly. This study examined hypertrophy, hyperplasia, and apoptosis, three basic aspects of tissue growth, in livers of Sprague-Dawley and Wistar rats made diabetic by iv injection of streptozotocin 8, 30, or 90 days previously. Immunohistochemical measurement of proliferating cell nuclear antigen revealed that hepatic DNA labeling indices were similar in normal control animals and diabetic rats 30 or 90 days post diabetic induction, but were reduced to 45 to 50% of control in insulin-treated diabetic animals, perhaps due to altered receptor activity or to partial insulin resistance, as reported previously. Flow cytometry indicated a 613% increase in diploid hepatocytes in the livers of diabetic rats 30 days after the onset of diabetes, compared to control. Diabetic livers contained 29% fewer tetraploid cells, 81% fewer octaploid cells, and 20% more binucleated hepatocytes than normal controls. At 90 days, the overall smaller size of hepatocytes in diabetic tissue was evidenced by more cells per area. Insulin treatment prevented some of these changes, but did not restore ploidy to a normal distribution. Mitosis, while 300% of normal at 8 days after streptozotocin injection, was reduced to 25% of normal after 90 days of diabetes. The morphological evidence of apoptosis was decreased by 23% to 76% in the diabetic liver, and was reversed but not normalized by insulin treatment. This study indicates that the hepatomegaly observed in streptozotocin-induced experimental diabetes may be due primarily to early hyperplasia, and later decreased apoptosis.

Effect of lindane and phenobarbital on cyclooxygenase-2 expression and prostanoid synthesis by Kupffer cells.

Prostaglandins (PGs) have been implicated in tumor promotion. In this study, we investigated the effect of the hepatic tumor promoters lindane and phenobarbital (PB) on the PG metabolism of Kupffer cells in vitro and in vivo, in particular on the expression of cyclooxygenase (COX), the leading enzyme in prostanoid synthesis. Exposure of primary cultures of Kupffer cells to lindane for 1 h stimulated the production of the PGs PGE(2) and PGD(2) markedly (up to 50-fold) and that of PGF(2alpha) by >3-fold. This effect was accompanied by an increase in the COX-2 protein, as demonstrated by western blotting. Similarly, PB, which shares several effects with lindane in rat liver, also clearly induced COX-2. Lindane and PB affected the PG synthesis in vitro and in vivo in Kupffer cells of rats that had been treated with the two compounds for 56 days. Kupffer cells, which were isolated at days 2, 5 and 56 of the treatment, showed a significant increase in the levels of COX-2 mRNA and protein. Total COX activity was increased approximately 2-fold and 3- to 5-fold in Kupffer cell homogenates of PB- and lindane-treated animals, respectively, compared with the untreated controls. These results suggest that paracrine mechanisms may contribute to the tumor-promoting activity of lindane and PB, stimulating the production of PGs by Kupffer cells.

DNA adduct formation in mammalian cell cultures by polycyclic aromatic hydrocarbons (PAH) and nitro-PAH in coke oven emission extract.

Mammalian cells in culture were used to study the genotoxic potential of coke oven emissions constituting a complex mixture of chemicals. For this purpose, particle extracts and some polycyclic aromatic and nitroaromatic hydrocarbons (PAH and nitro-PAH) occurring in these mixtures were assayed for DNA adduct formation using the -postlabeling technique. In primary cultures of rat hepatocytes, benzo[a]pyrene (B[a]P), benz[a]anthracene (B[a]A) and benzo[b]fluoranthene (B[k]F) caused DNA adduct levels in the range of 1 adduct/108 nucleotides. 4-Nitropyrene (4-NP), 6-nitrochrysene (6-NC), 3-nitrofluoranthene (3-NF) caused DNA adduct levels that were by one to two orders of magnitude higher. The crude particle extract and its fractions differing in acidity and polarity induced the formation of DNA reactive material within diagonal radioactive zones (DRZ) on the autoradiograms. On a weight base, the neutral aromatic fraction contributed by more than 80% to the total adduct level in hepatocytes. To examine whether the PAH- and nitro-PAH-DNA derived adducts can be further differentiated, hepatocyte cultures were preincubated with 2,3,7,8-tetrachloro-p-dioxin (TCDD) to induce the activity of cytochrome P450 1A1. TCDD pretreatment strongly increased the levels of PAH-DNA adducts, whereas, the levels of nitro-PAH adducts were markedly decreased. NCI-H322 cells, a human lung tumor cell line derived from Clara cells, exhibited PAH-DNA adduct levels between 10 and 100, and nitro-PAH-DNA adducts at levels between 0.2 to about 30 adducts per 108 nucleotides, respectively. In contrast to hepatocytes, incubations with extractable organic matter (EOM) and the neutral aromatic EOM fraction displayed several distinct spots in the chromatograms of NCI-H322 cells. The major spot was assigned by cochromatography to be identical with the major DNA adduct formed by incubation with B[a]P alone. In V79NH cells, a Chinese hamster lung cell line expressing nitro-PAH activating enzymes, but virtually no cytochrome P450 activity, PAH-derived DNA adducts were not detectable. Nitro-PAH-derived DNA adducts, however, were formed at levels between 10 and 300 adducts/108 nucleotides. The slightly and the moderately polar EOM fraction caused the formation of distinct adduct spots suggesting the occurrence of nitro-PAH in these fractions. GC/MS analyses revealed the presence of twelve PAH in the aromatic fraction, at a total amount of about 10% (w/w), and of four nitro-PAH in the slightly polar and the acidic fraction amounting to about 0.2% (w/w). In conclusion, our results indicate that PAH and nitro-PAH contribute to the genotoxicity of coke oven emissions. Using DNA adduct analysis in rat hepatocytes (+/-pretreatment with TCDD) and in NCI-H322 and in V79NH cells offers a promising approach to determine the genotoxic activity of PAH and nitro-PAH in any complex environmental samples.

Inhibition of transforming growth factor-beta1 and UV light-induced apoptosis by prostanoids in primary cultures of rat hepatocytes.

Treatment of rat hepatocytes cultured in collagen gel with transforming growth factor-beta1 (TGFbeta1) or with UV light strongly increased the frequency of apoptotic nuclei within 24 h; at doses of 0.5 ng/ml TGFbeta1 or 90 J/m2 UV light about 17 and 22% apoptotic nuclei were determined, respectively. DNA of the treated cells showed internucleosomal DNA fragmentation. Already the presence of the cytokine for only 1 h significantly induced apoptosis. The prostanoids PGI2, PGD2, and PGE1 decreased the frequency of apoptotic nuclei in a dose-dependent manner by up to 70 to 80% and suppressed internucleosomal DNA fragmentation. In contrast, PGE2 and PGF2alpha elicited a smaller protective effect and arachidonic acid had none. In the case of PGE1 it was shown that the prostaglandin was most effective when added together with TGFbeta1 or within 2 h before or after treatment with this cytokine. An early increase of the tumor suppressor gene product p53 is thought to play a decisive role in UV light-induced apoptosis. However, this increase in p53 was not affected by the strong cytoprotective prostacyclin PGI2. Our findings show a marked antiapoptotic activity of the prostanoids PGE1, PGI2, and PGD2 and raise the question of whether these prostanoids may influence apoptosis in pathological processes in the liver.

Formation of DNA adducts by cyproterone acetate and some structural analogues in primary cultures of human hepatocytes.

Recently, we have shown that the therapeutically-used progestin and antiandrogen cyproterone acetate (CPA) causes the formation of high levels of DNA adducts in rat hepatocytes and rat liver [J. Topinka, U. Andrae, L.R. Schwarz, T. Wolff, Cyproterone acetate generates DNA adducts in rat liver and in primary rat hepatocyte cultures, Carcinogenesis 14 (1993) 423-427: J. Topinka, B. Binkova, L.R. Schwarz, T. Wolff, Cyproterone acetate is an integral part of hepatic DNA adducts induced by this steroidal drug, Carcinogenesis 17 (1996) 167-169; S. Werner, J. Topinka, T. Wolff, L.R. Schwarz, Accumulation and persistence of DNA adducts of the synthetic steroid cyproterone acetate in rat liver, Carcinogenesis 16 (1995) 2369-2372; J. Topinka, B. Binkova, H.K. Zhu, U. Andrae, I. Neumann, L.R. Schwarz, S. Werner, T. Wolff, DNA damaging activity of the cyproterone acetate analogues chlormadinone acetate and megestrol acetate in rat liver, Carcinogenesis 16 (1995) 1483-1487]. Its structural analogues, chlormadinone acetate (CMA) and megestrol acetate (MGA) were much less active in this respect [J. Topinka, B. Binkova, H.K. Zhu, U. Andrae, I. Neumann, L.R. Schwarz, S. Werner, T. Wolff, DNA damaging activity of the cyproterone acetate analogues chlormadinone acetate and megestrol acetate in rat liver, Carcinogenesis 16 (1995) 1483-1487]. In the present study we addressed the question whether these compounds and two further analogues, medroxyprogesterone acetate (MPA) and progesterone, induce the formation of DNA adducts in primary cultures of human hepatocytes. Incubation of CPA with human hepatocytes from two male and four female donors induced the formation of significant levels of CPA-DNA adducts detectable by 32P-postlabeling. The by far most prevalent DNA adduct is most likely identical with adduct A formed in CPA-treated rats. DNA binding was found even at 0.03 microM CPA, the lowest concentration used. The maximum effect occurred at about 10 microM in 5 of the 6 cell preparations. At this concentration 480 and 2690 adducts x 10(-9) nucleotides were detected in hepatocytes of the two male donors and 1072, 816, 613 and 659 adducts x 10(-9) nucleotides in the hepatocytes of the four female donors after an exposure of 6 h with CPA. Extending the incubation time to 20 h resulted in a roughly three-fold higher binding. CMA and MGA were assayed in two of the liver cell preparations from the female donors. At a concentration of 20 microM and an incubation time of 6 h, DNA adduct levels for CMA were 21 and 43% and for MGA 31 and 65% of the levels observed with 20 microM CPA. In contrast, DNA binding of MPA amounted to less than 1% of that observed with CPA and DNA binding of the natural occurring progestin progesterone was below the level of detection. The results point to a genotoxic risk associated with the therapeutic use of CPA and possibly of CMA and MGA. Furthermore, the findings suggest that the significant genotoxicity observed with CPA, MGA and CMA is associated with the presence of the double bond in position 6-7 of the steroid, which is absent in MPA and progesterone.

Phase II metabolism of benzene.

The hepatic metabolism of benzene is thought to be a prerequisite for its bony marrow toxicity. However, the complete pattern of benzene metabolites formed in the liver and their role in bone marrow toxicity are not fully understood. Therefore, benzene metabolism was studied in isolated rodent hepatocytes. Rat hepatocytes released benzene-1,2-dihydrodiol, hydroquinone (HQ), catechol (CT), phenol (PH), trans-trans-muconic acid, and a number of phase II metabolites such as PH sulfate and PH glucuronide. Pretreatment of animals with 3-methylcholantrene (3-MC) markedly increased PH glucuronide formation while PH sulfate formation was decreased. Likewise, V79 cells transfected with the 3-MC-inducible rat UGT1.6 cDNA showed a considerable rate of PH and HQ glucuronidation. In addition to inducing glucuronidation of phenols, 3-MC treatment (reported to protect rats from the myelotoxicity of benzene) resulted in a decrease of hepatic CYP2E1. In contrast, pretreatment of rats with the CYP2E1-inducer isopropanol strongly enhanced benzene metabolism and the formation of phenolic metabolites. Mouse hepatocytes formed much higher amounts of HQ than rat hepatocytes and considerable amounts of 1,2,4-trihydroxybenzene (THB) sulfate and HQ sulfate. In conclusion, the protective effect of 3-MC in rats is probably due to a shift from the labile PH sulfate to the more stable PH glucuronide, and to a decrease in hepatic CYP2E1. The higher susceptibility of mice toward benzene may be related to the high rate of formation of the myelotoxic metabolite HQ and the semistable phase II metabolites HQ sulfate and THB sulfate.

Steroidal drug cyproterone acetate is activated to DNA-binding metabolites by sulfonation.

The antiandrogenic and gestagenic steroid cyproterone acetate (CPA) has been widely used in human therapy. There is currently a debate about the safety of CPA, since it proved to be genotoxic in rat liver and human hepatocytes [I. Neumann et al., Carcinogenesis (Lond.), 13: 373-378, 1992, J. Topinka et al., Carcinogenesis (Lond.), 14: 423-427, 1993, L. R. Schwarz et al., Biological Reactive Intermediates: V. Basic Mechanistic Research in Toxicology and Human Risk Assessment. pp. 243-251, 1996; A. Martelli et al., Carcinogenesis (Lond.), 16: 1265-1269, 1995]. Little is known about the metabolic pathways of activation of CPA to genotoxic metabolites. Using rat hepatocytes and subcellular fractions of female rat liver, we have examined whether sulfoconjugation plays an essential role in the activation of CPA to DNA-binding metabolites which are detectable with 32P-postlabeling. Incubation of hepatocyte cultures with 30 microM CPA for 6 h caused the formation of several DNA adducts; the total adduct level amounted to about 12,400 adducts/10(9) nucleotides. When the cells were incubated in sulfate-free medium to prevent the synthesis of the cosubstrate of sulfonation, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), formation of all CPA-DNA adducts was greatly reduced, amounting to only 5% of that determined in the presence of sulfate (810 microM). Activation of CPA is likely to be catalyzed by hydroxysteroid sulfotransferase(s), because the specific substrate dehydroepiandrosterone almost completely inhibited DNA-binding of CPA. Our assumption that sulfonation plays a decisive role in the bioactivation of CPA is further supported by the results obtained with an in vitro system consisting of calf thymus DNA, various subcellular liver fractions, and the cofactor PAPS, NADPH, or NADH. Significant DNA binding only occurred when cytosol and both PAPS and the reduced pyridine nucleotides were present. The DNA adduct spot obtained was chromatographically identical to the adduct spot A detected in isolated liver cells, suggesting that the CPA-DNA adduct formed in vivo and in vitro is identical. Cytosol is known to contain not only sulfotransferases but also reductases. Thus, the requirement for NADPH or NADH suggests that in addition to sulfotransferase(s), reductases are involved in the activation of CPA. We propose that bioactivation of CPA involves reduction of the keto group at C-3 followed by sulfonation of the hydroxysteroid. The resulting sulfoconjugate is most likely unstable and supposed to generate a reactive carbonium ion.

Suppression of agonist induced Ca2+ oscillations in cultured hepatocytes by nafenopin: possible involvement of protein kinase C.

The alpha 1-agonist phenylephrine (5 microM) induces an increase in the free cytosolic Ca2+ concentration, followed by repetitive transients of the cytoplasmic Ca2+ concentration, in single Fura-2 loaded hepatocytes. The tumor promoting, hypolipidemic drug nafenopin suppressed the cellular Ca2+ response to phenylephrine. The effect of nafenopin on the Ca2+ increase and Ca2+ oscillations was largely prevented by the specific protein kinase C inhibitor Gö 6976. This finding suggests involvement of protein kinase C in the action of nafenopin on phenylephrine induced Ca2+ mobilization.

Phenobarbital transiently stimulates uptake of 2-aminoisobutyric acid in hepatocytes.

Phenobarbital (PB) is a classical inducer of drug metabolizing enzymes and known to stimulate liver growth transiently in rodents. Previous studies have shown that regenerative liver growth after a partial hepatectomy is accompanied by the induction of the amino acid transport system A. In the present study we investigated whether amino acid transport is also increased by treatment of rats with PB. Na(+) -dependent hepatic uptake of the non-metabolizable amino acid 2-aminoisobutyric acid (AIB), which proceeds largely via transport system A, was studied in isolated hepatocytes from PB treated and untreated rats. Uptake of AIB (100 microM) was maximally induced (2.5-fold) 8 h after the beginning of PB treatment. Within 4 days, transport rates decreased to values similar to those determined in hepatocytes from untreated animals, despite the continuation of PB treatment. In contrast, induction of the PB-inducible cytochromes P450 2B1/2 was markedly increased during the entire experiment, as determined with the isoenzyme-selective substrate pentoxyresorufin. Kinetic analysis of AIB uptake revealed a "high" and a "low" affinity transport system. It is most likely that the high affinity system represents amino acid transport system A. Treatment with PB increased the V(max) value but did not affect the apparent Km value of the high affinity system. The present data suggest that the hepatic mitogen PB transiently induces amino acid transport system A.

Cyproterone acetate is an integral part of hepatic DNA adducts induced by this steroidal drug.

We have recently shown that cyproterone acetate (CPA), an active component of some antiandrogenic drugs, induces the formation of DNA adducts detectable by the 32P-DNA postlabelling technique in rat liver. The postlabelling technique, however, does not provide evidence for the chemical nature of the adducts observed. To ascertain whether the CPA-induced adducts do contain CPA, we have incubated tritiated CPA with cultured hepatocytes from female rats, digested the DNA to 3'-monophosphonucleosides, extracted the DNA adducts formed into butanol and phosphorylated the adducts in the extract with unlabelled ATP. One major and one minor 3H-labelled adduct spot were detectable on the TLC chromatograms. The spots appeared at positions identical to those observed in the 32P-postlabelling experiments with unlabelled CPA. Furthermore, the ratio of 3H activity for the major versus the minor adduct spot was 11.9 +/- 1.8, which agreed with the corresponding ratio for the 32P activities, which was 13.2 +/- 3.5. These findings indicate that the CPA-induced DNA adducts, which we have previously detected by 32P-postlabelling do contain CPA or CPA metabolites.

Benzene metabolism in rodent hepatocytes: role of sulphate conjugation.

1. Hepatocytes isolated from the adult male NMRI mouse or Wistar rat were incubated for 1 h with 0.5 mM 14C-benzene, the supernatant was separated from the cells, and analysed for benzene metabolites. Separately, formation of sulphate conjugates during benzene metabolism was studied in hepatocytes in the presence of 35S-sulphate. In addition sulphate conjugation of the benzene metabolites hydroquinone and 1,2,4-trihydroxybenzene was investigated in mouse liver cytosol supplemented with 3'-phosphoadenosine-5'-phospho-35S-sulphate. 2. Two novel metabolites, not detectable in rat hepatocyte incubations, were found in mouse hepatocytes, and were identified as 1,2,4-trihydroxybenzene sulphate and hydroquinone sulphate. Formation of the 35S-labelled conjugates could be demonstrated in incubations of mouse liver cytosol with hydroquinone or 1,2,4-trihydroxybenzene supplemented with 3'-phosphoadenosine-5'-phospho-35S-sulphate, and in mouse hepatocytes incubated with benzene and 35S-sulphate. 3. In comparison with hepatocytes from the Wistar rat, hepatocytes from the NMRI mouse were almost three times more effective in metabolizing benzene. The higher formation of hydroquinone, and the formation of trihydroxybenzene sulphate and hydroquinone sulphate, mainly contributed to the higher rate of benzene metabolism. 4. In conclusion, qualitative and quantitative differences in benzene metabolism may contribute to the higher susceptibility of mouse towards the myelotoxic and leucaemogenic action of benzene.

Accumulation and persistence of DNA adducts of the synthetic steroid cyproterone acetate in rat liver.

Cyproterone acetate (CPA) is a synthetic steroid which is widely used in antiandrogenic and gestagenic drugs. We have recently shown that CPA induces DNA adducts in cultured rat hepatocytes and in rat liver (1). In the present investigation, we studied the persistence and accumulation of CPA-derived DNA adducts in the liver of rats using the 32P-postlabeling technique. To study the persistence of CPA-DNA adducts, rats were treated with a single oral dose of 10 (female rats) or 100 mg CPA/kg body wt (male rats). Four DNA adducts were detected in the liver of both gender. In female rats, maximal total DNA adduct levels of 3.40 +/- 0.04 adducts/10(6) nucleotides were observed after 1 week. Eleven weeks later, 40% of the adducts determined after 1 week were still detectable. In male rats, maximal hepatic DNA adduct levels of approximately 98 +/- 3/10(9) nucleotides were attained after 2 weeks. The adduct level decreased during the following 4 weeks to approximately 40% of the earlier observed maximal level. To study the accumulation of the CPA-DNA adducts, rats were treated daily with a low oral dose of 50 micrograms CPA/kg body wt for 42 days. During this treatment period, the level of the four adducts increased continuously from approximately 10 to approximately 380 adducts/10(9) nucleotides in the liver of female rats. DNA adducts were formed at much lower levels in male rats; only one type of DNA adduct was detectable, the level of which increased to approximately 6 adducts/10(9) nucleotides after 42 days. In conclusion, CPA induces DNA adducts in rat liver; binding of the steroid is much higher in female compared to male rats. The CPA-DNA adducts show a high persistence and as a consequence of their long half life, CPA-DNA adducts accumulate significantly in the liver of rats.