[Show abstract][Hide abstract] ABSTRACT: In an attempt to examine the interaction of man-made mineral fibres with benzo[a]pyrene (B[a]P), homozygous X-lacI transgenic F344 rats were intratracheally treated with rock (stone) wool RWI and glass wool MMVF 10 fibres together with B[a]P. To analyze the induction of gene mutations by fibres and B[a]P in lung, single doses of 1 and 2 mg fibres/animal or multiple doses of 2 mg fibres/animal were administered weekly on 4 consecutive weeks (total dose 8 mg/animal). B[a]P (10 mg/animal) was administered either simultaneously with fibres (for single dose treatment with fibres) or together with the last fiber treatment (for multiple dose treatment with fibres). Animals were scarified 4 weeks after the last treatment. Benzo[a]pyrene administered simultaneously with RW1 fibres exhibited a strong synergistic effect on mutagenicity, the observed mutant frequency (MF) being more than three-fold higher than the net sum of the MF induced after separate administration of both agents. Our data suggest that DNA adducts induced by simultaneous B[a]P and fiber treatment lead to a strong increase in mutatant frequencies.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 04/2006; 595(1-2):167-73. DOI:10.1016/j.mrfmmm.2005.11.003 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The potential of two asbestos substitute mineral fibres--rock (stone) wool RW1 and glass wool MMVF10--to induce gene mutations, DNA strand breaks, inflammation and oxidative stress has been studied in rats. Male homozygous lamda-lacI transgenic F344 rats were intratracheally instilled with single doses of 1 and 2 mg/animal of fibres or with multiple doses of 2 mg/animal administered weekly on four consecutive weeks (8 mg in total). Exposure to RW1 fibres for 16 weeks significantly increased mutant frequency (MF) in the lung in a dose-dependent manner, while MMVF10 fibres did not exhibit any increase of MF at any dose. RW1 fibres gave a significant increase of MF at a dose of 1 mg. Four weeks after instillation, neither the single nor the multiple doses significantly increased MF for both fibre types. To investigate mechanisms for induction of mutations, other genotoxicity markers and parameters of inflammatory and oxidative damage were determined in relation to MF. A weak correlation of mutagenicity data with other genotoxicity parameters studied was observed. DNA strand breaks as measured by comet assay were increased in alveolar macrophages and lung epithelial cells of RW1 and MMVF10 treated rats. RWl fibres caused more extensive lung inflammation as measured by release of neutrophils into broncho-alveolar lavage fluid than MMVF10 fibres. The effects were observed 16 weeks post-exposure, indicating a persistence of the pathogenic process during the exposure period. Only minor differences in the extent of inflammatory processes were observed between the doses of 2 mg and 4 x 2 mg, suggesting that any threshold for inflammation lies below the dose of 2 mg. With the exception of the highest dose of MMVF10 fibres after 16 weeks of exposure, no significant increase of oxidative damage as measured by levels of malondialdehyde in lung tissue was observed. MMVF10 fibres caused weaker inflammation in the lung of rats and did not exhibit any mutagenic effect. We conclude that a weak but chronic inflammation (more likely than acute inflammation or direct oxidative damage) in the lung tissue of fibre treated rats characterized by moderate influx of inflammatory cells into BAL is probably responsible for the observed mutagenic effect of RW1 fibres.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 04/2006; 595(1-2):174-83. DOI:10.1016/j.mrfmmm.2005.11.001 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In order to get more insight into the mechanism of asbestos-related lung cancer, the mutagenic potential of asbestos was examined in vivo in rat lung. Groups of five transgenic lambda-lacI (Big Blue) rats were intratracheally instilled with single doses of 1 or 2mg, or with four weekly doses of 2mg, per animal of the amosite asbestos. Sixteen weeks after instillation, the mutation frequency was found to be increased in lung DNA by 2-fold at doses of 2 mg (P = 0.035) and of 4 x 2 mg (P = 0.007) amosite. No significant changes were observed after 4 weeks of exposure. In separate experiments, wild-type F344 rats were treated by the same regimen as described above and markers of inflammation, genotoxicity, cell proliferation and lung tissue damage were analysed. Our results indicate a weak but persistent inflammation and cell proliferation which possibly plays a major role in the observed mutagenic effect.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 10/2004; 553(1-2):67-78. DOI:10.1016/j.mrfmmm.2004.06.023 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To study the suspected mechanism of the interaction between tobacco smoking and asbestos exposure in the modulation of cancer risk, the mutagenic potential of asbestos in combination with the tobacco smoke carcinogen benzo[a]pyrene (B[a]P) was examined in vivo in the rat lung. B[a]P was administered intratracheally in one set of experiments, or by two daily intraperitoneal injections in another set of experiments, to lambdalacI transgenic rats, together with 1, 2 or 4 x 2 mg amosite in one experiment. In the first experiment, the combined action of amosite and B[a]P caused a synergistic (superadditive) increase of mutation frequency in the lung, as compared to groups treated only with asbestos or B[a]P. In the second experiment, i.p. treatment with B[a]P did not significantly alter the mutation frequency induced by amosite, neither after 4 nor after 16 weeks of exposure. The B[a]P-DNA adduct levels were unaffected by amosite co-treatment in both experiments. We assume that the synergistic increase of mutation frequency after intratracheal treatment was due to the mitogenic activities of B[a]P and of amosite. In conclusion, our findings indicate that a weak and delayed mutagenic effect of amosite in rat lung observed in another study was strongly enhanced by the concomitant action of B[a]P. The striking enhancement effect of B[a]P may provide a basis for understanding the suspected synergism of smoking on asbestos carcinogenesis.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 10/2004; 553(1-2):79-90. DOI:10.1016/j.mrfmmm.2004.06.025 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In order to probe for the existence of a no-effect levels for mutations induced by multiple dose treatment with cyproterone acetate (CPA), female lacI-transgenic Big Blue rats were treated daily for 3 weeks with oral doses of 5.0, 1.0 or 0.2mg/kg CPA b.w., respectively. The dose of 5mg/kg CPA b.w. ineffective as a single dose (see part I) increased the mutation frequency by 2.5-fold. Daily treatment with 1.0 and with 0.2 mg/kg CPA b.w. for 3 weeks, however, was not effective indicating that the 1 mg dose represents a no-effect level for multiple dose treatment. The finding that a total dose of 21 x 5 = 105 mg/kg CPA b.w. caused 50% less DNA adducts, but a mutation frequency three-fold higher (data extrapolated from part I) than observed after daily treatment with 5mg/kg CPA b.w. for 21 days points to a crucial role of cell proliferation in mutagenesis by CPA. Our present results, in combination with previous findings, offer a basis to estimate the risk to develop mutations in human liver following treatment with CPA. Previous studies revealed that CPA-DNA adducts are formed in human hepatocytes at lower levels than in those of female rats [Mutat. Res. 395 (1997) 179; Cancer Res. 56 (1996) 4391]. Moreover, an in vitro-study indicated that human hepatocytes in culture do not respond to the mitogenic effect of CPA, while rat hepatocytes did [Cancer Res. 51 (1991) 1143]. We conclude that the risk of humans to develop mutations under treatment with CPA is substantially lower than in the female rat.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 07/2004; 550(1-2):101-8. DOI:10.1016/j.mrfmmm.2004.02.002 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The anti-androgen and progestagen cyproterone acetate (CPA) is known to cause liver tumors in rats. The drug has been identified recently as a mutagen in the liver of female transgenic lambdalacI (Big Blue) rats at high doses after an expression time of 6 weeks. A dose of 50 mg CPA/kg BW, however, did not increase the mutation frequency (MF) of controls indicating a no-effect level of mutagenicity [Carcinogenesis 19 (1998) 241]. The present study was performed to assess the existence of a no-effect level of mutagenicity. In order to figure out conditions of maximum response, the time course of the MF was determined after administration of a single dose of 100 mg CPA/kg BW to female Big Blue rats. The MF showed a strong initial rise to a maximum 2 weeks after CPA administration accompanied by a corresponding increase of cell proliferation and of DNA adduct levels. Thereafter, the MF decreased within further 2 weeks to one third of the maximum level which was maintained for another 4 weeks. The DNA adduct levels decreased only by 15% during this time period suggesting that mutated hepatocytes were eliminated predominantly. A dose dependence curve determined at a fixation time of 2 weeks revealed a no-effect level of 5 mg CPA/kg BW for mutagenicity. In conclusion, our findings indicate that the length of the observation period may be a critical determinant for the outcome of a mutagenesis study in rat liver. Furthermore, the existence of a no-effect level for the mutagenicity of CPA in rat liver was confirmed. However, it has to be clarified whether the dose of 5 mg CPA/kg BW corresponding to the "transient" type of mutations or the previous dose of 50 mg CPA/kg BW related to a "permanent" type of mutations is more relevant for the assessment of the genotoxic risk.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 07/2004; 550(1-2):89-99. DOI:10.1016/j.mrfmmm.2004.02.003 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In order to get more insight into the mechanism of asbestos-related lung cancer, the mutagenic potential of asbestos was examined in vivo in rat lung. Groups of five transgenic -lacI (Big Blue TM) rats were intratracheally instilled with single doses of 1 or 2 mg, or with four weekly doses of 2 mg, per animal of the amosite asbestos. Sixteen weeks after instillation, the mutation frequency was found to be increased in lung DNA by 2-fold at doses of 2 mg (P = 0.035) and of 4 × 2 mg (P = 0.007) amosite. No significant changes were observed after 4 weeks of exposure. In separate experiments, wild-type F344 rats were treated by the same regimen as described above and markers of inflammation, genotoxicity, cell proliferation and lung tissue damage were analysed. Our results indicate a weak but persistent inflammation and cell proliferation which possibly plays a major role in the observed mutagenic effect. (J. Topinka).
[Show abstract][Hide abstract] ABSTRACT: 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.
Advances in Experimental Medicine and Biology 02/2001; 500:687-96. DOI:10.1007/978-1-4615-0667-6_101 · 2.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 08/2000; 469(1):83-93. · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 12/1998; 419(1-3):91-105. DOI:10.1016/S1383-5718(98)00127-2 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The gestagenic and antiandrogenic drug cyproterone acetate (CPA) is mitogenic, tumorigenic and induces DNA-adducts and DNA-repair synthesis in rat liver. Thus CPA is expected to be mutagenic. However in vitro mutagenicity test systems were negative. To examine whether CPA induces mutations in rat liver, the in vivo mutation assay based on Big Blue transgenic F344 rats was employed. Single oral doses of 25, 50, 75, 100 and 200 mg CPA/kg b.w. respectively were administered to female Big Blue rats. Six weeks after treatment, liver DNA was assayed for mutations. At the highest dose, 200 mg CPA/kg b.w., the frequency of (17 +/- 4) x 10(-6) spontaneous mutations was increased to a maximum of (80 +/- 8) x 10(-6) mutations. One-hundred and 75 mg CPA/kg b.w. resulted in mutation frequencies of (35 +/- 5) and (27 +/- 5) x 10(-6), respectively. The mutation frequency at doses of 50 and 25 mg CPA/kg b.w. was similar to that of vehicle treated controls. Statistical analysis of the dose-effect relationship revealed that it was not possible to decide whether a threshold dose exists or not. DNA adducts were analyzed by the 32P-postlabelling technique. The total level of the major and the two minor adducts observed in the autoradiograms increased between doses of 25 to 75 mg CPA/kg b.w. to a maximum of approximately 12,000 +/- 3000 adducts per 10(9) nucleotides. The level did not further increase significantly with 100 and 200 mg CPA/kg b.w. After CPA treatment no preneoplastic liver foci were observed. However, single glutathione-S-transferase placental form (GST-P) positive hepatocytes were observed and the frequency was dependent on the dose. These cells are not supposed to represent initiated cells, since they occurred only transiently after 6 weeks and disappeared thereafter completely. In conclusion, our results demonstrate that CPA is mutagenic in vivo. The mutation frequency increased at high CPA doses, when the increase of the DNA adduct formation had already ceased. This suggests that the mitogenic activity of CPA is required to express the mutations.
[Show abstract][Hide abstract] ABSTRACT: 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.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 01/1998; 395(2-3):179-87. DOI:10.1016/S1383-5718(97)00167-8 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Cancer Research 11/1996; 56(19):4391-7. · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.