The anticancer drug ellipticine forms covalent DNA adducts, mediated by human cytochromes P450, through metabolism to 13-hydroxyellipticine and ellipticine N2-oxide

Ellipticine is an antineoplastic agent, the mode of action of which is considered to be based on DNA intercalation and inhibition of topoisomerase II. We found that ellipticine also forms the cytochrome P450 (CYP)-mediated covalent DNA adducts. We now identified the ellipticine metabolites formed by human CYPs and elucidated the metabolites responsible for DNA binding. The 7-hydroxyellipticine, 9-hydroxyellipticine, 12-hydroxyellipticine, 13-hydroxyellipticine, and ellipticine N(2)-oxide are generated by hepatic microsomes from eight human donors. The role of specific CYPs in the oxidation of ellipticine and the role of the ellipticine metabolites in the formation of DNA adducts were investigated by correlating the levels of metabolites formed in each microsomal sample with CYP activities and with the levels of the ellipticine-derived deoxyguanosine adducts in DNA. On the basis of this analysis, formation of 9-hydroxyellipticine and 7-hydroxyellipticine was attributable to CYP1A1/2, whereas production of 13-hydroxyellipticine and ellipticine N(2)-oxide, the metabolites responsible for formation of two major DNA adducts, was attributable to CYP3A4. Using recombinant human enzymes, oxidation of ellipticine to 9-hydroxyellipticine and 7-hydroxyellipticine by CYP1A1/2 and to 13-hydroxyellipticine and N(2)-oxide by CYP3A4 was corroborated. Homologue modeling and docking of ellipticine to the CYP3A4 active center was used to explain the predominance of ellipticine oxidation by CYP3A4 to 13-hydroxyellipticine and N(2)-oxide.     

DNA adduct formation by the anticancer drug ellipticine in human leukemia HL-60 and CCRF-CEM cells

Ellipticine induces formation of two DNA adducts in leukemia HL-60 and CCRF-CEM cells, identical with deoxyguanosine adducts generated by ellipticine metabolites 13-hydroxyellipticine and 12-hydroxyellipticine in vitro and in vivo. The ellipticine cytotoxicity to HL-60 (IC(50)=0.64microM) and CCRF-CEM cells (IC(50)=4.7microM) correlates with levels of DNA adducts. The different expressions of enzymes activating ellipticine in cells explain this finding. While cytochrome P450 1A1 and cyclooxygenase-1 are expressed in both cells, HL-60 cells express also high levels of another activator, myeloperoxidase. The results suggest the adduct formation as a new mode of antitumor action of ellipticine for leukemia.     

DNA adduct formation by the anticancer drug ellipticine in rats determined by 32P postlabeling

Ellipticine is a potent antineoplastic agent whose mode of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine also forms covalent DNA adducts in vitro and that the formation of the major adduct is dependent on the activation of ellipticine by cytochrome P450 (CYP). Here, we investigated the capacity of ellipticine to form DNA adducts in vivo. Male Wistar rats were treated with ellipticine, and DNA from various organs was analyzed by (32)P postlabeling. Ellipticine-specific DNA adduct patterns, similar to those found in vitro, were detected in most test organs. Only DNA of testes was free of the ellipticine-DNA adducts. The highest level of DNA adducts was found in liver (19.7 adducts per 10(7) nucleotides), followed by spleen, lung, kidney, heart and brain. One major and one minor ellipticine-DNA adducts were found in DNA of all these organs of rats exposed to ellipticine. Besides these, 2 or 3 additional adducts were detected in DNA of liver, kidney, lung and heart. The predominant adduct formed in rat tissues in vivo was identical to the deoxyguanosine adduct generated in DNA by ellipticine in vitro as shown by cochromatography in 2 independent systems. Correlation studies showed that the formation of this major DNA adduct in vivo is mediated by CYP3A1- and CYP1A-dependent reactions. The results presented here are the first report showing the formation of CYP-mediated covalent DNA adducts by ellipticine in vivo and confirm the formation of covalent DNA adducts as a new mode of ellipticine action.     

Activation of 3-nitrobenzanthrone and its metabolites by human acetyltransferases,sulfotransferases and cytochrome P450 expressed in Chinese hamster V79 cells

3-nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and ambient air pollution. 3-aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA) and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) have been identified as 3-NBA metabolites. Recently we found that 3-NBA and its metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) form the same DNA adducts in vivo in rats. In order to investigate whether human cytochrome P450 (CYP) enzymes (i.e., CYP1A2), human N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) contribute to the metabolic activation of 3-NBA and its metabolites, we developed a panel of Chinese hamster V79MZ-h1A2 derived cell lines expressing human CYP1A2 in conjunction with human NAT1, NAT2, SULT1A1 or SULT1A2, respectively. Cells were treated with 0.01, 0.1 or 1 microM 3-NBA, or its metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA). Using both enrichment versions of the (32)P-postlabeling assay, nuclease P1 digestion and butanol extraction, essentially 4 major and 2 minor DNA adducts were detected in the appropriate cell lines with all 4 compounds. The major ones were identical to those detected in rat tissue; the adducts lack an N-acetyl group. Human CYP1A2 was required for the metabolic activation of 3-ABA and 3-Ac-ABA (probably via N-oxidation) and enhanced the activity of 3-NBA (probably via nitroreduction). The lack of acetylated adducts suggests N-deacetylation of 3-Ac-ABA and N-Ac-N-OH-ABA. Thus, N-hydroxy-3-aminobenzanthrone (N-OH-ABA) appears to be a common intermediate for the formation of the electrophilic arylnitrenium ions capable of reacting with DNA. Human NAT1 and NAT2 as well as human SULT1A1 and SULT1A2 strongly contributed to the high genotoxicity of 3-NBA and its metabolites. Moreover, N,O-acetyltransfer reactions catalyzed by human NATs leading to the corresponding N-acetoxyester may be important in the bioactivation of N-Ac-N-OH-ABA. As human exposure to 3-NBA is likely to occur primarily via the respiratory tract, expression of CYPs, NATs and SULTs in respiratory tissues may contribute significantly and specifically to the metabolic activation of 3-NBA and its metabolites. Consequently, polymorphisms in these genes could be important determinants of lung cancer risk from 3-NBA.     

Comparison of cytochrome P450- and peroxidase-dependent metabolic activation of the potent carcinogen dibenzo[a,l]pyrene in human cell lines: formation of stable DNA adducts and absence of a detectable increase in apurinic sites

The potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) has been reported to form both stable and depurinating DNA adducts upon activation by cytochrome P450 enzymes and/or cellular peroxidases. Only stable DB[a,l]P-DNA adducts were detected in DNA after reaction of DB[a,I]P-11,12-diol-13,14-epoxides in solution or cells in culture. To determine whether DB[a,l]P can be activated to metabolites that form depurinating adducts in cells with either high peroxidase (human leukemia HL-60 cell line) or cytochrome P450 activity (human mammary carcinoma MCF-7 cell line), cultures were treated with DB[a,l]P for 4 h, and the levels of stable adducts and apurinic (AP) sites in the DNA were determined. DNA samples from DB[a,l]P-treated HL-60 cells contained no detectable levels of either stable adducts or AP sites. MCF-7 cells exposed to 2 microM DB[a,l]P for 4 h contained 4 stable adducts per 10(6) nucleotides, but no detectable increase in AP sites. The results indicate that metabolic activation of DB[a,l]P by cytochrome P450 enzymes to diol epoxides that form stable DNA adducts, rather than one-electron oxidation catalyzed either by cytochrome P450 enzymes or peroxidases to form AP sites, is responsible for the high carcinogenic activity of DB[a,l]P.     

Idoxifene derivatives are less reactive to DNA than tamoxifen derivatives, both chemically and in human and rat liver cells

The drug tamoxifen shows evidence of genotoxicity, and induces liver tumours in rats. Covalent DNA adducts have been detected in the liver of rats treated with tamoxifen, and these arise through metabolism at the alpha-position to give an ester which reacts with DNA. (E)-1-(4-iodophenyl)-2-phenyl-1-[4-(2-pyrrolidinoethoxy)phenyl]-but-1-en e (idoxifene) is an analogue of tamoxifen in which formation of DNA adducts is greatly reduced; we could not detect any adducts in the DNA of cultured rat hepatocytes treated with 10 microM idoxifene, after analysis by the 32P-post-labelling method. The metabolite (Z)-4-(4-iodophenyl)-4-[4-(2-pyrrolidinoethoxy)phenyl]-3-phenyl-3-but en-2-ol (alpha-hydroxyidoxifene) gave adducts in rat hepatocytes, but far fewer than the corresponding tamoxifen metabolite. In human hepatocytes, neither idoxifene nor tamoxifen induced detectable levels of DNA adducts. We prepared the alpha-acetoxy ester of idoxifene as a model for the ultimate reactive metabolite formed in rat liver. It was less reactive than alpha-acetoxytamoxifen, as might be expected on mechanistic grounds. It reacted with DNA in the same way, to give adducts which were probably N2-alkyldeoxyguanosines, but to a lower extent. All these results indicate that idoxifene is much less genotoxic than tamoxifen, and should therefore be a safer drug.     

Bioactivation of 3-aminobenzanthrone, a human metabolite of the environmental pollutant 3-nitrobenzanthrone: evidence for DNA adduct formation mediated by cytochrome P450 enzymes and peroxidases

3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen found in diesel exhaust and ambient air pollution. The main metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), was detected in the urine of salt mining workers occupationally exposed to diesel emissions. We evaluated the role of hepatic cytochrome P450 (CYP) enzymes in the activation of 3-ABA in vivo by treating hepatic cytochrome P450 oxidoreductase (POR)-null mice and wild-type littermates intraperitoneally with 0.2 and 2mg/kg body weight of 3-ABA. Hepatic POR-null mice lack POR-mediated CYP enzyme activity in the liver. Using the (32)P-postlabelling method, multiple 3-ABA-derived DNA adducts were observed in liver DNA from wild-type mice, qualitatively similar to those formed in incubations using human hepatic microsomes. The adduct pattern was also similar to those formed by the nitroaromatic counterpart 3-NBA and which derive from reductive metabolites of 3-NBA bound to purine bases in DNA. DNA binding by 3-ABA in the livers of the null mice was undetectable at the lower dose and substantially reduced (by up to 80%), relative to wild-type mice, at the higher dose. These data indicate that POR-mediated CYP enzyme activities are important for the oxidative activation of 3-ABA in livers, confirming recent results indicating that CYP1A1 and -1A2 are mainly responsible for the metabolic activation of 3-ABA in human hepatic microsomes. No difference in DNA binding was found in kidney and bladder between null and wild-type mice, suggesting that cells in these extrahepatic organs have the metabolic capacity to oxidize 3-ABA to species forming the same 3-ABA-derived DNA adducts, independently from the CYP-mediated oxidation in the liver. We determined that different model peroxidases are able to catalyse DNA adduct formation by 3-ABA in vitro. Horseradish peroxidase (HRP), lactoperoxidase (LPO), myeloperoxidase (MPO), and prostaglandin H synthase (PHS) were all effective in activating 3-ABA in vitro, forming DNA adducts qualitatively similar to those formed in vivo in mice treated with 3-ABA and to those found in DNA reacted with N-hydroxy-3-aminobenzanthrone (N-OH-ABA). Collectively, these results suggest that both CYPs and peroxidases may play an important role in metabolizing 3-ABA to reactive DNA adduct forming species.     

Further characterization of the DNA adducts formed in rat liver after the administration of tamoxifen, N-desmethyltamoxifen or N, N-didesmethyltamoxifen.

The present study compares the formation of DNA adducts, determined by (32)P-postlabelling, in the livers of rats given tamoxifen and the N-demethylated metabolites N-desmethyltamoxifen and N, N-didesmethyltamoxifen. Results show that after 4 days treatment (0.11 mmol/kg i.p.), similar levels of DNA damage were seen after treatment with either tamoxifen or N-desmethyltamoxifen [109 +/- 40 (n = 3) and 100 +/- 33 (n = 4) adducts/10(8) nucleotides, respectively], even though the concentration of tamoxifen in the livers of tamoxifen-treated rats was about half that of N-desmethyltamoxifen in the N-desmethyltamoxifen-treated animals (51 +/- 16 and 100 +/- 8 nmol/g, respectively). Administration of N, N-didesmethyltamoxifen to rats resulted in a 5-fold lower level of damage (19 adducts/10(8) nucleotides, n = 2). Following (32)P-postlabelling and HPLC, hepatic DNA from rats treated with tamoxifen and its metabolites showed distinctive patterns of adducts. Treatment of rats with N,N-didesmethyltamoxifen gave a major product that co-eluted with one of the minor adduct peaks seen in the livers of rats given tamoxifen. Following dosing with N-desmethyltamoxifen, the major product co-eluted with one of the main peaks seen following treatment of rats with tamoxifen. This suggests that tamoxifen can be metabolically converted to N-desmethyltamoxifen prior to activation. However, analysis of the (32)P-postlabelled products from the reaction between alpha-acetoxytamoxifen and calf thymus DNA showed two main peaks, the smaller one of which ( approximately 15% of the total) also co-eluted with that attributed to N-desmethyltamoxifen. This indicates that N-desmethyltamoxifen and N,N-didesmethyltamoxifen are activated in a similar manner to tamoxifen leading to a complex mixture of adducts. Since an HPLC system does not exist that can fully separate all these (32)P-postlabelled adducts, care has to be taken when interpreting results and determining the relative importance of individual adducts and the metabolites they are derived from in the carcinogenic process.     

Metabolism of benzo[a]pyrene by cultured tracheobronchial tissues from mice,rats, hamsters,bovines and humans

The metabolism of benzo[a]pyrene (BP)⁴by cultured tracheobronchial tissues from different species - human, bovine, hamster, rat and mouse - has been investigated. The total metabolism, as measured by both organic solvent-extractable and water-soluble metabolites of BP, was substantial in the respiratory tract from humans and from animal species susceptible to the carcinogenic action of BP. The ratio of organic-extractable metabolites to water-soluble metabolites was greater than one in hamster, human and C57BI/6N mouse, but less than one in rat, bovine and DBA/2N mouse, suggesting that determination of both activation and deactivation pathways are important in assessing carcinogenic risk of a chemical. Sulphate esters and glutathione conjugates were the major water-soluble metabolites in all animal species; tetrols and diols were the major organic extractable metabolites. The level of trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, the proximate carcinogenic form of BP, was three times higher in C57BI/6N than in DBA/2N mouse trachea. Trans-9,10-dihydro-9,10-dihydroxybenzo[a]pyrene was the major metabolite formed by cultured hamster trachea. The binding levels of BP to cellular DNA were quite similar in all tissues, although slightly higher binding was observed in hamster trachea. Wide inter-individual variation in the binding of BP to DNA was seen in tissues from outbred species. The major BP-DNA adducts in all animal species were formed by interaction of benzo[a]pyrene diol-epoxide with the 2-amino group of deoxyguanosine. Both stereoisomeric forms of (±)-(7β,8α)-dihydroxy-(9α, 10α)-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE I) reacted with deoxyguanosine, the (7R)-form being the most reactive. No difference in the relative distribution of the various adducts was seen between the species except in the CD rat, where BPDE-deoxyadenosine adducts accounted for 20% of the total modification. In cultured hamster trachea the persistence of the different adducts was similar. In conclusion, the metabolism of BP is qualitatively similar in tracheobronchial tissues from both humans and animal species in which BP has been experimentally shown to be carcinogenic.     

Species- and length of exposure-dependent differences in the benzo(a)pyrene:DNA adducts formed in embryo cell cultures from mice, rats, and hamsters

The activation of benzo(a)pyrene (BaP) to DNA-binding metabolites in early-passage embryo cell cultures prepared from various species of rodents was investigated by exposing cells from mice (BALB/c and Sencar), rats (Wistar and Fischer 344), and Syrian hamsters to [3H]BaP for various lengths of time. The BaP:DNA adducts containing cis-vicinal hydroxyl groups such as those formed from 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BaPDE) were separated from the other types of BaP:DNA adducts by immobilized boronate chromatography, and the individual adducts were analyzed by high-performance liquid chromatography. A number of BaP:DNA adducts were present in the DNA from the cultures from all three species after 5 h of BaP treatment. After a 24-h exposure to BaP, the mouse and hamster embryo cell DNA contained a large amount of the adduct formed by reaction of (+)-anti-BaPDE with the 2-amino group of deoxyguanosine (dGuo) and a small amount of a 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:dGuo adduct. A large number of BaP:DNA adducts derived from 7 beta, 8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene and other unidentified BaP metabolites were present in rat embryo cell cultures at all times. Neither the Fischer 344 nor the Wistar rat embryo cell cultures had a significant amount of (+)-anti-BaPDE:dGuo adduct after 5 h of BaP treatment, and in the Wistar rat cells larger amounts of other adducts were present even after a 96-h exposure to BaP. In cell cultures from all three species the proportion of (+)-anti-BaPDE:dGuo adduct increased as the length of time of exposure to BaP increased. There are major differences in the metabolic activation of BaP to DNA binding metabolites in embryo cells from various species of rodents. However, the variations between cell cultures from different strains of rats or mice are not as great as the variations between cell cultures from different species. The time-dependent alterations in the BaP:DNA adducts indicate that analysis after various lengths of time of exposure to BaP is essential to characterize accurately the pathways of metabolic activation of BaP in cells from various species and tissues.     

Species differences in the formation of benzo(a)pyrene-DNA adducts in rodent and human endometrium

The formation of adducts of benzo(a)pyrene metabolites on DNA was investigated in endometrial tissue from humans, hamsters, mice, and rats. anti-Benzo(a)pyrene-7,8-diol-9,10-epoxide was the predominant adduct identified in all the species studied. The amount of (+)-anti-benzo(a)pyrene-7,8-diol-9,10-epoxide bound to DNA from human endometrium was approximately 3 times higher than to DNA from hamster tissue. Among the three animal species examined, the level of this adduct was highest in hamsters and lowest in rats. The high pressure liquid chromatography profiles of adducts formed in endometrium from humans and hamsters were similar, but the specific activity (pmol/mg DNA) of each adduct formed was different. syn-7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene adduct was present in humans, hamsters, and rats but was not detected in mouse endometrium. There was an unidentified adduct present only in rat tissue. Rats had the lowest level of total DNA-bound radioactivity and the largest percentage of this total eluted as an uncharacterized radioactive peak that eluted with water (53%). These results demonstrate that endometrial tissues from humans and three rodent species differ with regard to the quantities and proportions of benzo(a)pyrene-DNA adducts formed from benzo(a)pyrene.     

Covalent binding of 1,2-dihaloalkanes to DNA and stability of the major DNA adduct, S-[2-(N7-guanyl)ethyl]glutathione

The major DNA adduct formed from the carcinogen ethylene dibromide (1,2-dibromoethane, EDB) is S-[2-(N7-guanyl)ethyl]glutathione, resulting from the reaction of guanyl residues with the half-mustard S-(2-bromoethyl)glutathione, which is generated by glutathione S-transferase-catalyzed conjugation of EDB with glutathione. The half-life of the alkylating species [putative S-(2-bromoethyl)glutathione or the derived episulfonium ion] was estimated to be less than 10 s. However, the stability was enough for approximately half of the alkylating metabolites to leave isolated rat hepatocytes before reacting with nucleic acids. Treatment of isolated rat hepatocytes with diethylmaleate decreased covalent binding of EDB to DNA, but treatment with 1-phenylimidazole did not, consistent with the view that conjugative metabolism is of greater importance than oxidation with regard to DNA binding. When EDB was administered to rats in vivo, only one major adduct, S-[2-(N7-guanyl)ethyl]glutathione, was formed in liver or kidney. S-[2-(N7-Guanyl)ethyl]glutathione was found in liver and kidney DNA of rats treated with 1,2-dichloroethane, but other adducts were also present. The gamma-glutamyl transpeptidase inhibitor AT-125 [L-(alpha-(5S)-alpha-amino-S-chloro-4,5-dihydro-5-isoxazoleacetic acid] did not affect the level of EDB bound to DNA by glutathione-fortified rat kidney homogenates or bound to liver or kidney DNA in vivo. The in vitro half-life of S-[2-(N7-guanyl)ethyl]glutathione in calf thymus DNA was 150 h; the half-life of the adduct in rat liver, kidney, stomach, and lung was between 70 and 100 h. Isolated S-[2-(N7-guanyl)ethyl]glutathione did not react with DNA to form new adducts. These results provide a further basis for understanding the carcinogenic action of 1,2-dihaloethanes.     

Syntheses of DNA adducts of two heterocyclic amines, 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) and 2-amino-9H-pyrido[2,3-b]indole (AalphaC) and identification of DNA adducts in organs from rats dosed with MeAalphaC

2-Amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (AalphaC) are mutagenic and carcinogenic heterocyclic amines formed during ordinary cooking. MeAalphaC and AalphaC are activated to mutagenic metabolites by cytochrome P450-mediated N-oxidation to the corresponding N2-OH derivatives. The proximate mutagenic N2-OH derivatives of MeAalphaC and AalphaC did not react with deoxynucleosides or DNA. However, upon acetylation with acetic anhydride both reacted with 2'-deoxyguannosine and 3'-phospho-2'-deoxyguanosine, resulting in one adduct each, but not with other nucleosides or nucleotides. The adducts were identified as N2-(2'-deoxyguanosin-8-yl)-MeAalphaC, N2-(2'-deoxyguanosin-8-yl)-AalphaC, N2-(3'-phospho-2'-deoxyguanosin-8-yl)-MeAalphaC and N2-(3'-phospho-2'-deoxyguanosin-8-yl)-AalphaC by comparison with adducts of known structure obtained by reaction of the parent amines with acetylated guanine N3-oxide. N2-OH-MeAalphaC and N2-OH-AalphaC reacted with calf thymus DNA after addition of acetic anhydride. 32P-postlabelling analysis of modified DNA showed one major adduct co-migrating with N2-(3',5'-diphospho-2'-deoxyguanosin-8-yl)-MeAalphaC and N2-(3',5'-diphospho-2'-deoxyguanosin-8-yl)-AalphaC, respectively. Some minor adducts presumed to be undigested oligomers were also detected. 32P-postlabelling analysis of DNA from several organs of rats dosed orally with MeAalphaC showed that in vivo N2-(2'-deoxyguanosin-8-yl)-MeAalphaC also was the major adduct formed. Relative adduct level in DNA isolated from the liver of the rats was about 50.40 adducts/10(9) nt. The adduct levels were approximately 4-fold lower in the colon and the heart and approximately 12-fold lower in the kidney of the rats.     

Quantification of DNA and hemoglobin adducts of 3,4-epoxy-1,2-butanediol in rodents exposed to 3-butene-1,2-diol

1,3-Butadiene (BD) is a confirmed rodent carcinogen and a suspect human carcinogen that forms mutagenic epoxide metabolites during biotransformation. Species differences in the roles of individual DNA reactive intermediates in BD mutagenicity and carcinogenicity are not completely understood. Evidence suggests that 1,2:3,4-diepoxybutane (DEB) is responsible for the mutagenic effect induced by exposures to low concentrations of BD in mice and that metabolites of 3-butene-1,2-diol (BD-diol) are involved in the mutagenicity at high exposures in both mice and rats. Two reactive metabolites, 3,4-epoxy-1,2-butanediol (EB-diol) and hydroxymethylvinyl ketone (HMVK), are formed during the biotransformation of BD-diol and could potentially be involved in BD-diol associated mutagenicity. To examine the role of EB-diol in BD-diol mutagenicity we have evaluated the dosimetry of N7-(2,3,4-trihydroxybutyl)guanine (THB-Gua) and N-(2,3,4-trihydroxybutyl)valine (THB-Val) in female B6C3F1 mice and female F344 rats exposed by inhalation to 0, 6, 18 and 36 p.p.m. BD-diol for 4 weeks (6 h/day x 5 days/week). Results showed higher levels of both THB-Gua and THB-Val in mice than in rats. An evaluation of THB-Gua adducts showed virtually no differences between liver and lung for either species, suggesting that EB-diol is stable and is freely circulated. The data also indicated that THB adduct formation began to plateau around 18 p.p.m. in both species. Most importantly, the shape of the dose-response curve for THB adduct formation mimicked the one observed for hypoxanthine-guanine phosphoribosyltransferase (Hprt) mutation frequency. This showed that THB adducts, which are not thought to be responsible for causing the mutations, are good quantitative indicators of mutagenicity in rodents exposed to BD-diol. Although the potential contribution of HMVK still needs to be evaluated, the data suggest that EB-diol is responsible, at least in part, for BD-diol associated mutagenicity in rodents.     

Relationship between content and activity of cytochrome P450 and induction of heterocyclic amine DNA adducts in human liver samples in vivo and in vitro.

This study was designed to estimate a correlation between metabolic activation phenotypes and formation of DNA adducts by heterocyclic amines (HCA) in 15 liver samples from healthy donors. The correlation between the amount of endogenous DNA adducts and the content of cytochrome P450 in human liver samples in vivo was statistically significant at r(2) = 0.71 and P < 0.005. Furthermore, the isolated human liver microsomes were treated in vitro with two HCAs, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-9H-pyrido[2,3-b]indole (A alpha C), which have been recognized to induce two DNA adducts: 3',5'-diphosphate-N-(2'-deoxyguanosin-8-yl)-PhIP (3',5'-pdGp-C8-PhIP) and 3',5'-diphosphate-N-(2'-deoxyguanosin-8-yl)-A alpha C (3',5'-pdGp-C8-A alpha C). The correlations between the amount of DNA adducts induced by both compounds in vitro and the content of cytochrome P450 in human microsomes are statistically significant at r(2) = 0.69 and r(2) = 0.62 (P < 0.001), respectively. Furthermore, the level of DNA adducts after treatment with PhIP and A alpha C correlated with the activities of three isozymes of cytochrome P450: CYP1A1, CYP1A2, and CYP3A4. Therefore, three chemical inhibitors were used in the experiments: ellipticine against CYP1A1, furafylline against CYP1A2, and troleandomycin against CYP3A4. The highest inhibition levels in the formation of 3',5'-pdGp-C8-PhIP and 3',5'-pdGp-C8-A alpha C adducts were estimated to occur in the presence of furafylline at 56% and 69%, respectively. Ellipticine was involved in the inhibition of 40% of 3',5'-pdG-C8-PhIP adducts and in only 18% of the inhibition of 3',5'-pdGp-C8-A alpha C adducts. Troleandomycin did not significantly inhibit the formation of 3',5'-pdGp-C8-PhIP adducts under these conditions, but it inhibited the formation of 31% of the 3',5'-pdGpC8-A alpha C adducts. We conclude that the formation of DNA adducts can be used as a relevant marker of interindividual variability in the metabolic activation of HCAs in humans.     

Analysis of diepoxide-specific cyclic N-terminal globin adducts in mice and rats after inhalation exposure to 1,3-butadiene

1,3-Butadiene is an important industrial chemical used in the production of synthetic rubber and is also found in gasoline and combustion products. It is a multispecies, multisite carcinogen in rodents, with mice being the most sensitive species. 1,3-Butadiene is metabolized to several epoxides that form DNA and protein adducts. Previous analysis of 1,2,3-trihydroxybutyl-valine globin adducts suggested that most adducts resulted from 3-butene-1,2-diol metabolism to 3,4-epoxy-1,2-butanediol, rather than from 1,2;3,4-diepoxybutane. To specifically examine metabolism of 1,3-butadiene to 1,2;3,4-diepoxybutane, the formation of the 1,2;3,4-diepoxybutane-specific adduct N,N-(2,3-dihydroxy-1,4-butadiyl)-valine was evaluated in mice treated with 3, 62.5, or 1250 ppm 1,3-butadiene for 10 days and rats exposed to 3 or 62.5 ppm 1,3-butadiene for 10 days, or to 1000 ppm 1,3-butadiene for 90 days, using a newly developed immunoaffinity liquid chromatography tandem mass spectrometry assay. In addition, 2-hydroxy-3-butenyl-valine and 1,2,3-trihydroxybutyl-valine adducts were determined. The analyses of several adducts derived from 1,3-butadiene metabolites provided new insight into species and exposure differences in 1,3-butadiene metabolism. Mice formed much higher amounts of N,N-(2,3-dihydroxy-1,4-butadiyl)-valine than rats. The formation of 2-hydroxy-3-butenyl-valine and N,N-(2,3-dihydroxy-1,4-butadiyl)-valine was similar in mice exposed to 3 or 62.5 ppm 1,3-butadiene, whereas 2-hydroxy-3-butenyl-valine was 3-fold higher at 1250 ppm. In both species, 1,2,3-trihydroxybutyl-valine adducts were much higher than 2-hydroxy-3-butenyl-valine and N,N-(2,3-dihydroxy-1,4-butadiyl)-valine. Together, these data show that 1,3-butadiene is primarily metabolized via the 3-butene-1,2-diol pathway, but that mice are much more efficient at forming 1,2;3,4-diepoxybutane than rats, particularly at low exposures. This assay should also be readily adaptable to molecular epidemiology studies on 1,3-butadiene-exposed workers.     

Mechanism of lower genotoxicity of toremifene compared with tamoxifen

An increased incidence of endometrial cancer has been reported in breast cancer patients taking tamoxifen (TAM) and in healthy women participating in the TAM chemoprevention trials. Because TAM-DNA adducts are mutagenic and detected in the endometrium of women treated with TAM, TAM adducts are suspected to initiate the development of endometrial cancer. Treatment with TAM has been known to promote hepatocarcinoma in rats, but toremifene (TOR), a chlorinated TAM analogue, did not. TAM adducts are primarily formed via sulfonation of the alpha-hydroxylated TAM metabolites. To explore the mechanism of the lower genotoxicity of TOR, the formation of DNA adducts induced by TOR metabolites was measured using (32)P-postlabeling/ high-performance liquid chromatography analysis and compared with that of TAM metabolites. When alpha-hydroxytoremifene was incubated with DNA, 3'-phosphoadenosine 5'-phosphosulfate, and either rat or human hydroxysteroid sulfotransferase, the formation of DNA adducts was two orders of magnitude lower than that of alpha-hydroxytamoxifen. alpha-hydroxytoremifene was a poor substrate for rat and human hydroxysteroid sulfotransferases. In addition, the reactivity of alpha-acetoxytoremifene, a model activated form of TOR, with DNA was much lower than that of alpha-acetoxytamoxifen. Thus, TOR is likely to have lower genotoxicity than TAM. TOR may be a safer alternative by avoiding the development of endometrial cancer.     

Metabolic activation of 6-nitrochrysene in explants of human bronchus and in isolated rat hepatocytes

It has previously been shown that 6-nitrochrysene can be activated to electrophilic species capable of reacting with DNA through metabolic pathways that form N-hydroxy-6-aminochrysene or trans-1,2-dihydroxy-1,2-dihydro-6-aminochrysene as critical intermediates. Since the lung is a known target tissue for the carcinogenic action of polycyclic nitroaromatic hydrocarbons, we investigated the metabolism and DNA binding of [3H]6-nitrochrysene in 11 specimens of human bronchus. Analysis of medium from [3H]6-nitrochrysene-treated explants indicated the presence of trans-9,10-dihydroxy-9,10-dihydro-6-nitrochrysene (0.04-330 pmol/mg epithelial DNA), trans-1,2-dihydroxy-1,2-dihydro-6-nitrochrysene (12-1700 pmol/mg epithelial DNA), 6-aminochrysene (1.6-2200 pmol/mg epithelial DNA), and trans-1,2-dihydroxy-1,2-dihydro-6-aminochyrsene (3.6-610 pmol/mg epithelial DNA). Both the levels and the relative proportions of these metabolites varied widely in explants from different individuals. The amount of DNA recovered and the level of DNA modification were sufficient for adduct analysis in eight of the 11 cases for which metabolite data were obtained. Five additional bronchial specimens for which metabolite data were not obtained were also analyzed for carcinogen-DNA adducts. The levels of binding varied from 0.06 to 30.5 pmol [3H]6-nitrochrysene bound/mg DNA (two adducts per 10(8) nucleotides-10 adducts per 10(6) nucleotides). HPLC analyses of enzymatic hydrolysates of the explant DNA indicated that 11 of 13 cases contained adducts with retention times identical to those of adducts derived from trans-1,2-dihydroxy-1,2-dihydro-6-aminochrysene or N-hydroxy-6-aminochrysene. The adduct derived from trans-1,2-dihydroxy-1,2-dihydro-6-aminochrysene was the major adduct detected in eight of 13 cases. The reasons for the variation in metabolism and adduct formation observed in [3H]6-nitrochrysene-treated explants of bronchus from different donors are not known but may reflect differences in the activities of enzymes responsible for the metabolism of this compound. The influence of induction of drug metabolizing enzymes on the activation pathway of 6-nitrochrysene in an intact cell system was tested using rat hepatocytes. 6-Nitrochrysene was incubated with freshly isolated hepatocytes from rats that were either untreated or pretreated with phenobarbital, 3-methylcholanthrene or Aroclor 1254. Although the levels of adducts were similar in all cases, the pattern of DNA adducts formed in these hepatocytes was dependent on the nature of the pretreatment of the rats. As previously reported, hepatocytes from untreated rats contained adducts derived from N-hydroxy-6-aminochrysene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential induction of N(2),3-ethenoguanine in rat brain and liver after exposure to vinyl chloride

Although vinyl chloride (VC) clearly induces hepatic angiosarcoma in humans and rodents, a causal association with brain tumors has not been definitively established with the available epidemiological and experimental evidence. Because VC acts by genotoxic mechanisms, DNA adduct formation is thought to be a sensitive biomarker of early events in carcinogenesis. Adult male Sprague Dawley rats were exposed to 0 or 1100 ppm VC for 1 or 4 weeks (6 h/day, 5 days/week) by inhalation. Male weanlings were similarly exposed for 5 days. Another group of male adults was exposed to 1100 ppm [(13)C(2)]VC in a nose-only inhalation apparatus for 5 days (6 h/day). A sensitive gas chromatography high-resolution mass spectrometry assay was used to measure the major promutagenic DNA adduct, N(2),3-ethenoguanine (N(2),3-epsilonG), in rat brain and hepatocyte (HEP) DNA. The respective concentrations of N(2),3-epsilonG in control rat brain DNA at 1 and 4 weeks were 5.0 +/- 0.9 and 5.6 +/- 1.1 N(2),3-epsilonG/10(8) unmodified guanine. There was no change in N(2),3-epsilonG in adult rat brain after exposure to 1100 ppm VC for 1 or 4 weeks. In HEPs from the same animals, these adduct concentrations increased from 5.5 +/- 1.4 to 55 +/- 2.0 N(2),3-epsilonG/10(8) unmodified guanine after a 1-week exposure and from 3.0 +/- 0.3 to 110 +/- 20 N(2),3-epsilonG/10(8) unmodified guanine after a 4-week exposure. When weanlings were exposed to 1100 ppm VC for 5 days, there was a statistically significant (P = 0.04) increase in N(2),3-epsilonG in brain from 1.5 +/- 0.2 to 4.4 +/- 1.1 N(2),3-epsilonG/10(8) unmodified guanine. Weanlings exposed to 1100 ppm VC had an even greater increase in N(2),3-epsilonG in HEPs from 1.6 +/- 0.1 to 97 +/- 5.0 N(2),3-epsilonG/10(8) unmodified guanine. [(13)C(2)]N(2),3-epsilonG was not detected in brain DNA from adult rats exposed to 1100 ppm [(13)C(2)]VC for 5 days but was present in HEP DNA at 55 +/- 4.0 [(13)C(2)]N(2),3-epsilonG/10(8) unmodified guanine. The concentrations of the endogenous adduct in both organs were unchanged after this exposure. 7-(Oxoethyl)guanine (OEG), the major DNA adduct formed by VC, was reduced to 7-(2-hydroxyethyl)guanine and measured by liquid chromatography-electrospray ionization-tandom mass spectrometry in brain and HEP DNA from rats exposed to 1100 ppm VC for 1 week. Whereas 4.0 +/- 0.8 OEG/10(6) unmodified guanine were present in HEP DNA from VC-exposed rats, no adducts were detectable in brain DNA (detection limit, 0.3 OEG/10(6) unmodified guanine). These findings indicate that the genotoxic metabolite of VC is not formed in or transported to adult rat brain. Thus, it is unlikely that N(2),3-epsilonG or other VC-induced promutagenic DNA adducts play a significant role in initiating carcinogenesis in adult rat brain after exposure to VC. The data for weanling rats are less clear. Whereas a small increase in N(2),3-epsilonG in the brains of weanlings was found after exposure to 1100 ppm VC, the resulting adduct concentration was similar to that measured in unexposed adults. Future exposures of weanling rats to the stable isotopically labeled compound will be necessary to conclusively determine whether this increase was due to VC.     

Apoptosis and age-dependant induction of nuclear and mitochondrial etheno-DNA adducts in Long-Evans Cinnamon (LEC) rats: enhanced DNA damage by dietary curcumin upon copper accumulation

Long-Evans Cinnamon (LEC) rats, a model for human Wilson's disease, develop chronic hepatitis and liver tumors owing to accumulation of copper and induced oxidative stress. Lipid peroxidation (LPO)-induced etheno-DNA adducts in nuclear- and mitochondrial-DNA along with apoptosis was measured in LEC rat liver. Levels of etheno-DNA adducts (1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine) increased with age reaching a peak at 8 and 12 weeks in nuclear and mitochondrial DNA, respectively. This is the first demonstration that etheno-DNA adducts are also formed in mitochondrial DNA. Apoptosis was assessed by TUNEL+ cells in liver sections. CD95L RNA expression was also measured by in situ hybridization in the same sections. The highest nuclear DNA adduct levels coincided with a reduced apoptotic rate at 8 weeks. Mitochondrial-DNA adducts peaked at 12 weeks that coincided with the highest apoptotic rate, suggesting a link of etheno-DNA adducts in mitochondrial DNA to apoptosis. The DNA damage in liver was further enhanced and sustained by 0.5% curcumin in the diet. Treatment for 2 weeks elevated etheno-DNA adducts 9- to 25-fold in nuclear DNA and 3- to 4-fold in mitochondrial-DNA, providing a plausible explanation as to why in our earlier study [Frank et al. (2003) Mutat. Res., 523-524, 127-135], curcumin failed to prevent liver tumors in LEC rats. Our results also confirm the reported in vitro DNA damaging potential of curcumin in the presence of copper ions by reactive oxygen species. LPO-induced adduct formation in nuclear and mitochondrial DNA appear as early lesions in LEC rat liver carcinogenesis and are discussed in relation to apoptotic events in the progression of malignant disease.