Mammalian peroxidases activate anticancer drug ellipticine to intermediates forming deoxyguanosine adducts in DNA identical to those found in vivo and generated from 12-hydroxyellipticine and 13-hydroxyellipticine

Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation, inhibition of topoisomerase II and cytochrome P450-mediated formation of covalent DNA adducts. This is the first report on the molecular mechanism of ellipticine oxidation by peroxidases (human myeloperoxidase, human and ovine cyclooxygenases, bovine lactoperoxidase, horseradish peroxidase) to species forming ellipticine-DNA adducts. Using NMR spectroscopy, the structures of 2 ellipticine metabolites were identified; the major product is the ellipticine dimer, in which the 2 ellipticine skeletons are connected via N(6) of the pyrrole ring of one ellipticine molecule and C9 in the second one. The minor metabolite is ellipticine N(2)-oxide. Using (32)P-postlabeling and [(3)H]-labeled ellipticine, we showed that ellipticine binds covalently to DNA after its activation by peroxidases. The DNA adduct pattern induced by ellipticine consisted of a cluster of up to 4 adducts. The 2 adducts are indistinguishable from the 2 major adducts generated between deoxyguanosine in DNA and either 13-hydroxy- or 12-hydroxyellipticine or in rats treated with ellipticine, or if ellipticine was activated with human hepatic and renal microsomes. The results presented here are the first characterization of the peroxidase-mediated oxidative metabolites of ellipticine and we have proposed species, 2 carbenium ions, ellipticine-13-ylium and ellipticine-12-ylium, as reactive species generating 2 major DNA adducts seen in vivo in rats treated with ellipticine. The study forms the basis to further predict the susceptibility of human cancers to ellipticine.     

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 environmental contaminant 3-nitrobenzanthrone after intratracheal instillation in rats

3-Nitrobenzanthrone (3-NBA) is an environmental pollutant and suspected human carcinogen found in emissions from diesel and gasoline engines and on the surface of ambient air particulate matter; human exposure to 3-NBA is likely to occur primarily via the respiratory tract. In our study female Sprague Dawley rats were treated by intratracheal instillation with a single dose of 0.2 or 2 mg/kg body weight of 3-NBA. Using the butanol enrichment version of the (32)P-postlabeling method, DNA adduct formation by 3-NBA 48 hr after intratracheal administration in different organs (lung, pancreas, kidney, urinary bladder, heart, small intestine and liver) and in blood was investigated. The same adduct pattern consisting of up to 5 DNA adduct spots was detected by thin layer chromatography in all tissues and blood and at both doses. Highest total adduct levels were found in lung and pancreas (350 +/- 139 and 620 +/- 370 adducts per 10(8) nucleotides for the high dose and 39 +/- 18 and 55 +/- 34 adducts per 10(8) nucleotides for the low dose, respectively) followed by kidney, urinary bladder, heart, small intestine and liver. Adduct levels were dose-dependent in all organs (approximately 10-fold difference between doses). It was demonstrated by high performance liquid chromatography (HPLC) that all 5 3-NBA-derived DNA adducts formed in rats after intratracheal instillation are identical to those formed by other routes of application and are, as previously shown, formed from reductive metabolites bound to purine bases. Although total adduct levels in the blood were much lower (41 +/- 27 and 9.5 +/- 1.9 adducts per 10(8) nucleotides for the high and low dose, respectively) than those found in the lung, they were related to dose and to the levels found in lung. These results show that uptake of 3-NBA by the lung induces high levels of specific DNA adducts in several organs of the rat and an identical adduct pattern in DNA from blood. Therefore, 3-NBA-DNA adducts present in the blood are useful biomarkers for exposure to 3-NBA and may help to assess the effective biological dose in humans exposed to it.     

Covalent DNA damage in tissues of cigarette smokers as determined by 32P-postlabeling assay

Covalent DNA addition products (adducts) formed by the reaction of chemical carcinogens or their metabolites with DNA are critically involved in the initiation of chemical carcinogenesis and may serve as molecular markers and dosimeters for environmental carcinogen exposures. Using a highly sensitive 32P-postlabeling assay for DNA adduct analysis, we studied DNA damage elicited by cigarette smoke in tissues of smokers. A multitude of characteristic smoking-induced, presumably aromatic DNA adducts were found to occur in a dose- and time-dependent manner in the lung, bronchus, and larynx of smokers with cancer of these organs and to decline only slowly after cessation of smoking. Low levels of adducts appeared to persist for up to 14 years in the lungs of exsmokers with high previous exposures. These results corroborate data of epidemiological studies showing that the lung cancer risk and mortality of smokers increase with the intensity and duration of smoking and decline only slowly after cessation of smoking. Tissue distribution studies in autopsy samples revealed the presence of smoking-associated DNA lesions also in the kidney, bladder, esophagus, heart, ascending aorta, and liver. The most extensive DNA damage was found in lung and heart, i.e., 1 aromatic adduct in about 10(7) DNA nucleotides. Our results suggest that cigarette smoking-induced DNA adduct formation is causally related to cancer in the target organs.     

DNA adduct formation by the ubiquitous environmental contaminant 3-nitrobenzanthrone in rats determined by (32)P-postlabeling.

Diesel exhaust is known to induce tumors in animals and is suspected of being carcinogenic in humans. Of the compounds found in diesel exhaust and in airborne particulate matter, 3-nitrobenzanthrone (3-NBA), is a particularly powerful mutagen. We investigated the capacity of 3-NBA to form DNA adducts in vivo that could be used as agent-specific biomarkers of exposure. Female Sprague-Dawley rats were treated orally with 2 mg/kg body weight of 3-NBA, and DNA from various organs was analyzed by (32)P-postlabeling. High levels of 3-NBA-specific adducts were detectable in all organs. Both enrichment versions nuclease P1 digestion and n-butanol extraction resulted in patterns consisting of either 3 or 4 adducts remarkably similar in all tissues examined. The highest level of DNA adducts was found in the small intestine (38 adducts per 10(8) nucleotides) followed by forestomach, glandular stomach, kidney, liver, lung and bladder. To provide information on the nature of the adducts formed in vivo in rats, DNA adducts were cochromatographed in 2 independent systems with standardized deoxyguanosine adducts and deoxyadenosine adducts produced by reaction of 3-NBA in the presence of xanthine oxidase with deoxyribonucleoside 3'-monophosphates in vitro. In both systems, each of the rat adducts comigrated either with a deoxyguanosine or a deoxyadenosine-derived 3-NBA adduct. Our results demonstrate that 3-NBA binds covalently to DNA after metabolic activation, forming multiple DNA adducts in vivo, all of which are products derived from reductive metabolites bound to the purine bases (deoxyguanosine 60% and deoxyadenosine 40%).     

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.     

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.     

Species and organ differences in DNA adduct formation and repair after treatment with 4-hydroxyaminoquinoline 1-oxide

4-Hydroxyaminoquinoline 1-oxide (4HAQO) demonstrates obvious organotropic and species specificity in its carcinogenesis and the present investigation concerns 4HAQO DNA adduct formation and repair as studied in various organs of four animal species (rats, mice, guinea pigs and hamsters). Three hours after an iv injection of 10 mg per kg body weight of tritium-labeled 4HAQO, the major organs were removed and used for assessment of label incorporation in the DNA. The results showed that the DNA binding levels generally correlated well with the reported species and organ specificity of 4HAQO tumorigenesis. For example, rats showed highest DNA binding in the pancreas and kidney, major target organs. The levels of DNA binding in the liver were invariably low in all 4 animal species, in agreement with the lack of hepatocarcinogenicity associated with 4HAQO exposure. A clear relationship between DNA adduct formation and carcinogen dose was also found after treatment of mice with 4HAQO at doses of 1, 5, 10 and 20 mg per kg body weight in all tissues (pancreas, kidney and lung) except for the liver. Comparison of DNA repair processes in rats, a highly susceptible species, and hamsters, a resistant species in terms of 4HAQO carcinogenicity, revealed highest formation and slowest removal of adducts in the target organs of the rat. In the hamster organs and the rat lung and liver, DNA adduct formation was generally low and in the case of elevation in the initial phase, quickly removed.     

Species and Organ Differences in DNA Adduct Formation and Repair after Treatment with 4-Hydroxyaminoquinoline 1-Oxide

4-Hydroxyaminoquinoline 1-oxide (4HAQO) demonstrates obvious organotropic and species specificity in its carcinogenesis and the present investigation concerns 4HAQO DNA adduct formation and repair as studied in various organs of four animal species (rats, mice, guinea pigs and hamsters). Three hours after an iv injection of 10 mg per kg body weight of tritium-labeled 4HAQO, the major organs were removed and used for assessment of label incorporation in the DNA. The results showed that the DNA binding levels generally correlated well with the reported species and organ specificity of 4HAQO tumorigenesis. For example, rats showed highest DNA binding in the pancreas and kidney, major target organs. The levels of DNA binding in the liver were invariably low in all 4 animal species, in agreement with the lack of hepatocarcinogenicity associated with 4HAQO exposure. A clear relationship between DNA adduct formation and carcinogen dose was also found after treatment of mice with 4HAQO at doses of 1, 5, 10 and 20 mg per kg body weight in all tissues (pancreas, kidney and lung) except for the liver. Comparison of DNA repair processes in rats, a highly susceptible species, and hamsters, a resistant species in terms of 4HAQO carcinogenicity, revealed highest formation and slowest removal of adducts in the target organs of the rat. In the hamster organs and the rat lung and liver, DNA adduct formation was generally low and in the case of elevation in the initial phase, quickly removed.     

Identification of a genotoxic mechanism for the carcinogenicity of the environmental pollutant and suspected human carcinogen o-anisidine

2-methoxyaniline (o-anisidine) is an industrial and environmental pollutant and a bladder carcinogen for rodents. The mechanism of its carcinogenicity was investigated with 2 independent methods, 32P-postlabeling and 14C-labeled o-anisidine, to show that o-anisidine binds covalently to DNA in vitro after its activation by human hepatic microsomes. We also investigated the capacity of o-anisidine to form DNA adducts in vivo. Rats were treated i.p. with o-anisidine (0.15 mg/kg daily for 5 days) and DNA from several organs was analyzed by 32P-postlabeling. Two o-anisidine-DNA adducts, identical to those found in DNA incubated with o-anisidine and human microsomes in vitro, were detected in urinary bladder (4.1 adducts per 10(7) nucleotides), the target organ, and, to a lesser extent, in liver, kidney and spleen. These DNA adducts were identified as deoxyguanosine adducts derived from a metabolite of o-anisidine, N-(2-methoxyphenyl)hydroxylamine. This metabolite was identified in incubations with human microsomes. With 9 human hepatic microsomal preparations, we identified the specific CYP catalyzing the formation of the o-anisidine metabolites by correlation studies and by examining the effects of CYP inhibitors. On the basis of these analyses, oxidation of o-anisidine was attributed mainly to CYP2E1. Using recombinant human CYP (in Supersomes) and purified CYPs, the participation of CYP2E1 in o-anisidine oxidation was confirmed. In Supersomes, CYP1A2 was even more efficient in oxidizing o-anisidine than CYP2E1, followed by CYP2B6, 1A1, 2A6, 2D6 and 3A4. The results, the first report on the potential of the human microsomal CYP enzymes to activate o-anisidine, strongly suggest a carcinogenic potential of this rodent carcinogen for humans.     

Human hepatic and renal microsomes, cytochromes P450 1A1/2, NADPH:cytochrome P450 reductase and prostaglandin H synthase mediate the formation of aristolochic acid-DNA adducts found in patients with urothelial cancer

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Understanding which human enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual's susceptibility to this plant carcinogen. Using the (32)P postlabeling assay, we examined the ability of microsomal samples from 8 human livers and from 1 human kidney to activate AAI, the major component of the plant extract AA, to metabolites forming adducts in DNA. Microsomes of both organs generated DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(deoxyadenosin-N(6)-yl)aristolactam I, 7-(deoxyguanosin-N(2)-yl)aristolactam I and 7-(deoxyadenosin-N(6)-yl)aristolactam II were identified as AA-DNA adducts formed from AAI by all human hepatic and renal microsomes. To define the role of human microsomal enzymes in the activation of AAI, we investigated the modulation of AAI-DNA adduct formation by cofactors and selective inhibitors of microsomal reductases, cytochrome P450 (CYP) enzymes, NADPH:CYP reductase and NADH:cytochrome b(5) reductase. We also determined whether the activities of CYP and NADPH:CYP reductase in different human hepatic microsomal samples correlated with the levels of AAI-DNA adducts formed by the same microsomal samples. On the basis of these studies, we attribute most of the activation of AAI in human hepatic microsomes to CYP1A2. In contrast to human hepatic microsomes, in human renal microsomes NADPH:CYP reductase is more effective in AAI activation. In addition, prostaglandin H synthase is another enzyme activating AAI in renal microsomes. The results demonstrate for the first time the potential of microsomal enzymes in human liver and kidney to activate AAI by nitroreduction.     

32P-postlabeling assay in mice of transplacental DNA damage induced by the environmental carcinogens safrole, 4-aminobiphenyl, and benzo(a)pyrene

Transplacental exposure of fetuses to carcinogens is known to induce tumors in the offspring, often with a high incidence and short latency. While covalent adduction of DNA appears to be essential for tumor initiation, little is known about the binding of carcinogens to the DNA of fetal tissues. A sensitive 32P-postlabeling method enabled us to study the binding of the environmental carcinogens safrole (600 mumol/kg p.o.), 4-aminobiphenyl (800 mumol/kg), and benzo(a)pyrene (200 mumol/kg) to the DNA of various maternal and fetal tissues after administration of test carcinogens to pregnant ICR mice on day 18 of gestation. The results show that these carcinogens bound to the DNA of maternal and fetal liver, lung, kidney, heart, brain, intestine, skin, maternal uterus, and placenta, with organ-specific quantitative and qualitative differences. It was possible for the first time to analyze DNA adduct patterns in minute amounts of tissue, for example those available from fetal heart. The covalent binding index (mumol adducted nucleotides per mol of DNA nucleotides/mumol carcinogen administered per g body weight) 24 h after safrole treatment was estimated for the different organs and ranged from 0.1 to 247 and 0.1 to 5.8 for maternal and fetal DNA, respectively. Covalent binding index values of 0.2 to 13 and 0.1 to 0.3 for maternal and fetal DNA, respectively, were found for 4-aminobiphenyl. Benzo(a)pyrene treatment yielded covalent binding index values of 0.6 to 6.5 and 0.3 to 0.7 for maternal and fetal DNA, respectively. In both maternal and fetal tissues, safrole exhibited preferential binding to liver DNA. 4-Aminobiphenyl bound preferentially to DNA of maternal liver and kidney but showed no preference among fetal tissues. Benzo(a)pyrene exhibited weak tissue preference in both maternal and fetal organs. For all of the compounds studied, the fetal adduct levels were generally lower than the corresponding maternal adduct levels, especially when the level of maternal adduction was high. The major finding was that several carcinogens of diverse structure or their metabolites readily crossed the placenta and gave rise to DNA adducts in fetal organs. The resulting DNA damage in rapidly proliferating tissues may play a critical role in transplacental carcinogenesis.     

大白菜抑制大鼠体内致癌物PhIP-DNA加合物形成及其可能的作用机理

目的研究大白菜（ｂｒａｓｓｉｃａｃｈｉｎｅｎｓｉｓ）对结肠致癌物２－氨基－１－甲基－６－苯基咪唑［４,５－ｂ］吡啶（ＰｈＩＰ）致癌的预防作用及机理。方法雄性ＳＤ大鼠喂饲基础饲料或掺有大白菜粉（２０％）混合饲料１０天后,经口摄入ＰｈＩＰ（１０ｍｇ／ｋｇ）。以３２Ｐ－后标记方法分析动物结肠粘膜、心、肺和肝中ＰｈＩＰ－ＤＮＡ加合物含量,并测定参与ＰｈＩＰ代谢的细胞色素Ｐ４５０（ＣＹＰ）１Ａ１和１Ａ２以及谷胱甘肽转硫酶（ＧＳＴ）活性。结果经喂饲含大白菜饲料的大鼠,其结肠、心、肺、肝等器官中,ＰｈＩＰ－ＤＮＡ加合物含量显著低于喂饲基础饲料的动物（Ｐ＜０．０１）,抑制率结肠为８２．３％、心脏为６０．６％、肺为４８．４％、肝脏为４８．９％。大白菜对参与ＰｈＩＰ解毒的ＣＹＰ１Ａ１和ＧＳＴ有显著的诱导作用。与对照组比较,喂饲大白菜大鼠的肝ＣＹＰ１Ａ１活性增加８０．６％（Ｐ＜０．０１）,肝和肺细胞浆ＧＳＴ活性分别增加１８．２％和３５．６％（Ｐ＜０．０５）。结论食用大白菜可有效抑制ＰｈＩＰ－ＤＮＡ加合物形成,其作用机理可能是诱导解毒酶     

Immunocytochemical visualization of DNA adducts in mouse tissues and human white blood cells following treatment with benzo[a]pyrene or its diol epoxide. A quantitative approach

The formation and stability of benzo[a]pyrene DNA adducts were studied in tissues of BALB/c mice exposed to benzo[a]pyrene (B[a]P). The DNA adducts were visualized with an immunocytochemical peroxidase staining technique using an antiserum specific for the major B[a]P-derived adduct in DNA [(+/-)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE-N2-dG)]. The nuclear staining density was measured by microdensitometry. When mice were treated with an increasing dose of B[a]P the nuclear staining increased in the tissues studied (lung, heart and kidney). A linear relationship was found between the immunocytochemical nuclear staining signal and the actual DNA adduct level in the lung as measured by 32P-postlabeling. Maximum adduct formation was found 5 days after a single i.p. injection of B[a]P. Adduct levels decreased gradually after 7 days, but even after 61 days a slight specific staining was still present, suggesting that not all adducts had disappeared at that time. As judged from the disappearance of [3H]thymidine from prelabeled DNA the loss of adducts from the lung was not a result of DNA repair but one of cell turnover. In human white blood cells B[a]P-derived adducts could be detected after in vitro incubation with the reactive metabolite of B[a]P (BPDE). Dose-response studies demonstrated a positive relationship between BPDE-DNA adduct formation, the immunocytochemical staining signal and the BPDE concentration in the culture medium.     

Comparison of DNA adduct formation by aristolochic acids in various in vitro activation systems by 32P-post-labelling: evidence for reductive activation by peroxidases

Aristolochic acid I (AAI) and aristolochic acid II (AAII), the two major components of the carcinogenic plant extract aristolochic acid (AA), are known to be mutagenic and to form DNA adducts in vivo. According to the structures of the major DNA adducts identified in animals and humans, nitroreduction is the crucial pathway in the metabolic activation of these naturally occurring nitroarenes to their ultimate carcinogenic species. Using the nuclease P1-enhanced version of the 32P-post-labelling assay we investigated the formation of DNA adducts by AAI and AAII in different in vitro activation systems in order to determine the most suitable in vitro system mimicking target tissue activation. Although DNA adducts resulting from oxidative activation of AAs have not yet been identified both reductive and oxidative in vitro systems were employed. In vitro incubations were conducted under standardized conditions (0.3 mM AAs; 4 mM dNp as calf thymus DNA) using rat liver microsomes, xanthine oxidase (a mammalian nitroreductase), horseradish peroxidase, lactoperoxidase and chemical reduction by zinc. Enzymatic incubations were performed under aerobic and anaerobic conditions. A combination of two independent chromatographic systems (ion-exchange chromatography and reversed-phase HPLC) with reference compounds was used for the identification of DNA adducts detected by the 32P-post-labelling assay. The two known major adducts of AAI or AAII found in vivo were generated by all in vitro systems except for incubations with AAII and horseradish peroxidase where two unknown adducts predominated. Irrespective of the in vitro activation system used, the majority of adduct spots obtained were identified as the previously characterized four AA-DNA adducts: dA-AAI, dA-AAII, dG-AAI and dG-AAII. This indicates that both reductive and peroxidative activation of AAI or AAII resulted in chromatographically indistinguishable DNA adducts. Thus, peroxidase mediated activation of AAs led to the formation of the same adducts that had been observed in vivo and upon reductive activation in several in vitro systems. Quantitative analyses of individual adducts formed in the various in vitro systems revealed relative adduct labelling (RAL) values over a 100,000-fold range from 4 in 10(3) for activation of AAII to deoxyadenosine adducts by zinc to only 3 in 10(8) for activation of AAII by lactoperoxidase. The extent of DNA modification by AAI was higher than by AAII in all enzymatic in vitro systems. Only activation by zinc resulted in higher total binding to exogenous DNA by AAII than by AAI. Aerobic incubations with rat liver microsomes generated AAI- and AAII-DNA adduct profiles reproducing profiles in target tissue (forestomach) of rats, thus providing the most appropriate activation among the in vitro systems tested.      

DNA adduct and tumor formations in rats after intratracheal administration of the urban air pollutant 3-nitrobenzanthrone

3-Nitrobenzanthrone (3-NBA) has been isolated from diesel exhaust and airborne particles and identified as a potent direct-acting mutagen in vitro and genotoxic agent in vivo. In order to evaluate the in vivo toxicity and carcinogenicity of 3-NBA in a situation corresponding to inhalation, a combined short-term and lifetime study with intratracheal (i.t.) instillation in female F344 rats was performed. DNA adduct formation, as a marker for the primary effect and analyzed by 32P-HPLC after single instillation, showed a few major DNA adducts and a rapid increase with a peak after 2 days, followed by a decline. No DNA adducts above the background level were observed after 16 days. The highest DNA adduct formation was observed in lung [approximately 250 DNA adducts/10(8) normal nucleotides (NN)] closely followed by kidney (approximately 200 DNA adducts/10(8) NN), whereas liver contained only 12% (approximately 30 DNA adducts/10(8) NN) of the levels of DNA adducts found in lung. In the tumor study, squamous cell carcinomas were found after 7-9 months in the high-dose group (total dose of 2.5 mg 3-NBA) and after 10-12 months in the low-dose group (total dose of 1.5 mg 3-NBA). The fraction of squamous cell carcinoma out of the total amount of tumors observed at the end of experiment at 18 months, corresponded to 3/16 and 11/16 in the low- and high-dose group, respectively. A single case of adenocarcinoma was also observed in each group. In the control group, no tumors were observed during the entire study of 18 months. In addition, a few cases of squamous metaplasia were also observed in the lung in both dose groups but not in the controls. In conclusion, 3-NBA forms DNA adducts in the lung immediately after i.t. administration but almost all DNA adducts were eliminated after 16 days. Tumor formation in two dose groups was observed in a dose-dependent manner with squamous cell carcinomas as the predominant tumor type at high exposure.     

DNA modification by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in rats

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant mutagenic heterocyclic amine by weight in cooked foods. This mutagen was found to produce DNA adducts in all ten tested organs of rats using the 32P-postlabeling method. The level of DNA adducts in the pancreas, kidney and liver increased dose-dependently and feeding time-dependently up to four weeks. When diet containing 0.05% PhIP was given to rats for four weeks, levels of PhIP-DNA adducts were relatively high in the lung, pancreas and heart, being around 20 per 10(7) nucleotides, and lowest in the liver, being 2.20 per 10(7) nucleotides. Thus, PhIP showed a unique feature in the formation of DNA adducts compared to other mutagenic and carcinogenic heterocyclic amines, which produce the highest level of DNA adducts in the liver.