Identification of the major metabolites and DNA adducts formed from 2-nitropyrene in vitro

In contrast to 1-nitropyrene (1-NP), which is the most abundant nitropolycyclic aromatic hydrocarbon in numerous environmental sources, 2-nitropyrene (2-NP) has been detected only in the ambient air and not in direct emissions. Thus, 2-NP can be used as an indicator for monitoring human exposure to nitropolynuclear aromatic hydrocarbons in ambient air. Therefore, it is essential to determine the possible metabolic pathways of 2-NP. The metabolism of 2-NP by rat liver 9000 g supernatant was investigated. Under aerobic conditions, ring oxidation to 6-hydroxy-2-nitropyrene and nitroreduction to 2-aminopyrene (2-AP) were observed. When incubations were carried out in an atmosphere of nitrogen, 2-AP was the only metabolite detected. These results are consistent with those observed with 1-NP. In vitro metabolic activation of 2-NP to DNA adducts catalyzed by xanthine oxidase was also examined. Two adducts were characterized as N-(deoxyguanosin-8-yl)-2-aminopyrene and N-(deoxyadenosin-8-yl)-2-aminopyrene. The presence of deoxyadenosine adduct, which is derived from the nitroreduction pathway, may contribute to the powerful direct-acting mutagenicity of 2-NP.     

A cyclophosphamide/DNA phosphoester adduct formed in vitro and in vivo

The antitumor activity of cyclophosphamide is thought to be due to the alkylating activity of phosphoramide mustard, a metabolite of cyclophosphamide. Reaction of 2'-deoxyguanosine 3'-monophosphate and phosphoramide mustard resulted in the formation of several adducts that could be detected by high performance liquid chromatography (HPLC). One of these adducts, isolated and purified by HPLC, could be detected by 32P postlabeling. This product was identified by UV, nuclear magnetic resonance, and mass spectrometry and by acid, base, and enzymatic hydrolysis to be 2'-deoxyguanosine 3'-monophosphate 2-(2-hydroxyethyl)aminoethyl ester. A combination of HPLC fractionation of digested DNA and 32P postlabeling was used to detect this adduct in calf thymus DNA incubated in vitro with metabolically activated cyclophosphamide and in DNA from the liver of mice treated with cyclophosphamide. In these DNA samples the adduct occurred at a level of 1/10(5) and 1/3 x 10(7) nucleotides, respectively.     

Identification of the major serum albumin adduct formed by 4-aminobiphenyl in vivo in rats

Serum albumin was isolated from rats at 27 h after administration of the carcinogen [2,2'-3H]-4-aminobiphenyl. Pronase digestion of the purified albumin yielded a mixture of radiolabeled materials which was resolved into 5 major components by reverse-phase liquid chromatography. From detailed UV, 1H-NMR, and mass spectral analyses, four of these were determined to be 4-aminobiphenyl, 4'-hydroxy-4-acetylaminobiphenyl, and two other metabolites, all of which are presumed to be non-covalently associated with the serum albumin. The fifth component, however, resulted from covalent bond formation and was identified as a tetrapeptide containing 3-tryptophanyl-4-acetylaminobiphenyl, the amino acid sequence of which was H2N-ala-trp-ala-val. Since rat serum albumin contains only a single tryptophan residue in a hydrophobic drug binding site, its high selectivity for carcinogen binding suggests a unique role for this protein in the detoxification and/or transport of ultimate carcinogenic metabolites.     

Co-chromatography of a tamoxifen epoxide-deoxyguanylic acid adduct with a major DNA adduct formed in the livers of tamoxifen-treated rats

Tamoxifen is a potent liver carcinogen in rats and has beenshown to form covalent DNA adducts in the livers of severalspecies of rodent. We have shown previously by ³²P-postlabelling(Carcinogenesis, 13, 2197–2203) that >85% of the totaladducts detected and resolved by multi-directional TLC migrateas a single spot. In the present study, this material was furtheranalysed by reverse-phase HPLC and resolved into two approximatelyequal components. Tamoxifen 1,2-epoxide, a postulated metaboliteof tamoxifen, was reacted with DNA and polydeoxyribonucleotidesand the products analysed. ³²P-Postlabelling revealed threemajor adduct spots on TLC which comigrated with the three majoradduct spots seen with DNA from livers of tamoxifen-treatedrats. Moreover, the major epoxide adduct, which contained guanineas the modified base, eluted on HPLC as a single major peakcoincident with one of the major peaks derived from the liverDNA of tamoxifen-treated rats. These results demonstrate that{small tilde}40% of the tamoxifen-DNA adducts formed in vivoare chromatographically indistinguishable with the major productof the reaction of tamoxifen epoxide with guanine residues inDNA and provide important clues to the mechanism of activationof tamoxifen to a genotoxic carcinogen.     

Identification of the major adducts formed by reaction of 5-methylchrysene anti-dihydrodiol-epoxides with DNA in vitro

5-Methylchrysene is metabolically converted to the bay-region dihydrodiol-epoxides, trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene (DE-I), in which the methyl group and the epoxide ring are in the same bay region, and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-5-methylchrysene (DE-II). Previous studies have indicated that DE-I is more important in 5-methylchrysene carcinogenesis than is DE-II. Both DE-I and DE-II were individually reacted with calf thymus DNA in vitro. The DNA was enzymatically hydrolyzed to deoxyribonucleosides, and the modified deoxyribonucleosides were separated by chromatography on Sephadex LH-20 and analyzed by high-performance liquid chromatography. One major adduct and seven minor adducts were formed from each dihydrodiol-epoxide. The major adduct was, in each case, characterized by its pH-dependent partition coefficient, stability to base, mass spectrum, ultraviolet spectrum, and nuclear magnetic resonance spectrum as a deoxyguanosine derivative resulting from addition of the exocyclic amino group of deoxyguanosine to the benzylic carbon of the epoxide ring of the dihydrodiol-epoxide. The results of this study show that the major DNA adducts formed from 5-methylchrysene via DE-I and DE-II are structurally similar.     

Identification of the DNA adduct formed by metabolism of 1,8-dinitropyrene in Salmonella typhimurium

The incubation of [³H]1,8-dinitropyrene with Salmonella typhimuriumTA98NR followed by isolation of the DNA from these cells, hydrolysisof the DNA to nucleosides, butanol extraction of the hydrolysateand purification by reversed-phase liquid chromatography affordeda single product. Calf thymus DNA, after treatment with N-hydroxyl-1-amino-8-nitropyrene,was hydrolyzed, extracted and purified in a similar fashionto give a single compound which was shown to be the deoxyguanosinederivative 1-N-(2'-deoxyguanosin-8-yl)-amino-8-nitropyrene bya combination of proton n.m.r. and u.v.-vis. spectroscopy andfast atom bombardment mass spectrometry. The DNA adducts formedin vivo and in vitro exhibited identical chromatographic andchemical behavior. Under acidic or basic conditions the in vivoand in vitro adducts were converted to identical products. Reductionof the adduct gave a new, highly fluorescent product that hada fluorescent emission spectrum identical to that of 1,8-diaminopyrene.     

Isolation and characterization of the major serum albumin adduct formed by aflatoxin B1 in vivo in rats

Aflatoxin B₁ (AFB₁) was shown to react primarily with one ormore lysine residues in serum albumin (SA), accounting for morethan half of the total binding to this protein. The radioactivityassociated with SA following administration of [U-¹⁴C]AFB₁ torats was cleared with a half-life of 2.5 days, which is notsignificantly different from the half-life of unmodified albuminin the normal rat. The product isolated from a Pronase digestof in vivo-modified SA was identical by chromatographic retentiontime and u.v. and mass spectroscopy to the synthetic productobtained by the acylasecatalyzed deacetylation of the reactionproduct of N-acetyl-L-lysine with 8,9-dihydro-8,9-dibromo-AFB₁.The latter was characterized by u.v., fluorescence, 500 MHz¹H-n.m.r. and fast atom bombardment mass spectrometry. The spectraldata strongly support a structure in which the terminal dihydrofuranring of AFB₁ has been converted to a pyrrolinone ring. It isproposed that the initial adduct is formed by condensation ofthe dialdehyde tautomer of 8,9-dihydro-8,9-di-hydroxy-AFB₁,with the -amino group of lysine, to form a Schiff base, andthat the Schiff base undergoes an Amadori rearrangement to an-amino ketone. The pyrrolinone ring is formed by condensationof the amino group with the remaining aldehyde to yield thefinal product. The purified product was relatively stable butwas shown to decompose significantly under the conditions usedto isolate it from modified SA.     

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.     

Liver microsomal metabolism of the environmental carcinogen 3-nitrofluoranthene. I. Phenolic metabolites

The liver microsomal metabolism of the environmental carcinogen3-nitrofluoranthene has been investigated. Five phenolic metaboliteswere isolated and characterized by their mass and NMR spectraas: 3-nitrofluoranthen-1-ol, 3-nitrofluoranthen-6-ol, 3-nitrofluoranthen-(7or 10)-ol, 3-nitrofluoranthen-8-ol and 3-nitrofluoranthen-9-ol.While the microsomes isolated from Sprague-Dawley rat liverand C57B16 mouse liver preferentially metabolized the 3-nitrofluorantheneto 3-nitrofluoranthen-8-ol and 3-nitro-fluoranthen-9-ol, themicrosomes from Hartley guinea-pig liver preferentially metabolized3-nitrofluoranthene to 3-nitrofluoranthen-6-ol. 3-Nitrofluoranthen-1-olwas a minor metabolite in each of the incubations. The resultsindicate the presence of the nitro group on fluoranthene directsthe metabolism away from the C1–C5 positions, and moretowards the C6–C10 positions.     

Isolation and characterization of the major fluoranthene-hemoglobin adducts formed in vivo in the rat

The binding of fluoranthene (FA) to hemoglobin was studied both in vitro and in vivo in the rat. The in vitro binding of microsomally activated FA to rat hemoglobin appeared to involve the fluoranthene 2,3-dihydrodiol-1,10b-epoxides. Three classes of hemoglobin adducts were observed in rats chronically administered FA in the diet. Based on high pressure liquid chromatography retention times, UV and mass spectral evidence, and behavior upon cis-diol affinity chromatography, the major class of globin adducts formed in vivo was demonstrated to result from binding of syn and anti isomers of FA 2,3-dihydrodiol-1,10b-epoxides to beta-cysteine-125 of rat hemoglobin. These adducts represented at least 41% of the total binding to globin. A minor class of adducts (12% of the total binding) appeared to involve the binding of an unidentified FA metabolite to the same cysteine residue of the protein. A substantial portion of FA binding to rat hemoglobin in vivo (29%) involved metabolic pathways which were not duplicated by simple in vitro systems. That portion of the binding to globin has not been characterized.     

Effects of human intestinal flora on mutagenicity of and DNA adduct formation from food and environmental mutagens

Although the intestinal flora is believed to have a critical role in carcinogenesis, little is known about the role of the human intestinal flora on the effects of mutagens in vivo. The aim of the present study was to address a possible role of the human intestinal flora in carcinogenesis, by exploiting human-flora-associated (HFA) mice. The capacity of human faeces to activate or inactivate 2-amino-3-methyl-3H:-imidazo[4,5-f]quinoline (IQ) and 2-nitrofluorene was determined using the Ames assay. Human faecal suspensions that were active in this regard were then selected and orally inoculated into germfree NMRI mice to generate HFA mice. HFA, germfree, conventionalized and conventional mice were administered IQ, 2-amino-9H:-pyrido[2,3-b]indole (2-amino-alpha-carboline; AAC) and 2-nitrofluorene. The activity of human intestinal flora against mutagens could be transferred into the mice. In comparing germfree mice and mice harbouring an intestinal flora, the presence of a flora was essential for the activities of faeces against mutagens. After administration of IQ and 2-nitrofluorene, DNA adducts were observed in the mice with a flora, while adducts were extremely low or absent in germfree animals. DNA adducts after AAC treatment were higher in germfree mice in some tissues including colon than in mice with bacteria. Differences in DNA adduct formation were also observed between HFA mice and mice with mouse flora in many tissues. These results clearly indicate that the intestinal flora have an active role in DNA adduct formation and that the role is different for the different chemicals to which the animals are exposed. The results also demonstrate that the human intestinal flora have different effects from the mouse flora on DNA adduct formation as well as in vitro metabolic activities against mutagens. Studies using HFA mice could thus provide much-needed information on the role of the human intestinal flora on carcinogenesis in vivo.     

Identification of N-(deoxyguanosin-8-yl)-2-amino-3,4-dimethylimidazo[4,5-f]quinoline (dG-C8-MeIQ) as a major adduct formed by MeIQ with nucleotides in vitro and with DNA in vivo

The N-hydroxylamine of a carcinogenic heterocyclic amine, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline(MeIQ), was reacted with four 2'-deoxynucleoside 3'-monophosphatesafter O-acetylation. ³²P-Postlabeing analysis demonstrated thatthe adduct was formed with only the guanine nucleotide, andthe structure of the compound in the obtained adduct spot wasdetermined to be N-(deoxyguanosin-8-yl)-MeIQ 3',5'-diphosphate(3',5'-pdGp-C8-MeIQ). DNA samples from livers of mice fed MelQwere also ³²P labeled under standard conditions and additionallytreated with nuclease P1 and phosphodiesterase I. A single adductspot was obtained and the structure of the adduct was identifiedas 5'-pdG-C8-MeIQ. Thus, MelQ binds at the C-8 position of guaninein vitro and in vivo, like other heterocyclic amines.     

DNA adduct formation by o-phenylphenol metabolite in vivo and in vitro.

[U-14C]o-Phenylphenol (OPP) was found to bind covalently to calf thymus DNA during a 60 min incubation in the presence of microsomes, but not in their absence, indicating that metabolic conversion of the parent compound, OPP, to an activated form is essential. Postlabeling analysis with bladder DNA of rats fed a diet containing 2% OPP for 13 weeks revealed one major adduct on TLC. In an in vitro postlabeling experiment with calf thymus DNA, both of the major metabolites of OPP, phenylhydroquinone (PHQ) and phenylbenzoquinone (PBQ), formed adducts, but no adducts were observed with OPP. The chemical structure responsible for adduct formation is thought to be the PHQ semiquinone radical intermediate formed during interconversion between PHQ and PBQ. When the oligonucleotides, pd(A)12-18, pd(C)12-18, pd(G)12-18 and pd(T)12-18, were used in vitro, only pd(G)12-18 gave TLC-detectable adducts on treatment with PHQ and PBQ. The covalent binding appears to be rather specific to guanine residues. These results suggest that covalent binding of the OPP metabolite is one of the underlying events in OPP-induced carcinogenesis in rats.     

32P-HPLC suitable for characterization of DNA adducts formed in vitro by polycyclic aromatic hydrocarbons and derivatives

Analysis of DNA adducts demands both high sensitivity and goodresolution. A high-performance liquid chromato-graphy methodfor ³²P-postlabeled DNA adducts (³²P-HPLC) was used to investigateDNA adduct formation from 38 polycyclic hydrocarbons and biphenylsin vitro. The ³²P-HPLC method proved to be useful for separation,detection and characterization of DNA adducts from most of thesubstances. The in vitro method used to form the DNA adducts,with calf thymus DNA, nucleotide 3'-phosphates and metabolicactivation through S-9 liver homo-genate, gave poor quantitativereproducibility. However, the results showed that the ³²P-HPLCmethod was suitable for characterizing DNA adducts from manysubstances. From 35 of the tested substances 365 DNA and nucleotide3'-phospate adducts were detected and characterized concerningretention times. Of the adducts, 171 were detected in DNA and39 of them from five substances were characterized concerningtarget nucleotides. The retention time library built can beused in future analyses of DNA with complex patterns of DNAadducts.     

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%).     

DNA adduct formation by the environmental contaminant 3-nitrobenzanthrone in V79 cells expressing human cytochrome P450 enzymes

Diesel exhaust is known to induce tumours in animals. Of the compounds found in diesel exhaust 3-nitrobenzanthrone (3-NBA) is particularly a powerful mutagen. Recently we showed that 3-NBA is genotoxic in vivo in rats by forming specific DNA adducts derived from nitroreduction. In this study a panel of genetically engineered V79 Chinese hamster cell lines expressing various human cytochrome P450 (CYP) enzymes (CYP1A1, CYP3A4) and/or human NADPH:CYP oxidoreductase (CYPOR) was used to identify CYP enzymes involved in the metabolic activation of 3-NBA. We analyzed the formation of specific DNA adducts by 32P-postlabelling after exposing cells to 1 microM 3-NBA. A similar pattern with a total of four distinct 3-NBA-DNA adducts was found in all cells, identical to those detected previously in DNA from rats treated with 3-NBA in vivo. Total adduct levels ranged from 75 to 132 using nuclease P1 and from 103 to 220 adducts per 10(8) nucleotides, using butanol enrichment. Comparison of DNA binding between different V79MZ derived cells revealed that human CYPOR and CYP3A4 were involved in the metabolic activation of 3-NBA. Furthermore, dose-dependent high adduct levels were detected after exposure to 0.01, 0.1 or 1 microM 3-NBA in the subclone V79NH which exhibits high activities of nitroreductase and N,O-acetyltransferase. Our results suggest that nitroreduction is the major pathway in the human bioactivation of 3-NBA. Moreover, acetylation of the initially formed N-hydroxy arylamine intermediates may contribute to the high genotoxic potential of 3-NBA.     

Factors modulating the formation of DNA adduct by aflatoxin B1 in vitro

Forty-two compounds belonging to various chemical groups havebeen tested for their ability to suppress formation of aflatoxinB₁-DNA adduct mediated by microsome in vitro. While many ofthese compounds have either marginal or no modulating effect,some have been identified as effective inhibitors. The stronginhibition of DNA adduct formation by retinoids (retinol, retinal,retinoic acid and retinyl acetate), riboflavin, riboflavin 5'-phosphate,flavin adenine dinudeotide, Cu²⁺, 7,8-benzoflavone, disulfiram,butylated hydroxyanisole, butylated hydroxytoluene and phenothiazinesuggests that these agents may have potential anticardnogenicactivity against aflatoxin B₁.     

Carcinogenicity in rats of the mutagenic compounds 1-nitropyrene and 3-nitrofluoranthene

1-Nitropyrene and 3-nitrofluoranthene are present in diesel exhaust, in pollutants in air, and were also present in certain xerographic toners and copies. Their carcinogenicities were studied in male F344/DuCrj rats by subcutaneous injection. Sarcomas, mainly malignant fibrous histiocytomas at the site of injection were induced in 8 to 17 (47%) rats by 1-nitropyrene and in 4 of 10 (40%) rats by 3-nitrofluoranthene. Some tumors were serially transplantable in the same strain of rats.     

Characterization of the major DNA adduct formed by the food mutagen 2-amino-3-methyl-9H-pyrido[2, 3-b] indole (MeA{alpha}C) in primary rat hepatocytes

Cooking of proteinous food results in the formation of heterocyclicamines. Among these, 2-amino-3-methyl-9H-pyrido[2, 3-b]indole(MeA     

DNA adduct formation of aristolochic acid I and II in vitro and in vivo

Aristolochic acid I (AA I) and aristolochic acid II (AA II),the two main ingredients of the carcinogenic plant extract aristolochicacid (AA), are metabolized to reactive intermediates which bindcovalently to DNA in vitro and in vivo. DNA adduct formationwas analysed by the ³²P-postlabelling assay. In in vitro incubationswith rat liver 9000 g supernatant (S9) and calf thymus DNA (CT-DNA),AA I showed an identical pattern of DNA adducts on thin-layerchromatograms under aerobic and anaerobic conditions, whereasAA II gave rise to DNA adduct formation only anaerobically.The anaerobically obtained DNA adduct pattern by AA II in vitrowas similar to the AA I adduct patterns. Aristolactams I andII, the metabolites of AA I and AA II formed under anaerobicconditions, did not form DNA adducts in the presence of S9 mixand CT-DNA. Incubations with xanthine oxidase, known to enzymaticallyreduce aromatic nitro groups, also activated AA I and AA IIto reactive intermediates, producing almost identical adductpatterns as obtained by S9 mix-mediated metabolism. Activationof AA I by S9 mix in the presence of poly(dG) resulted in theformation of two adducts, one of which was shown to be chromatographicallyindistinguishable from an adduct obtained by reaction with CT-DNA.For the in vivo studies AA I and AA II were administered orallyto male Wistar rats, and DNA from liver, brain, oesophagus,stomach lining, forestomach lining, kidney and bladder was analysedfor DNA adducts by ³²P-postlabelling. The adduct patterns inDNA from forestomach and kidney—target tissues of AA—andDNA from non-target tissues like stomach lining and liver weresimilar to the patterns obtained from the in vitro incubations.In the bladder (also a target tissue) only AA II gave rise toDNA adduct formation. These findings suggest that DNA adductformation by AA I and AA II does not directly correlate withthe initiation of the carcinogenic process and subsequent tumourformation in target tissues in the rat.