32P-postlabeling analysis of 1-nitropyrene-DNA adducts in female Sprague-Dawley rats

The ³²P-postlabeling technique was used to qualitatively establishthe pattern of DNA adduct formation in mammary tissue and liverfollowing administration of 1-nitropyrene to female Sprague-Dawleyrats. 1-Nitropyrene (100 mg/kg b.w.) was administered by gavagein trioctanoin and the rats were sacrificed 24 h later. DNAwas isolated from mammary fat pads and liver, enzymaticallyhydrolyzed to deoxy ribonucleoside-3'-monophosphates and thenconverted to [5'-³²P]3',5'-bisphosphates. The polyethyleneimine-cellulose(PEI-cellulose) TLC ³²P-fingerprints revealed the presence ofmultiple putative adducts in the mammary fat pads and in thelivers. To investigate the role of nitroreduction in the formationof these adducts, calf thymus DNA was incubated with [³H]1-nitropyrenein vitro in the presence of xanthine oxidase. The DNA was isolatedand analyzed by the ³²P-postlabeling technique. A major adductspot was detected and confirmed as N-(deoxyguanosin-8-yl)-1-amino-pyrene.This adduct cochromatographed with a minor in vivo adduct ofDNA obtained from mammary fat pads and livers. However, themajor adducts detected in vivo did not appear to originate fromsimple nitroreduction of 1-nitropyrene. The results of thisstudy suggest that other metabolic pathways, such as ring oxidation,or ring oxidation followed by nitroreduction, may be responsiblefor the putative 1-nitropyrene—DNA adducts observed inmammary fat pads and livers of female Sprague-Dawley rats.     

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.     

Structural characterization of the major adducts formed by reaction of 4,5-epoxy-4,5-dihydro-1-nitropyrene with DNA.

The only available marker of DNA adducts formed from 1-nitropyrene (1-NP) and DNA, N-(deoxyguanosin-8-yl)-1-aminopyrene, is derived from the nitroreduction pathway. Our studies, as well as those of others, have indicated that multiple DNA adducts are formed from 1-NP in vivo and in vitro. Thus the need for additional DNA adduct markers was apparent. Therefore, it was our goal to characterize the DNA adducts formed from 4,5-epoxy-4,5-dihydro-1-nitropyrene, a metabolite of 1-NP. The epoxide was incubated with calf thymus DNA (pH 5.4). The DNA was enzymatically hydrolyzed to deoxyribonucleosides which were analyzed by reverse phase HPLC. Three major peaks were obtained in yields less than 5%. The structural assignment of these adducts was made by comparison of their proton nuclear magnetic resonance spectra with those of cis- and trans-4,5-dihydro-4.5-dihydroxy-1-nitropyrene, and by long range coupling constants, decoupling experiments, D2O exchange, partitions and acid hydrolysis. Two adducts result from trans and one from cis addition of the N2-exocyclic amino group of deoxyguanosine to the C5-benzylic carbon of the epoxide ring. This is the first report that describes the structure of the DNA adducts formed with a ring-oxidized metabolite of 1-NP. On the basis of this finding we suggest that K-region oxides of 1-NP may be responsible for the formation of the putative 1-NP-DNA adducts in vivo.     

Acetyl coenzyme A-dependent binding of carcinogenic and mutagenic dinitropyrenes to DNA

Dinitropyrenes are mutagenic and carcinogenic environmental pollutants found in diesel emissions and urban air particulates. In Salmonella typhimurium these compounds appear to be activated to mutagens by sequential nitroreduction and acetylation. We have examined whether or not similar activation pathways occur with mammalian nitroreductases and acetylases. When rat liver cytosol, NADPH and calf thymus DNA were incubated with [4,5,9,10(-3)H]1-nitropyrene, [4,5,9,10(-3)H]1,3-, 1,6- or 1,8-dinitropyrene very low levels of nitrated pyrene binding with DNA were detected. Addition of acetyl coenzyme A (AcCoA) to these incubations increased the binding of dinitropyrenes 20- to 40-fold while the binding of 1-nitropyrene was not affected. The extent of AcCoA-dependent binding of dinitropyrenes reflected the amount of nitroreduction, as measured by aminonitropyrene formation. However, the increase in binding of dinitropyrenes to DNA in the presence of AcCoA did not occur with dog liver cytosol which is known to be deficient in N-acetylases. These results suggest that cytosolic nitroreductases catalyze the formation of N-hydroxy arylamine intermediates which in the case of dinitropyrenes are converted to reactive N-acetoxy arylamines by cytosolic AcCoA-dependent acetylases.     

Formation of DNA adducts in vitro and in Salmonella typhimurium upon metabolic reduction of the environmental mutagen 1-nitropyrene

The polycyclic nitroaromatic hydrocarbon 1-nitropyrene is an environmental pollutant, a potent bacterial mutagen, and a carcinogen. Xanthine oxidase, a mammalian nitroreductase, catalyzed the in vitro metabolic activation of this compound to DNA-bound adducts. Maximum adduct formation occurred at pH 5.5 to 6.0 and was increased by the addition of catalase to the incubation medium. DNA binding from 1-nitropyrene was inhibited by hydrogen peroxide, L-ascorbate, and glutathione. Enzymatic hydrolysis of the modified DNA and subsequent analysis by high-pressure liquid chromatography indicated the presence of one major and two minor adducts. The major adduct was characterized by mass spectrometry and nuclear magnetic resonance spectroscopy as N-(deoxyguanosin-8-yl)-1-aminopyrene. The minor adducts appear to be decomposition products of the major adduct. When Salmonella typhimurium TA1538 was incubated with 1-nitropyrene, a strong correlation was found between the extent of DNA binding and the frequency of induced histidine reversions. Analysis of the bacterial DNA indicated one major adduct which had chromatographic properties and pKaS identical to those of N-(deoxyguanosin-8-yl)-1-aminopyrene. These data indicate that N-hydroxy-1-aminopyrene is probably the mutagenic and DNA-binding species formed during the metabolic reduction of 1-nitropyrene.     

Carcinogen-DNA adduct formation in the lungs and livers of preweanling CD-1 male mice following administration of [3H]-6-nitrochrysene, [3H]-6-aminochrysene, and [3H]-1,6-dinitropyrene

6-Nitrochrysene (NC) and 6-aminochrysene (AC) have been shown to be potent lung and liver carcinogens when administered in multiple i.p. doses to preweanling mice. 1,6-Dinitropyrene has been shown to be a strong hepatocarcinogen but a weak lung carcinogen in this same bioassay. We have examined carcinogen-DNA adduct profiles in the target tissues of preweanling male CD-1 mice following administration of single or multiple doses of these compounds. Depending on the tissue and the dosing schedule, the total level of DNA modification in animals dosed with [3H]NC was 2- to 9-fold higher than in animals dosed with [3H]AC. Regardless of the dosing schedule, DNA isolated from the lungs and livers of both [3H]NC- and [3H]AC-treated preweanling male mice contained a single major and chromatographically identical adduct. This major adduct, which accounted for as much as 90% of the total carcinogen-DNA adducts in enzymatic hydrolysates from treated animals, was chromatographically distinct from the major C8-purine-substituted adducts formed from the reaction of N-hydroxy-AC with calf thymus DNA. In contrast to the results obtained with NC and AC, the major carcinogen-DNA adduct formed in the livers of mice treated with [3H]-1,6-dinitropyrene was found to cochromatograph with 1-N-(deoxyguanosin-8-yl)amino-6-nitropyrene, a product derived from N-hydroxy-1-amino-6-nitropyrene. Since NC and its nitro-reduced derivative, AC, yielded an identical carcinogen-DNA adduct in vivo and this adduct was not derived from N-hydroxy-AC, we conclude that the metabolic activation of NC in the neonatal mouse must involve some previously undescribed combination of ring-oxidation and nitro-reduction pathways. This activation pathway could be an important factor in determining the potency of NC and AC as carcinogens in this bioassay system.     

The binding of N-hydroxy-2-acetylaminofluorene to DNA and repair of the adducts in primary rat hepatocyte cultures

Primary cultures of rat hepatocytes were exposed to [ring-³H]-N-hydroxy-2-acetylaminofluorene(N-OH-AAF) for 4 h, and the RNA and DNA nucleoside adducts wereisolated and identified by h.p.l.c. The DNA adducts were shownto be N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF),N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxy-guanosin-8-yl)-2-acetylaminofluorene(dG-N2-AAF), while the RNA adducts were N-(guanosin-8-yl)-2-acetyl-aminofluorene,and N-(guanosin-8-yl)-2-aminofluorene. The removal of theseadducts was measured up to 38 h following the cessation of exposureof the hepatocytes to N-OH-AAF. The dG-C8-AAF adduct was removedwith a half-life of approximately 10 h, while dG-N²-AAF anddG-C8-AF remained constant for 14 h, followed by a slow rateof removal. The dG-C8-AAF adduct initially composed about 60%of the total DNA adducts of primary hepatocytes in contrastto the 20% found in liver in vivo. The formation of the 3 DNAadducts and the different rates of repair indicate that primarycultured rat hepatocytes may be a valuable system to study initiationof liver carcinogenesis by N-OH-AAF.     

Comparison between DNA adduct formation and tumorigenesis in livers and bladders of mice chronically fed 2-acetylaminofluorene

Female BALB/c mice continuously fed 2-acetylaminofluorene (AAF) develop liver and bladder tumors. The incidence of liver tumors is linearly related to the carcinogen concentration in the diet, while the tumor response in the bladder is markedly non-linear. In the current experiments, liver and bladder DNA adducts were measured in female BALB/c mice fed several different concentrations of AAF for 28 days. The adduct concentrations were then compared to the previously reported incidences of neoplastic and preneoplastic lesions in these tissues. In initial experiments, mice were fed either 30 or 150 mg [ring-3H]AAF/kg diet for 21 days. Liver DNA adducts were identified by HPLC, which indicated the presence of one major adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF). This adduct was also the major product detected by 32P-postlabeling in liver and bladder DNA from mice fed the same concentrations of AAF for 28 days. Radioimmunoassays, conducted with an antibody specific for dG-C8-AF, showed that steady-state concentrations of dG-C8-AF were obtained at 28 days of AAF feeding; thus, this time point was used to determine the relationship between the dose of AAF and the adduct levels. In mice fed nine concentrations of AAF (5-150 mg AAF/kg diet), the adduct concentrations after 28 days of feeding were linearly related to dose in both the liver and bladder, with the adduct concentration being approximately 3-fold greater in the bladder. These results indicate that a linear correlation exists between the hepatic concentration of dG-C8-AF and the liver tumor incidence. In the bladder however, a linear relationship was not observed, which suggests that additional tissue-specific factors, such as toxicity, are essential components for tumorigenesis in this tissue.     

Oxidative microsomal metabolism of 1-nitropyrene and DNA-binding of oxidized metabolites following nitroreduction

1-Nitropyrene is an environmental mutagen and carcinogen whichundergoes both oxidative and reductive metabolism. We have previouslyshown that nitroreduction to N-hydroxy-1-aminopyrene leads tothe formation of arylamine-DNA adducts. In the present study,we have investigated the oxidative metabolism of 1-nitropyreneand the subsequent binding of ring-oxidized metabolites to DNA.In vitro incubations were conducted using hepatic microsomesfrom uninduced rats or from rats pretreated with phenobar-bital,Aroclor 1254, 3-methykholanthrene, or 3-methyl-cholanthreneand trans-stilbene oxide. H.p.l.c. analysis of the incubationmixtures indicated the presence of the previously reported metabolites,1-aminopyrene, 3-, 6-, and 8-hydroxy-1-nitropyrene, and 1-nitropyrenetrans-4, 5-dihydrodiol. In addition, 1-nitropyrene 4, 5-oxide,1-nitropyrene 9, 10-oxide, 1-nitropyrene ira/is-9, 10-dihydrodioland 1-pyrenol were identified. The formation of both K-regiondihydrodiols could be increased by transstHbene oxide inductionof microsomal epoxide hydrase. Formation of the K-region epoxideswas greatest using phenobarbital- and Aroclor-induced microsomesand increased with increasing oxygen tension, while 1-pyrenolformation was highest in 3-methylcholanthrene-induced microsomalincubations and was not affected by the oxygen tension. Whencalf thymus DNA was added to the microsomal incubations, similarlevels of DNA-binding occurred in incubations conducted underoxygen, air, argon or anaerobic conditions. H.p.l.c. analysisof the enzymatically hydrolyzed DNA indicated the presence ofmultiple DNA adducts with the major product coeluting with AKdeoxy-guanosin-8-yl)-l-aminopyrene.The K-region oxides bound directly to DNA to give adducts similarto the minor products detected in the microsomal incubations.Incubation of the K-region oxides with the nitroreductase, xanthineoxidase, increased the DNA-binding and resulted in an additionalad-duct which coeluted with AKdeoxyguanosin-8-yl)-l-amino pyrene.3-Hydroxy-l-nitropyrene bound extensively to DNA upon nitroreductionby rat liver cytosol or xanthine oxidase, while 6- and 8-hydroxy-l-nitropyrenebound only slightly. None of these oxidized metabolites wasactivated to DNA-binding species by cytosolic nitroreductionfollowed by AcCoA-dependent acetylation. The fact that oxidizedmetabolitesof 1-nitropyrene are reduced to DNA-binding derivatives moreeasily than 1-nitropyrene itself may be important in vivo where1-nitropyrene has been shown to be readily oxidized.     

Carcinogen-DNA adducts in cultures of rat and human hepatocytes.

Exposure to chemical carcinogens can often be identified by detection of DNA adduct lesions. Primary cultures of isolated rat and human hepatocytes were exposed to 2-acetyl-aminofluorene (AAF), 4-aminobiphenyl (ABP), or benzo[a]pyrene (BP). The isolated DNA from the exposed cells was analyzed using the 32P-post-labeling assay. A greater total of carcinogen-DNA adducts, 2-12-fold, were observed in human hepatocytes than rat hepatocytes at the same concentrations. The predominant DNA adducts for each carcinogen were the same between rat and human cells. The N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) was the major AAF-DNA adduct. The N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) was the major ABP-DNA adduct. In the rat N2-[10 beta-(7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene)yl] deoxyguanosine (dG-N2-BP) and two unidentified adducts were nearly equivalent in amount, while the major BP-DNA adducts in the humans was the dG-N2-BP. The rat hepatocyte in vitro results are comparable to the predominant adducts found with rats exposed in vivo. The two different cultures of human hepatocytes demonstrated qualitative and quantitative differences in specific DNA adducts from rat hepatocytes. This study and others using human hepatocyte cultures demonstrate that this in vitro system can provide useful information for assessing human carcinogenic hazards.     

Formation of DNA adducts in mouse tissues after intratracheal instillation of 1-nitropyrene

1-Nitropyrene (1-NP), a ubiquitous environmental pollutant,is a mammalian mutagen and causes cancer in animals. The abilityof the lung, liver and kidney to form 1-NP-DNA adducts was determinedin adult male B6C3F₁ mice following a single intratracheal instillationof 1-NP. 1-NP-DNA adducts were isolated and characterized inmouse lung, liver and kidney by HPLC analysis of the enzymaticallydigested DNA. Multiple DNA adducts were present in lung, liverand kidney at 1 day after administration. One of the major adductsin lung (20% of the total eluted radioactivity) coeluted withthe synthetic marker, N-(deoxyguanosin-8-yl)-1-amino-pyrene(C8-dG-AP). This adduct (10% of total eluted radio-activity)and others were still present in the lung at 28 days after administrationof 1-NP. One of the adducts in liver and kidney DNA digestsalso coeluted with C8-dG-AP. Treatment of the adducts with 0.3M NaOH resulting in earlier eluting peaks containing radioactivity,indicative of an imidazole ring-opening adduct. A portion ofthe original peak of radioactivity that coeluted with C8-dG-APand other adducts, however, was not affected by 0.3 M NaOH.Thus, the chromatographic properties and chemical behavior ofthe adducts formed in vivo suggest that one of the adducts inthe lung is C8-dG-AP which is formed by nitroreduction of 1-NP.Other adducts may be formed via ring-oxidation followed in someinstances by nitroreduction. These data indicate that DNA adductsof 1-NP metabolites may be formed in the lung (a primary sitefor inhaled particles), liver and kidney following inhalationof airborne particles containing 1-NP.     

Role of the intestinal microflora in the formation of DNA and haemoglobin adducts in rats treated with 2-nitrofluorene and 2-aminofluorene by gavage

The role of the intestinal microflora in the metabolic activationof nitroarenes and arylamines was studied in female Wistar ratsthat received a dose of 1 mmol/kg 2-aminofluorene (2-AF) insunflower oil by gavage. Another group received the same doseof 2-nitrofluorene (2-NF). A third group of animals was usedas controls. Germfree (GF) rats, GF rats with a rat microflora(RM) and GF rats with a human microflora (HM) were treated.After treatment with 2-AF significant differences were observedin the formation of haemoglobin (Hb) adducts and DNA adducts.The 2-AF-Hb adduct level (mean ± SD) observed in GF rats(0.57 ± 0.13 µmol/g Hb) was considerably lowerthan that observed in RM rats (5.1 ± 0.6) and in HM rats(6.2 ± 13). DNA adduct levels showed the opposite pattern:levels of adducts co-migrating with deoxyguanosin-8-yl-aminofluorene(dG-C8-AF) in liver tissue were higher in GF rats (4.6 ±1.4 fmol/µg DNA) as compared to RM rats (2.6 ±0.04) or HM rats (2.0 ± 0.7). In lung tissue and whiteblood cells a similar influence of the intestinal microfloraon DNA adduct levels was observed. These results suggest thatthe intestinal microflora cleaves conjugates of 2-AF or N-hydroxy-2-AF,thus facilitating entero-hepatic recirculation of these compoundsand enhancing the formation of reactive intermediates bindingto Hb. The latter is not observed for DNA adduct formation,indicating that most of these adducts have been formed aftera single passage through the liver. After treatment with 2-NF,Hb and DNA adduct levels were much lower. An adduct spot wasobserved that was not presentin rats that received 2-AF. InGF animals only very low levels of DNA adducts co-migratingwith dG-C8-AF or deoxyguanosin-8-yl-acetyl-aminofluorene andno Hb adducts were observed, indicating that the metabolic activityof the microflora is an essential step in both Hb and DNA adductformation.     

Dinitropyrene metabolism,DNA adduct formation,and DNA amplification in an SV40-transformed chinese hamster embryo cell line

The environmental pollutants 1,6-dinitropyrene (1,6-DNP) and 1,8-dinitropyrene (1,8-DNP) are strongly carcinogenic in a number of animal models. These DNPs are metabolized by nitroreduction to N-hydroxy arylamine derivatives that either directly or after acetylation bind to cellular DNA. In the experiments reported here, we examined whether DNA adduct formation by 1,6-DNP and 1,8-DNP was associated with amplification of specific DNA sequences, a process that may be causally related to tumorigenesis. CO60 cells, an SV40-transformed Chinese hamster embryo cell line, were incubated with 2.5 or 50 ng/mL [4,5,9,10-³H]1,6-DNP for 5 h. Highpressure liquid chromatographic analysis of organic extracts of the medium indicated the presence of 1-acetylamino-6-nitropyrene, suggesting that these cells are capable of nitroreduction and acetylation. ³²P-Postlabeling analysis of DNA isolated from cells exposed to 1.0 or 2.5 ng/mL 1,6-DNP revealed dose-related formation of N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene. A similar adduct, presumably N-(deoxyguanosin-8-yl)-1-amino-8-nitropyrene, was detected after incubations with 1,8-DNP. DNA isolated from analogous experiments was slot-blotted onto nylon membranes and hybridized with ³²P-labeled SV40, c-fos, or β-actin DNA probes. β-Actin was not amplified and c-fos was amplified only a small amount; however, there was dose-related amplification of SV40 sequences, whose levels were in some instances approximately 20 times that observed in solvent-treated controls. These data indicate that DNA adduct formation by 1,6-DNP and 1,8-DNP is associated with the amplification of certain DNA sequences, a response that may be related to the tumorigenic potential of these compounds.     

DNA adduct formation in liver following the administration of [3H]2-nitrofluorene to rats in vivo.

The formation of RNA and DNA adducts by the environmental pollutant 2-nitrofluorene (2-NF) has been investigated in rat liver in vivo. The adduct pattern was studied after trifluoroacetic acid hydrolysis of DNA or RNA, followed by analysis of the adducts by HPLC. This was also done by enzymatic hydrolysis of DNA, followed by 32P-postlabeling. Both after oral and i.v. administration of [3H]2-NF, one major adduct was found. This adduct did not co-migrate with one of the known adducts of 2-(acetyl)-aminofluorene, N-deoxyguanosin-8-yl-2-aminofluorene (dG-C8-AF), which could have been formed after nitroreduction of 2-NF. 32P-Postlabeling revealed that two minor adducts were also formed, one of which was dG-C8-AF. The observation that the major adduct was also formed after i.v. administration of 2-NF to bile duct-catheterized rats makes a role for the intestinal microflora in the formation of this adduct very unlikely. In vitro experiments with inhibitors of the enzyme epoxide hydrolase indicated that epoxidation of 2-NF may play a role in the microsomal bioactivation of this compound.     

Metabolism and DNA binding of 2-nitropyrene in the rat.

2-Nitropyrene (2-NP), a contaminant of ambient air, is a potent bacterial mutagen in the Ames assay and induces leukemia/lymphoma in female Sprague-Dawley rats. To understand the mechanistic basis for its tumorigenic activity, it is essential to elucidate the metabolic pathways of 2-NP in vivo. Such knowledge will also assist in developing analytical methods for monitoring human exposure to nitropolynuclear aromatic hydrocarbons in ambient air. Thus, 2-nitro[U-4,5,9,10-14C]pyrene was synthesized and administered to male F344 rats by intragastric gavage at a dose of 30 mg (0.4 mCi/mM)/kg body weight. During the first 48 h, 57.5% of the dose was eliminated in the feces and 9.7% was eliminated in the urine. Correspondingly, after 168 h, 58.9 and 10.6% were excreted in feces and urine, respectively. Fecal metabolites (isolated amounts) included 6-hydroxy-2-acetylaminopyrene (19.5%), 6-hydroxy-2-aminopyrene (10.4%), 2-aminopyrene (10.0%), 2-acetylaminopyrene (0.8%), and unmetabolized 2-nitropyrene (10.0%). 6-Hydroxy-2-acetylaminopyrene, 6-hydroxy-2-aminopyrene, and 2-aminopyrene were identified as their acetyl derivatives by comparison of their chromatographic retention times, mass spectra, and UV spectra to those of synthetic standards. Urinary metabolites included 6-hydroxy-2-acetylaminopyrene (2.0%); glucuronide conjugates were tentatively identified (3.2%). The results of this study indicate that nitroreduction and ring oxidation are metabolic pathways in vivo. For DNA binding studies, rats were treated with 2-nitro[4,5,9,10-3H]pyrene [1.6 mg (598 mCi/mM)/kg body weight]. The levels of binding (pM bound/mg DNA) were as follows: 1.3, liver; 1.14, mammary tissue; 0.65, lung; 1.67, kidney; and 1.8, bladder. Upon high-performance liquid chromatographic analysis of the DNA hydrolysate (liver, mammary, and kidney), approximately 2.0% of the radioactivity coeluted with the synthetic markers derived from nitroreduction, N-(deoxyguanosin-8-yl)-2-aminopyrene and N-(deoxyadenosin-8-yl)-2-aminopyrene. Thus, simple nitroreduction of 2-NP does not significantly contribute to the total DNA binding of 2-NP metabolites in vivo. The significance of each pathway for the tumorigenic effects of 2-NP remains to be examined.     

Cytotoxicity, cellular transformation and DNA adducts in normal human diploid fibroblasts exposed to 1-nitrosopyrene, a reduced derivative of the environmental contaminant, 1-nitropyrene

Treatment of normal human diploid fibroblasts with 0.3 —22 µM 1-nitrosopyrene resulted in a dose-dependent decreasein relative cloning efficiency and an increase in anchorage-independentgrowth in soft agar. When compared to previous experiments,1-nitrosopyrene was 10- to 20-fold more cytotoxic and 5- to10-fold more potent at inducing morphological transformationthan 1-nitropyrene. Incubation of the fibroblasts with 8 µM1-nitropyrene in the presence of xanthine oxidase, a mammaliannitroreductase, resulted in the formation of one major DNA adduct,N-(deoxyguanosin-8-yI)-1-aminopyrene, at a level of 1.1 adductsper 10⁶ nucleo-tides. Fibroblasts treated with 1-nitrosopyreneformed the same DNA adduct; however, only a 0.3 µM concentrationwas required to give 0.7 adducts per 10⁶ nucleotides in thefibro-blast DNA. These data indicate that a limiting step inthe cellular toxicity and transformation of normal human diploidfibroblasts by 1-nitropyrene is the initial reduction to 1-nitrosopyrene.     

Immunocytological detection of AAF-DNA adducts in HeLa cell nuclei

Acetylaminofluorene-DNA adducts (AAF-DNA) were detected in the nuclei of HeLa cells exposed to N-acetoxy-2-acetylaminofluorene (N-Ac-AAF), using an immunocytological technique and specific antibodies directed against AAF modified DNA. The proportion of cells exhibiting specific nuclear immunoreactivity was dose-dependent. The time course of disappearance of adduct specific nuclear immunoreactivity was compared with removal of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) and other adducts.     

DNA binding by 1-nitropyrene and 1,6-dinitropyrene in vitro and in vivo: effects of nitroreductase induction

1-Nitropyrene, the predominant nitropolycyclic aromatic hydrocarbonfound in diesel exhaust,is a mutagen and tumorigen. 1,6-Dinitropyreneis present in diesel exhaust in much smaller quantities thanl-nitropyrene, but is much more mutagenic and carcinogenic.In an attempt to understand this difference in biological potencies,we have compared the extent of DNA binding by these two nitropyrenesin vivo. We have also determined the effect of 1-nitropyrenepretreatment upon the induction of nitroreductases and the subsequentDNA binding by both 1-nitropyrene and 1,6-dinitropyrene. CovalentDNA binding by 1-nitropyrene could not be detected in vivo;however, 1,6-dinitropyrene formed N-(deoxyguano-sin-8-yl)-1-amino-6-nitropyreneas the major DNA adduct in rat liver, kidney, urinary bladderand mammary gland, with the highest levels being found in thebladder. The capability of liver microsomes to catalyze theoxidative metabolism of 1-nitropyrene was unchanged after treatingrats with 8 mg/kg 1-nitropyrene. Cytochrome P-450, NADPH-cytochromeP-450 reductase and cytochrome b₅ levels were also unchanged,while slight increases were detected in NADH-cytochrome b₅ reductaseand epoxide hydrase activities. Liver cytosolic and microsomalnitroreductase activities toward both 1-nitro-pyrene and 1,6-dinitropyrenewere increased 2-fold, and cytosolic nitrosoreductase activitytoward 1-nitrosopyrene and 1-nitro-6-nitrosopyrene was elevatedby {small tilde}20%. DNA binding of both 1-nitropyrene and 1,6-dinitropyrenein vitro was 2-fold higher when using cytosol from 1-nitropyrene-pretreatedrats. However, pretreatment of rats with l-nitropyrene onlyslightly increased the amount of in vivo DNA binding by 1,6-dinitropyreneexcept in kidney where there was a 60% increase. These resultsindicate that although nitroreduction is involved in DNA adductformation by 1,6-dinitropyrene, additional factors (e.g. O-acetylation)limit the extent of DNA binding in vivo.     

Role of TP53 in repair of N-(deoxyguanosin-8-yl)-4-aminobiphenyl adducts in human transitional cell carcinoma of the urinary bladder

The global genomic repair of DNA adducts was examined in human papillary transitional cell carcinoma (TCC) cell lines after exposure to N:-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), the proximate carcinogenic metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP). (32)P-post-labeling analysis of TCC cultures exposed to N-OH-AABP revealed a major adduct, identified as the 3',5'-bisphosphate derivative of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP). The amount of adduct formation in TCC10 was dependent upon the dose and the duration of exposure and ranged between 1 and 5 adducts/10(7) nucleotides. To test if p53 regulates repair of the dG-C8-ABP adduct in genomic DNA, an isogeneic set of cell lines was obtained by infection of the TCC10 cultures with a retroviral construct expressing a trans-dominant mutant of p53, namely a Val-->Ala mutation at codon 143. The TDM143-TCC10 line expressing the mutant form of p53 was selected. The rate of repair of dG-C8-ABP was compared between TCC10 and TDM143-TCC10 cultures after treatment with 15 microM N-OH-AABP. The rate of disappearance of the adduct was monitored over a period of time after chemical treatment. (32)P-post-labeling analysis of dG-C8-ABP in parental TCC10 showed its rapid removal, the majority of adducts disappearing within 48 h. In contrast to TCC10, TDM143-TCC10 was relatively slower in removal of dG-C8-ABP. After 24 h DNA repair TDM143-TCC10 showed an approximately 3-fold greater amount of dG-C8-ABP compared with TCC10. These results imply that p53 plays a role in the repair of ABP adducts and that in p53 null cells the unrepaired DNA damage could cause accumulation of mutations, which might contribute to increased genomic instability and neoplastic progression.     

Evidence for N-(deoxyguanosin-8-yl)-1-aminopyrene as a major DNA adduct in female rats treated with 1-nitropyrene

[³H]1-Nitropyrene was administered at a dose of 25 mg/kg byi.p. injection to female Wistar rats. Animals were killed 24hlater and DNA was isolated from kidney, liver and mammary gland,enzymically hydrolysed and analysed by reversephase h.p.l.c.A major adduct peak was detected in DNA from each of the threeorgans. Enzymic hydrolysates of DNA, which had been reactedin vitro with 1-nitropyrene in the presence of xanthine oxidase,were similarly analysed by h.p.l.c. One major adduct peak wasobtained which had the same retention time as the in vivo product.Confirmatory evidence that the in vivo adduct and the in vitroadduct were structurally similar was obtained from the determinationof the pH-dependent solvent partitioning profiles. Further,treatment of the in vivo adduct from liver, kidney or mammarygland DNA hydrolysates and the in vitro adduct with sodium hydroxideresulted in the formation of a more polar product which elutedearlier on h.p.l.c. This behaviour is consistent with scissionof the imidazole ring of a deoxyguanosine adduct.The major DNAadduct formed in vitro following xanthine oxidase reductionof 1-nitropyrene has previously been identified by others asN-(deoxyguanosin-8-yl)-1-amino-pyrene. The present data suggestthat the in vivo 1-nitro-pyrene-DNA adduct has the same structure.