2-Aminofluorene metabolism and DNA adduct formation by mononuclear leukocytes from rapid and slow acetylator mouse strains

Following exposure of mice to the arylamine carcinogen 2-aminofluorene,DNA-carcinogen adducts can be found in the target tissues liverand bladder, and also in circulating leukocytes. Evidence ispresented here that mouse mononuclear leukocytes (MNL) are capableof metabolizing 2-aminofluorene to DNA-binding metabolites whichgive rise to the adducts found in the MNL. Both lymphocytesand monocytes were able to acetylate arylamines during 18 hof culture. The degree of acetytation was determined by theN-acetyltransferase genotype of the mice as shown through useof acetylator congenic strains which differ only in the Nat-2gene. Cultured MNL from rapid acetylator mice (C57BL/6J andA.B6-Nat¹) produced about twice as much N-acetylaminofluorenefrom 2-aminofluorene and 6- to 8-fold as much N-acetyl-p-amino-benzoicacid from p-aminobenzoic acid as cells from slow acetylatormice (B6.A-Nat⁵ and A/J). Other differences in arylamine metabolismby MNL in culture were observed and shown to be due to geneticfactors, currently unidentified, other than N-acetyltransferase.DNA adduct formation following incubation of MNL with the arylaminecarcinogen 2-aminofluorene was related to both acetylation capacityand to other genetic metabolic factors in the mouse genome.MNL from rapid acetylator mice with the C57BL/6J background(B6) had 3-fold the DNA adduct levels of cells from the correspondingslow acetylator congenic (B6.A-Nat^$). Similarly, MNL from rapidacetylator mice with the A/J background (A.B6-Nat^r) had twicethe DNA adduct levels of those from their corresponding slowcongenic (A). Adduct levels in MNL from C57BL/6J were nearlythe same as those of MNL from A/J, again indicating the involvementof loci other than acetylation in DNA adduct formation. Thefinding of genetically dependent arylamine carcinogen metabolismand DNA adduct formation in cultured MNL suggests the possibilityof using cultured MNL for assessing individual susceptibilityto arylamine-induced DNA damage.     

DNA adduct levels in congenic rapid and slow acetylator mouse strains following chronic administration of 4-aminobiphenyl.

4-Aminobiphenyl (4-ABP) is a human and mouse bladder carcinogen. Epidemiological studies have shown that individuals with a slow acetylator phenotype, especially those exposed to high levels of carcinogenic aromatic amines, show an increased susceptibility to bladder cancer. In order to determine if a slow acetylator phenotype results in increased DNA damage, congenic mouse strains C57BL/6J and B6.A-Nat(s), which differ genetically at the acetyltransferase (EC 2.3.1.5) locus as homozygous rapid (Natr/Natr) and homozygous slow (Nat(s)/Nat(s)) acetylators respectively, were continuously administered 4-ABP.HCl (55-300 p.p.m.) in their drinking water for 28 days. The levels of covalently bound N-(deoxyguanosin-8-yl)-4-ABP-DNA adducts, which are believed to be critical for the initiation of tumors, were quantitated in the liver and bladder by 32P-postlabeling analysis. The levels of the hepatic DNA adduct increased with dose in both sexes, but were independent of the mouse acetylator genotype. At comparable doses, however, the levels of DNA adducts were 2-fold higher in the liver of the female as compared to the male animals. The DNA adducts also increased with dose in bladder of the male mice, but in contrast to the liver, the adduct levels were approximately 2-fold lower in the bladder DNA of the female mice. Also in contrast to the liver, the levels of bladder DNA adducts were significantly higher (P < or = 0.03) in the phenotypic rapid acetylator females compared to the slow acetylators at both 75 and 150 p.p.m. doses; the median levels of adducts were 10-20% higher in the phenotypic slow acetylator male bladders compared to their rapid acetylator counterparts. The results of these studies are consistent with the increased carcinogenicity of 4-ABP to the liver of female mice and the bladder of male mice. They further suggest that factors other than acetylator phenotype limit the extent of DNA adduct formation from 4-ABP in these mice.     

2-Aminofluorene-DNA adduct formation in acetylator congenic mouse lines

The effect of the acetylator polymorphism on hepatic 2-aminofluorene-DNAadduct formation in mice was studied using two recent developmentsfrom our laboratory. Acetylator congenic mouse lines differingfrom their parental inbred lines in N-acetyltransferase activitywere used to separate the effect of the N-acetyltransferasepolymorphism from effects of differences in other geneticallypolymorphic enzymes. DNA adduct formation was used as an indicatorof arylamine induced DNA damage. Adduct formation was measuredby HPLC analysis of 3 nucleotides from hepatic DNA of treatedanimals. At a high dose (60 mg/kg) of 2-aminofluorene for a3 h exposure, rapid acetylator mice (C57BL/6J) accumulated twicethe adducts of slow acetylators (A/J). In acetylator congenicmice this difference increased so that rapid acetylators withthe slow background (A.B6-Nat¹) had 5- to 7-times the DNA damageof the slow acetylator congenic with the rapid background (B6.A-Nat⁵).It was also found that within each mouse line examined, femaleshad higher levels of adduct formation than males. Acetylatorcongenic mouse lines were useful in distinguishing the effectof acetylator genes from the total genetic background. Similarly,congenics were useful in demonstrating the contribution thatenzymes other than N-acetyltransferase make to differences inadduct formation in inbred mouse lines.     

2-aminofluorene-hepatic DNA adducts in congenic mouse lines differing in Ah responsiveness

The influence of beta-naphthoflavone (BNF) pretreatment on 2-aminofluorene (2-AF)-hepatic DNA adduct formation was evaluated in Ah-responsive and non-responsive congenic mouse lines through use of HPLC analysis of 32P-postlabeled nucleotides. C57BL/6J (B6) mice were used as an example of BNF-responsive mice while B6.D-Ahd, a line congenic with B6, was used as the non-responsive line. Induction at the Ah locus with BNF increased adduct levels in hepatic DNA in B6 mice but not in B6.D mice 3 h after a 60 mg/kg i.p. dose of 2-AF. The slow acetylator counterparts of B6 and B6.D, namely B6.A and B6.A.D-NatsAhd (a new congenic line produced from B6.A and B6.D), had lower adduct levels than the rapid acetylators before induction. Although adduct levels in B6.A and B6.A.D were increased following BNF induction, the level of adducts remained below those of induced B6 mice. In the three lines that responded to BNF induction, male mice had a greater relative increase in hepatic DNA adduct levels than females. For all four lines, with or without BNF pretreatment, greater adduct levels were found in the females. These results imply that responsiveness to aromatic hydrocarbon induction, as well as rapid acetylation, may be risk factors in hepatic DNA damage following arylamine exposure. Female mice appear to be more susceptible to such damage than males.     

Determinants of 4-aminobiphenyl-DNA adducts in bladder cancer biopsies

Exposure to 4-aminobiphenyl (4-ABP) is an important determinant of urinary bladder cancer in humans. We have analyzed by gas chromatography-mass spectrometry the DNA adducts of 4-ABP in 75 bladder cancer biopsies. The purpose was to understand whether smoking, N-acetyltransferase 2 (NAT2) polymorphism, diet or tumor grade were determinants of 4-ABP-DNA levels. 4-ABP-DNA adducts were above the detection limit of 0.1 fmol/microg DNA for 37/75 patients. Overall the level of adducts was 2.7 +/- 0.7 (mean +/- SE) fmol/microg DNA (86 +/- 22 adducts/10(8) normal nucleotides, mean +/- SE). A strong association with grade was observed. In the group of patients with detectable 4-ABP-DNA adducts the odds ratio for having a tumor grade of 2 or 3 was respectively 4.3 (95% CI 0.8-21.9) and 6 (1.3-27.5), compared with grade 1. A non-statistically significant association was found between adduct levels and the deduced slow acetylator phenotype in grades 2 and 3. The intake of fruit and vegetables produced a lower frequency of detectable adducts, though the association was not statistically significant. Detectable 4-ABP-DNA adducts were clearly associated with current smoking in higher tumor grades (grade 3 versus grades 1 + 2, odds ratios 10.4; 95% CI 1.7-63.1). Overall, our findings indicate that higher levels of DNA adducts characterize more invasive tumors (higher tumor grades). This seems to be facilitated by smoking and contrasted by the intake of fruit and vegetables.     

Comparative 32P-analysis of cigarette smoke-induced DNA damage in human tissues and mouse skin

Previous studies using a highly sensitive 32P-postlabeling assay for the analysis of carcinogen/mutagen-induced DNA damage have shown the presence of tobacco smoking-related DNA adducts in human placenta (Everson, R.B., Randerath, E., Santella, R.M., Cefalo, R.C., Avitts, T. A., and Randerath, K., Science (Wash. DC), 231: 54-57, 1986). The occurrence of such adducts in smokers' bronchus and larynx is reported here. Since the chemical nature of these adducts could not be characterized by direct methods due to the extremely low levels of individual adducts (less than 0.03 fmol per microgram DNA), we have sought an experimental animal model for studying the formation of tobacco-related DNA adducts. Because cigarette smoke condensate is known to initiate tumors in mouse skin, ICR mice were treated topically with cigarette tar equivalent to 1.5, 3, 6, and 9 cigarettes for 0.4, 3, 5, and 7 days, respectively, and skin DNA was isolated 1 day after the last treatment. When DNA from exposed mice was analyzed by the 32P-postlabeling assay, 12 distinct 32P-labeled DNA adduct spots, as well as a diagonal radioactive zone, which presumably reflected the presence of incompletely resolved adducts, were noted on polyethyleneimine-cellulose TLC fingerprints. One derivative in particular (adduct 1) was seen to increase rapidly during the early treatment phase and also to persist to 8 days after treatment. The prominent adduct 1 was observed in the same location on the fingerprints of DNA samples from smokers. Cochromatography experiments suggested identity of human and mouse DNA adduct 1. Similarly, several other human and mouse adducts (adducts 3, 5, 6, and 9) appeared identical, and the diagonal radioactive zone was also present on DNA adduct maps from smokers. While absolute levels of individual human adducts were too low to be accurately quantitated, semiquantitative estimation of total tobacco-related aromatic DNA adducts in the human specimens gave values of 1 adduct in (1.7-2.9) X 10(7) nucleotides (0.10-0.18 fmol per micrograms DNA), with adduct 1 constituting 8.5-14% of the total. On the basis of these results, it appears now feasible to determine the chemical origin of smoking-induced DNA adducts in human tissues by preparation of authentic 32P-labeled reference adducts from animals treated with characterized subfractions of cigarette tar, 32P-postlabeling, and cochromatography of 32P-labeled human and animal adducts.     

N-acetyltransferase 2 phenotype but not NAT1*10 genotype affects aminobiphenyl-hemoglobin adduct levels.

Aminobiphenyls (ABPs) in tobacco have been implicated in bladder cancer etiology in smokers. N-Acetylation of ABPs in the liver, predominantly by the N-acetyltransferase 2 (NAT2) isozyme, represents a detoxification pathway, whereas O-acetylation of N-hydroxy-ABPs in the bladder, predominantly by the N-acetyltransferase 1 (NAT1) isozyme, represents a bioactivation pathway. We and others have demonstrated that NAT2 phenotype affects 3- and 4-ABP-hemoglobin adduct levels (higher levels in slow acetylators), which are considered valid biomarkers of the internal dose of ABP to the bladder. We have also shown that NAT1 genotype (NAT1*10 allele) is associated with increased DNA adduct levels in urothelial tissue and higher risk of bladder cancer among smokers. It is not known whether NAT1*10 genotype influences ABP-hemoglobin adduct levels. Therefore, we assessed 403 primarily non-Hispanic white residents of Los Angeles County for their NAT2 acetylator phenotype, NAT1*10 acetylator genotype, and 3- and 4-ABP-hemoglobin adduct levels. Eighty-two subjects were current tobacco smokers of varying intensities. Tobacco smokers had significantly higher mean 3- and 4-ABP-hemoglobin adduct levels relative to nonsmokers. The levels increased with increased amounts smoked per day (two-sided, P < 0.0001 in all cases). With adjustment for NAT1 genotype and race, the smoking-adjusted geometric mean level of 3-ABP-hemoglobin adducts in NAT2 slow acetylators was 47% higher than that in NAT2 rapid acetylators (P = 0.01). The comparable value for 4-ABP-hemoglobin adducts was 17% (P = 0.02). In contrast, no association between NAT1*10 genotype and 3- or 4 ABP-hemoglobin adduct levels was observed after adjustment for NAT2 phenotype, smoking, and race. The present study suggests that the impact of the NAT1*10 genotype on 3- and 4-ABP-hemoglobin adducts is noninformative on the possible association between NAT1 activity and bladder cancer risk.     

DNA adducts in different tissues of smokers and non-smokers

Purified DNA from human lung, liver, bladder, pancreas, breast and cervix has been analysed for DNA adducts using the nuclease P1 modification of the 32P post-labelling technique. Tissues were obtained at autopsy from 13 men and 6 women. Relatives were asked to provide information on smoking history for deceased subjects. All tissues examined except the breast had detectable adducts. In lung, bladder and pancreatic tissue a characteristic pattern of adducts was seen which has previously been reported as typical of cigarette-smoke-induced damage. Smokers and former smokers tended to have higher adduct levels than non-smokers in the tissues examined but this was only significant for the lung. There appeared to be considerable variation in adduct levels among smokers which could not be accounted for by duration or daily consumption level. Certain smokers had high adduct levels in all tissues examined, whilst in others high levels were only seen in some tissues. All cervical samples examined had detectable adducts. These results confirm the finding that cigarette smoking is associated with DNA damage in the lung and suggest that similar damage may be related to tobacco-induced neoplasms of other tissues.     

Role of metabolism on the DNA binding of MeIQx in mice and bacteria

We report the effects of several inducers of P450 metabolizing enzymes on DNA adduct formation by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in C57BL/6 mice. We also examined the role of N:O-acetylation and the nitrenium ion in the genotoxicity of MeIQx, since these have been implicated in the activation of other aminoimidazoazaarenes (AIA) to DNA reactive species. Mice were given phenobarbital (PB), Aroclor 1254, beta-naphthoflavone (BNF) or corn oil, i.p., followed 3-5 days later with oral administration of MeIQx. Induction of Aroclor and BNF produced DNA with 8-fold more adducts than either the corn oil-alone or PB-treated animals. Both corn oil-alone and PB-treated animals were similar. Four major adducts were found in all cases with no differences among inducers as judged by co-chromatography. Azido-MeIQx induced calf-thymus-DNA adducts produced identical adduct profiles to those seen for the mouse DNA. Similar adduct profiles were obtained from Salmonella TA98, and the nitroreductase deficient strains (TA98NR and TA98/1,8-DNP6) exposed to MeIQx in the presence of Aroclor-induced-mouse-liver S9. Adduct frequencies in TA98/1,8-DNP6 were significantly lower than in TA98 and TA98NR. The data described in this report demonstrate that induction quantitatively increases adduct numbers but does not affect the types of DNA damage. These data also suggest that the same DNA reactive intermediates are formed in vivo as in vitro and support the hypothesis that the metabolism of MeIQx involves the P450I family of isozymes, N:O-acetyltransferases and possibly a nitrenium ion. The application of radioanalytic scanners for quantitation of 32P-postlabelling adduct maps is described.     

2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline-induced DNA adduct formation and mutagenesis in DNA repair-deficient Chinese hamster ovary cells expressing human cytochrome P4501A1 and rapid or slow acetylator N-acetyltransferase 2.

2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) is one of the most potent and abundant mutagens in the western diet. Bioactivation includes N-hydroxylation catalyzed by cytochrome P450s followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). In humans, NAT2*4 allele is associated with rapid acetylator phenotype, whereas NAT2*5B allele is associated with slow acetylator phenotype. We hypothesized that rapid acetylator phenotype predisposes humans to DNA damage and mutagenesis from MeIQx. Nucleotide excision repair-deficient Chinese hamster ovary cells were constructed by stable transfection of human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. CYP1A1 and NAT2 catalytic activities were undetectable in untransfected Chinese hamster ovary cell lines. CYP1A1 activity did not differ significantly (P > 0.05) among the CYP1A1-transfected cell lines. Cells transfected with NAT2*4 had 20-fold significantly higher levels of sulfamethazine N-acetyltransferase (P = 0.0001) and 6-fold higher levels of N-hydroxy-MeIQx O-acetyltransferase (P = 0.0093) catalytic activity than cells transfected with NAT2*5B. Only cells transfected with both CYP1A1 and NAT2*4 showed concentration-dependent cytotoxicity and hypoxanthine phosphoribosyl transferase mutagenesis following MeIQx treatment. Deoxyguanosine-C8-MeIQx was the primary DNA adduct formed and levels were dose dependent in each cell line and in the following order: untransfected < transfected with CYP1A1 < transfected with CYP1A1 and NAT2*5B < transfected with CYP1A1 and NAT2*4. MeIQx DNA adduct levels were significantly higher (P < 0.001) in CYP1A1/NAT2*4 than CYP1A1/NAT2*5B cells at all concentrations of MeIQx tested. MeIQx-induced DNA adduct levels correlated very highly (r2 = 0.88) with MeIQx-induced mutants. These results strongly support extrahepatic activation of MeIQx by CYP1A1 and a robust effect of human NAT2 genetic polymorphism on MeIQx-induced DNA adducts and mutagenesis. The results provide laboratory-based support for epidemiologic studies reporting higher frequency of heterocyclic amine-related cancers in rapid NAT2 acetylators.     

Chromosome site-specific immunohistochemical detection of DNA adducts in N-acetoxy-2-acetylaminofluorene--exposed Chinese hamster ovary cells

In these studies a polyclonal antiserum elicited against a carcinogen-DNA adduct was used to explore the localization of DNA adducts in metaphase chromosomes of cultured cells. Morphological visualization of the adduct N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) in Chinese hamster ovary (CHO) cells exposed to the direct-acting carcinogen N-acetoxy-2-acetylaminofluorene (N-Ac-AAF) was accomplished by indirect immunofluorescence with an anti-G-C8-AF antiserum. At the same time the pattern of chromosomal DNA replication was determined by replicative incorporation of bromodeoxyuridine (BrdUrd) and chromosomal staining with anti-BrdUrd. Visualization of DNA in chromosomes was accomplished with Hoechst 33258 dye. When synchronized CHO cells were exposed to N-Ac-AAF for 0.5 h during early S phase, the chromosomal pattern of dG-C8-AF adduct formation was not random. Metaphase chromosome spreads from cells exposed to N-Ac-AAF in different experiments contained certain chromosome regions that had a consistently high adduct concentration. The regions of high DNA damage corresponded to the regions active in DNA synthesis when BrdUrd and the carcinogen were given simultaneously in early S phase. In addition, the patterns of high adduct concentration and replicative synthesis shifted when the carcinogen and BrdUrd were given simultaneously during late S phase. Thus, the stage of cell cycle in which adducts are induced is an important factor in the specific location of the highest concentrations of this type of DNA lesion.     

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.     

Correlation of DNA adduct formation and riddelliine-induced liver tumorigenesis in F344 rats and B6C3F1 mice [Cancer Lett. 193 (2003) 119-125

Riddelliine is a naturally occurring pyrrolizidine alkaloid that induces liver hemangiosarcomas in male and female F344 rats and male B6C3F1 mice. We previously reported that eight dehydroretronecine (DHR)-derived DNA adducts were formed in liver DNA of rats treated with riddelliine. In order to examine the relationship between DNA adduct levels and the incidence of hemangiosarcomas, we have measured DHR-derived DNA adduct levels in purified rat and mouse liver endothelial cells, the cells of origin for the hemangiosarcomas. F344 rats and B6C3F1 mice were treated by gavage 5 days per week for 2 weeks with riddelliine at 1.0 mg/kg for rats and 3.0 mg/kg for mice. One, 3, 7, and 28 days after the last dose, liver parenchymal and endothelial cell fractions were isolated, and the quantities of DHR-derived DNA adducts were determined by 32P-postlabeling/HPLC. The DHR-derived DNA adduct levels in the endothelial cells were significantly greater than in the parenchymal cells. The DNA adduct levels in rat endothelial cells were greater than in the mouse endothelial cells. These results indicate that the levels of riddelliine-induced DNA adducts in specific populations of liver cells correlate with the preferential induction of liver hemangiosarcomas by riddelliine.     

Analysis of DNA adducts in rats exposed to pentachlorophenol

Pentachlorophenol (PCP) is a widely used biocide that has been reported to be hepatocarcinogenic in mice. Its effects in rats are equivocal, but the liver clearly is not a target organ for carcinogenesis. The carcinogenic effects of PCP in mice may relate to reactive oxygen species generated during metabolism. PCP is known to increase the hydroxyl radical-derived DNA lesion, 8-oxodeoxyguanosine (ohdG), in the liver of exposed mice. To investigate whether the generation of oxidative DNA damage and direct DNA adducts may explain the species difference in carcinogenicity, we have analyzed ohdG in hepatic DNA from PCP-exposed rats. Rats were exposed acutely to PCP for 1 or 5 days. Tissues also were obtained from a 27 week interim sacrifice of the 2 year National Toxicology Program carcinogenesis bioassay. We used HPLC with electrochemical array detection for ohdG analysis. Single or 5 day exposure to PCP (up to 120 or 60 mg/kg/day, respectively) did not increase ohdG. Dietary exposure to 1000 p.p.m. PCP (equivalent to 60 mg/kg/day) for 27 weeks induced a 2-fold increase in ohdG (1.8 versus 0.91x10(-6) in controls). In parallel, formation of direct DNA adducts was analyzed by 32P-post-labeling following nuclease P1 adduct enrichment. We detected two major DNA adducts with relative adduct labeling of 0.78x10(7) adducts per total nucleotides. One of these adducts was found to co-migrate with the adduct induced by the metabolite, tetrachloro-1,4-benzoquinone. We observed differences in DNA adduct formation between acute and chronic studies, with acute studies not inducing any detectable amount of DNA adducts. These results indicated that chronic, but not acute exposure to PCP increased ohdG and direct adducts in hepatic DNA. As the same exposure conditions that enhanced ohdG did not produce liver cancer in rats, the generation of reactive oxygen species, oxidative DNA damage and direct DNA adducts is not sufficient for the induction of hepatocarcinogenesis by PCP in the rat.     

Formation and removal of specific acetylaminofluorene-DNA adducts in mouse and human cells measured by radioimmunoassay.

Rabbit antiserum prepared against N-(guanosin-8-yl)-acetylaminofluorene was utilized in radioimmunoassay to detect formation and removal of C-8 adducts from the DNA of cultured cells exposed to N-acetoxy-2-acetylaminoflorene. The assay was able to quantitate both acetylated and deacetylated C-8 adducts between 0.5 and 5 pmol while the N2 adduct, 3-(deoxyguanosin-N2-yl)acetylaminofluorene, was not detected below 160 pmol. By varying the proportions of acetylated and deacetylated C-8 adducts in the radioimmunoassay, a series of standard curves were developed from which the relative proportion of each adduct could be determined in unknown mixtures. DNA from mouse epidermal cells and human skin fibroblasts exposed to N-acetoxy-2-acetylaminofluorene in culture contained only 3 and 5% respectively, of the C-8 adduct in the acetylated form. Quantitation by radioimmunoassay of total C-8 adducts bound to DNA yielded values approximately 25% lower than total carcinogen binding determined by radiolabeling. When removal of C-8 adducts was followed over a 23-hr, carcinogen-free culture period, mouse and human cells removed 40 and 50%, respectively, of bound acetylated and deacetylated C-8 adducts. These studies demonstrate the versatility of radioimmunoassay as a molecular probe for studies of chemical carcinogens.     

Correlation of DNA adduct formation and riddelliine-induced liver tumorigenesis in F344 rats and B6C3F(1) mice

Riddelliine is a naturally occurring pyrrolizidine alkaloid that induces liver hemangiosarcomas in male and female F344 rats and male B6C3F(1) mice. We previously reported that eight dehydroretronecine (DHR)-derived DNA adducts were formed in liver DNA of rats treated with riddelliine. In order to examine the relationship between DNA adduct levels and the incidence of hemangiosarcomas, we have measured DHR-derived DNA adduct levels in purified rat and mouse liver endothelial cells, the cells of origin for the hemangiosarcomas. F344 rats and B6C3F(1) mice were treated by gavage 5 days per week for 2 weeks with riddelliine at 1.0 mg/kg for rats and 3.0 mg/kg for mice. One, 3, 7, and 28 days after the last dose, liver parenchymal and endothelial cell fractions were isolated, and the quantities of DHR-derived DNA adducts were determined by (32)Ppostlabeling/HPLC. The DHR-derived DNA adduct levels in the endothelial cells were significantly greater than in the parenchymal cells. The DNA adduct levels in rat endothelial cells were greater than in the mouse endothelial cells. These results indicate that the levels of riddelliine-induced DNA adducts in specific populations of liver cells correlate with the preferential induction of liver hemangiosarcomas by riddelliine.     

Effect of a complex environmental mixture from coal tar containing polycyclic aromatic hydrocarbons (PAH) on the tumor initiation, PAH-DNA binding and metabolic activation of carcinogenic PAH in mouse epidermis.

Human exposure to polycyclic aromatic hydrocarbons (PAH) occurs through complex mixtures such as coal tar. The effect of complex PAH mixtures on the activation of carcinogenic PAH to DNA-binding derivatives and carcinogenesis were investigated in mice treated topically with NIST (National Institute of Standards and Technology) Standard Reference Material 1597 (SRM), a complex mixture of PAH extracted from coal tar, and either additional benzo[a]pyrene (B[a]P) or dibenzo[a,l]pyrene (DB[a,l]P). In an initiation-promotion study using 12-O-tetradecanoylphorbol-13-acetate as the promoter for 25 weeks, the SRM and B[a]P co-treated mice had a similar incidence of papillomas per mouse compared with the group exposed to B[a]P alone as the initiator. PAH-DNA adduct analysis of epidermal DNA by 33P-post-labeling and reversed-phase high-performance liquid chromatography found the SRM co-treatment led to a significant decrease in the total level of DNA adducts and B[a]P-DNA adducts to less than that observed in mice treated with B[a]P alone at 6, 12 and 72 h exposure. After 24 and 48 h exposure, there was no significant difference in the levels of adducts between these groups. In the DB[a,l]P initiation-promotion study, the co-treated group had significantly fewer papillomas per mouse than mice treated with DB[a,l]P alone as initiator. Averaging over the times of exposure gave strong evidence that mice co-treated with SRM and DB[a,l]P had a significantly lower level of PAH-DNA adducts than mice treated with DB[a,l]P alone. Western immunoblots showed that both cytochrome P450 (CYP) 1A1 and 1B1 were induced by the SRM. These results are consistent with the hypothesis that two major factors determining the carcinogenic activity of PAH within a complex mixture are (i) the persistence of certain PAH-DNA adducts as well as total adduct levels, and (ii) the ability of the components present in the mixture to inhibit the activation of carcinogenic PAH by the induced CYP enzymes.     

Epidemiological models of carcinogenesis: the example of bladder cancer.

Epidemiological studies have clearly shown that smokers have an increased risk of bladder cancer. Chemical, biochemical, and molecular investigations indicate that such risk might be due to aromatic amines which are present in tobacco smoke. In particular, collaborative studies have shown that smokers have increased levels of hemoglobin-4-aminobiphenyl adducts in their blood and that these levels show a dose-response relationship and an association with the most carcinogenic variety of tobacco, air-cured or black tobacco. Adduct concentrations were also modulated by the genetically based slow acetylator phenotype. In addition, investigations in dogs and humans have described a DNA adduct in bladder biopsies and in exfoliated bladder cells that is a derivative of 4-aminobiphenyl. This paper summarizes the epidemiological, biochemical, and molecular evidence concerning the possible mechanisms of bladder cancer induction in smokers and in occupationally exposed workers. The case of bladder cancer is an example of integration between epidemiological studies, mathematical modeling, and laboratory investigations aiming at the elucidation of mechanisms of carcinogenesis.     

Initiation of hepatocarcinogenesis in infant male B6C3F1 mice by N-hydroxy-2-aminofluorene or N-hydroxy-2-acetylaminofluorene depends primarily on metabolism to N-sulfooxy-2-aminofluorene and formation of DNA-(deoxyguanosin-8-yl)-2-aminofluorene adducts

Previous work from this laboratory provided strong evidencethat N-sulfooxy-2-aminofluorene is the major ultimate electro-philicand carcinogenic metabolite of N-hydroxy-2-acetyl-aminofluorene(N-hydroxy-AAF) in the livers of infant male B6C3F₁ (C57BL/6Jx C3H/HeJ F₁ mice. Over 90% of the hepatic DNA adducts in thesemice consisted of N-(deoxyguan-osin-8-yl)-2-aminofluorene [N-(dGuo-8-yl)]and<10% were deoxyguanosinyl adducts containing 2-acetylaminofluor-ene(AAF) residues. In the present study hepatic DNA adduct formationand tumor initiation by N-hydroxy-2-aminofluor-ene (N-hydroxy-AF)were examined in these mice. N-(dGuo-8-yl)-AF was the only adductdetected in the hepatic DNA; the level at 9 h after a singlei.p. dose of 0.04 or 0.06 µmol/g body wt of [³H]N-hydroxy-AFwas 1.0 or 1.7 pmol/mg DNA. Pre-treatment with a single i.p.dose (0.04 µmol/g body wt) of the sulfotransferase inhibitorpentachlorophenol (PCP) decreased the DNA adduct level by >80%.Similar levels of this adduct were found by ³²P-postlabelinganalysis of DNA from mice treated with unlabeled N-hydroxy-AF.The liver DNA of in-fant male brachyinorphic B6C3F₂ mice [deficientin 3'-phos-phoadenosine-5'-phosphosulfate (PAPS)] containedonly 0.3 pmol/mg DNA of N-(dGuo-8-yl)-AF after an i.p. doseof 0.06 µmol of N-hydroxy-AF/g body wt, while their phenotypi-callynormal (PAPS-sufficient) male littermates had 1.9 pmol/mg DNA.A single i.p. dose of 0, 0.015, 0.03, 0.06 or 0.12 µmol/body wt of N-hydroxy-AF in infant male B6C3F mice induced by10 months an average of 0.2, 2.5, 7, 11 or 14 hepatomas/mouse.Pretreatment with PCP reduced the liver tumor multiplicity ateach dose level by >80%. Essen-tially the same average tumormultiplicities and inhibitions of tumor formation by PCP pretreatmentwere obtained following injections of N-hydroxy-AF or N-hydroxy-AAFat the three lower dose levels. Collectively these data stronglyindicated that N-sulfooxy-2-aminofluorene is the major ultimate electrophilic and carcinogenic metabolite of N-hydroxyAF in the livers of infant male B6C3F₁ mice. Furthermore, sinceonly N-(dGuo-8-yl)-AF adducts were found in the he atic DNAthese lesions appear to be critical in the initiation of hepatocarcinogenesisin these mice by N-hydroxy-AF.     

Dose-related differences in DNA adduct levels in rodent tissues following skin application of complex mixtures from air pollution sources

Dose-related differences in the binding of DNA reactive intermediates for three environmentally important complex mixture particulate extracts and a well-studied carcinogen, benzo[a]pyrene (BaP), were examined in female C-57 mice following multiple topical treatments ranging from 1 to 120 mg/mouse. Particulate extracts from coke oven, coal soot and diesel exhaust were selected as model complex mixtures based on short-term mutagenicity assays, animal bioassays for carcinogenicity or epidemiological studies, where increased incidences of lung cancer in exposed populations were detected. Positive and negative control animals were treated with 1.2 mg BaP or acetone respectively. DNA was isolated from skin, lung and liver 24 h following the last application and analyzed for DNA adducts using the nuclease P1 version of the 32P-postlabeling assay. Each of the particulate extracts produced distinct patterns of DNA adducts. A diagonal zone of radioactivity, presumably representing multiple putative DNA adducts, was observed for coke-oven, coal-soot- and diesel-modified DNA samples. One adduct, common to all three complex-mixture-modified DNA samples, co-migrated with the major BaP adduct observed following treatment with BaP alone. Based on the BaP concentration for each of the extracts it seems unlikely that this adduct is derived from BaP alone. It is possible that an adduct is formed with chromatographic properties similar to the major BaP-derived adduct detected in mice treated with BaP alone. This adduct was detected in all tissues examined and represented approximately 12-34% of the total number of adducts detected within the diagonal radioactive zone for all coke-oven- and coal-soot-exposed tissues (skin, lung and liver). In contrast, this adduct represented 49-67% of the total radioactivity recovered from the diagonal zone of DNA isolated from lungs of animals exposed to diesel extract. The highest total number of adducts resulted from the metabolism of coke oven extract followed by coal soot and diesel treatments respectively. A dose-dependent increase in adduct formation was observed for all tissues in the diesel- and coal-soot-treatment mice. Liver and lung, but not skin, DNA adduct levels increased in a dose-dependent manner in the coke-oven-treated mice. The percentage of dose administered, detected as DNA adducts increased in all tissues as the dose decreased for all three complex mixtures. These data have important implications for risk assessment of these complex mixtures.