Differences in the covalent binding of benzo[a]pyrene, safrole, 1'-hydroxysafrole, and 4-aminobiphenyl to DNA of pregnant and non-pregnant mice

The effects of pregnancy on the covalent binding of several carcinogens to DNA were investigated in mice. Non-pregnant or timed-pregnant (18th day of gestation) ICR mice of similar age were treated with benzo[a]pyrene (BP, 200 mumol/kg), safrole (600 mumol/kg), 1'-hydroxysafrole (400 mumol/kg), 4-aminobiphenyl (4-ABP, 800 mumol/kg), or trioctanoin (4 ml/kg) per os. Tissue DNA adduct levels at 24 h after carcinogen treatment were analyzed via a 32P-postlabeling assay. Pregnancy lowered the binding of the ultimate carcinogenic metabolite of BP, 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE I), to liver and lung DNA by 29-41%, but not the binding of other metabolites. The binding of safrole and its proximate carcinogen, 1'-hydroxysafrole, to liver and kidney DNA was increased 2.3-3.5 fold. Pregnancy decreased the binding of 4-ABP to liver DNA by approximately 18% but increased its binding to kidney DNA by 67%. The results suggest that exposure to some genotoxic compounds, especially those requiring conjugation reactions for metabolic activation, may be more hazardous during pregnancy than in the non-pregnant state.     

Use of the 32P-postlabelling assay to study transplacental carcinogens and transplacental carcinogenesis

The formation of DNA adducts represents a key step in the postnatal initiation of the carcinogenic process. Little is known as yet about the role of prenatally induced adducts in transplacental carcinogenesis in offspring. Measurement of transplacental DNA damage in fetal organs of experimental animals has been difficult in the past because of the small amounts of DNA available and low adduct levels. In principle, these difficulties have been overcome by the recent development of a highly sensitive 32P-postlabelling assay which can be applied to a large number of DNA adducts of diverse structure and requires only microgram amounts of DNA for analysis. In this assay, tissue DNA is degraded to mononucleotides; these are enzymatically 32P-labelled via T4 polynucleotide kinase-catalysed [32P]phosphate transfer from [gamma--32P]ATP, to form 5'--32P-labelled 3',5'-bisphosphate derivatives; the labelled products are separated into normal and adducted [32P]nucleotides and quantified by thin-layer chromatography, autoradiography and scintillation (Cerenkov) counting. This technique allows the detection and quantitation of one adduct in 10(8)-10(10) DNA nucleotides (approximately 1-100 adducts/mammalian genome) using a 10-micrograms DNA sample and has been applied in studies of adduct formation from transplacental carcinogens in fetal and adult rodent tissues. In this paper, we review application of 32P-postlabelling to DNA adducts formed with transplacental or suspected transplacental carcinogens in fetal and maternal tissues. The carcinogens studied include diethylstilboestrol (DES), benzo[a]pyrene, safrole, 4-aminobiphenyl and 4-nitroquinoline-1-oxide, as well as cigarette smoke condensate. In DNA of DES-exposed hamsters, one major and several minor adduct spots were observed, which were absent from vehicle controls. A characteristic adduct, which resembled the major hamster adduct chromatographically, was detected in all exposed mouse tissue, except fetal kidney. Chronic administration of low doses of DES to male Syrian hamsters led to an entirely different pattern of adducts in kidney DNA, the target organ of carcinogenesis. These adducts did not contain covalently bound oestrogen moieties and appeared to be formed indirectly: oestrogen appeared to induce or enhance the synthesis of an endogenous electrophilic metabolite reacting with DNA. Thus, multiple mechanisms exist by which DES can damage DNA. Additional work using 32P-postlabelling has shown that non-hormonal genotoxicants (e.g., benzo[a]pyrene, safrole, 4-aminobiphenyl, 4-nitroquinoline-1-oxide) and cigarette smoke condensate given to pregnant mice can induce specific DNA adduct profiles in fetal tissues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of covalent DNA modifications and micronucleated erythrocytes by 4-nitroquinoline 1-oxide in adult and fetal mice

Pregnancy and development are known to modify carcinogenesis. Little is known about the mechanism for the modulation. These studies investigated the relative sensitivity of nonpregnant, pregnant, and fetal mice to the induction of covalent DNA modifications and micronucleated erythrocytes by 4-nitroquinoline 1-oxide (4-NQO). Our results revealed that 4-NQO was bound to guanine nucleotides of DNA in all maternal and fetal organs tested. The adduct levels ranged from 2-60 base modifications per 10(9) DNA bases when 4-NQO was administered s.c. Overall, 4-NQO bound preferentially to DNA of the maternal tissues compared with that of the corresponding fetal tissues, with the exception of the liver. The adduct levels in maternal and fetal organs fell into 3 distinct levels. The greatest binding was in maternal lungs and pancreas (the target organs for carcinogenesis). The lowest binding levels were in maternal liver and all fetal organs studied. Gestation age at the time of 4-NQO treatment did not produce a significant effect on the amounts of adduct formation in the tissues examined, with the exception of placenta and bone marrow. Chronic treatment did not affect binding preference. At the cellular level, 4-NQO treatment induced twice the frequency of micronucleated erythrocytes in the bone marrow of pregnant mice compared with the nonpregnant mice and fetal liver, on a mg/kg basis. However, the polychromatic erythrocytes of fetal liver were more sensitive than those of adult bone marrow to the induction of micronuclei, when adduct levels were taken into account. A positive correlation of organotropsim between 4-NQO-induced DNA adducts and carcinogenicity was observed for maternal tissues, but not for fetal tissues. Fetal tissues, overall, lack the enzymes to metabolically activate 4-NQO. Fetal cells elicit greater biological responses, compared with adult cells, at equal adduct levels. This study reveals that the effective doses in maternal and fetal tissues may differ and, therefore, will be a better basis for further understanding the molecular mechanism of transplacental carcinogenesis.     

Effects of route of administration on tissue distribution of DNA adducts in mice: comparison of 7H-dibenzo(c,g)carbazole, benzo(a)pyrene, and 2-acetylaminofluorene

The environmental pollutant 7H-dibenzo(c,g)carbazole (DBC) has been shown to be a potent carcinogen in various mouse tissues, but displays an unusual degree of hepatocarcinogenicity. We have previously reported that in accord with this activity, mouse liver is the target organ for DBC-DNA binding, with total levels being up to 2700 times greater than in extrahepatic tissues after s.c. administration. To elaborate on this finding, we have directly compared the tissue distribution of DNA damage by three diverse aromatic carcinogens, DBC, benzo(a)pyrene (BP), and 2-acetylaminofluorene (AAF). Following a single topical, p.o., or s.c. administration of 80 mumol/kg of test compound to male BALB/c mice, a 32P-postlabeling assay showed the total number of DBC adducts in liver DNA to be 11-138 times that in kidney, lung, or skin DNA. The degree of hepatic adduction varied as a function of the route of administration, with the highest occurring after topical application and the lowest after s.c. injection. The tissue preference for AAF and BP adducts varied with the route of administration and was much less than for DBC adducts, except that topical application of BP gave DNA adduct levels in skin that were 91-218 times greater than in other tissues. For a given tissue and route of administration, DNA adduction by DBC was 1.7- to 950-fold greater than that by BP and AAF, except in skin where the level of DNA adducts from BP was 3 to 4 times that from DBC. We conclude that (a) DBC exhibits an exceptional and unique preference for liver DNA adduction after different routes of administration; (b) DBC is more potent overall than BP or AAF in causing tissue DNA damage; and (c) for each of the three carcinogens, the route of exposure is a much less important factor than the nature of the carcinogen in determining the tissue distribution of covalent DNA damage.     

Differential effect of gestation stage on benzo(a)pyrene-induced micronucleus formation and/or covalent DNA modifications in mice

Benzo(a)pyrene (BP), an environmental carcinogen, binds ubiquitously to the DNA of maternal and fetal tissues (Lu et al., Cancer Res., 46: 3046-3054, 1986). These studies further investigated the effect of gestation age on the induction of genetic damage by BP. Timed-pregnant ICR mice were treated with one dose of BP on various days of gestation and sacrificed 24-120 h after treatment. At the molecular level, BP covalently bound to the DNA of adult bone marrow and fetal liver of mice at all gestation ages. Compared to the nonpregnant mice (adduct level = 15 adducts/10(8) bases), the adduct levels in the pregnant adult bone marrow were decreased up to 50% during early gestation (days 3-9) and then increased steadily to a 4-fold excess over nonpregnant values during late gestation (days 15-18). In the fetal liver, adduct levels exhibited little variation (3-4 adducts/10(8) bases) between days 11 and 15 of gestation and then increased sharply to 14 adducts/10(8) bases after day 16. At the cellular level, a higher percentage of polychromatic RBCs from adult and fetal mice after BP treatment contained micronuclei (MN) than controls (solvent or untreated). Bone marrow from pregnant mice exhibited greater increases in the formation of MN during early gestation (days 3-9) relative to late gestation (days 15-18), compared to the nonpregnant mice. In the fetuses, the amounts of MN formed were higher than those found in the adult nonpregnant or maternal mice, but these amounts decreased with gestation progression. Thus, MN induction with gestation progression differed from DNA adduction in adults and fetuses. In addition, the dose and time responses of MN formation also differed from those of covalent DNA modifications, when analyzed in the bone marrow of pregnant mice treated on gestation day 5. Collectively, our results showed that pregnancy and development modulate different types of genetic damage in different ways. Fetal tissues may be more sensitive than maternal tissues to genetic damage. Factors in addition to DNA adduct formation may be responsible for MN induction.     

Modulation of benzo[a]pyrene-induced covalent DNA modifications in adult and fetal mouse tissues by gestation stage

In these studies, we investigated the influence of gestation age on the induction of covalent DNA modifications by benzo[a]pyrene (B[a]P). Timed-pregnant ICR mice were given a single treatment of B[a]P (80 mg/kg, p.o.) on different days of gestation, killed 24 h later and analyzed for the presence of B[a]P-induced DNA adducts using the P1 nuclease version of the 32P-postlabeling method. Our results showed that B[a]P bound to embryonic, placental, fetal and maternal DNA throughout gestation with gestation-stage dependency. Overall, B[a]P bound less to maternal DNA during organogenesis and placentation compared to other stages of gestation and to the non-pregnant stage. The ontogenesis of B[a]P-induced DNA adducts in fetal tissues exhibited organ specificity that had two different types of profiles. With advancing gestation age, one type (lung, carcass and placenta) exhibited a steady linear increase, and the other type [gastrointestinal tract (GIT) and skin] a biphasic increase. In the fetal and maternal organs, adduct levels peaked 2 days before parturition. Over the course of gestation, fetal adduct levels were 70-100% of adult levels in the skin, 7-12% in the GIT, 25-40% in the liver and 15-80% in the lung. The adduct levels in many fetal organs exhibited little relationship to placental adduct levels throughout gestation. Collectively, our results indicate that: (i) transplacental DNA damage induced by B[a]P is determined mainly by fetal competence in metabolic activation and/or detoxification of B[a]P; and (ii) events occurring during placentation and organogenesis inhibit B[a]P binding to maternal tissues.     

Elevated mitochondrial cisplatin-DNA adduct levels in rat tissues after transplacental cisplatin exposure

Although there is evidence that the toxic effects of cis- diamminedichloroplatinum(II) (cisplatin) include morphologically abnormal mitochondria, direct demonstrations of mitochondrial DNA damage by this chemotherapeutic agent have rarely been reported. Here we show that, in rats exposed to a single dose of cisplatin during gestation, cisplatin-DNA binding levels in both maternal and fetal liver and brain mitochondrial DNA are higher than those observed in genomic DNA. Pregnant F344/NCr rats were injected i.p. with either 5 or 15 mg cisplatin/kg body wt at 18 days of gestation and killed 24 h later. Cisplatin-DNA adducts were determined by dissociation-enhanced lanthanide fluoroimmunoassay using a cisplatin-DNA standard modified in the same range as the biological samples. Values for genomic cisplatin- DNA adducts in multiple maternal and fetal tissues have been presented elsewhere. Here, genomic DNA adduct levels for liver, brain, kidney and placenta are reported again for comparison with mitochondrial DNA adduct levels in the same tissues. In maternal and fetal brain, mitochondrial DNA adduct levels were approximately 7- to 50-fold higher than genomic DNA adduct levels, and in fetal liver they were approximately 2- to 16-fold higher than genomic DNA adduct levels. These studies demonstrate extensive cisplatin-DNA adduct formation in brain and liver mitochondria of fetal rats exposed transplacentally and suggest that mitochondrial DNA in some organs may be a particular target for cisplatin genotoxicity.      

Ethylnitrosourea-induced transplacental carcinogenesis in the mouse: tumor response, DNA binding, and adduct formation

We have confirmed previous results which suggest that transplacental exposure of fetal mice to carcinogens does not cause an increase in tumor incidence as they mature unless treatment occurs after midorganogenesis. In C3HeB/FeJ mice we found a negligible increase in tumor incidence and multiplicity following transplacental exposure to the direct-acting carcinogen ethylnitrosourea (ENU) on gestation day 10, but significant increases in lung and liver tumor incidence following exposure on days 13 or 15 or in adults. To explore the possibility that this observed difference is due to differences in the biodistribution of the carcinogen or its interaction with cellular macromolecules, the level of covalent binding between ENU and fetal and maternal DNA following an i.p. injection of a dose of 50 mg/kg of tritium-labeled ENU was measured 30 min after its injection into pregnant females on days 10, 13, and 15 of gestation. The DNA from fetal and maternal lung, liver, and brain was isolated and the amount of covalent binding estimated from the dpm/mg DNA recovered. Samples of DNA were hydrolyzed and chromatographed to determine that the bound tritium was associated with ENU-DNA adducts and not as a product of DNA synthesis. The level of binding of ENU to fetal DNA was equivalent at all gestation days studied but was significantly less than maternal tissues. Binding to the DNA of maternal liver was 4-fold greater than to fetal DNA while maternal lung and brain DNA were bound at intermediate levels. We conclude that the lack of carcinogenic response to ENU documented here, in fetal mice exposed early in gestation (day 10), is not due to differences in ENU binding to fetal DNA during development.     

Dose-response relationships for the binding of benzo(a)pyrene metabolites to DNA and protein in lung, liver, and forestomach of control and butylated hydroxyanisole-treated mice

In this study, the formation of benzo(a)pyrene (BP) metabolite:DNA adducts in lung, liver, and forestomach of control and butylated hydroxyanisole (BHA)-treated (5 mg/g diet) female A/HeJ mice was examined as a function of BP dose (p.o.), ranging from 2 to 1351 mumol/kg. The major identified adduct in each tissue at each dose was the (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDEI):deoxyguanosine adduct. A 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct, a(-)-BPDEI:deoxyguanosine adduct, and an unidentified adduct were also observed. In lung and liver of untreated animals, the dose-response curves for BPDEI:DNA adduct levels were sigmoidal. In forestomach, there was no indication of saturation of DNA binding over the BP dose range examined. The dose-response curves became linear as BP dose approached zero and thus, no threshold dose existed below which binding of BPDEI to DNA did not occur, at least in lung, liver, and forestomach of these mice. In forestomach, the dose-response curve for BPDEI:DNA adducts in BHA-treated mice, 0.5% of diet for 2 weeks, was parallel to the curve for control animals and thus, the inhibition (45%) of adduct formation is independent of BP dose. In contrast, BHA treatment diminished the curvilinear nature of the dose-response curves for BPDE adducts in lung and liver. The inhibition of BPDEI:DNA adduct formation by BHA in lung and liver was dose dependent. The inhibition of lung (68%) and liver (82%) adduct formation was highest at a BP dose of 270 mumol/kg. As the BP dose approached zero, the inhibition of BPDEI:DNA adduct formation by BHA decreased with BP dose and approached values of approximately 40% (lung) and 55% (liver). The dose dependency of the binding of BP metabolites to protein was also examined. BPDEI:DNA adduct concentrations ranged from 2 to 10% of protein binding concentrations in liver of untreated animals, from 3 to 7% in forestomach, and from 5 to 7% in lung. The dose-response curves for protein binding of BP metabolites in lung and liver from BHA-treated animals were essentially parallel to those in control animals and thus, the inhibition of protein binding by BHA treatment had no dose dependency in these organs. No consistent BHA effect was observed on the amount of binding of BP metabolites to forestomach protein.     

N-methylation reduces the DNA-binding activity of 7H-dibenzo[c,g]carbazole ~300-fold in mouse liver but only ~2-fold in skin: possible correlation with carcinogenic activity

N-methyl-dibenzo[c,g]carbazole (MeDBC) lacks the potent hepatocarcinogenicactivity in mice characteristic for 7H-dibenzo[c,g]carbazole(DBC), while both compounds are local carcinogens, leading topapilloma and carcinoma formation in skin after topical application.Because DNA binding is considered an essential step in the initiationof chemical carcinogenesis, the DNA adduction by MeDBC was comparedwith that by DBC in mouse liver and skin via a ³²P-post-labelingtechnique. Both compounds elicited chromatographically similaradducts in liver; however, the extent of total DNA binding ofDBC was 343- and 265-fold greater than that of MeDBC 24 h aftertopical and i.p. administration, respectively, of a 37 µmol/kgdose. In skin, the adduct pattern elicited by either compoundafter topical application was dif ferent from that seen in liver,and three of four adducts derived from MeDBC were chromatographicallydistinct from those produced by DBC. Quantitative analysis revealedthat total adduction in skin by DBC was 2.3-fold higher thanby MeDBC. When the adduct levels were compared between liverand skin, topically applied MeDBC bound preferentially to skinversus liver DNA by a factor of 10, while the opposite was truefor DBC. These data are in agreement with the car cinogenicityreported for DBC and MeDBC and support the hypothesis that theextent of covalent DNA modification by these compounds is associatedwith their biological activity. We conclude that an unsubstitutednitrogen is essential for the genotoxic activity of DBC in liverbut not skin. The results also demonstrate the potential ofthe ³²P-postlabeling assay in predicting the organotropism ofclosely related carcinogens.     

DNA adduct formation and persistence in rat tissues following exposure to the mammary carcinogen dibenzo[a,l]pyrene.

Dibenzo[a,l]pyrene (DBP), an environmentally significant polycyclic aromatic hydrocarbon (PAH), is one of the most potent carcinogens with greater carcinogenicity in rodent mammary glands and skin than 7,12-dimethylbenz[a]anthracene or benzo[a]pyrene, respectively. In this study, we have examined the formation and persistence of stable DNA adducts in rats administered a carcinogenic intramammillary (i.m.) dose of DBP (0.25 micromol/gland). 32P-post-labeling analysis of mammary epithelial DNA 6 h, and 2, 5 and 14 days post-treatment produced one major (approximately 30%) and at least six minor adducts. Non-target tissue DNA (lung, heart, bladder and pancreas) also showed essentially the same adduct pattern as did mammary DNA, except liver which resulted in four additional adduct spots. The mammary DNA was most adducted (2640 +/- 532 adducts/10(9) nucleotides) on day 5 while the other tissue DNAs had 10- to 65-fold lower adduct levels (lung > liver > heart > bladder > pancreas). Adduct levels continued to increase at all time points examined for all tissues, except mammary tissue which showed a decline ( approximately 40%) on day 14. Chromatographic comparison with adducts formed in vitro by reaction of syn- and anti-DBP-11, 12-diol-13,14-epoxides (DBPDEs) with DNA and individual nucleotides indicated that the in vivo adducts were deoxyadenosine- and deoxyguanosine-derived, formed by interaction with both the anti- and syn-isomers; the adenine-derived adducts comprised 60-75% of the total adducts. However, the liver-specific DNA adducts (nos 8-11) were not derived from any of the DBPDE isomers. Our data show: (i) significantly higher DBP-DNA adduction in mammary tissue as compared with non-target tissues, which is consistent with its mammary carcinogenicity; (ii) adenine is highly reactive towards DBP metabolites as has been observed for many other PAHs; and (iii) the peak binding of DBP with DNA was shifted beyond 14 days for the non-target tissues by i.m. route of exposure.     

Detection of polycyclic aromatic hydrocarbon-DNA adducts in white blood cells of foundry workers

Iron foundry workers, exposed to high levels of polycyclic aromatic hydrocarbons (PAHs), silica, and metal fumes and dusts, are at elevated risk of lung cancer. Benzo(a)pyrene and a number of structurally related PAHs are metabolically activated to diol epoxides (e.g., 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a) pyrene) which are mutagenic, carcinogenic in experimental animals, and form covalent adducts with DNA. The levels of these adducts were measured in an enzyme-linked immunosorbent assay using a polyclonal anti-benzo(a)pyrene diol epoxide-I-DNA antibody which cross-reacts with DNA modified by diol epoxides of structurally related PAHs. DNA was analyzed from peripheral blood cells of 35 Finnish foundry workers and 10 controls. Workers were classified as having low (less than 0.05 micrograms/m3), medium (0.05-0.2 micrograms/m3), or high (greater than 0.2 micrograms/m3) exposure to benzo(a)pyrene (as an indicator of PAH). When adjustment was made for cigarette smoking and time since vacation, benzo(a)pyrene exposure was significantly related to adduct levels (P = 0.0001). Each of the three exposure groups had significantly elevated adduct levels compared to controls. Among the exposed workers, the low group differed significantly from the high and medium categories. This study supports the usefulness of monitoring adduct formation in a population occupationally exposed to carcinogens.     

32P-post-labelling analysis of DNA adducts formed in the livers of animals treated with safrole, estragole and other naturally-occurring alkenylbenzenes. I. Adult female CD-1 mice

The binding of a series of alkenylbenzenes to liver DNA of adultfemale CD-1 mice, isolated 24 h after i.p. administration ofnon-radioactive test compound (2 or 10 mg/mouse), was investigatedby a modified ³²P-post-labelling assay. The known hepatocarcinogens,safrole, estragole and methyleugenol, exhibited the strongestbinding to mouse-liver DNA (1 adduct in 10 000–15 000DNA nucleotides or 200–300 pmol adduct/mg DNA after administrationof a 10 mg dose), while several related compounds, which havenot been shown thus far to be carcinogenic in rodent bioassays,bound to mouse-liver DNA at 3–200x lower levels. The lattercompounds included allylbenzene, anethole, myristicin, parsleyapiol, dill apiol and elemicin. Eugenol did not bind. Low bindingto mouse-liver DNA was also observed for the weak hepatocarcinogen,isosafrole. Two main ³²P-labelled adducts, which appeared tobe guanine derivatives, were detected for each of the bindingchemicals on thin-layer chromatograms. The loss of safrole adductsfrom liver DNA was biphasic: a rapid loss during the first week(t_frac12;{small tilde}3 days) was followed by a much slowerdecline up to 20 weeks after treatment (t_frac12;{small tilde}2.5months). Adducts formed by reaction of 1'-acetoxysafrole, amodel ultimate carcinogen, with mouseliver DNA in vitro werechromatographically identical to safrole-DNA adducts formedin vivo. Pretreatment with pentachlorophenol, a known inhibitorof sulphotransferases, inhibited the binding of safrole to mouse-liverDNA, providing further evidence that the metabolic activationof the allylbenzenes proceeds by the formation of 1'-hydroxyderivatives as proximate carcinogens and 1'-sulphoöxy derivativesas ultimate carcinogens.     

4-Aminobiphenyl hemoglobin adducts in fetuses exposed to the tobacco smoke carcinogen in utero

Maternal-fetal exchange of a potent tobacco-related human carcinogen, 4-aminobiphenyl, was studied in smoking (n = 14) and nonsmoking (n = 38) pregnant women. N-Hydroxy-4-aminobiphenyl, the active metabolite of 4-aminobiphenyl, forms chemical addition products (adducts) with hemoglobin. Levels of 4-aminobiphenyl hemoglobin adducts were measured in maternal-fetal paired blood samples obtained from smoking and nonsmoking women during labor and delivery. Carcinogen-hemoglobin adducts were detected in all maternal and fetal blood samples. Levels of such adducts were significantly higher (P less than .001) in maternal and fetal blood samples from smokers: the mean 4-aminobiphenyl hemoglobin adduct level was 92 +/- 54 pg/g of hemoglobin in blood samples from fetuses of smokers, and 17 +/- 13 pg/g of hemoglobin in blood samples from fetuses of nonsmokers; the mean maternal 4-aminobiphenyl hemoglobin adduct level was 183 +/- 108 pg/g of hemoglobin in smokers, and 22 +/- 8 pg/g of hemoglobin in nonsmokers. Fetal carcinogen-adduct levels were consistently lower than maternal levels: the mean maternal to fetal ratio was 2.4 +/- 1.1 in smokers and 1.9 +/- .98 in nonsmokers. Fetal 4-aminobiphenyl hemoglobin adduct levels were strongly associated (correlation coefficient [r2] = .51, P = .002) with maternal 4-aminobiphenyl hemoglobin adduct levels when paired samples from smoking mothers were analyzed. A measure of third-trimester tobacco smoke exposure based on number of cigarettes smoked per day, amount of each cigarette smoked, and depth of inhalation was associated (r2 = .59, P = .029) with maternal 4-aminobiphenyl levels but not with fetal 4-aminobiphenyl levels. This study demonstrates that a potent tobacco-related carcinogen, 4-aminobiphenyl, or its active metabolite, N-hydroxy-4-aminobiphenyl, crosses the human placenta and binds to fetal hemoglobin in concentrations that are significantly higher in smokers than in nonsmokers.     

Covalent binding of benzo[a]pyrene to macromolecules in lung and liver of rats following intratracheal instillation

Benzo[a]pyrene (BP) was administered intratracheally to male Sprague-Dawley rats at a dose of 25 micrograms/kg. Covalent binding of BP metabolites to DNA, RNA and protein in lung and liver was analyzed 6 h after administration. In both lung and liver, relative amounts bound based on mass of each macromolecule decreased in the order RNA greater than protein greater than DNA. For DNA and RNA, but not protein, binding in liver exceeded that in lung. Types of DNA adducts in lung and liver were analyzed using boronate chromatography followed by HPLC. Several adducts were formed between BP metabolites and DNA in lung, and the adduct present in greatest amounts was that formed between deoxyguanosine and (+)-anti-BP diol epoxide (BPDE:dGuo). Only 2 adducts were distinguishable in liver. BPDE:dGuo was detected, though the predominant adduct, as yet unidentified, did not contain cis-vicinal hydroxyl groups.     

Immunohistochemical demonstration of carcinogen-DNA adducts in target and non-target tissues of rats given a prostate carcinogen, 3,2'-dimethyl-4-aminobiphenyl

An immunohistochemical procedure was applied which allows accurate localization of DNA lesions within organs and tissues of rats given 3,2'-dimethyl-4-aminobiphenyl (DMAB) using polyclonal antibodies against DMAB-DNA adducts. Dose-related nuclear staining was observed in organs regardless of DMAB-carcinogenic organotropism. In the male accessory sex organs, the lateral lobe of the prostate, a non-target site, demonstrated a similar staining intensity to that found for the ventral prostate and seminal vesicle, target sites. Orchiectomy and pretreatment with ethinyl estradiol resulted in a moderate to slight decrease in binding in the accessory sex organs. No observable decrease in staining intensity was evident in most organs 168 h after the administration of DMAB. These findings suggest that DNA adduct formation itself is not necessarily sufficient for tumor induction.     

Reaction of 7,12-dimethylbenz(a)anthracene with DNA of fetal and maternal rat tissues in vivo.

Pregnant BD-IX rats (21st day of gestation) received a single IV injection (15 mg/kg) of tritiated 7,12-dimethylbenz(a)anthracene (DMBA), A DOSE KNOWN TO INduce a high incidence of nervous-system tumors in the offspring. The animals were killed 12 h later and hydrocarbon-deoxyribonucleoside products from DNA of maternal and fetal tissues were separated on Sephadex LH-20 columns eluted with a 20-100% methanol gradient. Concentrations of the major DMBA-DNA adduct varied considerably, with highest values in maternal intestine, liverand lung, followed by spleen, kidney and brain. In fetal intestine and liver, concentrations were 34% and 16% lower than in the respective maternal organs whereas the reaction with cerebral DNA was 2 1/2 times higher in fetuses than in the pregnant mother. This indicates that there is no significant placental barrier to DMBA or DMBA metabolites involved in DNA binding and that rat fetuses participate in the metabolic formation of the ultimate carcinogen.     

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 and persistence of novel benzo(a)pyrene adducts in rat lung, liver, and peripheral blood lymphocyte DNA

Male CD rats were injected with single i.p. doses of benzo(a)pyrene (B(a)P), and peripheral blood lymphocytes (PBLs), livers, and lungs were removed at various times after administration. DNA adducts were analyzed in each tissue by 32P postlabeling with nuclease P1 enhancement. Sister chromatid exchange frequencies were concomitantly measured in cultured whole blood. B(a)P-DNA adducts were observed in all three tissues from animals sacrificed between 1 and 56 days after injection. Maximal adduction levels occurred at about 4 days after administration, followed by a gradual loss of adducts over the period examined. The apparent half-lives of total DNA adducts were 15 days in liver, 17 days in PBLs, and 22 days in lung. Induced sister chromatid exchanges were linearly related to the amount of DNA adducts remaining in the PBLs at the time of harvest up to 56 days and were significantly elevated above concurrent controls up to 14 days. One of the major adducts found in each tissue was N2-(10 beta-[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a) pyrene]yl)deoxyguanosine. An additional novel major adduct was found in the liver DNA and is derived from the further metabolism of B(a)P-trans-7,8-dihydrodiol. A second major novel B(a)P adduct was found in the DNA of lung tissues and accounts for about 40% of the total adducts present. Experimental evidence suggests that this adduct is derived from a metabolic pathway that includes the formation of 9-hydroxy-B(a)P.     

A comparison of covalent DNA binding of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in respiratory tissues from human, rat and mouse

In vivo and in vitro covalent DNA binding was investigated in an attempt to explain the higher susceptibility of A/J mouse lung and Fischer-344 rat trachea to 7,12-dimethylbenz[a]anthracene (DMBA) as compared to benzo[a]pyrene (BP), and to evaluate the relative susceptibility of the human respiratory tract to these compounds. After in vivo administration of either BP or DMBA to A/J mice covalent DNA binding was higher in the liver than in the lungs. Forty-eight hours after administration, but not before, binding of DMBA was higher than that of BP in both organs. In vitro studies using cultured explants of both human and A/J mouse peripheral lung, as well as human bronchus and Fischer-344 rat trachea, revealed that covalent DNA binding of DMBA to mouse lung and rat trachea were similar and that both were significantly higher than that of BP to these organs. Binding of BP and DMBA was similar in both human tissues and did not differ from BP binding in the animal tissues. Enzymatic hydrolysis and HPLC separation of the DNA-hydrocarbon adducts revealed that patterns of adducts in human and mouse peripheral lung were similar and qualitatively resembled known patterns in other target and non-target tissues. It is concluded that the higher susceptibility of the mouse lung and rat trachea to DMBA as compared to BP may be related to the higher covalent DNA binding of the former and that the relative carcinogenic risk of the human respiratory tract after exposure to DMBA may be the same as that after BP exposure.