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.     

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.     

Age-, tissue-, and Ah genotype-dependent differences in the binding of 3-methylcholanthrene and its metabolite(s) to mouse DNA

The induction of transplacental carcinogenesis by 3-methylcholanthrene (MC) in mice is determined, in part, by the genotype at the Ah locus. The relationship of Ah genotype and MC-induced DNA adducts was tested by comparing the response of pregnant and fetal C57BL/6 mice (Ahb Ahb; responsive to the induction of MC metabolism) and DBA/2mice (Ahd Ahd; nonresponsive). On day 17 of gestation (day 1 = presence of vaginal plug), C57BL/6 mice were treated i.p. with 100 mg/kg MC and DBA/2 mice with 30 mg/kg. Mice were sacrificed 24 h later and the tissues were analyzed for the presence of DNA adducts using the P1 nuclease version of the 32P-postlabeling method. With a 3.3-fold difference in administered dose, the total adduct levels in fetal DNA were (a) similar in both strains with the exception of liver, for which C57BL/6 mice had more adducts; (b) higher in the lung than skin, liver, or thymus; and (c) only 1/4 to 1/14 of the adult levels. Maternal DBA/2DNA contained more adducts in the thoracic lymph nodes and liver but fewer in the placenta and lung, compared to maternal C57BL/6 DNA. More adducts were detected in lung DNA than liver DNA in C57BL/6 mice. In contrast, these levels were similar in DBA/2 mice. When the difference in dose administered was considered in conjunction with this, less MC bound to DNA of C57BL/6 than DBA/2 mice overall. To identify adducts, oxidized metabolites of MC, 1-hydroxy-, 2-hydroxy-, 9,10-dihydrodiol-, or 3-methoxymethyl-MC, were topically applied to the dorsal skin of both strains. All of these metabolites produced adducts. Approximately 14 different adduct spots were detected. The two most abundant adducts were produced by 1-hydroxy-, 2-hydroxy-, and 9,10-dihydrodiol-MC. One of these also contained a 3-hydroxymethyl group. Several adducts did not contain the 9,10-dihydroxy group. The adducts derived from 3-methoxymethyl-MC were consistently found in greater abundance in DNA from C57BL/6 tissues, compared with DBA/2. Thus, oxidation of the 3-methyl group may be enhanced by Ah-dependent induction of MC metabolism. Together, these results suggest that the individual and total adduct levels are influenced by the genotype at the Ah locus, the route of administration, and the metabolite(s) with tissue and age specificity.     

Persistence of benzo(a)pyrene metabolite:DNA adducts in lung and liver of mice

The persistence of benzo(a)pyrene (BP) metabolite:DNA adducts has been studied in lung and liver of A/HeJ and C57BL/6J mice after a dose of BP (6 mg/mouse) which induces pulmonary adenomas in A/HeJ mice but not in C57BL/6J mice. BP is not a hepatic carcinogen in either strain. Following p.o. administration of [3H]BP, animals were killed at times ranging from 10 hr to 28 days, and BP metabolite:DNA adducts were analyzed by high-pressure liquid chromatography. The major adduct identified in each tissue was the (+)-7 beta-8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct. A 7 beta, 8 alpha-dihydroxy-9 beta,10 beta,epoxy-7,8,9, 10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct, a (-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct, and an unidentified adduct were also observed. The disappearance of (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydro-BP adduct in A/HeJ mice followed first-order kinetics over the time period examined, with a half-life of 18 and 9 days in lung and liver, respectively. The decay of this adduct in C57BL/6J mice was biphasic in both tissues. Our data on cell turnover suggest that there is active removal of adducts in liver, but that normal DNA turnover can account for the partial or possibly total observed disappearance of adducts in lung. These results suggest that the tissue specificity for BP-induced neoplasia in A/HeJ mice may be related to the relative persistence of adducts and high cell turnover rates in lung. In contrast, the results on formation and persistence of adducts and cell turnover do not provide an explanation for the strain difference in susceptibility to BP-induced pulmonary adenomas. It was also shown that the rates of removal of BP metabolite:DNA adducts in A/HeJ mice are not significantly different at a 500-fold lower BP dose.     

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)     

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

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

Persistence, gestation stage-dependent formation and interrelationship of benzo[a]pyrene-induced DNA adducts in mothers, placentae and fetuses of Erythrocebus patas monkeys

Since DNA adducts have been detected in the placentae of pregnantwomen who smoke cigarettes, the importance of these adductsas biomarkers of fetal exposure and risk has been evaluatedusing a non-human primate as a model. Pregnant Erythrocebuspatas monkeys on days 50, 100 or 150 of gestation (term = 160± 5 days) were treated once with 5–50 mg/kg benzo[a]pyrene(B[a]P), p.o. Fetuses were removed by Cesarean section 1–50days after treatment and analyzed for DNA adducts by the nucleaseP₁ version of the ³²P-postlabeling method. B[a]P induced highlevels of DNA adducts in all fetal organs, placentae and maternallivers in all three trimesters of gestation. DNA adduct levelswere higher in mid-gestation compared to early and late gestation.The major adduct detected was 10ß-(deoxyguanosin)-N²-yl-7ß,8     

Formation and persistence of DNA adducts in the liver of brown bullheads exposed to benzo[a]pyrene

The formation and persistence of benzo[a]pyrene (BP)-DNA adducts in the liver of brown bullheads (Ictalurus nebulosus) treated with the hydrocarbon (20 mg/kg body wt, i.p.) was investigated using the 32P-postlabeling assay. The highest level of covalent binding of BP to liver DNA (188 fmol BP adducts/mg DNA) was observed 25-30 days following treatment. After 70 days, the adduct level in liver DNA had declined to approximately 26% of the maximum adduct level. One major BP-DNA adduct and several minor ones were detected in the liver. The major adduct co-chromatographed with anti-BP-7,8-diol-9,10-epoxide-deoxyguanosine (anti-BPDE-dGuo) adduct. The data suggest that brown bullheads metabolically activate BP by the same mechanism as the mammalian systems susceptible to carcinogenic effects of the hydrocarbon.     

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.     

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.     

Increased formation of hepatic N2-ethylidene-2'-deoxyguanosine DNA adducts in aldehyde dehydrogenase 2-knockout mice treated with ethanol

N2-ethylidene-2'-deoxyguanosine (N2-ethylidene-dG) is a major DNA adduct induced by acetaldehyde. Although it is unstable in the nucleoside form, it is relatively stable when present in DNA. In this study, we analyzed three acetaldehyde-derived DNA adducts, N2-ethylidene-dG, N2-ethyl-2'-deoxyguanosine (N2-Et-dG) and alpha-methyl-gamma-hydroxy-1,N2-propano-2'-deoxyguanosine (alpha-Me-gamma-OH-PdG) in the liver DNA of aldehyde dehydrogenase (Aldh)-2-knockout mice to determine the influence of alcohol consumption and the Aldh2 genotype on the levels of DNA damage. In control Aldh2+/+ mice, the level of N2-ethylidene-dG adduct in liver DNA was 1.9 +/- 0.7 adducts per 10(7) bases and was not significantly different than that of Aldh2+/- and -/- mice. In alcohol-fed mice (20% ethanol for 5 weeks), the adduct levels of Aldh2+/+, +/- and -/- mice were 7.9 +/- 1.8, 23.3 +/- 4.0 and 79.9 +/- 14.2 adducts per 10(7) bases, respectively, and indicated that adduct level was alcohol and Aldh2 genotype dependent. In contrast, an alcohol- or Aldh2 genotype-dependent increase was not observed for alpha-Me-gamma-OH-PdG, and N2-Et-dG was not detected in any of the analyzed samples. In conclusion, the risk of formation of N2-ethylidene-dG in model animal liver in vivo is significantly higher in the Aldh2-deficient population and these results may contribute to our understanding of in vivo adduct formation in humans.     

4-Aminobiphenyl induces liver DNA adducts in both neonatal and adult mice but induces liver mutations only in neonatal mice

The mechanisms underlying the susceptibility of neonatal mice to genotoxic carcinogens were investigated by analyzing the DNA adducts and mutations induced in the livers of neonatal and adult Big Blue transgenic mice by 4-aminobiphenyl (4-ABP), a potent human and rodent carcinogen. Neonatal and adult mice were treated with a regimen of 4-ABP known to induce tumors in neonatal mice. Animals were sacrificed 1 day after the last treatment for DNA adduct analysis and 8 weeks after the last treatment for analysis of lacI and cII mutant frequency (MF). N-(Deoxyguanosin-8-yl)-4-ABP was the major DNA adduct identified in the livers of the 4-ABP-treated mice and levels of this adduct were significantly higher in treated animals than in the controls for both the neonates and adults. Adduct levels for adult females (44.0 +/- 4.8 adducts/10(6) nucleotides) were higher than in neonatal females (25.9 +/- 2.2 adducts/10(6) nucleotides), while adduct levels in adult males (13.5 +/- 2.0 adducts/10(6) nucleotides) were lower than in neonatal males (33.8 +/- 4.1 adducts/10(6) nucleotides). 4-ABP treatment significantly increased the liver cII MFs in both sexes of neonatal mice but not in adult mice. Sequence analysis of cII mutant DNA revealed that 4-ABP induced a unique spectrum of mutations in neonatal mice, characterized by a high frequency of G:C-->T:A transversion, while the mutation spectrum in 4-ABP-treated adults was similar to that of control mice. Our results indicate that DNA adduct formation by 4-ABP depends as much on sex as it does on age, whereas the conversion of DNA adducts into mutations differed with animal age. These observations suggest that neonates are more sensitive than adults to genotoxic carcinogens because the relatively high levels of cell division in the developing animal facilitate the conversion of DNA damage into mutation. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html     

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.     

DNA adduct formation in the bone marrow of B6C3F1 mice treated with benzene

We used P₁-enhanced ³²P-postlabeling to investigate DNA adductformation in the bone marrow of B6C3F1 mice treated intraperitoneallywith benzene (BZ). No adducts were detected in the bone marrowof controls or mice treated with various doses of BZ once aday. After twice-daily treatment with BZ, 440 mg/kg, for 1 to7 days, one major and two minor DNA adducts were detected. Therelative adduct levels ranged from 0.06–1.46x10^–7.In vitro treatment of bone marrow from B6C3F1 mice with variousdoses of hydroquinone (HQ) for 24 h also produced three DNAadducts. These adducts were the same as those formed after invivo treatment of bone marrow with BZ. Co-chromatography experimentsindicated that the principal DNA adduct detected in the bonemarrow of B6C3F1 mice was the same as that detected in HL-60cells treated with HQ. This finding suggests that HQ may bethe principal metabolite of BZ leading to DNA adduct formationin vivo. DNA adduct 2 corresponds to the DNA adduct formed inHL-60 cells treated with 1,2,4-benzenetriol. DNA adduct 3 remainsunidentified. After a 7-day treatment with BZ, 440 mg/kg twicea day, the number of cells per femur decreased from 1.6x10⁷to 0.85xlO⁷, indicating myelotoxicity. In contrast, administrationof BZ once a day produced only a small decrease in bone marrowcellularity. These studies demonstrate that metabolic activationof BZ leads to the formation of DNA adducts in the bone marrow.Further investigation is required to determine the role of DNAadducts and other forms of DNA damage in the myelotoxic effectsof exposure to BZ.     

Benzo(a)pyrene-DNA adduct formation and removal in mouse epidermis in vivo and in vitro: relationship of DNA binding to initiation of skin carcinogenesis

An antiserum specific for the major benzo(a)pyrene (BP) adduct formed with deoxyguanosine in vivo has been used by enzyme-linked immunosorbent assay to monitor the formation and removal of DNA-bound products in BALB/c mouse epidermis exposed topically to initiating doses of BP and in BALB/c mouse keratinocytes exposed in vitro to BP or its activated derivatives. In mouse epidermal DNA, formation of antibody-recognizable products increased proportionally between doses of 50 and 250 nmol of BP, giving 2.3 to 6.0 fmol/micrograms of DNA, respectively, and reached a plateau of 10 to 11 fmol/micrograms of DNA at doses between 1000 and 1500 nmol. Antibody-recognizable adducts comprised roughly one-half of the total BP-DNA binding, since a 250-nmol dose of [3H]BP yielded 6 fmol/micrograms of DNA by enzyme-linked immunosorbent assay and 12.9 fmol/micrograms of DNA by radiolabeling. Removal of trans-(7R)-N2-(10-[7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]-yl)-deoxyguanosine adducts was monitored in epidermal DNA of mice exposed to 500 nmol of BP and, although no correction was approximated for DNA turnover in the skin, about one-half of the adducts formed by 24 hr were removed 3 days later, and only 10% remained at the end of a week. BP-DNA binding and removal were also studied in cultured mouse keratinocytes, where proliferating basal cells and terminally differentiating cells can be selectively studied by modulating the Ca2+ concentration of the medium. BP dose-response studies showed that, in cells of different maturation states, BP-DNA adduct levels were similar. Adduct formation greater than 10 to 11 fmol/micrograms (the highest obtained in vivo) was associated with extensive cytotoxicity and cell death. The kinetics of adduct removal was followed in culture under conditions in which dilution by DNA synthesis or cell loss could be monitored. Results of these experiments suggested that initial removal of BP-DNA adducts was more rapid in the differentiating population although, in both populations, 50% of the adduct was removed by 24 hr. The formation of foci resistant to Ca2+-induced terminal differentiation has been associated previously with carcinogen treatment in cultured keratinocytes. Exposure to BP or the antidiol-epoxide, at concentrations producing low cytotoxicity, yielded frequencies of differentiation-altered foci proportional to the dose of the compound used and to the number of DNA adducts formed.     

Benzo[a]pyrene-induced DNA damage in mouse fetal tissues

We have studied the occurrence and persistence of DNA damagein the hepatic and pulmonary tissues of fetal, newborn and adultCD1 mice exposed to selected doses of benzo[a]pyrene (BP) byutilizing the alkaline elution technique. Firstly 12-, 15- and18-day pregnant and 1-, 7- and 82 to 85-day-old mice were treatedi.p. with 10 mg/kg BP and the DNA fragmentation evaluated 4h later. This approach indicated that, among the ages considered,15-day-old fetuses were the most sensitive to BP genotoxicity.Therefore we concentrated on this intrauterine stage and evaluatedthe role of the maternal and fetal environment on the inductionand the kinetics of disappearance of DNA damage by BP. BP atthe dose levels of 0, 2 and 10 mg/kg was injected i.p. intopregnant females or directly into single fetuses and the fetallivers and lungs recovered 2, 4, 24 and 48 h later. Accordingto the above protocol other 12-day-pregnant mice were treatedi.p. with 500 mg/kg arochlor and their 15-day-old fetuses directlyinjected with the same doses of BP. The results showed thatthe maximum DNA damage is present at 4 h following BP treatmentand it almost disappeared at 48 h irrespective of the routeof BP administration. However, the decrease was not uniformand while at 48 h the lesion reached the control level in theliver, it remained sightly higher in the lung. The effects weremarkedly magnified in the arochlor-induced groups where theintrafetal injection of BP caused an average 2-fold increaseand an earlier appearance of DNA damage in both liver and lungcompared with uninduced animals. The amplified BP activity inducedby arochlor was particularly evident in the lung where at 48h there was still a significant amount of DNA damage. Sincethe lung is a preferential site of transplacental carcinogeniceffects in CD1 mice, our results favor the conclusion that acorrelation exists between DNA damage and tumor induction inthe fetuses of this mouse strain.     

SHORT COMMUNICATION: Detection of DNA adducts in the white blood cells of B6C3F1 mice treated with benzene

We have employed the P₁-enhanced ³²P-postlabeling procedureto detect the formation DNA of adducts in the white blood cells(WBC) of B6C3F1 mice treated by i.p. injection with benzene.Treatment twice a day with 440 mg/kg benzene for 1–7 daysresulted in the formation of one major (adduct 1) and one minor(adduct 2) DNA adduct in the WBCs of mice. The same DNA adductpattern was also found in the bone marrow (BM) of benzene treatedmice. The relative adduct levels were dependent upon both benzenedose from 100–440 mg/kg and treatment time from 1 to 7days. The relative adduct levels ranged between 0.11 and 1.33adducts in 10⁷ nucleotides for WBCs and 0.16–1.21 adductsin 107 nucleotides for BM. Following treatment with benzene,the levels of DNA adducts formed in WBCs were significantlycorrelated with the levels of DNA adducts formed in BM (r² =0.97, P <0.001). Our results suggest that measurement ofDNA adducts in WBCs may be an indicator of DNA adduct formationin BM following BZ exposure.     

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.