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.     

Persistence of DNA adducts in rat liver and kidney after multiple doses of the carcinogen N-hydroxy-2-acetylaminofluorene

Male and female Sprague-Dawley rats were treated by gastric intubation either 1, 2, 3, or 4 times at biweekly intervals with 10-mg/kg doses of the hepatocarcinogen of N-[ring-3H]-hydroxy-2-acetylaminofluorene. Then either 1 or 14 days following the last treatment, the concentrations of 2-aminofluorene and 2-acetylaminofluorene adducts in liver and kidney DNA were established. 2-Acetylaminofluorene adducts were found in male rat liver DNA only. The C-8-acetylated adduct, N-(deoxyguanosin-8-yl)-2-acetylaminofluorene, was detected only on the day following treatment at a concentration between 1.0 and 2.4 pmol/mg DNA. A second acetylated adduct, 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, was found at both 1 and 14 days after treatment and, as a result, increased in concentration with repeated doses, from 0.2 pmol/mg DNA after one dose to 2.8 pmol/mg DNA after four treatments. The major adduct detected in male rat liver and the only adduct found in female rat liver and in kidney from either sex was N-(deoxyguanosin-8-yl)-2-aminofluorene. This adduct was slowly lost from the DNA and therefore increased in concentration with repetitive treatments as follows: male liver, 4.0 to 9.4 pmol/mg DNA; female liver, 11.4 to 30.6 pmol/mg DNA; male kidney, 1.1 to 1.8 pmol/mg DNA; and female kidney, 1.8 to 17.7 pmol/mg DNA. These data indicate that certain DNA adducts can accumulate in both target and non-target tissues and therefore suggest that persistence of DNA adducts per se is not sufficient for tumor induction.     

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.     

Activation of 4-hydroxytamoxifen and the tamoxifen derivative metabolite E by uterine peroxidase to form DNA adducts: Comparison with DNA adducts formed in the uterus of Sprague-Dawley rats treated with tamoxifen

Daily intraperitoneal treatment of female Sprague-Dawley ratswith either 5, 10 or 20 mg/kg tamoxifen (TAM) for 1 weeks increasedthe level of peroxidase activity in the uterus 2- to 10-foldcompared to the control level. Using uterine extracts preparedfrom control and TAM treated animals, we investigated the activationof 4-hydroxytamoxifen (4-HO-TAM) and (E, Z)-1, 2-dipheny-1-(4-hydroxyphenyl)-but-1-ene(cis/trans-metabolite E) to form DNA adducts. Activation of4-HO-TAM by uterine extracts prepared from either control orTAM-treated rats produced one major (a) and Two minor DNA (band c) adducts. A similar activation of cis/trans-metaboliteE produced two adducts (d and e). There was good correlationbetween levels of uterine peroxidase activity and levels ofDNA adducts formed by 4-HO-TAM and cis/trans-metabolite E. Activationof 4-HO-TAM and cis/trans-metabolite E with horseradish peroxidase(HRP) produced the same adducts as observed by activation withuterine extract Treatment of Sprague-Dawley rats with 5 and10 mg/kg for 7 days produced eleven DNA adducts in the liverwith no adducts detected in the uterus. However, treatment ofrats with 20 mg/kg of TAM for 7 days produced the same adductpattern in the liver and also one major adduct (1) in the uteruswith a relative adduct level of 6.4 ±4.1x10^–9.Tamoxifen-DNA adduct 1 detected both in the liver and in theuterus of treated rats was similar to adducts produced by activationof 4-HO-TAM with either uterine extract or HRP. The resultsof these studies suggest a general model whereby the tamoxifenmetabolite 4-HO-TAM is further activated in the uterus by peroxidaseenzymes to form DNA adducts.     

Analysis of 7-methylbenz[a]anthracene-DNA adducts formed in SENCAR mouse epidermis by 32P-postlabeling

The present study has analysed the DNA adducts formed in SENCAR mouse epidermis following topical application of 7-methylbenz[a]anthracene (7- MBA). Mice were treated with 400 nmol of 7-MBA, which represents an initiating dose of this hydrocarbon for SENCAR mice. DNA adducts were analysed 24 h after topical application of the hydrocarbon by 32P- postlabeling coupled with either HPLC analysis or an improved TLC procedure giving better resolution of DNA adducts through the use of a D6 solvent [isopropanol:4N NH4OH (1:1)] following D5. Twenty-four hours after topical application of 400 nmol 7-MBA, the level of total covalent binding was 0.37 +/- 0.07 pmol/mg DNA as determined by 32P- postlabeling. This level of binding correlated well with the relative tumor initiating activity of this hydrocarbon compared to 7,12- dimethylbenz[a]anthracene (6.4 +/- 0.01 pmol/mg DNA) and dibenz[a,j]anthracene (0.03 +/- 0.01 pmol/mg DNA). Analysis of the 32P- labeled 3',5'-diphosphodeoxyribonucleosides by HPLC and TLC revealed the presence of deoxyguanosine (dGuo) and deoxyadenosine (dAdo) adducts formed from both the anti- and syn-bay-region diol-epoxides of 7-MBA (anti- and syn-7-MBADEs). The major DNA adduct derived from 7-MBA in mouse epidermis was tentatively identified as (+) anti-7-MBADE-trans-N2- dGuo. In addition, a minor dGuo adduct derived from the bay-region syn- diol-epoxide of 7-MBA was detected as well as a minor dAdo adduct from this diol-epoxide. Another minor dAdo adduct was also detectably present which arose from either the anti- or syn-diol epoxide. Furthermore, several unidentified DNA adducts were present in both HPLC and TLC chromatograms of DNA samples from 7-MBA-treated mice. These results are discussed in terms of the role of specific 7-MBA-DNA adducts in tumor initiation by this hydrocarbon.      

Formation of C8-modified deoxyguanosine and C8-modified deoxyadenosine as major DNA adducts from 2-nitropyrene metabolism mediated by rat and mouse liver microsomes and cytosols

2-Nitropyrene, the geometric isomer of the most studied nitropolycyclic aromatic hydrocarbon (nitro-PAH), 1-nitropyrene, is an environmental contaminant detected in ambient air and a potent direct-acting mutagen. Its metabolic activation leading to the formation of DNA adducts was studied. The activated metabolite, N-hydroxy-2-aminopyrene, was prepared and reacted with calf thymus DNA. Upon enzymatic hydrolysis of the DNA, the resulting nucleosides were separated by HPLC, and the adducts were characterized by mass and proton NMR spectral analysis. Both N-(deoxyguanosin-8-yl)-2-aminopyrene and N-(deoxyadenosin-8-yl)-2-aminopyrene, in a 5:2 ratio, were identified. These adducts were then utilized as standards to identify the DNA adducts formed from reaction of [3H]2-nitropyrene with DNA mediated by liver microsomes and cytosols of mouse and rat. In all cases, both adducts were formed. The quantities of the two adducts formed in each system were: mouse liver microsomes (11.3 pmol [3H]2-nitropyrene/mg DNA), rat liver microsomes (23), mouse liver cytosol (11.4) and rat liver cytosol (5.1). Thus, these adducts were formed in highest yield from rat liver microsomes and the lowest from rat liver cytosol. The deoxyguanosine/deoxyadenosine adduct ratio was higher from rat and mouse liver microsomes (7.8:9.2) than from rat and mouse liver cytosols (2.5:3.1). Our results represent the first direct demonstration of a C8-deoxyadenosine adduct being formed as a major product from the reaction of a nitro-PAH metabolite with DNA.     

DNA adduct formation by 7,12-dimethylbenz[a]anthracene and its noncarcinogenic 2-fluoro analogue in female Sprague-Dawley rats

The potent polycyclic aromatic hydrocarbon 7,12 dimethylbenz[a]anthracene (DMBA) bound to the DNA of numerous organs of the female outbred Sprague-Dawley rat after iv administration under a regimen known to produce a high yield of mammary adenocarcinomas. The maximum DNA binding levels observed following iv administration of 5 mg (20 mumol) DMBA range from approximately 12 mumol hydrocarbon/mol deoxyribonucleic for the liver to approximately 5 mumol hydrocarbon/mol deoxyribonucleotide for the mammary gland, the target tissue. Administered under identical conditions, the noncarcinogenic analogue 2-fluoro-7,12-dimethylbenz[a]anthracene (2F-DMBA) bound to the DNA at levels of about 5-10% that of DMBA (i.e., 0.3-1.6 mumol/mol deoxyribonucleotide). Chromatographic analysis of the hydrocarbon-deoxyribonucleoside adducts produced showed that for DMBA at least two major types of identifiable adducts were observed in all tissues examined, the major one being that resulting from the reaction of a DMBA bay-region diol-epoxide. The other adduct type resulted from the binding of an analogous diol-epoxide of a DMBA metabolite, 7-hydroxymethyl-12-methylbenz[a]anthracene. Adducts from 2F-DMBA were observed on high-pressure liquid chromatography but were in quantities insufficient for characterization. The finding of higher levels of chromatographically identical DMBA-DNA adducts in the nontarget (liver) tissue than in the target tissue indicated that adduct formation per se was not a sufficient stimulus for the cancer induction. However, the failure of the structurally similar 2F-DMBA, which produced only very low levels of DNA adducts, to induce mammary cancer implies that certain levels of carcinogen-DNA adducts may be necessary for carcinogenesis.     

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.     

Formation of 7-(4-oxobutyl)guanine in hepatic DNA of rats treated with N-nitrosopyrrolidine.

We have reported previously the formation of two structurally distinct exocyclic guanine adducts (adducts 1 and 6) in liver DNA of F344 rats treated with N-nitrosopyrrolidine (NPYR). In this study, we detected and characterized a previously unidentified guanine adduct in liver DNA of NPYR-treated rats. The structure of this adduct was established as 7-(4-oxobutyl)guanine (adduct 2) by comparison with the synthetic standard and confirmed by NaBH4 reduction to 7-(4-hydroxybutyl)guanine. The level of adduct 2 in liver DNA of F344 rats treated with 450 mg/kg of NPYR by i.p. administration was 643 +/- 9 mumol/mol guanine, approximately one-third of the level of adduct 1. This study is the first to demonstrate the in vivo formation of a formylalkyl-substituted guanine adduct by a nitrosamine.     

Genotoxic potential of tamoxifen and analogues in female Fischer F344/n rats, DBA/2 and C57BL/6 mice and in human MCL-5 cells.

Chronic administration of tamoxifen to female rats causes hepatocellular carcinomas. We have investigated damage to liver DNA caused by the administration of tamoxifen to female Fischer F344/N rats or C57B1/6 or DBA/2 mice using 32P-postlabelling. Following the administration of tamoxifen for 7 days (45 mg/kg/day) and extraction of hepatic DNA, up to 7 radiolabelled adduct spots could be detected after PEI-cellulose chromatography of the 32P-labelled DNA digests. Tamoxifen caused a time-dependent increase in the level of adduct detected up to a value of at least 1 adduct/10(6) nucleotides after 7 days dosing. A dose response relationship was demonstrated over the range of 5-45 mg/kg/day (0.013-0.12 mmol/kg/day). On cessation of dosing there was a loss of adducts from the liver DNA. These adducts were not detected in DNA from vehicle-dosed controls or in DNA from kidney, lung, spleen, uterus or peripheral lymphocytes. Pyrrolidinotamoxifen caused a similar level of adduct formation as tamoxifen. In contrast, no significant adduct formation could be detected in liver DNA from rats given droloxifene or toremifene. Mice given tamoxifen (45 mg/kg/day for 4 days) showed levels of adducts in the liver which were 30-40% of those present in rats. Exposure of rat hepatocytes to tamoxifen in vitro, resulted in induction of unscheduled DNA synthesis, when preparations from rats which had been pretreated with tamoxifen in vivo were used. No such increase could be detected in hepatocytes from control rats, suggesting tamoxifen may induce enzymes responsible for its own activation. Tamoxifen induced a significant increase in micronucleus formation in a dose dependent manner in cultures of MCL-5 cells, a human cell line that expresses 5 different human cytochrome P450 isoenzymes, as well as epoxide hydrolase.     

Formation of benzo(a)pyrene/DNA adducts and their relationship to tumor initiation in mouse epidermis

The tumorigenicity of benzo(a)pyrene [B(a)P] applied topically as a skin tumor initiator in Sencar mice and the formation of epidermal B(a)P/deoxyribonucleoside adducts were compared over a similar range of doses (50 to 1600 nmol). The tumor-initiating activity of B(a)P, its covalent binding to mouse epidermal DNA, and the formation of the major hydrocarbon/deoxyribonucleoside adduct showed approximately parallel dose-response curves. The major hydrocarbon/deoxyribonucleoside adduct formed cochromatographed with marker adducts of (N2-(10S-[7R,8S,9R-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]y) deoxyguanosine while other minor adducts also were observed. The disappearance of DNA-bound products in the epidermis was followed for 21 days after an initiating dose of B(a)P (100 nmol) was applied topically to the mice. The half-lives of the B(a)P/deoxyribonucleoside adducts and the total radioactivity bound to the DNA were 4.5 and 5.5 days, respectively. However, in spite of the loss of measurable DNA-bound material, the tumor yield was unchanged regardless of whether promotion was begun 7 or 21 days after initiation. The results suggest a possible causal relationship between B(a)P/deoxyribonucleoside adduct formation and papilloma formation in mouse skin.     

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.     

Metabolic activation of the carcinogen 6-hydroxymethylbenzo[a]pyrene: formation of an electrophilic sulfuric acid ester and benzylic DNA adducts in rat liver in vivo and in reactions in vitro

Hydroxylation of meso-methyl groups with subsequent formation of reactive electrophilic esters has been proposed as a possible activation pathway in the metabolism, DNA binding and carcinogenicity of some methyl-substituted polycyclic aromatic hydrocarbons. Some data in vitro have been reported in support of this concept. In this study, sulfotransferase activity for 6-hydroxymethylbenzo[a]pyrene (HMBP) in rat and mouse liver cytosols was demonstrated to mediate formation of benzylic adducts from this hydrocarbon with guanosine and with deoxyguanosine and deoxyadenosine in DNA. These benzylic adducts were also obtained from reactions of synthetic 6-sulfooxymethylbenzo[a]pyrene (SMBP) with individual (deoxy)ribonucleosides or DNA. The structure of the major DNA adduct formed from HMBP and SMBP was determined from NMR spectroscopy to be N2-(benzo[a]pyrene-6-methylenyl)-deoxyguanosine. Low levels of a deoxycytidine adduct were also obtained from DNA reacted with SMBP. Covalent modification of DNA by acetyl-CoA- and ATP-dependent activation of HMBP also produced the identical benzylic adducts, but the amounts were smaller than those obtained in the sulfotransferase-mediated reaction. The i.p. administration of HMBP to rats resulted in the formation of a hepatic DNA adduct. After enzymatic hydrolysis to the nucleoside level, this DNA adduct was chromatographically identical with the deoxyguanosine adduct formed in the above in vitro reactions. This adduct accounted for approximately 20-30% of total HMBP residues bound to hepatic DNA and its formation was significantly inhibited by pretreatment of rats with dehydroepiandrosterone, an inhibitor of the sulfotransferase activity for HMBP. The i.p. administration of comparable doses of SMBP to rats led to the formation of much larger amounts of the adducts with the guanine, adenine, and cytosine bases in the liver DNA. The data indicate that the sulfotransferase activity in the rat liver for HMBP plays a major role in the benzylic DNA adduct formation from this hydrocarbon in rat liver in vivo.     

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.     

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.     

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.     

Effect of aspirin and indomethacin on the formation of benzo(a)pyrene-induced pulmonary adenomas and DNA adducts in A/HeJ mice

Previous results in various in vitro systems suggest that prostaglandin endoperoxide synthetase (PES) could serve as either an alternative or an additional enzyme to the cytochrome P-450-dependent monooxygenases for the formation of mutagenic, cell-transforming, and DNA-binding metabolites of carcinogens. To test this hypothesis in vivo, we examined the effect of PES inhibitors on benzo(a)pyrene (BP)-induced pulmonary adenoma and BP metabolite:DNA adduct formation in A/HeJ mice. Animals were treated with a dosage regimen of aspirin which inhibited PES but had no effect on the cytochrome P-450-dependent oxidation of 7,8-dihydrodihydroxybenzo(a)pyrene. Aspirin did not significantly alter the number of pulmonary adenomas per mouse at either dose of BP (6.0 and 3.0 mg per mouse, administered twice, 2 weeks apart). In addition, aspirin treatment did not depress the in vivo formation of BP metabolite:DNA adducts in lung or liver at either dose of BP (6.0 and 0.06 mg/mouse). The lower dose of BP was used in the adduct study to assess the effect of aspirin at a more environmental exposure level of BP. Treatment with indomethacin, another PES inhibitor, also did not reduce the pulmonary BP metabolite:DNA adduct levels. The failure of PES inhibitors to reduce the number of pulmonary adenomas and BP metabolite:DNA adduct levels suggests that cooxidation of BP during prostaglandin biosynthesis may not play a significant role in BP-induced pulmonary adenomas.     

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.     

32P-Postlabeling Analysis of DNA Adducts in Rats during Estrogen-induced Hepatocarcinogenesis and Effect of Tamoxifen on DNA Adduct Level

DNA adduct formation in the liver, pancreas, kidneys and uterus in ethynylestradiol (EE)-induced carcinogenesis and the effect of tamoxifen (TAM) on DNA adduct formation were evaluated in female Wistar JCL rats using the ³²P-postlabeling method. Hyperplastic nodules were noted in the liver of all rats 4 months after the first oral administration of 0.075 mg of EE, and hepatocellular carcinoma was detected in 8.1% of rats treated with EE for 12 months. DNA adducts increased in the liver for 4 months, reaching a level of 7.3 adducts/10⁷ nucleotides and decreasing thereafter. Formation of DNA adducts was also noted in the pancreas and kidney, but the adduct levels were lower than those in the liver. TAM inhibited estrogen receptors (ER) in liver tissues and completely suppressed the development of hyperplastic nodules or hepatocellular carcinoma but did not affect DNA adduct formation in the liver. In this model, therefore, EE is considered to cause mutations of hepatocytes due to DNA adduct formation without mediation by ER and to induce initiated cells to develop into hepatocellular carcinoma in the presence of ER-mediated hormonal activities.     

DNA adduct formation and removal in specific liver cell populations during chronic dietary administration of 2-acetylaminofluorene

The concentration of DNA adducts in specific hepatic cell typeshas been determined in F344 rats fed 0.02% 2-acetyl-aminofluorene(AAF) for 28 days followed by control diet for an additional28 days. In animals killed at 28 days of AAF feeding, the majorDNA adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene, was presentin each cell type in the order: hepatocytes (282? 28 fmol/µgDNA) > whole liver (232 ? 33 fmol/µg DNA) > nonparenchymalcells (128 ? 30 fmol/µg DNA) > bile duct fraction (60?12 fmol/µg DNA). After an additional 28 days on controldiet, the adduct level in each cell fraction was 30–40fmol/µg DNA. Adduct removal was biphasic in whole liver,hepatocytes and nonparenchymal cells, with a fast phase apparentuntil the adduct concentration reached 60 fmol/µg DNA.In whole liver and hepatocytes this level was obtained in approximatelyseven days, and in nonparenchymal cells the fast phase was completein about two days. Adduct removal in the bile duct fractionexhibited only a single slow phase. At the end of the AAF feeding,hepatocytes accounted for 86% of the total liver DNA adducts.After an additional 28 days on control diet, hepatocyte adductsstill contributed a major fraction (67%) of the total persistentadduct population. Thus, hepatocytes, the target cell for AAF-inducedhepatic tumors, dominate the adduct formation and removal profileobserved in whole liver.