Hepatic DNA adduct formation in rats treated with benzo[f]quinoline

The formation of hepatic DNA adducts in male Sprague-Dawley rats following i.p. administration of benzo[f]quinoline (BfQ) was examined using a 32P-post-labeling assay. BfQ exhibited a low binding (11-27 amol adducts/microgram DNA) to liver DNA. Two BfQ-nucleoside adducts (one major and one minor) were detected. The BfQ-DNA adducts formed in vivo were chromatographically distinct from the adducts formed by the reaction of calf thymus DNA in vitro with BfQ-5,6-oxide, syn-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydroBfQ, anti-9 alpha,10 beta-dihydroxy-7 alpha,8 alpha-epoxy-7,8,9,10-tetrahydroBfQ, or anti-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydroBfQ-N- oxide. These results suggest that the bay-region diol epoxide of BfQ, unlike the bay-region diol epoxide derivatives of polynuclear aromatic hydrocarbons, is not involved in the covalent binding of BfQ to 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.     

DNA adduct formation in mice treated with ochratoxin A.

Several authors have reported the occurrence of renal and hepatic tumours in mice and rats exposed to ochratoxin A in long-term studies. The compound was not mutagenic, however, in various microbial and mammalian gene mutation assays, either with or without metabolic activation. Contradictory results were obtained for induction of unscheduled DNA synthesis and sister chromatid exchange. We showed previously that ochratoxin A causes DNA damage, manifested as single-strand breaks in mouse spleen cells and in vivo. These findings, which suggest that ochratoxin A is weakly genotoxic to mammalian cells, prompted us to search for DNA adducts using a modified 32P-postlabelling method, the sensitivity of which was improved by treatment with nuclease P1. DNA was isolated from liver, kidney and spleen excised from mice 24, 48 and 72 h after oral treatment with ochratoxin A at 0.6, 1.2 and 2.5 mg/kg body weight. Several adducts were found in the DNA of the three organs, the levels varying greatly. After administration of 2.5 mg/kg body weight, 40 adducts per 10(9) nucleotides were found in kidney DNA and 7 adducts per 10(9) nucleotides in liver after 72 h. The levels of most of the adducts increased from 24 to 72 h, but those of others diminished after 24 or 48 h. Adducts were found in spleen only at 24 and 48 h. These results confirm the genotoxicity of ochratoxin A.     

DNA adduct formation by tamoxifen with rat and human liver microsomal activation systems

Using microsomal preparations from rat and human liver, we investigatedthe activation of the anti-estrogen compound tamoxifen (TMX)to form DNA adducts. Pretreatment of rats with phenobarbitalincreased DNA adduct formation by microsomal activation of TMX3- to 6-fold, depending on the cofactors used. When reducednicotinamide-adenine dinucleotide phosphate (NADPH) was usedas a cofactor in human and rat microsomal activation systems,the relative DNA adduct levels were 2.9 and 5.2 x 10^–8respectively and 1-3 TMX-DNA adducts were detected by ²³P-postlabeling;DNA adduct 1 was the same in both microsomal systems. When cumenehydroperoxide (CuOOH) was used as a cofactor, activation ofTMX produced four major DNA adducts and several minor DNA adductsin both rat and human liver microsomes; the relative adductlevels were 11.1 and 23.1 xlO^–8 respectively. TMX-DNAadducts 1, 4, 5 and 6 were similar in both human and rat microsomalsystems with CuOOH as the cofactor. The TMX-DNA adducts formedwith NADPH as the cofactor were clearly different from thoseformed with CuOOH as the cofactor, which implies that the metabolitesleading to the individual DNA adducts were different. Additionof a P450 inhibitor, either n-octylamine or     

Microsomal and peroxidase activation of 4-hydroxy-tamoxifen to form DNA adducts: comparison with DNA adducts formed in Sprague-Dawley rats treated with tamoxifen

Using rat liver microsomal preparations and peroxidase enzymes,we have investigated the formation of DNA adducts by the antiestrogencompound tamoxifen (TAM) and its metabolite 4-hydroxy-tamoxifen(4-OH-TAM). When reduced nicotinamide-adenine dinucleotide phosphate(NADPH) was used as a cofactor in microsomal activation of either4-OH-TAM or TAM, one DNA adduct and relative DNA adduct levelsof 4.6 and 3.1x10^–8, respectively were detected by 32P-postlabeling.The DNA adduct produced by microsomal activation of 4-OH-TAMand TAM was the same. With cumene hydroperoxide (CuOOH) as thecofactor for the microsomal activation of either 4-OH-TAM orTAM, three to six DNA adducts were produced; the relative adductlevels were 8.0 and 20.6x10^–8, respectively. Comparisonof the DNA adduct patterns produced by 4-OH-TAM and TAM showedthat they were distinct However one of the DNA adducts (a) producedby microsomal activation of 4-OH-TAM using CuOOH was the sameas adduct a produced by microsomal activation of 4-OH-TAM withNADPH. Activation of 4-OH-TAM with horseradish peroxidase resultedin the formation of a single DNA adduct and a relative adductlevel of 20.7x10^–8. Rechromatography analysis of thisDNA adduct showed that it was identical to that produced bymicrosomal activation of 4-OH-TAM with NADPH and one of theadducts produced using CuOOH as the cofactor. Ten DNA adductsand a relative adduct level of 15.3x10^–8 were detectedin the liver of female Sprague-Dawley rats treated daily with20 mg/kg of TAM for 7 days. The DNA adduct pattern in the liverof the treated animals was similar to that produced by microsomalactivation of TAM using CuOOH as the co-factor. The principalDNA adduct (no. 6) formed in the livers of rats treated withTAM was the same as the principal DNA adduct formed followingmicrosomal activation of TAM using CuOOH as a cofactor. TheDNA adduct formed following microsomal activation of eitherTAM or 4-OH-TAM using NADPH was also present as one of the adducts(1^*) formed in vivo following TAM treatment These studies demonstratethat 4-OH-TAM can be activated to form DNA adducts and thatit contributes to the formation of DNA adducts in the liverof rats treated with TAM.     

DNA adduct formation in rat, human and hamster pancreas treated with methylnitrosourea

The methylation of cellular macromolecules with dimethylnitrosamine (DMN) and methylnitrosourea (MNU) was studied in organ cultured rat, hamster and human pancreatic explants. At concentrations of DMN and MNU that caused similar methylation of protein in human explants DMN caused only 2.6% and 0.3% of the methylation of DNA and RNA that was produced by MNU. The DNA of explants treated with MNU was analyzed. The O6-methylguanine (O6-MeG)/7-methylguanine (7-MeG) ratio was greater in the hamster DNA than in DNA isolated from either rat or human. The time course of removal of methyl adducts from DNA was followed for 6 h after treatment with MNU. No decline in O6-MeG occurred during this period in hamster explants, although there was a decline in the content of 7-MeA and 3-MeA, whereas there was removal of O6-MeG in the DNA from human pancreas explants.     

Co-chromatography of a tamoxifen epoxide-deoxyguanylic acid adduct with a major DNA adduct formed in the livers of tamoxifen-treated rats

Tamoxifen is a potent liver carcinogen in rats and has beenshown to form covalent DNA adducts in the livers of severalspecies of rodent. We have shown previously by ³²P-postlabelling(Carcinogenesis, 13, 2197–2203) that >85% of the totaladducts detected and resolved by multi-directional TLC migrateas a single spot. In the present study, this material was furtheranalysed by reverse-phase HPLC and resolved into two approximatelyequal components. Tamoxifen 1,2-epoxide, a postulated metaboliteof tamoxifen, was reacted with DNA and polydeoxyribonucleotidesand the products analysed. ³²P-Postlabelling revealed threemajor adduct spots on TLC which comigrated with the three majoradduct spots seen with DNA from livers of tamoxifen-treatedrats. Moreover, the major epoxide adduct, which contained guanineas the modified base, eluted on HPLC as a single major peakcoincident with one of the major peaks derived from the liverDNA of tamoxifen-treated rats. These results demonstrate that{small tilde}40% of the tamoxifen-DNA adducts formed in vivoare chromatographically indistinguishable with the major productof the reaction of tamoxifen epoxide with guanine residues inDNA and provide important clues to the mechanism of activationof tamoxifen to a genotoxic carcinogen.     

Effect of N,N-didesmethyltamoxifen upon DNA adduct formation by tamoxifen and alpha-hydroxytamoxifen

Tamoxifen undergoes sequential metabolism to N-desmethyltamoxifen and N,N-didesmethyltamoxifen. Whereas N-desmethyltamoxifen is a major metabolite in humans, nonhuman primates, and rats, appreciable concentrations of N,N-didesmethyltamoxifen are formed in humans and nonhuman primates but not in rats. This difference in the extent of N,N-didesmethyltamoxifen formation may be important because it has been proposed that N,N-didesmethyltamoxifen inhibits the cytochrome P450 (CYP)-catalyzed alpha-hydroxylation of tamoxifen and resultant tamoxifen-DNA adduct formation. To test this hypothesis directly, we compared the extent of tamoxifen-DNA adduct formation in rats co-administered 27micromol N,N-didesmethyltamoxifen per kg body weight and either 27micromol tamoxifen per kg body weight or 27micromol alpha-hydroxytamoxifen per kg body weight daily for 7days. Female Sprague-Dawley rats treated with N,N-didesmethyltamoxifen had a 44% decrease (p >0.05) in CYP 3A2 content (the CYP isoform responsible for tamoxifen alpha-hydroxylation), an 18% decrease (p =0.010) in CYP 3A activity, and higher blood levels of tamoxifen and N-desmethyltamoxifen compared to rats treated with solvent. Total tamoxifen-DNA adduct levels were 4.1-fold higher (p <0.001) in rats given alpha-hydroxytamoxifen as compared to tamoxifen. N,N-Didesmethyltamoxifen treatment caused a 1.2-fold increase in total tamoxifen-DNA adduct levels with both tamoxifen and alpha-hydroxytamoxifen, a difference that was not significant. These results indicate that, with this experimental model, N,N-didesmethyltamoxifen does not impair the metabolism of tamoxifen to a reactive electrophile.     

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.     

Possible mechanisms for PhIP-DNA adduct formation in the mammary gland of female Sprague-Dawley rats

2-Amino-l-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP), themost abundant heterocyclic amine in fried beef, is a mammarygland carcinogen in rats. Using the ³²PP-postlabeling method,PhIP-DNA adduct levels were measured in mammary epithelial cellsisolated from female Sprague-Dawely rats given 10 daily dosesof PhIP (75 mg/ kg, p.o.) according to a protocol previouslyshown to induce mammary gland cancer. At 24 h, 48 h, 1 weekand 5 weeks after the last dose of PhIP, PhIP-DNA adduct levels[relative adduct labeling (RAL) x 10⁷, mean ± SD] were10.2 ± 0.7, 7.9 ± 2.7, 2.2 ± 0.6 and 0.9± 0.03 respectively. When isolated rat mammary epithelialcells (from untreated rats) were incubated in vitro with N-hydroxy-PhIP(45 µM, 1 h, 37°C), PhIP-DNA adducts were detectedin cell DNA (RAL =     

A mechanistic hypothesis for DNA adduct formation by tamoxifen following hepatic oxidative metabolism

A mechanism for the generation of electrophilic alkylating agentsfrom tamoxifen by hepatic     

Hprt lymphocyte mutant frequency in relation to DNA adduct formation in rats fed the hepatocarcinogen 2-acetylaminofluorene.

The lymphocyte hypoxanthine-guanine phosphoribosyltransferase (Hprt) assay is frequently used as a biomarker for the exposure of both humans and laboratory animals to potentially carcinogenic agents. To obtain information concerning the sensitivity of the rat Hprt lymphocyte assay toward aromatic amine carcinogens, male F344 rats were fed 0.02% 2-acetylaminofluorene (2-AAF) for 1 month and then returned to control diet for 2 months. At 4, 27, 48, 62, and 90 days after the initiation of 2-AAF-feeding, the frequency of mutants in the Hprt gene was determined. In addition, DNA was isolated from liver nuclei, spleen lymphocytes, bone marrow, and thymus, and DNA adducts were analyzed by 32P-postlabeling. 2-AAF feeding resulted in a significant induction of 6-thioguanine-resistant T-lymphocytes and the mutant frequency continued to increase after the 2-AAF feeding was stopped. The same major DNA adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene, was detected in liver, spleen lymphocytes, bone marrow, and thymus. DNA adduct levels were greatest in the tumor target tissue (liver) but occurred in all T-lymphocyte compartments, being highest in spleen lymphocytes. The DNA adduct levels were highest at the end of the 1-month 2-AAF feeding period and decreased rapidly in all tissues. The data indicate that the Hprt lymphocyte mutagenesis assay detects arylamine carcinogens, but with relatively low sensitivity.     

Hydrocortisone affects the N-acetylation of 2-aminofluorene and DNA-2-aminofluorene adduct formation in Sprague-Dawley rats

The effects of hydrocortisone on the in vivo acetylation of 2-aminofluorene (AF) and AF-DNA adducts in Sprague-Dawley rats were investigated. Pretreatment with hydrocortisone (50 mg/kg) 48 hours prior to the administration of AF (50 mg/kg) resulted in a 61% and 30% increase, respectively, in the urinary and fecal recovery of N-acetyl-2-aminofluorene (AAF) and a 36% increase in the metabolic clearance of AF to AAF. Hydrocortisone did not affect Michael's-Menten parameters for N-acetyltransferase (NAT) activity in blood, liver, lung and bladder. Similarly, the apparent value of Km for AF in the examined tissues was not affected by hydrocortisone. However, the apparent value of Vmax for liver NAT activity was significantly increased after hydrocortisone pretreatment. Following exposure of rats to AF with and without pretreatment with hydrocortisone, DNA-AF adducts were examined in the target tissue of liver and bladder and also in non-target tissue of lung and circulating leukocytes. The DNA-AF adducts in liver, bladder, lung and leukocytes were increased by pretreatment with hydrocortisone.     

DNA adduct formation in Salmonella typhimurium, cultured liver cells and in Fischer 344 rats treated with o-tolyl phosphates and their metabolites

2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is an electrophilicand a neurotoxic metabolite of o-tolyl phosphates. In a previouspaper we reported that 2-phenoxy-4H-1,3,2-benzodioxaphosphorin2-oxide is mutagenic in Salmonella typhimurium TA100 and formsDNA adducts in incubations with nucleotides, nucleosides andisolated DNA. In the present study we compare DNA adduct formationusing ³²P-post-labelling assays in 2-phenoxy-4H-1,3,2-benzodioxaphosphorin2-oxide-treated bacteria (S.typhimurium TA100) and hepatomacells with DNA adducts formed in liver, kidney, lung and heartof tri-o-tolyl phosphate-exposed Fischer 344 male rats. In bothbacteria and hepatoma cells two DNA adducts could be detectedafter treatment with 2-phenoxy-4H-1,3,2-benzodioxaphosphorin2-oxide. The minor adduct co-chromatographed with syntheticN³-(o-hydroxy-benzyl)deoxyuridine 3' monophosphate after postlabelling.The major DNA adduct was a cytidine adduct, most likely N³-(o-hydroxybenzyl)deoxycytidine3' monophosphate. Male Fischer 344 rats were treated orallyfor 10 days with tri-o-tolyl phosphate (50 mg/kg/day) and DNAwas isolated from liver, kidney, lung, heart, brain and testes1,4,7 and 28 days after giving the last dose. Analysis by ³²P-postlabellingrevealed that two adducts were present in the DNA isolated fromliver, kidney, lung and heart on the first day after givingthe last dose; DNA adducts were not detected in the brain andtestes. The adduct pattern after in vivo treatment with tri-o-tolylphosphate was identical with that found in bacteria and hepatomacells treated with 2-phenoxy-4H-1,3,2-benzo-dioxaphosphorin2-oxide, the major adduct being N³-(o-hydroxybenzyl)deoxycytidine3' monophosphate and the minor N³-(o-hydroxybenzyl)deoxyuridine3' monophosphate. Both DNA adducts persisted in the lungs forthe entire observation period, whereas in the kidney only thecytidine adduct could be detected 28 days after the last doseof tri-o-tolyl phosphate. In liver and heart the adducts weredetectable only on the first day after completion of the treatment.The results indicate that in addition to the well establishedneurotoxicity, some o-tolyl phosphates may have a carcinogenicpotential.     

In vivo genotoxicity and DNA adduct levels in the liver of rats treated with safrole

The induction of chromosome aberrations, sister chromatid exchanges (SCEs), and the formation of DNA adducts was studied in hepatocytes of F344 rats exposed in vivo to safrole. Hepatocytes were isolated 24 h after a single dose of safrole or five repeated doses (once a day) by gastric intubation and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after 48 h in culture. Safrole-DNA adducts were detected by a nuclease P1-enhanced 32P-post-labeling assay in isolated hepatocytes from the rats. While a single dose was not sufficient to induce detectable levels of chromosome aberrations at the time of assay, five repeated doses induced these changes with a maximum frequency of 13.4%, compared with the control value of 1.8%. Both a single dose and five repeated doses induced significant SCEs, to a maximum frequency of 0.81 SCEs per chromosome, while the control value was 0.59 SCEs per chromosome. Two major and two minor DNA adducts were detected after treatment with either a single dose or five repeated doses. The maximum amount of total DNA adducts was 89.8 DNA adducts/10(7) nucleotides. These results show that safrole is a genotoxic carcinogen in the rat liver in vivo and suggest that the cytogenetic effects of this compound may result from covalent DNA modification in the rat liver. This in vivo cytogenetic assay should provide a useful means of evaluation of the genotoxicity of hepatocarcinogens.      

DNA adduct formation in target tissues of Sprague-Dawley rats, CD-1 mice and A/J mice following tumorigenic doses of 1-nitropyrene

Recent reports have indicated that 1-nitropyrene is tumorigenic in laboratory animals. Since it is generally accepted that the covalent binding of carcinogens to DNA is causally related to tumorigenesis, we used 32P-postlabeling to examine the DNA adducts present in target tissues. 1-Nitropyrene (99.85-99.98% 1-nitropyrene, 0.15-0.02% 1,3-, 1,6- and 1,8-dinitropyrene by mass spectral analyses) was administered to Sprague-Dawley rats, CD-1 mice and A/J mice according to three tumorigenesis protocols. In DNA obtained from the injection site of Sprague-Dawley rats, two major adducts were observed. Based upon their chromatographic behavior and sensitivities to treatment with nuclease P1 and hydrazine, these adducts were identified as N-(deoxyguanosin-8-yl)-1-aminopyrene (dG-C8-AP) and N-(deoxyguanosin-8-yl)-1-amino-3-, 6- and/or 8-nitropyrene (dG-C8-ANP), which are adducts derived from the nitroreduction of 1-nitropyrene and dinitropyrenes respectively. In mammary gland DNA from Sprague-Dawley rats, two adducts were found. One of these had chromatographic characteristics and hydrazine and nuclease P1 sensitivities similar to dG-C8-AP, while the identity of the other adduct is presently unknown. The only DNA adduct detected in the livers of newborn CD-1 mice and the lungs of A/J mice was dG-C8-ANP. The presence of dG-C8-AP in the injection site and mammary gland of the Sprague-Dawley rats indicates that nitroreduction is involved in the metabolic activation of 1-nitropyrene in these tissues. Since an unidentified adduct was also found in the mammary gland, other pathways are important in this tissue. The presence of only dinitropyrene DNA adducts in the livers of CD-1 mice and lungs of A/J mice indicates that dinitropyrenes are activated very efficiently to electrophilic metabolites, to an extent far better than 1-nitropyrene.     

DNA adduct formation and induction of micronuclei and mutations in B6C3F1/Tk mice treated neonatally with acrylamide or glycidamide

Acrylamide, a food contaminant, is carcinogenic in experimental animals, with both genotoxic and nongenotoxic pathways being proposed. To obtain information regarding mechanisms of acrylamide tumorigenesis, we compared the extent of DNA adduct formation and induction of micronuclei and mutations in mice treated neonatally with acrylamide and its electrophilic metabolite glycidamide. Male and female B6C3F₁/Tk mice were treated intraperitoneally on postnatal days (PNDs) 1, 8 and 15 or PNDs 1–8 with 0.14 or 0.70 mmol acrylamide or glycidamide per kg body weight per day. One day after the final dose, B6C3F₁/Tk^+/+ mice were killed to measure DNA adduct levels and peripheral blood micronuclei. Three weeks after the last treatment, B6C3F₁/Tk^+/? mice were killed to assess the Hprt and Tk mutant frequencies in spleen lymphocytes. The levels of N7‐(2‐carbamoyl‐2‐hydroxyethyl)guanine, the major glycidamide‐DNA adduct, decreased in the order 0.70 mmol glycidamide > 0.70 mmol acrylamide > 0.14 mmol glycidamide ～ 0.14 mmol acrylamide. Only glycidamide increased the frequency of micronucleated reticulocytes and normochromatic erythrocytes. In mice treated on PNDs 1, 8 and 15, the Hprt mutant frequency was increased by 0.70 mmol glycidamide. In mice dosed on PNDs 1–8, 0.70 mmol glycidamide caused extensive mortality; each of the other treatments increased the Tk mutant frequency, whereas acrylamide increased the Hprt mutant frequency. These data suggest that the mutagenic response in neonatal mice treated on PNDs 1, 8 and 15 is due to glycidamide, whereas mutations resulting from dosing on PNDs 1–8 are due to another mechanism. © 2008 Wiley‐Liss, Inc.     

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/10(7) 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 in liver and kidney of male Syrian hamsters treated with estrogen and/or alpha-naphthoflavone

Chronic administration of estrogens to male Syrian hamsters induces kidney tumors. Co-administration of estrogen plus alpha-naphthoflavone (ANF) suppresses this kidney carcinogenesis but induces liver tumors instead. In an attempt to elucidate the mechanism of the switch from estrogen-induced kidney to liver carcinogenesis in response to ANF treatment, patterns of kidney and liver DNA adducts were investigated by 32P-postlabeling analysis and compared to controls. Chronic treatment of hamsters with ANF alone or in combination with estradiol resulted in a flavone-specific DNA adduct pattern in livers of these animals. These spots, adducts 1 and 2, on 32P-postlabeling maps were taken as evidence of covalent ANF-DNA modifications. The kidney-specific estrogen-induced indirect DNA adducts, observed previously in hamsters treated chronically with estrogen, occurred in renal but not hepatic DNA of animals treated with estradiol alone or in combination with ANF. Pretreatment of hamsters with ANF for 3 days decreased by 75-80% the hepatic and renal diethylstilbestrol (DES)-DNA adducts, which are formed after injection of a single large dose of DES. It is concluded from these changes in DNA adduct patterns and levels that estrogen quinone-DNA adduction may play an etiological role in estrogen-induced kidney cancer. The prevention of estrogen-induced kidney tumors by ANF co-treatment may be a consequence of the decrease in renal concentrations of these adducts in response to ANF. Hepatic concentrations of estrogen quinone-DNA adducts also decrease, but ANF-DNA adducts, observed only in liver, may assume an etiological role in the induction of hepatomas.     

Strong intensification of mouse hepatic tamoxifen DNA adduct formation by pretreatment with the sulfotransferase inhibitor and ubiquitous environmental pollutant pentachlorophenol

Although negative in assays for mutagenicity, the clinicallyimportant antiestrogen tamoxifen induces hepakic DNA adductformation in mice, rats and hamsters, as indicated by ³²P-postlabeling,and is a potent hepatocardnogen in rats. Both phenolic and alcoholicmetabolites of tamoxifen have been reported. As these metabolitesare potential candidates for sulfate coqjugation, we examinedwhether the sulfe transferase inhibitor pentachlorophenol, aubiquitous environmental contaminant, modulates hepatic tamoxifenadduct formation in vivo. Female ICR mice were given tamoxifen(45 mg/kg) daily per os for up to 4 days, with and without i.p.pretreatment with pentachloropheno1 (20 mg/kg) 1 h before dosingwith tamoxifen. At days 1,2 and 4, liver DNA wm analyzed 5 hafter tamoxifen administration by a modified monophosphate versionof the ³²P-postlabeling assay. At day 4, patachrophenol pretreatmentled to a large increase (13- to 17-fold) of the levels of fourtamoxifen adduct fractions, while two adducts appeared unaffected,resulting in an