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.     

Comparative tumorigenicity of 6-nitrochrysene and its metabolites in newborn mice

The tumorigenic activities of 6-nitrochrysene and its metaboliteswere evaluated in the newborn mouse model. Groups of mice weretreated with the appropriate compounds in DMSO by i.p. injectionson the 1st, 8th and 15th day of life. Seven hundred nmol/mouseof 6-nitrochrysene induced significant incidences and multiplicitiesof lung tumors in both sexes; only males were susceptible toliver tumor induction. At 100 nmol/mouse, 6-nitrochrysene hadsignificant tumori-genicity in both lung and liver but was lessactive than at the higher dose. Administration of 100 nmol/mouseof 6-nitrochrysene, only on day 1, caused about the same tumoryield as was observed after treatment with 700 nmol/mouse givenover 3 days. Among the metabolites of 6-nitrochrysene whichwere tested at 100 nmol/mouse, 6-nitrosochrysene and 6-aminochrysenewere significantly less active in the lung and in the liverthan 6-nitrochrysene. In the lung and trans-1, 2-dmydro-ld1hydxy-6-nitn chrysene and trans-l, 2-dihydro-l, 2-dihydroxy-6-aminochysenehad activities comparable to those observed in mice treatedwith equimolar doses of 6-nitrochrysene. In the liver, trans-l,2-dihydro-l, 2-dihydroxy-6-nitrochrysene was more active than6-nitrochrysene based on the number of tumors per mouse. Theseobservations support our hypothesis that 6-nitrochrysene ismetabolically activated by ring-oxidation to trans-1, 2-dihydro-1,2-dihydroxy-6-nitrochrysene, followed by nitro-reduction totrans-1, 2-dihydro-1, 2-dihydroxy-6-amino-chrysene and, finally,oxidation to a diol-apoxide.     

Carcinogen-DNA adduct formation in the lungs and livers of preweanling CD-1 male mice following administration of [3H]-6-nitrochrysene, [3H]-6-aminochrysene, and [3H]-1,6-dinitropyrene

6-Nitrochrysene (NC) and 6-aminochrysene (AC) have been shown to be potent lung and liver carcinogens when administered in multiple i.p. doses to preweanling mice. 1,6-Dinitropyrene has been shown to be a strong hepatocarcinogen but a weak lung carcinogen in this same bioassay. We have examined carcinogen-DNA adduct profiles in the target tissues of preweanling male CD-1 mice following administration of single or multiple doses of these compounds. Depending on the tissue and the dosing schedule, the total level of DNA modification in animals dosed with [3H]NC was 2- to 9-fold higher than in animals dosed with [3H]AC. Regardless of the dosing schedule, DNA isolated from the lungs and livers of both [3H]NC- and [3H]AC-treated preweanling male mice contained a single major and chromatographically identical adduct. This major adduct, which accounted for as much as 90% of the total carcinogen-DNA adducts in enzymatic hydrolysates from treated animals, was chromatographically distinct from the major C8-purine-substituted adducts formed from the reaction of N-hydroxy-AC with calf thymus DNA. In contrast to the results obtained with NC and AC, the major carcinogen-DNA adduct formed in the livers of mice treated with [3H]-1,6-dinitropyrene was found to cochromatograph with 1-N-(deoxyguanosin-8-yl)amino-6-nitropyrene, a product derived from N-hydroxy-1-amino-6-nitropyrene. Since NC and its nitro-reduced derivative, AC, yielded an identical carcinogen-DNA adduct in vivo and this adduct was not derived from N-hydroxy-AC, we conclude that the metabolic activation of NC in the neonatal mouse must involve some previously undescribed combination of ring-oxidation and nitro-reduction pathways. This activation pathway could be an important factor in determining the potency of NC and AC as carcinogens in this bioassay system.     

Formation and persistence of DNA adducts in organs of CD-1 mice treated with a tumorigenic dose of fluoranthene

Fluoranthene (FA) is tumorigenic to the lung when injected i.p.into CD-1 mice 1, 8 and 15 days after birth (Wang, J.-S. andBusby, W.F.Jr, Carcinogenesis, 14, 1871–1874, 1993). Levels,tissue distribution and persistence of FA-DNA adducts detectedby HPLC-³²P-postlabeling were investigated during the courseof lung tumorigenesis by FA.Anti-10b-N²-deoxyguanosin-1,2,3-trihydroxy-1,2,3,10b-tetrahydrofluoranthene(anti-FADE adduct) was consistently the major adduct in DNAsamples from lung, heart, liver and kidney of animals examinedat different time points from 2 h to 165 days after the lasttreatment with the tumorigenic dose (3.5 mg/mouse) of FA. Severalunidentified adducts were also detected. Lung, the target organfor FA tumorigenicity, contained higher levels of anti-FADEadduct than other tissues from 1–165 days after treatment.The anti-FADE adduct level decreased in a biphasic manner afterreaching maximum values at 2 h in heart and spleen plus thymusand 3 days in lungs, liver and kidneys. About 10% of the maximumamount of anti-FADE adduct remained in lung, liver and heart165 days after final FA treatment, at which time 44% of animalshad developed lung adenomas. Significant inter-litter variations,but no sex differences in adduct levels were observed. Theseresults indicated a positive correlation between anti-FADE adductlevel and persistence in relation to target organ specificityfor tumor formation.     

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.     

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.     

DNA adduct formation in B6C3F1 mice and Fischer-344 rats exposed to 1, 2, 3-trichloropropane

1, 2, 3-Trichloropropane (TCP) is a multispecies, multisitecarcinogen which has been found to be an environmental contaminantIn this study, we have characterized and measured DNA adductsformed in vivo following exposure to TCP. [¹⁴C]TCP was administeredto male B6C3F1 mice and Fischer-344 rats by gavage at dosesused in the NTP carcinogenesis bioassay. Both target and nontargetorgans were examined for the formation of DNA adducts. Adductswere hydrolyzed from DNA by neutral thermal or mild acid hydrolysis,isolated by HPLC, and detected and quanti-tated by measurementof radioactivity. The HPLC elution profile of radioactivitysuggested that one major DNA adduct was formed. To characterizethis adduct, larger yields were induced in rats by intraperitonealadministration of TCP (300 mg/kg). The DNA adduct was isolatedby HPLC based on coelution with the radiolabeled adduct, andcompared to previously identified adducts. The isolated adductcoeluted with S-[1-(hydroxymethyl)-2-(N⁷-guanyl)-ethyljglutathione,an adduct derived from the structurally related carcinogen 1,2-dibromo-3-chloropropane (DBCP). Analysis by electrospray massspectrometry suggested that the TCP-induced adduct and the DBCP-derivedadduct were identical. The ¹⁴C-labeled DNA adduct was distributedwidely among the organs examined. Adduct levels varied dependingon species, organ, and dose. In rat organs, adduct concentrationsfor the low dose ranged from 0.8 to 6.6 µmol per mol guanineand from 7.1 to 47.6 µmol per mol guanine for the highdose. In the mouse, adduct yields ranged from 0.32 to 28.1 µmolper mol guanine for the low dose and from 12.2 to 208.1 µmolper mol guanine for the high dose. The relationship betweenDNA adduct formation and organ-specific tumorigenesis was unclear.Although relatively high concentrations of DNA adducts weredetected in target organs, several nontarget sites also containedhigh adduct levels. Our data suggest that factors in additionto adduct formation may be important in TCP-induced carcinogenesis.     

Overexpression of beclin1 induced autophagy and apoptosis in lungs of K-ras mice

Beclin1, as a key molecule in controlling autophagy pathway, can activate both cell survival and cell death pathway. As a role of autophagy in cancer progression remains controversial, introduction of beclin1 to the lungs of K-ras^LA1 mice was performed via inhalation. Prolonged autophagy activation was induced by repeated exposure of lentivirus-beclin1, total of 8 times (2 times/week, 4 weeks). By the time of sacrifice, lungs were collected and analyzed for the therapeutic efficacy. Total numbers of tumors on the surface and histopathological tumor progression were reduced in the lungs of K-ras^LA1 mice. Successful delivery of beclin1 induced autophagy and apoptosis in the target organ, which were confirmed by following features; increased autophagic vacuoles in the cytosol, increased number of mitochondria with decreased mitochondrial 12S RNA, and increased protein levels of mitochondria-related apoptosis. Markers for cell proliferation and angiogenesis, PCNA and VEGF, which used for prediction of cancer prognosis, were significantly reduced after introduction of beclin1. Taken together, the results indicate that autophagy regulating gene, beclin1, can be a potential target for lung cancer gene therapy.     

Comparative mutational profiles of the environmental mammary carcinogen, 6-nitrochrysene and its metabolites in a lacI mammary epithelial cell line

The dietary and environmental agent, 6-nitrochrysene (6-NC) is a powerful mammary carcinogen and mutagen in rats. It is known to be metabolized by ring-oxidation, nitro-reduction and a combination of the two pathways. In order to determine the ultimate mutagenic metabolites, we have compared the previously determined mutational profile of 6-NC in rat mammary gland [T. Boyiri, et al. (2004) Carcinogenesis, 25, 637-643] with that of five of its known metabolites in the cII gene of lacI mammary epithelial cells in vitro. In vivo, 6-NC gives rise to three major mutations, AT > GC, AT > TA and GC > TA (in decreasing order) which comprise >70% of the mutations. The metabolite whose mutational profile was most similar to that of 6-NC in vivo was trans-1,2-dihydroxy-1,2-dihydro-N-hydroxy-6-aminochrysene (1,2-DHD-6-NHOH-C) which arises from both ring-oxidation and nitro-reduction. However, metabolites arising from either ring-oxidation or nitro-reduction alone exhibited some similarities to mutational profile of 6-NC. These results, taken in conjunction with previous data showing that the major DNA adducts in mammary tissue of rats treated with 6-NC are products of the reaction of 1,2-DHD-6-NHOH-C with guanine and adenine, make a strong case that 1,2-DHD-6-NHOH-C is the ultimate genotoxic metabolite from 6-NC.     

Aflatoxin B1 carcinogenesis and its relation to DNA adduct formation and adduct persistence in sensitive and resistant salmonid fish

Rainbow trout (Salmo gairdneri)and coho salmon (Oncorhyn-chuskisutch) were exposed to aflatoxin B1(AFB1) either by passiveembryo uptake or by dietary treatment after hatching and feedingonset. Trout exposed as embryos to an aqueous solution of 0.5p.p.m. AFB1 for 15 min showed a 62% tumor incidence 12 monthslater, whereas coho salmon exposed to a similar solution for30 min showed only a 9% incidence. The difference between salmonand trout response was even greater by dietary AFB1 treatment.Trout exposed for 4 weeks to 20 p.p.b. dietary AFB1 had a 62%tumor response 12 months later, whereas salmon exposed to 40p.p.b. dietary AFB1 for 4 weeks failed to develop tumors. A5% tumor incidence was observed in salmon 12 months after 3weeks exposure to 5000 p.p.b. dietary AFB1, a lethal dose fortrout. In addition to a lower tumor incidence when comparedto trout, the neoplastic response of salmon to AFB1 is to producebenign hepatic adenomas in contrast to the malignant hepatocellularcarcinomas seen in trout. AFB1 metabolism, DNA adduct formation,adduct persistence in vivo and in vitro and cytochrome P-450isozyme composition were compared in livers of trout and salmonto understand the role of metabolism and initiation in thisspecies difference. AFB1-DNA binding was 7–56 times greaterin trout than salmon liver at various times after AFB1 injection,20 times greater in embryos or in freshly isolated trout hepatocytepreparations after a 1 h incubation with aflatoxin Bl, and 18times greater in trout liver after a three week dietary (80p.p.b.) exposure. The major AFB1-DNA adduct was 8, 9-dihydro-8-(N⁷-guanyl)-9-hydroxyaflatoxinB1 in both species. Persistence of AFB1-DNA adducts in vivoin liver was high compared to mamalian systems, implying thatactive enzymatic removal of bulky DNA adducts is low in bothspecies and probably not a factor in their differential responseto aflatoxin. Species differences in other phase I and phaseII metabolism pathways and in AFB1 elimination were, overall,much less striking than those previously observed for troutfed inhibitors of aflatoxin carcinogenesis. Rates of bileeliminationof AFB1 detoxication products, and total excretion of aflatoxinsinto water after AFB1 exposure, were not significantly differentbetween trout and salmon. Since detoxication differences werenot observed, the species difference in AFB1-DNA binding appearsto reflect less efficient cytochrome P-450 metabolism of aflatoxinto the reactive 8, 9-epoxide in salmon, compared to trout. Insupport of this hypothesis, trout liver microsomes displayeda K_m (7.5 µM)for AFB1-DNA adduction in vitro that was7-fold lower than salmon (52 µM). Furthermore, immunoquantitationof various P-450 isozymes suggest that salmon liver microsomeshave much lower amounts of an isozyme immunochemically relatedto trout P-450 LM₂ which has previously been shown to be themajor isozyme catalyzing AFB1 8, 9-epoxidation. Other, post-initiationdifferences were not ruled out by these studies and may contributeto the differential response of rainbow trout and coho salmonto AFB1 hepatocarcino-genesis.     

On the analysis of 1-nitronaphthalene, 1-nitropyrene and 6-nitrochrysene in cigarette smoke

A method was developed for the analysis of 1-nitro-naphthalene,1-nitropyrene, and 6-nitrochrysene in main stream cigarettesmoke, using [¹⁴C]1-nitronaphthalene, [¹⁴C]1-nitropyrene, and[¹⁴C]6-nitrochrysene as tracers and internal standards. Cigarettesmoke condensate was collected and the appropriate fractionscontaining the labelled standards were obtained by a seriesof solvent partitions and column chromatography. Recovery ofinternal standards ranged from 60 to 70%. The fractions wereanalyzed by capillary gas chromatography with electron capturedetection and by combined capillary gas chromatography –mass spectrometry. 1-Nitronaphthalene (<10ng/cigarette),1-ni-tropyrene (<10ng/cigarette), and 6-nitrochrysene (<1ng/cigarette)were not detected in the mainstream smoke of the Universityof Kentucky 1R1 cigarette, a US commercial 85 mm non-filtercigarette, or a French commercial 70 mm non-filter cigarette.     

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.     

DNA adduct formation by secondary metabolites of cyclopenta[cd]pyrene in vitro.

In this study, calf thymus DNA was reacted in vitro with cyclopenta[cd]pyrene 3,4-epoxide (CPPE) or its metabolites, 3,4-dihydroCPP-3,4-diol (CPP-3,4-diol) and 4-hydroxy-3,4-dihydroCPP (4-OH-DCPP), activated with sulfotransferase. The adducts formed were analyzed by HPLC with fluorescence detection following enzymatic digestion of DNA to deoxynucleosides. We have shown previously that the major CPPE-reacted DNA adducts are cis-3-(deoxyguanosin-N2-yl)-4-hydroxy-3,4-dihydroCPP. Sulfotransferase activation of trans-CPP-3,4-diol yielded two adducts that were identical to the products resulting from the reaction of CPPE with DNA, while cis-CPP-3,4-diol gave very low covalent binding. Two adducts formed by sulfotransferase activation of 4-OH-DCPP were identical to the products of the reaction of synthetic 4-NaO3S-O-DCPP or sulfotransferase-activated 4-OH-DCPP with deoxyguanosine. These results indicate that guanine is the predominant site of CPP adduct formation in DNA, and that the 4-hydroxy-3-dGuo adducts can arise by reaction of DNA with either CPPE or sulfotransferase-activated trans-CPP-3,4-diol.     

Inositol and inositol hexaphosphate suppress cell proliferation and tumor formation in CD-1 mice

In previous studies, we have shown that inositol hexaphosphate(InsP₆), a constituent of cereal diet, inhibited azoxymethane-inducedexperimental large intestinal cancer (LIC) in Fischer 344 rats.We now report a similar antineoplastic action of InsP₆ in CD-1mice injected with 1,2-dimethylhydrazine (DMH). We had hypothesizedthat InsP₆ may bring about this effect by undergoing dephosphorylationto lower phosphorylated forms; the ready availability of Ins,to react with phosphates, may increase the total amount of thelower phosphorylated Ins and potentiate the action of InsP₆.LIC induced by DMH (15 mg/kg/week ? 13) in mice given a mixtureof 1% InsP₆ + 1% Ins show a significant reduction (P <0.005)in LIC prevalence over InsP₆ treatment. Surprisingly, Ins, anin vitro growth promoting agent also caused a significant (P< 0.001) suppression of LIC. InsP₆ ? Ins also showed a concomitantreduction in the mitotic rate in the non-neoplastic epithelium.Body weight data did not suggest any overt toxic effect of long-termadministration of InsP₆, Ins or InsP₆ ? Ins. Since InsP₆ isantineoplastic in two species of experimental animals, it should,in combination with Ins, be considered in our strategies forprevention of large intestinal cancer.     

Morphological transformation and DNA adduct formation by dibenz[a,h]anthracene and its metabolites in C3H10T1/2CL8 cells

The major routes of metabolic activation of dibenz[a,h]-anthracene(DBA) have been studied in transformable C3H10T1/2CL8 (C3H10T1/2)mouse embryo fibroblasts in culture. The morphological transformingactivities of three potential intermediates formed by metabolismof DBA by C3H10T1/2 cells, trans-3,4-dihydroxy-3,4-dihydro-DBA-(DBA-3,4-diol),trans-dihydroxy-3,4-dihydro-DBA-anti-1,2-oxide (DBA-3,4-diol-1,2-oxide)and DBA-5,6-oxide were determined. DBA-3,4-diol-1,2-oxide wasa strong morphological transforming agent giving a mean of 73%dishes with Type II or III foci and 1.63 Type II and HI fociper dish at 0.5 µg/ml. DBA-3,4-diol produced a mean of42% dishes with Type II or III foci and 0.81 Type II and IIIfoci per dish at 2.5 µg/ml. DBA gave a mean of 24% disheswith Type II or III foci and 0.29 Type II and III foci per dishat 2.5 µg/ml. DBA-5,6-oxide was found to be inactive.DNA adducts of DBA, DBA-3,4-diol, DBA-3,4-diol-1,2-oxide, DBA-1,4/2,3-tetroland DBA-5,6-oxide in C3H10T1/2 cells were analyzed by ³²P-postlabelingmethod. DBA gave 11 adducts, nine of which were observed inthe DNA of cells treated with DBA-3,4-diol and seven from cellstreated with DBA-3,4-diol-1,2-oxide. Two of these adducts thatappear in each of the treatment groups have been identifiedas the product of the interaction of DBA-3,4-diol-1,2-oxidewith 2-deoxyguanosine. Furthermore, there is evidence for DBA-DNAadducts in cells treated with DBA, DBA-3,4-diol and DBA-3,4-diol-1,2-oxidearising from metabolism to (+,-)-trans,trans-3,4,10,11-tetrahydroxy-3,4,10,11-tetrahydro-DBA(DBA-3,4,10,11-bis-diol). These results are based on co-migrationof C3H10T1/2 DNA adducts with skin DNA adducts formed aftertopical treatment of mice with DBA-3,4,10,11-bis-diol. In C3H10T1/2cells, DBA is metabolically activated through DBA-3,4-diol,which is further activated via the DBA-3,4-diol-1,2-oxide andDBA-3,4,10,11-bis-diol pathways. No evidence is provided forthe metabolism of DBA by the Kregion pathway.     

Aflatoxin B1-induced DNA adduct formation and p53 mutations in CYP450- expressing human liver cell lines

Epidemiological evidence has been supporting a relationship between dietary aflatoxin B1 (AFB1) exposure, development of human primary hepatocellular carcinoma (HCC) and mutations in the p53 tumor suppressor gene. However, the correlation between the observed p53 mutations, the AFB1 DNA adducts and their activation pathways has not been elucidated. Development of relevant cellular in vitro models, taking into account species and tissue specificity, could significantly contribute to the knowledge of cytotoxicity and genotoxicity mechanisms of chemical procarcinogens, such as AFB1, in humans. For this purpose a non-tumorigenic SV40-immortalized human liver epithelial cell line (THLE cells) which retained most of the phase II enzymes, but had markedly reduced phase I activities was used for stable expression of the human CYP1A2, CYP2A6, CYP2B6 and CYP3A4 cDNA. The four genetically engineered cell lines (T5-1A2, T5-2A6, T5-2B6 and T5-3A4) produced high levels of the specific CYP450 proteins and showed comparable or higher catalytic activities related to the CYP450 expression when compared to human hepatocytes. The T5-1A2, T5-2A6, T5-2B6 and T5-3A4 cell lines exhibited a very high sensitivity to the cytotoxic effects of AFB1 and were approximately 125-, 2-, 2- and 15-fold, respectively, more sensitive than the control T5-neo cells, transfected with an expressing vector which does not contain CYP450 cDNA. In the CYP450-expressing cells, nanomolar doses of AFB1-induced DNA adduct formation including AFB1-N7-guanine, -pyrimidyl and -diol adducts. In addition, the T5-1A2 cells showed AFM1-DNA adducts. At similar levels of total DNA adducts, both the T5-1A2 and T5-3A4 cells showed, at codon 249 of the p53 gene, AGG to AGT transversions at a relative frequency of 15x10(-6). In contrast, only the T5-3A4 cells showed CCC to ACC transversion at codon 250 at a high frequency, whereas the second most frequent mutations found in the T5-1A2 cells were C to T transitions at the first and second position of the codon 250. No significant AFB1-induced p53 mutations could be detected in the T5-2A6 cells. Therefore, the differential expression of specific CYP450 genes in human hepatocytes can modulate the cytotoxicity, DNA adduct levels and frequency of p53 mutations produced by AFB1.      

Carcinogenicity, DNA adduct formation and K-ras activation by 7H-dibenzo[c, g]carbazole in strain A/J mouse lung

N-Heterocyclic polynuclear aromatic hydrocarbons (NHA) are environmentalpollutants formed during the combustion of organic materials.7H-Dibenzo[c,g]carbazole (DBC) is a potent carcinogen in lung,liver and skin. We undertook these studies to determine whethertissue specificity for DBC lung carcinogenicity inthe strainA/J mouse is mirrored by formation of DBC-DNA adducts in lungtissue and whether these adducts are consistent with mutationpatterns in the K-ras gene. Strain A/J mice were given a singlei.p. injectionof DBC at doses of 0, 5, 10, 20 or 40 mg/kg andlevels of DNA adducts in the lung were monitored by ³²P-postlabelingon days 1,3,5,7,14 and 21. The remaining animals were sacrificed8 months after DBC treatment and lung tumor multiplicity andK-ras mutation patterns in the tumors were determined. The lungtumor response to DBC was dose related, with an average of 4.7± 1.2 tumors/mouse at 5 mg/kg and 48.1 ± 5.5 tumors/mouseat 40 mg/kg. As many as seven DBC-DNA adducts were observedin the lung. DNA binding levels in the lung were highest at40 mg/kg, with maximum binding at 5-7 days. At lower dose levelsthe maximum binding to DNA decreased and shifted to earliertime points. The DBC-DNA adduct in the lung with the highestlevel of binding at all dose levels was DBC-DNA adduct 3. Themajority of DBC-induced mutations in the K-ras gene in the lungwere A     

Factors modulating the formation of DNA adduct by aflatoxin B1 in vitro

Forty-two compounds belonging to various chemical groups havebeen tested for their ability to suppress formation of aflatoxinB₁-DNA adduct mediated by microsome in vitro. While many ofthese compounds have either marginal or no modulating effect,some have been identified as effective inhibitors. The stronginhibition of DNA adduct formation by retinoids (retinol, retinal,retinoic acid and retinyl acetate), riboflavin, riboflavin 5'-phosphate,flavin adenine dinudeotide, Cu²⁺, 7,8-benzoflavone, disulfiram,butylated hydroxyanisole, butylated hydroxytoluene and phenothiazinesuggests that these agents may have potential anticardnogenicactivity against aflatoxin B₁.     

Inhibition of polycyclic aromatic hydrocarbon-DNA adduct formation in epidermis and lungs of SENCAR mice by naturally occurring plant phenols

Naturally occurring plant phenols such as tannic acid, quercetin, myricetin, and anthraflavic acid are known to inhibit the mutagenicity of several bay-region diol-epoxides of polycyclic aromatic hydrocarbons (PAHs). The binding of bay-region diol-epoxides of PAHs to target tissue DNA is thought to be essential for the initiation of cancer by these compounds. In this study we investigated the effect of these plant phenols on PAH-DNA adduct formation in the epidermis and lung of SENCAR mice. In vitro addition of tannic acid, quercetin, myricetin, and anthraflavic acid (25 microM) to an incubation system containing epidermal microsomes prepared from either control or 3-methylcholanthrene-pretreated mice inhibited benzo(a)pyrene binding to calf thymus DNA by 63-64, 38-43, 36-37, and 27-33%, respectively. A single topical application of tannic acid, quercetin, myricetin, and anthraflavic acid at a dose of 400 mumol/kg body weight resulted in the inhibition of [3H]benzo(a)pyrene binding to epidermal DNA (48-73%) and protein (51-63%). The same dose of these plant phenols (400 mumol/kg) caused even greater inhibition of (+/-)-[3H]-7 beta,8 alpha-dihydroxy-7,8-dihydrobenzo(a)pyrene and [3H]-7,12-dimethybenz(a)anthracene binding to epidermal DNA and protein. The formation of (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene-deoxyguanosine adducts was substantially diminished in both epidermis (62-86%) and lungs (38-84%). These results indicate that tannic acid, quercetin, myricetin, and anthraflavic acid are potent inhibitors of carcinogen binding to epidermal and lung DNA and suggest that these plant phenols could prove useful in modifying the risk of tumor induction by PAHs such as benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene in these two tissues.     

Interindividual variation among humans in carcinogen metabolism, DNA adduct formation and DNA repair

Chemical carcinogens are generally activated enzymatkally toelectrophiles that form covalently bound carcinogen-DNA adducts.Detoxifying enzymes are competing with the activating enzymesfor these procarcinogenic chemical substrates. Wide person-to-personvariations in these two types of enzymatic activities are found.Repair rates of DNA damage caused by carcinogens also vary amongindividuals. These interindividual differences in the metabolismof chemical carcinogens and repair rates of carcinogen-inducedDNA damage reflect acquired and inherited host factors thatmay influence an individual's risk for development of cancer.