DNA damage in isolated hamster and rat pancreas cells by pancreatic carcinogens

N-Nitrosobis(2-oxopropyl)amine (BOP), N-nitrosobis(2-hydroxypropyl)-amine (BHP) and N-nitroso(2-hydroxypropyl-2-oxopropyl)amine (HPOP) are pancreatic carcinogens in the Syrian golden hamster (SGH) but do not cause pancreatic tumors in rats. In this study, the ability of these three compounds to induce DNA damage in isolated pancreas cells from both species was determined by alkaline elution analysis. BOP was highly potent in SGH cells, causing DNA damage at concentrations as low as 0.5 micrograms/ml, and HPOP, although less potent than BOP, also caused considerable damage. Isolated SGH pancreas cells are thus able to metabolize BOP and HPOP to DNA-damaging species. Of the three compounds tested, only HPOP at higher doses (25-100 micrograms/ml) induced DNA damage in isolated rat pancreas cells. BHP did not damage rat or SGH pancreas cell DNA at concentrations up to 100 micrograms/ml, apparently due to lack of uptake of this compound by the cells. The observed insensitivity to DNA damage in rat cells is consistent with the resistance of the rat pancreas to carcinogenesis by these three compounds. The sensitivity of SGH pancreas cells to BOP- and HPOP-induced DNA damage correlates with the high carcinogenicity of these compounds for the SGH pancreas.     

Inhibitory effect of esculin on oxidative DNA damage and carcinogenesis induced by N-nitrosobis(2-oxopropyl)amine in hamster pancreas

The effects of esculin, a natural coumarin compound, on the formation of 8-oxo-2'-deoxyguanosine (8-oxodG) and carcinogenesis induced by a chemical carcinogen, N-nitrosobis(2-oxopropyl)amine (BOP), were examined in the pancreas of female Syrian golden hamsters. Animals were given a diet containing esculin for 7 days, and killed 4~h after BOP treatment, and the contents of 8-oxodG were measured in the nuclear DNA of the pancreas. Esculin suppressed significantly the increase in the 8-oxodG content of hamster pancreas induced by BOP. Furthermore, the effect of esculin on the rapid production model experiment for pancreatic carcinogenesis using BOP was investigated. Esculin was given ad libitum as a 0.05% aqueous solution during either the initiation or promotion phases. The incidence of invasive tumors in animals given esculin during the initiation phase was significantly lower than in the control group, while the incidence in animals given esculin during the promotion phase showed no significant change. These results suggest that the intake of esculin has an inhibitory effect on BOP-induced oxidative DNA damage and carcinogenesis in hamster pancreas.     

Effect of green tea catechins on oxidative DNA damage of hamster pancreas and liver induced by N-Nitrosobis(2-oxopropyl)amine and/or oxidized soybean oil

It has been indicated that high fat diet is a risk factor of the pancreatic cancer by epidemiological studies. We examined whether the oxidized soybean oil (ox-oil) express the synergistic effect on the formation of 8-oxo-2'-deoxyguanosine (8-oxodG) in nuclear DNA of hamster pancreas induced by N-Nitrosobis(2-oxopropyl)amine (BOP) and whether the green tea catechins (GTC) suppressed it. Ox-oil was prepared by air oxidation, and the content of lipid hydroperoxide was 6.22 mg/ml. Hamsters were administered 0.3 ml of ox-oil/day orally for 4 weeks before BOP treatment. GTC was given ad libitum as a 0.1% aqueous solution. Four hours after subcutaneous administration of BOP, hamsters were sacrificed, and the contents of 8-oxodG were measured in nuclear DNA of pancreas and liver. The 8-oxodG content in the pancreas was increased by BOP and/or ox-oil administration. However, it was not suppressed by an intake of GTC. In the liver, though the content of 8-oxodG was increased by ox-oil, it tended to suppress the rise of 8-oxodG by a GTC intake. These results suggested that the long term intake of ox-oil might have the possibility to induce carcinogenesis in hamster pancreas and liver, and an intake of GTC might have the beneficial effect on liver.     

Suppression of 8-oxo-2'-deoxyguanosine formation and carcinogenesis induced by N-nitrosobis (2-oxopropyl)amine in hamsters by esculetin and esculin

Effects of esculetin (6,7-dihydroxycoumarin) and its glycoside, esculin, on 8-oxo-2'-deoxyguanosine (8-oxodG) formation and carcinogenesis induced by a chemical carcinogen, N-nitrosobis(2-oxopropyl)amine (BOP), were examined in the pancreas of female Syrian golden hamsters. Animals were administered esculetin by gastric intubation into the stomach 30 min before BOP administration or ingestion of a diet containing esculin for 7 days before BOP administration, and killed 1 or 4 h after BOP treatment, and the contents of thiobarbituric acid-reacting substrates (TBARS) and 8-oxodG in the pancreas were determined. Both compounds suppressed significantly the BOP-induced increases in 8-oxodG and TBARS contents in hamster pancreas. We further investigated the effect of esculin on pancreatic carcinogenesis by the rapid production model induced by augmentation pressure with a choline-deficient diet, ethionine, methionine and BOP. Esculin was given ad libitum as a 0.05% aqueous solution in either the initiation or promotion phases. The incidence of invasive tumors in animals given esculin during the initiation phase was significantly smaller than in the control group, while esculin given during the promotion phase showed no apparent effects. These results suggest that the intake of esculin has an inhibitory effect on BOP-induced oxidative DNA damage and carcinogenesis in hamster pancreas.     

p50 (NF-κB1) is an effector protein in the cytotoxic response to DNA methylation damage

The functional significance of the signaling pathway induced by O(6)-methylguanine (O(6)-MeG) lesions is poorly understood. Here, we identify the p50 subunit of NF-κB as a central target in the response to O(6)-MeG and demonstrate that p50 is required for S(N)1-methylator-induced cytotoxicity. In response to S(N)1-methylation, p50 facilitates the inhibition of NF-κB-regulated antiapoptotic gene expression. Inhibition of NF-κB activity is noted to be an S phase-specific phenomenon that requires the formation of O(6)-MeG:T mismatches. Chk1 associates with p50 following S(N)1-methylation, and phosphorylation of p50 by Chk1 results in the inhibition of NF-κB DNA binding. Expression of an unphosphorylatable p50 mutant blocks inhibition of NF-κB-regulated antiapoptotic gene expression and attenuates S(N)1-methylator-induced cytotoxicity. While O(6)-MeG:T-induced, p50-dependent signaling is not sufficient to induce cell death, this pathway sensitizes cells to the cytotoxic effects of DNA breaks.     

Similar repair of O6-methylguanine in normal and ataxia-telangiectasia fibroblast strains. Deficient repair capacity of lymphoblastoid cell lines does not reflect a genetic polymorphism

The ability of human fibroblast strains to repair the mutagenic DNA adduct O6-methylguanine (O6-MeG) induced by brief exposure to N-methyl-N'-nitroso-N-nitrosoguanidine (MNNG) was investigated. The repair reaction proceeded rapidly during the first hour after alkylation, followed by a slow, continuous phase of repair, and both processes were saturated by low doses of carcinogen. This was similar to what had previously been found in human lymphoblastoid lines. Three fibroblast strains from healthy donors and six strains from patients with ataxia telangiectasia were all proficient in their capacity to repair O6-MeG and had the same sensitivity to the cytotoxicity of MNNG and methyl methanesulphonate as normal cells. Three of these cell strains were derived from individuals whose lymphoblastoid lines were deficient in their ability to repair O6-MeG. These lymphoblastoid lines were also extremely hypersensitive to killing by methylating carcinogens. Because non-transformed cells from the same donors behaved normally with regard to both parameters, we concluded that the repair deficiency accompanied by carcinogen hypersensitivity of the lymphoblastoid lines does not indicate a genetic deficiency in the donor. These findings imply that lymphoblastoid lines may not always be the appropriate cell type for investigating genetic susceptibility to chemical mutagens.     

Suppression of 8-Oxo-2′-deoxyguanosine Formation and Carcinogenesis Induced by N-Nitrosobis(2-oxopropyl)amine in Hamsters by Esculetin and Esculin

Effects of esculetin (6,7-dihydroxycoumarin) and its glycoside, esculin, on 8-oxo-2′-deoxyguanosine (8-oxodG) formation and carcinogenesis induced by a chemical carcinogen, N-nitrosobis(2-oxopropyl)amine (BOP), were examined in the pancreas of female Syrian golden hamsters. Animals were administered esculetin by gastric intubation into the stomach 30?min before BOP administration or ingestion of a diet containing esculin for 7 days before BOP administration, and killed 1 or 4?h after BOP treatment, and the contents of thiobarbituric acid-reacting substrates (TBARS) and 8-oxodG in the pancreas were determined. Both compounds suppressed significantly the BOP-induced increases in 8-oxodG and TBARS contents in hamster pancreas. We further investigated the effect of esculin on pancreatic carcinogenesis by the rapid production model induced by augmentation pressure with a choline-deficient diet, ethionine, methionine and BOP. Esculin was given ad libitum as a 0.05% aqueous solution in either the initiation or promotion phases. The incidence of invasive tumors in animals given esculin during the initiation phase was significantly smaller than in the control group, while esculin given during the promotion phase showed no apparent effects. These results suggest that the intake of esculin has an inhibitory effect on BOP-induced oxidative DNA damage and carcinogenesis in hamster pancreas.     

Activation of carcinogenic N-nitrosopropylamines to mutagens by lung and pancreas S9 fractions from various animal species and man

The mutagenic potential of 7 carcinogenic N-nitrosopropylamines was examined by the Ames liquid incubation assay, using lung and pancreas 9000 × g supernatant (S9) fractions from rats, hamsters, mice, rabbits, monkeys and humans for metabolic activation. N-Nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP), N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitrosomethyl(2-oxopropyl)amine (MOP) showed positive mutagenicity in strain TA100 in the presence of lung S9 from each of the uninduced animals and humans. Besides the 3 N-nitrosopropylamines, N-nitrosomethyl(2-hydroxypropyl)amine (MHP) was also positive in the presence of lung S9 from polychlorinated biphenyl (PCB)-induced rats, hamsters and mice. On the other hand, in the presence of pancreas S9 from uninduced or PCB-induced animals, only HPOP and BOP showed positive mutagenicity. In contrast, N-nitrosobis(2-hydroxypropyl)amine (BHP), N-nitrosobis (2-acetoxypropyl)amine (BAP) and N-nitroso-2,6-dimethylmorpholine (NDMM) showed negative mutagenicity in the presence of lung and pancreas S9 from either uninduced or PCB-induced animals and humans. HPOP was a direct-acting mutagen, and lung and pancreas S9 from 5 animal species and man did not affect the activity. BOP was mutagenic even in the presence of bovine serum albumin. The mutagenic activation of MHP by lung S9 from PCB-induced rats, hamsters and mice was completely inhibited by preincubation in an atmosphere of carbon monoxide or by addition of cytochrome c or metyrapone to the S9 mixture, whereas 7,8-benzoflavone totally lacked this effect. However, that of MOP was insensitive to these inhibitors. These results of mutagenicity assay indicate that only the methyl derivatives of N-nitrosopropylamines, MHP and MOP are activated by the lung from 5 animal species and man, whereas the pancreas from all the tested animals did not activate the 7 N-nitrosopropylamines to mutagens, and that the phenobarbital-inducible major cytochrome P-450 in the lung of rodents is involved in the mutagenic activation of MHP.     

Repair of DNA O-alkylation damage by various human organs

Protein extracts from human adult liver, fetal liver, intestine, brain, kidney, lung and skin were tested against poly(dT)methylated X poly(dA), poly(dA)methylated X poly(dT) and methylated DNA. The suitability of various substrates was established in assays using E. coli extracts that removed O4-methylthymidine (O4-MedT), O2-MedT, and O6-methylguanine (O6-MeG). The human extracts efficiently removed O6-MeG and N3-methyladenine from methylated substrates. The adult liver exhibited low and fetal tissues negligible removal of O4-MedT. Only the liver showed limited removal of O2-MedT. The poor removal of the miscoding base O4-MedT by human organs could be an important factor in carcinogen induced mutagenesis, carcinogenesis and teratogenesis.     

The persistence of DNA damage in the pancreas of Syrian golden hamsters treated with N-nitrosobis(2-oxopropyl)amine

DNA damage was estimated in the liver, pancreas and salivary gland of Syrian hamsters given N-nitrosobis(2-oxopropyl)amine (BOP) by alkaline sucrose gradient centrifugation. A single BOP dose (10 mg/kg) produced in all 3 tissues extensive DNA damage that was largely repaired in the salivary gland by 4 weeks, while in the liver and pancreas, some DNA damage persisted until 4 weeks. When higher BOP doses (20 and 40 mg/kg) were used, considerable DNA damage was still evident in the pancreas, but not in the liver at 6 weeks. Greater damage persisted in hamsters given 40 mg/kg, compared with those administered 20 mg/kg.     

Mutation of V79 cells by N-dialkylnitrosamines after activation by hamster pancreas duct cells.

Pancreas duct epithelial cells (DEC), isolated from hamsters and cultured for up to 25 days, were able to metabolize N-nitrosobis(2-oxopropyl)amine (BOP) to species that were mutagenic in V79 cells. There was no decline in the nitrosamine-activating ability of DEC over the period of observation (25 d). DEC activated N-nitrosobis(2-hydroxypropyl)amine (BHP), N-nitrosodiethylamine (DEN), N-nitrosodimethylamine (DMN) and N-nitrosomethyl(2-oxopropyl)amine (MOP) and BOP in the same assay, although the mutation frequencies for BHP, DEN and DMN were barely different from that for the controls (4 +/- 1 mutants/10(6) cells). The mutation frequencies for a dose of 0.1 mM were BHP, 2 +/- 1; BOP, 113 +/- 7; DEN, 8 +/- 1; DMN, 5 +/- 2; and MOP, 18 +/- 3 (mutants/10(6) cells; means +/- SE). When hepatocytes were used the mutation frequencies were BHP, 3 +/- 1; BOP, 60 +/- 3; DEN, 8 +/- 2; DMN, 8 +/- 2; and MOP, 121 +/- 10. BOP was toxic to the DEC at doses above 0.1 mM. Experiments in which co-factors were omitted from the medium suggested that an isoform(s) of the cytochrome P-450 IIIA family was involved, directly or indirectly, in BOP activation.     

Repair of O(6)-methylguanine is not affected by thymine base pairing and the presence of MMR proteins

Methylation at the O(6)-position of guanine (O(6)-MeG) by alkylating agents is efficiently removed by O(6)-methylguanine-DNA methyltransferase (MGMT), preventing from cytotoxic, mutagenic, clastogenic and carcinogenic effects of O(6)-MeG-inducing agents. If O(6)-MeG is not removed from DNA prior to replication, thymine will be incorporated instead of cytosine opposite the O(6)-MeG lesion. This mismatch is recognized and processed by mismatch repair (MMR) proteins which are known to be involved in triggering the cytotoxic and genotoxic response of cells upon methylation. In this work we addressed three open questions. (1) Is MGMT able to repair O(6)-MeG mispaired with thymine (O(6)-MeG/T)? (2) Do MMR proteins interfere with the repair of O(6)-MeG/T by MGMT? (3) Does MGMT show a protective effect if it is expressed after replication of DNA containing O(6)-MeG? Using an in vitro assay we show that oligonucleotides containing O(6)-MeG/T mismatches are as efficient as oligonucleotides containing O(6)-MeG/C in competing for MGMT repair activity, indicating that O(6)-MeG mispaired with thymine is still subject to repair by MGMT. The addition of MMR proteins from nuclear extracts, or of recombinant MutSalpha, to the in vitro repair assay did not affect the repair of O(6)-MeG/T lesions by MGMT. This indicates that the presence of MutSalpha still allows access of MGMT to O(6)-MeG/T lesions. To elucidate the protective effect of MGMT in the first and second replication cycle after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment, MGMT transfected CHO cells were synchronized and MGMT was inactivated by pulse-treatment with O(6)-benzylguanine (O(6)-BG). Thereafter, the recovered cells were treated with MNNG and subjected to clonogenic survival assays. Cells which expressed MGMT in the first and second cell cycle were more resistant than cells which expressed MGMT only in the second (post-treatment) cell cycle. Cells which did not express MGMT in both cell cycles were most sensitive. This indicates that repair of O(6)-MeG can occur both in the first and second cell cycle after alkylation protecting cells from the killing effect of the lesion.     

Kinetic analysis of steps in the repair of damaged DNA by human O6-alkylguanine-DNA alkyltransferase

Rates of individual steps in the removal of alkyl groups from O6-methyl (Me) and -benzyl (Bz) guanine in oligonucleotides by human O6-alkylguanine DNA alkyltransferase (AGT) were estimated using rapid reaction kinetic methods. The overall reaction yields hyperbolic plots of rate versus AGT concentration for O6-MeG but linear plots for the O6-BzG reaction, which is approximately 100-fold faster. The binding of AGT and DNA (double-stranded 30-mer/36-mer complex) appears to be diffusion-limited. The rate of dissociation of the complex is approximately 25-fold slower (approximately 1 s(-1)) for DNA containing O6-MeG or O6-BzG than unmodified DNA. The fluorescent dC-analog 6-methylpyrrolo[2,3-d]pyrimidine-2(3H) one deoxyribonucleoside (pyrrolo dC), which pairs with G, was positioned opposite G, O6-MeG, or O6-BzG and used as a probe of the rate of base flipping. A rapid increase of fluorescence (k approximately 200 s(-1)) was observed with O6-MeG and O6-BzG and AGT but not with a Gly mutation at Arg128, which has been implicated in base flipping with crystal structures. Only weak and slower fluorescence changes were observed with G:pyrrolo dC or T:2-aminopurine pairs. These rate estimates were used in a kinetic model in which AGT binds and scans DNA rapidly, flips O6-alkylG residues, transfers the alkyl group in a chemical step that is rate-limiting in the case of O6-MeG but not O6-BzG, and releases the dealkylated DNA. The results explain the overall patterns of rates of alkyl group removal versus AGT concentration and the effects of the mutations, as well as the greater affinity of AGT for DNA with O6-alkylG lesions.     

Associations between smoking, GST genotypes and N7-methylguanine levels in DNA extracted from bronchial lavage cells

N7-Methylguanine (N7-MeG) DNA adducts are markers of human exposure to methylating agents including tobacco-specific nitrosamines (TSNAs). Repair of this adduct is poor, so levels in lung tissue should reflect variation in both intensity of exposure and in metabolism. N7-MeG adducts in lung DNA from bronchial lavage samples were measured to determine whether levels were higher in smokers than non-smokers, and if levels were modified by genetic variation in carcinogen-metabolising enzymes. Adducts were detected in 38 out of 44 DNA samples by 32P post-labelling of the N7-methyldeoxyguanosine-3'-monophosphate (N7-MedGp) isolated from DNA digests by two-stage HPLC. N7-MeG adduct levels were higher in smokers than in never smokers ((9.99 +/-20.3)x10(-7) versus (0.58+/-0.50)x10(-7) N7-MedGp/deoxyguanosine-3'-monophosphate (dGp); P=0.02) and intermediate in ex-smokers ((5.59+/-15.6)x10(-7) N7-MedGp/dGp). Adduct levels tended to be higher in individuals with GSTM1 null, GSTT1 null or GSTP1 ile/ile genotypes. When genotypes were combined, N7-MedGp levels among GSTM1 null/GSTT1 null individuals (n=6) were higher than among those having at least one wild-type allele of these two genes ((26.1+/-38.0)x10(-7) versus (2.73+/-4.07)x10(-7) N7-MedGp/dGp), although the results were not statistically significant (P=0.13). Adduct levels were highest in individuals with three unfavourable genotypes (GSTM1 null/GSTT1 null and GSTP1 ile/ile) compared with others ((74.5+/-13.1)x10(-7) versus (2.64+/-3.89)x10(-7) N7-MedGp/dGp, P=0.02). N7-MeG adduct levels in DNA isolated from lung tissue thus reflect exposure to cigarette smoke, and genetic variation in carcinogen-metabolising enzymes may modify these levels.     

Simultaneous determination of N7-alkylguanines in DNA by isotope-dilution LC-tandem MS coupled with automated solid-phase extraction and its application to a small fish model

In the present study, we report the development of a sensitive and selective assay based on LC (liquid chromatography)-MS/MS (tandem MS) to simultaneously measure N7-MeG (N7-methylguanine) and N7-EtG (N7-ethylguanine) in DNA hydrolysates. With the use of isotope internal standards (15N5-N7-MeG and 15N5-N7-EtG) and on-line SPE (solid-phase extraction), the detection limit of this method was estimated as 0.42 fmol and 0.17 fmol for N7-MeG and N7-EtG respectively. The high sensitivity achieved here makes this method applicable to small experimental animals. This method was applied to measure N7-alkylguanines in liver DNA from mosquito fish (Gambusia affinis) that were exposed to NDMA (N-nitrosodimethylamine) and NDEA (N-nitrosodiethylamine) alone or their combination over a wide range of concentrations (1-100 mg/l). Results showed that the background level of N7-MeG in liver of control fish was 7.89+/-1.38 mmol/mol of guanine, while N7-EtG was detectable in most of the control fish with a range of 0.05-0.19 mmol/mol of guanine. N7-MeG and N7-EtG were significantly induced by NDMA and NDEA respectively, at a concentration as low as 1 mg/l and increased in a dose-dependent manner. Taken together, this LC-MS/MS assay provides the sensitivity and high throughput required to evaluate the extent of alkylated DNA lesions in small animal models of cancer induced by alkylating agents.     

Carcinogen-mediated methotrexate resistance and dihydrofolate reductase amplification in Chinese hamster cells.

We have investigated different parameters characterizing carcinogen-mediated enhancement of methotrexate resistance in Chinese hamster ovary (CHO) cells and in simian virus 40-transformed Chinese hamster embryo (C060) cells. We show that this enhancement reflects dihydrofolate reductase (dhfr) gene amplification. The carcinogens used in this work are alkylating agents and UV irradiation. Both types of carcinogens induce a transient enhancement of methotrexate resistance which increases gradually from the time of treatment to 72 to 96 h later and decreases thereafter. Increasing doses of carcinogens decrease cell survival and increase the enhancement of methotrexate resistance. Enhancement was observed when cells were treated at different stages in the cell cycle, and it was maximal when cells were treated during the early S phase. These studies of carcinogen-mediated dhfr gene amplification coupled with our earlier studies on viral DNA amplification in simian virus 40-transformed cells demonstrate that the same parameters characterize the amplification of both genes. Possible cellular mechanisms responsible for the carcinogen-mediated gene amplification phenomenon are discussed.     

Ctrogenetic effects of mutagens/carcinogens after activation in a microsomal system in vitro I. Induction of chromosome aberrations and sister chromatid exchanges by diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) in CHO cells in the presence of rat-liver microsomes

A rat-liver microsomal system in vitro has been used to activate two indirectly acting carcinogens, DMN and DEN. On activation, both compounds were extremely potent in inducing chromosomal aberrations as well as sister chromatid exchanges in Chinese hamster cells. The implications of these findings and the potential utility of this technique to detect mutagens/carcinogens are discussed.     

Organ-specific DNA damage induced in mice by the organotropic carcinogens 4-nitroquinoline 1-oxide and dimethylnitrosamine.

The extent of DNA fragmentation induced in lung, kidney, and liver of mice injected with the chemical carcinogens 4-nitroquinoline 1-oxide (4NQO), dimethylnitrosamine (DMN) and the noncarcinogenic 4-aminoquinoline 1-oxide (4AQO) was estimated by the alkaline sucrose gradient technique. A floating of minced lung tissue pieces in the alkaline lysing solution on top of the gradients afforded a gentle method of lung DNA extraction. This technique minimized mechanical shearing of lung DNA and permitted comparisons to be made with liver and kidney DNA sedimentation patterns. The extent of DNA damage induced by 4NQO followed the order: lung, kidney, liver, while that induced by DMN followed the order: liver, kidney, lung. The sites of greatest DNA damage appeared to correlate with sites of high levels of DNA repair synthesis and the sites of tumor induction. No DNA damage was induced by the noncarcinogenic 4-aminoquinoline 1-oxide (4AQO).