DNA adduct formation and persistence in liver and extrahepatic tissues of northern pike (Esox lucius) following oral exposure to benzo[a]pyrene, benzo[k]fluoranthene and 7H-dibenzo[c,g]carbazole.

The formation and persistence of DNA adducts in liver, intestinal mucosa, gills and brain of juvenile northern pike (Esox lucius) following oral exposure to benzo[a]pyrene (BaP), benzo[k]fluoranthene (BkF) and 7H-dibenzo[c,g]carbazol (DBC) were analysed by 32P-postlabelling. The dosage was 25 micromol/kg body weight of each substance, administered on 5 occasions with an interval of 12-14 days. Sampling was carried out 9 days after the second treatment, and 9, 16, 33 and 78 days after the fifth treatment. Pikes were also fed with the substances singly for comparison of adduct patterns. A complex pattern of adducts was detected in all examined tissues from fish treated with the mixture. Total adduct levels were highest in intestine (347+/-17.4 nmol adducts/mol nucleotides, mean+/-SE), followed by liver (110+/-9.3), gills (69+/-6) and brain (14+/-4.2). In pike treated with BaP alone, one major adduct was detected in all examined tissues. This BaP-adduct made up approximately 50% of the total amount of adducts in the brain. Corresponding values in liver, intestine and gills were 23, 31 and 34%, respectively. One relatively weak BkF-adduct and at least 10 different DBC-adducts were detected in all analysed tissues. Total adduct level in the intestine declined to 29.4% of the maximum value 78 days after the last exposure, while there was no significant decline in adduct levels in liver, gills or brain. The results suggest that intestine is more susceptible to adduct formation than liver after oral exposure, and that adduct levels in the intestine represent ongoing or relatively recent exposure. DNA adducts in the other investigated tissues were much more persistent and may therefore accumulate during long-term exposure.     

32P-analysis of DNA adducts in somatic and reproductive tissues of rats treated with the anticancer antibiotic, mitomycin C

Mitomycin C (MMC) is a clinically used drug with mutagenic and antitumor activities, presumably elicited through its covalent binding to DNA, however, little is known about MMC binding to DNA in vivo. A 32P-postlabeling method that does not require radiolabeled test compounds was employed here to study the formation of DNA adducts in somatic and reproductive tissues of rats 24 h after an i.p. dose of 9 mg/kg MMC. Among 14 tissues studied in female rats, MMC-DNA adduct levels were within a 2-fold range in 11 tissues, i.e. bladder, colon, esophagus, heart, kidney, liver, lung, ovary, pancreas, small intestine and stomach (minimum levels of 9.6-21.9 adducts per 10(7) N). Three other tissues, i.e. brain, spleen and thymus, exhibited lower adduct levels (0.2 5.4 and 1.4 adducts, respectively, per 10(7) N). Liver DNA adduct levels were 32% lower in male than in female rats. Testicular DNA contained 2.5 adducts per 10(7) N, i.e. 5.3 times less than ovarian DNA. 32P-labeled adduct patterns were qualitatively similar among the different tissues and consisted of 10 adducts, one of which comprised 71 (+/- 5)% of the total. All these adducts were chromatographically identical to adducts formed by the reaction of chemically reduced MMC with DNA in vitro, demonstrating that metabolic activation of MMC occurred via reduction. Using homopolydeoxyribonucleotides modified with MMC, in vivo adducts were shown to be mostly (greater than 90%) guanine derivatives and small amounts of adenine, cytosine and thymine products. Most of the adducts appeared to be monofunctional derivatives of DNA nucleotides. Dose-dependent MMC-DNA adduct formation was determined in rat liver over an 82-fold range of MMC administered (0.11-9.0 mg/kg). The lowest dose level studied was 4.5 times lower than the recommended single dose for human cancer chemotherapy (20 mg/m2). Thus, these results predict that 32P-postlabeling methodology is suitable to monitor and quantify DNA adducts in tissue biopsies of patients receiving MMC chemotherapy.     

In vitro DNA and dGMP adducts formation caused by ochratoxin A

Ochratoxin A (OTA), a nephrotoxic and nephrocarcinogenic mycotoxin, leads to the formation of DNA adducts after administration to animals. This could be due to an epigenetic effect. In vitro assays can exclude an indirect effect, where the xenobiotic can generate, in vivo, endogenous reactive compounds which give adducts on DNA. Microsomes prepared from mice or rabbit kidney and liver, used as metabolic activators, were incubated in the presence of commercial salmon testes DNA and OTA, with NADPH or arachidonic acid used as cofactors. Upto 126 DNA adducts for 10(9) nucleotides were detected using the 32P postlabeling method after incubation with the mouse kidney system. Similar results were obtained with rabbit kidney microsomes. Using liver microsomes, the number of DNA adducts detected was much lower. When NADPH was used as a cosubstrate (to explore the cytochrome P450 metabolic pathways), with mice kidney microsomes, the adduct level was only 44% of the one obtained with arachidonic acid. These results lend support to the hypothesis of the preferential activation of OTA by the peroxidase activity of prostaglandin synthases and/or lipoxygenases to direct genotoxic metabolites, and are in agreement with the previously obtained results after in vivo treatment of mice. In order to identify the nucleotides of DNA modified by the OTA metabolites, dAMP, dGMP, dTMP and dCMP were used as substrates under the same conditions as with DNA. The adducts were found only on dGMP. The total adduct level was of 344 adducts per 10(9) nucleotides with the appearance of three major adducts in the presence of arachidonic acid. With NADPH, 271 adducts were obtained per 10(9) nucleotides, with again three major adducts, but only two of them were similar to two adducts obtained in the presence of arachidonic acid. Desferal (desferrioxamine B methanesulphonate), at a 50 microM concentration, did not reduce the adduct level. Adducts were also obtained when polydG, polydC and dG-p-dG were used as alternative substrates, whereas no adducts were obtained with polydA, polydT and polydC. The major adduct obtained after incubation of DNA with OTA, comigrated with the major adduct obtained with dGMP, in two chromatographic solvents. These results show that OTA is metabolized to genotoxic metabolite(s) which interact with the guanine residues of DNA.     

In vitro genotoxicity of PAH mixtures and organic extract from urban air particles part I: acellular assay

Acellular assay of calf thymus DNA ± rat liver microsomal S9 fraction coupled with ³²P-postlabelling was used to study the genotoxic potential of organic compounds bound onto PM10 particles collected in three European cities—Prague (CZ), Kosice (SK) and Sofia (BG) during summer and winter periods. B[a]P alone induced DNA adduct levels ranging from 4.8 to 768 adducts/10⁸ nucleotides in the concentration dependent manner. However, a mixture of 8 c-PAHs with equimolar doses of B[a]P induced 3.7–757 adducts/10⁸ nucleotides, thus suggesting the inhibition of DNA adduct forming activity by interaction among various PAHs. Comparison of DNA adduct levels induced by various EOMs indicates higher variability among seasons than among localities. DNA adduct levels for Prague collection site varied from 19 to 166 adducts/10⁸ nucleotides, for Kosice from 22 to 85 and for Sofia from 6 to 144 adducts/10⁸ nucleotides. Bioactivation with S9 microsomal fraction caused 2- to 7-fold increase in DNA adduct levels compared to −S9 samples, suggesting a crucial role of indirectly acting genotoxic EOM components, such as PAHs. We have demonstrated for the first time a significant positive correlation between B[a]P content in EOMs and total DNA adduct levels detected in the EOM treated samples (R = 0.83; p = 0.04). These results suggest that B[a]P content in EOM is an important factor for the total genotoxic potential of EOM and/or B[a]P is a good indicator of the presence of other genotoxic compounds causing DNA adducts. Even stronger correlation between the content of genotoxic compounds in EOMs and total DNA adduct levels detected (R = 0.94; p = 0.005) was found when eight c-PAHs were taken into the consideration. Our findings support a hypothesis that a relatively limited number of EOM components is responsible for a major part of its genotoxicity detectable as DNA adducts by ³²P-postlabelling.     

Dose responses for DNA adduct formation in tissues of rats and mice exposed by inhalation to low concentrations of 1,3-[2,3-[(14)C]-butadiene

Male Sprague-Dawley rats and B6C3F1 mice were exposed to either a single 6h or a multiple (5) daily (6h) nose-only dose of 1,3-[2,3-(14)C]-butadiene at exposure concentrations of nominally 1, 5 or 20 ppm. The aim was to compare the results with those from a similar previous study at 200 ppm. DNA isolated from liver, lung and testis of exposed rats and mice was analysed for the presence of butadiene related adducts, especially the N7-guanine adducts. Total radioactivity present in the DNA from liver, lung and testis was quantified and indicated more covalent binding of radioactivity for mouse tissue DNA than rat tissue DNA. Following release of the depurinating DNA adducts by neutral thermal hydrolysis, the liberated depurinated DNA adducts were measured by reverse phase HPLC coupled with liquid scintillation counting. The guanine adduct G4, assigned as N7-(2,3,4-trihydroxybutyl)- guanine, was the major adduct measured in liver, lung and testis DNA in both rats and mice. Higher levels of G4 were detected in all mouse tissues compared with rat tissue. The dose-response relationship for the formation of adduct G4 was approximately linear for all tissues studied for both rats and mice exposed in the 1-20 ppm range. The formation of G4 in liver tissue was about three times more effective for mouse than rat in this exposure range. Average levels of adduct G4 measured in liver DNA of rats and mice exposed to 5 x 6 h 1, 5 and 20 ppm 1,3-[2,3-(14)C]-butadiene were, respectively, for rats: 0.79 +/- 0.30, 2.90 +/- 1.19, 16.35 +/- 4.8 adducts/10(8) nucleotides and for mice: 2.23 +/- 0.71, 12.24 +/- 2.15, 48.63 +/- 12.61 adducts/10(8) nucleotides. For lung DNA the corresponding values were for rats: 1.02 +/- 0.44, 3.12 +/- 1.06, 17.02 +/- 4.07 adducts/10(8) nucleotides, and for mice: 3.28 +/- 0.32, 14.04 +/- 1.55, 42.47 +/- 13.12 adducts/10(8) nucleotides. Limited comparative data showed that the levels of adduct G4 formed in liver and lung DNA of mice exposed to a single exposure to butadiene in the present 20 ppm study and earlier 200 ppm study were approximately directly proportional across dose, but this was not observed in the case of rats. From the available evidence it is most likely that adduct G4 was formed from a specific isomer of the diol-epoxide metabolite, 3,4-epoxy-1,2-butanediol rather than the diepoxide, 1,2,3,4-diepoxybutane. Another adduct G3, possibly a diastereomer of N7-(2,3,4-trihydroxybutyl)-guanine or most likely the regioisomer N7-(1-hydroxymethyl-2,3-dihydroxypropyl)-guanine, was also detected in DNA of mouse tissues but was essentially absent in DNA from rat tissue. Qualitatively similar profiles of adducts were observed following exposures to butadiene in the present 20 ppm study and the previous 200 ppm study. Overall the DNA adduct levels measured in tissues of both rats and mice were very low. The differences in the profiles and quantity of adducts seen between mice and rats were considered insufficient to explain the large difference in carcinogenic potency of butadiene to mice compared with rats.     

Further evidence that eugenol does not bind to DNA in vivo

The naturally-occurring alkenylbenzene, eugenol, was examined for its ability to form DNA adducts in the livers of mice that had been treated with up to 10 mg of the compound. No adducts were detected by 32P-postlabelling with a limit of detection of 1 adduct in 10(9) nucleotides. Under these conditions adducts were readily detected in liver DNA from the structurally-related hepatocarcinogen safrole.     

Quantification of O6-methyl and O6-ethyl deoxyguanosine adducts in C57BL/6N/Tk+/- mice using LC/MS/MS

The carcinogenicity of many alkylating agents is derived from their ability to form persistent DNA adducts that induce mutations. This paper presents and validates methodology, based on LC with tandem mass spectrometry, for the separate or concurrent quantification by isotope dilution of O(6)-methyl-2'-deoxyguanosine (O(6)Me-dG) and O(6)-ethyl-2'-deoxyguanosine (O(6)Et-dG) DNA adducts. The limits of quantification were estimated to be < or =0.2 adducts/10(8) nucleotides for either adduct. This sensitivity permitted evaluation of adduct levels in livers from separate groups of untreated adult C57BL/6N/Tk(+/-) and C57BL/6N X Sv129 mice (undetectable to 5.5+/-6.7 O(6)Me-dG/10(8) nucleotides; undetectable to 0.04 O(6)Et-dG/10(8) nucleotides). Treatment of adult C57BL/6N/Tk(+/-) mice with equimolar doses (342micromol/kg body weight) of N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea produced adduct levels in liver of 1700+/-80 O(6)Me-dG/10(8) nucleotides and 260+/-60 O(6)Et-dG/10(8) nucleotides, respectively, when assessed 4h after dosing. These methods should be useful for evaluations of DNA adducts in relation to cellular processes that modify carcinogenic and toxicological responses in experimental animals and humans.     

Persistence of 7-(2-hydroxyethyl) guanine-DNA adducts in rats exposed to ethene by inhalation

Quantification of 7 2 hydroxyethyl guanine 7 HEG adduct in DNA of livers and lymphocytes of male Sprague-Dawley rats exposed to 300 ppm ethene by inhalation 12 h a day for three consecutive days was performed to evaluate the potential of ethene to produce DNA adducts in these tissues. The persistence of 7 HEG in livers and lymphocytes was studied in rats sacrificed 0, 1, 5, and 20 days after the last exposure. DNA samples from control and treated animals were analysed for 7 HEG and 7 methylguanine 7 MG adducts by thin layer chromatography TLC combined with a high pressure liquid chromatography HPLC assay. After a 3 day exposure to ethene, 7 HEG accumulated to a similar extent in liver and lymphocytes, with the mean adduct level of 7.0 0.7 adducts per 107 nucleotides in liver and 7.4 0.7 adducts per 107 nucleotides in lymphocytes of rats sacrificed immediately after cessation of exposure. The approximate half life of 7 HEG was 5 days in liver and 3 days in lymphocytes, which is consistent with the loss of adduct primarily by spontaneous depurination. In addition, the background levels of 7 HEG and 7 MG were determined in the liver and lymphocytes from the control rats.     

Relative sensitivity of 32P-postlabelling of DNA and the autoradiographic UDS assay in the liver of rats exposed to 2-acetylaminofluorene (2AAF)

Groups of male Alderley Park rats were dosed concomitantly with 2-acetylaminofluorene (2AAF) by gavage at doses between 0.01 mg/kg and 40 mg/kg, and livers sampled 2-72 h later. The liver of one group of animals was perfused to yield hepatocytes which were assayed in vitro for unscheduled DNA synthesis (UDS) via incorporation of tritiated thymidine and autoradiography. DNA was extracted from the livers of the other group and DNA adduct levels determined using the 32P-postlabelling technique. The major C-8 2-aminofluorene/guanosine adduct and 3 minor adducts were quantitated, enabling the relative sensitivity of the 2 techniques to be compared. A dose- and time-related UDS response was observed, which, at the most sensitive time-point (12 h) enabled DNA repair to be discerned at a dose level of 0.1-1 mg/kg of 2AAF, a response classified as formally positive at 5 mg/kg 2AAF. Only the C-8 adduct, as determined by 32P-postlabelling, was discernible at 0.01 mg/kg of 2AAF, although other adducts were visible on autoradiograms at higher dose levels. It is concluded that as part of a well-defined dose response, UDS can be discerned with confidence for doses of 2AAF between approximately 0.1 and 5 mg/kg, and DNA adducts for doses of 2AAF between approximately 0.01 and 1 mg/kg. Discernible UDS for 2AAF in the rat liver is apparent at approximately 13 DNA (total) adducts/10(8) nucleotides, or approximately 8 DNA (C-8) adducts/10(8) nucleotides. The presumed C-8 2-acetylaminofluorene/guanosine adduct, prepared by reaction of 2-acetoxy-2-acetylaminofluorene (2AAAF) with DNA, was a significant but unreliable marker of 2AAF/DNA adducts in the rat liver in vivo. DNA repair did not appear to remove DNA adducts selectively, and adducts remained in DNA when discernible DNA repair had ceased.     

DNA damage and repair in gastric cancer--a correlation with the hOGG1 and RAD51 genes polymorphisms

Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed 1 day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by ³²P-postlabeling and mutant frequency (MF) was determined using the λ Select-cII Mutation Detection System. Three major adducts (7-[deoxyadenosin-N⁶-yl]-aristolactam I, 7-[deoxyadenosin-N⁶-yl]-aristolactam II and 7-[deoxyguanosin-N²-yl]-aristolactam I) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10⁸ nucleotides in liver and 95–4598 adducts/10⁸ nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 × 10⁻⁶ in liver compared with the MFs of 78–1319 × 10⁻⁶ that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T → T:A transversion was the predominant mutation in AA-treated rats; whereas G:C → A:T transition was the main type of mutation in control rats. These results indicate that the AA treatment that eventually results in kidney tumors in rats also results in significant increases in DNA adduct formation and cII MF in kidney. Although the same treatment does not produce tumors in rat liver, it does induce DNA adducts and mutations in this tissue, albeit at lower levels than in kidney.     

Detection of 1,N2-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal after gavage of trans-4-hydroxy-2-nonenal or induction of lipid peroxidation with carbon tetrachloride in F344 rats

The 1,N2-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal (HNE-dGp-adducts) were quantitated in tissues of rats treated with trans-4-hydroxy-2-nonenal (HNE) or carbon tetrachloride, respectively, using a 32P-postlabeling method. The method development was based on chemically synthesized HNE-1,N2-propanodeoxyguanosine adduct standard, which was characterized by NMR and mass spectra. The adducts were enriched by Nuclease P1. They were subsequently reacted with gamma-32P-ATP to give the respective 3'-5'-bisphosphates, which were two-directionally separated on PEI-cellulose-TLC and quantitated by autoradiography. The labeling efficiency for the adduct standard was 27%, and the recovery of spiked amounts of adduct standard in the enzymatical procedure was about 80%. Internal standard was used to eliminate methodological variations. The determination of the limit of quantitation in DNA from rat tissues by spiking of HNE-dGp-adduct standard revealed a sensitivity of about 20 HNE-dGp-adducts/10(9) normal nucleotides. Background levels of HNE-dGp-adducts in tissues of rats including liver, kidney, lung, colon and forestomach were found in the range of 18-158 adducts/10(9) nucleotides with relatively high adduct levels in the liver and low adduct levels in kidney, lung and colon. These background levels were statistically significantly increased by the factor of 2 in liver, lung, colon and forestomach after induction of lipid peroxidation by carbon tetrachloride. The finding that background HNE-dGp-adduct levels may be in context with different metabolic activities of the tissues and the increase of HNE-dGp-adduct levels after application of carbon tetrachloride indicate that HNE-dGp-adducts are an endogenous lesion and that they are probably formed from radical initiated lipid peroxidation.     

Comparison of DNA binding between the carcinogen 2,6-dinitrotoluene and its noncarcinogenic analog 2,6-diaminotoluene

We used ³²P-postlabelling to compare DNA binding between the potent hepatocarcinogen 2,6-dinitrotoluene and its noncarcinogenic analog 2,6-diaminotoluene. The two compounds were compared to determine whether differences in DNA binding could partly explain the differences in their carcinogenicity. Fischer-344 rats were administered 1.2 mmol/kg of a compound by single i.p. injection and examined for DNA adduct formation in the liver. Four adducts were detected following administration of 2,6-dinitrotoluene, with a total adduct yield of 13.5 adducted nucleotides per 10⁷ nucleotides. Qualitatively identical adducts were also detected after treatment with the derivative 2-amino-6-nitrotoluene. Adduct yields from 2,6-dinitrotoluene were 30 times greater than from 2-amino-6-nitrotoluene. No adducts were observed following treatment with 2,6-diaminotoluene. 2,6-Dinitrotoluene and 2,6-diaminotoluene were also compared for qualitative differences in hepatotoxicity. 2,6-Dinitrotoluene produced extensive hemorrhagic necrosis in the liver, whereas no evidence of hepatocellular necrosis was detected following administration of the latter. The differences between the two compounds in both DNA binding and cytotoxicity were consistent with the differences in their carcinogenicity.     

Dose-dependent reduction of 3,2'-dimethyl-4-aminobiphenyl-derived DNA adducts in colon and liver of rats administered celecoxib

Colon cancer is second leading cause of cancer-related deaths in Western countries. Diet and smoking, which contain aromatic and heterocyclic amines, are major risk factors for colon cancer. Colorectal cancers have a natural history of long latency and therefore provide ample opportunities for effective chemoprevention. 3,2'-Dimethyl-4-aminobiphenyl (DMABP) is an experimental aromatic amine that causes cancer in rat colon and serves as an experimental model for arylamine and heterocyclic amine mutagens derived from diet and smoking. In this study, we investigated the effects of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor on DMABP-induced DNA adduct formation in rat liver and colon. Male F-344 rats (5-week old) were provided free access to modified AIN-76A rat chow containing 0 (control), 500, 1000, or 1500 ppm celecoxib. Two weeks later, the rats received a subcutaneous injection of 100mg/kg DMABP in peanut oil. Two days after DMABP treatment, the rats were killed and DMABP-derived adducts were analyzed in colon and liver DNA by butanol extraction-mediated (32)P-postlabeling. Two major DNA adducts, identified as dG-C8-DMABP and dG-N(2)-DMABP, were detected in liver and colon of rats treated with DMABP. These DNA adducts were diminished approximately 35-40% with 500 ppm and 65-70% with 1,000 ppm celecoxib. In the colon, no further decline in DNA adducts was observed at 1500 ppm. The same DMABP-DNA adducts also were detected in the liver and were also diminished by celecoxib treatment. The reduction in DMABP-DNA adduct levels in celecoxib-treated animals provides further support for celecoxib as a chemopreventive agent for colorectal cancer.     

Analyses of DNA adducts formed by ochratoxin A and aristolochic acid in patients with Chinese herbs nephropathy.

Chinese herbs nephropathy (CHN), a unique type of nephropathy has been associated with the intake of weight-reducing pills containing the Chinese herb Aristolochia fangchi. Moreover, an association between the use of A. fangchi and urothelial cancer in CHN patients has been reported indicating that aristolochic acid (AA) the major alkaloid of A. fangchi might be the causal agent. Similarities of CHN to the Balkan endemic nephropathy (BEN) have led to the hypothesis of a common etiological agent for both diseases. Evidence has accumulated that BEN is an environmentally-induced disease strongly associated with the fungal mycotoxin ochratoxin A (OTA). Both, AA and OTA are nephrotoxic and carcinogenic and induce the formation of DNA adducts. As OTA has been suspected as fungal contaminant in the herbal batches used for the preparation of the weight-reducing pills we analysed tissues from CHN patients by the 32P-postlabeling procedure for the presence of DNA adducts related to both OTA and AA exposure. Whereas, AA-specific DNA adducts were detected in all five urinary tract tissues from five patients (total RAL: 32-251 adducts per 10(9) nucleotides), OTA-related DNA adducts were detectable in two kidneys and one ureter only (total RAL: 1.5-3.7 adducts per 10(9) nucleotides). Thus, OTA-related DNA adduct levels were about 50 times lower than AA-DNA adduct levels. In female and male rats that were treated with the slimming regimen in the same way like the CHN patients except that the amount of Chinese herbs was 10 times higher, AA-DNA adducts were found in kidney tissues (total RAL ranging from 51 to 83 adducts per 10(9) nucleotides) but adducts derived from OTA were not observed. These results demonstrate that OTA-related DNA adducts do not play a key role in CHN or CHN-associated urothelial cancer.     

Monitoring the exposure of rats to 2-acetylaminofluorene by the estimation of mutagenic activity in excreta, sister-chromatid exchanges in peripheral blood cells and DNA adducts in peripheral blood, liver and spleen

The sensitivity of various methods suitable for biomonitoring the exposure to genotoxicants was compared in an animal model. The results were related to the presence of genotoxic effects in the target organ. Groups of male Wistar rats were given one oral dose of 0, 0.1, 10 or 200 mg 2-acetylaminofluorene (2-AAF)/5 ml dimethyl sulphoxide/kg body weight. Peripheral blood cells, excreta, liver and spleen were collected at different time intervals after dosing. Mutagenicity in urine and extracts of faeces was determined using the Ames test with Salmonella typhimurium TA98 with and without S9 and with and without beta-glucuronidase. Genotoxic effects were studied by measuring DNA-adduct formation in lymphocytes, liver and spleen, and sister-chromatid exchanges (SCEs) in lymphocytes. DNA adducts were measured with immunochemical techniques and postlabelling methods. Mutagenicity in urine and faeces, collected during the first 24 h after treatment, was detected at 2-AAF doses of 1 mg/kg b.w. and higher. At these doses DNA adducts also became apparent in the liver, the main target organ for tumour induction by 2-AAF. The adduct detected appeared to be the N-(deoxyguanosin-8-yl)-2-AAF adduct. There was no evidence of the presence of any other types of DNA adducts. At doses of 1 and 10 mg/kg b.w. no mutagenicity was detected in excreta collected during the second and third day after dosing. The DNA-adduct level in liver cells of the 1 mg/kg b.w. group was maximal 24 h after dosing. At 200 mg/kg b.w. a delay in excretion of mutagenicity with urine and faeces was seen and at 10 and 200 mg/kg b.w. the amount of DNA adducts continued to increase with time after dosing. At 24 and 48 h after treatment with 10 mg, the adduct levels were of the same order of magnitude as those found after the 20-fold higher dose. This points to overloading of the metabolizing system which in combination with the enterohepatic circulation, may lead to an increased retention of 2-AAF in the body. A slightly increased incidence of SCEs of doubtful significance was seen in lymphocytes, but only at the very high dose of 200 mg/kg b.w. No DNA adducts could be detected in blood lymphocytes or spleen cells at any of the dose levels applied, either with the immunochemical or with the postlabelling method.(ABSTRACT TRUNCATED AT 400 WORDS)     

32P-postlabelling analysis of DNA adducts from Fischer-344 rats administered 2,4-diaminotoluene.

Using 32P-postlabelling and thin layer chromatography, DNA adduct formation by the potent animal carcinogen 2,4-diaminotoluene in Fischer-344 rats was investigated. DNA from four different organs, liver, mammary gland, kidney and lung, were examined for adducts following single administration of this compound. DNA binding was detected in all four organs, with each producing one major and two minor adduct spots on autoradiograms. The adducts induced were qualitatively identical among the different organs, but quantitative differences were observed. The two target organs of 2,4-diaminotoluene induced carcinogenesis, the liver and mammary gland produced higher adduct yields, with levels up to 30-times higher than those for the two non-target organs. Since the liver is the principal target for 2,4-diaminotoluene induced carcinogenesis, we further examined DNA adducts from this site for the effects of different doses and time points. DNA binding in liver was detected following doses as low as 4.1 mumol/kg. At the highest concentration examined (2046 mumol/kg), the level of the major adduct was 29.2 adducted nucleotides per 10(7) total nucleotides. The yields for the two minor adducts were approximately one-tenth that for the major adduct. Following a 410 mumol/kg dose, DNA adduct removal over time was examined. DNA adduct removal exhibited biphasic kinetics, with a rapid initial phase followed by a slower rate of elimination. Up to 60% of maximum adduct levels persisted after 2 weeks. DNA binding by 2,4-diaminotoluene was also compared to that by its weakly carcinogenic analog, 2,4-dinitrotoluene. The two compounds produced identical adduct patterns, suggesting that they share common metabolites and adducts. Adduct yields from 2,4-dinitrotoluene, however, were lower. The results of our studies suggest that the differences in carcinogenic potency between 2,4-diaminotoluene and 2,4-dinitrotoluene, as well as the organotropic effects of 2,4-diaminotoluene may be explained, in part, by quantitative differences in the extent of DNA adduct formation.     

32P-postlabelling detection of aromatic DNA adducts in peripheral blood lymphocytes from aluminium production plant workers

Aluminium production plant workers are exposed to a great number of airborne polycyclic aromatic hydrocarbons and epidemiological studies suggest that these workers are at increased risk of lung and bladder cancer. Blood samples from 46 workers at 2 primary aluminium plants and from 29 occupationally unexposed control individuals were analysed. DNA was isolated from the peripheral blood lymphocytes and aromatic DNA adducts were detected by 32P-postlabelling assay using the nuclease P1 digestion procedure for the enrichment of the adducts. The total levels of DNA adducts of exposed individuals varied from the detection limit of about 0.5 adducts/10(8) nucleotides up to 7.1 adducts/10(8) nucleotides and control adduct levels were up to 2.42 adducts/10(8) nucleotides. There was no significant difference between the mean adduct levels of the control group and of the individuals of one plant. However, the mean DNA adduct level obtained from workers of the second plant was significantly higher than that of the controls (p less than 0.001) and of the first plant (p less than 0.01), respectively. This difference can be attributed to differences in the design of technology and different levels of exposure at the 2 plants. The results of this study encourage further investigations of the use of peripheral white blood cells as marker cells and of 32P-postlabelling analysis for monitoring occupational exposure to mixtures of environmental carcinogenic pollutants.     

Endogenous and background DNA adducts by methylating and 2-hydroxyethylating agents

Detection of 7-alkylguanine DNA adducts is useful to assess human exposure to and the resulting DNA damage caused by simple alkylating agents. The background 7-methylguanine (7-MG) and 7-hydroxyethylguanine (7-HEG) adduct levels were determined in human and rat tissues, using thin-layer chromatography (TLC) combined with high pressure liquid chromatography (HPLC). In addition, these two adduct levels were also compared in various tissues between smokers and non-smokers. The results demonstrated that the background level of 7-alkylguanine adducts in WBC and lung tissues of non-smokers was 2.9 and 4.0 adducts/107 nucleotides, respectively. In smokers with lung cancers 7-MG adduct level in lung samples (6.3+/-1.9 adducts/107 nucleotides) and in bronchus samples (6.1+/-1.5 adducts/107 nucleotides) was significantly higher than that in WBC samples (3.3+/-0.9 adducts/107 nucleotides). 7-HEG adduct levels obtained from the same individuals were 0.8+/-0.3 in lung, 1.0+/-0.8 in bronchus and 0.6+/-0.2 adducts/107 nucleotides in WBC, respectively. Animal studies showed that background levels of 7-MG (2.1-2.5 adducts/107 nucleotides) in control rats were approximately 2-4-fold higher than 7-HEG levels (0.6-0.9 adducts/107 nucleotides). After a 3-day exposure to 300 ppm ethene, 7-HEG adducts accumulated to a similar extent in different tissues of rats, with the mean adduct level of 5.6-7.0 in liver, 7.4 in lymphocytes and 5.5 adducts/107 nucleotides in kidney.     

Induction of aryl hydrocarbon hydroxylase and DNA adduct formation in parental and carcinogen transformed C3H/10T1/2 clone 8 cells by benzo[a]pyrene

C3H/10T1/2 clone 8 (10T1/2) cells possess aryl hydrocarbon hydroxylase (AHH) activity capable of metabolizing polycyclic aromatic hydrocarbons to ultimate carcinogenic forms. AHH activity in 10T1/2 cells was measured before and after culturing in the presence of benzo[a]pyrene (B[a]P), and compared to the AHH activity found in carcinogen-transformed 10T1/2 cell lines treated similarly. The cell lines were also examined for B[a]P-DNA adduct formation, using the 32P-postlabelling technique. Treatment of parental 10T1/2 cells with B[a]P was found to significantly increase AHH activity and produce substantial numbers of DNA adducts. In addition to a major B[a]P-DNA adduct, 5-6 minor DNA adducts were also detected. Relative to parental 10T1/2 cells, an aflatoxin B1-transformed 10T1/2 cell line (7SA) was found to have significantly depressed AHH activity. In addition, after treatment with B[a]P, 7SA cells had only 8% of the B[a]P-DNA adduct levels found in 10T1/2 cells. This system may provide an in vitro model for investigating mechanisms responsible for the depression of cytochrome P-450 activities by chemical carcinogens.