In in vitro assays using methylated DNAs as substrates, human liver fractions were shown to be able to catalyze the removal of 06-methylguanine. The amount of removal was proportional to the amount of protein added, and the loss of O6-methylguanine occurred with stoichiometric formation of guanine in the DNA and S-methylcysteine in protein. This indicates that human liver contains a protein similar to that previously found in bacteria exposed to ailcylating agents. This protein acts as a transmethylase, transferring the intact methyl group from 06-methylguanine in DNA to a cysteine residue on that protein. A similar activity is present in rodent liver, butit was found that human liver was about 10 times more active in carrying out this reaction. In contrast, there was no difference between the human and rat liver extracts in catalyzing the loss of another methylation product, 7-methylguanine, from alkylated DNA. The liver is the organ most likely to be alkylated after exposure to exogenous potential alkylating agents such as dimethylnitrosamine. The present results show that human liver has a significant capacity to repair 06-methylguanine in DNA, which has been implicated as a critical product in carcinogenesis and mutagenesis. Dimethylnitrosamine (Me2NNO) is known to be carcinogenic in many animal species (1). This effect has been associated with the capacity of the target tissues to metabolize Me2NNO into a mutagenic intermediate that reacts with DNA at various sites (2-4). Of the various DNA adducts formed, 06-methylguanine (MeGua) has been implicated in both mutagenesis and carcinogenesis (4-10). The persistence of06-alkylguanine in the DNA of a tissue correlates with the probability that that tissue will develop tumors after administration of alkylating agents, indicating that this adduct is a critical determinant in the initiation of carcinogenesis by nitrosamines such as Me2NNO (4, 9, 10). MeGua can be removed from DNA in Escherichia coli by a DNA repair system which transfers the methyl groups to a cysteine residue in protein (11,12). There is evidence that this system also occurs in rodent tissues, but it has not been fully characterized (4,(13)(14)(15)(16)(17). Humans are known to be exposed to nitrosamines, including Me2NNO (18,19), and Me2NNO is metabolized by human liver to generate the reactive intermediate that results in DNA alkylation (20,21).In the work presented here, we used an in vitro assay to demonstrate that human liver fractions can catalyze the removal of MeGua from DNA; we found that this capacity is about 10 times greater than that with comparable rat liver fractions. We have also shown that this human liver system transfers the methyl group from the 06 position of guanine to eysteine residue, regenerating guanine directly in the DNA. MATERIALS AND METHODS Chemicals. N-[3H]Methyl-N-nitrosourea (1.6 Ci/mmol; I Ci = 3.7 X 1010 becquerels) was obtained from New England Nuclear. All other biochemical reagents were obtained from Sigma.Tissues. The 10 samples of human liver (six m...
The radioactivity level in blood, bile, urine and contents of parts of the gastro-intestinal tract in rats was studied after subcutaneous administration of 3-H-1,2-dimethylhydrazine (3-H-SDMH) which induces colonic tumours. The alkylation of DNA, RNA and protein in the intestinal mucosa, liver and kidneys was estimated 1 h to 28 days after 3-H-SDMH treatment from the 3-H-incorporation into these macromolecules. Administration of 3-H-1,2-diethylhydrazine (3-H-SDEH) which does not induce intestinal tumours was made as a control. Fifteen to 30 min after 3-H-SDMH treatment, marked radioactivity was found in blood, bile, urine and in contents of all regions of gastro-intestinal tract. After 3-H-SDMH administration no label occurred in the contents of localized segments of gastro-intestinal tract although it was present in blood, bile and urine. 3-H-SDMH methylated DNA, RNA and proteins of intestinal mucosa, liver and kidney to a high degree. One hour after 3-H-SDMH treatment the incorporation of label into protein of intestinal mucosa was higher than into liver and kidneys. 3-H-SDEH did not alkylate macromolecules in these organs but did so in thymus, spleen and brain, which are target organs for this carcinogen. After total hepatectomy, 3-H-SDMH did not methylate macromolecules of the intestinal mucosa. The following mechanism for the carcinogenic effect of SDMH is suggested. A carcinogenic metabolite of SDMH forms, in the liver, a conjugate with glucuronic acid. This glucuronide enters the gut both with bile and directly via the circulation. Microbial beta-glucuronidase releases the active metabolite which, in turn, alkylates tissue macromolecules.
Results from previous experiments have indicated tha persistence of an increased cancer risk in subsequent generations following prenatal exposure to a chemical carcinogen. In the present experiment, the possible role of prezygotic events in determined cancer risk was investigated in the progeny of male rats treated with ethylnitrosourea (ENU) before mating with untreated females. Eight BDVI male rats were given a single i.p. dose of 80 mg/kg bw ENU and each rat was then caged at weeks 1, 2, 3 and 4 after treatment with three untreated females. Fertility was lower and preweaning mortality higher in the experimental group, as compared to controls, particularly at the 4th-week mating. Survival rates after weaning were similar in the progeny of treated males and controls, as was the total incidence of tumours. However, analysis of tumour incidence at the various organ sites showed an increased incidence of neurogenic tumours in the progeny of ENU-treated males, as compared to that of controls.
The levels of 3 DNA repair enzymes involved in alkylation and oxidative DNA damage repair in human peripheral blood leukocytes were measured in 20 smokers and 17 non-smokers. No differences in O6-alkylguanine-DNA-alkyltransferase (AGT) activity were found between the 2 groups and the AGT distribution within the population appeared to be unimodal. In contrast, the mean activities of both the methylpurine (MeP)- and the 2-6-diamino-4-hydroxy-5N formamidopyrimidine (FaPy)-DNA glycosylases were higher in the smokers, although only the difference between the MeP-DNA glycosylase means was statistically significant. The standard deviations of these 2 enzymes were also higher in the smokers. The MeP-DNA glycosylase activity showed a bimodal distribution when all subjects were considered. This may in part be due to the smoking habit; 83% of the subjects with enzyme activities higher than 500 fmoles/mg protein were current smokers, whilst 85% of the non-smokers had lower enzyme activities. However, if the smokers were considered separately, a bimodal distribution of this enzyme activity could still be observed. No strong correlation was observed between enzyme activity and age, although the slopes of the regression lines of enzyme activity on age were all negative. The relationship between enzyme activities was studied by bivariate distribution and a strong correlation was only found between the MeP-DNA glycosylase and the FaPy-glycosylase, with the highest values of both enzyme activities being observed in the smokers and the lowest in the non-smokers. Our results suggest that the activity of certain DNA repair enzymes can be modulated by environmental exposure.
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