7-ethylguanine and 6-ethylguanine

To investigate tissue-specific relations between DNA adducts and mutagenesis in vivo, lambda lacZ transgenic mice were treated i.p. with N-ethyl-N-nitrosourea (ENU), diethylnitrosamine (DEN), and ethyl methanesulphonate (EMS). In liver, bone marrow, and brain DNA from mice sacrificed at several time points after treatment O6-ethylguanine (O6-EtG) and N7-ethylguanine (N7-EtG) levels were determined as well as the mutant frequency (MF) in lacZ. In liver DNA of ENU- and DEN-treated mice, the bulk of O6-EtG was removed at 3 days after treatment, while the MF continued to increase thereafter. This suggests that O6-EtG is not the major premutagenic lesion in the liver. Indeed, sequence analysis of mutants showed only 24% GC-->AT transitions, consistent with the O6-EtG lesion, and 28% TA-->AT transversions, expected from O2-ethylthymine. In bone marrow after ENU treatment, a maximum mutation induction occurred at 3 days post-treatment, of which 43% were GC-->AT mutations and 22% were TA-->AT mutations. This suggests that in bone marrow O6-EtG may be a major premutagenic lesion at the 3-day time point. In liver and bone marrow, EMS treatment gave rise to a high level of N7-EtG and a low level of O6-EtG but no increase in MF. No adducts or mutation induction were observed in bone marrow of DEN-treated mice. No MF increase was observed in the brain of either ENU- or EMS-treated mice, although O6- and N7-adducts were present.

Topics: Animals; Bone Marrow; Brain; Diethylnitrosamine; DNA Adducts; Ethyl Methanesulfonate; Ethylnitrosourea; Female; Guanine; Lac Operon; Liver; Mice; Mice, Transgenic; Mutagens

The exposure-responses for several effects of limited exposures to diethylnitrosamine (DEN) in the livers of male Fischer 344 rats were measured and phenobarbital promotion was used to enhance expression of initiation of carcinogenesis. Five doses ranging from a cumulative total of 0.5 to 4 mmol DEN per kg body weight were given as weekly i.p. injections for 10 weeks. This was followed by 4 weeks recovery, after which the groups were maintained on either a basal diet or 0.05% phenobarbital (PB) to promote liver tumor development. All doses of DEN produced ethylation in liver DNA, which increased with dose. Indicative of toxicity, the centrilobular zone of glutamine synthetase-positive hepatocytes was reduced in relationship to exposure up to a cumulative exposure of 3 mmol/kg. The two lower exposures to DEN produced no increase in cell proliferation, whereas higher exposures resulted in marked increases, approximately 4-fold between 1.0 and 2.0 mmol/kg cumulative. At the end of the recovery period (14 weeks), hepatocellular altered foci (HAF), which expressed the placental form of glutathione S-transferase, were induced by all exposures, with an increase of approximately 4-fold between the exposures of 1.0 and 2.0 mmol/kg being the greatest. In rats maintained on basal diet or PB for 24 weeks after exposure, HAF increased further and with exposures of 2.0 mmol/kg and above, all rats developed hepatocellular carcinomas. With 1.0 mmol/kg, no liver tumor occurred in 12 rats without promotion, whereas in those given PB, two adenomas and two carcinomas were present in 12 rats. At the lowest exposure of 0.5 mmol/ kg, no tumor occurred in rats on basal diet, although HAF increased approximately 7-fold. With PB promotion, only one adenoma developed in 12 rats and HAF increased another 2-fold. Thus, the findings document non-linearity for some of the effects of DEN and a near no-effect level for initiation of promotable liver neoplasms at the lowest exposure in spite of a substantial induction of HAF.

Topics: Alkylating Agents; Alkylation; Animals; Cell Division; Diethylnitrosamine; DNA Adducts; DNA Damage; Dose-Response Relationship, Drug; Guanine; Liver Neoplasms; Male; Rats; Rats, Inbred F344

LacZ transgenic mice are suitable for short-term mutagenicity studies in vivo. Mutagenicity in these mice is determined in the lacZ transgene. Since the lacZ gene is of bacterial origin the question has been raised whether DNA-adduct formation and repair in the transgene are comparable to those in total genomic DNA. Mice were treated with N-ethyl-N-nitrosourea (ENU) and killed at several time points following treatment. Some mice were pretreated with O6-benzylguanine to inactivate the repair protein O6-alkylguanine-DNA alkyltransferase (AGT). O6-ethylguanine (O6-EtG) was determined in lacZ in liver and brain by means of a monoclonal antibody-based immunoaffinity assay. In addition, O6-EtG and N7-ethylguanine (N7-EtG) were assayed in total genomic DNA of liver and brain with an immunoslotblot procedure. In liver, the initial O6-EtG level in total genomic DNA was 1.6 times that in lacZ. The extent of repair of O6-EtG during the first 1.5 h after treatment was 2.1 times that in lacZ. At later time points, O6-EtG repair was the same. N7-EtG repair in genomic DNA was evident. In contrast to the liver, little repair of O6-EtG in total genomic and lacZ DNA occurred in the brain while N7-EtG was repaired. No initial difference in O6-EtG levels were found in lacZ and genomic brain DNA. These findings indicate that in the liver, total genomic DNA is more accessible than lacZ to ENU and/or the AGT protein, during the first 1.5 h following treatment. Because the difference in O6-EtG levels in the transgene and genomic DNA in the liver is restricted to the first 1.5 h after treatment, while the fixation of mutations occurs at later time points, O6-EtG-induced mutagenesis most likely is also very similar in both types of DNA.

Topics: Animals; Bacteriophage lambda; Brain; DNA; DNA Repair; Ethylnitrosourea; Female; Genome; Guanine; Immunoblotting; Lac Operon; Liver; Mice; Mice, Transgenic; Mutagenicity Tests; Mutagens; Polymerase Chain Reaction; Sensitivity and Specificity

Diethylnitrosamine exposure via the water resulted in the formation of 7-ethylguanine and O6-ethylguanine in rainbow trout liver DNA. The modified bases were quantitated by high-pressure liquid chromatography and fluorescence spectrophotometry. In vivo 7-ethylguanine and O6-ethylguanine levels were directly proportional to DEN concentrations between 62.5 and 250 ppm. 7-Ethylguanine and O6-ethylguanine levels were approximately directly proportional to duration of exposure to DEN between 0 and 6 hr under the conditions used, with less than proportionate increases thereafter. Removal of ethylguanines from liver DNA following a 24-hr exposure to 250 ppm DEN (a known carcinogenic regimen) was biphasic; 24% of the O6-ethylguanine and 32% of the 7-ethylguanine found immediately after exposure were removed in 12 hr but no significant decline was found over the period from 12 to 96 hr after exposure. Alkyl acceptor protein activity in trout liver was examined to assess the role of enzymatic repair in the observed loss of O6-ethylguanine in vivo. Although an O6-alkylguanine repair system similar to the alkyltransferase system reported in rodents was found in trout liver, only 4% of the O6-ethylguanine lost from DNA after exposure to 250 ppm DEN can be accounted for by activity of the alkyl acceptor protein. The high incidence of liver tumours observed in DEN-treated rainbow trout may be related to the rapid formation and substantial persistence of the promutagenic O6-ethylguanine in liver DNA.

Topics: Animals; Chromatography, High Pressure Liquid; Diethylnitrosamine; DNA; Dose-Response Relationship, Drug; Guanine; Liver; Trout; Water

Formation of O6-ethylguanine (O6EG) in trachea DNA was measured 12 h after acute i.p. administration of 50, 100 and 200 mg diethylnitrosamine (DEN)/kg body wt to Syrian golden hamsters. Purine bases were fractionated by h.p.l.c., and ethylated guanines were quantified optically by fluorescence spectrophotometry. Significant levels of this promutagenic base were found after the various single doses of DEN. O6EG in trachea DNA was also measured at various times up to 48 h after an acute i.p. dose of 200 mg DEN/kg body wt. The maximum level of O6EG was detected 12 h after DEN treatment. A rapid decrease in O6EG concentration was seen in trachea DNA between 12 and 24 h, but only a negligible decrease was detected from 24 to 48 h post-treatment. The formation and accumulation of O6EG in trachea, liver, lung and kidney DNA were determined in hamsters treated subchronically with DEN, 20 mg/kg body wt, s.c. twice weekly up to 8 weeks. O6EG accumulated to significant levels in trachea and liver DNA. 7-Ethylguanine did not accumulate in the DNA of any of the organs studied during subchronic DEN exposure. The formation and persistence of O6EG in trachea DNA of DEN-treated hamsters correlated with the sensitivity of this tissue to the carcinogenic action of DEN.

Topics: Alkylation; Animals; Cricetinae; Diethylnitrosamine; DNA; Guanine; Liver; Lung; Male; Mesocricetus; Trachea; Tracheal Neoplasms

The kinetics of formation and persistence of 7-ethylguanine (e7Gua) and O6-ethylguanine (O6eGua) were determined in rat liver and kidney DNA following i.p. injection with 12.5, 50, 100, or 200 mg DENA per kg body weight. The rate of ethylguanine formation in hepatic DNA was independent of carcinogen dose; however, the maximum level of DNA ethylation reached was linearly related to DENA dose. Persistence of O6eGua but not e7Gua in rat liver DNA appeared to be dose-dependent; the rate of decline in O6eGua concentration slowed as the dose of DENA increased. Ethylation of rat kidney DNA was quantifiable only following treatment with 200 mg DENA per kg body weight, and maximum concentrations of e7Gua and O6eGua were approximately ten times less than those in hepatic DNA of these animals. Nevertheless, elimination of e7Gua and O6eGua from DNA occurred at similar rates in these tissues. Whereas lung DNA from DENA-treated rats contained no detectable ethylguanines, both e7Gua and O6eGua were detected in lung DNA from treated hamsters. The half-life of e7Gua in hamster lung DNA was 28 h, while O6eGua persisted longer, exhibiting a half-life of 91 h. Only trace quantities of e7Gua and O6eGua were detected in hamster kidney DNA, precluding an accurate estimation of the kinetics of DNA alkylation in this tissue. The rate of formation of ethylguanines in hepatic DNA was faster in hamster than in rat, while maximum levels of e7Gua and O6eGua were similar in these two species. Persistence of both e7Gua and O6eGua was markedly different in hepatic DNA of rats and hamsters. e7Gua was eliminated at a faster rate in the hamster (half-life of 20 h), as compared to the rat (half-life of 35 h). Conversely, O6eGua persisted longer in hamster than in rat liver DNA; a half-life of 34 h was found for the hamster, compared to a half-life of 14 h for the rat. The half-lives of e7Gua and O6eGua in hepatic DNA of DENA-treated rats and hamsters were similar to those reported previously for m7Gua and O6mGua in these species, suggesting that the same enzymatic DNA repair systems act upon these structurally related DNA adducts. The formation and prolonged persistence of O6eGua in lung DNA of DENA-treated hamsters may be related to the sensitivity of this species to the induction of respiratory tract neoplasms following exposure to DENA.

Topics: Alkylation; Animals; Cricetinae; Diethylnitrosamine; DNA; Dose-Response Relationship, Drug; Guanine; Kinetics; Liver Neoplasms, Experimental; Lung Neoplasms; Male; Mesocricetus; Nitrosamines; Rats; Rats, Inbred Strains; Species Specificity

The dose-responses for N-nitroso-N-ethylurea (NEU)-induced mutagenesis in the hisG46 mutant, Salmonella typhimurium TA1535, and for the formation of O6-ethylguanine (O6-EtGua) and 7-ethylguanine in the DNA isolated from these cells were measured. Mutagenesis and O6-EtGua formation exhibited threshold-like behavior, whereas the formation of 7-ethylguanine was linear with dose. These results are consistent with a dependence of mutagenesis on O6-EtGua. There was no threshold in the production of O6-EtGua in isolated DNA treated with NEU. The failure of O6-EtGua to appreciably accumulate in the cellular DNA at low doses of NEU was attributed to a saturable, constitutive repair activity in the bacteria. Based on (i) the ratio of O6-EtGua in DNA to revertant fraction, (ii) published values for the size of the Salmonella genome and (iii) the target size and target bases (guanine-cytosine base pairs) for reversion of the hisG46 (missense) mutation, it was calculated that about 1/3 of the O6-EtGua's in the DNA led to mutations. Using the same calculations and data from previous experiments, a mutational efficiency for O6-methylguanine of 2/3 was obtained. Threshold-type responses in NEU-induced mutagenesis were observed in the other hisG46 mutants, TA100 and TA1975, but not in the frameshift mutant, TA98 where the dose response was linear. As TA98 contains the same DNA repair systems as TA100, frameshift mutations induced by NEU may result from DNA adducts produced linearly with dose.

Topics: DNA, Bacterial; Ethylnitrosourea; Guanine; Mutagenicity Tests; Mutagens; Mutation; Nitrosourea Compounds; Salmonella typhimurium

The extreme sensitivity of the developing rat brain to tumor induction by N-ethyl-N-nitrosourea (ENU) has been ascribed to the relatively inefficient repair of the presumed promutagenic lesion O6-ethylguanine from brain DNA. We have compared the brain of the newborn rat with liver, kidney and lung with respect to the repair of other types of DNA lesions that ENU induces, namely single strand breaks and alkali-labile lesions. The induction and repair or loss of these lesions has been analysed by alkaline sucrose gradient sedimentation using both mild as well as strong alkaline hydrolysis conditions. We found that ENU induces few lesions in the DNA of various organs that are detectable as breaks after mild alkaline hydrolysis. 24 h after ENU treatment such lesions are no longer detectable in brain DNA, while they are detectable in the DNA from other organs up to 10 days after ENU treatment. The number of ENU-induced lesions, detectable as breaks after strong alkaline hydrolysis, is far larger. These lesions were persistent in kidney DNA, disappeared slowly from liver DNA (t(1/2) = about 10 days), and more rapidly from brain and lung DNA (t(1/2) = 2-3 days). The latter result seems to be in contradiction with various reports that a large fraction of the ENU-induced alkali-labile lesions are stable in vivo. This difference between our results and those of others might be due to a difference between proliferating and non-proliferating cells. Whether the alkali-labile lesions are removed from brain and lung DNA by a specific repair mechanism or by other causes remains to be investigated.

Topics: Adenine; Alkylating Agents; Animals; Animals, Newborn; Brain; DNA; DNA Damage; DNA Repair; Ethylnitrosourea; Female; Guanine; Hydrogen-Ion Concentration; Kidney; Liver; Lung; Male; Rats; Rats, Sprague-Dawley; Sodium Hydroxide