Alkylation of DNA in rats by N-nitrosomethyl-(2-hydroxyethyl)amine: dose response and persistence of the alkylated lesions in vivo.

The in vivo alkylation of DNA by N-nitrosomethyl-(2-hydroxyethyl)amine (NMHEA) was examined in male and female F-344/N rats. NMHEA is a strong hepatocarcinogen in female rats when administered by gavage but a weaker hepatocarcinogen in male rats. Groups of 5 rats of each sex were treated by gavage with various doses of NMHEA dissolved in corn oil. After 4 h the animals were sacrificed and the livers, lungs, and kidneys were removed. The DNA from each liver was isolated and the neutral thermal and mild acid hydrolysates were separated by high-performance liquid chromatography. The alkylated guanines were quantified by fluorescence spectroscopy. NMHEA gives rise to four fluorescent alkylated guanines, 7- and O6-methylguanines, and 7- and O6-hydroxyethylguanines. The dose-response data revealed that all four lesions increased with dose. There was approximately 10x more methylation than hydroxyethylation at the 7 position of guanine. There was less O6 alkylation, but both methylation and hydroxyethylation were observed at all of the doses studied. The overall alkylation was the same in males and females at the 10- and 20-mg/kg doses, but at higher doses the females exhibited significantly higher levels of alkylation than males. The level of alkylation of DNA isolated from non-target tissues, lung, and kidney was low. The persistence of these lesions in vivo was studied at a dose of 25 mg/kg. Groups of five animals each were sacrificed at various times from 0 to 96 h. There was no significant difference between the sexes in persistence of any of the lesions in the liver. The 7-alkylguanines disappeared slowly over the observation period. 7-Methylguanine was present at 30% of the maximum level after 96 h, while 7-hydroxyethylguanine appeared to be more stable. The O6-alkylguanines were removed rapidly from the liver, being at base level by 48 h. The rapid removal of O6-hydroxyethylguanine suggests a repair process independent of O6-alkylguanine-DNA guanine alkyl transferase: an excision repair is postulated. In vitro alkylation of calf thymus DNA by N-nitrosomethyl-(2-tosyloxyethyl)amine, a surrogate for the putative O-sulfate conjugate of NMHEA, resulted in exclusive methylation of DNA-guanine at both the 7 and O6 positions; no hydroxyethylation was detected. In vitro alkylation of calf thymus DNA with 2-hydroxyethyl-ethylnitrosourea resulted in exclusive hydroxyethylation of DNA-guanine at the 7 and O6 positions.(ABSTRACT TRUNCATED AT 400 WORDS)

Methylation of liver DNA guanine in hamsters given hydrazine.

Formation of 7-methylguanine and O6-methylguanine in liver DNA was measured 24 hr after administration of various single oral doses of 15 to 120 mg hydrazine/kg body wt to Syrian golden hamsters, and at various times up to 96 hr after a single oral dose of 90 mg hydrazine/kg body wt. Formation of these bases in hamster liver DNA appeared similar to that reported earlier for rats and mice treated with necrogenic doses of hydrazine; however, persistence of O6-methylguanine in liver DNA following hydrazine administration was longer in hamster than in rat liver DNA. Administration of hydrazine sulfate in the drinking water of hamsters over a 9-week period resulted in accumulation of both 7-methylguanine and O6-methylguanine in liver DNA to the extent that about 2 out of every 10,000 guanine bases were methylated at each position on the base. Diethyl maleate pretreatment of hamsters depleted liver stores of glutathione and blocked DNA methylation in hydrazine-treated animals; however, buthionine sulfoximine, which also depleted glutathione stores, had no effect on the DNA methylation response to hydrazine poisoning in Sprague-Dawley rats. Hydrazine administration to hamsters and Swiss Webster mice resulted in more 7-methylguanine and O6-methylguanine in liver DNA than did administration of monomethylhydrazine, a proposed intermediate in the hydrazine-DNA methylation response. Even at the highest feasible dose of monomethylhydrazine to hamsters, no methylguanines could be detected, while these aberrant bases were readily quantifiable following hydrazine administration; thus, no evidence was obtained to support the proposal that monomethylhydrazine is an important intermediate in the methylation of DNA guanine in hydrazine-treated animals.

Tetraformyltrisazine and hydrazine-induced methylation of liver DNA guanine.

Hydrazine induces methylation of target-organ DNA guanine; the methylation mechanism was proposed to involve reaction of hydrazine with endogenous formaldehyde. One possible condensation product of hydrazine and formaldehyde is tetraformyltrisazine (TFT). TFT administered to Sprague-Dawley rats produced 7-methylguanine and O6-methylguanine in liver DNA at rates of formation and times to maximal methylguanine levels similar to those observed after hydrazine administration. TFT administration, however, resulted in greater amounts of methylguanines than did hydrazine on a molar basis, suggesting that TFT is perhaps a more proximal intermediate in hydrazine-induced methylation. The metabolic activation of TFT and hydrazine-plus-formaldehyde to methylating intermediates was detectable in in vitro systems containing as little as 0.2 mM TFT or 1 mM hydrazine-plus-formaldehyde, the lowest concentrations yet tested. The other major condensation product of hydrazine and formaldehyde, formalazine, also methylated liver DNA in vivo, but this polymer forms under conditions that would not be expected under in vivo administration of hydrazine. A novel pathway is proposed for the generation of a carbocation in hepatocytes exposed to hydrazine, consisting of condensation of hydrazine and formaldehyde to form TFT or formaldazine and/or formaldehyde hydroxymethylhydrazone and involves methylazomethanol as an intermediate.

The role of formaldehyde in hydrazine-induced methylation of liver DNA guanine.

Administration of the hepatotoxin and carcinogen, inorganic hydrazine, to rodents results in the formation of 7-methylguanine and O6-methylguanine in liver DNA; co-administration of [methyl-14C]methionine or [14C]formate with the hydrazine labels the methylguanines, suggesting involvement of the 1-carbon pool in the methylation process. The present study investigates the proposal that the methylation mechanism involves reaction of hydrazine with endogenous formaldehyde to yield formaldehyde hydrazone, which could be metabolized to the potent methylating agent diazomethane. Hamsters were pretreated with methanol, ethanol or cyanamide to alter the endogenous hepatic aldehyde levels prior to administration of hydrazine. Formaldehyde levels were refractory to the pretreatments; hepatic acetaldehyde levels were increased, but hydrazine administration under such conditions did not result in the formation of ethylated guanines in DNA. Methanol and ethanol inhibited hydrazine-induced methylation of DNA. Hydrazine incubated with liver S9 fraction and calf thymus DNA induced the formation of 7-methylguanine and O6-methylguanine when formaldehyde was present in the incubation system; substitution of formaldehyde with acetaldehyde in the incubation medium did not result in any detectable alkylation of DNA. Both liver microsomal and cytosolic fractions demonstrated heat-labile activity in supporting the hydrazine-induced methylation process. Tetraformyltrisazine, or a similar reaction product of hydrazine and formaldehyde, may be a more important intermediate than formaldehyde hydrazone in the hydrazine-induced methylation of DNA.

Recent findings on the metabolism of beta-hydroxyalkylnitrosamines.

beta-Hydroxynitrosamines appear to be refractory to alpha-oxidation, the common pathway of metabolism of simple dialkylnitrosamines. Some years ago, we postulated that nitrosamines bearing a hydroxyl in the beta position may be activated to alkylating agents by metabolic transformation to sulfate conjugates. Recent evidence has provided support for this hypothesis. A sulfate ester of N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (NHPOPA) has been found in the urine of hamsters treated with the nitrosamine. It has also been found that inhibition of sulfotransferases inhibited the development of DNA single-strand breaks in livers of rats treated with several beta-hydroxy-nitrosamines. Alkylation of rat liver DNA in vivo by N-nitroso(2-hydroxyethyl)methylamine (NHEMA) favoured methylation over 2-hydroxyethylation by a factor of 10. The methylation reaction was inhibited by sulfotransferase inhibitors. Thus, sulfation appears to be an important pathway for activation of beta-hydroxy-nitrosamines. There are, however, other pathways, such as the oxidation of the beta-hydroxyl group to a carbonyl, which may also result in the formation of electrophilic species capable of modifying cellular macromolecules.

Methylation of DNA guanine during the course of induction of liver cancer in hamsters by hydrazine or dimethylnitrosamine.

Hydrazine is carcinogenic to the mouse and rat, but three earlier studies have reported no carcinogenicity of hydrazine in the hamster. Administration of hydrazine to mice, rats and hamsters results in rapid methylation of liver DNA guanine for which endogenous formaldehyde appears to be the source of the methyl moiety. Hamsters were given hydrazine sulfate at 170, 340 and 510 mg/l in the drinking water for 2 years [average dose of 4.6, 8.3 and 10.3 mg hydrazine (free base)/kg body wt over the 2-year period], during which levels of methylation of DNA guanine in liver, kidney and lung, and histopathologic examinations of these tissues were carried out; dimethylnitrosamine, as a positive control, was administered at 10 mg/l in the drinking water (average dose of 1.1 mg/kg body wt over the 4-month measurement period). Both 7-methylguanine and O6-methylguanine were readily detectable at 6 months exposure in hamsters given hydrazine or dimethylnitrosamine; in hydrazine-treated animals only trace amounts of these bases could be detected after 12 months exposure; these bases were again detected in liver DNA at exposure times of 18 and 24 months. Hepatocellular carcinomas were observed in hamsters treated at the highest dose of hydrazine sulfate after 78 weeks of exposure; the incidence of liver cancer was dose-related over the course of the experiment: 32% for hamsters exposed to 510 mg hydrazine sulfate/l, 12% for 340 mg/l and none at 170 mg/l. Hamsters given dimethylnitrosamine developed high levels of 7-methylguanine and even higher levels of O6-methylguanine and both liver cholangiocellular carcinomas (73% incidence), as reported before, and hepatocellular carcinomas (27% incidence), a new finding. These results demonstrate for the first time that hydrazine is a liver carcinogen in the hamster and provide new information regarding the accumulation of DNA damage during the entire induction period for the carcinomas.