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Modulation of DNA adduct formation by pre-existing adducts was examined in synthetic oligonucleotides and genomic DNA (calf thymus); genotoxins studied were N-acetoxy-acetylaminofluorene (N-AcO-AAF), aminofluorene (AF), aflatoxin B1-8,9-epoxide (AFB1-8,9-epoxide), and dimethylsulfate (DMS). Oligodeoxynucleotides containing either guanine-C8-AAF (Gua-C8-AAF) or Gua-C8-AF adducts and a neighboring unadducted guanine (G) (target G), located 1, 2, or 4 nucleotides from the adduct, were reacted, as single- (ss) or double-stranded (ds) substrates, with dimethylsulfate (DMS) or AFB1-8,9-epoxide. A modified Maxam-Gilbert technique showed that the presence of the AAF adduct lowered the extent to which AFB1-8,9-epoxide, but not DMS, reacted with target G. Binding of AFB1-8,9-epoxide to the target G was attenuated (> or =5-fold) when the target was located immediately adjacent to an AAF, but not AF, adduct in ds-DNA. Reaction with AFB1-8,9-epoxide increased when the target G was located 2 or 4 nucleotides from the AAF adduct. Pretreatment of calf thymus DNA with AAF (0-1.8% nucleotides modified) reduced levels of Gua-N7-AFB1 adducts formed after subsequent treatment with AFB1-8,9-epoxide. Pretreatment of calf thymus DNA with AFB1 did not alter levels of adducts formed after subsequent treatment with N-AcO-AAF. The supposition that aflatoxin B1-binding to DNA may be altered by conformational changes in the helix, due to the presence of a pre-existing AAF adduct, is supported by the absence of an effect by AF and confirmation of local denaturation of the oligomer helix by use of chemical probes hydroxylamine and diethylpyrocarbonate. Nonetheless, the importance of changes in the nucleophilicity of neighboring nucleotides and local steric effects cannot be ruled out.

A synthetic DNA fragment was constructed to determine the effect of 5' and 3' neighbors of guanine runs on the binding of chemical carcinogens. Determinations were made on the relative intensity of reactivity between aflatoxin B1 or benzo(a)pyrene and methylnitrosourea or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea with various guanine positions in an endlabeled DNA fragment of known sequence. After reaction, the fragments were depurinated to produce strand breaks to allow Maxam and Gilbert sequencing for guanine positions. Relative reaction intensities were compared densitometrically. 3' neighbors exerted greater influence on carcinogen binding than did 5' neighbors, the influence extended only to the adjacent guanine and depended upon the chemical nature of the carcinogen. In addition, the presence of one carcinogen adduct in the guanine run influenced the formation of a subsequent adduct when the DNA was exposed to a second carcinogen, and this effect also depended on the nature of the second carcinogen. The results suggest that DNA adduct formation in the presence of multiple carcinogens is more complicated than an additive mechanism would suggest.

Hydrazine is acutely neurotoxic, hepatotoxic and nephrotoxic; it is also carcinogenic to liver and lung in rodents. Administration of hydrazine results in formation of 7-methylguanine and O6-methylguanine in target organ DNA of rats, mice, hamsters and guinea-pigs. It has been suggested that hydrazine reacts with endogenous formaldehyde to form a condensation product which could be metabolized to a methylating agent. Solutions of 0.50 mM hydrazine and formaldehyde have, upon mixing, NMR spectra (300 MHz) consistent with the formation of formaldehyde hydrazone but not other possible condensation products such as tetraformyltriazine or formaldehyde azine. These same solutions evidencing hydrazone formation, when incubated in an in vitro system containing post-mitochondrial (S9), microsomal, cytosolic or mitochondrial cell fractions, resulted in the methylation of DNA guanine; S9 was the most active fraction. Neither the P-450 monooxygenase nor flavin monooxygenase systems appeared to be important in hydrazine/formaldehyde-induced methylation of DNA. However, sodium azide, cyanamide and carbon monoxide all inhibited S9-supported DNA methylation. Bovine liver catalase, a heme-containing cytochrome, readily transformed hydrazine/formaldehyde to a methylating agent. The data support formation of formaldehyde hydrazone as the condensation product of hydrazine and formaldehyde which is rapidly transformed in various liver cell fractions, perhaps by catalase and/or catalase-like enzymes, to a methylating agent.

The formation and persistence of O6-methylguanine and 7-methylguanine in colon, kidney, and liver DNA were measured over a period of 25 weeks during 1,2-dimethylhydrazine (SDMH) carcinogenesis. Rats were given 14 weekly s.c. injections of 21 mg SDMH/kg body wt., and methylated guanines in DNA were determined quantitatively one week after each injection to measure the long-term accumulation of these aberrant bases. No accumulation of either base was seen in colon DNA, and no O6-methylguanine was seen to accumulate in liver DNA. The concentrations of O6-methylguanine and 7-methylguanine in kidney DNA did increase with reported administration of the carcinogen, but these bases were removed within six weeks after the last (14th) injection of SDMH. Large amounts of 7-methylguanine accumulated in liver DNA over the 14-week treatment period. The concentration of 7-methylguanine in liver and kidney DNA increased at rates greater than could be accounted for by using kinetic relationships determined in single-exposure studies. The concentration of O6-methylguanine in kidney DNA also increase at a rate greater than would be expected from calculations based on the rate of removal following a single administration of SDMH.

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.

Fischer 344 or Sprague Dawley rats were fasted overnight and given orally 30--90 mg hydrazine/kg body wt. The presence of 7-methylguanine and O6-methylguanine in liver DNA was demonstrated 5 and 24 h after hydrazine administration using two different analytical techniques. Methylation levels changed little with dose except for the highest dose (ca. LD50) at which the levels doubled. In a time-response study, rats were given 90 mg hydrazine/kg body wt. and killed 0.25 to 96 h later. Both 7-methylguanine and O6-methylguanine were detected quantitatively in liver DNA from rats as early as 15 min after hydrazine administration. After maximum levels of methylguanines had formed, 7-methylguanine was removed from DNA at a rate of approximately 50% in 47 h; the half-life of O6-methylguanine in liver DNA was approximately 13 hr. Three or four, but not one or two, daily administrations of 3 mg hydrazine/kg body wt. also produced detectable levels of 7-methylguanine in rat liver DNA. Neither 7-methylguanine or O6-methylguanine was detected in comparable amounts of liver DNA from control animals. The study confirms the observation that hydrazine administration results in the formation of methylated guanines in liver DNA.

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.