Activation of a β-Hydroxyalkylnitrosamine to Alkylating Agents: Evidence for the Involvement of a Sulfotransferase

N -Nitrosomethyl(2-hydroxyethyl)amine (NMHEA), when administered by gavage, is a strong liver carcinogen in F344 female rats, but a weak liver carcinogen in male rats. After repeated exposure to NMHEA, either in drinking water or by gavage, female rats accumulated higher levels of DNA-guanine adducts than did their male counterparts, suggesting a correlation with the observed disparity in carcinogenicity. NMHEA has been shown to alkylate rat liver DNA in vivo in a dose-dependent manner. Chemical investigations of NMHEA suggest that it becomes a strong electrophile when a good leaving group is substituted on the hydroxyl. We have proposed that NMHEA is activated to its ultimate carcinogenic form by conjugation with sulfate. The sulfate ester was postulated to undergo rapid cyclization to 3-methyl-1,2,3-oxadiazolinium ion, which has previously been found to be a potent methylating agent in vitro . The effect of sulfotransferase inhibitors on the DNA alkylation in rats by NMHEA was studied in vivo . Dichloronitrophenol, a powerful inhibitor of phenol sulfotransferase, had little effect on the methylation and O 6-hydroxyethylation of DNA guanine in female rats, while depressing the hydroxyethylation of the N-7 position of guanine. Dichloronitrophenol, however, dramatically enhanced the methylation of DNA in male rats. It also slightly inhibited the N -nitrosodimethylamine-induced methylation of DNA. On the other hand, propylene glycol, an alcohol sulfotransferase inhibitor, had a profound inhibitory effect on DNA methylation induced by NMHEA, very little effect on the formation of N 7-(2-hydroxyethyl)guanine, but a very strong effect on the O 6-hydroxyethylguanine lesions. NMHEA-induced alkylation was also studied in male and female brachymorphic mice, which are deficient in the ability to synthesize the sulfate donor 3′-phosphoadenosine 5′-phosphosulfate required for sulfotransferase activity, and their heterozygous siblings. No significant differences were seen between the heterozygous and brachymorphic mice in overall levels of alkylation, except in the case of 7-hydroxyethylation. In contrast to rats, male mice showed higher levels of formation of all DNA guanine adducts than did the females. However, propylene glycol was found to depress all the levels of alkylation in the brachymorphic mice, except for N 7-(2-hydroxyethyl)guanine, as was observed in rats. Additional DNA alkylation experiments in vitro showed that methylation occurred in a dose-dependent manner with added inorganic sulfate when NMHEA was activated by either freshly isolated primary hepatocytes or intact cultured hepatocytes. Methylation of calf thymus DNA by [ methyl -14C]NMHEA was independently demonstrated by the production of [14C]7-methylguanine in the presence of 3′-phosphoadenosine 5′-phosphosulfate and rat liver cytosol. These data support the hypothesis that methylation of DNA, at least in part, is due to an intermediate formed by the interaction of NMHEA with sulfate. The sulfotransferase enzyme responsible for NMHEA conjugation is not inhibited by dichloronitrophenol but is strongly inhibited by propylene glycol. 7-Hydroxyethylation of DNA must occur by a mechanism different from that of methylation or O 6-hydroxyethylation.