Activation of a ÃŸ-Hydroxyalkylnitrosamineto Alkylating Agents: Evidence for the Involvement of a Sulfotransferase1

yV-Nitrosomethyl(2-hydroxyethyl)amine (NMHEA), Â»henadminis tered 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, sug gesting a correlation with the observed disparity in carcinogenicity. NMHEA has been shown to alkylate rat liver DNA in vÃ¬voin 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. \Ve 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-l,2,3-oxadiazo-liiiiiini 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 Â¿V-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 jV7-(2-hydroxyethyl)guanine, but a very strong effect on the O6-hydroxyethylguanine lesions. NMHEA-induced alkylation was also stud ied in male and female brachymorphic mice, which are deficient in the ability to synthesize the sulfate donor 3'-phosphoadenosine 5'-phospho-sulfate 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 alkyla tion in the brachymorphic mice, except for Ar7-(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 |mefA>'/-'4C|NMHEA was independently demonstrated by the production of |'4C]7-methylguanine in the presence of 3'-phos-phoadenosine S'-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 differ ent from that of methylation or Â£>*-hydroxyethylation. activity of kinase. methylation cultured cell-free a added inorganic and the O '-alkylguan-ines was carried out by cation-exchange HPLC chromatography with fluorescence detection as previously described (2). Radioactivity was monitored using a model 1C Flo-1 beta radioactive flow detector (Radiomatic Instruments. Tampa, FL). The data were quantified by integration and comparison with calibrated standard solutions of guanine, 7-methyl- and hydroxyethylguanine, and O'-methyl- and hydroxyethylguanine (2). DNA guanine peaks were quantified from the mild acid hydrolysates, and the results are expressed as /jmol alkylated base/ mol guanine. The quantifiable detection limit for all the alkylated bases in this study was approximately 1 /amol base/mol guanine. Statistical Treatment of the Data. Analysis of covariance was used to test the significance of comparisons between data sets. Prior to analysis, some NMHEA alkylation data were transformed by taking their com mon (base 10) logarithms in order to satisfy homogeneity of variance and covariance. SEs were derived from the calculated mean square error for each data set. alkylation levels in brachymorphic