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Alkylation of DNA in Rats by N-Nitrosomethyl-(2-hydroxyethyl)amine: Dose Response and Persistence of the Alkylated Lesions in Vivo¹

Laboratory of Chemical and Physical Carcinogenesis, BRI-Basic Research Program, National Cancer Institute-Frederick Cancer Research Facility, Frederick, Maryland 21701

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. Incubation of the hydroxyethylated DNA in pH 7.4 buffer for various times up to 48 h revealed that the 7-hydroxyethylguanine was lost by slow depurination (t 52.5 h), but that O⁶-hydroxyethylguanine in DNA was completely stable under those conditions.

The results suggest that the difference in carcinogenicity of NMHEA in the two sexes may be connected to the higher level of alkylation in female rats as compared with males. The data also show that the O⁶-hydroxyethylguanine may be removed from DNA in vivo by a repair process other than one involving an alkyl transferase. The data also support the hypothesis that NMHEA may be activated to an alkylating agent by a mechanism which involves a conjugation of the hydroxyl group, perhaps sulfation.

¹ Sponsored by the National Cancer Institute, Department of Health and Human Services, under contract N01-C074101 with Bionetics Research, Inc., and N01-C023912 with Information Management Services, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government.

² To whom requests for reprints should be addressed, at National Cancer Institute, Frederick Cancer Research Facility, P.O. Box B, Frederick, MD 21701.

³ Staff member of Computer and Statistical Services, Information Management Services, Inc., National Cancer Institute, Frederick Cancer Research Facility.

Received 8/ 5/87. Revised 11/13/87. Accepted 12/10/87.

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