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)
Koepke
S R
SR
Laboratory of Chemical and Physical Carcinogenesis, National Cancer Institute-Frederick Cancer Research Facility, Maryland 21701.
Kroeger-Koepke
M B
MB
Bosan
W
W
Thomas
B J
BJ
Alvord
W G
WG
Michejda
C J
CJ
D000818:Animals; D002273:Carcinogens / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005260:Female; D006147:Guanine / Q000378:metabolism; D008297:Male; D008745:Methylation; D009602:Nitrosamines / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D012737:Sex Factors; D013997:Time Factors
D002273:Carcinogens; D009602:Nitrosamines; C055157:N-nitrosomethyl-(2-hydroxyethyl)amine; D006147:Guanine; D004247:DNA