Specific DNA alkylation damage and its repair in carcinogen-treated rat liver and brain.
The in vivo formation and repair of specific DNA lesions produced by alkylating agents of contrasting carcinogenic potencies were investigated. Male Sprague-Dawley rats were treated with direct-acting alkylating agents methylmethane sulfonate (MMS) or methylnitrosourea (MNU). The amounts of N-3-methyladenine (3-meA), N-7-methylguanine (7-meG), and methylphosphotriesters (mePTE) in the DNA of liver and brain were determined following selective removal of the methylated bases by enzyme 3-meA N-glycosylase from Micrococcus luteus and thermal depurination at neutral pH. Both enzyme- and heat-induced alkali-labile apurinic sites were converted to single-strand breaks on incubation with 0.1 M NaOH. The number of such sites was quantitated following centrifugation of the DNA in alkaline sucrose gradients, fluorescent detection of unlabeled DNA, and estimation of number-average molecular weight. The results show a carcinogen dose-dependent initial linear increase in the number of enzyme- and heat-induced DNA strand breakage in both liver and brain DNA. With a half-life of approximately 3 h, 3-meA was removed from the tissues, whereas 45 to 55% of 7-meG remained unrepaired at 48 h. The study of the alkylation damage induced by MNU treatment of rats showed that the kinetics of repair for 3-meA and 7-meG was similar to the MMS-treated tissues and that mePTE persisted over a 7-day period. The technique developed does not require the use of radiolabeled reagents of DNA and allows for the selective quantitation of DNA alkylation lesions like 3-meA and 7-meG in the presence of nitrosourea-induced phosphotriesters.
A A
AA
Wani
S M
SM
D'Ambrosio
D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D000477:Alkylating Agents / Q000494:pharmacology*; D000818:Animals; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D002273:Carcinogens / Q000494:pharmacology*; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D004260:DNA Repair; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008770:Methylnitrosourea / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D000477:Alkylating Agents; D002273:Carcinogens; C025946:3-methyladenine; D006147:Guanine; C008450:7-methylguanine; D008770:Methylnitrosourea; D004247:DNA; D008741:Methyl Methanesulfonate; D000225:Adenine