O6-methylguanine-DNA adducts in rat lymphocytes after in vivo exposure to N-nitrosodimethylamine (NDMA).
A non-invasive approach in immunopathological risk assessment was applied for analysis of the in vivo formation of DNA adducts. DNA methylation was studied in peripheral blood lymphocytes (PBLs) collected from Sprague-Dawley rats exposed to a single dose (75 mg/kg b.w.) of N-nitrosodimethylamine (NDMA). Three different techniques were applied for characterization and quantification of DNA adducts: (i) colloidal gold ultraimmunocytochemical localization of O6-methylguanosine (O6-meG)-DNA adducts, using affinity-purified, polyclonal antibody directed against O6-meG, (ii) quantitative assay using enzyme-linked immunosorbent assay (ELISA), amplified by the avidin-biotin (AB) system, and (iii) high-performance liquid chromatography (HPLC). The O6-meG-immunoreactive sites in PBLs seem to be concentrated in the nucleus. However, significant immunolabelling was also noted in the cytoplasm of the in vivo NDMA-exposed PBLs. Control preparations showed no specific gold immunolabelling. The O6-meG-DNA adduct formation in PBLs and hepatocytes, at 2-24 h following the exposure to NDMA, was analogous for both types of cells. The data showed high correlation for the ELISA and HPLC analytical methods. The data suggest an efficient O6-metG-DNA repair mechanism in lymphocytes, possibly analogous to the enzymatic repair of DNA adducts in liver cells.
Fadlallah
S
S
Département des Sciences Biologiques and TOXEN, Université du Québec à Montréal, Canada.
Lachapelle
M
M
Krzystyniak
K
K
Cooper
S
S
Denizeau
F
F
Guertin
F
F
Fournier
M
M
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000187:drug effects*; D018736:DNA Adducts / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D007124:Immunoenzyme Techniques; D008099:Liver / Q000378:metabolism; D008214:Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008745:Methylation; D009603:Nitroso Compounds / Q000633:toxicity*; D051381:Rats; D017207:Rats, Sprague-Dawley
D018736:DNA Adducts; D009603:Nitroso Compounds; C008651:4-nitrosodimethylaniline; D006147:Guanine; D004247:DNA; C008449:O-(6)-methylguanine