Preferential alkylation of mitochondrial deoxyribonucleic acid by N-methyl-N-nitrosourea

Abstract

The reaction of the carcinogen N-methyl-N-nitrosourea with mitochondrial DNA from various rat tissues was examined in vivo and in vitro. After incubation of isolated mitochondria or cell nuclei with N[(14)C]-methyl-N-nitrosourea in vitro and subsequent isolation and purification of the DNA the specific radioactivity of the mitochondrial DNA was 3-7 times that of the nuclear DNA. The incorporation of (14)C into embryonic mitochondrial DNA in vitro was about twice that into the liver mitochondrial DNA. Identical incorporation rates, however, were found for the reaction of isolated mitochondrial DNA or nuclear DNA respectively with N[(14)C]-methyl-N-nitrosourea. After intraperitoneal injection of 43.3-58.5mg of N[(14)C]-methyl-N-nitrosourea/kg body wt. to adult rats the labelling of the mitochondrial DNA was on average 5 times that of the nuclear DNA. A smaller specific labelling was observed for the ribosomal RNA, transfer RNA, and mitochondrial RNA as well as for the mitochondrial protein as compared with the mitochondrial DNA. After hydrolysis of the alkylated nucleic acids with hydrochloric acid, fractionation was carried out on Dowex 50 cation-exchange columns. In most experiments 70-80% of the input (14)C radioactivity was eluted in the 7-methylguanine fraction. The preferential alkylation of the mitochondrial DNA by N-methyl-N-nitrosourea in situ is discussed in connexion with the cytoplasmic-mutation hypothesis of carcinogenesis.

7-Methylguanine adducts in DNA are normally present at high levels and increase on aging: analysis by HPLC with electrochemical detection.

“…This excess of methylation in mtDNA compared to nDNA is presumably due to several factors. Numerous studies (34,(37)(38)(39) (2). This is presumably because the main source of oxidants, but not alkylating agents, is mitochondrial in origin.…”

Altering DNA base excision repair: Use of nuclear and mitochondrial‐targeted N‐methylpurine DNA glycosylase to sensitize astroglia to chemotherapeutic agents

“…Similar DNA repair enhancements were previously revealed in oxidation repair studies of primary rat astrocytes as well as a rat insulinoma cell line (Driggers et al, 1993;Hollensworth et al, 2000) and were attributed to a treatment-induced mtDNA repair adaptation that repairs not only 100% of the experimentally induced damage, but also preexisting endogenous damage. Relatively high levels of endogenous oxidative damage are present in mtDNA in large part because of its close proximity to the cell's main source of free radicals, the mitochondrial respiratory chain (Backer and Weinstein, 1980;Levy and Brabec, 1984;Niranjan et al, 1982;Rushmore et al, 1984;Wunderlich et al, 1970). MPG is known to excise various oxidative lesions (Dosanjh et al, 1994;Saparbaev and Laval, 1994); therefore, it is reasonable to conclude that heightened MPG activity increases the rate and extent to which both MNU generated as well as some preexisting endogenous DNA damages are repaired.…”

Do mitochondrial DNA fragments promote cancer and aging?

“…maintenance of ion homeostasis or ATP supply, have repeatedly been suggested to contribute to cellular transformation since tumor cell mitochondria can differ structurally and functionally from those of normal cells (see [811 for a review), but clear evidence in favour of this suggestion is lacking. There is, however, evidence that some chemical carcinogens primarily attack mitochondria [82][83][84][85][86][87][88][89][90][91]. It has also been proposed that mitochondria are the 'molecular clock' in eukaryotes [92] and that mitochondrial genetic damage is one of the fundamental mechanisms underlying aging [93,94].…”