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[¹⁴C]-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 ¹⁴C 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[¹⁴C]-methyl-N-nitrosourea. After intraperitoneal injection of 43.3–58.5mg of N[¹⁴C]-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 ¹⁴C 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.