Numerical changes in the mitochondrial DNA displacement loop in lung lesions induced by N-nitrosobis(2-hydroxypropyl)amine in rats

Abstract

Mutations in the mitochondrial DNA (mtDNA) displacement loop (D-loop) region were investigated to clarify the possible molecular mechanisms underlying the development of lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats, 6 weeks of age, were given 2000 ppm BHP in their drinking water for 12 weeks and then maintained without further treatment until sacrifice at 25 weeks. Genomic DNA was extracted from paraffin-embedded tissues and polymerase chain reaction (PCR)–single strand conformation polymorphism (SSCP) analysis, followed by nucleotide sequencing, was performed. Eleven out of 24 hyperplasias (45.6%), 8 out of 16 adenomas (50.0%), and 14 out of 21 adenocarcinomas (66.7%) showed numerical changes, in a polymeric C-tract at positions 16,086–16,092 of the mtDNA D-loop, with a one base insertion of cytosine increasing the length of the C-tract, from the seven nucleotides observed in normal lung tissues from non-BHP treated rats, to eight. These changes were all homoplasmic and no changes were found in lung lesions when the length of the C-tract in the normal lung tissues adjacent to the lesions was seven. These results suggest that alterations in the mtDNA D-loop may occur in an early phase of lung carcinogenesis induced in rats by BHP.