Mitochondrial DNA point mutations and a novel deletion induced.pdfVIP

Mutation Research 585 (2005) 127–136 Mitochondrial DNA point mutations and a novel deletion induced by direct low-LET radiation and by medium from irradiated cells James E.J. Murphy a,∗, Sharon Nugent a , Colin Seymour a,b,1, Carmel Mothersill a,1 a Radiation and Environmental Science Centre, Focas Institute, Dublin Institute of Technology, Kevin St. Dublin 8, Ireland b St. Luke’s Hospital, Rathgar, Dublin 6, Ireland Received 4 November 2004; received in revised form 19 April 2005; accepted 26 April 2005 Abstract Radiation damage incurred by nuclear DNA is well documented and interest is increasing in the properties of ‘bystander’ factor(s) and their ability to induce radiation-like damage in cells never exposed to radiation. ‘Bystander’ and direct low-LET radiation effects on the mitochondria, and more particularly the mitochondrial genome are less well understood. In this study HPV-G cells (a human keratinocyte cell line derived from human neonatal foreskin transfected with the HPV-16 virus) were exposed to either -radiation doses as low as 5 mGy and up to 5 Gy from a 60 Co teletherapy unit, or to growth medium taken from similarly irradiated cells, i.e. irradiated cell conditioned medium (ICCM). Mutation and deletion analysis was performed on mitochondrial DNA (mtDNA) 4–96 h after exposure. Primers flanking the so-called mitochondrial ‘common deletion’ were employed to assess its possible induction. Single-strand conformation polymorphism (SSCP) analysis was conducted to identify induced point mutations. The relative mitochondrial number per cell was analysed by semi-quantitative PCR (sqPCR). Results indicate the induction of a relatively novel deletion in the mitochondrial genome as early as 12 h after direct exposure to doses as low as 0.5 Gy and 24 h after exposure to 0.5-Gy ICCM. SSCP analysis identified