Mitochondrial DNA point mutations and a novel deletion induced by direct low-LET radiation and by medium from irradiated cells.
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 gamma-radiation doses as low as 5 mGy and up to 5 Gy from a 60Co 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 the induction of point mutations, in a non-consistent manner, in only the D-loop region of the mitochondrial genome and only in cells exposed to 5 Gy, and neither in cells exposed to lower doses of direct radiation nor in those exposed to ICCM. SqPCR also identified an increase in the number of mitochondria per cell after both exposure to low level gamma-radiation and ICCM, indicative of a possible mechanism to respond to mitochondrial stress by increasing the number of mitochondria per cell.
Radiation and Environmental Science Centre, Focas Institute, Dublin Institute of Technology, Kevin St. Dublin 8, Ireland. james.murphy@dit.ie
James E J
JE
Murphy
Sharon
S
Nugent
Colin
C
Seymour
Carmel
C
Mothersill
D001483:Base Sequence; D024201:Bystander Effect; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000009:adverse effects; D004249:DNA Damage / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000235:genetics / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D005720:Gamma Rays; D006801:Humans; D015603:Keratinocytes / Q000502:physiology / Q000528:radiation effects; D018499:Linear Energy Transfer; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D017384:Sequence Deletion
D017077:Culture Media, Conditioned; D004272:DNA, Mitochondrial