A COMPARISON OF CHANGES IN GENE EXPRESSION AND DNA DAMAGE: A STUDY OF THE EFFECTS OF AGE OF CELLS, AMOUNT AND LENGTH OF EXPOSURE TO COBALT CHROME PARTICLES

Abstract

Wear debris from worn cobalt chrome joint replacements causes an increase in chromosomal translocations and aneuploidy. In this study the relationship between the amount of DNA damage and the changes in gene expression was investigated in human fibroblasts after exposure to artificial cobalt chrome particles. The comparison was made with different doses of particles, at different time intervals and in fibroblasts of different ages, those that had completed 10 population doublings (10 PD fibroblasts) and those that had completed 35 population doublings (35 PD fibroblasts). The genes (TGF-©¬2, p38 MAPK, Integrin ¥â1, SOD1, Caspase 10, PURA, FRA-1 and VNR) were chosen after a previous screen with cDNA microarrays. The percentage of senescent cells was evaluated using an immunohistochemical assay for ¥â-galactosidase activity. The 35 PD fibroblasts showed significantly more ¥â-galactosidase activity than the 10 PD fibroblasts. The level of DNA damage, as detected with the alkaline comet assay, was greater at higher doses, at longer exposures (up to 24 hours) and in 10 PD fibroblasts. The expression of all the genes listed above was generally lower after exposure to cobalt chrome particles using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The reduction in gene expression, like the increase in DNA damage was greater at higher doses and at longer exposure times. After 24hr exposure the reduction in gene expression was greater in 10 PD fibroblasts compared to 35 PD fibroblasts. After 6hr exposure this was only true at higher doses of particles and the opposite was seen after a lower dose of particles. These results show that levels of gene expression of TGF-©¬2, p38 MAPK, Integrin ¥â1, SOD1, Caspase10, PURA, FRA-1 and VNR may be correlated with the level of DNA damage and that this depends on the dose and length of exposure and the age of the cells. This highlights the potential importance of these genes in the mutagenicity of cobalt chrome particles in human fibroblasts.