In vitro assessment of Benzo-a-Pyrene induced point mutations in the human HPRT gene
Shah, Ume Kulsoom Seager, Anna L Doak, Shareen H Johnson, George E Carmichael, Paul L Scott, Sharon J Scott, Andrew D Manshian, Bella Jenkins, Gareth JS
Benzo–a-Pyrene (BaP) is a well known environmental pollutant that has been shown to cause cancer in humans. It is a pro-carcinogen that requires metabolic activation (involving CYP 40 enzymes) to cause genotoxicity. Differences in the expression of key CYP enzymes appear to play an important role in the metabolic activation of BaP and the observed genotoxicity. In this work TK6, MCL5, and AHH-1 (human lymphoblastoid) cell lines with different CYP 450 activity were utilised to assess the role of metabolic activation on BaP induced genotoxicity. The higher expression of CYP1A1 enzymes in MCL5 cells was reflected in the increased cytotoxic and genotoxic responses seen in these cells, which was slightly greater than those observed for AHH-1. TK6 cells showed no difference in genotoxicity because of the absence of CYP1A1. HPRT forward mutation assay was used to assess the point mutations generated by BaP in MCL5 and AHH-1 cell lines and the mutants were selected by 6-thioguanine (6TG) resistance. Further investigations were performed to analyse the mutation spectra generated by BaP in these cell lines. A linear increase in the mutation frequency was observed in MCL5 cells following 4hr and 24hr exposures at 1-10µM dose range. No safe exposure levels could be established even at the lowest (1µM) dose following 4hr exposure in MCL5 cells. Most of the mutations observed in AHH-1 and MCL5 cells were base substitutions, specifically GCCG and GCTA transversion mutations within the exon 3 region of the hprt gene. A significant number of these alterations accounted for deletions, consisting of 30% tandem TG and ≈40% long (≥ base pair) deletions found in the MCL5 and AHH-1 cells consecutively. Further replicates are in progress to allow a more robust statistical analysis of the mutation spectra in the two cell lines.