How does p53 status influence cytotoxicity during regulatory in vitro mammalian cell genotoxicity tests?

The number of in vitro mammalian cell positives that do not correlate with follow-up invivo genotoxicity and carcinogenicity testing is of concern (Kirkland, et al, 2005). These misleading in vitro positives result in significant animal usage, increased cost or loss of compounds from development. Using p53 competent human cells provides more predictive data for the assessment of human hazard and risk with less misleading in vitro positives compared to traditionally used rodent cell lines that lack wild-type p53 function (Fowler, et al, 2012a). However, it remains unclear whether the species origin or p53 status of the cells impacts their ability to accurately predict genotoxicity in the in vitro mammalian cell tests. Cells lacking wild-type p53 may underestimate cytotoxicity with analysis of high concentrations genotoxiciy assessment, compared to a p53 functional cell line. Three closely related human lymphoblastoid cell lines that differ in their p53 status were tested; TK6 cells express wild-type p53, NH32 are p53 null and WTK1 overexpress mutant p53, similar to the commonly used rodent cells. Ethyl methanesulfonate (EMS), etoposide and paclitaxel (taxol) were tested according to regulatory guidelines (OECD, 2010) and cytotoxicity determined using relative populating doubling. Relative caspase-3/7 activity was also determined as a measure for apoptosis to aid interpretation of the cytotoxicity data. NH32 were sensitive to the cytotoxic effects of EMS compared to TK6 and WTK1. In contrast NH32 underestimated cytotoxicity with etoposide compared to TK6 and WTK1. A similar cytotoxic response was observed with all three cell lines with taxol; however cytotoxicity was observed at lower concentrations in TK6. The apoptotic response to each compound in WTK1 was significantly reduced compared to TK6, which demonstrate a typical wild-type p53 response. NH32 demonstrated similar levels of apoptosis to WTK1 following etoposide and taxol treatments but was more similar to TK6 with EMS. The results showed that p53 deficient cell lines do not consistently underestimate cytotoxicity and that cytotoxicity is drug specific, therefore other factors may be more relevant to the high number of in vitro positive in p53 compromised cells. An increase in mutability with loss of wild-type p53 function is discussed which lead to the increased sensitivity observed with the rodent cells lines (Fowler, et al, 2012a). Other differences between cells of human and rodent origin are also explored, identifying relevant factors in addition to p53 status.