Introductory Studies on the Viability of the HepG2 Cell Line as a Tool for Systematic Evaluation of the Genotoxic Potential of Primary Aromatic Amines

As life expectancies grow and cancer rates increase, genotoxicity testing has become essential for assessing the potential harm of xenobiotic chemicals that may affect genetic stability, leading to mutations and carcinogenesis. This rising importance has spurred interest in markers of DNA damage, including the phosphorylation of histone H2AX in response to double-strand DNA breaks. Recently, concerns have emerged about the potential genotoxicity of primary aromatic amines (PAA), which are key synthetic intermediates as well as potential cleavage and breakdown products of azo textile dyes. The discovery that microbiological activity could potentially break down azo dyes into constituent PAA has led to newly instituted of restrictions on their use and a race to gather data on the effects of this class of chemicals on human health. This thesis aimed to study the feasibility of applying a novel high-throughput in vitro assay to measure genotoxicity via the induction of γ-H2AX in HepG2 cells, as measured by automated fluorescence microscopy. Additionally, there was interest in comparing these results to data gathered in HaCaT cells, to see whether the change in cell line would have a positive effect on the sensitivity of the assay with regards to PAA treatment. The induction of γH2AX was also confirmed via western blotting, and the presence of metabolites of selected PAA was investigated via LC-MS. While there was a dose-response trend of γ-H2AX induction for HepG2 cells treated with the selected PAA, DCBD, further replicates would be needed to unequivocally decide if this cell line displays better sensitivity than HaCaT. While an Acetylated DCBD metabolite was measured via LC-MS in HaCaT cells, it was not seen in HepG2 samples. This could indicate that in HepG2 cells the metabolic response to PAA might not favor acetylation via N-Acetyltransferases, but rather other pathways, which might affect the rate of genotoxic activation of the substance.