| The hypothesis that GST expression is a key component of cellular defenses against cytotoxic and genotoxic damage by a wide range of electrophilic toxins is widely supported by evidence obtained from this lab and others. The view that our laboratory has evolved about GST protective efficacy includes kinetic considerations in the cell system, influx of toxin and metabolic activation rate, and availability of cellular support mechanisms, such as GSH supply, but key gaps in understanding the protective efficacy of GSTs include their function in intact cells in relation to what is known about their enzymology under ideal conditions, and the nature of competition between reactions of activation (ex., by CYP450s) and detoxification. Furthermore, the quantitative nature of this relationship is unknown.; Transgenic modeling with genetically engineered cultured cells was used to examine the activation and detoxification of polycyclic aromatic hydrocarbons. Various human GST isoforms were introduced into V79 cells previously stably transfected with human CYP1A1, CYP1B1, CYP1A2, or CYP3A4. Subsequently, cytotoxicity, mutagenicity, radiolabel, 32P-postlabeling, and HPLC assays were used to determine the toxicity of B[a]P and B[a]P-diols, DB[a,l]P and DB[a,l]P-diols, 3-OH-B[a]P, and AFB1, and what chemoprotective effects individual GST isozymes conferred against these compounds.; One of the major themes emerging from herein is that GST protection against one endpoint caused by an agent does not necessarily predict protection by the same isozyme against other cellular damage. In our system, hGSTP1 or hGSTM1-1, when co-expressed with hCYP1A1, confers strong protection against benzo[a]pyrene-induced cytotoxicity, while only conferring a moderate protection against mutagenicity. The relative level of GST expression can determine protection against toxicity of B[a]P. Interestingly, however, the presence of GST does not confer strong protection against bulk DNA adduct formation in our system. 32P-postlabeling studies revealed that the major adduct, N2-gua-BPDE, is nearly eliminated in cells co-expressing CYP1A1 and hGSTP1 and that this elimination does account for the slight reduction in bulk DNA adducts that we observe. (Abstract shortened by UMI.)... |
