Abstract:
The cytochrome P450 monoocygenases play a fascinating role as determinants of toxicity stemming from endogenous and foreign chemical exposure. In addition, certain natural and anthropogenic compounds, such as those present in plants, foods, or combustion by-products can dramatically alter the levels of expression of these genes, thereby modulating the metabolism of agents we are subsequently exposed to. The dramatic upregulation of the CYP2B genes upon exposure to Phenobarbital (PB) is one such example. This effect is potentially quite important and can be elicited by many dissimilar compounds that also produce a âPB-like' response.
PB-like inducers can up-regulate the expression of many genes, including the CYP2B subfamily members in mammalian liver. The mechanisms controlling the PB induction response are poorly understood. The aim of this dissertation research was to further characterize the molecular mechanisms underlying the PB induction process through studies involving the examination of post-translational modifications, functional characterization, and protein-DNA binding analysis. The utility of a baculovirus-mediated transfection technique to study CYP gene regulation also was examined.
The results demonstrate that despite the ability of cAMP to dramatically inhibit the PB induction process, PB exposures themselves do not alter the intracellular cAMP levels or PKA activity. Exposure of primary cultured rat hepatocyytes to baculovirus, an agent tested for its gene delivery potential, also resulted in a marked inhibition of the PB induction response. The mechanisms of this effect were examined and appear to involve baculovirus-induced alterations in cytokine expression. Further examination of post-translational modifications showed that the global histone deacetylase inhibitor, Trichostatain A (TSA), was without effect on PB mediated CYP2B gene activation.
Affinity purification studies were performed to examine protein-DNA interactions, as well as cooperative interactions between proteins, at the PB responsive unit of the CYP2B genes. Together with transgenic mice approaches, the results demonstrated that NF-1, an avidly bound factor in this region, is not a key determinant of the induction process.