Infections in humans and inflammatory stimuli in experimental animals cause an impairment of hepatic drug metabolism that is associated with decreases in the activities and levels of expression of many forms of cytochrome P450 (P450). In humans, this can result in elevated plasma levels of therapeutic agents, and subsequent drug-related toxicity. The investigators have also found recently that a subset of P450s, the CYP4A subfamily, are increased in the livers and kidneys of rats in two different models of inflammation. The goals of this project are to further characterize the induction of the P450 4A subfamily during inflammation, and to gain an understanding of the molecular mechanisms involved in both the induction of the P450 4A subfamily, and the decreases in other forms of P450, characterized by CYP2C11. The PI will identify transcription factors that are activated by inflammatory cytokines and stress hormones, and that bind to the CYP2C11 gene promoter and mediate its down-regulation during inflammation, using gel mobility shift assays and DNA footprinting. Chloramphenicol acetyltransferasae reporter gene constructs and site-directed mutagenesis will be used to determine the functional significance of the protein binding. Sphingolipid signaling pathways involved in cytokine down-regulation of CYP2C11 will be characterized in hepatocyte cultures by studying the structural specificity of sphingolipid effects on its expression, and by perturbing cellular sphingolipid metabolism with various pharmacological tools. The down-regulation of CYP2C11 by sphingolipids will be compared to the induction of acute phase protein genes and inducible nitric oxide synthase, to gain an understanding of how sphingolipids might regulate genes by different mechanisms. The PI will determine whether sphingoid molecules can modulate hepatic gene expression through the same response elements utilized by IL 1. The possible involvement of cytokine-activated transcription factor NF-kappaB will be examined using agents that block its activation. The induction of CYP4A by inflammatory mediators will be characterized in vivo by examining the time courses of the effects on CYP4A mRNAs, proteins and gene transcription, and by studying the sex- and strain specificities of the effects. The mechanism of CYP4A induction will be investigated by determining whether other genes regulated by the peroxisome proliferator activated receptor are induced under the same conditions, and by using gel mobility shift assays to determine directly whether the receptor is activated under inflammatory conditions. The PI will also investigate whether the CYP4A mRNAs can be induced by cytokines in cultured hepatocytes.
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