Date of Award

7-1-2005

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Department of Biological Sciences

First Advisor

Juarine Stewart, Ph.D.

Second Advisor

David Collart, Ph.D

Third Advisor

David Logan, Ph.D.

Abstract

This study showed that dihydroceramide and ceramide increased the expression of cytochrome (CYP) P450 when HepG2 cells were exposed to 3-methylcholanthrene (MC). Cytochrome P450s are a highly diversified set of heme proteins responsible for drug metabolism, blood homeostasis, cholesterol biosynthesis, and steriodogenesis. CYP1A1, the first P450 isoform to be named, is responsible for the metabolism and biotransformation of polycyclic aromatic hydrocarbons (PAH). 3-methylcholanthrene, an extremely hazardous PAH, has been shown to induce the CYP1A1 gene through the aryl hydrocarbon receptor (AhR) pathway. Past investigations have shown that the sphingolipid, C2-ceramide, induces CYP1A1 expression when HepG2 cells are exposed to 3-methylcholanthrene. Ceramide, the hydrophobic moiety of all complex sphingolipids, is known to act as a second messenger in the cell. Unlike ceramide, dihydroceramide, the precursor of an essential molecule needed for the formation of ceramide through the de novo pathway, has been reported to be biologically inactive. It is hypothesized that structurally different sphingolipids (ceramide and dihydroceramide) modulate the ability of MC to induce CYP1A1 expression by increasing the influx of MC into the cell. To test this hypothesis, CYP1A1 enzyme activity and protein amounts were assessed by the ethoxyresorufm-o-deethylase assay and Western Blot analysis, respectively. Through confocal microscopy it was found that the increased influx of MC was correlated to the increased expression of CYP1A1 in the presence of the sphingolipids when compared to MC treated cells alone. In addition to increased influx, it was found that ceramide and dihydroceramide also caused MC to quickly localize around the nucleus of the cells. An increased influx of MC in cells and localization to the nucleus can lead to the increased expression of CYP1A1 for the biotransformation ofPAHs. This dual effect can potentially enhance the chemical carcinogenesis process through increased production of the carcinogenic metobolites of these PAHs. Sphingolipid stimulation of CYP1A1 enzyme during the biotransformation of a PAH could then enhance the incidence of chemical carcinogenesis.

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