Polycyclic aromatic hydrocarbons (PAHs), complex organic molecules that are the by-products of carbon-based fuel combustion, are carcinogenic upon biotransformation in the body. Cytochrome P450 1A1 (CYP1A1) is part of the enzyme system primarily responsible for the metabolism and biotransformation of PAHs in humans. In HepG2 cells (a human hepatoma cell line), it has been established previously in this laboratory that 3-methylcholanthrene (MC), a PAH, induces CYP1A1 activity 20 to 30 fold over control values. Also in those studies, MC’s ability to induce this activity was significantly reduced by compounds that inhibit the synthesis of complex sphingolipids. However, addition of the synthetic short-chain sphingolipid C2-ceramide after inducer and inhibitor treatment restored CYP1A1 inducibility. This produced the current laboratory hypothesis that complex sphingolipids are involved in the signal transduction pathway used by MC to induce CYP1A1 activity.
This research investigated the role that sphingolipids play in the inducibility of CYP1A1 by MC. Initially, the induction of CYP1A1 was examined by MC as a transcriptional event in these cells. Western blot analysis was used to determine whether CYP1A1 protein amounts fluctuate with CYP1A1 activity per the treatments used. Further studies included quantitative real-time reverse transcriptase PCR to quantitate the amount of CYP1A1 mRNA that resulted from each treatment. It was found that the action of the inhibitor of sphingolipid synthesis seems to be dependent on the presence of a heavy metal. It was also found that C2-ceramide, the prototype complex sphingolipid used, causes synergistic induction of CYP1A1 enzyme activity and that this synergism apparently results from actions at the level of the gene. Thus, it can be concluded that complex sphingolipids are involved in the transcriptional regulation of CYP1A1 expression.
| Identifer | oai:union.ndltd.org:auctr.edu/oai:digitalcommons.auctr.edu:dissertations-4316 |
| Date | 01 December 2001 |
| Creators | Peters, Demia E. |
| Publisher | DigitalCommons@Robert W. Woodruff Library, Atlanta University Center |
| Source Sets | Atlanta University Center |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | ETD Collection for AUC Robert W. Woodruff Library |
Page generated in 0.0023 seconds