Complex Sphingolipid Involvement in the Expression of CYP1A1 Activity in MC-Exposed HepG2 CellsEPA Grant Number: U915823
Title: Complex Sphingolipid Involvement in the Expression of CYP1A1 Activity in MC-Exposed HepG2 Cells
Investigators: Peters, DeMia E.
Institution: Clark Atlanta University
EPA Project Officer: Broadway, Virginia
Project Period: August 1, 2000 through August 1, 2002
Project Amount: $62,155
RFA: Minority Academic Institutions (MAI) Fellowships for Graduate Environmental Study (2000) RFA Text | Recipients Lists
Research Category: Academic Fellowships , Ecological Indicators/Assessment/Restoration
The objectives of this research project are to experimentally determine: (1) whether the modulation of CYP1A1 activity by ISP1 is paralleled by modulation of CYP1A1 protein amount in treated cells; and (2) if sphingolipids act at the level of transcription by quantitating CYP1A1 mRNA levels and protein levels before and after treatment of cells with 3-methylcholanthrene (MC) alone and MC plus ISP1/FB1.
HepG2 cells will be grown in Dulbecco's Modified Eagle's Medium (DMEM), pH 7.4, supplemented with glucose, sodium bicarbonate, penicillin, streptomycin, and 10 percent fetal bovine serum, and maintained at 370?C in an atmosphere of 5 percent CO2. At confluency, DMEM without the serum supplement will be utilized and the cells will be treated. For treatment, the cells will be incubated with 5 mM MC (for maximal CYP1A1 induction); MC with 1 mM ISP1; or MC, ISP1 and 5 mM C2-ceramide. After the 18-hour treatment period, cells will be rinsed in ice-cold PBS, homogenized in 1 ml of 0.1 M sodium phosphate buffer (pH 7.6) and frozen in -700?C until assay. CYP1A1 will be assessed by monitoring one of its associated activities, ethoxyresorufin O-deethylase (EROD). Messenger RNA will be reverse transcribed using the polymerase chain reaction (PCR) apparatus to be utilized as a probe. A 631-base pair long CYP1A1 cDNA fragment (from 190 to 821) will be amplified by PCR using two oligonucleotides, 5'-sense: GGCCTGAAGAATCCACCAGGG and 3'-antisense: GGTAGGTAGCGAAGAATAGGG (56). This fragment will be digested with PstI and PvuII and subcloned into the PstI and SmaI sites of plasmid pT3T7a18. The plasmid will be purified and then sequenced to ensure that the cDNA is at the desired sequence. Once the desired probe has been sequenced, the RNA from the cells then will be analyzed by Northern blotting.
Investigations prior to this project showed that in HepG2 cells, 3-methylcholanthrene increased CYP1A1 activity 20- to 30-fold. However, this induction was significantly suppressed by inhibitors of sphingolipid synthesis (FB1 and ISP1). Neither of these compounds affected basal CYP1A1 activity. Induction of CYP1A1 activity by 3-methylcholanthrene was restored by addition of C2-ceramide to the cells. Therefore, these studies uncovered a requirement for complex sphingolipids during the induction of CYP1A1 by the PAH. The purpose of this study is to determine the apparent role that sphingolipids play in the signal transduction pathway used by MC to induce CYP1A1 activity.