Date

2014

Author

Evin, Emre

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Xenobiotic metabolism is the combination of phase I and phase II reactions which are named as modification and conjugation reactions, respectively. Most of the marketed drugs are metabolized by Cytochrome P450s (CYPs) known as phase I enzymes. CYP1A1 and CYP2E1 enzymes play role in activation/inactivation of many drugs and carcinogens. Phase II enzymes including NQO1 and GSTP1 are important for the elimination of toxic metabolites by reduction and conjugation reactions, respectively. Since in vitro studies are considered as the preliminary step for the discovery of new drug molecules or for the investigation of the effects of chemicals on the molecular basis, this study was aimed to show the best cell line model for studying CYP1A1, CYP2E1, NQO1 and GSTP1 enzymes playing role in drug and carcinogen metabolism. Therefore, protein and mRNA expression of these enzymes were analyzed in the HT29 and SW620 (colon); HEPG2 and HUH7 (liver); PNT1A and PC3 (prostate) cell lines. For this purpose, cell lines were grown as three replicates in 5% CO2 incubator followed by the protein extraction and RNA purification steps. Then, mRNA and protein expressions of the enzymes by cell lines were analyzed by qRT-PCR and Western blotting techniques, respectively. The results showed that all cell lines express CYP1A1 protein and relative protein expression is highest in the HT29 and SW620 (colon) cell lines. In addition, liver cell lines HEPG2 and HUH7 expressed highest CYP1A1 mRNA. CYP2E1 protein expression was found in all cell lines at relatively high levels except PC3. CYP2E1 mRNA expression was very high in HUH7 and HT29 cell lines. PNT1A and HEPG2 expressed NQO1 protein at relatively high levels while PC3 and HUH7 cell lines expressed NQO1 protein at significantly lower levels. NQO1 mRNA expression varies in cell lines and PNT1A showed the highest mRNA expression when compared with HUH7 cell line. HUH7 showed the highest GSTP1 protein expression whereas the HEPG2 did not show any GSTP1 protein expression. mRNA expression of GSTP1 in the descending order is PNT1A, HT29, SW620, PC3 and HUH7 while there is no mRNA expression in HEPG2 cell line. In conclusion, CYP1A1, CYP2E1, NQO1 and GSTP1 protein and mRNA expressions showed differences in different cell lines originated from colon, liver and prostate tissues. It was found that HT29 colon cell line is the best model among these cell lines to study all of four enzymes together since it expressed both protein and mRNA of these enzymes at relatively significant levels. However, if CYP1A1, CYP2E1, NQO1 and GSTP1 would be studied individually, HT29, HUH7, PNT1A and HT29 are the best models, respectively.

Subject Keywords

Xenobiotics., Cytochromes., Cell lines., Prostate, Colon (Anatomy)

URI

http://etd.lib.metu.edu.tr/upload/12617676/index.pdf
https://hdl.handle.net/11511/23850

Collections

Graduate School of Natural and Applied Sciences, Thesis