Date

2016

Author

Akkulak, Merve

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Vitamin D has long been known to function in the calcium homeostasis. Besides, it has widespread pleiotropic hormone actions. Therefore, studies on metabolism of vitamin D have attracted much attention in recent years. Vitamin D metabolism is regulated by cytochrome P450 enzymes (CYPs) such as CYP2R1, CYP27A1, CYP27B1 and CYP24A1. Phenolic compounds including quercetin and resveratrol are widely studied due to their important roles in human health as activators or inhibitors for enzymes catalyzing biochemical reactions. Although the interaction between these phenolic compounds and CYPs has been reported widely so far, there is no reported study about the role of quercetin and resveratrol on vitamin D metabolizing CYPs. This study was aimed to investigate the effects of quercetin and resveratrol on vitamin D metabolizing CYPs in human embryonic kidney cell line (HEK293) and human hepatocellular carcinoma cell line (HUH7). For this purpose, IC50 values of quercetin and resveratrol for HEK293 and HUH7 cells were determined by Alamar Blue assay. While IC50 values of quercetin were determined as 60.72 and 185.31 µM for HEK293 and HUH7 cells, IC50 values of resveratrol were determined as 34.36 and 131.93 µM for HEK-293 and HUH-7 cells, respectively. Furthermore, the effects of those phenolic compounds on protein and mRNA expressions of Vitamin D metabolizing CYPs were investigated by Western Blotting and q-RT-PCR, respectively. The results showed that quercetin treatment caused an increase in mRNA expression of CYP2R1 and CYP27A1 in HEK-293 cells. However, protein expressions of CYP2R1 and CYP27A1 in HEK-293 cells were decreased by quercetin treatment. In addition, CYP24A1 protein expression was increased in response to quercetin treatment for the same cell line. Resveratrol resulted an increase in mRNA expression of CYP27A1, CYP27B1 and CYP24A1 in HEK-293 cells. However, protein expressions of CYP27A1 and CYP27B1 in HEK293 cells were decreased by resveratrol treatment. In addition, CY24A1 protein expression was upregulated with resveratrol treatment in HEK293 cells. In HUH-7 cells, quercetin treatment caused an increase in mRNA expression of CYP2R1, CYP27B1 and CYP24A1. However, it did not significantly affect the protein expressions of CYP27B1 and CYP24A1 in HUH-7 cells. Resveratrol treatment caused an increase in mRNA expression of CYP2R1 and CYP24A1, whereas it caused decrease in mRNA expression of CYP27A1 and CYP27B1 in HUH-7 cells. Furthermore, resveratrol resulted a decrease in protein expression of CYP24A1. In this study, no CYP27A1 and CYP2R1 protein expressions were observed in HUH7 cells. All these results show that there is no significant correlation between mRNA transcript and protein levels. In conclusion, protein and mRNA expressions of vitamin D metabolizing CYPs were modulated differently depending on the type of phenolic compound and cell line.  

Subject Keywords

Vitamin D ., Quercetin., Resveratrol., Cytochrome P-450., Western immunoblotting.

URI

http://etd.lib.mtu.edu.tr/upload/12620426/index.pdf
https://hdl.handle.net/11511/26081

Collections

Graduate School of Natural and Applied Sciences, Thesis