mRNA Expression Of CYP2C11 Research Articles

Modulatory effects of Benjakul extract on rat hepatic cytochrome P450 enzymes

Benjakul, a traditional Thai formulation, has been used as a carminative and adaptogenic drug. It consists of five plants, Piper chaba Hunter, Piper sarmentosum Roxb., Piper interruptum Opiz., Plumbago indica Linn., and Zingiber officinale Roscoe, in equal ratios. Some individual herbs present in Benjakul were reported to modulate cytochrome P450 (CYP) enzymes. This study aimed to investigate the effects of Benjakul extract on the activities and mRNA expression levels of hepatic CYP2C11 and CYP3A1 in rats. Adult male rats were orally administered 200, 400, or 600 mg/kg BW Benjakul extract for 28 days. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine levels were assayed. CYP2C11 and CYP3A1 activities were analyzed using cytochrome P450 assay kits. The mRNA expression of CYP2C11 and CYP3A1 was measured using a quantitative real-time PCR assay. Benjakul treatment significantly increased the serum ALT and BUN levels. At doses of 200, 400, and 600 mg/kg BW, Benjakul treatment increased hepatic CYP3A1 activity and CYP3A1 mRNA expression. CYP2C11 mRNA expression was unchanged by treatment with Benjakul extract; however, treatment with the high and middle doses of Benjakul extract increased CYP2C11 activity. Treament with Benjakul extract induced CYP2C11 and CYP3A1 activity in rats. Concurrent use of Benjakul with conventional drugs should be considered to potentially induce herb-drug interactions.

Modulatory Effects of Benjakul Extract on Rat Hepatic Cytochrome P450 Enzymes

Benjakul, a traditional Thai formulation, has been used as a carminative and adaptogenic drug. It consists of five plants, Piper chaba Hunt, Piper sarmentosum Roxb, Piper interruptum Opiz., Plumbago indica Linn. and Zingiber officinale Roscoe, in an equal ratio. Some individual herbs present in Benjakul were reported to modulate cytochrome P450 (CYP) enzymes. This study was aimed to investigate the effects of Benjakul extract on the activities and mRNA expression levels of hepatic CYP2C11 and CYP3A1 in rats. Adult male rats were orally administered 200, 400, and 600 mg/kg BW Benjakul extract for 28 days. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine levels were assayed. The activities of CYP2C11 and CYP3A1 were analyzed using cytochrome P450 assay kits. The mRNA expression of CYP2C11 and CYP3A1 was measured using a quantitative real-time PCR assay. Benjakul treatment significantly increased the serum ALT and BUN levels. At the doses of 200, 400, 600 mg/kg BW, Benjakul treatment increased hepatic CYP3A1 activity and CYP3A1 mRNA expression. CYP2C11 mRNA expression was unchanged by Benjakul extract treatment; however, at the high and middle doses, Benjakul extract treatment increased CYP2C11 activity. Benjakul extract treatment induced CYP2C11 and CYP3A1 activity in rats. Concurrent use of Benjakul with conventional drugs should be considered to potentially induce an herb-drug interaction. Funding Information: This work was supported by a grant from Walailak University, grant number WU-IRG-63-0. Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The experimental protocol was approved by the Animal Ethics Committee of Walailak University.

Role of NF-κB in cytochrome P450 epoxygenases down-regulation during an inflammatory process in astrocytes

Cytochrome P450 (CYP) epoxygenases and their metabolic products, epoxyeicosatrienoic acids (EETs), have been proposed as important therapeutic targets in the brain. However, CYP expression can be modified by the presence of diverse pro-inflammatory cytokines and the subsequent activation of the NF-κB pathway. It has been indicated that CYP epoxygenases are down-regulated by inflammation in the heart, kidney and liver. However, up to this point, there has been no evidence regarding regulation of CYP epoxygenases during inflammation in the brain. Therefore, in order to explore the effects of inflammation and NF-κB activation in CYP2J3 and CYP2C11 regulation, rat primary astrocytes cultures were treated with LPS with and without IMD-0354 (selective NF-κB inhibitor). Cyp2j3 and Cyp2c11 mRNA expression was determined by qRT-PCR; protein expression was determined by immunofluorescence and by Western Blot and total epoxygenase activity was determined by the quantification of EETs by ELISA. NF-κB binding sites in Cyp2j3 and Cyp2c11 promoter regions were bioinformatically predicted and Electrophoretic Mobility Shift Assays (EMSA) were performed to determine if each hypothetic response element was able to bind NF-κB complexes. Results shown that LPS treatment is able to down-regulate astrocyte CYP2J3 and CYP2C11 mRNA, protein and activity. Additionally, we have identified NK-κB as the transcription factor involved in this regulation.

Decreased nuclear receptor activity mediates down-regulation of drug metabolizing enzymes in chronic kidney disease through epigenetic modulation

C kidney disease (CKD) is associated with a decreased expression and activity of several cytochrome P450 enzymes. This may result in drug-associated toxicity in CKD patients taking drugs that are metabolized by affected isozymes. The objective of this study was to determine the mechanism of hepatic drug metabolizing enzyme down-regulation in CKD. Hepatic CYP3A1, CYP3A2 and CYP2C11 mRNA expression were determined in rats with surgically induced CKD. Chromatin Immunoprecipitation (ChIP) was performed to determine nuclear receptor and epigenetic mediated differences in the promoter region of these enzymes. Hepatic CYP3A and CYP2C11 mRNA expression was significantly decreased in CKD rats compared to controls (P<0.05). RNA polymerase II binding to the CYP3A and CYP2C11 promoter regions was decreased in CKD rats (P<0.05). ChIP also revealed a decreased PXR binding to the CYP3A2 promoter in CKD rats (P<0.05). HNF4α binding to the CYP3A and CYP2C11 promoter regions was also decreased compared to controls (P<0.05). The decrease in PXR and HNF4α binding was concurrent with diminished histone 4 acetylation in the CYP3A2 promoter locus for nuclear receptor activation. The uremic toxin indoxyl sulfate also mediates a decrease in CYP3A expression. A novel mechanism of drug metabolizing enzyme regulation in CKD was demonstrated. The results show that decreased CYP3A and CYP2C11 mRNA expression is secondary to decreased PXR and HNF4α binding as a result of histone modulation in CKD. These data may partially explain why patients with CKD have a higher incidence of adverse medication events than patients with normal kidney function.

Decreased Nuclear Receptor Activity Mediates Down‐ Regulation of Drug Metabolizing Enzymes in Chronic Kidney Disease Through Epigenetic Modulation.

ObjectiveTo determine the mechanism of hepatic drug metabolizing enzyme down‐regulation in chronic kidney disease (CKD).MethodsHepatic CYP3A1, CYP3A2 and CYP2C11 mRNA expression were determined in rats with surgically induced CKD. Chromatin Immunoprecipitation (ChIP) was performed to determine nuclear receptor and epigenetic mediated differences in the promoter region of these enzymes.ResultsHepatic CYP3A and CYP2C11 mRNA expression was significantly decreased in CKD rats compared to controls (P&lt;0.05). RNA polymerase II binding to the CYP3A and CYP2C11 promoter regions was decreased in CKD rats (P&lt;0.05). ChIP also revealed a decreased PXR binding to the CYP3A2 promoter in CKD rats (P&lt;0.05). HNF4α binding to the CYP3A and CYP2C11 promoter regions was also decreased compared to controls (P&lt;0.05). The decrease in PXR and HNF4α binding was concurrent with diminished histone 4 acetylation in the CYP3A2 promoter locus for nuclear receptor activation.ConclusionsWe demonstrate a novel mechanism of drug metabolizing enzyme regulation in CKD. Our results show that decreased CYP3A and CYP2C11 mRNA expression is secondary to decreased PXR and HNF4α binding as a result of histone modulation in CKD. This research is supported by the Natural Sciences and Engineering Research Council.

Rosuvastatin suppresses the liver microsomal CYP2C11 and CYP2C6 expression in male Wistar rats

The aim was to investigate whether rosuvastatin affect rat cytochrome P450 (CYP) 2C11 and CYP2C6. CYP2C11 and CYP2C6 are considered as counterparts of human CYP2C9, which metabolizes many drugs including S-warfarin, diclofenac or ibuprofen.The male Wistar rats were fed standard laboratory diet (STD) or high cholesterol diet (HCD, 1% of cholesterol, 10% of lard fat) for 21 days.Rosuvastatin administration in STD (0.03% w/w) resulted in decreased mRNA expression of CYP2C11 as well as of CYP2C6 (here significant) and in a significant decrease of the respective protein expression as well as of the enzyme activity of both CYP2C forms. When rosuvastatin was administered in the HCD, the mRNA expression of both CYP2C forms was significantly lowered; the protein and activity parameters did not show significant changes.These results suggest that CYP2C11 as well as CYP2C6 expression and activity are negatively affected by rosuvastatin and may be modulated by high cholesterol high fat diet. Therefore, it should be taken into consideration that drugs metabolized by CYP2C9 in human could interact with rosuvastatin, as it has been already suggested for warfarin (rosuvastatin has increased its anticoagulant effect in human), and for telmisartan, sildenafil and glimepiride.

Cytochrome P450-2C11 mRNA Is Not Expressed in Endothelial Cells Dissected from Rat Renal Arterioles

Background: Cytochrome P450 (CYP) isoenzymes (CYP2C and CYP2J) are involved in the production of epoxyeicosatrienoic acids, which are postulated as endothelium-derived hyperpolarizing factors (EDHFs). We hypothesized that if CYP2C11 is involved in the EDHF-mediated responses, its mRNA should be expressed in endothelial cells. We, therefore, examined the mRNA expression of CYP2C11 in endothelial cells of renal arterioles. Methods: Laser microdissection was applied to isolate endothelial cells from the renal arterioles of 4 male and 4 female Wistar rats. As a positive control of CYP2C11 expression, hepatocytes were also dissected from these rats. RNA was isolated and real-time quantitative polymerase chain reaction (Q-PCR) analysis was applied. Results: Q-PCR analysis showed that CYP2C11 mRNA was not expressed in laser microdissected endothelial cells of renal arterioles of male and female rats. CYP2C11 mRNA expression was highly abundant in hepatocytes dissected from male livers, but in female livers hardly any CYP2C11 mRNA was detected. Conclusion: We have shown that endothelial cells can be dissected from small renal arterioles by laser microdissection to study the mRNA expression of specific genes by Q-PCR. Using this novel tool, we demonstrated that the CYP2C11 mRNA was not expressed in the endothelial cells of renal arterioles. Therefore, we speculate that CYP2C11 does not contribute to the EDHF-mediated responses in renal arterioles.

Characterization of recombinant CYP2C11: a vitamin D 25-hydroxylase and 24-hydroxylase

Studies were performed to further characterize the male-specific hepatic recombinant microsomal vitamin D 25-hydroxlase CYP2C11, expressed in baculovirus-infected insect cells, and determine whether it is also a vitamin D 24-hydroxylase. 25- and 24-hydroxylase activities were compared with those of 10 other recombinant hepatic microsomal cytochrome P-450 enzymes expressed in baculovirus-infected insect cells. Each of them 25-hydroxylated vitamin D2, vitamin D3, 1alpha-hydroxyvitamin D2 (1alphaOHD2), and 1alpha-hydroxyvitamin D3 (1alphaOHD3). CYP2C11 had the greatest activity with these substrates, except vitamin D3, which had the same activity as four of the other enzymes. The descending order of 25-hydroxylation by CYP2C11 was 1alphaOHD3 > 1alphaOHD2 > vitamin D2 > vitamin D3. Each of the recombinant cytochrome P-450 enzymes 24-hydroxylated 1alphaOHD2. CYP2C11 had the greatest activity. 24-Hydroxylation of 1alphaOHD3 was very low, and there was none with vitamin D3. Only CYP2C11 24-hydroxylated vitamin D2. Structures of vitamin D metabolites, including 24-hydroxyvitamin D2, 1,24(S)-dihydroxyvitamin D2, and 1,24-dihydroxyvitamin D3, were confirmed by HPLC and gas chromatography retention times and characteristic mass spectrometric fragmentation patterns. In male rats, hypophysectomy significantly reduced body weight, liver weight, hepatic CYP2C11 mRNA expression, and 24- and 25-hydroxylation of 1alphaOHD2. Expression of CYP2J3 and CYP2R1 mRNA did not change. In male rat hepatocytes, CYP2C11 mRNA expression and 24- and 25-hydroxylation were significantly reduced after culture for 24 h compared with uncultured cells. Expression of CYP2J3 and CYP2R1 either increased or did not change. It is concluded that CYP2C11 is a male-specific hepatic microsomal vitamin D 25-hydroxylase that hydroxylates vitamin D2, vitamin D3, 1alphaOHD2, and 1alphaOHD3. CYP2C11 is also a vitamin D 24-hydroxylase.

Differential regulation of endobiotic-oxidizing cytochromes P450 in vitamin A-deficient male rat liver.

1. The hepatic CYP4A-dependent omega-hydroxylation of arachidonic acid and CYP2C11-dependent 2alpha-/16alpha-hydroxylations of testosterone were decreased to 74 and 60% of respective control in microsomal fractions from vitamin A-deficient rats. Decreases in the rates of arachidonic acid omega-1-hydroxylation and testosterone 6beta-, 7alpha- and 17alpha-hydroxylations were less pronounced. 2. Corresponding decreases in microsomal CYP4A and CYP2C11 immunoreactive protein expression to 64 and 68% of respective control were observed in vitamin A-deficient rat liver. Expression of CYP3A proteins was unchanged from vitamin A-adequate control. 3. Northern analysis revealed a selective decrease in CYP4A2 mRNA expression in vitamin A-deficient rat liver to approximately 5% of control; expression of the related CYP4A1/4A3 mRNAs was not decreased. CYP2C11 mRNA expression was also decreased in vitamin A-deficient male rat liver to 39% of control levels. 4. Intake of the deficient diet containing all-trans-retinoic acid (ATRA) during the final week of the experiment restored CYP4A2 mRNA and CYP4A protein. Administration of exogenous androgen or episodic growth hormone was ineffective. In contrast, CYP2C11 expression was restored by ATRA and androgen, but not by growth hormone. 5. From these studies it emerges that CYP4A2, a fatty acid omega-hydroxylase in rat liver, is highly dependent on vitamin A for optimal expression, whereas CYP2C11 is indirectly down regulated by androgen deficiency resulting from vitamin A-deficiency. Altered CYP expression in vitamin A-deficiency provides insights into the relationship between dietary constituents and the intracellular formation of vasoactive eicosanoids as well as the clearance of androgenic steroids.

Lack of Effect of Medium Supplementation With Pyruvate and Hormones on Cytochrome P450-mediated Activity of Rat Hepatocytes in Primary Culture.

The loss of cytochrome P450 (CYP) -dependent activity continues to be a problem in the use of cultured hepatocytes in xenobiotic toxicity studies. It has been reported that the inclusion of pyruvate and various hormones in the culture medium improves the maintenance of various hepatic functions, including that of CYP2C11 mRNA expression. We have studied this further, by investigating the effects of the addition of pyruvate and hormones on various CYP-dependent enzyme activities and on the CYP-dependent toxicity of precocene II in rat hepatocyte cultures. No beneficial effects of this medium supplementation could be demonstrated.