Differential UGT1A1 induction by chrysin in primary human hepatocytes and HepG2 Cells
ABSTRACT Chrysin, a dietary flavonoid, has been shown to markedly induce UGT1A1 expression and activity in HepG2 and Caco-2 cell lines; thus, it has been suggested to have clinical utility in the treatment of UGT1A1-mediated deficiencies, such as unconjugated hyperbilirubinemia or the prevention of 7-ethyl-10-hydroxycamptothecin (SN-38) toxicity. However, little is known about its induction potential in a more physiologically relevant model system, such as primary hepatocyte culture. In this study, induction of UGT1A1 expression (mRNA, protein, and activity) was investigated in primary human hepatocyte cultures after treatment with chrysin and other prototypical inducers. Endogenous nuclear receptor-mediated UGT1A1 induction was studied using transient transfection reporter assays in primary human hepatocytes and HepG2 cells. Results indicated that induction of UGT1A1 expression was minimal in human hepatocytes treated with chrysin compared with that in HepG2 cells (1.2-versus 11-fold, respectively). Subsequent experiments to determine whether the differential response was due to its metabolic stability revealed strikingly different elimination rate constants between the two cell systems (half-life of 13 min in human hepatocytes versus 122 min in HepG2 cell suspensions). Further study demonstrated that UGT1A1 mRNA expression could be induced in human hepatocyte cultures by either increasing the chrysin dosing frequency or by modulating chrysin metabolism, suggesting that the differential induction observed in hepatocytes and HepG2 cells was due to differences in the metabolic clearance of chrysin. In conclusion, this study suggests that the metabolic stability of chrysin likely would limit its ability to induce UGT1A1 in vivo.
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- "Benchmark dose values obtained for individual genes, and signaling pathways were used to derive REP values that were then compared with the published 2005 WHO TEF values. The cell model employed, primary cultures of rat hepatocytes, is generally thought to better mimic in vivo hepatocyte function than commonly used immortalized cells (Guillouzo and Guguen- Guillouzo, 2008; LeCluyse et al., 1999; Smith et al., 2005). This makes primary hepatocytes a good model for studying a large number of chemical concentrations on hepatic transcription activity. "
ABSTRACT: Toxic equivalency factors (TEFs) for dioxin-like compounds (DLCs) are largely based on relative potency (REP) values derived from biochemical endpoints such as enzyme activity. As of yet, REPs based on gene expression changes have not been accounted for in the TEF values. In this study, primary rat hepatocytes were treated for 24 hours with 11 concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), or 2,3,7,8-tetrachlorodibenzofuran (TCDF) ranging from 0.00001-100 nM. Differential changes in gene expression were analyzed using analysis of variance to assess the relative contributions of concentration, congener, and the interaction between concentration and congener for each gene. A total of 3,283 genes showed significant changes with concentration (FDR < 0.05 and fold-change ± 1.5 in at least one concentration for one congener). Among these genes, 399 were significant for both concentration and congener effects indicating parallel concentration response curves with significant differences in potency. Only eight genes showed a significant concentration and congener interaction term indicating a minority of genes show non-parallel dose response curves among the three congeners. Benchmark dose (BMD) modeling was used to derive BMD values for induced individual genes and signaling pathways. The REP values for 4-PeCDF and TCDF were generally three- to five-fold lower than the WHO TEF values on both a gene and pathway basis. These findings suggest that the WHO TEF values may possibly over-predict the potency of these PCDD/F congeners, and demonstrate the importance of identifying functional pathways relevant to the toxicological MOA for establishing pertinent REPs.Toxicological Sciences 12/2013; 136(2):595-604. DOI:10.1093/toxsci/kft203 · 3.85 Impact Factor
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- "effects, better alternative to it includes, dietary flavones which do not have problem of side effects and are considered suitable to treat hyperbilirubinemia  . One of the important flavonoids belonging to this class is chrysin (5,7-dihydroxyflavone); chrysin has been shown to induce UGT1A1 expression in liver cells and greatly enhanced bilirubin glucuronidation in HepG2 cells  . Moreover, there are convincing evidences on chrysin's ability to induce UGT1A1 expression in tissue culture models . "
ABSTRACT: Accumulation of bilirubin, primarily because of its insolubility, has been found to be associated with liver diseases including jaundice. Free bilirubin is insoluble; its glucuronidation by bilirubin-UGT enzyme (UGT1A1) makes it soluble and eliminates it through urine and faeces. Taking CCl(4) induced rat liver dysfunction model, we demonstrated that suppression of UGT1A1 activity in rat liver increased serum bilirubin level which could be reversed by carlinoside (Cln), a flavone glycoside. Although Cln is a flavone compound, it escaped self-glucuronidation in the intestine and readily absorbed. Kinetic study of microsomal UGT1A1 from HepG2 cells suggested that Cln enhanced enzyme activity by increasing V(max) without altering K(m). This altered V(max) was found to be due to UGT1A1 overexpression by Cln which was observed in both HepG2 and rat primary hepatocytes. Since Nrf2 is the transcription factor of UGT1A1, we examined whether Cln effect on UGT1A1 overexpression is mediated through Nrf2. In Nrf2 knock-out cells, Cln could not elevate UGT1A1 activity indicating Nrf2 to be its target. Cln significantly increased Nrf2 gene expression in HepG2 cells which was subsequently localized in nuclear region. Results from ChIP assay showed that Cln markedly augmented Nrf2 binding to UGT1A1 promoter that consequently enhanced reporter activity. Our findings therefore show that Cln upregulated Nrf2 gene expression, increased its nuclear translocation and stimulated UGT1A1 promoter activity. Total outcome of these events brought about a significant increase of bilirubin glucuronidation. Cln therefore could be a worthy choice to intervene hyperbilirubinemia due to liver dysfunction.Biochemical pharmacology 07/2011; 82(9):1186-97. DOI:10.1016/j.bcp.2011.07.069 · 5.01 Impact Factor
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- "Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Valencia, CA) and reverse-transcribed using the High-Capacity cDNA Archive Kit (Applied Biosystems) per manufacturers' instructions. Primers and probes for CYP2B6, CYP3A4, UGT1A1, and MDR1 genes (Table 1) were designed using Primer Express version 2.0 (Applied Biosystems) and entered into the National Center for Biotechnology Information BLAST to ensure specificity as described previously (Maglich et al., 2002; Smith et al., 2005; Faucette et al., 2007; Li et al., 2008). The mRNA expression of CYP2B6, CYP3A4, UGT1A1, and MDR1 was normalized against that of human ␤-actin, which was detected using a predeveloped primer/probe mixture (Applied Biosystems). "
ABSTRACT: Methadone (MD) is the most established substance abuse pharmacotherapy of choice for the management of heroin dependence. To date, drug-drug interactions involving MD have been characterized asymmetrically among existing reports, which describe how other drugs affect the metabolic or pharmacokinetic profiles of MD; however, limited information is available regarding the potential for MD to influence similar fates of coadministered drugs. Moreover, little to no mechanistic evidence has been explored. Here, we show that MD induces hepatic drug-metabolizing enzymes (DMEs) through the activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). Real-time polymerase chain reaction analysis of human hepatocyte cultures revealed that MD induces the mRNA expression of CYP2B6, CYP3A4, UGT1A1, and multidrug resistance 1 in a concentration-related manner, with the maximal induction of CYP2B6 challenging that of the induction by rifampicin. Furthermore, MD-mediated induction of CYP2B6 and CYP3A4 proteins was observed in Western blot analysis. In cell-based reporter assays, MD significantly increased human (h) PXR-mediated CYP2B6 reporter activities but exhibited minimal effect on hCAR activation as a result of the constitutive activity of hCAR in HepG2 cells. Further studies revealed that treatment with MD resulted in significant nuclear accumulation of adenovirus/enhanced yellow fluorescent protein tagged-hCAR in human hepatocytes, which has been regarded as the initial step of CAR activation. Additional analysis of the two enantiomers of MD, R-(-)-MD (active) and S-(+)-MD (inactive), indicates the lack of stereoselectivity pertaining to MD-mediated DME induction. Overall, our results show that MD induces the hepatic expression of multiple DMEs by activating PXR- and CAR-mediated pathways.Drug metabolism and disposition: the biological fate of chemicals 07/2009; 37(9):1887-94. DOI:10.1124/dmd.109.027854 · 3.25 Impact Factor