Conflicting data exist on the effect of ginsenosides on transactivation of human glucocorticoid receptor α (herein referred to as glucocorticoid receptor), and relatively little is known regarding ...the effect of these chemicals on transrepression of this receptor. We investigated the effect of 20(S)-protopanaxadiol (PPD), PPD-type ginsenosides (Rb1, Rb2, Rc, Rd, Rh2, and Compound K), 20(S)-protopanaxatriol (PPT), and PPT-type ginsenosides (Re, Rf, Rg1, and Rh1) on glucocorticoid receptor binding, transactivation, and transrepression. Each ginsenoside was less efficacious than dexamethasone (positive control) in binding to the ligand-binding domain of glucocorticoid receptor. Among the ginsenosides investigated, Rh2 had the smallest IC50 value (15 ± 1µM), whereas it was 0.02 ± 0.01µM for dexamethasone. In contrast to dexamethasone, none of the ginsenosides influenced glucocorticoid receptor transactivation or transrepression in LS180 human colorectal adenocarcinoma cells, as assessed in a dual-luciferase reporter gene assay. Rh2 did not affect the endogenous mRNA level of tyrosine aminotransferase (marker for glucocorticoid receptor transactivation) or corticosteroid-binding globulin (marker for glucocorticoid receptor transrepression) in HepG2 human hepatocellular carcinoma cells. This chemical also did not alter the response by a glucocorticoid receptor agonist (dexamethasone or Compound A) in the dual-luciferase reporter gene assay or target gene expression assay. In conclusion, ginsenosides were less efficacious and less potent than dexamethasone in binding to the ligand-binding domain of glucocorticoid receptor. The number of glycosylated groups was associated with a decrease in receptor binding potency. PPD-type and PPT-type ginsenosides are not modulators of glucocorticoid receptor transactivation or transrepression in LS180 and HepG2 cells.
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The present study investigated the role of specific human cytochrome P450 (CYP) enzymes in the in vitro metabolism of valproic acid (VPA) by a complementary approach that used individual ...cDNA-expressed CYP enzymes, chemical inhibitors of specific CYP enzymes, CYP-specific inhibitory monoclonal antibodies (MAbs), individual human hepatic microsomes, and correlational analysis. cDNA-expressed CYP2C9*1, CYP2A6, and CYP2B6 were the most active catalysts of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation. The extent of 4-OH-VPA and 5-OH-VPA formation by CYP1A1, CYP1A2, CYP1B1, CYP2C8, CYP2C19, CYP2D6, CYP2E1, CYP4A11, CYP4F2, CYP4F3A, and CYP4F3B was only 1–8% of the levels by CYP2C9*1. CYP2A6 was the most active in catalyzing VPA 3-hydroxylation, whereas CYP1A1, CYP2B6, CYP4F2, and CYP4F3B were less active. Correlational analyses of VPA metabolism with CYP enzyme-selective activities suggested a potential role for hepatic microsomal CYP2A6 and CYP2C9. Chemical inhibition experiments with coumarin (CYP2A6 inhibitor), triethylenethiophosphoramide (CYP2B6 inhibitor), and sulfaphenazole (CYP2C9 inhibitor) and immunoinhibition experiments (including combinatorial analysis) with MAb-2A6, MAb-2B6, and MAb-2C9 indicated that the CYP2C9 inhibitors reduced the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by 75–80% in a panel of hepatic microsomes from donors with the CYP2C9*1/*1 genotype, whereas the CYP2A6 and CYP2B6 inhibitors had a small effect. Only the CYP2A6 inhibitors reduced VPA 3-hydroxylation (by ∼50%). The extent of inhibition correlated with the catalytic capacity of these enzymes in each microsome sample. Overall, our novel findings indicate that in human hepatic microsomes, CYP2C9*1 is the predominant catalyst in the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA, whereas CYP2A6 contributes partially to 3-OH-VPA formation.
Molybdenum disulfide (MoS2), which is composed of active edge sites and a catalytically inert basal plane, is a promising catalyst to replace the state‐of‐the‐art Pt for electrochemically catalyzing ...hydrogen evolution reaction (HER). Because the basal plane consists of the majority of the MoS2 bulk materials, activation of basal plane sites is an important challenge to further enhance HER performance. Here, an in situ electrochemical activation process of the MoS2 basal planes by using the atomic layer deposition (ALD) technique to improve the HER performance of commercial bulk MoS2 is first demonstrated. The ALD technique is used to form islands of titanium dioxide (TiO2) on the surface of the MoS2 basal plane. The coated TiO2 on the MoS2 surface (ALD(TiO2)‐MoS2) is then leached out using an in situ electrochemical activation method, producing highly localized surface distortions on the MoS2 basal plane. The MoS2 catalysts with activated basal plane surfaces (ALD(Act.)‐MoS2) have dramatically enhanced HER kinetics, resulting from more favorable hydrogen‐binding.
The catalytically inert basal plane of MoS2 is activated for the hydrogen evolution reaction (HER) by combining the atomic layer deposition (ALD) technique and an in situ electrochemical activation process. The basal plane activated MoS2 (ALD(Act.)‐MoS2) catalysts significantly improve the HER performance, resulting from more favorable hydrogen‐binding.
The pregnane X receptor (PXR) is a ligand-activated nuclear receptor that acts as a xenobiotic sensor, responding to compounds of foreign origin, including pharmaceutical compounds, environmental ...contaminants, and natural products, to induce transcriptional events that regulate drug detoxification and efflux pathways. As such, the PXR is thought to play a key role in protecting the host from xenobiotic exposure. More recently, the PXR has been reported to regulate the expression of innate immune receptors in the intestine and modulate inflammasome activation in the vasculature. In the current study, we report that activation of the PXR in primed macrophages triggers caspase-1 activation and interleukin-1
release. Mechanistically, we show that this response is nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3-dependent and is driven by the rapid efflux of ATP and P2X purinoceptor 7 activation following PXR stimulation, an event that involves pannexin-1 gating, and is sensitive to inhibition of Src-family kinases. Our findings identify a mechanism whereby the PXR drives innate immune signaling, providing a potential link between xenobiotic exposure and the induction of innate inflammatory responses.
Oxidative stress has been associated with valproic acid (VPA) treatment, and mitochondrial dysfunction has been implicated in the pathogenesis of VPA-idiosyncratic hepatotoxicity. The present study ...investigated the effect of VPA and the role of GSH on oxidative stress, mitochondrial membrane potential, and toxicity in freshly isolated rat hepatocytes. Hepatocytes were isolated from Sprague-Dawley rats, and total levels of glutathione (GSH) reduced by pretreatment with a combination of L-buthionine sulfoximine (2 mM) and diethylmaleate (0.5 mM) prior to VPA (0–1000 μg/ml) treatment. Oxidative stress was determined by measuring the levels of 15-F2t-isoprostane (15-F2t-IsoP) and 2′,7′-dichlorofluorescein (DCF). Mitochondrial membrane potential (Δψm) was determined by using the dual-fluorescent dye JC-1, and cell viability was evaluated by the water-soluble tetrazolium salt WST-1 assay. Exposure of rat hepatocytes to VPA (0–1000 μg/ml) resulted in a time- and dose-dependent increase in 15-F2t-IsoP and DCF fluorescence, and these levels were further elevated in GSH-reduced hepatocytes. In control hepatocytes, VPA had no effect on cell viability; however, significant cytotoxicity was observed in the glutathione-depleted hepatocytes treated with 1000 μg/ml VPA. The Δψm was only reduced in glutathione-reduced hepatocytes at 500 and 1000 μg/ml VPA. Our novel findings indicate that acute treatment of freshly isolated rat hepatocytes with VPA resulted in oxidative stress, which occurred in the absence of cytotoxicity, and that glutathione confers protection to hepatocytes against mitochondrial damage by VPA.
Cytochrome P450 2B6 (CYP2B6) is expressed predominantly in human liver. It catalyzes the oxidative biotransformation of various drugs, including bupropion, which is an antidepressant and a tobacco ...use cessation agent. Serious adverse effects of high dosages of bupropion have been reported, including the onset of seizure. As Ginkgo biloba extract may be consumed with bupropion or another CYP2B6 drug substrate, potential exists for an herb-drug interaction. Therefore, we investigated the effect of G. biloba extract and some of its chemical constituents (terpene trilactones and flavonols) on the in vitro catalytic activity of CYP2B6 as assessed by the bupropion hydroxylation assay with recombinant enzyme and hepatic microsomes. The amount of hydroxybupropion was quantified by a novel and validated ultraperformance liquid chromatography/mass spectrometry method. Enzyme kinetic analysis indicated that G. biloba extract competitively inhibited hepatic microsomal CYP2B6-catalyzed bupropion hydroxylation (apparent K(i) was 162 +/- 14 microg/ml). Bilobalide and ginkgolides A, B, C, and J were not responsible for the inhibition of CYP2B6 catalytic activity by the extract. Whereas the 3-O-glucoside and 3-O-rutinoside of quercetin, kaempferol, and isorhamnetin had no effect, the corresponding aglycones (10 and 50 microg/ml) decreased hepatic microsomal bupropion hydroxylation. The inhibition of CYP2B6 by kaempferol was competitive (apparent K(i) was 10 +/- 1 microg/ml). In summary, G. biloba extract and its flavonol aglycones are naturally occurring inhibitors of in vitro CYP2B6 catalytic activity and bupropion hydroxylation. Future studies are needed to investigate whether G. biloba extract interacts in vivo with bupropion or other clinically important CYP2B6 drug substrates.
Pregnane X receptor (PXR; NR1I2) is a ligand-activated transcription factor that plays a role not only in drug metabolism and transport but also in various other biological processes. Ginkgo biloba ...is a herbal medicine commonly used to manage memory impairment. Treatment of primary cultures of rat hepatocytes with G. biloba extract increases the mRNA expression of CYP3A23, which is a target gene for rat PXR. The present study was conducted to test the hypothesis that G. biloba extract activates PXR. Treatment of mouse PXR (mPXR) or human PXR (hPXR)-transfected HepG2 cells with G. biloba extract at 200 microg/ml increased mPXR and hPXR activation by 3.2- and 9.5-fold, respectively. Dose-response analysis showed a log-linear increase in hPXR activation by the extract over the range of 200 to 800 microg/ml. To determine whether G. biloba extract induces hPXR target gene expression, cultured LS180 human colon adenocarcinoma cells were treated for 72 h with the extract. G. biloba extract at 200, 400, and 800 microg/ml increased CYP3A4 mRNA expression by 1.7-, 2.4-, and 2.5-fold, respectively. The same concentrations of the extract increased CYP3A5 (1.3-3.6-fold) and P-glycoprotein (ABCB) 1 (2.7-6.4-fold) mRNA expression. At concentrations (5 and 10 microM) that did not down-regulate PXR gene expression and were not cytotoxic, L-sulforaphane (an hPXR antagonist) decreased CYP3A4, CYP3A5, and ABCB1 gene expression in cells treated with G. biloba extract. In summary, G. biloba extract activated mPXR and hPXR in a cell-based reporter gene assay and induced CYP3A4, CYP3A5, and ABCB1 gene expression in hPXR-expressing LS180 cells.
Ginkgo biloba extract activates pregnane X receptor (PXR), but how this occurs is not known. Therefore, we investigated the mechanism of PXR activation by the extract and the role of five individual ...terpene trilactones in the activation. In a cell-based reporter gene assay, G. biloba extract activated human PXR (hPXR), and at a concentration present in the extract, ginkgolide A, but not ginkgolide B, ginkgolide C, ginkgolide J, or bilobalide was partially responsible for the increase in hPXR activity of the extract. Likewise, in cultured human hepatocytes, only ginkgolide A contributed to the increase in hPXR target gene expression (CYP3A4 mRNA and CYP3A-mediated testosterone 6β-hydroxylation). The extract, but none of the terpene trilactones, bound to hPXR ligand-binding domain, as analyzed by a time-resolved fluorescence resonance energy transfer competitive binding assay. Only the extract and ginkgolide A recruited steroid receptor coactivator-1, as determined by a mammalian two-hybrid assay. Compared with hPXR, rat PXR (rPXR) was activated to a lesser extent by G. biloba extract. Similar to hPXR, only ginkgolide A contributed to rPXR activation by the extract. In contrast to the effect of G. biloba extract on PXR function, it did not affect hPXR expression. Overall, the main conclusions are that G. biloba extract is an hPXR agonist, and among the five terpene trilactones investigated, only ginkgolide A contributes to the actions of the extract. Our findings provide insights into the biological and chemical mechanisms of hPXR activation by G. biloba extract.
The naturally occurring SV23 splice variant of human constitutive androstane receptor (hCAR-SV23) is activated by di-(2-ethylhexyl)phthalate (DEHP), which is detected as a contaminant in fetal bovine ...serum (FBS). In our initial experiment, we compared the effect of dialyzed FBS, charcoal-stripped, dextran-treated FBS (CS-FBS), and regular FBS on the basal activity and ligand-activation of hCAR-SV23 in a cell-based reporter gene assay. In transfected HepG2 cells cultured in medium supplemented with 10% FBS, basal hCAR-SV23 activity varied with the type of FBS (regular>dialyzed>CS). DEHP increased hCAR-SV23 activity when 10% CS-FBS, but not regular FBS or dialyzed FBS, was used. With increasing concentrations (1–10%) of regular FBS or CS-FBS, hCAR-SV23 basal activity increased, whereas in DEHP-treated cells, hCAR-SV23 activity remained similar (regular FBS) or slightly increased (CS-FBS). Subsequent experiments identified a serum-free culture condition to detect DEHP activation of hCAR-SV23. Under this condition, artemisinin, artemether, and arteether increased hCAR-SV23 activity, whereas they decreased it in cells cultured in medium supplemented with 10% regular FBS. By comparison, FBS increased the basal activity of the wild-type isoform of hCAR (hCAR-WT), whereas it did not affect the basal activity of the SV24 splice variant (hCAR-SV24) or ligand activation of hCAR-SV24 and hCAR-WT by 6-(4-chlorophenyl)imidazo2,1-b1,3thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). The use of serum-free culture condition was suitable for detecting CITCO activation of hCAR-WT and hCAR-SV24. In conclusion, FBS leads to erroneous classification of pharmacological ligands of hCAR-SV23 in cell-based assays, but investigations on functional ligands of hCAR isoforms can be conducted in serum-free culture condition.
•FBS leads to erroneous pharmacological classification of hCAR-SV23 ligands.•Artemisinin, artemether, and arteether activate hCAR-SV23 in the absence of FBS.•Serum-free culture condition can be used for identifying ligands of hCAR isoforms.
There is a growing body of evidence that exposure to endocrine disrupting chemicals and to estrogenic compounds in particular can affect the testis and male fertility. In the present study, the ...constitutive expression of steroidogenic and non-steroidogenic cytochrome P450 (CYP) and related enzymes in adult rat testis, and their regulation by estradiol and bisphenol A, were investigated. CYP1B1, CYP2A1, NADPH-cytochrome P450 oxidoreductase (POR) and microsomal epoxide hydrolase (mEH) proteins, together with CYP17A1 and 3β-hydroxysteroid dehydrogenase (HSD3B), were detected by immunoblot analysis in testicular microsomes prepared from untreated adult Sprague Dawley rats. In contrast, CYP1A, CYP2B, CYP2E, CYP2D, CYP2C, CYP3A, and CYP4A enzymes were not detected. Immunofluorescence staining of cryosections of perfusion-fixed testes showed that CYP1B1, CYP2A1, CYP17A1, and HSD3B were expressed exclusively or mainly in interstitial cells, whereas mEH and POR protein staining was detected both in interstitial cells and in seminiferous tubules. Testicular CYP1B1 and CYP2A1 protein levels were decreased following treatment of adult rats with estradiol benzoate at 0.004, 0.04, 0.4, or 4 μmol/kg/day or bisphenol A at 400 or 800 μmol/kg/day, for 14 days, whereas expression of HSD3B was unaffected. Testicular CYP17A1, POR, and mEH protein expression was also downregulated at the three highest dosages of estradiol benzoate and at both dosages of bisphenol A. The present study is the first to establish the cellular localization of CYP1B1, mEH, and POR in rat testis and to demonstrate the suppressive effect of bisphenol A on testicular CYP1B1, CYP2A1, mEH, and POR protein levels.
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