Evaluation of Ginkgo biloba extract as an activator of human glucocorticoid receptor.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba, which is one of the most frequently used herbal medicines, is commonly used in the management of several conditions, including memory impairment. Previously, it was reported to decrease the expression of peripheral benzodiazepine receptor and the biosynthesis of glucocorticoids, thereby regulating glucocorticoid levels. However, it is not known whether Ginkgo biloba extract regulates the function of the glucocorticoid receptor. AIM OF THE STUDY: We determined whether Ginkgo biloba extract and several of its chemical constituents affect the activity of human glucocorticoid receptor (hGR). MATERIALS AND METHODS: A hGR-dependent reporter gene assay was conducted in HepG2 human hepatocellular carcinoma cells and hGR target gene expression assays were performed in primary cultures of human hepatocytes. RESULTS: Multiple lots and concentrations of the extract and several of its chemical constituents (ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, and bilobalide) did not increase hGR activity, as assessed by a cell-based luciferase reporter gene assay. The extract did not influence the expression of hGR target genes, including tyrosine aminotransferase (hTAT), constitutive androstane receptor (hCAR), or pregnane X receptor (hPXR), in primary cultures of human hepatocytes. Moreover, hGR antagonism by mifepristone (also known as RU486) did not attenuate the extent of induction of hCAR- and hPXR-regulated target genes CYP2B6 and CYP3A4 by Ginkgo biloba extract. CONCLUSION: Ginkgo biloba extract, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, and bilobalide are not activators of hGR. Furthermore, the extract does not influence the hGR-hCAR or the hGR-hPXR signaling pathway in primary cultures of human hepatocytes.

Isoform-selective activation of human constitutive androstane receptor by Ginkgo biloba extract: functional analysis of the SV23, SV24, and SV25 splice variants.

Naturally occurring splice variants of human constitutive androstane receptor (hCAR) exist, including hCAR-SV23 (insertion of amino acids SPTV), hCAR-SV24 (APYLT), and hCAR-SV25 (SPTV and APYLT). An extract of Ginkgo biloba was reported to activate hCAR-SV24 and the wild type (hCAR-WT). However, it is not known whether it selectively affects hCAR splice variants, how it activates hCAR isoforms, and which chemical is responsible for the effects of the extract. Therefore, we evaluated the impact of G. biloba extract on the functionality of hCAR-SV23, hCAR-SV24, hCAR-SV25, and hCAR-WT and compared it with that of phenobarbital, di-(2-ethylhexyl)phthalate (DEHP), 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO), and 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in cell-based reporter gene assays. Among the hCAR splice variants investigated, only hCAR-SV23 was activated by G. biloba extract, and this required cotransfection of a retinoid X receptor α (RXRα) expression plasmid. The extract activated hCAR-SV23 to a lesser extent than hCAR-WT, but ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, and bilobalide were not responsible for the effects of the extract. CITCO activated hCAR-SV23, hCAR-SV24, and hCAR-WT. By comparison, phenobarbital activated hCAR-WT, whereas DEHP activated hCAR-SV23, hCAR-SV24 (with exogenous RXRα supplementation), and hCAR-WT. TCPOBOP did not affect the activity of any of the isoforms. G. biloba extract and phenobarbital did not bind or recruit coactivators to the ligand-binding domains of hCAR-WT and hCAR-SV23, whereas positive results were obtained with the controls (CITCO for hCAR-WT and DEHP for hCAR-SV23). In conclusion, G. biloba extract activates hCAR in an isoform-selective manner, and hCAR-SV23, hCAR-SV24, and hCAR-WT have overlapping, but distinct, sets of ligands.

Human pregnane X receptor agonism by Ginkgo biloba extract: assessment of the role of individual ginkgolides.

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 6ß-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.

Modulation of CYP1B1 and CYP1A1 gene expression and activation of aryl hydrocarbon receptor by Ginkgo biloba extract in MCF-10A human mammary epithelial cells.

The aryl hydrocarbon receptor (AhR) signaling pathway regulates the production of CYP1B1 and CYP1A1, which catalyze the bioactivation of various procarcinogens. In the present study, we investigated the effect of Ginkgo biloba extract and some of its chemical constituents on CYP1B1 and CYP1A1 gene expression and AhR activity in cultured MCF-10A human mammary epithelial cells. Treatment of MCF-10A cells with noncytotoxic concentrations of G. biloba extract (25-300 microg/mL for 24 or 48 h) increased CYP1B1 and CYP1A1 mRNA expression, which was accompanied by an increase in CYP1-mediated ethoxyresorufin O-dealkylation activity. The inductive effects of G. biloba extract were attenuated by an AhR antagonist (3',4'-dimethoxyflavone). G. biloba extract (25-300 microg/mL) increased AhR-dependent reporter activity, as determined in MCF-10A cells transfected with an AhR-regulated luciferase reporter plasmid (pGudluc6.1). Bilobalide and ginkgolides A, B, C, and J were not responsible for the modulation of CYP1B1 and CYP1A1 gene expression or AhR activation by G. biloba extract. In contrast, quercetin increased CYP1B1 and CYP1A1 gene expression and activated AhR, whereas kaempferol and isorhamnetin suppressed constitutive CYP1B1 expression and antagonized AhR activation by benzo[a]pyrene. Overall, our findings provide an impetus for future investigations on the effect of G. biloba extract in CYP1-mediated chemical carcinogenesis.

Activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) by herbal medicines.

Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are transcription factors that control the expression of a broad array of genes involved not only in transcellular transport and biotransformation of many drugs, other xenochemicals, and endogenous substances, such as bile acid, bilirubin, and certain vitamins, but also in various physiological/pathophysiological processes such as lipid metabolism, glucose homeostasis, and inflammation. Ligands of PXR and CAR are chemicals of diverse structures, including naturally occurring compounds present in herbal medicines. The overall aim of this article is to provide an overview of our current understanding of the role of herbal medicines as modulators of PXR and CAR.

Inhibition of human CYP2B6-catalyzed bupropion hydroxylation by Ginkgo biloba extract: effect of terpene trilactones and flavonols.

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.

Identification of Ginkgo biloba as a novel activator of pregnane X receptor.

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.

Effect of Ginkgo biloba extract on oxidative metabolism of valproic acid in hepatic microsomes from donors with the CYP2C9*1/*1 genotype.

We investigated the effect of Ginkgo biloba extracts and some of its individual constituents on the oxidative metabolism of valproic acid (VPA) in hepatic microsomes from donors with the CYP2C9*1/*1 genotype. G. biloba extract decreased 4-ene-VPA, 3-OH-VPA, 4-OH-VPA, and 5-OH-VPA formation with mean (+/- SE) IC50 values of 340 +/- 40 microg/mL, 370 +/- 100 microg/mL, 180 +/- 30 microg/mL, and 210 +/- 20 microg/mL, respectively. This was associated with inhibition of not only CYP2C9*1, but also CYP2A6 and CYP2B6. Bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, and isorhamnetin-3-O-rutinoside were not responsible for the inhibition of VPA metabolism by the extract. When analyzed as the sum of the aglycone and total glycosides present in the extract, quercetin decreased 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation by 76%, 51%, and 70%, respectively, kaempferol decreased 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation by 65%, 46%, and 49%, respectively, and isorhamnetin decreased 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation by 29%, 26%, and 31%, respectively. The 3 aglycones did not affect 3-OH-VPA formation. In summary, G. biloba extract decreased hepatic microsomal formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA, but the effect was not due to the terpene trilactones or flavonol glycosides investigated in our study.

Ginkgolide A contributes to the potentiation of acetaminophen toxicity by Ginkgo biloba extract in primary cultures of rat hepatocytes.

The present cell culture study investigated the effect of Ginkgo biloba extract pretreatment on acetaminophen toxicity and assessed the role of ginkgolide A and cytochrome P450 3A (CYP3A) in hepatocytes isolated from adult male Long-Evans rats provided ad libitum with a standard diet. Acetaminophen (7.5-25 mM for 24 h) conferred hepatocyte toxicity, as determined by the lactate dehydrogenase (LDH) assay. G. biloba extract alone increased LDH leakage in hepatocytes at concentrations > or =75 mug/ml and > or =750 mug/ml after a 72 h and 24 h treatment period, respectively. G. biloba extract (25 or 50 mug/ml once every 24 h for 72 h) potentiated LDH leakage by acetaminophen (10 mM for 24 h; added at 48 h after initiation of extract pretreatment). The effect was confirmed by a decrease in [(14)C]-leucine incorporation. At the level present in a modulating concentration (50 mug/ml) of the extract, ginkgolide A (0.55 mug/ml), which increased CYP3A23 mRNA levels and CYP3A-mediated enzyme activity, accounted for part but not all of the potentiating effect of the extract on acetaminophen toxicity. This occurred as a result of CYP3A induction by ginkgolide A because triacetyloleandomycin (TAO), a specific inhibitor of CYP3A catalytic activity, completely blocked the effect of ginkgolide A. Ginkgolide B, ginkgolide C, ginkgolide J, quercetin, kaempferol, isorhamnetin, and isorhamnetin-3-O-rutinoside did not alter the extent of LDH leakage by acetaminophen. In summary, G. biloba pretreatment potentiated acetaminophen toxicity in cultured rat hepatocytes and ginkgolide A contributed to this novel effect of the extract by inducing CYP3A.

Contribution of CYP2C9, CYP2A6, and CYP2B6 to valproic acid metabolism in hepatic microsomes from individuals with the CYP2C9*1/*1 genotype.

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 approximately 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.

Distinct role of bilobalide and ginkgolide A in the modulation of rat CYP2B1 and CYP3A23 gene expression by Ginkgo biloba extract in cultured hepatocytes.

In the present study, primary cultures of rat hepatocytes were treated for 48 h with one of several extracts of Ginkgo biloba (10, 100, or 1000 microg/ml). Maximal increase in CYP2B1 and CYP3A23 mRNA levels was obtained at 100 microg/ml. This concentration of G. biloba extract also increased CYP3A2 and CYP3A18 mRNA expression in addition to CYP2B-mediated 7-benzyloxyresorufin O-dealkylation (BROD) and CYP3A-mediated testosterone 6beta-hydroxylation. In other experiments, cultured hepatocytes were treated for 48 h with bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, kaempferol, quercetin, isorhamnetin, or a flavonol diglycoside at a concentration that represented the level present in a 100 microg/ml concentration of an extract. Only bilobalide (2.8 microg/ml) increased CYP2B1 mRNA expression, and the -fold increase (7.9 +/- 0.5; mean +/- S.E.M.) was similar to that (8.3 +/- 1.7) by the extract. By comparison, only ginkgolide A (1.1 microg/ml) increased CYP3A23 mRNA expression, but the extent (2.6 +/- 0.5-fold) was less than the 5.3 +/- 1.7-fold increase by the extract. A greater concentration (5 microg/ml) of ginkgolide A was required to elevate CYP3A2 and CYP3A18 mRNA expression. Over the range of 1 to 5 microg/ml, bilobalide increased CYP2B1 mRNA and BROD, but not CYP3A23 mRNA or testosterone 6beta-hydroxylation, whereas ginkgolide A increased CYP3A23 mRNA and testosterone 6beta-hydroxylation, but not CYP2B1 mRNA or BROD. Overall, our novel results indicate a distinct role of bilobalide and ginkgolide A in the modulation of CYP2B1 and CYP3A23 gene expression and enzyme activities by G. biloba extract in primary cultures of rat hepatocytes.

Effect of Ginkgo biloba extract on procarcinogen-bioactivating human CYP1 enzymes: identification of isorhamnetin, kaempferol, and quercetin as potent inhibitors of CYP1B1.

In the present study, we investigated the effect of Ginkgo biloba extracts and some of its individual constituents on the catalytic activity of human cytochrome P450 enzymes CYP1B1, CYP1A1, and CYP1A2. G. biloba extract of known abundance of terpene trilactones and flavonol glycosides inhibited 7-ethoxyresorufin O-dealkylation catalyzed by human recombinant CYP1B1, CYP1A1, and CYP1A2, and human liver microsomes, with apparent Ki values of 2 +/- 0.3, 5 +/- 0.5, 16 +/- 1.4, and 39 +/- 1.2 microg/ml (mean +/- SE), respectively. In each case, the mode of inhibition was of the mixed type. Bilobalide, ginkgolides A, B, C, and J, quercetin 3-O-rutinoside, kaempferol 3-O-rutinoside, and isorhamentin 3-O-rutinoside were not responsible for the inhibition of CYP1 enzymes by G. biloba extract, as determined by experiments with these individual chemicals at the levels present in the extract. In contrast, the aglycones of quercetin, kaempferol, and isorhamentin inhibited CYP1B1, CYP1A1, and CYP1A2. Among the three flavonol aglycones, isorhamentin was the most potent in inhibiting CYP1B1 (apparent Ki = 3 +/- 0.1 nM), whereas quercetin was the least potent in inhibiting CYP1A2 (apparent Ki = 418 +/- 50 nM). The mode of inhibition was competitive, noncompetitive, or mixed, depending on the enzyme and the flavonol. G. biloba extract also reduced benzo[a]pyrene hydroxylation, and the effect was greater with CYP1B1 than with CYP1A1 as the catalyst. Overall, our novel findings indicate that G. biloba extract and the flavonol aglycones isorhamnetin, kaempferol, and quercetin preferentially inhibit the in vitro catalytic activity of human CYP1B1.

Lack of evidence for induction of CYP2B1, CYP3A23, and CYP1A2 gene expression by Panax ginseng and Panax quinquefolius extracts in adult rats and primary cultures of rat hepatocytes.

Treatment of rats with a single oral dose (10-30 mg/kg) of a crude Panax ginseng extract of unknown ginsenoside content has been reported to modestly increase hepatic microsomal cytochrome P450-mediated aminopyrine N-demethylation activity. In the present study, we compared the effect of P. ginseng and Panax quinquefolius extracts on rat hepatic CYP2B1, CYP3A23, and CYP1A2 gene expression. Adult male Sprague-Dawley rats (250-275 g) received, by oral gavage or i.p., P. ginseng extract [4% (w/w) total ginsenosides; 30 or 100 mg/kg/day for 1 or 4 days], P. quinquefolius extract [10% (w/w) total ginsenosides; 100 or 400 mg/kg/day for 21 consecutive days), or an equivalent volume (2 ml/kg) of the vehicle (0.9% NaCl or 0.3% carboxymethylcellulose) and were terminated 1 day after the last dose. P. ginseng and P. quinquefolius extracts did not affect body weight gain, absolute or relative liver weight, hepatic CYP2B1, CYP3A23, or CYP1A2 mRNA expression, or microsomal CYP2B-mediated 7-benzyloxyresorufin O-dealkylation (BROD) or CYP1A-mediated 7-ethoxyresorufin O-dealkylation (EROD) activity. In contrast, results from positive control experiments indicated that phenobarbital increased CYP2B1 mRNA and BROD activity, dexamethasone increased CYP3A23 mRNA, and beta-naphthoflavone increased CYP1A2 mRNA and EROD activity levels. Treatment of primary cultures of rat hepatocytes with either of the ginseng extracts (0.1-1000 microg/ml for 2 days) also did not affect CYP2B1 or CYP3A23 mRNA expression. Overall, our data indicate that P. ginseng and P. quinquefolius extracts do not increase rat hepatic CYP2B1, CYP3A23, or CYP1A2 gene expression.

Effect of Ginkgo biloba extract on rat hepatic microsomal CYP1A activity: role of ginkgolides, bilobalide, and flavonols.

The present study investigated the in vitro effect of Ginkgo biloba extracts and some of the individual constituents (ginkgolides, bilobalide, and flavonols such as kaempferol, quercetin, isorhamnetin, and their glycosides) on CYP1A-mediated 7-ethoxyresorufin O-dealkylation in hepatic microsomes isolated from rats induced with beta-naphthoflavone. G. biloba extract competitively inhibited CYP1A activity, with an apparent Ki value of 1.6 +/- 0.4 microg/mL (mean +/- SE). At the concentrations present in the G. biloba extracts, ginkgolides A, B, C, and J and bilobalide did not affect CYP1A activity, whereas kaempferol (IC50 = 0.006 +/- 0.001 microg/mL, mean +/- SE), isorhamnetin (0.007 +/- 0.001 microg/mL), and quercetin (0.050 +/- 0.003 microg/mL) decreased this activity. The monoglycosides (1 and 10 microg/mL) and diglycosides (10 microg/mL) of kaempferol and quercetin but not those of isorhamnetin also inhibited CYP1A activity. The order of inhibitory potency was kaempferol approximately equal to isorhamnetin > quercetin, and for each of these flavonols the order of potency was aglycone >> monoglycoside > diglycoside. In summary, G. biloba extract competitively inhibited rat hepatic microsomal CYP1A activity, but the effect was not due to ginkgolides A, B, C, or J, bilobalide, kaempferol, quercetin, isorhamnetin, or the respective flavonol monoglycosides or diglycosides.

Transcript profiling of cytochrome P450 genes in HL-60 human leukemic cells: upregulation of CYP1B1 by all-trans-retinoic acid.

All-trans-retinoic acid (ATRA) is used in the treatment of promyelocytic acute leukemia. The biotransformation of this drug is catalyzed by various cytochrome P450 (CYP) enzymes, but relatively little is known about the effect of ATRA on CYP enzyme expression in leukemic cells. In the present study, we conducted transcript profiling of CYP and related genes in cultured HL-60 human promyelocytic leukemic cells and determined the effect of ATRA on the expression of these genes. Reverse transcription-polymerase chain reaction (RT-PCR) analysis with a block-cycler indicated the presence of CYP1B1 but not CYP1A1, CYP2B6, CYP2C8, CYP2C9, CYP3A4, CYP3A5, or CYP26A1 transcript in cultured HL-60 cells. ATRA treatment (0.1-40 microM for 3 days) increased CYP1B1 mRNA levels by up to 3 fold, as determined by a quantitative real-time PCR method. The same ATRA treatment also resulted in the detection of CYP26A1 but not CYP1A1, CYP2B6, CYP2C8, CY2C9, CYP3A4, or CYP3A5 mRNA. Additional experiments showed that phenobarbital increased CYP2B6 mRNA expression and that pregnane X receptor (PXR) but not constitutive androstane receptor (CAR) was detected in HL-60 cells. Overall, our novel findings indicate the upregulation of CYP1B1 by ATRA in HL-60 human promyelocytic leukemic cells shown for the first time to express PXR but not CAR mRNA.

In vitro effect of standardized ginseng extracts and individual ginsenosides on the catalytic activity of human CYP1A1, CYP1A2, and CYP1B1.

Ginseng extract has been reported to decrease the incidence of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated tumorigenesis in mice. A potential mechanism for this effect by ginseng is inhibition of DMBA-bioactivating cytochrome P450 (P450) enzymes. In the present in vitro study, we examined the effect of a standardized Panax ginseng (or Asian ginseng) extract (G115), a standardized Panax quinquefolius (or North American ginseng) extract (NAGE), and individual ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1) on CYP1 catalytic activities, as assessed by 7-ethoxyresorufin O-dealkylation. G115 and NAGE decreased human recombinant CYP1A1, CYP1A2, and CYP1B1 activities in a concentration-dependent manner. Except for the competitive inhibition of CYP1A1 by G115, the mode of inhibition was the mixed-type in the other cases. A striking finding was that NAGE was 45-fold more potent than G115 in inhibiting CYP1A2. Compared with G115, NAGE also preferentially inhibited 7-ethoxyresorufin O-dealkylation activity in human liver microsomes. Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1, either individually or as a mixture and at the levels reflecting those found in an inhibitory concentration (100 microg/ml) of NAGE or G115, did not influence CYP1 activities. However, at a higher ginsenoside concentration (50 microg/ml), Rb1, Rb2, Rc, Rd, and Rf inhibited these activities. Overall, our in vitro findings indicate that standardized NAGE and G115 extracts, which were not treated with calf serum or subjected to acid hydrolysis, inhibited CYP1 catalytic activity in an enzyme-selective and extract-specific manner, but the effects were not due to Rb1, Rb2, Rc, Rd, Re, Rf, or Rg1.