Effects of quercetin on CYP450 and cytokines in Aroclor 1254 injured endometrial cells of the pregnant rats.

Polychlorinated biphenyls (PCBs) are widespread persistent residual environmental pollutants, which affect seriously the growth and reproductive alterations in humans and animals. Aroclor 1254 is a commercial mixture of PCBs. Quercetin is a flavonoid, which acts on estrogen receptors and causes the development of estrogen-related diseases. In this paper, the primary cultured endometrial cells in the pregnant rats were isolated and Aroclor 1254 was used to induce the injured endometrial cells model. The cells were treated with gradient quercetin, the viability of the endometrial cells, the expressions of CYP450, the contents of TNF-α, IL-6, estradiol (E2), and progesterone (P4) were measured. It showed that the viability of the cultured endometrial cells, the expression of CYP1A1 and CYP2B1, and the contents of TNF-α, E2, and IL-6 in the injured endometrial cells increased with the treatment of quercetin. It shows that quercetin has protective effect on the injured endometrial cells in the pregnant rats, this provide a basis on herbal medicine protection for animal reproductive diseases caused by environmental endocrine disruptors.

Increased exposure of vitamin A by Chrysanthemum morifolium Ramat extract in rat was not via induction of CYP1A1, CYP1A2, and CYP2B1.

The aim of this study was to investigate the effect of Chrysanthemum morifolium Ramat (CM) extract on the pharmacokinetics of retinol and activities of cytochrome P450s (CYP450s) related to retinoid metabolism. Rats were treated with CM extract for 15 d. Plasma concentrations of retinol were measured following oral administration of retinol (45 mg/kg). Basal levels of retinol and retinoic acid in serum and liver were also measured. 7-Ethoxyresorufin-O-deethylase activity, phenacetin-O-deethylase activity, and 7-pentoxyresorufin-O-deethylase activities were used to assay the activities of CYP1A1, CYP1A2, and CYP2B1 in hepatic microsomes of rats, respectively. Protein expressions of the 3 CYP450s were measured by western blot. Our studies demonstrated that CM extract dose-dependently increased basal level of retinol in serum. In pharmacokinetic experiment, CM extract dose-dependently increased plasma concentrations of retinol after oral administration of retinol to rats treated with CM extract. But activities and expressions of CYP1A1, CYP1A2, and CYP2B1 in hepatic microsomes of rats were also induced by CM extract.

Green tea extract increases cyclophosphamide-induced teratogenesis by modulating the expression of cytochrome P-450 mRNA.

The effects of green tea extract (GTE) on the fetal development and external, visceral and skeletal abnormalities induced by cyclophosphamide were investigated in rats. Pregnant rats were daily administered GTE (100mg/kg) by gavage for 7 d, from the 6th to 12th day of gestation, and intraperitoneally administered with cyclophosphamide (11mg/kg) 1h after the final treatment. On the 20th day of gestation, maternal and fetal abnormalities were determined by Cesarian section. Cyclophosphamide was found to reduce fetal and placental weights without increasing resorption or death. In addition, it induced malformations in live fetuses; 94.6%, 41.5% and 100% of the external (skull and limb defects), visceral (cleft palate and ureteric dilatation) and skeletal (acrania, vertebral/costal malformations and delayed ossification) abnormalities. When pre-treated with GTE, cyclophosphamide-induced body weight loss and abnormalities of fetuses were remarkably aggravated. Moreover, repeated treatment with GTE greatly increased mRNA expression and activity of hepatic cytochrome P-450 (CYP) 2B, which metabolizes cyclophosphamide into teratogenic acrolein and cytotoxic phosphoramide mustard, while reducing CYP3A expression (a detoxifying enzyme). The results suggest that repeated intake of GTE may aggravate cyclophosphamide-induced body weight loss and malformations of fetuses by modulating CYP2B and CYP3A.

Regional specificity in deltamethrin induced cytochrome P450 expression in rat brain.

Oral administration of deltamethrin (5 mg/kg x7 or 15 or 21 days) was found to produce a time-dependent increase in the mRNA expression of xenobiotic metabolizing cytochrome P450 1A1 (CYP1A1), 1A2 and CYP2B1, 2B2 isoenzymes in rat brain. RT-PCR studies further showed that increase in the mRNA expression of these CYP isoenzymes observed after 21 days of exposure was region specific. Hippocampus exhibited maximum increase in the mRNA expression of CYP1A1, which was followed by pons-medulla, cerebellum and hypothalamus. The mRNA expression of CYP2B1 also exhibited maximum increase in the hypothalamus and hippocampus followed by almost similar increase in midbrain and cerebellum. In contrast, mRNA expression of CYP1A2 and CYP2B2, the constitutive isoenzymes exhibited relatively higher increase in pons-medulla, cerebellum and frontal cortex. Immunoblotting studies carried out with polyclonal antibody raised against rat liver CYP1A1/1A2 or CYP2B1/2B2 isoenzymes also showed increase in immunoreactivity comigrating with CYP1A1/1A2 or 2B1/2B2 in the microsomal fractions isolated from hippocampus, hypothalamus and cerebellum of rat treated with deltamethrin. Though the exact relationship of the xenobiotic metabolizing CYPs with the physiological function of the brain is yet to be clearly understood, the increase in the mRNA expression of the CYPs in the brain regions that regulate specific brain functions affected by deltamethrin have further indicated that modulation of these CYPs could be associated with the various endogenous functions of the brain.

Cytochrome P450 gene induction in rats ex vivo assessed by quantitative real-time reverse transcriptase-polymerase chain reaction (TaqMan).

Drug-induced changes in expression of cytochrome P450 (P450) genes are a significant issue in the preclinical development of pharmaceuticals. For example, preclinically, P450 induction can affect safety studies by reducing the systemic exposure of a compound undergoing toxicological evaluation, thus limiting the exposure that can be safely investigated in patients. Therefore, the induction potential of candidate drugs has been studied as part of the drug development process, typically using protein and/or catalytic end points. However, measuring changes in the levels of mRNA using TaqMan technology offers the opportunity to investigate this issue with the advantages of better dynamic range and specific enzyme identification. Here, we describe the TaqMan application to study ex vivo the P450 gene induction in the rat. Initially, livers from rats dosed with the prototypic P450 inducers beta-napthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX), and clofibric acid (CLO) were analyzed for mRNA levels of CYP1A1, 1A2, 2B1, 2B2, 2E1, 3A2, 3A23, and 4A1 and compared with control animals. The maximum fold induction of mRNA varied: 2500-fold for CYP1A1 with BNF, 680-fold for CYP2B1 with PB, 59-fold for CYP3A23 with DEX, and 16-fold for CYP4A1 with CLO. This method was then applied to estimate the inductive potential of putative drug candidates undergoing rodent toxicological evaluation. We present a summary of these data that demonstrates the sensitivity and specificity of the TaqMan assay to distinguish between inducers and noninducers and that offers a highly specific alternative to the quantification of drug effects on P450 expression using immunodetection and substrate metabolism.

CAR expression and inducibility of CYP2B genes in liver of rats treated with PB-like inducers.

The expression of the CAR gene and inducibility of CYP2B protein in the liver of male Wistar rats treated with phenobarbital (PB) and triphenyldioxane (TPD) were investigated. To clarify the role of phosphorylation/dephosphorylation in these processes, rats were treated with inhibitors of Ca(2+)/calmodulin-dependent kinase II (W7) or protein phosphatases PP1 and PP2A (OA) before induction. Constitutive expression of the CAR gene in livers of untreated rats was detected by multiplex RT-PCR. Treatment with W7 resulted in a 2.8-fold induction of CAR gene expression, whereas OA led to a 2.4-fold decrease of the mRNA level. The same results were obtained for CYP2B genes expression, which were increased by W7 treatment (two-fold) and decreased by OA (2.3-fold). PB-induction did not lead to significant alteration in the level of CAR gene expression, although CYP2B genes expression was enhanced two-fold over control values. TPD caused a two-fold increase of both CAR and CYP2B mRNA levels. Both inducers reduced the effects of inhibitors on CAR gene expression. Results of EMSA showed that PB, TPD or W7 alone induced formation of complexes of NR1 with nuclear proteins. Appearance of the complexes correlated with an increase in CYP2B expression, and their intensities were modulated by the protein kinase inhibitors. Thus, our results demonstrate that constitutive expressions of CAR as well as CYP2B during induction are regulated by phosphorylation/dephosphorylation processes.

[Effects of ginkgolides on gene expression of hepatic cytochrome P-450 in rats].

OBJECTIVE: To observe the effects of ginkgolides on gene expression of hepatic cytochrome P-450 in rats. METHOD: Sprague-Dawley rats were administered ginkgolides (100 mg x kg(-1) body weight) through oral gavage once daily for four consecutive days. The level of gene expression in liver tissues was analyzed by competitive reverse transcription-polymerase chain reaction (competitive RT-PCR). RESULT: A single and prospective band of CYP1A1, CYP1A2, CYP2B1/B2, CYP2C11, CYP2E1, CYP4A1 and cyclophilin was observed after polymerase chain reaction (PCR) when the reactive system of reverse transcription (RT) had no target RNA, which confirmed the competitor had a specific capacity to bind to the CYP or cyclophilin primer. CYP1A1 mRNA was not dectectable in the livers of untreated control rats and ginkgolides-treated rats. The levels of CYP2C11 and CYP2E1 were not changed by ginkgolides treatment. In contrast, the levels of gene expression for CYP1A2 and CYP2B1/B2 were decreased, however, the levels of gene expression for CYP3A1 and CYP4A1 in ginkgolides group were distinctly increased compared with the control. CONCLUSION: A specific effect of ginkgolides on cytochrome P-450 gene expression was observed in this investigation. Ginkgolides had various effects on different cytochrome P-450 isoforms.

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 strain and diet upon constitutive and chemically induced activities of several xenobiotic-metabolizing enzymes in rats.

The response of animals in toxicity studies reflects a complex interaction of a number of variables, some intrinsic to a particular study design and others resulting from the treatment itself. The influences of strain and diet upon constitutive and benzo(a)pyrene (B(a)P) induced activities of several hepatic Phase I and II enzymes were studied in a multifactoral design. Male and female CDF and Crl:CD rats were fed a standard rodent diet ad libitum, a 75% of ad libitum restricted feeding regimen or a phytoestrogen-free diet for approximately 3 weeks. During the last five days of the study, rats were administered either corn oil (vehicle) or 15 mg/kg/day B(a)P via oral gavage. The constitutive activities of hepatic CYP1A1, CYP1A2, CYP2B1/2, and mixed isoforms of UDP-glucuronosyl transferase, sulfotransferase, and glutathione-S-transferase varied significantly by feeding regimen and strain. Responses to B(a)P administration were also observed to be influenced by diet and strain in a manner similar to that observed for constitutive activities. These findings point out the potentially significant interactions of relatively commonly encountered variables that may affect results of hazard testing, especially when employing near metabolically saturating dosages of test chemicals.

Pretreatment with Ginkgo biloba extract weakens the hypnosis action of phenobarbital and its plasma concentration in rats.

In a previous study, we found that orally administered Ginkgo biloba extract (GBE) induced hepatic cytochrome P450 (CYP) in rats, especially the CYP2B type. This fact suggested that GBE influenced the availability and safety of drugs that were metabolized via CYP2B type enzymes. To confirm this possibility, in this study we examined the effect of feeding a 0.1, 0.5 and 1.0% GBE diet for 2 weeks on the pharmacokinetics and pharmacological action of phenobarbital, which is known to be metabolized by CYP2B in Wistar rats. The feeding of GBE markedly shortened the sleeping time in rats. Furthermore, the maximal phenobarbital plasma concentration (Cmax) and the 24-h area under the curve (AUC0-24) were decreased in rats fed GBE. These findings indicate that GBE reduces the therapeutic potency of phenobarbital via enhancement of cytochrome P450 expression, and raises the possibility that GBE and drug interactions may occur clinically.

Ginkgo biloba extract markedly induces pentoxyresorufin O-dealkylase activity in rats.

We examined the effect of Ginkgo biloba extract (GBE) on hepatic drug-metabolizing enzymes, particularly cytochrome P450 (CYP), in rats. Rats were fed a GBE-containing diet or received GBE by intragastric gavage. The concentration of CYP and activity of various CYP enzymes in the liver were increased in a dose- and time-dependent manner. Significant increases in the concentration and activities of CYP enzymes were detected on day 1 of feeding of a 0.5% GBE diet and after administration of 10 mg GBE/kg body weight for 5 days by intragastric gavage. Among the CYP enzymes, the activity of pentoxyresorufin O-dealkylase (PROD), a CYP2B enzyme, was especially markedly increased. The induction of CYP2B enzyme by GBE was confirmed by Western blot analysis. Addition of GBE to a CYP assay system in vitro caused concentration-dependent inhibition of various CYP enzyme activities. The inhibition was more marked for the microsomal enzymes from GBE-treated rats than for those from control rats and more marked against PROD activity among the CYP enzymes tested. When the inhibition of various CYP enzymes activities by GBE in vitro was compared, no marked difference was observed between rat and human hepatic microsomal enzymes. These results indicate that excess intake of GBE induces CYP enzymes, particularly PROD, and may modify the efficacy of drugs taken simultaneously.

Protective effects of aged garlic extract against bromobenzene toxicity to precision cut rat liver slices.

Precision-cut liver slices from phenobarbital-treated rats were incubated for up to 8 h with the industrial solvent and hepatotoxin bromobenzene at a final concentration of 1 mM. Phenobarbital pretreatment potentiates bromobenzene hepatotoxicity by inducing those P450 isoforms responsible for the formation of the active hepatotoxin, namely bromobenzene-3,4-oxide. A reduction in cell viability was indicated by a decrease in the K+, ATP and glutathione content of the slices and the increased release of the intracellular enzymes, lactate dehydrogenase and alanine aminotransferase, into the medium. Furthermore, levels of lipid peroxidation as judged by the formation of thiobarbituric acid reactive substances, were increased approximately 5-fold. Aged garlic extract (AGE) at concentrations of 1-5% (v/v) reduced the toxicity of bromobenzene in a concentration-dependent manner as judged by all of the parameters of viability studied, with the exception of lipid peroxidation which was reduced to control levels even at the lowest concentration of garlic extract used. AGE was found to cause partial inhibition of cytochrome P450 when assayed as both 7-ethoxycoumarin O-deethylase and 7-pentoxyresorufin O-depentylase activities, but even the highest concentration used inhibited both activities by less than 50%. It is suggested that the hepatoprotective effects of AGE are due primarily to the reduced glutathione-sparing properties of its constituents, most probably its organosulphur compounds.

Piperine inhibits aflatoxin B1-induced cytotoxicity and genotoxicity in V79 Chinese hamster cells genetically engineered to express rat cytochrome P4502B1.

We have investigated the potential of piperine for inhibiting the activity of cytochrome P4502B1 and protecting against aflatoxin B1 (AFB1) in V79MZr2B1 (r2B1) cells, i.e. V79 Chinese hamster cells engineered for the expression of rat CYP4502B1. The cells were found to contain high activities of 7-methoxycoumarin demethylase (MOCD). Piperine inhibited MOCD in preparations of r2B1 cells with an IC50 of approximately 10 microM. The cells in culture dealkylated 7-methoxycoumarin (MOC) to 7-OH-coumarin linearly, at least for 12 h, where piperine produced concentration-dependent inhibition with IC50 < 30 microM. The time required for maximal inhibition was approximately 8 h with both 30 and 60 microM concentrations of piperine used. AFB1 at 0.1-20 microM caused a concentration dependent decrease in the amount of DNA and an increase in the formation of micronuclei (MN). The mycotoxin at 10 microM reduced DNA by approximately 30% and increased MN appearance by 20-fold against the background level of 7 MN per 500 nuclei. Piperine at 60 microM completely counteracted cytotoxicity and formation of MN by 10 microM AFB1 and reduced the toxic effects of 20 microM AFB1 by > 50%. The results suggest that: (i) Piperine is a potent inhibitor of rat CYP4502B1 activity; (ii) AFB1 is activated by r2B1 cells to cytotoxic and genotoxic metabolites; and (iii) piperine counteracts CYP4502B1 mediated toxicity of AFB1 in the cells and might, therefore, offer a potent chemopreventive effect against procarcinogens activated by CYP4502B1.