ABSTRACT
BACKGROUND AND OBJECTIVES: Gambogenic acid (GNA), which possesses diverse antitumor activities both in vitro and in vivo, is regarded as a potential anticancer compound. Cytochrome P450 (CYP) enzymes play an important role in the metabolism of most xenobiotics; constitutive androstane receptor (CAR), a nuclear receptor that might be activated by xenobiotics and associated with the expression of some CYPs. In this study, we determined the effect of GNA on multiple rat liver CYP isoforms (CYP1A2, 2B1, and 2E1) and CAR as well as the potential of GNA to interact with co-administered drugs. METHODS: Male SD rats were randomly divided into the control, and the low (5 mg/kg)-, medium (25 mg/kg)-, and high- (100 mg/kg) dose GNA groups. After the intragastric administration of GNA for 14 consecutive days, a cocktail method was adopted to evaluate the activities of CYP1A2, 2B1, and 2E1. The liver expression of CYP1A2, 2B1, and 2E1 and CAR was analyzed by Western blotting (WB) and quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR). RESULTS: The 14-day administration of GNA significantly increased both the mRNA and protein expressions and the activity of CYP2E1. Additionally, the mRNA and protein expressions of CYP1A2 were clearly induced, while only the high GNA dose increased the activity of liver CYP1A2. Moreover, the mRNA expression levels of CYP2B1 and CAR were increased, but their protein levels and the activity parameters of CYP2B1 did not show significant changes. CONCLUSIONS: The obtained results suggest that the CYP1A2 and CYP2E1 enzymes could be induced in rats after treatment with GNA. Therefore, when GNA is administrated with other drugs, potential drug-drug interactions (DDI) mediated by CYP1A2 and CYP2E1 induction should be taken into consideration.
Subject(s)
Cytochrome P-450 CYP1A2/biosynthesis , Cytochrome P-450 CYP2B1/biosynthesis , Cytochrome P-450 CYP2E1/biosynthesis , Phenacetin/pharmacokinetics , Receptors, Cytoplasmic and Nuclear/biosynthesis , Xanthenes/pharmacology , Animals , Bupropion/blood , Bupropion/pharmacokinetics , Chlorzoxazone/blood , Chlorzoxazone/pharmacokinetics , Constitutive Androstane Receptor , Cytochrome P-450 CYP1A2/metabolism , Cytochrome P-450 CYP2B1/metabolism , Cytochrome P-450 CYP2E1/metabolism , Cytochrome P-450 Enzyme Inducers/blood , Dose-Response Relationship, Drug , Drug Interactions , Liver/metabolism , Male , Phenacetin/blood , RatsABSTRACT
The hepatic cytochrome P450 (CYP450) enzyme superfamily is one of the most important drug-metabolizing enzyme systems, which is responsible for the metabolism of a large number of clinically relevant medications used in traumatic brain injury (TBI) therapy. Modification of CYP450 expression may have important influences on drug metabolism and lead to untoward effects on those with narrow therapeutic windows. However, the impact of blast-induced TBI (bTBI) on the expression of CYP450 has received little attention. The subfamilies of CYP1A, 2B, 2D, and 3A account for about 85 % of all human drug metabolism of clinical significance. Therefore, in this study, we investigated the expressions of hepatic CYP1A2, CYP2B1, CYP2D1, and CYP3A2 in rats suffering bTBI. Meanwhile, we also measured some important cytokines in serum after injury, and calculated the correlation between these cytokines and the expressions of CYP1A2, CYP2B1, CYP2D1, and CYP3A2. The results showed that bTBI could significantly reduce mRNA expressions of CYP1A2, CYP2D1, and CYP3A2 at the early stage and induce the expressions from 48 h to 1 week after injury. The protein expressions of these CYP450s had all been downregulated from 24 to 48 h post- injury, and then began to elevate at 48 h after bTBI. The cytokines, IL-1ß, IL-2, IL-6, and TNF-α, increased significantly in the early phase, and began to reduce at the delayed phase of bTBI. The serum levels of IL-1ß, IL-6, and TNF-α but not IL-2 were significantly negative correlated with the mRNA expressions of CYP2B1 and CYP2D1 and the proteins expressions of CYP1A2, CYP2B1, CYP2D1, and CYP3A2. In conclusion, our work has, for the first time, indicated that bTBI has significant impact on the expressions of CYP1A2, CYP2B1, CYP2D1, and CYP3A2, which may be related to the cytokines induced by the injury.
Subject(s)
Brain Injuries, Traumatic/enzymology , Cytochrome P-450 CYP1A2/biosynthesis , Cytochrome P-450 CYP2B1/biosynthesis , Cytochrome P-450 CYP3A/biosynthesis , Cytochrome P450 Family 2/biosynthesis , Liver/enzymology , Animals , Brain Injuries, Traumatic/pathology , Cytochrome P-450 CYP1A2/genetics , Cytochrome P-450 CYP2B1/genetics , Cytochrome P-450 CYP3A/genetics , Cytochrome P450 Family 2/genetics , Gene Expression Regulation, Enzymologic , Male , Microsomes, Liver/enzymology , Rats , Rats, Sprague-DawleyABSTRACT
Cytochrome P450 (CYP) in the brain plays an essential role in the local metabolism of various compounds, including clinically used drugs, toxins, and endogenous substances. In the present study, we compared the expression profiles of mRNAs for several CYP subtypes in the brain between male and female rats. The expression of CYP1A2, CYP2B1, and CYP2D2 in females was significantly higher than that in males. On the other hand, the expression level of the other CYP subtypes examined in the male brain was similar to that in the female brain. These results strongly suggest that marked gender differences exist in the expression profiles of some CYP subtypes in rat brain.
Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Brain Chemistry/genetics , Cytochrome P-450 CYP2B1/genetics , Cytochromes/genetics , Animals , Aryl Hydrocarbon Hydroxylases/biosynthesis , Cytochrome P-450 CYP1A2 , Cytochrome P-450 CYP2B1/biosynthesis , Cytochromes/biosynthesis , Female , Gene Expression Regulation, Enzymologic , Male , Polymerase Chain Reaction , Rats , Rats, Wistar , Sex CharacteristicsABSTRACT
INTRODUCTION: Hepatocyte-Kupffer cell (KC) co-cultures represent a promising approach for in vitro modeling of complex interactions between parenchymal and non-parenchymal cells in the liver, responsible for drug-induced liver injury (DILI). In this study we aimed to compare hepatocyte monocultures with hepatocyte-KC co-cultures regarding some basic liver functions associated with the chemical defense system. These pathways involve transporters and enzymes the function of which is highly sensitive towards hepatotoxic events. METHODS: CYP2B1/2 induction and the biliary and sinusoidal elimination of bilirubin (B) and taurocholate (TC) were studied in rat hepatocyte sandwich cultures compared with rat hepatocyte-KC sandwich co-cultures of 1:0, 6:1, 2:1 and 1:1 cell combinations representing the physiologic and pathologic conditions of the liver. RESULTS: KCs decreased phenobarbital inducibility of CYP2B1/2 in a cell ratio dependent manner and activation of KCs by lipopolisacharide (LPS) amplified this effect. Similarly, KCs decreased the transport of B and its glucuronides (BG) in both sinusoidal and canalicular directions resulting in its intracellular accumulation. In contrast, the uptake and the efflux of TC were greater in the co-cultures than in the hepatocyte monocultures. Immuno-labelling of sodium-dependent taurocholate transporter (Ntcp) revealed increased expression of the transporter in the presence of KCs. DISCUSSION: Here we presented that KCs have a direct impact on some hepatocyte functions suggesting that the co-culture model may be more suitable for drug related hepatotoxicity studies than hepatocyte monocultures.
Subject(s)
Aryl Hydrocarbon Hydroxylases/biosynthesis , Bilirubin/metabolism , Cytochrome P-450 CYP2B1/biosynthesis , Hepatocytes/enzymology , Kupffer Cells/enzymology , Models, Biological , Steroid Hydroxylases/biosynthesis , Taurocholic Acid/metabolism , Animals , Biological Transport , Chemical and Drug Induced Liver Injury/enzymology , Chemical and Drug Induced Liver Injury/metabolism , Coculture Techniques , Drug Interactions , Enzyme Induction , Hepatocytes/drug effects , Kupffer Cells/drug effects , Lipopolysaccharides/pharmacology , Male , Metabolic Detoxication, Phase I , Rats , Rats, WistarABSTRACT
The medium-term multiorgan initiation-promotion chemical bioassay (diethylnitrosamine, methyl-nitrosourea, butyl-hydroxybutylnitrosamine, dihydroxypropylnitrosamine, dimethylhydrazine [DMBDD]) with the Fischer 344 rat was proposed as an alternative to the conventional 2-year carcinogenesis bioassay for regulatory purposes. The acronym DMBDD stands for the names of five genotoxic agents used for initiation of multiorgan carcinogenesis. The Brazilian Agency for the Environment officially recognized a variation of this assay (DMBDDb) as a valid method to assess the carcinogenic potential of agrochemicals. Different from the original protocol, this DMBDDb is 30-week long, uses Wistar rats and two positive control groups exposed to carcinogenesis promoters sodium phenobarbital (PB) or 2-acetylaminofluorene (2-AAF). This report presents the experience of an academic laboratory with the DMBDDb assay and contributes to the establishment of this alternative DMBDD bioassay in a different rat strain. Frequent lesions observed in positive groups to evaluate the promoting potential of pesticides and the immunohistochemical expressions of liver cytochrome P450 (CYP) 2B1/2B2 and CYP1A2 enzymes were assessed. Commonly affected organs were liver, kidney, intestines, urinary bladder, and thyroid. PB promoting activity was less evident than that of 2-AAF, especially in males. This study provides a repository of characteristic lesions occurring in positive control animals submitted to a modified alternative 2-stage multiorgan protocol for carcinogenesis in Wistar rat.
Subject(s)
2-Acetylaminofluorene/toxicity , Carcinogenicity Tests/methods , Carcinogens/toxicity , Neoplasms, Experimental/chemically induced , Phenobarbital/toxicity , Precancerous Conditions/chemically induced , Animals , Aryl Hydrocarbon Hydroxylases/biosynthesis , Biological Assay , Cytochrome P-450 CYP1A2/biosynthesis , Cytochrome P-450 CYP2B1/biosynthesis , Female , Liver/drug effects , Liver/enzymology , Liver/pathology , Male , Neoplasms, Experimental/enzymology , Organ Size/drug effects , Organ Specificity , Precancerous Conditions/enzymology , Rats, Wistar , Steroid Hydroxylases/biosynthesisABSTRACT
Cytochrome P450 (CYP) is an important enzyme that can act on xenobiotic substances such as toxic chemicals or drugs. Phenobarbital (PB) has been widely used to induce CYP2B activity to investigate the drug-drug interaction of CYP2B substrate drugs. Leelamine is a diterpene compound, and is the current focus of efforts to develop a treatment for diabetes. In this study, we identified the selective and potent inductive effect of leelamine on CYP2B at doses of 5, 10, or 20 mg/kg in male ICR mice for 1 or 3 days. In liver, the activity of CYP2B significantly increased 3.6-fold after treatment with leelamine, compared to vehicle-treated group. Activities of benzyloxyresorufin O-dealkylase and pentoxyresorufin O-dealkylase significantly increased 6.3- and 5.3-fold, respectively, with a single treatment of 20 mg/kg leelamine for 1 day. Furthermore, immunoblot analysis showed that significantly and dose-dependently increased CYP2B10 protein levels in liver. However, PCR results showed that there were no significant changes in the CAR and CYP2B mRNA levels after leelamine treatment. Accordingly, we suggest that leelamine is a novel substitute of PB for the selective induction of CYP2B activity in vivo.
Subject(s)
Cytochrome P-450 CYP2B1/biosynthesis , Diterpenes/chemistry , Diterpenes/pharmacology , Liver/drug effects , Liver/enzymology , Animals , Body Weight/drug effects , Body Weight/physiology , Cytochrome P-450 Enzyme Inducers/chemistry , Cytochrome P-450 Enzyme Inducers/pharmacology , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Enzyme Activation/physiology , Enzyme Induction/drug effects , Enzyme Induction/physiology , Male , Mice , Mice, Inbred ICRABSTRACT
Xenobiotics may interfere with the hypothalamic-pituitary-thyroid endocrine axis by inducing enzymes that inactivate thyroid hormones and thereby reduce the metabolic rate. This induction results from an activation of xeno-sensing nuclear receptors. The current study shows that benzo[a]pyrene, a frequent contaminant of processed food and activator of the arylhydrocarbon receptor (AhR) activated the promoter and induced the transcription of the nuclear receptor constitutive androstane receptor (CAR, NR1I3) in rat hepatocytes. Likewise, phenobarbital induced the AhR transcription. This mutual induction of the nuclear receptors enhanced the phenobarbital-dependent induction of the prototypic CAR target gene Cyp2b1 as well as the AhR-dependent induction of UDP-glucuronosyltransferases. In both cases, the induction by the combination of both xenobiotics was more than the sum of the induction by either substance alone. By inducing the AhR, phenobarbital enhanced the benzo[a]pyrene-dependent reduction of thyroid hormone half-life and the benzo[a]pyrene-dependent increase in the rate of thyroid hormone glucuronide formation in hepatocyte cultures. CAR ligands might thus augment the endocrine disrupting potential of AhR activators by an induction of the AhR.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/agonists , Benzo(a)pyrene/toxicity , Endocrine Disruptors/toxicity , Hepatocytes/drug effects , Phenobarbital/toxicity , Receptors, Aryl Hydrocarbon/agonists , Receptors, Cytoplasmic and Nuclear/agonists , Thyroid Hormones/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cells, Cultured , Constitutive Androstane Receptor , Cytochrome P-450 CYP2B1/biosynthesis , Cytochrome P-450 Enzyme Inducers/toxicity , Dose-Response Relationship, Drug , Drug Synergism , Enzyme Induction , Glucuronides/metabolism , Glucuronosyltransferase/biosynthesis , Half-Life , Hepatocytes/metabolism , Male , Promoter Regions, Genetic/drug effects , Proteolysis , Rats , Rats, Wistar , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription, Genetic/drug effects , Transcriptional Activation/drug effects , Transfection , Up-RegulationABSTRACT
Peroxisome proliferator activated receptor α (PPARα) ligands, fibrates used to control hyperlipidemia. We demonstrated CYP2B induction by clofibric acid (CFA) however, the mechanism was not clear. In this study, HepG2 cells transfected with expression plasmid of mouse constitutive androstane receptor (CAR) or PPARα were treated with CFA, phenobarbital (PB) or TCPOBOP. Luciferase assays showed that CFA increased CYP2B1 transcription to the same level as PB, or TCPOBOP in HepG2 transfected with mouse CAR But failed to induce it in PPARα transfected cells. CYP2B expressions were increased with PB or CFA in Wistar female rats (having normal levels of CAR) but not in Wistar Kyoto female rats (having low levels of CAR). The induction of CYP2B by PB or CFA was comparable to nuclear CAR levels. CAR nuclear translocation was induced by CFA in both rat strains. This indicates that fibrates can activate CAR and that fibrates-insulin sensitization effect may occur through CAR, while hypolipidemic effect may operate through PPARα.
Subject(s)
Aryl Hydrocarbon Hydroxylases/biosynthesis , Clofibric Acid/administration & dosage , Cytochrome P-450 CYP2B1/biosynthesis , PPAR alpha/biosynthesis , Receptors, Cytoplasmic and Nuclear/genetics , Steroid Hydroxylases/biosynthesis , Animals , Constitutive Androstane Receptor , Gene Expression Regulation, Enzymologic/drug effects , Hep G2 Cells , Humans , Insulin/metabolism , Liver/drug effects , Liver/enzymology , Mice , PPAR alpha/genetics , Phenobarbital/administration & dosage , Pyridines/administration & dosage , RatsABSTRACT
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.
Subject(s)
/toxicity , Cytochrome P-450 CYP1A1/biosynthesis , Cytochrome P-450 CYP2B1/biosynthesis , Quercetin/administration & dosage , Animals , Endometrium/cytology , Endometrium/drug effects , Female , Gene Expression Regulation/drug effects , Humans , Interleukin-6/biosynthesis , Pregnancy , Progesterone/biosynthesis , Rats , Receptors, Estrogen/drug effects , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
AIMS: In this study, we determined the expression level of miRNAs and the induction of CYP1A1 and CYP2B1 in the livers and ovaries of female Wistar rats treated with DDT, benzo[a]pyrene (BP), and 3-methylcholanthrene (MC). This study compared CYP1A/2B induction and miRNA expression levels to cast light on a possible role of miRNA in the tissue-specific induction of CYPs. MAIN METHODS: The induction of CYP1A1/2B1 enzymes was detected by ethoxy-, pentoxyresorufin O-dealkylation and Western blot analysis. The CYP1A1/2B1 gene expression was determined by RT-real time PCR. Relative levels of expression for selected in silico miR species were determined by real time PCR with small nuclear U6 RNA employed as a reference gene. KEY FINDINGS: After bioinformatic analysis, miR-21, 221, 222, and 429 were chosen as potential post-transcriptional regulators of rat CYP1A and CYP2B. It was shown that miR-21, 221, 222, and 429 expression levels decreased in the liver of DDT-, BP-, and MC-treated rats, whereas increases were observed in CYP1A1 and CYP2B1 mRNA expression levels and protein content, and EROD and PROD activities. Conversely, a tendency for elevated levels of miRNAs in the ovaries of inducer-treated female rats was observed. In the ovaries, a high level of CYP1A1 and CYP2B1 mRNA expression was observed, although protein content and enzyme activity were not visible. SIGNIFICANCE: These data suggest a potential involvement of miRNA in the post-transcriptional regulation of CYP1A and CYP2B in the livers and ovaries of chemically induced rats.
Subject(s)
Cytochrome P-450 CYP1A1/biosynthesis , Cytochrome P-450 CYP2B1/biosynthesis , DDT/toxicity , MicroRNAs/biosynthesis , Polycyclic Aromatic Hydrocarbons/toxicity , Animals , Female , Liver/drug effects , Liver/metabolism , MicroRNAs/genetics , Ovary/drug effects , Ovary/metabolism , Rats , Rats, WistarABSTRACT
Stroke is a neurological condition and may cause changes in hepatic drug-metabolizing enzymes. Hepatic CYP2B is involved in the metabolism of a variety of centrally active substances. The purpose of this study was to investigate the possible down-regulation mechanism of hepatic CYP2B after acute stroke. Using a rat model of acute stroke induced by middle cerebral artery occlusion, we studied the influence of brain ischemia/reperfusion (I/R) injury on CYP2B expression. Effects of 3,5,3'-triiodo-L-thyronine (T3) treatment on constitutive androstane receptor (CAR) and thyroid hormone receptors (TRs, including TRα and TRß) proteins were detected in Huh7 cells. We found dramatic decreases in the levels of plasma free triiodthyronine, free thyroxine and hepatic CYP2B expression. Both CAR and retinoid X receptor alpha (RXRα) were significantly dissociated from the phenobarbital-responsive enhancer module (PBREM) of the CYP2B1 promoter in the early stages of the acute stroke. The levels of the polymer of TRs, CAR, and RXRα were time-dependently decreased after brain I/R injury. T3 regulated the CAR expression at the transcriptional level, and facilitated the translocation of TRα/ß proteins as well as the binding of TRs, RXRα, and CAR to PBREM region. The reduction of thyroid hormone levels after a brain I/R injury may be the initial trigger for the down-regulation of hepatic CYP2B1 via induction of the dissociation of CAR from the TRs and from the PBREM region. Our data suggest that patients with acute ischemic stroke may have a decreased CYP2B-mediated metabolism of exogenous and endogenous compounds because of the low level of thyroid hormones.
Subject(s)
Cytochrome P-450 CYP2B1/antagonists & inhibitors , Disease Models, Animal , Down-Regulation/physiology , Receptors, Cytoplasmic and Nuclear/physiology , Receptors, Thyroid Hormone/physiology , Stroke/genetics , Stroke/metabolism , Thyroid Hormones/deficiency , Animals , Cell Line, Tumor , Constitutive Androstane Receptor , Cytochrome P-450 CYP2B1/biosynthesis , Humans , Male , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Microsomes, Liver/pathology , Rats , Rats, Wistar , Stroke/enzymology , Thyroid Hormones/metabolismABSTRACT
Polychlorinated biphenyls (PCBs) are among the most ubiquitously detectable 'persistent organic pollutants'. In contrast to 'dioxinlike' (DL) PCBs, less is known about the molecular mode of action of the larger group of the 'non-dioxinlike' (NDL) PCBs. Owing to the life-long exposure of the human population, a carcinogenic, i.e., tumor-promoting potency of NDL-PCBs has to be considered in human risk assessment. A major problem in risk assessment of NDL-PCBs is dioxin-like impurities that can occur in commercially available NDL-PCB standards. In the present study, we analyzed the induction of CYP2B1 and CYP3A1 in primary rat hepatocytes using a number of highly purified NDL-PCBs with various degrees of chlorination and substitution patterns. Induction of these enzymes is mediated by the nuclear xenobiotic receptors CAR (Constitutive androstane receptor) and PXR (Pregnane X receptor). For CYP2B1 induction, concentration-response analysis revealed a very narrow window of EC50 estimates, being in the range of 1-4µM for PCBs 28 and 52, and between 0.4 and 1µM for PCBs 101, 138, 153 and 180. CYP3A1 induction was less sensitive to NDL-PCBs, the most pronounced induction being achieved at 100µM with the higher chlorinated congeners. Using okadaic acid and small interfering RNAs targeting CAR and PXR, we could demonstrate that CAR plays a major role and PXR a minor role in NDL-PCB-driven induction of CYPs, both effects showing no stringent structure-activity relationship. As the only obvious relevant determinant, the degree of chlorination was found to be positively correlated with the inducing potency of the congeners.
Subject(s)
Cytochrome P-450 Enzyme System/biosynthesis , Environmental Pollutants/toxicity , Hepatocytes/enzymology , Polychlorinated Biphenyls/toxicity , Receptors, Cytoplasmic and Nuclear/drug effects , Receptors, Steroid/drug effects , Animals , Binding, Competitive/drug effects , Blotting, Western , Cell Survival/drug effects , Cells, Cultured , Constitutive Androstane Receptor , Cytochrome P-450 CYP2B1/biosynthesis , Cytochrome P-450 CYP3A/biosynthesis , Enzyme Induction/drug effects , Enzyme Inhibitors/pharmacology , Gene Expression/drug effects , Hepatocytes/drug effects , Immunohistochemistry , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Okadaic Acid/pharmacology , Pregnane X Receptor , Primary Cell Culture , RNA, Small Interfering/pharmacology , Rats , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Steroid/antagonists & inhibitorsABSTRACT
Chalepensin, a furanocoumarin, is present in several medicinal Rutaceae plants and causes a mechanism-based inhibition of human and mouse cytochrome P450 (P450, CYP) 2A in vitro. To address the in vivo effect, we investigated the effects of chalepensin on multiple hepatic P450 enzymes in C57BL/6JNarl mice. Oral administration of 10 mg/kg chalepensin to mice for 7 days significantly decreased hepatic coumarin 7-hydroxylation (Cyp2a) and increased 7-pentoxyresorufin O-dealkylation (Cyp2b) activities, whereas activities of Cyp1a, Cyp2c, Cyp2e1, and Cyp3a were not affected. Without affecting its mRNA level, the decreased Cyp2a activity was accompanied by an increase in the immunodetected Cyp2a5 protein level. In chalepensin-treated mice, microsomal Cyp2a5 was less susceptible to ATP-fortified cytosolic degradation than that in control mice, resulting in the elevated protein level. The in vitro inactivation through NADPH-fortified pre-incubation with chalepensin also protected microsomal Cyp2a5 against protein degradation. Using cell-based reporter systems, chalepensin at a concentration near unbound plasma concentration activated mouse constitutive androstane receptor (CAR), in agreement with the observed induction of Cyp2b. These findings revealed that suicidal inhibition of Cyp2a5 and the CAR-mediated Cyp2b9/10 induction concurrently occurred in chalepensin-treated mice.
Subject(s)
Aryl Hydrocarbon Hydroxylases/antagonists & inhibitors , Cytochrome P-450 CYP2B1/biosynthesis , Furocoumarins/pharmacology , Steroid Hydroxylases/antagonists & inhibitors , Animals , Aryl Hydrocarbon Hydroxylases/biosynthesis , Blotting, Western , Cell Survival/drug effects , Cells, Cultured , Constitutive Androstane Receptor , Cytosol/drug effects , Cytosol/enzymology , Cytosol/metabolism , Genes, Reporter , Isoenzymes/drug effects , Isoenzymes/metabolism , Male , Mice , Mice, Inbred C57BL , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Mixed Function Oxygenases/metabolism , NADP/metabolism , Plasmids/genetics , Polymerase Chain Reaction , RNA/isolation & purification , Receptors, Cytoplasmic and Nuclear/drug effects , Ruta/chemistry , Steroid Hydroxylases/biosynthesisABSTRACT
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.
Subject(s)
Chrysanthemum/chemistry , Cytochrome P-450 CYP1A1/biosynthesis , Cytochrome P-450 CYP2B1/biosynthesis , Cytochromes/biosynthesis , Drugs, Chinese Herbal/pharmacology , Liver/drug effects , Vitamin A/metabolism , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Chlorogenic Acid/analysis , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP1A2 , Cytochrome P-450 CYP2B1/metabolism , Cytochromes/metabolism , Dose-Response Relationship, Drug , Drugs, Chinese Herbal/administration & dosage , Drugs, Chinese Herbal/chemistry , Enzyme Induction/drug effects , Flavonoids/analysis , Flowers/chemistry , Kinetics , Liver/enzymology , Liver/metabolism , Male , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Random Allocation , Rats , Rats, Sprague-Dawley , Tretinoin/blood , Tretinoin/metabolism , Vitamin A/administration & dosage , Vitamin A/bloodABSTRACT
The aims of the present study were to determine cytochrome P450 enzyme activity in six strains of experimental rodents (n = 5/sex/species): ICR, C57BL/6 and DBA/2 mice; Sprague Dawley and Wistar rats; and Dunkin Hartley guinea pigs. After animals were treated with the typical inducers ß-naphthoflavone (BNF), dexamethasone (DEX) and phenobarbital (PB), the levels of O-dealkylation of ethoxyresorufin (EROD), methoxyresorufin (MROD), pentoxyresorufin (PROD) and benzyloxyresorufin (BROD) activity were determined using responsive catalytic reactions to study CYP1A1, CYP1A2 and CYP2B, respectively. A maximal induction of EROD and MROD was found in BNF-treated animals from all strains (2.4- to 15.1-fold) except DBA/2 (0.9- to 1.8-fold). C57BL/6 mice had the strongest BNF-induced EROD (15.1-fold) and MROD (8.3-fold) activities. No differences in BNF-induced EROD and MROD activities were observed between males and females. However, the EROD activity of Wistar rats and the MROD activity of Sprague Dawley rats were higher in males than females. DEX induced PROD activity only in mice (1.3- to 7.1-fold), but not in rats and guinea pigs (0.2- to 1.1-fold). However, induction of BROD activity was found in DEX-treated mice and rats (1.5 to 12.5-fold), but not in guinea pigs (0.3 to 0.4-fold). PB caused a significant elevation of PROD (1.7- to 10.4-fold) and BROD (31- to 13.2-fold) activities in all the animals. PB-induced BROD activity was higher in females than males in Sprague Dawley rats. These observations strongly suggest that the choice of experimental animal strain, species and inducer is of critical importance for studies of drug metabolism and interaction.
Subject(s)
Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP2B1/metabolism , Cytochrome P-450 Enzyme System/metabolism , Liver/enzymology , Animals , Cytochrome P-450 CYP1A1/biosynthesis , Cytochrome P-450 CYP2B1/biosynthesis , Cytochrome P-450 Enzyme System/biosynthesis , Dexamethasone/pharmacology , Enzyme Induction , Female , Guinea Pigs , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Inbred ICR , Phenobarbital/pharmacology , Rats , Rats, Sprague-Dawley , Rats, Wistar , Species Specificity , beta-Naphthoflavone/pharmacologyABSTRACT
Primary hepatocytes are widely used in investigating drug metabolism and its toxicological effects. N-Nitrosodiethylamine (NDEA)-induced genotoxicity and cytotoxicity was used in primary cultures of female rat hepatocytes in the presence of phenobarbital (PB). PB pre-treatment (1mM) increased the number of necrotic (2-fold) and apoptotic cells (4-fold) after NDEA treatment (0.21-105 µg/mL). The mitotic indices and the number of micronucleated cells decreased, thus suggesting cytotoxicity. An increased number of chromosomal aberrations were observed after pre-treatment with PB. NDEA-treatment (0.21-21 µg/mL) induced expression of the CYP2B1 and CYP2B2 mRNA and PB treatment alone induced ~6-fold and ~2-fold increases of CYP2B1 and CYP2B2 mRNA, respectively. NDEA treatment following PB exposure increased CYP2B1 mRNA expression under all tested concentrations and also increased CYP2B2 expression at 21 and 105 µg/mL. Our data suggest that the alteration of CYP2B1/2 expression by PB increased the cytotoxicity and genotoxicity of NDEA leading to the final genotoxic metabolite.
Subject(s)
Carcinogens/toxicity , Cytochrome P-450 Enzyme System/biosynthesis , Diethylnitrosamine/toxicity , Hepatocytes/drug effects , Mutagens/toxicity , Phenobarbital/toxicity , Up-Regulation/drug effects , Animals , Aryl Hydrocarbon Hydroxylases/biosynthesis , Aryl Hydrocarbon Hydroxylases/genetics , Cell Death/drug effects , Cells, Cultured , Chromosome Aberrations/drug effects , Cocarcinogenesis , Cytochrome P-450 CYP2B1/biosynthesis , Cytochrome P-450 CYP2B1/genetics , Cytochrome P-450 Enzyme System/genetics , Enzyme Induction/drug effects , Female , Hepatocytes/pathology , Micronuclei, Chromosome-Defective/drug effects , Mitotic Index , RNA, Messenger/metabolism , Rats , Steroid Hydroxylases/biosynthesis , Steroid Hydroxylases/geneticsABSTRACT
Chokeberry is a rich source of procyanidins known to have several types of biological activity including anticarcinogenic potential in experimental models. In this study we examined the effect of chokeberry juice on the hepatic and mammary gland carcinogen metabolizing enzyme expression altered by the polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene (DMBA). Sprague-Dawley rats were gavaged with chokeberry juice (8 ml/kg b.w.) for 28 consecutive days. DMBA was administered i.p. on the 27th and the 28th days. Pretreatment with chokeberry juice reduced the activity of CYP1A1 and increased that of CYP2B involved in metabolic activation/detoxication of DMBA in rat liver, as well as expression and activity of phase II enzymes. Chokeberry juice had no effect on these parameters in the mammary gland and DMBA induced DNA damage in rat blood cells. These results together with our earlier observations indicate that metabolic alterations induced by chokeberry feeding are tissue specific and depend on the class of carcinogen.
Subject(s)
9,10-Dimethyl-1,2-benzanthracene/toxicity , Carcinogens/toxicity , Fruit , Liver/drug effects , Mammary Glands, Animal/drug effects , Photinia , Animals , Comet Assay , Cytochrome P-450 CYP1A1/biosynthesis , Cytochrome P-450 CYP2B1/biosynthesis , Cytochrome P-450 Enzyme System/biosynthesis , DNA Damage/drug effects , Female , Glutathione Transferase/biosynthesis , Isoenzymes/biosynthesis , Liver/enzymology , Mammary Glands, Animal/enzymology , NAD(P)H Dehydrogenase (Quinone)/biosynthesis , Rats , Rats, Sprague-DawleyABSTRACT
Previously, we demonstrated that cytochrome P450 2B1 (CYP2B1) can generate reactive oxygen species in puromycin aminonucleoside (PAN)-induced nephrotic syndrome, an animal model of minimal-change disease in humans. In this study we found that overexpression of CYP2B1 in rat glomerular epithelial cells in vitro significantly increased PAN-induced reactive oxygen species generation, cytotoxicity, cell death, and collapse of the actin cytoskeleton. All of these pathological changes were markedly attenuated by siRNA-induced CYP2B1 silencing. The cellular CYP2B1 protein content was significantly decreased whereas its mRNA level was markedly increased, suggesting regulation by protein degradation rather than transcriptional inhibition in the PAN-treated glomerular epithelial cells. This degradation of CYP2B1 was accompanied by the induction of heme oxygenase-1, an important indicator of heme-induced oxidative stress. In PAN-treated CYP2B1-silenced glomerular epithelial cells the induction of heme oxygenase-1 and caspase-3 activity were significantly decreased. Further, cleavage of the stress-induced pro-apoptotic endoplasmic reticulum-specific pro-caspase-12 was prevented in the silenced cells. Our results support a pivotal role of CYP2B1 for reactive oxygen species production in the endoplasmic reticulum in PAN-induced cytotoxicity.
Subject(s)
Cytochrome P-450 CYP2B1/genetics , Epithelial Cells/drug effects , Gene Silencing , Kidney Glomerulus/cytology , Kidney Glomerulus/drug effects , Puromycin Aminonucleoside/toxicity , Animals , Cells, Cultured , Cytochrome P-450 CYP2B1/biosynthesis , Gene Expression , RatsABSTRACT
The constitutive androstane receptor (CAR; NR1I3) is a key transcriptional factor that regulates genes encoding drug-metabolizing enzymes and drug transporters. However, studies on regulation of CAR target genes via up- or down-regulation of CAR are limited. In this study, we examined the effects of PPARalpha agonists (ciprofibrate, bezafibrate, fenofibrate and WY14643) on the expression of CAR and its target gene CYP2B1/2 in rat primary hepatocytes. Results from real-time PCR analysis showed that CAR and CYP2B1/2 mRNAs exhibit increases in response to all PPARalpha agonists studied (5 to 10-folds of control). Pretreatment of cells with cycloheximide, an inhibitor of protein synthesis, completely suppressed increase in CYP2B1/2 mRNA in response to ciprofibrate, suggesting that protein synthesis is required in this process. In addition, the induction of CAR by ciprofibrate on the protein level was observed with nuclear extracts as well as total cell lysates. These results indicate that CYP2B1/2 mRNAs are induced by PPARalpha agonists and that this effect is accompanied by increase in the expression of CAR gene at both mRNA and nuclear protein levels. Activated PPARalpha may increase functional CAR protein, which can induce the expression of CAR target genes such as CYP2B.
Subject(s)
Aryl Hydrocarbon Hydroxylases/biosynthesis , Cytochrome P-450 CYP2B1/biosynthesis , Gene Expression Regulation, Enzymologic , PPAR alpha/agonists , Receptors, Cytoplasmic and Nuclear/biosynthesis , Steroid Hydroxylases/biosynthesis , Animals , Aryl Hydrocarbon Hydroxylases/genetics , Cell Culture Techniques , Clofibric Acid/analogs & derivatives , Clofibric Acid/antagonists & inhibitors , Clofibric Acid/pharmacology , Constitutive Androstane Receptor , Cycloheximide/pharmacology , Cytochrome P-450 CYP2B1/genetics , Drug Interactions , Enzyme Induction/drug effects , Enzyme Induction/genetics , Fibric Acids , Hepatocytes/drug effects , Hepatocytes/metabolism , Male , Rats , Rats, Sprague-Dawley , Receptors, Cytoplasmic and Nuclear/genetics , Steroid Hydroxylases/geneticsABSTRACT
Age-related changes in hepatic expression and activity of cytochrome P450 (CYP) were investigated in male rats aged 3 (weanling), 12 (young), 26 (adult), and 104 (old) weeks. Levels of microsomal protein, total CYP, and cytochrome b(5) increased fully after puberty. CYP1A1 was detected only in 3-week-old rats, and CYP1A2, CYP2B1, and CYP2E1 were maximally expressed at 3 weeks but decreased at 12 and 26 weeks. CYP2C11 and CYP3A2 increased markedly after puberty and decreased with aging. Ethoxyresorufin-O-deethylase, methoxyresorufin-O-demethylase, pentoxyresorufin-O-depenthylase, and p-nitrophenol hydroxylase activities were at their highest in 3-week-old rats, and midazolam hydroxylase activity was at a maximum in 12-week-old rats but decreased with aging. The present results show that increasing age caused significant alterations in hepatic expression/activity of CYP isoforms in an isoform-specific manner. These results suggest that age-related changes in hepatic CYP isoforms may be an important factor for deciding the efficacy and safety of xenobiotics.