CYP1A1 Levels Research Articles

222 ANTIFUNGAL AGENTS AS AGRICULTURAL PRODUCTS, FENHEXAMID, FLUDIOXONIL, AND CYPRODINIL, INDUCED THE EXPRESSION OF CYTOCHROME P450 FAMILY AND CELL CYCLE-RELATED GENES IN ESTROGEN RECEPTOR EXPRESSING BG-1 OVARIAN CANCER CELLS

Fenhexamid, fludioxonil, and cyprodinil are antifungal agents used in agricultural applications, which are present at measurable amounts in fruits and vegetables. The induction of CYP gene expression including CYP1A1 and CYP1B1 is mediated by the transformation of polycyclic aromatic hydrocarbons (PAHs), and modulated by aryl hydrocarbon receptor (AhR). CYP1A1 is expressed in the liver, pancreas, thymus, uterus, and small intestine. CYP1B1 is abundant in the prostate, breast, and uterus. Expression levels of CYP1A1 and CYP1B1 indicate PAH-induced immunotoxicity, oxidative stress, and activation of environmental carcinogens. In this study, the ability of cell viability was examined as MTT assay by pesticides; fenhexamid, fludioxonil and cyprodinil. In addition, expression levels of mRNA and protein of AhR, CYP1A1, and Cyclin D1 were analysed by RT-PCR and Western blot analysis in BG-1 ovarian cancer cells with oestrogen receptors (ER). To evaluate the ability of cell viability, BG-1 cells were cultured with a negative control (0.1% DMSO), 17β-oestradiol (E2; 1 × 10–9 M), fenhexamid, fludioxonil or cyprodinil (10–5–10–8 M). To evaluate the expression levels of mRNA and protein, BG-1 cells were cultured with a negative control (0.1% DMSO), 17β-oestradiol (E2; 10–9 M) and these pesticides (10–5 M). As results, E2 as a positive control markedly increased BG-1 cell viability ~5 times compared to a negative control (P < 0.05). In addition, treatments with these pesticides increased BG-1 cell viability at the concentrations of 10–8 and 10–5 M about from 1.5 to 2 times, respectively (P < 0.05). When respective treatment co-treated with ICI 182 780, an ER antagonist, BG-1 cell viability was reversed to the level of a negative control. The mRNA expression of CYP1A1 was increased by E2, fenhexamid, and cyprodinil in a time-dependent manner but not by fludioxonil, while its level was reversed in the presence of ICI 182 780. In parallel with their transcriptional levels, protein levels of CYP1A1 and cyclin D1 were induced by E2 and these pesticides, while the level of AhR was not altered by E2 and these pesticides. Taken together, these results imply that the pesticides, fenhexamid, fludioxonil, and cyprodinil, may have disruptive effects on ER expressing cells or tissues by alteration of CYP1A1 and cyclin D1 via an ER-dependent pathway.

213 DIOXIN SIGNIFICANTLY ENHANCED THE TUMOR MASS FORMATION IN A XENOGRAFTED MOUSE MODEL TRANSPLANTED WITH BG-1 OVARIAN CANCER CELLS

Previous studies suggest that environmental factors, such as high levels of meat consumption, caffeine, cigarette smoking, and endocrine disrupting chemicals (EDC), may enhance a high risk of ovarian cancer. Cytochrome P450 (CYP) 1A1 may play a major role in metabolic activation of procarcinogens to carcinogens. For example, polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons (HAHs) occur by pyrolysis of fossil fuels and flow into the body by organic matter, such as tobacco leaves and contaminated water by pesticides. 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD) is a commonplace pollutant and promoter of carcinogenesis as the most potent substance. In this study, we examined the effects of 17β-oestradiol (E2), TCDD, and TCDD in the presence of E2 on the expressions of CYP1A1, CYP1B1, and aryl hydrocarbon receptor (AhR) by RT–PCR analysis and Western blot analysis. In addition, the cell viability by TCDD and E2 were examined in BG-1 human ovarian cancer cells by MTT assay. To evaluate the cell viability, BG-1 cells were cultured with a negative control (0.1% DMSO), E2 (1 × 10–9 M) or TCDD (10–6–10–8 M). E2 markedly increased BG-1 cell viability ~5 times, and TCDD also induced BG-1 cell viability highest at the concentration of 1 × 10–8 M compared to a control (0.1% DMSO) (P < 0.05). When respective treatment was co-treated with ICI 182 780, an ER antagonist, BG-1 cell viability was reversed to the level of a negative control. Although the mRNA expression of CYP1B1 was not altered by E2, TCDD, or TCDD plus E2, the transcriptional level of CYP1A1 appeared to be increased by E2 and TCDD in a time-dependent manner. When the cells were treated with TCDD plus E2, the mRNA level of CYP1A1 was more greatly increased than by only TCDD or E2 treatment. In xenograft mouse models transplanted with BG-1 cells, E2 treatment significantly increased the tumour mass formation ~6 times, and TCDD also induced tumour formation ~4 times compared to a vehicle (0.1% DMSO in PBS) during 8 weeks in this xenograted mouse model. In addition, TCDD plus E2 treatment also greatly induced ovarian tumour formation compared to only E2 treatment in this mouse model. Taken together, these results indicate that TCDD may induce ovarian cancer cell growth via CYP1A1 gene expression and have disruptive effect in oestrogen receptor (ER) expressing cells or tissues.

Endocrine disruption in human placenta: expression of the dioxin-inducible enzyme, CYP1A1, is correlated with that of thyroid hormone-regulated genes.

Thyroid hormone (TH) is essential for normal development; therefore, disruption of TH action by a number of industrial chemicals is critical to identify. Several chemicals including polychlorinated biphenyls are metabolized by the dioxin-inducible enzyme CYP1A1; some of their metabolites can interact with the TH receptor. In animals, this mechanism is reflected by a strong correlation between the expression of CYP1A1 mRNA and TH-regulated mRNAs. If this mechanism occurs in humans, we expect that CYP1A1 expression will be positively correlated with the expression of genes regulated by TH. The objective of the study was to test the hypothesis that CYP1A1 mRNA expression is correlated with TH-regulated mRNAs in human placenta. One hundred sixty-four placental samples from pregnancies with no thyroid disease were obtained from the GESTE study (Sherbrooke, Québec, Canada). Maternal and cord blood TH levels were measured at birth. The mRNA levels of CYP1A1 and placental TH receptor targets [placental lactogen (PL) and GH-V] were quantitated by quantitative PCR. CYP1A1 mRNA abundance varied 5-fold across 132 placental samples that had detectable CYP1A1 mRNA. CYP1A1 mRNA was positively correlated with PL (r = 0.64; P < .0001) and GH-V (P < .0001, r = 0.62) mRNA. PL and GH-V mRNA were correlated with each other (r = 0.95; P < .0001), suggesting a common activator. The mRNAs not regulated by TH were not correlated with CYP1A1 expression. CYP1A1 mRNA expression is strongly associated with the expression of TH-regulated target gene mRNAs in human placenta, consistent with the endocrine-disrupting action of metabolites produced by CYP1A1.

Evaluation of in vitro inhibition and induction of cytochrome P450 activities by hydrolyzed ginkgolides

Ethnopharmacological relavanceThe extracts of Ginkgo biloba leaves are effective in treating cerebral infarction, of which ginkgolides have been demonstrated to be the active ingredients. The purpose of this study was to determine whether hydrolyzed ginkgolides would cause potential drug–drug interactions (DDI) during its clinical use via inhibition or induction of the major human cytochrome P450s (CYPs). Materials and methodsThe inhibition (direct and metabolism-dependent inhibiton on CYP activities) and induction (mRNA expression level and activity of CYPs) by the hydrolyzed ginkgolides were evaluated in human liver microsomes and cryopreserved human hepatocytes, respectively. ResultsWithin 0.1 to 10μg/mL, the hydrolyzed ginkgolides showed negligible direct inhibition against CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4m (midazolam as substrate) and 3A4t (testosterone as substrate), with IC50 values determined to be >10μg/mL (concentrations expressed as the sum of equivalent concentrations of ginkgolide A, B and K). For the metabolism-dependent inhibition studies, the preincubation of 30min did not substantially alter the IC50 values when compared with the corresponding values in the direct inhibition studies. The activities and mRNA expression levels for CYP1A2 and 2B6 within each drug-treated group (0.1, 1 and 10μg/mL) were not affected after the 48-h incubation. For CYP3A4, the activity and mRNA expression level were not altered when incubated with 0.1 and 1μg/mL of hydrolyzed ginkgolides. When incubated with hydrolyzed ginkgolides at 10μg/mL, the relative activity and relative mRNA expression level of CYP3A4 remarkably increased to 4.59±3.67 and 17.2±9.16-fold of the corresponding vehicle control values, respectively. ConclusionsThe hydrolyzed ginkgolides is not likely to cause DDI via inhibition of the major human CYPs. However, the CYP3A4 induction might be clinically relevant.

Effects of cytokines on CYP3A4 expression and reversal of the effects by anti-cytokine agents in the three-dimensionally cultured human hepatoma cell line FLC-4

The expression of hepatic cytochrome P450 (CYP) enzymes is altered under pathological conditions with increased levels of cytokines. In this study, we analyzed the effects of cytokines (interleukin [IL]-1β, IL-6 and tumor necrosis factor α) on the expression of CYP3A4 using newly introduced three-dimensionally cultured human hepatocarcinoma FLC-4 cells. The mRNA level of CYP3A4 was significantly decreased by IL-1β, IL-6 and tumor necrosis factor-α. Formation of α-hydroxytriazolam catalyzed by CYP3A was decreased by IL-1β and IL-6. Pre-treatment with IL-6 enhanced the cytotoxic effects of gefitinib and paclitaxel. In addition, tocilizumab and IL-1 receptor antagonist restored the decreased expression of CYP3A4 mRNA by IL-6 and IL-1β, respectively. These results obtained by using three-dimensionally cultured FLC-4 cells are consistent with results obtained by using primary human hepatocytes and results of clinical studies. Therefore, three-dimensionally cultured FLC-4 cell system may be a promising cellular tool to assess the effects of cytokines on CYP3A4 expression.

Cytochrome P450 3 A Expression in Pigs Livers After 24-Hour Preservation in Biolasol Solution Depending on the Type of Transgenesis

BackgroundAn insufficient number of organs for transplantation shows the need for the development of new technologies. Xenotransplantation might be the answer. ObjectiveTo determine if the type of transgenesis influences the level of CYP3A4, which takes an active part in xenobiotics metabolism in livers after 24-hour storage, depending on the kind of solution used for preservation. Materials and methodsThe experiment was carried out on 30 livers of Polish White Landrace divided into 5 groups depending on transgene type. The following human genes were transferred: α1,2-fucosyltransferase (groups I and II), α-galactosidase (III), combined α1,2-fucosyltransferase/α-galactosidase transgene (IV), and livers without modification (V). The livers were perfused and subsequently stored for 24 hours in Ringer's solution (group I) or Biolasol solution (II-V). Reperfusion/reflush was performed. CYP3A29 isomer concentration was analyzed in liver specimens collected twice: 30 minutes after perfusion and 30 minutes after reperfusion/reflush. Expression of mRNA CYP3A29 was marked using RT-PCR analysis and of protein CYP3A29 using Western blotting technique. ResultsThe most significant decrease in protein CYP3A29 expression after 24-hour preservation was observed in group I (55.88% decrease), while the least significant was observed in group IV (10.44% decrease). mRNA expression evaluation was similar: the most significant decrease was observed in group I (87.8% decrease) and the least significant in group III (4.6% decrease). Conclusionα1,2-Fcosyltransferase transgene seems to influence mRNA and protein CYP3A expression in case of liver grafting and preservation for transplantation. CYP3A expression was also influenced by the kind of preservation solution used.

Modulating potential of L-sulforaphane in the expression of cytochrome p450 to identify potential targets for breast cancer chemoprevention and therapy using breast cell lines.

The L-sulforaphane (SFN) component of broccoli sprout showed anticancer activity in several preclinical studies including breast cancer. Estrogens are considered major risk factors in breast carcinogenesis. The aim of this study was to evaluate the effect of SFN on the expression of cytochrome P450 involved in the estrogen metabolism in breast cancer cell lines MCF7 and MDA-MB-231 and in non-tumorigenic MCF10A cell line. The expression of CYP19, CYP1A1, 1A2, 1B1 was determined at the transcript and protein levels. There were found some remarkable differences in the effect of SFN at a dose of 5 µmol/L on CYP19 expression: in ER(+) MCF7 significant reduction, in ER(-) MDA-MB-231 an increased expression and unchanged expression in MCF10A cell line. The effect of SFN on CYPs (1A1, 1A2, 1B1) involved in estrogen catabolism was to a lesser extent dependent on breast cell line. The slightly reduced CYP1A1 protein level was observed in all cell lines tested. An increased level of CYP1A2 and decreased level of CYP1B1 expression were found in MCF10A. These results indicate that the naturally occurring L isomer of SFN may affect the expression of P450s involved in estrogen metabolism. This effect may contribute to the anticancer activity of SFN in breast tissue.

Acute and chronic effects of IL-22 on acetaminophen-induced liver injury.

Acetaminophen (APAP)-induced liver injury (AILI) accounts for half of the acute liver failure cases in the United States. A better understanding of the underlying mechanisms of AILI is necessary for the development of novel antidotes. We found that pretreatment with IL-22 protected mice from APAP-mediated hepatotoxicity. The protection was dependent on STAT3, as IL-22 failed to reduce APAP hepatotoxicity in liver-specific STAT3 knockout mice. In contrast to the acute exposure to IL-22, the endogenous chronic overexpression of IL-22 in IL-22 transgenic (TG) mice or IL-22 adenovirus treatment for 6 wk resulted in a markedly increased susceptibility to AILI. Furthermore, the hepatic expression levels of cytochrome 2E1 (Cyp2E1) and Cyp1A2 were much higher in IL-22TG mice. Ablation of Cyp2E1 but not hepatic STAT3 abolished AILI and protein-adduct formation in IL-22TG mice. Finally, hepatic expression of HNF-1α, a transcriptional factor that is known to control Cyp2E1 expression, was elevated in IL-22TG mice compared with wild-type mice. Upregulation of hepatic Cyp2E1 was only observed in mice with constitutive overexpression of IL-22 but not with short-term treatment with one dose of IL-22 or multiple doses of IL-22 for 2 wk. In conclusion, short-term acute IL-22 exposure protects mice against AILI through STAT3 activation; however, chronic constitutive overexpression of IL-22 exacerbates AILI by increasing Cyp2E1 and toxic reactive APAP metabolite production. These findings may not only enhance our understanding of the effects of chronic inflammation on AILI in patients with liver disease, but are also helpful to identify novel therapeutic targets for the treatment of AILI.

Altered CYP2C9 activity following modulation of CYP3A4 levels in human hepatocytes: an example of protein-protein interactions.

Cytochrome P450 (P450) protein-protein interactions resulting in modulation of enzyme activities have been well documented using recombinant isoforms. This interaction has been less clearly demonstrated in a more physiologic in vitro system such as human hepatocytes. As an expansion of earlier work (Subramanian et al., 2010), in which recombinant CYP2C9 activity decreased with increasing levels of CYP3A4, the current study modulated CYP3A4 content in human hepatocytes to determine the impact on CYP2C9. Modulation of CYP3A4 levels in situ was enabled by the use of a long-term human hepatocyte culture model (HepatoPac) shown to retain phenotypic hepatocyte function over a number of weeks. The extended period of culture allowed time for knockdown of CYP3A4 protein by small interfering RNA (siRNA) with subsequent recovery, as well as upregulation through induction with a recovery period. CYP3A4 gene silencing resulted in a 60% decrease in CYP3A4 activity and protein levels with a concomitant 74% increase in CYP2C9 activity, with no change in CYP2C9 mRNA levels. Upon removal of siRNA, both CYP2C9 and CYP3A4 activities returned to pre-knockdown levels. Importantly, modulation of CYP3A4 protein levels had no impact on cytochrome P450 reductase activities or levels. However, the possibility for competition for limiting reductase cannot be ruled out. Interestingly, lowering CYP3A4 levels also increased UDP-glucuronosyltransferase 2B7 activity. These studies clearly demonstrate that alterations in CYP3A4 levels can modulate CYP2C9 activity in situ and suggest that further studies are warranted to evaluate the possible clinical consequences of these findings.

Higher expression of cytochrome P450 3A4 in human mesenchymal and adipose-derived stem cells than in dermal fibroblasts: With emphasis on the correlation with basal pregnane X receptor expression

Among the cytochrome P 450 (CYP) enzymes, CYP3A4 is most abundantly existed and metabolizes most of the drugs. We have shown human dermal fibroblasts (HDFs) express low levels of CYPs, but could be engineered to express much higher levels to be used as drug metabolism prediction. To seek better cell source for engineering, the basal mRNA expression and enzyme activities of CYP3A4 in HDFs (n=5), bone marrow-derived mesenchymal stem cells (BMMSCs, n=7), and adipose-derived stem cells (ADSCs, n=3) were compared. The basal CYP3A4 expression and activity in BMMSCs and ADSCs were significantly higher than in HDFs. However, the coefficient of variation of CYP3A4 expression was very high in each cell type. Comparing the expression levels of 4 regulators glucocorticoid receptor, pregnane X receptor (PXR), hepatocyte nuclear factor 4α, and hydrocarbon receptor nuclear translocator in the total 15 cell samples, we found the only basal PXR expression was significantly correlated with CYP3A4 expression. The gender and age of patients were not significant determinants in the expressions of PXR and CYP3A4. In conclusion, BMMSCs and ADSCs expressed higher levels of CYP3A4 than HDFs and the expression of CYP3A4 might be mainly determined by basal PXR expression.

Chemical and biological characterization of air particulate matter 2.5, collected from five cities in China

Fifteen polycyclic aromatic hydrocarbons (PAHs) in PM2.5 samples collected in five different cities (Hong Kong (HK), Guangzhou (GZ), Xiamen (XM), Xi'an (XA) and Beijing (BJ)) in China in the winter 2012–12 were analyzed by gas chromatography-mass spectrometry. The biological effects of organic extracts were assayed using the human bronchial epithelial cells BEAS-2B. All sixteen priority PAHs can be found in the PM2.5 samples of XA and BJ, but not in HK, GZ and XM, demonstrating the differential spatial source and distribution of PAHs. Our results showed that the total PAHs ranged from 3.35 to 80.45 ng/m3 air, leading by BJ, followed by XA, XM, GZ and HK. In the cell culture study, transcript levels of pro-inflammatory cytokine interleukin-6 (IL-6), CYP1A1 and CYP1B1 were found to be induced in the treatment. The cells exposed to extracts from XA and BJ demonstrated significant migratory activities, indicating a sign of increase of tumorigenicity.

Up-regulation on cytochromes P450 in rat mediated by total alkaloid extract from Corydalis yanhusuo.

BackgroundYanhusuo (Corydalis yanhusuo W.T. Wang; YHS), is a well-known traditional Chinese herbal medicine, has been used in China for treating pain including chest pain, epigastric pain, and dysmenorrhea. Its alkaloid ingredients including tetrahydropalmatine are reported to inhibit cytochromes P450 (CYPs) activity in vitro. The present study is aimed to assess the potential of total alkaloid extract (TAE) from YHS to effect the activity and mRNA levels of five cytochromes P450 (CYPs) in rat.MethodsRats were administered TAE from YHS (0, 6, 30, and 150 mg/kg, daily) for 14 days, alanine aminotransferase (ALT) levels in serum were assayed, and hematoxylin and eosin-stained sections of the liver were prepared for light microscopy. The effects of TAE on five CYPs activity and mRNA levels were quantitated by cocktail probe drugs using a rapid chromatography/tandem mass spectrometry (LC-MS/MS) method and reverse transcription-polymerase chain reaction (RT-PCR), respectively.ResultsIn general, serum ALT levels showed no significant changes, and the histopathology appeared largely normal compared with that in the control rats. At 30 and 150 mg/kg TAE dosages, an increase in liver CYP2E1 and CYP3A1 enzyme activity were observed. Moreover, the mRNA levels of CYP2E1 and CYP3A1 in the rat liver, lung, and intestine were significantly up-regulated with TAE from 6 and 30 mg/kg, respectively. Furthermore, treatment with TAE (150 mg/kg) enhanced the activities and the mRNA levels of CYP1A2 and CYP2C11 in rats. However, the activity or mRNA level of CYP2D1 remained unchanged.ConclusionsThese results suggest that TAE-induced CYPs activity in the rat liver results from the elevated mRNA levels of CYPs. Co-administration of prescriptions containing YHS should consider a potential herb (drug)–drug interaction mediated by the induction of CYP2E1 and CYP3A1 enzymes.

Dual-color fluorescence imaging to monitor CYP3A4 and CYP3A7 expression in human hepatic carcinoma HepG2 and HepaRG cells.

Human adult hepatocytes expressing CYP3A4, a major cytochrome P450 enzyme, are required for cell-based assays to evaluate the potential risk of drug-drug interactions caused by transcriptional induction of P450 enzymes in early-phase drug discovery and development. However, CYP3A7 is preferentially expressed in premature hepatoblasts and major hepatic carcinoma cell lines. The human hepatocellular carcinoma cell line HepaRG possesses a high self-renewal capacity and can differentiate into hepatic cells similar to human adult hepatocytes in vitro. Transgenic HepaRG cells, in which the expression of fluorescent reporters is regulated by 35 kb regulatory elements of CYP3A4, have a distinct advantage over human hepatocytes isolated by collagenase perfusion, which are unstable in culture. Thus, we created transgenic HepaRG and HepG2 cells by replacing the protein-coding regions of human CYP3A4 and CYP3A7 with enhanced green fluorescent protein (EGFP) and DsRed reporters, respectively, in a bacterial artificial chromosome vector that included whole regulatory elements. The intensity of DsRed fluorescence was initially high during the proliferation of transgenic HepaRG cells. However, most EGFP-positive cells were derived from those in which DsRed fluorescence was extinguished. Comparative analyses in these transgenic clones showed that changes in the total fluorescence intensity of EGFP reflected fold changes in the mRNA level of endogenous CYP3A4. Moreover, CYP3A4 induction was monitored by the increase in EGFP fluorescence. Thus, this assay provides a real-time evaluation system for quality assurance of hepatic differentiation into CYP3A4-expressing cells, unfavourable CYP3A4 induction, and fluorescence-activated cell sorting-mediated enrichment of CYP3A4-expressing hepatocytes based on the total fluorescence intensities of fluorescent reporters, without the need for many time-consuming steps.

Quantitative analysis of cytochrome P450 isoforms in human liver microsomes by the combination of proteomics and chemical probe-based assay

Cytochrome P450 (CYP) is one of the most important drug-metabolizing enzyme families, which participates in the biotransformation of many endogenous and exogenous compounds. Quantitative analysis of CYP expression levels is important when studying the efficacy of new drug molecules and assessing drug-drug interactions in drug development. At present, chemical probe-based assay is the most widely used approach for the evaluation of CYP activity although there are cross-reactions between the isoforms with high sequence homologies. Therefore, quantification of each isozyme is highly desired in regard to meeting the ever-increasing requirements for carrying out pharmacokinetics and personalized medicine in the academic, pharmaceutical, and clinical setting. Herein, an absolute quantification method was employed for the analysis of the seven isoforms CYP1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1 using a proteome-derived approach in combination with stable isotope dilution assay. The average absolute amount measured from twelve human liver microsomes samples were 39.3, 4.3, 54.0, 4.6, 10.3, 3.0, and 9.3 (pmol/mg protein) for 1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1, respectively. Importantly, the expression level of CYP3A4 showed high correlation (r = 0.943, p < 0.0001) with the functional activity, which was measured using bufalin-a highly selective chemical probe we have developed. The combination of MRM identification and analysis of the functional activity, as in the case of CYP3A4, provides a protocol which can be extended to other functional enzyme studies with wide application in pharmaceutical research.

Ivermectin exposure leads to up-regulation of detoxification genes in vitro and in vivo in mice

The biodisposition of the antiparasitic drug ivermectin in host and parasite is decisive for its efficacy and strongly depends on the efflux by ATP-Binding Cassette (ABC) transporters and on its biotransformation by cytochromes P450. The purpose of this study was to evaluate, in vitro and in vivo, the ivermectin ability in modulating the expression of the most important genes involved in drug detoxification. Gene expression of ABC transporters and cytochromes was evaluated by RT-qPCR in murine hepatic and intestinal cell lines exposed to increasing ivermectin doses, and in liver and intestine of mice orally administered with single or repeated therapeutic doses of ivermectin (0.2mg/kg). Plasma, brain, liver and intestinal concentrations of ivermectin and its main metabolite were measured by HPLC in ivermectin-treated mice. In hepatocyte cell line, ivermectin up-regulated expression of Abcb1a, Abcb1b, Abcc2, Cyp1a1, Cyp1a2, Cyp2b10; while Abcb1a, Abcb1b, Abcg2, Cyp1a1, Cyp1a2, Cyp2b10 and Cyp3a11 levels were induced in intestinal cell line. In mice, repeated administration of ivermectin induced the expression of Abcb1a, Abcc2, Cyp1a1 and Cyp2b10 in intestine while only Cyp3a11 was induced in liver. Compared with single administration, repeated ivermectin administration lowered plasma, liver and intestine drug concentration, while increasing main metabolite content in plasma and intestine. These findings can be regarded as a warning that repeated ivermectin exposure is able to induce detoxification systems in mammals that may lead to subtherapeutic drug concentration. This may also be an important consideration in the assessment of drug−drug interaction and toxicity for other ABC transporters and CYP450s substrates.

Expression profile of CYP1A1 and CYP1B1 enzymes in endometrial tumors.

The cytochrome P450 CYP1A1 and CYP1B1 enzymes are phase I extrahepatic enzymes involved in the activation of pro-carcinogenic compounds to carcinogenic metabolites. Although differential overexpression of CYP1A1 and CYP1B1 has been documented at the messenger RNA (mRNA) and protein level, studies that have examined CYP1 expression by enzyme activity assays are limited. In the current study, the expression of CYP1A1 and CYP1B1 was investigated in a panel of human tumors of endometrial origin by quantitative reverse transcriptase PCR (qRT-PCR), Western blotting, and enzyme activity assays. The data revealed that approximately 36 % (5/14) and 43 % (6/14) of the endometrial tumors overexpressed CYP1A1 and CYP1B1 mRNA, whereas in 57 % of the endometrial tumors, CYP1 mRNA levels were downregulated. The mean mRNA levels of CYP1B1 and CYP1A1 in endometrial tumors did not show a significant difference compared to normal tissues (p > 0.05). Western blotting confirmed the qRT-PCR results and CYP1A1 and CYP1B1 proteins were shown to be downregulated in 7/14 (50 %) of the tumors and overexpressed in 4/14 (29 %) of the tumors. As regards to enzyme activity, 21 % (3/14) of the endometrial samples revealed elevated CYP1 activity levels across the tumor counterparts. Overall, the data suggest a putative downregulation of CYP1A1 and CYP1B1 expression in endometrial tumors, whereas overexpression of active CYP1 enzymes in 21 % of the tumors highlights the potential use of the latter enzymes as chemotherapeutic targets in endometrial cancer.

Ecotoxicological status of seven sperm whales (Physeter macrocephalus) stranded along the Adriatic coast of Southern Italy

ABSTRACT In December 2009 seven male sperm whales (Physeter macrocephalus) stranded along the Adriatic coast of Southern Italy. Genetic and photo‐identification data indicated that these specimens belonged to the Mediterranean Sea population. Complete necropsies were performed on the three best preserved animals. Blubber and muscle samples were collected from all seven specimens for contaminants analysis (DDTs, PCBs and PAHs) and biomarker responses (CYP1A1 and CYP2B) were evaluated from the three better preserved animals using western blot analysis. Fibroblast cell cultures from two specimens were set up by culturing cell lines from integument tissue, to explore the susceptibility of these marine mammals to different xenobiotic compounds. The levels of CYP1A1 and CYP2B, proteins that are induced by some POPs (DDTs and PCBs) and PAHs were detected using the indirect immunofluorescence assay. The results showed relatively high levels of contaminants, in particular from organochlorine xenobiotics, and strong responses of CYP1A1 and CYP2B reflecting toxicological stress of these sperm whales. This is not considered to be their cause of death, but may have contributed to lowering the defences of their immune system. Copyright © 2014 John Wiley &amp; Sons, Ltd.

Modulation of CYP1A1 and CYP1A2 hepatic enzymes after oral administration of Chios mastic gum to male Wistar rats.

Chios mastic gum (CMG), a resin derived from Pistacia lentiscus var. chia, is known since ancient times for its pharmacological activities. CYP1A1 and CYP1A2 enzymes are among the most involved in the biotransformation of chemicals and the metabolic activation of pro-carcinogens. Previous studies referring to the modulation of these enzymes by CMG have revealed findings of unclear biological and toxicological significance. For this purpose, the modulation of CYP1A1 and CYP1A2 enzymes in the liver of male Wistar rats following oral administration of CMG extract (CMGE), at the levels of mRNA and CYP1A1 enzyme activity, was compared to respective enzyme modulation following oral administration of a well-known bioactive natural product, caffeine, as control compound known to involve hepatic enzymes in its metabolism. mRNA levels of Cyp1a1 and Cyp1a2 were measured by reverse transcription real-time polymerase chain reaction and their relative quantification was calculated. CYP1A1 enzyme induction was measured through the activity of ethoxyresorufin-O-deethylase (EROD). The results indicated that administration of CMGE at the recommended pharmaceutical dose does not induce significant transcriptional modulation of Cyp1a1/2 and subsequent enzyme activity induction of CYP1A1 while effects of the same order of magnitude were observed in the same test system following the administration of caffeine at the mean daily consumed levels. The outcome of this study further confirms the lack of any toxicological or biological significance of the specific findings on liver following the administration of CMGE.

Bioactive components of Glycyrrhiza uralensis mediate drug functions and properties through regulation of CYP450 enzymes.

Glycyrrhiza uralensis (G. uralensis) is a common medicinal plant that has mainly been used to modulate the pharmaceutical activity of herbal medicines. Although G. uralensis has been shown to affect the expression and activity of the key metabolic enzyme cytochrome P450 (CYP450), the detailed mechanism of this process has yet to be elucidated. The present study aimed to elucidate the effects of bioactive components of G. uralensis on different isoforms of CYP450 and determine the ability of these components to modulate drug properties. In the present study, mRNA levels of CYP1A2, CYP2D6, CYP2E1, and CYP3A4 were investigated by quantitative polymerase chain reaction (qPCR) in HepG2 cells following treatment with the major bioactive compounds of G. uralensis. The activity of CYP450 enzymes was investigated in human liver microsomes using the cocktail probe drug method, and the metabolites of specific probes were detected by UPLC‑MS/MS. The effects of G. uralensis on CYP450 were assessed using bioinformatics network analysis. Several compounds from G. uralensis had various effects on the expression and activity of multiple CYP450 isoforms. The majority of the compounds analysed the inhibited expression of CYP2D6 and CYP3A4. Several CYP isoforms were differentially modulated depending on the specific compound and dose tested. In conclusion, the present study suggested that G. uralensis influenced the expression and activity of CYP450 enzymes. Therefore, caution should be taken when G. uralensis is co‑administered with drugs that are known to be metabolized by CYP450. This study contributed to the knowledge of the mechanisms by which this medicinal plant, commonly known as licorice, modulates drug efficacy.

6-shogaol, a major compound in ginger, induces aryl hydrocarbon receptor-mediated transcriptional activity and gene expression.

Xenobiotics are usually detoxified by drug-metabolizing enzymes and excreted from the body. The expression of many of drug-metabolizing enzymes is regulated by the aryl hydrocarbon receptor (AHR). Some substances in vegetables have the potential to be AHR ligands. To search for vegetable components that exhibit AHR-mediated transcriptional activity, we assessed the activity of vegetable extracts and identified the active compounds using the previously established stable AHR-responsive HepG2 cell line. Among the hot water extracts of vegetables, the highest activity was found in ginger. The ethyl acetate fraction of the ginger hot water extract remarkably induced AHR-mediated transcriptional activity, and the major active compound was found to be 6-shogaol. Subsequently, the mRNA levels of AHR-targeting drug-metabolizing enzymes (CYP1A1, UGT1A1, and ABCG 2) and the protein level of CYP1A1 in HepG2 cells were shown to be increased by 6-shogaol. This is the first report that 6-shogaol can regulate the expression of detoxification enzymes by AHR activation.