Cytochrome P450 1A1 mRNA Research Articles

Arecoline inhibits the 2,3,7,8-tetrachlorodibenzo- p-dioxin-induced cytochrome P450 1A1 activation in human hepatoma cells

In the present study, we investigated the effect of arecoline, a major areca nut alkaloid, on the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced activation of cytochrome P4501A1 (CYP1A1) in a human hepatoma cell line Huh-7. We treated Huh-7 cells with 10 nM TCDD in the presence of different concentrations of arecoline (50–300 μM). Our results indicated that arecoline attenuated the TCDD-induced CYP1A1 enzyme activation with an inhibitory effect on cell proliferation. By using real-time RT-PCR, we demonstrated that arecoline inhibited the TCDD-induced activations of CYP1A1 and AhR repressor (AhRR) mRNA expression in a similar pattern. Our results revealed that arecoline inhibited AhR mRNA expression with no direct effect on CYP1A1 enzyme activity. Therefore, in our present study, the observed inhibitory effect of arecoline on CYP1A1 activation was not due to the up-regulation of AhRR or direct inhibitory effect on CYP1A1. Taken together, here we have demonstrated that arecoline attenuates the TCDD-induced CYP1A1 activation mainly via down-regulation of AhR expression in human hepatoma cells, suggesting the possible involvement of arecoline in the AhR-mediated metabolism of environmental toxicants in liver.

Same-sample analysis of ethoxyresorufin- O-deethylase activity and cytochrome P4501A mRNA abundance in chicken embryo hepatocytes

Inducibility of the cytochrome P4501A4 (CYP1A4) enzyme, measured as ethoxyresorufin- O-deethylase (EROD) activity, has been used as a biomarker for sensitivity to the effects of dioxin-like compounds in avian species. Here, we present a quantitative reverse transcriptase-PCR (Q-PCR) method for assessing this biomarker response at the level of messenger RNA (mRNA) expression. The method was validated for use in fresh samples as well as samples that have been analyzed for EROD activity previously. Concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on CYP1A4 and CYP1A5 mRNA abundance were detected in fresh and post-EROD hepatocyte cultures. Although the quality of the RNA obtained from post-EROD samples was low, quantification of the CYP1A mRNA response to TCDD was not compromised. Several benefits of evaluating CYP1A mRNA expression in addition to EROD activity were noted. The CYP1A mRNA bioassay may provide more accurate estimates for the potency of environmental mixtures of contaminants and has a very low detection limit. When working with hepatocytes cultured from wild or endangered species, our approach can help to circumvent the problem of small sample size by maximizing the amount of data obtained from each sample.

Effects of Tryptophan Photoproducts in the Circadian Timing System: Searching for a Physiological Role for Aryl Hydrocarbon Receptor

The aryl hydrocarbon receptor (AhR) mediates adverse effects of dioxins, but its physiological role remains ambiguous. The similarity between AhR and canonical circadian clock genes suggests potential involvement of AhR in regulation of circadian timing. Photoproducts of tryptophan (TRP), including 6-formylindolo[3,2-b]carbazole (FICZ), have high affinity for AhR and are postulated as endogenous ligands. Although TRP photoproducts activate AhR signaling in vitro, their effects in vivo have not been investigated in mammals. Because TRP photoproducts may act as transducers of light, we examined their effects on the circadian clock. Intraperitoneal injection of TRP photoproducts or FICZ to C57BL/6J mice dose dependently induced AhR downstream targets, cytochrome P4501A1 (CYP1A1) and cytochrome P4501B1 mRNA expression, in liver. c-fos mRNA, a commonly used marker for light responses, was also induced with FICZ, and all responses were AhR dependent. A rat-immortalized suprachiasmatic nucleus (SCN) cell line, SCN 2.2, was used to examine the direct effect of TRP photoproducts on the molecular clock. Both TRP photoproducts and FICZ-increased CYP1A1 expression and prolonged FICZ incubation altered the circadian expression of clock genes (Per1, Cry1, and Cry2) in SCN 2.2 cells. Furthermore, FICZ inhibited glutamate-induced phase shifting of the mouse SCN electrical activity rhythm. Circadian light entrainment is critical for adjustment of the endogenous rhythm to environmental light cycle. Our results reveal a potential for TRP photoproducts to modulate light-dependent regulation of circadian rhythm through triggering of AhR signaling. This may lead to further understanding of toxicity of dioxins and the role of AhR in circadian rhythmicity.

Internal dose-effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in gonadotropin-primed weanling rat model

Single sc injection of 5 IU equine chorionic gonadotropin (eCG) induces ovulation in weanling female rats 3 days later. It has been shown that treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 24 h before eCG injection reduces eCG-stimulated ovarian hypertrophy and inhibits ovulation. The present study intended to compare internal dose-effects of TCDD between these endpoints and representative endpoints for TCDD toxicity, such as weights of the liver and thymus, in weanling female rats given orally 0, 1, 4 or 16 microg/kg TCDD 24 h before eCG injection on postnatal day 25. Measurement of plasma TCDD concentrations by ELISA at 6, 72 and 96 h after TCDD revealed that significant levels of TCDD were maintained in systemic circulation until 96 h (on the day of induced ovulation) with the highest level at 6 h after TCDD treatment. Ovarian TCDD concentrations varied similarly and tended to be higher than those in the thymus at all time points, whereas hepatic concentrations of TCDD were the highest among the tissues. Although > or = 4 microg/kg TCDD affected the weights of the thymus and liver, no differences were observed in ovarian weights at any time point or in ovulation between corn oil-treated and TCDD-treated groups. Furthermore, ovarian levels of representative mRNAs in follicles were not affected by TCDD treatment. Since TCDD increased the amount of cytochrome P450 1A1 mRNA in the ovary, the administered TCDD stimulated the aryl hydrocarbon receptor-signaling pathway. From these results, we concluded that thymus weights of weanling female rats responded to TCDD at a lower internal dose as compared with that ovarian hypertrophy and follicular growth from early antral stage to ovulation would respond to.

Toxic effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in developing red seabream ( Pagrus major) embryo: An association of morphological deformities with AHR1, AHR2 and CYP1A expressions

The toxicity of dioxins such as 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) is mainly mediated by the aryl hydrocarbon receptor (AHR), which regulates the multiple target genes including cytochrome P4501A (CYP1A). In general, bony fishes, which possess at least two distinct AHRs are one of the most sensitive vertebrates to TCDD in early life stage. However, the physiological and toxicological roles of piscine multiple AHRs are not fully understood, especially in marine fish. To understand which AHR is responsible for TCDD toxicity in a marine fish species, we characterized the early life stage toxicity related to the expression of AHRs and CYP1A in red seabream ( Pagrus major). The embryos at 10 h post-fertilization (hpf) were treated with 0–100 μg/L TCDD for 80 min waterborne exposure. TCDD dose-dependently elicited developmental toxicities including mortality, yolk sac edema, retarded body growth, spinal deformity, reduced heart rate, shortened snout, underdeveloped fin, heart, and lower jaw. Intriguingly, hemorrage and pericardium edema, typical TCDD developmental defects noticed in other fish species, were not found in red seabream until test termination. The EC egg50 s for yolk sac edema, underdeveloped fin, and spinal deformity were 170, 240, and 340 pg/g, respectively. The LC egg50 was 360 pg/g embryo, indicating that this species is one of the most sensitive fishes to TCDD toxicity. The expression levels of rsAHR1, rsAHR2 and CYP1A mRNAs were also determined in different developmental stages. The rsAHR2 mRNA expression dose-dependently increased following TCDD exposure, while rsAHR1 mRNA level was not altered. Level of rsAHR2 mRNA measured by two-step real-time PCR was 30 times higher than rsAHR1 in embryos treated with the highest dose. Temporal patterns of rsAHR2 and CYP1A mRNAs were similar in TCDD-treated embryos, representing a significant positive correlation between rsAHR2 and CYP1A mRNA levels, but not between rsAHR1 and CYP1A. In comparison of temporal trends of TCDD-induced AHRs and CYP1A expression, and developmental toxicities, the highest expression of rsAHR2 and CYP1A mRNA were detected prior to the appearance of maximal incidence of TCDD toxic manifestations. These results suggest that rsAHR2 may be dominantly involved in the transcriptional regulation of CYP1A, and several TCDD defects are dependent on the alteration of rsAHR2 and/or rsAHR2-CYP1A signaling pathway that is controlled through their expression levels.

Structural and functional characterization of microcystin detoxification-related liver genes in a phytoplanktivorous fish, Nile tilapia ( Oreochromis niloticus)

Liver genes related to phase I and phase II detoxification, as well as inhibition of reactive oxygen species (ROS) production, were cloned, and their response to microcystin-LR (MC-LR) and lipopolysaccharide (LPS) exposure via intraperitoneal injection, was determined in a phytoplanktivorous fish, Nile tilapia ( Oreochromis niloticus). The cloned full-length cDNA of tilapia soluble glutathione S-transferase (sGST) was classified as alpha-class GST based on their amino acid sequence identity with other species. The tilapia sGST clone was 861 bp in length, and contained a 25 bp 5′-UTR, a 167 bp 3′-UTR and an open reading frame of 669 bp, encoding a polypeptide of 222 amino acids. Using genome walker method, a 366 bp 5′-flanking sequence of tilapia sGST gene was further obtained, and the possible regulatory elements were identified. Partial cDNA sequences of glutathione peroxidase (GPX) and uncoupling protein 2 (UCP2) were also obtained by PCR using degenerate primers from tilapia liver. To study the transcriptional response of liver genes to microcystin treatment, tilapia were respectively exposed to a single 50 μg kg − 1 body weight (bwt) dose of pure MC-LR, a single 2 mg kg − 1 bwt dose of LPS and a co-exposure MC-LR and LPS (50 μg kg − 1 bwt + 2 mg kg − 1 bwt), and were then sacrificed at 24 h post-exposure. Using beta-actin as external control, a significant increase (about 80%) in sGST mRNA expression was found in response to the MC-LR exposure after 24 h ( P < 0.05), indicating the importance of sGST in microcystin detoxification. A slight decrease of sGST mRNA expression was observed in the liver of tilapia, exposed to LPS and MC-LR + LPS. It seems that the LPS response element (LPSRE), identified in the promoter region of tilapia sGST gene, may be functional at a rather low level. In contrast, the levels of cytochrome P450 1A (CYP1A) mRNA expression were found to keep unchanged to either MC-LR, or LPS, or MC-LR + LPS treatment, indicating that unlike the phase II enzyme (sGST), the phase I enzyme (CYP1A) might not play an important role in the detoxification process of microcystins. Although not significant, the mRNA expression level of GPX tended to increase in the liver of tilapia exposed to both MC-LR and LPS ( P > 0.05). In addition, a significant increase in UCP2 mRNA expression was observed in the liver of tilapia exposed to LPS ( P < 0.05), as well as an obvious but not significant increase in MC-LR exposure group. We suggest that phase II detoxification enzyme, instead of phase I detoxification enzyme, might be responsible for the strong tolerance of the phytoplanktivorous fish to microcystins, and hepatocyte proteins coping with oxidative stress (GPX and UCP2), might also have some auxiliary effect. In addition, the rather low and insignificant response of tilapia sGST gene to the inhibitory effect of LPS exposure, might possibly be critical to the phytoplanktivorous fish to utilize toxic blue-green algae.

Epigenetic Inactivation of the Dioxin-Responsive Cytochrome P4501A1 Gene in Human Prostate Cancer

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin) is a toxic environmental contaminant that works through dioxin response elements (DRE) to activate gene expression. We tested the hypothesis that cancer-related epigenetic changes suppress dioxin activation of the cytochrome P4501A1 (CYP1A1) gene. 5-Aza-2'-deoxycytidine (5-aza-CdR), an inhibitor of DNA methylation, increases TCDD-inducible CYP1A1 mRNA expression in cancerous LNCaP cells but not in noncancerous PWR-1E and RWPE-1 cells (all human prostate cell lines). Bisulfite DNA sequencing shows that the TCDD-responsive CYP1A1 enhancer is highly methylated in LNCaP cells but not in RWPE-1 cells. In vivo footprinting experiments reveal that unmethylated DRE sites do not bind protein in response to TCDD in LNCaP cells, whereas inducible DRE occupancy occurs in RWPE-1 cells. Pretreatment of LNCaP cells with 5-aza-CdR partially restores TCDD-inducible DRE occupancy, showing that DNA methylation indirectly suppresses DRE occupancy. Chromatin immunoprecipitation experiments reveal that LNCaP cells lack trimethyl histone H3 lysine 4, a mark of active genes, on the CYP1A1 regulatory region, whereas this histone modification is prevalent in PWR-1E and RWPE-1 cells. We also analyzed CYP1A1 enhancer methylation in human prostate tissue DNA. We do not detect CYP1A1 enhancer methylation in 30 DNA samples isolated from noncancerous prostate tissue. In contrast, 11 of 30 prostate tumor DNA samples have detectable CYP1A1 enhancer methylation, indicating that it is hypermethylated in prostate tumors. This is the first report that shows that CYP1A1 is aberrantly hypermethylated in human prostate cancer and has an altered, inaccessible chromatin structure that suppresses its dioxin responsiveness.

Indole-3-carbinol alters placental cytochrome P450 1A1 and P-glycoprotein levels in rats: A potential role in intensifying fetal intrauterine growth-retardation produced by tobacco smoke

To investigate the deleterious effects and possible mechanism of prenatal indole-3-carbinol (I3C) treatment on normal and tobacco-induced intrauterine growth restriction (IUGR) in rats, prenatal development toxicity in rats was studied. Expression of rat placental cytochrome P4501A1 ( CYP1A1) and P-glycoprotein (Pgp), including mdr1a and mdr1b, were detected using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Results showed that prenatal oral I3C had no significant effects on corpora lutea counts, implantation or the number of live, dead and resorbed fetuses in normal rats. Fetal malformations, sex ratio, neonatal body weights and physical developmental indices were also unchanged after prenatal I3C treatment. However, the offspring in the tobacco+I3C (4 mg kg −1) group showed lower average body weights (3.98±0.29 g) than tobacco control (4.48±0.11 g), and body and tail lengths lagged significantly behind those of the tobacco-smoke exposure only group. Expression of placental CYP1A1 mRNA by RT-PCR was not detected in the normal group, but was detected in the I3C, tobacco and tobacco+I3C groups. The level of CYP1A1 mRNA expression in the tobacco+I3C group was higher than in tobacco control. The level of mdr1a mRNA increased significantly in the I3C group when compared to normal control, and no obvious difference was detected between tobacco and tobacco+I3C groups. Expression of mdr1b mRNA was increased in the I3C and tobacco+I3C groups compared to their respective controls. Immunohistochemistry results showed that placental Pgp expression was enhanced in the I3C, tobacco and tobacco+I3C groups when compared to the normal control. The results suggest that prenatal oral I3C had no developmental toxicity but intensified fetal IUGR produced by prenatal tobacco-smoke exposure in rats. Up-regulations of placental CYP1A1 and Pgp by I3C might underlie the toxic mechanism.

Regulation of theCYP1A1Gene by 2,3,7,8-Tetrachlorodibenzo-p-dioxin but Not by β-Naphthoflavone or 3-Methylcholanthrene Is Altered in Hepatitis C Virus Replicon-Expressing Cells

Exposure to hepatitis C virus (HCV) can lead to the development of cirrhosis and hepatocellular carcinoma. To examine the effects of long-term HCV infection on hepatic cytochrome P450 1A1 (CYP1A1) expression and function, we used a human hepatoma cell line expressing the HCV subgenomic replicon (Huh.8) to evaluate CYP1A1 induction by the aryl hydrocarbon receptor (AhR) ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this study, we demonstrate that the induction of CYP1A1 expression in Huh.8 cells by TCDD but not by beta-naphthoflavone or 3-methylcholanthrene was significantly diminished. TCDD exposure of Huh.8 cells resulted in greater than 55% suppression of CYP1A1 transcription compared with the parent cell line Huh7, whereas protein levels and enzyme activities were further diminished. Suppression of CYP1A1 mRNA expression in TCDD-treated Huh.8 cells was partially reversed after pretreatment with the antioxidants N-acetylcysteine and nordihydroguaiaretic acid, suggesting a role for oxidative stress. Induced CYP1A1 message, protein, and enzyme activity were partially restored in an Huh7 cell line expressing the HCV replicon containing a deletion in the nonstructural protein NS5A. Furthermore, adenoviral expression of NS5A in Huh7 partially suppressed TCDD-induced CYP1A1 protein and enzyme activity, implicating this protein in the mechanism of suppression. These findings demonstrate that TCDD-mediated AhR signaling is impaired in hepatocytes in which HCV is present and that NS5A alone or in the presence of other nonstructural proteins of the subgenomic replicon is in part responsible.

Evidence for cytochrome P450 3A expression and catalytic activity in rat blood lymphocytes

Freshly isolated peripheral blood lymphocytes from control rats were found to catalyze the N-demethylation of erythromycin, known to be mediated by cytochrome P450 3A (CYP3A) isoenzymes in rat liver. Pretreatment of rats with dexamethasone (100 mg/kg × 3 days, i.p.), a CYP3A inducer, resulted in 3–4-fold increase in the activity of erythromycin demethylase (EMD) in freshly isolated peripheral blood lymphocytes. This increase in the enzyme activity was found to be associated with an increase in the rate of the reaction and affinity of the substrate towards the enzyme. Significant inhibition of the EMD activity on in vitro addition of ketoconazole, a specific CYP3A inhibitor in liver and polyclonal antibody raised against rat liver CYP3A have suggested that EMD activity in blood lymphocytes is catalyzed primarily by CYP3A isoenzymes. Further, immunoblot analysis with polyclonal antibody raised against rat liver CYP3A revealed significant immunoreactivity, co-migrating with the liver isoenzyme, indicating constitutive expression of CYP3A in blood lymphocytes. Pretreatment with dexamethasone was found to significantly increase the expression of CYP3A protein in freshly isolated rat blood lymphocytes, as observed with liver. Likewise, significant CYP3A mRNA detected in control rat blood lymphocytes has further demonstrated constitutive expression of CYP3A isoenzymes in blood lymphocytes. Furthermore, several fold increase in CYP3A mRNA expression following pretreatment with dexamethasone showed similarities in the regulation of CYP3A isoenzymes in rat blood lymphocytes with the liver enzyme. The data suggest that the blood lymphocytes can be used to monitor tissue expression of CYP3A isoenzymes and validate the suitability of lymphocytes as surrogates of CYP status in less accessible target tissues.

Small Interfering RNA-Mediated Silencing of Cytochrome P450 3A4 Gene

RNA interference (RNAi) is a specific and powerful tool used to manipulate gene expression and study gene function. The cytochrome P450 3A4 (CYP3A4) can metabolize more than 50% of drugs. In the present study, we investigated whether vector-expressed small interfering RNAs (siRNAs) altered the CYP3A4 expression and function using the Chinese hamster cell line (V79) overexpressing CYP3A4 (CHL-3A4). Three different siRNA oligonucleotides (3A4I, 3A4II, and 3A4III) were designed and tested for their ability to interfere with CYP3A4 gene expression. Our study demonstrated that transient transfection of CHL-3A4 cells with the 3A4III siRNAs, but not 3A4I and II, significantly reduced CYP3A4 mRNA levels by 65% and protein expression levels by 75%. All these siRNAs did not affect the expression of CYP3A5 at both mRNA and protein levels in V79 cells overexpressing CYP3A5. Transfection of CHL-3A4 cells with 3A4III siRNAs significantly diminished the cytotoxicity of two CYP3A4 substrate drugs, cyclophosphamide and ifosfamide, in CHL-3A4 cells, with the IC50 increased from 55 to 210 microM to >1000 microM. Nifedipine at 5.78, 14.44, and 28.88 microM was significantly (P < 0.01) depleted by approximately 100, 40, and 22%, respectively, in S9 fractions from CHL-3A4 cells compared with parental CHL-pIC19h cells. In addition, transfection of the CHL-3A4 cells with vectors expressing the 3A4III siRNAs almost completely inhibited CYP3A4-mediated nifedipine metabolism. This study demonstrated, for the first time, the specific suppression of CYP3A4 expression and function using vector-based RNAi technique. The use of RNAi is a promising tool for the study of cytochrome P450 family function.

The persistent DDT metabolite, 1,1‐dichloro‐2,2‐bis(p‐chlorophenyl)ethylene, alters thyroid hormone‐dependent genes, hepatic cytochrome P4503A, and pregnane × receptor gene expressions in atlantic salmon (Salmo salar) parr

The present study investigated the effects of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) on the thyroid and steroid-metabolizing system in Atlantic salmon (Salmo salar) parr. Fish were exposed to waterborne DDE and thyroxine (T4), both singly and in combination, for 5 d. Thyroid-stimulating hormone (TSHbeta), T4 deiodinase (T4ORD), thyroid receptors (TRalpha and TRbeta), and insulin-like growth factor type 1 receptor (IGF-1R) were analyzed using quantitative (real-time) polymerase chain reaction in liver, brain, and kidney, whereas cytochrome P4503A (CYP3A) and pregnane X receptor (PXR) mRNA levels were analyzed only in the liver. Exposure to DDE and T4, both singly and in combination, inhibited TSHbeta expression in the brain. The DDE induced TSHbeta in the liver, and T4 inhibited TSHbeta in the liver and kidney, both singly and in combination with DDE. The DDT-metabolite DDE induced T4ORD expression in the kidney and liver, and combined exposure with T4 inhibited T4ORD expression in the brain, kidney, and liver. The IGF-1R and TRalpha expressions were induced by DDE and T4 singly in the brain, whereas combined exposure with both compounds did not affect IGF-1R and TRd transcript levels. Whereas T4 inhibited TRbeta expression in the liver, exposure to DDE, both singly and in combination with T4, induced TRbeta transcript levels in the liver. Exposure to T4 and DDE, both singly and in combination, resulted in a parallel pattern of CYP3A and PXR mRNA induction in the liver. These results indicate that DDE alters thyroid hormone-dependent genes and hepatic CYP3A and PXR levels. The hepatic modulation of CYP3A and PXR transcript levels by DDE represents a novel aspect of DDE toxicity that, to our knowledge, has not been demonstrated previously in fish. Therefore, the present study demonstrates some possible physiological and endocrine consequences from exposure to endocrine-disrupting chemicals for salmon parr during smoltification.

Hypericum perforatum: Which Constituents may Induce Intestinal MDR1 and CYP3A4 mRNA Expression?

In vitro and in vivo studies suggest that extracts of St John's wort (Hypericum perforatum, L .; SJWE) interact with various drugs, by enhancing their elimination, due to induction of intestinal and hepatic cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp), the gene product of multidrug resistance gene 1 (MDR1/ABCB1). The aim of our study was to identify the major constituents responsible for this induction and their relative importance. Therefore, plant extracts were investigated that vary in these constituents with respect to their effect on mRNA expression of MDR1/CYP3A4. First, different pure constituents of Hypericum perforatum L . were investigated. Secondly, diverse SJWE with different concentrations of hyperforin, quercitrin and hypericin were investigated. The concentrations of hyperforin, hypericin, and quercitrin in the plant extracts were determined by HPLC, and an "artificial extract" containing the same mixture of these constituents was investigated. Different plant extracts, pure constituents or "artificial extracts" were applied to the human colon carcinoma-derived cell line (LS180) and the induction of MDR1 and CYP3A4 expression was analyzed by quantitative RT-PCR. MDR1 and CYP3A4 mRNA expression were both induced by single constituents of SJW such as hypericin and hyperforin in a concentration of 10 microM. Additionally, CYP3A4 mRNA expression was induced by quercitrin. SJW extracts containing hyperforin induced significantly MDR1 mRNA expression, whereas no CYP3A4 induction was observed after treatment with any of the investigated SJWE. These effects could be mimicked by "artificial extracts" containing the same compositions of hyperforin, hypericin and quercitrin as the plant extracts.

Expression of mRNA for several enzymes of xenobiotic detoxification in normal and spontaneously transformed mesothelial cells and mesothelioma cells of rats

Expression of mRNA for the mdr1 gene, cytochrome P450 isoforms 1A1 and 1B1, Ah receptor, and ARNT protein regulating the concentration of cytochrome P450 mRNA was compared in normal and spontaneously transformed mesothelial cells and mesothelioma cells from rats. Expression of cytochrome P450 1A1 and 1B1 mRNA decreased in transformed mesothelial and mesothelioma cells compared to normal mesothelial cells. mRNA for the mdr1 gene was undetected in normal mesothelial cells. Expression of mRNA for the Ah receptor and ARNT protein did not differ in cultured cells.

Cytochrome P450 2A5 expression in a G6PD‐deficient mouse model of oxidative stress

Murine hepatic cytochrome P450 2A5 (CYP2A5), unlike most CYP enzymes, is upregulated during hepatitis and hepatotoxicity. To determine if oxidative stress is responsible for this induction, glucose-6-phosphate dehydrogenase (G6PD)-deficient mice, that are more susceptible to oxidative stress, were treated with the prototypical CYP2A5 inducer pyrazole. Compared to normal mice, G6PD-deficient mice had 3, 1.6, and 5 times greater induction of CYP2A5 mRNA, protein, and activity, respectively. Involvement of oxidative stress was determined using markers of oxidative damage and expression of oxidant-responsive genes. Although no significant elevation in protein carbonyl groups or glutathione peroxidase and superoxide dismutase transcripts were observed, NADPH quinone oxidoreductase 1, which protects against oxidative stress and redox cycling, was increased by pyrazole. Glutathione-S-transferase (GST) alpha, mu, and pi mRNA levels were also elevated by pyrazole, with GST pi showing 1.9 times greater induction in G6PD-deficient mice. Glucose-regulated protein 78, an endoplasmic reticulum stress protein, showed a 1.7 fold greater response to pyrazole in deficient mice. These results suggest that conditions leading to upregulation of CYP2A5 are complex and factors other than oxidative stress may be involved in regulation of CYP2A5. Supported by the Natural Sciences and Engineering Research Council of Canada.

Characterization of the aryl hydrocarbon receptor repressor and a comparison of its expression in Atlantic tomcod from resistant and sensitive populations

Atlantic tomcod from the Hudson River, USA, are resistant to cytochrome P4501A1 (CYP1A1) mRNA induction and early life stage toxicities induced by coplanar polychlorinated biphenyls (PCBs) or tetrachlorodibenzo-p-dioxins but not polycyclic aromatic hydrocarbons. We sought to determine if basal expression or inducibility of aryl hydrocarbon receptor repressor (AHRR) mRNA is higher in tomcod from the resistant Hudson River population than in those from sensitive populations. Tomcod AHRR cDNA was characterized and its expression quantified in different tissues and life stages of tomcod from the Hudson River, Miramichi River, Canada (sensitive), and among environmentally exposed tomcod from these two sources and the St. Lawrence River, Canada. Phylogenetic analysis revealed that tomcod AHRR falls within the clade of other vertebrate aryl hydrocarbon receptors (AHRs) but is most closely related to the four previously identified AHRR genes. Induction of AHRR mRNA was observed in all tissues of PCB77-treated juvenile tomcod of Miramichi River descent, and expression differed among tissues and was significantly related to levels of CYPIAI mRNA expression. Aryl hydrocarbon receptor repressor mRNA was similarly inducible in F2 embryos of Miramichi and Hudson River descent by benzo[a]pyrene but less by PCB77 in Hudson River offspring. A significant, positive correlation was observed between CYP1A1 mRNA and AHRR mRNA concentrations in environmentally exposed tomcod from the three rivers. We conclude that differences in basal expression or inducibility of AHRR mRNA are not the mechanistic basis of resistance but that levels of AHRR often mirror those of CYP1A1, suggesting that a common AHR pathway-related mechanism may modulate expression of both genes.

MDR- and CYP3A4-mediated drug–herbal interactions

According to recent epidemiological reports, almost 40% of American population use complimentary and alternative medicine (CAM) during their lifetime. Patients detected with HIV or cancer often consume herbal products especially St. John's wort (SJW) for antidepressants in combination with prescription medicines. Such self-administered herbal products along with prescribed medicines raise concerns of therapeutic activity due to possible drug–herbal interactions. P-glycoprotein (P-gp) and cytochrome P450 3A4 (CYP3A4) together constitute a highly efficient barrier for many orally absorbed drugs. Available literature, clinical reports and in vitro studies from our laboratory indicate that many drugs and herbal active constituents are substrates for both P-gp and CYP3A4. Results from clinical studies and case reports indicate that self-administered SJW reduce steady state plasma concentrations of amitriptyline, cyclosporine, digoxin, fexofenadine, amprenavir, indonavir, lopinavir, ritonavir, saquinavir, benzodiazepines, theophyline, irinotecan, midazolan and warfarin. This herbal agent has been also reported to cause bleeding and unwanted pregnancies when concomitantly administered with oral contraceptives. Most of these medicinal agents and SJW are substrates for P-gp and/or CYP3A4. In vitro studies from our laboratory suggest that short-term exposure with pure herbal agents such as hypericin, kaempferol and quercetin or extract of SJW resulted in higher uptake or influx of ritonavir and erythromycin. Hypericin, kaempferol and quercetin also caused a remarkable inhibition of cortisol metabolism with the percent intact cortisol values of 64.58%, 89.6% and 90.1%, respectively, during short-term in vitro experiments. Conversely, long-term exposure of herbal agents (hyperforin, kaempferol and quercetin) showed enhanced expression of CYP3A4 mRNA in Caco-2 cells. In another study, we observed that long-term exposure of hypericin, kaempferol, quercetin and silibinin resulted in higher MDR-1 mRNA expression in Caco-2 cells. Therefore, herbs can pharmacokinetically act as inhibitors or inducers. Medicinal agents that are substrates P-gp-mediated efflux and/or CYP-mediated metabolism are likely to be potential candidates for drug–herbal interactions. The duration of exposure of cells/healthy volunteers/animals to herbals appears to be critical for drug–herbal interaction. An increase in plasma drug concentration is possible during concomitant administration of SJW and prescribed drugs. In contrast, prolonged intake of herbal supplement followed by drug administration may result in subtherapeutic concentrations. Therefore, clinical implications of such drug herbal interactions depend on a variety of factors such as dose, frequency and timing of herbal intake, dosing regimen, route of drug administration and therapeutic range. In vitro screening techniques will play a major role in identifying possible herb–drug interactions and thus create a platform for clinical studies to emerge. Mechanisms of drug–herbal interaction have been discussed in this review article.

Cytochrome P450 expression—induction profile and chemically mediated alterations of the WIF‐B9 cell line

WIF-B9 is a hybrid cell line obtained by fusion of rat hepatoma cells (Fao) and human fibroblasts (WI38). It exhibits the structural and functional characteristics of differentiated hepatocytes, including active bile canaliculi. The aim of the present study was to characterize the WIF-B9 cell line as a model for analysing drug-induced hepatic effects. The drug metabolism potential of WIF-B9 cells was identified by studying the rat and human CYP (cytochrome P450) mRNA constitutive expression profile and induction potential after exposure to reference inducers. The morphological alterations provoked by chemical entities were also characterized. Competitive reverse transcriptase-PCR revealed that four rat (1A1, 2B1/2, 2E1 and 4A1) and four human (1A1, 2Cs, 2D6 and 2E1) CYP mRNA isoforms were constitutively expressed in WIF-B9 cells. The rat CYP forms were expressed at levels 2-4 orders of magnitude higher than the human forms. Exposure for 20-72 h to increasing concentrations of CYP reference inducers (beta-naphthoflavone, 3-methyl cholanthrene, dexamethasone, phenobarbital, clofibrate and pregnenolone 16alpha-carbonitrile) revealed that the rat CYP 1A1, 1A2, 3A1, 3A2 and 4A1 and human CYP 1A1 and 2Cs mRNAs were inducible. Rat CYP 1A1 and 1A2 were the most inducible isoforms since they were overexpressed up to 100-fold after 20-48 h of treatment with beta-naphthoflavone. Human CYP 1A1 and 2Cs mRNAs were induced 3-fold after 48 h of treatment with phenobarbital. Other mechanisms involved in hepatotoxicity were explored using microscopy and immunofluorescence. The WIF-B9 cell line exhibited fragmentation and dilatation of bile canaliculi upon exposure to erythromycin, and to isoniazid and cytochalasins, respectively. Monensin promoted cell depolarization and cytoplasmic granulation. Ethionine promoted cytoplasmic vacuolation and dilatation of the Golgi structures. These results indicate that the CYP expression and induction profiles and the morphological features of WIF-B9 cells allow prediction in vitro of the induction and hepatotoxicity profiles of chemical entities.

Gene Expression Patterns in Estrogen (Nonylphenol) and Aryl Hydrocarbon Receptor Agonists (PCB-77) Interaction Using Rainbow Trout (Oncorhynchus Mykiss) Primary Hepatocyte Culture

It was previously reported that in vivo exposure of fish to combined aryl hydrocarbon receptor agonist (AhR; 3,3′,4,4′-tetrachlorobiphenyl,PCB-77) and estrogen receptor agonist (ER; nonylphenol. NP) resulted in potentiation and inhibition (depending on dose ratio, sequential order of exposure, and seasonal changes) of NP-induced responses by PCB-77. The experiments described in this report extend this study by testing whether the effects of PCB-77 on NP-induced ER signaling are mediated through AhR-induced transcriptional suppression of target genes. Trout hepatocytes were isolated by a two-step collagenase perfusion method. After 48-h culture, hepatocytes were exposed to 5 or 10 µM nonylphenol (NP) singly and in combination with PCB-77 at 0.1, 1, and 10 µM. Cells were harvested after 96-h exposure and processed for RNA isolation. Gene expression patterns were quantified using real-time polymerase chain reaction (PCR) with specific primer sets and by Northern blot. Exposure of cells to NP caused significant elevation of ERα, ERβ, Vtg, and Zrp mRNA expressions, while combined exposure with PCB-77 concentration inhibited NP-induced ERs and their target gene expressions. Exposure of trout hepatocytes to PCB-77 alone caused a rapid induction of cytochrome P-450 (CYP) 1A1 mRNA, and combined exposure with NP caused significant reduction in PCB-77 induced CYP1A1 gene expression. Exposure of cells to PCB-77 concentrations induced significant reduction in AhRα mRNA (except 1 µM PCB-77, which caused the induction of AhRα mRNA levels). AhRβ mRNA levels in the cells were inhibited after 96-h exposure to PCB-77, while combined exposure with 5 µM NP restored the PCB-77-inhibited AhRβ mRNA levels to baseline. Taken together, the overall results in this study show that PCB-77 suppresses the gene expression of the ERs and their target genes by transcription mechanism(s). The roles of AhRs in mediating these responses seem to involve the ligand-activated AhR transcriptional induction of CYP1A1. In addition to their frequently described functions as activators of metabolic potentiation and detoxification of various foreign chemicals, data presented in the present study point to other endogenous functions of AhRs that need to be studied further.

Sulindac regulates the aryl hydrocarbon receptor-mediated expression of Phase 1 metabolic enzymes in vivo and in vitro

Sulindac, a widely used non-steroidal anti-inflammatory drug (NSAID), has been shown to inhibit chemically induced carcinogenesis in animal models. In the present study, we have investigated the molecular mechanism by which sulindac affects the activity and expression of the enzymes that mediate the initial detoxification steps of many environmental carcinogens, the cytochromes P450 1A1, 1A2 and 1B1. Sulindac treatment of Sprague-Dawley rats resulted in a dose-dependent increase in hepatic cytochrome P450 (CYP) enzyme activity and in the expression of hepatic CYPs 1A1 and 1B1 mRNA. In the HepG2 human liver cancer cell line, sulindac caused a sustained, dose-dependent increase in CYP enzyme activity. Sulindac treatment resulted in a profound, dose-dependent increase in CYP 1A1 mRNA and a modest increase in 1A2 mRNA. The increase in CYP 1A1 mRNA induced by sulindac was, like enzyme activity, sustained for several days after the initial treatment. Sulindac induced the transcription of the CYP1A1 gene, as measured by the level of heterogeneous nuclear 1A1 RNA and by actinomycin D chase experiment. Since the transcription of CYP1A1 is under the control of the aryl hydrocarbon receptor (AhR), we examined the ability of sulindac to activate the receptor. Sulindac bound to the AhR, as measured by ligand-binding assay, and induced the binding of the AhR with the xenobiotic-responsive element present in the promoter region of the CYP1A1 gene. These results are the first demonstration that NSAIDs modulate carcinogen metabolic enzymes and provide a novel mechanism to explain the established chemopreventive activity of sulindac.