P450 1A1 mRNA Expression Research Articles

Omega-3 Polyunsaturated Fatty Acids Protect Against Cigarette Smoke-Induced Oxidative Stress and Vascular Dysfunction.

In cigarette smokers endothelial dysfunction, measured by flow-mediated dilation (FMD), precedes cardiovascular disease (CVD) and can be improved by supplementation with n - 3 polyunsaturated fatty acids (PUFAs). We developed a mouse model of cigarette smoke (CS)-induced endothelial dysfunction that resembles impaired FMD observed in human cigarette smokers and investigated the mechanism by which n - 3 PUFAs mediate vasoprotection. We hypothesized that loss of nitric oxide (NO)-dependent vasodilation in CS-exposed mice would be prevented by dietary n - 3 PUFAs via a decrease in oxidative stress. C57BL/6 mice were fed a chow or n - 3 PUFA diet for 8 weeks and then exposed to mainstream CS or filtered air for 5 days, 2 h/day. Mesenteric arterioles were preconstricted with U46619 and dilated by stepwise increases in pressure (0-40 mmHg), resulting in increases in flow, ± inhibitor of NO production or antioxidant, Tempol. Markers of oxidative stress were measured in lung and heart. CS-exposed mice on a chow diet had impaired FMD, resulting from loss of NO-dependent dilation, compared with air exposed mice. Tempol restored FMD by normalizing NO-dependent dilation and increasing NO-independent dilation. CS-exposed mice on the n - 3 PUFA diet had normal FMD, resulting from a significant increase in NO-independent dilation, compared with CS-exposed mice on a chow diet. Furthermore, n - 3 PUFAs decreased two CS-induced markers of oxidative stress, 8-epiprostaglandin-F2α levels and heme oxygenase-1 mRNA, and significantly attenuated CS-induced cytochrome P4501A1 mRNA expression. These data demonstrate that dietary n - 3 PUFAs can protect against CS-induced vascular dysfunction via multiple mechanisms, including increasing NO-independent vasodilation and decreasing oxidative stress.

Hop (Humulus lupulus L.) Extract and 6-Prenylnaringenin Induce P450 1A1 Catalyzed Estrogen 2-Hydroxylation.

Humulus lupulus L. (hops) is a popular botanical dietary supplement used by women as a sleep aid and for postmenopausal symptom relief. In addition to its efficacy for menopausal symptoms, hops can also modulate the chemical estrogen carcinogenesis pathway and potentially protect women from breast cancer. In the present study, an enriched hop extract and the key bioactive compounds [6-prenylnarigenin (6-PN), 8-prenylnarigenin (8-PN), isoxanthohumol (IX), and xanthohumol (XH)] were tested for their effects on estrogen metabolism in breast cells (MCF-10A and MCF-7). The methoxyestrones (2-/4-MeOE1) were analyzed as biomarkers for the nontoxic P450 1A1 catalyzed 2-hydroxylation and the genotoxic P450 1B1 catalyzed 4-hydroxylation pathways, respectively. The results indicated that the hop extract and 6-PN preferentially induced the 2-hydroxylation pathway in both cell lines. 8-PN only showed slight up-regulation of metabolism in MCF-7 cells, whereas IX and XH did not have significant effects in either cell line. To further explore the influence of hops and its bioactive marker compounds on P450 1A1/1B1, mRNA expression and ethoxyresorufin O-dealkylase (EROD) activity were measured. The results correlated with the metabolism data and showed that hop extract and 6-PN preferentially enhanced P450 1A1 mRNA expression and increased P450 1A1/1B1 activity. The aryl hydrocarbon receptor (AhR) activation by the isolated compounds was tested using xenobiotic response element (XRE) luciferase construct transfected cells. 6-PN was found to be an AhR agonist that significantly induced XRE activation and inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced XRE activity. 6-PN mediated induction of EROD activity was also inhibited by the AhR antagonist CH223191. These data show that the hop extract and 6-PN preferentially enhance the nontoxic estrogen 2-hydroxylation pathway through AhR mediated up-regulation of P450 1A1, which further emphasizes the importance of standardization of botanical extracts to multiple chemical markers for both safety and desired bioactivity.

Relationship of blood aryl hydrocarbon receptor mRNA and cytochrome P450 1A1 mRNA expression with corrected QT interval among residents exposed to arsenic via drinking water

To investigate the relationship between blood aryl hydrocarbon receptor(AhR)mRNA and cytochrome P450(CYP)1A1 mRNA expression and corrected QT interval among residents exposed to arsenic via drinking water. Arsenic exposure area in Bayannao'er city of Nei Monggol Autonomous Region was selected as the survey point, and the residents living more than 10 years in this area were investigated from December 2012 to January 2015.A total of 233 residents were divided into four groups according to drinking water arsenic concentration (ranged from 0.8 to 824.7 μg/L): control group (drinking water arsenic concentration <10.0 μg/L, n=55), low exposure group (drinking water arsenic concentration 10.0-99.9 μg/L, n=47), middle exposure group (drinking water arsenic concentration 100.0-199.9 μg/L, n=45), high exposure group (drinking water arsenic concentration ≥200.0 μg/L, n=86). Epidemiological investigation was performed.Real-time PCR technology was used to detect the expression levels of blood AhR mRNA and CYP1A1 mRNA, and the relationship between expression levels of blood AhR mRNA and CYP1A1 mRNA and corrected QT interval was analyzed. (1) Blood AhR mRNA and CYP1A1 mRNA expression levels were similar among control group, low exposure group and middle exposure group (all P>0.05) while blood AhR mRNA (3.18×10(-3)(2.42×10(-3), 4.45×10(-3)) vs.2.30×10(-3)(1.53×10(-3), 3.20×10(-3)), P<0.05) and CYP1A1 mRNA (1.58×10(-3)(0.80×10(-3), 2.73×10(-3))vs.1.00×10(-3)(0.59×10(-3), 2.09×10(-3)), P<0.05) expression levels were significantly higher in high dose group than in control group.(2) AhR mRNA expression level was similar between residents with longer corrected QT interval and residents with normal corrected QT interval (2.89×10(-3)(1.90×10(-3), 3.71×10(-3)) vs.2.58×10(-3)(1.85×10(-3), 3.57×10(-3)), P>0.05). CYP1A1 mRNA expression level was significantly higher in residents with longer corrected QT interval than in residents with normal corrected QT interval (1.50×10(-3)(0.78×10(-3), 2.63×10(-3)) vs.1.16×10(-3)(0.64×10(-3), 2.36×10(-3)), P<0.05). (3) AhR mRNA expression level was similar between residents with sinus tachycardia or bradycardia or normal heart rate(2.89×10(-3)(1.71×10(-3), 4.45×10(-3)) vs.2.64×10(-3)(1.86×10(-3), 3.52×10(-3)), P>0.05). CYP1A1 mRNA expression level was significantly higher in residents with sinus tachycardia or bradycardia than in residents with normal heart rate (1.47×10(-3)(0.87×10(-3), 2.77×10(-3)) vs.1.24×10(-3)(0.64×10(-3), 2.31×10(-3)), P<0.05). Long-term exposure to arsenic is associated with upregulated blood AhRmRNA and CYP1A1 mRNA expression.Blood CYP1A1 mRNA expression, but not AhRmRNA expression, is associated with prolonged corrected QT interval.

Towards science-based sediment quality standards—Effects of field-collected sediments in rainbow trout (Oncorhynchus mykiss)

Sediments can act as long-term sinks for environmental pollutants. Within the past decades, dioxin-like compounds (DLCs) such as polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) have attracted significant attention in the scientific community. To investigate the time- and concentration-dependent uptake of DLCs and PAHs in rainbow trout (Oncorhynchus mykiss) and their associated toxicological effects, we conducted exposure experiments using suspensions of three field-collected sediments from the rivers Rhine and Elbe, which were chosen to represent different contamination levels. Five serial dilutions of contaminated sediments were tested; these originated from the Prossen and Zollelbe sampling sites (both in the river Elbe, Germany) and from Ehrenbreitstein (Rhine, Germany), with lower levels of contamination. Fish were exposed to suspensions of these dilutions under semi-static conditions for 90 days. Analysis of muscle tissue by high resolution gas chromatography and mass spectrometry and of bile liquid by high-performance liquid chromatography showed that particle-bound PCDD/Fs, PCBs and PAHs were readily bioavailable from re-suspended sediments. Uptake of these contaminants and the associated toxicological effects in fish were largely proportional to their sediment concentrations. The changes in the investigated biomarkers closely reflected the different sediment contamination levels: cytochrome P450 1A mRNA expression and 7-ethoxyresorufin-O-deethylase activity in fish livers responded immediately and with high sensitivity, while increased frequencies of micronuclei and other nuclear aberrations, as well as histopathological and gross pathological lesions, were strong indicators of the potential long-term effects of re-suspension events.Our study clearly demonstrates that sediment re-suspension can lead to accumulation of PCDD/Fs and PCBs in fish, resulting in potentially adverse toxicological effects. For a sound risk assessment within the implementation of the European Water Framework Directive and related legislation, we propose a strong emphasis on sediment-bound contaminants in the context of integrated river basin management plans.

Activation of the aryl hydrocarbon receptor by the widely used Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2).

Small molecular weight protein kinase inhibitors are frequently used tools to unravel the complex network of cellular signal transduction under certain physiological and pathophysiological conditions.4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2) is a widely used compound to block the activity of Src family kinases, the major group of non-receptor tyrosine kinases, which trigger multiple cellular signaling pathways. Here, we show that PP2 induces cytochrome P450 1A1 mRNA expression and enzyme activity in a dose-dependent manner in human HepG2 hepatoma cells and NCTC 2544 keratinocytes. By means of reporter gene assays, RNA interference, electrophoretic mobility shift assay, and competitive ligand-binding assay, we further demonstrate that PP2 is a ligand for the aryl hydrocarbon receptor (AHR), an intracellular chemosensor that regulates xenobiotic metabolism, environmental stress responses, and immune functions. Upon ligand-dependent activation, the AHR translocates into the nucleus and dimerizes with the AHR nuclear translocator (ARNT) to modulate the expression of its target genes. In addition, AHR activation is frequently accompanied by an activation of the tyrosine kinase c-Src, resulting in stimulation of cell-surface receptors and downstream signal transduction. As PP2 activates the AHR/ARNT pathway by simultaneously blocking c-Src-mediated alternative signaling routes, this compound may be a suitable tool to study the contribution of the different AHR-dependent signaling pathways to biological processes and adverse outcomes. On the other hand, the unexpected property of PP2 to stimulate AHR/ARNT signaling should be carefully taken into account in future investigations in order to avoid a false interpretation of experimental results and molecular interrelations.

Combined effects of Iranian heavy crude oil and bacterial challenge (Streptococcus iniae) on biotransformation and innate immune responses in rockfish (Sebastes schlegeli).

To clarify the effects of spilled crude oil on fish bacterial disease resistance, rockfish (Sebastes schlegeli) were exposed to Iranian Heavy crude oil (IHCO) and Streptomyces iniae in combination. Hepatic biotransformation enzymes (ethoxyresorufin O-de-ethylase, glutathione-S-transferase) and plasma biochemical parameters (glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and glucose) in fish exposed to IHCO were not significantly different from those in unexposed fish. The level of biliary 1-OH-pyrene and cytochrome P4501A mRNA expression increased in a dose-dependent manner with IHCO exposure. The interferon stimulated gene 15, interleukin-1beta and cathepsin L were increased significantly in the liver in IHCO-exposed fish, but not dose-dependently, but the granulocyte colony stimulating factor was not related to IHCO exposure. The percentage mortality in fish following a single exposure to S. iniae was positively correlated with IHCO exposure concentration. We concluded that IHCO exposure exacerbates fish mortality following environmental bacterial infection.

Regulation of reproduction- and biomarker-related gene expression by sex steroids in the livers and ovaries of adult female western mosquitofish (Gambusia affinis)

To assess the adverse toxicological effects of steroid hormones on western mosquitofish (Gambusia affinis), 180 adult females were exposed to individual or binary combinations of progesterone (1μg/L), testosterone (1μg/L) and 17β-estradiol (1μg/L) for eight days. The expression patterns of vitellogenin, estrogen receptor, androgen receptor, metallothionein, and cytochrome P450 1A genes in mosquitofish varied according to tissue as well as the specificity of steroids. Treatment by progesterone or testosterone alone inhibited target gene expression in the livers. The expression levels of both vitellogenin A and vitellogenin B mRNAs were up-regulated by17β-estradiol, and a parallel induction of estrogen receptor α mRNA expression was also observed in the livers. In addition, 17β-estradiol treatment alone suppressed androgen receptor α, metallothionein and cytochrome P450 1A mRNA expression in the livers. In general, multiple hormone treatments had different effects on target gene expression compared with corresponding hormone alone. The results demonstrate that steroid hormones cause multiple biological responses including the expression of vitellogenin, estrogen receptor and androgen receptor mRNA in the hormone signaling pathways and the expression of metallothionein and cytochrome P450 1A mRNA in the xenobiotic signaling pathway.

Changes of cytochrome P4501A mRNA expression and physiology responses in the olive flounder, Paralichthys olivaceus, exposed to benzo[a]pyrene

Benzo[a]pyrene (BaP) is generated by the incomplete combustion of organic substances such as oil and coal, and is a widespread organic environmental contaminant in terrestrial and aquatic ecosystems. To determine the effects of BaP on organisms, we investigated its time- and dose-related effects on the levels of cytochrome P4501A (P4501A) mRNA in the liver and gills of the olive flounder (Paralichthys olivaceus) using quantitative polymerase chain reaction (QPCR) and measured the plasma glucose, cortisol, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels. The full-length olive flounder P4501A cDNA consists of 1566 nucleotides and encodes a 521-amino-acid protein. In the liver and gills, the expression of P4501A mRNA was highest 6 h after exposure to both 10 and 30 µg l−1 BaP, and then decreased. In addition, the plasma parameters increased with exposure. These results suggest that P4501A plays an important role in the detoxification of BaP, which stressed the olive flounder. Therefore, these physiological parameters may be indicators of BaP-induced stress responses.

High dose of commercial products of kava ( Piper methysticum) markedly enhanced hepatic cytochrome P450 1A1 mRNA expression with liver enlargement in rats

Commercial products containing the kava plant ( Piper methysticum), known to have the anxiolytic activity, are banned in several European countries and Canada because of the suspicion of a potential liver toxicity. In some reports, kava and kavalactones (major constituents of kava) inhibited activities of cytochrome P450 (CYP) isoforms including CYP1A2. On the other hand, a few studies showed that administration of kava to rats moderately increased CYP1A2 proteins in the liver. CYP1A isoforms are likely responsible for the metabolic activation of potent carcinogenic environmental toxins such as aflatoxins, benzo[ a]pyrene, and others. On these bases, we have investigated the effects of administration of commercial kava products on gene expression of hepatic CYP1A isoforms in rats. A high dose (equivalent to approximately 380 mg kavalactones/kg/day; 100 times of the suggested dosage for human use) of two different types of kava products for 8 days significantly increased liver weights. CYP1A2 mRNA expression was moderately increased (2.8–7.3 fold). More importantly, the high dose of kava markedly enhanced CYP1A1 mRNA expression (75–220 fold) as well as ethoxyresorufin O-deethylase activities and CYP1A1 immunoreactivities. Thus, no observed adverse effect levels of kavalactones would be lower than 380 mg/kg/day. When the safety factor of kavalactones is assumed to be 100, a value most often used upon the risk analysis of chemicals and designed to account for interspecies and intraspecies variations, a number of kava product users likely ingest more kavalactones than acceptable daily intakes. Based on overall evidence, we should pay considerable attention to the possibility that kava products induce hepatic CYP1A1 expression in human especially in sensitive individuals.

Diesel exhaust particles suppress expression of sex steroid hormone receptors in TM3 mouse Leydig cells

Murine Leydig TM3 cells, derived from mouse testis, were exposed to diesel exhaust particles (DEP) and DEP components for 24 h. mRNA expression of estrogen receptor (ER) α and CYP1A1 were then measured by real-time PCR. Exposure to DEP (10 μg/ml) reduced ERα mRNA expression by approximately 50% and increased P450 1A1 mRNA expression by approximately fivefold. Cycloheximide (CHX), an inhibitor of protein synthesis, abolished the DEP-induced effects on ERα mRNA expression. Thus, protein synthesis mediated the DEP-induced reduction of ERα mRNA expression.

Chronic fuel oil toxicity in American mink ( Mustela vison): systemic and hematological effects of ingestion of a low-concentration of bunker C fuel oil

Petroleum oil enters the coastal marine environment through various sources; marine mammals such as sea otters that inhabit this environment may be exposed to low concentrations of petroleum hydrocarbons through ingestion of contaminated prey. The inability to perform controlled studies in free-ranging animals hinders investigations of the effects of chronic petroleum oil exposure on sea otter morbidity and mortality, necessitating the development of a reliable laboratory model. We examined the effects of oral exposure to 500 ppm bunker C fuel oil over 113–118 days on American mink, a species phylogenetically related to the sea otter. Hematological parameters and organs were examined for fuel oil-associated changes. Hepatic cytochrome P4501A1 mRNA expression and fecal cortisol concentrations were also measured. Ingestion of fuel oil was associated with a decrease in erythrocyte count, hemoglobin concentration (Hgb), hematocrit (HCT), and an increase in mean corpuscular volume (MCV). Total leukocytes were elevated in the fuel oil group from increases in neutrophils, lymphocytes, and monocytes. Significant interactions between fuel oil and antigen challenge were found for erythrocyte parameters, monocyte and lymphocyte counts. Liver and adrenal weights were increased although mesenteric lymph node weights were decreased in the fuel oil group. Hepatic cytochrome P4501A1 mRNA was elevated in the fuel oil group. Fecal cortisol concentration did not vary between the two groups. Our findings show that fuel oil exposure alters circulating leukocyte numbers, erythrocyte homeostasis, hepatic metabolism and adrenal physiology and establish a framework to use mink as a model for sea otters in studying the systemic effects of marine contaminants.

Induced CYP1A mRNA, protein and catalytic activity in the liver of feral fish, leaping mullet, Liza saliens

In this study, we examined whether levels of P4501A mRNA expression were naturally induced in feral fish, Liza saliens, and whether CYP1A protein levels and associated enzyme activity, EROD, were also increased. Induction of mRNA was measured using a nucleic acid hybridization technique. For the hybridization studies, a new 33-mer oligonucleotide probe 5′-dCTC ATC CAG CTT CCT GTC CTC GCA GTG ATC AAT-3′ was designed, which corresponded to the totally conserved amino acid motif of CYP1A protein from positions 291 to 301 among the various fish species. Results of Northern blot analysis revealed that RNA isolated from the liver of mullet collected from the highly contaminated region of Izmir Bay with a dissolved and dispersed petroleum hydrocarbon content of 12.45 μg l −1 gave a strong hybridization signal, whereas only a weak hybridization signal was detected in the liver RNA of fish caught from the reference site containing less than 1 μg l −1 of petroleum hydrocarbons. Similarly, fish from the contaminated site had approximately 80 times more EROD activity than the feral fish captured from the reference site. Studies using polyclonal antibodies produced against purified mullet CYP1A also showed the similar trend. In conclusion, feral leaping mullet caught from contaminated water displayed induction of CYP1A at three levels of expression, namely, mRNA, apoprotein and catalytic activity.

Hemodynamic Dysfunction and Cytochrome P4501A mRNA Expression Induced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin during Embryonic Stages of Lake Trout Development

Lake trout embryos exposed to [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) manifest toxicity after hatching by subcutaneous edema of the yolk sac, pericardial edema, meningeal edema, subcutaneous hemorrhages, and a marked congestion of blood flow in various vascular beds culminating in death. Our objective was to determine if this syndrome was associated temporally with morphologic lesions in the vascular endothelium, increased vascular permeability, and cytochrome P4501A (CYP1A) mRNA induction. Lake trout embryos exposed as fertilized eggs to TCDD were found to exhibit marked reductions in perfusion of the peripheral vasculature during the early sac fry stage of development (stage F19), which consistently preceded other gross lesions and mortality observed later in sac fry development (stage F210). This reduction in blood flow was manifested as severe capillary congestion and hemoconcentration in certain vascular beds. Transmission electron microscopic (TEM) examination of endothelial cells in these vascular beds failed to reveal cellular necrosis at hatching (stage E58) and throughout sac fry development (stages F19–F210). Rather, only subtle ultrastructural changes in endothelial cells were found consisting of increased vacuolation, separation of intercellular junctions, and cytoplasmic blebbing, consistent with the TCDD dose and time course for developmental cardiovascular toxicity, which began to manifest itself in some embryos approximately 1 week prior to hatching (E58). To assess permeability of yolk sac vasculature to certain constituents in blood, sac fry (stage F210) were analyzed for the presence of plasma proteins, granulocytes, and serum creatine kinase activity in yolk sac subcutaneous edema fluid from control and TCDD-exposed treatment groups. TCDD dose- and time-related increases in yolk sac edema volume, plasma protein content of edema fluid, granulocyte concentration, and creatine kinase activity in the fluid were observed in midstage and late stage of sac fry development (stage F210). Thus, yolk sac subcutaneous edema fluid is an ultrafiltrate of blood and results from increased vascular permeability. In contrast to the changes in vascular blood flow and permeability induced by TCDD during stages F19 and F210 of sac fry development, respectively, CYP1A mRNA levels were induced by TCDD as early as the 10-somite embryo (stage E25). TCDD also caused a dose-related increase in CYP1A mRNA levels in sac fry at hatching (stage E58) and throughout sac fry development (stages F19–F210). We conclude that subtle, ultrastructural changes in vascular endothelial cells consistently precede increases in vascular permeability and sac fry mortality; however, induction of CYP1A mRNA occurs prior to any observable morphological lesions, changes in vascular permeability, or sac fry mortality.

Expression of Functional Aromatic Hydrocarbon Receptor and Aromatic Hydrocarbon Nuclear Translocator Proteins in Murine Bone Marrow Stromal Cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) acting through the aromatic hydrocarbon receptor (AhR) and its dimerization partner, the AhR nuclear translocator protein (arnt), elicits numerous toxicological effects including immunosuppression and thymic atrophy. Previous work has shown that TCDD alters bone marrow prothymocyte populations. These effects could be mediated at the lymphocyte level directly and/or through effects on bone marrow stromal cells, a population important in the support of lymphopoiesis. The purpose of this study was to characterize AhR and arnt expression in three murine bone marrow stromal cell lines (S17, M2-10B4, and BMS2) and in primary stromal cell cultures. Immunoblot analysis detected AhR protein in M2-10B4 and BMS2 cells. AhR protein was also detected in the primary cultures. Arnt protein could be detected in all cell cultures. Electrophoretic mobility shift assays detected TCDD-dependent dioxin-responsive element (DRE) binding in all three cell lines. DNA binding was sequence-specific and dependent on AhR, as demonstrated by the addition of unlabeled DRE DNA or of anti-AhR antibody. Results obtained with the primary cultures paralleled those seen with the stromal cell lines. The ED50for induction of TCDD-dependent DRE binding in M2-10B4 cells was 0.21 nM. TCDD treatment did not induce stromal P4501A1 mRNA expression but did increase P4501B1 mRNA levels in all three cell lines and in the primary cultures. These results indicate that murine bone marrow stromal cells express AhR and arnt proteins. Furthermore, these proteins are functional in terms of their DRE-binding ability and potential to regulate mRNA levels in a gene-specific fashion.

Regulation of Dioxin Receptor Function by Omeprazole

The intracellular dioxin (aryl hydrocarbon) receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) and related environmental pollutants and functions as a ligand-activated transcription factor. In this study we have examined the effects on dioxin receptor function of a potentially novel ligand, omeprazole, which is widely clinically used as a gastric anti-ulcer drug. In primary human hepatocytes omeprazole potently induced cytochrome P4501A1 mRNA expression, whereas this effect was not detected in mouse primary hepatocytes. In human hepatoma cells omeprazole was found to induce transcription of reporter genes via the xenobiotic response element that is recognized by the ligand-activated dioxin receptor. In contrast, the human dioxin receptor was not activated by omeprazole upon expression in a receptor-deficient mouse hepatoma cell line. In a reconstituted yeast (Saccharomyces cerevisiae) model system, however, both the mouse and human dioxin receptors were potently activated by omeprazole. Although omeprazole failed to displace dioxin in in vitro ligand binding assays, a residue within the ligand binding domain that is critical for dioxin binding in vitro was also critical for omeprazole responsiveness in vivo. Consistent with this observation, both omeprazole and dioxin responsiveness of the dioxin receptor was inhibited in mutant yeast cells expressing low levels of the molecular chaperone hsp90 that is critical for ligand binding activity. The sulfoxide group that is essential for formation of a planar conversion product of omeprazole was found to be critical for dioxin receptor activation. Taken together, these data suggest that omeprazole represents a precursor for a novel class of dioxin receptor agonists that are bona fide dioxin receptor ligands but generated in a strictly species-specific manner.

Monooxygenation, cytochrome P4501A1 and P4501A1-mRNA in rat liver slices exposed to beta-naphthoflavone and dexamethasone in vitro

Precision-cut liver slices (0.5 mm) were incubated at 30 degrees C in a modified William's Medium E for up to 48 hrs. During the incubation, K+ and GSH/GSSG concentrations did not decrease. Cytochrome P450-dependent dealkylation rates of 7-ethoxycoumarin (ECOD), 7-allyloxycoumarin (ACOD) and 7-ethoxyresorufin (EROD) decreased to 1/3, 1/2 or did not change at all, respectively, after a 48 hrs incubation period. Exposure of the slices to 25 microM beta-naphthoflavone (beta NF) resulted in about 3 times higher monooxygenation rates. An exposure to a combination beta NF and dexamethasone (10(-6)M) caused a marked induction (6 times higher rates) after 48 hrs. Simultaneously an increase in P4501A1 content was observed. P4501A1-mRNA expression (measured by RT-PCR) was distinctly increased following beta NF exposure for 6 or 24 hrs. DMSO (0.2%) and dexamethasone alone modified monooxygenation rates, but did not have significant effects on P4501A1 content or, in the case of DMSO, P4501A1 gene expression (for dexamethasone not determined). Liver slices are a useful and simple tool for the detection of a beta NF-like induction within a few hours after preparation of the slices.

Expression of 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible cytochrome P4501A1 in human splenic lymphocyte cultures

The induction of cytochrome P4501A1 (P4501A1) and P4501A1-specific 7-ethoxyresorufin O -deethylase (EROD) activity by 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) was investigated in human splenic lymphocytes cultures. EROD activity was induced by TCDD in mitogen (phytohemagglutinin and pokeweed mitogen) stimulated blast cells but not in the resting cells. TCDD markedly induced EROD activity in a dose- and time-dependent manner. The expression of P4501A1 mRNA was increased by TCDD in mitogen-stimulated cells as detected by Northern blot analysis. These findings support the conclusion that TCDD induced the expression of P4501A1 gene, resulting in increased EROD activity in mitogen-stimulated human splenic lymphocytes cultures.

Expression of 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible cytochrome P4501A1 in human splenic lymphocyte cultures

The induction of cytochrome P4501A1 (P4501A1) and P4501A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in human splenic lymphocytes cultures. EROD activity was induced by TCDD in mitogen (phytohemagglutinin and pokeweed mitogen) stimulated blast cells but not in the resting cells. TCDD markedly induced EROD activity in a dose- and time-dependent manner. The expression of P4501A1 mRNA was increased by TCDD in mitogen-stimulated cells as detected by Northern blot analysis. These findings support the conclusion that TCDD induced the expression of P4501A1 gene, resulting in increased EROD activity in mitogen-stimulated human splenic lymphocytes cultures.

Retinoic acid antagonizes basal as well as coal tar and glucocorticoid-induced cytochrome P4501A1 expression in human skin.

Cytochrome P4501A1 is known to be expressed in skin and thus has been implicated in the pathogenesis of skin cancer due to certain environmental carcinogens. Retinoic acid (RA) has been used in chemoprevention of certain skin and other epithelial cancers. Therefore, we used Northern and Western analysis to determine the effect of externally applied RA on basal P4501A1 expression. RA reduced basal levels of P4501A1 mRNA and protein by 68 (n = 14, P = 0.005) and 75% (n = 7, P = 0.04) respectively. RA application also reduced basal levels of P4501A2 (another P4501A1 subfamily member) mRNA by 93% (n = 7, P = 0.001) as determined by reverse transcription-polymerase chain reaction. Interestingly, P4501A1 mRNA expression induced by coal tar or glucocorticoid (clobetasol) was reduced 46 (n = 10, P = 0.003) and 69% (n = 5, P < 0.05) respectively by RA co-application. Downregulation of basal P4501A1 expression and antagonism of coal tar mediated P4501A1 induction by RA may be a mechanism involved in chemo-prevention of skin and other epithelial cancer by RA.