Induction Of Cytochrome P4501A mRNA Research Articles

Structure-activity relationships for alkyl-phenanthrenes support two independent but interacting synergistic models for PAC mixture potency

Multiple lines of evidence at whole animal, cellular and molecular levels implicate polycyclic aromatic compounds (PACs) with three rings as drivers of crude oil toxicity to developing fish. Phenanthrene (P0) and its alkylated homologs (C1- through C4-phenanthrenes) comprise the most prominent subfraction of tricyclic PACs in crude oils. Among this family, P0 has been studied intensively, with more limited detail available for the C4-phenanthrene 1-methyl-7-isopropyl-phenanthrene (1-M,7-IP, or retene). While both compounds are cardiotoxic, P0 impacts embryonic cardiac function and development through direct blockade of K+ and Ca2+ currents that regulate cardiomyocyte contractions. In contrast, 1-M,7-IP dysregulates aryl hydrocarbon receptor (AHR) activation in developing ventricular cardiomyocytes. Although no other compounds have been assessed in detail across the larger family of alkylated phenanthrenes, increasing alkylation might be expected to shift phenanthrene family member activity from K+/Ca2+ ion current blockade to AHR activation. Using embryos of two distantly related fish species, zebrafish and Atlantic haddock, we tested 14 alkyl-phenanthrenes in both acute and latent developmental cardiotoxicity assays. All compounds were cardiotoxic, and effects were resolved into impacts on multiple, highly specific aspects of heart development or function. Craniofacial defects were clearly linked to developmental cardiotoxicity. Based on these findings, we suggest a novel framework to delineate the developmental toxicity of petrogenic PAC mixtures in fish, which incorporates multi-mechanistic pathways that produce interactive synergism at the organ level. In addition, relationships among measured embryo tissue concentrations, cytochrome P4501A mRNA induction, and cardiotoxic responses suggest a two-compartment toxicokinetic model that independently predicts high potency of PAC mixtures through classical metabolic synergism. These two modes of synergism, specific to the sub-fraction of phenanthrenes, are sufficient to explain the high embryotoxic potency of crude oils, independent of as-yet unmeasured compounds in these complex environmental mixtures.

A novel approach to predict the comprehensive EROD potency: Mechanism-based curve fitting of CYP1A1 activity by PAHs

Cytochrome P450 1A1 (CYP1A1) plays critical roles in polycyclic aromatic hydrocarbon (PAH) toxicity, including DNA adduction and ROS generation. Therefore, CYP1A1 activity quantified by the 7-ethoxyresorufin-O-deethylase (EROD) assay (named EROD potency) has been considered a typical biomarker of PAH exposure and toxicity. The EROD dose–response curve always presents a biphasic style, increasing at low concentrations and decreasing at high concentrations of PAHs, but relative effect potency (REP) commonly used in PAH risk assessment is only involved in the increasing phase. In this study, a full bell-shaped EROD curve fitting formula Eq. (1) was obtained by considering both CYP1A1 mRNA induction and enzyme inhibition to completely assess the EROD potency of PAHs. Correspondingly, in silico models of QSAR and docking methods successfully predicted the full EROD curves of PAHs, and the structure-activity relationship indicated that PAHs with heavy molecular weight and large diameter showed stronger EROD potency. Further EROD potency with predicted curve parameters (EC50,ind and area index) was confirmed by the reported REP (R2 = 0.697–0.977) and experimental data from human and mouse cells (R2 = 0.700–0.804). This study provides a novel curve fitting for the EROD dose–response relationship and a prediction model for PAH EROD potency.

Serotonin is an endogenous regulator of intestinal CYP1A1 via AhR

Aryl hydrocarbon receptor (AhR) is a nuclear receptor that controls xenobiotic detoxification via induction of cytochrome P450 1A1 (CYP1A1) and regulates immune responses in the intestine. Metabolites of L-tryptophan activate AhR, which confers protection against intestinal inflammation. We tested the hypothesis that serotonin (5-HT) is an endogenous activator of AhR in intestinal epithelial cells. Treatment of Caco-2 monolayers with 5-HT induced CYP1A1 mRNA in a time- and concentration-dependent manner and also stimulated CYP1A1 activity. CYP1A1 induction by 5-HT was dependent upon uptake via serotonin transporter (SERT). Antagonism of AhR and knockdown of AhR and its binding partner aryl hydrocarbon receptor nuclear translocator (ARNT) attenuated CYP1A1 induction by 5-HT. Activation of AhR was evident by its nuclear translocation after 5-HT treatment and by induction of an AhR-responsive luciferase reporter. In vivo studies showed a dramatic decrease in CYP1A1 expression and other AhR target genes in SERT KO ileal mucosa by microarray analysis. These results suggest that intracellular accumulation of 5-HT via SERT induces CYP1A1 expression via AhR in intestinal epithelial cells, and SERT deficiency in vivo impairs activation of AhR. Our studies provide a novel link between the serotonergic and AhR pathways which has implications in xenobiotic metabolism and intestinal inflammation.

Toxicity and cytochrome P450 1A mRNA induction by 6-formylindolo[3,2-b]carbazole (FICZ) in chicken and Japanese quail embryos

The tryptophan derivative formylindolo[3,2-b]carbazole (FICZ) binds with high ligand affinity to the aryl hydrocarbon receptor (AHR) and is readily degraded by AHR-regulated cytochrome P450 family 1 (CYP1) enzymes. Whether in vivo exposure to FICZ can result in toxic effects has not been examined and the main objective of this study was to determine if FICZ is embryotoxic in birds. We examined toxicity and CYP1 mRNA induction of FICZ in embryos from chicken (Gallus domesticus) and Japanese quail (Coturnix japonica) exposed to FICZ (2–200μgkg−1) by yolk and air sac injections. FICZ caused liver toxicity, embryo mortality, and CYP1A4 and CYP1A5 induction in both species with similar potency. This is in stark contrast to the very large difference in sensitivity of these species to halogenated AHR agonists. We also exposed chicken embryos to a low dose of FICZ (4μgkg−1) in combination with a CYP inhibitor, ketoconazole (KCZ). The mixture of FICZ and KCZ was lethal while FICZ alone had no effect at 4μgkg−1. Furthermore, mixed exposure to FICZ and KCZ caused stronger and more long-lasting hepatic CYP1A4 induction than exposure to each compound alone. These findings indicate reduced biotransformation of FICZ by co-treatment with KCZ as a cause for the enhanced effects although additive AHR activation is also possible. To conclude, FICZ is toxic to bird embryos and it seems reasonable that the toxicity by FICZ involves AHR activation. However, the molecular targets and biological events leading to hepatic damage and mortality are unknown.

Mixture effects between different azoles and β-naphthoflavone on the CYP1A biomarker in a fish cell line

The cytochrome P450 1A (CYP1A) biomarker response was studied in the Poeciliopsis lucida hepatocellular carcinoma (PLHC-1) cell line, which represents a good model for studies on aryl hydrocarbon receptor (AhR) – CYP1A signaling. The PLHC-1 cells were exposed to the prototypical CYP1A inducer and AhR agonist β-naphthoflavone (BNF) in combination with different azoles. Two imidazoles (clotrimazole and prochloraz) and two benzimidazoles (nocodazole and omeprazole) were used. Exposure to clotrimazole, prochloraz and nocodazole resulted in 2–4 fold induction of the CYP1A-mediated ethoxyresorufin-O-deethylase (EROD) activities at 24 and 48h, whereas exposure to the omeprazole for 48h had no effect on the EROD activity. Clotrimazole, nocodazole and prochloraz also acted as inhibitors of EROD activities in situ in PLHC-1 cells (IC50=1.3–7.7μM), whereas omeprazole had no effect on this activity (IC50=72μM). Exposure to 10μM prochloraz resulted in 3-fold induction of CYP1A mRNA and exposure to 10μM nocodazole resulted in 16-fold induction of CYP1A mRNA levels at 24h compared to controls. In the mixture experiments, more-than-additive mixture effects between BNF and the azoles clotrimazole, prochloraz and nocodazole on EROD activities were evident, with nocodazole showing the strongest mixture effect. The presence of nocodazole increased the response to BNF up to 200-fold on CYP1A mRNA and up to 16-fold on EROD activities and prolonged the effect of BNF exposure on EROD activities by 24h or longer. This suggests that azoles that are inhibitors and/or competing substrates for the CYP1A enzymes can cause increased sensitivity to exposures to chemicals that depend on CYP1A metabolism for their elimination in situations of mixed chemical exposures. The results also suggest that the EROD biomarker response can be significantly affected in azole-contaminated areas. The responsiveness of the EROD biomarker to BNF exposure was studied in PLHC-1 that had been pre-treated with nocodazole for 5 or 24h at concentrations that are known to disassemble microtubules at 24h in these cells. Pre-treatment of PLHC-1 cells with nocodazole for either 5 or 24h had no effect on the responsiveness to BNF exposure, which implies that the EROD activity can be induced in cells with disassembled microtubules.

Biochemical responses, morphometric changes, genotoxic effects and CYP1A expression in the armored catfish Pterygoplichthys anisitsi after 15 days of exposure to mineral diesel and biodiesel

Despite being considered friendlier to the environment, biodiesel fuel can be harmful to aquatic organisms, especially when combined with petroleum diesel fuel. In this work we evaluated the effects of mineral diesel fuel containing increasing concentrations of biodiesel (5% and 20%, namely B5 and B20) and pure biodiesel (B100), at concentrations of 0.001 and 0.01mLL−1, after 15 days of exposure, in armored catfish (Pterygoplichtys anisitsi). Toxicity tests were also performed to estimate LC50 values (96h) for each compound. Biotransformation enzymes [ethoxyresorufin-O-deethylase (EROD), and glutathione S-transferase (GST)] as well as oxidative stress markers (superoxide dismutase, SOD, catalase, CAT, glutathione peroxidase, GPx, and the level of lipid peroxidation) were measured in liver and gills after treatment. Genotoxic effects were also accessed in erythrocytes using the comet assay and by evaluating the frequency of micronuclei formation. Further, the mRNA of cytochrome P450 1A (CYP1A) was also measured in liver. Mortality was not observed even exposure to concentrations as high as 6.0mLL−1. EROD and GST activities were increased after B5 and B20 treatments; however, CYP1A mRNA induction was not observed. SOD and CAT activities were decreased, but GPx was significantly higher for all treatments in gills. There were no significant changes in lipid peroxidation, but genotoxicity markers revealed that all treatments increased comet scores. Fuels B5 and B20 increased micronuclei frequency. Our results indicate that despite being less toxic, biodiesel may cause sublethal alterations in fish that may alter long term health.

Chemopreventive Role of Metformin during DMBA–induced Breast Carcinogenesis in MCF10A Cells through CYP1A1‐AhR Signaling Pathway

Recent studies have established that metformin (MET), an oral hypoglycemic drug, possesses antioxidant activity and effective against different types of cancer. Although several postulated mechanisms were reported, the role of the cytochrome P450 1A1 (CYP1A1)‐aryl hydrocarbon receptor (AhR) signaling pathway in the protective effect of MET against breast cancer has not been reported before. Therefore, we examined the capacity of MET to inhibit CYP1A1 gene expression and DNA adducts formation in human breast cells MCF10A by dimethylbenz[a]anthracene (DMBA), a well‐known breast chemical carcinogen. Our results showed that DMBA‐induced DNA adduct was completely prevented by MET as evidenced by decrease in DNA repair genes and 8‐hydroxy‐2′‐deoxyguanosine level. Importantly, MET inhibited the induction of CYP1A1 mRNA, protein and activity levels by DMBA, suggesting a direct role of CYP1A1. Mechanistically, this was strongly correlated with MET ability to inhibit AhR‐dependent luciferase activity, but there was no effect on AhR activation, as evidenced by binding assay, suggesting that MET is not an AhR ligand. In conclusion, the present work provides the first evidence that MET inhibits the DMBA‐mediated carcinogenicity by inhibiting the expression of CYP1A1 through an AhR ligand‐independent mechanism. This work was supported by the Deanship of Scientific Research grant no. RGP‐VPP‐141.

Posttranslational mechanisms modulating the expression of the cytochrome P450 1A1 gene by methylmercury in HepG2 cells: A role of heme oxygenase-1

Recently we demonstrated the ability of mercuric chloride (Hg2+) in human hepatoma HepG2 cells to significantly decrease the TCDD-mediated induction of Cytochrome P450 1A1 (CYP1A1) mRNA, protein, and catalytic activity levels. In this study we investigated the effect of methylmercury (MeHg) on CYP1A1 in HepG2 cells. For this purpose, cells were co-exposed to MeHg and TCDD and the expression of CYP1A1 mRNA, protein, and catalytic activity levels were determined. Our results showed that MeHg did not alter the TCDD-mediated induction of CYP1A1 mRNA, or protein levels; however it was able to significantly decrease CYP1A1 catalytic activity levels in a concentration-dependent manner. Importantly, this inhibition was specific to CYP1A1and was not radiated to other aryl hydrocarbon receptor (AhR)-regulated genes, as MeHg induced NAD(P)H:quinone oxidoreductase 1 mRNA and protein levels. Mechanistically, the inhibitory effect of MeHg on the induction of CYP1A1 coincided with an increase in heme oxygenase-1 (HO-1) mRNA levels. Furthermore, the inhibition of HO-1 activity, by tin mesoporphyrin, caused a complete restoration of MeHg-mediated inhibition of CYP1A1 activity, induced by TCDD. In addition, transfection of HepG2 cells with siRNA targeting the human HO-1 gene reversed the MeHg-mediated inhibition of TCDD-induced CYP1A1. In conclusion, this study demonstrated that MeHg inhibited the TCDD-mediated induction of CYP1A1 through a posttranslational mechanism and confirms the role of HO-1 in a MeHg-mediated effect.

Benzo(a)pyrene induces hepatic AKR1A1 mRNA expression in tilapia fish (Oreochromis niloticus)

AKR1A1 or aldehyde reductase is a member of the aldo-keto reductases superfamily that is evolutionarily conserved among species. AKR1A1 is one of the five AKRs (AKR1A1 and 1C1-1C4) implicated in the metabolic benzo(a)pyrene (BaP) activation to reactive BaP 7,8-dione. BaP is a polycyclic aromatic hydrocarbon (PAH) widely distributed in aquatic ecosystems and its metabolic activation is necessary to produce its toxic effects. Although the presence of AKR1A1 in fish has been reported, its tissue distribution in tilapia (Oreochromis niloticus) and AKR1A1 inducibility by BaP are not known yet. Moreover, cytochrome P4501A (CYP1A) mRNA expression in fish has been used as a PAH biomarker of effect. Therefore, BaP effects on AKR1A1 and CYP1A gene expressions in tilapia, a species of commercial interest, were investigated by real-time RT-PCR. A partial AKR1A1 cDNA was identified, sequenced and compared with AKR1A1 reported sequences in the GenBank DNA database. Constitutive AKR1A1 mRNA expression was detected mainly in liver, similarly to that of CYP1A. BaP exposure resulted in statistically significant AKR1A1 and CYP1A mRNA induction in liver (20- and 120-fold, respectively) at 24 h. On the other hand, ethoxyquin (EQ) was used as control inducer for AKR1A1 mRNA. Interestingly, EQ also induced CYP1A mRNA levels in tilapia liver. Our results suggest that teleost AKR1A1, in addition to CYP1A, are inducible by BaP. The mechanism of AKR1A1 induction by BaP and its role in fish susceptibility to BaP toxic effects remains to be elucidated.

Analysis of the CYP1A1 mRNA Dose-Response in Human Keratinocytes Indicates that Relative Potencies of Dioxins, Furans, and PCBs Are Species and Congener Specific

Reports indicate that toxic equivalency factors (TEFs) based primarily on rodent data do not accurately predict in vitro human responsiveness to certain dioxin-like chemicals (DLCs). To investigate this in cells responsive to dioxins and relevant to chloracne, normal human epidermal keratinocytes were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and several DLCs, each with a TEF value of 0.1, representing three classes of congeners. We estimated half maximal effective concentration (EC50)-based donor-specific relative potency (REP) values for cytochrome P450 1A1 (CYP1A1) messenger RNA (mRNA) induction for TCDD, 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (HxCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,6,7,8-hexachlorodibenzofuran (HxCDF), and 3,3',4,4',5-pentachlorobiphenyl (PCB 126). We also determined EC50-based population-level REP values (n = 4) for CYP1A1 mRNA induction for TCDD, HxCDF, and PCB 126. Furthermore, an alternative factor, the relative threshold factor (RTF) based on the low end (threshold) of the dose-response curve, was calculated. Our results demonstrated that HxCDF had a population-based REP value of 0.98, 9.8-fold higher than its assigned TEF value of 0.1. Conversely, PCB 126 had an REP value of 0.0027 and an RTF of 0.0022, 37-fold and 45-fold less than its assigned TEF of 0.1, respectively. The REP values for HxCDD and TCDF were 0.24 and 0.10, respectively, similar to their assigned value of 0.1. Therefore, although the DLCs tested in the current study all possessed the same assigned TEF value of 0.1, congener-specific differences in REPs and RTFs were observed for human keratinocytes. These congener-specific discrepancies are likely because of differences in interspecies factors that have yet to be defined.

Harman induces CYP1A1 enzyme through an aryl hydrocarbon receptor mechanism

Harman is a common compound in several foods, plants and beverages. Numerous studies have demonstrated its mutagenic, co-mutagenic and carcinogenic effects; however, the exact mechanism has not been fully identified. Aryl hydrocarbon receptor (AhR) is a transcription factor regulating the expression of the carcinogen-activating enzyme; cytochrome P450 1A1 (CYP1A1). In the present study, we examined the ability of harman to induce AhR-mediated signal transduction in human and rat hepatoma cells; HepG2 and H4IIE cells. Our results showed that harman significantly induced CYP1A1 mRNA in a time- and concentration-dependent manner. Similarly, harman significantly induced CYP1A1 at protein and activity levels in a concentration-dependent manner. Moreover, the AhR antagonist, resveratrol, inhibited the increase in CYP1A1 activity by harman. The RNA polymerase inhibitor, actinomycin D, completely abolished the CYP1A1 mRNA induction by harman, indicating a transcriptional activation. The role of AhR in CYP1A1 induction by harman was confirmed by using siRNA specific for human AhR. The ability of harman to induce CYP1A1 was strongly correlated with its ability to stimulate AhR-dependent luciferase activity and electrophoretic mobility shift assay. At post-transcriptional and post-translational levels, harman did not affect the stability of CYP1A1 at the mRNA and the protein levels, excluding other mechanisms participating in the obtained effects. We concluded that harman can directly induce CYP1A1 gene expression in an AhR-dependent manner and may represent a novel mechanism by which harman promotes mutagenicity, co-mutagenicity and carcinogenicity.

Transcriptional and posttranscriptional regulation of CYP1A1 by vanadium in human hepatoma HepG2 cells

We recently demonstrated that V(5+) downregulates 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated induction of Cyp1a1 mRNA, protein, and catalytic activity levels in Hepa 1c1c7 cells through transcriptional mechanism. Therefore, it is important to investigate whether similar changes occur in humans. For this purpose, we examined the effect of V(5+) (as ammonium metavanadate, NH(4)VO(3)) on the expression of aryl hydrocarbon receptor (AhR)-regulated gene; cytochrome P450 1A1 (CYP1A1) at each step of the AhR signal transduction pathway in human hepatoma HepG2 cells. Our results show a significant reduction in TCDD-mediated induction of CYP1A1 mRNA, protein, and activity levels after V(5+) treatment in a dose-dependent manner. Investigating the effect of co-exposure to V(5+) and TCDD at transcriptional levels revealed that V(5+) significantly inhibited TCDD-mediated induction of AhR-dependent luciferase reporter gene expression. Looking at the posttranscriptional level, V(5+) did not affect CYP1A1 mRNA stability, thus eliminating the possible role of V(5+) in modifying CYP1A1 gene expression through this mechanism. On the other hand, at the posttranslational level, V(5+) was able to significantly decrease CYP1A1 protein half-life contributing to the inconsistency between catalytic activity and transcriptional level. Importantly, we showed that V(5+) did not significantly alter the heme oxygenase-1 mRNA level, thus eliminating any possibility that V(5+) might have decreased CYP1A1 activity through affecting its heme content. This study demonstrates for the first time that V(5+) downregulates the expression of CYP1A1 at the transcriptional, posttranscriptional and posttranslational mechanisms in the human hepatoma HepG2 cells.

Arsenite down-regulates cytochrome P450 1A1 at the transcriptional and posttranslational levels in human HepG2 cells

Aryl hydrocarbon receptor (AhR) ligands, typified by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), and metals, typified by arsenite (As(III)), are environmental cocontaminants, and their molecular interaction may disrupt the coordinated regulation of the carcinogen activating enzyme cytochrome P450 1A1 (CYP1A1). Therefore, in this study we examined the effects of coexposure to As(III) and TCDD on the expression of CYP1A1 in HepG2 cells. Our results showed that As(III) caused a dose-dependent decrease in TCDD-mediated induction of CYP1A1 mRNA, protein, and catalytic activity levels. As(III) significantly inhibited TCDD-mediated induction of AhR-dependent luciferase reporter gene expression without altering CYP1A1 mRNA stability. In addition, As(III) increased heme oxygenase-1 (HO-1) mRNA, which coincided with a further decrease in the CYP1A1 catalytic activity levels. When a competitive HO-1 inhibitor, tin mesoporphyrin, was applied to HepG2 cells or the cells were transfected with siRNA for HO-1 there was a partial restoration of the inhibition of TCDD-mediated induction of CYP1A1 catalytic activity. Treatment of cells with heme or hemoglobin partially restored the As(III)-mediated inhibition of CYP1A1 catalytic activity. On the other hand, cobalt protoporphyrin increased HO-1 mRNA, with a concomitant decrease in CYP1A1 activity, without affecting CYP1A1 mRNA, which was reversed by HO-1 siRNA transfection. This study demonstrates that As(III) down-regulates CYP1A1 through transcriptional and posttranslational mechanisms. In addition, HO-1 is involved in the As(III)-mediated down-regulation of CYP1A1 at the catalytic activity level.

Response of CYP1A Gene expression in fish liver of catfish (Ariopsis felis) from Gulf of Mexico and their relationship with the genetic variability

We determined the hepatic Cytochrome P4501A (CYP1A) mRNA and Ethoxyresorufin-O-deethylase (EROD) activities in the fish, Ariopsis felis, from highly polluted to relatively pristine regions in the southwest Gulf of Mexico and their relationship with the genetic polymorphisms of the same fish. We hypothesized that a high genetic variation reflects interindividual variability in levels of CYP1A mRNA underlying the pathway culminating in EROD induction caused by the environmental contaminants. Catfish from Laguna de Mecoacan exhibited marked induction of CYP1A mRNA and high levels of hepatic EROD activities, whereas fish from Laguna de Celestun showed no induction of CYP1A mRNA and moderately low levels of EROD activities. In contrast, the similarity index considering all samples varied from 0.4 to 0.87, showing a wide range of variation. A dendrogram showed a clear grouping of fish collected from the Laguna de Terminos, Rio Coatzacoalcos and Laguna de Celestun, with discrete subgroups according to region. In contrast, fish from Laguna de Mecoacan were grouped together completely separate from the rest of the fish. Despite the low number of fish from Mecoacan (a high bootstrap support was observed in this group), the results indicated a significant genetic variability in comparison with the other ecosystems included. The differential level of expression of CYP1A and the EROD activity observed among the ecosystems analyzed could be due to the high range of genetic variation, with special emphasis on fish collected in Mecoacan where it is possible to find a subspecies of Ariopsis felis.

Potent Modification of Inducible CYP1A1 Expression by Flavonoids

The present study examined modifications of β-naphthoflavone (β-NF)-induced cytochrome P450 1A1 (CYP1A1) expression by flavonoids in mouse hepatocytes in primary culture. Some flavonoids (apigenin, chrysin, flavone, flavanone, galangin, luteolin, and naringenin) by themselves induced CYP1A1 mRNA expression, especially flavone which was even more effective than β-NF. The effect on β-NF-induced CYP1A1 mRNA expression was varied, namely additive, suppressive, or both. An additive effect was observed after combined treatment with flavanone, naringenin, and chrysin, whereas kaempferol, myricetin, and quercetin decreased CYP1A1 levels. Apigenin, chrysin, galangin, luteolin, and morin synergistically enhanced β-NF-induced CYP1A1 expression at 24 h, but considerably suppressed it at 9 h. The structure-activity relationship of flavonoids affecting CYP1A1 expression as inducers or inhibitors is discussed. The present observations suggest the need to reveal the mechanism by which CYP1A1 expression is modified by flavonoids for risk assessment, since CYP1A1 activates environmental carcinogenic polycyclic hydrocarbons and flavonoids are major constituents in food.

Molecular cloning and β-naphthoflavone-induced expression of a cytochrome P450 1A ( CYP1A) gene from an anadromous river pufferfish, Takifugu obscurus

In recent years, there has been a decline in the wild populations of river pufferfish, Takifugu obscurus. Besides overexploitation for commercial purposes, environmental pollution is believed to have contributed to its decline. However, almost no information exists about genes involved in metabolism of xenobiotics by this species. Nevertheless, there is interest in fugu fishes, since they possess the smallest genome among vertebrates. We cloned and characterized the full-length cDNA sequence of a cytochrome P450 1A ( CYP1A) gene from T. obscurus. Phylogenic relationship of T. obscurus CYP1A was also compared to other fish species. The tissue distribution and time-dependant induction of CYP1A mRNA were studied by real-time PCR in T. obscurus exposed to an aryl hydrocarbon receptor (Ahr) agonist, β-naphthoflavone (BNF). The greatest basal expression in livers of control as well as BNF-treated individuals. However, brain, gill, gonad, intestine, and kidney also expressed CYP1A. Muscles expressed the least CYP1A. The results of the time-course study revealed induction in brain and gills after 6 h and at 12 h in most tissues. Except for gills, all other organs retained induced expression of CYP1A mRNA up to 96 h.

Comparison of hepatic and extra hepatic induction of cytochrome P4501A by graded doses of aryl hydrocarbon receptor agonists in Atlantic tomcod from two populations

Atlantic tomcod Microgadus tomcod from the Hudson River, New York, are exposed to high levels of polycyclic aromatic hydrocarbons (PAHs) and bioaccumulate mixtures of polychlorinated biphenyls (PCBs), polychlorinated dibenzo- p-dioxins and polychlorinatedfurans (PCDD/Fs). Previous studies demonstrated that hepatic cytochrome P4501A (CYP1A) mRNA was not inducible in tomcod from the Hudson River treated with single doses of PCB77 or 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), but was inducible with PAHs. In this study, we sought to determine if CYP1A mRNA was inducible with higher doses of these and other halogenated aromatic hydrocarbons (HAHs) in Hudson River tomcod and if decreased sensitivity to gene inducibility occurs across all tissues. Tomcod from the Hudson River and the cleaner Miramichi River, New Brunswick, were treated individually with graded doses of TCDD and coplanar PCBs (PCB77, PCB81, PCB126, PCB169) and profiles of hepatic CYP1A mRNA expression were compared between the two populations. CYP1A mRNA inducibility was also compared in multiple tissues of tomcod from the two rivers that were treated with PCB77. Additionally, hepatic CYP1A mRNA was characterized in Miramichi River tomcod treated with pairs of PCB congeners that included aryl hydrocarbon receptor (AHR) agonists and antagonists. Hepatic CYP1A mRNA was significantly inducible by all agonists in tomcod from the Miramichi River and TCDD and two of four PCBs in tomcod from the Hudson River. CYP1A mRNA was also significantly inducible in four of five tissues of tomcod from the Miramichi River but only in liver of Hudson River tomcod. In summary, CYP1A mRNA inducibility was approximately two orders of magnitude less sensitive in tomcod from the Hudson River than in those from the Miramichi River. But when achieved, maximum levels of CYP1A expression were similar in tomcod from the two populations. Co-administration of PCB126 and PCB77 did not produce significantly greater CYP1A mRNA induction than administration of PCB126 alone and co-administration of mono- ortho-substituted PCB105 significantly decreased CYP1A mRNA inducibility by PCB77. These results indicate that CYP1A mRNA expression is significantly inducible by HAHs in tomcod from the Hudson River and suggest that all components of the AHR pathway are present and functional, but that the pathway is less sensitive to activation than in tomcod from the Miramichi River. Our results also indicate that CYP1A mRNA levels in environmentally exposed fish may not reflect additive tissue burdens of PCB congeners.

Induction of CYP1A mRNA in Carp (Cyprinus carpio) from the Kalamazoo River Polychlorinated Biphenyl-Contaminated Superfund Site and in a Laboratory Study

The Kalamazoo River Superfund site in Michigan is contaminated with polychlorinated biphenyls (PCBs), which were heavily discharged into the river from several paper companies as part of the deinking process in the 1950s through 1970s. We characterized biomarkers of chronic PCB exposure in a resident fish population using real-time reverse transcriptase-polymerase chain reaction to examine mRNA expression levels of multiple genes in carp (Cyprinus carpio) liver from PCB contaminated and reference sites in the Kalamazoo River. We also measured these same genes in juvenile carp exposed to dietary PCBs for 4 months. Kalamazoo River carp had significantly increased levels of cytochrome P450 1A (CYP1A) mRNA as did carp fed PCBs in the laboratory. No significant mRNA upregulation occurred in the specific oxidative stress genes (gamma-glutamylcysteine synthetase and magnesium superoxide dismutase) and metabolic genes (phosphoenolpyruvate carboxykinase and nucleolin) examined. These data are consistent with the idea that carp from the Kalamazoo River Superfund Site are responding to PCB exposure via upregulation of CYP1A independent of activation of the oxidative stress response genes normally thought to be co-regulated with CYP1A.

Induction of cytochrome P450-1A by the equine estrogen equilenin, a new endogenous aryl hydrocarbon receptor ligand

Equilenin is one of 10 kinds of estrogens that are found in pregnant mares’ urine. It has been used extensively for estrogen replacement therapy in postmenopausal women. Typical inducers of the cytochrome P4501A1 (CYP1A1), such as TCDD, benzo(a)pyrene (B(a)P) and 3-methylcholanthrene, have a planar molecular structure in common and bind to the aryl hydrocarbon receptor (AhR). The structure of equilenin differs from classic estrogens by the presence of two additional double bonds in ring B of the steroid nucleus, and it is planar. This structural similarity of equilenin to the typical AhR agonist prompted us to investigate the capability of equilenin to induce CYP1A1 expression. Administration of equilenin to two mouse strains (C57BL and DBA) that exhibit different degrees of responsiveness to an Ah-receptor agonist and showed that equilenin was capable of dose-dependently increasing both the ethoxyresorufin O-deethylase activity and CYP1a proteins in both strains of mice. Equilenin also induced CYP1A1 mRNA in treated HepG2 cell lines and transcriptional activity in an XRE-directed luciferase reporter gene. Competitive binding studies using C57BL AhR indicated equilenin weakly displaced 3H-B(a)P from AhR. Together, these data show that equilenin, an equine steroid hormone, served as an AhR ligand in the present study.

Evidence of Spatially Extensive Resistance to PCBs in an Anadromous Fish of the Hudson River

Populations of organisms that are chronically exposed to high levels of chemical contaminants may not suffer the same sublethal or lethal effects as naive populations, a phenomenon called resistance. Atlantic tomcod (Microgadus tomcod) from the Hudson River, New York, are exposed to high concentrations of polycyclic aromatic hydrocarbons (PAHs) and bioaccumulate polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs). They have developed resistance to PCBs and PCDDs but not to PAHs. Resistance is largely heritable and manifests at early-life-stage toxic end points and in inducibility of cytochrome P4501A (CYP1A) mRNA expression. Because CYP1A induction is activated by the aryl hydrocarbon receptor (AHR) pathway, as are most toxic responses to these compounds, we sought to determine the geographic extent of resistance to CYP1A mRNA induction by PCBs in the Hudson River tomcod population. Samples of young-of-the-year tomcod were collected from seven locales in the Hudson River, extending from the Battery at river mile 1 (RM 1) to RM 90, and from the Miramichi River, New Brunswick, Canada. Laboratory-reared offspring of tomcod adults from Newark Bay, in the western portion of the Hudson River estuary, were also used in this study. Fish were partially depurated in clean water and intraperitoneally injected with 10 ppm coplanar PCB-77, 10 ppm benzo[a]pyrene (BaP), or corn oil vehicle, and levels of CYP1A mRNA were determined. CYP1A was significantly inducible by treatment with BaP in tomcod from the Miramichi River, from laboratory-spawned offspring of Newark Bay origin, and from all Hudson River sites spanning 90 miles of river. In contrast, only tomcod from the Miramichi River displayed significantly induced CYP1A mRNA expression when treated with PCB-77. Our results suggest that the population of tomcod from throughout the Hudson River estuary has developed resistance to CYP1A inducibility and probably other toxicities mediated by the AHR pathway. Tomcod from the Hudson River may represent the most geographically expansive population of vertebrates with resistance to chemical pollutants that has been characterized.