Functional characterization of zebrafish cytochrome P450 1 family proteins expressed in yeast.

BACKGROUND: Zebrafish express five cytochrome P450 1 genes: CYP1A, CYP1B1, CYP1C1, CYP1C2, inducible by aryl hydrocarbon receptor agonists, and CYP1D1, a constitutively expressed CYP1A-like gene. We examined substrate selectivity of CYP1s expressed in yeast. METHODS: CYP1s were expressed in W(R) yeast, engineered to over-express P450 reductase, via pYES/DEST52 and via pYeDP60. Microsomal fractions from transformed yeast were examined for activity with fluorogenic substrates, benzo[a]pyrene and testosterone. Modeling and docking approaches were used to further evaluate sites of oxidation on benzo[a]pyrene and testosterone. RESULTS: CYP1s expressed in yeast dealkylated ethoxy-, methoxy-, pentoxy- and benzoxy-resorufin (EROD, MROD, PROD, BROD). CYP1A and CYP1C2 had the highest rates of EROD activity, while PROD and BROD activities were low for all five CYP1s. The relative rates of resorufin dealkylation by CYP1C1, CYP1C2 and CYP1D1 expressed via pYeDP60 were highly similar to relative rates obtained with pYES/DEST52-expressed enzymes. CYP1C1 and CYP1C2 dealkylated substituted coumarins and ethoxy-fluorescein-ethylester, while CYP1D1 did not. The CYP1Cs and CYP1D1 co-expressed with epoxide hydrolase oxidized BaP with different rates and product profiles, and all three produced BaP-7,8,9,10-tetrol. The CYP1Cs but not CYP1D1 metabolized testosterone to 6ß-OH-testosterone. However, CYP1D1 formed an unidentified testosterone metabolite better than the CYP1Cs. Testosterone and BaP docked to CYP homology models with poses consistent with differing product profiles. CONCLUSIONS: Yeast-expressed zebrafish CYP1s will be useful in determining further functionality with endogenous and xenobiotic compounds. GENERAL SIGNIFICANCE: Determining the roles of zebrafish CYP1s in physiology and toxicology depends on knowing the substrate selectivity of these enzymes.

The cytochrome P450 2AA gene cluster in zebrafish (Danio rerio): expression of CYP2AA1 and CYP2AA2 and response to phenobarbital-type inducers.

The cytochrome P450 (CYP) 2 gene family is the largest and most diverse CYP gene family in vertebrates. In zebrafish, we have identified 10 genes in a new subfamily, CYP2AA, which does not show orthology to any human or other mammalian CYP genes. Here we report evolutionary and structural relationships of the 10 CYP2AA genes and expression of the first two genes, CYP2AA1 and CYP2AA2. Parsimony reconstruction of the tandem duplication pattern for the CYP2AA cluster suggests that CYP2AA1, CYP2AA2 and CYP2AA3 likely arose in the earlier duplication events and thus are most diverged in function from the other CYP2AAs. On the other hand, CYP2AA8 and CYP2AA9 are genes that arose in the latest duplication event, implying functional similarity between these two CYPs. A molecular model of CYP2AA1 showing the sequence conservation across the CYP2AA cluster reveals that the regions with the highest variability within the cluster map onto CYP2AA1 near the substrate access channels, suggesting differing substrate specificities. Zebrafish CYP2AA1 transcript was expressed predominantly in the intestine, while CYP2AA2 was most highly expressed in the kidney, suggesting differing roles in physiology. In the liver CYP2AA2 expression but not that of CYP2AA1, was increased by 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and, to a lesser extent, by phenobarbital (PB). In contrast, pregnenolone 16α-carbonitrile (PCN) increased CYP2AA1 expression, but not CYP2AA2 in the liver. The results identify a CYP2 subfamily in zebrafish that includes genes apparently induced by PB-type chemicals and PXR agonists, the first concrete in vivo evidence for a PB-type response in fish.

Role of zebrafish cytochrome P450 CYP1C genes in the reduced mesencephalic vein blood flow caused by activation of AHR2.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes various signs of toxicity in early life stages of vertebrates through activation of the aryl hydrocarbon receptor (AHR). We previously reported a sensitive and useful endpoint of TCDD developmental toxicity in zebrafish, namely a decrease in blood flow in the dorsal midbrain, but downstream genes involved in the effect are not known. The present study addressed the role of zebrafish cytochrome P450 1C (CYP1C) genes in association with a decrease in mesencephalic vein (MsV) blood flow. The CYP1C subfamily was recently discovered in fish and includes the paralogues CYP1C1 and CYP1C2, both of which are induced via AHR2 in zebrafish embryos. We used morpholino antisense oligonucleotides (MO or morpholino) to block initiation of translation of the target genes. TCDD-induced mRNA expression of CYP1Cs and a decrease in MsV blood flow were both blocked by gene knockdown of AHR2. Gene knockdown of CYP1C1 by two different morpholinos and CYP1C2 by two different morpholinos, but not by their 5 nucleotide-mismatch controls, was effective in blocking reduced MsV blood flow caused by TCDD. The same CYP1C-MOs prevented reduction of blood flow in the MsV caused by ß-naphthoflavone (BNF), representing another class of AHR agonists. Whole-mount in situ hybridization revealed that mRNA expression of CYP1C1 and CYP1C2 was induced by TCDD most strongly in branchiogenic primordia and pectoral fin buds. In situ hybridization using head transverse sections showed that TCDD increased the expression of both CYP1Cs in endothelial cells of blood vessels, including the MsV. These results indicate a potential role of CYP1C1 and CYP1C2 in the local circulation failure induced by AHR2 activation in the dorsal midbrain of the zebrafish embryo.

Cytochrome P4501A biomarker indication of oil exposure in harlequin ducks up to 20 years after the Exxon Valdez oil spill.

Hydrocarbon-inducible cytochrome P4501A (CYP1A) expression was measured, as ethoxyresorufin-O-deethylase (EROD) activity, in livers of wintering harlequin ducks (Histrionicus histrionicus) captured in areas of Prince William Sound, Alaska, USA, oiled by the 1989 Exxon Valdez spill and in birds from nearby unoiled areas, during 2005 to 2009 (up to 20 years following the spill). The present work repeated studies conducted in 1998 that demonstrated that in harlequin ducks using areas that received Exxon Valdez oil, EROD activity was elevated nearly a decade after the spill. The present findings strongly supported the conclusion that average levels of hepatic EROD activity were higher in ducks from oiled areas than those from unoiled areas during 2005 to 2009. This result was consistent across four sampling periods; furthermore, results generated from two independent laboratories using paired liver samples from one of the sampling periods were similar. The EROD activity did not vary in relation to age, sex, or body mass of individuals, nor did it vary strongly by season in birds collected early and late in the winter of 2006 to 2007, indicating that these factors did not confound inferences about observed differences between oiled and unoiled areas. We interpret these results to indicate that harlequin ducks continued to be exposed to residual Exxon Valdez oil up to 20 years after the original spill. This adds to a growing body of literature suggesting that oil spills have the potential to affect wildlife for much longer time frames than previously assumed.

Role of AHR2 in the expression of novel cytochrome P450 1 family genes, cell cycle genes, and morphological defects in developing zebra fish exposed to 3,3',4,4',5-pentachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Halogenated agonists for the aryl hydrocarbon receptor (AHR), such as 3,3',4,4',5-pentachlorobiphenyl (PCB126) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause developmental toxicity in fish. AHR dependence of these effects is known for TCDD but only presumed for PCB126, and the AHR-regulated genes involved are known only in part. We defined the role of AHR in regulation of four cytochrome P450 1 (CYP1) genes and the effect of PCB126 on cell cycle genes (i.e., PCNA and cyclin E) in zebra fish (Danio rerio) embryos. Basal and PCB126-induced expression of CYP1A, CYP1B1, CYP1C1, and CYP1C2 was examined over time as well as in relation to cell cycle gene expression and morphological effects of PCB126 in developing zebra fish. The four CYP1 genes differed in the time for maximal basal and induced expression, i.e., CYP1B1 peaked within 2 days postfertilization (dpf), the CYP1Cs around hatching (3 dpf), and CYP1A after hatching (14-21 dpf). These results indicate developmental periods when the CYP1s may play physiological roles. PCB126 (0.3-100nM) caused concentration-dependent CYP1 gene induction (EC50: 1.4-2.7nM, Lowest observed effect concentration [LOEC]: 0.3-1nM) and pericardial edema (EC50: 4.4nM, LOEC: 3nM) in 3-dpf embryos. Blockage of AHR2 translation significantly inhibited these effects of PCB126 and TCDD. PCNA gene expression was reduced by PCB126 in a concentration-dependent manner, suggesting that PCB126 could suppress cell proliferation. Our results indicate that the four CYP1 genes examined are regulated by AHR2 and that the effect of PCB126 on morphology in zebra fish embryos is AHR2 dependent. Moreover, the developmental patterns of expression and induction suggest that CYP1 enzymes could function in normal development and in developmental toxicity of PCB126 in fish embryos.

Cytochrome P4501A is induced in endothelial cell lines from the kidney and lung of the bottlenose dolphin, Tursiops truncatus.

Marine mammals respond to the presence of polycyclic and planar halogenated aromatic hydrocarbons (PAH or PHAH) with the induced expression in endothelium of cytochrome P4501A1, regulated through the aryl hydrocarbon receptor (AHR) transcription factor. Physiological responses in other animals, such as edema and inflammation indicate that the endothelium may be compromised by exposure to AHR agonists, which are ubiquitous in the marine environment. In other mammals and fish the cellular and molecular consequences of exposure to AHR agonists have been elucidated in cultured endothelial cells. We have cultured and characterized cetacean endothelial cells (EC) and used them in induction studies. Endothelial cells were cultured from the lung and kidney of the bottlenose dolphin, Tursiops truncates, and exposed to the AHR agonists beta-naphthoflavone (betaNF) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). betaNF (1-3 microM) induced significant increases in CYP1A1 (O-deethylation of 7-ethoxyresorufin to resorufin; EROD) activity to 3.6 and 0.92 pmol/mg/min in lung and kidney EC, respectively. TCDD was more potent than betaNF, and more efficacious, with maximum induction of CYP1A1 activity of 10.1 and 15.2 pmol/mg/min in lung and kidney EC at 3-10 nM TCDD. The differential response indicates that the lung and kidney endothelial cells in culture retain the ability to respond in a selective manner to specific stimuli. Both the molecular mechanisms of induction and the physiological consequences, especially in the vasculature, of toxicant exposure can be studied in this system.

Evaluating cytochrome p450 in lesser scaup (Aythya affinis) and tree swallow (Tachycineta bicolor) by monooxygenase activity and immunohistochemistry: possible nonlethal assessment by skin immunohistochemistry.

Six-month-old lesser scaup (Aythya affinis) and nestling tree swallows (Tachycineta bicolor) were injected intraperitoneally with beta-naphthoflavone (BNF) in corn oil or in vehicle alone. Liver samples were taken and stored at -80 degrees C until microsome preparation and monooxygenase assay. Skin samples were placed in buffered formalin for subsequent immunohistochemical (IHC) analysis for cytochrome P4501A (CYP1A). Lesser scaup treated with BNF at 20 or 100 mg/kg body weight showed approximately 6- to 18-fold increases in four monooxygenases (benzyloxyresorufin-O-dealkylase, ethoxyresorufin-O-dealkylase, methoxyresorufin-O-dealkylase, and pentoxyresorufin-O-dealkylase). No IHC response was observed for CYP1A in the skin of vehicle-injected ducks, whereas in the skin from BNF-treated ducks, the positive IHC response was of similar magnitude for both dose levels of BNF. Tree swallows injected with BNF at 100 mg/kg, but not at 20 mg/kg, showed significant increases (approximately fivefold) in hepatic microsomal O-dealkylase activities. Cytochrome P4501A was undetectable by IHC response in skin from corn oil-treated swallows, but positive IHC responses were observed in the skin of one of five swallows at 20 mg/kg and four of five swallows at 100 mg/kg. Although these data do not allow construction of significant dose-response curves, the IHC responses for CYP1A in skin support the possible use of this nonlethal approach for biomonitoring contaminant exposure of birds. In addition, the CYP1A signal observed at the bases of emerging feathers suggest that these might provide less invasive sampling sites for IHC analysis of CYP1A.

Plant lignan secoisolariciresinol suppresses pericardial edema caused by dioxin-like compounds in developing zebrafish: Implications for suppression of morphological abnormalities.

Dioxins and dioxin-like compounds (DLCs) enter the body mainly through diet and cause various toxicological effects through activation of the aryl hydrocarbon receptor (AhR), a ligand activated transcription factor. Some plant extracts and phytochemicals are reported to suppress this transformation. However, most of these reports have been from in vitro experiments and few reports have been from in vivo experiments. In addition, there has been no report of foodstuffs that effectively prevent AhR-associated morphological abnormalities such as deformities caused by dioxins and DLCs in vivo. In this study, we show that secoisolariciresinol (SECO), a natural lignan-type polyphenolic phytochemical found mainly in flaxseed, has a rescuing effect, actually suppressing morphological abnormalities (pericardial edema) in zebrafish embryos exposed to 3,3',4,4',5-pentachlorobiphenyl (PCB126), a dioxin-like PCB congener. Importantly, the rescuing effect of SECO was still evident when it was applied 16 h after the beginning of exposure to PCB126. This study suggests that SECO may be useful as a natural suppressive agent for morphological abnormalities caused by dioxins and DLCs.

Cytochrome p4501a induced differentially in endothelial cells cultured from different organs of Anguilla rostrata.

Endothelial cells are a structural barrier and an active regulator of many bodily processes. Cytochrome P4501A (CYP1A) activity is induced in the endothelium of teleosts and mammals exposed to lipophilic xenobiotics, such as polycyclic aromatic hydrocarbons, and can have significant consequences for endothelial functions. We exposed cultures of characterized endothelial cells from the heart, kidney, and rete mirabile of the eel, Anguilla rostrata, to aryl hydrocarbon receptor (AhR) agonists. In heart endothelial cells, the maximum response (based on O-deethylation of 7-ethoxyresorufin to resorufin [EROD] activity) to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 113 pmol/mg/min, was at 1 nM TCDD and the peak response to beta-napthoflavone (betaNF), 135 pmol/mg/min, was at 3 microM betaNF. The maximum response to TCDD in the kidney endothelial cells is 12 pmol/mg/min at 0.3 nM TCDD. The rete mirabile capillary endothelial cells responded minimally or not at all to exposure to TCDD and betaNF. Both the heart and kidney endothelial cells (but not the rete mirabile capillary cells) have a low level of EROD activity (12.7 and 5.2 pmol/mg/min, respectively) in untreated or dimethylsulfoxide-treated cells. The robust response of the heart endothelial cells to induction and the lack of response in the rete mirabile capillary endothelial cells indicate that these cells are a good resource to use to investigate the physiological consequences of AhR agonist exposure and CYP1A induction in different areas of the vasculature.

Role of pregnane X receptor and aryl hydrocarbon receptor in transcriptional regulation of pxr, CYP2, and CYP3 genes in developing zebrafish.

Ligand-activated receptors regulate numerous genes, and mediate effects of a broad set of endogenous and exogenous chemicals in vertebrates. Understanding the roles of these transcription factors in zebrafish (Danio rerio) is important to the use of this non-mammalian model in toxicological, pharmacological, and carcinogenesis research. Response to a potential agonist for the pregnane X receptor (Pxr) [pregnenolone (PN)] was examined in developing zebrafish, to assess involvement of Pxr in regulation of selected genes, including genes in cytochrome P450 subfamilies CYP2 and CYP3. We also examined interaction of Pxr and the aryl hydrocarbon receptor (Ahr) signaling pathways. Pregnenolone caused a dose-dependent increase in mRNA levels of pxr, ahr2, CYP1A, CYP2AA1, CYP2AA12, CYP3A65, and CYP3C1, most of which peaked at 3 µM PN. The well-known Ahr agonist 3,3',4,4',5-pentachlorobiphenyl (PCB126) also upregulated expression of pxr, ahr2, CYP1A, CYP2AA12, CYP3A65, and CYP3C1 in a dose-dependent manner. Inhibition of pxr translation by morpholino antisense oligonucleotides (MO) suppressed PN-induced expression of pxr, ahr2, CYP3A65, and CYP3C1 genes. Levels of CYP2AA1 and CYP2AA12 mRNA were increased in the control-MO group exposed to PN; this was prevented by knocking down Pxr. Similarly, Ahr2-MO treatment blocked PCB126-induced mRNA expression of pxr, CYP1A, CYP2AA12, CYP3A65, and CYP3C1. The present study shows self-regulation of pxr by PN in developing zebrafish. Selected zebrafish CYP1, CYP2 (including several CYP2AAs) and CYP3 genes appear to be under the regulation of both Pxr and Ahr2.

Regulation of pregnane-X-receptor, CYP3A and P-glycoprotein genes in the PCB-resistant killifish (Fundulus heteroclitus) population from New Bedford Harbor.

Killifish survive and reproduce in the New Bedford Harbor (NBH) in Massachusetts (MA), USA, a site severely contaminated with polychlorinated biphenyls (PCBs) for decades. Levels of 22 different PCB congeners were analyzed in liver from killifish collected in 2008. Concentrations of dioxin-like PCBs in liver of NBH killifish were ∼400 times higher, and the levels of non-dioxin-like PCBs ∼3000 times higher than in killifish from a reference site, Scorton Creek (SC), MA. The NBH killifish are known to be resistant to the toxicity of dioxin-like compounds and to have a reduced aryl hydrocarbon receptor (AhR) signaling response. Little is known about the responses of these fish to non-dioxin-like PCBs, which are at extraordinarily high levels in NBH fish. In mammals, some non-dioxin-like PCB congeners act through nuclear receptor 1I2, the pregnane-X-receptor (PXR). To explore this pathway in killifish, a PXR cDNA was sequenced and its molecular phylogenetic relationship to other vertebrate PXRs was determined. Killifish were also collected in 2009 from NBH and SC, and after four months in the laboratory they were injected with a single dose of either the dioxin-like PCB 126 (an AhR agonist) or the non-dioxin-like PCB 153 (a mammalian PXR agonist). Gills and liver were sampled three days after injection and transcript levels of genes encoding PXR, cytochrome P450 3A (CYP3A), P-glycoprotein (Pgp), AhR2 and cytochrome P450 1A (CYP1A) were measured by quantitative PCR. As expected, there was little effect of PCB exposure on mRNA expression of AhR2 or CYP1A in liver and gills of NBH fish. In NBH fish, but not in SC fish, there was increased mRNA expression of hepatic PXR, CYP3A and Pgp upon exposure to either of the two PCB congeners. However, basal PXR and Pgp mRNA levels in liver of NBH fish were significantly lower than in SC fish. A different pattern was seen in gills, where there were no differences in basal mRNA expression of these genes between the two populations. In SC fish, but not in NBH fish, there was increased mRNA expression of branchial PXR and CYP3A upon exposure to PCB126 and of CYP3A upon exposure to PCB153. The results suggest a difference between the two populations in non-AhR transcription factor signaling in liver and gills, and that this could involve killifish PXR. It also implies possible cross-regulatory interactions between that factor (presumably PXR) and AhR2 in liver of these fish.

Expression of P-glycoprotein and cytochrome p450 1A in intertidal fish (Anoplarchus purpurescens) exposed to environmental contaminants.

Whether P-glycoproteins (P-gps) like those which confer multidrug resistance in tumor cell lines are important in adaptation to chemicals in natural populations of vertebrates exposed to contaminant mixtures is the focus of this study. P-gp expression was examined in the intertidal fish high cockscomb blenny (Anoplarchus purpurescens) exposed to crude oil or pulp mill effluent. The relationship between P-gp expression and cytochrome p450 1A (CYP1A) induction also was investigated. Immunohistochemical (IHC) analysis revealed that levels of P-gp expression in the bile canaliculi were three- to five-fold greater in oil exposed fish than in control fish. Levels of P-gp expression were highly correlated with hepatic CYP1A levels previously measured in these fish. In fish from sites near pulp mills, P-gp expression in freshly caught fish did not correlate with proximity to pulp mills. However, hepatic P-gp expression levels in freshly caught fish were 14-fold higher than in fish from those sites that were depurated in clean water for 6 weeks. CYP1A levels were also elevated in liver of freshly caught as compared with depurated fish. Expression of neither CYP1A nor P-gp was elevated in depurated fish exposed to sediment and food from within the original pulp mill effluent stream. Depurated fish, which were injected with the aryl hydrocarbon receptor (AHR) agonist ss-naphthoflavone (BNF) showed an expected induction of CYP1A but no induction of P-gp. These results suggest that in blennies, unlike CYP1A, P-gp expression is not regulated by the AHR pathway; although P-gp and CYP1A both may be induced by some compounds in petroleum and unidentified xenobiotics at field sites. While our data indicate that CYP1A and P-gp are not coordinately regulated, these proteins may play complementary roles in cellular detoxification. Thus the elevation of P-gp activity may be an important mechanism of multixenobiotic resistance for organisms, such as intertidal fish, which are commonly exposed to anthropogenic contaminants and naturally occurring toxins.

Exposure to hydrocarbons 10 years after the Exxon Valdez oil spill: evidence from cytochrome P4501A expression and biliary FACs in nearshore demersal fishes.

Three biomarkers of hydrocarbon exposure, CYP1A in liver vascular endothelium, liver ethoxyresorufin O-deethylase (EROD), and biliary fluorescent aromatic compounds (FACs), were examined in the nearshore fishes, masked greenling (Hexagrammos octogrammus) and crescent gunnel (Pholis laeta), collected in Prince William Sound, Alaska, 7-10 years after the Exxon Valdez oil spill (EVOS). All biomarkers were elevated in fish collected from sites originally oiled, in comparison to fish from unoiled sites. In 1998, endothelial CYP1A in masked greenling from sites that were heavily oiled in 1989 was significantly higher than in fish collected outside the spill trajectory. In 1999, fishes collected from sites adjacent to intertidal mussel beds containing lingering Exxon Valdez oil had elevated endothelial CYP1A and EROD, and high concentrations of biliary FACs. Fishes from sites near unoiled mussel beds, but within the original spill trajectory, also showed evidence of hydrocarbon exposure, although there were no correlations between sediment petroleum hydrocarbon and any of the biomarkers. Our data show that 10 years after the spill, nearshore fishes within the original spill zone were still exposed to residual EVOS hydrocarbons.

The transcriptional response to oxidative stress during vertebrate development: effects of tert-butylhydroquinone and 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Oxidative stress is an important mechanism of chemical toxicity, contributing to teratogenesis and to cardiovascular and neurodegenerative diseases. Developing animals may be especially sensitive to chemicals causing oxidative stress. The developmental expression and inducibility of anti-oxidant defenses through activation of NF-E2-related factor 2 (NRF2) affect susceptibility to oxidants, but the embryonic response to oxidants is not well understood. To assess the response to chemically mediated oxidative stress and how it may vary during development, zebrafish embryos, eleutheroembryos, or larvae at 1, 2, 3, 4, 5, and 6 days post fertilization (dpf) were exposed to DMSO (0.1%), tert-butylhydroquinone (tBHQ; 10 µM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 2 nM) for 6 hr. Transcript abundance was assessed by real-time qRT-PCR and microarray. qRT-PCR showed strong (4- to 5-fold) induction of gstp1 by tBHQ as early as 1 dpf. tBHQ also induced gclc (2 dpf), but not sod1, nqo1, or cyp1a. TCDD induced cyp1a but none of the other genes. Microarray analysis showed that 1477 probes were significantly different among the DMSO-, tBHQ-, and TCDD-treated eleutheroembryos at 4 dpf. There was substantial overlap between genes induced in developing zebrafish and a set of marker genes induced by oxidative stress in mammals. Genes induced by tBHQ in 4-dpf zebrafish included those involved in glutathione synthesis and utilization, signal transduction, and DNA damage/stress response. The strong induction of hsp70 determined by microarray was confirmed by qRT-PCR and by use of transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) under control of the hsp70 promoter. Genes strongly down-regulated by tBHQ included mitfa, providing a molecular explanation for the loss of pigmentation in tBHQ-exposed embryos. These data show that zebrafish embryos are responsive to oxidative stress as early as 1 dpf, that responsiveness varies with development in a gene-specific manner, and that the oxidative stress response is substantially conserved in vertebrate animals.

Identification and expression of multiple CYP1-like and CYP3-like genes in the bivalve mollusk Mytilus edulis.

Various sequencing projects over the last several years have aided the discovery of previously uncharacterized invertebrate sequences, including new cytochrome P450 genes (CYPs). Here we present data on the identification and characterization of two CYP1-like and three CYP3-like genes from the bivalve mollusk Mytilus edulis, and assess their potential as biomarkers based on their responses to several known vertebrate aryl hydrocarbon receptor (AHR) agonists. Quantitative real-time PCR was used to measure CYP transcript levels in digestive gland, labial palps, adductor muscle, gill, foot, and different regions of the mantle. Levels of both CYP1-like genes were highest in digestive gland, whereas labial palps had the highest expression levels of the three CYP3-like genes followed by digestive gland and outer margin of the mantle. Mussels were exposed by injection to the AHR agonists, ß-naphthoflavone (BNF; 25 µg g(-1)), 3,3',4,4',5-polychlorinated biphenyl (PCB126; 2 µg g(-1)), or 6-formylindolo[3,2-b]carbazole (FICZ; 0.1 µg g(-1)), or to Aroclor 1254 (a mixture of PCBs; 50 µg g(-1)) for 24 h, followed by CYP expression analysis. There was no statistically significant change in expression of either of the CYP1-like genes after exposure to the various AHR agonists. The CYP3-like-1 gene was significantly up-regulated by BNF in gill tissues and the CYP3-like-2 gene was up-regulated in digestive gland by PCB126 and in gill tissue by BNF. These results suggest that distinct mechanisms of CYP gene activation could be present in M. edulis, although the importance of the CYP1-like and CYP3-like genes for xenobiotic and endogenous lipids biotransformation requires additional investigation.

Cytochrome p450 1 genes in birds: evolutionary relationships and transcription profiles in chicken and Japanese quail embryos.

BACKGROUND: Cytochrome P450 1 (CYP1) genes are biomarkers for aryl hydrocarbon receptor (AHR) agonists and may be involved in some of their toxic effects. CYP1s other than the CYP1As are poorly studied in birds. Here we characterize avian CYP1B and CYP1C genes and the expression of the identified CYP1 genes and AHR1, comparing basal and induced levels in chicken and quail embryos. METHODOLOGY/PRINCIPAL FINDINGS: We cloned cDNAs of chicken CYP1C1 and quail CYP1B1 and AHR1. CYP1Cs occur in several bird genomes, but we found no CYP1C gene in quail. The CYP1C genomic region is highly conserved among vertebrates. This region also shares some synteny with the CYP1B region, consistent with CYP1B and CYP1C genes deriving from duplication of a common ancestor gene. Real-time RT-PCR analyses revealed similar tissue distribution patterns for CYP1A4, CYP1A5, CYP1B1, and AHR1 mRNA in chicken and quail embryos, with the highest basal expression of the CYP1As in liver, and of CYP1B1 in eye, brain, and heart. Chicken CYP1C1 mRNA levels were appreciable in eye and heart but relatively low in other organs. Basal transcript levels of the CYP1As were higher in quail than in chicken, while CYP1B1 levels were similar in the two species. 3,3',4,5,5'-Pentachlorobiphenyl induced all CYP1s in chicken; in quail a 1000-fold higher dose induced the CYP1As, but not CYP1B1. CONCLUSIONS/SIGNIFICANCE: The apparent absence of CYP1C1 in quail, and weak expression and induction of CYP1C1 in chicken suggest that CYP1Cs have diminishing roles in tetrapods; similar tissue expression suggests that such roles may be met by CYP1B1. Tissue distribution of CYP1B and CYP1C transcripts in birds resembles that previously found in zebrafish, suggesting that these genes serve similar functions in diverse vertebrates. Determining CYP1 catalytic functions in different species should indicate the evolving roles of these duplicated genes in physiological and toxicological processes.

Structural features of cytochrome P450 1A associated with the absence of EROD activity in liver of the loricariid catfish Pterygoplichthys sp.

The Amazon catfish genus Pterygoplichthys (Loricariidae, Siluriformes) is closely related to the loricariid genus Hypostomus, in which at least two species lack detectable ethoxyresorufin-O-deethylase (EROD) activity, typically catalyzed by cytochrome P450 1 (CYP1) enzymes. Pterygoplichthys sp. liver microsomes also lacked EROD, as well as activity with other substituted resorufins, but aryl hydrocarbon receptor agonists induced hepatic CYP1A mRNA and protein suggesting structural/functional differences in Pterygoplichthys CYP1s from those in other vertebrates. Comparing the sequences of CYP1As of Pterygoplichthys sp. and of two phylogenetically related siluriform species that do catalyze EROD (Ancistrus sp., Loricariidae and Corydoras sp., Callichthyidae) showed that these three proteins share amino acids at 17 positions that are not shared by any fish in a set of 24 other species. Pterygoplichthys and Ancistrus (the loricariids) have an additional 22 amino acid substitutions in common that are not shared by Corydoras or by other fish species. Pterygoplichthys has six exclusive amino acid substitutions. Molecular docking and dynamics simulations indicate that Pterygoplichthys CYP1A has a weak affinity for ER, which binds infrequently in a productive orientation, and in a less stable conformation than in CYP1As of species that catalyze EROD. ER also binds with the carbonyl moiety proximal to the heme iron. Pterygoplichthys CYP1A has amino acid substitutions that reduce the frequency of correctly oriented ER in the AS preventing the detection of EROD activity. The results indicate that loricariid CYP1As may have a peculiar substrate selectivity that differs from CYP1As of most vertebrate.

Cytochrome P4501A biomarker indication of the timeline of chronic exposure of Barrow's goldeneyes to residual Exxon Valdez oil.

We examined hepatic EROD activity, as an indicator of CYP1A induction, in Barrow's goldeneyes captured in areas oiled during the 1989 Exxon Valdez spill and those from nearby unoiled areas. We found that average EROD activity differed between areas during 2005, although the magnitude of the difference was reduced relative to a previous study from 1996/1997, and we found that areas did not differ by 2009. Similarly, we found that the proportion of individuals captured from oiled areas with elevated EROD activity (≥ 2 times unoiled average) declined from 41% in winter 1996/1997 to 10% in 2005 and 15% in 2009. This work adds to a body of literature describing the timelines over which vertebrates were exposed to residual Exxon Valdez oil and indicates that, for Barrow's goldeneyes in Prince William Sound, exposure persisted for many years with evidence of substantially reduced exposure by 2 decades after the spill.

Distinct roles of two zebrafish AHR repressors (AHRRa and AHRRb) in embryonic development and regulating the response to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The aryl hydrocarbon receptor (AHR) repressor (AHRR), an AHR-related basic helix-loop-helix/Per-AHR nuclear translocator-Sim protein, is regulated by an AHR-dependent mechanism and acts as a transcriptional repressor of AHR function. Resulting from a teleost-specific genome duplication, zebrafish have two AHRR genes (AHRRa and AHRRb), but their functions in vivo are not well understood. We used antisense morpholino oligonucleotides (MOs) in zebrafish embryos and a zebrafish liver cell line (ZF-L) to characterize the interaction of AHRRs and AHRs in normal embryonic development, AHR signaling, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. Zebrafish embryos exposed to TCDD (2 and 8nM) during early development showed strong induction of CYP1A, AHRRa, and AHRRb at 48 and 72 hours post-fertilization (hpf). An MO targeting AHR2 inhibited TCDD-induced expression of CYP1A, AHRRa, and AHRRb by 84-95% in 48 hpf embryos, demonstrating a primary role for AHR2 in mediating AHRR induction. Dual MO knockdown of both AHRRs in ZF-L cells enhanced TCDD induction of CYP1A, but not other CYP1 genes. In embryos, dual knockdown of AHRRs, or knockdown of AHRRb alone, enhanced the induction of CYP1A, CYP1B1, and CYP1C1 by TCDD and decreased the constitutive expression of Sox9b. In contrast, knockdown of AHRRa did not affect Sox9b expression or CYP1 inducibility. Embryos microinjected with each of two different MOs targeting AHRRa and exposed to dimethyl sulfoxide (DMSO) displayed developmental phenotypes resembling those typical of TCDD-exposed embryos (pericardial edema and lower jaw malformations). In contrast, no developmental phenotypes were observed in DMSO-exposed AHRRb morphants. These data demonstrate distinct roles of AHRRa and AHRRb in regulating AHR signaling in vivo and suggest that they have undergone subfunction partitioning since the teleost-specific genome duplication.

New cytochrome P450 1B1, 1C2 and 1D1 genes in the killifish Fundulus heteroclitus: Basal expression and response of five killifish CYP1s to the AHR agonist PCB126.

Knowledge of the complement of cytochrome P450 (CYP) genes is essential to understanding detoxification and bioactivation mechanisms for organic contaminants. We cloned three new CYP1 genes, CYP1B1, CYP1C2 and CYP1D1, from the killifish Fundulus heteroclitus, an important model in environmental toxicology. Expression of the new CYP1s along with previously known CYP1A and CYP1C1 was measured by qPCR in eight different organs. Organ distribution was similar for the two CYP1Cs, but otherwise patterns and extent of expression differed among the genes. The AHR agonist 3,3',4,4',5-pentachlorobiphenyl (PCB126) (31 pmol/g fish) induced expression of CYP1A and CYP1B1 in all organs examined, while CYP1C1 was induced in all organs except testis. The largest changes in response to PCB126 were induction of CYP1A in testis (approximately 700-fold) and induction of CYP1C1 in liver (approximately 500-fold). CYP1B1 in liver and gut, CYP1A in brain and CYP1C1 in gill also were induced strongly by PCB126 (> 100-fold). CYP1C1 expression levels were higher than CYP1C2 in almost all tissues and CYP1C2 was much less responsive to PCB126. In contrast to the other genes, CYP1D1 was not induced by PCB126 in any of the organs. The organ-specific response of CYP1s to PCB126 implies differential involvement in effects of halogenated aromatic hydrocarbons in different organs. The suite of inducible CYP1s could enhance the use of F. heteroclitus in assessing aquatic contamination by AHR agonists. Determining basal and induced levels of protein and the substrate specificity for all five CYP1s will be necessary to better understand their roles in chemical effects and physiology.