Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on preimplantation mouse embryos

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent environmental contaminant that can exert developmental toxicity. To investigate the stage-specific effects of TCDD on preimplantation embryos, we exposed mouse embryos to TCDD at different stages (1-, 2-, and 8-cell) and collected them at different stages of development (the 1- or 2-, 8-cell, and blastocyst stage, respectively). Semiquantitative RT-PCR revealed increased constitutive gene expression of the arylhydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt) at the 1-cell stage, decreased expression at the 2- to 8-cell stage, and increased expression again at the blastocyst stage, and addition of TCDD to media did not affect their mRNA levels. Interestingly, no cytochrome P4501A1 (CYP1A1) mRNA was detected in embryos at the 1-, 2-, and 8-cell stages after exposure to 10 nM TCDD for 12 or 24 h, whereas CYP1A1 mRNA was significantly increased at the blastocyst stage in response to TCDD, and its induction was found to be concentration-dependent on TCDD exposure from 0.01 to 10 nM for 24 h. In addition, no significant differences in development rate of preimplantation embryos, cell number of blastocyst embryos, or apoptotic indices, such as TUNEL-positive cell number or Bax/Bcl-2 expression ratios were observed at the blastocyst stage between TCDD-exposed groups and non-exposed group. These results suggest that the sensitivity to TCDD differs with the embryonic stage, which may reflect an ability of embryos to adapt to environmental stressors, such as dioxins.     

Role of aryl hydrocarbon receptor in mesencephalic circulation failure and apoptosis in zebrafish embryos exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent and potent developmental toxicant in various animals, with developing fish being the most sensitive organisms. Although the expression of aryl hydrocarbon receptor (AHR) as well as the partner molecule, AHR nuclear translocator (ARNT) in the brain has been reported, the effect of TCDD on the brain remains to be clarified in detail. Previously, we reported local circulation failure and apoptosis in dorsal midbrain caused by TCDD in developing zebrafish. In the present experiments, we investigated the effects of morpholino antisense oligos against aryl hydrocarbon receptor 2 (zfAHR2) (AHR2-MO) on toxicological endpoints caused by TCDD in developing zebrafish. AHR2-MO but not its negative homologue (4mis-AHR2-MO) improved TCDD-evoked circulation failure in mesencephalic vein and reduced the occurrence of apoptosis in dorsal midbrain, with concomitant inhibition of CYP1A induction in vascular endothelium. Injection of bovine serum albumin (BSA) into the general circulation, followed by immunohistochemistry with anti-BSA, showed that TCDD raised vascular permeability to albumin in dorsal midbrain, which was blocked by AHR2-MO and N-acetlycystein. In the absence of TCDD, development of embryos injected with AHR2-MO appeared normal at least until 60 h after fertilization. It is concluded that AHR2 activation in the vascular endothelium of the zebrafish embryo midbrain is involved in the mesencephalic circulation failure and apoptosis elicited by TCDD. This is the further evidence that vascular endothelium is the target of TCDD in relation to local circulation failure and apoptosis in dorsal midbrain.     

Characterization of CYP1A1 and CYP1A3 gene expression in rainbow trout (Oncorhynchus mykiss)

The rainbow trout CYP1A1 and CYP1A3 genes share 96% amino acid identity and have similar enzymatic activity. The expression of CYP1A1 and 1A3 genes was investigated in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rainbow trout tissues, sac fry and cell lines. Both CYP1A1 and CYP1A3 were induced by TCDD in all the tissues examined. While CYP1A3 gene was expressed constitutively at higher levels than CYP1A1 in trout intestine, preferential expression of CYP1A1 occurred in trout liver, heart, kidney and trout sac fry. In rainbow trout gonad (RTG)-2 and rainbow trout hepatoma (RTH)-149 cell lines, CYP1A1 was constitutively expressed and induced by exposure to TCDD, but CYP1A3 message was not detected, even after TCDD treatment. Quantitative analysis of CYP1A genes expression in rainbow trout liver revealed that TCDD induced CYP1A1 expression more than 50-fold and CYP1A3 RNA levels increased at least 100-fold over untreated fish. The cell- and tissue-specific expression indicates that these closely related CYP1A genes are independently regulated and that negative regulation may play a role in CYP1A3 gene expression.     

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.     

Benzo(a)pyrene decreases brain and ovarian aromatase mRNA expression in Fundulus heteroclitus

The higher molecular weight polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BaP) are typically associated with genotoxicity, however, newer evidence suggests that these compounds may also act as endocrine system disruptors. We hypothesized that altered expression of the P450 enzyme aromatase genes could be a target for reproductive or developmental dysfunction caused by BaP exposure. Aromatase is at least partially responsible for estrogen homeostasis by converting androgens into estrogens. In fish, there are two isoforms of aromatase, a predominantly ovarian form, CYP19A1, and a brain form, CYP19A2. CYP19 mRNA expression was measured following BaP exposure (0, 10, 100 microg/L waterborne for 10 or 15 days) in Fundulus adults, juveniles and embryos by in situ hybridization. The CYP19A1 expression was significantly decreased after BaP exposure in the 3-month-old Fundulus immature oocytes, but BaP did not affect CYP19A1 expression at any stage in adult oocytes. In embryo brains, BaP significantly decreased CYP19A2 compared to controls by 3.6-fold at 14 days post-fertilization. In adults, CYP19A2 expression was decreased significantly in the pituitary and hypothalamus (81% and 85% of controls, respectively). Promoter regions of Fundulus CYP19s were cloned, and putative response elements in the CYP19A1 and CYP19A2 promoters such as CRE, AhR and ERE may be involved in BaP-mediated changes in CYP19 expression. In order to compare the mechanism of BaP-mediated inhibition with that of a known aromatase inhibitor, fish were also exposed to fadrozole (20 and 100 microg/L). Fadrozole did not significantly decrease the mRNA expression in embryos or adult Fundulus. However, aromatase enzyme activity was significantly decreased in adult ovary and brain tissues. These studies provide a greater molecular understanding of the mechanisms of action of BaP and its potential to impact reproduction or development.     

Developmental and tissue-specific expression of AHR1, AHR2, and ARNT2 in dioxin-sensitive and -resistant populations of the marine fish Fundulus heteroclitus.

Fundulus heteroclitus is a well-characterized marine fish model for studying aryl hydrocarbon toxicity. The F. heteroclitus population in New Bedford Harbor (NBH), a Superfund site in southeastern Massachusetts, exhibits heritable resistance to the toxic effects of planar halogenated aromatic hydrocarbons (PHAHs), including 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs). To investigate the role of the aryl hydrocarbon receptor (AHR) signal transduction pathway in PHAH resistance, we measured the relative levels of AHR1, AHR2, and ARNT2 mRNA in whole embryos at different developmental stages and in dissected tissues of adults, comparing expression of these genes in NBH fish with fish from a reference site (Scorton Creek, MA [SC]). Expression of both AHR1 and AHR2 mRNA increased during development, achieving maximum levels prior to hatching. Maximal embryonic expression of AHR1 was delayed relative to AHR2. Whole NBH and SC embryos exhibited no discernable differences in expression of these genes. As we have previously observed, adult SC fish expressed AHR2 and ARNT2 mRNA in all tissues examined, while AHR1 was expressed predominantly in brain, heart, and gonads. In contrast, AHR1 mRNA was widely expressed in NBH fish, appearing with unusual abundance in gill, gut, kidney, liver, and spleen. This AHR1 expression pattern was not observed in the lab-reared progeny of NBH fish, demonstrating that constitutive AHR1 expression in gill, gut, kidney, liver, and spleen is not a heritable phenotype. Furthermore, widespread AHR1 expression was not induced in reference-site fish by TCDD or PCB mixtures, suggesting that aberrant AHR1 expression is not simply a normal physiological response of contaminant exposure. These results identify ubiquitous AHR1 expression as an attribute unique to feral NBH F. heteroclitus, and they represent a first step in determining the regulatory mechanisms underlying this expression pattern and its possible role in TCDD resistance.     

Testing methods for developmental neurotoxicity of environmental chemicals

Lake trout embryos exposed to [(3)H]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 F(1)9), which consistently preceded other gross lesions and mortality observed later in sac fry development (stage F(2)10). 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 E(5)8) and throughout sac fry development (stages F(1)9-F(2)10). 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 (E(5)8). To assess permeability of yolk sac vasculature to certain constituents in blood, sac fry (stage F(2)10) 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 F(2)10). 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 F(1)9 and F(2)10 of sac fry development, respectively, CYP1A mRNA levels were induced by TCDD as early as the 10-somite embryo (stage E(2)5). TCDD also caused a dose-related increase in CYP1A mRNA levels in sac fry at hatching (stage E(5)8) and throughout sac fry development (stages F(1)9-F(2)10). 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.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin toxicity in the zebrafish embryo: local circulation failure in the dorsal midbrain is associated with increased apoptosis.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on local circulation and apoptosis in the midbrain were investigated in zebrafish (Danio rerio) embryos during early development. Embryos were exposed to TCDD from 24 h post fertilization (hpf) until observation, in water maintained at 28.5 degrees C. TCDD decreased blood flow in the mesencephalic vein, the only vessel perfusing the dorsal midbrain of the embryo. At 50 hpf, blood flow was maximally reduced in this vessel and gradually returned to the control level at 60 hpf. In contrast, blood flows in the trunk and in other vessels of the head of the embryo did not significantly change until 72 hpf. Furthermore, TCDD exposure caused apoptosis in the midbrain at 60 hpf, and the TCDD dose response relationship for this effect was similar to that for reduced blood flow in the mesencephalic vein at 50 hpf. The effects of TCDD on apoptosis in the midbrain, but not on blood flow, were abolished by Z-VAD-FMK, a general caspase inhibitor. TCDD effects on both endpoints were mimicked by beta-naphthoflavone (BNF), an aryl hydrocarbon receptor (AHR) agonist, and almost abolished by concomitant exposure to TCDD and alpha-naphthoflavone (ANF), an AHR antagonist. Concomitant exposure to TCDD and either an inhibitor of cytochrome P450 (CYP) (SKF525A or miconazole) or an antioxidant (N-acetylcysteine or ascorbic acid) inhibited these effects of TCDD. The incidence of apoptosis in the midbrain was inversely related to blood flow in this brain region following these various treatments and graded TCDD exposure concentrations (r = -0.91). The same range of TCDD exposure concentrations that reduced blood flow and increased apoptosis in the midbrain greatly enhanced CYP1A mRNA expression and immunoreactivity at 50 hpf in endothelial cells of blood vessels including the mesencephalic vein and the heart, but not the brain parenchyma. Taken together, these results suggest that TCDD induces apoptosis in the midbrain of the zebrafish embryo secondary to local circulation failure, which could be related to AHR activation, induction of CYP1A, and oxidative stress.     

TCDD induces CYP1A4 and CYP1A5 in chick liver and kidney and only CYP1A4, an enzyme lacking arachidonic acid epoxygenase activity, in myocardium and vascular endothelium

The toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other Ah receptor ligands, species differences in sensitivity and the relationship of CYP1A induction to the toxicity, are poorly understood. Ah receptor ligands induce formation of CYP1A1 and 1A2 in mammals and of a different set of enzymes, CYP1A4 and 1A5, in chicks. We examined induction by TCDD of CYP1A4 and 1A5 mRNA and protein in chick embryo liver, heart, kidney, lung, intestine, bursa, spleen, thymus, brain, and muscle by in situ hybridization and immunohistochemistry and verified the histochemical findings by CYP-specific assays, 7-ethoxyresorufin deethylase for CYP1A4 and arachidonic acid epoxygenation for CYP1A5. CYP1A4 alone was extensively induced in the cardiovascular system, in cardiac myocytes, in perivascular cells having the same location as impulse-conducting Purkinje cells, and like CYP1A1, in vascular endothelium in every organ examined. Unlike mammalian CYP1A, CYP1A4 and 1A5 were both substantially induced in kidney proximal tubules as well as liver, and neither enzyme was induced in kidney glomeruli or lung or brain parenchymal cells. The findings demonstrate (a) a route for CYP1A4 to affect cardiac function, (b) that vascular endothelium is a major site of CYP1A induction across species, and (c) that CYP1A induced in heart or endothelial cells cannot affect cardiac or vascular function via generation of arachidonic acid epoxides because the CYP1A enzymes induced in those organs are not arachidonic acid epoxygenases. Further, the specificity of CYP1A induction sites and of the catalytically active enzymes induced at each site support a significant role for CYP1A induction in Ah receptor ligand toxicity and species differences in sensitivity.     

Modulation of human cytochrome P450 1B1 expression by 2,4,3',5'-tetramethoxystilbene.

We have previously shown that 2,4,3',5'-tetramethoxystilbene (TMS), a synthetic trans-stilbene analog, is one of the most potently selective inhibitors of recombinant human cytochrome P450 1B1 (CYP1B1) in vitro. In the present studies, the effects of TMS on CYP1B1 expression were investigated in human cancer cells. TMS significantly inhibited CYP1-mediated 7-ethoxyresorufin O-deethylation activity in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced MCF-7 cells or lung microsomes of Sprague-Dawley rats treated with 7,12-dimethylbenz[a]anthracene. TCDD-stimulated CYP1B1 protein and mRNA expression was significantly suppressed by TMS in a concentration-dependent manner in MCF-7, MCF-10A, and HL-60 cells. Whereas TMS down-regulated TCDD-induced CYP1B1 gene expression, the levels of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator mRNA expression were not changed by TMS treatment. In human cancer cells, TMS induced apoptotic cell death, and the cytotoxic effects of TMS were significant when the cells were incubated with TCDD. CYP1B1 was able to convert TMS to a metabolite(s) when incubated with NADPH. Metabolic activation of TMS by CYP1B1 induced by TCDD may mediate cellular toxicity of TMS in human cancer cells because the sensitivity to TMS in MCF-7 cells treated with TCDD was more significant than in HL-60 cells treated with TCDD. Taken together, our results indicate that TMS acts as a strong modulator of CYP1B1 gene expression as well as a potent selective inhibitor in vitro. The ability of TMS to induce apoptotic cell death in tumor cells, as well as CYP1B1 inhibition, may contribute to its usefulness for cancer chemoprevention.     

AhR, ARNT, and CYP1A1 mRNA quantitation in cultured human embryonic palates exposed to TCDD and comparison with mouse palate in vivo and in culture.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is developmentally toxic in many species and induces cleft palate in the C57BL/6N mouse embryo. Palatogenesis in mouse and human embryos involves homologous processes at the morphological, cellular, and molecular levels. In organ culture, mouse and human palates respond similarly to TCDD. The present study quantitates the expression of AhR, ARNT, and CYP1A1 mRNA in human embryonic palates in organ culture. Palatal tissues were exposed to 1 x 10(-10), 1 x 10(-9), or 1 x 10(-8) M TCDD or control medium and sampled at 0, 2, 4, and 6 hours for quantitative RT-PCR using a synthetic RNA internal standard. Similar measurements of CYP1A1 gene expression were collected for mouse palates cultured in this model. In human palates, AhR expression correlated with ARNT and CYP1A1 mRNA expression. TCDD induction of CYP1A1 was time- and concentration-dependent. The expression of these genes presented a uniform and continuous distribution across the group of embryos, with no subset of either high or low expressors/responders. The ratio of AhR to ARNT was approximately 4:1. AhR mRNA increased during the culture period in both treated and control subjects; however, ARNT expression was relatively constant. TCDD did not alter either AhR or ARNT expression in a consistent dose- or time-related manner. Comparison of human and mouse data showed a high correlation across species for the induction of CYP1A1. Human embryos expressed approximately 350 times less AhR mRNA than the mouse, and in earlier studies it was shown that human palates required 200 times more TCDD to produce the same effects. When the morphological, cellular, and molecular responses to TCDD between mouse and human are compared, it seems highly unlikely that human embryos could be exposed to sufficient TCDD to achieve changes in palatal differentiation that would lead to cleft palate.     

Histological analysis of acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in zebrafish

Previous studies have demonstrated that acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by injection leads to inhibition of caudal fin regeneration in zebrafish. Since the TCDD exposure in these studies is systemic, it is possible that pathology in organs other than the fin could result in inhibition of fin regeneration. Therefore, histopathology of adult zebrafish (Danio rerio) organs was characterized following abdominal cavity injection of a TCDD dose (70ng/g). The most pronounced histopathologic changes 5 days post-injection included lipidosis and hypertrophy of liver hepatocytes and hypertrophy of gill lamellae. Effects of TCDD exposure on immunolocalization of the zebrafish aryl hydrocarbon receptor nuclear translocator (ARNT2), the heterodimer partner of the aryl hydrocarbon receptor (AHR2), and an AHR regulated gene cytochrome P450 1A (CYP1A) was also determined. ARNT2 was immunolocalized to the gastrointestinal tract, gill lamellae, kidney, ventricle of the heart, caudal fin, brain and liver of zebrafish. TCDD exposure had no measurable effect on ARNT2 abundance or localization. CYP1A was immunolocalized in TCDD exposed fish as a biomarker for cells with an activated AHR pathway. CYP1A was not detected in any tissue from vehicle exposed fish. Significant TCDD-dependent induction of CYP1A was detected in the proximal tubules of the kidney, in liver hepatocytes and in the gastrointestinal tract of TCDD exposed fish. Significant but lower TCDD-dependent CYP1A expression was evident in the gill, caudal fin and ventricle of the heart. Overall, TCDD exposure in adult zebrafish leads to histopathology similar to that reported in other fish species, and it appears unlikely that the histopathology in these organs completely explains the inhibition of fin regeneration.     

Hepatic versus extrahepatic expression of CYP3A30 and CYP3A56 in adult killifish (Fundulus heteroclitus)

Members of the CYP3A subfamily represent the largest portion of CYP proteins in liver and intestine in vertebrates. The CYP3A enzymes are involved in metabolic clearance of numerous chemically diverse compounds including toxins, carcinogens, pesticides, therapeutic drugs, dietary products and hormones. Most studies of CYP3A have been performed in mammals, whereas relatively little is known of that in non-mammalian species. We have investigated CYP3A expression in the marine and estuarine killifish (Fundulus heteroclitus). We isolated a novel CYP3A cDNA sequence, denoted CYP3A56, from killifish intestine. The CYP3A56 sequence shared 98% nucleotide and amino acid sequence identity with CYP3A30, previously isolated from killifish liver. We hypothesize that a recent gene duplication event has occurred within the killifish CYP3A subfamily. The CYP3A30 and CYP3A56 genes were co-expressed in liver, intestine, gill, kidney, spleen, brain and ovary in several individuals. The hepatic versus extrahepatic CYP3A30/56 mRNA expression was analyzed in adult killifish of both sexes, using conventional as well as real-time semi-quantitative RT-PCR. Tissues expressing CYP3A30/56 mRNA were in a descending order of magnitude: liver>intestine>gill>spleen>kidney>brain. Furthermore, inter-individual differences (up to 18%) in CYP3A30/56 mRNA expression were evident in killifish, in particular in extrahepatic organs. For comparison, CYP3A protein expression levels were determined using polyclonal antibodies (PAb) against rainbow trout (Oncorhynchus mykiss) CYP3A. Sexually dimorphic expression of hepatic and extrahepatic CYP3A30/56 mRNA and CYP3A proteins was observed in killifish. For example, males displayed up to 2.5-fold higher CYP3A protein expression compared with females. In agreement with CYP3A30/56 mRNA analysis, highest CYP3A protein levels were observed in liver and intestine. Low CYP3A protein levels were seen in gill, kidney and spleen. Cellular localization of CYP3A protein expression was investigated using immunohistochemistry analysis and PAb against rainbow trout CYP3A. Strong CYP3A protein staining was seen in intestinal enterocytes, in gill filaments and in renal tubular epithelial cells. Moderate CYP3A staining was seen in hepatocytes in the liver, whereas mild staining was observed in hematopoietic cells in the spleen and in follicles in the ovary. Thus, similar to mammals, CYP3A expression in fish is prominent in the digestive- and respiratory tracts and may be important for the first-pass metabolism of xenobiotics. Moreover, CYP3A expression also is evident in brain and ovary in killifish, which suggests a role for CYP3A enzymes in biotransformation of xenobiotics and fine-tuning levels of steroid hormones in situ in extrahepatic organs.     

CYP1C1 messenger RNA expression is inducible by benzo[a]pyrene in Fundulus heteroclitus embryos and adults.

CYP1C is the newest member of the CYP1 family of P450s; however, its physiological significance, inducers, and metabolic functions are unknown. Two full-length alleles of Fundulus heteroclitus CYP1C1 complementary DNA were cloned. The 529 amino acid protein shared the highest amino acid identity with Stenotomus chrysops CYP1C1 (81%). To investigate whether the carcinogen benzo[a]pyrene (BaP) was a CYP1C1 inducer, we used real-time PCR to quantitatively measure tissue- and sex-specific expression of both CYP1C1 and CYP1A messenger RNAs (mRNAs) in BaP-exposed adult fish. CYP1C1 mRNA expression was constitutively higher than CYP1A in brain, spleen, eye, and gonad, while CYP1A was higher in gastrointestinal tract (GI), heart, gill, and liver. Kidney had equal but high expression of both CYP1s. There were sex differences in constitutive CYP1 expression in the GI, liver, gill, and eye. BaP exposure caused induction of CYP1C1 expression in female and male heart (31- and 17-fold), gill (seven- and four-fold), and liver (six- and five-fold), respectively. Embryo CYP1 expression was constitutively highest at 2 weeks posthatch, and whole embryos expressed 3- to 15-fold more CYP1C1 mRNA compared to CYP1A. BaP, 10 microg/l for 10 days, caused induction of both genes at 120 and 240 h postfertilization. Our results suggest that teleost CYP1C, in addition to CYP1A, is inducible by BaP, has a broad tissue distribution, and should be further investigated for its role in carcinogen bioactivation.     

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 β-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.     

Cellular distribution and induction of CYP1A following exposure of gilthead seabream, Sparus aurata, to waterborne and dietary benzo(a)pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin: an immunohistochemical approach

The present study aimed to investigate cellular expression of cytochrome P4501A (CYP1A) protein in the seabream, Sparus aurata, exposed to one of two CYP1A-inducing contaminants, benzo(a)pyrene (B(a)P) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Male adult fish were exposed to several concentrations of TCDD or B(a)P either via water or via food. Fish were sampled after 0, 5, 10, 15 or 20 days of treatment and the time- and concentration-dependent induction of CYP1A protein in cells and tissues was studied using immunohistochemistry. A general site of CYP1A induction was the vascular endothelium. Aqueous exposures resulted in elevation of CYP1A immunoreactivity in gill pillar cells, heart endothelium, renal tubular epithelium, hepatocytes, and gut mucosal epithelium. In contrast, dietary exposure resulted in strong CYP1A immunostaining in gut epithelium but in only mild to moderate staining elsewhere. Both B(a)P and TCDD induced CYP1A in similar cellular response patterns in most organs examined, although TCDD generally led to higher staining intensity and frequency (i.e. the number of CYP1A-positive cells within an organ), an effect that is likely to be related to compound-specific differences in induction potency, metabolism and penetration. Contaminant-specific staining patterns were observed in the gills, where TCDD exposure evoked CYP1A immunostaining in the endothelial pillar cells, while B(a)P induced CYP1A staining in the branchial epithelial cells. This work points to the importance of immunohistochemical identification of cell-specific CYP1A responses in assessing the toxicology of CYP1A-inducing xenobiotics.