The chlorinated AHR ligand 3,3',4,4',5-pentachlorobiphenyl (PCB126) promotes reactive oxygen species (ROS) production during embryonic development in the killifish (Fundulus heteroclitus)

Exposure to dioxin-like chemicals that activate the aryl hydrocarbon receptor (AHR) can result in increased cellular and tissue production of reactive oxygen species (ROS). Little is known of these effects during early fish development. We used the fish model, Fundulus heteroclitus, to determine if the AHR ligand and pro-oxidant 3,3',4,4',5-pentachlorobiphenyl (PCB126) can increase ROS production during killifish development, and to test a novel method for measuring ROS non-invasively in a living organism. The superoxide-sensitive fluorescent dye, dihydroethidium (DHE), was used to detect in ovo ROS production microscopically in developing killifish exposed to PCB126 or vehicle. Both in ovo CYP1A activity (ethoxyresorufin-o-deethylase, EROD) and in ovo ROS were induced by PCB126. In ovo CYP1A activity was inducible by PCB126 concentrations as low as 0.003 nM, with maximal induction occurring at 0.3 nM PCB126. These PCB126 concentrations also significantly increased in ovo ROS production in embryonic liver, ROS being detectable as early as 5 days post-fertilization. These data demonstrate that the pro-oxidant and CYP1A inducer, PCB126, increases both CYP1A activity and ROS production in developing killifish embryos. The superoxide detection assay (SoDA) described in this paper provides a semi-quantitative, easily measured, early indicator of altered ROS production that can be used in conjunction with simultaneous in ovo measurements of CYP1A activity and embryo development to explore functional relationships among biochemical, physiological and developmental responses to AHR ligands.     

New CYP1 genes in the frog Xenopus (Silurana) tropicalis: induction patterns and effects of AHR agonists during development

The Xenopus tropicalis genome shows a single gene in each of the four cytochrome P450 1 (CYP1) subfamilies that occur in vertebrates, designated as CYP1A, CYP1B1, CYP1C1, and CYP1D1. We cloned the cDNAs of these genes and examined their expression in untreated tadpoles and in tadpoles exposed to waterborne aryl hydrocarbon receptor agonists, 3,3′,4,4′,5-pentachlorobiphenyl (PCB126), β-naphthoflavone (βNF), or indigo. We also examined the effects of PCB126 on expression of genes involved in stress response, cell proliferation, thyroid homeostasis, and prostaglandin synthesis. PCB126 induced CYP1A, CYP1B1, and CYP1C1 but had little effect on CYP1D1 (77-, 1.7-, 4.6- and 1.4-fold induction versus the control, respectively). βNF induced CYP1A and CYP1C1 (26- and 2.5-fold), while, under conditions used, indigo tended to induce only CYP1A (1.9-fold). The extent of CYP1 induction by PCB126 and βNF was positively correlated to the number of putative dioxin response elements 0-20 kb upstream of the start codons. No morphological effect was observed in tadpoles exposed to 1 nM-10 μM PCB126 at two days post-fertilization (dpf) and screened 20 days later. However, in 14-dpf tadpoles a slight up-regulation of the genes for PCNA, transthyretin, HSC70, Cu-Zn SOD, and Cox-2 was observed two days after exposure to 1 μM PCB126. This study of the full suite of CYP1 genes in an amphibian species reveals gene- and AHR agonist-specific differences in response, as well as a much lower sensitivity to CYP1 induction and short-term toxicity by PCB126 compared with in fish larvae. The single genes in each CYP1 subfamily may make X. tropicalis a useful model for mechanistic studies of CYP1 functions.     

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.     

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.     

Effects of 3,3',4,4',5-pentachlorobiphenyl, a coplanar polychlorinated biphenyl congener, on cultured neonatal mouse testis.

3,3',4,4',5-Pentachlorobiphenyl (PCB126), a congener with a planar configuration, has been established to have relatively strong toxicities similar to those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via aryl hydrocarbon receptors. We investigated the effects of this coplanar PCB on mammalian early spermatogenesis and steroidogenesis in a mouse neonatal testicular organ culture system. Testes collected from newborn mice were subjected to organ culture in medium containing 0, 10, 100 or 1000 nM PCB126. Histochemical analysis revealed that the BrdU-labeling indices of both spermatogenic cells and Sertoli cells were unchanged in all testis specimens exposed to the coplanar PCB. CYP1A1 and steroidogenic enzymes (P450scc, P450c17, 3beta-HSD and 17beta-HSD) mRNA levels were determined by semiquantitative RT-PCR. The CYP1A1 mRNA level in cultured testis was significantly increased by PCB126 in a dose-dependent manner. Although mRNA levels of 3beta-HSD and 17beta-HSD were unchanged, the P450scc mRNA level was significantly down-regulated by PCB126 in a dose-dependent manner. In contrast, the P450c17 mRNA level was significantly higher in 1000 nM PCB126-exposed testis than in control testis. These results suggest that the coplanar PCB does not alter the proliferative activity of spermatogenic cells and Sertoli cells in neonatal testis, but that it directly affects the expression of steroidogenic enzyme genes.     

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.     

A comparative study on the effects of 2,3,7,8,-tetrachlorodibenzo-p-dioxin polychlorinated biphenyl126 and estrogen in human bronchial epithelial cells

Epidemiological studies on 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) exposure indicated high incidences of pulmonary dysfunctions and lung cancer. Animal studies also demonstrated lung cancer development in female, but not in male, rats exposed to TCDD. Such effects, however, have not been reported in polychlorinated biphenyls (PCB) exposure. In our present study, we have investigated the effects of TCDD and PCB126, with or without cotreatment with 17 beta-estradiol (E2), on a human bronchial epithelial cell line BEAS-2B. We found that treatment with either TCDD or PCB126 alone reduced cell numbers as well as thymidine incorporation. Cell death, however, was only detected in PCB126-, but not TCDD-, treated cultures. The TCDD-induced cell reduction, therefore, could not be contributed to cell death. Meanwhile, because TCDD- and PCB126-enhanced CYP1A1 and CYP1B1 expressions were significantly reduced by the AhR antagonist and CYP1 inhibitor alpha-naphthoflavone (ANF), this indicated that the effects of TCDD and PCB126 were AhR and cytochrome p450 1 dependent. We also found that while E2 itself did not alter CYP1A1 and CYP1B1 expressions, cotreatment of E2 with TCDD or PCB126 would significantly enhance TCDD-, but not PCB126-, induced toxicity. We further demonstrated that in the presence of E2, 1 nM TCDD increased the production of E2 metabolites, 2-methoxyestradiol (2-MeOE2) and 4-methoxyestradiol (4-MeOE2). PCB126, however, only increased 2-MeOE2 formation without significant induction of 4-MeOE2. We believe that these metabolites, especially 4-MeOE2, interacted with TCDD to further suppress cell growth. Our data provided the first demonstration on the enhancement of TCDD-induced toxicity in human lung cells via interaction with estrogen.     

Relative contributions of affinity and intrinsic efficacy to aryl hydrocarbon receptor ligand potency

Models of receptor action are valuable for describing properties of ligand-receptor interactions and thereby contribute to mechanism-based risk assessment of receptor-mediated toxic effects. In order to build such a model for the aryl hydrocarbon receptor (AHR), binding affinities and CYP1A induction potencies were measured in PLHC-1 cells and were used to determine intrinsic efficacies for 10 halogenated aromatic hydrocarbons (HAH): 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7, 8-tetrachlorodibenzofuran (TCDF), and eight polychlorinated biphenyls (PCB). TCDD, TCDF, and non-ortho-substituted PCBs 77, 81, 126, and 169 behaved as full agonists and displayed high-intrinsic efficacy. In contrast, the mono- and di-ortho-substituted PCBs bound to the AHR but displayed lower or no intrinsic efficacy. PCB 156 was a full agonist, but with an intrinsic efficacy 10- to 50-fold lower than non-ortho-substituted PCBs. PCB 118 was a very weak partial agonist. PCBs 105 and 128 were shown to be competitive antagonists in this system. The model was then used to predict CYP1A induction by binary mixtures. These predictions were tested with binary mixtures of PCB 126, 128, or 156 with TCDD. Both PCB 156 (a low-intrinsic efficacy agonist) and PCB 128 (a competitive antagonist) inhibited the response to TCDD, while the response to TCDD and PCB126 was additive. These data support the following conclusions: 1) only 1-2% of the receptors in the cell need be occupied to achieve 50% of maximal CYP1A induction by one of the high-intrinsic efficacy agonists, demonstrating the existence of "spare" receptors in this system; 2) the insensitivity of fish to ortho-substituted PCBs is due to both reduced affinity and reduced intrinsic efficacy compared to non-ortho-substituted PCBs; 3) PCB congeners exhibit distinct structure-affinity and structure-efficacy relationships. Separation of AHR ligand action into the properties of affinity and intrinsic efficacy allows for improved prediction of the behavior of complex mixtures of ligands, as well as mechanistic comparisons across species and toxic endpoints.     

Developmental toxicity,EROD, and CYP1A mRNA expression in zebrafish embryos exposed to dioxin‐like PCB126

Dioxin‐like PCB126 is a persistent organic pollutant that causes a range of syndromes including developmental toxicity. Dioxins have a high affinity for aryl hydrocarbon receptor (AhR) and induce cytochrome P4501A (CYP1A). However, the role of CYP1A activity in developmental toxicity is less clear. To better understand dioxin induced developmental toxicity, we exposed zebrafish (Danio rerio) embryos to PCB126 at concentrations of 0, 16, 32, 64, and 128 μg L^?1 from 3‐h post‐fertilization (hpf) to 168 hpf. The embryonic survival rate decreased at 144 and 168 hpf. The fry at 96 hpf displayed gross developmental malformations, including pericardial and yolk sac edema, spinal curvature, abnormal lower jaw growth, and non‐inflated swim bladder. The pericardial and yolk sac edema rate significantly increased and the heart rate declined from 96 hpf compared with the controls. PCB126 did not alter the hatching rate. To elucidate the mechanism of PCB126‐induced developmental toxicity, we conducted ethoxyresorufin‐O‐deethylase (EROD) in vivo assay to determine CYP1A enzyme activity, and real‐time PCR to study the induction of CYP1A mRNA gene expression in embryo/larval zebrafish at 24, 72, 96, and 132 hpf. In vivo EROD activity was induced by PCB126 at 16 μg L^?1 concentration as early as 72 hpf but significant increases were observed only in zebrafish exposed to 64 and 128 μg L^?1 doses (p < 0.005) at 72, 96, and 132 hpf. Induction of CYP1A mRNA expression was significantly upregulated in zebrafish exposed to 32 and 64 μg L^?1 at 24, 72, 96, and 132 hpf. Overall, the severe pericardial and yolk sac edema and reduced heart rate suggest that heart defects are a sensitive endpoint, and the general trend of dose‐dependent increase in EROD activity and induction of CYP1A mRNA gene expression provide evidence that the developmental toxicity of PCB126 to zebrafish embryos is mediated by activation of AhR. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 201–210, 2016.     

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.     

Effect of TCDD on mRNA expression of genes encoding bHLH/PAS proteins in rat hypothalamus

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) brings about a wide variety of toxic and biochemical effects via an AH receptor (AHR)-mediated signalling pathway. Wasting syndrome and acute lethality are TCDD-induced endpoints showing a striking sensitivity difference between two rat strains, TCDD-sensitive Long-Evans (Turku/AB) (L-E) and TCDD-resistant Han/Wistar (Kuopio) (H/W). These rat strains were used to study hypothalamic effects of TCDD on expression of genes encoding AHR-regulated bHLH/PAS proteins potentially involved in molecular pathogenesis of the wasting syndrome. In addition, two well-established target genes of TCDD, CYP1A1 and CYP1A2 were also examined. Quantitative RT-PCR was used to measure mRNA levels in hypothalamus, which is a major center of food intake and body weight regulation. At both 6 and 96 h after a single dose of 50 microg/kg TCDD, significant elevations were found in mRNA levels of AHR repressor (AHRR), CYP1A1 and CYP1A2, but not those of AHR, ARNT or ARNT2. Likewise, TCDD (100 microg/kg) did not alter the expression of SIM1, implicated in the suppressive impact of TCDD on food intake, nor that of PER2, involved in regulation of circadian rhythms. Differences between H/W and L-E rats appeared in constitutive levels of AHR and ARNT and in TCDD-induced levels of CYP1A2, AHRR, AHR and ARNT, which all were about two- to four-fold lower in H/W rats. Thus, although the changes found do not account for the wasting syndrome, expression of all principal genes of the AHR-signalling pathway in rat hypothalamus make it a candidate target for TCDD.     

Effects of natural and synthetic estrogens and various environmental contaminants on vitellogenesis in fish primary hepatocytes: comparison of bream (Abramis brama) and carp (Cyprinus carpio).

Interaction of environmental estrogens with the estrogen receptor (ER) has been shown in various fish species. Our objective was to compare the sensitivity of bream (Abramis brama) to (xeno-)estrogens with that of the carp (Cyprinus carpio), by measuring the effects of 17beta-estradiol (E2), estrone (E1), ethynylestradiol (EE2), bisphenol A (BPA), nonylphenol (NP), methoxychlor (MXCL), and halogenated aromatic hydrocarbons (HAHs) such as polychlorinated biphenyls (PCB126, PCB118), 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD), and 2,3,4,7,8-pentachlorodibenzofuran (PCDF) on vitellogenesis in primary hepatocytes. Comparing the EC50 values in bream hepatocytes: EE2 (0.1-0.2 microM) < E1 (0.6-0.2 microM) < E2 (1.9 microM) with those of carp hepatocytes EE2 (0.03-0.06 microM) < E2 (0.3 microM) approximately E1 (0.2-0.3 microM) we found differences in sensitivity and ranking of the estrogenic potency of E2 and E1, indicating interspecies differences. Exposure to BPA, NP, MXCL, and HAHs did not or only weakly induce vitellogenesis. Bream hepatocytes coexposed to E2 and TCDD, PCB126 or PCDF showed a concentration-dependent inhibition of E2-induced vitellogenesis. IC50 (concentration of a compound that elicits 50% inhibition of E2-induced vitellogenesis) values determined in bream were: TCDD (0.02-0.09 nM) < PCB126 (0.35-0.1 nM) < PCDF (2.0-0.1) and in carp were: TCDD (0.01 nM) < PCB126 (0.4 nM). PCB118 showed no (anti-)estrogenic response. IC50 values and benchmark-concentration for TCDD and PCB126 in bream and carp hepatocytes were in the same range, indicating similar sensitivity to these compounds. Due to their anti-estrogenic capacity with benchmark-concentrations in the pM range TCDD, PCDF, and PCB126 may form a potential hazard for the reproductive success of fish species by inhibition of vitellogenesis.