An aryl hydrocarbon receptor odyssey to the shores of toxicology: the Deichmann Lecture, International Congress of Toxicology-XI.

The science of toxicology is devoted, in large part, to understanding mechanisms of toxicity so that we can more accurately assess the risk posed by exposure to xenobiotic agents and, perhaps, intervene in the toxicologic process to mitigate harm. Dioxin-like chemicals continue to be of great concern as environmental toxicants. About 30 years ago the aryl hydrocarbon receptor (AHR) was discovered as a specific binding site for 2,3,7,8-tetrachlorodibenzo-p-dioxin. This giant step led to our current view that essentially all toxic effects of dioxins are AHR-mediated. The AHR serves as the archetype for understanding toxicity mediated by other soluble receptors. The fact that toxicity is receptor-mediated has important implications, especially for dose-response relationships. In laboratory animals genetic differences in AHR gene structure lead to profound differences in responsiveness to dioxin-like chemicals. Humans, however, exhibit relatively few AHR polymorphisms and these seem to exert only modest effects on downstream events. Dioxin toxicity is fundamentally due to AHR-mediated dysregulation of gene expression. Our current challenging goal is to determine which dysregulated genes underlie specific forms of dioxin toxicity. Mapping AHR-mediated gene expression in a variety of biological systems may help explain why dramatic differences in susceptibility to dioxin toxicity exist among laboratory species and why humans appear to be relatively resistant to adverse effects of dioxins.     

Effects of anthocyanins on the AhR–CYP1A1 signaling pathway in human hepatocytes and human cancer cell lines

Anthocyanins are plant pigments occurring in flowers and berry fruits. Since a phenomenon of food–drug interactions is increasingly emerging, we examined the effects of 21 major anthocyanins and the extracts from 3 food supplements containing anthocyanins on the aryl hydrocarbon receptor (AhR)–cytochrome P450 CYP1A1 signaling pathway in human hepatocytes and human hepatic HepG2 and intestinal LS174T cancer cells. Pelargonidin-3-O-rutinoside (PEL-2) and cyanidin-3,5-O-diglucoside (CYA-3) dose-dependently activated AhR, as revealed by gene reporter assay. PEL-2 and CYA-3 induced CYP1A1 mRNA but not protein in HepG2 and LS174T cells. Neither compounds induced CYP1A1 mRNA and protein in four different primary human hepatocytes cultures. The effects of PEL-2 and CYA-3 on AhR occurred by ligand-dependent and ligand-independent mechanisms, respectively, as demonstrated by ligand binding assay. In a direct enzyme inhibition assay, none of the antocyanins tested inhibited the CYP1A1 marker activity to less than 50% even at 100 μM concentration. PEL-2 and CYA-3 at 100 μM inhibited CYP1A1 to 79% and 65%, respectively. In conclusion, with exception of PEL-2 and CYA-3, there were no effects of 19 major anthocyanins and 3 food supplements containing anthocyanins on AhR–CYP1A1 signaling, implying zero potential of these compounds for food–drug interactions with respect to AhR–CYP1A1 pathway.     

2,3,7,8-tetrachlorodibenzo-p-dioxin inhibits zebrafish caudal fin regeneration.

Adult zebrafish completely regenerate their caudal fins following partial amputation. Fin regrowth can easily be monitored in vivo and regenerating tissues can be used to study this dynamic developmental process. In this study we determined that fin regeneration is significantly affected by exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Zebrafish caudal fins were partially amputated, and the fish received intraperitoneal (ip) injection of 2.8, 14, or 70 ng/g weight TCDD or vehicle control. By 7 days postamputation, fish exposed to the highest concentration of TCDD regenerated 15% of their fin compared to 65% regrowth in control fish. To determine if this effect was stage specific, zebrafish were exposed to 70 ng/g TCDD on 1, 2, 3, or 4 days postamputation. Fin regeneration was significantly inhibited at all time points following TCDD exposure. TCDD exposure also induced hyperpigmentation in de novo tissue. Zebrafish were dosed with BrdU, following fin amputation and TCDD exposure, to study changes in cell proliferation. By 4 days postamputation, cell proliferation rates were significantly lower in TCDD-exposed fish. TCDD toxicity is mediated through the aryl hydrocarbon receptor (AHR), and RT-PCR experiments confirmed AHR2, ARNT2b, and TCDD-dependent CYP1A expression in the regenerating tissue. These results demonstrate that zebrafish caudal fin regeneration is a unique model to investigate molecular mechanism(s) of TCDD toxicity.     

Coplanar polychlorinated biphenyls activate the aryl hydrocarbon receptor in developing tissues of two TCDD-responsive lacZ mouse lines.

In utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can have an immediate impact on developmental processes that then lead to long-term deficits in function. To define the specific tissues affected by TCDD during development, we developed a lacZ-reporter gene mouse model driven by activation of the aryl hydrocarbon receptor (AhR). Exposure to TCDD on gestational day (GD) 14 results in strong activation of the lacZ transgene in numerous tissues including fore and hind paws, ear, and genital tubercle. Experiments were conducted to examine the ability of alternative AhR ligands to activate our model system. The coplanar polychlorinated biphenyl congeners 3,4,5,3',4'-pentachlorobiphenyl (PCB126) and 3,4,3',4'-tetrachlorobiphenyl (PCB77) both induced staining in fetal tissues identical to that observed following TCDD exposure. Exposure of fetuses to the PCB mixture Aroclor 1254 and the non-coplanar congener 2,3,6,2',5'-pentachlorobiphenyl (PCB95) did not result in any activation of the lacZ transgene. In addition to the testing of alternative ligands, another line of reporter mice was generated to determine the potential influence of the site of insertion of the lacZ transgene on the reported observations. Both TCDD and the coplanar PCBs induced a similar pattern of staining in the new line as compared to that observed in the original lacZ reporter mouse line. The ability of AhR ligands, other than TCDD, to activate the AhR-mediated transgene, in combination with the insertion-site independence of the response, strengthens the data previously derived from this model and increases the utility of this system for investigations examining AhR-mediated events during development.     

Inhibitory Effects of Vitamin A on TCDD-induced Cytochrome P-450 1A1 Enzyme Activity and Expression

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an extremely potentenvironmental contaminant that produces a wide range of adversebiological effects, including the induction of cytochrome P4501A1(CYP1A1) that may enhance the toxic effects of TCDD. Severalstudies indicated that concurrent supplementation of vitaminA could reduce the toxicity, and potentially inhibit CYP1A1activity (measured as ethoxyresorufin-O-deethylase [EROD] activity).In the present study, we investigated the in vivo effects ofvitamin A on EROD activities and the expression of CYP1A1 inthe liver of TCDD-treated mice. In Experiment I, the mice weregiven a single oral dose of 40 µg TCDD/kg body weightwith or without the continuous administration of 2500 IU vitaminA/kg body weight/day, and were killed on day 1, 3, 7, 14, or28. In Experiment II, the mice were given daily an oral doseof 0.1 µg TCDD/kg body weight with or without supplementof 2000 IU vitamin A/kg body weight, and were killed on day14, 28, or 42. In both experiments, TCDD caused liver damageand increase in relative liver weights, augmented the EROD activitiesand CYP1A1 expression, and increased the aryl hydrocarbon receptor(AhR) mRNA expression, but did not alter the AhR nuclear translocator(ARNT) mRNA expression. CYP1A1 mRNA expression and AhR mRNAexpression showed a similar time course. The liver damage inTCDD + vitamin A–treated mice was less severe than thatin TCDD-treated mice. EROD activities, CYP1A1 expression, andAhR mRNA expression in vitamin A + TCDD–treated mice werelower than those in TCDD-treated mice, indicating that supplementationof vitamin A might attenuate the liver damage caused by TCDD.     

Aryl hydrocarbon receptor‐mediated effects of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin on glucose‐stimulated insulin secretion in mice

Epidemiological and laboratory studies suggested that exposure to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) affects glucose homeostasis and increases the incidence of type 2 diabetes mellitus. To evaluate the effects of TCDD on insulin secretion from islets of Langerhans (islets), we designed in vivo, ex vivo and in vitro experiments. For the in vivo experiment, male C57BL/6J and aryl hydrocarbon receptor (AhR)‐null mice were injected intraperitoneally with TCDD (10 μg kg^?1 b.w.), fasted for 12 h and administered glucose 24 h post‐administration. TCDD exposure significantly decreased the plasma insulin concentration at 60 and 120 min after a glucose challenge in C57BL/6J mice but not in AhR‐null mice. In contrast, the plasma glucose concentration was not changed by TCDD exposure in both C57BL/6J and AhR‐null mice. For the ex vivo experiment, we isolated islets 24 h after TCDD administration and determined the glucose‐stimulated insulin secretion from the islets. The insulin secretion level was found to be significantly decreased by TCDD exposure at 60 min after glucose treatment. For the in vitro experiment, islets harvested from untreated C57BL/6J mice were exposed to 0.1, 1, 10 or 100 nM TCDD for 24 h and analyzed for glucose‐stimulated insulin secretion. Insulin secretion from the islets remained unchanged regardless of TCDD dose. In conclusion, TCDD exposure impaired the second phase of glucose‐stimulated secretion of insulin from the islets via the AhR signaling pathway. Copyright © 2009 John Wiley & Sons, Ltd.     

Response of the incisor tooth to 2,3,7,8-tetrachlorodibenzo-p-dioxin in a dioxin-resistant and a dioxin-sensitive rat strain.

Dioxins are ubiquitous environmental pollutants that afflict developing teeth. To find out if the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the continuously erupting rat incisor is associated with the sensitivity to TCDD acute lethality and to see the histological basis for any macroscopic findings, we exposed 25 resistant Han/Wistar (Kuopio; H/W) and 20 sensitive Long-Evans (Turku/AB; L-E) female rats to total doses of 0.17, 1.7, 17, and 170 (only H/W rats) micro g/kg TCDD. Each dose group comprised five animals. The treatment was started when the rats were 10 weeks old and continued for 20 weeks. The exposure time covered two life cycles of the incisor. Stereomicroscopic examination of the dissected mandibles showed color defects and pulpal perforation of the lower incisors at 17 and 170 micro g/kg TCDD. Tissue sections revealed odontoblastic and pulpal cell death and the consequent arrest of dentin formation at the incisal tooth end at the same doses. H/W rat incisors were affected closer to the germinative tooth end at 170 than at 17 micro g/kg TCDD, resulting in a larger perforation. In accordance with the enamel discoloration, the postsecretory enamel organ underwent, albeit inconsistently, precocious squamous metaplasia with pronounced proliferation. Thus, both the mesenchymal and, to a lesser extent, epithelial elements of the forming tooth were affected dose-dependently at relatively high doses of TCDD. Similar responses in both strains implied that the impaired formation of the incisor tooth, at least of its mesenchymal elements, is not associated with the differential resistance of H/W and L-E rats to TCDD acute lethality.     

Application of the ethoxyresorufin-O-deethylase (EROD) assay to mixtures of halogenated aromatic compounds

The ethoxyresorufin-O-deethylase (EROD) assay monitors the induction of the xenobiotic-metabolizing enzyme cytochrome P-450 (CYP) 1A1 and is a widely used biomarker for exposure of wildlife to substances that bind the aryl hydrocarbon (Ah) receptor. In this work the induction of EROD activity by single compounds and binary mixtures in primary rat hepatocytes was compared with the predictions of a kinetic model involving mixtures of inducers. The inducing agents were also examined for their ability to activate the Ah receptor to its DNA-binding form and for their ability to act as competitive inhibitors for CYP 1A1. Xenobiotics that failed to activate the Ah receptor did not induce EROD activity. Competitive inhibition for CYP 1A1 between the xenobiotic and 7-ethoxyresorufin caused EROD activity to fall at high xenobiotic concentrations. Competition for a limited number of Ah receptor sites depressed the EROD activity of a strong inducer such as 2,3,7,8-tetrachlorodibenzo-p-dioxin at high concentrations of a weak inducer. Application of the kinetic model to the example of a mixture of low concentrations of dibenzo-p-dioxins and much higher concentrations of polychlorinated biphenyls indicated that EROD assays often seriously underestimate the true potency of an environmental sample. Hence the EROD assay cannot be used for determining dioxin equivalent concentrations using the toxic equivalence factor approach.     

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.     

Toxicogenomic profile of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the murine fetal heart: modulation of cell cycle and extracellular matrix genes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and similar environmental contaminants have been demonstrated to be potent cardiovascular teratogens in developing piscine and avian species. In the present study, we investigated the effects of TCDD on gene expression during murine cardiovascular development. C57Bl6N pregnant mice were dosed with 1.5, 3.0, or 6.0 microg TCDD/kg on gestational day (GD) 14.5, and microarray analysis was used to characterize the global changes in fetal cardiac gene expression on GD 17.5. TCDD significantly altered expression of a number of genes involved in xenobiotic metabolism, cardiac homeostasis, extracellular matrix production/remodeling, and cell cycle regulation. Interestingly, while the AhR-responsive genes Cyp1A1, Cyp1B1, Ugt1a6, and Ahrr, were all induced by TCDD in the fetal murine heart, other AhR-responsive genes, Cyp1a2, Nqo1, and Gsta1, were not. Quantitative real-time polymerase chain reactions confirmed the changes in expression of several G1/S-type cyclins and extracellular matrix-related genes. These results demonstrate the global changes in cardiac gene expression that result from TCDD exposure of the fetal murine heart and implicate genes involved in cell cycle and extracellular matrix regulation in TCDD-induced cardiac teratogenicity and functional deficits.     

In utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure: effects on fetal and adult cardiac gene expression and adult cardiac and renal morphology.

The mouse heart is a target of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during fetal development, and microarray analysis demonstrates significant changes in expression of cardiac genes involved in extracellular matrix (ECM) remodeling. We tested the hypothesis that developmental TCDD exposure would disrupt cardiac ECM expression and be associated with changes in cardiac morphology in adulthood. In one study, time-pregnant C57BL/6 mice were dosed with corn oil or 1.5, 3.0, or 6.0 microg TCDD/kg on gestation day (GD) 14.5 and sacrificed on GD 17.5, when changes in fetal cardiac mRNA expression were analyzed using quantitative PCR. TCDD induced mRNA expression of genes associated with ECM remodeling (matrix metalloproteinase 9 and 13, preproendothelin-1 [preproET-1]), cardiac hypertrophy (atrial natriuretic peptide, beta-myosin heavy chain, osteopontin), and aryl hydrocarbon receptor (AHR) activation (cytochrome P4501A1, AHR repressor). Further, all TCDD-induced changes required the AHR since gene expression was not altered in AHR knockout fetuses. In a second study, time-pregnant mice were treated with corn oil or 6.0 microg TCDD/kg on GD 14.5, and male offspring were assessed for changes in cardiac gene expression and cardiac and renal morphology at 3 months. All TCDD-induced changes in cardiac gene expression observed fetally, except for preproET-1, remained induced in the hearts of adult male offspring. Adult male offspring of TCDD-exposed dams also displayed cardiac hypertrophy, decreased plasma volume, and mild hydronephrosis. These results demonstrate that in utero and lactational TCDD exposures alter cardiac gene expression and cardiac and renal morphology in adulthood, which may increase the susceptibility to cardiovascular dysfunction.     

Heart malformation is an early response to TCDD in embryonic zebrafish.

The zebrafish (Danio rerio) has become an attractive vertebrate model for studying developmental processes, and is emerging as a model system for studying the mechanisms by which toxic compounds perturb normal development. When exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) shortly after fertilization, zebrafish embryos exhibit pericardial edema and reduced blood flow by 72 h post fertilization (hpf). To better understand the progression of dioxin toxicity in zebrafish, we have examined the effects of TCDD on heart development. At 72 hpf, TCDD-treated embryos exhibited altered looping, with the atria positioned distinctly posterior to the ventricles, contrary to the looping of control hearts, where the two chambers lied side by side. Moreover, the ventricles in dioxin-exposed hearts became more compact, and the atria elongated in comparison to controls. These defects are not secondary to pericardial edema because they were observed when edema formation was suppressed with osmotic support. In addition to morphological changes, TCDD produced functional deficits in the developing hearts, including blood regurgitation and a striking ventricular standstill that became prevalent by 120 hpf. We also assessed the effect of TCDD on the heart size using stereological measurements, which demonstrated significant reduction in heart tissue volume at 72 hpf. Perhaps our most significant finding was a decrease in the total number of cardiomyocytes in TCDD-exposed embryos by 48 hpf, one day prior to observable effects on peripheral blood flow. We conclude that the developing heart is an important target for TCDD in zebrafish.     

Aryl hydrocarbon receptor knockout rats are insensitive to the pathological effects of repeated oral exposure to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin

Sustained activation of the aryl hydrocarbon receptor (AHR) is believed to be the initial key event in AHR receptor‐mediated tumorigenesis in the rat liver. The role of AHR in mediating pathological changes in the liver prior to tumor formation was investigated in a 4‐week, repeated‐dose study using adult female wild‐type (WT) and AHR knockout (AHR‐KO) rats treated with 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD). Beginning at 8 weeks of age, AHR‐KO and WT rats were dosed by oral gavage with varying concentrations of TCDD (0, 3, 22, 100, 300 and 1000 ng kg^?1 day^?1). Lung, liver and thymus histopathology, hematology, serum chemistry and the distribution of TCDD in liver and adipose tissue were examined. Treatment‐related increases in the severity of liver and thymus pathology were observed in WT, but not AHR‐KO rats. In the liver, these included hepatocellular hypertrophy, bile duct hyperplasia, multinucleated hepatocytes and inflammatory cell foci. A loss of cellularity in the thymic cortex and thymic atrophy was observed. Treatment‐related changes in serum chemistry parameters were also observed in WT, but not AHR‐KO rats. Finally, dose‐dependent accumulation of TCDD was observed primarily in the liver of WT rats and primarily in the adipose tissue of AHR‐KO rats. The results suggest that AHR activation is the initial key event underlying the progression of histological effects leading to liver tumorigenesis following TCDD treatment. Copyright © 2015 John Wiley & Sons, Ltd.