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The physiological functions of the aryl hydrocarbon receptor (AHR) are only beginning to unfold. Studies in wildtype and AHR knockout (AHRKO) mice have recently disclosed that AHR activity is required for obesity and steatohepatitis to develop when mice are fed with a high-fat diet (HFD). In addition, a line of AHRKO mouse has been reported to accumulate retinoids in the liver. Whether these are universal manifestations across species related to AHR activity level is not known yet. Therefore, we here subjected wildtype and AHRKO male rats (on Sprague-Dawley background) to HFD feeding coupled with free access to 10% sucrose solution and water; controls received a standard diet and water. Although the HFD-fed rats consumed more energy throughout the 24-week feeding regimen, they did not get overweight. However, relative weights of the brown and epididymal adipose tissues were elevated in HFDfed rats, while that of the liver was lower in AHRKO than wildtype rats. Moreover, the four groups exhibited diet-or genotype-dependent differences in biochemical variables, some of which suggested marked dissimilarities from AHRKO mice. Expression of pro-and anti-inflammatory genes was induced in livers of HFD-fed AHRKO rats, but histologically they did not differ from others. HFD reduced the hepatic concentrations of retinyl palmitate, 9-cis-4oxo-13,14-dihydroretinoic acid and (suggestively) retinol, whereas AHR status had no effect. Hence, the background strain/line of AHRKO rat is resistant to diet-induced obesity, and AHR does not modulate this or liver retinoid concentrations. Yet, subtle AHR-dependent differences in energy balance-related factors exist despite similar weight development. (c) 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

Phytoestrogens have been widely praised for their health-promoting effects, whereas synthetic environmental estrogens are considered a toxicological risk to human health. The aim of this study was therefore to compare in vitro the estrogenic, cytotoxic, and genotoxic profiles of three common estrogen-like endocrine-disrupting chemicals: the phytoestrogens 8-prenylnaringenine (8-PN) and genistein and the synthetic xenoestrogen tartrazine. As assessed by a yeast bioreporter assay and estrogen-dependent proliferative response in human mammary gland adenocarcinoma cell line (MCF-7), 8-PN showed the highest estrogen-like activity of the three compounds, followed by tartrazine and genistein. After 24-h incubation on MCF-7 cells, all three compounds exhibited low cytotoxicity in the lactate dehydrogenase assay and no genotoxicity in the micronucleus assay. These results demonstrate that 8-PN, genistein and tartrazine possess variable estrogenic activity but display little cellular toxicity in short-term tests in vitro. No difference between phytoestrogens and a synthetic xenoestrogen could be established. Supplementary Information The online version contains supplementary material available at 10.1007/s11356-021-12629-y.

Commercially processed meat and fish products are common sources of human exposure to chemical food mutagens. In this study, we investigated the mutagenic potential of 20 different commercially processed meat and fish products (7 product types with 2-3 lots of each), along with the presence of four principal polycyclic aromatic hydrocarbons (PAHs) (benzo[a]pyrene [BaP], benzo[b]fluoranthene [BbF], benzo[a]anthracene [BaA] and chrysene [CHR]) in them. Sample extraction was carried out by an accelerated solvent extraction method, while the concentrations of the 4 PAHs were determined by gas chromatography-tandem mass spectrometry (GC-MS/MS). The mutagenic potential of food extracts was assessed by the standard plate incorporation assay (Ames test) using two strains ofSalmonellatyphimurium (TA 100 and TA 98) both in the presence and the absence of metabolic activation (S9-mix). The results show that in the majority of food items investigated, PAH levels were below the limit of quantification (0.78 mu g/kg), except for smoked fish (0.8-15 mu g/kg for the 3 lots), one batch of which even exceeded the maximum limits for both the sum of the 4 PAHs (44 mu g/kg vs. 30 mu g/kg) and BaP (8.2 mu g/kg vs. 5.0 mu g/kg). Furthermore, all three batches of smoked fish were also found to be mutagenic on both strains ofSalmonella, both in the presence and the absence of metabolic activation. Overall, the data from both assays were in a fairly good agreement with one another, suggesting that PAHs are major contributors to mutagenicity of processed food products and the set maximum levels for PAHs are usually protective against food mutagenicity, although food samples harboring PAHs at levels approaching the maximum limits may exhibit mutagenic potential. Since the number of samples investigated was relatively small, further studies are warranted to verify the conclusions.

8-prenylnaringenin (8-PN) is a prenylated flavonoid, occurring, in particular, in hop, but also in other plants. It has proven to be one of the most potent phytoestrogens in vitro known to date, and in the past 20 years, research has unveiled new effects triggered by it in biological systems. These findings have aroused the hopes, expectations, and enthusiasm of a “wonder-drug” for a host of human diseases. However, the majority of 8-PN effects require such high concentrations that they cannot be reached by normal dietary exposure, only pharmacologically; thus, adverse impacts may also emerge. Here, we provide a comprehensive and up-to-date review on this fascinating compound, with special reference to the range of beneficial and untoward health consequences that may ensue from exposure to it.

In rats, direct exposure to TCDD causes myriad toxicities. Exposed rats experience hepatotoxicity, wasting syndrome and immune suppression, amongst others. "Inherited exposure", as occurs in the F3 generation of directly exposed F0 animals, has also been shown to cause toxicity: both male and female F3 rats demonstrate an increased incidence of adult onset disease, females also display reproductive abnormalities and increased incidence of ovarian diseases while males show increased incidence of kidney disease and an altered sperm epigenome. Here, we explore the hepatic transcriptomic profile of male and female F3 Sprague-Dawley rats bred through the paternal germ line from F0 dams exposed to a single dose of TCDD (0, 30, 100, 300 or 1000 ng/kg body weight) by oral gavage. We hypothesize that RNA transcripts with altered abundance in livers of unexposed F3 progeny of treated F0 Sprague-Dawley rats may result from epigenetic modifications to the genome. We further survey patterns of differential methylation within male F3 rat testis. Female F3 rats demonstrated more TCDD-mediated hepatic transcriptomic changes than males, with differences primarily in the lowest dose group. In testis from male F3 rats, multiple olfactory receptors displayed patterns of differential methylation. Hypermethylation of Egfr and Mc5r among testes from TCDD lineage rats was observed, but without corresponding changes in hepatic mRNA abundance. Further studies examining these differences in other tissue types are warranted.

Dioxins and related compounds are common environmental contaminants. Although their levels have gone down, they are still of concern, in particular regarding developmental toxicity. The adverse effects of these compounds are mediated by the aryl hydrocarbon receptor (AHR), whose canonical signaling pathway has been unveiled in fair detail. The alternative (non-genomic) pathways are much more obscure. AHR has also proven to be a master regulator of numerous physiological phenomena, which has led to the search of selective AHR modulators with low toxicity. Papers of this Special Issue address the developmental toxicity of dioxins and related compounds as well as selective modulators of AHR and both its canonical and alternative signaling pathways. In addition, new optical and stereoscopic methods for the detection of dioxins are presented. As a whole, this Special Issue provides an up-to-date view on a wide variety of aspects related to dioxin toxicity mechanisms from both original research articles and reviews. Non

The aryl hydrocarbon receptor (AHR) mediates many toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, the AHR alone does not explain the widely different outcomes among organisms. To identify the other factors involved, we evaluated three transgenic mouse lines, each expressing a different rat AHR isoform (rWT, DEL, and INS) providing widely different resistance to TCDD toxicity, as well as C57BL/6 and DBA/2 mice which exhibit a similar to tenfold divergence in TCDD sensitivity (exposures of 5-1000 mu g/kg TCDD). We supplement these with whole-genome sequencing, together with transcriptomic and proteomic analyses of the corresponding rat models, Long-Evans (L-E) and Han/Wistar (H/W) rats (having a similar to 1000-fold difference in their TCDD sensitivities; 100 mu g/kg TCDD), to identify genes associated with TCDD-response phenotypes. Overall, we identified up to 50% of genes with altered mRNA abundance following TCDD exposure are associated with a single AHR isoform (33.8%, 11.7%, 5.2% and 0.3% of 3076 genes altered unique to rWT, DEL, C57BL/6 and INS respectively following 1000 mu g/kg TCDD). Hepatic Pxdc1 was significantly repressed in all three TCDD-sensitive animal models (C57BL/6 and rWT mice, and L-E rat) after TCDD exposure. Three genes, including Cxxc5, Sugp1 and Hgfac, demonstrated different AHRE-1 (full) motif occurrences within their promoter regions between rat strains, as well as different patterns of mRNA abundance. Several hepatic proteins showed parallel up- or downward alterations with their RNAs, with three genes (SNRK, IGTP and IMPA2) showing consistent, strain-dependent changes. These data show the value of integrating genomic, transcriptomic and proteomic evidence across multi-species models in toxicologic studies.

IMA-08401 (C2) is a novel aryl hydrocarbon receptor (AHR) agonist and selective AHR modulator (SAHRM) that is structurally similar to laquinimod (LAQ). Both compounds are converted to the AHR-active metabolite DELAQ (IMA-06201) in vivo. SAHRMs have been proposed as therapeutic options for various autoimmune disorders. Clinical trials on LAQ have not reported any significant toxic outcomes and C2 has shown low toxicity in rats, however, their functional resemblance to the highly toxic AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) raises questions. Here, we characterize the hepatic transcriptomic changes induced by acute (single-dose) and subacute exposure (repeated dosing for 5 days followed by a 5-day recovery period) to C2 in Sprague-Dawley rats. Exposure to C2 leads to activation of the AHR, as shown by altered transcription of Cyp1a1. We identify a heightened response early after exposure that drops off by day 10. Acute exposure to C2 leads to changes to transcription of genes involved in antiviral and antibacterial responses, which highlights the immunomodulator effects of this AHR agonist. Subacute exposure causes an oxidative stress response in the liver, the consequences of which require further study on target tissues such as the CNS and immune system, both of which may be compromised in this patient population.

Alternative splicing is a co-transcriptional mechanism that generates protein diversity by including or excluding exons in different combinations, thereby expanding the diversity of protein isoforms of a single gene. Abnormalities in this process can result in deleterious effects to human health, and several xenobiotics are known to interfere with splicing regulation through multiple mechanisms. These changes could lead to human diseases such as cancer, neurological disorders, autoimmune diseases, and developmental disorders. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant generated as a byproduct of various industrial activities. Exposure to this dioxin has been linked to a wide range of pathologies through the alteration of multiple cellular processes. However, the effects of TCDD exposure on alternative splicing have not yet been studied. Here, we investigated whether a single po. dose of 5 μg/kg or 500 μg/kg TCDD influence hepatic alternative splicing in adult male C57BL/6Kou mouse. We identified several genes whose alternative splicing of precursor messenger RNAs was modified following TCDD exposure. In particular, we demonstrated that alternative splicing of Cyp1a1, Ahrr, and Actn1 was significantly altered after TCDD treatment. These findings show that the exposure to TCDD has an impact on alternative-splicing, and suggest a new avenue for understanding TCDD-mediated toxicity and pathogenesis.

The mediator of dioxin toxicity, aryl hydrocarbon receptor (AHR), has also important physiological functions. Selective AHR modulators (SAHRMs) share some effects of dioxins, except for their marked toxicity. We recently characterised toxicologically two novel SAHRMs, prodrugs IMA-08401 and IMA-07101 in rats, demonstrating that they are far less deleterious than the most toxic AHR-agonist, TCDD. Here, we analysed the in vitro toxicity and in silico AHR binding of the respective active, deacetylated metabolites, IMA-06201 (N-ethyl-N-phenyl-5-chloro-1,2-dihydro-4-hydroxy-1-methyl-2-oxo-quinoline-3-carboxamide) and IMA-06504 (N-(4-trifluoromethylphenyl)-1,2-dihydro-4-hydroxy-5-methoxy-1-methyl-2-oxo-quinoline-3-carboxamide). In H4IIE rat hepatoma cells, IMA-06201 and IMA-06504 induced CYP1A1 with comparable potencies and efficacies to those of TCDD. They had little effect on cell viability as assessed by LDH leakage and MTT reduction assays, and were not mutagenic in the Ames test, but IMA-06504 elicited a maximally 2.7-fold increase in micronuclei. Molecular docking simulations showed that similar to TCDD, they occupy the central region of AHR ligand binding cavity. Hence, while showing low to negligible in vitro toxicity, these novel SAHRMs bind to the AHR qualitatively in a similar fashion to TCDD, and appear comparably powerful AHR agonists. Combined with our earlier results demonstrating that they seem considerably less toxic in vivo than TCDD, these compounds are thus highly interesting new SAHRMs.

The aryl hydrocarbon receptor (AHR) mediates the toxicity of dioxins, but also plays important physiological roles. Selective AHR modulators, which elicit some effects imparted by this receptor without causing the marked toxicity of dioxins, are presently under intense scrutiny. Two novel such compounds are IMA-08401 (N-acetyl-N-phenyl-4-acetoxy-5-chloro-1,2-dihydro-1-methyl-2-oxo-quinoline-3-carboxamide) and IMA-07101 (N-acetyl-N-(4-trifluoromethylphenyl)-4-acetoxy-1,2-dihydro-5-methoxy-1-methyl-2-oxo-quinoline-3-carboxamide). They represent, as diacetyl prodrugs, AHR-active metabolites of the drug compounds laquinimod and tasquinimod, respectively, which are intended for the treatment of autoimmune diseases and cancer. Here, we toxicologically assessed the novel compounds in Sprague-Dawley rats, after a single dose (8.75-92.5 mg/kg) and 5-day repeated dosing at the highest doses achievable (IMA-08401: 100 mg/kg/day; and IMA-07101: 75 mg/kg/day). There were no overt clinical signs of toxicity, but body weight gain was marginally retarded, and the treatments induced minimal hepatic extramedullary haematopoiesis. Further, both the absolute and relative weights of the thymus were significantly decreased. Cyp1a1 gene expression was substantially increased in all tissues examined. The hepatic induction profile of other AHR battery genes was distinct from that caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The only marked alterations in serum clinical chemistry variables were a reduction in triglycerides and an increase in 3-hydroxybutyrate. Liver and kidney retinol and retinyl palmitate concentrations were affected largely in the same manner as reported for TCDD. In vitro, the novel compounds activated CYP1A1 effectively in H4IIE cells. Altogether, these novel compounds appear to act as potent activators of the AHR, but lack some major characteristic toxicities of dioxins. They therefore represent promising new selective AHR modulators. (C) 2017 Elsevier Inc. All rights reserved.

Background 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent congener of the dioxin class of environmental contaminants. Exposure to TCDD causes a wide range of toxic outcomes, ranging from chloracne to acute lethality. The severity of toxicity is highly dependent on the aryl hydrocarbon receptor (AHR). Binding of TCDD to the AHR leads to changes in transcription of numerous genes. Studies evaluating the transcriptional changes brought on by TCDD may provide valuable insight into the role of the AHR in human health and disease. We therefore compiled a collection of transcriptomic datasets that can be used to aid the scientific community in better understanding the transcriptional effects of ligand-activated AHR. Results Specifically, we have created a datasets package – TCDD.Transcriptomics – for the R statistical environment, consisting of 63 unique experiments comprising 377 samples, including various combinations of 3 species (human derived cell lines, mouse and rat), 4 tissue types (liver, kidney, white adipose tissue and hypothalamus) and a wide range of TCDD exposure times and doses. These datasets have been fully standardized using consistent preprocessing and annotation packages (available as of September 14, 2015). To demonstrate the utility of this R package, a subset of “AHR-core” genes were evaluated across the included datasets. Ahrr, Nqo1 and members of the Cyp family were significantly induced following exposure to TCDD across the studies as expected while Aldh3a1 was induced specifically in rat liver. Inmt was altered only in liver tissue and primarily by rat-AHR. Conclusions Analysis of the “AHR-core” genes demonstrates a continued need for studies surrounding the impact of AHR-activity on the transcriptome; genes believed to be consistently regulated by ligand-activated AHR show surprisingly little overlap across species and tissues. Until now, a comprehensive assessment of the transcriptome across these studies was challenging due to differences in array platforms, processing methods and annotation versions. We believe that this package, which is freely available for download (http://labs.oicr.on.ca/boutros-lab/tcdd-transcriptomics) will prove to be a highly beneficial resource to the scientific community evaluating the effects of TCDD exposure as well as the variety of functions of the AHR. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3446-z) contains supplementary material, which is available to authorized users.

2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) is an aromatic, long-lived environmental contaminant. While the pathogenesis of TCDD-induced toxicity is poorly understood, it has been shown that the aryl hydrocarbon receptor (AHR) is required. However, the specific transcriptomic changes that lead to toxic outcomes have not yet been identified. We previously identified a panel of 33 genes that respond to TCDD treatment in two TCDD-sensitive rodent species. To identify genes involved in the onset of hepatic toxicity, we explored 25 of these in-depth using liver from two rat strains: the TCDD-resistant Han/Wistar (H/W) and the TCDD-sensitive Long–Evans (L–E). Time course and dose–response analyses of mRNA abundance following TCDD insult indicate that eight genes are similarly regulated in livers of both strains of rat, suggesting that they are not central to the severe L–E-specific TCDD-induced toxicities. The remaining 17 genes exhibited various divergent mRNA abundances between L–E and H/W strains after TCDD treatment. Several genes displayed a biphasic response where the initial response to TCDD treatment was followed by a secondary response, usually of larger magnitude in L–E liver. This secondary response was most often an exaggeration of the original TCDD-induced response. Only cytochrome b5 type A (microsomal) (Cyb5a) had equivalent TCDD sensitivity to the prototypic AHR-responsive cytochrome P450, family 1, subfamily a, polypeptide 1 (Cyp1a1), while six genes were less sensitive. Four genes showed an early inter-strain difference that was sustained throughout most of the time course (atypical chemokine receptor 3 (Ackr3), collagen, type XVIII, alpha 1 (Col18a1), Cyb5a and glutamate dehydrogenase 1 (Glud1)), and of those genes examined in this study, are most likely to represent genes involved in the pathogenesis of TCDD-induced hepatotoxicity in L–E rats. Electronic supplementary material The online version of this article (doi:10.1007/s00204-016-1720-0) contains supplementary material, which is available to authorized users.

Aims: Environmental pollutants appearing in wastewater, bottled mineral water, tap water, and bottled drinking water are potential, but yet poorly characterized, sources of human exposure to endocrine disrupting chemicals globally. Here, we investigated the current situation in the most densely populated region in Finland. Methods: Influent and effluent bi-monthly samples from a major wastewater treatment plant in Helsinki were obtained over a preceding 2-year period at two time-points (in 2011 and 2014). Equivalent samples from a household water purification plant (located in the same region) were also analyzed, together with various brands of bottled still and mineral water as well as tap water from residential buildings. Samples were obtained in one liter sterile containers, extracted by solid-phase extraction method, and their estrogenic potential determined by a yeast bioluminescent assay. Results: The estrogenic activities of influent samples from the wastewater treatment plant in Helsinki were generally low (from less than limit of detection to 0.7 ng/L estrogen equivalent quantities (EEQ)), except in March and August 2011, when relatively high levels (14.0 and 7.8 ng/L EEQ, respectively) were obtained. Meanwhile, no estrogenic activity was recorded in any of the treated effluent samples from the wastewater treatment plant, influent and effluent samples from the drinking water plant, as well as tap water, bottled still, and mineral waters. Conclusions: These findings indicate that the purification method applied in Helsinki wastewater treatment plant, activated sludge with mechanical, chemical and biological purification steps, is effective in reducing estrogenic activity, and that tap or bottled waters are not a significant source of these compounds to the population in this region.

Processed foods are an insufficiently characterized source of chemical mutagens for consumers. Here, we evaluated the genotoxicity of selected food products in Finland. Mutagenicity was determined by the standard plate incorporation assay followed by methylcellulose overlay and treat-and-wash assays, using the Salmonella strains TA 100 and 98 with and without metabolic activation. Generally, the mutagenic activity of food samples was low, but exhibited lot-wise variation. Cold cuts of cold-smoked beef, grilled turkey, and smoked chicken (a single batch of each) were mutagenic in all three assays with the TA 100 strain with and without metabolic activation, indicating the mutagenic effect was not secondary to histidine release from the food products. However, none of the food extracts showing mutagenic potential induced DNA damage in vitro using the Comet Assay. Our findings imply that in Finland today, there are still products the production methods of which should be refined to reduce the potential risk of mutagenicity to consumers.

Food and drinking water are poorly delineated sources of human exposure to chemical food mutagens and endocrine-disrupting chemicals. In this study, we investigated the presence of mutagens and chemicals exhibiting estrogenic activity in the daily diet of Nigerians, using in vitro assays. Commercially processed foods or snacks and various brands of pure water sachets were extracted by solid-phase extraction and liquid-liquid extraction, respectively. Mutagenicity was determined by the conventional Ames test and two complementary assays on two strains of Salmonella (TA 100 and TA 98), while the estrogenic activity was assessed by a yeast bioluminescent assay, using two recombinant yeast strains (Saccharomyces cerevisiae BMAEREluc/ERα and S. cerevisiae BMA64/luc). A third of the food varieties investigated (chin-chin, hamburger, suya and bean cake) were mutagenic in all three assays, either in the presence or absence of S9 mix. Of the packed water samples, five out of the sixteen investigated (31%), were found to be estrogenic, with estradiol and bisphenol A equivalents ranging from 0.79 to 44.0 ng/L and 124.2 to 1,000.8 ng/L, respectively. Hence, although the current situation in Nigeria does not appear to be substantially worse than, e.g., in Europe, regular monitoring is warranted in the future.

In haem degradation, haem oxygenase-1 (HO-1) first cleaves haem to biliverdin, which is reduced to bilirubin by biliverdin IXα reductase (BVR-A). The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic accumulation of biliverdin in moderately TCDD-resistant line B (Kuopio) rats. Using line B and two TCDD-sensitive rat strains, the present study set out to probe the dose-response and biochemical mechanisms of this accumulation. At 28 days after exposure to 3-300 μg/kg TCDD in line B rats, already the lowest dose of TCDD tested, 3 μg/kg, affected serum bilirubin conjugates, and after doses ≥100 μg/kg, the liver content of bilirubin, biliverdin and their conjugates (collectively 'bile pigments') as well as HO-1 was elevated. BVR-A activity and serum bile acids were increased only by the doses of 100 and 300 μg/kg TCDD, respectively. Biliverdin conjugates correlated best with biliverdin suggesting it to be their immediate precursor. TCDD (100 μg/kg, 10 days) increased hepatic bilirubin and biliverdin levels also in TCDD-sensitive Long-Evans (Turku/AB; L-E) rats. Hepatic bilirubin and bile acids, but not biliverdin, were increased in feed-restricted L-E control rats. In TCDD-sensitive line C (Kuopio) rats, 10 μg/kg of TCDD increased the body-weight-normalized biliary excretion of bilirubin. Altogether, the results suggest that at acutely toxic doses, TCDD induces the formation of bilirubin in rats. However, concurrently, TCDD seems to hamper the quantitative conversion of biliverdin to bilirubin in line B and L-E rats' liver. Biliverdin conjugates are most likely formed as secondary products of biliverdin.

Dioxins are ubiquitous and persistent environmental contaminants whose background levels are still reason for concern. There is mounting evidence from both epidemiological and experimental studies that paternal exposure to the most potent congener of dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), can lower the male/female ratio of offspring. Moreover, in laboratory rodents and zebrafish, TCDD exposure of parent animals has been reported to result in reduced reproductive performance along with other adverse effects in subsequent generations, foremost through the paternal but also via the maternal germline. These impacts have been accompanied by epigenetic alterations in placenta and/or sperm cells, including changes in methylation patterns of imprinted genes. Here, we review recent key studies in this field with an attempt to provide an up-to-date picture of the present state of knowledge to the reader. These studies provide biological plausibility for the potential of dioxin exposure at a critical time-window to induce epigenetic alterations across multiple generations and the significance of aryl hydrocarbon receptor (AHR) in mediating these effects. Currently available data do not allow to accurately estimate the human health implications of these findings, although epidemiological evidence on lowered male/female ratio suggests that this effect may take place at realistic human exposure levels.

Background 2,3,7,8–tetrachlorodibenzo-p-dixion (TCDD) is the most potent of the dioxin congeners, capable of causing a wide range of toxic effects across numerous animal models. Previous studies have demonstrated that males and females of the same species can display divergent sensitivity phenotypes to TCDD toxicities. Although it is now clear that most TCDD-induced toxic outcomes are mediated by the aryl hydrocarbon receptor (AHR), the mechanism of differential responses to TCDD exposure between sexes remains largely unknown. To investigate the differential sensitivities in male and female mice, we profiled the hepatic transcriptomic responses 4 days following exposure to various amounts of TCDD (125, 250, 500 or 1000 μg/kg) in adult male and female C57BL/6Kuo mice. Results Several key findings were revealed by our study. 1) Hepatic transcriptomes varied significantly between the sexes at all doses examined. 2) The liver transcriptome of males was more dysregulated by TCDD than that of females. 3) The alteration of “AHR-core” genes was consistent in magnitude, regardless of sex. 4) A subset of genes demonstrated sex-dependent TCDD-induced transcriptional changes, including Fmo3 and Nr1i3, which were significantly induced in livers of male mice only. In addition, a meta-analysis was performed to contrast transcriptomic profiles of various organisms and tissues following exposure to equitoxic doses of TCDD. Minimal overlap was observed in the differences between TCDD-sensitive or TCDD-resistant models. Conclusions Sex-dependent sensitivities to TCDD exposure are associated with a set of sex-specific TCDD-responsive genes. In addition, complex interactions between the aryl hydrocarbon and sex hormone receptors may affect the observable differences in sensitivity phenotypes between the sexes. Further work is necessary to better understand the roles of those genes altered by TCDD in a sex-dependent manner, and their association with changes to sex hormones and receptors. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1840-6) contains supplementary material, which is available to authorized users.

Background Quantitative real-time PCR (qPCR) is the “gold-standard” technique for measuring mRNA abundances. To correctly compare samples and generate biologically valid results, qPCR data usually require comprehensive normalization to account for sample content variation between reactions. The most common normalization approaches use one or more endogenous controls (reference or house-keeping genes) to adjust the measured levels of experimental genes appropriately. Ideal reference genes are those that display minimal variation across experimental conditions, and thus can vary widely across different biological systems. In particular, toxicogenomic studies of transcriptionally-disruptive toxins, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), require careful consideration of reference genes. Results We examined seven candidate reference genes in 199 mice varying in genotype and time/dose of TCDD exposure. We assessed gene-stability in four ways: (1) the variance of the raw Cq values across biological replicates, (2) the fold-change from basal mRNA levels following treatment, (3) the inter- and intra-group stability evaluated using the NormFinder algorithm, (4) the comparative ΔCq method for each candidate gene. Univariate analyses showed Hprt and Eef1a1 are the two most stable individual reference genes. It has been suggested that using multiple genes would produce a more consistent normalization factor; multivariate analysis was performed using NormFinder. In general, stability increased with the number of genes used, but specific gene-combinations synergized. Conclusions We have validated seven reference genes for use in analyzing mRNA abundances in mouse models of TCDD toxicity. The use of multiple reference genes increases stability, providing more consistent normalization and more reliable results. The number of reference genes used should be maximized, based on experimental capabilities (platform, sample availability, etc.). Our results show the benefit of validating reference genes using multiple methods prior to generating large biological datasets.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant that produces myriad toxicities in most mammals. In rodents alone, there is a huge divergence in the toxicological response across species, as well as among different strains within a species. But there are also significant differences between males and females animals of a single strain. These differences are inconsistent across model systems: the severity of toxicity is greater in female rats than males, while male mice and guinea pigs are more sensitive than females. Because the specific events that underlie this difference remain unclear, we characterized the hepatic transcriptional response of adult male and female C57BL/6 mice to 500 mu g/kg TCDD at multiple time-points. The transcriptional profile diverged significantly between the sexes. Female mice demonstrated a large number of altered transcripts as early as 6 h following treatment, suggesting a large primary response. Conversely, male animals showed the greatest TCDD-mediated response 144 h following exposure, potentially implicating significant secondary responses. Nr1i3 was statistically significantly induced at all time-points in the sensitive male animals. This mRNA encodes the constitutive androstane receptor (CAR), a transcription factor involved in the regulation of xenobiotic metabolism, lipid metabolism, cell cycle and apoptosis. Surprisingly though, changes at the protein level (aside from the positive control, CYP1A1) were modest, with only FMO3 showing clear induction, and no genes with sex-differences. Thus, while male and female mice show transcriptional differences in their response to TCDD, their association with TCDD-induced toxicities remains unclear. (C) 2015 The Authors. Published by Elsevier Inc.

Gene expression measurements with quantitative real-time polymerase chain reaction (PCR) have a major hindrance of not directly measuring RNA and, thus, needing a reverse transcription (RT) step to first produce complementary DNA (cDNA). However, few studies have assessed the robustness of the RT step, and almost none has compared the factual performance of RT and real-time PCR enzymes. Here, we examined the variability of RT and PCR reactions and compared enzyme reproducibility by reverse transcribing identical RNA with eight RT enzymes and then amplifying the cDNA produced by one of them with six real-time PCR enzymes. The same four reference genes were measured in both experiments, and the data were analyzed with Bayesian multilevel models. Reproducibility and the efficiency of the RT enzymes, excluding one, varied moderately, but RT was always less precise than PCR; four PCR enzymes performed in an excellent manner. The transcription efficiencies of two of the measured reference genes (Actb and Sdha) lacked covariance with the general RT efficiency and showed poor reproducibility. In conclusion, most variation in quantitative real-time RT-PCR (RT-qPCR) emanates from the RT phase, and gene-related factors seem to be the primary determinants of this variation, discouraging the use of control genes in RT-qPCR normalization without prior information of their RT robustness.

The biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been the subject of intense study for decades. It is now clear that essentially all TCDD-induced toxicities are mediated by DNA–protein interactions involving the Aryl Hydrocarbon Receptor (AHR). Nevertheless, it remains unknown which AHR target genes cause TCDD toxicities. Several groups, including our own, have developed rodent model systems to probe these questions. mRNA expression profiling of these model systems has revealed significant inter-species heterogeneity in rodent hepatic responses to TCDD. It has remained unclear if this variability also exists within a species, amongst rodent strains. To resolve this question, we profiled the hepatic transcriptomic response to TCDD of diverse rat strains (L-E, H/W, F344 and Wistar rats) and two lines derived from L-E×H/W crosses, at consistent age, sex, and dosing (100μg/kg TCDD for 19h). Using this uniquely consistent dataset, we show that the majority of TCDD-induced alterations in mRNA abundance are strain/line-specific: only 11 genes were affected by TCDD across all strains, including well-known dioxin-responsive genes such as Cyp1a1 and Nqo1. Our analysis identified two novel universally dioxin-responsive genes as well as 4 genes induced by TCDD in dioxin-sensitive rats only. These 6 genes are strong candidates to explain TCDD-related toxicities, so we validated them using 152 animals in time-course (0 to 384h) and dose–response (0 to 3000μg/kg) experiments. This study reveals that different rat strains exhibit dramatic transcriptional heterogeneity in their hepatic responses to TCDD and that inter-strain comparisons can help identify candidate toxicity-related genes.

Basal divergences in circadian feeding and drinking patterns among different rat strains may bias comparative physiological and pharmacological studies. To examine this issue in detail, Long-Evans (Turku/AB) (L-E) and Han/Wistar (Kuopio) (H/W) rats were monitored with an automated system for seven consecutive days after a habituation period. For the analysis, days were split into hours or into four diurnal periods, and the chronologically repetitive nature of the data was taken into account. While L-E rats displayed two distinct peaks in feeding and drinking that coincided with the light shifts, H/W rats exhibited their lowest consummatory activity at the morning light shift. Further differences were recorded in a number of variables including numbers of meals and drinking episodes as well as their durations. These findings reveal surprisingly wide inter-strain differences. Hence, one should be careful in generalizing ingestive behavioural data obtained from a single rat strain to the entire species.

In rats, 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) causes anorexia that may lead to fatal wasting but has hitherto been poorly characterized. Therefore, we studied in-depth feeding and drinking behaviors of TCDD-sensitive L–E rats for 5 (100 μg/kg; lethal dose) or 10 (10 μg/kg; sublethal) days and of TCDD-resistant H/W rats for 14 (100 or 1000 μg/kg; both sublethal) days postexposure to TCDD. The 1000-fold higher resistance of H/W rats to acute lethality of TCDD results from a mutation in their AH receptor (AHR). We split days into four (morning, daytime, evening, and night) or two (light/dark) circadian periods and took the repeated nature of the data into account. In L–E rats at 100 μg/kg, the feed intake dropped precipitously, due to reduced meal sizes. In H/W rats, the hypophagia remained moderate and stemmed from a reduced meal frequency. While the suppression in L–E rats peaked during the morning (at 100 μg/kg), the main effects in H/W rats were seen during the constant light or dark phases. Furthermore, chronologic data analysis revealed alterations in consecutive feeding and drinking patterns. Thus, striking differences were found between these strains in the timing and structure of consummatory behaviors, suggesting involvement of the AHR in these behaviors.

Aversion to novel food items was studied in male rats and mice after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure using chocolate consumption as an indicator. The correlation of this phenomenon with susceptibility to acute toxicity and CYP1A1 induction was examined by determining the dose-response of chocolate aversion in differently dioxin-sensitive rat lines after TCDD (0.01-10 μg/kg). Furthermore, the dependence of this behavioral alteration on the AH receptor (AHR) was studied employing AHR-deficient and wild-type mice. We offered chocolate for both species as a novel food item immediately after the exposure, and it was available with standard rodent chow for 3 days. The ED₅₀ value for the extremely resistant rat line A (LD₅₀) value > 10,000 μg/kg) was 0.36 μg/kg, for the semi-resistant line B (LD₅₀) value 830 μg/kg) 1.07 μg/kg and for the TCDD-sensitive line C (LD₅₀ value 40 μg/kg) 0.34 μg/kg. Interestingly, the ED₅₀ values for chocolate aversion were very similar to those for CYP1A1 induction in these rat lines. Findings on AHR-deficient and wild-type mice implied the involvement of the AHR in this intriguing response, which may thus represent a mechanism to restrict exposure to potentially toxic dietary substances causing hepatic induction of drug-metabolizing enzymes.

A conspicuous but scantly studied feature of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity is avoidance of unfamiliar foodstuffs, which seems to be one of the very few exquisitely sensitive behavioural effects in adult laboratory animals. Here we characterized this peculiar response further after low doses of TCDD. The time-course of the novelty avoidance, the role of nutriment form and dependence of the aversion on the time lag between TCDD exposure and the presentation of a novel food item was determined using rats with different sensitivities to lethality of TCDD. Rats were offered chocolate, liquid nutriment or familiar feed with an unfamiliar texture and the consumptions were measured for varying periods. Aversion to a novel food item (chocolate) emerged within 5.5 h after TCDD exposure. A lag of a week or more between TCDD exposure and the presentation of chocolate abolished the avoidance whereas simultaneous presentation of chocolate with TCDD treatment rendered the rats oblivious to the chocolate's presence for over 40 days. Rats avoided also liquid nutriments when these were coupled with TCDD administration but this faded much sooner than chocolate aversion. Even a change in feed texture at the exposure was able to elicit the response. However, habituation was found to interfere with the aversion. These findings indicate that temporal proximity to TCDD exposure is a requisite for the avoidance response which emerges rapidly and may linger on for extended periods, but is not strictly confined to any specific food type. The molecular mechanisms of this tantalizing behavioural alteration remain to be determined.

Dioxins are ubiquitous environmental contaminants that have attracted toxicological interest not only for the potential risk they pose to human health but also because of their unique mechanism of action. This mechanism involves a specific, phylogenetically old intracellular receptor (the aryl hydrocarbon receptor, AHR) which has recently proven to have an integral regulatory role in a number of physiological processes, but whose endogenous ligand is still elusive. A major acute impact of dioxins in laboratory animals is the wasting syndrome, which represents a puzzling and dramatic perturbation of the regulatory systems for energy balance. A single dose of the most potent dioxin, TCDD, can permanently readjust the defended body weight set-point level thus providing a potentially useful tool and model for physiological research. Recent evidence of response-selective modulation of AHR action by alternative ligands suggests further that even therapeutic implications might be possible in the future.

The AHR locus encodes the aryl hydrocarbon receptor (AHR), a transcriptional regulator of multiple drug-metabolizing enzymes and mediator of toxicity of dioxin-like chemicals. The Han/Wistar (Kuopio) rat strain (H/W) is remarkably resistant to lethal effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) because of a point mutation in the exon/intron 10 boundary in AHR genomic structure that leads to use of 3 alternative cryptic splice sites, potentially creating 3 alternative transcripts and 2 protein products. The deletion variant (DV), which lacks 43 amino acids in the transactivation domain, has the highest intrinsic transactivation activity in vitro; amino acids 766 to 783 suppress transactivation function. However, DV expression levels in H/W rats in vivo are low in liver, lung, thymus, kidney, and testis; insertion variant mRNAs (IVs) are the dominant mRNA forms in H/W rats in which wild-type AHR mRNA is undetectable. In dioxin-sensitive rat strains and lines that are homozygous for wild-type AHR alleles, wild-type AHR mRNA is the most abundant transcript but some IV transcripts are detectable. TCDD treatment in vivo increases transcript levels for both the DV and IVs in H/W rats and increases wild-type transcript levels in dioxin-sensitive rats but does not alter which transcript forms are expressed. In silico modeling indicates that the DV mRNA has lost considerable secondary structure, whereas at the protein level, the transactivation domain of the IV in the dioxin-resistant H/W rat has greater alpha-helical content and a more hydrophobic terminus than wild-type AHR, which may produce a protein conformation that is less amenable to interaction with other regulatory proteins.

One characteristic feature of acute 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity is dramatic interspecies and interstrain variability in sensitivity. This complicates dioxin risk assessment for humans. However, this variability also provides a means of characterizing mechanisms of dioxin toxicity. Long-Evans (Turku/AB) rats are orders of magnitude more susceptible to TCDD lethality than Han/Wistar (Kuopio) rats, and this difference constitutes a very useful model for identifying mechanisms of dioxin toxicity. We adopted a proteomic approach to identify the differential effects of TCDD exposure on liver protein expression in Han/Wistar rats as compared with Long-Evans rats. This allows determination of which, if any, protein markers are indicative of differences in dioxin susceptibility and/or responsible for conferring resistance. Differential protein expression in total liver protein was assessed using two-dimensional gel electrophoresis, computerized gel image analysis, in-gel digestion, and mass spectrometry. We observed significant changes in the abundance of several proteins, which fall into three general classes: (i) TCDD-independent and exclusively strain-specific (e.g. isoforms of the protein-disulfide isomerase A3, regucalcin, and agmatine ureohydrolase); (ii) strain-independent and only dependent on TCDD exposure (e.g. aldehyde dehydrogenase 3A1 and rat selenium-binding protein 2); (iii) dependent on both TCDD exposure and strain (e.g. oxidative stress-related proteins, apoptosis-inducing factor, and MAWD-binding protein). By integrating transcriptomic (microarray) data and genomic data (computational search of regulatory elements), we found that protein expression levels were mainly controlled at the level of transcription. These results reveal, for the first time, a subset of hepatic proteins that are differentially regulated in response to TCDD in a strain-specific manner. Some of these differential responses may play a role in establishing the major differences in TCDD response between these two strains of rats. As such, our work is expected to lead to new insights into the mechanism of TCDD toxicity and resistance.

Conventional biochemical and molecular techniques identified previously several genes whose expression is regulated by the aryl hydrocarbon receptor (AHR). We sought to map the complete spectrum of AHR-dependent genes in male adult liver using expression arrays to contrast mRNA profiles in Ahr-null mice (Ahr(-/-)) with those in mice with wild-type AHR (Ahr(+)(/)(+)). Transcript profiles were determined both in untreated mice and in mice treated 19 h earlier with 1000 microg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Expression of 456 ProbeSets was significantly altered by TCDD in an AHR-dependent manner, including members of the classic AHRE-I gene battery, such as Cyp1a1, Cyp1a2, Cyp1b1, and Nqo1. In the absence of exogenous ligand, AHR status alone affected expression of 392 ProbeSets, suggesting that the AHR has multiple functions in normal physiology. In Ahr(-/-) mice, only 32 ProbeSets exhibited responses to TCDD, indicating that the AHR is required for virtually all transcriptional responses to dioxin exposure in liver. The flavin-containing monooxygenases, Fmo2 and Fmo3, considered previously to be uninducible, were highly induced by TCDD in an AHR-dependent manner. The estrogen receptor alpha as well as two estrogen-receptor-related genes (alpha and gamma) exhibit AHR-dependent expression, thereby extending cross-talk opportunities between the intensively studied AHR and estrogen receptor pathways. p53 binding sites are over-represented in genes down-regulated by TCDD, suggesting that TCDD inhibits p53 transcriptional activity. Overall, our study identifies a wide range of genes that depend on the AHR, either for constitutive expression or for response to TCDD.

Mechanistic toxicology has predominantly been focused on adverse effects that are caused by reactive metabolites or by reactive oxygen species. However, many important xenobiotics exert their toxicity, not by generating reactive products, but rather by altering expression of specific genes. In particular, some environmental contaminants target nuclear receptors that function as regulators of transcription. For example, binding of xenobiotic chemicals to steroid receptors is a principle mechanism of endocrine disruption. The aryl hydrocarbon receptor (AHR) mediates toxicity of dioxin-like compounds. In mice, a polymorphism in the AHR ligand-binding domain reduces binding affinity by about 10-fold in the DBA/2 strain compared with the C57BL/6 strain; consequently, dose-response curves for numerous biochemical and toxic effects are shifted about one log to the right in DBA/2 mice. In the Han/Wistar (Kuopio) (H/W) rat strain, a polymorphism causes a deletion of 38 or 43 amino acids from the AHR transactivation domain. This deletion is associated with a greater than 1000-fold resistance to lethality from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Genes in the conventional AH gene battery (e.g. CYP1A1, CYP1A2, CYP1B1, ALDH3A1, NQO1 and UGT1A1) remain responsive to TCDD in H/W rats despite the large deletion. However, the deletion may selectively alter the receptor's ability to dysregulate specific genes that are key to dioxin toxicity. We are identifying these genes using an expression array approach in dioxin-sensitive vs. dioxin-resistant rat strains and lines. Polymorphisms exist in the human AH receptor, but thus far they have not been shown to have any substantial effect on human responses to AHR-ligands.

Polychlorinated dibenzo-p-dioxins (PCDDs) are highly toxic environmental contaminants, and 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) is the most potent dioxin. Dioxins bind specifically to the cytosolic aryl hydrocarbon receptor (AHR), which is a ligand-activated transcription factor, and a majority of toxic effects of dioxins are mediated via AHR. We have recently demonstrated that TCDD disrupts bone modeling and decreases bone mechanical strength, and that partial resistance to these effects is related to an altered transactivation domain in AHR structure. In order to better understand the effects of dioxins on bone, we studied the presence and precise localization of AHR and also the number and activity of osteoclasts after TCDD treatments. Total RNA was extracted from mixed bone cell population cultures and expression of AHR mRNA was studied using RT-PCR. Bone cells expressed a considerable amount of AHR mRNA. To see which bone cells express AHR, immunostainings were performed in primary rat bone cell cultures, pure human osteoclast cultures and histological sections from AHR knockout and wild type bones. Immunostaining revealed a strong expression of AHR both in osteoclasts and osteoblasts with an especially prominent stain in bone resorbing osteoclasts. Effects of dioxin on primary bone cells were evaluated after TCDD treatment in the pit formation assay. The activity of osteoclasts was not affected measured as the percentage of active osteoclasts and the actual area of resorbed bone. These data indicate that even though TCDD-treated bones show decreased mechanical strength and size, this is not a direct result from increased osteoclastic bone resorption.

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 μg/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 μg/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.

Dioxins are persistent environmental contaminants that cause multiple disorders in laboratory animals, including teratogenesis. In mice, the most important teratogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are hydronephrosis and cleft palate. Aryl hydrocarbon receptor (AHR) mediates most of the TCDD-induced effects, but modulation of these effects by other factors such as epidermal growth factor receptor (EGFR) has been propounded. TCDD changes the expression of both EGF and its receptor EGFR, which may be one step in the pathway leading to cleft palate and hydronephrosis. In the present study, the importance of EGFR in TCDD-induced teratogenicity was evaluated. Heterozygous EGFR(+/-)-mice were mated and pregnant females exposed to 1.5-106.0 microg/kg TCDD on gestation day (GD) 10 and killed on GD 18. The fetuses were studied for cleft palate, hydronephrosis, and open eyes. There was no marked difference among the three genotypes in sensitivity to cleft palate or hydronephrosis, but in EGFR(-/-)-mice frequency of the open eye malformation decreased dose-dependently. In conclusion, EGFR signaling is not required for TCDD-induced cleft palate or hydronephrosis but TCDD appears to counteract the effect of EGFR deficiency on eye opening.

Exposure to environmental dioxins via mother’s milk may be one causative factor of mineralization defects in children’s teeth. A prerequisite for the completion of enamel mineralization is the removal of enamel matrix. To test the hypothesis that dioxins interfere with enamel maturation, we administered lactating Han/Wistar rats a single dose of 2,3,7,8-tetrachlorodibenzo -p-dioxin (TCDD; 50 or 1000 μg/kg) on the day after delivery and analyzed tissue sections of the pup heads at post-natal days (Pn) 9 and 22. By Pn22, the first and second molars of the exposed pups, but not controls, showed retention of enamel matrix. Predentin was thicker than normal. Immunostaining for the aryl hydrocarbon/dioxin receptor (AhR) and cytochrome P4501A1 (CYP1A1) in ameloblasts and odontoblasts was reduced, suggesting that TCDD interferes with tooth mineralization via AhR. Extinction of AhR may lead to abolition of CYP1A1 expression as a sign of impaired dental cell function.

Previous studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can arrest molar tooth development in rats after in utero and lactational exposure, and that the sensitive stage is temporally restricted. To define the stage in which TCDD is able to arrest tooth development and the cellular background of the effect, mouse embryonic molar tooth explants including various early developmental stages from initiation to late cap stage were exposed to TCDD in organ culture. TCDD did not inhibit morphogenesis of the first molar teeth including the early bud-staged E12 first molars, but the teeth were smaller than in control cultures. Accordingly, the second molars underwent morphogenesis in the presence of TCDD when explanted at E15 when they were at the bud stage. TCDD arrested their development when explanted at E14 when they had not yet reached the early bud stage. Immunohistochemical localization of incorporated bromodeoxyuridine in cultured E14 teeth showed that TCDD did not affect cell proliferation. Localization of apoptosis by terminal deoxynucleotidyl transferase (TdT)-mediated nick end labeling (TUNEL) method revealed that TCDD enhanced apoptosis of dental epithelial cells, especially in the dental lamina of both the first and second molars, and in the inner dental epithelium at the cusp tips of the first molars. Thus, TCDD can arrest tooth development in vitro if the exposure starts at the initiation stage, whereas exposure at later stages leads to smaller tooth size and deformation of cuspal morphology. TCDD interferes with tooth development by stimulating apoptosis in those cells of the dental epithelium, which are predetermined to undergo apoptosis during normal development.

Most of the biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated by the bHLH/PAS protein AH receptor (AHR). For regulation of gene activities, AHR dimerizes with another member of the bHLH/PAS protein family, AHR nuclear translocator (ARNT). A substrain of Wistar rats, Han/Wistar (Kuopio) (H/W), is about 1000-fold more resistant to the acute lethality of TCDD than other strains, exemplified by Long-Evans (Turku/AB) (L-E); the LD50 values for these two strains are9600 and 10-20 microg/kg, respectively. Previous studies have demonstrated that the major reason for the exceptional TCDD resistance of H/W rats lies in their AHR, which is remodeled at its C-terminal transactivation domain, but there appears to be another contributing gene product. The present study set out to compare the primary structure of ARNT and the closely related ARNT2 proteins in H/W and L-E rats by cDNA cloning. To our surprise, we found several isoforms of these proteins only one of which has previously been reported in rats. All of the isoforms appeared to arise from alternative splicing. For ARNT, isoforms with deletions at exon 5, 3(') end of exon 6 or 5(') end of exon 11, or with an insertion at 5(') end of exon 20 were discovered. There was also interindividual variation in the number of glutamine-encoding codons at 5(') end of exon 16. The most exciting new variant was revealed for ARNT2, because the insertion found at 5(') end of exon 19 disrupts the functionally critical transactivation domain in the protein, implying a dominant negative role for this isoform. The relative expression levels of the variants did not differ in the two rat strains, nor did TCDD modify the ratios, suggesting that the variants do not contribute to TCDD resistance. However, the regulation of ARNT and ARNT2 activities may be more intricate than previously assumed.

Background Research on the aryl hydrocarbon receptor (AHR) has largely focused on variations in toxic outcomes resulting from its activation by halogenated aromatic hydrocarbons. But the AHR also plays key roles in regulating pathways critical for development, and after decades of research the mechanisms underlying physiological regulation by the AHR remain poorly characterized. Previous studies identified several core genes that respond to xenobiotic AHR ligands across a broad range of species and tissues. However, only limited inferences have been made regarding its role in regulating constitutive gene activity, i.e. in the absence of exogenous ligands. To address this, we profiled transcriptomic variations between AHR-active and AHR-less-active animals in the absence of an exogenous agonist across five tissues, three of which came from rats (hypothalamus, white adipose and liver) and two of which came from mice (kidney and liver). Because AHR status alone has been shown sufficient to alter transcriptomic responses, we reason that by contrasting profiles amongst AHR-variant animals, we may elucidate effects of the AHR on constitutive mRNA abundances. Results We found significantly more overlap in constitutive mRNA abundances amongst tissues within the same species than from tissues between species and identified 13 genes (Agt, Car3, Creg1, Ctsc, E2f6, Enpp1, Gatm, Gstm4, Kcnj8, Me1, Pdk1, Slc35a3, and Sqrdl) that are affected by AHR-status in four of five tissues. One gene, Creg1, was significantly up-regulated in all AHR-less-active animals. We also find greater overlap between tissues at the pathway level than at the gene level, suggesting coherency to the AHR signalling response within these processes. Analysis of regulatory motifs suggests that the AHR mostly mediates transcriptional regulation via direct binding to response elements. Conclusions These findings, though preliminary, present a platform for further evaluating the role of the AHR in regulation of constitutive mRNA levels and physiologic function. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-1053) contains supplementary material, which is available to authorized users.

The aryl hydrocarbon receptor (AHR) mediates toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and regulates expression of several genes such as CYP1A1. Little is known about what regulates expression of the AHR itself. We tested the ability of TCDD to alter in vivo expression of its own receptor in rat strains that are susceptible to TCDD lethality [Long-Evans (Turku AB) (L-E) and Sprague Dawley (SD)] and in a rat strain that is remarkably resistant to TCDD lethality [Han/Wistar (Kuopio) (H/W)]. Rats were administered a single, intragastric dose of 5 or 50 μg/kg of TCDD. Hepatic cytosol, nuclear extract, and RNA were prepared at 1, 4, and 10 days after TCDD exposure. AHR expression was assessed at three levels: ligand binding function, immunoreactive protein and mRNA. TCDD at 5 μg/kg produced a 2- to 3-fold increase in cytosolic AHR in all strains; 50 μg/kg produced depletion at day 1 followed by recovery in SD and H/W but not L-E rats. Both the increase in AHR above basal levels and the recovery from initial depletion were accompanied by elevations in steady-state AHR mRNA, suggesting a pre-translational mechanism for AHR regulation by its own ligand. This up-regulation in vivo is in contrast to the sustained depletion of AHR caused by TCDD in cell culture. There was no clear relationship between AHR regulation and strain sensitivity; thus, the large inherent strain differences in susceptibility to TCDD lethality probably are not explained by differential regulation of AHR by TCDD.

Polychlorinated dibenzodioxins are environmental contaminants commonly produced as a by-product of industrial processes. The most potent of these, 2,3,7,8-tetrachlorodibenzo-rho-dioxin (TCDD), is highly lipophilic, leading to bioaccumulation. White adipose tissue (WAT) is a major site for energy storage, and is one of the organs in which TCDD accumulates. In laboratory animals, exposure to TCDD causes numerous metabolic abnormalities, including a wasting syndrome. We therefore investigated the molecular effects of TCDD exposure on WAT by profiling the transcriptomic response of WAT to 100 mu g/kg of TCDD at 1 or 4 days in TCDD-sensitive Long-Evans (Turku/AB; L-E) rats. A comparative analysis was conducted simultaneously in identically treated TCDD-resistant Han/Wistar (Kuopio; H/W) rats one day after exposure to the same dose. We sought to identify transcriptomic changes coinciding with the onset of toxicity, while gaining additional insight into later responses. More transcriptional responses to TCDD were observed at 4 days than at I day post-exposure, suggesting WAT shows mostly secondary responses. Two classic AHR-regulated genes, Cyp1a1 and Nqo1, were significantly induced by TCDD in both strains, while several genes involved in the immune response, including Ms4a7 and Fl1a1 were altered in L-E rats alone. We compared genes affected by TCDD in rat WAT and human adipose cells, and observed little overlap. Interestingly, very few genes involved in lipid metabolism exhibited altered expression levels despite the pronounced lipid mobilization from peripheral fat pads by TCDD in L-E rats. Of these genes, the lipolysis-associated Lpin1 was induced slightly over 2-fold in L-E rat WAT on day 4. (C) 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license.

Interactions of diet and diet-induced obesity, and the characteristic wasting syndrome caused by 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) were studied in TCDD-resistant Han/Wistar and TCDD-sensitive Long–Evans rats. The rats were made obese by feeding them either a high-energy diet (consisting of chocolate, cheese, and chow) or force feeding. TCDD reduced body weight in a parallel manner in lean and obese rats. The high-energy diet diminished the body weight loss and increased the survival time in L-E rats after a lethal dose of TCDD, while energy supplement with high-fat/low-protein food had an opposite effect. In conclusion, diet-induced obesity and TCDD had additive effects on body weight. Dietary manipulations were able to modify the weight loss and survival time after TCDD. Fat seems to have a negative impact, while carbohydrate or protein may have a positive impact in this respect. The results are in agreement with a view that TCDD-exposed rats have a negative fat balance favoring fat loss.

2,3,7,8-Tetrachlorodibenzo- p -dioxin (TCDD), 1 2 the most toxic congener of dioxins, exhibits wide sensitivity differences between a sensitive Long–Evans (L-E) rat and a resistant Han/Wistar (H/W) rat. The sensitivity is determined probably by two autosomal genes and it is highly end point dependent. The difference is more than 1000-fold for acute toxicity and negligible for CYP1A1 induction. The rat strains were recently shown to have differences in the size of AH receptor (AHR), which mediates most effects of TCDD. In the present study, the rat strains were crossed and the resistant alleles of genes determining TCDD sensitivity were segregated to new rat lines. Selection was based on AHR phenotype determined by Western blot and resistance to TCDD lethality. Two genes determining resistance were found: the Ahr and a novel gene designated “ B .” In homozygous rats, the H/W type Ahr hw allele prevented TCDD lethality up to 2000 μg/kg or more, and the H/W type “ B hw ” allele also increased resistance to TCDD lethality but to a lesser extent. Heterozygous rats were only slightly more resistant to acute lethality than the respective sensitive homozygous rats. CYP1A1 induction was similar irrespective of the Ahr and “ B ” genotypes, but a substantial increase in serum bilirubin seen after low doses in sensitive rats occurred only after large doses in “ B hw/hw ” and not at all in Ahr hw/hw rats. In conclusion, the Ahr hw allele is a major determinant of the exceptional resistance of H/W rats to TCDD lethality. There is also an additional gene, whose function remains to be characterized, conferring limited resistance to TCDD toxicity. These two H/W rat-derived alleles are separately expressed in the new rat lines created.