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2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread, persistent, and highly toxic environmental pollutant. The most TCDD-sensitive and the most TCDD-resistant rat strains (Long-Evans [Turku/AB] and Han/Wistar [Kuopio], respectively) were crossbred to separate the alleles of two genes (Ahrand an unidentified gene “B”) mediating resistance against TCDD toxicity. During crossbreeding, a new type of toxicity in livers of both sexes was detected, characterized macroscopically by intense dark green to black color and swelling that appeared most frequently after a large dose (300 μg/kg or more as a single intragastric dose) and a follow-up period of more than three weeks. Therefore, studies were undertaken to identify the causative pigment chemically and to examine the hepatotoxicity histologically. The pigment fractions were separated by thin layer chromatography and then analyzed by HPLC and electrospray mass spectrometry. The pigment was found to consist of biliverdin and several biliverdin-related compounds. In liver histopathology carried out on male rats, progressive sinusoidal distension and hepatic peliosis with membrane-bound cysts were seen. The clinical manifestations of pigment accumulation were recorded most often in intermediately resistant rat lines such as line B (homozygous for the geneB), but never occurred in rats expressing only the Han/Wistar (Kuopio)-type Ah receptor with an altered transactivation domain structure. [ABSTRACT FROM PUBLISHER]

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 HFD-fed 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-4-oxo-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. [ABSTRACT FROM AUTHOR]

Reduced gluconeogenesis due to decreased activity of key gluconeogenic enzymes in liver, together with feed refusal, has been suggested to play an important role in 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced lethality in rats. This study was carried out to further analyse the toxicological significance of reduced gluconeogenesis by comparing dose-responses and time-courses of effects of TCDD on the activity of phosphoenolpyruvate carboxykinase (PEPCK) in liver, liver glycogen concentration as well as plasma concentrations of glucose and amino acids in both genders of TCDD-sensitive Long-Evans (L-E) rats and TCDD-resistant Han/Wistar (H/W) rats. A dose-dependent decrease in PEPCK activity was observed in H/W rats, but in L-E rats the activity was not decreased. However, TCDD impaired the strong increase in liver PEPCK activity observed in pair-fed controls of the L-E strain. Liver glycogen concentrations were severely decreased in L-E rats and moderately in H/W rats. This effect seems to be secondary to reduced feed intake, since a similar decrease was seen in pair-fed controls. Decreases in plasma glucose concentrations were also more profound in L-E rats than in H/W rats, but pair-fed controls were generally less affected. Circulating concentrations of amino acids were markedly increased in TCDD-treated L-E rats, which is likely to reflect increased mobilization of amino acids and their decreased metabolism in liver. Reduction of liver PEPCK activity cannot account for the sensitivity difference of these two strains of rats in terms of mortality. Nevertheless, the response of both strains of TCDD-treated rats regarding gluconeogenesis is different from that seen in pair-fed controls and suggesting that impairment of this pathway contributes to the development of the wasting syndrome. [ABSTRACT FROM AUTHOR]

We compared the effects of 2,3,7,8-tetra (TCDD), 1,2,3,7,8-penta (PeCDD), 1,2,3,4,7,8-hexa (HxCDD) and 1,2,3,4,6,7,8-heptachlorodibenzo- p-dioxin (HpCDD) on brain serotonin metabolism, plasma tryptophan and liver tryptophan pyrrolase activity in two rat strains, TCDD-sensitive Long-Evans ( Turku AB; L-E) and TCDD-resistant Han/Wistar ( Kuopio; H/W). Previously it was shown that L-E rats exhibit the expected rank order of potency for CDDs in terms of acute toxicity with TCDD being the most potent, followed by PeCDD, HxCDD and HpCDD. In contrast, to H/W rats HxCDD was the most toxic and TCDD the least toxic of these congeners. In the present study, the CDDs decreased body weight in L-E rats in the following order of potency: TCDD>PeCDD>HxCDD>HpCDD. The same rank order was recorded for elevations in brain tryptophan and plasma free tryptophan concentrations as well as for inhibition of the main hepatic tryptophan metabolizing enzyme, tryptophan pyrrolase. By contrast, in H/W rats HxCDD was the most effective congener in producing loss of body weight, followed by HpCDD, PeCDD and TCDD. This was also true of changes in tryptophan homeostasis. These findings imply that in TCDD-susceptible L-E and TCDD-resistant H/W rats the potency of dioxin congeners in inducing acute toxicity highly correlates with their ability to disrupt tryptophan homeostasis. However, there may not be a direct causal relationship between body weight loss and altered tryptophan homeostasis, because the magnitudes of these two phenomena were not consistently parallel across the dioxin congeners tested. [ABSTRACT FROM AUTHOR]

We have previously reported that in rats 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) lethality is associated (although not necessarily causally) with changes in brain serotonin (5-HT) metabolism. In the present study, we have examined whether this holds for other species by comparing the effect of TCDD in the most TCDD-susceptible and the most TCDD-resistant species, guinea pigs and hamsters, respectively. Body weight gain of guinea pigs exposed to TCDD (0.3-2.7 μg/kg) diminished dose dependently, while the effect was marginal in hamsters (900-4600 μg/kg). Brain 5-hydroxyindoleacetic acid (the main metabolite of brain 5-HT), brain tryptophan (the precursor amino acid of 5-HT), and plasma free and total tryptophan were not affected at any dose in guinea pigs. In contrast, 4 days after exposure, the levels of plasma free and total tryptophan were consistently increased in hamsters. These, as well as brain tryptophan, were still elevated 10 days after exposure. TCDD did not affect plasma glucose level in either species. Liver glycogen was decreased in a dose-dependent manner in TCDD-treated guinea pigs as well as in their pair-fed controls on day 10. There was no change in liver glycogen in hamsters. The activity of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase was only depressed in hamsters by all doses of TCDD. We conclude that changes in tryptophan metabolism or in carbohydrate homeostasis cannot explain the wide interspecies differences in susceptibility to the acute lethality of TCDD, although they may correlate with some aspects of its toxicity in certain species. [ABSTRACT FROM AUTHOR]

Target Tissue Morphology and Serum Biochemistry following 2,3,7,8-Tetrachlorodibenzo--dioxin (TCDD) Exposure in a TCDD-Susceptible and a TCDD-Resistant Rat Strain. POHJANVIRTA, R., KULJU, T., MORSELT, A. F. W., TUOMINEN, R., JUVONEN, R., ROZMAN, K.,MÄNNISTÖ, P., COLLAN, Y., SAINIO, E.-L., AND TUOMISTO, J. (1989). . 12, 698–712. The mode of action of the highly toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is unknown. It was recently discovered that two strains of rat, Long-Evans (L-E) and Han/Wistar (H/W), differ widely in susceptibility to TCDD. Employing this strain divergence as a probe, the present study set out to assess the role of various biochemical and morphological effects in TCDD lethality. In the main experiment, the rats were treated once ip with 0, 5, 50, or (H/W) 500 μg/kg TCDD and killed 1 to 16 days postexposure. Several target organs were evaluated by light microscopy and a number of serum lipid and carbohydrate parameters as well as a few major regulatory hormones were analyzed. The results demonstrated that most alterations caused by TCDD were essentially similar in both strains. TCDD reduced circulating thyroxine to a slightly greater extent and more permanently in the sensitive L-E strain. Moreover, a highly significant interaction on thyroid-stimulating hormone was found among strain, dose. and time. Serum concentrations of corticosterone and free fatty acids were increased only in the L-E rats given 50 μg/kg TCDD, i.e., at an apparent LDl00 dose level for this strain. Yet, the most striking interstrain difference was seen in the liver which was distinctly affected after Day 4 in L-E rats given 50 μg/kg TCDD but only marginally affected in rats from any H/W group. The lesion, while showing no necrotic cell changes, was suggestive of plasma membrane damage, possibly reflecting the production of free radicals. The relation of the findings to possible mechanisms of TCDD action is discussed. [ABSTRACT FROM PUBLISHER]

A comparative study was carried out in the most TCDD-resistant [Han/Wistar (H/W), LD50 > 3000 μg/kg] and the most TCDD-susceptible [Long-Evans (L-E), LD50 about 10 μg/kg] rat strain to assess the significance of kinetic factors in TCDD toxicity. Young adult males of both strains were administered 5 μg/kg (1.9 μCi/kg) 14C-TCDD intraperitoneally. Four rats per strain were killed at 4 hr, 1, 4, 8, 16, and 32 days after exposure. A total of 22 tissues along with blood and serum were sampled for liquid scintillation counting. From half of the animals, daily urine and faeces were also analyzed. In addition, 3 rats per strain were given 50 μg/kg (19 μCi/kg) 14C-TCDD and prepared for whole-body autoradiography after 1, 4 or 8 days. The livers of two rats per strain killed at 4 hr, 4 or 16 days, and the excreta from two rats of both strains collected on days 1-4, 5-8, 13-16, and 29-32 after exposure were analyzed for metabolites of TCDD by high pressure liquid chromatography. The label was mainly excreted in faeces as metabolites of TCDD, and the half-life of elimination was 20.8 (L-E) or 21.9 (H/W) days. A very similar overall distribution pattern was observed in both strains irrespective of dose, and the liver was the major site of accumulation. Practically all liver 14C-activity was found as the parent compound. Moderate strain-related differences were observed in the thyroid, thymus, prostate, adrenals, and brown and white fat, where lower values were recorded in H/W rats. Site-dependent variation was detected in the brain. The results do not support the view that TCDD metabolism or disposition would have a major impact on the strain difference in TCDD lethality. [ABSTRACT FROM AUTHOR]

A salient sign of fatal 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) intoxication is dramatic body weight loss accompanied by hypophagia. Yet, the nature of this wasting syndrome is unknown. As all of the current leptogenic (weight reducing) drugs exert their action by affecting aminergic neurotransmission, this study set out to screen the reversibility of TCDD-induced anorexia with the following agents modulating aminergic neurotransmission: amphetamine, amperozide, chlordiazepoxide, clonidine, haloperidol, morphine, PCPA, phenoxybenzamine, reserpine and sotalol. In addition, dexamethasone, indomethacin, and insulin were included in the drug battery. The agents were administered subcutaneously to adult male Long-Evans rats over a period lasting from 3 to 14 days. Half of each drug group was concomitantly exposed to a lethal dose of TCDD (20 μg/kg). None of the regimens were able to mitigate the wasting syndrome. TCDD proved to markedly diminish the nocturnal feed intake while practically sparing daytime feed consumption. Insulin increased the daytime feeding of TCDD-exposed rats, and the termination of treatment resulted in almost total aphagia in this group. Amphetamine, dexamethasone, PCPA, and reserpine caused weight loss in drug control rats and aggravated the action of TCDD. However, clonidine had no effect on the weight of control rats but accelerated weight decline in TCDD-cotreated animals. TCDD seemed to have a somewhat minor influence on drinking than on feeding. Clonidine stimulated water intake in controls but not in TCDD-exposed rats. These results suggest that aminergic neurotransmission is not specifically or crucially affected by TCDD, but further studies are needed to confirm this conclusion. [ABSTRACT FROM AUTHOR]

Adult male Han/Wistar rats were administered 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) intraperitoneally at doses ranging from 125 to 1400 μg/kg and monitored for 39 to 48 days. Two rats succumbed in the course of the experiment: one in the group receiving 625 and one dosed 1000 μg/kg. Body weights of the animals decreased by 20 to 30% during the first 10 to 14 days and became stable thereafter. Feed consumption decreased to 1/3-1/2 of control levels by Day 4 (calculated per metabolic body mass) and returned gradually to starting values by about 4 weeks after dosing. Water intake displayed a triphasic pattern: at first it was slightly increased (Days 1 to 3), then reduced (on Days 4 to 12) and finally increased again throughout the remainder of the test period. The absolute and/or relative weights of thymus, testicles, ventral prostate and interscapular brown fat were significantly decreased at termination. These results indicate that the LD50-value for TCDD in the male, adult Han/Wistar rat is substantially above 1400 μg/kg, and that suppression of appetite is the principal phenomenon responsible for TCDD-induced body weight reduction. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A foreword to the journal "Acta Veterinaria Scandinavica" is presented.

Western blotting is a well-established, inexpensive and accurate way of measuring protein content. Because of technical variation between wells, normalization is required for valid interpretation of results across multiple samples. Typically this involves the use of one or more endogenous controls to adjust the measured levels of experimental molecules. Although some endogenous controls are widely used, validation is required for each experimental system. This is critical when studying transcriptional-modulators, such as toxicants like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).To address this issue, we examined hepatic tissue from 192 mice representing 47 unique combinations of strain, sex, Ahr-genotype, TCDD dose and treatment time. We examined 7 candidate reference proteins in each animal and assessed consistency of protein abundance through: 1) TCDD-induced fold-difference in protein content from basal levels, 2) inter- and intra- animal stability, and 3) the ability of each candidate to reduce instability of the other candidates. Univariate analyses identified HPRT as the most stable protein. Multivariate analysis indicated that stability generally increased with the number of proteins used, but gains from using >3 proteins were small. Lastly, by comparing these new data to our previous studies of mRNA controls on the same animals, we were able to show that the ideal mRNA and protein control-genes are distinct, and use of only 2–3 proteins provides strong stability, unlike in mRNA studies in the same cohort, where larger control-gene batteries were needed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

The aryl hydrocarbon receptor (AHR) is a widely expressed ligand-dependent transcription factor that mediates cellular responses to dioxins and other planar aromatic hydrocarbons. Ahr-null mice are refractory to the toxic effects of dioxin exposure. Although some mechanistic aspects of AHR activity are well understood, the tissue specificity of AHR effects remains unclear, both during development and following administration of exogenous ligands. To address the latter issue, we defined and compared transcriptional responses to dioxin exposure in the liver and kidney of wild-type and Ahr-null adult C57BL/6J mice treated with either 2,3,7,8-tetrachlorodibenzo-p-dioxin or corn-oil vehicle. In both tissues, essentially all effects of dioxin on hepatic mRNA levels were mediated by the AHR. Although 297 genes were altered by dioxin exposure in the liver, only 17 were changed in the kidney, including a number of well-established AHR target genes. Ahr genotype had a large effect in both tissues, profoundly remodeling both the renal and hepatic transcriptomes. Surprisingly, a large number of genes were affected by Ahr genotype in both tissues, suggesting the presence of a basal AHR gene battery. Alterations of the renal transcriptome in Ahr-null animals were associated with perturbation of specific functional pathways and enrichment of specific DNA motifs. Our results demonstrate the importance of intertissue comparisons, highlight the basal role of the AHR in liver and kidney, and support a role in development or normal physiology. [ABSTRACT FROM PUBLISHER]

Abstract: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic accumulation of biliverdin and its monoglucuronide in moderately TCDD-resistant line B rats, but not in highly TCDD-resistant line A rats. In the mammalian heme degradation process, heme is cleaved to biliverdin by the rate-limiting enzyme heme oxygenase-1 (HO-1). Subsequently, biliverdin IXα reductase (BVRA) catalyzes the reduction of biliverdin to bilirubin. In heme biosynthesis, the rate-limiting enzyme is δ-aminolevulinic acid synthetase 1 (ALAS1). The effect of TCDD on HO-1, BVRA and ALAS1 was studied at the levels of mRNA (all three enzymes), protein expression (HO-1), and enzymatic activity (BVRA, liver only) in order to determine whether the accumulation of biliverdin could be due to their altered expression. In both lines A and B, 300μg/kg TCDD transiently repressed hepatic HO-1 mRNA on day 2 but induced HO-1 protein expression at later time-points; however, the impact emerged earlier (day 14 vs. day 35) in line B rats. In spleen, TCDD repressed HO-1 mRNA and protein expression in lines A and B through days 2–35, but did not affect its mRNA levels in TCDD-sensitive L-E rats (10 days after 100μg/kg). In all rat strains/lines, there was a strong repression of ALAS1 and a moderate induction of BVRA mRNA in liver, but mostly not in spleen. Hepatic BVRA activity was increased in lines A and B on day 14. At 5 weeks, it was still elevated in line A but reduced to 51% of control in line B. The results suggest that hepatic heme degradation is induced by TCDD in rats; however, this does not alone explain the accumulation of biliverdin in line B rats. Other factors such as the late repression of BVRA found here and possibly oxidative stress may be important contributors to biliverdin accumulation in these rats. [Copyright &y& Elsevier]

Despite great interspecies differences in adult 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) sensitivity, the toxic potency of TCDD is similar across species in fetal mortality. Han/Wistar (Kuopio; H/W) rats are exceptionally resistant to acute toxicity of TCDD, but show sensitivity to embryotoxicity and teratogenicity. The resistance of adult H/W rats to acute TCDD toxicity is based on a point mutation in the transactivation domain of the aryl hydrocarbon receptor (AHR) and to an unknown gene “B”. This study investigated the time course of postnatal development of resistance to TCDD and the significance of genotypic variation in resistance development. H/W, line A (a new line with the H/W-type mutated AHR), and line B rats (a line with normal AHR but moderately resistant because of gene “B”) were exposed to a single dose of TCDD 2–56 days after birth. H/W and line A rats received 1000 μg/kg; male and female B rats received 200 and 100 μg/kg, respectively. Survival was monitored for 42 days. Interestingly, although TCDD ceased growth and weight gain in all TCDD groups, the younger dosed animals did not seem to reach the body weight of the older dosed animals even in 100 days. The survival results after 42 days showed that line A rats are fairly resistant to TCDD immediately after birth, and their full TCDD resistance develops during the first week of life. The moderate resistance of line B rats develops approximately at the time of weaning. This difference in the time course of resistance development suggests that there are basic differences in pathways mediating resistance in lines A and B rats. [Copyright &y& Elsevier]

The aryl hydrocarbon receptor repressor (AHRR) is a negative regulator of AH receptor (AHR), which mediates most of the toxic and biochemical effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR has been shown to be the major reason for the exceptionally wide (ca. 1000-fold) sensitivity difference in acute toxicity of TCDD between two rat strains, sensitive Long–Evans (Turku/AB) (L–E) and resistant Han/Wistar (Kuopio) (H/W), but there is another, currently unknown contributing factor involved. In the present study, we examined AHRR structure and expression in these rat strains to find out whether AHRR could be this auxiliary factor. Molecular cloning of AHRR coding region showed that consistent with AHRR proteins in other species, the N-terminal end of rat AHRR is highly conserved, but PAS B and Q-rich domains are severely truncated or lacking. Identical structures were recorded in both strains. Next, the time-, dose-, and tissue-dependent expression of AHRR was determined using quantitative real-time RT-PCR. In liver, AHRR expression was very low in untreated rats, but it increased rapidly after TCDD exposure (100 μg/kg). Testis exhibited the highest constitutive expression of AHRR, whereas kidney, spleen, and heart showed the highest induction of AHRR in response to TCDD treatment. Again, no marked differences were found between H/W and L–E rats, implying that AHRR is not the auxiliary contributing factor to the strain difference in TCDD sensitivity. However, simultaneous measurement of CYP1A1 mRNA reinforced the view that AHRR is an important determinant of tissue-specific responsiveness to TCDD. [Copyright &y& Elsevier]

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 are >9600 and 10–20 μg/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. [Copyright &y& Elsevier]

• Circulating REOH and ATRA-concentrations were lower in AHRKO than WT mice. • TCDD had no effect on retinoid concentrations in AHRKO mice. • TCDD reduced liver REPA and CORA concentrations in WT mice. • TCDD elevated serum and kidney REOH and liver ATRA concentrations in WT female mice. • TCDD reduced renal CORA concentration in WT male mice. Young adult wild-type and aryl hydrocarbon receptor knockout (AHRKO) mice of both sexes and the C57BL/6J background were exposed to 10 weekly oral doses of 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD; total dose of 200 μg/kg bw) to further characterize the observed impacts of AHR as well as TCDD on the retinoid system. Unexposed AHRKO mice harboured heavier kidneys, lighter livers and lower serum all- trans retinoic acid (ATRA) and retinol (REOH) concentrations than wild-type mice. Results from the present study also point to a role for the murine AHR in the control of circulating REOH and ATRA concentrations. In wild-type mice, TCDD elevated liver weight and reduced thymus weight, and drastically reduced the hepatic concentrations of 9- cis -4-oxo-13,14-dihydro-retinoic acid (CORA) and retinyl palmitate (REPA). In female wild-type mice, TCDD increased the hepatic concentration of ATRA as well as the renal and circulating REOH concentrations. Renal CORA concentrations were substantially diminished in wild-type male mice exclusively following TCDD-exposure, with a similar tendency in serum. In contrast, TCDD did not affect any of these toxicity or retinoid system parameters in AHRKO mice. Finally, a distinct sex difference occurred in kidney concentrations of all the analysed retinoid forms. Together, these results strengthen the evidence of a mandatory role of AHR in TCDD-induced retinoid disruption, and suggest that the previously reported accumulation of several retinoid forms in the liver of AHRKO mice is a line-specific phenomenon. Our data further support participation of AHR in the control of liver and kidney development in mice. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 ~ tenfold divergence in TCDD sensitivity (exposures of 5-1000 μg/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 ~ 1000-fold difference in their TCDD sensitivities; 100 μg/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 μg/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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • IMA-06201 and IMA-06504 are selective AHR modulators. • As CYP1A1 inducers, they appear comparable to TCDD in potency and efficacy. • They also show low to negligible cytotoxicity and mutagenicity in vitro. • Based on in silico modelling, their binding to the AHR resembles that of TCDD. • Therefore, they have potential as drug compound candidates and research tools. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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/tcddtranscriptomics) 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Polychlorinated dibenzodioxins are environmental contaminants commonly produced as a by-product of industrial processes. The most potent of these, 2,3,7,8-tetrachlorodibenzo- ρ -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 μg/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 1 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 F13a1 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 μg/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. [ABSTRACT FROM AUTHOR]