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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.

Background: Obesity is increasingly taking an important stage as a cause of death worldwide, and interventions with a good cost‐effectiveness ratio are needed. Psychotria densinervia is one of these natural products with health benefits. Objective. The present study evaluated the cytotoxicity, antiadipogenic, low‐density lipoprotein (LDL), oxidation inhibitory activities, and acute toxicity of Psychotria densinervia hydroethanolic leaf and bark extracts. Methods: The cytotoxicity evaluation of the extracts (62.5, 125, 250, and 500 μg/mL) using the MTT assay and the antiadipogenic activity (25, 50, 100, and 200 μg/mL) using oleic acid were carried out in SW‐872 cells. Copper sulfate (CuSO4)‐induced oxidation was used in the evaluation of the effect of extracts (0.25, 0.5, and 1 mg/mL) against LDL oxidation. The oral acute toxicity evaluation of a single dose of 2000 mg/kg of the extracts was performed in Wistar albino rats weighing 127 ± 2 g. Results: The leaf and bark extracts did not show any sign of cytotoxicity at the tested concentrations. The best antiadipogenic activity was observed by the standard orlistat (38.45 ± 1.70 μg/mL), followed by the leaf extract (IC50: 41.47 ± 0.50 μg/mL) and the least the bark extract (IC50: 107.50 ± 0.90 μg/mL). At a concentration of 1 mg/mL, the leaf extract presented an oxidation lag time of 130 min, which was higher and better than that of the bark extract (120 min). Quercetin (standard) presented an oxidation lag time longer than 3 h. The oral acute toxicity evaluation did not show any signs of toxicity indicating that the LD50 was greater than 2000 mg/kg. Conclusion: Based on the results obtained, the P. densinervia hydroethanolic leaf extract possesses a better antioxidant and antiadipogenic activities than the bark extract. [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.

Background2,3,7,8-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.ResultsSpecifically, 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.ConclusionsAnalysis 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.

Phytotoxic compounds isolated and identified from different plants have the ability to use as plant‐based herbicides. Phytotoxic chemicals may be essential to weed management and environmental protection in order to reduce the indiscriminate use of synthetic pesticides. It has been reported that Elaeocarpus floribundus plant possesses phytotoxic compounds. The leaf extracts of this species demonstrated significant growth inhibition against the tested plants (dicot plant lettuce and plant monocot timothy) and inhibition was dose‐ and species‐dependent pattern. Two phytotoxic compounds were separated using different purifications methods and identified as compounds 1 and 2. All phytotoxic compounds displayed potent growth limitation against the tested species (cress). The compound concentrations needed for the inhibition of 50% growth (IC50 value) of tested species ranged from 1.06 to 8.53 µM (micromolar). Findings of this research suggest that these compounds might be responsible for the phytotoxicity of Elaeocarpus floribundus plant. The results of this study may be helpful for the development of natural herbicide to control weeds. [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.

Background2,3,7,8-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.ResultsSeveral 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.ConclusionsSex-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.

BackgroundResearch 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.ResultsWe 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.ConclusionsThese findings, though preliminary, present a platform for further evaluating the role of the AHR in regulation of constitutive mRNA levels and physiologic function.

Crossbreeding aims to increase the productivity of local or indigenous animals by introducing exotic breeds. This study aims to assess the effects of crossbreeding using 50% Boer bucks crossed with Ethiopian indigenous Woyto‐Guji goats and to evaluate farmers' perceptions towards crossbred kids. Data were collected from five purposively selected districts in southern Ethiopia. Personal interviews, focus group discussions (FGDs), and field observations were employed to gather information on the goat production systems and farmers' perceptions. The mean ± SD of goat flock size in the study area were 7.31 ± 5.89 heads per household (HH) with larger flock sizes observed in the Alaba zone (10.32 ± 6.56). Goats in the studied areas were primarily kept for income generation. The average age at first kidding was 11.3 ± 1.3 months. Relatively better management practices were observed for crossbred goats in the Alaba, Loko Abaya, and Gurage zones. Respondent farmers highly appreciated Boer crossbred goats due to their superior perception of attractive coat color (4.39 times greater, P < 0.001), docile behavior (3.59 times greater, P < 0.001), fast growth rate (1.64 times greater, P < 0.05), and market preference (5.81 times greater, P < 0.001). However, susceptibility to disease and drought was considered as drawbacks of crossbred kids in the studied areas. It was also reported that crossbreed goats fetched better prices than indigenous goats of a similar age group and under the same management system. All visited farmers expressed a strong interest in crossbreeding. Based on these findings, it can be concluded that Boer crossbred goats perform well in southern Ethiopia. Therefore, the continued production of crossbred kids can be disseminated to these areas. Additionally, it is suggested to consider the interests of goat producers in the remaining areas. Integrated improved management systems need to be implemented to enhance the survival of crossbred kids. Sustainable training programs should be organized for goat keepers, focusing on aspects such as crossbreeding, minimizing inbreeding, buck rotation, and improved feeding and management practices. [ABSTRACT FROM AUTHOR]

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.

Rat is a major model organism in toxicogenomics and pharmacogenomics. Hepatic mRNA profiles after treatment with xenobiotic chemicals are used to predict and understand drug toxicity and mechanisms. Surprisingly, neither inter- and intra-strain variability of mRNA abundances in control rats nor the heritability of rat mRNA abundances yet been established. We address these issues by studying five populations: the popular Sprague-Dawley strain, sub-strains of Long-Evans and Wistar rats, and two lines derived from crosses between the Long-Evans and Wistar sub-strains. Using three independent techniques--variance analysis, linear modelling, and unsupervised pattern recognition--we characterize extensive intra- and inter-strain variability in mRNA levels. We find that both sources of variability are non-random and are enriched for specific functional groups. Specific transcription-factor binding-sites are enriched in their promoter regions and these genes occur in "islands" scattered throughout the rat genome. Using the two lines generated by crossbreeding we tested heritability of hepatic mRNA levels: the majority of rat genes appear to exhibit directional genetics, with only a few interacting loci. Finally, a comparison of inter-strain heterogeneity between mouse and rat orthologs shows more heterogeneity in rats than mice; thus rat and mouse heterogeneity are uncorrelated. Our results establish that control hepatic mRNA levels are relatively homogeneous within rat strains but highly variable between strains. This variability may be related to increased activity of specific transcription-factors and has clear functional consequences. Future studies may take advantage of this phenomenon by surveying panels of rat strains.

BackgroundThe major toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) appear to result from dysregulation of mRNA levels mediated by the aryl hydrocarbon receptor (AHR). Dioxin-like chemicals alter expression of numerous genes in liver, but it remains unknown which lie in pathways leading to major toxicities such as hepatotoxicity, wasting and lethality. To identify genes involved in these responses we exploited a rat genetic model. Rats expressing an AHR splice-variant lacking a portion of the transactivation domain are highly resistant to dioxin-induced toxicities. We examined changes in hepatic mRNA abundances 19 hours after TCDD treatment in two dioxin-resistant rat strains/lines and two dioxin-sensitive rat strains/lines.ResultsResistant rat strains/lines exhibited fewer transcriptional changes in response to TCDD than did rats with wildtype AHR. However, well-known AHR-regulated and dioxin-inducible genes such as CYP1A1, CYP1A2, and CYP1B1 remained fully responsive to TCDD in all strains/lines. Pathway analysis indicated that the genes which respond differently to TCDD between sensitive and resistant rats are mainly involved in lipid metabolism, cellular membrane function and energy metabolism. These pathways previously have been shown to respond differently to dioxin treatment in dioxin-sensitive versus dioxin-resistant rats at a biochemical level and in the differential phenotype of toxicologic responses.ConclusionThe transactivation-domain deletion in dioxin-resistant rats does not abolish global AHR transactivational activity but selectively interferes with expression of subsets of genes that are candidates to mediate or protect from major dioxin toxicities such as hepatotoxicity, wasting and death.

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.

BackgroundMouse and rat models are mainstays in pharmacology, toxicology and drug development -- but differences between strains and between species complicate data interpretation and application to human health. Dioxin-like polyhalogenated aromatic hydrocarbons represent a major class of environmentally and economically relevant toxicants. In mammals dioxin exposure leads to a broad spectrum of adverse affects, including hepatotoxicity of varying severity. Several studies have shown that dioxins extensively alter hepatic mRNA levels. Surprisingly, though, analysis of a limited portion of the transcriptome revealed that rat and mouse responses diverge greatly (Boverhof et al. Toxicol Sci 94:398-416, 2006).ResultsWe employed oligonucleotide arrays to compare the response of 8,125 rat and mouse orthologs. We confirmed that there is limited inter-species overlap in dioxin-responsive genes. Rat-specific and mouse-specific genes are enriched for specific functional groups which differ between species, conceivably accounting for species-specificities in liver histopathology. While no evidence for the involvement of copy-number variation was found, extensive inter-species variation in the transcriptional-regulatory network was identified; Nr2f1 and Fos emerged as candidates to explain species-specific and species-independent responses, respectively.ConclusionOur results suggest that a small core of genes is responsible for mediating the similar features of dioxin hepatotoxicity in rats and mice but non-overlapping pathways are simultaneously at play to result in distinctive histopathological outcomes. The extreme divergence between mouse and rat transcriptomic responses appears to reflect divergent transcriptional-regulatory networks. Taken together, these data suggest that both rat and mouse models should be used to screen the acute hepatotoxic effects of drugs and toxic compounds.

Background: Phytoestrogens have been praised for their beneficial health effects, whereas synthetic xenoestrogens have been connected to ailments. Aims: To ascertain whether the toxicities of natural and synthetic estrogens differ, we examined the potent phytoestrogen 8-prenylnaringenin (8-PN), the common synthetic xenoestrogen tartrazine, and the physiological estrogen 17β-estradiol (E2). Methods: These three compounds were tested for cytotoxicity, cell proliferation and genotoxicity in human HepG2 and rat H4IIE hepatoma cells. Results: All three estrogens elicited cytotoxicity at high concentrations in both cell lines. They also inhibited cell proliferation, with E2 being the most effective. They all tended to increase micronuclei formation. Conclusion: Natural estrogens were no less toxic than a synthetic one. [ABSTRACT FROM AUTHOR]