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 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.
Resistant 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.
The 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 acute toxicity of the ubiquitous environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) varies widely among species and strains. Previous studies in rats have established that ...females are approximately 2-fold more sensitive to TCDD lethality than males. However, there is a surprising gap in the literature regarding possible gender-related sensitivity differences in mice. In the present study, by using three substrains of TCDD-sensitive C57BL/6 mice and transgenic mice on this background, we demonstrated that: 1) in contrast to the situation in rats, female mice are the more resistant gender; 2) the magnitude of the divergence between male and female mice depends on the substrain, but can amount to over 10-fold; 3) AH receptor protein expression levels or mutations in the primary structure of this receptor are not involved in the resistance of female mice of a C57BL/6 substrain, despite their acute LD50 for TCDD being over 5000μg/kg; 4) transgenic mice that globally express the rat wildtype AH receptor follow the mouse type of gender difference; 5) in gonadectomized mice, ovarian estrogens appear to enhance TCDD resistance, whereas testicular androgens seem to augment TCDD susceptibility; and 6) the gender difference correlates best with the severity of liver damage, which is also reflected in hepatic histopathology and the expression of pro-inflammatory cytokines, especially IL-6. Hence, the two closely related rodent species most often employed in toxicological risk characterization studies, rat and mouse, represent opposite examples of the influence of gender on dioxin sensitivity, further complicating the risk assessment of halogenated aromatic hydrocarbons.
► In contrast to rats, male mice are more sensitive to TCDD toxicity than female mice. ► The resistance of female C57BL/6Kuo mice matches or exceeds that of male DBA/2 mice. ► The resistance of female C57BL/6Kuo mice is not based on AHR structure or abundance. ► Both androgens and estrogens appear to influence TCDD sensitivity. ► TCDD sensitivity correlates best with the severity of lesions in the liver.
Phytoestrogens have been praised for their beneficial health effects, whereas synthetic xenoestrogens have been connected to ailments.
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).
These three compounds were tested for cytotoxicity, cell proliferation and genotoxicity in human HepG2 and rat H4IIE hepatoma cells.
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.
Natural estrogens were no less toxic than a synthetic one.
Despite several decades of research, the complete mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other xenobiotic agonists of the aryl hydrocarbon receptor (AHR) cause toxicity ...remains unclear. While it has been shown that the AHR is required for all major manifestations of toxicity, the specific downstream changes involved in the development of toxic phenotypes remain unknown. Here we examine a panel of 13 genes that are AHR-regulated in many species and tissues. We profiled their hepatic mRNA abundances in two rat strains with very different sensitivities to TCDD: the TCDD-sensitive Long–Evans (Turku/AB; L–E) and the TCDD-resistant Han/Wistar (Kuopio; H/W). We evaluated doses ranging from 0 to 3000μg/kg at 19h after TCDD exposure and time points ranging from 1.5 to 384h after exposure to 100μg/kg TCDD. Twelve of 13 genes responded to TCDD in at least one strain, and seven of these showed statistically significant inter-strain differences in the time course analysis (Aldh3a1, Cyp1a2, Cyp1b1, Cyp2a1, Fmo1, Nfe2l2 and Nqo1). Cyp2s1 did not respond to TCDD in either rat strain. Five genes exhibited biphasic responses to TCDD insult (Ahrr, Aldh3a1, Cyp1b1, Nfe2l2 and Nqo1), suggesting a secondary event, such as association with additional transcriptional modulators. Of the 12 genes that responded to TCDD during the dose–response analysis, none had an ED50 equivalent to that of Cyp1a1, the most sensitive gene in this study, while nine genes responded to doses at least 10–100 fold higher, in at least one strain (Ahrr (L–E), Aldh3a1 (both), Cyp1a2 (both), Cyp1b1 (both), Cyp2a1 (L–E), Inmt (both), Nfe2l2 (L–E), Nqo1 (L–E) and Tiparp (both)). These data shed new light on the association of the AHR target genes with TCDD toxicity, and in particular the seven genes exhibiting strain-specific differences represent strong candidate mediators of Type-II toxicities.
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•NanoString measured hepatic mRNA molecules following TCDD treatment.•TCDD-sensitive Long–Evans and TCDD-resistant Han/Wistar rats were compared.•Time courses and dose responses were analyzed for AHR-core gene changes.•7 genes displayed inter-strain mRNA differences at times after TCDD exposure.•2 of the AHR-core genes had significant inter-strain differences in their TCDD ED50.
Mouse 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).
We 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.
Our 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.
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.
Previous studies have shown that several aryl hydrocarbon receptor (AHR) agonists, including β-naphthoflavone (BNF), elicit avoidance of novel food items in rodents, with this behavioral response ...displaying a similar dose-response to hepatic induction of CYP1A1. The avoidance has been found to bear substantial similarity to conditioned taste avoidance/aversion (CTA). The present study set out to confirm the indispensability of AHR in the avoidance response, to verify whether vagal afferent fibers are involved in it, and to see if AHR signaling might interfere with the effect of the classic trigger of CTA, LiCl. To this end, globally AHR deficient (AHRKO) or vagotomized wildtype rats were treated by gavage with 60 mg/kg BNF or ip with 0.15 M LiCl (4 ml/kg), and presented with chocolate which was either novel or familiar to them. Both the avoidance response and Cyp1a1 induction were missing in AHRKO rats. In contrast, Ahr+/− rats exhibited them in full, save for a single outlier. Total subdiaphragmatic vagotomy failed to interfere with the avoidance of novel or familiar chocolate or induction of Cyp1a1. After LiCl administration, male AHRKO rats showed a significantly mitigated suppression of chocolate consumption compared with wildtype animals (~60% vs. ~10% of control chocolate intake, respectively). A similar tendency was seen in females, but they were less responsive to LiCl. These findings corroborate AHR as a prerequisite of the BNF-induced novel food avoidance, prove vagal afferents unlikely mediators of this response, and imply an unforeseen involvement of AHR signaling in the thoroughly-characterized CTA instigated by LiCl.
•AH receptor knockout (AHRKO) rats do not exhibit BNF-induced novel food avoidance.•The response is preserved in heterozygous AHRKO rats.•Vagotomy does not interfere with this behavioral response in wildtype rats.•Also conditioned taste aversion triggered by LiCl is attenuated in AHRKO rats.
Dioxin-like chemicals are well known for their ability to upregulate expression of numerous genes via the AH receptor (AHR). However, recent transcriptomic analyses in several laboratories indicate ...that dioxin-like chemicals or AHR genotype itself also can downregulate levels of mRNAs encoded by numerous genes. The mechanism responsible for such downregulation is unknown. We hypothesized that microRNAs (miRNAs), which have emerged as powerful negative regulators of mRNA levels in several systems, might be responsible for mRNA downregulation in dioxin/AHR pathways. We used two miRNA array platforms as well as quantitative reverse transcriptase–polymerase chain reaction to measure miRNA levels in wild-type (WT) versus Ahr-null mice, in dioxin-sensitive Long-Evans (L-E; Turku/AB) rats versus dioxin-resistant Han/Wistar (H/W; Kuopio) rats and in rat 5L and mouse Hepa-1 hepatoma cells in culture. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vivo caused few changes in miRNA levels in mouse or rat livers, and those changes that were statistically significant were of modest magnitude. Hepatoma cells in culture also exhibited few changes in miRNA levels in response to TCDD. AHR genotype had little effect on hepatic miRNA levels, either in constitutive expression or in response to TCDD—only a few miRNAs differed in expression between Ahr-null mice compared to mice with WT AHR or between L-E rats (that have WT AHR) compared to H/W rats (whose AHR has a large deletion in the transactivation domain). It is unlikely that mRNA downregulation by dioxins is mediated by miRNAs, nor are miRNAs likely to play a significant role in dioxin toxicity in adult rodent liver.
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