Abstract The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxicity of dioxins, but also plays important physiological roles, which are only beginning ...to unfold. Previous studies have surprisingly unveiled that low doses of the potent AHR agonist TCDD induce a strong and persistent avoidance of novel food items in rats. Here, we further examined the involvement of the AHR in the avoidance response in Sprague-Dawley rats with three established AHR agonists: 6-formylindolo(3,2-b)carbazole (FICZ), β-naphthoflavone (BNF) and benzo a pyrene (BaP); with a novel selective AHR modulator (C2); and with an activator of another nuclear receptor, CAR: 2,4,6-tryphenyldioxane-1,3 (TPD). As sensitive indices of AHR or CAR activity, we used Cyp1a1 and Cyp2b1 gene expression, as they are, respectively, the drug-metabolizing enzymes specifically regulated by them. We further attempted to address the roles played by enhanced neophobia and conditioned taste aversion (CTA) in the avoidance behaviour. All AHR agonists triggered practically total avoidance of novel chocolate, but the durations varied. Likewise, acutely subtoxic doses of C2, differing by 25-fold, all elicited a similar outcome. In contrast, TPD did not influence chocolate consumption at all. If rats were initially accustomed to chocolate for 6 h after single FICZ or BNF exposure, avoidance was still clearly present two weeks later when chocolate was offered again. Hence, the avoidance response appears to specifically involve the AHR instead of being triggered by induction of intestinal or hepatic nuclear receptor signalling in general. It is also shared by both endogenous and exogenous AHR activators. Moreover, this behavioural change in rats seems to contain elements of both CTA and enhanced neophobia, but further clarification of this is still required.
Flavin-containing monooxygenases (FMOs) are important in detoxication but generally are considered not to be inducible by xenobiotics. Our recent microarray studies revealed induction of FMO2 and ...FMO3 mRNAs by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in liver of mice with wild-type aryl hydrocarbon receptor (AHR) but not in Ahr-null mice. The aim of the present study was to delineate mechanisms of FMO regulation. In adult male mice, basal FMO3 mRNA is low but was induced 6-fold at 4 h and 6000-fold at 24 h. The ED50 was approximately 1 microg/kg for FMO2 and FMO3, similar to that for the classic AHR-regulated gene, Cyp1a1. In adult female mice basal FMO3 mRNA is high and was not induced at 4 h but was elevated 8-fold at 24 h. FMO5 mRNA was significantly down-regulated by TCDD in both male and female adult mice. Juvenile mice show no sex difference in response to TCDD; FMO3 was induced 4 to 6-fold by TCDD in both sexes. Chromatin immunoprecipitation demonstrated recruitment of AHR and aryl hydrocarbon nuclear translocator proteins to Fmo3 regulatory regions, suggesting that induction by TCDD is a primary AHR-mediated event. Although FMO2 and FMO3 mRNAs were highly induced by TCDD in adult males, overall FMO catalytic activity increased only modestly. In contrast to the striking up-regulation of FMO2 and FMO3 in mouse liver, TCDD has little effect on FMO mRNA in rat liver. However, FMO2 and FMO3 mRNAs were highly induced in transgenic mice that express wild-type rat AHR, indicating that lack of induction in rat is not due to an incompetent AHR in this species.
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 (100μg/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.
In this study we investigated the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the p53 response to DNA damaging agents. Pre-treatment of rats with TCDD attenuated the p53 liver response to ...diethylnitrosamine (DEN) and reduced levels of p53 and Ser15 phosphorylated p53. In addition, there were more slowly migrating p53 species, forming a ladder, which suggests an increased ubiquination of p53 in TCDD-pre-treated rats. Terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labelling analysis indicated decreased apoptosis rates in the livers of these rats. Studies on aryl hydrocarbon receptor (AhR) knockout mice and their wild-type littermates confirmed this effect in AhR +/+ but not in AhR −/− mice, indicating that this effect may be AhR-mediated. Quantitative RT-PCR analysis revealed no increased mRNA levels in TCDD-treated rats, but immunohistological studies indicated that TCDD modulated Mdm2 protein levels, and in particular, increased nuclear levels in rat hepatocytes in situ. In vitro studies employing HepG2 cells confirmed the in vivo data. Thus, TCDD increased basal levels of Mdm2 protein, but not mRNA, and attenuated the p53 response to a variety of genotoxic and cytotoxic agents. The increase in Mdm2 protein levels was accompanied by rapid and highly sensitive phosphorylation of Mdm2 at Ser166, which has been associated to active Mdm2. In summary, TCDD is a potent inhibitor of p53 that may influence the liver's ability to handle genotoxic agents in a safe way, and may play a role in TCDD-induced carcinogenesis.
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.
AHR in energy balance regulation Pohjanvirta, Raimo
Current opinion in toxicology,
February 2017, 2017-02-00, Letnik:
2
Journal Article
Recenzirano
Recent studies on mice genetically modified at the Ahr locus and fed on high-fat diet have revealed a novel physiological role for the AHR in energy balance. Globally impaired function of the ...receptor counteracts the development of obesity by increasing energy expenditure, which appears to occur mostly in the skeletal muscle and brown adipose tissue. On the other hand, global and tissue-specific loss of AHR signaling can have opposite effects on liver fat content and their impact on insulin sensitivity is also context-dependent. As tryptophan metabolites are key AHR activators, these findings suggest that the AHR may act as a protein sensor enabling adequate protein intake from low-protein diets by allowing calorie overfeeding without resultant obesity.
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One characteristic feature of acute 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity is dramatic interspecies and interstrain variability in sensitivity. This complicates dioxin risk assessment ...for humans. However, this variability also provides a means of characterizing mechanisms of dioxin toxicity. Long-Evans (Turku/AB) rats are orders of magnitude more susceptible to TCDD lethality than Han/Wistar (Kuopio) rats, and this difference constitutes a very useful model for identifying mechanisms of dioxin toxicity. We adopted a proteomic approach to identify the differential effects of TCDD exposure on liver protein expression in Han/Wistar rats as compared with Long-Evans rats. This allows determination of which, if any, protein markers are indicative of differences in dioxin susceptibility and/or responsible for conferring resistance. Differential protein expression in total liver protein was assessed using two-dimensional gel electrophoresis, computerized gel image analysis, in-gel digestion, and mass spectrometry. We observed significant changes in the abundance of several proteins, which fall into three general classes: (i) TCDD-independent and exclusively strain-specific (e.g. isoforms of the protein-disulfide isomerase A3, regucalcin, and agmatine ureohydrolase); (ii) strain-independent and only dependent on TCDD exposure (e.g. aldehyde dehydrogenase 3A1 and rat selenium-binding protein 2); (iii) dependent on both TCDD exposure and strain (e.g. oxidative stress-related proteins, apoptosis-inducing factor, and MAWD-binding protein). By integrating transcriptomic (microarray) data and genomic data (computational search of regulatory elements), we found that protein expression levels were mainly controlled at the level of transcription. These results reveal, for the first time, a subset of hepatic proteins that are differentially regulated in response to TCDD in a strain-specific manner. Some of these differential responses may play a role in establishing the major differences in TCDD response between these two strains of rats. As such, our work is expected to lead to new insights into the mechanism of TCDD toxicity and resistance.
The AHR locus encodes the aryl hydrocarbon receptor (AHR), a transcriptional regulator of multiple drug-metabolizing enzymes and
mediator of toxicity of dioxin-like chemicals. The Han/Wistar (Kuopio) ...rat strain (H/W) is remarkably resistant to lethal
effects of 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) because of a point mutation in the exon/intron 10 boundary in AHR genomic structure that leads to use of 3 alternative cryptic splice sites, potentially creating 3 alternative transcripts
and 2 protein products. The deletion variant (DV), which lacks 43 amino acids in the transactivation domain, has the highest
intrinsic transactivation activity in vitro; amino acids 766 to 783 suppress transactivation function. However, DV expression
levels in H/W rats in vivo are low in liver, lung, thymus, kidney, and testis; insertion variant mRNAs (IVs) are the dominant
mRNA forms in H/W rats in which wild-type AHR mRNA is undetectable. In dioxin-sensitive rat strains and lines that are homozygous
for wild-type AHR alleles, wild-type AHR mRNA is the most abundant transcript but some IV transcripts are detectable. TCDD treatment in vivo
increases transcript levels for both the DV and IVs in H/W rats and increases wild-type transcript levels in dioxin-sensitive
rats but does not alter which transcript forms are expressed. In silico modeling indicates that the DV mRNA has lost considerable
secondary structure, whereas at the protein level, the transactivation domain of the IV in the dioxin-resistant H/W rat has
greater α-helical content and a more hydrophobic terminus than wild-type AHR, which may produce a protein conformation that
is less amenable to interaction with other regulatory proteins.
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