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BMC genomics 11
Aryl hydrocarbon receptor (AHR)-regulated transcriptomic changes in rats sensitive or resistant to major dioxin toxicities.
BACKGROUND: 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, b... expand abstractut 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. RESULTS: 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. CONCLUSION: 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. collapse abstract
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Toxicological sciences : an official journal of the Society of Toxicology 2009 Oct; 112(1)
Dioxin-dependent and dioxin-independent gene batteries: comparison of liver and kidney in AHR-null mice.
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. ... expand abstractAlthough 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. collapse abstract
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Toxicology and applied pharmacology 2009 Apr; 236(2)
Transgenic mouse lines expressing rat AH receptor variants--a new animal model for research on AH receptor function and dioxin toxicity mechanisms.
Han/Wistar (Kuopio; H/W) rats are exceptionally resistant to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity mainly because of their mutated aryl hydrocarbon receptor (AHR) gene. In H/W rats, altered splicing of the AHR mRNA generates two AHR pro... expand abstractteins: deletion (DEL) and insertion (INS) variants, with the INS isoform being predominantly expressed. To gain further insight into their functional properties, cDNAs of these and rat wild-type (rWT) isoform were transferred into C57BL/6J-derived mice by microinjection. The endogenous mouse AHR was eliminated by selective crossing with Ahr-null mice. A single mouse line was obtained for each of the three constructs. The AHR mRNA levels in tissues were generally close to those of C57BL/6 mice in INS and DEL mice and somewhat higher in rWT mice; in testis, however, all 3 constructs exhibited marked overexpression. The transgenic mouse lines were phenotypically normal except for increased testis weight. Induction of drug-metabolizing enzymes by TCDD occurred similarly to that in C57BL/6 mice, but there tended to be a correlation with AHR concentrations, especially in testis. In contrast to C57BL/6 mice, the transgenics did not display any major gender difference in susceptibility to the acute lethality and hepatotoxicity of TCDD; rWT mice were highly sensitive, DEL mice moderately resistant and INS mice highly resistant. Co-expression of mouse AHR and rWT resulted in augmented sensitivity to TCDD and abolished the natural resistance of female C57BL/6 mice, whereas mice co-expressing mouse AHR and INS were resistant. Thus, these transgenic mouse lines provide a novel promising tool for molecular studies on dioxin toxicity and AHR function. collapse abstract
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BMC genomics 9
Transcriptomic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in liver: comparison of rat and mouse.
BACKGROUND: 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 aromati... expand abstractc 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). RESULTS: 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. CONCLUSION: 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. collapse abstract
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Drug metabolism and disposition: the biological fate of chemicals 2008 Nov; 36(12)
Aryl hydrocarbon receptor-dependent induction of flavin-containing monooxygenase mRNAs in mouse liver.
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 (TCD... expand abstractD) 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. collapse abstract
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Toxicology 2008 Sep; 250(2-3)
Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on heme oxygenase-1, biliverdin IXalpha reductase and delta-aminolevulinic acid synthetase 1 in rats with wild-type or variant AH receptor.
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 cl... expand abstracteaved to biliverdin by the rate-limiting enzyme heme oxygenase-1 (HO-1). Subsequently, biliverdin IXalpha reductase (BVRA) catalyzes the reduction of biliverdin to bilirubin. In heme biosynthesis, the rate-limiting enzyme is delta-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, 300 microg/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 100 microg/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. collapse abstract
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Archives of toxicology 2008 Oct; 82(11)
Patterns of dioxin-altered mRNA expression in livers of dioxin-sensitive versus dioxin-resistant rats.
Dioxins exert their major toxicologic effects by binding to the aryl hydrocarbon receptor (AHR) and altering gene transcription. Numerous dioxin-responsive genes previously were identified both by conventional biochemical and molecular techniques and... expand abstract by recent mRNA expression microarray studies. However, of the large set of dioxin-responsive genes the specific genes whose dysregulation leads to death remain unknown. To identify specific genes that may be involved in dioxin lethality we compared changes in liver mRNA levels following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in three strains/lines of dioxin-sensitive rats with changes in three dioxin-resistant rat strains/lines. The three dioxin-resistant strains/lines all harbor a large deletion in the transactivation domain of the aryl hydrocarbon receptor (AHR). Despite this deletion, many genes exhibited a "Type-I" response-that is, their responses were similar in dioxin-sensitive and dioxin-resistant rats. Several genes that previously were well established as being dioxin-responsive or under AHR regulation emerged as Type-I responses (e.g. CYP1A1, CYP1A2, CYP1B1 and Gsta3). In contrast, a relatively small number of genes exhibited a Type-II response-defined as a difference in responsiveness between dioxin-sensitive and dioxin-resistant rat strains. Type-II genes include: malic enzyme 1, ubiquitin C, cathepsin L, S-adenosylhomocysteine hydrolase and ferritin light chain 1. In silico searches revealed that AH response elements are conserved in the 5'-flanking regions of several genes that respond to TCDD in both the Type-I and Type-II categories. The vast majority of changes in mRNA levels in response to 100 microg/kg TCDD were strain-specific; over 75% of the dioxin-responsive clones were affected in only one of the six strains/lines. Selected genes were assessed by quantitative RT-PCR in dose-response and time-course experiments and responses of some genes were assessed in Ahr-null mice to determine if their response was AHR-dependent. Type-II genes may lie in pathways that are central to the difference in susceptibility to TCDD lethality in this animal model. collapse abstract
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Toxicology and applied pharmacology 2008 Jun; 230(1)
Genome-wide effects of acute progressive feed restriction in liver and white adipose tissue.
Acute progressive feed restriction (APFR) represents a specific form of caloric restriction in which feed availability is increasingly curtailed over a period of a few days to a few weeks. It is often used for control animals in toxicological and pha... expand abstractrmacological studies on compounds causing body weight loss to equalize weight changes between experimental and control groups and thereby, intuitively, to also set their metabolic states to the same phase. However, scientific justification for this procedure is lacking. In the present study, we analyzed by microarrays the impact on hepatic gene expression in rats of two APFR regimens that caused identical diminution of body weight (19%) but differed slightly in duration (4 vs. 10 days). In addition, white adipose tissue (WAT) was also subjected to the transcriptomic analysis on day-4. The data revealed that the two regimens led to distinct patterns of differentially expressed genes in liver, albeit some major pathways of energy metabolism were similarly affected (particularly fatty acid and amino acid catabolism). The reason for the divergence appeared to be entrainment by the longer APFR protocol of peripheral oscillator genes, which resulted in derailment of circadian rhythms and consequent interaction of altered diurnal fluctuations with metabolic adjustments in gene expression activities. WAT proved to be highly unresponsive to the 4-day APFR as only 17 mRNA levels were influenced by the treatment. This study demonstrates that body weight is a poor proxy of metabolic state and that the customary protocols of feed restriction can lead to rhythm entrainment. collapse abstract
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Toxicological sciences : an official journal of the Society of Toxicology 2007 Sep; 99(2)
microRNAs in adult rodent liver are refractory to dioxin treatment.
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 al... expand abstractso 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. collapse abstract
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Molecular pharmacology 2007 Sep; 72(4)
Aryl hydrocarbon receptor splice variants in the dioxin-resistant rat: tissue expression and transactivational activity.
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... expand abstract effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) because of a point mutation in the exon/intron 10 boundary in AHR genomic structure that leads to use of 3 alternative cryptic splice sites, potentially creating 3 alternative transcripts and 2 protein products. The deletion variant (DV), which lacks 43 amino acids in the transactivation domain, has the highest intrinsic transactivation activity in vitro; amino acids 766 to 783 suppress transactivation function. However, DV expression levels in H/W rats in vivo are low in liver, lung, thymus, kidney, and testis; insertion variant mRNAs (IVs) are the dominant mRNA forms in H/W rats in which wild-type AHR mRNA is undetectable. In dioxin-sensitive rat strains and lines that are homozygous for wild-type AHR alleles, wild-type AHR mRNA is the most abundant transcript but some IV transcripts are detectable. TCDD treatment in vivo increases transcript levels for both the DV and IVs in H/W rats and increases wild-type transcript levels in dioxin-sensitive rats but does not alter which transcript forms are expressed. In silico modeling indicates that the DV mRNA has lost considerable secondary structure, whereas at the protein level, the transactivation domain of the IV in the dioxin-resistant H/W rat has greater alpha-helical content and a more hydrophobic terminus than wild-type AHR, which may produce a protein conformation that is less amenable to interaction with other regulatory proteins. collapse abstract
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Toxicology 2007 Jun; 235(1-2)
Differences in acute toxicity syndromes of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin in rats.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent congener of polychlorinated dibenzo-p-dioxins. The potency of 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) is only 10% of that of TCDD for typical aryl hydrocarbon receptor (AHR)-mediate... expand abstractd effects. Acute lethality, macroscopic effects, and liver toxicity of TCDD and HxCDD were compared in male rats of the strain Han/Wistar (Kuopio; H/W), and of the lines A and B. The latter two rat lines originate from crossbreeding of H/W and Long-Evans (Turku/AB) rats. H/W and line A rats are highly resistant to acute toxicity of TCDD due to an altered AHR, while line B rats are moderately resistant due to H/W-type alleles of another, yet unidentified gene contributing to TCDD resistance ("gene B"). The rats received 200-10,000 microg/kg of either TCDD or HxCDD intragastrically and were monitored for 46 days. In all rats, the highest dose of HxCDD (10,000 microg/kg) reduced body weight more effectively than an identical dose of TCDD. Only HxCDD (10,000 microg/kg) caused gastrointestinal hemorrhage, pale (fatty) livers and death by day 15 in H/W and line A rats. In line B rats, HxCDD caused pronounced hepatic fatty degeneration, whereas TCDD induced hepatic accumulation of biliverdin and its derivatives. Both congeners induced sinusoidal distension in liver. In H/W and line A rats, the estimated LD(50) values were >10,000 microg/kg and 2000-10,000 microg/kg for TCDD and HxCDD, respectively; for line B rats they were 480 microg/kg and 1000-2000 microg/kg, respectively. Thus, HxCDD was more potent than TCDD in inducing acute mortality in H/W and line A rats, contrary to what is predicted by toxic equivalency factor (TEF) values. In line B, the expected rank order of potencies prevailed. These findings suggest that in addition to the canonical AHR-mediated toxic pathways, HxCDD possesses an AHR-independent mechanism of toxicity, whose main manifestations are rapid body weight loss, mortality, fatty liver and gastrointestinal hemorrhage. collapse abstract
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Basic & clinical pharmacology & toxicology 2006 Mar; 98(4)
Assessment by c-Fos immunostaining of changes in brain neural activity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and leptin in rats.
The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes multiple effects in laboratory animals. One of these is a wasting syndrome (a dramatic loss of body weight over 2-5 weeks) whose mechanism is still largely unknown. We explo... expand abstractited the over 1000 times difference in TCDD sensitivity between Long-Evans (Turku/AB); (L-E) and Han/Wistar (Kuopio); (H/W) rats to reveal brain areas that might be activated by a single dose of TCDD (50 microg/kg) given 24 hr previously. Leptin (1.3 mg/kg intraperitoneally 2 hr before tissue harvest) was used as a reference compound, as its neural pathway for decreasing food intake in the control of energy homeostasis is fairly well known. Serial sections of the brains were immunostained with an antibody for the activity marker c-Fos, and selected areas -- primarily in the hypothalamus -- were analysed with a computer-assisted microscope. Given alone, TCDD did not elicit any major alterations in c-Fos protein levels in the hypothalamic nuclei at the early time-point studied (24 hr after administration), neither in pooled data nor in individual strains. The control substance leptin proved that the method is valid as it increased the number of c-Fos-immunopositive cells in the hypothalamic ventromedial and arcuate nuclei. Although the present findings are not suggestive of a primary role for the hypothalamus in the wasting syndrome, a time-course study covering also the feeding-active dark hours is warranted for their verification. collapse abstract
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Molecular & cellular proteomics : MCP 2006 Apr; 5(5)
Differential expression profiling of the hepatic proteome in a rat model of dioxin resistance: correlation with genomic and transcriptomic analyses.
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... expand abstract 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. collapse abstract
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Chemico-biological interactions 2006 Mar; 160(2)
Evaluation of various housekeeping genes for their applicability for normalization of mRNA expression in dioxin-treated rats.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is an extremely sensitive, convenient and rapid method to measure mRNA levels in cells and tissues, and is gaining popularity in toxicology. To correct for sample-to-sample variat... expand abstraction, normalization of the expression data is required. The conventional way to perform normalization is to select a reference gene whose expression is believed to remain stable across all experimental conditions, then relate the concentrations of gene(s) of interest to those of this housekeeping gene. Since recent evidence shows that some housekeeping genes are actually not as refractory to experimental manipulations as previously thought, we validated a large number (18) of commonly used housekeeping genes for acute toxicity studies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an extremely potent environmental toxin known to regulate a wide variety of genes. Microarray and qRT-PCR analyses coherently demonstrated that about 50% of the housekeeping genes examined were responsive to TCDD in rat liver with the magnitudes of change up to nearly 10-fold. Extension of the study to spleen and hypothalamus verified that phosphoglycerate kinase 1 (Pgk1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) retained their basal expression levels in all experimental settings, although body weight loss-generated repression may mask a slight induction of GAPDH by TCDD in liver. These findings show that normalization genes for qRT-PCR must be carefully validated in advance, especially if the study involves a potent modifier of gene expression. collapse abstract
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Journal of biochemical and molecular toxicology 19(3)
Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and leptin on hypothalamic mRNA expression of factors participating in food intake regulation in a TCDD-sensitive and a TCDD-resistant rat strain.
An acutely toxic dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to a drastically and permanently reduced feed intake and wasting by an unknown mechanism. We focused on the possible interference of TCDD with hypothalamic factors known to tak... expand abstracte part in the regulation of eating and metabolism, utilizing the over 1000-fold TCDD-sensitivity difference between Long-Evans (Turku/AB; L-E) and Han/Wistar (Kuopio) rats. The mRNA expression of 18 hypothalamic factors (including NPY, AgRP, and CART) was measured by quantitative RT-PCR at 6, 24 and 96 h after TCDD administration. The effects of TCDD were compared with those of leptin and with feed restriction employing a TCDD dose that elicited a severe reduction of feed intake in L-E rats. TCDD mainly modified expression of orexigenic factors causing an initial suppression followed by reversal to enhanced expression by 96 h. The latter was also seen in feed-restricted controls. In contrast, leptin altered both orexigenic and anorexigenic factor mRNAs in a more even manner and its effects were clustered at 6 h. The transient nature of feeding-promoting factor suppression does not strongly support a key role for this phenomenon in TCDD-induced wasting syndrome. However, the fact that TCDD mainly affected orexigenic factors and the temporal differences in response found between the rat strains warrant further research. collapse abstract
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Molecular pharmacology 2005 Dec; 69(1)
Aryl hydrocarbon receptor regulates distinct dioxin-dependent and dioxin-independent gene batteries.
Conventional biochemical and molecular techniques identified previously several genes whose expression is regulated by the aryl hydrocarbon receptor (AHR). We sought to map the complete spectrum of AHR-dependent genes in male adult liver using expres... expand abstractsion arrays to contrast mRNA profiles in Ahr-null mice (Ahr(-/-)) with those in mice with wild-type AHR (Ahr(+)(/)(+)). Transcript profiles were determined both in untreated mice and in mice treated 19 h earlier with 1000 microg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Expression of 456 ProbeSets was significantly altered by TCDD in an AHR-dependent manner, including members of the classic AHRE-I gene battery, such as Cyp1a1, Cyp1a2, Cyp1b1, and Nqo1. In the absence of exogenous ligand, AHR status alone affected expression of 392 ProbeSets, suggesting that the AHR has multiple functions in normal physiology. In Ahr(-/-) mice, only 32 ProbeSets exhibited responses to TCDD, indicating that the AHR is required for virtually all transcriptional responses to dioxin exposure in liver. The flavin-containing monooxygenases, Fmo2 and Fmo3, considered previously to be uninducible, were highly induced by TCDD in an AHR-dependent manner. The estrogen receptor alpha as well as two estrogen-receptor-related genes (alpha and gamma) exhibit AHR-dependent expression, thereby extending cross-talk opportunities between the intensively studied AHR and estrogen receptor pathways. p53 binding sites are over-represented in genes down-regulated by TCDD, suggesting that TCDD inhibits p53 transcriptional activity. Overall, our study identifies a wide range of genes that depend on the AHR, either for constitutive expression or for response to TCDD. collapse abstract
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Toxicology and applied pharmacology 2005 Aug; 207(2 Suppl)
Toxicological implications of polymorphisms in receptors for xenobiotic chemicals: the case of the aryl hydrocarbon receptor.
Mechanistic toxicology has predominantly been focused on adverse effects that are caused by reactive metabolites or by reactive oxygen species. However, many important xenobiotics exert their toxicity, not by generating reactive products, but rather ... expand abstractby altering expression of specific genes. In particular, some environmental contaminants target nuclear receptors that function as regulators of transcription. For example, binding of xenobiotic chemicals to steroid receptors is a principle mechanism of endocrine disruption. The aryl hydrocarbon receptor (AHR) mediates toxicity of dioxin-like compounds. In mice, a polymorphism in the AHR ligand-binding domain reduces binding affinity by about 10-fold in the DBA/2 strain compared with the C57BL/6 strain; consequently, dose-response curves for numerous biochemical and toxic effects are shifted about one log to the right in DBA/2 mice. In the Han/Wistar (Kuopio) (H/W) rat strain, a polymorphism causes a deletion of 38 or 43 amino acids from the AHR transactivation domain. This deletion is associated with a greater than 1000-fold resistance to lethality from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Genes in the conventional AH gene battery (e.g. CYP1A1, CYP1A2, CYP1B1, ALDH3A1, NQO1 and UGT1A1) remain responsive to TCDD in H/W rats despite the large deletion. However, the deletion may selectively alter the receptor's ability to dysregulate specific genes that are key to dioxin toxicity. We are identifying these genes using an expression array approach in dioxin-sensitive vs. dioxin-resistant rat strains and lines. Polymorphisms exist in the human AH receptor, but thus far they have not been shown to have any substantial effect on human responses to AHR-ligands. collapse abstract
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Toxicology and applied pharmacology 2006 Apr; 212(3)
Interference by 2,3,7,8-tetrachlorodibenzo-p-dioxin with cultured mouse submandibular gland branching morphogenesis involves reduced epidermal growth factor receptor signaling.
Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to mouse embryonic teeth, sharing features of early development with salivary glands in common, involves enhanced apoptosis and depends on the expression of epidermal growth factor (EGF) receptor... expand abstract. EGF receptor signaling, on the other hand, is essential for salivary gland branching morphogenesis. To see if TCDD impairs salivary gland morphogenesis and if the impairment is associated with EGF receptor signaling, we cultured mouse (NMRI) E13 submandibular glands with TCDD or TCDD in combination with EGF or fibronectin (FN), both previously found to enhance branching morphogenesis. Explants were examined stereo-microscopically and processed to paraffin sections. TCDD exposure impaired epithelial branching and cleft formation, resulting in enlarged buds. The glands were smaller than normal. EGF and FN alone concentration-dependently stimulated or inhibited branching morphogenesis but when co-administered with TCDD, failed to compensate for its effect. TCDD induced cytochrome P4501A1 expression in the glandular epithelium, indicating activation of the aryl hydrocarbon receptor. TCDD somewhat increased epithelial apoptosis as observed by terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labeling method but the increase could not be correlated with morphological changes. The frequency of proliferating cells was not altered. Corresponding to the reduced cleft sites in TCDD-exposed explants, FN immunoreactivity in the epithelium was reduced. The results show that TCDD, comparably with EGF and FN at morphogenesis-inhibiting concentrations, impaired salivary gland branching morphogenesis in vitro. Together with the failure of EGF and FN at morphogenesis-stimulating concentrations to compensate for the effect of TCDD this implies that TCDD toxicity to developing salivary gland involves reduced EGF receptor signaling. collapse abstract
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Life sciences 2005 Aug; 77(12)
Bone resorption by aryl hydrocarbon receptor-expressing osteoclasts is not disturbed by TCDD in short-term cultures.
Polychlorinated dibenzo-p-dioxins (PCDDs) are highly toxic environmental contaminants, and 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) is the most potent dioxin. Dioxins bind specifically to the cytosolic aryl hydrocarbon receptor (AHR), which is a liga... expand abstractnd-activated transcription factor, and a majority of toxic effects of dioxins are mediated via AHR. We have recently demonstrated that TCDD disrupts bone modeling and decreases bone mechanical strength, and that partial resistance to these effects is related to an altered transactivation domain in AHR structure. In order to better understand the effects of dioxins on bone, we studied the presence and precise localization of AHR and also the number and activity of osteoclasts after TCDD treatments. Total RNA was extracted from mixed bone cell population cultures and expression of AHR mRNA was studied using RT-PCR. Bone cells expressed a considerable amount of AHR mRNA. To see which bone cells express AHR, immunostainings were performed in primary rat bone cell cultures, pure human osteoclast cultures and histological sections from AHR knockout and wild type bones. Immunostaining revealed a strong expression of AHR both in osteoclasts and osteoblasts with an especially prominent stain in bone resorbing osteoclasts. Effects of dioxin on primary bone cells were evaluated after TCDD treatment in the pit formation assay. The activity of osteoclasts was not affected measured as the percentage of active osteoclasts and the actual area of resorbed bone. These data indicate that even though TCDD-treated bones show decreased mechanical strength and size, this is not a direct result from increased osteoclastic bone resorption. collapse abstract
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Toxicology 2005 Feb; 208(1)
Effect of TCDD on mRNA expression of genes encoding bHLHPAS proteins in rat hypothalamus.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) brings about a wide variety of toxic and biochemical effects via an AH receptor (AHR)-mediated signalling pathway. Wasting syndrome and acute lethality are TCDD-induced endpoints showing a striking sensitivi... expand abstractty difference between two rat strains, TCDD-sensitive Long-Evans (Turku/AB) (L-E) and TCDD-resistant Han/Wistar (Kuopio) (H/W). These rat strains were used to study hypothalamic effects of TCDD on expression of genes encoding AHR-regulated bHLH/PAS proteins potentially involved in molecular pathogenesis of the wasting syndrome. In addition, two well-established target genes of TCDD, CYP1A1 and CYP1A2 were also examined. Quantitative RT-PCR was used to measure mRNA levels in hypothalamus, which is a major center of food intake and body weight regulation. At both 6 and 96 h after a single dose of 50 microg/kg TCDD, significant elevations were found in mRNA levels of AHR repressor (AHRR), CYP1A1 and CYP1A2, but not those of AHR, ARNT or ARNT2. Likewise, TCDD (100 microg/kg) did not alter the expression of SIM1, implicated in the suppressive impact of TCDD on food intake, nor that of PER2, involved in regulation of circadian rhythms. Differences between H/W and L-E rats appeared in constitutive levels of AHR and ARNT and in TCDD-induced levels of CYP1A2, AHRR, AHR and ARNT, which all were about two- to four-fold lower in H/W rats. Thus, although the changes found do not account for the wasting syndrome, expression of all principal genes of the AHR-signalling pathway in rat hypothalamus make it a candidate target for TCDD. collapse abstract
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Carcinogenesis 2004 Dec; 26(1)
TCDD activates Mdm2 and attenuates the p53 response to DNA damaging agents.
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... expand abstract 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. collapse abstract
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Biochemical and biophysical research communications 2004 Aug; 321(3)
Dioxin-responsive AHRE-II gene battery: identification by phylogenetic footprinting.
We identified a set of genes that respond to dioxins through the recently discovered AHRE-II ("XRE-II") enhancer element. A total of 36 genes containing AHRE-II motifs conserved across human, mouse, and rat gene orthologs were identified by genome-wi... expand abstractde transcription-factor binding-site searches and phylogenetic footprinting. Microarray experiments on liver from rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin revealed statistically significant changes in mRNA levels for 13 of these 36 genes after three hours and 15 genes after 19h. The set of responsive genes was functionally characterized by ontological analysis and found to be enriched in ion-channels and transporters. Our identification of 36 putatively AHRE-II-regulated genes highlights the regulatory versatility of the aryl hydrocarbon receptor (AHR) and the ability of the AHR and its dimerization partner, ARNT, to act both as a ligand-activated transcription-factor (on AHRE-I) and as a ligand-activated coactivator (on AHRE-II). Collectively, these results demonstrate that the AHRE-II induction mechanism is employed by multiple genes and provide the first categorization of the gene battery of a ligand-activated coactivator. collapse abstract
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Biological chemistry 385(3-4)
Simultaneous exposure of rats to dioxin and carbon monoxide reduces the xenobiotic but not the hypoxic response.
Aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor-1alpha (HIF-1alpha) are conditionally regulated transcription factor subunits that form heterodimeric complexes with their common partner, AhR nuclear translocator (ARNT/HIF-1beta). Whereas... expand abstract the environmentally toxic compound 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD) initiates the trans-activation activity of AhR:ARNT/HIF-1beta, hypoxic exposure stabilizes HIF-1alpha and functionally activates the HIF-1alpha:ARNT/HIF-1beta complex. To analyze a possible crosstalk between these two pathways in vivo, rats were given dioxin orally and/or were exposed to carbon monoxide (CO), causing functional anemia. We found that exposure to CO inhibited the xenobiotic response while dioxin application had no significant negative impact on hypoxia-mediated gene transcription. collapse abstract
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Toxicology letters 2004 May; 150(3)
Effects of epidermal growth factor receptor deficiency and 2,3,7,8-tetrachlorodibenzo-p-dioxin on fetal development in mice.
Dioxins are persistent environmental contaminants that cause multiple disorders in laboratory animals, including teratogenesis. In mice, the most important teratogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are hydronephrosis and cleft... expand abstract palate. Aryl hydrocarbon receptor (AHR) mediates most of the TCDD-induced effects, but modulation of these effects by other factors such as epidermal growth factor receptor (EGFR) has been propounded. TCDD changes the expression of both EGF and its receptor EGFR, which may be one step in the pathway leading to cleft palate and hydronephrosis. In the present study, the importance of EGFR in TCDD-induced teratogenicity was evaluated. Heterozygous EGFR(+/-)-mice were mated and pregnant females exposed to 1.5-106.0 microg/kg TCDD on gestation day (GD) 10 and killed on GD 18. The fetuses were studied for cleft palate, hydronephrosis, and open eyes. There was no marked difference among the three genotypes in sensitivity to cleft palate or hydronephrosis, but in EGFR(-/-)-mice frequency of the open eye malformation decreased dose-dependently. In conclusion, EGFR signaling is not required for TCDD-induced cleft palate or hydronephrosis but TCDD appears to counteract the effect of EGFR deficiency on eye opening. collapse abstract
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Toxicology and applied pharmacology 2004 Mar; 196(1)
Postnatal development of resistance to short-term high-dose toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in TCDD-resistant and -semiresistant rats.
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 toxicit... expand abstracty 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 microg/kg; male and female B rats received 200 and 100 microg/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. collapse abstract
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