Raimo Pohjanvirta

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 expression 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 μg/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 α as well as two estrogen-receptor-related genes (α and γ) 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.

Target tissue morphology and serum biochemistry following 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in a TCDD-susceptible and a TCDD-resistant rat strain.

The mode of action of the highly toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is unknown. It was recently discovered that two strains of rat, Long-Evans (L-E) and Han/Wistar (H/W), differ widely in susceptibility to TCDD. Employing this strain divergence as a probe, the present study set out to assess the role of various biochemical and morphological effects in TCDD lethality. In the main experiment, the rats were treated once ip with 0,5,50, or (H/W) 500 micrograms/kg TCDD and killed 1 to 16 days postexposure. Several target organs were evaluated by light microscopy and a number of serum lipid and carbohydrate parameters as well as a few major regulatory hormones were analyzed. The results demonstrated that most alterations caused by TCDD were essentially similar in both strains. TCDD reduced circulating thyroxine to a slightly greater extent and more permanently in the sensitive L-E strain. Moreover, a highly significant interaction on thyroid-stimulating hormone was found among strain, dose, and time. Serum concentrations of corticosterone and free fatty acids were increased only in the L-E rats given 50 micrograms/kg TCDD, i.e., at an apparent LD100 dose level for this strain. Yet, the most striking interstrain difference was seen in the liver which was distinctly affected after Day 4 in L-E rats given 50 micrograms/kg TCDD but only marginally affected in rats from any H/W group. The lesion, while showing no necrotic cell changes, was suggestive of plasma membrane damage, possibly reflecting the production of free radicals. The relation of the findings to possible mechanisms of TCDD action is discussed.

Hepatic Ah-receptor levels and the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatic microsomal monooxygenase activities in a TCDD-susceptible and -resistant rat strain

Previous studies have shown that in two inbred strains of mice, straightforward correlations exist among the number of hepatic Ah-receptors, enzyme inducibility by TCDD, and lethality of TCDD. Here, studies were conducted in two strains of rats (Han/Wistar and Long-Evans) which differ widely in susceptibility to the lethal effects of TCDD, to determine if these are general phenomenona in TCDD toxicity. The total number of specific binding sites (Ah-receptors) for [3H]TCDD proved to be approximately equal in the livers of both rat strains. Likewise, no notable difference was detected in the effect of TCDD on the activities of 7-ethoxyresorufin O-deethylase, 7-ethoxycoumarin O-deethylase, and ethylmorphine N-demethylase or on the amount of cytochrome P-450 in hepatic microsomal fractions. Immunoblot analysis was carried out with monoclonal antibodies (Mabs). Mab 1-7-1 directed against rat liver 3-methylcholanthrene (MC)-inducible P-450 recognized forms P-450c and P-450d in TCDD-treated rats in a dose-dependent fashion and to a similar extent in both strains. In contrast, Mab 2-66-3 (against phenobarbital-inducible P-450) did not recognize any proteins in either strain, confirming the conclusion that TCDD elicits a MC-type induction of hepatic cytochrome P-450 in both strains of rats. Thus, it seems that the correlations observed in mice do not hold in rats and therefore should not be generalized. The parameters measured in the present study are causally unrelated to the mechanism of lethal action of TCDD in these rat strains.

Dioxins, the aryl hydrocarbon receptor and the central regulation of energy balance

Dioxins are ubiquitous environmental contaminants that have attracted toxicological interest not only for the potential risk they pose to human health but also because of their unique mechanism of action. This mechanism involves a specific, phylogenetically old intracellular receptor (the aryl hydrocarbon receptor, AHR) which has recently proven to have an integral regulatory role in a number of physiological processes, but whose endogenous ligand is still elusive. A major acute impact of dioxins in laboratory animals is the wasting syndrome, which represents a puzzling and dramatic perturbation of the regulatory systems for energy balance. A single dose of the most potent dioxin, TCDD, can permanently readjust the defended body weight set-point level thus providing a potentially useful tool and model for physiological research. Recent evidence of response-selective modulation of AHR action by alternative ligands suggests further that even therapeutic implications might be possible in the future.

Transcriptomic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in liver: Comparison of rat and mouse

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.

Exposure to 2,3,7,8-tetrachlorodibenzo-para-dioxin leads to defective dentin formation and pulpal perforation in rat incisor tooth

A single dose of 1000 micrograms 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg body weight was given to TCDD-resistant (Han/Wistar) young adult male rats. Changes in the skulls and the continuously erupting incisor teeth were evaluated 16 weeks after the administration of TCDD. The skulls of the experimental rats (N = 11) were significantly smaller than those of the control rats (N = 11), and the upper and lower incisor teeth of all experimental rats were significantly thinner than the control rat teeth. The pulps of the lower incisors of all experimental rats were lingually exposed to the oral cavity at their incisal ends. Also in 3 cases the pulps of the upper incisors were exposed, but never in the control rats. Whereas the labial surfaces of the incisors of the control rats were brown, those of the experimental rat teeth appeared greyish and mottled. Histological examination revealed that the pulp chambers in the incisal halves of the affected teeth were larger than normal, at the expense of the thickness of dentin. Towards the incisal tooth ends, odontoblasts gradually lost their polarity and the pulp tissue became necrotic. A dentin zone next to the pulp chambers was irregular. Lingual tapering of the teeth was pronounced, which gave them a mesiodistally flattened appearance. The superficial zone of the otherwise regular enamel was poorly pigmented. In conclusion, a single injection of TCDD was shown to impair normal growth of the skull and incisor tooth formation in rats. The small size of the incisors, their aberrant shape and the defective dentin (and enamel) formation could be mediated by vitamin A metabolism, known to be interfered with by TCDD.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on liver phosphoenolpyruvate carboxykinase (PEPCK) activity, glucose homeostasis and plasma amino acid concentrations in the most TCDD-susceptible and the most TCDD-resistant rat strains.

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

In vivo up-regulation of aryl hydrocarbon receptor expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a dioxin-resistant rat model,

The aryl hydrocarbon receptor (AHR) mediates toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and regulates expression of several genes such as CYP1A1. Little is known about what regulates expression of the AHR itself. We tested the ability of TCDD to alter in vivo expression of its own receptor in rat strains that are susceptible to TCDD lethality [Long-Evans (Turku AB) (L-E) and Sprague Dawley (SD)] and in a rat strain that is remarkably resistant to TCDD lethality [Han/Wistar (Kuopio) (H/W)]. Rats were administered a single, intragastric dose of 5 or 50 microg/kg of TCDD. Hepatic cytosol, nuclear extract, and RNA were prepared at 1, 4, and 10 days after TCDD exposure. AHR expression was assessed at three levels: ligand binding function, immunoreactive protein and mRNA. TCDD at 5 microg/kg produced a 2- to 3-fold increase in cytosolic AHR in all strains; 50 microg/kg produced depletion at day 1 followed by recovery in SD and H/W but not L-E rats. Both the increase in AHR above basal levels and the recovery from initial depletion were accompanied by elevations in steady-state AHR mRNA, suggesting a pre-translational mechanism for AHR regulation by its own ligand. This up-regulation in vivo is in contrast to the sustained depletion of AHR caused by TCDD in cell culture. There was no clear relationship between AHR regulation and strain sensitivity; thus, the large inherent strain differences in susceptibility to TCDD lethality probably are not explained by differential regulation of AHR by TCDD.

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 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.

Biochemical effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds on the central nervous system

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds are an important class of environmental contaminants which induce several types of biochemical alterations. Their effects have been most thoroughly characterized in the liver, especially regarding the Ah receptor-mediated induction of xenobiotic metabolizing enzymes. The behavioral signs exhibited by animals exposed to TCDD (progressive anorexia and body weight loss) suggest a role for the central nervous system (CNS) in TCDD toxicity. At lethal doses, TCDD affects the metabolism of serotonin, a neurotransmitter able to modulate food intake in the brain. This effect is associated with an elevated concentration of free tryptophan in the plasma. There does not appear to be any major changes in catecholaminergic neurotransmitter systems in TCDD-treated rats. Cytochrome P-450 related enzyme activities are induced by TCDD in the brain. As is the case in the liver, this induction does not correlate with susceptibility to TCDD lethality in rats. The involvement of the CNS in TCDD toxicity is still obscure. Elucidation of this role as well as the mechanism of TCDD-induced wasting may well advance our understanding of the regulation of food intake and body weight.