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.
•The genotoxicity of selected commercial food products in Finland was evaluated.•Some food products showed potential as sources of both direct and indirect mutagens.•The mutagenic impact was not due ...to histidine release.•No DNA damage was detected in HepG2 cells by food extracts with mutagenic potential.•Genotoxicity was not confined to a particular food type or method of processing.
Processed foods are an insufficiently characterized source of chemical mutagens for consumers. Here, we evaluated the genotoxicity of selected food products in Finland. Mutagenicity was determined by the standard plate incorporation assay followed by methylcellulose overlay and treat-and-wash assays, using the Salmonella strains TA 100 and 98 with and without metabolic activation. Generally, the mutagenic activity of food samples was low, but exhibited lot-wise variation. Cold cuts of cold-smoked beef, grilled turkey, and smoked chicken (a single batch of each) were mutagenic in all three assays with the TA 100 strain with and without metabolic activation, indicating the mutagenic effect was not secondary to histidine release from the food products. However, none of the food extracts showing mutagenic potential induced DNA damage in vitro using the Comet Assay. Our findings imply that in Finland today, there are still products the production methods of which should be refined to reduce the potential risk of mutagenicity to consumers.
Aims: Environmental pollutants appearing in wastewater, bottled mineral water, tap water, and bottled drinking water are potential, but yet poorly characterized, sources of human exposure to ...endocrine disrupting chemicals globally. Here, we investigated the current situation in the most densely populated region in Finland. Methods: Influent and effluent bi-monthly samples from a major wastewater treatment plant in Helsinki were obtained over a preceding 2-year period at two timepoints (in 2011 and 2014). Equivalent samples from a household water purification plant (located in the same region) were also analyzed, together with various brands of bottled still and mineral water as well as tap water from residential buildings. Samples were obtained in one liter sterile containers, extracted by solid-phase extraction method, and their estrogenic potential determined by a yeast bioluminescent assay. Results: The estrogenic activities of influent samples from the wastewater treatment plant in Helsinki were generally low (from less than limit of detection to 0.7 ng/L estrogen equivalent quantities (EEQ))3 except in March and August 2011, when relatively high levels (14.0 and 7.8 ng/L EEQ, respectively) were obtained. Meanwhile, no estrogenic activity was recorded in any of the treated effluent samples from the wastewater treatment plant, influent and effluent samples from the drinking water plant, as well as tap water, bottled still, and mineral waters. Conclusions: These findings indicate that the purification method applied in Helsinki wastewater treatment plant, activated sludge with mechanical, chemical and biological purification steps, is effective in reducing estrogenic activity, and that tap or bottled waters are not a significant source of these compounds to the population in this region.
Mounting evidence of the effects of endocrine-disrupting chemicals (EDCs) in humans has led to assaying a vast array of food items (processed or packaged) as possible sources of human exposure to ...estrogens. In this study, we investigated the current situation in this respect of different food supplements and beer brands. Eleven food supplements and 24 beer brands were obtained from Helsinki, Finland. Sample preparation was carried out by established methods while estrogenic activities were assessed by a yeast bioluminescent assay, using two recombinant yeast strains (Saccharomyces cerevisiae BMAEREluc/ERα and S. cerevisiae BMA64/luc). All the food supplements as well as 81% of the beer samples tested were found to be estrogenic, with estradiol equivalent concentrations of food supplements and beer brands ranging from 7.5 to 11.5 µg/ml and from below detection limits to 43.6 ng/ml, respectively. The estrogenic activities detected in beer samples were not dependent on the beer's alcoholic content, the country of production, or the size of the production brewery. The results of our study imply that both food supplements and beers can be a significant source of human exposure to estrogens. Therefore, further studies and regular surveillance are warranted.
2,3,7,8-Tetrachlorodibenzo- p -dioxin (TCDD) is the most potent dioxin. There are exceptionally wide inter- and intraspecies differences in sensitivity
to TCDD toxicity with Han/Wistar (H/W) ( Kuopio ...) rats being the most resistant mammals tested. A peculiar feature of H/W rats is that despite their unresponsiveness to the
acute lethality of TCDD, their sensitivity to other biological impacts of TCDD (e.g., CYP1A1 induction) is preserved. The
biological effects of TCDD are mediated by the aryl hydrocarbon receptor (AhR). We recently found that the AhR of H/W rats
(about 98 kDa) is smaller than the receptor in other rat strains (106 kDa). In the present study, molecular cloning and sequencing
of the H/W rat AhR revealed that the reason for its smaller size is a deletion/insertion-type change at the 3â² end of exon
10 in the receptor cDNA. This change emanates from a single point mutation at the first nucleotide of intron 10, resulting
in altered mRNA splicing. At the protein level, the mutation leads to a total loss of either 43 or 38 amino acids (with altered
sequence for the last seven amino acids in the latter case) toward the carboxyl-terminal end in the trans -activation domain of the AhR. H/W rats also harbor a point mutation in exon 10 that will cause a Val-to-Ala substitution
in codon 497, but this occurs in a variable region of the AhR. These findings suggest that there is a relatively small region
in the AhR trans -activation domain that may be capable of providing selectivity to its function.
Most of the biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-
p-dioxin (TCDD) are mediated by the bHLH/PAS protein AH receptor (AHR). For regulation of gene activities, AHR dimerizes with ...another member of the bHLH/PAS protein family, AHR nuclear translocator (ARNT). A substrain of Wistar rats, Han/Wistar (
Kuopio) (H/W), is about 1000-fold more resistant to the acute lethality of TCDD than other strains, exemplified by Long–Evans (
Turku/AB) (L–E); the LD50 values for these two strains are >9600 and 10–20
μg/kg, respectively. Previous studies have demonstrated that the major reason for the exceptional TCDD resistance of H/W rats lies in their AHR, which is remodeled at its C-terminal transactivation domain, but there appears to be another contributing gene product. The present study set out to compare the primary structure of ARNT and the closely related ARNT2 proteins in H/W and L–E rats by cDNA cloning. To our surprise, we found several isoforms of these proteins only one of which has previously been reported in rats. All of the isoforms appeared to arise from alternative splicing. For ARNT, isoforms with deletions at exon 5, 3
′ end of exon 6 or 5
′ end of exon 11, or with an insertion at 5
′ end of exon 20 were discovered. There was also interindividual variation in the number of glutamine-encoding codons at 5
′ end of exon 16. The most exciting new variant was revealed for ARNT2, because the insertion found at 5
′ end of exon 19 disrupts the functionally critical transactivation domain in the protein, implying a dominant negative role for this isoform. The relative expression levels of the variants did not differ in the two rat strains, nor did TCDD modify the ratios, suggesting that the variants do not contribute to TCDD resistance. However, the regulation of ARNT and ARNT2 activities may be more intricate than previously assumed.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD),11Some of these results were presented at the 37th Annual Meeting of the Society of Toxicology, held in Seattle, Washington, March 1–5, 1998 (abstract ...326).22Abbreviations used: AHR, aryl hydrocarbon receptor;Ahr,AHR gene; “B,” an unknown dioxin resistance gene; EROD, ethoxyresorufin-O-deethylase; H/W, Han/Wistar; hw, H/W type; L-E, Long–Evans; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TNT, Tris–NaCl–Tween 20 buffer; wt, wild typethe most toxic congener of dioxins, exhibits wide sensitivity differences between a sensitive Long–Evans (L-E) rat and a resistant Han/Wistar (H/W) rat. The sensitivity is determined probably by two autosomal genes and it is highly end point dependent. The difference is more than 1000-fold for acute toxicity and negligible for CYP1A1 induction. The rat strains were recently shown to have differences in the size of AH receptor (AHR), which mediates most effects of TCDD. In the present study, the rat strains were crossed and the resistant alleles of genes determining TCDD sensitivity were segregated to new rat lines. Selection was based on AHR phenotype determined by Western blot and resistance to TCDD lethality. Two genes determining resistance were found: theAhrand a novel gene designated “B.” In homozygous rats, the H/W typeAhrhwallele prevented TCDD lethality up to 2000 μg/kg or more, and the H/W type “Bhw” allele also increased resistance to TCDD lethality but to a lesser extent. Heterozygous rats were only slightly more resistant to acute lethality than the respective sensitive homozygous rats. CYP1A1 induction was similar irrespective of theAhrand “B” genotypes, but a substantial increase in serum bilirubin seen after low doses in sensitive rats occurred only after large doses in “Bhw/hw” and not at all inAhrhw/hwrats. In conclusion, theAhrhwallele is a major determinant of the exceptional resistance of H/W rats to TCDD lethality. There is also an additional gene, whose function remains to be characterized, conferring limited resistance to TCDD toxicity. These two H/W rat-derived alleles are separately expressed in the new rat lines created.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) brings about a wide spectrum of toxic and biochemical changes, most of which are mediated by the AH receptor (AHR). Recent cloning of the AHR from the two ...most TCDD-resistant laboratory animals, Han/Wistar (Kuopio) rats and hamsters, suggested a critical role for the C-terminal transactivation domain structure in TCDD sensitivity. Here we cloned the AHR from the most TCDD-susceptible species, guinea pig. The N-terminus of its AHR was highly similar to that in the resistant animals. However, the C-terminal Q-rich subdomain was only about half the size of this subunit in the hamster AHR. There was a distinct correlation across published mammalian species between the number of glutamine residues in the Q-rich subdomain and sensitivity to the acute lethality of TCDD. The closest homolog of the Guinea pig receptor turned out to be the human AHR, which may be relevant for dioxin risk assessment.
The mediator of dioxin toxicity, aryl hydrocarbon receptor (AHR), has also important physiological functions. Selective AHR modulators (SAHRMs) share some effects of dioxins, except for their marked ...toxicity. We recently characterised toxicologically two novel SAHRMs, prodrugs IMA-08401 and IMA-07101 in rats, demonstrating that they are far less deleterious than the most toxic AHR-agonist, TCDD. Here, we analysed the in vitro toxicity and in silico AHR binding of the respective active, deacetylated metabolites, IMA-06201 (N-ethyl-N-phenyl-5-chloro-1,2-dihydro-4-hydroxy-1-methyl-2-oxo-quinoline-3-carboxamide) and IMA-06504 (N-(4-trifluoromethylphenyl)-1,2-dihydro-4-hydroxy-5-methoxy-1-methyl-2-oxo-quinoline-3-carboxamide). In H4IIE rat hepatoma cells, IMA-06201 and IMA-06504 induced CYP1A1 with comparable potencies and efficacies to those of TCDD. They had little effect on cell viability as assessed by LDH leakage and MTT reduction assays, and were not mutagenic in the Ames test, but IMA-06504 elicited a maximally 2.7-fold increase in micronuclei. Molecular docking simulations showed that similar to TCDD, they occupy the central region of AHR ligand binding cavity. Hence, while showing low to negligible in vitro toxicity, these novel SAHRMs bind to the AHR qualitatively in a similar fashion to TCDD, and appear comparably powerful AHR agonists. Combined with our earlier results demonstrating that they seem considerably less toxic in vivo than TCDD, these compounds are thus highly interesting new SAHRMs.
•IMA-06201 and IMA-06504 are selective AHR modulators.•As CYP1A1 inducers, they appear comparable to TCDD in potency and efficacy.•They also show low to negligible cytotoxicity and mutagenicity in vitro.•Based on in silico modelling, their binding to the AHR resembles that of TCDD.•Therefore, they have potential as drug compound candidates and research tools.
Changes in the perinatal testosterone surge have been related to demasculinization of the central nervous system and androgen-dependent growth of the reproductive organs in male mammals. Earlier ...reports suggest that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) interferes with androgen production, but the perinatal effects have remained elusive. In the present study we explored in utero-effects of TCDD (0.05, 0.1, 0.5, and 1.0 μg/kg), introduced on day 13.5 of pregnancy, on prenatal (day 19.5 post-conception p.c.) testosterone (T) surge and pituitary luteinizing hormone (LH) production in TCDD-resistant Han/Wistar (H/W) and TCDD-sensitive Long-Evans (L-E) rats. To elucidate estrogenic effects on T and LH production, Sprague-Dawley (S-D) fetuses with previously known DES-sensitivity were exposed in utero to diethylstilbestrol (DES, 100–300 μg/kg) on days 13.5, 15.5, and 17.5 p.c. For comparison, H/W fetuses that responded to TCDD treatments were exposed to DES at concentration of 100 μg/kg. It was found that TCDD has a stimulatory effect on testicular T synthesis in the H/W fetuses and that their circulating T concentrations increased significantly. The effect was not seen in the inbred L-E fetuses, which throughout the study showed considerably low testicular T levels. Pituitary LH concentrations also increased in the H/W fetuses exposed to TCDD. Effects of TCDD (1.0 μg/kg) in the H/W fetuses could be confirmed in vitro by human chorionic gonadotropin (hCG) stimulation assay showing the highest response rate in the TCDD exposed testes. Stimulation of cyclic AMP (adenosine-3′, 5′-cyclic monophosphatecAMP) production was not considerably altered by in utero TCDD exposure. A significant depression in testicular and plasma T content was seen in the DES-exposed S-D and H/W fetuses, but pituitary LH levels did not alter considerably. In the presence of hCG, DES-exposed testes showed lower in vitro T and cAMP production rates compared to the untreated testes. TCDD (1.0 μg/kg) increased and DES decreased the male body weight gain, but the changes were not sex-dependent. It is concluded that TCDD may increase the amplitude of the prenatal testosterone surge in male rats by stimulating pituitary LH production and enhancing the sensitivity of the fetal testis to LH. DES, on the contrary, apparently impairs testicular steroidogenesis and pituitary function.