Primary structure and inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) of aryl hydrocarbon receptor repressor in a TCDD-sensitive and a TCDD-resistant rat strain

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 (100microg/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.     

Preferential inducibility of CYP1A1 and CYP1A2 by TCDD: differential regulation in primary human hepatocytes versus transformed human cells

Cytochrome P4501A1 (CYP1A1) induction, a marker of aryl hydrocarbon (Ah) receptor activation, has been associated with carcinogenicity of the environmental agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Consistently, we show that TCDD treatment led to induction of CYP1A1 in responsive human cancer cell lines including HepG2, LS174T, and MCF-7, as determined by Western blotting and CYP1A form-selective R-warfarin 6- and 8-hydroxylation. TCDD, however, preferably induced CYP1A2, not CYP1A1, in primary human hepatocytes. Such CYP1A form-preferred induction at the protein level was apparently uncorrelated with non-preferred mRNA induction in any cells studied. Moreover, while both genes were up-regulated by TCDD in primary hepatocytes and HepG2 cells, the induction of CYP1A1 and CYP1A2 at the mRNA level was distinguishable, indicated by the marked differences in activation kinetics and the response to the protein synthesis inhibitors, anisomycin and cycloheximide. Furthermore, formation of total benzo(a)pyrene (BaP)-DNA adducts was not altered following BaP exposure in TCDD-treated primary hepatocytes, whereas significantly elevated, in a CYP1A1-dependent manner, in the treated HepG2 cells. Taken together, our findings, demonstrating the complexities of TCDD-associated human Ah receptor function and differential regulations of CYP 1A enzymes, suggest clearly the need for caution when extrapolating data obtained in cell-based models.     

Characteristic expression of aryl hydrocarbon receptor repressor gene in human tissues: organ-specific distribution and variable induction patterns in mononuclear cells

To investigate the expression of aryl hydrocarbon receptor repressor (AhRR) and related molecules in various tissues and the effects of aromatic hydrocarbons (AHs) on their expression, we developed a reliable technique of quantification of human AhRR as well as aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT) and cytochrome P450 1A1 (CYP1A1) mRNA by real-time TaqMan PCR method. First, we examined the expression of these genes in human adult or fetal tissues. The levels of AhRR expression were extremely high in testis, very high in lung, ovary, spleen and pancreas from adults, whereas those were low in those from fetuses. On the other hand, CYP1A1 expression was extremely high in lung, and AhR and ARNT were ubiquitously expressed in almost all tissues. Second, we compared the expression levels of these genes in mononuclear cells (MNCs) from various sources. Comparison of the basal expression levels of these genes in MNCs demonstrated that MNCs from umbilical cord blood showed higher AhRR or CYP1A1 expression than those from adults. The induction of AhRR or CYP1A1 expression by 3-methylcholanthrene (3-MC) was observed in MNCs from adults but not from umbilical cord blood. Consequently, there existed characteristic differences in the basal levels of AhRR and CYP1A1 expression in MNCs, as well as in their inducibility by 3-MC among MNCs from various types of human bloods. These results will provide basic information for a possible application of AhRR and CYP1A1 measurements to evaluate AH exposure in vivo.     

Recombinant expression of aryl hydrocarbon receptor for quantitative ligand-binding analysis

Recombinant expression of the aryl hydrocarbon receptor (AhR) yields small amounts of ligand-binding-competent AhR. Therefore, Spodoptera frugiperda (Sf9) cells and baculovirus have been evaluated for high-level and functional expression of AhR. Rat and human AhR were expressed as soluble protein in significant amounts. Expression of ligand-binding-competent AhR was sensitive to the protein concentration of Sf9 extract, and coexpression of the chaperone p23 failed to affect the yield of functional ligand-binding AhR. The expression system yielded high levels of functional protein, with the ligand-binding capacity (B_max) typically 20-fold higher than that obtained with rat liver cytosol. Quantitative estimates of the ligand-binding affinity of human and rat AhR were obtained; the K_d for recombinant rat AhR was indistinguishable from that of native rat AhR, thereby validating the expression system as a faithful model for native AhR. The human AhR bound TCDD with significantly lower affinity than the rat AhR. These findings demonstrate high-level expression of ligand-binding-competent AhR, and sufficient AhR for quantitative analysis of ligand binding.     

Dioxin-induced adseverin expression in the mouse thymus is strictly regulated and dependent on the aryl hydrocarbon receptor

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a ligand for the ubiquitous, intracellular aryl hydrocarbon receptor (AhR), up-regulates the actin-modulating protein adseverin in mouse lymphoid tissues, a response that may be correlated to the immunotoxicity of TCDD. Here, by using chimeric mice with TCDD-responsive (AhR(+/+)) hematopoietic cells and TCDD-unresponsive (AhR(minus sign/minus sign)) thymic stroma, or the reverse, we show that TCDD-induced expression of adseverin in thymus is dependent on AhR expression in hematopoietic cells but not in stroma. The use of fetal thymic organ cultures also indicates that TCDD-induced expression of adseverin is confined to the thymocytes. The thymic stroma showed no induction of adseverin expression after TCDD exposure, although TCDD clearly activated the AhR in these cells, as indicated by the induction of CYP1A1. Adseverin was not induced in the thymus of normal adult C57BL/6 mice exposed to beta-estradiol or dexamethasone, two other agents, which also cause thymic atrophy. This further supports that adseverin induction is a specific gene regulatory effect by TCDD on thymocytes.     

Identification of novel splice variants of ARNT and ARNT2 in the rat

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 microg/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.     

Novel cDNA sequences of aryl hydrocarbon receptors and gene expression in turtles (Chrysemys picta and Pseudemys scripta) exposed to different environments

Reproductive changes have been observed in painted turtles from a site with known contamination located on Cape Cod, MA, USA. We hypothesize that these changes are caused by exposure to endocrine-disrupting compounds and that genes involved in reproduction are affected. The aryl hydrocarbon receptor (AHR) is an orphan receptor that is activated by environmental contaminants. AHR mRNA was measured in turtles exposed to soil collected from a contaminated site. Adult turtles were trapped from the study site (Moody Pond, MP) or a reference site and exposed to laboratory environments containing soil from either site. The red-eared slider was used to assess neonatal exposure to soil and water from the sites. The environmental exposures occurred over a 13-month period. Juveniles showed an age-dependent increase in brain AHR1. Juvenile turtles exposed to the MP environment had elevated gonadal AHR1. Adult turtles exposed to the MP environment showed significantly decreased brain AHR2. The painted turtle AHR is the first complete reptile AHR cDNA sequence. Phylogenetic analysis of the painted turtle AHR showed that it clusters with other AHR2s. Partial AHR1 and partial AHR2 cDNA sequences were cloned from the red-eared slider. MEME analysis identified 18 motifs in the turtle AHRs, showing high conservation between motifs that overlapped functional regions in both AHR isoforms.     

Generation of a 'humanized' hCYP1A1_1A2_Cyp1a1/1a2(-/-)_Ahrd mouse line harboring the poor-affinity aryl hydrocarbon receptor

Herein, we describe generation of the hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahr^d mouse line, which carries human functional CYP1A1 and CYP1A2 genes in the absence of mouse Cyp1a1 and Cyp1a2 genes, in a (>99.8%) background of the C57BL/6J genome and harboring the poor-affinity aryl hydrocarbon receptor (AHR) from the DBA/2J mouse. We have characterized this line by comparing it to our previously created hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahr^b1 line—which carries the same but has the high-affinity AHR of the C57BL/6J mouse. By quantifying CYP1A1 and CYP1A2 mRNA in liver, lung and kidney of dioxin-treated mice, we show that dose-response curves in hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahr^d mice are shifted to the right of those in hCYP1A1_1A2_Cyp1a1/1a2(−/−)_Ahr^b1 mice—similar to, but not as robust as, dose-response curves in DBA/2J versus C57BL/6J mice. This new mouse line is perhaps more relevant than the former to human risk assessment vis-à-vis human CYP1A1 and CYP1A2 substrates, because poor-affinity rather than high-affinity AHR occurs in the vast majority of the human population.     

Curcumin and its derivatives inhibit 2,3,7,8,-tetrachloro-dibenzo-p-dioxin-induced expression of drug metabolizing enzymes through aryl hydrocarbon receptor-mediated pathway

Abstract

Certain dioxins, including 2,3,7,8,-tetrachloro-dibenzo-p-dioxin (TCDD), are exogenous ligands for an aryl hydrocarbon receptor (AhR) and induces various drug-metabolizing enzymes. In this study, we examined the effect of curcumin on expression of drug-metabolizing enzymes through the AhR and NF-E2 related factor 2 (Nrf2) pathways. Curcumin dose-dependently inhibited TCDD-induced expression of phase I enzyme cytochrome P450 1A1 (CYP1A1) and phase II enzymes NAD(P)H:quinone oxidoreductase-1 (NQO1) and heme oxygenase 1 (HO-1) but not tert-butyl hydroquinone-induced NQO1 and HO-1, suggesting that curcumin inhibited only AhR pathway, but not Nrf2 one directly. Furthermore, we used 14 curcumin derivatives and obtained the correlation between hydrophobicity of the compounds and suppressive effect against AhR transformation. Results from the quantitative structure active correlative analysis indicated that methoxy groups and β-diketone structure possessing keto-enol tautomerism in curcumin were necessary to inhibit AhR transformation, and the addition of methyl and methoxy group(s) to the curcumin increased the inhibition effect.