Treated With 2,3,7,8-tetrachlorodibenzo-p-dioxin Research Articles

Systematic identification of molecular mechanisms for aryl hydrocarbon receptor mediated neuroblastoma cell migration.

Tumor cell migration is affected by the aryl hydrocarbon receptor (AhR). However, the systematic molecular mechanisms underlying AhR-mediated migration of human neuroblastoma cells are not fully understood. To address this issue, we performed an integrative analysis of mRNA and microRNA (miR) expression profiles in human neuroblastoma SK-N-SH cells treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent agonist of AhR. The cell migration was increased in a time- and concentration- dependent manner, and was blocked by AhR antagonist (CH223191). A total of 4,377 genes were differentially expressed after 24-hour-treatment with 10-10 M TCDD, of which the upregulated genes were significantly enriched in cell migration-related biological pathways. Thirty-four upregulated genes, of which 25 were targeted by 78 differentially expressed miRs, in the axon guidance pathway were experimentally confirmed, and the putative dioxin-responsive elements were present in the promoter regions of most genes (79%) and miRs (82%) in this pathway. Furthermore, two promigratory genes (CFL2 and NRP1) induced by TCDD was reversed by blockade of AhR. In conclusion, AhR-mediated mRNA-miR networks in the axon guidance pathway may represent a potential molecular mechanism of dioxin-induced directional migration of human neuroblastoma cells.

Transporter Gene Regulation in Sandwich Cultured Human Hepatocytes Through the Activation of Constitutive Androstane Receptor (CAR) or Aryl Hydrocarbon Receptor (AhR).

The induction potentials of ligand-activated nuclear receptors on metabolizing enzyme genes are routinely tested for new chemical entities. However, regulations of drug transporter genes by the nuclear receptor ligands are underappreciated, especially in differentiated human hepatocyte cultures. In this study, gene induction by the ligands of constitutive androstane receptor (CAR) and aryl hydrocarbon receptor (AhR) was characterized in sandwich-cultured human hepatocytes (SCHH) from multiple donors. The cells were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), omeprazole (OP), 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) and phenobarbital (PB) for three days. RNA samples were analyzed by qRT-PCR method. As expected, CITCO, the direct activator, and PB, the indirect activator of CAR, induced CYP3A4 (31 and 40-fold), CYP2B6 (24 and 28-fold) and UGT1A1 (2.9 and 4.2-fold), respectively. Conversely, TCDD and OP, the activators of AhR, induced CYP1A1 (38 and 37-fold), and UGT1A1 (4.3 and 5.0-fold), respectively. In addition, OP but not TCDD induced CY3A4 by about 61-fold. Twenty-four hepatic drug transporter genes were characterized, and of those, SLC51B was induced the most by PB and OP by about 3.3 and 6.5 fold, respectively. Marginal inductions (about 2-fold) of SLC47A1 and SLCO4C1 genes by PB, and ABCG2 gene by TCDD were observed. In contrast, SLC10A1 gene was suppressed about 2-fold by TCDD and CITCO. While clinical relevance of SLC51B gene induction or SLC10A1 gene suppression warrants further investigation, the results verified that the assessment of transporter gene inductions are not required for new drug entities, when a drug does not remarkably induce metabolizing enzyme genes by CAR and AhR activation.

142 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) regulates the expression of AhR-related factors(AhR, ARNT, CYP1A1), and cytokines in peripheral blood mononuclear cells and changes in differentiation of CD4+ T cells from healthy subjects, patients with atopic dermatitis and psoriasis

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, important for xenobiotic metabolism, which binds to various endogenous and exogenous ligands in the skin. Therefore, we aim to evaluate the role of the AhR and its ligands in chronic inflammatory skin disease such as psoriasis (PS) and atopic dermatitis (AD). PS and AD patients and healthy volunteers were recruited. Blood samples were taken from three groups for isolation of peripheral blood mononuclear cells (PBMCs) and CD4+ T cell. Each isolated PBMCs and CD4+ T cells were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 6-for-mylindolo[3,2-b] carbasole (FICZ) under various concentrations and time duration. In PBMCs, after treatment with TCDD and FICZ, the expression of AhR-related factors and interleukin (IL)-22 were increased in PS and AD patients. IL-17 was increased in PS patients but remained stable in AD patients. In the CD4+T cells, after treatment with TCDD and FICZ, the expression of AhR-related factors and interleukin (IL)-17, IL-22 were increased in PS and AD patients. In the CD4+T cells, compared to the control group, CCR10+ cells (marker for Th22 cells) and CD161+CCR10+ cells (marker for Th17 and Th22 cells) were significantly increased in PS patients and CCR10+ cells were significantly increased in AD patients of the TCDD- treated group. In conclusion, CD4+T cells of AD and PS patients presented higher expression of AhR, CYP1A1, IL-17 and IL-22. Additionally, CD4+T cells of AD and PS patients tend to show features of Th17 cells or Th22 cells. These results suggest that AD and PS patients may be affected more sensitively to AhR ligands and activation of AhR may contribute to the disease development in AD and PS.

CARM1 is involved in CYP1A1 gene expression as a transcriptional coactivator

Protein arginine methyltransferases (PRMTs) have been proposed as a transcriptional coactivators in the induction of CYP1A1. In this study, we investigated the change of AhR and CYP1A1 expression by inhibition of CARM1 (also known as PRMT4) to show the involvement of protein arginine methylation in CYP1A1 gene expression. Also, we tested the hypothesis that CARM1 recruitment to the CYP1A1 promoter and subsequent histone arginine methylation are required for the activation of transcription. We found that treatment of HepG2 cells with silencing RNA targeting CARM1 or an arginine methyltransferase inhibitor (AMI-5) significantly decreased the CYP1A1 gene expression level. Furthermore, treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which increases histone arginine methylation in histone H3R17, also caused increases in CARM1 and H3R17 signals in chromatin immunoprecipitation assays. These results indicate that CARM1 is recruited to the CYP1A1 promoter region as a coactivator, and histone arginine methylation makes XRE (xenobiotic responsive element) regions more accessible for transcription.

다이옥신계 TCDD(2,3,7,8-tetrachlorodibenzo-p-dioxin)에 노출된 흰쥐의 간 기능, 항산화효소 활성도 및 면역관련 인자에 대한 키토산 올리고머의 섭취량에 따른 효과

다이옥신계 TCDD(2,3,7,8-tetrachlorodibenzo-p-dioxin)에 노출된 흰쥐의 간 기능, 항산화효소 활성도 및 면역관련 인자에 대한 키토산 올리고머의 섭취량에 따른 효과

Aryl hydrocarbon receptor knockout rats are insensitive to the pathological effects of repeated oral exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Sustained activation of the aryl hydrocarbon receptor (AHR) is believed to be the initial key event in AHR receptor-mediated tumorigenesis in the rat liver. The role of AHR in mediating pathological changes in the liver prior to tumor formation was investigated in a 4-week, repeated-dose study using adult female wild-type (WT) and AHR knockout (AHR-KO) rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Beginning at 8 weeks of age, AHR-KO and WT rats were dosed by oral gavage with varying concentrations of TCDD (0, 3, 22, 100, 300 and 1000 ng kg(-1) day(-1) ). Lung, liver and thymus histopathology, hematology, serum chemistry and the distribution of TCDD in liver and adipose tissue were examined. Treatment-related increases in the severity of liver and thymus pathology were observed in WT, but not AHR-KO rats. In the liver, these included hepatocellular hypertrophy, bile duct hyperplasia, multinucleated hepatocytes and inflammatory cell foci. A loss of cellularity in the thymic cortex and thymic atrophy was observed. Treatment-related changes in serum chemistry parameters were also observed in WT, but not AHR-KO rats. Finally, dose-dependent accumulation of TCDD was observed primarily in the liver of WT rats and primarily in the adipose tissue of AHR-KO rats. The results suggest that AHR activation is the initial key event underlying the progression of histological effects leading to liver tumorigenesis following TCDD treatment. Copyright © 2015 John Wiley & Sons, Ltd.

Effects of Artemisia capillaris extract on disorders of hepatic functions and lipid metabolism in rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

This study was conducted in order to investigate the effects of Artemisia capillaris (AC) extract on disorders of hepatic functions and lipid metabolism induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an endocrine disrupter, using male rats (SD, five weeks old) for a period of three weeks. These 37 animals were divided into four groups. AC extract was added as 1.5% or 3% levels to basal diets, respectively. TCDD (40 ug/kg B.W) was administered by intraperitoneal injection into rats after a week from the beginning of the experiment. AC extract alleviated the increase of rat’s relative liver weights induced by TCDD. Thymuses of all rats treated with TCDD were apparently shrunken by approximately 80%. Levels of white blood cells (WBC), red blood cells, hemoglobin, and hematocrits were significantly increased by treatment with TCDD, however, WBC tended to decrease by AC extract diets. In hepatic function, the elevation of glutamic oxalacetic transaminase activities by TCDD treatment was diminished by AC extract diets. Serum HDL-cholesterol levels were significantly elevated by AC extract diets. The apparent increase of triglyceride levels of rat livers induced by TCDD was significantly suppressed in the AC extract diet groups. Hepatic cytosolic catalase activities significantly decreased by treatment with TCDD showed a recovering trend by AC extract diets. In histochemical observation, the fat droplets and apoptosis of hepatocytes treated with TCDD were markedly alleviated by AC extract diets. These results indicated that AC could exert recovering effects on some disorders of hepatic functions, lipids metabolism, and antioxidant activities resulting from TCDD treatment.

Comparison of TCDD-elicited genome-wide hepatic gene expression in Sprague–Dawley rats and C57BL/6 mice

Although the structure and function of the AhR are conserved, emerging evidence suggests that downstream effects are species-specific. In this study, rat hepatic gene expression data from the DrugMatrix database (National Toxicology Program) were compared to mouse hepatic whole-genome gene expression data following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). For the DrugMatrix study, male Sprague–Dawley rats were gavaged daily with 20μg/kg TCDD for 1, 3 and 5days, while female C57BL/6 ovariectomized mice were examined 1, 3 and 7days after a single oral gavage of 30μg/kg TCDD. A total of 649 rat and 1386 mouse genes (|fold change|≥1.5, P1(t)≥0.99) were differentially expressed following treatment. HomoloGene identified 11,708 orthologs represented across the rat Affymetrix 230 2.0 GeneChip (12,310 total orthologs), and the mouse 4×44K v.1 Agilent oligonucleotide array (17,578 total orthologs). Comparative analysis found 563 and 922 orthologs differentially expressed in response to TCDD in the rat and mouse, respectively, with 70 responses associated with immune function and lipid metabolism in common to both. Moreover, QRTPCR analysis of Ceacam1, showed divergent expression (induced in rat; repressed in mouse) functionally consistent with TCDD-elicited hepatic steatosis in the mouse but not the rat. Functional analysis identified orthologs involved in nucleotide binding and acetyltransferase activity in rat, while mouse-specific responses were associated with steroid, phospholipid, fatty acid, and carbohydrate metabolism. These results provide further evidence that TCDD elicits species-specific regulation of distinct gene networks, and outlines considerations for future comparisons of publicly available microarray datasets.

Hepatoprotective potential of astaxanthin against 2,3,7,8-tetrachlorodibenzo-p-dioxin in cultured rat hepatocytes

The purpose of this study was to evaluate the effect of carotenoid astaxanthin (ASTA) on cultured primary rat hepatocytes treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the cell viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT), lactate dehydrogenase (LDH) activity, 8-oxo-2-deoxyguanosine (8-OH-dG), total antioxidant capacity (TAC), and total oxidative stress (TOS) levels, and liver micronucleus rates. ASTA (2.5, 5, and 10 µM) was added to cultures alone or simultaneously with TCDD (5 and 10 µM) for 48 h. The results of MTT and LDH assays showed that both doses of TCDD caused significant decrease in cell viability. Also, TCDD significantly increased TOS and decreased TAC level in rat hepatocytes. On the basis of increasing doses, the dioxin caused significant increase in micronucleated hepatocytes) and 8-OH-dG level as compared to control culture. The presence of ASTA with TCDD minimized its effects on primary hepatocytes cultures and DNA damages.

Expression of aryl hydrocarbon receptor in human epidermis, hair follicles and sebaceous glands and its significance

Objective To observe the expression of aryl hydrocarbon receptor (AhR) in human epidermis,hair follicles and sebaceous glands and the activation of AhR by dioxin.Methods Full-thickness skin specimens were obtained from 3 volunteers receiving cosmetic facial surgery.Indirect immunofluorescence and immunohistochemistry were used to detect the expression of AhR in these skin specimens,as well as on cultured human HaCaT keratinocytes and SZ95 sebocytes.Western blot was performed to detect the expressions of AhR protein on HaCaT cells and SZ95 sebocytes after 3-day treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).Results AhR protein was found to be expressed in human epidermis,hair follicles and sebaceous glands,and the expression was stronger in the stratum spinosum and stratum granulosum than in the basal layer,in the outer and central layer than in the middle layer of sebaceous lobuli,in hair follicle outer root sheath and hair cuticles than in the inner root sheath.There was a high expression of AhR in the nucleus and cytoplasm of HaCaT and SZ95 cells.A significant decrease was observed in the expression of AhR in both HaCaT cells and SZ95 cells after treatment with TCDD for 3 days,hinting that AhR was activated by TCDD.Conclusions AhR is highly expressed in human epidermis,hair follicles and sebaceous glands,and can be activated by TCDD,suggesting that dioxin may induce the abnormal differentiation of epidermis,hair follicles and sebaceous glands via AhR singaling pathway. Key words: follicle; Cell line

Activation of the Aryl Hydrocarbon Receptor by TCDD Inhibits Mammary Tumor Metastasis in a Syngeneic Mouse Model of Breast Cancer

Treatment with aryl hydrocarbon receptor (AhR) agonists can slow or reverse the growth of primary mammary tumors in rodents, which has fostered interest in developing selective AhR modulators for treatment of breast cancer. However, the major goal of breast cancer therapy is to inhibit metastasis, the primary cause of mortality in women with this disease. Studies conducted using breast cancer cell lines have demonstrated that AhR agonists suppress proliferation, invasiveness, and colony formation in vitro; however, further exploration using in vivo models of metastasis is warranted. To test the effect of AhR activation on metastasis, 4T1.2 mammary tumor cells were injected into the mammary gland fat pad of syngeneic Balb/c mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Primary tumor growth was monitored for 4 weeks, at which time metastasis was determined. TCDD treatment suppressed metastasis by approximately 50%, as measured both in the lung and in mammary glands at sites distant from the primary tumor. Primary tumor growth was not suppressed by TCDD exposure nor was proliferation of 4T1.2 cells affected by TCDD treatment in vitro. Taken together, these results suggest that the protective effect of AhR activation was selective for the metastatic process and not simply the result of a direct decrease in tumor cell proliferation or survival at the primary site. These observations in immunologically intact animals warrant further investigation into the mechanism of the protective effects of AhR activation and support the promise for use of AhR modulators to treat breast cancer.

Effects of vitamin E on reproductive hormones and testis structure in chronic dioxin-treated mice

The purpose of this study was to investigate the effects of vitamin E on reproductive hormones and testis structure in mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Five experimental groups of a combination of TCDD and vitamin E were designed as follows: 0 ng/kg/d and 0 mg/kg/d (control group), 100 ng/kg/d and 0 mg/kg/d (Group I), 100 ng/kg/d and 20 mg/kg/d (Group II), 100 ng/kg/d and 100 mg/kg/d (Group III), and 100 ng/kg/d and 500 mg/kg/d (Group IV) respectively. Vitamin E and TCDD were given by oral gavage for 7 weeks. The results demonstrated that TCDD decreased the levels of brain gonadotropin releasing hormone (GnRH), testis luteinizing hormone (LH) and follicle stimulating hormone (FSH), serum testosterone and testis spermatozoa number, and damaged testis structure. Vitamin E at 20 mg/kg alleviated the decrease of GnRH; vitamin E at 20, 100, and 500 mg/kg antagonized the decline of LH and FSH; vitamin E at 20 and 100 mg/kg reversed the decrease of testosterone and spermatozoa number; and vitamin E at 100 mg/kg decreased the damage of the testis structure caused by TCDD. The results indicate that vitamin E antagonizes the reproductive endocrine toxicity and alleviates the changes in testicular structure caused by TCDD.

Analysis of the CYP1A1 mRNA Dose-Response in Human Keratinocytes Indicates that Relative Potencies of Dioxins, Furans, and PCBs Are Species and Congener Specific

Reports indicate that toxic equivalency factors (TEFs) based primarily on rodent data do not accurately predict in vitro human responsiveness to certain dioxin-like chemicals (DLCs). To investigate this in cells responsive to dioxins and relevant to chloracne, normal human epidermal keratinocytes were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and several DLCs, each with a TEF value of 0.1, representing three classes of congeners. We estimated half maximal effective concentration (EC50)-based donor-specific relative potency (REP) values for cytochrome P450 1A1 (CYP1A1) messenger RNA (mRNA) induction for TCDD, 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (HxCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,6,7,8-hexachlorodibenzofuran (HxCDF), and 3,3',4,4',5-pentachlorobiphenyl (PCB 126). We also determined EC50-based population-level REP values (n = 4) for CYP1A1 mRNA induction for TCDD, HxCDF, and PCB 126. Furthermore, an alternative factor, the relative threshold factor (RTF) based on the low end (threshold) of the dose-response curve, was calculated. Our results demonstrated that HxCDF had a population-based REP value of 0.98, 9.8-fold higher than its assigned TEF value of 0.1. Conversely, PCB 126 had an REP value of 0.0027 and an RTF of 0.0022, 37-fold and 45-fold less than its assigned TEF of 0.1, respectively. The REP values for HxCDD and TCDF were 0.24 and 0.10, respectively, similar to their assigned value of 0.1. Therefore, although the DLCs tested in the current study all possessed the same assigned TEF value of 0.1, congener-specific differences in REPs and RTFs were observed for human keratinocytes. These congener-specific discrepancies are likely because of differences in interspecies factors that have yet to be defined.

The effects of Aryl Hydrocarbon Receptor activation by TCDD on IL-12R signaling and generation of regulatory T cells (47.9)

Abstract We have previously shown that allo-reactive donor CD4+ T cells are converted into CD25High Foxp3-negative regulatory T cells during an acute Graft-vs-Host Response (aGVHR) in mice treated with 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). Activation of the Aryl Hydrocarbon Receptor (AhR) in donor CD4+ T cells is required for full suppression of aGVHR and generation of regulatory T cells. CD4+ T cells exposed to TCDD during aGVHR exhibit a significant increase in IL-12Rb2 mRNA and protein as well as phosphorylated-STAT4 expression. We used IL-12Rb2-deficient (KO) donor T cells to determine whether increased IL-12R signaling was important for the generation of TCDD-induced regulatory T cells. CD25 protein expression was not changed on IL-12Rb2 KO cells compared to wild-type control (WT), however significantly fewer IL-12Rb2 KO T cells expressed phosphorylated-STAT4. A marked decrease in expression levels of phosphorylated-STAT1 and STAT5 was also observed in both WT and IL-12Rb2 KO donor T cells from TCDD-treated mice compared to vehicle control. Although IL-12Rb2 deficiency does not appear to affect the generation of donor-derived CD25High Foxp3-negative regulatory T cells during aGVHR in TCDD-treated mice, current studies focus on the effects of TCDD and altered IL-12R signaling on Foxp3-positive regulatory T cell generation.

Effect of vitamin E on reproductive function in the mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin*

The study was conducted to determine the effects of vitamin E on reproductive performance in mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The 75 female and 40 male mice were randomly assigned into five groups. The levels of both TCDD and vitamin E given by gavage were 0 and 0 (Control group), 100 and 0 (experimental group I), 100 and 20 (experimental group II), 100 and 100 (experimental group III), and 100 ng/kg/day and 500 mg/kg/day (experimental group IV), respectively. Males and females were mixed to mate at the ratio of 1:2 after 4-week experiment. The gavage treatments were continued until the end of gestation in female mice after mating. The results showed that the litter number, survival rate, and body weight at birth of offspring in experimental group I had significantly decreased, and the females' pregnancy rate and pup sex ratio in experimental group I had the decreasing tendencies when compared with the control group. The litter number in experimental group III, survival rate in experimental group II and III, body weight at birth in experimental group III and IV exhibited significant increase compared with experimental group I. The female pregnancy rate in both experimental group III and IV recovered to 100%, but there were no significant differences when compared with experimental group I. The pups' sex ratio had a gradually increasing tendency with increase of vitamin E level, but there was no significant difference among experimental group I-IV. The results suggest that TCDD could induce reproductive toxicity in mice, whereas vitamin E alleviated adverse effects on reproductive performance in mice caused by TCDD.

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 also 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.

Cobaltous chloride and hypoxia inhibit aryl hydrocarbon receptor-mediated responses in breast cancer cells

The aryl hydrocarbon receptor (AhR) is expressed in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Treatment with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) induces CYP1A1 protein and mRNA levels and also activates inhibitory AhR-ERα crosstalk associated with hormone-induced reporter gene expression. In ZR-75 cells grown under hypoxia, induction of these AhR-mediated responses by TCDD was significantly inhibited. This was not accompanied by decreased nuclear AhR levels or decreased interaction of the AhR complex with the CYP1A1 gene promoter as determined in a chromatin immunoprecipitation assay. Hypoxia-induced loss of Ah-responsiveness was not associated with induction of hypoxia-inducible factor-1α or other factors that sequester the AhR nuclear translocation (Arnt) protein, and overexpression of Arnt under hypoxia did not restore Ah-responsiveness. The p65 subunit of NFκB which inhibits AhR-mediated transactivation was not induced by hypoxia and was primarily cytosolic in ZR-75 cells grown under hypoxic and normoxic conditions. In ZR-75 cells maintained under hypoxic conditions for 24 h, BRCA1 (an enhancer of AhR-mediated transactivation in breast cancer cells) was significantly decreased and this contributed to loss of Ah-responsiveness. In cells grown under hypoxia for 6 h, BRCA1 was not decreased, but induction of CYP1A1 by TCDD was significantly decreased. Cotreatment of ZR-75 cells with TCDD plus the protein synthesis inhibitor cycloheximide for 6 h enhanced CYP1A1 expression in cells grown under hypoxia and normoxia. These results suggest that hypoxia rapidly induces protein(s) that inhibit Ah-responsiveness and these may be similar to constitutively expressed inhibitors of Ah-responsiveness (under normoxia) that are also inhibited by cycloheximide.

Internal dose-effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in gonadotropin-primed weanling rat model

Single sc injection of 5 IU equine chorionic gonadotropin (eCG) induces ovulation in weanling female rats 3 days later. It has been shown that treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 24 h before eCG injection reduces eCG-stimulated ovarian hypertrophy and inhibits ovulation. The present study intended to compare internal dose-effects of TCDD between these endpoints and representative endpoints for TCDD toxicity, such as weights of the liver and thymus, in weanling female rats given orally 0, 1, 4 or 16 microg/kg TCDD 24 h before eCG injection on postnatal day 25. Measurement of plasma TCDD concentrations by ELISA at 6, 72 and 96 h after TCDD revealed that significant levels of TCDD were maintained in systemic circulation until 96 h (on the day of induced ovulation) with the highest level at 6 h after TCDD treatment. Ovarian TCDD concentrations varied similarly and tended to be higher than those in the thymus at all time points, whereas hepatic concentrations of TCDD were the highest among the tissues. Although > or = 4 microg/kg TCDD affected the weights of the thymus and liver, no differences were observed in ovarian weights at any time point or in ovulation between corn oil-treated and TCDD-treated groups. Furthermore, ovarian levels of representative mRNAs in follicles were not affected by TCDD treatment. Since TCDD increased the amount of cytochrome P450 1A1 mRNA in the ovary, the administered TCDD stimulated the aryl hydrocarbon receptor-signaling pathway. From these results, we concluded that thymus weights of weanling female rats responded to TCDD at a lower internal dose as compared with that ovarian hypertrophy and follicular growth from early antral stage to ovulation would respond to.

Blocking Expression of AHR2 and ARNT1 in Zebrafish Larvae Protects Against Cardiac Toxicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin

The zebrafish (Danio rerio) has become an attractive vertebrate model for studying developmental processes, and is emerging as a model system for studying the mechanisms by which xenobiotic compounds perturb normal development. Embryos treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) shortly after fertilization exhibit a range of adverse effects on the heart: an early reduction in cardiac myocyte number, followed by a change in heart looping and morphology, with an apparent compaction of the ventricle and overall decrease in heart size. These changes are accompanied by impaired cardiac function including a decrease in cardiac output and eventually irreversible ventricular standstill. The mechanisms involved in mediating effects of TCDD on the heart remain unknown. However, it is widely accepted that aryl hydrocarbon receptor (AHR) activation mediates endpoints of TCDD toxicity in vertebrates. In zebrafish, there are multiple forms of AHR and AHR nuclear translocator protein (ARNT) raising the question about whether different endpoints of TCDD toxicity are mediated by different components of the AHR/ARNT pathway. To address this question we used morpholino oligonucleotide technology to specifically block the expression of zfAHR2, zfARNT1, zfARNT2, and zfCYP1A, and assessed the previously described effects of TCDD on heart morphology, size, and function in the developing morphants. We report that blocking zfAHR2 and zfARNT1 expression provided protection against the TCDD-mediated alteration in heart morphology, reduced cardiac myocyte number, decreased cardiac output and ventricular standstill in zebrafish larvae, while the zfarnt2 and zfcyp1a morpholinos did not block the TCDD-induced cardiac toxicity.

In vivo – in vitro toxicogenomic comparison of TCDD-elicited gene expression in Hepa1c1c7 mouse hepatoma cells and C57BL/6 hepatic tissue

BackgroundIn vitro systems have inherent limitations in their ability to model whole organism gene responses, which must be identified and appropriately considered when developing predictive biomarkers of in vivo toxicity. Systematic comparison of in vitro and in vivo temporal gene expression profiles were conducted to assess the ability of Hepa1c1c7 mouse hepatoma cells to model hepatic responses in C57BL/6 mice following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).ResultsGene expression analysis and functional gene annotation indicate that Hepa1c1c7 cells appropriately modeled the induction of xenobiotic metabolism genes in vivo. However, responses associated with cell cycle progression and proliferation were unique to Hepa1c1c7 cells, consistent with the cell cycle arrest effects of TCDD on rapidly dividing cells. In contrast, lipid metabolism and immune responses, representative of whole organism effects in vivo, were not replicated in Hepa1c1c7 cells.ConclusionThese results identified inherent differences in TCDD-mediated gene expression responses between these models and highlighted the limitations of in vitro systems in modeling whole organism responses, and additionally identified potential predictive biomarkers of toxicity.