Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on murine heart development: alteration in fetal and postnatal cardiac growth, and postnatal cardiac chronotropy.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related chemicals are potent cardiovascular teratogens in developing piscine and avian species. In the present study we investigated the effects of TCDD on murine cardiovascular development. Pregnant mice (C57Bl6N) were dosed with 1.5-24 microg TCDD/kg on gestation day (GD) 14.5. At GD 17.5, fetal mice exhibited a dose-related decrease in heart-to-body weight ratio that was significantly reduced at a maternal dose as low as 3.0 microg TCDD/kg. In addition, cardiocyte proliferation was reduced in GD 17.5 fetal hearts at the 6.0-microg TCDD/kg maternal dose. To determine if this reduction in cardiac weight was transient, or if it continued after birth, dams treated with control or 6.0 microg TCDD/kg were allowed to deliver, and heart weight of offspring was determined on postnatal days (P) 7 and 21. While no difference was seen on P 7, on P 21 pups from TCDD-treated litters showed an increase in heart-to-body weight ratio and in expression of the cardiac hypertrophy marker atrial natriuretic factor. Additionally, electrocardiograms of P 21 offspring showed that the combination of in utero and lactational TCDD exposure reduced postnatal heart rate but did not alter cardiac responsiveness to isoproterenol stimulation of heart rate. These results demonstrate that the fetal murine heart is a sensitive target of TCDD-induced teratogenicity, resembling many of TCDD-induced effects observed in fish and avian embryos, including reduced cardiocyte proliferation and altered fetal heart size. Furthermore, the combination of in utero and lactational TCDD exposure can induce cardiac hypertrophy and bradycardia postnatally, which could increase the risk of cardiovascular disease development.     

Coplanar polychlorinated biphenyls activate the aryl hydrocarbon receptor in developing tissues of two TCDD-responsive lacZ mouse lines.

In utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can have an immediate impact on developmental processes that then lead to long-term deficits in function. To define the specific tissues affected by TCDD during development, we developed a lacZ-reporter gene mouse model driven by activation of the aryl hydrocarbon receptor (AhR). Exposure to TCDD on gestational day (GD) 14 results in strong activation of the lacZ transgene in numerous tissues including fore and hind paws, ear, and genital tubercle. Experiments were conducted to examine the ability of alternative AhR ligands to activate our model system. The coplanar polychlorinated biphenyl congeners 3,4,5,3',4'-pentachlorobiphenyl (PCB126) and 3,4,3',4'-tetrachlorobiphenyl (PCB77) both induced staining in fetal tissues identical to that observed following TCDD exposure. Exposure of fetuses to the PCB mixture Aroclor 1254 and the non-coplanar congener 2,3,6,2',5'-pentachlorobiphenyl (PCB95) did not result in any activation of the lacZ transgene. In addition to the testing of alternative ligands, another line of reporter mice was generated to determine the potential influence of the site of insertion of the lacZ transgene on the reported observations. Both TCDD and the coplanar PCBs induced a similar pattern of staining in the new line as compared to that observed in the original lacZ reporter mouse line. The ability of AhR ligands, other than TCDD, to activate the AhR-mediated transgene, in combination with the insertion-site independence of the response, strengthens the data previously derived from this model and increases the utility of this system for investigations examining AhR-mediated events during development.     

Comparative microarray analysis of basal gene expression in mouse Hepa-1c1c7 wild-type and mutant cell lines.

Hepa-1c1c7 wild-type and benzo[a]pyrene-resistant derived mutant cell lines have been used to elucidate pathways and mechanisms involving the aryl hydrocarbon receptor (AhR). However, there has been little focus on other biological processes which may differ between the isolated lines. In this study, mouse cDNA microarrays representing 4858 genes were used to examine differences in basal gene expression between mouse Hepa-1c1c7 wild-type and c1 (truncated Cyp1a1 protein), c4 (AhR nuclear translocator, ARNT, deficient), and c12 (low AhR levels) mutant cell lines. Surprisingly, c1 mutants exhibited the greatest number of gene expression changes compared to wild-type cells, followed by c4 and c12 lines, respectively. Differences in basal gene expression were consistent with cell line specific variations in morphology, mitochondrial activity, and proliferation rate. MTT and direct cell count assays indicate both c4 and c12 mutants exhibit increased proliferative activity when compared to wild-type cells, while the c1 mutants exhibited decreased activity. This study further characterizes Hepa-1c1c7 wild-type and mutant cells and identifies significant differences in biological processes that should be considered when conducting comparative mechanistic studies with these lines.     

Mechanisms of interaction of endocrine-disrupting chemicals with glutamate-evoked secretion of gonadotropin-releasing hormone.

In previous studies, we detected a dichlorodiphenyltrichloroethane (DDT) derivative in the serum of children with sexual precocity after migration from developing countries. Recently, we reported that DDT stimulated pulsatile gonadotropin-releasing hormone (GnRH) secretion and sexual maturation in the female rat. The aim of this study was to delineate the mechanisms of interaction of endocrine-disrupting chemicals including DDT with GnRH secretion evoked by glutamate in vitro. Using hypothalamic explants obtained from 15-day-old female rats, estradiol (E2) and DDT caused a concentration-related increase in glutamate-evoked GnRH release while p,p'-dichlorodiphenyldichloroethene and methoxychlor had no effect. The effective DDT concentrations in vitro were consistent with the serum concentrations measured in vivo 5 days after exposure of immature rats to 10 mg/kg/day of o,p'-DDT. Bisphenol A induced some stimulatory effect, whereas no change was observed with 4-nonylphenol. The o,p'-DDT effects in vitro were prevented partially by a selective antagonist of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of glutamate receptors. A complete prevention of o,p'-DDT effects was caused by an estrogen receptor (ER) antagonist as well as an aryl hydrocarbon receptor (AHR) antagonist and inhibitors of protein kinases A and C and mitogen-activated kinases. While an intermittent incubation with E2 caused no change in amplification of the glutamate-evoked GnRH release for 4 h, continuous incubation with E2 or o,p'-DDT caused an increase of this amplification after 3.5 h of incubation. In summary, DDT amplifies the glutamate-evoked GnRH secretion in vitro through rapid and slow effects involving ER, AHR, and AMPA receptor mediation.     

In vivo kinetics of trichloroacetate in male Fischer 344 rats.

Trichloroacetate (TCA) is a toxicologically important metabolite of the industrial solvents trichloroethylene and tetrachloroethylene, and a by-product of the chlorination of drinking water. Tissue disposition and elimination of 14C-TCA were investigated in male Fischer 344 rats injected iv with 6.1, 61, or 306 micromol TCA/kg body weight. Blood and tissues were collected at various time points up to 24 h. No metabolites were observed in plasma, urine, or tissue extracts. Overall TCA kinetics in tissues were similar at all doses. Based on similar terminal elimination rate constants, tissues could be divided into three classes: plasma, RBC, muscle, and fat; kidney and skin; and liver, small intestine, and large intestine. Nonextractable radiolabel, assumed to be biologically incorporated metabolites in both liver and plasma, increased with time, peaking at 6-9 h postinjection. The fraction of the initial dose excreted in the urine at 24 h increased from 67% to 84% as the dose increased, whereas fecal excretion decreased from 7% to 4%. The cumulative elimination of TCA as CO2 at 24 h decreased from 12% to 8% of the total dose. Two important kinetic processes were identified: a) hepatic intracellular concentrations of TCA were significantly greater than free plasma concentrations, indicating concentrative transport at the hepatic sinusoidal plasma membrane, and b) TCA appears to be reabsorbed from urine postfiltration at the glomerulus, either in the renal tubules or in the bladder. These processes have an impact on the effective tissue dosimetry in liver and kidney and may play an important role in TCA toxicity.     

UPAN对去卵巢大鼠神经元退行性变的作用

目的　通过观察UPAN对卵巢切除大鼠学习、记忆功能 ,海马神经元超微结构以及有关蛋白质表达的影响来证实UPAN对神经元退行性变有改善作用。方法　♀大鼠 ,行双侧卵巢切除手术造模 ,并于 6wk后皮下注射UPAN进行治疗 ,12wk后行水迷宫实验 ,至 13wk开始行灌注固定 ,取脑组织作超薄切片进行电镜分析 ;作冰冻切片进行雌激素受体 (ERα)、糖元合成激酶 (GSK 3)、细胞周期动态激酶(CDK 5 )、蛋白磷酸酯酶 (PP 1、PP2A及PP 2B)免疫组化染色。结果　水迷宫检测卵巢切除组大鼠较正常组游完全程所须时间明显延长及错误反应次数明显增多 ,UPAN治疗组结果与正常组接近。去卵巢组大鼠海马神经元超微结构明显损害 ,ERα表达增高 ,GSK 3、CDK 5、PP 1、PP2A及PP 2B表达增高 ,但以蛋白激酶GSK 3和CDK 5增高最为明显。使用UPAN治疗后能明显改善海马神经元超微结构的损害 ,使上述有关蛋白质的表达恢复到正常水平。结论　去卵巢大鼠存在着学习、记忆功能障碍 ,海马神经元超微结构以及有关蛋白质的表达出现异常改变。UPAN能明显改善其学习、记忆功能 ,维持海马神经元超微结构的完整性以及使有关蛋白质表达恢复到正常水平。     

Physiological role of the aryl hydrocarbon receptor in mouse ovary development.

The aryl hydrocarbon receptor (AhR) regulates the toxicity of environmental contaminants such as 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). As the physiological role of the AhR in the ovary is unknown, the purpose of this study was to test the hypothesis that the AhR regulates the appearance and numbers of ovarian follicles. Ovaries were harvested from AhR-deficient (AhRKO) and wild-type mice on gestational day 18 (GD 18) and postnatal days (PND) 2-3, 8, 32-35, and 53. Complete serial sections of ovaries were evaluated histologically for the presence of germ cells and follicles. On GD 18, there was no difference in the number of germ cells per ovary between AhRKO and wild-type fetuses. However, by PND 2-3, AhRKO mice had significantly more fully formed primordial follicles (AhRKO = 38,440 +/- 3632 versus wild-type = 21,120 +/- 2688) and fewer single germ cells than wild-type mice (AhRKO = 12,696 +/- 1192 vs. wild-type = 18,160 +/- 720). On PND 8 and 32-35, there was no difference in the number of follicles between AhRKO and wild-type mice but by PND 53, AhRKO mice had significantly fewer antral follicles than wild-type (AhRKO = 3416 +/- 480 vs. wild-type = 6776 +/- 1024). Taken together, these results suggest that the AhR may play a role in the formation of primordial follicles and the regulation of antral follicle numbers.     

In utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure: effects on fetal and adult cardiac gene expression and adult cardiac and renal morphology.

The mouse heart is a target of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during fetal development, and microarray analysis demonstrates significant changes in expression of cardiac genes involved in extracellular matrix (ECM) remodeling. We tested the hypothesis that developmental TCDD exposure would disrupt cardiac ECM expression and be associated with changes in cardiac morphology in adulthood. In one study, time-pregnant C57BL/6 mice were dosed with corn oil or 1.5, 3.0, or 6.0 microg TCDD/kg on gestation day (GD) 14.5 and sacrificed on GD 17.5, when changes in fetal cardiac mRNA expression were analyzed using quantitative PCR. TCDD induced mRNA expression of genes associated with ECM remodeling (matrix metalloproteinase 9 and 13, preproendothelin-1 [preproET-1]), cardiac hypertrophy (atrial natriuretic peptide, beta-myosin heavy chain, osteopontin), and aryl hydrocarbon receptor (AHR) activation (cytochrome P4501A1, AHR repressor). Further, all TCDD-induced changes required the AHR since gene expression was not altered in AHR knockout fetuses. In a second study, time-pregnant mice were treated with corn oil or 6.0 microg TCDD/kg on GD 14.5, and male offspring were assessed for changes in cardiac gene expression and cardiac and renal morphology at 3 months. All TCDD-induced changes in cardiac gene expression observed fetally, except for preproET-1, remained induced in the hearts of adult male offspring. Adult male offspring of TCDD-exposed dams also displayed cardiac hypertrophy, decreased plasma volume, and mild hydronephrosis. These results demonstrate that in utero and lactational TCDD exposures alter cardiac gene expression and cardiac and renal morphology in adulthood, which may increase the susceptibility to cardiovascular dysfunction.     

Methoxychlor induces proliferation of the mouse ovarian surface epithelium.

While the pesticide methoxychlor (MXC) has a variety of adverse effects on the female reproductive system, the effects of MXC on the ovarian surface epithelium (OSE) are unknown. Thus, this study tested the hypothesis that MXC alters the growth of the OSE. Mouse OSE cells were isolated by enzymatic digestion and cultured with vehicle, 3 microM of MXC, or 3 microM of 2,2-bis[p-hydroxyphenyl]-1,1,1,-trichloroethane (HPTE) for 14 days. After culture, proliferation and apoptosis were assessed by measurement of cell density, immunohistochemistry, and real-time polymerase chain reaction. Cell density was 66% greater for MXC-treated cells and 95% greater for HPTE-treated cells than controls (p < or = 0.05). The estrogen receptor blocker ICI 182,780 abolished MXC- and HPTE-induced increases in cell density. Proliferating cell nuclear antigen (PCNA) staining was positive in only 22 +/- 2.3% of controls, compared to 35 +/- 2.4% of MXC-treated cells and 40 +/- 2.4% of HPTE-treated cells (p < or = 0.05). The cell cycle regulators, cyclinD2 and cdk4, were significantly increased in MXC- and HPTE-treated cells compared to controls. The ApopTag assay demonstrated apoptotic cells in 4.8 +/- 0.45% of controls, 2.2 +/- 0.56% of MXC-treated cells, and 2.1 +/- 0.33% of HPTE-treated cells (p < or = 0.005). Expression of bcl-2 was significantly increased in MXC- and HPTE-treated cells, while bax was decreased in MXC- and HPTE-treated cells compared to controls. Collectively, these data indicate that MXC and HPTE stimulate OSE cell growth by increasing proliferation and inhibiting apoptosis. Further, since ICI 182,780 blocked MXC- and HPTE-induced OSE growth, these data suggest that the effects of MXC and HPTE on the OSE are mediated by estrogen receptors.     

Role of the modulation of CYP1A1 expression and activity in chemoprevention

As one of the main extra‐hepatic cytochrome P450 (CYP) enzymes, CYP1A1 has been comprehensively investigated for its ability to metabolize both exogenous and endogenous compounds into their carcinogenic derivatives. These derivatives are linked to cancer initiation and progression. The compound benzo‐a‐pyrene (BaP), a copious and noxious compound present in coal tar, automobile exhaust fumes, cigarette smoke and charbroiled food, is metabolised by CYP1A1 and has been studied in great detail. Other compounds reliant on the same enzyme for their activation include 7,12 dimethylbenz(a)anthracene (DMBA) and heterocyclic amine, 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP). This review takes an in‐depth look at a number of phytochemicals, plant extracts and a few synthetic compounds that have been researched and deemed potential chemopreventives via their interaction with the activity and expression of CYP1A1. It will also review a useful active site model of CYP1A1. Based on inhibitors of CYP1A1 that have demonstrated in vivo use as chemopreventors, CYP1A1 is a useful initial target for screening compounds with such potential, with the use of rapid in vitro and/or in silico assessments. Chemoprevention is a means by which healthy tissues are protected via the prevention, inhibition or reversal of carcinogenesis. This review focuses on one important pathway of carcinogenesis and identifies the important role that CYP1A1 plays in that pathway. It is hoped that highlighting the importance of such a key target, will help revive further research into and application of inhibitors of CYP1A1 towards generating improved chemopreventors. Copyright © 2014 John Wiley & Sons, Ltd.     

Correlation between gene expression of aryl hydrocarbon receptor (AhR), hydrocarbon receptor nuclear translocator (Arnt), cytochromes P4501A1 (CYP1A1) and 1B1 (CYP1B1), and inducibility of CYP1A1 and CYP1B1 in human lymphocytes.

The relationships between gene expression of aryl hydrocarbon receptor (AhR), aryl hydrocarbon receptor nuclear translocator (Arnt), cytochromes P4501A1 (CYP1A1), 1B1 (CYP1B1), CYP1A1, and the inducibility of CYP1A1 and CYP1B1 were determined in 32 cultivated human lymphocytes. Cytochrome P450 induction was performed by incubating lymphocytes with benzanthracene. The relative gene expression levels were determined by quantitative real-time RT-PCR assay. We found that gender is an important confounding factor for gene expression in cultivated lymphocytes. AhR, CYP1A1 and CYP1B1 levels in noninduced lymphocytes were significantly higher in female nonsmokers than in male nonsmokers (p < 0.05). Nevertheless, CYP1A1 and CYP1B1 inducibility was lower in female nonsmokers. CYP1A1 inducibility was higher in male smokers than in male nonsmokers (p < 0.05). After controlling for gender and cigarette smoking, AhR levels positively correlated with CYP1B1 levels and CYP1A1 inducibility (p < 0.01 and p = 0.03, respectively). Arnt levels also correlated with CYP1B1 levels in induced lymphocytes (p < 0.01). However, AhR levels were negatively correlated with CYP1B1 inducibility. These data indicate that AhR expression associates with individual variation of CYP1A1 inducibility and CYP1B1 expression in cultivated lymphocytes. Furthermore, gender and cigarette smoking are important confounding factors for gene expression levels in cultivated lymphocytes.     

Effect of aqueous tobacco smoke extract and shear stress on PECAM-1 expression and cell motility in human uterine endothelial cells.

Tobacco smoke constituents have several adverse effects on endothelial cells. Exposure to tobacco smoke during pregnancy is associated with adverse effects on pregnancy outcome possibly related to endothelial dysfunction. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is an important regulator of endothelial function. This study tests the idea that an aqueous extract of cigarette smoke alters the expression of PECAM-1 in uterine endothelial cells. Human uterine microvascular endothelial cells were cultured in cigarette smoke-conditioned medium (CSM) under arterial physiological flow conditions (shear or frictional stress in the range 7.5-15 dyne/cm(2)) and the expression of PECAM-1 was assessed by immunofluorescence microscopy and Western blotting. Thick reticular PECAM-1-associated bands found at cell-cell junctions in static cultures became significantly thinner or disappeared when the cells were exposed to shear stress or to CSM for 24 h. This diminution at cell junctions was accompanied by increased punctate cytoplasmic/cell surface staining. Under shear stress conditions, PECAM-1 was equally distributed between cell surface and intracellular sites. In contrast, when cells were exposed to both shear stress and CSM, PECAM-1 was predominantly localized to the cell surface. It was shown that shear stress increased endothelial cell migration and that CSM abrogated this effect. These results suggest that, under shear stress conditions, PECAM-1 is not predominantly concentrated at intercellular junctions in uterine endothelial cells. Exposure of cells to unidentified soluble components of cigarette smoke leads to alterations in PECAM-1 distribution that may cause endothelial dysfunction. If this occurs in vivo it could contribute to the adverse effects on pregnancy outcome associated with exposure to cigarette smoke.     

Toxicogenomic profile of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the murine fetal heart: modulation of cell cycle and extracellular matrix genes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and similar environmental contaminants have been demonstrated to be potent cardiovascular teratogens in developing piscine and avian species. In the present study, we investigated the effects of TCDD on gene expression during murine cardiovascular development. C57Bl6N pregnant mice were dosed with 1.5, 3.0, or 6.0 microg TCDD/kg on gestational day (GD) 14.5, and microarray analysis was used to characterize the global changes in fetal cardiac gene expression on GD 17.5. TCDD significantly altered expression of a number of genes involved in xenobiotic metabolism, cardiac homeostasis, extracellular matrix production/remodeling, and cell cycle regulation. Interestingly, while the AhR-responsive genes Cyp1A1, Cyp1B1, Ugt1a6, and Ahrr, were all induced by TCDD in the fetal murine heart, other AhR-responsive genes, Cyp1a2, Nqo1, and Gsta1, were not. Quantitative real-time polymerase chain reactions confirmed the changes in expression of several G1/S-type cyclins and extracellular matrix-related genes. These results demonstrate the global changes in cardiac gene expression that result from TCDD exposure of the fetal murine heart and implicate genes involved in cell cycle and extracellular matrix regulation in TCDD-induced cardiac teratogenicity and functional deficits.     

Differential expression patterns of Wnt and beta-catenin/TCF target genes in the uterus of immature female rats exposed to 17alpha-ethynyl estradiol.

To characterize the effects of an estrogen receptor (ER) agonist on the gene expressions in the uterus, immature female rats were administered once orally with 17alpha-ethynyl estradiol (EE, 3 mug/kg), a potent ER agonist. We focused on four categories of sex steroid hormone receptor genes: well-known estrogen target genes, Wnt genes, and beta-catenin/T-cell factor (TCF) target genes. ERalpha, ERbeta, progesterone receptor, and androgen receptor mRNAs were all downregulated at 24 and/or 48 h after EE administration. Complement C3 and insulin-like growth factor 1 mRNAs were markedly induced after EE administration. Although the time courses of Wnt4, Wnt5a, and Wnt7a mRNA status varied until 12 h after EE administration, all of them were simultaneously downregulated at 24 and 48 h. The remarkable downregulation of Wnt7a mRNA in response to EE was considered to be important to understand the various uterine phenomena affected by ER agonists. In the beta-catenin/TCF target genes, the downregulation of anti-Mullerian hormone type 2 receptor and bone morphogenetic protein 4 mRNA after EE administration appeared to be closely related to the downregulation of Wnt7a. The upregulation of cyclin D1 and follistatin mRNA at the early phase after EE administration was considered to have been affected by the upregulation of Wnt4. These results indicate that an ER agonist influences not only the mRNA expression of sex steroid hormone receptor genes and well-known estrogen target genes but also Wnt genes (Wnt4, Wnt5a, Wnt7a) and beta-catenin/TCF target genes in the uterus of immature rats, indicating that their molecules are the potential players affected by estrogenic stimuli.     

Activation of aryl hydrocarbon receptor by benzo[a]pyrene increases interleukin 33 expression and eosinophil infiltration in a mouse model of allergic airway inflammation

We recently demonstrated that benzo[a]pyrene (BaP), the aryl hydrocarbon receptor (AhR) ligand, directly contributes to aggravation of cutaneous allergy in a mouse model of allergic dermatitis. The present study aimed to determine whether BaP-induced AhR activation results in development of airway inflammation. Initially, the potential for a direct relationship between BaP-induced AhR activation and airway inflammation was investigated in vivo, using a mouse model of type 2 helper T cell (Th2) hapten toluene-2,4-diisocyanate (TDI)-induced airway inflammation. Mice were orally administered BaP at 48, 24, and 4 h before the final allergen challenge. Oral administration of BaP showed a significant increase in lung inflammation and eosinophil infiltration. While expression of Th2 cytokines such as interleukin 4 (IL-4) and IL-13 was not affected by exposure to BaP, AhR activation significantly increased IL-33 expression. To confirm the in vivo results, in vitro experiments were performed using the human eosinophilic leukemia cell line (EOL-1), human bronchial epithelial cell line (BEAS-2B), and human lung adenocarcinoma epithelial cell line (A549). Results indicated that pre-treatment with BaP increased expression of IL-8 in house dust mite-activated EOL-1, BEAS-2B, and A549 cells. In addition, IL-33 levels in BEAS-2B cells were significantly increased after BaP exposure. Our findings indicated that BaP-induced AhR activation is involved in the pro-inflammatory response in respiratory allergy, and that this effect may be mediated by increased IL-33 expression and eosinophil infiltration.