Modulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated myelotoxicity by thyroid hormones

Although binding by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the Ah receptor is a prerequisite for toxicity, the events responsible for subsequent TCDD effects are essentially unknown. Several lines of evidence have indicated that thyroid hormones share common molecular properties with TCDD and can modulate its toxicity. In the present studies we employed suppression of murine bone marrow hematopoiesis by TCDD as an in vitro model to study the relationship between thyroid hormones and TCDD toxicity. Supraphysiological levels of thyroid hormone mimicked TCDD myelotoxicity, in that both were inhibited by a common antagonist, 1-NH2-3,7,8-trichlorodibenzo-p-dioxin. Furthermore, myelotoxicity by both TCDD and thyroid hormone segregated with the Ah locus in congenic mice. These data provide evidence of a relationship between TCDD and thyroid hormones in that hormonal activity may help regulate TCDD toxicity.     

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in C57B1/6 mice

Three-month-old male $\text{C57B1}\text{6}$ mice were given single oral doses of 0, 100, 150, or 200 μg of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg. The LD50 was 114 μ/kgg. In mice that died, depletion of the thymus and spleen were consistently found and edema and terminal hemorrhages occurred frequently. In a second experiment, 4-month-old male mice were dosed po with 0, 0.2, 1.0, 5.0 or 25 μg/kg, once a week for 2 or 6 weeks. Some deaths and growth retardation occurred in the 25 μg/kg dose group. Significantly increased liver and decreased thymus weights were found in the 1, 5 and 25 μg/kg dose groups. Total neutrophils were increased significantly, whereas hemoglobin values and mean corpuscular hemoglobin concentrations were decreased significantly after 6 doses of 25 μg/kg. Total serum protein and α-, β-, and γ-globulins were significantly decreased. TCDD was porphyrogenic. The hepatic porphyria was probably associated with liver damage. Degenerative and necrotic changes in the liver were essentially centrilobular and were accompanied by cellular infiltrates and ceroid pigment deposition. Proliferation of bile duct and bile duct epithelial cells occurred. Lipid accumulation was centrilobulary localized in the mice receiving 0.2 μg/kg, was more pronounced in the mice of the intermediate dose levels, and involved hepatocytes throughout the lobule in the 25 μg/kg dose group.     

Factors influencing the induction of DNA single strand breaks in rats by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to induce lipid peroxidation and DNA damage in rat hepatic nuclei was investigated. In addition, the role of iron in hepatic DNA damage was examined. Female Sprague-Dawley rats were treated with a single dose of 25--100 micrograms TCDD/kg orally, and sacrificed 3-14 days after treatment. Liver nuclei were isolated, and DNA single strand breaks (DNA-SSB) and lipid peroxidation were determined. Lipid peroxidation was assessed by measuring the content and production of thiobarbituric acid reactive substances (TBARS) while DNA-SSB were determined by the alkaline elution technique. The results demonstrate that TCDD dose and time-dependent increases in hepatic nuclear TBARS content and production occur in conjunction with an increase in DNA-SSB. The administration of the dithiolthione antioxidant oltipraz (30 mg/kg/day for 10 days) resulted in a significant decrease (47%) in the incidence of TCDD-induced DNA-SSB. Clofibrate administration (100 mg/kg/day for 12 days) and pair feeding had no effect on TCDD-induced DNA-SSB. The incubation of hepatic microsomes and mitochondria from TCDD-treated rats with nuclei from untreated animals for one hr at 37 degrees C resulted in enhanced DNA damage which was abolished by the addition of 0.10 mM desferrioxamine (DFX). Incubation with cytosol had no significant effect. Incubation of 0.10 mM Fe2+ or Fe3+ with isolated hepatic nuclei from untreated rats produced significant increases in DNA-SSB, which were abolished by the addition of 0.10 mM DFX to the incubation medium. TCDD may produce an increased bioavailability of iron which leads to enhanced DNA single strand breaks and lipid peroxidation in hepatic nuclei.     

Methoxychlor suppresses the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible CYP1A1 expression in murine Hepa-1c1c7 cells

Methoxychlor (MXC) is a pesticide that was developed as a replacement for dichlorodiphenyltrichloroethane (DDT). The influence of MXC on CYP1A1 expression or the functions of mouse hepatoma Hepa-1clc7 remain unclear. Cultured Hepa-1c1c7 cells were treated with MXC with or without 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to assess the role of MXC on CYP1A1 expression. MXC alone did not affect CYP1A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity. In contrast, TCDD-inducible EROD activities were markedly reduced upon concomitant treatment with TCDD and MXC in a concentration-dependent manner. Treatment with ICI 182.780, an estrogen-receptor antagonist, did not affect the suppressive effects of MXC on TCDD-inducible EROD activity. TCDD-inducible CYP1A1 mRNA levels were markedly suppressed upon treatment with TCDD and MXC, and this is consistent with their effects on EROD activity. A transient transfection assay using dioxin-response element (DRE)-linked luciferase and an electrophoretic mobility shift assay revealed that MXC reduced the transformation of the aryl hydrocarbons (Ah) receptor to a form capable of specifically binding to the DRE sequence in the promoter region of the CYP1A1 gene. These results suggest that the downregulation of CYP1A1 gene expression by MXC in Hepa-1c1c7 cells might be an antagonism of the DRE binding potential of the nuclear Ah receptor but is not mediated through the estradiol receptor.     

Aryl hydrocarbon receptor-dependent induction of loss of mitochondrial membrane potential in epididydimal spermatozoa by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant known to exhibit toxic effects on the male reproductive system, including the epididymus and spermatozoa. However, the mechanism(s) that mediate dioxin toxicity in spermatozoa remain unclear. The aim of the present study was to investigate whether exposure to TCDD would cause a loss in mitochondrial membrane potential (Deltapsi(m)) in spermatozoa and whether such an effect is mediated by the Ah receptor (AhR). Exposure of C57BL/6 male mice to TCDD at concentrations of 0.1-50 microg/kg for 24 h caused a dose-dependent loss of Deltapsi(m) in epididymal spermatozoa compared to spermatozoa from vehicle-treated mice. However, this effect was not apparent in spermatozoa from AhR knockout (KO) mice. Exposure of spermatozoa from C57BL/6 mice to 1 nM or 5 nM TCDD in vitro also induced loss of Deltapsi(m). TCDD-exposed C57BL/6 mice failed to exhibit changes in the morphology of testes and epididymus, and did not show any increase in number of apoptotic germ cells. In addition, comparison of reactive oxygen species (ROS) production in spermatozoa from vehicle- and TCDD-treated mice indicated that exposure to TCDD resulted in elevated ROS levels in the spermatozoa from TCDD-treated mice. Moreover, blockade of ROS production by pretreatment with ROS scavenger N-acetylcysteine (NAC) mitigated the loss of Deltapsi(m) following TCDD exposure. Taken together, these data suggest that direct exposure of spermatozoa to TCDD triggers loss of Deltapsi(m) that is mediated by AhR-dependent production of ROS.     

Alteration of the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by estradiol and tamoxifen

We have hypothesized that part of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is mediated by interaction with the estrogen receptor complex. The experiments reported here investigate the interactions of TCDD with agonists and antagonists of the estrogen receptor. CD-1 female mice were observed for 2 months after treatment with various combinations of corn oil, estradiol, or tamoxifen, and/or TCDD in corn oil on 3 consecutive days. Estradiol had little effect on acute TCDD lethality but increased severity of TCDD-induced ascites and antagonized TCDD-induced uterine suppression. Severe liver damage did occur in TCDD and estradiol:TCDD treatment groups. Tamoxifen, a competitive inhibitor and a mixed agonist of the mouse estrogen receptor, antagonized the estrogenic effects of estradiol and estradiol:TCDD. Tamoxifen or tamoxifen:TCDD treatment greatly slowed body weight gain in comparison to controls and estrogen-treated animals. While the dose of tamoxifen used was otherwise non-toxic, tamoxifen greatly increased toxicity of TCDD as measured by time to death and percent lethality while having no effect on relative liver weight or relative uterine weight changes induced by TCDD. These findings are consistent with the hypothesis that a portion of the toxicity of TCDD is manifest through activity of the estrogen receptor complex.     

A cytosolic binding protein for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the uterus and deciduoma of rats

The cytosol fraction of the uterus of proestrous rats and of deciduoma specifically binds 3H-2,3,7,8-tetrachlorodibenzo-p-dioxin (3H-TCDD). The 3H-TCDD binding protein has a sedimentation coefficient of 9 S in low ionic 10-40% sucrose density gradients. The binding of 3H-TCDD in the 9 S region is abolished by a 500-fold molar excess of unlabeled benzo[a]pyrene, 3-methylcholanthrene or by a 100-fold molar excess of unlabeled TCDD. Incubation of the binding protein with TCDD in amounts in excess of 500 nM causes aggregation of the TCDD binding protein. Neither estradiol, progesterone, testosterone, dihydrotestosterone nor cortisol competed with TCDD in binding to this 9 S protein. The decidual tissue contains two binding components for TCDD as shown by Scatchard analysis. One of the components has a high affinity for TCDD (Kd = 1.68 nM) and is saturable. The number of binding sites is about 75 fmol/mg protein. The TCDD binding protein eluted through a DEAE-cellulose column using a gradient of 0.25 M KCl. The binding of estradiol and progesterone to their respective receptors was not affected by TCDD or by other polycyclic aromatic hydrocarbons as shown by sucrose density gradients and by microtiter competition assays. These results suggest that TCDD acts by binding to its own receptor system in the target tissue and not by competing with estrogen or progesterone for binding to their receptors. The possible role of the receptor in teratogenesis is discussed.     

Topical transduction of superoxide dismutase mediated by HIV-1 Tat protein transduction domain ameliorates 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice

A domain (RKKRRQRRR) derived from HIV-1 Tat is one of the most efficient protein transduction domains (PTD) for delivering macromolecules including proteins into cells and tissues. Antioxidant enzymes such as superoxide dismutase (SOD) and catalase are major cellular defenses against oxidative stress which results in various diseases including skin inflammation. In this study, we examined the effect of SOD fused with HIV-1 Tat PTD (Tat-SOD) on TPA-induced skin inflammation in mice. Topical application of Tat-SOD to mice ears 1h after TPA application once a day for 3 days dose-dependently inhibited TPA-induced ear edema in mice. Topical application on mice ears of Tat-SOD also suppressed TPA-induced expression of proinflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6 as well as cyclooxygenase-2 (COX-2) and production of PGE(2). Furthermore, topical application of Tat-SOD resulted in significant reduction in activation of NF-kappaB and mitogen-activated protein kinases (MAPK) in the mice ears treated with TPA. These data demonstrates that Tat-SOD inhibits TPA-induced inflammation in mice by reducing the levels of expression of proinflammatory cytokines and enzymes regulated by the NF-kappaB and MAPK and can be used as a therapeutic agent against skin inflammation related to oxidative damage.     

Increased permeability of the rat biliary tree by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment and protection by hepatoactive agents

Male Sprague-Dawley rats were treated ip on Day 0 with 0, 20, 50, or 100 micrograms/kg of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and biliary tree permeability was evaluated on Days 2, 4, 7, 10, 14, or 20 by segmented retrograde intrabiliary injection of [3H]sucrose or [14C]mannitol. Seven days after 100 micrograms/kg TCDD, the percentage recovery in bile of both [3H]sucrose (73.9 +/- 4.2 vs 27.6 +/- 7.6, control vs TCDD, means +/- SE) and [14C]mannitol (22.7 +/- 2.2 vs 12.1 +/- 2.2) was decreased, demonstrating that the permeabilities of both the intracellular (canalicular) and paracellular pathways were increased. Seven days after 50 micrograms/kg TCDD, the recovery of [3H]sucrose was decreased (73.5 +/- 5.4 vs 39.0 +/- 2.8) but the recovery of [14C]mannitol was not (25.5 +/- 1.5 vs 22.9 +/- 1.9). Thus, an increase in paracellular permeability is obtained at a lower dose of TCDD. In rats treated with 100 micrograms/kg TCDD on Day 0, co-treatment with chlordecone (15 mg/kg/day on Days 2-6) or thyroxine (50 micrograms/kg on Day 2) had no effect. Pregnenolone-16 alpha-carbonitrile (75 mg/kg/day on Days 4-6) treatment increased [14C]mannitol recovery in both TCDD and control groups; its effect must not be specific. Methimazole given in drinking water (0.5%) on Days -7 through 7 reversed the increased permeability effects of TCDD (100 micrograms/kg) without affecting the biliary tree permeability ([14C]mannitol recovery) of control animals.     

Involvement of tumor necrosis factor (TNF)-alpha in phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin edema in mice

Topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse ear induced a prolonged skin inflammation. Histological analysis revealed that the early stage (approximately 3 h) and later stage (6-24 h) of the skin reaction are characterized by dermal edema and cell accumulation, respectively. Topical application with TPA also induced increase in the level of TNF-alpha and prostagrandin E2 (PGE2) at the application site. The increase of TNF-alpha was transient with a peak at approximately 5 h, followed by a gradual elevation of PGE2 level in the skin. An in vitro study with human keratinocytes as well as immunohistochemical analysis suggested that TNF-alpha induction in the skin might be produced by epidermis treated with TPA. Administration of a cyclooxygenase inhibitor indomethacin inhibited the later stage of the TPA-induced edema. In contrast, TNF-alpha antagonist etanercept inhibited exclusively the early stage of the reaction. Taken together, these data demonstrate that the prolongation of the skin inflammation induced by TPA may be due to the sequential production of proinflammatory mediators such as eicosanoids and cytokines, and show for the first time the importance of TNF-alpha in the TPA-induced dermatitis especially at the stage where dermal edema is significant.     

Repeated dose toxicity and relative potency of 1,2,3,4,6,7-hexachloronaphthalene (PCN 66) 1,2,3,5,6,7-hexachloronaphthalene (PCN 67) compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for induction of CYP1A1, CYP1A2 and thymic atrophy in female Harlan Sprague-Dawley rats

In this study we assessed the relative toxicity and potency of the chlorinated naphthalenes 1,2,3,4,6,7-hexachloronaphthalene (PCN 66) and 1,2,3,5,6,7-hexachloronaphthalene (PCN 67) relative to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Chemicals were administered in corn oil:acetone (99:1) by gavage to female Harlan Sprague-Dawley rats at dosages of 0 (vehicle), 500, 1500, 5000, 50,000 and 500,000ng/kg (PCN 66 and PCN 67) and 1, 3, 10, 100, and 300ng/kg (TCDD) for 2 weeks. Histopathologic changes were observed in the thymus, liver and lung of TCDD treated animals and in the liver and thymus of PCN treated animals. Significant increases in CYP1A1 and CYP1A2 associated enzyme activity were observed in all animals exposed to TCDD, PCN 66 and PCN 67. Dose response modeling of CYP1A1, CYP1A2 and thymic atrophy gave ranges of estimated relative potencies, as compared to TCDD, of 0.0015-0.0072, for PCN 66 and 0.00029-0.00067 for PCN 67. Given that PCN 66 and PCN 67 exposure resulted in biochemical and histopathologic changes similar to that seen with TCDD, this suggests that they should be included in the WHO toxic equivalency factor (TEF) scheme, although the estimated relative potencies indicate that these hexachlorinated naphthalenes should not contribute greatly to the overall human body burden of dioxin-like activity.     

Cell death and impairment of glucose-stimulated insulin secretion induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the beta-cell line INS-1E

The aim of this research was to characterize 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity on the insulin-secreting beta-cell line INS-1E. A sharp decline of cell survival (below 20%) was observed after 1 h exposure to TCDD concentrations between 12.5 and 25 nM. Ultrastructurally, beta-cell death was characterized by extensive degranulation, appearance of autophagic vacuoles, and peripheral nuclear condensation. Cytotoxic concentrations of TCDD rapidly induced a dose-dependent increase in intracellular calcium concentration. Blocking calcium entry by EGTA significantly decreased TCDD cytotoxicity. TCDD was also able to rapidly induce mitochondrial depolarization. Interestingly, 1 h exposition of INS-1E cells to very low TCDD concentrations (0.05-1 nM) dramatically impaired glucose-stimulated but not KCl-stimulated insulin secretion. In conclusion, our results clearly show that TCDD exerts a direct beta-cell cytotoxic effect at concentrations of 15-25 nM, but also markedly impairs glucose-stimulated insulin secretion at concentrations 20 times lower than these. On the basis of this latter observation we suggest that pancreatic beta-cells could be considered a specific and sensitive target for dioxin toxicity.     

Effects of thyroid hormones on the induction of cleft palate by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in C57BL/6N mice

The induction of cleft palate by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) administered with thyroid hormones triiodothyronine (T3) or thyroxine (T4) was investigated in C57BL/6N mice. Timed-pregnant mice were treated with vehicle, TCDD, T3, T4, TCDD plus T3, or TCDD plus T4 on Days 10 to 13 of gestation. No cleft palates were observed in any control fetuses in this study, nor have there been any cleft palates in 1193 fetuses or 154 control litters in the past 24 months. TCDD (3 micrograms/kg/day) caused about 8% cleft palates per litter, while T3 (120, 240, 480 micrograms/kg/day) and T4 (625, 1250, 2500 micrograms/kg/day) resulted in no more than 1.2% cleft palates per litter in any of the treatment groups and the incidence was not dose related. The combination of TCDD (3 micrograms/kg/day) plus T3 at 120, 240, and 480 micrograms/kg/day resulted in 15.9, 20.6, and 31.4% cleft palates per litter, respectively. TCDD plus T4 at 625, 1250 and 2500 micrograms/kg/day caused 15.1, 22.9, and 27.2% cleft palates per litter. No cleft palates were observed when large doses of T3 were given in combination with T4. These data demonstrated that coadministration of T3 or T4 with TCDD increased the incidence of cleft palate to incidences greater than expected from the separate administration of the hormones plus TCDD.     

Oxidative stress in female B6C3F1 mice following acute and subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly persistent trace environmental contaminant and is one of the most potent toxicants known to man. Hassoun et al. (1998, Toxicol. Sci. 42, 23-27) reported an increase in the production of reactive oxygen species (ROS) in the brain of female B6C3F1 mice following subchronic exposure to TCDD at doses as low as 0.45 ng/kg/day. In the present study, oxidative stress was characterized in liver, spleen, lung, and kidney following subchronic (0.15-150 ng/kg; 5 days/week for 13 weeks, po) or acute exposure (0.001-100 microg/kg, po) to TCDD in order to investigate the interaction between tissue concentration and time for production of ROS. Seven days following acute administration of TCDD, mice were sacrificed; they demonstrated increases in liver superoxide anion production (SOAP) and thiobarbituric acid reactive substances (TBARS) at doses of 10 and 100 microg/kg, associated with hepatic TCDD concentrations of 55 and 321 ng/g, respectively. Liver obtained from mice following subchronic TCDD exposure demonstrated an increase in SOAP and TBARS above controls at doses of 150 ng/kg/day with liver TCDD concentration of only 12 ng/g. Interestingly, glutathione (GSH) levels in lung and kidney following sub-chronic TCDD exposure were decreased at the low dose of 0.15 ng/kg/day. This effect disappeared at higher TCDD doses. The data suggest that higher tissue TCDD concentrations are required to elicit oxidative stress following acute dosing than with subchronic TCDD exposure. Therefore, the mechanism of ROS production following TCDD exposure does not appear to be solely dependent upon the concentration of TCDD within the tissue. In addition, very low doses of TCDD that result in tissue concentrations similar to the background levels found in the human population produced an effect on an oxidative stress endogenous defense system. The role of this effect in TCDD-mediated toxicity is not known and warrants further investigation.     

Celiprolol inhibits mitogen-activated protein kinase and endothelin-1 and transforming growth factor-beta(1) gene in rats

We evaluated the cardioprotective effects of long-term treatment with celiprolol (for 5 weeks), a specific beta(1)-adrenoceptor antagonist with a weak beta(2)-adrenoceptor agonist action, on endothelin-1 and transforming growth factor (TGF)-beta(1) expression and cardiovascular remodeling in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Upregulated preproendothelin-1, endothelin ET(A) receptor, TGF-beta(1), c-fos, and type I collagen expression and extracellular signal-regulated kinase activities were suppressed by celiprolol. Celiprolol effectively inhibited vascular lesion formation such as medial thickness and perivascular fibrosis. These observations suggested that extracellular signal-regulated kinase and c-fos gene pathway may contribute to the cardiovascular remodeling of DOCA rats, and that cardioprotective effects of celiprolol on cardiovascular remodeling may be mediated, at least in part, by suppressed expression of endothelin-1 and TGF-beta(1).