2,3,7,8-Tetrachlorodibenzo-p-dioxin induces a proteomic pattern that defines cleft palate formation in mice

Objective

To identify the differential protein pattern in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced cleft palates using a proteomic approach.

Methods

At gestation day (GD) 12, TCDD (64 g/kg; n = 30) or corn oil control (n = 30) was given to time-pregnant C57BL/6J mice by gavage. The anatomical, histological, proteomic changes in the palates of the fetal mice were studied on GD18. Total protein was extracted from the palate tissue and examined by 2-dimensional gel electrophoresis (2-DE). Spots differentially expressed between the two groups were selected for analysis by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The proteins were identified by data searching in the Mascot database.

Results

In TCDD group, the incidence of cleft palate was 100%. Ten differential protein spots with the largest fold change were selected for further identification by mass spectrometry, 7 showed significantly higher volumes and 3 showed significantly lower volumes in TCDD palates than the control palates (all p < 0.05). Peroxiredoxin-1 were robustly up-regulated in the cleft palate group, as well proteins linked to energy metabolism, cell migration, and apoptosis.

Conclusions

Peroxiredoxin-1 protein may be associated with cleft palate in mice induced by TCDD. The embryo mouse palate tissues energy metabolism cells migration/apoptosis related proteins have the disorder.     

Common differentially expressed proteins were found in mouse cleft palate models induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin and retinoic acid

Cleft palate(CP) is a widely studied congenital malformation. However, its etiology and pathogenesis still remain unclear. Proteins are fundamental molecules that participate in every biological process within cells. In this study, we established CP mouse models induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and retinoic acid (RA), using proteomics technology isobaric tags for relative and absolute quantitation (iTRAQ) to investigate the key proteins in the formation of CP. Pregnant mice were given a gavage of TCDD 28μg/kg or retinoic acid 80mg/kg of body weight or equivalent corn oil at gestational day 10.5(GD10.5) and sacrificed at GD 17.5. Foetal mice were recorded and collected for further detection. Western blot was performed to verify the iTRAQ results. Eventually, we obtained 18 common differentially expressed proteins in TCDD group and RA group compared with normal control, 17 up-regulated and 1 down-regulated. 14-3-3sigma and Annexin A1 were up-regulated in experimental groups at GD17.5, which was consistent with Western blot. We speculated that the common differentially expressed proteins might be one of the molecular mechanisms in the formation of cleft palate.     

Induction of cell-cycle arrest by all-trans retinoic acid in mouse embryonic palatal mesenchymal (MEPM) cells.

all-trans retinoic acid (atRA), the oxidative metabolite of vitamin A, is essential for normal embryonic development. Also, high levels of atRA are teratogenic in many species and can effectively induce cleft palate in the mouse. Most cleft palate resulted from the failed fusion of secondary palate shelves, and maintenance of the normal cell proliferation is important in this process of shelf growth. To clarify the mechanism by which atRA causes cleft palate, we investigated the effect of atRA on proliferation activity and cell cycle distribution in mouse embryonic palatal mesenchymal (MEPM) cells. atRA inhibited the growth of MEPM cells by inducing apoptosis in a dose-dependent manner. atRA also caused a G1 block in the cell cycle with an increase in the proportion of cells in G0/G1 and a decrease in the proportion of cells in S phase, as determined by flow cytometry. We next investigated the effects of atRA on molecules that regulate the G1 to S phase transition. These studies demonstrated that atRA inhibited expression of cyclins D and E at the protein level. Furthermore, atRA treatment reduced phosphorylated Rb and decreased cdk2 and cdk4 kinase activity. These data suggest that atRA had antiproliferative activity by modulating G1/S cell cycle regulators and by inhibition of Rb phosphorylation in MEPM cells, which might account for the pathogenesis of cleft palate induced by retinoic acid.     

Toxic interaction of specific polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin: Increased incidence of cleft palate in mice

The induction of cleft palate in $\text{C57BL}\text{6N}$ mice is an extremely reproducible and sensitive indicator of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. This endpoint was used to look for potential interactions between two polychlorinated biphenyl (PCB) congeners and TCDD. Both 2,3,4,5,3′,4′-hexachlorobiphenyl (HCB) and 2,4,5,2′,4′,5′-HCB are of relatively low toxic potency, but their biological properties differ. Pregnant mice were treated with TCDD and either HCB on gestation Days 10 through 13, and the fetuses examined for the presence of cleft palate and renal abnormalities on gestation Day 18. At a dose of TCDD which caused a low level of cleft palate, moderate hydronephrosis was observed. No renal or palatal anomalies were detected after 2,4,5,2′,4′,5′-HCB treatment, and the combination of this isomer with TCDD had no effect on the incidence of TCDD-induced cleft palate. 2,3,4,5,3′,4′-HCB caused mild renal toxicity, but no cleft palate. However, treatment of pregnant mice with a combination of TCDD and 2,3,4,5,3′,4′-HCB resulted in a 10-fold increase in the incidence of cleft palate. Thus, the toxicity of compounds such as TCDD may be enhanced by compounds of relatively low acute toxicity such as selected PCBs. The widespread environmental occurrence of such combinations suggests a need for further evaluation of the mechanism of this interaction.     

Limited PCB antagonism of TCDD-induced malformations in mice.

C57BL/6N mice used to model induction of cleft palate and kidney malformations in offspring following maternal treatment with TCDD, were dosed on gestation day (gd) 9 with 2,2',4,4',5,5'-hexachlorobiphenyl (HCB) (62.5, 125, 250, 500, 1000 mg/kg) and/or gd 10 with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (15 or 18 micrograms/kg) to investigate the potential protective effects of HCB against TCDD-induced teratogenicity. Maternal body weight gain was increased by combinations of 15 micrograms TCDD/kg and 125-500 mg HCB/kg and decreased at doses of 15 micrograms TCDD/kg + 1000 HCB mg/kg. At the doses used in this study, there was no effect of either compound on number of live or dead offspring. Fetal body weight was slightly decreased in all groups dosed with greater than or equal to 250 mg HCB/kg. HCB did not induce cleft palate at a dose of 1000 mg/kg, but did induce increases in hydronephrosis and hydroureter at 500 and 1000 mg/kg. Combinations of HCB and TCDD decreased the incidence of cleft palate induced by TCDD alone, but only at doses of 15 micrograms TCDD/kg combined with 125-500 mg HCB/kg. The antagonism of hydronephrosis (incidence and severity) appeared over a narrower dose range (15 micrograms TCDD/kg + 500 mg HCB/kg). HCB induced increases (3-fold) in ethoxyresorufin-O-deethylase (EROD) activity at doses of 500 and 1000 mg/kg, suggesting that the limited antagonism of TCDD teratogenicity by HCB could be under the control of the Ah-receptor.     

甲基亚硝基胍致小鼠腭裂的分子机制

目的探讨小鼠腭裂发生的分子机制。方法以甲基亚硝基胍(MNNG)为受试物诱导腭裂动物模型,采用抑制消减杂交技术从全基因组水平上对MNNG致腭裂相关差异表达基因进行筛选。结果得到MNNG逆向表达片断27个,正向差异表达片断14个。经测序和GeneBank比对后得到已知基因9条,余者为未知功能基因或未知基因。结论Gpc3可能通过调节腭发生过程中某些基因干扰间充质细胞正常增殖;Ptprs的表达抑制影响了某种腭突发生中细胞信号转导通路;腱生蛋白的抑制表达可能使细胞粘连力的去除失败或者由于基质中MMPs的表达降低而使腭中缝消除受阻;抑或由EgfrPtprsTnC协同作用而导致腭裂发生。还可能影响细胞对营养物质的摄取而引起Rps25的上调诱发过度的细胞凋亡而在腭裂发生中发挥作用。     

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.     

2,2',4,4',5,5'-hexachlorobiphenyl as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist in C57BL/6J mice

At doses as high as 750 to 1000 mumol/kg, 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP) did not cause fetal cleft palate, suppress the splenic plaque-forming cell response to sheep red blood cells, or induce hepatic microsomal ethoxyresorufin O-deethylase (EROD) in C57BL/6J mice. Despite the lack of activity of HCBP in eliciting any of these aryl hydrocarbon (Ah) receptor-mediated responses, competitive binding studies indicated that HCBP competitively displaced 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) from the murine hepatic cytosolic receptor. Cotreatment of C57BL/6J mice with TCDD (3.7 nmol/kg) and HCBP or 4,4'-diiodo-2,2',5,5'-tetrachlorobiphenyl (I2-TCBP) (400 or 1000 mumol/kg) showed that both compounds partially antagonized TCDD-mediated cleft palate and immunotoxicity (i.e., suppression of the splenic plaque-forming cell response to sheep red blood cells), and HCBP antagonized TCDD-mediated hepatic microsomal EROD induction. Thus, HCBP and I2-TCBP, like the commercial polychlorinated biphenyl mixture Aroclor 1254, were partial antagonists of TCDD action in C57BL/6J mice; however, it was also apparent from the results that Aroclor 1254 was the more effective antagonist at lower doses. Using [3H]TCDD, it was also shown that some of the effects of HCBP on TCDD-mediated cleft palate may be due to the decreased levels of TCDD found in the fetal palates after cotreatment with TCDD and HCBP. 4,4'-[125I2]diiodo-2,2',5,5'-tetrachlorobiphenyl ([125I2]TCBP) of high specific activity (3350 Ci/mmol) was synthesized and used to investigate the direct binding of this compound to the murine hepatic Ah receptor or other cytosolic proteins. No direct specific binding was observed between 125I2-TCBP and any cytosolic proteins using a sucrose density gradient assay procedure. These results contrasted with previous studies with Aroclor 1254 that suggested that this mixture acted as a competitive Ah receptor antagonist.     

Retinoic acid and 2,3,7,8-tetrachlorodibenzo-p-dioxin selectively enhance teratogenesis in C57BL/6N mice

TCDD is one of the most toxic man-made compounds and an extremely potent teratogen in mice. Many of its toxic symptoms resemble those seen during vitamin A deficiency. Vitamin A and its derivatives, such as alltrans-retinoic acid (RA), are also teratogenic in mice, as well as many other species. Both TCDD and RA produce cleft palate in susceptible strains of mice. However, while TCDD produces hydronephrosis, RA does not, and TCDD does not produce limb bud defects while RA does. To determine whether TCDD and RA would enhance or antagonize the teratogenic effects of the other compound, C57BL/6N dams were treated po on Gestation Day (gd) 10 or 12 with 10 ml corn oil/kg containing TCDD (0-18 micrograms/kg), RA (0-200 mg/kg), or combinations of the two chemicals. Dams were killed on gd 18 and toxicity and teratogenicity assessed. Coadministration of TCDD and RA had no effect on maternal or fetal toxicity beyond what would be expected by either compound alone. Cleft palate was induced by RA at lower doses on gd 10 than on gd 12, but by TCDD at lower doses on gd 12 than on gd 10. Sensitivity to TCDD-induced hydronephrosis was similar on both gd 10 and 12. The limb bud defects were only observed when RA was administered on gd 10, not when given on gd 12. No other soft tissue or skeletal malformations were related to administration of TCDD or RA. No effect of TCDD was observed on the incidence or severity of limb bud defects induced by RA, nor did RA influence the incidence or severity of hydronephrosis induced by TCDD. However, the incidence of cleft palate was dramatically enhanced by coadministration of the xenobiotic and vitamin. On both gd 10 and 12, the dose-response curves for cleft palate induction were parallel, suggesting some similarities in mechanism between the two compounds. However, combination treatment resulted in a synergistic response that varied with the stage of development and was tissue specific.     

Antiteratogenic effect of resveratrol in mice exposed in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin

The effect of resveratrol, an aryl hydrocarbon receptor antagonist, on the teratogenicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated. Pregnant C57BL/6J mice were orally administered resveratrol (50 mg/kg) for 6 consecutive days, from gestational day (GD) 8 to GD13, followed by an oral challenge with TCDD (14 mug/kg) on GD12. TCDD caused severe fetal malformations including cleft palate (40.7%), renal pelvic dilatation (100%, mean score 3.060), and ureteric dilatation (100%, mean score 3.210) and tortuosity (95.1%). Resveratrol significantly reduced both the incidence of TCDD-induced cleft palate to 18.4% and the degrees of renal pelvic and ureteric dilatations caused by TCDD. The results suggest that pretreatment with resveratrol might bring a beneficial outcome for reducing the incidence and severity of fetal malformations caused by TCDD exposure in utero.     

Effects of epidermal growth factor receptor deficiency and 2,3,7,8-tetrachlorodibenzo-p-dioxin on fetal development in mice

Dioxins are persistent environmental contaminants that cause multiple disorders in laboratory animals, including teratogenesis. In mice, the most important teratogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are hydronephrosis and cleft palate. Aryl hydrocarbon receptor (AHR) mediates most of the TCDD-induced effects, but modulation of these effects by other factors such as epidermal growth factor receptor (EGFR) has been propounded. TCDD changes the expression of both EGF and its receptor EGFR, which may be one step in the pathway leading to cleft palate and hydronephrosis. In the present study, the importance of EGFR in TCDD-induced teratogenicity was evaluated. Heterozygous EGFR^+/−-mice were mated and pregnant females exposed to 1.5–106.0 μg/kg TCDD on gestation day (GD) 10 and killed on GD 18. The fetuses were studied for cleft palate, hydronephrosis, and open eyes. There was no marked difference among the three genotypes in sensitivity to cleft palate or hydronephrosis, but in EGFR^−/−-mice frequency of the open eye malformation decreased dose-dependently. In conclusion, EGFR signaling is not required for TCDD-induced cleft palate or hydronephrosis but TCDD appears to counteract the effect of EGFR deficiency on eye opening.     

Teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the expression of EGF and/or TGF-alpha.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure produces hydronephrosis and cleft palate in mice. These responses are correlated with disruption of expression of epidermal growth factor (EGF) receptor ligands, primarily EGF and transforming growth factor-alpha (TGF-alpha), and altered epithelial cell proliferation and differentiation. This research examined the role of these growth factors in TCDD-induced teratogenicity by using wild type (WT) and knockout (-/-) mice that do not express EGF, TGF-alpha, or both EGF and TGF-alpha. Pregnant females were weighed on GD 12 and dosed by gavage with either corn oil or TCDD at 24 microg/kg, 5 ml/kg. On GD 17.5, the maternal parameters evaluated included body weight, body weight gain, liver weight (absolute and adjusted for body weight). The number of implantations, live and dead fetuses, early or late resorptions, the proportion of males, fetal body weight, fetal absolute and relative liver weight, placenta weight, incidence of cleft palate, and the severity and incidence of hydronephrosis were recorded. TCDD did not affect maternal weight gain, fetal weight, or survival, but maternal and fetal liver weights and liver-to-body weight ratios were increased in all genotypes. The WT and TGF-alpha (-/-), but not the EGF (-/-) and EGF + TGF-alpha (-/-) fetuses, developed cleft palate after exposure to 24 microg TCDD/kg. Hydronephrosis was induced by TCDD in all genotypes, with the incidence in EGF + TGF-alpha (-/-) fetuses comparable to that of the WT. The incidence and severity of this defect was substantially increased in EGF (-/-) and TGF-alpha (-/-). In conclusion, this study demonstrated that expression of EGF influences the induction of cleft palate by TCDD. Also, EGF and TGF-alpha are not required for the induction of hydronephrosis, but when either is absent the response of the fetal urinary tract to TCDD is enhanced.     

Teratogenicity of tcdd and the congener 3,3′,4,4′-tetrachloroazoxybenzene in sensitive and non-sensitive mouse strains after reciprocal blastocyst transfer

The teratogenic sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and its congeners is straindependent in the mouse. Reciprocal blastocyst transfer was performed between sensitive (NMRI) and nonsensitive (DBA) strains to show if there is a direct effect on embryonic tissues or if damage is secondary to maternal effects. Treatment with TCDD (30 μg/kg body weight) and 3,3′,4,4′-tetrachloroazoxybenzene (TCAOB) (8 mg/kg) on day 12 caused cleft palate in 75–100% of all NMRI fetuses, irrespective of whether they had remained in their own (NMRI) dams or as aliens in DBA dams. Only 1 out of 25 alien NMRI fetuses in DBA dams did not develop cleft palate. Under the same conditions, none of the 24 DBA fetuses in NMRI dams had cleft palate, although 89% of their NMRI littermates were affected. Also, no DBA fetuses having developed in their own dams had cleft palate.     

Teratogenic potency of 2,3,4,7,8-pentachlorodibenzofuran and of three mixtures of polychlorinated dibenzo-p-dioxins and dibenzofurans in mice. Problems with risk assessment using TCDD toxic-equivalency factors

The potency of 2,3,4,7,8-pentachlorodibenzofuran (P5CDF) and of three defined 2,3,7,8-TCDD-free mixtures of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs/PCDFs) to induce cleft palates in NMRI mice was studied. The data were compared with a dose-response curve for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The slope of the dose-response curve for P5CDF was the same as for TCDD. However, application of the International-TCDD-Toxic-Equivalency (I-TE) factor (NATO/CCMS 1988) of 0.5 overestimated the potency of the pentachlorinated congener about 2.5-fold under these experimental conditions, suggesting 0.2 as a TE factor. When assessing the cleft palate frequency on the basis of I-TEs and the weight of the substances, the potencies of the two PCDF mixtures studied were also clearly overestimated. This result was not substantially changed when using the TE factor of 0.2 for P5CDF. For the PCDD mixture studied, the cleft palate-inducing potency found largely agreed with the prediction when applying the I-TE factors. According to our data, the use of TE factors as calculated by the UBA/BGA (1985) or the NATO/CCMS (1988) are both conservative when attempting to assess the cleft palate incidence induced by PCDF mixtures in mice.     

Characterization of the Peak Period of Sensitivity for the Induction of Hydronephrosis in C57BL/6N Mice following Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin

Characterization of the Peak Period of Sensitivity for the Inductionof Hydronephrosis in C57BL/6N Mice following Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin.COUTURE, L. A., HARRIS, M. W., AND BIRNBAUM, L. S. (1990). Fundam.Appl. Toxicol. 15, 142–150. 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) is an extremely potent teratogen in mice. Hydronephrosisand cleft palate are the most sensitive measures of teratogenicityin mice following exposure to TCDD and other structurally relatedpolyhalogenated aromatic hydrocarbons. Despite a relativelylong half-life, investigators have identified a critical windowfor the induction of cleft palate in C57BL/6N mice. To characterizethe critical period for renal teratogenesis, pregnant C57BL/6Nmice were treated once by gavage with 0–24 µg TCDD/kgbody wt on Gestation Day (GD) 6, 8, 10, 12, or 14. All damswere killed on GD 18, and the fetuses were examined for thepresence of hydronephrosis and cleft palate. Maternal liver-to-bodyweight ratios were significantly elevated above controls onall days, while maternal weight gain was unaffected. Fetal mortalitywas increased relative to controls only at 24 µg TCDD/kgon GD 6. There was no significant difference in fetal body weightsbetween control and TCDD-treated fetuses. The incidence of cleftpalate increased in a dose-related fashion from GD 6 to GD 12,and identification of GD 12 as the critical window for inductionof clefting of the hard palate was confirmed. Hydronephrosiswas observed at all dose levels, regardless of exposure day,and the incidence was close to 100% at 3 µg TCDD/kg andhigher doses on GD 12 and earlier. At all doses on GD 14, boththe incidence and severity of hydronephrosis were decreasedrelative to all other days. There was a dose-related increasein the severity of the renal lesion on each day, but betweenGD 6 and 12 severity was constant Thus, while palatal sensitivityto TCDD increased with gestational age between GD 6 and 12,there was no difference among these days in development of hydronephrosis.The data suggest, however, that on GD 14 the urinary tract maybe less sensitive to TCDD.     

Teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in CF-1 mice

The effect of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) on the developing embryo and fetus of CF-1 mice has been evaluated. Pregnant CF-1 mice were given TCDD by oral gavage on Days 6 through 15 of gestation at dosages of 0, 0.001, 0.01, 0.1, 1, and 3 μg/kg/day. Little or no maternal toxicity was observed at any dosage. Cleft palate and dilated renal pelvis were found at 3.0 μg/kg/day. Cleft palate was found at 1.0 μg/kg/day. No malformations were found at the intermediate dosages of 0.1 or 0.01 μg/kg/day. Teratogenic effects observed, i.e., cleft palate and dilated renal pelvis, were comparable to those seen in studies utilizing other strains of mice. The incidence of malformations was not statistically significant at the 0.1-μg/kg/day dosage and below. The experimental nonteratogenic dosage for TCDD in the developing embryo and fetus of CF-1 mice was estimated to be 0.1 μg/kg/day.     

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist in C57BL/6 mice

6-Methyl-1,3,8-trichlorodibenzofuran (MCDF), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and TCDD plus MCDF were administered to C57BL/6 mice and their effects on several aryl hydrocarbon (Ah) receptor-mediated responses including hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction, immunotoxicity and teratogenicity were determined. MCDF did not induce hepatic microsomal AHH and EROD at doses up to 500 mumol/kg, however, co-administration of MCDF (50 mumol/kg) with a dose of TCDD which elicited a submaximal induction response (i.e. ED80-100, 15 nmol/kg) resulted in some small but significant inhibition of the induction of hepatic microsomal AHH and EROD (14 and 17%, respectively) compared to that observed with TCDD alone. Co-administration of TCDD and other doses of MCDF (10, 100, 200 or 500 mumol/kg) did not effect the induction response. These results were in contrast to the effectiveness of MCDF as an antagonist of the induction of AHH and EROD by TCDD in the rat (up to 50% inhibition of monooxygenase induction). Administration of MCDF (4, 20 and 40 mumol/kg) to C57BL/6 mice caused some inhibition of the splenic plaque-forming cell response to sheep erythrocytes only at the highest dose (26% decrease); the interaction of MCDF (4, 20 and 40 mumol/kg) and an immunotoxic dose of TCDD (3.7 nmol/kg) resulted in significant protection from the immunotoxic effects of TCDD at the 2 higher dose levels of MCDF. Similarly, MCDF (400 mumol/kg) did not cause cleft palate in mice but at this dose level MCDF afforded some protection from TCDD (20 micrograms/kg)-mediated cleft palate in mice. However, studies utilizing [3H]TCDD suggested that the protective effects may be due to modulation of TCDD reaching the palate in the co-treated animals (MCDF plus TCDD). Although both MCDF and Aroclor 1254 were both weak Ah receptor agonists in C57BL/6 mice, the former compound was much less effective as a TCDD antagonist. The observed species-specific effects for these 2 TCDD antagonists may be related species-dependent differences in receptor structure and receptor-ligand (i.e. agonist or antagonist) interactions.     

Rat embryonic palatal shelves respond to TCDD in organ culture

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a highly toxic environmental contaminant, is teratogenic in mice, inducing cleft palate (CP) and hydronephrosis at doses which are not overtly maternally or embryo toxic. Palatal shelves of embryonic mice respond to TCDD, both in vivo and in organ culture, with altered differentiation of medial epithelial cells. By contrast, in the rat TCDD produces substantial maternal, embryonic, and fetal toxicity, including fetal lethality, with few malformations. In this study the possible effects of maternal toxicity on induction of cleft palate were eliminated by exposure of embryonic rat palatal shelves in organ culture. The shelves were examined for specific TCDD-induced alterations in differentiation of the medial cells. On Gestation Day (GD) 14 or 15 palatal shelves from embryonic F344 rats were placed in organ culture for 2 to 3 days (IMEM:F12 medium, 5% FBS, 0.1% DMSO) containing 0, 1 x 10(-8), 1 x 10(-9), 1 x 10(-10), or 5 x 10(-11) M TCDD. The medial epithelial peridermal cells degenerated on shelves exposed to control media or 5 x 10(-11) M TCDD. Exposure to 10(-10), 10(-9), and 10(-8) M TCDD inhibited this degeneration in 20, 36, and 60% of the shelves, respectively, and was statistically significant at the two highest doses. A normally occurring decrease in [3H]TdR incorporation was inhibited in some GD 15 shelves cultured with 10(-10) and 10(-9) M TCDD. The medial cells of TCDD-exposed shelves continued to express high levels of immunohistochemically detected EGF receptors. The altered differentiation of rat medial epithelium is similar to that reported for TCDD-exposed mouse medial cells in vivo and in vitro. However, in order to obtain these responses, the cultured rat shelves require much higher concentrations of TCDD than the mouse shelves. Thus TCDD induces the same effects at a cellular level in medial epithelium of rats and mice, but cleft palate is not seen in rats because the level required to produce the cellular effects would result in maternal and embryonic toxicity including fetal lethality.     

Teratogenic effects of 2,3,7,8-tetrabromodibenzo-p-dioxin and three polybrominated dibenzofurans in C57BL/6N mice

Brominated flame retardants involved in many industrial uses contain polybrominated dibenzo-p-dioxins (PBDDs) and dibenzofurans (PBDFs) as contaminants. The levels of these contaminants can be dramatically increased by combustion. These chemicals are closely related in structure to the polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), of which 2,3,7,8-tetrachloridibenzo-p-dioxin (TCDD) is the most toxic isomer. TCDD and related PCDFs are potent mouse teratogens inducing cleft palate and hydronephrosis at doses below those at which overt maternal and embryo/fetal toxicity occurs. This study examines the teratogenic effects of 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD), 2,3,7,8-tetrabromodibenzofuran (TBDF), 1,2,3,7,8-pentabromodibenzofuran (1PeBDF), and 2,3,4,7,8-pentabromodibenzofuran (4PeBDF) in C57BL/6N mice treated on gestation day (gd) 10 and examined on gd 18. Pregnant dams were treated with 0-4000 micrograms of each congener per kilogram body weight in 10 ml corn oil/kg. Dose selection was based on the relative toxicity of the chlorinated isomers. Maternal toxicity and developmental toxicity were assessed, and the hard palate and kidney, the target organs for the teratogenic effects of TCDD and related compounds, were examined for structural abnormalities. While the maternal liver weight increased at all dose levels examined for all four compounds, there was no evidence of any maternal toxicity. Embryo/fetal mortality was increased only at greater than or equal to 500 microgram TBDF/kg, while fetal weight increased in a dose-related manner following exposure to TBDD and TBDF. All compounds produced hydronephrosis (HN) at doses below that at which cleft palate (CP) occurred. The incidence of HN was significantly increased above background levels at the following doses (micrograms/kg): TBDD, 3; TBDF, 25; 1PeBDF, 500; 4PeBDF, 400. The LOELs (micrograms/kg) for CP were: TBDD, 48; TBDF, 200; 1PeBDF, 4000; 4PeBDF, 2400. The cleft palate incidence for all four brominated compounds and TCDD could be fit to a common slope, compatible with the concept that these chemicals all exert their teratogenic effects through a common mechanism. The potency of these chemicals, relative to TCDD as 1 for the induction of cleft palate, is TBDD, 0.24; TBDF, 0.10; 1PeBDF, 0.004; and 4PeBDF, 0.005. Previous studies from our laboratory had determined that the chlorinated dibenzofuran isomers had relative potencies of 0.05 (TCDF), 0.03 (1PeCDF), and 0.09 (4PeCDF). Thus, bromination decreases the teratogenic activity of TBDD relative to TCDD and of both 1- and 4PeBDF relative to the chlorinated isomers. However, substitution of bromines for chlorines increases the potency of TBDF relative to TCDF.(ABSTRACT TRUNCATED AT 400 WORDS)