2,3,7,8-Tetrachlorodibenzo-p-dioxin alters embryonic palatal medial epithelial cell differentiation in vitro

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in mice, inducing cleft palate and hydronephrosis. After exposure in vivo, TCDD specifically alters differentiation of embryonic palatal medial epithelial cells. In this study, the palatal epithelial cell response to TCDD is determined in vitro. C57BL/6N palatal shelves were placed in organ culture on gestation day (GD) 12 in Richter's improved modified Eagle's medium:Ham's F12 medium (1:1) with 1% fetal bovine serum for 3 or 4 days. Medium contained 0.1% dimethylsulfoxide and TCDD at 0, 10(-13), 10(-12), 10(-11), 10(-10), and 10(-9) M, with some doses at 5 x 10(-11), 7.5 x 10(-11), and 5 x 10(-12) M. Epithelial cell responses to TCDD occurred over a narrow range of concentrations, with maximal response at 5 x 10(-11) M. Cytotoxicity was detected at 1 x 10(-10) M. At a stage when control medial cells ceased proliferation and EGF receptors were not detected immunohistochemically. TCDD-exposed medial cells incorporated [3H]thymidine and high levels of epidermal growth factor receptors were detected. TCDD prevented programmed cell death of medial peridermal cells, and induced a shift in the differentiation of medial cells toward an oral-like phenotype. The responses to TCDD observed after exposure in vitro were indistinguishable from previously reported effects observed after exposure in vivo. In the present study, the distribution of TCDD in the fetus after exposure in vivo was examined. The levels of exposure to TCDD are similar for in vitro and in vivo exposure routes. The levels of TCDD in 1 x 10(-11) to 1 x 10(-10) M solutions (3 to 32 pg/ml) were comparable to levels observed in fetal tissues after in vivo exposure on GD 11 to 30 microns/kg [3H]TCDD, where the palatal shelf contained 1.4 to 3.5. pg TCDD, representing 0.0003% of the total dose. In vivo, TCDD was detected in the GD 11 embryo 3 hr postexposure and the TCDD was equally distributed between the embryonic head and body. At 72 hr postexposure, 0.035% of the total dose was in fetal tissues, and 1% of the TCDD in the fetus was found in the palatal shelf. The present study shows that the palatal epithelium responds to TCDD in vitro in a manner comparable to that observed after in vivo exposure, and that the response occurs at a concentration comparable to in vivo levels in the fetus. The availability of an in vitro system will facilitate studies of TCDD toxicity that are difficult or impossible to perform in vivo, such as comparisons of TCDD effects between species, including human tissues.     

TCDD alters medial epithelial cell differentiation during palatogenesis

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widely distributed, persistent environmental contaminant that is teratogenic in mice, where it induces hydronephrosis and cleft palate. The incidence of clefting has been shown to be dose dependent after exposure on either gestation Day (GD) 10 or 12, although the embryo is more susceptible on GD 12. TCDD-exposed palatal shelves meet but do not fuse, and programmed cell death of the medial epithelial cells is inhibited. The mechanism of action through which TCDD alters the program of medial cell development has not been examined in earlier studies, and it is not known whether the mechanism is the same regardless of the dose or developmental stage of exposure. In this study, C57BL/6N mice, a strain sensitive to TCDD, were dosed orally on GD 10 or 12 with 0, 6, 12, 24, or 30 micrograms/kg body wt, in 10 ml corn oil/kg. Embryonic palatal shelves were examined on GD 14, 15, or 16. The degree of palatal closure, epithelial surface morphology, and cellular ultrastructure, the incorporation of [3H]TdR, the expression of EGF receptors, and the binding of 125I-EGF were assessed. After exposure on GD 10 or 12, TCDD altered the differentiation pathway of the medial epithelial cells. The palatal shelves were of normal size and overall morphology, but fusion of the medial epithelia of the opposing shelves did not occur. TCDD prevented programmed cell death of the medial peridermal cells. The expression of EGF receptors by medial cells continued through Day 16 and the receptors were able to bind ligand. The medial cells differentiated into a stratified, squamous, keratinizing epithelium. The shift in phenotype to an oral-like epithelium occurred after exposure on either GD 10 or 12. At the lower dose (6 micrograms/kg), fewer cleft palates were produced, but those shelves which did respond had a fully expressed shift in differentiation. Whether the exposure begins on GD 10 or 12 or whether the dosing level produces only a few cleft palates within a litter, TCDD produced cleft palate by altering the differentiation program of the medial cells.     

TCDD-induced altered expression of growth factors may have a role in producing cleft palate and enhancing the incidence of clefts after coadministration of retinoic acid and TCDD

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in mice, inducing cleft palate and hydronephrosis at doses which are not overtly maternally toxic or embryotoxic. After TCDD exposure the palatal shelves of normal size come into contact, but fail to fuse due to altered differentiation of the medial epithelial cells. These cells continue to express EGF receptors, proliferate, and differentiate into an oral-like stratified squamous epithelium. The present study examines the effect of TCDD on the expression of growth factors which are believed to regulate differentiation and proliferation in the palate. This study also examined the combined effect of TCDD and retinoic acid (RA), since in teratology studies coadministration of these agents results in an enhancement of cleft palate incidence. Embryos were exposed in vivo on Gestation Day (GD) 10 or 12 to TCDD ot TCDD + RA and the palatal shelves were dissected on GD 14-16. Growth factor expression was determined immunohistochemically using antibodies to TGF-alpha, EGF, TGF-beta 1, or TGF-beta 2. The growth factors displayed specific spatial and temporal expression in the palatal shelves. TCDD reduced the expression of TGF-alpha, EGF, and TGF-beta 1 in epithelial and mesenchymal cells. The degree of reduction was generally greater after exposure on GD 10 to TCDD alone or in combination with RA when compared to that on GD 12. The abnormal proliferation and differentiation of TCDD-exposed medial cells may be a response to reduced expression of EGF and TGF-alpha. Low levels of these factors may be related to the previously observed elevated levels of EGF receptors in medial cells. In other systems, low levels of ligand have resulted in upregulation of the EGF receptor. Continued proliferation and altered differentiation could also be attributable to decreased levels of TGF-beta 1, a factor inhibitory to epithelial proliferation. Since TGF-beta 1 stimulates mesenchymal growth and TGF-alpha and EGF stimulate epithelial proliferation, the formation of small shelves after exposure to TCDD + RA on GD 10 may be due to the severe reduction in these factors. Only a slight to moderate reduction in growth factor expression occurs after exposure to TCDD + RA on GD 12 and in this case shelves of normal size form. Since TCDD and RA appear to act in part through pathways that involve TGF-beta 1, in vitro experiments were designed to examine the involvement of TGF-beta 1 in TCDD teratogenicity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on fetal mouse urinary tract epithelium in vitro

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produces hydronephrosis by altering the differentiation and proliferation of ureteric epithelial cells in the fetal C57BL/6N mouse urinary tract. This study tests the hypothesis that the late fetal urinary tract epithelial cells respond to TCDD with increased proliferation and that the responses do not require contributions from other maternal or fetal tissues. This was achieved by exposing late gestation fetal urinary tract cells to TCDD in an in vitro model. Isolated ureteric cells from gestation day (GD) 18 fetal ureters were plated in medium supplemented with trace elements, a complex mixture of lipids, a defined mixture of purified hormones and growth factors. Both epithelial and mesenchymal cells remain viable under these conditions. The cultures were exposed to 0.1% dimethylsulfoxide (DMSO), 1x10(-8), 1x10(-9) or 1x10(-10) M TCDD. Exposure to 1x10(-10) M TCDD did not affect the cultures, while 1x10(-8) and 1x10(-9) M TCDD supported epithelial, but not mesenchymal, cell survival and stimulated epithelial cell proliferation and differentiation. The TCDD-exposed cells expressed high levels of keratin and little or no vimentin, confirming that the cells, which survive and differentiate are epithelial. However, after continuous exposure to epidermal growth factor (EGF), the TCDD-induced stimulation of ureteric epithelial growth could not be detected. In conclusion, this study demonstrates that late gestational ureteric cells respond to TCDD in vitro with the stimulation of epithelial cell growth and differentiation.     

Cellular alterations and enhanced induction of cleft palate after coadministration of retinoic acid and TCDD

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and retinoic acid (RA) are both teratogenic in mice. TCDD is a highly toxic, stable environmental contaminant, while RA is a naturally occurring form of vitamin A. Exposure to TCDD induces hydronephrosis and cleft palate, and exposure to RA induces limb defects and cleft palate. Teratology studies previously have shown that the incidence of clefting is higher after exposure to RA + TCDD than would be observed for the same doses of either compound given alone. This study examines the cellular effects which result in cleft palate, after po administration on gestation Day (GD) 10 or 12 of RA + TCDD in corn oil (10 ml/kg total volume). Exposure on GD 10 to 6 micrograms TCDD + 40 mg RA/kg inhibited early growth of the shelves and clefting was due to a failure of shelves to meet and fuse. This effect on mesenchyme was observed in previous studies to occur after exposure on GD 10 to 40 mg/kg RA alone, but not after TCDD alone. After exposure on GD 12 to 6 micrograms TCDD + 80 mg RA/kg, clefting was due to a failure of shelves to fuse after making contact, because the medial cells differentiated into an oral-like epithelium. This response was observed in previous studies to occur after exposure to TCDD alone, but RA alone on GD 12 resulted in differentiation toward nasal-like cells. The interaction between TCDD and RA results in RA-like clefting after exposure on GD 10 and TCDD-like clefting after exposure on GD 12, and this clefting occurs at higher incidences than would occur after the same levels of either agent alone. After exposure on either GD 10 or 12 to RA + TCDD, the programmed cell death of the medial cells does not occur, and these cells continue to express EGF receptors and to bind 125I-EGF. The effects of RA and TCDD may involve modulation of the cells responses to embryonic growth and differentiation factors.     

AhR, ARNT, and CYP1A1 mRNA quantitation in cultured human embryonic palates exposed to TCDD and comparison with mouse palate in vivo and in culture.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is developmentally toxic in many species and induces cleft palate in the C57BL/6N mouse embryo. Palatogenesis in mouse and human embryos involves homologous processes at the morphological, cellular, and molecular levels. In organ culture, mouse and human palates respond similarly to TCDD. The present study quantitates the expression of AhR, ARNT, and CYP1A1 mRNA in human embryonic palates in organ culture. Palatal tissues were exposed to 1 x 10(-10), 1 x 10(-9), or 1 x 10(-8) M TCDD or control medium and sampled at 0, 2, 4, and 6 hours for quantitative RT-PCR using a synthetic RNA internal standard. Similar measurements of CYP1A1 gene expression were collected for mouse palates cultured in this model. In human palates, AhR expression correlated with ARNT and CYP1A1 mRNA expression. TCDD induction of CYP1A1 was time- and concentration-dependent. The expression of these genes presented a uniform and continuous distribution across the group of embryos, with no subset of either high or low expressors/responders. The ratio of AhR to ARNT was approximately 4:1. AhR mRNA increased during the culture period in both treated and control subjects; however, ARNT expression was relatively constant. TCDD did not alter either AhR or ARNT expression in a consistent dose- or time-related manner. Comparison of human and mouse data showed a high correlation across species for the induction of CYP1A1. Human embryos expressed approximately 350 times less AhR mRNA than the mouse, and in earlier studies it was shown that human palates required 200 times more TCDD to produce the same effects. When the morphological, cellular, and molecular responses to TCDD between mouse and human are compared, it seems highly unlikely that human embryos could be exposed to sufficient TCDD to achieve changes in palatal differentiation that would lead to cleft palate.     

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.     

Developmental stage specificity and dose response of secalonic acid D-induced cleft palate and the absence of cytotoxicity in developing mouse palate

Incidence of cleft palate (CP) in full-term mouse fetuses was evaluated following administration of 25 mg/kg of the mycotoxin, secalonic acid D (SAD), to groups of female mice on each of Days 10, 11, 12, 13, 14, or 15 of pregnancy. Although the highest numerical incidence (45.3%) of cleft palate resulted following SAD exposure on Day 12 of pregnancy, and the response tapered off to 16.9% on Day 10 and 0% on Day 15 of pregnancy, similar responses were produced also following exposures on Days 11 (38.4%) and 13 (39.9%) of pregnancy. Maternal exposure to doses of 0, 15, 20, 25, or 30 mg/kg of SAD, given on Day 12 of pregnancy indicated that although fetuses in the 30-mg/kg group had the highest incidence (51.9%) of CP, the effect was associated with increased resorptions and decreased fetal weights. The 25-mg/kg dose was optimally teratogenic (45.3% cleft palate) and maximally tolerable with neither an increase in resorptions nor a decrease in fetal body weights. Cytotoxicity of the optimally teratogenic dose of SAD (25 mg/kg given ip) on Day 12 of pregnancy was evaluated as a possible mechanism of SAD teratogenicity using indices such as mesenchymal cell density, mitotic index, and the uptake of [3H]thymidine in the developing palatal shelves. No evidence of SAD cytotoxicity was obtained in palatal shelves indicating a possible role for nonlethal cellular effects of SAD in the pathogenesis of CP. These studies also suggest the suitability of the maternal 25-mg/kg dose of SAD to study cellular biochemical effects in the developing embryo without the complicating influence of cytotoxic effects.     

Teratological studies on the TCDD congener 3,3′,4,4′-tetrachloroazoxybenzene in sensitive and nonsensitive mouse strains: Evidence for direct effect on embryonic tissues

The teratogenicity of 3,3,4,4-tetrachloroazoxybenzene (TCAOB), a TCDD congener, was studied in Ah-responsive (C57BL and NMRI) and non-responsive (DBA/2J and AKR/NBom) strains of mice. In the responsive strains, the TCAOB produced cleft palate and hydronephrosis in 50–90% of the offspring at a dose level of 6–8 mg/kg b.w. in the absence of apparent maternal toxicity. Day 11 was shown to be the day of highest sensitivity (palatal closure occurs at day 14) in the C57BL strain. Higher doses (16 mg/kg b.w.) produced high rate of fetal death both in responsive (C57BL; 60%) and non-responsive (DBA; 40%) strains. These doses induced cleft palate in 95% of the surviving C57BL fetuses but failed to do so in the DBA strain. The non-sensitivity of the DBA and AKR strains appeared to segregate as a dominant trait. Backcrosses between NMRI x DBA F₁ generation and NMRI showed an intermediate sensitivity. It was shown that the genotype of the embryo was of ultimate importance for the development of cleft palate. There appeared however to be an additional host (maternal) factor as well, because the offspring of NMRI females mated with NMRI x DBA F₁ males showed a higher rate of cleft palate as compared to those of the crossing between NMRI x DBA F₁ females and NMRI males. Light and scanning electron microscopy indicated that the apical epithelial cells of the secondary palates failed to follow the normal pattern of programmed cell death, suggesting a similar mechanism of pathogenesis as previously described for TCDD.     

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)     

Teratogenicity of three polychlorinated dibenzofurans in C57BL/6N mice

Polychlorinated dibenzofurans (PCDFs) are widespread environmental contaminants which have been detected in human tissues and implicated in several poisoning incidents. Their toxic effects are similar to those observed with other related halogenated aromatic hydrocarbons such as TCDD. The teratogenic effects of three PCDFs, 1,2,3,7,8-pentachlorodibenzofuran (1-PeCDF), 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), and 1,2,3,4,7,8-hexachlorodibenzofuran (HCDF), were assessed in C57BL/6N mice. Pregnant mice were exposed on Gestation Days 10-13 to 10 ml corn oil/kg containing PCDFs. The dams were killed on Gestation Day 18 and maternal and fetal toxicity were assessed. All three compounds were highly teratogenic, with very steep and parallel dose-response curves for the two diagnostic indicators of dioxin-like teratogenicity, hydronephrosis, and cleft palate. 4-PeCDF was the most teratogenic with an ED50 of 36 micrograms/kg for cleft palate and 7 micrograms/kg for hydronephrosis. 4-PeCDF was approximately 4 times as potent as 1-PeCDF and 10 times as potent as HCDF. The teratogenic responses occurred at a dose below that where any obvious maternal or fetal toxicity was detected. Thus, these three compounds cause teratogenic responses similar to those seen with TCDD but are only 1/10 to 1/100 as potent.     

Teratogenic potency of TCDD, TCDF and TCDD-TCDF combinations in C57BL/6N mice

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibenzofuran (TCDF) cause the same spectrum of fetal anomalies in C57BL/6N mice. Pregnant dams were treated with TCDD, TCDF and combinations of the 2 compounds on gestation day 10, and examined for maternal and fetal effects on day 18. The fetal kidneys were the most sensitive target for teratogenicity. The dose response for cleft palate induction fit the probit model for both compounds, suggesting that TCDD was approximately 30 times more potent than TCDF. The interaction between these 2 compounds was consistent with a model for additive toxicity.     

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.     

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.     

Suppressive effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on vascular remodeling that takes place in the normal labyrinth zone of rat placenta during late gestation.

The maintenance of the placental vasculature is essential for sustaining normal fetal growth. On the basis of our previous observation that fetal death was accompanied by placental hypoxia upon exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (R. Ishimura et al., 2002a, Toxicol. Appl. Pharmacol. 185, 197-206), we here investigated the effects of TCDD on the placenta, focusing on the development of the labyrinth zone. Holtzman rats were administered a single oral dose of 1.6 mug of TCDD/kg body weight or an equivalent volume of vehicle (control) on gestation day (GD) 15, and placental tissues were analyzed on GD20. Immunohistochemical staining showed that the exposure to TCDD decreased the size of maternal blood sinusoids and caused the constriction of fetal capillaries in the placenta. In contrast, we found that vascular remodeling occurred in the labyrinth zone of normal rat placenta; that is, the vascular development in the normal labyrinth zone during the late gestation (from GD16 to GD20) showed dilated maternal blood sinusoids and fetal capillaries accompanied by a decrease in thickness and the apoptosis of trophoblasts. The present results demonstrate that this remodeling is suppressed by TCDD, which is further supported by the decreased expression level of Tie2 mRNA, the gene which is associated with vascular remodeling, and also by the decrease in the number of apoptotic trophoblasts in TCDD-exposed rats. The present study provided a new finding on the development of the vasculature in the labyrinth zone during the late gestation under normal conditions and showed the inhibition of vascular remodeling in TCDD-exposed rats.     

Cell cycle proteins in normal and chemically induced abnormal secondary palate development: a review

Cell cycle progression and thus proper cell number is essential for normal development of organs and organisms. Craniofacial tissues including the secondary palate are vulnerable to disruption of cell cycle progression and proliferation by many chemicals including mycotoxin, secalonic acid D (SAD), glucocorticoids, retinoic acid and 2,3,7,8-tetrachlorodibenzodioxin. Induction of cleft palate (CP) by SAD in mice occurs from a reduction in the size of developing palatal shelves. This is associated with an inhibition of proliferation of murine and human embryonic palatal mesenchymal (MEPM and HEPM) cells as well as a G1/S block of cell cycle. In murine embryonic palates and HEPM cells, SAD inhibited G1/S-phase-specific cyclin-dependent kinase (CDK)2 activity, reduced the level of cyclin E and increased the level of the CDK2 inhibitor, p21. These results, together with those from other laboratories, suggest that common cell cycle protein targets (biomarkers), relevant to the pathogenesis of CP by multiple chemical exposures, that can form the basis for the diagnosis and the development of preventive strategies, are likely to exist.     

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.     

Effect of D,L-alpha-difluoromethyl ornithine on cleft palate induced by the TCDD congener, 3,3',4,4'-tetrachloroazoxybenzene, in the fetuses of mice

The importance of inhibition of ornithine decarboxylase (ODC) activity in murine embryonic tissues for the teratogenic action of the TCDD congener 3,3',4,4'-tetrachloroazoxybenzene (TCAOB), has been studied. When D,L-alpha-difluoromethyl ornithine (DFMO), an inhibitor of ODC activity, was coadministered with TCAOB, it decreased the frequency of cleft palate compared with TCAOB alone. Fetal death induced by TCAOB was not affected by DFMO treatment. It is suggested that the mechanism of cleft palate induced by TCAOB may involve ODC stimulation. However fetal death induced by the latter compound may involve another mechanism, that may not be related to the mechanism of cleft palate induction.     

3,3'4,4'-Tetrachlorobiphenyl in pregnant mice: embryotoxicity, teratogenicity, and toxic effects on the cultured embryonic thymus

3,3',4,4'-Tetrachlorobiphenyl (TCB) is a known ligand of the Ah-receptor. When TCB was given to Ah-responsive C57BL/6 mice at gestation day 11, 12 or 13, a pattern of embryotoxic effects similar to those of TCDD was produced. This pattern included death and resorptions of the conceptus (peak sensitivity at day 11), as well as characteristic malformations such as cleft palate, dilated kidney pelvis (peak sensitivity day 12), and thymus hypoplasia (peak sensitivity day 13). The ED50 for cleft palate induction was found to be about 100 mg/kg, as compared to 30 micrograms/kg for TCDD (earlier results). The binding affinity of TCB for the Ah-receptor has been reported to be two orders of magnitude lower than that of TCDD. When TCB was introduced into a thymus organ culture (thymi taken from day-14 embryos), the lymphoid cell development was inhibited with an approximate EC50 of 5 X 10(-8) M. This is approximately 100 times higher than that of TCDD and in good agreement with the receptor binding affinities of both compounds. The difference in in vivo toxicity between TCB and TCDD can be explained by a more rapid metabolism and excretion of TCB.