Biomedical subjects

B D Abbott

Publications and source records attributed to B D Abbott.

At least 19 recordsLinked to original sources

PubMed1997

Effects of TCDD on Ah receptor, ARNT, EGF, and TGF-alpha expression in embryonic mouse urinary tract.

Prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces hydronephrosis in C57BL/6N mice. The etiology of this response involves TCDD-induced hyperplasia of ureteric epithelium, which occludes the ureteric lumen, blocking the flow of urine. The present study localizes and examines the effects of TCDD on the expression of the Ah receptor (AhR), the Ah receptor nuclear translocator (ARNT), epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha) in the epithelial cells of the developing urinary tract, particularly the ureteric bud derivatives (ureter and tubules). Pregnant C57BL/6N mice were dosed on gestation day (GD) 10 with either corn oil or TCDD at 12 micrograms/kg; a dose of 24 micrograms/kg is expected to induce 100% hydronephrosis. The metanephric urinary tract is morphologically detectable as early as GD 12; thus, embryos were removed on GD 12, 13, and 14, and the lower dorsal torso was prepared for immunohistochemistry or in situ hybridization. Regardless of treatment, the expression of both AhR and ARNT increased in epithelial cells of the ureter and AhR increased in the metanephric tubules from GD 12-14. In situ hybridization localized the expression of AhR and ARNT mRNAs to these derivatives of the ureteric bud and levels of mRNA increased throughout the developmental period examined. There were no significant effects of TCDD treatment on expression of AhR, while TCDD significantly decreased levels of ARNT in tubules on GD 14. The epithelial cells of the ureter and tubules expressed TGF-alpha and EGF. EGF increased from GD 12 to 13 in the tubules and ureter, but there was no difference from GD 13 to 14. Treatment with TCDD reduced TGF-alpha significantly only in tubules on GD 13. TCDD exposure significantly decreased EGF in ureter and tubule cells on both GD 13 and 14. In summary, the epithelial cells of the embryonic mouse urinary tract expressed AhR, ARNT, EGF, and TGF-alpha in developmentally dependent patterns. These proteins are involved in the regulation of embryonic cell proliferation during normal urinary tract development and are probably involved in the hyperplastic response to TCDD.

Animals↗

PubMed1996

Rapid distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to embryonic tissues in C57BL/6N mice and correlation with palatal uptake in vitro.

2,3,7,8-Tetracholoridbenzo-p-dioxin (TCDD) is a developmentally toxic environmental contaminant capable of inducing cleft palate and hydronephrosis in embryonic C57BL/6N mice. In this study, the disposition of TCDD was determined in pregnant C57BL/6N mice in the 24 hr immediately following oral administration on Gestation Day (GD) 12. TCDD was detected in maternal blood, liver, and fat and in the placenta, embryonic liver, and palate within 30 min after dosing on GD 12. The levels peaked in blood and placenta at 3 hr and in the other tissues at 8 hr. Levels of TCDD decreased slightly after 8 hr in embryonic liver and palate. In vitro systems were used to study the mechanisms of action of TCDD and in these models exposure is typically reported as concentration of TCDD in the culture medium. The present study is the first to allow a direct comparison of the level of TCDD in embryonic tissue after in vivo and in vitro exposures. Uptake of TCDD was determined in embryonic palatal organ culture and tissue levels were then expressed in comparable units for both in vivo and in vitro exposures. The data provide new information on distribution in the pregnant mouse and the embryo and also show that the palatal organ culture model provides a reasonable dosimetric representation of in utero exposure.

Administration, Oral↗

PubMed1995

Review of the interaction between TCDD and glucocorticoids in embryonic palate.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant that produces adverse biological effects including developmental toxicity and teratogenesis. In the mouse embryo, TCDD induces cleft palate and hydronephrosis. The synthetic glucocorticoid, hydrocortisone (HC), induces cleft palate and a potent, synergistic interaction has been observed between TCDD and HC in C57BL/6N embryonic mice. The morphology and etiology of TCDD- and HC-induced clefts are distinctly different with formation of small palatal shelves following HC exposure and failure of normally-sized shelves to fuse after TCDD treatment. Each exposure also alters expression of several growth factors. When EGF, TGF alpha, EGF receptor, and the TGF beta's are considered as a combinatorial, interacting set of regulators, TCDD and HC each produce a unique pattern of increased and/or decreased expression across the set. The interaction of HC and TCDD results in a cleft palate whose etiology most closely resembles that observed after HC exposure, i.e. small palatal shelves. HC+TCDD-exposure also produces a pattern of growth factor expression which closely resembles that seen after HC. Both TCDD and HC act through receptor-mediated mechanisms and each compound has its own receptor. The Ah receptor (AhR) binds TCDD and the glucocorticoid receptor (GR) binds HC. On gestation day (GD) 14, in the embryonic palate exposed to TCDD, the AhR was downregulated and the GR expression increased. Conversely, following HC exposure, the GR was downregulated and AhR levels were elevated. HC+TCDD produced increased expression of both receptors and this pattern would be predicted to produce HC-like clefts as the GR-mediated responses would result in small palatal shelves. The observed cross-regulation of the receptors is believed to be important in the synergistic interaction between TCDD and HC for the induction of cleft palate.

Abnormalities, Drug-Induced↗

PubMed1995

Cell death in rat and mouse embryos exposed to methanol in whole embryo culture.

Methanol induces developmental toxicity in rats and mice producing exencephaly, cleft palate, cervical skeletal defects, reduced body weight, and increased embryo/fetal death. Exposure to methanol in whole embryo culture also induces developmental retardation, dysmorphogenesis, and embryo lethality. In the present study, cultured rat and mouse embryos were exposed to methanol and subsequently observed for morphological effects and increased cell death using modified Feulgen staining which allowed nuclei throughout the embryo to be examined in situ. Growth and developmental scores were reduced by methanol in both rat and mouse embryos and the mouse embryos were affected at lower concentrations when compared to the rat. Methanol increased cell death in specific regions of both rat and mouse embryos, including the forebrain, the visceral arches, otic and optic placodes. These regions form derivatives which manifest morphological abnormalities following exposure in vivo. Methanol did not increase cell death in the neuroepithelium or neural folds and neural tube defects cannot be explained by excess cell death. The results of this study suggest that increased cell death in specific regions of the exposed embryos has a role in producing cranial malformations, abnormalities of the eye and ear, and cleft palate.

Animals↗

PubMed1995

Developmental expression of two members of a new class of transcription factors: I. Expression of aryl hydrocarbon receptor in the C57BL/6N mouse embryo.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor with a basic region/helix-loop-helix (bHLH) motif. AhR has been sequenced and the functional domains defined and there is information on the formation of complexes with other peptides and interactions with DNA, although these areas continue to be investigated. AhR mediates many biological effects such as developmental toxicity, including induction of cleft palate and hydronephrosis. This regulatory protein is expressed in embryonic liver and has been immunohistochemically localized in cells of human and mouse secondary palate. The expression of AhR in embryonic tissues and its ability to disrupt development suggests a significant role for this protein in development. The present study examines the pattern of AhR expression in the C57BL/6N mouse embryo from gestation days (GD) 10-16, using in situ hybridization and immunohistochemical analysis. AhR mRNA was localized with 35S-RNA antisense riboprobe (cAh1 probe, 1.8 Kb amino terminal DNA). AhR protein was localized with purified monoclonal antibody (RPT-9) raised against the N-terminal peptide sequence. AhR mRNA and protein were expressed in GD 10-13 neuroepithelium, and as development progressed the levels in brain decreased. GD 10-12 embryos also showed AhR in branchial arches, heart, somites, and liver. AhR protein and mRNA in heart were highest at GD 10-11 and decreased with age. In liver, AhR mRNA and protein levels increased and nuclear localization became more pronounced with gestational age. In GD 14-16 embryos levels in liver and adrenal were highest, but AhR was present in ectoderm, bone, and muscle. AhR expression was specific for both cell type, organ/tissue, and developmental stage, suggesting that this novel ligand-activated transcriptional regulator may be important in normal embryonic development.

Animals↗

PubMed1995

Developmental expression of two members of a new class of transcription factors: II. Expression of aryl hydrocarbon receptor nuclear translocator in the C57BL/6N mouse embryo.

The aryl hydrocarbon receptor (AhR) and the AhR nuclear translocator protein (ARNT) are basic-helix-loop-helix (bHLH) proteins involved in transcriptional regulation. The AhR is a ligand-activated partner of the ARNT protein. Both proteins are required to transcriptionally regulate gene expression. ARNT must be complexed to AhR to permit binding to the regulatory DNA sequence. The AhR-ligand complex is known to mediate a range of biological responses, such as developmental toxicity, induction of cleft palate, and hydronephrosis. AhR and ARNT are expressed in human embryonic palatal cells and AhR was recently shown to have a specific developmental pattern of expression in the mouse embryo. In the present study, expression of ARNT is characterized in C57Bl/6N mouse embryos from gestation day (GD) 10-16 using immunohistochemistry and in situ hybridization. Af affinity purified antibody against human ARNT (1.1 micrograms/ml) was detected with an avidin-biotin-peroxidase complex. ARNT mRNA was localized with a 35S-RNA probe from pBM5/NEO-M1-1. Specific spatial and temporal patterns of ARNT expression emerged and mRNA and protein expression correlated. The GD 10-11 embryos showed highest levels of ARNT in neuroepithelial cells of the neural tube, visceral arches, otic and optic placodes, and preganglionic complexes. The heart also had significant expression of ARNT with strong nuclear localization. After GD11, expression in heart and brain declined. In GD 12-13 embryos expression was highest in the liver where expression increased from GD 12-16. At GD 15-16 the highest levels of ARNT occurred in adrenal gland and liver, although ARNT was also detected in submandibular gland, ectoderm, tongue, bone, and muscle. In all of these tissues ARNT was cytoplasmic as well as nuclear, except in some of the cortical adrenal cells in which ARNT was strongly cytoplasmic with little or no nuclear localization. These specific patterns of ARNT expression, which differ in certain tissues from the expression of AhR, suggest that ARNT may have additional roles in normal embryonic development.

Animals↗

PubMed1995

Morphology of the transepicondylar axis and its application in primary and revision total knee arthroplasty.

A morphologic anatomic study was done of the lower extremity to investigate various relationships of the transepicondylar axis (TEA). Thirteen cadaver specimens were dissected and mounted to a metal frame with a pin passing through the TEA. The center of the knee was determined as the depth of the anterior intercondylar groove. The ratio of the upper leg to lower leg measured from femoral head center and ankle center to TEA was 1.02. The mean distance of the TEA to the joint line was 3.08 cm medial and 2.53 cm lateral. The mean femoral angle comparing the TEA to mechanical axis was 0.61 degrees varus. The mean tibial angle comparing the TEA to the mechanical axis was 0.4 degrees varus in extension and 0.43 degrees in flexion, with no significant difference in the lower extremity angle with flexion (P < .01). The TEA is an important landmark that, from this study, is virtually perpendicular to the mechanical axis of the lower extremity and parallels the knee flexion axis. Femoral component rotation and joint line positioning in total knee arthroplasty can be determined using the TEA.

Biomechanical Phenomena↗

PubMed1995

Elevated plasma transforming growth factor-beta 1 levels in breast cancer patients decrease after surgical removal of the tumor.

OBJECTIVE: The authors determined whether untreated breast cancer patients have elevated plasma levels of transforming growth factor-beta 1 (TGF-beta 1). SUMMARY BACKGROUND DATA: Increased plasma TGF-beta 1 levels recently were found after chemotherapy in patients with advanced breast cancer. However, it currently is unknown whether this elevation in plasma TGF-beta 1 is caused by chemotherapy-induced normal tissue damage or whether it results from the presence of the tumor. METHODS: An enzyme-linked immunosorbent assay was used to measure plasma TGF-beta 1 levels in 26 newly diagnosed breast cancer patients before and after definitive surgery. Patients were grouped by postoperative tumor status: 1) negative lymph nodes (group 1); 2) positive lymph nodes (group 2); and 3) overt residual disease (group 3). The site of TGF-beta 1 production in the tumors was localized by immunohistochemistry and in situ hybridization. RESULTS: Plasma TGF-beta 1 levels were elevated preoperatively in 81% of the patients; TGF-beta 2 and TGF-beta 3 were undetectable. The preoperative TGF-beta 1 levels in the three patient groups were similar; however, the postoperative plasma TGF-beta 1 levels differed by disease status. The mean plasma TGF-beta 1 level in group 1 (n = 12) normalized after surgery (19.3 +/- 3.2 vs. 5.5 +/- 1.0 ng/mL, p < 0.001). In contrast, the mean plasma TGF-beta 1 levels remained above normal in both group 2 (n = 9) and group 3 (n = 5) after surgery. Transforming growth factor-beta 1 expression was found to be preferentially increased in the tumor stroma. CONCLUSIONS: Breast tumors result in increased plasma TGF-beta 1 levels in 81% of patients. After surgical removal of the primary tumor, the plasma TGF-beta 1 level normalizes in the majority of patients; persistently elevated levels correlate with the presence of lymph node metastases or overt residual tumor. These findings suggest that the usefulness of TGF-beta 1 as a potential plasma marker for breast tumors deserves further study.

Biomarkers, Tumor↗

PubMed1994

Effects of methanol on embryonic mouse palate in serum-free organ culture.

Methanol has widespread applications in industry and manufacturing and is under consideration as an alternative automotive fuel. Human exposure to methanol would be expected to increase if applications expand in coming years. Methanol has been shown to be a reproductive and developmental toxicant in the rodent, producing cleft palate in the CD-1 mouse. Developmental toxicity has also been demonstrated in vitro for rat and mouse embryos in whole embryo culture. The present study examines the developmental toxicity of methanol in the palate using a serum-free organ culture model. Gestation day 12 CD-1 mouse embryos were dissected and mid-craniofacial tissues were cultured in BGJ medium at 37 degrees C for 4 days with medium changes at 24 hr intervals. Cultures were exposed to methanol from 0-20 mg/ml for 6 hr, 12 hr, 1 or 4 days. Some cultures were exposed to ethanol for 4 days at doses ranging from 0-15 mg/ml. All cultures were gassed with a 50% O2, 5% CO2, and 45% N2 upon addition of fresh medium and prior to the addition of alcohol. Following organ culture the craniofacial explants were examined for effects on morphology, fusion, proliferation, and growth. Incidence and completeness of palatal fusion decreased with increasing exposure. Depending on the concentration and duration of methanol exposure, the medial epithelium either degenerated completely or remained intact in unfused palates and either condition would interfere with fusion. Cellular proliferation appeared to be a specific and sensitive target for methanol as craniofacial tissues responded to methanol with reduction in total DNA content at an exposure that did not affect total protein. However, both DNA and protein decreased with increasing exposure to methanol. Incorporation of thymidine decreased significantly after 4 day exposure and autoradiography of 3H-thymidine (TdR) demonstrated exposure-dependent reduction in proliferation of palatal mesenchymal cells. Ethanol decreased fusion score, total protein, and DNA, but 3H-TdR/DNA was not significantly changed. In general the ethanol was more potent than methanol for inhibition of protein and DNA synthesis and palatal fusion. This study demonstrated that methanol can selectively affect specific sensitive cell populations and has effects on proliferation and cell fate.

Animals↗

PubMed1994

Immunohistochemical double-staining for Ah receptor and ARNT in human embryonic palatal shelves.

The aryl hydrocarbon receptor (AhR) and the AhR nuclear translocator protein (ARNT) are basic-helix-loop-helix-PAS (HLH) proteins involved in transcriptional regulation. Polycyclic aromatic halogenated chemicals, of which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent, bind to the AhR. In the cellular cytoplasm, the AhR exists as a complex with the heat shock protein HSP90 and other small peptides. This complex dissociates following ligand binding and then the ligand-bound AhR binds ARNT. The ligand-AhR-ARNT complex interacts with a specific, nuclear DNA sequence, the dioxin response element (DRE), altering transcription of a regulated gene. Studies in hepatoma cell lines indicate that both proteins are required for regulation of transcription. In this study, AhR and ARNT were localized immunohistochemically in human embryonic palatal cells and specific patterns of expression were seen for each protein. A double-staining protocol revealed that epithelial cells expressed both AhR and ARNT, but in mesenchyme and nasal spine cartilage individual cells were identified which expressed either AhR or ARNT. This heterogeneous pattern may be a means of suppressing transcriptional regulation and also suggests the existence of other, unidentified basic-helix-loop-helix partner(s). The heterogeneous expression pattern may also reflect a complex role for these HLH proteins as transcriptional regulators of embryonic development.

Aryl Hydrocarbon Receptor Nuclear Translocator↗

PubMed1994

Early events following maternal exposure to 5-fluorouracil lead to dysmorphology in cultured embryonic tissues.

The chemotherapeutic agent 5-fluorouracil (5-FU) is teratogenic in a number of species, yet the mechanism(s) of its developmental toxicity are not fully understood. Administration of 5-FU to the pregnant CD rat on day 14 of gestation results in dose-dependent growth retardation and numerous malformations in near-term fetuses, including hindlimb defects and cleft palate. Following treatment, a number of rapid biochemical and cellular alterations are detectable in embryonic hindlimbs, craniofacial and other tissues, which include thymidylate synthetase (TS) inhibition and altered cell cycle progression. In order to assess the importance of these early events in 5-FU-induced dysmorphogenesis, embryonic mid-facial tissues and hindlimbs were dissected 3 or 6 hr after administration of 5-FU to the dam and placed in explant culture. After 5 days in culture, craniofacial explants were evaluated morphologically for palatal closure and growth was assessed by measuring total protein and DNA content. Hindlimb explants were stained for cartilage using alcian blue to evaluate development of the digits. Craniofacial explants cultured at either 3 or 6 hr after exposure exhibited dose-dependent growth retardation and defects of palatal fusion at the end of the culture period. Deficits in protein and DNA content were similar to those in craniofacial tissues that continued to develop in utero after treatment, although morphological defects in cultured explants did not correlate well with the incidence of cleft palate in vivo. Dose-dependent deficits in metatarsal and phalanx development were observed in hindlimb explants dissected either 3 or 6 hr after maternal treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcian Blue↗

PubMed1994

Ah receptor in embryonic mouse palate and effects of TCDD on receptor expression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent member of a family of halogenated aromatic hydrocarbons which are widespread environmental contaminants. In animals the adverse biological effects of TCDD include carcinogenesis, reproductive toxicity, immune function alteration, hyperkeratosis, hepatotoxicity, thymic involution, and teratogenesis. In the mouse embryo, TCDD induces cleft palate through a mechanism which involves altered differentiation and proliferation of the palatal cells, resulting in the failure of opposing shelves to fuse. Cleft palate induction by TCDD requires the Ah receptor. This study examines the expression of the Ah receptor in secondary palate of control and TCDD-exposed C57BL/6N embryos using in situ hybridization, Northern blots, and immunohistochemistry. Ah receptor protein expression was significantly higher in epithelial versus mesenchymal cells, and regional differences in expression within the epithelium were statistically significant. TCDD exposure was shown to downregulate Ah receptor mRNA and protein throughout the palatal shelf and this occurred at both the teratogenic dose and the dose which was not sufficient to produce cleft palate. This study represents the first demonstration of the tissue and cellular localization of the Ah receptor, raising questions about the extrapolation of results from cultured tumor cells to those observed in vivo.

Animals↗

PubMed1994

Interactive regulation of Ah and glucocorticoid receptors in the synergistic induction of cleft palate by 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a wide-spread environmental contaminant that produces adverse biological effects including carcinogenesis, reproductive toxicity, immune dysfunction, hyperkeratosis, hepatotoxicity, thymic involution and teratogenesis. In the mouse embryo, TCDD induces cleft palate and hydronephrosis. Glucocorticoids are endogenous steroid compounds that have an important role in development, but are teratogenic at pharmacological doses. The synthetic glycocorticoid, hydrocortisone (HC), induces cleft plate and a potent, synergistic interaction has been observed between TCDD and HC. Both TCDD and HC act through receptor-mediated mechanisms and each compound has its own receptor, the Ah receptor (AhR) and the glucocorticoid receptor (GR), respectively. The morphology and etiology of TCDD- and HC-induced clefts are distinctly different, as HC clefting is due to formation of small palatal shelves, while TCDD-treated shelves fail to fuse due to effects on epithelial cell proliferation and differentiation. The present study examines the expression of AhR and GR in the embryonic palate following exposure to TCDD, HC, and HC + TCDD. C57BL/6N pregnant mice were treated with HC (25 or 100 mg/kg/day GD10-13, sc), TCDD (3 micrograms/kg/day GD10-13, or 24 micrograms/kg GD10, orally), or HC + TCDD (25 mg/kg/day sc and 3 micrograms/kg/day orally, GD10-13). Craniofacial tissues were collected from the embryos on GD14 and examined for AhR and GR expression using in situ hybridization. Northern blots, and immunohistochemistry. We found that in the embryonic palate exposed to TCDD, the AhR was downregulated and the GR expression increased. Conversely, following HC exposure, the GR was downregulated and AhR levels were elevated. HC + TCDD produced increased expression of both receptors. Effects on AhR appeared to be regulated at the transcriptional level, as both protein and mRNA were altered in similar directions. The observed cross-regulation of the receptors is believed to be important in the synergistic interaction between TCDD and HC for the induction of cleft palate.

Animals↗

PubMed1993

Effects of 5-fluorouracil on embryonic rat palate in vitro: fusion in the absence of proliferation.

5-Fluorouracil (5-FU) inhibits the enzyme thymidylate synthetase (TS) which results in inhibition of DNA synthesis. 5-FU is teratogenic in many species, inducing cleft palate, limb, and tail defects. In the present study, gestation day (GD) 14 embryonic rat craniofacial explants were exposed to 5-FU in organ culture with increasing concentrations and durations of exposure. Palates exposed to 5-FU were morphologically abnormal and craniofacial shape, size, and palatal fusion pattern were affected with the severity of effects dependent on concentration and duration of exposure. Cleft palate was induced in vitro as opposing palates overlapped in a narrowed oral cavity. Palates exposed to higher levels of 5-FU were growth inhibited, but fused even though proliferation ceased and few cells were available to participate in elevation and fusion. This was demonstrated as a biphasic concentration-response profile for palatal fusion in which 0.05 to 0.15 micrograms 5-FU/ml produced decreasing rates of palatal fusion, while exposure to 0.15 to 3.0 micrograms/ml resulted in progressively increasing rates of fusion. The effects of 5-FU were detected biochemically as a reduction in TS activity which was concentration and time dependent during the first 12 hours, with a return to control levels by 24 hours. During the first day, 5-FU did not alter protein levels, but DNA levels significantly decreased at the high concentration, 2.0 micrograms/ml. After 5 days in culture, both DNA and protein decreased with increasing 5-FU concentration and duration of exposure. Also by the end of the culture period, 3H-TdR incorporation had decreased in a concentration dependent manner. It is concluded that progressive inhibition of proliferation and growth in organ culture results in two different morphological outcomes: cleft palate resulting from a narrowed oral cavity and increased incidence of anterior palatal fusion under conditions of strong growth reduction. This study demonstrates that elevation and fusion can occur in the absence of growth and proliferation. Based on these observations, severe inhibition of growth or proliferation would not necessarily be sufficient to induce cleft palate.

Animals↗

PubMed1992

Comparisons of the effects of TCDD and hydrocortisone on growth factor expression provide insight into their interaction in the embryonic mouse palate.

Cleft palate (CP) can be induced in embryonic mice by a wide range of compounds, including glucocorticoids and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Hydrocortisone (HC), a glucocorticoid, retards embryonic growth producing small palatal shelves, while TCDD exposure blocks the fusion of normally sized shelves. TCDD induction of CP involves altered differentiation of the medial epithelial cells. Recent studies indicate that growth factors such as EGF, TGF-alpha, TGF-beta 1, and TGF-beta 2 are involved in palatogenesis, regulating proliferation, differentiation, and extracellular matrix production. A synergism has been observed between HC and TCDD in which doses too low to induce CP alone are able to produce greater than 90% incidence when coadministered. In the present study a standard teratology protocol was performed in C57BL/6N mice to examine the synergism at doses lower than those previously published. Data from this study indicate synergistic interactions at doses as low as 3 micrograms TCDD/kg + 1 mg HC/kg. This extreme sensitivity suggests the involvement of a receptor-mediated mechanism possibly resulting in altered regulation of gene expression. Mechanisms of interaction were further studied by comparing growth of the shelves, fate of the medial epithelium, and expression of growth factor mRNAs and peptides. Pregnant mice were dosed on GDs 10-13 with HC (100 mg/kg sc) or with HC (25 mg/kg sc) + TCDD (3 micrograms/kg orally), doses producing 30% and 99% CP, respectively. The interaction between HC and TCDD results in a small HC-like palate, rather than the morphology typical of TCDD-induced clefting. Both compounds inhibited programmed cell death of the medial epithelium, which instead differentiated into an oral-like epithelium. The alterations in growth factor expression after HC or HC + TCDD were similar. Expression of EGF, TGF-beta 1, TGF-beta 2, and EGF receptor increased in specific palatal regions. Increased levels of mRNA were observed only for TGF-beta 1. The effect of TCDD alone on growth factor expression differ from those seen with HC or HC + TCDD. These divergent effects on growth factor expression may contribute to the differences in shelf size and thus to the different mechanisms of HC and TCDD clefting. Thus the synergism between HC and TCDD may involve similar and potentially additive effects on regulators of proliferation and differentiation in the palate, but additional contributing factors cannot be excluded.

Animals↗

PubMed1992

Embryonic palatal responses to teratogens in serum-free organ culture.

This study examines development of rat, mouse, and human embryonic palates in submerged, serum-free organ culture. The concentration-response profiles for retinoic acid (RA), triamcinolone (TRI), hydrocortisone (HC), dexamethasone (DEX), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were examined and the mechanisms of clefting in vitro were compared to observed in vivo responses. Craniofacial regions were dissected on gestational day (GD) 12 for mice and GD 14 for rat, and cultured for 3-4 days in Bigger's BGJb medium in flasks flushed with 50% O2, 45% N2, 5% CO2. Growth and fusion of secondary palates were scored under a dissecting microscope. In serum-free control medium, mouse and rat palatal fusion occurred within the 4-day culture period. Supplementing with fetal bovine serum (FBS) in excess of 1% interfered with growth and fusion in control medium. RA significantly inhibited fusion of mouse and rat palates at 5 x 10(-9) and 1 x 10(-10) M, respectively, with RA-induced clefting related to abnormal proliferation and differentiation of medial epithelia. In contrast, glucocorticoid-induced clefting was due to concentration-dependent inhibition of shelf growth. TRI significantly inhibited fusion at 4 x 10(-5) M, and 1 x 10(-4) M DEX or HC, inhibited fusion of 19 and 42% of shelves, respectively. The response rate for DEX in the presence of 1% FBS was increased (42% unfused). TCDD clefting was due to altered medial epithelial differentiation and 1 x 10(-8) M TCDD affected 36% of CD-1 mouse, 23% of C57BL/6N mouse, and 47% of F344 rat palates. When the medium was supplemented with 1% FBS, selenium, transferrin, and additional glutamine, the response of C57BL/6N embryos increased to 75%. This rate is similar to that reported for Trowell's-type cultures with IMEM:F12 medium and 1% FBS. The increased responsiveness to DEX or TCDD in the presence of serum suggests that an unknown factor in serum may be required for full activity. Three human embryonic palatal explants (GD 52 or 53) were cultured for 3-6 days and fused during culture. The present study demonstrates that serum-free organ culture supports development of mouse, rat, and human palatal explants. The present study demonstrates the capacity of this organ culture system to model palatogenesis for several species, and to distinguish between various mechanisms of clefting as presented through selected model compounds. This model should be useful for exploring mechanisms of activity at a cellular and molecular level.

Abnormalities, Drug-Induced↗

PubMed1991

TCDD exposure of human embryonic palatal shelves in organ culture alters the differentiation of medial epithelial cells.

The highly toxic, polychlorinated aromatic compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) occurs as a contaminant throughout the environment. Epidemiology studies of populations accidentally exposed to TCDD have failed to identify TCDD as a human teratogen, but these studies are limited by the small numbers of exposed pregnancies and imprecise estimates of exposure. TCDD is highly teratogenic in mice, inducing cleft palate and hydronephrosis. TCDD exposure in vivo of embryonic mice alters the differentiation and expression of growth factors in the medial epithelial palatal cells. These alterations also occur in rat and mouse palates exposed to TCDD in organ culture. In the present study, human embryonic palatal shelves were cultured in the rodent organ culture system. In order to achieve in vitro the developmental stage at which fusion would normally occur, GD 52 shelves were cultured for 4 days, GD 53 shelves were cultured for 3 days, and GD 54 shelves were cultured for 3 days. Three of four palatal shelves exposed to 5 x 10(-11) M TCDD were identical to their homologous controls (right shelf cultured with control medium; left shelf cultured with TCDD-containing medium). TCDD at 1 x 10(-7) M produced cytotoxicity detected by transmission electron microscopy (TEM). Exposure to 1 x 10(-8) M TCDD resulted in continued incorporation of thymidine ([3H]-TdR detected autoradiographically) by palatal medial cells, failure of the medial peridermal cells to degenerate as observed by scanning electron microscopy (SEM), and differentiation into a stratified, squamous epithelium. These alterations are identical to those induced by TCDD in vitro in rat and mouse palatal cells. The main difference between these species is the level of TCDD required to elicit the responses. Cultured mouse palates respond to 5 x 10(-11) M TCDD with altered medial cell differentiation, and 1 x 10(-10) M TCDD is cytotoxic. The rat shelves respond with altered differentiation at 1 x 10(-8) M and cytotoxicity at 1 x 10(-7) M. All the human shelves respond at 1 x 10(-8) M TCDD with altered differentiation, 1 out of 4 responded at 5 x 10(-11) M, and cytotoxicity occurred at 1 x 10(-7) M. The present data suggest human embryonic palates are less sensitive than those of the C57BL/6N mouse, and that exposure to high levels of TCDD would be required to elicit altered differentiation in the palatal shelf.

Analysis of Variance↗

PubMed1990

Induction of ethoxyresorufin-O-deethylase and inhibition of glucocorticoid receptor binding in skin and liver of haired and hairless HRS/J mice by topically applied 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The biochemical changes associated with the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been reported to include alterations in glucocorticoid and epidermal growth factor receptors and mixed function oxidase (MFO) induction. TCDD induces MFO activity in skin of both haired and hairless HRS/J mice. However, epidermal hyperplasia and hyperkeratosis are produced only in the skin of hairless mice. Therefore, since steroid and growth factor responses are implicated in cell proliferation and differentiation, these mice constitute a model system for assessing the possible roles of glucocorticoid and epidermal growth factor receptors in the toxicity of TCDD. The effect of dermal TCDD application (12 micrograms/kg in 100 microliters acetone) on ethoxyresorufin-O-deethylase (EROD) activity, glucocorticoid receptor binding and epidermal growth factor receptors in liver and skin of hairless and haired mice was determined. No differences existed in the basal number of cytosolic glucocorticoid receptors (Bmax) or the apparent equilibrium binding constants (Kd) in control liver, dorsal skin and abdominal skin of male and female hairless mice and haired male mice. Seven days after topical application of TCDD, decreases of approximately 38% were observed in the hepatic Bmax of the glucocorticoid receptors in both haired and hairless mice. However, in dorsal skin, TCDD decreased Bmax by approximately 40% in hairless mice but only 18% in haired mice. The dexamethasone-glucocorticoid receptor complex from both liver and skin of control and TCDD treated mice had similar sedimentation co-efficients in sucrose density gradients. TCDD had no effect on the Kd of glucocorticoid receptors of skin or liver in haired and hairless mice. No difference was observed in the time-dependent increases in hepatic EROD activity between haired and hairless mice after dermal application of TCDD. However, the maximum induction of EROD activity in microsomes from the skin of haired mice was only 60% of the activity observed in hairless animals. The induction of EROD by TCDD did not correlate temporally with the decrease in glucocorticoid receptor binding. The application of TCDD to the skin of hairless mice resulted in epidermal hyperplasia and dermal keratinization, while little change was observed in the general morphology of the skin of haired mice following dermal application. The application of TCDD had no effect on the incidence and distribution of epidermal growth factor receptors in skin of haired and hairless mice as determined immunohistochemically. Thus, the biochemical effects of TCDD are not only strain dependent, but tissue specific. Furthermore, decreases in glucocorticoid and epidermal growth factor receptors do not appear to be general markers of TCDD toxicity.

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