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

doi:10.1016/0041-008X(90)90337-T

Toxicology and Applied Pharmacology

Volume 106, Issue 3, December 1990, Pages 418-432

https://doi.org/10.1016/0041-008X(90)90337-T Get rights and content

Abstract

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 or TCDD + RA and the palatal shelves were dissected on GD 14–16. Growth factor expression was determined immunohistochemically using antibodies to TGF-α, EGF, TGF-β1, or TGF-β2. The growth factors displayed specific spatial and temporal expression in the palatal shelves. TCDD reduced the expression of TGF-α, EGF, and TGF-β1 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-α. 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-β1, a factor inhibitory to epithelial proliferation. Since TGF-β1 stimulates mesenchymal growth and TGF-α 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-β1, in vitro experiments were designed to examine the involvement of TGF-β1 in TCDD teratogenicity. Shelves from untreated embryos were removed on GD 12, placed in organ culture, and exposed to TCDD or TCDD + TGF-β1 for 3 days. In vitro, TGF-β1 eliminated some of the effects of TCDD on epithelial cells. A specific spatial and temporal expression of TGF-α, TGF-β1, EGF, and TGF-β2 appears to be important for normal palatogenesis, and the disruption of this pattern by TCDD correlates with altered proliferation and differentiation in the medial region, ultimately resulting in cleft plate.

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The role of MEG3 in the proliferation of palatal mesenchymal cells is related to the TGFβ/Smad pathway in TCDD inducing cleft palate
2021, Toxicology and Applied Pharmacology
Citation Excerpt :
When the ability of mesenchymal cells to proliferate is inhibited by TCDD, the palatal shelves may not grow to their normal sizes for contact, leading to the development of CP. In addition, TGF-β1 could stimulate the mesenchymal growth after exposure to TCDD in vivo (Abbott and Birnbaum, 1990). We also found that the expression of the growth factors TGF-β1 was reduced in mesenchymal cells at GD14, which may not fully promote the proliferation of mesenchymal cells.
Cleft palate (CP) is a common birth defect with a high incidence of occurrence in humans. The 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic halogenated aromatic hydrocarbon, with a strong CP effect on mice. Increasing recent evidences have shown that long-noncoding RNAs (lncRNAs) play an important role in several diseases, including CP. However, there is a paucity of studies on the role of lncRNA MEG3 in the occurrence and development of TCDD-induced CP. In this study, the relationship between MEG3 and the proliferation of palatal mesenchymal cells and the underlying molecular mechanism were studied by establishing fetal CP with TCDD (64 μg/kg) in C57BL/6N mice. The results revealed that MEG3 was highly expressed during the critical period of CP formation and that the fetal mesenchymal proliferation was significantly inhibited at certain critical periods in the mice receiving TCDD. In addition, we noted a possibility of a crosstalk between MEG3 and the TGF-β/Smad pathway, such that the inhibition of the TGF-β/Smad pathway was induced by TCDD. Cumulatively, our study suggests that TCDD-induced CP may be caused by MEG3 inhibition of the proliferation of palatal mesenchymal cells involving the TGFβ/Smad pathway, which may provide a novel perspective to understand the pathogenesis of CP.
Distinguishing mode of action of compounds inducing craniofacial malformations in zebrafish embryos to support dose-response modeling in combined exposures
2020, Reproductive Toxicology
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Both the disturbance of the retinoic acid (RA) balance and activation of the aryl hydrocarbon receptor (AhR; predominantly AhR-2) are long-established causes of developmental craniofacial malformations [9,10], and could therefore be included as major interactions in the pathway network (Fig. 1). In this line, inhibition of the RA metabolizing enzyme Cyp26 through triazoles and disruption of AhR-signalling through AhR ligands (dioxin-like acting compounds) can be understood as a cause of developmental craniofacial malformations in zebrafish [8,11,12] and rodents [9,13]. Molecular pathways for both MOAs are well described, including the role of inhibition of Cyp26A and B in causing a disbalance of RA and subsequent effects on regulation of hox(n) genes [14], and effects on EGF/TGFβ3 signaling following AhR activation [15].
Knowledge on mode-of-action (MOA) is required to understand toxicological effects of compounds, notably in the context of risk assessment of mixtures. Such information is generally scarce, and often complicated by the existence of multiple MOAs per compound. Here, MOAs related to developmental craniofacial malformations were derived from literature, and assembled in a MOA network. A selection of gene expression markers was based on these MOAs. Next, these markers were verified by qPCR in zebrafish embryos, after exposure to reference compounds. These were: triazoles for inhibition of retinoic acid (RA) metabolism, AM580 and CD3254 for selective activation of respectively RA-receptor (RAR) and retinoid-X-receptor (RXR), dithiocarbamates for inhibition of lysyl oxidase, TCDD for activation of the aryl-hydrocarbon-receptor (AhR), VPA for inhibition of histone deacetylase (HDAC), and PFOS for activation of peroxisome proliferator-activated receptor-alpha (PPARα). Next, marker gene profiles for these reference compounds were used to map the profiles of test compounds to known MOAs. In this way, 2,4-dinitrophenol matched with the TCDD and RAR profiles, boric acid with RAR, endosulfan with PFOS, fenpropimorph with dithiocarbamates, PCB126 with AhR, and RA with triazoles and RAR profiles. Prochloraz showed no match. Activities of these compounds in ToxCast assays, and in silico analysis of binding affinity to the respective targets showed limited concordance with the marker gene expression profiles, but still confirmed the complex MOA profiles of reference and test compounds. Ultimately, this approach could be used to support modeling of mixture effects based on upfront knowledge of (dis)similarity of MOAs.
Defining embryonic developmental effects of chemical mixtures using the embryonic stem cell test
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The embryonic stem cell test (EST) was applied to evaluate dose addition in combined exposures of teratogenic compounds in the EFSA-defined cumulative assessment group “craniofacial malformations”, which was one of the selected cases in the EU-H2020 project “EuroMix”. Test compounds were selected through reported effects in rodents, and represented a wide variety of chemical families and modes of action (MOA), including triazoles to inhibit CYP26; (synthetic) retinoids, to activate RAR/RXR; valproic acid, to inhibit histone deacetylase; dithiocarbamates, to disrupt extracellular matrix formation; dioxin (-like) compounds, to activate the aryl hydrocarbon receptor; 17alpha-ethynylestradiol, to activate the estrogen receptor; 5-fluorouracil, to disrupt DNA-synthesis; MEHP and PFOS, to activate peroxisome proliferation activated receptors; and methyl mercury, to induce oxidative stress and inhibit protein function. The EST appeared particularly useful to evaluate differentiation-inhibiting effects of compounds targeting early processes in craniofacial development, possibly related to the early fate of neural crest cells. Mixtures, designed as equipotent concentrations of two compounds with similar or dissimilar MOA, and single compounds showed overlapping dose-responses. This observation is consistent with dose addition in the EST in all studied binary mixtures, irrespective of MOA, and thereby supports the application of dose-addition as a default in cumulative risk assessment.
Dose addition in chemical mixtures inducing craniofacial malformations in zebrafish (Danio rerio) embryos
2020, Food and Chemical Toxicology
Citation Excerpt :
A first compound with dissimilar MOA is TCDD. TCDD is a teratogen in mice, affecting palatal formation and development due to disturbance of TGF-α, EGF, TGF-β1 and TGF-β2 developmental pathways (Abbott and Birnbaum, 1990). Next, the dithiocarbamate thiram is known to induce palatal and skeletal malformation in mice, hamsters and rabbits (Robens, 1969).
A challenge in cumulative risk assessment is to model hazard of mixtures. EFSA proposed to only combine chemicals linked to a defined endpoint, in so-called cumulative assessment groups, and use the dose-addition model as a default to predict combined effects. We investigated the effect of binary mixtures of compounds known to cause craniofacial malformations, by assessing the effect in the head skeleton (M-PQ angle) in 120hpf zebrafish embryos. We combined chemicals with similar mode of action (MOA), i.e. the triazoles cyproconazole, triadimefon and flusilazole; next, reference compounds cyproconazole or triadimefon were combined with dissimilar acting compounds, TCDD, thiram, VPA, prochloraz, fenpropimorph, PFOS, or endosulfan. These mixtures were designed as (near) equipotent combinations of the contributing compounds, in a range of cumulative concentrations. Dose-addition was assessed by evaluation of the overlap of responses of each of the 14 tested binary mixtures with those of the single compounds. All 10 test compounds induced an increase of the M-PQ angle, with varying potency and specificity. Mixture responses as predicted by dose-addition did not deviate from the observed responses, supporting dose-addition as a valid assumption for mixture risk assessment. Importantly, dose-addition was found irrespective of MOA of contributing chemicals.
Retinoic acid co-treatment aggravates severity of dioxin-induced skin lesions in hairless mice via induction of inflammatory response
2018, Biochemical and Biophysical Research Communications
Citation Excerpt :
All these findings suggest that TCDD and retinoids may have antagonistic properties in vivo. At the same time, the combination of RA and TCDD was shown to produce a strong synergistic effect in cleft palate induction during mouse embryonic development [12]. Furthermore, similarly to dioxin, the retinoids possess immunosuppressive, carcinogenic, and teratogenic effects in animals.
Exposure to toxic halogenated polyaromatic hydrocarbons, of which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent, induces diverse skin pathologies in humans, including chloracne, hyperkeratosis, hamartomas, etc. While the toxic effects of TCDD have been extensively studied, effective approaches to their treatment are still lacking. Retinoids are commonly used in therapy of acneiform skin diseases. In vitro, retinoids elicit antagonistic effects on keratinocyte differentiation and proliferation, as compared to TCDD, suggesting their potential in treatment of TCDD-induced skin lesions. Nevertheless, the modulation of TCDD activity in skin by retinoids in vivo was never reported. We have used N-TERT keratinocyte cell line and hairless (hr) mice to determine if retinoic acid (RA) can lessen or reverse TCDD-induced effects in vitro and in vivo. RA co-treatment suppressed TCDD-induced changes in the expression of differentiation-associated genes and N-TERT keratinocyte viability in vitro. However, in hairless mice (in vivo), RA/TCDD co-treatment produced more severe effects, than treatment with either of the two compounds individually. RA/TCDD co-application to mouse skin strongly stimulated keratinocyte proliferation, resulting in dramatic epidermal hyperplasia. It has also led to massive immune cell infiltration into the dermis, and increased mRNA expression of inflammation markers, including IL1β, IL6 and S100A7. Thus, retinoids not only appeared ineffective in treatment of TCDD-induced skin lesions in hairless mice, but also resulted in their exaggeration. These in vivo results question previous cell culture-based claims that RA may reduce TCDD-induced skin effects and caution against the reliance on in vitro data in TCDD toxicology research.
Dioxin-induced retardation of development through a reduction in the expression of pituitary hormones and possible involvement of an aryl hydrocarbon receptor in this defect: A comparative study using two strains of mice with different sensitivities to dioxin
2014, Toxicology and Applied Pharmacology
We have previously revealed that treating pregnant rats with 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) reduces the expression of gonadotropins and growth hormone (GH) in the fetal and neonatal pituitary. A change in gonadotropin expression impairs the testicular expression of steroidogenic proteins in perinatal pups, and imprint defects in sexual behavior after reaching maturity. In this study, we examined whether TCDD also affects the expression of gonadotropin and GH in mice using C57BL/6J and DBA/2J strains which express the aryl hydrocarbon receptor (Ahr) exhibiting a different affinity for TCDD. When pregnant C57BL/6J mice at gestational day (GD) 12 were given oral TCDD (0.2–20 μg/kg), all doses significantly attenuated the pituitary expression of gonadotropin mRNAs in fetuses at GD18. On the other hand, in DBA/2J mice, a much higher dose of TCDD (20 μg/kg) was needed to produce a significant attenuation. Such reduction in the C57BL/6J strain continued until at least postnatal day (PND) 4. In agreement with this, TCDD reduced the testicular expression of steroidogenic proteins in C57BL/6J neonates at PND2 and 4, although the same did not occur in the fetal testis and ovary. Furthermore, TCDD reduced the perinatal expression of GH, litter size and the body weight of newborn pups only in the C57BL/6J strain. These results suggest that 1) also in mice, maternal exposure to TCDD attenuates gonadotropin-regulated steroidogenesis and GH expression leading to the impairment of pup development and sexual immaturity; and 2) Ahr activation during the late fetal and early postnatal stages is required for these defects.

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²: Current address: Director, Environmental Toxicology Division (MD-68), Health Effects Research Laboratory, US EPA, Research Triangle Park, NC 27711.

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Regular articleTCDD-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

Abstract

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

Toxicol. Appl. Pharmacol.

Dev. Biol.

Dev. Biol.

Retinoic acid alters EGF receptor expression during palatogenesis

Development

Retinoic acid induced alterations in the expression of growth factors in embryonic mouse palatal shelves

Teratology

Etiology of retinoic acid-induced cleft palate varies with the embryonic stage

Teratology

Retinoids and EGF alter embryonic mouse palatal epithelial and mesenchymal cell differentiation in organ culture

J. Craniofacial Genet. Dev. Biol.

The role of TGF-β in mouse development

Ann. N.Y. Acad. Sci.

Regulation of epithelial cell proliferation by transforming growth factors

J. Cell. Biochem.

Epidermal growth factor and transforming growth factor-α

Brit. Med. Bull.

Epidermal growth factor regulates the expression of its own receptor

Regular article
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