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.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin up-regulates stathmin 1 in the eye: Suggestive evidence for the involvement of stathmin 1 in accelerated eye opening in mice induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin

When pregnant mice were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the average time to eye opening in the offspring was shortened by about a day. How acceleration of eye opening by TCDD occurs remains unknown. To reveal the underlying mechanisms of the accelerated eye opening, pregnant mice were intraperitoneally injected with corn oil or TCDD at GD (gestation day) 11, and tissues around the eye of neonatal mice were subject to proteome analysis and RT-PCR. Upon TCDD administration, translationally controlled tumor protein (TCTP) and 60S acidic ribosomal protein p2 (RLA2) were reduced, while stathmin 1(STMN1) was increased, at both protein and mRNA levels. One hypothetical mechanism for eye opening is the proliferation of corneal epithelial cells before eye opening. STMN1, but not TCTP and RLA2, was up-regulated in immortalized human corneal epithelial cells (HCE-T) by TCDD, which promoted proliferation of HCE-T probably by accelerating the G1/S transition. Down-regulation of STMN1 by the antisense oligonucleotide technology inhibited proliferation of HCE-T, suggesting that STMN1, of which expression is enhanced by TCDD, may be involved in accelerated eye opening, probably by stimulating proliferation of corneal epithelial cells.     

Involvement of apoptotic cell death and cell cycle perturbation in retinoic acid-induced cleft palate in mice

Retinoic acid (RA), a metabolite of vitamin A, plays a key role in a variety of biological processes and is essential for normal embryonic development. On the other hand, exogenous RA could cause cleft palate in offspring when it is given to pregnant animals at either the early or late phases of palatogenesis, but the pathogenetic mechanism of cleft palate caused by excess RA remains not fully elucidated. The aim of the present study was to investigate the effects of excess of RA on early palatogenesis in mouse fetuses and analyze the teratogenic mechanism, especially at the stage prior to palatal shelf elevation. We gave all-trans RA (100 mg/kg) orally to E11.5 ICR pregnant mice and observed the changes occurring in the palatal shelves of their fetuses. It was found that apoptotic cell death increased not only in the epithelium of the palatal shelves but also in the tongue primordium, which might affect tongue withdrawal movement during palatogenesis and impair the horizontal elevation of palatal shelves. In addition, RA was found to prevent the G(1)/S progression of palatal mesenchymal cells through upregulation of p21(Cip1), leading to Rb hypophospholylation. Thus, RA appears to cause G(1) arrest in palatal mesenchymal cells in a similar manner as in various cancer and embryonic cells. It is likely that apoptotic cell death and cell cycle disruption are involved in cleft palate formation induced by RA.     

Blocking expression of AHR2 and ARNT1 in zebrafish larvae protects against cardiac toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The zebrafish (Danio rerio) has become an attractive vertebrate model for studying developmental processes, and is emerging as a model system for studying the mechanisms by which xenobiotic compounds perturb normal development. Embryos treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) shortly after fertilization exhibit a range of adverse effects on the heart: an early reduction in cardiac myocyte number, followed by a change in heart looping and morphology, with an apparent compaction of the ventricle and overall decrease in heart size. These changes are accompanied by impaired cardiac function including a decrease in cardiac output and eventually irreversible ventricular standstill. The mechanisms involved in mediating effects of TCDD on the heart remain unknown. However, it is widely accepted that aryl hydrocarbon receptor (AHR) activation mediates endpoints of TCDD toxicity in vertebrates. In zebrafish, there are multiple forms of AHR and AHR nuclear translocator protein (ARNT) raising the question about whether different endpoints of TCDD toxicity are mediated by different components of the AHR/ARNT pathway. To address this question we used morpholino oligonucleotide technology to specifically block the expression of zfAHR2, zfARNT1, zfARNT2, and zfCYP1A, and assessed the previously described effects of TCDD on heart morphology, size, and function in the developing morphants. We report that blocking zfAHR2 and zfARNT1 expression provided protection against the TCDD-mediated alteration in heart morphology, reduced cardiac myocyte number, decreased cardiac output and ventricular standstill in zebrafish larvae, while the zfarnt2 and zfcyp1a morpholinos did not block the TCDD-induced cardiac toxicity.     

Antiteratogenic effects of alpha-naphthoflavone on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposed mice in utero

The effects of α-naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist, on the reproductive toxicity and teratogenicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were investigated. Pregnant C57BL/6J mice were orally administered α-naphthoflavone either once on gestational day 12 (GD12; 50 μg/kg) or for 6 days (GD8–GD13; 5 mg/kg/day) followed by an oral challenge with TCDD (14 μg/kg) on GD12. Cesarean section was performed on GD18 for the evaluation of maternal and fetal toxicities. TCDD caused severe fetal malformations including cleft palate (43.7%) and renal pelvic and ureteric dilatations (100%). The administration of α-naphthoflavone either in a single treatment or 6-days remarkably reduced the incidence of cleft palate to 27.6% and 26.5%, respectively. In addition, the degree of renal pelvic and ureteric dilatations caused by TCDD were significantly attenuated by repeated treatment of α-naphthoflavone. These results suggest that AhR antagonists such as α-naphthoflavone could be promising candidates for reducing the incidence and severity of fetal malformations caused by TCDD exposure in utero.     

Elevated binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene to the Ah receptor in hepatic cytosols from phenobarbital-treated rats and mice

Binding of 3-methylcholanthrene (MC),2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and other “MC-type” inducers to cytosolic Ah receptor sites is the first specific step in induction of aryl hydrocarbon hydroxylase (AHH; cytochrome P₁?450) by these compounds. [₃H]TCDD and [₃H]MC were used as radioligands to quantitate and characterize Ah receptor in hepatic cytosols from genetically “responsive” C57BL/6J mice, genetically “nonresponsive” DBA/2J mice, and AHH-inducible Sprague-Dawley rats. Injection of 50–100 mg/kg of phenobarbital (PB) for 3 days more than doubled the concentration of Ah receptor in hepatic cytosol from Sprague-Dawley rats. In C57BL/6J mice, PB injection at 25 mg/kg × 3 days significantly increased (P < 0.01) the Ah receptor concentration in hepatic cytosol. No cytosolic Ah receptor was detectable in hepatic cytosol from untreated DBA/2J mice, nor did any Ah receptor appear after PB treatment in this “nonresponsive” strain. Although PB significantly elevated Ah receptor in hepatic cytosols of responsive rodents, many previous studies have shown that the maximal level of AHH activity in animals given PB and an “MC-type” inducer simultaneously is additive rather than synergistic. Ah receptor concentrations can be doubled by PB treatment without doubling the subsequent AHH-induction response to “MC-type” compounds. Thus, the cytosolic Ah receptor concentration per se may not be the primary determinant of a given tissue's maximal capacity for AHH induction by “MC-type” compounds.     

DNA fragmentation induced by all‐trans retinoic acid and its steroidal analogue EA‐4 in C2C12 mouse and HL‐60 human leukemic cells in vitro

We have recently shown that retinoic acid induces micronucleation mainly via chromosome breakage (Alakhras et al. Cancer Lett 2011; 306: 15–26). To further study retinoic acid clastogenicity and evaluate DNA damaging potential we investigated the ability of (a) all‐trans retinoic acid and its steroidal analogue EA‐4 to induce DNA fragmentation by using Comet assay under alkaline unwinding and neutral condition electrophoresis, and (b) the retinoids under study to induce small (0–1 kb) DNA fragments. Two cell lines, C2C12 mouse cells and HL‐60 human leukemic cells were used in this study. We found that all‐trans retinoic acid and its steroidal analogue EA‐4 (a) provoke DNA migration due to DNA fragmentation as it is shown by the increased values of Comet parameters, and (b) induce significantly small‐size fragmented genomic DNA as indicated by the quantification of necrotic/apoptotic small DNA segments in both cell systems. A different response between the two cell lines was observed in relation to retinoid ability to increase the percentage of DNA in the tail as well as break DNA in to small fragments. Our findings confirm the ability of retinoic acid to provoke micronucleation by disrupting DNA into fragments, among which small pieces of double‐stranded DNA up to 1 kb are identified. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparison between the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and six other compounds on the vitamin A storage,the UDP-glucuronosyltransferase and the aryl hydrocarbon hydroxylase activity in the rat liver

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrabromodibenzo-p-dioxin (TBrDD), 5-chloro-2-(2,4-dichlorophenoxy)phenol(3-Cl-predioxin) 4,5,6-trichloro-2-(2,4-dichlorophenoxy)phenol (5-Cl-predioxin), toxaphene, 3-methylcholanthrene (3-MC) and phenobarbital (PB) on the vitamin A storage, UDP-glucuronosyltransferase (UDPGT) and aryl hydrocarbon hydroxylase (AHH) activities in the liver of Sprague-Dawley rats was investigated. Vitamin A was determined as retinol by high pressure liquid chromatography. UDPGT was measured with p-nitrophenol as an aglycone and AHH with 3,4-benzopyrene as a substrate. Both in TCDD- and toxaphene-treated animals a reduced body weight gain was recorded, but no other overt signs of toxicity were seen in this study. Both the concentration and the total amount of hepatic retinol was significantly reduced in TCDD-, 3-MC-, PB- and TBrDD-treated animals. These compounds were also those which gave the most significant enzyme induction as regards the UDPGT and AHH activities. However, the reduction of hepatic retinol caused by these compounds did not correlate with the enzyme activities studied. When compared on a molecular basis, TCDD and TBrDD were in the order of several magnitudes more potent as reducers of hepatic retinol and likewise as enzyme inducers.     

Citral, an inhibitor of retinoic acid synthesis, attenuates the frequency and severity of branchial arch abnormalities induced by triazole-derivative fluconazole in rat embryos cultured in vitro

The clinically used antimycotic fluconazole (fluco) is teratogenic in rodents. Exposure in vitro to fluco, other investigated azoles (triadimefon, triadimenol, flusilazole, ketoconazole and imazalil) or retinoic acid (RA), is correlated to branchial arch abnormalities. Inhibition of RA degradation has been suggested as the azole-related mechanism. Citral is a RA synthesis inhibitor. E9.5 rat embryos were cultured for 48 h in normal serum or exposed in vitro to fluco 125 μM, citral 200 μM or co-exposed to the two molecules to test the hypothesis that citral attenuates fluco-related teratogenic effects. Some embryos were cultured for 12 extra hours, and cranial nerves immunodetected. Fluco induced typical abnormalities, including branchial arch and cranial nerve defects. The co-exposure to fluco + citral was significantly effective in reducing branchial arch and cranial nerve defects, supporting the hypothesis that citral balances the fluco-induced RA concentration increase. However, other fluco-related effects were unalterated by citral.     

Influence of retinoic acid on TBX1 expression in myocardial cells induced by Shh and Fgf8

The aim of this study was to explore the regulatory mechanism of retinoic acid (RA) on the TBX1 gene expression in myocardial cells. Ventricular cardiocytes were isolated from neonatal rats and cultured, and then treated with different concentrations of retinoic acid. The expression of Shh and Fgf8 at mRNA and protein levels in neonatal rat myocardial cells were measured by using RT-PCR and Western blot technique, respectively. There was basal expression of Shh and Fgf8 in the control group. When treated with 3 × 10⁻⁷ mol/L RA, we observed that the expression of Shh mRNA and protein in neonatal rat myocardial cells were up-regulated by 1.51 (P < 0.05) and 1.10 times (P < 0.05), respectively. In comparison with the control group, under the concentration of 5 × 10⁻⁷ mol/L RA, they were up-regulated by 2.21 (P < 0.05) and 2.38 times (P < 0.05) individually. Meanwhile, we could detect that the expression of Fgf8 mRNA and protein were up-regulated by 2.50 times (P < 0.05) and 80% (P < 0.05) separately compared with the control group after stimulation of 3 × 10⁻⁷ mol/L RA, and they were up-regulated by 3.48 (P < 0.05) and 2.04 times (P < 0.05) individually after stimulation of 5 × 10⁻⁷ mol/L RA. The results indicated that RA could induce the expression of Shh and Fgf8 in neonatal rat myocardial cells. At the same time, it has shown that Shh and Fgf8 were involved in the regulation process of RA on TBX1 expression.     

Nrf2 protects against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced oxidative injury and steatohepatitis

Previous studies demonstrate that Nrf2, a master regulator of antioxidative responses, is essential in mediating induction of many antioxidative enzymes by acute activation of the AhR. However, the role of Nrf2 in protecting against oxidative stress and DNA damage induced by sustained activation of the AhR remains unknown and was investigated herein. Tissue and blood samples were collected from wild-type (WT) and Nrf2-null mice 21 days after administration of a low-toxic dose (10 μg/kg ip) of TCDD. Only Nrf2-null mice lost body weight after TCDD treatment; however, blood levels of ALT were not markedly changed in either genotype, indicating a lack of extensive necrosis. Compared to livers of TCDD-treated WT mice, livers of TCDD-treated Nrf2-null mice had: 1) degenerated hepatocytes, lobular inflammation, marked fat accumulation, and higher mRNA expression of inflammatory and fibrotic genes; 2) depletion of glutathione, elevation in lipid peroxidation and marker of DNA damage; 3) attenuated induction of phase-II enzymes Nqo1, Gsta1/2, and Ugt2b35 mRNAs, but higher induction of cytoprotective Ho-1, Prdx1, Trxr1, Gclc, and Epxh1 mRNAs; 4) higher mRNA expression of Fgf21 and triglyceride-synthesis genes, but down-regulation of bile-acid-synthesis genes and cholesterol-efflux transporters; and 5) trend of induction/activation of c-jun and NF-kB. Additionally, TCDD-treated Nrf2-null mice had impaired adipogenesis in white adipose tissue. In conclusion, Nrf2 protects livers of mice against oxidative stress, DNA damage, and steatohepatitis induced by TCDD-mediated sustained activation of the AhR. The aggravated hepatosteatosis in TCDD-treated Nrf2-null mice is due to increased lipogenesis in liver and impaired lipogenesis in white adipose tissue.     

Ovariectomy aggravates convulsions and hippocampal γ-aminobutyric acid inhibition induced by cyclosporin A in rats

The possible cyclosporin A application for rheumatoid arthritis that develops preferentially in middle-aged women raises concerns about adverse effects of cyclosporin A, including neurotoxicity in patients with climacterium. The present study was aimed at elucidating the effect of cyclosporin A on the convulsive activity and γ-aminobutyric acid (GABA) neural activity of the hippocampus in ovariectomized rats, as a menopause/climacterium model. Ovariectomy markedly aggravated the effect of repeated administration of cyclosporin A (40 mg/kg, once a day for 5 or 6 days), convulsions and reduction of the basal GABA levels and aminooxyacetic acid-evoked GABA accumulation. These aggravations were blocked by estradiol replacement. The present findings demonstrated that ovariectomy increased the susceptibility to cyclosporin A-induced convulsions by accelerating an inhibitory action of cyclosporin A on GABA neural activity in the hippocampus, this being blocked by estrogen replacement. Menopause/climacterium is, therefore, included in the risk factors for cyclosporin A-induced neurotoxicity and this risk is lowered by estrogen replacement therapy.     

TCDD-induced cyclooxygenase-2 expression is mediated by the nongenomic pathway in mouse MMDD1 macula densa cells and kidneys

Cyclooxygenase-2 (Cox-2) plays a critical role in TCDD-induced hydronephrosis in mouse neonates. In this study we found that induction of Cox-2 by TCDD in MMDD1, a mouse macula densa cell line, is accompanied with a rapid increase in the enzymatic activity of cytosolic phospholipase A2 (cPLA2) as well as activation of protein kinases. Calcium serves as a trigger for such an action of TCDD in this cell line. These observations indicate that the basic mode of action of TCDD to induce the rapid inflammatory response in MMDD1 is remarkably similar to those mediated by the nongenomic pathway of aryl hydrocarbon receptor (AhR) found in other types of cells. Such an action of TCDD to induce Cox-2 in MMDD1 was not affected by “DRE decoy oligonucleotides” treatment or by introduction of a mutation on the DRE site of Cox-2 promoter, suggesting that this route of action of TCDD is clearly different from that mediated by the classical genomic pathway. An in vivo study with Ahr^nls mouse model has shown that TCDD-induces Cox-2 and renin expression in the kidneys of the Ahr^nls mice as well as Ahr^+/− mice, but not in the Ahr^−/− mice, indicating that this initial action of TCDD in mouse kidney does not require the translocation of AhR into the nucleus, supporting our conclusion that induction of Cox-2 by TCDD in mouse kidney is largely mediated by the nongenomic pathway of TCDD-activated AhR.     

Inhibition of C6 glioma cell proliferation by anandamide, 1-arachidonoylglycerol,and by a water soluble phosphate ester of anandamide: variability in response and involvement of arachidonic acid

It has previously been shown that the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) inhibit the proliferation of C6 glioma cells in a manner that can be prevented by a combination of capsazepine (Caps) and cannabinoid (CB) receptor antagonists. It is not clear whether the effect of 2-AG is due to the compound itself, due to the rearrangement to form 1-arachidonoylglycerol (1-AG) or due to a metabolite. Here, it was found that the effects of 2-AG can be mimicked with 1-AG, both in terms of its potency and sensitivity to antagonism by Caps and CB receptor antagonists. In order to determine whether the effect of Caps could be ascribed to actions upon vanilloid receptors, the effect of a more selective vanilloid receptor antagonist, SB366791 was investigated. This compound inhibited capsaicin-induced Ca(2+) influx into rVR1-HEK293 cells with a pK(B) value of 6.8+/-0.3. The combination of SB366791 and CB receptor antagonists reduced the antiproliferative effect of 1-AG, confirming a vanilloid receptor component in its action. 1-AG, however, showed no direct effect on Ca(2+) influx into rVR1-HEK293 cells indicative of an indirect effect upon vanilloid receptors. Identification of the mechanism involved was hampered by a large inter-experimental variation in the sensitivity of the cells to the antiproliferative effects of 1-AG. A variation was also seen with anandamide, which was not a solubility issue, since its water soluble phosphate ester showed the same variability. In contrast, the sensitivity to methanandamide, which was not sensitive to antagonism by the combination of Caps and CB receptor antagonists, but has similar physicochemical properties to anandamide, did not vary between experiments. This variation greatly reduces the utility of these cells as a model system for the study of the antiproliferative effects of anandamide. Nevertheless, it was possible to conclude that the antiproliferative effects of anandamide were not solely mediated by either its hydrolysis to produce arachidonic acid or its CB receptor-mediated activation of phospholipase A(2) since palmitoyltrifluoromethyl ketone did not prevent the response to anandamide. The same result was seen with the fatty acid amide hydrolase inhibitor palmitoylethylamide. Increasing intracellular arachidonic acid by administration of arachidonic acid methyl ester did not affect cell proliferation, and the modest antiproliferative effect of umbelliferyl arachidonate was not prevented by a combination of Caps and CB receptor antagonists.     

Stimulation of early gene induction and cell proliferation by lysophosphatidic acid in human amnion-derived WISH cells: role of phospholipase D-mediated pathway

Human-amniotic WISH cells express the lysophosphatidic acid (LPA) receptor, LPA(1), LPA(2) but not LPA(3). When WISH cells were stimulated with LPA, phospholipase D (PLD) activation was dramatically induced via a cytosolic calcium increase and protein kinase C activation. We also found that LPA stimulated two kinds of mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) and p38 kinase via PLD-dependent signaling pathways in WISH cells. In terms of the LPA-mediated functional modulation of WISH cells, we observed that LPA stimulates the induction of two early genes (c-Jun and c-Fos) and cellular proliferation in WISH cells. We examined the signaling pathways involved in LPA-mediated cellular responses. LPA-induced early gene induction was completely blocked by normal butanol (n-butanol) but not by t-butanol, suggesting that PLD activity is essentially required for the process. PD98059 (2'-amino-3'-methoxyflavone) but not SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole) also significantly blocked LPA-induced early gene induction, suggesting a crucial role for ERK. Pertussis toxin (PTX) did not affect on the LPA-induced early gene induction and ERK activation, ruling out the role of Gi/o protein(s) in the process. The cellular proliferation of WISH cells was also dramatically inhibited by n-butanol or PD98059. This study demonstrates the physiological role of LPA on the modulation of early gene induction and on WISH cell proliferation, and the crucial role played by PLD in the process.     

Activation of deoxycytidine kinase by protein kinase inhibitors and okadaic acid in leukemic cells

Deoxycytidine kinase (dCK) is a key enzyme in the deoxynucleoside salvage pathway and in the activation of numerous nucleoside analogues used in cancer and antiviral chemotherapy. Recent studies indicate that dCK activity might be regulated through reversible phosphorylation. Here, we report the effects of a large panel of protein kinase inhibitors on dCK activity in the B-leukemia cell line EHEB, both in basal conditions and in the presence of the nucleoside analogue 2-chloro-2'-deoxyadenosine (CdA) which induces activation of dCK. Except staurosporine and H-7 that significantly reduced the activation of dCK by CdA, no specific protein kinase inhibitor diminished basal dCK activity or its activation by CdA. In contrast, genistein, a general protein tyrosine kinase inhibitor, and AG-490, an inhibitor of JAK2 and JAK3, increased basal dCK activity more than two-fold. Two specific inhibitors of the MAPK/ERK pathway, PD-98059 and U-0126, also enhanced dCK activity. These data suggest that the JAK/MAPK pathway could be involved in the regulation of dCK. Moreover, we show that the activity of dCK, raised by CdA, can return to its initial level by treatment with protein phosphatase-2A (PP2A). Accordingly, dCK activity in intact cells increased upon incubation with okadaic acid (OA) at concentrations that should inhibit PP2A, but not protein phosphatase-1. Activation of dCK by protein kinase inhibitors and OA was also observed in CCRF-CEM cells and in chronic lymphocytic leukemia B-lymphocytes, suggesting a general mechanism of post-translational regulation of dCK, which could be exploited to enhance the activation of antileukemic nucleoside analogues.