Toxicological Sciences 66, 159-165 (2002)
Copyright © 2002 by the Society of Toxicology
REPRODUCTIVE AND DEVELOPMENTAL TOXICOLOGY |
Selective Inhibition of Murine Palatal Mesenchymal Cell Proliferation in Vitro by Secalonic Acid D
Department of Veterinary Biomedical Sciences, University of Missouri, 1600 E. Rollins, Columbia, Missouri 65211
Received June 7, 2001; accepted October 5, 2001
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ABSTRACT |
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Secalonic acid D (SAD), a teratogenic mycotoxin, induces cleft palate (CP) in the offspring of exposed mice by inhibiting palatal shelf growth. Since reduced proliferation, increased apoptosis, and/or decreased extracellular matrix (ECM) synthesis of palatal mesenchymal cells (PMC) can all contribute to smaller shelf size, the hypothesis that teratogenically relevant concentrations (0 to 120 µg/ml) of SAD will have adverse effects on one or more of these cellular processes was tested, using primary murine PMC cultures. Exposure to SAD resulted in significant and dose-dependent decreases in mesenchymal cell number, uptake of 3H-thymidine, and expression of proliferating cell nuclear antigen (PCNA). Trypan blue dye exclusion assay, however, revealed significant cell death only at higher doses, suggesting that the decrease in cell number at lower (more realistic) doses is likely a consequence of reduced cell proliferation and not cell death. Further, negative results in the DNA fragmentation analysis following SAD exposure suggested that cell death caused by higher levels of SAD was unrelated to apoptosis. Similarly, results of 3H-glucosamine uptake assay indicated inhibitory effect of SAD on accumulation of hyaluronic acid (HA) or sulfated glycosaminoglycans (sGAG) only at the highest dose tested. Also, SAD affected neither extracellular nor cell-associated fibronectin expression at any dose tested. Taken together, these data suggest that the pathogenesis of CP by SAD is likely a result of a reduction in the size of the palatal shelf caused by SAD-induced inhibition of mesenchymal cell proliferation.
Key Words: secalonic acid D; cleft palate; proliferation; glycosaminoglycans; fibronectin; proliferating cell nuclear antigen.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Palatal shelf elevation and fusion are the two events important in normal palate development. Proper shelf size is important for both of these events to occur. Small shelves generally fail to elevate fully, and invariably fail to make contact at the midline for fusion to be triggered. In addition, increase in size is critical if palatal shelves are to keep up with the growth in the width of the developing head. Mesenchymal cell number, determined by a balance between cell proliferation and apoptosis, is a major contributor to the shelf size. Although glucocorticoids (GC) are known to cause apoptosis in lymphoid and other cells (Cidlowski et al., 1996
; Planey and Litwack, 2000); examples of cleft palate (CP)-inducing agents also causing mesenchymal apoptosis are lacking. Agents such as GC (Nanda and Romeo, 1978; Salomon and Pratt, 1978), 5-fluorouracil and hadacidin (Abbott et al., 1993; Shah et al., 1991), retinoids (Yoshikawa et al., 1987) and cyclophosphamide (Shah, 1990) inhibit the growth of palatal shelves by reducing palatal mesenchymal cell number. This is associated with a decrease in DNA synthesis in vitro and/or in vivo. Palatal clefts result from exposure to all of these agents.
Accumulation of ECM components such as glycosaminoglycans (GAG), collagen, and fibronectin contribute to palatal shelf growth. This, in association with embryonic basal laminae and mesenchyme, plays an important role in tissue morphogenesis (Toole, 1981). Hyaluronic acid (HA), the nonsulfated GAG, is a major component of ECM in the developing facial region, including the palatal mesenchyme during shelf elevation (Pratt et al., 1973) whereas the sulfated GAGs (sGAG) such as chondroitin sulfate (CS) and heparan sulfate are minor contributors (Burk, 1983). Decreased synthesis of both HA and sGAG by Vitamin A (Kaye et al., 1979; Yoshikawa et al., 1987) and enhanced degradation of HA and CS by chlorcyclizine are associated with a failure of palatal shelf elevation and CP (Brinkley and Vickerman, 1982; Wilk et al., 1978). Decrease in palatal GAGs also occurs upon exposure to other CP-inducing agents: cortisone (Yoshikawa et al., 1986), salicylates (Larsson and Bostrom, 1965) and diazo-oxo-norleucine (Pratt et al., 1973). Fibronectin, a cell adhesion protein present as a polymeric fibrillar network, interacts with cells to provide signals that affect the morphology, motility, and gene expression of adherent cells (Johansson et al., 1997). Fibronectin is found throughout the palatal mesenchyme (Silver et al., 1981) with perinuclear and extracellular distribution (Kurisu et al., 1987). The fibronectin gene is responsive to both epidermal growth factor (EGF) and cAMP signaling pathways (Kreisberg et al., 1994; Silver et al., 1984).
SAD is a mycotoxin of potential significance in human and animal health and produces CP as the only malformation in fetal mice at doses relevant to human exposure (Reddy et al, 1981; Reddy and Reddy, 1991). More importantly, SAD has been used as a chemical model to study mechanisms of pathogenesis of environmentally induced CP in CD1 mice. The major palatal morphological deficit resulting from SAD exposure in mice is small shelves that fail to elevate. Biochemically, SAD affects palatal protein kinase A and C (PKA and PKC) signal transduction systems in vivo (Balasubramanian et al., 2000a; ElDeib and Reddy, 1988a), both of which together are known to regulate the cellular responses that contribute to shelf size. The present study was designed to use a primary palatal mesenchymal cell culture system to test the hypothesis that SAD-induced reduction in palatal shelf size is caused by reduced rate of mesenchymal cell proliferation, ECM synthesis, and/or increased rate of apoptosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials.
Secalonic acid D was extracted and purified as described by Reddy et al., (1979). Opti-minimum essential medium (Opti-MEM) and other tissue culture materials were purchased from Gibco-BRL (Life Technologies, Gaithesburg, MD). [3H-thymidine (20 Ci/ mmol) and [3H]-glucosamine (21.6 Ci/mmol) were obtained from NEN Biolabs (Boston, MA). Monoclonal antifibronectin antibody, cetylpyridinium chloride (CPC), papain, purified HA and CS, proteinase K, RNaseA, hyaluronidase, and other routine chemicals were from Sigma Chemicals (St. Louis, MO). Polyclonal antiproliferating cell nuclear antigen (PCNA) antibody was from Santa Cruz Biotech (Santa Cruz, CA).
Animals.
CD1 mice (Charles River, Wilmington, MA) were housed at 70° ± 2°F and humidity of 60 ± 5% in plastic cages with corncob bedding and water and chow (Purina, St Louis, MO) available ad libitum. Midpoint of a 2-h cohabitation resulting in a vaginal plug was considered as the beginning of gestation day (GD) zero. At the end of GD 13, pregnant females were sacrificed and the embryos collected. Our preliminary experiments indicated that the proliferation and protein phosphorylation pattern of PM cells was similar in both GD 12 and 13 embryos in response to SAD. Since PM cell proliferation is continuing on GD 13 in vivo and since a significant amount of SAD is still present in GD 13 palates (ElDeib and Reddy, 1988b), the results obtained from GD 13 embryonic PM cells are likely to be relevant to SAD-induced CP and applicable to GD 12 PM cells.
Cell culture and treatment.
Palatal mesenchymal cell cultures were established as described by Pisano and Greene (1999). Briefly, fetal secondary palatal shelves were dissected and collected in ice-cold, calcium/magnesium-free, phosphate-buffered saline (CMF-PBS). The palates were then finely minced and the mesenchymal cells were dissociated by incubation with 0.25% trypsin and 0.1% EDTA for 10 min. Cell number and density were determined and the cells were plated into 35-mm tissue culture dishes at a density of 2.5 x 104 cells/cm2 in O-MEM containing 5% fetal bovine serum, 2 mM glutamine, 55 µM mercaptoethanol, and antibiotic-antimycotic solution (150 U penicillin; 150 µg/ml streptomycin; 0.37 µg/ml amphotercin B). The cultures were grown at 37°C in a humidified atmosphere of 95% air–5% CO2, with media changed every alternate day. The cells were exposed to SAD (final concentration of 1.9, 7.5, 30 and 120 µg/ml) or vehicle (5% NaHCO3) once the cultures attained 
60% confluency (48 h). Following 48 h of incubation, the media were collected, the cell sheet washed with PBS, dissociated by incubating with 0.05% trypsin and 0.5% EDTA, neutralized with the medium, pelleted, and preserved at –80°C for further use.
Estimation of cell number and viability.
The standard counting technique with a hemocytometer, was used to estimate the total cell number and density. Cell viability determination was done by the trypan blue dye-exclusion technique. Briefly, 0.4% solution of trypan blue (to a final concentration of 0.2%) was added to a known volume of aliquot of the harvested cells, and the percent of stained and unstained cells was estimated by counting in a hemocytometer.
Measurement of DNA synthesis.
The control and SAD-treated groups were pulse labeled with [3H]-thymidine (2 µCi/ml) for 2 h before the completion of the 48-h SAD/vehicle exposure period. Thymidine incorporation was measured as trichloroacetic acid (TCA)-precipitable radioactivity as described previously (Hellman et al., 1984). Briefly, the harvested cells were washed twice with ice-cold CMF-PBS, then 3 times with ice-cold 5% TCA and, finally 2 times with absolute ethanol. The contents were then solubilized in 10 mM EDTA, pH 12.3 at 37°C for 20 min and neutralized with 0.77 M KH2PO4 (Welshons et al., 1993). Aliquots were drawn for measurement of radioactivity and estimation of protein content. Concentration of protein was estimated by the method of Bradford (1976). Radioactivity was measured by liquid scintillation counting, and the counts were expressed as DPM per unit of protein.
Analysis of DNA fragmentation.
The method of Viola et al.(2000) was used as described. The harvested cells were washed with PBS, pelleted, and incubated for 1 h at 50°C with lysis buffer containing 10 mM EDTA, 0.5% N-laurylsarcosine, and 0.5 mg/ml proteinase K in 50 mM Tris–HCl (pH, 8). Incubation was continued for 1 more h with RNase added to a concentration of 0.25 mg/ml. DNA was extracted from the lysate with phenol/chloroform twice and with phenol/chloroform/isoamylalcohol twice. The extract was diluted 2.5 times with Tris EDTA (TE) buffer and centrifuged at 13,000 x g for 10 min. The supernatant was collected and DNA precipitated with 0.1 volume of 3 M sodium acetate (pH, 5.5) and 2 volumes of ethanol. The precipitated DNA was dissolved in TE buffer and the concentration determined by absorbance at 260 nm. Sample buffer (1 mM EDTA, 1.49 mM bromophenol blue, 0.1% SDS, 25% glycerol), and 0.1% ethidium bromide were added to equal concentrations of DNA and were electrophoresed on 1.8% agarose gel with Tris-borate EDTA buffer. DNA was visualized under UV light.
Measurement of ECM synthesis.
Incorporation of [3H]-glucosamine into GAGs and selective fractionation techniques (Agren et al., 1994) were used to measure sGAGs and HA synthesis. Briefly, the cells were metabolically labeled with [3H]-glucosamine (3 µCi/ml) for 24 h followed by a 48-h exposure to SAD or vehicle. The culture medium was collected and aliquots of 0.5 ml were proteolysed with 0.5% papain in the presence of 5 mM EDTA and 5 mM cysteine-HCl at 60°C for 6 h. The samples were then boiled and one of the aliquots was further incubated with Streptomyces hyaluronidase (12 U/ml) at 37°C for 6 h. Precipitation of GAGs was accomplished with addition of, and incubation with 2% CPC in 20 mM NaCl for 3 h at room temperature. The precipitate was then collected on a 0.45 µm mixed cellulose ester filtration membrane (Millipore, Bedford, MA) by repeated washings with 1% CPC until no more radioactivity was detected in the filtrate. Radioactivity on the membrane containing the precipitate from papain digestion alone, representing the labeled total GAGs, and that on the membrane from the samples digested with papain and hyaluronidase, representing the labeled total GAGs less HA, were counted by liquid scintillation. The amount of labeled HA was calculated by subtracting the radioactivity of hyaluronidase-treated sample from untreated sample and expressed as DPM per million cells.
Western analyses of PCNA and fibronectin.
Extracts from the cellular portion were prepared by using a buffer containing 0.1 mM EDTA and 1% triton X-100 with protease inhibitors in 30 mM Tris-HCl (pH, 7.4). Equal quantities of either the media (100 µg protein) or the cellular fractions (30 µg protein) for analysis of fibronectin or 10 µg of cellular fraction for analysis of PCNA were separately electrophoresed on an 8% (fibronectin) or 12% (PCNA) polyacrylamide gel with SDS and then transferred onto nitrocellulose membrane. The membranes were subsequently blocked with 5% nonfat dry milk, washed with PBS-Tween, probed with antifibronectin (1:500) or anti-PCNA (1:1000) antibodies, and reacted with horse radish peroxidase-conjugated antimouse or antirabbit antibodies (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA) for fibronectin and PCNA, respectively. The bands were then visualized using enhanced chemiluminesence (NEN Life Science, Boston, MA).
Statistical analysis.
Data from at least 3 replicates for each parameter evaluated were analyzed for significance by one-way ANOVA using Dunnetts or Bonferroni's test at a p value of 
0.05.
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RESULTS |
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Secalonic acid D reduces palatal mesenchymal cell proliferation.
Mesenchymal cell number per well was significantly (p < 0.05) reduced at all concentrations of SAD in a dose-dependent fashion (Fig. 1
). Synthesis of DNA as measured by [3H]-thymidine uptake was significantly reduced at all doses of SAD compared with the control (Fig. 2). The expression of PCNA was also reduced in a similar manner (Fig. 3).
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Secalonic acid D-induced cytotoxicity is not mediated through apoptosis. To assess whether reduction in cell number is due to increased cell death, trypan blue dye exclusion analysis was carried out. The percentages of cells taking up the dye, indicative of cell death, increased with increase in concentration of SAD (Fig. 4
). However, the dye inclusion was significant only at the exposure concentrations of 7.5, 30, and 120 µg/ml of SAD, suggesting that the no-effect level was 1.9 µg/ml of SAD. To glean the role of apoptosis in SAD-induced cell death, DNA fragmentation analysis of cultured cells was carried out. No ladder formation, indicative of fragmented DNA, was evident in this analysis except for a very weak indication of fragmented DNA at the highest exposure dose of 120 µg/ml of SAD (Fig. 5).
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Secalonic acid D does not affect ECM synthesis.
Three major components of palatal ECM; sGAGs, HA, and fibronectin were quantified as a measure of ECM synthesis by cultured palatal mesenchymal cells. Metabolic labeling of PM cells with a precursor for glycosaminoglycans, [3H]-glucosamine, followed by selective fractionation, did not indicate any significant reduction in synthesis of either sGAGs (Fig. 6
) or HA (Fig. 7) except at the highest dose of 120 µg/ml of SAD, where a decrease in labeled sGAGs was observed (Fig. 6). Exposure to SAD at the 30 µg/ml level actually caused slight but significant increase in sGAG (Fig 6). Western blot analysis of the extracellular fluid (medium) and cellular extract did not indicate alterations in the quantity of fibronectin (Fig. 8.) in either of these components.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Based on the fact that the palatal cleft resulting from exposure to SAD is a consequence of smaller palatal shelves (ElDeib and Reddy, 1988a
), the objectives of the investigation in the present study were narrowed down to the 3 possibilities: reduced rate of proliferation, synthesis of ECM components, and increased apoptosis of palatal mesenchymal cells.
The fact that SAD reduced the mesenchymal cell number at all the concentrations tested, whereas cell death was induced only at higher doses, suggests that, at lower doses, the reduction in cell number by SAD is likely accounted for by reduced proliferation of mesenchymal cells. Data from [3H]-thymidine uptake analysis clearly supported this contention. Secalonic acid D affected proliferation of mesenchymal cells in vitro at as low as 1.9 µg/ml. The minimal inhibitory concentration of SAD is likely to be even lower. These concentrations are lower than those expected in vivo (30 µg/ml) if one assumes that the approximate median teratogenic dose (30 mg of SAD/kg body weight; Reddy et al, 1981) given to the dam distributes evenly and instantaneously. This suggests the relevance of the current findings to teratogenic effects of SAD in vivo. The cytostatic activity of SAD in cultured mouse leukemia L1210 cells and its antitumor activity in fibrosarcoma cells have been reported (Kurobane et al., 1987; Shimizu et al., 1983). The ability of SAD to inhibit cell proliferation has been suggested, by these authors, as a possible mechanism underlying these effects.
The reduced mesenchymal cell number at higher doses of SAD is likely due to a combination of reduced proliferation as well as increased cell death. Lack of SAD-induced changes in DNA analysis strongly suggests that SAD-induced cell death likely results from nonapoptotic mechanisms. Although, the exact mechanism of SAD-induced cytotoxicity is not known, the reported uncoupling of oxidative phosphorylation in mitochondria by SAD (Kawai et al., 1983) can explain this effect fully or partly.
Many agents capable of disrupting ECM synthesis and/or breakdown are capable of inducing CP in conjunction with smaller shelves that fail to elevate (Larsson and Bostrom, 1965; Pratt et al., 1973) clearly suggesting an important role for ECM molecules in palatogenesis. Lack of effects of SAD on the synthesis of any of the ECM components studied, even at concentrations capable of inducing cell death, however, suggests the lack of relevance of this mechanism to SAD-induced CP.
Taken together, the results indicate that reduction of shelf size and thus induction of CP by SAD is likely due to SAD-induced inhibition of mesenchymal proliferation and, to a lesser extent, nonapoptotic cell death but not due to altered synthesis of ECM or increased apoptosis. Placental transfer and the presence of SAD in the face and head (up to 0.25% of the total dose 24 h following dosing) of embryos from SAD-exposed mothers (ElDeib and Reddy, 1988b) suggests the possibility of a direct (as well as indirect via its effect on growth factors, etc.) effect of SAD and/or its metabolite(s) on the palatal mesenchyme.
The presence of many of the isozymes of protein kinase C (PKC) in the palate (Balasubramanian et al., 2000a) and the ability of epidermal growth factor (EGF) to activate PKC in murine embryonic palatal cells (Chepenik and Haystead, 1989) are well established. PKC is required for cell proliferation and differentiation (Musashi et al., 2000) and inhibition of activity of PKC (Murray et al., 1993) and suppression of activator protein-1 (Sugiura et al., 2000) can lead to inhibition of cell proliferation. A role for alterations in PKC signaling in SAD-induced inhibition of cell proliferation is suggested by SAD-induced disruption in the ontogeny and signaling of EGF (Reddy et al., 1998), by SAD-induced inhibition of the activity of PKC (Balasubramanian and Reddy, 2000) and by SAD-induced inhibition of the binding of AP-1 to its response element (Balasubramanian et al., 2000b) in the developing murine palate.
Secalonic acid D is also known to inhibit adenylate cyclase activity (Reddy et al., 1994), to alter cAMP levels (ElDeib and Reddy, 1988a), to inhibit protein kinase A (PKA) (unpublished) and to inhibit binding of the cAMP-responsive transcription factor CREB to cAMP response element (CRE; Umesh et al., 2000) in the developing murine palate. CREB and cAMP signaling also regulate cell proliferation (Della Fazia et al., 1997; Uyttersprot et al., 1999). Inhibition of this pathway is shown to be antiproliferative (Kruger et al., 1997; Sugiura et al., 2000). Many of the essential components required for cell proliferation such as cyclin A, a critical factor for DNA replication (Girard et al., 1991) and PCNA, an obligate processivity factor for DNA polymerase (Mathews et al., 1984; Lee et al., 1991) are responsive to cAMP signaling (Yoshizumi et al., 1997).
Proliferating cell nuclear antigen is synthesized during the late G1-early S phase of the cell cycle. It is most abundant during the S phase (preceding DNA synthesis) and rapidly declines during the G2/M phase (Kurki et al., 1986). With both CRE (Feuerstein et al., 1995) and AP-1 responsive (Liu et al., 1998) elements reported to be present in PCNA promoter, the effect of SAD on the expression of PCNA is likely a reflection of its effects on cAMP and PKC signaling pathways. The results of this study are consistent with the hypothesis that the inhibition of proliferation of mesenchymal cells, as indicated by reduction in expression of PCNA in vitro and in vivo (Umesh et al., 2000), is responsible for the smaller palatal shelves and thus CP in SAD-treated offspring. Further studies are required to identify the involvement of specific genes regulated by EGF-PKC-AP-1 and/or cAMP-PKA-CREB pathway(s) in the proliferative function of palatal mesenchymal cells and the effects of SAD on their function.
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NOTES |
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1 To whom correspondence should be addressed. Fax: (573) 884-6890. Email: reddyc{at}missouri.edu.
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