ToxSci Advance Access originally published online on September 7, 2006
Toxicological Sciences 2006 94(2):342-350; doi:10.1093/toxsci/kfl101
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The Effects of Anticholinergic Insecticides on Human Mesenchymal Stem Cells
* Biomedical Tissue Research Group, Department of Biology, University of York, Heslington, York, YO10 5YW, United Kingdom
Department Oral Biology and Department of Psychiatry, Albert Szent-Györgyi Center for Medical and Pharmaceutical Sciences, Faculty of Medicine, University of Szeged, Szeged, Hungary, 3000CA
1 To whom correspondence should be addressed. Fax: +44 (0) 1904-328659. E-mail: pg5{at}york.ac.uk.
Received May 28, 2006; accepted August 28, 2006
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ABSTRACT |
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Mesenchymal stem cells (MSCs) are located primarily in the bone marrow and are characterized by their capacity to differentiate into mesenchymal lineages such as bone, fat, and cartilage in response to appropriate signals. Several signaling mechanisms act to control MSC survival, proliferation, and differentiation, and failure or disruption of these signaling pathways can lead to degenerative disease or neoplasia. Organophosphate (OP) and carbamate pesticides, which are used in large amounts in agriculture to control insects, are designed to disrupt acetylcholine signaling by inhibiting the enzyme acetylcholinesterase (AChE). Effects of OP and carbamate pesticides on the human central nervous system have been well documented. However, AChE is broadly distributed, and the effects of anticholinergic insecticides on nonnervous tissue have received little attention. In the present study we found that human MSCs express AChE, which makes these cells potential targets for AChE inhibiting agents. We therefore examined the effects of an OP pesticide, chlorpyrifos, and a carbamate, carbofuran, on MSC characteristics. It was found that micromolar concentrations of these anticholinergic insecticides had no effect on MSC survival or proliferation but limited MSC differentiation capacity by inhibiting osteogenic differentiation. These results demonstrate that exposure to micromolar concentrations of OP and carbamate pesticides may affect tissue turnover and pathophysiology by interfering with MSC regulation.
Key Words: acetylcholine; acetylcholinesterase; carbofuran; chlorpyrifos; mesenchymal stem cell; osteogenesis.
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INTRODUCTION |
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Worldwide agriculture relies heavily on the use of pesticides to control insects and other pests. Commonly used pesticides are the organophosphate (OP) and carbamate pesticides. These pesticides are not only used in agriculture but have several applications for household use, such as in insect and lawn sprays and pet shampoos. They are designed to disrupt insects' nervous systems by inhibiting the enzyme acetylcholinesterase (AChE). AChE catalyzes the degradation of acetylcholine (ACh) and is required to terminate cholinergic signaling in neuronal synapses.
The widespread use of OP and carbamate pesticides means we face a continuous risk of acute or chronic exposure to these compounds, which can have several adverse effects. The symptoms of acute exposure range from nausea and diarrhea to paralysis and even death, depending on the exposure dose (O'Malley, 1997
). Chronic effects of OP and carbamate pesticides are less well documented and more difficult to study. Exposure to AChE inhibiting insecticides has been suggested to contribute to impaired visuomotor performance and depression in exposed subjects (Rosenstock et al., 1991; Ruckart et al., 2004; Steenland et al., 2000). However, ACh signaling is not limited to the nervous system and plays important roles in nonnervous tissues (Grando et al., 2003; Wessler et al., 2001). Therefore, environmental AChE inhibitors may have broad-ranging effects on cell and tissue function. Although a clear connection between anticholinergic insecticides and cancer is not established (Gulf War and Health, 2003), there is accumulating evidence of an association between chronic OP pesticide exposure and the incidence of a range of cancers (Clavel et al. 1996; Mills and Yang, 2003; Zahm and Blair, 1992). Furthermore, a suspected link between OP pesticides and reduced bone formation in humans has been reported (Compston et al., 1999). The expression of high levels of AChE in bone-forming osteoblasts and their progenitors would support an effect of AChE inhibitors on these cells (Genever et al., 1999; Grisaru et al., 1999; Inkson et al., 2004).
Mesenchymal stem cells (MSCs) are multipotent cells that are capable of self-renewal and rapid expansion. They reside in the bone marrow and at other sites of the body, such as in adipose tissue (Zuk et al., 2001), the skin (Toma et al., 2001), and hair follicles (Hoogduijn et al., 2006). Following the appropriate stimuli they give rise to various mesenchymal lineages such as osteoblasts, adipocytes, chondrocytes, and myocytes (Jiang et al., 2002; Toma et al., 2001). MSC proliferation and differentiation are controlled by the coordinated action of several signaling molecules, such as Wnts (Boland et al., 2004; De Boer et al., 2004a,b; Etheridge et al., 2004), bone morphogenic proteins (Diefenderfer et al., 2003; Sammons et al., 2004), Notch (De Jong et al., 2004), and probably many others that have not yet been examined in detail.
In the present study, we examined the possibility that human MSCs express AChE and that AChE inhibitors may act on MSCs to affect their proliferation and differentiation characteristics by interfering with ACh signaling. We have investigated the effects of two commonly used anticholinergic insecticides, the OP pesticide chlorpyrifos and the carbamate carbofuran, on the survival, proliferation, and differentiation of human MSCs.
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MATERIALS AND METHODS |
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Cell culture.
Human MSCs were isolated from femoral heads of patients undergoing hip replacement operations following informed consent (Harrogate District Hospital). Bone marrow was given a random number, not identifiable with the donor, and removed from the femoral heads, rinsed with medium, and the obtained cell suspension was transferred to a culture flask containing
-minimal essential medium (-MEM) (Invitrogen, Paisley, United Kingdom), supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen), and 15% fetal bovine serum (FBS) (Invitrogen). After 3–4 days of culture, all nonadherent cells were removed. The adherent cells started rapid proliferation and were subcultured upon confluency using 0.25% trypsin/EDTA solution (Invitrogen). MSCs were used for differentiation assays before reaching passage 5. Culture medium was refreshed twice a week, and the cultures were maintained at 37°C, 5% CO2, and 95% air atmosphere.
Flow cytometry analysis.
Flow cytometry was used to detect the expression of a panel of markers that are associated with MSCs (Stro-1+, CD29+, CD44+, CD73+, CD105+, and CD166+) (Etheridge et al. 2004). Cells were removed from the culture flask by incubation in PBS containing 5mM EDTA and 0.2% BSA (washing buffer) and incubated with 100 times diluted antibodies against CD29, CD73, CD105 (all BD Biosciences Pharmingen, San Diego, CA), or Stro-1 (R&D systems, Abingdom, United Kingdom) in washing buffer for 30 min on ice. The cells were then washed and incubated with a secondary FITC-conjugated antimouse antibody (Sigma-Aldrich, Poole, United Kingdom) for another 30 min on ice in the dark. Antibodies against CD44 and CD166 (BD Biosciences Pharmingen) were conjugated with a fluorescent label and incubated in a 10 times dilution in buffer on ice in the dark for 30 min. The cells were then washed and fluorescence measured with a CyAn flow cytometer (Dako Cytomation, Ely, United Kingdom). Fluorescence was plotted on a logarithmic scale.
Treatment with carbofuran and chlorpyrifos.
Cells were exposed to 0.1, 1, or 10µM carbofuran or chlorpyrifos (Riedel-de Haën, Hanover, Germany) over different time periods. Chlorpyrifos-oxon (Chem Service, West Chester, PA), an analogue of chlorpyrifos that is formed via metabolic conversion of chlorpyrifos by cytochrome P450, was used to compare its inhibition of AChE with that of chlorpyrifos. Stock solutions of the pesticides were made up in methanol and equal amounts of methanol (0.1%) were added to control cultures. For longer exposure times, medium and pesticides were refreshed twice weekly.
Western blot analysis.
Following treatment of confluent MSCs with carbofuran or chlorpyrifos for 48 h, cells were lysed in cell lysis buffer (Cell Signaling, Danvers, MA), and protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, IL,). Denaturing loading buffer was added to the cell lysates, which were then heated to 95°C for 5 min before loading 10 µg of protein per well on a 10% SDS-polyacrylamide gel. Gels were run at 150 V, and proteins were transferred to a nitrocellulose membrane (Amersham Biosciences, Little Chalfont, United Kingdom) at 200 A. The membranes were then blocked in a 4% skimmed milk solution and subsequently probed with a 1:10,000 dilution of a mouse monoclonal antibody against human AChE (Affinity, Nottingham, United Kingdom, cat no. A27320) or a 1:5000 dilution of a mouse monoclonal antibody against GAPDH (ImmunoChemical, Long Beach, CA, cat no. RGM2) for 1 h at room temperature. An additional mouse monoclonal antihuman AChE antibody (Abcam, Cambridge, United Kingdom, cat no. ab23455) was also used, in this case, at a dilution of 1:2000. After washing, the membranes were incubated with horseradish peroxidase–labeled goat antimouse IgG antibodies (1:5000, Sigma-Aldrich) for 1 h at room temperature, washed, incubated with ECL reagent, and exposed to hyperfilm (both Amersham Biosciences).
Measurement of AChE activity.
The effects of the pesticides on AChE activity were measured using an Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit (Molecular Probes, Paisley, United Kingdom) following the manufacturer's instructions. Purified AChE (0.1 U/ml) or MSC extracts were preincubated with 0.1, 1, or 10µM carbofuran, chlorpyrifos, or chlorpyrifos-oxon for 30 min on ice.
AChE activities in MSCs were measured after harvesting the cells in PBS with 0.1% Triton X-100. AChE activities in the cell extracts were determined in the presence of 0.1mM tetraisopropyl pyrophosphoramide (Sigma Chemical St Louis, MO) to inhibit butyrylcholinesterase. We used the radiometric method of Johnson and Russell (Johnson and Russell et al., 1975) to measure AChE activities. The substrate of the reaction was acetylcholine iodide ([acetyl-3H], specific activity 1.48 GBq/mmol, NEN Life Sciences Products, Inc., Boston, MA). Reactions were terminated by the addition of 10µM of the specific AChE inhibitor BW284C51 (Sigma Chemical).
Toxicity assay.
MSCs were seeded in 96-well plates at 3000 cells/cm2 and allowed to grow for 3–21 days. The MTT assay was used to determine viable cell numbers (Mosmann, 1983). Briefly, medium was replaced with 100 µl fresh medium before the assay, and 25 µl of 5 mg/ml MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan) (Sigma-Aldrich) in PBS was added and incubated for 2 h at 37°C. 100 µl lysis buffer (10% SDS and 0.01M HCl in dH2O) was then added, and absorbance was read at 405 nm using a Dynex MRX II plate reader (Dynex Technologies Worthing, United Kingdom).
Proliferation assay.
MSC proliferation was determined by measuring the incorporation of bromodeoxyuridine (BrdU) (Sigma-Aldrich) in DNA of cells in the S-phase of the cell cycle by immunofluorescent staining. MSCs were seeded at approximately 40% confluency and treated for 48 h with pesticide, followed by 4 h incubation with 10µM BrdU. The cells were then washed one time in PBS and fixed in 100% ice-cold methanol for 5 min. After rehydration in PBS, the cells were incubated in 4M HCl two times for 15 min to denature the double-stranded DNA, washed three times in PBS, and incubated for 1 h with a 200 times dilution of a mouse monoclonal anti-BrdU antibody (Bio Cell Consulting, Rheinach, Switzerland), followed by a 1 in 200 dilution of a FITC-conjugated secondary antibody (Sigma-Aldrich). To calculate the percentage of dividing MSCs, BrdU-positive cells were scored from a total of on average 164 cells per experiment. Three experiments using MSCs from three different donors were carried out.
Differentiation assays.
Osteogenic differentiation was induced by culturing 90% confluent MSC cultures for up to 18 days in -MEM supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin, 15% heat-inactivated FBS (60°C, 30 min), 5mM ß-glycerophosphate (Sigma-Aldrich), 50 µg/ml L-ascorbic acid phosphate (Sigma-Aldrich), and 10nM dexamethasone (Sigma-Aldrich).
To induce adipogenic differentiation, 90% confluent MSCs were cultured in -MEM supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin, 15% heat-inactivated FBS, 50 µg/ml L-ascorbic acid phosphate, 10nM dexamethasone, 500µM isobutylmethylxanthine (Sigma-Aldrich), and 60µM indomethacin (Sigma-Aldrich) for 14 days.
Detection of osteogenic differentiation.
Osteogenic differentiation was detected by alkaline phosphatase enzyme histochemistry followed by von Kossa staining to detect mineralization after 18 days of culture. Cells were incubated in 0.1M Tris buffer of pH 9.2 containing 1 mg/ml Fast red (Sigma-Aldrich) and 0.2 mg/ml naphthol AS-MX phosphate (Sigma-Aldrich) to detect alkaline phosphatase activity as pink staining. The cells were then fixed in 4% paraformaldehyde for 5 min, and mineralization was detected by von Kossa staining. Briefly, after two washes with PBS and one with dH20, the cells were incubated in 1% silver nitrate on a light box for 10–60 min. The cells were then incubated for 5 min in 2.5% Na2S2O3, washed three times with dH2O, and images taken.
Quantitative determination of alkaline phosphatase activity was performed by measuring the absorbance of the reaction product from the conversion of paranitrophenol phosphate (PNP) (Sigma-Aldrich) by alkaline phosphatase. Cells were fixed in 95% ethanol for 5min and incubated in a buffer containing 20mM NaHCO3, 3mM MgCl2, and 1 mg/ml PNP, pH 9.5 for 10–30 min. The buffer was then transferred to a 96-well plate, and absorbance was measured at 405 nm using a Dynex MRX II plate reader.
Detection of adipogenic differentiation.
Differentiated adipogenic cells were stained with oil red O to detect lipid. Cells were fixed in 60% isopropanol for 1 min and incubated in 0.3% oil red O (Sigma-Aldrich) solution in 60% isopropanol for 30 min. After three washes in PBS, the cells were imaged.
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RESULTS |
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AChE Expression in MSCs
MSCs isolated from the bone marrow of three different donors were analyzed by western blot analysis for AChE expression. Cultured human MSCs expressed AChE with a molecular weight of 68 kD, corresponding to the size of neuronal AChE (Fig. 1). Expression of AChE was not affected by exposure to 1µM carbofuran or chlorpyrifos for 48 h. Exposure of MSCs to 1 or 0.1µM pesticide for 4 or 7 days did also not affect AChE expression (results not shown).
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Inhibition of AChE Activity by Carbofuran and Chlorpyrifos
To determine the effect of carbofuran and chlorpyrifos on AChE activity, 0.1 U/ml AChE was incubated with 0.1, 1, or 10µM pesticide for 30 min on ice. 0.1µM of the carbamate carbofuran caused an 84% reduction in AChE activity compared to control levels, with 1 and 10µM, causing further inhibitory effects (Fig. 2A). The OP pesticide chlorpyrifos was less potent and reduced AChE activity to 59% of control levels at a 10µM concentration. OP pesticides are metabolized in the body by cytochrome P450 to form more potent compounds (Sams et al., 2000
; Tang et al., 2001). The metabolized product of chlorpyrifos, chlorpyrifos-oxon, reduced AChE activity to 21, 4, and 7% of control levels at 0.1, 1, or 10µM concentrations, respectively. Incubation of crude MSC extracts of two different donors for 30 min with carbofuran and chlorpyrifos-oxon also significantly inhibited AChE activity, albeit with a lower potency than seen with purified AChE (Fig. 2B). The inhibitory effect of chlorpyrifos was not significant compared to controls.
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Toxicity Effects of Carbofuran and Chlorpyrifos on MSCs
The addition of carbofuran and chlorpyrifos to the culture medium did not affect MSC morphology. Even after 4 weeks of exposure to 1µM carbofuran or chlorpyrifos, there were no changes in gross cellular morphology (Fig. 3A). The pesticides furthermore did not affect the survival of MSCs. A MTT assay was carried out in sixfold on MSCs from three different donors to determine viable cell numbers, and there was no significant change in MSC numbers after 3, 7, 10, 14, or 21 days of culture in the presence of 0.1, 1, or 10µM carbofuran or chlorpyrifos compared to untreated MSCs (Fig. 3B).
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Effects of Carbofuran and Chlorpyrifos on Proliferation
Proliferation of MSCs from three donors was measured by determining BrdU incorporation into the DNA of the cells. After 4 h incubation with BrdU, on average, 164 cells per experiment were analyzed, and 7.7% of the MSCs showed positive immunofluorescence staining for BrdU. BrdU incorporation did not change significantly after treatment with 10µM carbofuran or chlorpyrifos for 48 h, with 6.8 and 6.9% of the cells showing positive staining, respectively (Fig. 4).
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Effects of Carbofuran and Chlorpyrifos on the Expression of MSC Cell Surface Markers
MSCs are characterized by the expression of the cell surface markers CD29, CD44, CD73, CD105, CD166, and Stro-1. The expression of each marker was tested on MSCs from at least three different donors, and flow cytometry demonstrated that the expression of CD29, CD44, CD73, CD105, CD166, and Stro-1 was on average 86, 48, 64, 78, 44, and 57%, respectively (Table 1). Exposure of MSCs to 0.1, 1, or 10µM carbofuran or chlorpyrifos for periods of 24 h up to 14 days did not affect the expression of these proteins (results not shown). Differentiation of MSCs would normally be expected to lead to a decrease in the expression of MSC markers. This was demonstrated by 10 days induction of osteogenic differentiation of MSCs, which resulted in a decrease in the expression of CD29, CD44, CD73, CD105, CD166, and Stro-1 to 42, 47, 32, 36, 33, and 37%, respectively (Table 1). The addition of 1µM carbofuran during osteogenic differentiation of MSCs elevated the expression of Stro-1 by 42–79% of the cells, compared to osteogenic differentiation without carbofuran. Chlorpyrifos (1µM) increased Stro-1 expression by 18–55%. The expression of CD29, CD44, CD73, CD105, and CD166 was largely unaffected by the addition of carbofuran or chlorpyrifos during osteogenic differentiation.
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AChE Expression and Activity in MSCs during Osteogenic Differentiation and the Effect of Carbofuran and Chlorpyrifos
AChE protein levels were tested in MSCs from three donors, and there were no changes in AChE expression levels after 3, 7, or 18 days of osteogenic differentiation (Fig. 5A). There was, however, an increase in AChE activity levels during osteogenic differentiation with a peak activity at day 3 (Fig. 5B). Carbofuran and chlorpyrifos (1µM), however, significantly reduced peak AChE activities in MSCs undergoing osteogenic differentiation to 54 and 68% of control levels, respectively (Fig. 5C).
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Effects of carbofuran and chlorpyrifos on osteogenic differentiation of MSCs.
Osteogenic differentiation of MSCs is characterized by an up regulation of alkaline phosphatase and by the deposition of mineral, which can be visualized by von Kossa staining. In the presence of 0.1, 1, and 10µM carbofuran or chlorpyrifos, osteogenic differentiation was markedly inhibited after 18 days, as demonstrated by reduced alkaline phosphatase activity and decreased mineralization (Fig. 6A). Quantitative measurement of alkaline phosphatase activity of MSCs from three donors showed that 0.1, 1, and 10µM concentrations of carbofuran or chlorpyrifos had little effect on alkaline phosphate activity after 3 days of osteogenic differentiation compared to untreated controls (Fig. 6B). After 7 days, this reduction in alkaline phosphatase activity was significant, causing a 160–270% decrease compared to controls. After 14 days of osteogenic differentiation, treatment with 0.1, 1, and 10µM carbofuran and 0.1µM chlorpyrifos induced significant reductions in alkaline phosphatase activity, causing up to 190% decreases in activity.
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The inhibitory effect of treatment with anticholinergic insecticides on osteogenic differentiation of MSCs was reversible. Cells that were pretreated with 1µM carbofuran or chlorpyrifos for 4 weeks before initiating osteogenic differentiation maintained alkaline phosphatase activity relative to untreated MSCs (results not shown).
AChE expression and activity in MSCs during adipogenic differentiation.
Adipogenic differentiation of MSCs did not significantly affect AChE levels in MSCs after 3, 7, 12, and 18 days of differentiation (Fig. 7A). AChE activity was, however, markedly up regulated in MSCs from three different donors after 3 days of adipogenic differentiation and remained elevated up to day 12, after which AChE levels returned to basal levels (Fig. 7B).
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Effects of carbofuran and chlorpyrifos on adipogenic differentiation of MSCs.
Under control conditions, there were a limited number of cells in the MSC cultures that contained lipid-filled vesicles, which were stained with oil red O (Fig. 8A). Adipogenic differentiation of MSCs for 14 days was characterized by an increase in the number and size of lipid-filled vesicles (Fig. 8B). The presence of 0.1, 1, or 10µM concentrations of carbofuran or chlorpyrifos during adipogenic differentiation did not notably change oil red O staining intensity in three MSC cultures examined (Figs. 8C–H).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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AChE is a widely expressed enzyme that is present at high levels on cell surfaces and in the extracellular space (Wessler et al., 1998
). Its main function is to hydrolyze rapidly ACh to terminate cholinergic signaling. In addition to its cholinergic enzyme activity, AChE may have noncholinergic functions and appears, for example, to play a role in cell adhesion (Bigbee and Sharma, 2004). Due to its importance in controlling ACh signaling and its widespread distribution in neural, but also nonneural tissues, it is not surprising that inhibition of AChE by agricultural pesticides leads to significant cellular and organ dysregulation.
We have previously shown that AChE is expressed by osteoblasts (Genever et al., 1999; Inkson et al., 2004), and in the present study, we demonstrated that AChE is present on osteoblast precursor cells, MSCs. This suggests that pesticides that target cholinergic signaling may affect bone development and remodeling via their effects on MSCs. We have examined the effects of a carbamate (carbofuran) and an OP (chlorpyrifos) pesticide on bone marrow–derived MSC phenotype, survival, proliferation, and differentiation. It was demonstrated that exposure to 1µM concentrations of carbofuran or chlorpyrifos did not affect AChE expression levels in MSCs. However, AChE enzymatic activity was significantly reduced by micromolar concentrations of carbofuran. This was especially evident on purified AChE, whereas the inhibition of ACh breakdown was smaller in MSC extracts, probably due to interference of other enzymes. The OP pesticide chlorpyrifos was less potent in inhibiting AChE activity. Many OP pesticides, however, are metabolized by hepatocytes, and also other cell types such as keratinocytes and bone marrow stromal cells (Baron et al., 2001), to form biologically more active structures (Sams et al., 2000), which in many cases enhances their potency several fold (Poet et al., 2003; Tang et al., 2001). Chlorpyrifos is metabolized by cytochrome P450 to the superpotent chlorpyrifos-oxon (Sams et al., 2000). We have recently found that MSCs express cytochrome P450 isoforms 26, 2C9, and 3A4 (Hoogduijn, unpublished data), suggesting that they are capable of converting chlorpyrifos and other OP pesticides to their more potent oxon forms. This idea was supported by data in the present study that showed significant AChE inhibitory effects of chlorpyrifos after 3 days of incubation with MSCs. These findings indicate that care should be taken when predicting OP pesticide toxicity on MSCs, as noneffective concentrations of anticholinergic insecticides become toxic once metabolized by cytochrome P450.
The biological effects of carbofuran and chlorpyrifos on undifferentiated MSCs were limited. There were no consistent effects on MSC morphology or proliferation, and up to 10µM concentrations of pesticide did not induce toxicity in MSCs derived from over 90% of donors. MSCs from an occasional donor, however, showed cell death in response to 10µM concentrations of anticholinergic insecticides, indicating that there may be donor-specific differences in sensitivity to OP and carbamate pesticides.
The most striking effects of carbofuran and chlorpyrifos on MSCs were on osteogenic differentiation. During in vitro osteogenic differentiation of MSCs, AChE levels remained unchanged, but AChE activity increased threefold over the first 3 days. Inhibition of AChE activity by carbofuran and chlorpyrifos lead to an inhibition of osteogenic differentiation, which was demonstrated by reduced alkaline phosphatase activities and deposition of calcified nodules. The variability of alkaline phosphatase activities shown in Figure 6 depends in the first place on differences in potency of MSC cultures from different donors to upregulate the activity of this enzyme during osteogenic differentiation. Furthermore, as differentiation is interrelated with proliferation, the variation in cell number between different cultures after treatment with carbofuran and chlorpyrifos as shown in Figure 3 may be responsible for further variation in alkaline phosphate activities. In addition to a reduction in alkaline phosphatase activity, treatment with carbofuran or chlorpyrifos tended to maintain expression of the MSC marker Stro-1, which is expressed by preosteoblastic cells but normally gradually lost in the osteoblastic maturation process. The enduring expression of Stro-1 in MSCs undergoing osteogenic differentiation in the presence of carbofuran of chlorpyrifos indicates that the cells maintain a more primitive phenotype. These in vitro results would support a suspected association between chronic exposure to OP pesticides and reduced bone formation in agricultural workers (Compston et al., 1999). Interestingly, our results suggest that the effects of OP and carbamate pesticides on osteogenic differentiation are reversible. MSCs that were pretreated with 1µM carbofuran or chlorpyrifos for 4 weeks before osteogenic induction showed no reduction or delay in osteogenic differentiation compared to untreated controls, and this would suggest that these pesticides, potentially by inhibiting AChE, act as a temporary brake for osteogenic differentiation.
The results of the present study demonstrate that exposure of MSCs to relatively low concentrations of anticholinergic insecticides reduce osteogenic differentiation of MSCs. This is potentially mediated via an inhibition of the enzymatic activity of AChE by these pesticides, which would result in increased ACh levels in the extracellular space. Furthermore, other, noncholinergic mechanisms of action of the pesticides have been suggested (Costa, 2005), and these may play an additional role in the biological effects of OP and carbamate pesticides on MSCs. Two messages arise from this research. In the first place, pesticides that target the ACh signaling system may play a role in MSC differentiation, and the role of ACh signaling in MSC regulation warrants further research. Secondly, it is the first time that effects of OP and carbamate pesticides on MSCs have been reported, and these findings should be taken into account when treating and preventing OP and carbamate toxicity.
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SUPPLEMENTARY DATA |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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NOTES |
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2 Present address: Erasmus Medical Center, Transplantation Laboratory Internal Medicine, 3000 Dr Rotterdam, Netherlands.
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ACKNOWLEDGMENTS |
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This work was supported by a European Commission grant (QLK4-CT-2002-02264).
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