* INSERM UMR-S 461, Faculté de Pharmacie Paris XI, 5 rue J.-B. Clément, 92296 Châtenay-Malabry Cedex, France; and Laboratoire de Toxicologie, Faculté de Pharmacie, Université Saint-Joseph, Beirut, Lebanon
Received December 18, 2003; accepted February 27, 2004
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ABSTRACT |
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Key Words: ochratoxin A; Bcl-xL; mitochondria; lymphocyte; apoptosis.
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INTRODUCTION |
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OTA has been shown to be immunosuppressive in vivo and in vitro (Muller et al., 1995, 1999
; Stormer and Lea, 1995
). Several studies in mice have shown that OTA treatment resulted in depletion of lymphoid cells and suppression of the antibody response (Creppy et al., 1983b
; Muller et al., 1995
). OTA also induced macrophage activation (Boorman et al., 1984
), altered natural killer cell activity (Luster et al., 1987
), and downregulated lymphocyte proliferation of murine and human origins (Prior and Sisodia, 1982
; Stormer and Lea, 1995
; Thuvander et al., 1995
). Concentrations as low as 5 ng OTA/kg body weight have been shown to suppress immune responses in mice (Haubeck et al., 1981
).
Apoptosis is an important process in a wide variety of different biological systems and also in chemical-induced cell death (Cohen, 1997). The immune system is now recognized as a target organ for many xenobiotics such as drugs and chemicals, which are able to trigger unwanted apoptosis or to alter the regulation of programmed cell death. Reducing the number of immune-competent cells after xenobiotic treatment can lead to immunosuppressive effects, resulting in an increased susceptibility to tumors or infectious diseases. Many experimental works have dealt with the influence of xenobiotics on the immune system. Glucocorticoids used as immunosuppressive and anti-inflammatory agents are known to provoke apoptosis of thymocytes and activated T cells (Perrin-Wolff et al., 1995
). Low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affect thymocyte development, and the maturation of CD4+ CD8+ double-positive cells is skewed toward CD8 single-positive cells (Nohara et al., 2000
). Fungal toxins have been also reported to have immunotoxic effects (Bondy and Pestka, 2000
). The trichothecenes, notably T-2 and HT-2 toxins, provoke a rapid and strong apoptosis of human lymphoid cells (Holme et al., 2003
; Shifrin and Anderson, 1999
).
OTA immunotoxicity has been explored to different degrees in multiple species, including rodents, poultry, and pigs (Bondy and Pestka, 2000). Among the possible mechanisms leading to immunosuppression, a decrease in the number of lymphocytes due to direct cytotoxicity or to apoptosis has been previously described with xenobiotics. Ochratoxin A has been shown to induce apoptosis in different cellular models. These observations prompted us to examine if the immunosuppressive effects of OTA could be due to lymphocyte apoptosis. Our results show that OTA induces a dose-dependent apoptosis of human peripheral blood lymphocytes through a mitochondrial pathway leading to caspase activation. In addition, we observe that OTA provokes a decrease of Bcl-xL expression that may be a trigger for OTA-induced apoptosis.
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MATERIALS AND METHODS |
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Cell treatment.
OTA (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide (DMSO, Sigma) to make a stock solution of 50 mM, from which final concentrations were prepared. Appropriate concentrations of OTA or DMSO (control) were added to cell suspension and incubated at 37°C in 5% CO2 for indicated times. N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk; 50 µM; Bachem, Weil am Rhein, Germany), dissolved in DMSO, was added 1 h before the addition of OTA.
Measurement of apoptosis.
Cells were incubated for various times with different concentrations of OTA, washed with PBS, and permeabilized with ethanol by incubation overnight at 20°C. The cells were then washed with PBS, treated with RNase (20 µg/ml) and stained with propidium iodide (PI, 50 µg/ml). Apoptosis was determined by the quantification of DNA hypodiploidy using flow cytometry. Data acquisition was performed using Cellquest® software (Becton Dickinson).
Assessment of mitochondrial transmembrane potential (m).
To determine changes in the inner mitochondrial transmembrane potential (m), we used DiOC6(3) (80 nM), a fluorochrome known to incorporate into all cells driven by the
m. PI (5 µg/ml), which enters cells with damaged plasma membrane, was also used to differentiate apoptosis from necrosis. Cells treated with OTA were labeled with DiOC6(3) at 37°C for 10 min, and then PI was added at 4°C. The cells were scored immediately by cytofluorometric analysis (Becton Dickinson). Quadrants were fixed using the untreated control. Early apoptotic cells are in the lower left quadrant, and the late apoptotic cells undergoing secondary necrosis are in the upper left quadrant (Fig. 4A).
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RT-PCR.
Total cellular RNA was isolated from 5 x 106 cells using the Trizol® reagent (Invitrogen, Paisley, UK). The amount of RNA recovered was measured by spectrophotometry. cDNA was synthesized by reverse transcriptase from 2 µg total RNA using 4 µM oligo(dT) primer, 2 µM of dNTP (Amersham, Piscataway, NJ), 20 U of Rnasin (Promega, Madison, WI), and 2 U of AMV reverse transcriptase (Promega) in a total volume of 25 µl.
mRNA expression levels for bcl-xL were determined using PCR. The oligonucleotide primer sequences were as follow: bcl-xL sense, 5'-AGGATACAGCTGGAGTCAGT-3'; bcl-xL antisense, 5'-ACCTGCATCTCCTTGTCTAC-3'. ß-actin cDNA amplification was used as an internal control. The ß-actin sense primer was 5'-GGGTCAGAAGGATTCCTATG-3', and the antisense primer was 5'-GGTCTCAAACATGATCTGGG-3'; 5 µl of the cDNA was used for amplification by PCR. Amplification of cDNAs was performed using 50 pmol primers, 200 µM dNTP, and 1 U Taq DNA polymerase (Qbiogene, Illkirch, France) with reaction buffer. An initial 5-min denaturation was carried out. Cycles of denaturation, annealing and extension were then performed for 30 s at 94°C, 60 s at 60°C, and 60 s at 72°C for bcl-xL, and 30 s at 95°C, 30 s at 55°C, and 30 s at 72°C for ß-actin. PCR reactions were performed at various cycle numbers to ensure that the results obtained were within the linear range of the amplification curve. PCR reactions were then performed at 30 cycles for bcl-xL and 23 cycles for ß-actin. We used the expression ratio (bcl-xL/ß-actin)OTA/(bcl-xL/ß-actin)control as measured by densitometry to evaluate bcl-xL gene expression.
Measurement of bcl-xL mRNA stabilization.
Kit 225 cells were untreated or treated with 5 µM OTA for 14 h before the addition of 1 µg/ml actinomycin D to arrest transcription. The kinetics of bcl-xL mRNA degradation in the control and OTA-treated cells were assessed using RT-PCR analysis.
Statistics.
The data are presented as mean values ± SE. For statistical analysis, the dependence between response (the percentage of subG1 cells, time or doses of OTA) was assessed by linear regressions through values of slope or interactions. Comparisons between slopes were made by parallelism tests between adjusted lines or by interaction assessments. For Figure 7, ANOVA was performed because the data were in a 3 x 2 x 2 factorial design. Statistical difference was achieved if p < 0.05.
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RESULTS |
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OTA Triggers Apoptosis and Mitochondrial Membrane Permeabilization in Kit 225 Cells
To investigate the mechanisms of OTA-induced apoptosis in lymphoid cells, we used a human lymphoid cell line, Kit 225, which is more amenable to transfection than primary cells. We first investigated the apoptotic pathway in this cell line after OTA treatment. Figure 5A shows the dose-response for OTA-induced apoptosis in Kit 225 cells as measured by subG1 cells quantification. The percentage of subG1 cells increased with the time of exposure (24 and 48 h) and the concentration of OTA (5 and 10 µM). To confirm that induction of apoptosis in this model involved the mitochondria, cells were simultaneously stained with DiOC6(3) and PI. OTA induced an early drop in m that increased with time (Fig. 5B). Caspase-9 and caspase-3 were also activated in this cell line by OTA (data not shown). These results indicate that, as is the case for PBMC, OTA-induced cell death in Kit 225 cells implicates mitochondrial perturbations, thus validating this cell line for mechanistic evaluation.
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However, in Kit 225-bcl-xL clones although a significant protection from OTA-induced apoptosis was present, we still observed apoptosis, suggesting that overexpression of Bcl-xL did not fully protect the cells over time. When we evaluated the protein level of exogenous Bcl-xL (Bcl-xL-Myc) in OTA-treated cells, our results showed a significant decrease, demonstrating that Bcl-xL-Myc was also lost over time (Fig. 7C).
OTA Treatment Did Not Affect bcl-xL Gene Transcription
As a first step to unveil the cause of Bcl-xL decrease, we asked whether Bcl-xL was cleaved by caspases, as previously described in other models (Clem et al., 1998). The failure of z-VAD.fmk to prevent endogenous Bcl-xL decrease in Kit 225 cells treated with OTA (Fig. 8A) implies that Bcl-xL is probably not a substrate for caspases in these cells.
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OTA Induces Bcl-xL Decrease in PBMC
To further corroborate the role of Bcl-xL in OTA-induced apoptosis, we investigated whether the loss of Bcl-xL was confined to Kit 225 cells or whether it was also found in PBMC. As observed in Kit 225 cells, we noted a significant decrease in the Bcl-xL protein level at different concentrations such as 5 µM OTA at 24 h, whereas Bcl-2 expression was not modified, confirming the observation in a relevant model (Fig. 9).
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DISCUSSION |
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To elucidate the mechanism by which OTA triggers apoptosis, we used two types of cells: PBMC and Kit 225 cells. These cells undergo apoptosis in a time- and dose-dependent manner. In addition, low doses of OTA such as 0.5 µM triggered apoptosis in PBMC after 4 days of incubation with OTA, suggesting that chronic exposure to low doses of OTA may expose humans to inadvertent immunosuppression. Seegers et al. have also reported that OTA induced DNA degradation associated with apoptosis, but this observation was made in phytohemagglutinin-stimulated human lymphocytes (Seegers et al., 1994).
In our models, OTA was shown to induce apoptosis via the activation of caspase-9 and caspase-3. Other examples of xenobiotics capable of activating the caspase pathway similarly to OTA have been described previously. For instance, acetaminophen was shown to induce a caspase-dependent apoptosis in PBMC by triggering the activation of caspase-3, 9, and 8 independently of Fas with mitochondria as a primary target (Boulares et al., 2002). Silica can also initiate caspase-9 and caspase-3 activation in a macrophage cell line by destabilizing mitochondrial integrity (Thibodeau et al., 2003
).
Mitochondria are organelles that play a central role in the apoptotic process. In fact, several pro-apoptotic signals target the mitochondria and culminate in MMP (Green and Reed, 1998; Kroemer and Reed, 2000
), leading to the release of cytochrome c, formation of the apoptosome, and activation of caspase-9 and consequently caspase-3 (Li et al., 1997
).
m loss was found in cells incubated with OTA, implying that mitochondria play a role in the mechanism of OTA-induced apoptosis. These data suggested that
m loss after OTA treatment preceded the caspase activation and subsequently was responsible for the induction of apoptosis. Moreover, the
m loss triggered by OTA was not inhibited by z-VAD treatment (data not shown), suggesting that caspases are not implicated in the onset of
m loss in opposition with what was reported in other models (Finucane et al., 1999
; Ricci et al., 2003
).
Bcl-2 and Bcl-xL are anti-apoptotic proteins that have been first described to potently inhibit dexamethasone or -irradiation-induced apoptosis and also cytokine deprivation-induced apoptosis (for a review, see Kroemer et al., 1998
). It has been suggested that both proteins inhibit apoptosis at the level of mitochondrial function by preventing cytochrome c efflux to the cytosol, thus inhibiting caspase-9 activation (Hu et al., 1998
; Vander Heiden et al., 1997
). Since in previous reports a decline in the expression of Bcl-2 and Bcl-xL was thought to be responsible for the onset of apoptosis, we investigated the possible role of the anti-apoptotic proteins, Bcl-2 and Bcl-xL, in OTA-induced apoptosis. Herein, OTA triggers the decrease of Bcl-xL protein, while Bcl-2 level was not affected. When Bcl-xL was overexpressed in Kit 225 cells, both OTA-induced apoptosis and
m loss were strongly inhibited. This effect was also found in Kit 225 cells overexpressing Bcl-2 (data not shown). Moreover, pro-apoptotic Bax, largely cytosolic in healthy cells, was found to translocate to mitochondria in Kit 225 cells treated with OTA at 24 and 48 h (data not shown). All these data suggest that OTA induced a perturbation in the equilibrium between pro and anti-apoptotic proteins of the Bcl-2 family.
Bcl-xL has been shown to delay apoptosis induced by various stimuli (Boise et al., 1993; Fang et al., 1994
; Gonzalez-Garcia et al., 1994
; Shiraiwa et al., 1996
) and to play a stronger protective role against apoptosis than Bcl-2 in certain circumstances (Gottschalk et al., 1994
). Bcl-xL has also been reported to control apoptosis mediated by nitric oxide and reactive oxygen species (Shimizu et al., 1995
). In macrophages, Bcl-xL, but not Bcl-2, was able to regulate their susceptibility against nitric oxide toxicity (Lakics et al., 2000
; Okada et al., 1998
). Recent studies have demonstrated that the down-regulation of Bcl-xL by different treatments such as retinoic acid (Fujimura et al., 2003
), honokiol (Yang et al., 2002
) and arsenic trioxide (Hyun Park et al., 2003
) was correlated with apoptosis, while Bcl-2 level remained unchanged.
The rapid loss of Bcl-xL protein after OTA treatment appeared to be unrelated to transcription inhibition, since bcl-xL mRNA in Kit 225 cells was not downregulated over time compared to untreated cells. Furthermore, OTA had no effect on bcl-xL mRNA stabilization compared to control cells. These results would suggest that OTA could activate a proteolytic mechanism leading to Bcl-xL protein decrease. Indeed, in Kit 225 cells overexpressing exogenous Bcl-xL under the control of the CMV promoter, we have also shown a decrease in exogenous Myc-tagged Bcl-xL likely due to proteolytic activity induced by OTA treatment. The failure of z-VAD.fmk to prevent endogenous Bcl-xL breakdown in Kit 225 cells also suggested that Bcl-xL was not cleaved by caspases in our model and that other proteases may be involved in the proteolysis of Bcl-xL. Recent studies, however, pointed out that noncaspase proteases such as cathepsins, calpains, and the proteasome complex can contribute to cell death and also cleave death-related substrates including caspases (Egger et al., 2003; Jaattela and Tschopp, 2003
). Bcl-xL decrease has been previously documented upon treatment by xenobiotics such as retinoic acid (Fujimura et al., 2003
). However, in this case, retinoic acid clearly inhibits bcl-xL mRNA expression by suppression of transcription. Other mycotoxins such as T-2 and HT-2 toxins have been shown to induce apoptosis in HL-60 cells due to the activation of both caspase-9 and caspase-8 and consequently caspase-3. However, Bcl-2 and Bcl-xL levels were not altered in these models (Holme et al., 2003
).
OTA has been shown to inhibit both DNA and protein synthesis under certain conditions. High concentrations of OTA (above 100 µM) inhibited phenylalanyl-tRNA synthetase (Baudrimont et al., 1997) and, consequently, general protein synthesis (Creppy et al., 1979
, 1983a
). We do not believe that inhibition of protein synthesis could account for the observed decreased Bcl-xL level after OTA treatment of human lymphocytes. Indeed, we did not observe any changes in the protein level of other proteins such as Bcl-2. Moreover, the preincubation of cells with L-phenylalanine, a competitive inhibitor of OTA for this effect, did not protect cells from OTA-induced apoptosis (data not shown). This is consistent with the results of Bruinink and Sidler who reported the inability of L-phenylalanine to protect neuronal cells from toxicity due to OTA (Bruinink and Sidler, 1997
).
In conclusion, OTA induces apoptosis by disrupting mitochondrial function in human T-lymphocytes. Our work demonstrates for the first time that Bcl-xL loss due to OTA accompanies lymphocyte demise and may be the trigger for the apoptotic process. These data also indicate that OTA-induced apoptosis is dose- and time-dependent, and low concentrations of OTA provoke m loss and cell death after a 4-day exposure. These observations have to be integrated with the fact that OTA has a long elimination half-life (Studer-Rohr et al., 2000
), and exposure to low doses upon a long period may lead to immunotoxic effects. Additional research is needed to elucidate the mechanism(s) responsible for OTA-induced apoptosis leading to Bcl-xL decrease.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at INSERM UMR-S 461, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry, France. Fax: 0033 1 46835496. E-mail: marc.pallardy{at}cep.u-psud.fr
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