Chem. Res. Toxicol. 2005, 18, 825-833
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Beauvericin Activates Ca2+-Activated Cl- Currents and Induces Cell Deaths in Xenopus Oocytes via Influx of Extracellular Ca2+ Chih-Yung Tang,† Yi-Wen Chen,† Guey-Mei Jow,‡ Cheng-Jen Chou,§ and Chung-Jiuan Jeng*,‡ Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan; School of Medicine, Fu Jen Catholic University, Hsin-Chuang, Taipei County, Taiwan; and National Research Institute of Chinese Medicine, Taipei, Taiwan Received September 24, 2004
Beauvericin is a mycotoxin that infects a wide variety of cereal grains. The toxicological importance of beauvericin is implicated by its cytotoxicity in animal and human cells, which has been suggested to result from an increase in intracellular Ca2+ concentration ([Ca2+]i). Despite the fact that beauvericin may activate extracellular Ca2+ influx, beauvericin-induced cell deaths has been suggested to be exclusively due to Ca2+ release from internal Ca2+ stores. We endeavored to elucidate the mechanism of beauvericin-induced [Ca2+]i increase by studying the effects of beauvericin in Xenopus oocytes. By applying a -140-mV prepulse prior to a series of test pulses, we found that beauvericin induced small inward currents at -140 mV, followed by outwardly rectifying currents that displayed an apparent reversal potential close to the expected equilibrium potential of Cl-. Both the inward and outward currents induced by beauvericin were blocked by niflumic acid, a specific blocker for Ca2+-activated Cl- currents (ICl,Ca). Removal of extracellular Ca2+, as well as perfusion of lanthanide, abrogated beauvericininduced currents. Beauvericin also displayed prominent cytotoxic effects in Xenopus oocytes in a dose-dependent manner. In the absence of extracellular Ca2+, cytotoxicity-induced by 10 and 30 µM, but not 50 µM, of beauvericin was significantly diminished. Our results are consistent with the idea that beauvericin induces extracellular Ca2+ influx, which in turn activates ICl,Ca and contributes to beauvericin-induced cell deaths in Xenopus oocytes.
Introduction Beauvericin is a depsipeptide that was first identified in a soil-borne entomopathogenic (insect-pathogenic) fungus Beauveria bassiana, which was recognized as the causative agent for heavy losses of the European sericulture in the 16th and 17th centuries (1, 2). Nowadays, beauvericin is considered to be a putative mycotoxin (toxic fungal metabolite) that may affect human and animal health, since it is also produced by many species of the fungus Fusarium that infect important cereal grains such as corn, rice, and wheat (3-7). The potential mycotoxic role of beauvericin is exemplified by results from in vitro studies using cell lines. For instance, beauvericin induces significant cell deaths in insect, murine, and human tumor cell lines (8-14). Beauvericin is a potent and specific cholesterol acyltransferase inhibitor in rat liver microsomes (15). Whether this enzyme inhibition property may result in cytotoxic effects, however, has yet to be verified. In mammalian cell lines, cell deaths caused by beauvericin have been suggested to involve a Ca2+-dependent pathway, in which beauvericin induces a significant increase in intracellular Ca2+ concentration ([Ca2+]i)1 that leads to a combination of cellular apoptotic and necrotic responses (10, 12). * To whom correspondence should be addressed. Phone: +886-22905-2088; fax: +886-2-2905-2096; e-mail:
[email protected]. † National Taiwan University. ‡ Fu Jen Catholic University. § National Research Institute of Chinese Medicine.
The mechanism of beauvericin-induced [Ca2+]i increase, however, remains inconclusive. For instance, what is the source of Ca2+ fluxes leading to a [Ca2+]i increase? Beauvericin-induced apoptotic changes such as DNA fragmentation have been demonstrated to take place in the complete absence of extracellular Ca2+ (12), suggesting that beauvericin triggers release of Ca2+ from internal Ca2+ stores. In fact, beauvericin has since been regarded as an apoptotic agent that releases Ca2+ exclusively from endoplasmic reticulum (11). On the other hand, on the basis of its chemical structure (see Figure 6A, left panel), beauvericin has long been considered a potential ionophore permeable to or capable of transporting cations such as K+, Na+, and Ca2+ (16-19), raising the possibility that beauvericin may confer a pathway to extracellular Ca2+ influx via plasma membrane. Since the majority of the evidence for ionophoric properties of beauvericin is based on reconstitution studies in liposomes or lipid bilayers, a direct link between induction of extracellular Ca2+ influx and the cytotoxic effect of beauvericin has not yet been demonstrated. In this report, we investigate the mechanism underlying beauvericin-induced [Ca2+]i increase by studying the effects of beauvericin in Xenopus oocytes. Our results indicate that application of beauvericin induces a sig1 Abbreviations: [Ca2+] , intracellular Ca2+ concentration; I i Cl,Ca, Ca2+-activated Cl- currents; I-V, current-voltage.
10.1021/tx049733d CCC: $30.25 © 2005 American Chemical Society Published on Web 04/14/2005
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nificant amount of extracellular Ca2+ influx that manifests the activation of Ca2+-activated Cl- currents (ICl,Ca). We also provide evidence suggesting that extracellular Ca2+ influx contributes to the cytotoxic effects of beauvericin in Xenopus oocytes.
Materials and Methods Preparation of Xenopus Oocytes. Adult female Xenopus laevis (African Xenopus Facility, Knysna, South Africa) were anesthetized by immersion in Tricaine (1.5 g/liter). Ovarian follicles were removed from Xenopus frogs, cut into small pieces, and incubated in ND96 solution [(in mM) 96 NaCl, 2 KCl, 1.8 MgCl2, 1.8 CaCl2, and 5 HEPES, pH 7.2]. To remove follicular membrane, Xenopus oocytes were incubated in Ca2+-free ND96 containing collagenase (2 mg/mL) on an orbital shaker (∼200 rpm) for about 60-90 min at room temperature. After several washes with Ca2+- and collagenase-free ND96, oocytes were transferred to ND96. Stage V-VI Xenopus oocytes were then selected and stored at 16 °C in ND96 supplemented with 50 mg/mL gentamycin. Electrophysiology and Data Analysis. For functional studies, Xenopus oocytes were transferred to a recording bath containing normal Ringer solution of the following composition (in mM): 115 NaCl, 3 KCl, 1.8 CaCl2, and 10 HEPES (pH 7.2 with NaOH). The bath volume was about 200 µL. For Ca2+-free bath solution, BaCl2 was added to normal Ringer solution to replace CaCl2. Cl--free bath solution consisted of (in mM) 115 Na-Aspartate, 3 K-Aspartate, 1.8 Ca(OH)2, and 10 HEPES (pH 7.2 with NaOH). For low Cl- bath solution, 60 mM NaCl was used to replace part of Na-Aspartate in the Cl--free bath solution. Borosilicate electrodes (0.1-1 MΩ) used in voltage recording and current injecting were filled with 3 M KCl. Ionic currents through oocytes were acquired using the conventional two-electrode voltage clamp technique with an OC-725C oocyte clamp (Warner, Hamden, CT). Data were filtered at 1 kHz (OC725C oocyte clamp) and digitized at 100 µs per point using the Digidata 1332A/pCLAMP 8.0 data acquisition system (Axon Instruments, Foster City, CA). The holding potential was set at -35 mV, and test pulses of different durations and amplitudes were applied as specified in the result section. All recordings were performed at room temperature (20-22 °C). Data analyses were performed via built-in analytical functions of the pCLAMP 8.0 software. Peak current amplitudes measured at each test voltage were plotted against voltage (current-voltage curve, I-V curve). Apparent reversal potentials (Vrev) of recorded currents were then determined from I-V curves. All values were presented as mean ( SEM. Pharmacological Agents. Beauvericin and bassiatin were purified from the insect-body portion of fungus Codyceps cicadae as previously reported (20). Purified beauvericin and bassiatin were dissolved in dimethyl sulfoxide (DMSO) at the concentration of 12.5 mM and 30 mM, respectively, as the stock solution. Stock solution was stored at -20 °C until being diluted to the desired concentration on the day of experiment. Final DMSO concentration was always less than 0.5%. All other chemicals were purchased from Sigma (Sigma-Aldrich, St. Louis, MO). Assessment of the Viability of Xenopus Oocytes. To evaluate the cytotoxicity of beauvericin and bassiatin, Xenopus oocytes were incubated at room temperature in 10-mL solution in glass scintillation vials (30 oocytes/vial) covered with aluminum foil. After a specified duration of incubation, the appearances of Xenopus oocytes were visually inspected and were classified as healthy, spotty, or dead state according to the standard depicted in Figure 7 On the basis of the normalization of data collected from three to five repetitions of experiments, oocyte viability in each type of solution was quantitatively expressed as the relative percentage of each of the three states of appearance. All values were presented as mean ( SEM.
Tang et al.
Results Beauvericin Activates Endogenous Cl- Currents in Xenopus Oocytes. Using the conventional twoelectrode voltage clamp system, we studied the effects of beauvericin on endogenous currents in Xenopus oocytes. From a holding potential of -35 mV, we applied 1-s prepulses ranging from -140 to +60 mV, followed by a 1 s, +60 mV test pulse (Figure 1A, top panel). During the depolarizing and hyperpolarizing prepulses, beauvericin displayed no significant effects on Xenopus oocytes, although upon hyperpolarization to -140 mV, a small inward current was observed in the presence of beauvericin (Figure 1A, session a). During the +60 mV test pulse following prepulses, significant outward currents, however, were observed in Xenopus oocytes perfused with beauvericin (Figure 1A, session b). The development of beauvericin-induced outward currents at +60 mV was clearly associated with prior hyperpolarizing prepulses, since the amplitude of outward currents increased as prepulses became more hyperpolarized (Figure 1A, bottom panel). Significant outward currents began to appear as the prepulse potential became more negative to -80 mV, and the amplitude of the outward current increased by about 2-fold as the prepulse potential shifted from -80 to -140 mV. The presence of outward currents was not due to DMSO, in which stock beauvericin was prepared, as no significant outward currents were observed in Xenopus oocytes perfused with up to 1% DMSO in the Ringer solution (data not shown). To further understand the nature of beauvericininduced outward currents, we applied 1 s, -140 mV prepulses prior to a series of 1-s test pulses ranging from -140 to +60 mV (Figure 1B, top panel). In the presence of beauvericin, depolarizing test pulses activated prominent outward currents, while small or no inward currents were elicited by hyperpolarizing test pulses (Figure 1B, third panel). The plot of peak current amplitudes versus test pulses also demonstrated that, following a -140 mV prepulse, beauvericin-induced currents showed an outwardly rectifying I-V relationship with an apparent reversal potential of about -30.2 ( 7.5 mV (n ) 12) (Figure 1B, bottom panel). The expected equilibrium potential of Cl- for Xenopus oocytes incubated in Ringer solution is -25 mV (21). When we lowered external Clconcentration to 60 mM, the apparent reversal potential of beauvericin-induced outwardly rectifying currents shifted to about 4.8 ( 3.6 mV (n ) 5), which is close to the calculated reversal potential of 0 mV, on the basis of the estimation that the intracellular Cl- concentration in oocytes is about 62 mM (21). Furthermore, beauvericin-induced currents were absent in Cl--free (aspartatebased) solution (data not shown). These observations are consistent with the idea that the beauvericin-induced outwardly rectifying currents may involve Cl- as charge carriers. The amplitudes of beauvericin-induced outward currents following a -140 mV prepulse varied among different Xenopus oocytes. For example, in the presence of 10 µM beauvericin, the peak current amplitudes at +40 mV ranged from less than 1 µA to over 10 µA, with a mean current amplitude of about 3.0 µA (Figure 1C). Increasing the concentration of beauvericin up to 50 µM, however, did not significantly increase the peak current amplitudes, while decreasing the concentration down to 1 µM did reduce the average current amplitude down to
Beauvericin Induces Extracellular Ca2+ Influx
Figure 1. Effects of beauvericin on endogenous currents in Xenopus oocytes. (A) Xenopus oocytes were initially incubated in normal Ringer solution (control), followed by a perfusion of 10 µM beauvericin in Ringer solution. The holding potential was -35 mV. The voltage clamp protocol comprised 1-s prepulses ranging from -140 to +60 mV (top panel, session a), followed by a 1 s +60 mV test pulse (top panel, session b). In the presence of beauvericin, the -140 mV prepulse induced a small inward current (third panel, session a), and the ensuing +60 mV test pulses activated significant outward currents (third panel, session b). Bottom panel: peak amplitudes of outward currents at +60 mV in the absence (open circle) or presence (filled circle) of 10 µM beauvericin were plotted against corresponding prepulse potentials that preceded the +60 mV test pulse. (B) The voltage clamp protocol comprised 1-s prepulses to -140 mV, followed by 1-s test pulses ranging from -140 to +60 mV. In the presence of beauvericin, significant outward but not inward currents were observed in response to test pulses. Bottom panel: peak current amplitudes were plotted against corresponding test pulse potentials in the absence (open circle) or presence (filled circle) of 10 µM beauvericin. (C) Mean peak outward current amplitudes at +40 mV (following a 1 s, -140 mV prepulse) measured from Xenopus oocytes 10-20 min after the perfusion of indicated concentrations of beauvericin. The numbers in parentheses refer to the total number of oocytes tested. The mean amplitude for 0.5, 1, 3, 5, 10, 30, and 50 µM beauvericin, as well as normal Ringer (control) solution was (mean ( SEM) 0.6 ( 0.2, 1.3 ( 0.2, 1.6 ( 0.6, 2.1 ( 0.4, 3.0 ( 0.5, 2.6 ( 0.6, 2.9 ( 0.6, and 0.5 ( 0.1 µA, respectively. The mean current amplitude in the presence of 1, 3, 5, 10, 30, or 50 µM beauvericin was significantly larger than that for the control solution (Student’s t-test, p < 0.01).
about 1.3 µA (Figure 1C), suggesting that the current induction effect saturated at about 10 µM beauvericin. In comparison, for Xenopus oocytes perfused with control solution, the mean current amplitudes at +40 mV were
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about 0.5 µA (Figure 1C). The kinetics of outward currents also exhibited considerable variations among different Xenopus oocytes. For outward currents at +40 mV, the rising time constant was about 18-52 ms, and the decay time constant was about 360-580 ms. Upon perfusion of beauvericin, the outward currents at +40 mV usually became visible within 5 min and reached their maximal amplitudes about 10-15 min after drug wash-in. The time course of the development of beauvericin-induced currents appeared accelerated as the concentration of beauvericin increased. Beauvericininduced currents were sustained for the length of recording, which typically lasted for 30 min or longer. Beauvericin-induced currents remained virtually the same after washing out beauvericin with normal Ringer solution, suggesting that beauvericin effects in Xenopus oocytes were irreversible or minimally reversible. As mentioned above, in the presence of beauvericin, small inward currents were observed upon hyperpolarization to -140 mV (Figure 1). Given that the -140 mV prepulse was required for the activation of the outwardly rectifying currents, the next question to ask was whether and how the initial inward currents were related to the ensuing outwardly rectifying currents. To address this issue, we varied the pulse duration of the -140 mV prepulse, which was followed by a depolarizing test pulse to +40 mV. As shown in Figure 2, the maximal outward current was recorded when the depolarizing test pulse was applied during the peak of the inward current. Minimal outward currents were observed if the +40 mV test pulse was applied either prior to the appearance of or after the termination of the inward current. The peak amplitudes of inward currents at -140 mV were usually at least 4-fold lower than those for the ensuing outward currents at +40 mV, which may explain why inward currents were not always observed. In addition, the inward and outward currents shared virtually an identical time course in the decay process (Figure 2A), although the exact kinetics as determined by simple exponential fittings varied among different batches of Xenopus oocytes, ranging from about 330 to 650 ms (compare Figures 2-5). These results suggest that the outward currents at +40 mV are equivalent to the tail currents of the preceding inward currents at -140 mV. The foregoing close correlation between the initial hyperpolarization-induced inward current and the subsequent outward current upon depolarization suggests that the two components of beauvericin-induced currents may actually be mediated by the same type of endogenous channel in Xenopus oocytes that displays a strong outward rectification and a reversal potential close to that of a Cl- channel. One candidate is the Ca2+-activated Clcurrent (ICl,Ca) that has been extensively studied in Xenopus oocytes (22, 23). When ICl,Ca in Xenopus oocytes is activated by submaximal [Ca2+]i increase (
