Clioquinol-induced increase and decrease in the intracellular Zn2+ level in rat thymocytes.

AIMS: Clioquinol is emerging as a potential therapy for some diseases, such as Alzheimer disease and cancer. This agent is a lipophilic chelator of Zn(2+). In this study, the effect of clioquinol on the intracellular Zn(2+) level was examined in order to gain insights into the toxicological profile of clioquinol. MAIN METHODS: The effect of clioquinol was estimated using a flow cytometer and FluoZin-3, a fluorescent indicator for Zn(2+), in rat thymocytes. KEY FINDINGS: Clioquinol, at concentrations ranging from 10 to 300 nM, augmented FluoZin-3 fluorescence in a concentration-dependent manner. However, the effect induced by 1 µM clioquinol was less than that by 300 nM clioquinol. Removal of extracellular Zn(2+), using the membrane impermeable Zn(2+)-chelator diethylenetriamine-N,N,N',N″,N″-pentaacetic acid (DTPA), abolished the clioquinol-induced augmentation of FluoZin-3 fluorescence. Clioquinol did not augment Fluo-3 fluorescence, an indicator of intracellular Ca(2+), in the presence of DTPA. The results suggested that clioquinol caused an extracellular Zn(2+)-dependent increase in the intracellular Zn(2+) concentration. However, in the presence of DTPA, clioquinol at micromolar concentrations (1-10 µM) attenuated FluoZin-3 fluorescence in a concentration-dependent manner. Clioquinol even at 10 µM did not affect FluoZin-3 fluorescence under cell-free condition. The concentration-response relationship for the clioquinol induced change in Zn(2+) level appeared to be bell-shaped. These results indicate that micromolar concentrations of clioquinol, without chelated Zn(2+), decrease intracellular Zn(2+) concentration. SIGNIFICANCE: The effect of clioquinol on the intracellular Zn(2+) level varies, depending on the extracellular Zn(2+) concentration and the clioquinol concentration. Clioquinol may therefore exert various types of Zn(2+)-dependent cytotoxicity.

A comparison of the cellular actions of polaprezinc (zinc-L-carnosine) and ZnCl2.

AIMS: Zinc supplementation has been proven to be beneficial for the prevention of some health problems. Many zinc supplements are used for medical and nutritional purposes. However, it is difficult to distinguish between them in terms of their cellular actions. We compared the cellular actions of polaprezinc (zinc-l-carnosine) with those of ZnCl(2) in order to determine whether polaprezinc has greater zinc-related actions than ZnCl(2). MAIN METHODS: Cellular actions of polaprezinc and ZnCl(2) were estimated by flow-cytometric techniques with appropriate fluorescent probes in rat thymocytes. KEY FINDINGS: Both agents had almost equal stimulatory effects on the intracellular Zn(2+) level and cellular level of nonprotein thiol in a similar concentration-dependent manner. However, the increase in cell lethality caused by ZnCl(2) under severe oxidative stress was significantly greater than that caused by polaprezinc. SIGNIFICANCE: There are various zinc supplements, for example, zinc gluconate, zinc picolinate, and zinc methionine. However, the differences in their cellular actions have not been elucidated to date. Such studies could distinguish between zinc supplements.

Pharmacokinetics of ampicillin-sulbactam and the renal function-based optimization of dosing regimens for prophylaxis in patients undergoing cardiovascular surgery.

UNLABELLED: Surgical site infections are a major cause of postoperative morbidity and mortality in cardiovascular surgery. Proper antibiotic prophylaxis can reduce the rate of such infections, but the concentration of antibiotic must be maintained at an adequate level throughout the operation. This study aimed to use renal function to determine the most appropriate timing for intraoperative repeated dosing of ampicillin-sulbactam, a commonly used prophylactic antibiotic, to maintain adequate concentrations throughout the course of surgery. The mean volume of distribution, elimination rate constant, elimination half-life, and total clearance of ampicillin were 13.2 l, 0.652 h⁻¹, 1.32 h, and 8.45 l/h, respectively. A statistically significant (P < 0.0001) correlation (r = 0.771) was observed between the total clearance of ampicillin and creatinine clearance of the patients. Plasma concentrations of ampicillin were simulated with the pharmacokinetic parameters obtained. We developed a nomogram for adjusting the dosing interval according to renal function and predicted ampicillin trough concentrations. We revealed the best dosage and dosing interval for cardiovascular surgery by analyzing the perioperative pharmacokinetics of ampicillin-sulbactam administered prophylactically. We suggest that the dosage and dosing interval for ampicillin-sulbactam should be adjusted to optimize treatment efficacy and safety, on the basis of the MIC90 of methicillin-sensitive Staphylococcus aureus (MSSA) in each institution. TRIAL REGISTRATION: UMIN000007356.

Nanomolar concentrations of zinc pyrithione increase cell susceptibility to oxidative stress induced by hydrogen peroxide in rat thymocytes.

Zinc pyrithione is used as an antifouling agent. However, the environmental impacts of zinc pyrithione have recently been of concern. Zinc induces diverse actions during oxidative stress; therefore, we examined the effect of zinc pyrithione on rat thymocytes suffering from oxidative stress using appropriate fluorescent probes. The cytotoxicity of zinc pyrithione was not observed when the cells were incubated with 3 µM zinc pyrithione for 3 h. However, zinc pyrithione at nanomolar concentrations (10 nM or more) significantly increased the lethality of cells suffering from oxidative stress induced by 3 mM H(2)O(2). The application of zinc pyrithione alone at nanomolar concentrations increased intracellular Zn(2+) level and the cellular content of superoxide anions, and decreased the cellular content of nonprotein thiols. The simultaneous application of nanomolar zinc pyrithione and micromolar H(2)O(2) synergistically increased the intracellular Zn(2+) level. Therefore, zinc pyrithione at nanomolar concentrations may exert severe cytotoxic action on cells simultaneously exposed to chemicals that induce oxidative stress. If so, zinc pyrithione leaked from antifouling materials into surrounding environments would be a risk factor for aquatic ecosystems. Alternatively, zinc pyrithione under conditions of oxidative stress may become more potent antifouling ingredient.

Anti-microbial and therapeutic effects of modified Burow's solution on refractory otorrhea.

OBJECTIVE: Burow's solution, which contains 13% aluminum acetate, has been shown to be effective against chronic otitis media. Since the preparation of Burow's solution is time-consuming, its rapid preparation method has been recently developed. In this study, we evaluated the therapeutic effects of the modified Burow's solution on refractory otorrhea in patients with chronic suppurative otitis and its anti-microbial activity in vitro. METHODS: Fourteen ears of 12 patients with chronic otitis media, granular myringitis, otitis externa and postoperative mastoid cavity problems were treated topically with cotton swab/ball soaked with modified Burow's solution or its four-fold diluted ear drops once a week. We then examined the antimicrobial spectrum of modified Burow's solution against clinical bacterial isolates from otorrhea and laboratory bacterial strains in vitro. RESULTS: In all ears, refractory otorrhea disappeared after 1-17 weeks treatment of modified Burow's solution with a mean of 5.4 weeks without apparent side-effects such as ototoxicity. Modified Burow's solution inactivated all Gram positive bacteria within 5min except Enterococcus species, all Gram negative bacteria including Pseudomonas aeruginosa within 30s and Candida albicans within 2min. In addition, modified Burow's solution inactivated MRSA completely within 5min, while 80.6% of MRSA survived even a 20-min contact with 0.3% ofloxacin. CONCLUSION: These findings indicate that modified Burow's solution, in addition to bearing a broad antimicrobial activity, is as effective as the original Burow's solution in the treatment of chronic suppurative otitis.

Putative role of intracellular Zn(2+) release during oxidative stress: a trigger to restore cellular thiol content that is decreased by oxidative stress.

Although the ability of zinc to retard the oxidative process has been recognized for many years, zinc itself has been reported to induce oxidative stress. In order to give some insights into elucidating the role of intracellular Zn(2+) in cells suffering from oxidative stress, the effects of N-ethylmaleimide (NEM) and ZnCl(2) on cellular thiol content and intracellular Zn(2+) concentration were studied by use of 5-chloromethylfluorescein diacetate (5-CMF-DA) and FluoZin-3 pentaacetoxymethyl ester (FluoZin-3-AM) in rat thymocytes. The treatment of cells with NEM attenuated 5-CMF fluorescence and augmented FluoZin-3 fluorescence in a dose-dependent manner. These NEM-induced phenomena were observed under external Zn(2+)-free conditions. Results suggest that NEM decreases cellular thiol content and induces intracellular Zn(2+) release. Micromolar ZnCl(2) dose-dependently augmented both FluoZin-3 and 5-CMF fluorescences, suggesting that the elevation of intracellular Zn(2+) concentration increases cellular thiol content. Taken together, it is hypothesized that intracellular Zn(2+) release during oxidative stress is a trigger to restore cellular thiol content that is decreased by oxidative stress.

Cytometric analysis on cytotoxicity of curcumin on rat thymocytes: Proapoptotic and antiapoptotic actions of curcumin.

Curcumin exhibits various pharmacological actions including anti-inflammatory, anti-infectious, and anticancer actions. Furthermore, the supplements containing curcumin are supplied for persons consuming alcoholic beverage. A primary criterion for an ingredient ingested by general population is that it exerts no harmful effect. In this study, we examined the effect of curcumin on rat thymocytes to see if curcumin exerts cytotoxicity on normal cells. The incubation with 10 µM curcumin for 24h increased the population of dead cells while it was not the case for 5 µM or less. Curcumin at 5-10 µM increased the populations of shrunken cells and the cells positive to annexin V, phenomena for early stage of apoptosis. However, the incubation with 10 µM curcumin suppressed the increase in population of cells with hypodiploid DNA, a phenomenon for late stage of apoptosis. Thus, curcumin at 10 µM may show both proapoptotic and antiapoptotic actions. The simultaneous incubation with 5 µM, but not 3 µM, curcumin and 0.5% ethanol increased the population of shrunken cells. It is likely that curcumin at 5 µM or more exerts cytotoxic action on normal cells although many studies show some anticancer actions of curcumin at 10 µM or more on cancer cells.

Low micromolar zinc exerts cytotoxic action under H2O2-induced oxidative stress: excessive increase in intracellular Zn2+ concentration.

The ability of zinc to retard oxidative processes has been recognized for many years. However, zinc is cytotoxic under certain oxidative stress. In this study, we investigated the effect of H2O2 on intracellular Zn2+ concentration of rat thymocytes and its relation to the cytotoxicity. Experiments were cytometrically performed by the use of fluorescent probes, propidium iodide, FluoZin-3-AM, and 5-chloromethylfluorescein diacetate. ZnCl2 potentiated cytotoxicity of H2O2 while TPEN, a chelator for intracellular Zn2+, attenuated it. Results suggested an involvement of intracellular Zn2+ in the cytotoxicity of H2O2. H2O2 at concentrations of 30microM or more (up to 1000microM) significantly increased intracellular Zn2+ concentration. There were two mechanisms. (1) H2O2 decreased cellular content of nonprotein thiols, possibly resulting in release of Zn2+ from thiols as cellular Zn2+ binding sites. (2) H2O2 increased membrane Zn2+ permeability because external ZnCl2 application further elevated intracellular Zn2+ concentration. Micromolar H2O2 may induce excessive elevation of intracellular Zn2+ concentration that is harmful to cellular functions. However, the incubation with micromolar ZnCl2 alone increased cellular content of nonprotein thiols, one of the factors protecting cells against oxidative stress. Though zinc is generally considered to be protective with its antioxidant property, this study reveals the toxic effect of zinc even in micromolar range under oxidative stress induced by H2O2.

Zinc at clinically-relevant concentrations potentiates the cytotoxicity of polysorbate 80, a non-ionic surfactant.

Polysorbate 80, a non-ionic surfactant, is used in the formula of water-insoluble anticancer agents for intravenous application. In our recent studies, this surfactant decreased cellular thiol content and the chemicals decreasing cellular thiol content increased intracellular Zn(2+) concentration. In this study using rat thymocytes, the effect of polysorbate 80 on FluoZin-3 fluorescence, an indicator for intracellular Zn(2+), and the influence of ZnCl(2) on cytotoxicity of polysorbate 80 were examined in order to test the possibility that Zn(2+) is involved in cytotoxic action of polysorbate 80. The surfactant at concentrations of 10 microg/ml or more significantly augmented FluoZin-3 fluorescent in a concentration-dependent manner, indicating an increase in intracellular Zn(2+) concentration. The increase by polysorbate 80 was also observed after removing extracellular Zn(2+), suggesting an intracellular Zn(2+) release. The simultaneous application of polysorbate 80 (30 microg/ml) and ZnCl(2) (10-30 microM) significantly increased cell lethality. The simultaneous application of ZnCl(2) accelerated the process of cell death induced by polysorbate 80 and the combination increased oxidative stress. Results may indicate that the cytotoxicity of polysorbate 80 at clinical concentrations is modified by micromolar zinc. Although there is no clinical report that polysorbate 80 and zinc salt are simultaneously applied to human as far as our knowledge, it may be speculated that zinc induces some diverse actions in cancer treatment with water-insoluble anticancer agent including nanoparticle drug of which the solvent is polysorbate 80.

Micromolar Zn(2+) potentiates the cytotoxic action of submicromolar econazole in rat thymocytes: possible disturbance of intracellular Ca(2+) and Zn(2+) homeostasis.

Econazole, one of imidazole antifungals, has been reported to exhibit an inhibitory action on Mycobacterium tuberculosis and its multidrug-resistant strains under in vitro and ex vivo conditions. There is a chemotherapeutic potential of econazole against tuberculosis. We have revealed that Zn(2+) at micromolar concentrations potentiates the cytotoxicity of imidazole antifungals by increasing membrane Zn(2+) permeability. It is reminiscent of a possibility that econazole exhibits harmful action on human in the presence of Zn(2+) at a physiological range when the agent is systemically administered. Because it is necessary to characterize the cytotoxic action of econazole in the presence of Zn(2+), we have cytometrically examined the effects of econazole, ZnCl(2), and their combination on rat thymocytes. ZnCl(2) at concentrations ranging from 1 microM to 30 microM significantly increased the lethality induced by 10 microM econazole in a concentration-dependent manner. Econazole at a sublethal concentration of 1 microM significantly augmented the intensity of side scatter in the presence of micromolar ZnCl(2), suggesting the change in an intracellular circumstance by the combination of econazole and ZnCl(2). Econazole at 0.3 microM or more in the presence of ZnCl(2) increased the intensity of Fluo-3 fluorescence, an indicator for intracellular Ca(2+). Furthermore, the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn(2+), was also augmented by econazole at 0.1 microM or more in the presence of ZnCl(2). Results suggest that the combination of submicromolar econazole with micromolar ZnCl(2) may increase the intracellular concentration of Ca(2+) and Zn(2+), leading to disturbance of intracellular Ca(2+) and Zn(2+) homeostasis that triggers cytotoxic action.

Increase in intracellular Zn2+ concentration by thimerosal in rat thymocytes: intracellular Zn2+ release induced by oxidative stress.

Thimerosal (TMR), an ethylmercury-containing preservative in pharmaceutical products, was recently reported to increase intracellular Zn(2+) concentration. Therefore, some health concerns about the toxicity of TMR remain because of physiological and pathological roles of Zn(2+). To reveal the property of TMR-induced increase in intracellular Zn(2+) concentration, the effect of TMR on FluoZin-3 fluorescence, an indicator of intracellular Zn(2+), of rat thymocytes was examined. TMR at concentrations ranging from 0.3 microM to 10 microM increased the intensity of FluoZin-3 fluorescence in a concentration-dependent manner under external Ca(2+)- and Zn(2+)-free condition. The threshold concentration was 0.3-1 microM. The increase in the intensity was significant when TMR concentration was 1 microM or more. N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a chelator for intracellular Zn(2+), completely attenuated the TMR-induced augmentation of FluoZin-3 fluorescence. Hydrogen peroxide (H(2)O(2)) and N-ethylmaleimide, reducing cellular thiol content, significantly increased FluoZin-3 fluorescence intensity and decreased 5-chloromethylfluorescein (5-CMF) fluorescence intensity, an indicator for cellular thiol. The correlation coefficient between TMR-induced augmentation of FluoZin-3 fluorescence and attenuation of 5-CMF fluorescence was -0.882. TMR also attenuated the 5-CMF fluorescence in the presence of TPEN. Simultaneous application of H(2)O(2) and TMR synergistically augmented the FluoZin-3 fluorescence. It is suggested that TMR increases intracellular Zn(2+) concentration via decreasing cellular thiol content.

Zn(2+), derived from cell preparation, partly attenuates Ca(2+)-dependent cell death induced by A23187, calcium ionophore, in rat thymocytes.

A23187, a calcium ionophore, is used to induce Ca(2+)-dependent cell death by increasing intracellular Ca(2+) concentration ([Ca(2+)](i)) under in vitro condition. Since this ionophore also increases membrane permeability of metal divalent cations such as Zn(2+) and Fe(2+) rather than Ca(2+), trace metal cations in cell suspension may affect Ca(2+)-dependent cell death induced by A23187. Therefore, the effects of chelators for divalent metal cations, EDTA and TPEN, on the A23187-induced cytotoxicity were cytometrically examined in rat thymocytes. The cytotoxicity of A23187 was attenuated by 1mM EDTA while it was augmented by 50 microM EDTA and 10 microM TPEN. These changes were statistically significant. The A23187-induced increase in Fluo-3 fluorescence intensity, a parameter for [Ca(2+)](i), was significantly reduced by 1mM EDTA while it was not the case for 50 microM EDTA and 10 microM TPEN. The intensity of FluoZin-3 fluorescence, a parameter for [Zn(2+)](i), increased by A23187 was respectively reduced by 50 microM EDTA and 10 microM TPEN. It is suggested that the attenuation of A23187-induced cytotoxicity by 1mM EDTA is due to the chelation of extracellular Ca(2+) and Zn(2+) while the augmentation by 50 microM ETDA or 10 microM TPEN is due to the chelation of extracellular Zn(2+). The Tyrode's solution without thymocytes contained 32.4 nM of zinc while it was 216.9 nM in the cell suspension. In conclusion, trace Zn(2+), derived from cell preparation, partly attenuates the Ca(2+)-dependent cell death induced by A23187.

Antimicrobial effects of drugs against multidrug-resistant Pseudomonas aeruginosa.

We evaluated the effects of antimicrobial drugs on four strains of Pseudomonas aeruginosa that are resistant to eight widely used antipseudomonal drugs (piperacillin, piperacillin-tazobactam, imipenem, meropenem, ceftazidime, aztreonam, amikacin, ciprofloxacin) and colistin. In the killing test, colistin (2 microg/ml) was the most effective, followed by aztreonam (48 microg/ml), piperacillin-tazobactam (192-4 microg/ml), piperacillin (192 microg/ml), and a three drug combination of azetreonam (16 microg/ml), ceftazidime (16 microg/ml), and amikacin (4 microg/ml). Six hours after drug addition, colistin (2 microg/ml), aztreonam (48 microg/ml), piperacillin-tazobactam (192-4 microg/ml), piperacillin (192 microg/ml), and the above three drug combination had bacteriostatic effects on all four strains. Colistin, three time breakpoint of aztreonam, piperacillin, or piperacillin-tazobactam, and the three drug combination of aztreonam, ceftazidime, and amikacin were effective in vitro.

Imidazole antifungals, but not triazole antifungals, increase membrane Zn2+ permeability in rat thymocytes Possible contribution to their cytotoxicity.

The use of zinc as a nutritional supplement has become common in many countries. Since zinc has diverse actions, it may be difficult to predict its synergistic and/or antagonistic action in simultaneous presence of drug(s). The combination of imidazole antifungals, but not triazole antifungals, with 3-30 microM ZnCl2 significantly increased the lethality of rat thymocytes. Since intracellular Zn2+ exerts various actions on the process of cell death, there is a possibility that imidazole antifungals, but not triazole antifungals, increases concentration of intracellular Zn2+ ([Zn2+]i). To test the possibility, we examined the effects of imidazole and triazole antifungals on [Zn2+]i of rat thymocytes in absence and presence of extracellular Zn2+ by the use of FluoZin-3, a fluorescent Zn2+ indicator. Imidazole antifungals (clotrimazole, econazole, and oxiconazole) increased the [Zn2+]i in the presence of extracellular Zn2+ while it was not the case for triazole antifungals (itraconazole and fluoconazole). Thus, it is suggested that imidazole antifungals increase the membrane permeability of Zn2+. The potency order in the augmentation of FluoZin-3 fluorescence by imidazole antifungals in the presence of extracellular Zn2+ was the same as that in their cytotoxic action. Therefore, the cytotoxic action of imidazole antifungals may be related to their action on membrane Zn2+ permeability.

Some characteristics of quercetin-induced cytotoxicity on rat thymocytes under in vitro condition.

Quercetin, a flavonoid found in fruits and vegetables, exerts beneficial effects that contribute to human health. Therefore, quercetin preparation is expected as complementary or alternative medicine used by general population. The plausible criterion for such medicines is to exert no toxic action on normal cells. In this study, the effects of quercetin on normal cells were examined using rat thymocytes in RPMI-1640 medium. Significant cytotoxic actions of quercetin were observed at 30 microM. Quercetin increased the populations of propidium-stained cells, shrunken cells, annexin V-positive cells, and the cells with hypodiploidal DNA. Thus, the type of cell death induced by quercetin was apoptosis. Z-VAD-FMK, a pan-inhibitor for caspases, partly attenuated the process of quercetin-induced apoptosis. It can be suggested that plasma concentration of quercetin should be below 30 microM after the digestion when quercetin preparation as complementary or alternative medicine is used.

Synergic cytotoxic action induced by simultaneous application of zinc and clotrimazole in rat thymocytes.

In our previous study, the application of clotrimazole, an antifungal drug, with CdCl(2) or PbCl(2) significantly increased cell lethality of rat thymocytes, even though their individual concentrations were ineffective in affecting the viability. This observation prompted us to study the case for the combination of clotrimazole and ZnCl(2) because the use of zinc as a nutritional supplement has become common. Their combination induced very potent cytotoxic action on rat thymocytes with "bell-shape" dose-response relation. An acceleration of apoptotic process by the combination was suggested for the mechanism. The present result may provide a new insight into toxicological characteristics of clotrimazole.

Propofol, an anesthetic possessing neuroprotective action against oxidative stress, promotes the process of cell death induced by H2O2 in rat thymocytes.

Propofol (2,6-diisopropylphenol) is a general anesthetic possessing a neuroprotective action against oxidative stress produced by H2O2. H2O2 induces an exposure of phosphatidylserine on outer surface of cell membranes, resulting in change in membrane phospholipid arrangement, in rat thymocytes. Since propofol is highly lipophilic, the agent is presumed to interact with membrane lipids and hence to modify the cell vulnerability to H2O2. Therefore, to test the possibility, we have examined the effect of propofol on rat thymocytes simultaneously incubated with H2O2. Although propofol (up to 30 microM) alone did not significantly affect the cell viability, the agent at 10 microM started to increase the population of dead cells in the presence of 3 mM H2O2 and the significant increase was observed at 30 microM. Propofol at clinically relevant concentrations (10-30 microM) facilitated the process of cell death induced by H2O2 in rat thymocytes. However, propofol protected rat brain neurons against the oxidative stress induced by H2O2 under same experimental condition. Therefore, the action of propofol may be dependent on the type of cells.

Microbial contamination of nebulization solution and its measures.

We evaluated the microbial contamination of nebulization solutions in medication cups from a total of 76 ultrasonic nebulizers in use in 10 hospitals. In addition, an interview survey was given to nurses to evaluate the disinfection methods of these ultrasonic nebulizers. Of a total of 76 nebulization solution samples, 11 (14.5%) were contaminated with 10-10(2) colony-forming units (CFU)/ml and 9 (11.8%) with 10(3)-10(5) CFU/ml. The major contaminants were glucose non-fermentative bacilli such as Burkholderia cepacia, CDC gr.IV C-2, and Sphingomonas paucimobilis. Comparison of microbial contamination between the frequencies of disinfection showed a significantly lower number of contaminated samples when the cups were disinfected once daily than when disinfected once at intervals of 2-7 d (p=0.00037). In addition, comparison between the presence and absence of preservatives contained in the nebulization solution showed a significantly lower number of contaminated samples in the presence, rather than in the absence, of preservatives (p=0.00001). These results show that disinfection of ultrasonic nebulizers at 24-h intervals is desirable. In particular, when nebulization solutions not containing preservatives are used, disinfection at 24-h intervals is indispensable.

Tri-n-butyltin-induced blockade of store-operated calcium influx in rat thymocytes.

Tri-n-butyltin (TBT), one of environmental pollutants, disturbs intracellular Ca(2+) homeostasis by increasing intracellular Ca(2+) concentration ([Ca(2+)]i). Effect of TBT on oscillatory change in [Ca(2+)]i (Ca(2+) oscillation) of rat thymocytes was examined using a laser microscope with fluo-3-AM in order to further elucidate the TBT toxicity related to intracellular Ca(2+). The Ca(2+) oscillation was completely attenuated by 300nM TBT. Since store-operated Ca(2+) channels are involved in the generation of Ca(2+) oscillation, the action of TBT on an increase in [Ca(2+)]i by Ca(2+) influx through store-operated Ca(2+) channels was examined. The increase in [Ca(2+)]i by the store-operated Ca(2+) influx was not affected by 3nM TBT. However, TBT at 10nM or more significantly reduced the increase in [Ca(2+)]i. It is likely that TBT attenuates the Ca(2+) oscillation by reducing the Ca(2+) influx through store-operated Ca(2+) channels.

Modification of vulnerability to dodecylbenzenesulfonate, an anionic surfactant, by calcium in rat thymocytes.

We have previously reported that cremophor EL, a nonionic surfactant, at clinical concentrations significantly decreases the cell viability of rat thymocytes with phosphatidylserine-exposed (PS-exposed) membranes under in vitro condition. It is reminiscent of a possibility that sodium dodecylbenzenesulfonate (DCBS), an anionic surfactant world-widely used for detergents, also affects the cells in the similar manner. To test the possibility, the effect of DCBS on rat thymocytes has been examined using a flow cytometer with fluorescent probes. Exposure of PS on outer surface of cell membranes was induced by A23187, a calcium ionophore to increase intracellular Ca(2+) concentration ([Ca(2+)](i)). DCBS at 1µg/mL (2.87µM) significantly decreased the viability of cells with PS-exposed membranes, but not with intact membranes. DCBS also significantly decreased the viability of cells exposed to H(2)O(2), an oxidative stress increasing the [Ca(2+)](i). On the other hand, the decrease in extracellular Ca(2+) concentration ([Ca(2+)](e)) increased the cell vulnerability to DCBS and vice versa. Intact membrane lipid bilayer and extracellular Ca(2+) are required to maintain membrane integrity. Therefore, the change of membrane property by manipulation of [Ca(2+)](i) and [Ca(2+)](e) is one of causes for the augmentation of DCBS cytotoxicity.