Zinc-related actions of sublethal levels of benzalkonium chloride: Potentiation of benzalkonium cytotoxicity by zinc.

Benzalkonium chloride (BZK) is a common preservative used in pharmaceutical and personal care products. ZnCl2 was recently reported to significantly potentiate the cytotoxicity of some biocidal compounds. In the present study, therefore, we compared the cytotoxic potency of BZK and then further studied the Zn2+-related actions of the most cytotoxic agent among BZK, using flow cytometric techniques with appropriate fluorescent probes in rat thymocytes. Cytotoxicity of benzylcetyldimethylammonium (BZK-C16) was more potent that those of benzyldodecyldimethylammonium and benzyldimethyltetradecylammonium. ZnCl2 (1-10 µM) significantly potentiated the cytotoxicity of BZK-C16 at a sublethal concentration (1 µM). The co-treatment of cells with 3 µM ZnCl2 and 1 µM BZK-C16 increased the population of both living cells with phosphatidylserine exposed on membrane surfaces and dead cells. BZK-C16 at 0.3-1.0 µM elevated intracellular Zn2+ levels by increasing Zn2+ influx, and augmented the cytotoxicity of 100 µM H2O2. Zn2+ is concluded to facilitate the toxicity of BZK. We suggest that the toxicity of BZK is determined after taking extracellular (plasma) and/or environmental Zn2+ levels into account.

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.

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.

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.

Cremophor EL augments the cytotoxicity of hydrogen peroxide in lymphocytes dissociated from rat thymus glands.

The pharmaceutical uses of cremophor EL, a non-ionic surfactant, are similar to those of polysorbate 80. In our previous study, polysorbate 80 exerted some adverse actions on rat thymocytes under in vitro condition. Therefore, the effects of cremophor EL on thymic lymphocytes were examined using a flow cytometer with appropriate fluorescent dyes. Cremophor EL at 10 microg/ml or more (up to 300 microg/ml) concentration-dependently decreased cellular content of glutathione. The cell viability of thymocytes under control condition was 95.4 +/- 1.2% (n = 7, mean +/- S.D.). The incubation of thymocytes with 300 microg/ml cremophor EL or 3 mM hydrogen peroxide for 2 h, respectively, decreased the cell viability to 90.8 +/- 2.8% or 91.2 +/- 2.6%. However, the simultaneous incubation with cremophor EL and hydrogen peroxide decreased the cell viability to 28.7 +/- 8.2%. Cremophor EL at 100 microg/ml accelerated the process of cell death induced by hydrogen peroxide. Results suggest that cremophor EL increases the susceptibility to oxidative stress. Cremophor EL at clinically relevant concentrations may increase the therapeutic potential of some anticancer agents to produce oxidative stress.

Flow-cytometric analysis on adverse effects of polysorbate 80 in rat thymocytes.

The effects of polysorbate 80, a non-ionic surfactant widely used in pharmaceutical products, on rat thymocytes were examined to reveal its toxic property at the cellular level. Polysorbate 80 at concentrations of 1-100 microg/ml did not significantly affect the cell viability. This surfactant at 30 microg/ml or more augmented the intensity of fluo-3 fluorescence, indicating the increase in intracellular Ca(2+) concentration. Such an augmentation of fluo-3 fluorescence by polysorbate 80 was not seen under the Ca(2+)-free condition, suggesting that polysorbate 80 increased membrane Ca(2+) permeability. The concentration-dependent polysorbate 80 at 10 microg/ml or more attenuated the intensity of 5-chloromethylfluorescein, indicating a decrease in cellular content of glutathione by polysorbate 80. Furthermore, the agent at 1 microg/ml or more attenuated the intensity of bis-(1,3-dibutylbarbituric acid) trimethine oxonol fluorescence, being independent from the changes in membrane potential. This phenomenon indicates that polysorbate 80 at 1 microg/ml or more may attenuate the incorporation of anionic compounds into the membranes. It can be suggested that polysorbate 80 modifies some of membranes and intracellular physiological parameters without affecting the cell viability.