Bisabololoxide A, one of the constituents in German chamomile extract, attenuates cell death induced by calcium overload.

Bisabololoxide A (BSBO), main constituents in German chamomile extract, is responsible for antipruritic effect. In previous study, the incubation with 30-100 µM BSBO for 24 h exerted cytotoxic and proapoptotic effects on rat thymocytes. To further characterize BSBO cytotoxicity, the effect on the cells suffering from calcium overload by calcium ionophore A23187 was examined. A23187 induced Ca(2+) -dependent cell death. Contrary to our expectation, 1-10 µM BSBO inhibited A23187-induced increase in cell lethality of rat thymocytes. BSBO attenuated A23187-induced increases in populations of shrunken living cells, phosphatidylserine-exposed living cells, and dead cells, without affecting the increase in intracellular Ca(2+) concentration and the Ca(2+) -dependent hyperpolarization. The effect of BSBO on A23187-treated cells may be unique because the activation of Ca(2+) -dependent K(+) channels is required for cell shrinkage, externalization of phosphatidylserine, and cell death in some cells. The cell death induced by A23187 was not inhibited by Z-VAD-FMK, a pan-inhibitor of caspases. Thus, the cell death may be a necrosis with some features observed during an early stage of apoptosis. These results suggest that BSBO at low micromolar concentrations is cytoprotective against calcium overload.

Bisabololoxide A, one of the main constituents in German chamomile extract, induces apoptosis in rat thymocytes.

German chamomile (Matricaria recutita L.), one of the popular ingredients in herbal teas, has been traditionally used for medicinal purposes. Bisabololoxide A (BSBO) is one of the main constituents in this herb. BSBO is supposed to be principle in some bioactivities of German chamomile such as anti-inflammatory, gastrointestinal, and antipruritic actions. Although the use of German chamomile has spread, the information related to toxicity of BSBO is very limited. In present study, the cytotoxic effect of micromolar BSBO was cytometrically examined on rat thymocytes by using appropriate fluorescent dyes. When the cells were incubated with BSBO for 24 h, BSBO at concentrations of 30 microM or more significantly increased populations of dead cells, shrunken cells, and cells with phosphatidylserine exposed on membrane surface. Both cell shrinkage and externalization of membrane phosphatidylserine are general features in an early stage of apoptosis. In addition, BSBO significantly increased population of cells containing hypodiploid DNA, and the increase was completely attenuated by Z-VAD-FMK, a pan-inhibitor for caspases, indicating an involvement of caspase activation. Thus, it is likely that the type of cell death induced by BSBO is apoptosis. The significant changes in cellular parameters of rat thymocytes by BSBO were not observed when the concentration was 10 microM or less. Furthermore, the short incubation (3 h) of cells even with 30-100 microM BSBO did not significantly affect the cells. Therefore, it may be suggested that BSBO is practically safe when German chamomile is conventionally used.

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.

Extract of Ginkgo biloba leaves attenuates kainate-induced increase in intracellular Ca2+ concentration of rat cerebellar granule neurons.

In order to reveal one of possible mechanisms for neuronal protective action of extract of Ginkgo biloba leaves (EGBL), the effect of EGBL on kainate- and KCl-induced increases in intracellular Ca(2+) concentration ([Ca(2+)]i) of rat cerebellar neurons was examined using a confocal laser microscope with appropriate fluorescent probes. EGBL at 3 microg/ml started to attenuate kainate-induced increase of [Ca(2+)]i and further increase in EGBL concentration (up to 30 microg/ml) concentration-dependently and significantly inhibited the kainate response. The complete inhibition by EGBL was observed in some neurons when the concentration was 10-30 microg/ml. The kainate-induced increase in [Ca(2+)]i was mainly due to Ca(2+) influx through voltage-dependent Ca(2+) channel opened by membrane depolarization via activation of kainate receptor-channel. However, the increase in [Ca(2+)]i by KCl was not significantly affected by EGBL at concentrations where the kainate response was greatly inhibited. EGBL consisting of flavone glycosides and terpene lactones is known to be an antioxidant. Furthermore, in this study, it is shown that EGBL exerts an inhibitory action on kainate receptor (a subtype of glutamate receptor). Since some of neurodegenerative diseases are due to cell death induced by glutamate excitotoxicity and oxidative stress, EGBL may be very suitable for preventing and/or treating such diseases.