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Alginetin is the major product formed from pentoses and hexurionic acids. Alginetin is producted by cooking process of food including pection, a naturally-occurring polysacharride found in many plants. However, the biological interaction and toxicity of alginetin are not known at all. The aim of the present study was to investigate the cellular actions of alginetin on rat thymic lymphocytes. The effects of alginetin on the cell were examined using flow cytometry with fluorescent probes. Alginetin increased cellular content of non-protein thiols ([NPT]i) and elevated intracellular Zn2+ levels ([Zn2+]i). Chelation of intracellular Zn2+ reduced the effect of alginetin on [NPT]i, and chelation of external Zn2+ almost completely diminished alginetin-induced elevation of [Zn2+]i, indicating that alginetin treatment increased Zn2+ influx. Increased [NPT]i and [Zn2+]i levels in response to alginetin were positively correlated. Alginetin protected cells against oxidative stress induced by hydrogen peroxide and Ca2+ overload by calcium ionophore. It is considered that the increases in [NPT]i and [Zn2+]i are responsible for the cytoprotective activity of alginetin because NPT attenuates oxidative stress and Zn2+ competes with Ca2+. Alginetin may be produced during manufacturing of jam, which may provide additional health benefits of jam., Competing Interests: The authors have declared that no competing interests exist.

Tertiary butylhydroquinone (TBHQ) is a food additive and has various beneficial actions under in vitro and in vivo experimental conditions. Therefore, it is necessary to collect additional data on the toxicity of TBHQ in order to avoid adverse effects during clinical applications. Changes in plasma membrane potential are associated with changes in physiological functions even in non-excitable cells such as lymphocytes. Thus, compounds that affect membrane potential may modify some lymphocytic functions. The effect of TBHQ on plasma membrane potential was examined in rat thymocytes using flow cytometric techniques. Treatment of rat thymocytes with TBHQ caused hyperpolarization and then depolarization. The TBHQ-induced hyperpolarization was due to the activation of Ca 2+ -dependent K + channels. TBHQ elevated intracellular Ca 2+ levels. The depolarization by TBHQ was caused by a nonspecific increase in membrane ionic permeability. Both the sustained depolarization and elevation of intracellular Ca 2+ level by TBHQ are thought to be adverse for thymocytes because such changes disturb membrane and intracellular signaling. The thymus is most active during neonatal and pre-adolescent periods. If TBHQ exerts adverse actions on thymocytes, it may result in an immunotoxic effect in neonates and adolescents., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Hydroxyhydroquinone (HHQ) is generated during coffee bean roasting. A cup of coffee contains 0.1-1.7 mg of HHQ. The actions of HHQ on mammalian DNA were examined because HHQ is a metabolite of benzene, which causes leukemia. Currently, information on the cellular actions of HHQ is limited. We examined the effects of sublethal levels of HHQ on the concentration of intracellular Ca 2+ in rat thymic lymphocytes by using a flow cytometric technique with fluorescent probes. HHQ at 10 μM or more significantly elevated intracellular Ca 2+ levels by increasing the membrane permeability of divalent cations, resulting in hyperpolarization via the activation of Ca 2+ -dependent K + channels. HHQ-induced changes in the intracellular Ca 2+ concentration and membrane potential may affect the cell functions of lymphocytes. HHQ-reduced coffee may be preferable in order to avoid the possible adverse effects of HHQ., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cetylpyridinium chloride (CPC) is an antimicrobial agent used in many personal care products, with subsequent release into the environment. Since CPC is found at low concentrations in river and municipal wastewater, its influence on wildlife is of concern. Therefore, in this study, we used flow cytometry to examine the effects of sublethal concentrations of CPC on rat thymic lymphocytes in order to characterize the cellular actions of CPC at low concentrations in the presence and absence of H 2 O 2 -induced oxidative stress. CPC treatment increased the population of living cells with phosphatidylserine exposed on the outer surface of their plasma membranes (a marker of early stage apoptosis), elevated intracellular Zn 2+ levels, and decreased the cellular content of nonprotein thiols. CPC also potentiated the cytotoxicity of H 2 O 2 . Our results suggest that, even at environmentally relevant sublethal concentrations, CPC exerts cytotoxic effects under oxidative stress conditions by increasing intracellular Zn 2+ concentration and decreasing the cellular content of nonprotein thiols. These findings indicate that, under some in vitro conditions, CPC is bioactive at environmentally relevant concentrations. Therefore, CPC release from personal care products into the environment may need to be regulated to avoid its adverse effects on wildlife., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Chlorhexidine (CHX) is an antibacterial agent used in various types of pharmaceutical products. Therefore, CHX is easily found around us. Owing to its positive charge, the electrochemical property of cell membranes was assumed to be a key point of cytotoxic action of CHX. Depolarization of membranes attenuated the cytotoxic action of CHX in rat thymic lymphocytes. CHX interfered with annexin V binding to membranes. Manipulations to induce exposure of phosphatidylserine on the outer membrane surface augmented the cytotoxic action of CHX, indicating that changes in the electrochemical property of membranes affected the cytotoxic action of CHX. Hence, CHX might kill cells physiologically undergoing apoptosis, resulting instead in necrotic cell death. However, the threshold CHX concentration in this in vitro study was slightly higher than blood CHX concentrations observed clinically. Therefore, these results may support the safety of CHX use although CHX possesses unique cytotoxic actions described in this study., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Previous studies on the cytotoxicity of arachidonic acid (ARA) elucidated the involvement of oxidative stress and Ca(2+). In the present study, the Zn(2+)-related cytotoxicity of ARA was studied by a flow cytometric technique with appropriate fluorescent probes in rat thymocytes. Addition of 10 μM ZnCl2 enhanced the increase in cell lethality induced by 10 μM ARA. The removal of Zn(2+) by Zn(2+) chelators attenuated the ARA-induced increase in cell lethality. Thus, Zn(2+) is suggested to be involved in ARA cytotoxicity. ARA at 3-10 μM elevated intracellular Zn(2+) level. The Zn(2+) chelators attenuated the ARA-induced increase in intracellular Zn(2+) level while ARA significantly increased intracellular Zn(2+) level in the presence of 3 μM ZnCl2, suggesting the involvement of external Zn(2+). Zn(2+) reportedly exerts cytotoxic action under oxidative stress induced by hydrogen peroxide, via an excessive increase in intracellular Zn(2+) levels. Since ARA induces oxidative stress, the simultaneous administration of zinc and ARA may be harmful., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Dithiocarbamate fungicides are used as alternative antifouling agents to highly toxic organotin antifouling agents, such as tri-n-butyltin and triphenyltin. There are some concerns regarding their environmental and health risks. It has been shown that tri-n-butyltin increases intracellular Zn(2+) levels of mammalian lymphocytes. Therefore, we examined the effects of dithiocarbamate fungicides (Ziram, Thiram, and Zineb) on rat thymic lymphocytes using a flow-cytometric technique to elucidate how these fungicides affect intracellular Zn(2+) levels. We further determined whether the agents increase intracellular Zn(2+) and/or Ca(2+), because both Zn(2+) and Ca(2+) are intracellular signals in lymphocytes, and excessive increases in their intracellular concentrations can have adverse effects. Dithiocarbamate fungicides increased intracellular Zn(2+) levels, without affecting intracellular Ca(2+) levels. Ziram was the most potent compound, increasing intracellular Zn(2+) levels via Zn(2+) influx. Ziram (1μM) greatly decreased the cellular nonprotein thiol content, and Zn(2+) chelators attenuated the Ziram-induced decrease. Ziram increased the population of annexin V-positive cells in a Zn(2+)-dependent manner. Therefore, we propose that dithiocarbamate fungicides induce Zn(2+) influx, resulting in an excessive elevation of intracellular Zn(2+) levels, leading to the induction of apoptosis. This study gives a basic insight into the mechanisms of dithiocarbamate fungicide-induced adverse events., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Tolylfluanid, a phenylsulfamide fungicide, is one of the many pesticides that are frequently detected in crops. Therefore, its health risk is a concern. Micromolar concentrations of tolylfluanid induce chromosomal aberrations and micronuclei in mammalian lymphocytes. The findings prompted us to study the cellular actions of tolylfluanid and another frequently detected pesticide, dichlofluanid, at submicromolar and micromolar concentrations. Of the cellular actions of chemicals, the action on cellular Ca(2+) homeostasis is important since Ca(2+) is involved in cell signaling and death. Consequently, in this study, the effects of phenylsulfamide fungicides were examined on rat thymocytes by using fluorescent probes in order to further characterize the cellular actions of phenylsulfamide fungicides. Both phenylsulfamide fungicides exhibited biphasic, early and late, increase in intracellular Ca(2+) levels. The early phase was dependent on intracellular Ca(2+) release and increased membrane Ca(2+) permeability. The late phase was owing to Ca(2+) influx via activation of store-operated Ca(2+) channels and the further increase of membrane ionic permeability. Voltage-dependent Ca(2+) channels were not involved. The increases in intracellular Ca(2+) levels by phenylsulfamide fungicides were observed at drug concentrations of 0.1 μM or more (up to 10 μM). Thus, it is plausible that micromolar concentrations of phenylsulfamide fungicides deregulate intracellular Ca(2+) homeostasis in rat thymocytes. Both phenylsulfamide fungicides at 10 μM promoted the transition from intact living cells to living cells with phosphatidylserine-exposed membranes. This was not the case for phenylsulfamide fungicides at 3 μM. The potency of tolylfluanid was similar to that of dichlofluanid. Although the information on residual concentrations of tolylfluanid and dichlofluanid is very limited, their residual concentrations do not reach micromolar levels. It is unlikely that humans will develop adverse effects on exposure to phenylsulfamide fungicides under present environmental conditions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Many effects of sesamol, an antioxidative constituent of sesame oil, have been reported for human health benefits due to its antioxidative action. However, we recently isolated two cytotoxic products, trimer and tetramer of sesamol, from oxidation of sesamol by an assay-guided purification. In this study, we have revealed some cytotoxic characteristics of these products in rat thymocytes and human leukemia K562 cells. Incubation of cells with trimer or tetramer at 10-30 microM for 24h significantly increased cell lethality and population of rat thymocytes containing hypodiploid DNA, suggesting cell death with DNA fragmentation, while it was not the case for 30 microM sesamol. The cytotoxic action of tetramer was more potent than that of trimer in rat thymocytes when their concentrations were 10-30 microM. The incubation of cells with 10 microM tetramer for 24h increased the population of cells with exposed phosphatidylserine, the activity of caspases, and the nick of DNA. These results indicate tetramer-induced apoptosis. In K562 cells, the incubation with tetramer at 10 microM for 72 h significantly inhibited the growth without affecting the lethality. However, tetramer at 30 microM significantly increased cell lethality. It is likely that tetramer exerts more cytotoxic action on normal non-proliferative cells (rat thymocytes) rather than proliferative cancer cells (human leukemia K562 cells). It may be necessary to consider the condition for preservation of sesamol and the safety of products from in vivo oxidation of sesamol for human health., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Since clotrimazole, known as an antifungal drug, exerts diverse actions on cellular functions, it is expected that clotrimazole can be used for other purposes. This antifungal drug protects the cells overloaded with Ca(2+) by A23187, a calcium ionophore. Therefore, the agent may prevent the cells from death induced by heavy metals such as CdCl(2), PbCl(2), or HgCl(2) that are respectively proposed to increase intracellular Ca(2+) concentration. To test this possibility, we have examined the effect of clotrimazole on the cells simultaneously treated with CdCl(2), PbCl(2), or HgCl(2) using rat thymocytes and a flow cytometer with fluorescent probes. The simultaneous application of clotrimazole and CdCl(2) significantly decreased cell viability, even though the concentrations of both were ineffective at affecting the viability. The significant decrease in cell viability was not due to the inhibition of Ca(2+)-ATPase and Ca(2+)-dependent K(+) channels that were induced by clotrimazole. The simultaneous application increased the population of cells with phosphatidylserine exposed on membrane surface, indicating the change in asymmetrical distribution of membrane phospholipids. Furthermore, the cytotoxicity induced by the combination of clotrimazole and CdCl(2) under nominally Ca(2+)-free condition was more profound than that under normal Ca(2+) condition. Therefore, the membrane may be a target for the cytotoxic action of clotrimazole and CdCl(2) that were simultaneously applied. It is also the case for PbCl(2), but not the case for HgCl(2). It is concluded that clotrimazole can modulate the cytotoxicity of some heavy metals.

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.

4-Vinylcatechol (4VC) has been identified as an aroma compound in roasted foods, especially coffee. It is also a component in traditional herbal medicines. This compound may be subconsciously ingested through foods and herbs. Recent experimental evidence has shown that 4VC possesses an antioxidative action. However, the antioxidative action of 4VC at cellular levels is not well characterized. The effects of 4VC (0.1–100 µM) were examined on rat thymic lymphocytes without and with oxidative stress induced by 300 µM hydrogen peroxide (H2O2). Cell treatment with 100 µM 4VC alone for 4 h significantly increased the population of dead cells. Thus, 4VC at 100 µM or above elicits cytotoxicity. However, 4VC at sublethal concentrations (1–10 µM) significantly attenuated the H2O2-induced increase in cell lethality in a concentration-dependent manner. While application of 10 µM 4VC slowed the process of cell death induced by H2O2, 4VC did not antagonize the H2O2-induced reduction of cellular nonprotein thiols. Although 4VC at 10 µM did not affect intracellular Ca2+ and Zn2+ levels, the agent potentiated the H2O2-induced increases in these levels. These actions of 10 µM 4VC are adverse to the cells under the oxidative stress. However, 10 µM 4VC partly attenuated the cell death induced by 100 nM A23187, a calcium ionophore. There are conflicting actions of 4VC at 1–100 µM on the cells under oxidative stress although the agent is used for an antioxidant. Thus, caution is required when using 4VC as a therapeutic agent. [ABSTRACT FROM AUTHOR]

Coffee contains hydroxyhydroquinone (HHQ). HHQ is one of the by-products released during bean roasting. Therefore, it is important to elucidate the bioactivity of HHQ to predict its beneficial or adverse effects on humans. We studied zinc-dependent and independent actions of commercially procured synthetic HHQ in rat thymocytes using flow cytometric techniques with propidium iodide, FluoZin-3-AM, 5-chloromethylfluorescein diacetate, and annexin V-FITC. HHQ at 1050 µM elevated intracellular Zn2+ levels by releasing intracellular Zn2+. HHQ at 10 µM increased cellular thiol content in a zinc-dependent manner. However, HHQ at 30–50 µM reduced cellular thiol content. Although the latter actions of HHQ (30–50 µM) were suggested to increase cell vulnerability to oxidative stress, HHQ at 0.3–100 µM significantly protected cells against oxidative stress induced by H2O2. The process of cell death induced by H2O2 was delayed by HHQ, although both H2O2 and HHQ increased the population of annexin V-positive living cells. However, HHQ at 10–30 µM promoted cell death induced by A23187, a calcium ionophore. HHQ at 10–30 µM exerted contrasting effects on cell death caused by oxidative stress and Ca2+ overload. Because HHQ is considered to possess diverse cellular actions, coffee with reduced amount of HHQ may be preferable to avoid potential adverse effects. [ABSTRACT FROM AUTHOR]

Many businesses thrive by producing health supplements from agricultural products, as exemplified by the production of functional (or health) foods using plants traditionally cultivated in rural areas. Dyes, such as indican, indigo, indoxyl, and indirubin, present in dye plants, possess antibacterial, antifungal, and antiproliferative activities. However, these effects may also lead to cytotoxicity. Thus, studies on normal mammalian cells are necessary to identify cytotoxicity and prevent adverse effects of functional foods that contain these dyes. In this study, the effects of indican, indigo, indoxyl, and indirubin were evaluated by flow cytometry using appropriate fluorescent probes in rat thymic lymphocytes. Among the dyes analyzed, indirubin exerted distinct cellular activities. Treatment with indirubin (10–30 μM) increased the population of shrunken dead cells. The side scatter, but not forward scatter, increased in indirubin-treated living cells. It increased the population of annexin V-bound living and dead cells and that of dead cells without annexin V. Indirubin elevated intracellular Ca2+, but not Zn2+ levels. The cellular content of superoxide anions increased and that of glutathione decreased. Indirubin depolarized the cellular plasma and mitochondrial membranes. It did not potentiate or attenuate the cytotoxicity of A23187 (Ca2+ overload) and H2O2 (oxidative stress). The results suggested that indirubin induces both apoptotic and non-apoptotic cell death. It may be difficult to predict and prevent the adverse effects of indirubin due to its diverse activities on normal mammalian cells. Therefore, indirubin should be removed from products that contain dye plant extracts. [ABSTRACT FROM AUTHOR]

Abstract: In our previous study, the application of clotrimazole, an antifungal drug, with CdCl2 or PbCl2 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 ZnCl2 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. [Copyright &y& Elsevier]

Abstract N -(3-oxododecanoyl)- l -homoserine-lactone (ODHL), a quorum sensing molecule, affects intracellular Zn2+ concentration ([Zn2+]i) and cellular levels of nonprotein thiols ([NPT]i) of rat thymic lymphocytes, both of which are assumed to affect cell vulnerability to oxidative stress. Therefore, it is interesting to examine the effects of ODHL on the cells under oxidative stress. ODHL augmented the cytotoxicity of H 2 O 2 , but not calcium ionophore A23187. ODHL potentiated the H 2 O 2 -induced elevation of [Zn2+]i, wherein, it greatly attenuated the H 2 O 2 -induced increase in intracellular Ca2+ concentration. ODHL did not affect [NPT]i in the presence of H 2 O 2. Therefore, we conclude that the elevation of [Zn2+]i is involved in the ODHL-induced potentiation of H 2 O 2 cytotoxicity. Our findings suggest that ODHL modifies cell vulnerability to oxidative stress in host cells. Highlights • N -(3-oxododecanoyl)- l -homoserine-lactone (ODHL) potentiates H 2 O 2 cytotoxicity. • ODHL does not potentiate the cytotoxicity of calcium ionophore A23187. • ODHL further augments H 2 O 2 -induced elevation of intracellular Zn2+ level. • ODHL greatly attenuates H 2 O 2 -induced increase in intracellular Ca2+ level. • Zn2+ contributes to ODHL-induced potentiation of H 2 O 2 cytotoxicity. [ABSTRACT FROM AUTHOR]

Abstract Azoxystrobin, a broad-spectrum fungicide, has been increasingly used in the agricultural industry. In Japan in 2018, azoxystrobin at five times the normal limit was detected in a shipment of Australian barley that had been used in food products. Therefore, the effects of azoxystrobin need to be carefully examined to predict potential adverse reactions in humans. In this study, the effects of azoxystrobin on the membrane potential and intracellular Ca2+ levels of thymocytes have been photochemically examined using flow cytometry. Azoxystrobin hyperpolarized plasma membrane potential. This hyperpolarization appeared to be due to the activation of Ca2+-dependent K+ channels, as both the removal of extracellular Ca2+ and addition of charybdotoxin attenuated the observed hyperpolarization. In the presence of quinine, an anti-malarial drug that blocks Ca2+-dependent K+ channels, azoxystrobin depolarized the membranes instead. Azoxystrobin increased intracellular Ca2+ levels in a concentration-dependent manner through the influx of extracellular Ca2+ and intracellular release of Ca2+, as confirmed by reduction in azoxystrobin-induced response in the absence of extracellular Ca2+. It appears likely that azoxystrobin at micromolar concentrations modifies membrane ion permeability in thymocytes. Since changes in membrane potential and intracellular Ca2+ levels occur during typical physiological lymphocyte responses, azoxystrobin may disturb lymphocyte function. Highlights • Azoxystrobin hyperpolarizes the membranes of rat thymocytes. • The hyperpolarization is due to activation of Ca2+-dependent K+ channels. • Azoxystrobin depolarizes the membranes in the presence of quinine. • These effects of azoxystrobin are probably harmful for lymphocytes. [ABSTRACT FROM AUTHOR]

Highlights • Captan at sublethal levels induces adverse effects on rat lymphocytes. • Captan raises intracellular Ca2+ level by increasing membrane Ca2+ permeability. • Captan raises intracellular Zn2+ level by increasing intracellular Zn2+ release. • Captan raises and lowers cellular thiol content as captan concentration increases. • Captan increases cell vulnerability to oxidative stress. Abstract Captan, a phthalimide fungicide, is considered to be relatively nontoxic to mammals. There is a possibility that captan affects membrane and cellular parameters of mammalian cells, resulting in adverse effects, because of high residue levels. To test the possibility, we examined the effects of captan on rat thymic lymphocytes using flow-cytometry with appropriate fluorescent probes. Treatment with 10 and 30 μM captan induced apoptotic and necrotic cell death. Before cell death occurred, captan elevated the intracellular concentrations of Ca2+ and Zn2+ and decreased the concentration of cellular thiol compounds. These captan-induced phenomena are shown to cause cell death and are similar to those caused by oxidative stress. Captan also elevated the cytotoxicity of hydrogen peroxide. Results indicate that 10 and 30 μM captan cause cytotoxic effects on mammalian cells. Despite no report on the significant environmental toxicity hazard of captan in humans, it may exhibit adverse effects, described above, on wild organisms. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effects of dibromoacetonitrile (DBAN), a by-product in water bacterial control, at sublethal concentrations on rat thymocytes, by using a cytometric technique with appropriate fluorescent dyes. By using this method, the possibility that DBAN induces cellular actions related to oxidative stress was assessed. DBAN reduced the content of cellular nonprotein thiols under Zn 2+ -free conditions. It elevated the intracellular level of Zn 2+ , being independent from external Zn 2+ . DBAN increased cell vulnerability to the cytotoxic action of hydrogen peroxide. These actions of DBAN were likely related to oxidative stress. DBAN is formed by the reaction of bromides and chlorinated oxidants during water disinfection. Hydrolysis of 2,2-dibromo-3-nitrilopropionamide, an antimicrobial used in hydraulic fracturing fluids for production of shale gas and oil, produces DBAN. Therefore, the concern regarding the levels of DBAN in industrial water systems is necessary to avoid the environmental risk to humans and wild mammals. [ABSTRACT FROM AUTHOR]

Chlorothalonil, a polychlorinated aromatic fungicide, is considered non-toxic to small mammals. However, chlorothalonil inactivates sulfhydryl enzymes and depletes cellular glutathione. Chlorothalonil increases intracellular Zn 2+ concentration ([Zn 2+ ]i) in mammalian cells possibly because intracellular Zn 2+ is released via zinc-thiol/disulfide interchange. The effects of chlorothalonil at sublethal concentrations on the cellular content of nonprotein thiols ([NPT]i) and [Zn 2+ ]i were examined using flow cytometry in rat thymocytes. Low concentrations (0.3–1 μM) of chlorothalonil increased, but high concentrations (3–10 μM) decreased [NPT]i. These effects of chlorothalonil were partly attenuated by an intracellular Zn 2+ chelator. Chlorothalonil at 0.3–10 μM increased [Zn 2+ ]i in a concentration-dependent manner, which was largely dependent on the release of intracellular Zn 2+ . Both the decrease in [NPT]i and increase in [Zn 2+ ]i increase the vulnerability of cells to oxidative stress. Chlorothalonil at 1–10 μM potentiated the cytotoxicity of H 2 O 2 (300 μM). It was also the case for 10 μM pentachloronitrobenzene, but not 10 μM pentachlorophenol. In conclusion, chlorothalonil at low (sublethal) micromolar concentrations is cytotoxic to mammalian cells under oxidative stress. [ABSTRACT FROM AUTHOR]

To study the adverse effects of N-(3-oxododecanoyl)- l -homoserine-lactone (ODHL), a quorum sensing molecule, on mammalian host cells, its effect on membrane potential was examined in rat thymic lymphocytes using flow cytometric techniques with a voltage-sensitive fluorescent probe. As 3–300 μM ODHL elicited hyperpolarization, it is likely that it increases membrane K + permeability because hyperpolarization is directly linked to changing K + gradient across membranes, but not Na + and Cl − gradients. ODHL did not increase intracellular Ca 2+ concentration. ODHL also produced a response in the presence of an intracellular Zn 2+ chelator. Thus, it is unlikely that intracellular Ca 2+ and Zn 2+ are attributed to the response. Quinine, a non-specific K + channel blocker, greatly reduced hyperpolarization. However, because charybdotoxin, tetraethylammonium chloride, 4-aminopyridine, and glibenclamide did not affect it, it is pharmacologically hypothesized that Ca 2+ -activated K + channels, voltage-gated K + channels, and ATP-sensitive K + channels are not involved in ODHL-induced hyperpolarization. Although the K + channels responsible for ODHL-induced hyperpolarization have not been identified, it is suggested that ODHL can elicit hyperpolarization in mammalian host cells, disturbing cellular functions. [ABSTRACT FROM AUTHOR]

Ziram, a dithiocarbamate fungicide, protects various vegetables and fruits against infections by fungus. Recently, there have been increasing anxieties about the risks in the use of dithiocarbamate fungicides. Our previous studies showed that Zn 2+ was a determinant of Ziram cytotoxicity. In addition, Zn 2+ is linked to H 2 O 2 cytotoxicity. Therefore, in this study, we aimed to test the hypothesis that Ziram could augment the cytotoxicity of H 2 O 2 by examining the changes induced by Ziram in some cellular parameters in rat thymic lymphocytes subjected to H 2 O 2 -induced oxidative stress using flow-cytometric methods with fluorescent dyes. Ziram significantly attenuated H 2 O 2 -induced cell death at sublethal concentrations. However, in the cells under oxidative stress elicited by H 2 O 2 , Ziram promoted the changing over from intact cells to living cells with exposed phosphatidylserine (PS) on plasma membranes, whereas it inhibited the transition from PS-exposed living cells to dead cells. Ziram significantly augmented H 2 O 2 actions, including reduction of cellular glutathione levels and elevation of intracellular Zn 2+ concentrations. Conversely, it attenuated H 2 O 2 -induced depolarization of mitochondrial membrane potential. Ziram at sublethal concentrations seems to exhibit promotive and suppressive actions on the process of cell death caused by H 2 O 2 . Ziram increased the number of living cells with exposed PS, a phenomenon characteristic of early stages of apoptosis. Thus, it is concluded that Ziram exhibits pseudo-cytoprotective actions against H 2 O 2 -induced oxidative stress. Ziram at sublethal concentrations exerts promotive and suppressive actions on the process of cell death caused by oxidative stress. [ABSTRACT FROM AUTHOR]

Benzalkonium chloride (BZK) is a common preservative used in pharmaceutical and personal care products. ZnCl 2 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 Zn 2+ -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. ZnCl 2 (1–10 μM) significantly potentiated the cytotoxicity of BZK-C16 at a sublethal concentration (1 μM). The co-treatment of cells with 3 μM ZnCl 2 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 Zn 2+ levels by increasing Zn 2+ influx, and augmented the cytotoxicity of 100 μM H 2 O 2 . Zn 2+ is concluded to facilitate the toxicity of BZK. We suggest that the toxicity of BZK is determined after taking extracellular (plasma) and/or environmental Zn 2+ levels into account. [ABSTRACT FROM AUTHOR]

Abstract: 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 Zn2+ concentration. In this study using rat thymocytes, the effect of polysorbate 80 on FluoZin-3 fluorescence, an indicator for intracellular Zn2+, and the influence of ZnCl2 on cytotoxicity of polysorbate 80 were examined in order to test the possibility that Zn2+ is involved in cytotoxic action of polysorbate 80. The surfactant at concentrations of 10μg/ml or more significantly augmented FluoZin-3 fluorescent in a concentration-dependent manner, indicating an increase in intracellular Zn2+ concentration. The increase by polysorbate 80 was also observed after removing extracellular Zn2+, suggesting an intracellular Zn2+ release. The simultaneous application of polysorbate 80 (30μg/ml) and ZnCl2 (10–30μM) significantly increased cell lethality. The simultaneous application of ZnCl2 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. [Copyright &y& Elsevier]

Abstract: We have previously revealed that thimerosal, an organomercurial preservative, increases intracellular Zn2+ concentration in rat thymocytes. Because thimerosal contains ethylmercury that confers the toxicity, it is a possibility that methylmercury (MetHg), an environmental pollutant, also increases intracellular Zn2+ concentration. This possibility was tested by measuring intracellular Zn2+ level with FluoZin-3, a fluorescent probe for intracellular Zn2+. MetHg at concentrations ranging from 100nM to 1μM significantly increased the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn2+ concentration, under external Ca2+- and Zn2+-free condition in a concentration-dependent manner. TPEN, a chelator for intracellular Zn2+, completely diminished the MetHg-induced augmentation of FluoZin-3 fluorescence. MetHg at 100nM or more significantly decreased the intensity of 5-chlormethylfluorescein fluorescence, an indicator for cellular thiol content. Such MetHg-induced changes in the fluorescence were correlated with a coefficient of −0.917. Taken together, it is suggested that submicromolar MetHg releases Zn2+ from intracellular thiol, resulting in the increase in intracellular Zn2+ concentration. However, it is unlikely that MetHg at critical maternal blood concentration (27nM) affects intracellular Zn2+ homeostasis. [Copyright &y& Elsevier]

Abstract: Effect of tri-n-butyltin (TBT), an environmental pollutant, on intracellular Zn2+ concentration was tested in rat thymocytes to reveal one of cytotoxic profiles of TBT at nanomolar concentrations using a flow cytometer and appropriate fluorescent probes. TBT at concentrations of 30nM or more (up to 300nM) significantly increased the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn2+ concentration, under external Ca2+- and Zn2+-free condition. Chelating intracellular Zn2+ completely attenuated the TBT-induced augmentation of FluoZin-3 fluorescence. Result suggests that nanomolar TBT releases Zn2+ from intracellular store site. Oxidative stress induced by hydrogen peroxide also increased the FluoZin-3 fluorescence intensity. The effects of TBT and hydrogen peroxide on the fluorescence were additive. TBT-induced changes in the fluorescence of FluoZin-3 and 5-chloromethylfluorescein, an indicator for cellular thiol content, were correlated with a coefficient of −0.962. Result suggests that the intracellular Zn2+ release by TBT is associated with TBT-induced reduction of cellular thiol content. However, chelating intracellular Zn2+ potentiated the cytotoxicity of TBT. Therefore, the TBT-induced increase in intracellular Zn2+ concentration may be a type of stress responses to protect the cells. [Copyright &y& Elsevier]

Triphenyltin (TPT), previously used as an agricultural fungicide and industrial antifoulant, is now considered an environmental pollutant. The effect of TPT on human health is concerning due to its presence as a contaminant in seafood. In this study, the changes in intracellular Zn2+ concentration ([Zn2+]i) and cellular content of nonprotein thiols ([NPT]i) induced by triphenyltin chloride (TPTCH), were measured in rat thymic lymphocytes. This was studied by flow-cytometry using the fluorescent probes FluoZin-3-AM and 5-chloromethylfluorescein diacetate (5-CMF-DA). Incubation with TPTCH, at 0.1 μM or more (up to 3 μM), increased [Zn2+]i in a concentration-dependent manner. The TPTCH-induced elevation in [Zn2+]i was due to the increase in membrane Zn2+ permeability and intracellular Zn2+ release. Incubation with TPTCH at 0.3 μM significantly increased [NPT]i levels, whereas the addition of an intracellular Zn2+ chelator had no effect on the same. TPT at higher concentrations (1 or 3 μM) reduced [NPT]i. TPT may disturb intracellular Zn2+ signaling in lymphocytes that disturbs cellular functions. Unlabelled Image • Triphenyltin chloride elevates intracellular Zn2+ levels in lymphocytes. • Triphenyltin chloride increases membrane Zn2+ permeability and intracellular Zn2+ release. • Electrophilic triphenyltin chloride is more potent than nucleophilic triphenyltin hydride. • Triphenyltin, an environmental pollutant, disrupts intracellular Zn2+ homeostasis. [ABSTRACT FROM AUTHOR]