Activation of the VPAC2 Receptor Impairs Axon Outgrowth and Decreases Dendritic Arborization in Mouse Cortical Neurons by a PKA-Dependent Mechanism.

Clinical studies have shown that microduplications at 7q36.3, containing VIPR2, confer significant risk for schizophrenia and autism spectrum disorder (ASD). VIPR2 gene encodes the VPAC2 receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Lymphocytes from patients with these mutations exhibited higher VIPR2 gene expression and VIP-induced cAMP responsiveness, but mechanisms by which overactive VPAC2 signaling may lead to these psychiatric disorders are unknown. We have previously found that repeated administration of a selective VPAC2 receptor agonist Ro25-1553 in the mouse during early postnatal development caused synaptic alterations in the prefrontal cortex and sensorimotor gating deficits. In this study, we aimed to clarify the effects of VPAC2 receptor activation on neurite outgrowth in cultured primary mouse cortical neurons. Ro25-1553 and VIP caused reductions in total numbers and lengths of both neuronal dendrites and axons, while PACAP38 facilitated elongation of dendrites, but not axons. These effects of Ro25-1553 and VIP were blocked by a VPAC2 receptor antagonist PG99-465 and abolished in VPAC2 receptor-deficient mice. Additionally, Ro25-1553-induced decreases in axon and dendritic outgrowth in wild-type mice were blocked by a protein kinase A (PKA) inhibitor H89, but not by a PKC inhibitor GF109203X or a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor U0126. PACAP38- induced facilitation of dendritic outgrowth was blocked by U0126. These results suggest that activation of the VPAC2 receptor impairs neurite outgrowth and decreases branching of cortical neurons by a PKA-dependent mechanism. These findings also imply that the VIPR2-linkage to mental health disorders may be due in part to deficits in neuronal maturation induced by VPAC2 receptor overactivation.

Impaired extinction of cued fear memory and abnormal dendritic morphology in the prelimbic and infralimbic cortices in VPAC2 receptor (VIPR2)-deficient mice.

The structurally related neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been implicated in stress regulation and learning and memory. Several bodies of research have shown the impact of the PACAP specific receptor PAC1 on fear memory, but the roles of other PACAP receptors in regulating fear stress responses remain to be elucidated. Here we aimed to investigate the effects of genetic deletion of VIPR2 encoding the VPAC2 receptor, which binds both VIP and PACAP, on fear-related memory and on dendritic morphology in the brain regions of the fear circuitry. Male VPAC2 receptor knockout (VPAC2-KO) and littermate wild-type control mice were subjected to Pavlovian fear conditioning paradigm. VPAC2-KO mice displayed normal acquisition of fear conditioning, contextual and cued fear memory, but impaired extinction of cued fear memory. Morphological analyses revealed reductions in cell body size and total branch number and length of apical and basal dendrites of prelimbic cortex neurons in VPAC2-KO mice. In addition, Sholl analysis indicated that the amount of dendritic material distal to the soma was decreased, while proximal dendritic material was increased. In the infralimbic cortex, the amount of apical dendritic material proximal to the soma was increased in VPAC2-KO mice, while other indices of morphology did not differ. Finally, there were no differences in dendritic morphology in basolateral amygdala neurons between genotypes. These findings suggest that the VPAC2 receptor plays an important role in the fear extinction processes and the regulation of the dendritic morphology in the prelimbic and infralimbic cortices.

Prenatal Exposure to Histone Deacetylase Inhibitors Affects Gene Expression of Autism-Related Molecules and Delays Neuronal Maturation.

Valproic acid (VPA) is a multi-target drug and an inhibitor of histone deacetylase (HDAC). We have previously demonstrated that prenatal exposure to VPA at embryonic day 12.5 (E12.5), but not at E14.5, causes autism-like behavioral abnormalities in male mouse offspring. We have also found that prenatal VPA exposure causes transient histone hyperacetylation in the embryonic brain, followed by decreased neuronal cell numbers in the prefrontal and somatosensory cortices after birth. In the present study, we examined whether prenatal HDAC inhibition affects neuronal maturation in primary mouse cortical neurons. Pregnant mice were injected intraperitoneally with VPA (500 mg/kg) and the more selective HDAC inhibitor trichostatin A (TSA; 500 µg/kg) at E12.5 or E14.5, and primary neuronal cultures were prepared from the cerebral cortices of their embryos. Prenatal exposure to VPA at E12.5, but not at E14.5, decreased total number, total length, and complexity of neuronal dendrites at 14 days in vitro (DIV). The effects of VPA weakened at 21 DIV. Exposure to TSA at E12.5, but not at E14.5, also delayed maturation of cortical neurons. In addition, real-time quantitative PCR revealed that the prenatal exposure to TSA decreased neuroligin-1 (Nlgn1), Shank2, and Shank3 mRNA levels and increased contactin-associated protein-like 2 mRNA level. The delay in neuronal maturation was also observed in Nlgn1-knockdown cells, which were transfected with Nlgn1 siRNA. These findings suggest that prenatal HDAC inhibition causes changes in gene expression of autism-related molecules linked to a delay of neuronal maturation.

Decreased expression of hippocampal Na⁺/Ca²âº exchanger isoform-1 by pentylenetetrazole kindling in mice.

Previous studies have shown that inhibitors of the Na(+)/Ca(2+) exchanger (NCX) attenuate seizure activity in drug-induced epilepsy models, but the role of NCX in epilepsy is not fully understood. The present study examined the effects of pentylenetetrazole (PTZ)-induced kindling on the mRNA expression of NCX isoforms (NCX1, NCX2 and NCX3) in mouse brain. Chronic administration of PTZ at 40mg/kg resulted in kindling seizure development. It caused decreases in the mRNA levels of NCX1 and NCX2, but not NCX3, in the hippocampus. Changes in NCX isoform expression levels were not observed in the prefrontal cortex or striatum. Acute PTZ at 40mg/kg, which caused little seizure activity, also decreased NCX2, but not NCX1 mRNA levels in the hippocampus. These results suggest that down-regulation of hippocampal NCX1 expression is associated with PTZ-induced kindling seizure development.

Chronic treatment with valproic acid or sodium butyrate attenuates novel object recognition deficits and hippocampal dendritic spine loss in a mouse model of autism.

We recently showed that prenatal exposure to valproic acid (VPA) in mice causes autism-like behavioral abnormalities, including social interaction deficits, anxiety-like behavior and spatial learning disability, in male offspring. In the present study, we examined the effect of prenatal VPA on cognitive function and whether the effect is improved by chronic treatment with VPA and sodium butyrate, histone deacetylase inhibitors. In addition, we examined whether the cognitive dysfunction is associated with hippocampal dendritic morphological changes. Mice given prenatal exposure to VPA exhibited novel object recognition deficits at 9 weeks of age, and that the impairment was blocked by chronic (5-week) treatment with VPA (30 mg/kg/d, i.p.) or sodium butyrate (1.2g/kg/d, i.p.) starting at 4 weeks of age. In agreement with the behavioral findings, the mice prenatally exposed to VPA showed a decrease in dendritic spine density in the hippocampal CA1 region, and the spine loss was attenuated by chronic treatment with sodium butyrate or VPA. Furthermore, acute treatment with sodium butyrate, but not VPA, significantly increased acetylation of histone H3 in the hippocampus at 30 min, suggesting the difference in the mechanism for the effects of chronic VPA and sodium butyrate. These findings suggest that prenatal VPA-induced cognitive dysfunction is associated with changes in hippocampal dendritic spine morphology.

Methylphenidate and venlafaxine attenuate locomotion in spontaneously hypertensive rats, an animal model of attention-deficit/hyperactivity disorder, through α2-adrenoceptor activation.

Recent clinical studies have shown that serotonin-norepinephrine reuptake inhibitors such as venlafaxine and duloxetine are effective against symptoms of attention-deficit/hyperactivity disorder such as inattention, oppositionality, and hyperactivity. We have recently found that these serotonin-norepinephrine reuptake inhibitors, like methylphenidate, reduced the hyperactivity in spontaneously hypertensive rats (SHR), an animal model of attention-deficit/hyperactivity disorder. The present study investigated whether the α2-adrenoceptor and the dopamine-D1 receptor are involved in the behavioral effects of methylphenidate and venlafaxine in SHR. Adolescent male SHR showed greater horizontal locomotion in a familiar open field than male Wistar Kyoto and Wistar rats, and methylphenidate (0.3 mg/kg) and venlafaxine (30 mg/kg) reduced horizontal locomotion in SHR, but not Wistar Kyoto or Wistar rats. The effects of methylphenidate and venlafaxine were blocked by idazoxan (an α2-adrenoceptor antagonist), but not by SCH23390 (a dopamine-D1 receptor antagonist). These findings suggest that the α2-adrenoceptor plays a key role in the effects of methylphenidate and venlafaxine on enhanced locomotion in SHR.

Effect of overexpression of the brain-specific Na(+)/Ca(2+) exchanger splice variant NCX1.5 on NO cytotoxicity in HEK293 cells.

Nitric oxide (NO) induces cytotoxicity in neuronal and glial cells via activation of the Na(+)/Ca(2+) exchanger (NCX). This study examined the role of the predominant brain-specific NCX splice variant NCX1.5 in NO-induced cytotoxicity in the HEK293 cell expression system. Cells were transfected with the plasmid construct pcDNA3.1/V5-His containing full-length rat NCX1.5 cDNA. There was no difference in the cytotoxic effects of the NO donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine between control and transfected cells. These results suggest that NO cytotoxicity is not dependent on NCX1.5.

Yttrium decreases the intracellular Zn2+ concentration in rat thymocytes by attenuating a temperature-sensitive Zn2+ influx.

Yttrium is used in the production of various electronic devices because the alloy it contains enhances or modifies the properties of other elements. In order to study the cytotoxic action of yttrium, the effect of yttrium chloride (YCl(3)) on the intracellular Zn(2+) level was examined in rat thymocytes using a flow cytometer with FluoZin-3-AM and propidium iodide. The application of YCl(3) significantly decreased the intensity of the FluoZin-3 fluorescence, suggesting a decrease in the intracellular Zn(2+) level or quenching of the FluoZin-3 fluorescence by Y(3+). However, since Y(3+) did not attenuate the FluoZin-3 fluorescence under cell-free conditions, the latter suggestion was ruled out. Rat thymocytes possess a temperature-sensitive membrane pathway that carries Zn(2+) into the cells. The application of YCl(3) attenuated the FluoZin-3 fluorescence augmented by externally applied ZnCl(2) in a concentration-dependent manner. This suggested that Y(3+) inhibited the Zn(2+) influx, resulting in the decrease in the intracellular Zn(2+) level. Yttrium may induce dyshomeostasis of intracellular Zn(2+), leading to some cytotoxic actions.

Triclosan, an environmental pollutant from health care products, evokes charybdotoxin-sensitive hyperpolarization in rat thymocytes.

The effects of triclosan, an environmental pollutant from household items and health care products, on membrane potential and intracellular Ca(2+) concentrations of rat thymocytes were examined by a flow cytometry with fluorescent probes, di-BA-C(4) and fluo-3-AM, because triclosan is often found in humans and wild animals. Triclosan at a concentration of 3 µM decreased the intensity of di-BA-C(4) fluorescence, indicating the triclosan-induced hyperpolarization. The application of charybdotoxin, a specific inhibitor of Ca(2+)-dependent K(+) channels, and the removal of external Ca(2+) eliminated the triclosan-attenuation of di-BA-C(4) fluorescence. Furthermore, triclosan augmented the fluo-3 fluorescence under normal Ca(2+) condition, indicating that triclosan increased intracellular Ca(2+) concentration. These results suggest that triclosan induces membrane hyperpolarization by increasing intracellular Ca(2+) concentration that activates Ca(2+)-dependent K(+) channels. Since the change in membrane potential of lymphocytes influence cellular immune functions, triclosan may exert adverse actions on immune system in human and wild animals.

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.

Some characteristics of membrane Cd²+ transport in rat thymocytes: an analysis using Fluo-3.

Although cadmium-induced apoptosis of lymphocytes is one of common features in the immunotoxicity of cadmium, the membrane pathway for intracellular cadmium accumulation is not fully elucidated. To characterize membrane Cd(2+) transport of rat thymocytes, the change in intracellular Cd(2+) concentration under various conditions was examined by the use of Fluo-3, a fluorescent probe for monitoring the change in intracellular concentration of divalent metal cations. The membrane Cd(2+) transport was estimated by the augmentation of Fluo-3 fluorescence induced by bath application of CdCl(2). Lowering temperature strongly suppressed the augmentation of Fluo-3 fluorescence by CdCl(2), suggesting that the metabolic process can be involved in membrane Cd(2+) transport. External acidification (decreasing pH) and membrane depolarization by adding KCl attenuated the augmentation, indicating the requirement of electrochemical driving force for membrane Cd(2+) transport into the cells. Bath application of CaCl(2) and ZnCl(2) equally decreased the augmentation, suggesting their competition with Cd(2+) at the membrane transport. The augmentation by CdCl(2) was lesser in the cells treated with N-ethylmaleimide inducing chemical depletion of cellular thiols. The result suggests the contribution of sulfhydryl groups to membrane Cd(2+) transport. Taken together, it is suggested that the cells possess a temperature-sensitive membrane Cd(2+) pathway, driven by electrochemical gradient of Cd(2+) and transmembrane potential, with competitive binding site. Based on the characteristics described above, it is unlikely that the membrane Cd(2+) transport in rat thymocytes is attributed to a single transport system although it has characteristics that are similar to those of divalent cation transporter 1.

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.

Cytotoxic action of bisabololoxide A of German chamomile on human leukemia K562 cells in combination with 5-fluorouracil.

German chamomile (Matricaria recutita L.) is a popular ingredient in herbal teas. In previous study, micromolar bisabololoxide A, one of main constituents in German chamomile, exerted cytotoxic action on rat thymocyte, a normal non-proliferative cell. This result prompted us to study the effect of bisabololoxide A on proliferative cancer cells and to seek the possibility of its use with 5-fluorouracil, an anticancer agent. In this study, the effect of micromolar bisabololoxide A on human leukemia K562 cells was cytometrically examined. Although the incubation of K562 cells with 10 µM bisabololoxide A for 72h did not significantly increase the percentage populations of dead cells and shrunken cells, the inhibitory action on the growth was obviously observed. It was not the case for the concentrations of less than 5 µM. The threshold concentration of bisabololoxide A to exert the cytotoxic action on K562 cells was ascertained to be 5-10 µM. Bisabololoxide A at 5-10 µM did not exert cytotoxic action on normal non-proliferative cells (rat thymocytes) in our previous study. Since the antiproliferative action of micromolar bisabololoxide A on cancerous cells was expected to be beneficial to cancer treatment, the modification of antiproliferative action of 5-fluorouracil (3-30 µM) by bisabololoxide A was studied. The combination of 5-fluorouracil and bisabololoxide further inhibited the growth of K562 cells although the additive inhibition of growth by bisabololoxide A became smaller as the concentration of 5-fluorouracil increased. Therefore, it is suggested that the simultaneous application of German chamomile containing bisabololoxide A may reduce the dose of 5-fluorouracil.

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.

Apoptosis-inducing action of two products from oxidation of sesamol, an antioxidative constituent of sesame oil: a possible cytotoxicity of oxidized antioxidant.

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.

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.

Methylmercury elicits intracellular Zn2+ release in rat thymocytes: its relation to methylmercury-induced decrease in cellular thiol content.

We have previously revealed that thimerosal, an organomercurial preservative, increases intracellular Zn(2+) 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 Zn(2+) concentration. This possibility was tested by measuring intracellular Zn(2+) level with FluoZin-3, a fluorescent probe for intracellular Zn(2+). MetHg at concentrations ranging from 100 nM to 1 microM significantly increased the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn(2+) concentration, under external Ca(2+)- and Zn(2+)-free condition in a concentration-dependent manner. TPEN, a chelator for intracellular Zn(2+), completely diminished the MetHg-induced augmentation of FluoZin-3 fluorescence. MetHg at 100 nM 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 Zn(2+) from intracellular thiol, resulting in the increase in intracellular Zn(2+) concentration. However, it is unlikely that MetHg at critical maternal blood concentration (27 nM) affects intracellular Zn(2+) homeostasis.

Tri-n-butyltin increases intracellular Zn(2+) concentration by decreasing cellular thiol content in rat thymocytes.

Effect of tri-n-butyltin (TBT), an environmental pollutant, on intracellular Zn(2+) 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 30 nM or more (up to 300 nM) significantly increased the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn(2+) concentration, under external Ca(2+)- and Zn(2+)-free condition. Chelating intracellular Zn(2+) completely attenuated the TBT-induced augmentation of FluoZin-3 fluorescence. Result suggests that nanomolar TBT releases Zn(2+) 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 Zn(2+) release by TBT is associated with TBT-induced reduction of cellular thiol content. However, chelating intracellular Zn(2+) potentiated the cytotoxicity of TBT. Therefore, the TBT-induced increase in intracellular Zn(2+) concentration may be a type of stress responses to protect the cells.