Rat Thymocytes Research Articles

Cytometric Analysis of Cytotoxicity of Polyphenols and Related Phenolics to Rat Thymocytes: Potent Cytotoxicity of Resveratrol to Normal Cells

The chemopreventive and chemotherapeutic actions of polyphenols and related phenolics have received considerable attention since these compounds induce apoptosis in several types of cancer cells in vitro. A plausible criterion for the use of such compounds is that they should not exert any toxic effect on normal cells. However, information about the toxicity of polyphenols and related phenolics to normal cells is limited. In this study, the effects of polyphenols and related phenolics on rat thymocytes were examined by flow cytometric analysis with appropriate fluorescent probes. The compounds examined in this study were caffeic acid, rosmarinic acid, chlorogenic acid, (+)-catechin, 6-gingerol, sesamol, resveratrol, and eugenol. Of these, resveratrol was the most cytotoxic on rat thymocytes incubated for 24 hrs with 100 microM of this compound. Resveratrol at a concentration of 10 microM or more (up to 100 microM) led to a significant dose-dependent increase in the population of dead cells, shrunken living cells, annexin V-positive cells and cells with hypodiploidal DNA. In the presence of benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-FMK), a pan-inhibitor of caspases, the resveratrol-induced increase in the population of cells with hypodiploidal DNA was partially inhibited. Overall, it is suggested that resveratrol at a concentration of 10 microM or more induces apoptosis in normal cells as well as cancer cells (previously reported elsewhere). Thus, at concentrations that are suitable for chemopreventive and chemotherapeutic actions, resveratrol may exert a cytotoxic effect on normal cells.

Protective effects of C60 fullerenes on staurosporine‐, cytosine arabinoside‐ and hydrogen peroxide‐induced cell death of normal but not of transformed T lymphocytes

AbstractC60 fullerenes are carbon nanospheres known to exert a protective activity in several models of apoptotic cell death. Both normal rat thymocytes and human T‐cell lymphoma Jurkat cells were treated with staurosporine, cytosine arabinoside and hydrogen peroxide as agents that trigger cell death. Activation of caspase‐3 as apoptosis marker was demonstrated in cells incubated for 4 h in the presence of these agents at concentrations causing 50 % cell viability reduction. Addition of C60 fullerenes (10‐5 M) protected normal T cells but not human T lymphoma cells from apoptosis induced either by staurosporine, cytosine arabinoside or hydrogen peroxide. These data demonstrate selectivity of fullerenes C60 biological effects in cultures of normal and transformed T cells.

The influence of the microstructure of high noble gold-platinum dental alloys on their corrosion and biocompatibility in vitro

The aim of this work was to compare the microstructures of two high noble experimental Au-Pt alloys with similar composition with their corrosion and biocompatibility in vitro. We showed that Au-Pt II alloy, composed of 87.3 wt.% Au, 9.9 wt.% Pt, 1.7 wt.% Zn and 0.5 wt.% Ir + Rh + In, although possessing better mechanical properties than the Au-Pt I alloy (86.9 wt.% Au, 10.4 wt.% Pt, 1.5 wt.% Zn and 0.5 wt.% Ir + Rh + In), exerted higher adverse effects on the viability of L929 cells and the suppression of rat thymocyte functions, such as proliferation activity, the production of Interleukin-2 (IL-2), expression of IL-2 receptor and activation — induced apoptosis after stimulation of the cells with Concanavalin-A. These results correlated with the higher release of Zn ions in the culture medium. As Zn2+, at the concentrations which were detected in the alloy’s culture media, showed a lesser cytotoxic effect than the Au-Pt conditioning media, we concluded that Zn is probably not the only element responsible for alloy cytotoxicity. Microstructural characterization of the alloys, performed by means of scanning electron microscopy in addition to energy dispersive X-ray and X-ray diffraction analyses, showed that Au-Pt I is a two-phase alloy containing a dominant Au-rich α1 phase and a minor Pt-rich α2 phase. On the other hand, the Au-Pt II alloy additionally contained three minor phases: AuZn3, Pt3Zn and Au1.4Zn0.52. The highest content of Zn was identified in the Pt3Zn phase. After conditioning, the Pt3Zn and AuZn3 phases disappeared, suggesting that they are predominantly responsible for Zn loss, lower corrosion stability and subsequent lower biocompatibility of the Au-Pt II alloy.

A sensitive and specific ELISA detects methionine sulfoxide-containing apolipoprotein A-I in HDL

Oxidized HDL has been proposed to play a key role in atherogenesis. A wide range of reactive intermediates oxidizes methionine residues to methionine sulfoxide (MetO) in apolipoprotein A-I (apoA-I), the major HDL protein. These reactive species include those produced by myeloperoxidase, an enzyme implicated in atherogenesis. The aim of the present study was to develop a sensitive and specific ELISA for detecting MetO residues in HDL. We therefore immunized mice with HPLC-purified human apoA-I containing MetO(86) and MetO(112) (termed apoA-I(+32)) to generate a monoclonal antibody termed MOA-I. An ELISA using MOA-I detected lipid-free apoA-I(+32), apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in buffer or human plasma. Detection was concentration dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radical or metal ions, or LDL and methionine-containing proteins other than apoA-I modified by 2e-oxidants. Because the ELISA we have developed specifically detects apoA-I containing MetO in HDL and plasma, it should provide a useful tool for investigating the relationship between oxidized HDL and coronary artery disease.

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 μM to 30 μM significantly increased the lethality induced by 10 μM econazole in a concentration-dependent manner. Econazole at a sublethal concentration of 1 μM 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 μM 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 μM 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.

Zn2+, derived from cell preparation, partly attenuates Ca2+-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.

Involvement of calcium-independent phospholipase A2 in regulation of Ca2+ signals induced by a calmodulin inhibitor in rat thymocytes

Previous studies have shown that micromolar concentrations of calmodulin inhibitor calmidazolium induce fast activation of nonselective Ca2+ channels in plasma membranes of Ehrlich ascites carcinoma cells (Zinchenko, V.P., Kasymov, V.A., Li, V.V., and Kaimachnikov, N.P., Biofizika (Rus.), 2005, vol. 50 (6), pp. 1055–1069). In order to detect this type of Ca2+ channels in other cells and to establish common regulatory mechanisms, we studied calmidazolium effects on rat thymocytes. It was found that calmidazolium induces biphasic increases in Ca2+ content in cytosol of rat thymocytes due to Ca2+ entry from external medium and reflects the activity of nonselective Ca2+ channels permeable for Mn2+ and Ni2+ ions. The rate and the amplitude of the fast phase are decreased, while those of the slow phase are increased in the presence of specific inhibitors of Ca2+-independent phospholipase A2 (bromoenol lactone and palmitoyl trifluoromethyl ketone). The rate and the amplitude of the fast phase are also inhibited by arachidonic acid and the lipoxygenase inhibitor nordihydroguaiaretic acid, while the Ca2+-dependent phospholipase A2 inhibitor bromophenacyl bromide, the cyclooxygenase inhibitor indomethacin, the specific store-operated Ca2+ channel inhibitor gadolinium and the phospholipase C inhibitor U73122 have no such effect. The rate of the fast phase only slightly depends on temperature, while that of the slow phase shows a strong temperature dependence and increases with a rise in temperatures (Q10 = 2). The amplitude of the fast phase of the Ca2+ signal increases with a decrease of temperatures due to prolongation of the maximum activity of the Ca2+ channel. The data obtained suggest that iPLA2 is an intermediate link in the activation of calmidazolium-induced nonselective Ca2+ channels. The iPLA2 products lysophospholipids and arachidonic acid activate and inhibit Ca2+ channels, respectively. The fact that these compounds manifest different affinities for Ca2+ channels shed additional light on the mechanisms of biphasic Ca2+ elevation in thymus cell cytosol and prolongation of the active state of Ca2+ channels at low temperatures.

Oral Antibiotics Attenuate Bowel Segment Reversal–Induced Alterations in Subpopulation and Function of Peripheral Blood Leukocytes, Thymocytes, and Splenocytes in Massive Bowel‐Resected Rats

The authors previously demonstrated that oral antibiotics significantly attenuated inflammatory response, improved intestinal bacterial overgrowth, and augmented anabolic response in massive bowel-resected rats with bowel-segment reversal. Herein, the effects of oral antibiotics on immune functions were investigated. Male Wistar rats were subjected to a sham operation or a 70% small bowel resection with or without a 3-cm small bowel segment reversal. Thereafter, half numbers of animals with bowel resection and reversal were orally administered clindamycin plus amoxicillin (50 plus 50 mg/kg/day) for 3 weeks. Age-matched nonsurgical rats were included as references. Peripheral blood, spleen, and thymus were collected for analyzing immunocyte subpopulations and function. Bowel resection significantly decreased weight gain, thymic weight, and splenic helper-T, suppressor-T, and mature-B cells but significantly increased splenic macrophage distribution and concanvavalin A-stimulated splenocytic proliferation and cytokine production, such as tumor-necrosis factor (TNF)-alpha, interferon-gamma, and interleukin (IL)-2 (1-way ANOVA, P<.05). Bowel segment reversal further decreased circulating suppressor-T cells but increased circulating natural killer cells and spontaneous splenocytic TNF-alpha production. Segment reversal-induced elevations in bacterial numbers of gut content, circulating white blood cell, nitric oxide, IL-6, splenocytic interferon-gamma and IL-2 production, reductions in T-thymocytic distribution, and alterations in thymocytic interferon-gamma and IL-2 production were attenuated by oral antibiotics. Moreover, oral antibiotics significantly increased splenocytic granulocytes and spontaneous thymocytic proliferation. Oral antibiotics might augment the beneficial effects of bowel segment reversal on anabolism via attenuating bacterial overgrowth and inflammatory response, and restore subpopulation and function of splenocytes and thymocytes in massive bowel-resected rats.

Thiorphan-Induced Survival and Proliferation of Rat Thymocytes by Activation of Akt/Survivin Pathway and Inhibition of Caspase-3 Activity

The activity of substance P (SP) in the rat thymus seems to be tightly controlled by its bioavailability. In this study, we provide evidence for the expression of the SP-degrading enzyme, neutral endopeptidase (NEP)/CD10, by rat thymocyte subsets, and we illustrate its involvement in the in vivo SP/neurokinin-1 receptor (NK(1)R)-mediated regulation of thymocyte survival and proliferation. NEP/CD10 was expressed at both mRNA and protein levels on a substantial portion (45.5%) of CD5(+) thymocytes, namely on the CD4(+)CD8(+) (double positive; DP) and CD4(+) subsets. Continuous administration of thiorphan, a specific NEP/CD10 inhibitor, by means of miniosmotic pumps, enhanced rat thymocyte preprotachykinin-A (PPT-A) and NK(1)R mRNA expression as well as SP and NK(1)R protein levels in an NK(1)R-dependent manner. Thiorphan increased CD10(+)CD4(+) and CD10(+)DP thymocyte numbers, and an NK(1)R antagonist, (S)1-{2-[3(3-4-dichlorophenyl)-1-(3-iso-propoxyphenylacetyl)-piperidine-3-yl]ethyl}-4-pheny-1-azoniabicyclo[2.2.2]octane, chloride (SR140333), abrogated these stimulatory effects. In addition, the NEP/CD10 inhibitor stimulated interleukin (IL)-2 production, IL-2 receptor alpha chain expression, and concanavalin A-induced proliferation of CD5(+) thymocytes, and it inhibited spontaneous and NK(1)R-dependent thymocyte apoptosis. The thiorphan-protective antiapoptotic and proliferative effects involved the activation of Akt serine-threonine kinase, subsequent up-regulation of survivin mRNA, down-regulation of procaspase-3 mRNA levels, and suppression of caspase-3 activity, which were inhibited by SR140333 and mimicked by exogenous SP administration. Overall, our findings suggest that by controlling SP availability, NEP/CD10 negatively regulates thymocyte homeostasis and development.

Effects of FK506 on rat thymus: time-course analysis by immunoperoxidase technique and flow cytofluorometry

The effect of administration of FK506 at 1 mg/kg body weight for 14 days on rat lymphoid tissues, especially the thymus, and recovery after discontinuation of treatment, were investigated by the immunoperoxidase technique and flow cytofluorometry using monoclonal antibodies OX6, OX7, OX8, OX18 and W3/25, reactive with rat lymphocytes. Marked reduction of the thymic medulla upon treatment was clearly demonstrated by staining with OX18 and OX6. Changes produced by FK506 were also observed in the cortical area of the thymus, and were especially marked in the subcapsular area and around blood vessels. Eventually, the thymic cortex appeared patchy, this change being maximal 14 days after the start of administration. Obvious restitution of the thymic medulla was evident about 14 days after withdrawal of FK506. Flow cytometric analysis of the thymus showed that the percentages of cells labelled positively with OX7, OX8 and W3/25 were increased with FK506 treatment, and recovered to the normal level soon after withdrawal. Furthermore, the peak of fluorescence intensity of OX7+, OX8+ and W3/25+ cells showed a temporary shift to the right during FK506 treatment; however, the peak of fluorescence intensity of OX18+ cells showed a temporary shift to the left. Treatment with FK506 also produced a significant change in 3H-thymidine uptake by thymocyte. These results suggest that FK506 may inhibit the proliferation, maturation and differentiation of thymocytes. However, thymocytes prepared from FK506-treated rats and labelled with FITC behaved similarly to rat thymocytes in normal recipient rats. This suggests that during FK506 treatment thymocytes may retain their potential for peripheral mobilization.

Immunolocalization of TGF-β2 in the rat thymus during late stages of prenatal development

The aim of this study was to investigate the immunolocalization of transforming growth factor beta (TGF-beta2) in rat thymic stromal cells and thymocytes and investigate the roles of TGF-beta2 in thymopoiesis during the late stages of fetal development. Twelve adult pregnant female Wistar rats weighing 250-270 g were used in this study. The rats were killed by cervical dislocation on gestation days 16 (GD16), 18 (GD18) and 20 (GD20). Fetal thymus glands were prepared and examined by an immunohistochemical technique to reveal binding of an anti-TGF-beta2 rabbit polyclonal antibody. The thymic primordium was surrounded with a connective tissue capsule at GD16 and at this stage TGF-beta2 immunoreactivity was not observed. At GD18, the connective tissue capsule had formed septa which subdivided the tissue into lobules and at this stage TGF-beta2 immunolocalization was detected in the capsule and in thymocytes. Lobulation was more evident at GD20 and TGF-beta2 immunoreactivity of thymocytes was more extensive than on GD18. Results indicate that TGF-beta2 may play an important role in the organization or development of thymocytes in the late stages of thymopoiesis.

Possible use of quercetin, an antioxidant, for protection of cells suffering from overload of intracellular Ca 2+: A model experiment

Quercetin is known to protect the cells suffering from oxidative stress. The oxidative stress elevates intracellular Ca 2+ concentration, one of the phenomena responsible for cell death. Therefore, we hypothesized that quercetin would protect the cells suffering from overload of intracellular Ca 2+. To test the hypothesis, the effects of quercetin on the cells suffering from oxidative stress and intracellular Ca 2+ overload were examined by using a flow cytometer with appropriate fluorescence probes (propidium iodide, fluo-3-AM, and annexin V-FITC) and rat thymocytes. The concentrations (1–30 μM) of quercetin to protect the cells suffering from intracellular Ca 2+ overload by A23187, a calcium ionophore, were similar to those for the cells suffering from oxidative stress by H 2O 2. The cell death respectively induced by H 2O 2 and A23187 was significantly suppressed by removal of external Ca 2+. Furthermore, quercetin greatly delayed the process of Ca 2+-dependent cell death although it did not significantly affect the elevation of intracellular Ca 2+ concentration by H 2O 2 and A23187, respectively. It is concluded that quercetin can protect the cells from oxidative injury in spite of increased concentration of intracellular Ca 2+. Results suggest that quercetin is also used for protection of cells suffering from overload of intracellular Ca 2+.

Mitochondrial uncoupling protein 1 expression in thymocytes

Using an antibody specific and selective to mitochondrial uncoupling protein 1 (UCP1) peptide, this study confirms the observation that UCP 1 is present in thymocytes isolated from UCP 1 wild-type, but not UCP 1 knock-out mice. UCP 1 is also shown to be present in thymocytes isolated from rat. It was also demonstrated that an antibody raised to the full-length UCP 1 protein appears to be non-specific for UCP 1, as it detects protein in UCP 1 wild-type and UCP 1 knock-out mice, protein in mitochondria isolated from brown adipose tissue of both UCP 1 wild-type and UCP 1 knock-out mice, as well as detecting protein in mitochondria isolated from rat spleen, kidney, skeletal muscle and liver, tissues that do not express UCP 1. We were also able to show that CIDEA, a soluble protein with a suggested role in regulating UCP 1 function, is equally abundant in thymocytes from UCP 1 wild-type and UCP 1 knock-out mice. Taken together our data demonstrate that (a) UCP 1 is present in rat and mouse thymocytes, (b) that the antibody to full-length UCP 1 is not specific for UCP 1 and (c) that the absence of UCP 1 does not affect native expression of CIDEA in thymocytes.

Imidazole antifungals, but not triazole antifungals, increase membrane Zn 2+ 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 μM ZnCl 2 significantly increased the lethality of rat thymocytes. Since intracellular Zn 2+ exerts various actions on the process of cell death, there is a possibility that imidazole antifungals, but not triazole antifungals, increases concentration of intracellular Zn 2+ ([Zn 2+] i). To test the possibility, we examined the effects of imidazole and triazole antifungals on [Zn 2+] i of rat thymocytes in absence and presence of extracellular Zn 2+ by the use of FluoZin-3, a fluorescent Zn 2+ indicator. Imidazole antifungals (clotrimazole, econazole, and oxiconazole) increased the [Zn 2+] i in the presence of extracellular Zn 2+ while it was not the case for triazole antifungals (itraconazole and fluoconazole). Thus, it is suggested that imidazole antifungals increase the membrane permeability of Zn 2+. The potency order in the augmentation of FluoZin-3 fluorescence by imidazole antifungals in the presence of extracellular Zn 2+ was the same as that in their cytotoxic action. Therefore, the cytotoxic action of imidazole antifungals may be related to their action on membrane Zn 2+ permeability.

Therapeutic Reactive Oxygen Generation

An increase of the intracellular reactive oxygen species (ROS) concentration leads to the development of oxidative stress and, thus, to the damage of cell components. The cause-and-effect relations between these processes have not been fully established yet. The ability of photo excited supramolecular composites containing fullerenes C60 immobilized at nanosilica particles to generate reactive oxygen species (ROS) in cells of two types (rat thymocytes, and transformed cells of ascite Erlich carcinoma, EAC, and leucosis L1210) is demonstrated. The damaging effect of photo excited C60-composites are shown, which appeared to be selective and manifested in transformed cells, but not in thymocytes. It has been shown that after the irradiation of aqueous solutions or cell suspensions in the presence of fullerene C60, the generation of reactive oxygen species is observed. It has been shown that the influence of photo excited fullerene C60 on metabolic processes depends on the composition of C60-containing complex and on the type of the cells. The damaging effects of photo excited fullerene C60-containing composites were demonstrated to be selective. The data presented suggest that the application of fullerene C60-containing composites for the selective activation of ROS-dependent death program in certain types of tumor cells is very promising.

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.

In vitro cytostatic and immunomodulatory properties of the medicinal mushroom Lentinula edodes

Lentinula edodes, known as "shiitake" is one of the widely used medicinal mushrooms in the Orient. Antitumour activity of extracts of this mushroom has been widely demonstrated in animals and humans. However, this activity was shown to be host mediated and not by direct cytotoxic activity to cancer cells. This study demonstrates cytotoxic and cell growth inhibitory (cytostatic) effect of aqueous extracts of the mushroom on MCF-7 human breast adenocarcinoma cell line using an MTT cytotoxicity assay. Such effect was demonstrated with fruit body and mycelial extracts, the difference being that there was no significant suppression on normal cells with the latter. Furthermore mycelial extracts did not induce any cytostatic effect in both cancer and normal cell lines based on a DNA synthesis assay. The significant suppression of the proliferation of cancer cells was reflected by the comparatively low IC(50) values and the simultaneous higher respective values on normal fibroblast cells. The immunostimulatory activity of both fruit body and mycelial extracts was tested by the lymphocyte transformation test (LTT), which is based on the capacity of active immunomodulators to augment the proliferative response of rat thymocytes to T mitogens in vitro. Both fruit body and mycelial preparations were able to enhance the proliferation of rat thymocytes directly and act as co-stimulators in the presence of the T-mitogen PHA. Interestingly both extracts, similarly to zymosan showed SI(comit)/SI(mit) ratios of about 2, indicating adjuvant properties. Overall L. edodes aqueous extracts have demonstrated direct inhibition of the proliferation of human breast cancer cells in vitro and immunostimulatory properties in terms of mitogenic and co-mitogenic activity in vitro.

An endogenous calcium-dependent, caspase-independent intranuclear degradation pathway in thymocyte nuclei: Antagonism by physiological concentrations of K + ions

Calcium ions have been implicated in apoptosis for many years, however the precise role of this ion in the cell death process remains incomplete. We have extensively examined the role of Ca 2+ on nuclear degradation in vitro using highly purified nuclei isolated from non-apoptotic rat thymocytes. We show that these nuclei are devoid of CAD (caspase-activated DNase), and DNA degradation occurs independent of caspase activity. Serine proteases rather than caspase-3 appear necessary for this Ca 2+-dependent DNA degradation in nuclei. We analyzed nuclei treated with various concentrations of Ca 2+ in the presence of both a physiological (140 mM) and apoptotic (40 mM) concentration of KCl. Our results show that a 5-fold increase in Ca 2+ is required to induce DNA degradation at the physiological KCl concentration compared to the lower, apoptotic concentration of the cation. Ca 2+-induced internucleosomal DNA degradation was also accompanied by the release of histones, however the apoptotic-specific phosphorylation of histone H2B does not occur in these isolated nuclei. Interestingly, physiological concentrations of K + inhibit both Ca 2+-dependent DNA degradation and histone release suggesting that a reduction of intracellular K + is necessary for this apoptosis-associated nuclear degradation in cells. Together, these data define an inherent caspase-independent catabolic pathway in thymocyte nuclei that is sensitive to physiological concentrations of intracellular cations.

Cytometric Analysis of the Cytotoxic Action of Adenosine 5'-Monophosphate on Rat Thymocytes

The cascade of adenosine 5'-monophosphate-activated protein kinase (AMPK) is known to be a sensor of cellular energy charge. In this context, a paradigm for obesity treatment via the activation of AMPK has attracted the suppliers of complementary medicines and supplements. It is possible that products that increase the concentration of adenosine 5'-monophosphate (AMP) in the body would be efficacious in reducing body weight. However, since there is currently little information concerning the toxicity of AMP, the cytotoxic action of AMP on rat thymocytes was examined by flow cytometry. Incubation of cells with AMP at a concentration of 30 μM or more for 24 hr significantly increased the populations of dead cells, shrunken cells, and cells containing hypodiploidal DNA in a concentration-dependent manner. Z-VAD-FMK, a pan-inhibitor of caspases, attenuated the AMP-induced changes in cell populations. It is concluded that AMP at a concentration of 30 μM or more exerts a cytotoxic action, which is dependent on the activation of caspases.

In Vitro and In Vivo Evaluation of Various Carbonyl Compounds against Cyanide Toxicity with Particular Reference to Alpha-Ketoglutaric Acid

Cyanide is a rapidly acting neurotoxin that necessitates immediate, vigorous therapy. The commonly used treatment regimen for cyanide includes the intravenous administration of sodium nitrite (SN) and sodium thiosulphate (STS). Due to many limitations of these antidotes, a search for more effective, safer molecules continues. Cyanide is known to react with carbonyl compounds to form the cyanohydrin complex. The present study addresses the efficacy of several carbonyl compounds and their metabolites or nutrients with alpha-ketoglutaric acid (A-KG), citric acid, succinic acid, maleic acid, malic acid, fumaric and oxaloacetic acid, glucose, sucrose, fructose, mannitol, sorbitol, dihydroxyacetone, and glyoxal (5 or 10 mM; −10 min) against toxicity of potassium cyanide (KCN; 10 mM) in rat thymocytes in vitro. Six hours after KCN, cell viability measured by MTT assay and crystal violet dye exclusion revealed maximum cytoprotection by A-KG, followed by oxaloacetic acid. A-KG also resolved the leakage of intracellular lactate dehydrogenase, loss in nuclear integrity (propidium iodide staining), and altered mitochondrial membrane potential (rhodamine 123 assay) as a result of cyanide toxicity. Protection Index (ratio of LD50 of KCN in protected and unprotected animals; PI) of all the compounds (oral; 1.0 g/kg; −10 min) determined in male mice, revealed that maximum protection was afforded by A-KG (7.6 PI), followed by oxaloacetic acid (6.4 PI). Comparative evaluation of various salts of A-KG alone or with STS (intraperitoneal; 1.0 g/kg; −15 min) showed that maximum protection was conferred by disodium anhydrous salt of A-KG, which also significantly prevented the inhibition of brain cytochrome oxidase caused by 0.75 LD50 KCN. This study indicates the potential of A-KG as alternative cyanide antidote.