The Effect of Dual Bioactive Compounds Artemisinin and Metformin Co-loaded in PLGA-PEG Nano-particles on Breast Cancer Cell lines: Potential Apoptotic and Anti-proliferative Action.

The most prevalent malignancy among women is breast cancer. Phytochemicals and their derivatives are rapidly being recognized as possible cancer complementary therapies because they can modify signaling pathways that lead to cell cycle control or directly alter cell cycle regulatory molecules. The phytochemicals' poor bioavailability and short half-life make them unsuitable as anticancer drugs. Applying PLGA-PEG NPs improves their solubility and tolerance while also reducing drug adverse effects. According to the findings, combining anti-tumor phytochemicals can be more effective in regulating several signaling pathways linked to tumor cell development. The point of the study was to compare the anti-proliferative impacts of combined artemisinin and metformin on cell cycle arrest and expression of cyclin D1 and apoptotic genes (bcl-2, Bax, survivin, caspase-7, and caspase-3), and also hTERT genes in breast cancer cells. T-47D breast cancer cells were treated with different concentrations of metformin (MET) and artemisinin (ART) co-loaded in PLGA-PEG NPs and free form. The MTT test was applied to assess drug cytotoxicity in T47D cells. The cell cycle distribution was investigated using flow cytometry and the expression levels of cyclin D1, hTERT, Bax, bcl-2, caspase-3, and caspase-7, and survivin genes were then determined using real-time PCR. The findings of the MTT test and flow cytometry revealed that each state was cytotoxic to T47D cells in a time and dose-dependent pattern. Compared to various state of drugs (free and nano state, pure and combination state) Met-Art-PLGA/PEG NPs demonstrated the strongest anti-proliferative impact and considerably inhibited the development of T-47D cells; also, treatment with nano-formulated forms of Met-Art combination resulted in substantial downregulation of hTERT, Bcl-2, cyclin D1, survivin, and upregulation of caspase-3, caspase-7, and Bax, in the cells, as compared to the free forms, as indicated by real-time PCR findings. The findings suggested that combining an ART/MET-loaded PLGA-PEG NP-based therapy for breast cancer could significantly improve treatment effectiveness.

Therapeutic Effects of Hydrogen Gas Inhalation on Trimethyltin-Induced Neurotoxicity and Cognitive Impairment in the C57BL/6 Mice Model.

Oxidative stress (OS) is one of the causative factors in the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease (AD) and cognitive dysfunction. In the present study, we investigated the effects of hydrogen (H2) gas inhalation in trimethyltin (TMT)-induced neurotoxicity and cognitive dysfunction in the C57BL/6 mice. First, mice were divided into the following groups: mice without TMT injection (NC), TMT-only injection group (TMT only), TMT injection + lithium chloride-treated group as a positive control (PC), and TMT injection + 2% H2 inhalation-treated group (H2). The TMT injection groups were administered a single dosage of intraperitoneal TMT injection (2.6 mg/kg body weight) and the H2 group was treated with 2% H2 for 30 min once a day for four weeks. Additionally, a behavioral test was performed with Y-maze to test the cognitive abilities of the mice. Furthermore, multiple OS- and AD-related biomarkers such as reactive oxygen species (ROS), nitric oxide (NO), calcium (Ca2+), malondialdehyde (MDA), glutathione peroxidase (GPx), catalase, inflammatory cytokines, apolipoprotein E (Apo-E), amyloid ß (Aß)-40, phospho-tau (p-tau), Bcl-2, and Bcl-2- associated X (Bax) were investigated in the blood and brain. Our results demonstrated that TMT exposure alters seizure and spatial recognition memory. However, after H2 treatment, memory deficits were ameliorated. H2 treatment also decreased AD-related biomarkers, such as Apo-E, Aß-40, p-tau, and Bax and OS markers such as ROS, NO, Ca2+, and MDA in both serum and brain. In contrast, catalase and GPx activities were significantly increased in the TMT-only group and decreased after H2 gas treatment in serum and brain. In addition, inflammatory cytokines such as granulocyte colony-stimulating factors (G-CSF), interleukin (IL)-6, and tumor necrosis factor alpha (TNF-α) were found to be significantly decreased after H2 treatment in both serum and brain lysates. In contrast, Bcl-2 and vascular endothelial growth factor (VEGF) expression levels were found to be enhanced after H2 treatment. Taken together, our results demonstrated that 2% H2 gas inhalation in TMT-treated mice exhibits memory enhancing activity and decreases the AD, OS, and inflammatory-related markers. Therefore, H2 might be a candidate for repairing neurodegenerative diseases with cognitive dysfunction. However, further mechanistic studies are needed to fully clarify the effects of H2 inhalation on TMT-induced neurotoxicity and cognitive dysfunction.

Fucoidan-Rich Substances from Ecklonia cava Improve Trimethyltin-Induced Cognitive Dysfunction via Down-Regulation of Amyloid ß Production/Tau Hyperphosphorylation.

Ecklonia cava (E. cava) was investigated to compare the effect of polyphenol and fucoidan extract and mixture (polyphenol:fucoidan = 4:6) on cognitive function. The ameliorating effect of E. cava was evaluated using the Y-maze, passive avoidance and Morris water maze tests with a trimethyltin (TMT)-induced cognitive dysfunction model, and the results showed that the fucoidan extract and mixture (4:6) had relatively higher learning and memory function effects than the polyphenol extract. After a behavioral test, the inhibitory effect of lipid peroxidation and cholinergic system activity were examined in mouse brain tissue, and the fucoidan extract and mixture (4:6) also showed greater improvements than the polyphenol extract. Mitochondrial activity was evaluated using mitochondrial reactive oxygen species (ROS) content, mitochondrial membrane potential (MMP, ΔΨm), adenosine triphosphate (ATP) content, and mitochondria-mediated protein (BAX, cytochrome C) analysis, and these results were similar to the results of the behavioral tests. Finally, to confirm the cognitive function-related mechanism of E. cava, the amyloid-ß production and tau hyperphosphorylation-medicated proteins were analyzed. Based on these results, the improvement effect of E. cava was more influenced by fucoidan than polyphenol. Therefore, our study suggests that the fucoidan-rich substances in E. cava could be a potential material for improving cognitive function by down-regulating amyloid-ß production and tau hyperphosphorylation.

Neuroprotective effect of clavulanic acid on trimethyltin (TMT)-induced cytotoxicity in PC12 cells.

Trimethyltin (TMT) is a short-chain trialkyltin with various applications in industry. In addition, it is a known neurotoxin, producing significant and selective neurodegeneration in the limbic system of both human and animals. Recently, effect of clavulanic acid (CA) in nervous system has been mentioned. Therefore, in this study, the role of CA in TMT-induced toxicity in PC12 cells was evaluated. For this study, PC12 cells were cultured and exposed to different concentrations of CA for 24 h. Then, TMT (20 µM) was added to cells. After that, MTT test was performed to assay cytotoxicity. Reactive oxygen species production (ROS) was determined using 2,7-dichlorofluorescein diacetate (DCFH-DA) method. Additionally, the levels of Bax, Bcl-2, caspase-3, CERB and p-CREB proteins were evaluated using Western blot analysis. The exposure of PC12 cells to TMT reduced cell viability, increased intracellular ROS production, elevated Bax/Bcl-2 ratio and enhanced the expression of caspase-3 (Pro and cleaved forms) protein. Pretreatment of cells with CA before TMT, significantly reduced ROS generation, diminished upregulation of proapoptotic Bax protein and attenuated caspase-3 protein expression. In conclusion, CA exhibited significant neuroprotective effects against neurotoxicity of TMT mainly throughout reduction of ROS production and regulation of proteins, which are involved in apoptosis pathway.

Anti-inflammatory effects of Ginkgo biloba extract against trimethyltin-induced hippocampal neuronal injury.

BACKGROUND: Despite the immense neuromodulatory potentials of Ginkgo biloba extract as a memory enhancer, its underlying mechanism seems inadequate particularly with regard to its anti-inflammatory properties. AIM: The objective of the present study is to investigate the protective potentials of Ginkgo biloba extract (GBE) against hippocampal neuronal injury induced by trimethyltin (TMT), a potent neurotoxicant. METHODS: Male SD rats were administered trimethyltin (8.5 mg kg-1 b.wt) single intraperitoneal (i.p.) injection, followed by Ginkgo biloba extract (100 mg kg-1 b.wt i.p) for 21 days. RESULTS: The co-administration of GBE with TMT showed marked improvement in cognitive functions. Concomitantly, there was a significant decrease in oxidative stress as evident by reduction in MDA and total ROS levels. In addition, there was a marked suppression of astrocyte activation (GFAP), transcription factor NFκB and proinflammatory cytokines (TNF-α, IL-1α, 1L-6), which were found to be elevated by TMT administration. Histopathological observations showed remarkable improvement in hippocampal neuronal injury in the conjunctive group. CONCLUSION: Therefore, it is suggested that Ginkgo biloba extract is an effective agent against trimethyltin-induced hippocampal neuronal loss owing to its antioxidative as well as anti-inflammatory properties.

Enhanced expression of immediate-early genes in mouse hippocampus after trimethyltin treatment.

Immediate-early genes (IEGs) are transiently and rapidly activated in response to various cellular stimuli. IEGs mediate diverse functions during pathophysiologic events by regulating cellular signal transduction. We investigated the temporal expression of several IEGs, including c-fos, early growth response protein-1 (Egr-1), and activity-regulated cytoskeleton-associated protein (Arc), in trimethyltin (TMT)-induced hippocampal neurodegeneration. Mice (7 weeks old, C57BL/6) administered TMT (2.6mg/kg intraperitoneally) presented severe neurodegenerative lesions in the dentate gyrus (DG) and showed behavioral seizure activity on days 1-4 post-treatment, after which the lesions and behavior recovered spontaneously over time. c-fos, Egr-1, and Arc mRNA and protein levels significantly increased in the mouse hippocampus after TMT treatment. Immunohistochemical analysis showed that nuclear c-fos expression increased mainly in the DG, whereas nuclear Egr-1 expression was increased extensively in cornu ammonis (CA) 1, CA3, and the DG after TMT treatment. Increased Arc levels were detected in the cellular somata/dendrites of the hippocampal subregions after TMT treatment. Therefore, we suggest that increased IEGs are associated with TMT-induced pathological events in mouse hippocampus.

Trimethyltin chloride inhibits neuronal cell differentiation in zebrafish embryo neurodevelopment.

Trimethyltin chloride (TMT) is a neurotoxicant widely present in the aquatic environment, primarily from effluents of the plastic industry. It is known to cause acute neuronal death in the limbic-cerebellar system, particularly in the hippocampus. However, relatively few studies have estimated the effects of TMT toxicity on neurodevelopment. In this study, we confirmed the dose-dependent effects of TMT on neurodevelopmental stages through analysis of morphological changes and fluorescence assays using HuC-GFP and olig2-dsRed transgenic zebrafish embryos. In addition, we analyzed the expression of genes and proteins related to neurodevelopment. Exposure of embryos to TMT for 4 days post fertilization (dpf) elicited a concentration-related decrease in body length and increase in axial malformation. TMT affected the fluorescent CNS structure by decreasing pattern of HuC-GFP and olig2-dsRed transgenic zebrafish. In addition, it significantly modulated the expression patterns of Sonic hedgehog a (Shha), Neurogenin1 (Ngn1), Embryonic lethal abnormal vision like protein 3 (Elavl3), and Glial fibrillary acidic protein (Gfap). The overexpression of Shha and Ngn1, and downregulation of Elavl3 and Gfap, indicate repression of proneural cell differentiation. Our study demonstrates that TMT inhibits specific neurodevelopmental stages in zebrafish embryos and suggests a possible mechanism for the toxicity of TMT in vertebrate neurodevelopment.

Effect of Chaenomeles sinensis Extract on Choline Acetyltransferase Activity and Trimethyltin-Induced Learning and Memory Impairment in Mice.

The aim of this study was to search for a novel choline acetyltransferase (ChAT) activator from plants traditionally grown in Korea. An ethanol extract from Chaenomeles sinensis Koehne showed the highest ChAT-activating effect in vitro in an assay that used human neuroblastoma cells and [(14)C]acetyl-CoA. The active compound was speculated to be stearic acid methyl ester (SAME). In an in vivo experiment, C. sinensis extract and SAME improved trimethyltin (TMT)-induced deficits in learning and memory in mice as assessed by a Y-maze behavioral test and a passive avoidance test. The C. sinensis extract might attenuate the TMT-induced brain disorder. This study suggests that SAME from C. sinensis might be useful in the treatment of Alzheimer's disease.

Trimethyltin-Induced Microglial Activation via NADPH Oxidase and MAPKs Pathway in BV-2 Microglial Cells.

Trimethyltin (TMT) is known as a potent neurotoxicant that causes neuronal cell death and neuroinflammation, particularly in the hippocampus. Microglial activation is one of the prominent pathological features of TMT neurotoxicity. Nevertheless, it remains unclear how microglial activation occurs in TMT intoxication. In this study, we aimed to investigate the signaling pathways in TMT-induced microglial activation using BV-2 murine microglial cells. Our results revealed that TMT generates reactive oxygen species (ROS) and increases the expression of CD11b and nuclear factor-κB- (NF-κB-) mediated nitric oxide (NO) and tumor necrosis factor- (TNF-) α in BV-2 cells. We also observed that NF-κB activation was controlled by p38 and JNK phosphorylation. Moreover, TMT-induced ROS generation occurred via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in BV-2 cells. Interestingly, treatment with the NADPH oxidase inhibitor apocynin significantly suppressed p38 and JNK phosphorylation and NF-κB activation and ultimately the production of proinflammatory mediators upon TMT exposure. These findings indicate that NADPH oxidase-dependent ROS generation activated p38 and JNK mitogen-activated protein kinases (MAPKs), which then stimulated NF-κB to release proinflammatory mediators in the TMT-treated BV-2 cells.

Glial activation with concurrent up-regulation of inflammatory mediators in trimethyltin-induced neurotoxicity in mice.

Trimethyltin (TMT), a potent neurotoxic chemical, causes dysfunction and neuroinflammation in the brain, particularly in the hippocampus. The present study assessed TMT-induced glial cell activation and inflammatory cytokine alterations in the mouse hippocampus, BV-2 microglia, and primary cultured astrocytes. In the mouse hippocampus, TMT treatment significantly increased the expression of glial cell markers, including the microglial marker ionized calcium-binding adapter molecule 1 and the astroglial marker glial fibrillary acidic protein. The expression of M1 and M2 microglial markers (inducible nitric oxide synthase [iNOS] and CD206, respectively) and pro-inflammatory cytokines (interleukin [IL]-1ß, IL-6 and tumor necrosis factor [TNF]-α) were significantly increased in the mouse hippocampus following TMT treatment. In BV-2 microglia, iNOS, IL-1ß, TNF-α, and IL-6 expression increased significantly, whereas arginase-1 and CD206 expression decreased significantly after TMT treatment in a time- and concentration-dependent manner. In primary cultured astrocytes, iNOS, arginase-1, IL-1ß, TNF-α, and IL-6 expression increased significantly, whereas IL-10 expression decreased significantly after TMT treatment in a time- and concentration-dependent manner. These results indicate that significant up-regulation of pro-inflammatory signals in TMT-induced neurotoxicity may be associated with pathological processing of TMT-induced neurodegeneration.

Synthesis and characterization of a novel o-tolyltelluronic trimethyltin ester and its cytotoxic assessment in vitro.

A new arenetelluronic triorganotin ester, namely (Me3Sn)4[o-Me-PhTe(µ-O)(OH)O2)]2 (1) has been prepared by the reaction of o-tolyltelluronic acid and Me3SnCl in the presence of potassium hydroxide. The complex was fully characterized by elemental analysis, FT-IR, NMR ((1)H, (13)C, (119)Sn) spectroscopy and X-ray crystallography. Structure analysis revealed that the complex crystallized as Sn4Te2 units and a 1D linear chain was formed by intermolecular C-HO interactions. Cytotoxic assessments showed that the complex can induce apoptotic cell death via accumulation of ROS, collapse of the MMP and activating caspase-3. The results indicated that ROS is crucial to the cytotoxicity induced by the complex.

Exposure to an organometal compound stimulates adipokine and cytokine expression in white adipose tissue.

OBJECTIVE: White adipose tissue (WAT) is now considered a defined tissue capable of interactions with other organ systems. WAT role in elevating the level of systemic chronic inflammation suggests that alterations in this tissue as the result of disease or environmental factors may influence the development and progression of various obesity-related pathologies. This study investigated WAT cell-specific responses to an organometal compound, trimethyltin (TMT), to determine possible contribution to induced inflammation. METHODS: Human primary mature adipocytes and macrophage differentiated THP-1 cells were cultured in TMT presence and relative toxicities and different adipokine levels were determined. The inflammatory response was examined in TMT presence for primary cells from obese ob/ob mice WAT, and after TMT injection in ob/ob mice. RESULTS: Both adipocytes and macrophages were resistant to cell death induced by TMT. However, adipocytes cultured in TMT presence showed increased expression of TNFα and IL-6, and modified leptin levels. In macrophage cultures, TMT also increased TNFα and IL-6, while MCP-1 and MIP-1α were decreased. In vivo, a single injection of TMT in ob/ob mice, elevated TNFα, MIP-1α and adiponectin in WAT. CONCLUSIONS: Elevation of the inflammatory related products can be induced by chemical exposure in adipocytes and macrophages, as well as murine WAT. These data suggest that numerous factors, including a systemic chemical exposure, can induce an inflammatory response from the WAT. Furthermore, when characterizing both chemical-induced toxicity and the progression of the chronic inflammation associated with elevated WAT content, such responses in this target tissue should be taken into consideration.

Apoptotic and necrotic action mechanisms of trimethyltin in human hepatoma G2 (HepG2) cells.

In evaluating the cytotoxic effects and the mechanisms of the apoptotic and necrotic actions of trimethyltin chloride (TMT) on human hepatoma G2 (HepG2) cells, the present study focused on the involvement of antiproliferation, DNA damage, cell death, apoptosis-related proteins, and p53-dependent transcriptional activity. Twenty-four hour TMT treatments (4-64 microM) induced apoptosis and necrosis in HepG2 cells. Thirty-two micromolar and higher concentration significantly increases cell death. DNA damage was observed at 8 microM. Additionally, TMT increased the activity of cellular caspase-3 and the release of mitochondrial cytochrome c in a concentration-dependent manner. Our data demonstrated that the Bcl-2 family of proteins was involved in the apoptotic process but that p53 expression level was not affected. The results of luciferase reporter assay indicated that TMT-induced apoptosis seemed to adopt a transcription-dependent route, by activating p53 target genes such as PUMA and p21.

The type 1 interleukin 1 receptor is not required for the death of murine hippocampal dentate granule cells and microglia activation.

Alterations in inflammatory process, neuronal death, and glia response have been observed under manipulation of interleukin-1 (IL-1) and subsequent signaling through the type 1 IL-1 receptor (IL-1R1). To investigate the influence of IL-1R1 activation in the pathophysiology of a chemical-induced injury to the murine hippocampus, we examined the level and pattern of neuronal death and neuroinflammation in male weanling mice exposed to trimethyltin hydroxide (2.0 mg TMT/kg, i.p.). Dentate granule cell death occurred at 6 h post-TMT as detected by active caspase 3 immunostaining and presence of lectin positive microglia. The severity of neuronal death and microglia response increased by 12-24 h with elevations in mRNA levels for TNFalpha and IL-1alpha. In IL-1R1 null (IL-1R1-/-) mice, the pattern and severity of neuronal death at 24 or 72 h post-TMT was similar as compared to wildtype (WT) mice. In both groups, mRNA levels for TNFalpha and MIP-1alpha were elevated, no significant change was seen in either IL-1alpha or IL-1beta, and the early activation of microglia, including their ability to progress to a phagocytic phenotype, was maintained. Compared to WT mice, IL-1R1-/- mice displayed a limited glial fibrillary acidic protein (GFAP) astrocytic response, as well as a preferential induction in mRNA levels of Fas signaling components. Cumulatively, these results indicate that IL-1R1 activation is not necessary for TMT-induced death of dentate granule neurons or local activation of microglia; however, IL-1R1 signaling is involved in mediating the structural response of astrocytes to injury and may regulate apoptotic mechanisms via Fas signaling components.

In vivo acute treatment with trimethyltin chloride causes neuronal degeneration in the murine olfactory bulb and anterior olfactory nucleus by different cascades in each region.

Our earlier study demonstrated that in vivo acute treatment with trimethyltin chloride (TMT) produces severe neuronal damage in the dentate gyrus and cognition impairment in mice. In the present study, we assessed whether TMT was capable of causing neuronal degeneration in the olfactory bulb (OB) and anterior olfactory nucleus (AON) of the mouse brain. An intraperitoneal injection of TMT at the dose of 2.8 mg/kg led to a dramatic increase in the number of degenerating cells, which were reactive with antibody against single-stranded DNA, in the granule cell layer (GCL) of the OB and AON 1 day and 2 days later, respectively. TMT treatment produced a marked translocation of phospho-c-Jun-N-terminal kinase from the cytoplasm to the nucleus in the AON. Expectedly, a marked increase in phospho-c-Jun-positive cells was seen in the AON after the treatment. In addition to the AON, the mitral cell layer of the olfactory bulb showed the presence of phospho-c-Jun-positive cells after the treatment. However, the GCL had no cells positive for either phospho-c-Jun-N-terminal kinase or phospho-c-Jun at any time after the treatment with TMT. Similarly, TMT-induced nuclear translocation of the lysosomal enzyme deoxyribonuclease II was seen in the AON, but not in the GCL. On the other hand, TMT elicited the expression of activated caspase 3 in the GCL but not in the AON. Taken together, our results suggest that TMT is capable of causing neuronal degeneration in the murine OB and AON through different cascades in the two structures.

Lack of trimethyltin (TMT)-induced elevation of plasma corticosterone in PACAP-deficient mice.

Accumulating evidence implicates pituitary adenylate cyclase-activating polypeptide (PACAP) in a number of stress responses. By using PACAP-deficient mice, PACAP has been shown to have an in vivo role in the regulation of the sympathoadrenal axis, but a role in regulating the hypothalamo-pituitary-adrenal (HPA) axis has not been fully addressed. To elucidate the role of endogenous PACAP in HPA axis regulation during pathological conditions, mice lacking the Adcyap1 gene encoding the neuropeptide PACAP (Adcyap1-/-) were injected with trimethyltin (TMT), a neurotoxin known to induce neuronal damage and several systemic responses including elevated plasma corticosterone levels. In wild-type controls, TMT induced transient decreases in water and food intake, with a concomitant decrease in body weight; however, no significant changes were observed in Adcyap1-/- mice. Basal corticosterone levels were not significantly different between the mutant and wild-type mice. TMT induced a marked elevation of plasma corticosterone above basal levels in wild-type mice but no significant increase was seen in Adcyap1-/- mice. The present article suggests that PACAP is involved in the corticosterone release in some pathological conditions but not in the basal state.

Effects of in vivo treatment of rats with trimethyltin chloride on respiratory properties of rat liver mitochondria.

Liver mitochondria isolated from rats treated in vivo with trimethyltin chloride show stimulation of respiration using glutamate/malate as substrate, and a transient inhibition on rates of respiration using palmitoyl-L-carnitine as substrate. This phenomenon was observed with both ADP- and FCCP-stimulated respiration. In contrast, rates of respiration by liver mitochondria isolated from rats treated in vivo with trimethyltin chloride, following prior treatment with clofibrate, were inhibited when glutamate/malate was respiratory substrates. With palmitoyl-L-carnitine no effect of trimethyltin chloride was observed. In vitro treatment of rat liver mitochondria, or of rat liver homogenates, led to the expected, powerful inhibition of respiration. The synthesis of ATP by liver mitochondria isolated from rats treated in vivo with trimethyltin chloride was not inhibited compared to mitochondria isolated from control rats. Similarly, ATP synthesis by mitochondria isolated from rats treated with clofibrate, before treatment with trimethyltin chloride, was not inhibited. We, therefore, conclude that the powerful inhibitory effects of trimethyltin found in vitro, is not expressed in vivo during the first 36 hr following administration. In vivo treatment of rats with trimethyltin chloride caused a marked increase in hepatic levels of taurine and glycine, while levels of glutathione and glutamine were diminished. This is consistent with an enhanced oxidative stress in the liver. Our findings lead to the conclusion that increased oxidative stress, rather than inhibition of the mitochondrial ATPase, is a likely major cause of the in vivo toxic effects due to trimethyltin chloride.

Neurotoxicant-induced elevation of adrenomedullin expression in hippocampus and glia cultures.

Adrenomedullin (AM), a vasoactive peptide first isolated from pheochromocytoma, has been reported to be present in neurons in the central nervous system and in tumors of neural and glial origin. In this study, we investigated AM expression both in the hippocampus and in glial cell cultures using a chemical-induced model of injury. An acute intraperitoneal injection of the organometal trimethyltin (TMT) results in neurodegeneration of the hippocampal CA3-4 pyramidal cell layer. Within 4 days of injection, sparse, punctate staining for AM and lectin was evident in the CA3-4 region; by 10 days, a minimal level of CA3-4 neuronal degeneration was evident, with an increase in glial fibrillary acidic protein (GFAP)-positive astrocytes throughout the hippocampus. Degeneration progressed in severity until 30 days post-TMT, with distinct positive immunoreactivity for AM in the CA4 region. mRNA levels for tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, GFAP, and AM in the hippocampus were increased over control levels within 4 days following TMT. In cultured glial cells, a 6 hr exposure to TMT (10 microM) produced a morphological response of the cells and increased immunoreactivity for vimentin, GFAP, and AM. mRNA levels for TNFalpha, IL-1alpha, GFAP, vimentin, and AM were elevated within 3-6 hr of exposure. In culture, neutralizing antibodies to IL-1alpha and TNFalpha were effective in inhibiting the TMT-induced elevation of AM mRNA. These data suggest an interaction between the proinflammatory cytokines and glia response in the regulation of AM in response to injury.

N-chlorotaurine, a novel endogenous antimicrobial agent: tolerability testing in a mouse model.

OBJECTIVE: To investigate the tolerability of N-chlorotaurine, a new antimicrobial agent, by application to the middle ear in a mouse model. METHODS: Five BALB/c mice were each injected through the tympanic membrane with 5 microL of 0.1%, 1.0%, and 10% N-chlorotaurine and compared with animals in which 0.9% isotonic sodium chloride solution, 0.2% gentamicin sulfate, and 0.25% trimethyltin chloride were instilled. Auditory brainstem responses to clicks were evaluated repeatedly between 4 and 75 days after injection, and histologic investigations of the inner ear were performed subsequently. Three additional groups of mice were injected with isotonic sodium chloride solution, 1.0% N-chlorotaurine, and 0.25% trimethyltin, and brainstem responses to tone bursts of 8, 16, and 32 kHz were tested. In addition, the middle ear was examined histologically. RESULTS: Mice treated with isotonic sodium chloride solution, 0.1% N-chlorotaurine, and 0.2% gentamicin sulfate did not show changes in response threshold. Treatment with 1.0% and 10% N-chlorotaurine caused a reversible increase in auditory brainstem response threshold by 20 dB 4 days after application because of local irritation around the perforation of the tympanic membrane. In contrast, 0.25% trimethyltin showed a permanent elevation of auditory brainstem response threshold of 10 to 15 dB and a scattered loss of outer hair cells predominantly in the apical turn. No alterations of the inner ear were observed in the other treatment groups. The mucous membrane of the middle ear remained unaffected in all test groups. CONCLUSION: Application of N-chlorotaurine to the middle ear is well tolerated without adverse effects and may be a useful new endogenous antimicrobial agent for local treatment of otologic infections.

Trimethyltin reduces ATP levels and MTT reduction in the LRM55 rat astrocytoma cell line.

The LRM55 rat astrocytoma cell line was used to study the time and concentration effects of trimethyltin (TMT) exposure on intracellular adenosine triphosphate (ATP) levels, formazan production from (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) release. TMT concentrations of > or =50 microM produced a delayed increase in extracellular LDH from approximately 20% at 24 h to almost 70%, at 72 h. Twenty-four hours before cell lysis was detectable ATP levels decreased to less than 30% and formazan production declined to 70% (50 microM), 31% (100 microM), and 21% (200 microM) of control values. Concentrations of TMT (5 and 10 microM) that produced little or no LDH release also decreased ATP levels (62 and 49% of control, respectively) and formazan production (63 and 52% of control, respectively) by 48 h. These data support the hypothesis that TMT exposure interferes with energy production and that this event likely contributes to the delayed cell death seen in this cell line. Moreover, the declines in ATP and formazan production can occur at subtoxic concentrations in LRM55 cells.