Attenuation Of Cytotoxicity Research Articles

Inhibitory effects of a water-soluble jujube polysaccharide against biofilm-forming oral pathogenic bacteria

Oral diseases caused by infectious pathogens raises significant concerns in public health. In the light of side effects of current antibiotics therapy and growing drug resistance of pathogenic bacteria, natural products have become attractive alternatives for antibiotics agents in dental practice. This current study investigated the effects of polysaccharides extracted from Zizyphus jujuba Mill. on three major oral biofilm-forming pathogenic bacteria including caries-inducing Streptococcus mutans, lesions-causing MRSA, and periodontitis-related Porphyromonas gingivalis, as well as general oral microbiota. Our results demonstrated that jujube polysaccharide prepared in this study was mainly composed by galacturonic acid with an average molecular weight 242 kDa, which were further characterized for structural features by FT-IR spectra and NMR spectroscopy analysis. This jujube polysaccharide was shown to exhibit remarkable inhibitory effects against all the tested oral bacterial pathogens through various mechanisms including growth inhibition, biofilm prevention and disruption, intervention of bacterial infection (adhesion and invasion), attenuation of cytotoxicity, modulation of excessive inflammatory response of LPS-stimulated and MRSA-infected macrophages as well as positive regulation of oral microbiota. The present study paves the way to explore jujube polysaccharides for the prevention and treatment of oral infectious diseases.Graphic Abstract.

Protective effects of Thai silk sericins and their related mechanisms on UVA-induced phototoxicity and melanogenesis: Investigation in primary melanocyte cells using a proteomic approach

UV radiation causes excess production of melanin as a result of hyperpigmentation and skin disorders. Silk sericin exhibited bioactivities to skin and inhibited UV-induced phototoxicity and melanogenesis in skin cells; however, the mechanism related to sericin against UV-induced melanogenesis has not been investigated. This study aimed to investigate the protective effects of Thai silk sericins against UVA-induced phototoxicity and melanogenesis and their related mechanisms. Thai silk sericins exhibited cytoprotective effects against UV-induced toxicity in human primary melanocytes by attenuation of cytotoxicity, intracellular ROS generation, and mitochondrial potential impairment. Pre- and post-treatment with sericin significantly inhibited melanin synthesis and tyrosinase activity against UVA exposure. In addition, sericin S2 could reduce the basal melanin content in zebrafish embryos. The proteomic analysis demonstrated that Thai silk sericins altered the protein expression in melanocytes especially proteins related to stress, inflammatory, cytokine stimulation, cell proliferation, and cell survival processes that contribute to cytoprotective effect and inhibitory effect on melanogenesis of sericin. Moreover, we demonstrated the novel mechanism of Thai silk sericins in inhibiting UVA-induced melanogenesis via increasing BMP4 expression in MAPK/ERK signaling pathway. These evidences support the potential use of Thai silk sericins in prevention of hyperpigmentation in skin disorders especially after UVA exposure.

Permeabilized Cryopreserved Human Hepatocytes as an Exogenous Metabolic System in a Novel Metabolism-Dependent Cytotoxicity Assay for the Evaluation of Metabolic Activation and Detoxification of Drugs Associated with Drug-Induced Liver Injuries: Results with Acetaminophen, Amiodarone, Cyclophosphamide, Ketoconazole, Nefazodone, and Troglitazone.

We report here a novel in vitro experimental system, the metabolism-dependent cytotoxicity assay (MDCA), for the definition of the roles of hepatic drug metabolism in toxicity. MDCA employs permeabilized cofactor-supplemented cryopreserved human hepatocytes (MetMax Human Hepatocytes, MMHH), as an exogenous metabolic activating system, and human embryonic kidney 293 (HEK293) cells, a cell line devoid of drug-metabolizing enzyme activity, as target cells for the quantification of drug toxicity. The assay was performed in the presence and absence of cofactors for key drug metabolism pathways known to play key roles in drug toxicity: NADPH/NAD+ for phase 1 oxidation, uridine 5'-diphosphoglucuronic acid (UDPGA) for uridine 5'-diphospho-glucuronosyltransferase (UGT) mediated glucuronidation, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) for cytosolic sulfotransferase (SULT) mediated sulfation, and glutathione (GSH) for glutathione S-transferase (GST) mediated GSH conjugation. Six drugs with clinically significant hepatoxicity, resulting in liver failure or a need for liver transplantation: acetaminophen, amiodarone, cyclophosphamide, ketoconazole, nefazodone, and troglitazone were evaluated. All six drugs exhibited cytotoxicity enhancement by NADPH/NAD+, suggesting metabolic activation via phase 1 oxidation. Attenuation of cytotoxicity by UDPGA was observed for acetaminophen, ketoconazole, and troglitazone, by PAPS for acetaminophen, ketoconazole, and troglitazone, and by GSH for all six drugs. Our results suggest that MDCA can be applied toward the elucidation of metabolic activation and detoxification pathways, providing information that can be applied in drug development to guide structure optimization to reduce toxicity and to aid the assessment of metabolism-based risk factors for drug toxicity. GSH detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites, a key property of drugs with idiosyncratic hepatotoxicity. SIGNIFICANCE STATEMENT: Application of the metabolism-dependent cytotoxicity assay (MDCA) for the elucidation of the roles of metabolic activation and detoxification pathways in drug toxicity may provide information to guide structure optimization in drug development to reduce hepatotoxic potential and to aid the assessment of metabolism-based risk factors. Glutathione (GSH) detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites that may be applied toward the evaluation of idiosyncratic hepatotoxicity.

Remifentanil Suppresses Oxidative Stress and Inflammation Induced by Glutamate via Activation of PPARγ/HO-1 Signaling Pathway in Hippocampal Neuronal Cells

Excitotoxicity caused by glutamate severely damages the central nervous system, contributing to the progress of neurodegenerative diseases. Remifentanil is an ultra-short acting synthetic α-opioid receptor agonist and it protects the body against oxidative stress. Oxidative stress is a causative factor for neuronal cell death, contributing to the pathogenesis of neurological diseases. More importantly, remifentanil has been confirmed to have neuroprotective effects on cerebral ischemia. Hence, the aim of the present study was to investigate the molecular mechanism underlying the effect of remifentanil on glutamate (Glu)-induced oxidative stress and inflammation in hippocampal cells. In present study, the cell viability was detected via CCk-8 assay. The cell apoptosis was evaluated by tunel assay. Western blot was performed for measurement of protein expression level. Generation of ROS level was detected by the ROS Activity Assay Kit (KA3842, Abnova) and DCF-DA staining method. MDA and SOD levels were detected by corresponding kits. The results from the present study suggested that remifentanil enhanced cell viability, reduced cell apoptosis rate and prevented oxidative stress in glutamate-induced HT22 cells. The PPARγ/HO-1 pathway was activated by remifentanil. After inhibition of PPARγ/HO-1 pathway, the anti-apoptosis and anti-oxidative stress effects of remifentanil were abolished. In conclusion, remifentanil has anti-apoptosis and anti-oxidative stress effects on glutamate-induced HT22 Cells via PPARγ/HO-1 pathway. Hence, remifentanil is a promising agent for attenuation of cytotoxicity induced by glutamate, providing a new strategy for treatment of excitotoxicity caused by glutamate in the central nervous system.

Cultivation of hMSCs in Human Plasma Prevents the Cytotoxic and Genotoxic Potential of ZnO-NP In Vitro

Zinc oxide nanoparticles (ZnO-NPs) are commonly used for industrial applications. Consequently, there is increasing exposure of humans to them. The in vitro analysis of cytotoxicity and genotoxicity is commonly performed under standard cell culture conditions. Thus, the question arises of how the results of genotoxicity and cytotoxicity experiments would alter if human plasma was used instead of cell culture medium containing of fetal calf serum (FCS). Human mesenchymal stem cells (hMSCs) were cultured in human plasma and exposed to ZnO-NPs. A cultivation in expansion medium made of DMEM consisting 10% FCS (DMEM-EM) served as control. Genotoxic and cytotoxic effects were evaluated with the comet and MTT assay, respectively. hMSC differentiation capacity and ZnO-NP disposition were evaluated by histology and transmission electron microscopy (TEM). The protein concentration and the amount of soluble Zn2+ were measured. The cultivation of hMSCs in plasma leads to an attenuation of genotoxic and cytotoxic effects of ZnO-NPs compared to control. The differentiation capacity of hMSCs was not altered. The TEM showed ZnO-NP persistence in cytoplasm in both groups. The concentrations of protein and Zn2+ were higher in plasma than in DMEM-EM. In conclusion, the cultivation of hMSCs in plasma compared to DMEM-EM leads to an attenuation of cytotoxicity and genotoxicity in vitro.

Activating Transcription Factor 3 regulates in part the enhanced tumour cell cytotoxicity of the histone deacetylase inhibitor M344 and cisplatin in combination

BackgroundActivating Transcription Factor (ATF) 3 is a key regulator of the cellular integrated stress response whose expression has also been correlated with pro-apoptotic activities in tumour cell models. Combination treatments with chemotherapeutic drugs, such as cisplatin, and histone deacetylase (HDAC) inhibitors have been demonstrated to enhance tumour cell cytotoxicity. We recently demonstrated a role for ATF3 in regulating cisplatin-induced apoptosis and others have shown that HDAC inhibition can also induce cellular stress. In this study, we evaluated the role of ATF3 in regulating the co-operative cytotoxicity of cisplatin in combination with an HDAC inhibitor.ResultsThe HDAC inhibitor M344 induced ATF3 expression at the protein and mRNA level in a panel of human derived cancer cell lines as determined by Western blot and quantitative RT-PCR analyses. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatments also enhanced the cytotoxic effects of cisplatin in these cancer cell lines. The mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways and dependent on ATF4, a known regulator of ATF3 expression. ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) as well as chromatin immunoprecipitation (ChIP) assays were utilized in determining the mechanistic induction of ATF3 by M344. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells.ConclusionThis study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.

Vulnerability of glial cells to hydrogen peroxide in cultured hippocampal slices

Massive production of free radicals (FR) has been associated with a variety of pathological conditions in the central nervous system (CNS). We have used the FR generating compound hydrogen peroxide (H 2O 2) in organotypic hippocampal slice cultures to model oxidative injury in the brain. Necrotic cell death was monitored for up to 48 h using propidium iodide (PI) and confocal microscopy. A 1 h exposure to H 2O 2 (0.5–2.5 mM) caused a dose-dependent, and region specific cell death in hippocampal slice cultures. Glial cells demonstrated a high degree of vulnerability to H 2O 2. During the initial 3 h post-injury period, regions of the slice where glial cell bodies predominated showed massive cell death. The majority of neurons in the pyramidal layers were spared, though at later time points they appeared damaged as well. Carboxy-dichlorofluorescein imaging revealed a corresponding early increase in ROS generation in glial cells compared to pyramidal neurons. Immunohistochemistry of PI labeled slices identified astrocytes as the cells most sensitive to H 2O 2 toxicity. In dissociated cell cultures of hippocampal astrocytes and neurons, astrocytes also exhibited a significantly higher sensitivity to H 2O 2 than neurons. Hydrogen peroxide-induced cytotoxicity in all regions of the hippocampal slice culture was significantly attenuated by pre-treatment with antioxidants (α-tocopherol and glutathione), and was not prevented by blockade of Ca 2+ influx, or NMDA channel activation. Cyclosporin A, an inhibitor of mitochondrial permeability transition, reduced cytotoxicity in glial areas by more than 50%, while in the CA2–CA3 pyramidal layers a much smaller, but still significant, attenuation of cytotoxicity was observed. Our results suggest that mitochondria are primary targets of H 2O 2 toxicity, particularly in astrocytes.

Universal and mutation-resistant anti-enteroviral activity: potency of small interfering RNA complementary to the conserved cis-acting replication element within the enterovirus coding region

The promising potential of RNA interference-based antiviral therapies has been well established. However, the antiviral efficacy is largely limited by genomic diversity and genetic instability of various viruses, including human enterovirus B (HEB). In this work, the first evidence supporting the anti-HEB activity of the small interfering RNA (siRNA) targeting the highly conserved cis-acting replication element (CRE) within virus coding region 2C is presented. HeLa cells pre-treated with siRNA complementary to the conserved sequence of the loop region of CRE(2C) were effectively rescued from the cytopathic effects of HEBs. Downregulation of virus replication and attenuation of cytotoxicity were consistently observed in various reference strains and clinical isolates. Cells treated with this siRNA were resistant to the emergence of viable escape mutants and showed sustained antiviral ability. Collectively, the data suggest that the siRNA based on the disordered structure within the highly conserved cis-acting coding region has potential as a universal, persistent anti-HEB agent. The same strategy can be successfully applied to the development of siRNA with consistent antiviral effects in other virus groups possessing similar RNA elements.

Cytochrome P450 2E1 null mice provide novel protection against cisplatin-induced nephrotoxicity and apoptosis

Reactive oxygen metabolites (ROM) are important mediators of cisplatin-induced nephrotoxicity and apoptosis. The site and source of generation of these metabolites are not well defined. Cytochrome P450 (CYP) are heme-containing enzymes that can generate ROM during the oxidative metabolism of exogenous and endogenous compounds. CYP2E1 was identified and localized to the kidney proximal tubule. There is evidence to suggest that CYP2E1 is involved in the generation of ROM. The current study was performed utilizing CYP2e1 null mice (CYP2e1-/-). Cisplatin nephrotoxicity was induced in mice by single intraperitoneal injection of cisplatin and animals were sacrificed 72 hours later. Renal function was assessed and various biochemical tests were performed, including histologic studies. CYP2e1-/- demonstrated marked functional and histologic protection against cisplatin-induced renal injury. Incubation of CYP2e1-/- kidney slices with cisplatin resulted in significant decrease in the generation of ROM and attenuation of cytotoxicity as compared to that of wild-type mice (CYP2e1+/+). Cisplatin-induced apoptosis was also markedly reduced in the CYP2e1-/- mice. Direct incubation of cisplatin with the microsomes isolated from CYP2e1-/- kidney cortex produced significant decrease in the generation of hydrogen peroxide, catalytic iron content, and hydroxyl radical formation compared to CYP2e1+/+ microsomes. Our results thus demonstrate a pivotal role of CYP2E1 in cisplatin-induced nephrotoxicity and apoptosis. We postulate that the interaction of cisplatin with CYP2E1 results in the generation of ROM that causes renal injury and initiates apoptosis.