Impact of capsaicin concentration evoking coughs on clinical variables in patients with asthma.

BACKGROUND: Inhaled capsaicin (8-methyl-N-vanillyl-6-nonenamide) has been used to induce cough in a safe and dose-dependent manner. Chronic cough is associated with an increased sensitivity to inhaled capsaicin in patients with asthma. The aim of this study was to evaluate clinical impact of capsaicin provocation test for chronic cough, and to find relationship between capsaicin concentration producing coughs and clinical variables in patients with asthma. Methods: 385 patients with chronic cough [capsaicin provocation test (+, n = 152)] vs. [capsaicin provocation test (-, n = 233)] who has done with capsaicin provocation test recruited and evaluated by asthma diagnosis and clinical variables. Asthma diagnoses were based on the Global Initiative for Asthma guidelines. Results: Capsaicin positivity was more prevalent in patient with asthma diagnosis than in patients without asthma diagnosis (129/304 vs. 24/81, p = 0.037). Capsaicin positivity was more prevalent in female patients than in male patients (123/271 = 45.4% vs. 30/114 = 26.3%, p = 0.001). Capsaicin concentration producing coughs correlated with smoke amount (r = 0.126, p = 0.014). Capsaicin positivity was more prevalent in nonsmoker patients than in smoker patients (133/295 = 45.1% vs. 20/90 = 22.2%, p = 0.001). Capsaicin concentration producing coughs negatively correlated with methacholine PC20 (4 mg mL-1, p = 0.037), (16 mg mL-1, p = 0.069) and (20 mg mL-1, p = 0.045). Capsaicin concentration producing coughs correlated with BMI (r = 0.120, p = 0.019). Capsaicin concentration producing coughs negatively correlated with FEV1/FVC % pred. (r = -0.137, p = 0.007). There was no relationship between capsaicin concentration producing coughs and age, IgE, and atopy. Conclusions: Capsaicin test for asthma diagnosis should be considered for variable clinical factors. Key message Cough in asthmatic patients is not only common and troublesome but also predicts disease severity and poor prognosis. The capsaicin cough challenge test is a simple and reproducible provocation method for assessing cough susceptibility in patients with cough. Capsaicin test for asthma diagnosis should be considered for variable clinical factors.

Coix lacryma-jobi var. ma-yuen Stapf sprout extract has anti-metastatic activity in colon cancer cells in vitro.

BACKGROUND: Coix lacryma-jobi var. ma-yuen (Rom.Caill.) Stapf has been used in China as an herbal medicine. Many studies of this plant have reported anti-proliferative and apoptotic activities on human cancer cell lines. Therefore, this study of the anti-metastatic effect of Coix lacryma-jobi var. ma-yuen Stapf sprout extract (CLSE) in colorectal cancer cells may provide a scientific basis for exploring anti-cancer effects of edible crops. METHODS: To evaluate the effect of CLSE on cell proliferation and signaling, we performed a Cell Counting Kit-8 (CCK-8) assay in HCT116 cells and used western blot analysis. Furthermore, scratch-wound healing, transwell migration, matrigel invasion, and adhesion assays were conducted to elucidate the anti-metastatic effects of CLSE under hypoxic conditions in colon cancer cells. RESULTS: First, CLSE decreased deferoxamine (DFO)-induced migration of colon cancer cells by 87%, and blocked colon cancer cell migration by 80% compared with hypoxia control cells. Second, CLSE treatment resulted in a 54% reduction in hypoxia-induced invasiveness of colon cancer cells, and 50% inhibition of adhesive potency through inactivation of the extracellular signal-regulated kinase (ERK) 1/2 and protein kinase b (AKT) pathways. Third, conditioned medium collected from CLSE-treated HCT116 cells suppressed tube formation of human umbilical vein endothelial cells (HUVECs) by 91%. CONCLUSIONS: CLSE inhibited migration, invasion, and adhesion of colon cancer cells and tube formation by HUVECs via repression of the ERK1/2 and AKT pathways under hypoxic conditions. Therefore, CLSE may be used to treat patients with colon cancer.

Trifloxystrobin induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in HaCaT, human keratinocyte cells.

As the outermost layer of the body, the skin plays an important role in exposure to pesticides, which could have negative impacts on human health. Trifloxystrobin is a widely used fungicide of the strobilurin class, however, there is little information regarding the skin contact-associated toxic mechanism. Therefore, the present study was performed in order to identify the skin toxicity mechanism of trifloxystrobin using HaCaT (keratinocyte of human skin) cells. Following 24 or 48 h treatment, cell viability, and subsequent Annexin V-FITC/propidium iodide assay, TUNEL assay and Western blotting were performed to investigate the cell death mechanism of trifloxystrobin. Exposure to trifloxystrobin resulted in diminished viability of HaCaT cells in both a time- and concentration-dependent manner. The cell death was derived through apoptotic pathways in the HaCaT cells. Furthermore, we explored the effect of trifloxystrobin on TRAIL-mediated extrinsic apoptosis using siRNA transfection. Knockdown of death receptor 5 suppressed trifloxystrobin-provoked apoptosis. These results indicate that trifloxystrobin induces TRAIL-mediated apoptosis and has an inhibitory effect in keratinocytes that can interfere with the barrier function and integrity of the skin. This could be proposed as a mechanism of skin toxicity by trifloxystrobin and considered in the management of pesticide exposure.

Removal of SDS from biological protein digests for proteomic analysis by mass spectrometry.

BACKGROUND: Metal-organic frameworks (MOFs - MIL-101) are the most exciting, high profiled developments in nanotechnology in the last ten years, and it attracted considerable attention owing to their uniform nanoporosity, large surface area, outer-surface modification and in-pore functionality for tailoring the chemical properties of the material for anchoring specific guest moieties. MOF's have been particularly highlighted for their excellent gas storage and separation properties. Recently biomolecules-based MOF's were used as nanoencapsulators for antitumor and antiretroviral controlled drug delivery studies. However, usage of MOF material for removal of ionic detergent-SDS from biological samples has not been reported to date. Here, first time we demonstrate its novel applications in biological sample preparation for mass spectrometry analysis. METHODS: SDS removal using MIL-101 was assessed for proteomic analysis by mass spectrometry. We analysed removal of SDS from 0.5 % SDS solution alone, BSA mixture and HMEC cells lysate protein mixture. The removal of SDS by MIL-101 was confirmed by MALDI-TOF-MS and LC-MS techniques. RESULTS: In an initial demonstration, SDS has removed effectively from 0.5 % SDS solution by MIL-101via its binding attraction with SDS. Further, the experiment also confirmed that MIL-101 strongly removed the SDS from BSA and cell lysate mixtures. CONCLUSIONS: These results suggest that SDS removal by the MIL-101 method is a practical, simple and broad applicable in proteomic sample processing for MALDI-TOF-MS and LC-MS analysis.

Meloxicam inhibits fipronil-induced apoptosis via modulation of the oxidative stress and inflammatory response in SH-SY5Y cells.

Oxidative stress and inflammatory responses have been identified as key elements of neuronal cell apoptosis. In this study, we investigated the mechanisms by which inflammatory responses contribute to apoptosis in human neuroblastoma SH-SY5Y cells treated with fipronil (FPN). Based on the cytotoxic mechanism of FPN, we examined the neuroprotective effects of meloxicam against FPN-induced neuronal cell death. Treatment of SH-SY5Y cells with FPN induced apoptosis via activation of caspase-9 and -3, leading to nuclear condensation. In addition, FPN induced oxidative stress and increased expression of cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNF-α) via inflammatory stimulation. Pretreatment of cells with meloxicam enhanced the viability of FPN-exposed cells through attenuation of oxidative stress and inflammatory response. FPN activated mitogen activated protein kinase (MAPK) and inhibitors of MAPK abolished FPN-induced COX-2 expression. Meloxicam also attenuated FPN-induced cell death by reducing MAPK-mediated pro-inflammatory factors. Furthermore, we observed both nuclear accumulation of p53 and enhanced levels of cytosolic p53 in a concentration-dependent manner after FPN treatment. Pretreatment of cells with meloxicam blocked the translocation of p53 from the cytosol to the nucleus. Together, these data suggest that meloxicam may exert anti-apoptotic effects against FPN-induced cytotoxicity by both attenuating oxidative stress and inhibiting the inflammatory cascade via inactivation of MAPK and p53 signaling.

Pulmonary Proteome and Protein Networks in Response to the Herbicide Paraquat in Rats.

Paraquat (PQ) has been one of the most widely used herbicides in the world. PQ, when ingested, is toxic to humans and may cause acute respiratory distress syndrome. To investigate molecular perturbation in lung tissues caused by PQ, Sprague Dawley male rats were fed with PQ at a dose of 25 mg/kg body weight for 20 times in four weeks. The effects of PQ on cellular processes and biological pathways were investigated by analyzing proteome in the lung tissues in comparison with the control. Among the detected proteins, 321 and 254 proteins were over-represented and under-represented, respectively, in the PQ-exposed rat lung tissues in comparison with the no PQ control. All over- and under-represented proteins were subjected to Ingenuity Pathway Analysis to create 25 biological networks and 38 pathways of interacting protein clusters. Over-represented proteins were involved in the C-jun-amino-terminal kinase pathway, caveolae-mediated endocytosis signaling, cardiovascular-cancer-respiratory pathway, regulation of clathrin-mediated endocytosis, non-small cell lung cancer signaling, pulmonary hypertension, glutamate receptor, immune response and angiogenesis. Under-represented proteins occurred in the p53 signaling pathway, mitogen-activated protein kinase signaling pathway, cartilage development and angiogenesis inhibition in the PQ-treated lungs. The results suggest that PQ may generate reactive oxygen species, impair the MAPK/p53 signaling pathway, activate angiogenesis and depress apoptosis in the lungs.

Chlorpyrifos induces NLRP3 inflammasome and pyroptosis/apoptosis via mitochondrial oxidative stress in human keratinocyte HaCaT cells.

Chlorpyrifos (CPF) has been widely used around the world as a pesticide for both agricultural and residential application. Although various studies have reported toxicity and health-related effects from CPF exposure, the molecular mechanism of CPF toxicity to skin has not been well-characterized. The present study determined the potential mechanism involved in skin toxicity of CPF using the HaCaT human skin keratinocyte cell line. After treating to HaCaT cells, CPF triggered reactive oxygen species (ROS) generation and mitochondrial oxidative stress. We focused on NLRP3 inflammasome, known to induce innate immune response. We used mitochondrial ROS (mROS) scavenger mitoTEMPO to demonstrate a role for mROS in NLRP3 inflammasome and programmed cell death induced by CPF. Our results showed that CPF provoked NLRP3 inflammasome and pyroptosis/apoptosis via an increase of mROS in HaCaT cells. This study proposes that CPF induces innate immune response and skin inflammation through activating the NLRP3 inflammasome in skin epithelial cells. CPF may lead to cutaneous disease conditions and antioxidants could be proposed for therapy against skin exposure to CPF.

Comparison of international guidelines of dermal absorption tests used in pesticides exposure assessment for operators.

The number of farmers who have suffered from non-fatal acute pesticide poisoning has been reported to vary from 5.7% to 86.7% in South Korea since 1975. Absorption through the skin is the main route of exposure to pesticides for farmers who operate with them. Several in vitro tests using the skins of humans or animal and in vivo tests using laboratory animals are introduced for the assessment of human dermal absorption level of pesticides. The objective of this study is to evaluate and compare international guidelines and strategies of dermal absorption assessments and to propose unique approaches for applications into pesticide registration process in our situation. Until present in our situation, pesticide exposure level to operator is determined just using default value of 10 as for skin absorption ratio because of data shortage. Dermal absorption tests are requested to get exposure level of pesticides and to ultimately know the safety of pesticides for operators through the comparison with the value of AOEL. When the exposure level is higher than AOEL, the pesticide cannot be approved. We reviewed the skin absorption test guidelines recommended by OECD, EFSA and EPA. The EPA recommends assessment of skin absorption of pesticides for humans through the TPA which includes all the results of in vitro human and animal and animal in vivo skin absorption studies. OECD and EFSA, employ a tiered approach, which the requirement of further study depends on the results of the former stage study. OECD guidelines accept the analysis of pesticide level absorbed through skin without radioisotope when the recovery using the non-labeled method is within 80~120%. Various factors are reviewed in this study, including the origin of skin (gender, animal species and sites of skin), thickness, temperature and, etc., which can influence the integrity of results.

Potential autophagy enhancers protect against fipronil-induced apoptosis in SH-SY5Y cells.

Oxidative stress created by environmental toxicants activates several signaling pathways. Autophagy is one of the first lines of defense against oxidative stress damage. The autophagy pathway can be induced and up-regulated in response to intracellular reactive oxygen species (ROS). Recently, we reported that fipronil (FPN)-induced mitochondria-dependent apoptosis is mediated through ROS in human neuroblastoma SH-SY5Y cells. In this study, we explored the role of autophagy to prevent FPN neurotoxicity. We investigated the modulation of FPN-induced apoptosis according to autophagy regulation. FPN activated caspase-9 and caspase-3, and induced nuclear fragmentation and condensation, all of which indicate that FPN-induced cell death was due to apoptosis. In addition, we observed FPN-induced autophagic cell death by monitoring the expression of LC3-II and Beclin-1. Exposure to FPN in SH-SY5Y cells led to the production of ROS. Treatment with N-acetyl-cysteine (NAC) effectively blocked both apoptosis and autophagy. Interestingly, pretreatment with rapamycin, an autophagy inducer, significantly enhanced the viability of FPN-exposed cells; the enhancement of cell viability was partially due to alleviation of FPN-induced apoptosis via a decrease in levels of cleaved caspase-3. However, pretreatment with 3-methyladenine (3MA) a specific inhibitor for autophagy, remarkably strengthened FPN toxicity and further induced activation of caspase-3 in these cells. Our studies suggest that FPN-induced cytotoxicity is modified by autophagy regulation and that rapamycin is neuroprotective against FPN-induced apoptosis through enhancing autophagy.