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Background: Resveratrol, a natural antioxidant polyphenol, has the functions of anti-inflammation, anti-cancer, liver protection and cardioprotection. Microorganism biotransformation-produced resveratrol (MBR) product shows higher purity than the natural source of resveratrol and costs less than the chemically synthesized resveratrol. The aim of the present study was to investigate the protective effects of MBR in hamsters treated with a high-fat diet (HFD). Methods: MBR was obtained by the fermentative process of piceid. Hamsters were randomly divided into four groups: HFD plus oral administration of MBR 0 (C), 5 (L), 20 (M) or 50 mg/kg (H), respectively. After six-week of treatment, hamsters were sacrificed, and tissues were collected for further analysis. Results: MBR at these three dosages did not influence the appetite or growth of the hamsters. Liver enzymes, blood glucose, total cholesterol, triglyceride, and liver weight were significantly reduced in the MBR groups than in the control group. Additionally, high-density lipoprotein-cholesterol (HDL-C) was also elevated in all MBR groups. On the other hand, serum low-density lipoprotein-cholesterol (LDL-C) was decreased in the MBR groups. Triglyceride (TG) in liver tissue and fatty liver level were lower in group H. Memory-associated proteins, phosphorylation of calmodulin-dependent protein kinase II (p-CaMK II) and synaptophysin (SYP), were increased in the brains of MBR groups. Conclusion: The high yield- and short procedure-produced MBR has the potential to protect animals fed with HFD from hyperlipidemia, hepatic steatosis, hyperglycemia, and synaptic impairment, which might be beneficial for patients with these types of diseases.
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Fígado Gorduroso , Hiperlipidemias , Animais , Antioxidantes/farmacologia , Biotransformação , Glicemia/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/farmacologia , HDL-Colesterol , LDL-Colesterol , Cricetinae , Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Hiperlipidemias/tratamento farmacológico , Hiperlipidemias/etiologia , Fígado , Polifenóis/metabolismo , Polifenóis/farmacologia , Resveratrol/farmacologia , Sinaptofisina/metabolismo , TriglicerídeosRESUMO
4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), a major active metabolite of bisphenol A (BPA), is generated in the mammalian liver. Some studies have suggested that MBP exerts greater toxicity than BPA. However, the mechanism underlying MBP-induced pancreatic ß-cell cytotoxicity remains largely unclear. This study demonstrated the cytotoxicity of MBP in pancreatic ß-cells and elucidated the cellular mechanism involved in MBP-induced ß-cell death. Our results showed that MBP exposure significantly reduced cell viability, caused insulin secretion dysfunction, and induced apoptotic events including increased caspase-3 activity and the expression of active forms of caspase-3/-7/-9 and PARP protein. In addition, MBP triggered endoplasmic reticulum (ER) stress, as indicated by the upregulation of GRP 78, CHOP, and cleaved caspase-12 proteins. Pretreatment with 4-phenylbutyric acid (4-PBA; a pharmacological inhibitor of ER stress) markedly reversed MBP-induced ER stress and apoptosis-related signals. Furthermore, exposure to MBP significantly induced the protein phosphorylation of JNK and AMP-activated protein kinase (AMPK)α. Pretreatment of ß-cells with pharmacological inhibitors for JNK (SP600125) and AMPK (compound C), respectively, effectively abrogated the MBP-induced apoptosis-related signals. Both JNK and AMPK inhibitors also suppressed the MBP-induced activation of JNK and AMPKα and of each other. In conclusion, these findings suggest that MBP exposure exerts cytotoxicity on ß-cells via the interdependent activation of JNK and AMPKα, which regulates the downstream apoptotic signaling pathway.
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Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células Secretoras de Insulina/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fenóis/toxicidade , Animais , Sobrevivência Celular , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ratos , Transdução de SinaisRESUMO
BACKGROUND: With advancement of hemodialysis (HD) technique, late fistula failure (LFF) remains a problem significantly affecting life quality of patients. We attempt to identify factors affecting LFF in patients on chronic HD in Taiwan from the National Health Insurance Research Database. METHODS: This case-control study enrolled patients over 18 years old and who received regular HD for more than 3 months. LFF was defined as the first fistula failure episode beyond 3 months of chronic HD. We analyzed characteristics, comorbidities and medicine and investigated the association factors of LFF by logistic regression model. A trend test was conducted for risk in different provider levels. Sensitivity tests were conducted to test consistency. RESULTS: Of 1558 patients recruited, 772 (49.6%) were identified as LFF cases and 786 were identified as controls. The data showed that patients with diabetes mellitus (DM) had 42% increased rate of LFF. Patients receiving more than 10 HD sessions per month had a 90% increased rate of LFF; patients receiving chronic HD in private clinics had a 49% reduction rate of LFF. There were no significant differences in age, dialysis frequency, and comorbidities among different provider levels. There was a significant trend of risk reduction of the event from medical centers, regional hospitals, district hospitals, to private clinics. The sensitivity tests revealed similar results. CONCLUSIONS: The factors associated with LFF include DM and receiving more HD sessions; on the contrary, receiving HD in private clinics is associated with less risk of LFF.
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Derivação Arteriovenosa Cirúrgica/métodos , Falência Renal Crônica/epidemiologia , Falência Renal Crônica/terapia , Vigilância da População , Diálise Renal/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Derivação Arteriovenosa Cirúrgica/efeitos adversos , Estudos de Casos e Controles , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/epidemiologia , Diabetes Mellitus/terapia , Feminino , Fístula , Humanos , Falência Renal Crônica/diagnóstico , Masculino , Pessoa de Meia-Idade , Vigilância da População/métodos , Diálise Renal/efeitos adversos , Fatores de Risco , Taiwan/epidemiologia , Falha de Tratamento , Adulto JovemRESUMO
Molybdenum (Mo), a well-known toxic environmental and industrial pollutant, causes adverse health effects and diseases in humans and has received attention as a potential risk factor for DM. However, the roles of Mo in the mechanisms of the toxicological effects in pancreatic ß-cells are mostly unclear. In this study, the results revealed dysfunction of insulin secretion and apoptosis in the pancreatic ß-cell-derived RIN-m5F cells and the isolated mouse islets in response to Mo. These effects were accompanied by a mitochondria-dependent apoptotic signals including a decreased in the MMP, an increase in cytochrome c release, and the activation of caspase cascades and PARP. In addition, ER stress was triggered as indicated by several key molecules of the UPR. Furthermore, exposure to Mo induced the activation of ERK1/2, JNK, AMPKα, and GSK3-α/ß. Pretreatment with specific pharmacological inhibitors (in RIN-m5F cells and isolated mouse islets) of JNK (SP600125) and AMPK (Compound C) or transfection with si-RNAs (in RIN-m5F cells) specific to JNK and AMPKα effectively prevented the Mo-induced apoptosis and related signals, but inhibitors of ERK1/2 and GSK3-α/ß (PD98059 and LiCl, respectively) did not reverse the Mo-induced effects. Additionally, both the inhibitors and specific si-RNAs could suppress the Mo-induced phosphorylation of JNK and AMPKα each other. Taken together, these results suggest that Mo exerts its cytotoxicity on pancreatic ß-cells by inducing dysfunction and apoptosis via interdependent JNK and AMPK activation downstream-regulated mitochondrial-dependent and ER stress-triggered apoptosis pathways.
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Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Janus Quinases/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Molibdênio/farmacologia , Animais , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , RNA Interferente Pequeno , Transdução de Sinais/efeitos dos fármacosRESUMO
From 1986 to the present, the popular research model organism Caenorhabditis elegans has been thought to completely lack DNA methylation and seems to have lost DNA methylation enzymes from its genomes. In the present study, we report the development of a sensitive and selective assay based on LC-MS/MS to simultaneously measure 5-methyl-2'-deoxycytidine (5-mdC) and 5-hydroxymethyl-2'-deoxycytidine (5-hmdC) in DNA hydrolysates. With the use of isotope internal standards ([2H3]5-mdC and [2H3]5-hmdC) and online solid-phase extraction, the detection limits of 5-mdC and 5-hmdC were estimated to be 0.01 and 0.02 pg respectively, which correspond to a 0.000006% and 0.00001% methylation and hydroxymethylation level. This method was applied to investigate whether DNA methylation/hydroxymethylation exists in C. elegans. The present study for the first time demonstrates that 5-mdC is present in C. elegans genomic DNA (0.0019-0.0033% of cytosine methylated) using LC-MS/MS, whereas another epigenetic modification, 5-hmdC, is not detectable. Furthermore, we found that C. elegans DNA was hypo- or hyper-methylated in a dose-dependent manner by the DNA methyltransferase (DNMT)-inhibiting drug decitabine (5-aza-2'-deoxycytidine) or cadmium respectively. Our data support the possible existence of an active DNA-methylation mechanism in C. elegans, in which unidentified DNMTs could be involved. The present study highlights the importance of re-evaluating the evolutionary conservation of DNA-methylation machinery in nematodes which were traditionally considered to lack functional DNA methylation.
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Caenorhabditis elegans/metabolismo , Citosina/análogos & derivados , Metilação de DNA , DNA/metabolismo , Desoxicitidina/análogos & derivados , Marcação por Isótopo/métodos , Espectrometria de Massas em Tandem/métodos , 5-Metilcitosina/análogos & derivados , Animais , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Cádmio/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Cromatografia Líquida , Citosina/metabolismo , Metilação de DNA/efeitos dos fármacos , Decitabina , Desoxicitidina/metabolismo , Sistemas On-Line , Reprodutibilidade dos Testes , Extração em Fase SólidaRESUMO
In a previous study, treatment at higher concentrations of arsenic trioxide or co-exposure to arsenic trioxide and humic acid was found to be inhibited cell growth of cervical cancer cells (SiHa cells) by reactive oxygen species generation. However, treatment at lower concentrations slightly increased cell viability. Here, we investigate the enhancement of progression effects of environmentally relevant concentration of humic acid and arsenic trioxide in SiHa cell lines in vitro and in vivo by measuring cell proliferation, migration, invasion, and the carcinogenesis-related protein (MMP-2, MMP-9, and VEGF-A) expressions. SiHa cells treated with low concentrations of humic acid and arsenic trioxide alone or in co-exposure significantly increased reactive oxygen species, glutathione levels, cell proliferation, scratch wound-healing activities, migration abilities, and MMP-2 expression as compared to the untreated control. In vivo the tumor volume of either single drug (humic acid or arsenic trioxide) or combined drug-treated group was significantly larger than that of the control for an additional 45 days after tumor cell injection on the back of NOD/SCID mice. Levels of MMP-2, MMP-9, and VEGF-A, also significantly increased compared to the control. Histopathologic effects of all tumor cells appeared round in cell shape with high mitosis, focal hyperkeratosis and epidermal hyperplasia in the skin, and some tumor growth in the muscle were observed. Our results may indicate that exposure to low concentrations of arsenic trioxide and humic acid is associated with the progression of cervical cancer. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1121-1132, 2016.
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Proliferação de Células/efeitos dos fármacos , Substâncias Húmicas/toxicidade , Óxidos/toxicidade , Animais , Apoptose/efeitos dos fármacos , Trióxido de Arsênio , Arsenicais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Glutationa/metabolismo , Células HeLa , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transplante Heterólogo , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Fator A de Crescimento do Endotélio VascularRESUMO
A 62-year-old male undergoing peritoneal dialysis (PD) for over two years presented with sudden bloody peritoneal dialysate, but no other symptoms. Laboratory tests indicated anemia, and a computed tomographic scan revealed a 4.4 cm tumor in the liver with hemoperitoneum, leading to a diagnosis of ruptured hepatocellular carcinoma (HCC), stage IIIB T4N0M0. The patient underwent a successful laparoscopic segmentectomy, and PD was resumed after a month of hemodialysis without complications. This case underscores the importance of considering malignancy in PD patients presenting with hemoperitoneum, as timely detection of HCC can significantly improve prognosis.
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Background: Hemostatic abnormality has contributed to vascular access thrombosis in patients with chronic kidney disease (CKD). Previous studies have demonstrated that far-infrared radiation (FIR) therapy can maintain the patency and maturity of arteriovenous fistulas of patients undergoing hemodialysis (HD). However, prolonged access bleeding is observed once FIR is conducted at the end of dialysis. FIR can block the binding of platelet and von Willebrand factor (vWF), a predictor of hemostatic abnormality and vascular access thrombosis. However, clinical studies exploring FIR and vWF are sparse. Methods: We recruited 20 HD patients, 21 CKD patients, and 20 controls to examine the alteration of vWF and a disintegrin and metalloproteinase with thrombospondin type 1 repeats 13 (ADAMTS13) following a single 40-min session of FIR therapy. In addition, the alteration of these factors in the HD group was examined following a 40-min FIR session thrice a week for 3 months. Results: A decreasing trend in the vWF activity-antigen ratio of participants in all groups following a single FIR session was observed. In addition, the ratio in the HD group was significantly lower following 3 months of FIR therapy. The subgroup analysis revealed a consistent trend and multiple regression analysis showed that participants not taking hydroxymethylglutaryl-coenzyme A reductase inhibitor, diabetes mellitus, and higher hemoglobin levels were the significant factors. The alteration of the vWF activity-antigen ratio correlated moderately to that of ADAMTS13 antigen and activity. Conclusion: FIR may alter the ratio of ultra-large vWF multimers through ADAMTS13, contributing to inhibiting platelet-endothelium interactions of CKD patients.
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Mercury is a toxic heavy metal that is an environmental and industrial pollutant throughout the world. Mercury exposure leads to many physiopathological injuries in mammals. However, the precise toxicological effects of mercury on pancreatic islets in vivo are still unclear. Here, we investigated whether mercuric compounds can induce dysfunction and damage in the pancreatic islets of mice, as well as the possible mechanisms involved in this process. Mice were treated with methyl mercuric chloride (MeHgCl, 2 mg/kg) and mercuric chloride (HgCl(2), 5 mg/kg) for more than 2 consecutive weeks. Our results showed that the blood glucose levels increased and plasma insulin secretions decreased in the mice as a consequence of their exposure. A significant number of TUNEL-positive cells were revealed in the islets of mice that were treated with mercury for 2 consecutive weeks, which was accompanied by changes in the expression of the mRNA of anti-apoptotic (Bcl-2, Mcl-1, and Mdm-2) and apoptotic (p53, caspase-3, and caspase-7) genes. Moreover, plasma malondialdehyde (MDA) levels increased significantly in the mice after treatment with mercuric compounds for 2 consecutive weeks, and the generation of reactive oxygen species (ROS) in the pancreatic islets also markedly increased. In addition, the mRNA expression of genes related to antioxidation, including Nrf2, GPx, and NQO1, were also significantly reduced in these islets. These results indicate that oxidative stress injuries that are induced by mercuric compounds can cause pancreatic islets dysfunction and apoptosis in vivo.
Assuntos
Apoptose/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Cloreto de Mercúrio/toxicidade , Compostos de Metilmercúrio/toxicidade , Animais , Glicemia/análise , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Insulina/sangue , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Malondialdeído/sangue , Cloreto de Mercúrio/química , Compostos de Metilmercúrio/química , Camundongos , Camundongos Endogâmicos ICR , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Pleural effusions (PEs) are common in clinical practice and can be due to many different underlying diseases such as cancer, congestive heart failure, or pneumonia. An accurate differential diagnostic categorization is essential, as the treatment and prognosis of PEs largely depend on its cause. In this study, we tested the hypothesis that nitrite and nitrate concentrations in PEs are associated with the inflammation and infection conditions. We therefore measured the nitrite and nitrate levels in 143 PE samples using a sensitive liquid chromatography-tandem mass spectrometry method and investigated their diagnostic potential in differentiating PEs. The results showed that nitrite concentrations and nitrite/nitrate ratios were higher in exudates than in transudates (NO2-: 2.12 vs. 1.49 µM; NO2-/NO3-: 23.3 vs. 14.0). Both the nitrite concentrations and the nitrite/nitrate ratios were positively correlated with the three Light's criteria. Moreover, the receiver operating characteristic curve analysis revealed that the nitrite/nitrate ratio with an area under the curve of 0.71 could be a potential diagnostic biomarker in separating infectious PEs (IPEs) from other types of PEs. Taken together, the nitrite/nitrate ratio not only reflected the statuses of inflammation, but also the nitrate reduction by pathogenic bacteria infection in the pleural cavity. The nitrite/nitrate ratio could be a better biomarker in the differential diagnosis of PEs than the nitrite concentration alone.
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The COVID-19 pandemic has infected more than 180 million people and caused more than 3.95 million deaths worldwide. In addition to personal hygiene, augmented cleaning, social distancing, and isolation, vaccine development and immunization are essential for this highly contagious disease. Patients with maintenance hemodialysis (MHD) have a greater risk of COVID-19 infection owing to their comorbidities, defective immunity, and repeated crowded in-center dialysis settings. However, many patients hesitate to get vaccinated because of their misunderstandings. The Efficacy of COVID-19 vaccination has been intensively discussed in the general population, whereas the data concerning the effectiveness of vaccination in MHD patients are relatively scanty. Nevertheless, those limited publications can provide some valuable information. Overall, lower and more delayed antibody responses following COVID-19 vaccination were observed in patients with MHD than in healthy controls in the settings of different populations, vaccines and dosage, definitions of the immune response, and antibody detection timepoints. Younger age, previous COVID-19 infection, and higher serum albumin level were positively associated with antibody formation, whereas older age and receiving immunosuppressive therapy were unfavorable factors. However, it remains uncertain between the elicited antibodies following vaccination and the genuine protection against COVID-19 infection. Patients with MHD should make their COVID-19 vaccination a priority in addition to other protective measures. More studies focusing on different vaccines, non-humoral immune responses, and risk-benefit analyses are warranted.
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Bisphenol A (BPA) is recognized as a harmful pollutant in the worldwide. Growing studies have reported that BPA can cause adverse effects and diseases in human, and link to a potential risk factor for development of neurodegenerative diseases (NDs). 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), which generated in the mammalian liver after BPA exposure, is a major active metabolite of BPA. MBP has been suggested to exert greater toxicity than BPA. However, the molecular mechanism of MBP on the neuronal cytotoxicity remains unclear. In this study, MBP exposure significantly reduced Neuro-2a cell viability and induced apoptotic events that MBP (5-15 µM) exhibited greater neuronal cytotoxicity than BPA (50-100 µM). The mitochondria-dependent apoptotic signals including the decrease in mitochondrial membrane potential (MMP) and the increase in cytosolic apoptosis-induced factor (AIF), cytochrome c release, and Bax protein expression were involved in MBP (10 µM)-induced Neuro-2a cell death. Exposure of Neuro-2a cells to MBP (10 µM) also triggered endoplasmic reticulum (ER) stress through the induction of several key molecules including glucose-regulated protein (GRP)78, C/EBP homologous protein (CHOP), X-box binding protein (XBP)-1, protein kinase R-like ER kinase (PERK), eukaryotic initiation factor 2α (eIF2α), inositol-requiring enzyme(IRE)-1, activation transcription factor(AFT)4 and ATF6, and caspase-12. Pretreatment with 4-PBA (an ER stress inhibitor) and specific siRNAs for GRP78, CHOP, and XBP-1 significantly suppressed the expression of these ER stress-related proteins and the activation of caspase-12/-3/-7 in MBP-exposed Neuro-2a cells. Furthermore, MBP (10 µM) exposure dramatically increased the activation of extracellular regulated protein (ERK)1/2 and decreased Akt phosphorylation. Pretreatment with PD98059 (an ERK1/2 inhibitor) and transfection with the overexpression of activation of Akt1 (myr-Akt1) effectively suppressed MBP-induced apoptotic and ER stress-related signals. Collectively, these results demonstrate that MBP exposure exerts neuronal cytotoxicity via the interplay of ERK activation and Akt inactivation-regulated mitochondria-dependent and ER stress-triggered apoptotic pathway, which ultimately leads to neuronal cell death.
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Apoptose/efeitos dos fármacos , Compostos Benzidrílicos/toxicidade , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fenóis/toxicidade , Animais , Compostos Benzidrílicos/administração & dosagem , Linhagem Celular Tumoral , Citocromos c/efeitos dos fármacos , Relação Dose-Resposta a Droga , Chaperona BiP do Retículo Endoplasmático , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismo , Neurônios/patologia , Fenóis/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Mercury is a well-known highly toxic metal. In this study, we characterize and investigate the cytotoxicity and its possible mechanisms of inorganic mercury in pancreatic beta-cells. Mercury chloride (HgCl2) dose-dependently decreased the function of insulin secretion and cell viability in pancreatic beta-cell-derived HIT-T15 cells and isolated mouse pancreatic islets. HgCl2 significantly increased ROS formation in HIT-T15 cells. Antioxidant N-acetylcysteine effectively reversed HgCl2-induced insulin secretion dysfunction in HIT-T15 cells and isolated mouse pancreatic islets. Moreover, HgCl2 increased sub-G1 hypodiploids and annexin-V binding in HIT-T15 cells, indicating that HgCl2 possessed ability in apoptosis induction. HgCl2 also displayed several features of mitochondria-dependent apoptotic signals including disruption of the mitochondrial membrane potential, increase of mitochondrial cytochrome c release and activations of poly (ADP-ribose) polymerase (PARP) and caspase 3. Exposure of HIT-T15 cells to HgCl2 could significantly increase both apoptotic and necrotic cell populations by acridine orange/ethidium bromide dual staining. Meanwhile, HgCl2 could also trigger the depletion of intracellular ATP levels and increase the LDH release from HIT-T15 cells. These HgCl2-induced cell death-related signals could be significantly reversed by N-acetylcysteine. The intracellular mercury levels were markedly elevated in HgCl2-treated HIT-T15 cells. Taken together, these results suggest that HgCl2-induced oxidative stress causes pancreatic beta-cell dysfunction and cytotoxicity involved the co-existence of apoptotic and necrotic cell death.
Assuntos
Apoptose/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Cloreto de Mercúrio/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Acetilcisteína/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Antioxidantes/farmacologia , Western Blotting , Morte Celular/efeitos dos fármacos , Linhagem Celular , Separação Celular , Cricetinae , DNA/biossíntese , DNA/genética , Citometria de Fluxo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , L-Lactato Desidrogenase/metabolismo , Camundongos , Membranas Mitocondriais/efeitos dos fármacos , Necrose , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Determination of the modulation of nitrite and nitrate levels in biological samples usually poses a major challenge, owing to their high background concentrations. To effectively investigate the variation of nitrite/nitrate in vivo, in this study, we developed a15N-labelled nitrite/nitrate tracer analysis using LC-MS/MS following the derivatization with 2,3-diaminonaphthalene. This method allows for the determination of 15N-labelled nitrite/nitrate as 15N-2,3-naphthotriazole (15N-NAT) that can efficiently differentiate newly introduced nitrite/nitrate from the background nitrite/nitrate in biological matrices. We also investigated the contribution of background 14N-NAT isotopomers to 15N-NAT, which has long been overlooked in the literature. Our results indicated that the contribution of background 14N-NAT isotopomers to 15N-NAT is significant. Such contribution is constant (~2.2% under positive ion mode and 1.1% under negative ion mode), and does not depend upon the concentration of 14N-NAT or the sample matrix measured. An equation has been therefore developed, for the first time, to correct the contribution of background 14N-NAT isotopomers to 15N-NAT. With the proposed 15N-labelled nitrite/nitrate tracer analysis, the amount and percentage distribution of 15NO2- and 15NO3-, both in urine and feces, after oral administration of 15N-labelled nitrite/nitrate are clearly demonstrated. The excretions of 15NO2- and 15NO3- were significantly increased with the increasing dose implying that the dietary nitrite/nitrate intake is an important source in urine/feces. The present method allows for the simple, reliable and accurate quantification of 15NO2- and 15NO3-, and it should also be useful to trace the biotransformation of nitrite and nitrate in vivo.
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Naftalenos/farmacocinética , Nitratos/metabolismo , Nitritos/metabolismo , Isótopos de Nitrogênio/farmacocinética , Triazóis/farmacocinética , Administração Oral , Animais , Transporte Biológico , Biotransformação , Cromatografia Líquida , Fezes , Radicais Livres , Masculino , Camundongos , Camundongos Endogâmicos ICR , Naftalenos/urina , Espectrometria de Massas em Tandem , Triazóis/administração & dosagem , Triazóis/urinaRESUMO
BACKGROUND: Cardiovascular (CV) disease contributes to nearly half of the mortalities in patients with end-stage renal disease. Patients who received prehemodialysis arteriovenous access (pre-HD AVA) creation had divergent CV outcomes. METHODS: We conducted a population-based cohort study by recruiting incident patients receiving HD from 2001 to 2012 from the Taiwan National Health Insurance Research Database. Patients' characteristics, comorbidities, and medicines were analyzed. The primary outcome of interest was major adverse cardiovascular events (MACEs), defined as hospitalization due to acute myocardial infarction, stroke, or congestive heart failure (CHF) occurring within the first year of HD. Secondary outcomes included MACE-related mortality and all-cause mortality in the same follow-up period. RESULTS: The patients in the pre-HD AVA group were younger, had a lower burden of underlying diseases, were more likely to use erythropoiesis-stimulating agents but less likely to use renin-angiotensin-aldosterone system blockers. The patients with pre-HD AVA creation had a marginally lower rate of MACEs but a significant 35% lower rate of CHF hospitalization than those without creation (adjusted hazard ratio (HR) 0.65, 95% confidence interval (CI) [0.48-0.88]). In addition, the pre-HD AVA group exhibited an insignificantly lower rate of MACE-related mortality but a significantly 52% lower rate of all-cause mortality than the non-pre-HD AVA group (adjusted HR 0.48, 95% CI [0.39-0.59]). Sensitivity analyses obtained consistent results. CONCLUSIONS: Pre-HD AVA creation is associated with a lower rate of CHF hospitalization and overall death in the first year of dialysis.
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5-Fluorouracil (5-FU) is used in the treatment of head and neck cancer patients. However, adverse effects experienced such as mucositis and poor appetite may lead to interruption in chemotherapy. The aim of this study is to evaluate the efficacy of GMI, one fungal immunomodulatory protein found in Ganoderma microsporum, for mucositis induced by 5-FU in a mouse model. Mice were administered 5-FU intraperitoneally for 4 days per cycle for a total of 2 chemotherapy cycles. In addition, mice were pretreated with GMI or phosphate-buffered saline 3 days before 5-FU intraperitoneal injection and daily until day 14. On histological analysis, GMI prevented 5-FU-induced damage to the intestinal mucosa and tongue epithelium. We also demonstrated that GMI enhanced the cytotoxicity of 5-FU in 2 oral cancer cell lines, while GMI could not promote this effect in an oral normal cell. In conclusion, GMI alleviates 5-FU-induced damage and decelerates cell death in normal alimentary tract tissue.
Assuntos
Antimetabólitos Antineoplásicos/efeitos adversos , Fluoruracila/efeitos adversos , Ganoderma/metabolismo , Fatores Imunológicos/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosite/induzido quimicamente , Mucosite/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microsporum/efeitos dos fármacosRESUMO
Epidemiological studies have positively linked mercury exposure and neurodegenerative diseases (ND). Methylmercury (MeHg), an organic form of mercury, is a ubiquitous and potent environmental neurotoxicant that easily crosses the blood-brain barrier and causes irreversible injury to the central nervous system (CNS). However, the molecular mechanisms underlying MeHg-induced neurotoxicity remain unclear. Here, the present study found that Neuro-2a cells underwent apoptosis in response to MeHg (1-5 µM), which was accompanied by increased phosphatidylserine (PS) exposure on the outer cellular membrane leaflets, caspase-3 activity, and the activation of caspase cascades and poly (ADP-ribose) polymerase (PARP). Exposure of Neuro-2a cells to MeHg also triggered endoplasmic reticulum (ER) stress, which was identified via several key molecules (including: glucose-regulated protein (GRP)78, GRP94, C/EBP homologous protein (CHOP) X-box binding protein(XBP)-1, protein kinase R-like ER kinase (PERK), eukaryotic initiation factor 2α (eIF2α), inositol-requiring enzyme(IRE)-1, activation transcription factor(AFT)4, and ATF6. Transfection with GRP78-, GRP94-, CHOP-, and XBP-1-specific small interfering (si)RNA significantly suppressed the expression of these proteins, and attenuated cytotoxicity and caspase-12, -7, and -3 activation in MeHg-exposed cells. Furthermore, MeHg dramatically decreased Akt phosphorylation, and the overexpression of activation of Akt1 (myr-Akt1) could significantly prevent MeHg-induced Akt inactivation, as well as apoptotic and ER stress-related signals. Pretreatment with the antioxidant N-acetylcysteine (NAC) effectively prevented MeHg-induced neuronal cell reactive oxygen species (ROS) generation, apoptotic and ER stress-related signals, and Akt inactivation. Collectively, these results indicate that MeHg exerts its cytotoxicity in neurons by inducing ROS-mediated Akt inactivation up-regulated ER stress, which induces apoptosis and ultimately leads to cell death.
Assuntos
Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Compostos de Metilmercúrio/toxicidade , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Western Blotting , Caspase 3/metabolismo , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Camundongos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
A highly sensitive quantitative method based on LC-MS/MS was developed to directly measure 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 2'-deoxyguanosine (dG) in crude DNA hydrolysates. With the use of isotopic internal standards and online solid-phase extraction (SPE), this method has overcome the artifactual response often observed during electrospray ionization by optimizing the washing conditions of online SPE to remove excess dG and allows 8-oxodG and dG to be accurately and simultaneously monitored by mass spectrometry. The detection limit of this method was estimated as 1.8 fmol for 8-oxodG. With this method, we further investigated the artifactual oxidation that occurred during concentration and purification of the DNA hydrolysates, commonly used before sample analysis. Our results demonstrated that drying under vacuum or purification with C18 cartridges led to a significant increase in the measured 8-oxodG by 6.8-30 8-oxodG/10(6) dG. The artifactual formation of 8-oxodG can be reduced only by adding desferrioxamine (DFO) and not 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). However, DFO still failed to offer complete protection against oxidation during DNA hydrolysate concentration and purification. Therefore, to effectively prevent the artifacts formed during workup, the simplest approach is to use a direct measurement method involving an online enrichment/purification technique as proposed in this study.
Assuntos
Artefatos , Cromatografia Líquida/métodos , DNA/química , Desoxiguanosina/análogos & derivados , Espectrometria de Massas/métodos , 8-Hidroxi-2'-Desoxiguanosina , Animais , Óxidos N-Cíclicos/química , Desferroxamina/química , Desoxiguanosina/análise , Hidrólise , Camundongos , Sistemas On-Line , Oxirredução , Técnica de Diluição de Radioisótopos , Extração em Fase SólidaRESUMO
In the present study, we report the development of a sensitive and selective assay based on LC (liquid chromatography)-MS/MS (tandem MS) to simultaneously measure N7-MeG (N7-methylguanine) and N7-EtG (N7-ethylguanine) in DNA hydrolysates. With the use of isotope internal standards (15N5-N7-MeG and 15N5-N7-EtG) and on-line SPE (solid-phase extraction), the detection limit of this method was estimated as 0.42 fmol and 0.17 fmol for N7-MeG and N7-EtG respectively. The high sensitivity achieved here makes this method applicable to small experimental animals. This method was applied to measure N7-alkylguanines in liver DNA from mosquito fish (Gambusia affinis) that were exposed to NDMA (N-nitrosodimethylamine) and NDEA (N-nitrosodiethylamine) alone or their combination over a wide range of concentrations (1-100 mg/l). Results showed that the background level of N7-MeG in liver of control fish was 7.89+/-1.38 mmol/mol of guanine, while N7-EtG was detectable in most of the control fish with a range of 0.05-0.19 mmol/mol of guanine. N7-MeG and N7-EtG were significantly induced by NDMA and NDEA respectively, at a concentration as low as 1 mg/l and increased in a dose-dependent manner. Taken together, this LC-MS/MS assay provides the sensitivity and high throughput required to evaluate the extent of alkylated DNA lesions in small animal models of cancer induced by alkylating agents.