RESUMEN
BACKGROUND: Neuropathic pain is a common side-effect of chemotherapy. Although precise mechanisms are unclear, oxidative stress and mitochondrial damage are involved. We investigated whether the mitochondria targeted antioxidant, MitoVitE, provided better protection against paclitaxel-induced mitochondrial damage in rat dorsal root ganglion (DRG) cells, than a non-targeted form of vitamin E, Trolox. We also determined whether MitoVitE, compared with duloxetine, could limit paclitaxel-induced mechanical hypersensitivity in rats. METHODS: Mitochondrial function was measured in DRG cells exposed to paclitaxel with and without MitoVitE or Trolox. The effect of MitoVitE or Trolox on paclitaxel-induced cell killing in cancer cell lines was also determined. Rats received a cumulative dose of 8 mg kg-1 paclitaxel plus either MitoVitE (2 mg-1 kg day-1), duloxetine (10 mg kg-1 day-1) or vehicle control daily. Mechanical hind paw withdrawal thresholds were measured every two days. RESULTS: Paclitaxel caused loss of membrane potential in DRG cells. At 100 µM paclitaxel median [range] change was 61[44-78]%, P < 0.0001, which was ameliorated by MitoVitE (86[62-104]%) but not Trolox (46[46-57]%). Similarly, loss of metabolic activity and glutathione induced by paclitaxel (both P < 0.0001) were reduced by MitoVitE but not Trolox. Cytotoxicity of paclitaxel was not affected by co-exposure of ovarian cancer cells to either MitoVitE or Trolox, but was slightly reduced against breast cancer cells, in the presence of Trolox. Mean (SD) areas under the curve of withdrawal thresholds at 6 h after injection in rats given paclitaxel + control, or + MitoVitE (P < 0.0001) or + duloxetine (P < 0.0001) were 110 (5), 145 (10) and 156 (13) respectively. CONCLUSIONS: Paclitaxel affected mitochondrial function and glutathione in DRG cells, which was abrogated by MitoVitE but not Trolox, without decreasing cancer cell cytotoxicity. In rats, paclitaxel-induced mechanical hypersensitivity was ameliorated by MitoVitE treatment to an extent similar to duloxetine. These data confirm mitochondria as a mechanistic target for paclitaxel-induced damage and suggest mitochondria targeted antioxidants as future therapeutic strategies.
Asunto(s)
Hiperalgesia/prevención & control , Mitocondrias/efectos de los fármacos , Neuralgia/prevención & control , Compuestos Organofosforados/farmacología , Estrés Oxidativo/efectos de los fármacos , Paclitaxel/efectos adversos , Animales , Antineoplásicos Fitogénicos/efectos adversos , Antioxidantes/farmacología , Modelos Animales de Enfermedad , Técnicas In Vitro , Masculino , Ratas , Ratas Sprague-Dawley , Ubiquinona/farmacologíaRESUMEN
BACKGROUND: Mitochondrial oxidative stress has a role in sepsis-induced organ dysfunction. The endogenous mechanisms to initiate protective pathways are controlled by peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC1α) and nuclear factor erythroid 2-like 2 (NFE2L2). Activation of these pathways are potential therapeutic targets in sepsis. We used pharmacological activators to determine the effects on markers of mitochondrial damage and inflammation in human endothelial cells under conditions of sepsis. METHODS: Human endothelial cells were exposed to lipopolysaccharide plus peptidoglycan G to mimic a sepsis environment, with a range of concentrations of a selective synthetic agonist of silent information regulator-1 (SIRT-1) which activates PGC1α, or bis(2-hydroxy-benzylidene) acetone (2HBA) which activates NFE2L2, with and without inhibitors of these pathways. Cells were cultured for up to seven days and we measured mitochondrial membrane potential, metabolic activity, and density (as a marker of biogenesis), interkeukin-6 (to reflect inflammation) and glutathione (as a measure of antioxidant status). RESULTS: Under conditions mimicking sepsis, activation of the PGC1α and NFE2L2 pathways protected cells from LPS/PepG-induced loss of mitochondrial membrane potential (P=0.0002 and P=0.0009, respectively) and metabolic activity (P=0.05 and P<0.0001, respectively), and dampened interleukin-6 responses (P=0.003 and P=0.0001, respectively). Mitochondrial biogenesis (both P=0.0001) and glutathione (both P<0.0001) were also increased. These effects were blunted by the respective inhibitors. CONCLUSIONS: The development of organ dysfunction during human sepsis is linked to mitochondrial dysfunction, and so activation of PGC1α/NFE2L2 is likely to be beneficial. These pathways are attractive therapeutic targets for sepsis.
Asunto(s)
Antioxidantes/farmacología , Inflamación/metabolismo , Estrés Oxidativo/efectos de los fármacos , Sepsis/metabolismo , Acetona/análogos & derivados , Acetona/farmacología , Compuestos de Bencilo/farmacología , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Humanos , Interleucina-6/metabolismo , Mitocondrias/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Sirtuina 1/antagonistas & inhibidores , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Oxidative stress with dysregulated inflammation are hallmarks of sepsis. Zinc and selenium have important antioxidant functions, such that they could be important in patients with sepsis. We used an in vitro approach to assess the effect of zinc and selenium on oxidative stress, mitochondrial function, and inflammatory responses in conditions mimicking sepsis and related the findings to plasma concentrations and biomarkers in patients with and without sepsis. METHODS: Human endothelial cells were exposed to a range of zinc and selenium concentrations in conditions mimicking sepsis. Zinc, selenium, and a series of biomarkers of oxidative stress and inflammation were measured in plasma from critically ill patients with and without sepsis. RESULTS: Culturing cells with different concentrations of zinc caused altered zinc transporter protein expression and cellular zinc content, and selenium affected glutathione peroxidase 3 activity. Although zinc or selenium at physiological concentrations had no effect on interleukin-6 release in vitro, higher concentrations of the trace elements were associated with improved mitochondrial function. Plasma zinc and selenium concentrations were low in patients [zinc: median (range) 4.6 (2.1-6.5) µM in control patients without sepsis and 3.1 (1.5-5.4) µM in patients with sepsis, P=0.002; and selenium: 0.78 (0.19-1.32) µM in control patients and 0.42 (0.22-0.91) µM in sepsis patients, P=0.0009]. Plasma concentrations of interleukin-6, other biomarkers of inflammation, and markers of oxidative damage to proteins and lipids were elevated, particularly in patients with sepsis, and were inversely related to plasma zinc and selenium concentrations. CONCLUSIONS: Zinc and selenium concentrations were reduced in critically ill patients, with increased oxidative stress and inflammatory biomarkers, particularly in patients with sepsis. Oxidative stress as a result of suboptimal selenium and zinc concentrations might contribute to damage of key proteins. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov: registration number NCT01328509.
Asunto(s)
Antioxidantes/metabolismo , Inflamación/metabolismo , Estrés Oxidativo , Selenio/deficiencia , Sepsis/metabolismo , Zinc/deficiencia , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Células Cultivadas , Enfermedad Crítica , Células Endoteliales/efectos de los fármacos , Femenino , Glutatión Peroxidasa/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inflamación/sangre , Inflamación/patología , Interleucina-6/metabolismo , Masculino , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Selenio/sangre , Selenio/metabolismo , Sepsis/sangre , Sepsis/patología , Adulto Joven , Zinc/sangre , Zinc/metabolismoRESUMEN
BACKGROUND: Sepsis-induced organ failure is the major cause of death in critical care units, and is characterized by a massive dysregulated inflammatory response and oxidative stress. We investigated the effects of treatment with antioxidants that protect mitochondria (MitoQ, MitoE, or melatonin) in a rat model of lipopolysaccharide (LPS) plus peptidoglycan (PepG)-induced acute sepsis, characterized by inflammation, mitochondrial dysfunction and early organ damage. METHODS: Anaesthetized and ventilated rats received an i.v. bolus of LPS and PepG followed by an i.v. infusion of MitoQ, MitoE, melatonin, or saline for 5 h. Organs and blood were then removed for determination of mitochondrial and organ function, oxidative stress, and key cytokines. RESULTS: MitoQ, MitoE, or melatonin had broadly similar protective effects with improved mitochondrial respiration (P<0.002), reduced oxidative stress (P<0.02), and decreased interleukin-6 levels (P=0.0001). Compared with control rats, antioxidant-treated rats had lower levels of biochemical markers of organ dysfunction, including plasma alanine amino-transferase activity (P=0.02) and creatinine concentrations (P<0.0001). CONCLUSIONS: Antioxidants that act preferentially in mitochondria reduce mitochondrial damage and organ dysfunction and decrease inflammatory responses in a rat model of acute sepsis.
Asunto(s)
Antioxidantes/farmacología , Interleucina-6/metabolismo , Mitocondrias/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Sepsis/tratamiento farmacológico , Sepsis/fisiopatología , Enfermedad Aguda , Animales , Antioxidantes/uso terapéutico , Biomarcadores , Citocinas/biosíntesis , Escherichia coli , Pruebas de Función Renal , Lipopolisacáridos , Pruebas de Función Hepática , Masculino , Melatonina/farmacología , Melatonina/uso terapéutico , Insuficiencia Multiorgánica/fisiopatología , Insuficiencia Multiorgánica/prevención & control , Compuestos Organofosforados/uso terapéutico , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Sepsis/inducido químicamente , Staphylococcus aureus , Ubiquinona/análogos & derivados , Ubiquinona/uso terapéuticoRESUMEN
BACKGROUND: Development of organ dysfunction associated with sepsis is due in part to oxidative damage to mitochondria. Melatonin regulates the sleep-wake cycle and also has potent antioxidant activity. The aim of this study was to determine the effects of melatonin and other structurally related compounds on mitochondrial function, endogenous glutathione (GSH), and control of cytokine expression under conditions mimicking sepsis. METHODS: Human endothelial cells were treated with lipopolysaccharide (LPS) plus peptidoglycan G (PepG) to simulate sepsis, in the presence of melatonin, 6-hydroxymelatonin, tryptamine, or indole-3-carboxylic acid. Nuclear factor κB (NFκB) activation, interleukin (IL)-6 and IL-8, total glutathione, mitochondrial membrane potential, and metabolic activity were measured. RESULTS: LPS and PepG treatment resulted in elevated IL-6 and IL-8 levels preceded by activation of NFκB (all P<0.0001). Treatment with all four compounds resulted in lower IL-6 and IL-8 levels, and lower NFκB activation (P<0.0001). Loss of mitochondrial membrane potential and endogenous glutathione was seen when cells were exposed to LPS/PepG, but these were maintained in cells co-treated with melatonin, tryptamine, or 6-hydroxymelatonin (P<0.05), but not indole-3-carboxylic acid. Metabolic activity decreased after exposure to LPS/PepG and was maintained by melatonin and 6-hydroxymelatonin at the highest concentrations only. CONCLUSIONS: We have shown that in addition to melatonin, other structurally related indoleamine compounds have effects on NFκB activation and cytokine expression, GSH, mitochondrial membrane potential, and metabolic activity in endothelial cells cultured under conditions mimicking sepsis. Further work is needed to determine whether these compounds represent therapeutic approaches for disrupting the oxidative stress-inflammatory response signalling pathway in sepsis.
Asunto(s)
Antioxidantes/farmacología , Endotelio Vascular/efectos de los fármacos , Inflamación/fisiopatología , Melatonina/farmacología , Mitocondrias/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Glutatión/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/patología , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , FN-kappa B/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Sepsis/metabolismo , Sepsis/patología , Sepsis/fisiopatología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Pentraxin-3 (PTX3) may be a useful biomarker in sepsis, but its regulatory mechanisms are still unclear. Oxidative stress is well defined in patients with sepsis and has a role in regulation of inflammatory pathways which may include PTX3. We undertook an in vitro study of the effect of antioxidants on regulation of PTX3 in endothelial cells combined with a prospective observational pilot study of PTX3 in relation to markers of antioxidant capacity and oxidative stress in patients with sepsis. METHODS: Human endothelial cells were cultured with lipopolysaccharide 2 microg ml(-1), peptidoglycan G 20 microg ml(-1), tumour necrosis factor (TNF) alpha 10 ng ml(-1), interleukin-1 (IL-1) beta 20 ng ml(-1), or killed Candida albicans yeast cells plus either N-acetylcysteine (NAC) 25 mM, trolox 100 mM, or idebenone 1 microM. Plasma samples were obtained from 15 patients with sepsis and 11 healthy volunteers. RESULTS: PTX3 levels in plasma were higher in patients with sepsis than in healthy people [26 (1-202) ng ml(-1) compared with 6 (1-12) ng ml(-1), P=0.01]. Antioxidant capacity was lower in patients with sepsis than healthy controls [0.99 (0.1-1.7) mM compared with 2.2 (1.3-3.3) mM, P=0.01]. In patients with sepsis, lipid hydroperoxide levels were 3.32 (0.3-10.6) nM and undetectable in controls. We found no relationship between PTX3 and antioxidant capacity or lipid hydroperoxides. Cell expression of PTX3 increased with all inflammatory stimulants but was highest in cells treated with TNFalpha plus IL-1beta. PTX3 concentrations were lower in cells co-treated with antioxidants (all P<0.05), associated with lower nuclear factor kappaB expression for NAC and trolox (P<0.05). CONCLUSIONS: PTX3 expression is down-regulated in vitro by antioxidants. Plasma levels of PTX3 are elevated in sepsis but seem to be unrelated to markers of oxidant stress or antioxidant capacity.
Asunto(s)
Antioxidantes/farmacología , Proteína C-Reactiva/metabolismo , Sepsis/sangre , Componente Amiloide P Sérico/metabolismo , APACHE , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Biomarcadores/metabolismo , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Femenino , Humanos , Mediadores de Inflamación/farmacología , Peróxidos Lipídicos/sangre , Masculino , Persona de Mediana Edad , Estrés Oxidativo , Proyectos Piloto , Regulación hacia Arriba/efectos de los fármacos , Adulto JovenRESUMEN
BACKGROUND: Activated protein C (APC) therapy reduces mortality in high-risk patients with severe sepsis. The effects of APC on inflammatory responses have also been reported. Neutrophils are key cells involved in early host defence mechanisms in sepsis. We hypothesized that APC may have effects on neutrophil function. METHODS: Neutrophils were isolated from 10 healthy volunteers and incubated in the presence of lipopolysaccharide (LPS) with and without a range of therapeutically relevant concentrations of recombinant human APC. Respiratory burst activity was determined using flow-activated cell sorting (FACS) analysis. Apoptosis was determined using Annexin-V staining and FACS analysis. Cytokine bead array was used to simultaneously measure three key cytokines in culture supernatants: interleukin (IL)-1 beta, -6, and -8. For chemotaxis, neutrophil migration through a 5 microm membrane was measured in response to formyl-methyl-leucine-phenylalanine (FMLP) or IL-8 in the presence and absence of APC. RESULTS: Exposure to LPS resulted in significant increases in respiratory burst activity, IL-1 beta, -6, and -8 expression (all P<0.0001) and decreased the number of apoptotic cells (P<0.0001). The APC exposure resulted in a significant release of IL-6 (P=0.04) without affecting other cytokines. Respiratory burst and apoptosis were also unaffected by APC. Neutrophil chemotaxis in response to either FMLP or IL-8 was reduced by APC (P=0.005 and 0.007, respectively). CONCLUSIONS: This pilot study showed that APC treatment of human neutrophils results in a decreased IL-6 expression and chemotaxis, without affecting other cytokines, apoptosis, or respiratory burst activity.
Asunto(s)
Quimiotaxis de Leucocito/efectos de los fármacos , Interleucina-6/biosíntesis , Neutrófilos/efectos de los fármacos , Proteína C/farmacología , Adulto , Apoptosis/efectos de los fármacos , Células Cultivadas , Citocinas/biosíntesis , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Lipopolisacáridos/farmacología , Masculino , Persona de Mediana Edad , Activación Neutrófila/efectos de los fármacos , Neutrófilos/inmunología , Proteínas Recombinantes/farmacología , Estallido Respiratorio/efectos de los fármacosRESUMEN
Industrial alkylphenols in the environment may act as "xenoestrogens" to disrupt testicular development and decrease male fertility. Amongst possible targets for these compounds are testicular Sertoli cells, which nurture the developing sperm cells. We demonstrate that SERCA 2 and 3 Ca(2+) pumps are relatively abundant in rat testis microsomal membranes, and also in Sertoli, myoid, and TM4 cells (a Sertoli cell line). A number of estrogenic alkylphenols such as nonylphenol, octylphenol, bisphenol A, and butylated hydroxytoluene all inhibit testicular Ca(2+) ATPase in the low micromolar concentration range. These agents also mobilize intracellular Ca(2+) in intact TM4 cells in a manner consistent with the inhibition of ER Ca(2+) pumps. Alkylphenols dramatically decrease the viability of TM4 cells, an effect that is reversed by either a caspase inhibitor or by BAPTA, and is therefore consistent with Ca(2+)-dependent cell death via apoptosis. We postulate that alkylphenols disrupt testicular development by inhibiting ER Ca(2+) pumps, thus disturbing testicular Ca(2+) homeostasis.
Asunto(s)
Apoptosis , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Retículo Endoplásmico/efectos de los fármacos , Fenoles/farmacología , Testículo/efectos de los fármacos , Animales , ATPasas Transportadoras de Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Congéneres del Estradiol/farmacología , Técnicas In Vitro , Masculino , Ratas , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Células de Sertoli/citología , Células de Sertoli/efectos de los fármacos , Testículo/citología , Testículo/metabolismoRESUMEN
We have mapped sites of tamoxifen adduct formation, in the lacI gene using the polymerase STOP assay, following reaction in vitro with alpha-acetoxytamoxifen and horseradish peroxidase (HRP)/H(2)O(2) activated 4-hydroxytamoxifen. For both compounds, most adduct formation occurred on guanines. However, one adenine, within a run of guanines, generated a strong polymerase STOP site with activated 4-hydroxytamoxifen, and a weaker STOP site with alpha-acetoxytamoxifen at the same location. In Escherichia coli the lac I gene reacted with 4-hydroxytamoxifen was more likely to be mutated (2 orders of magnitude) than when reacted with alpha-acetoxytamoxifen, despite the greater DNA adduct formation by alpha-acetoxytamoxifen. This correlates with the greater predicted ability of activated 4-hydroxytamoxifen adducts to disrupt DNA structure than alpha-acetoxytamoxifen adducts. For lac I reacted with activated 4-hydroxytamoxifen, a hot spot of base mutation was located in the region of the only adenosine adduct. No mutational hot spots were observed with alpha-acetoxytamoxifen. Our data clearly shows a lack of correlation between gross adduct number, as assayed by (32)P-postlabeling and mutagenic potential. These data indicate the importance of minor adduct formation in mutagenic potential and further that conclusions regarding the mutagenicity of a chemical may not be reliably derived from the gross determination of adduct formation.