2--7--dichlorodihydrofluorescein and dihydroethidium

Oxidative stress is known to have a major impact on human sperm function and, as a result, there is a need to develop sensitive methods for measuring reactive oxygen species (ROS) generation by these cells. A variety of techniques have been developed for this purpose including chemiluminescence (luminol and lucigenin), flow cytometry (MitoSOX Red, dihydroethidium, 4,5-diaminofluorescein diacetate and 2',7'-dichlorodihydrofluorescein diacetate) and spectrophotometry (nitroblue tetrazolium). The relative sensitivity of these assays and their comparative ability to detect ROS generated in different subcellular compartments of human spermatozoa, have not previously been investigated. To address this issue, we have compared the performance of these assays when ROS generation was triggered with a variety of reagents including 2-hydroxyestradiol, menadione, 4-hydroxynonenal and arachidonic acid. The results revealed that menadione predominantly induced release of ROS into the extracellular space where these metabolites could be readily detected by luminol-peroxidase and, to a lesser extent, 2',7'-dichlorodihydrofluorescein. However, such sensitivity to extracellular ROS meant that these assays were particularly vulnerable to interference by leucocytes. The remaining reagents predominantly elicited ROS generation by the sperm mitochondria and could be optimally detected by MitoSOX Red and DHE. Examination of spontaneous ROS generation by defective human spermatozoa revealed that MitoSOX Red was the most effective indicator of oxidative stress, thereby emphasizing the general importance of mitochondrial dysregulation in the aetiology of defective sperm function.

Topics: Aldehydes; Arachidonic Acid; Estradiol; Estrogens, Catechol; Ethidium; Flow Cytometry; Fluoresceins; Humans; Leukocytes; Luminescence; Luminescent Measurements; Luminol; Male; Mitochondria; Oxidative Stress; Phenanthridines; Reactive Oxygen Species; Sensitivity and Specificity; Spectrophotometry; Spermatozoa; Vitamin K 3

Reactive oxygen species (ROS) produced during mitochondrial activity participate in the regulation of intracellular signaling pathways. However, there is only limited information concerning the role that ROS derived from the mitochondrial respiratory chain play in modulating neutrophil activity and participation in acute inflammatory processes. Because mitochondrial complex III is a major site of ROS formation, we examined whether selective complex III inhibition, through exposure of neutrophils to myxothiazol or antimycin A, would affect LPS-induced activation. Culture of neutrophils with antimycin A or myxothiazol resulted in increased intracellular levels of ROS, including superoxide and hydrogen peroxide (H(2)O(2)). Inhibition of complex III activity reduced LPS-induced degradation of IkappaB-alpha, nuclear accumulation of NF-kappaB, and proinflammatory cytokine production. The effects of antimycin A or myxothiazol appeared to be dependent on generation of H(2)O(2) since addition of pegylated catalase to neutrophils restored LPS-mediated IkappaB-alpha degradation and production of proinflammatory cytokines. Administration of myxothiazol to mice resulted in diminished mitochondrial complex III activity in the lungs and decreased severity of LPS-induced lung injury. These results indicate that inhibition of mitochondrial complex III diminishes Toll-like receptor 4-induced neutrophil activation through a mechanism dependent on H(2)O(2) generation and also reduces the severity of lung injury due to LPS exposure, a pathophysiologic process in which neutrophils play a major role.

Topics: Animals; Antimycin A; Cell Nucleus; Cytokines; Electron Transport Complex III; Ethidium; Fluoresceins; I-kappa B Proteins; Lipopolysaccharides; Lung Injury; Male; Methacrylates; Mice; Mitochondria; Neutrophil Activation; Neutrophils; NF-kappa B; NF-KappaB Inhibitor alpha; Oxidation-Reduction; Protein Transport; Reactive Oxygen Species; Thiazoles