5-5--6-6--tetrachloro-1-1--3-3--tetraethylbenzimidazolocarbocyanine and linsidomine

Nitrosative stress is produced by high levels of reactive nitrogen species (RNS). The RNS include peroxynitrite, a highly reactive free radical produced from a diffusion-controlled reaction between nitric oxide and superoxide anion. Peroxynitrite causes nitration and oxidation of lipids, proteins and DNA, and is thus considered an important pathogenic mechanism in various diseases. Although high levels of peroxynitrite are associated with astenozoospermia, few reports exist regarding the in vitro effect of high levels of this RNS on human sperm. The aim of this study was to evaluate the in vitro effect of nitrosative stress caused by peroxynitrite on the viability, motility and mitochondrial membrane potential of human spermatozoa. To do this, human spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a molecule that generates peroxynitrite. Incubations were done at 37°C for up to 4 h with SIN-1 concentrations between 0.2 and 1.0 mmol/l. Generation of peroxynitrite was confirmed using dihydrorhodamine 123 (DHR) by spectrophotometry and flow cytometry. Sperm viability was assessed by propidium iodide staining; sperm motility was analyzed by CASA, and the state of mitochondrial membrane potential (ΔΨm) by JC-1 staining. Viability and ΔΨm were measured by flow cytometry. The results showed an increase in DHR oxidation, demonstrating the generation of peroxynitrite through SIN-1. Peroxynitrite decreased progressive and total motility, as well as some sperm kinetic parameters. Mitochondrial membrane potential also decreased. These alterations occurred with no decrease in sperm viability. In conclusion, peroxynitrite-induced nitrosative stress impairs vital functions in the male gamete, possibly contributing to male infertility.

Topics: Adult; Benzimidazoles; Carbocyanines; Cell Survival; Fluorescent Dyes; Humans; Male; Membrane Potential, Mitochondrial; Molsidomine; Oxidative Stress; Peroxynitrous Acid; Rhodamines; Semen Analysis; Spectrometry, Fluorescence; Sperm Motility; Spermatozoa

Quantitative measurement of cellular oxidative stress (COS) and cytotoxicity are important to establish their significance in pathophysiologic conditions and disease states. So far, ample methods have been described to determine these processes based on spectrophotometric analysis. The application of simple, rapid, and sensitive fluorescence methods to determine the cytotoxicity and COS is described in the present chapter. Murine H9c2 cells were exposed to various free radical and non-free radical oxidants through use of diethylamine NONOate, 3-morpholinosydnonimine (SIN-1), and a synthetic preparation of peroxynitrite (PN). The viability of control and the treated H9c2 cells was measured based on the reduction of resazurin to resorufin which generates a fluorescent signal. The mitochondrial membrane potential was quantified by determining the cellular uptake of a fluorescent dye, (5,5('),6,6(')-tetrachloro-1,1(')-3,3(')-tetraethylbenzimidazolcarbocyanine iodide (JC-1)) and its segregation in the mitochondrial fraction. The intracellular GSH was determined by assaying the glutathione-S-transferase (GST)-catalyzed conjugation of GSH to monochlorobimane. This chapter describes the feasibility and potential of the above-described fluorescence approach as simple alternative methods to determine reactive oxygen and nitrogen species-induced cytotoxicity and oxidative stress using H9c2 cardiomyoblasts as a model system.

Topics: Animals; Antioxidants; Benzimidazoles; Carbocyanines; Cell Line; Cell Survival; Fluorescent Dyes; Glutathione; Hydrazines; Membrane Potential, Mitochondrial; Mice; Molsidomine; Myocytes, Cardiac; Nitric Oxide Donors; Oxidative Stress; Peroxynitrous Acid; Reactive Nitrogen Species; Reactive Oxygen Species