Cytotoxicity Of Ethyl Nitrite And Its Possible Mechanism

BackgroundEthyl nitrite (ENO) is an organic compound widely existing in foods, and it is also a semifinished product commonly used in organic chemistry. Although it have been known for hundreds of years, it's value in medicine has just been realized recently. This is greatly related to the discovery of the importance of nitric oxide in medicine.Nitric oxide (NO) is a vasodilator. Since Kinsella first reported in 1992 that inhaled nitric oxide (iNO) was an effective treatment for persistent pulmonary hypertension of the newborn(PPHN), iNO has been a hot topic in medicine. Thousands of hundreds of articles suggested iNO selectively relax pulmonary vascular smooth muscle and remarkably improve oxygenation in patients with pulmonary hypertension. However there is also considerable evidence to indicate that high concentration of NO can be toxic and that O₂-rich environments such as the lung during mechanical ventilation are particularly predisposed to its potentially mutagenic, proapoptotic and proinflammtory damage. These findings are well rationalized by the chemical reactions of NO with O₂ and reactive oxygen species, which yield higher oxides of nitrogen (NOx) that can cause inflammation,hemorrhage, and edema. Besides this, iNO therapy has some drawbacks including methemoglobinemia and a "rebound" increase in pulmonary artery pressure on discontinuing the gas.Unlike inhaled nitric oxide, endogenous nitric oxide synthase (NOSs) do not generate high levels of free NO in the respiratory system. Rather, it has been shown that the NO produced is complexed with thiols to form S-nitrosothiols(SNOs) including S-nitrosoglutathione ( GSNO ) ,S-nitrosocysteine ( CSNO ) and S-nitrosohemoglobin(SNO-Hb) in biological system, which are resistant to potentially toxic reaction with O2/O2".Realizing the difference between inhaled nitric oxide and endogenous NO and the advantages of SNOs, researchers have been exploring to find an ideal drug which would closely simulate endogenous NO by transforming NO groups into SNOs. Among them, Stamler research group selected ENO as a candidate gas. ENO fulfils following criteria: (1)resistance to decomposition in the presence of O₂;(2)reaction with glutathione to form GSNO;(3)limited ability to oxidize hemoglobin and (4)biocompatibility. In particular, ENO can be synthesized as a liquid but easily partitions into the gas phase because of its low boiling point(17℃).Up to the present, there are a few medical reports about ENO. These reports indicated that ENO can effectively attenuate pneumoperitoneum-induced blood flow decrease within the peritoneal cavity during laparoscopic procedures, they also indicate that inhalation of ENO can produce sustained improvements in arterial oxygenation in PPHN, and in a porcine model of lung injury, ENO inhalation provided immediate relief from hypoxic pulmonary vasoconstriction without affecting systemic hemodynamics. Further, in these studies, there was no rebound in cardiopulmonary hemodynamics or fall in oxygenation on stopping the drug, and there was no NOx detectable. All of these studies suggest ENO as a promising drug in medicine.Although the initial findings are encouraging, we just stride our first step on the new drug. As we know, there is no report about which dose ENO should be appropriate and safe to be used and there is also no article about the potential toxicity of ENO. We designed the experiments to investigate the possible cytotoxicity of ENO and to explore the possible mechanism with it. In the present study, we used microculture tetrzoalium assay (MTT) to investigate the V79 cell survival rate after exposure to different doses of ENO for different times, we also detected the lactic dehydrogenase (LDH) in culture medium after exposure to 0.0125% ENO for 4h. To explore the possible mechanism of cytotoxicity of ENO, we compared the cell survival rate between glutathione(GSH) depletion + ENO treatment group , GSH complementarity + ENO treatment group and ENO treatment group, and we also determined the nitrite/nitrate content in the culture...