Mechanism of oxidative stress from low levels of carbon monoxide.

The purpose of this study was to determine whether platelets and vascular endothelial cells would liberate nitric oxide free radical (NO)* and NO-derived oxidant species after exposure to carbon monoxide (CO) at concentrations up to 100 parts per million (ppm). We hypothesized that exposure to environmentally relevant concentrations of CO would increase production of agents that may be involved in human pathological processes, such as atherosclerosis. Platelets obtained from rats released NO when incubated with CO, but CO did not increase platelet nitric oxide synthase activity. Platelets released comparable NO levels when they were exposed to CO in vitro and when taken from rats that had been exposed to CO. Partial pressures of CO as low as 10 ppm could successfully compete with NO for intraplatelet binding sites in in vitro studies. We conclude that CO enhanced the release of NO from platelets because it inhibited NO sequestration by intraplatelet binding sites, and that this phenomenon can occur with exposure to CO concentrations found in the environment. Bovine pulmonary artery endothelial cells released NO in response to CO exposure. Carbon monoxide did not affect the transport of L-arginine across the plasma membrane or nitric oxide synthase activity; therefore, the mechanism appeared to be based on a disturbance of intracellular NO sequestration. Cells incubated with CO also released into the surrounding medium peroxynitrite, an NO-derived oxidant, based on oxidation of dihydrorhodamine 123 and p-hydroxyphenylacetic acid. Peroxynitrite-mediated oxidative stress to endothelial cells was identified as increased concentrations of nitrotyrosine in cell lysates, and by measuring the release of radioactive chromium. Carbon monoxide caused an acute injury when cells were continuously exposed for 4 hours, and a delayed injury when cells were exposed for 2 hours. Delayed injury was documented by leakage of radioactive chromium and by uptake of a vital fluorescent stain, ethidium homodimer-1, between 6 and 20 hours after CO exposure. Oxidative stress caused by CO exhibited several unique aspects because CO exposure did not alter the cellular content of reduced sulfhydryls nor did CO augment oxidative stress caused by superoxide, hydrogen peroxide, or a flux of NO. We concluded that concentrations of CO achieved in vivo when humans are exposed to CO concentrations found in the environment can cause endothelial cells to liberate NO and NO-derived oxidants, and that these products can adversely affect cell physiology.