Metabolism and detoxification of nitrite by trout hepatocytes

Nitrite (NO2-) is one of the most important toxicants to fish. Freshwater fish are especially sensitive, particularly salmonids. Nitrite uptake is thought to occur via the HCO3-, Cl- -exchanger at the gill epithelia with nitrite substituting for chloride. In this way freshwater fish accumulate nitrite in the blood up to 100-fold from the surrounding water. Another source, endogenous nitrite as a degradation product of nitric oxide, rarely leads to pharmacologically relevant concentrations. We developed a new method for the determination of nitrate (NO3-) in biological samples and used it to measure nitrite oxidation in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes which were found to detoxify nitrite to the almost non-toxic nitrate. Detoxification is inhibited by 0.05 mM bumetanide and 0.1 mM furosemide but not by SITS and DITS, suggesting the involvement of the Na+, K+, 2Cl- -cotransporter with nitrite or nitrate substituting for chloride. Oxidation of nitrite is strongly accelerated by 0.05 mM uric acid. The efficacy of this antioxidant suggests that similar reactions are involved as known for haemoglobin [33]. However, in the case of trout liver also membrane bound detoxificating activity can be observed which is also enhanced by uric acid. ATP concentrations remained constant in the hepatocytes during all experiments demonstrating that hepatocyte energy status was not influenced by nitrite oxidation. Thus nitrite resistance in fish is governed by at least two mechanisms, nitrite uptake and the rate of detoxification. It is unknown whether fish actually differ in their ability to distinguish between chloride and nitrite during branchial uptake, but evidence presented in this paper suggests a significant contribution of detoxification pathways to a possible nitrite tolerance of fish.