Intracellular Conversion of Environmental Nitrate and Nitrite to Nitric Oxide with Resulting Developmental Toxicity to the Crustacean Daphnia magna

Background: Nitrate and nitrite (jointly referred to herein as NO(x)) are ubiquitous environmental contaminants to which aquatic organisms are at particularly high risk of exposure. We tested the hypothesis that NO(x) undergo intracellular conversion to the potent signaling molecule nitric oxide resulting in the disruption of endocrine-regulated processes. Methodology/Principal Findings: These experiments were performed with insect cells (Drosophila S2) and whole organisms Daphnia magna. We first evaluated the ability of cells to convert nitrate (NO(3)(-)) and nitrite (NO(2)(-)) to nitric oxide using amperometric real-time nitric oxide detection. Both NO(3)(-) and NO(2)(-) were converted to nitric oxide in a substrate concentration-dependent manner. Further, nitric oxide trapping and fluorescent visualization studies revealed that perinatal daphnids readily convert NO(2)(-) to nitric oxide. Next, daphnids were continuously exposed to concentrations of the nitric oxide-donor sodium nitroprusside (positive control) and to concentrations of NO(3)(-) and NO(2)(-). All three compounds interfered with normal embryo development and reduced daphnid fecundity. Developmental abnormalities were characteristic of those elicited by compounds that interfere with ecdysteroid signaling. However, no compelling evidence was generated to indicate that nitric oxide reduced ecdysteroid titers. Conclusions/Significance: Results demonstrate that nitrite elicits developmental and reproductive toxicity at environmentally relevant concentrations due likely to its intracellular conversion to nitric oxide.