Abstract

Atrazine is a widely used triazine herbicide for controlling broadleaf and grassy weeds in the U.S. and is routinely detected in many surface and ground waters. This study was conducted to evaluate the effects of atrazine on the toxicity of selected organophosphate insecticides and gene expression using fourth-instar larvae of the aquatic midge, Chironomus tentans . Atrazine alone up to 1,000 μg/L, or parts per billion (ppb), did not show significant toxicity to the midges. However, atrazine concentrations from 1 to 1,000 ppb significantly enhanced dimethoate, disulfoton, or demeton- S-methyl toxicity when in combination. In contrast, atrazine at 10 ppb in combination with omethoate significantly decreased the insecticide toxicity. Biochemical analysis indicated that increased toxicity of dimethoate, disulfoton, and demeton-S-methyl in binary combination with atrazine correlated to increased inhibition of acetylcholinesterase. Cytochrome P450-dependent O-deethylation activity in atrazine-treated midges was 1.5-fold higher than that in untreated midges. Cytochrome P450 induction by atrazine may lead to increasing organophosphate toxicity by enhancing the oxidative activation of the insecticides into sulfoxide analogs with increased anticholinesterase activity. To better understand the molecular basis of atrazine's effects in midges, we utilized the genomic technique restriction fragment differential display (RFDD)-PCR to systematically compare gene expression profiles between atrazine-treated and untreated midges. The RFDD-PCR technique revealed various up- and down-regulated genes in atrazine-treated midges that are associated with hypoxia. Oxygen consumption was significantly enhanced in midges exposed to atrazine in a time- and concentration-dependent manner. Two down-regulated hemoglobin (Hb) genes were isolated from atrazine-treated midges using a C. tentans cDNA library. Concentration-dependent Hb expression was significantly decreased in atrazine-treated midges. Atrazine-induced hypoxic stress may result in the down-regulation of Hb genes as a feedback mechanism due to unavailable oxygen molecules to bind. Our study suggests that Hb in midges plays an important role in oxygen conservation when oxygen is abundant, but can be rapidly reduced in synthesis during hypoxic stress. This study provides new insights into the toxicological and physiological risk that atrazine imposes upon non-target, aquatic animals and may assist in identifying atrazine-specific genes used to evaluate non-target effects of atrazine and aquatic animal health.