ABSTRACT: Hormesis is a dose response phenomenon characterized by a stimulation of an organismal response at low doses of a chemical and inhibition of the response (toxicity) at a higher dose. In the present study, Daphnia magna were exposed to the energetic compound 2,4,6-trinitrotoluene (TNT) for 21 days, and the endpoints survival, growth (length and dry weight), and reproduction (number of neonates per survivor) were assesed, identifying hormetic responses at the lower treatment levels. In order to elucidate the mechanisms leading to hormesis, microarray analysis was performed at 0.004 (hormetic), 0.12 (sometimes hormetic), and 1.85 (toxic) mg/L TNT. Functional and transcriptional benchmark dose analyses performed on differentially expressed genes suggested the involvement of lipid metabolism in hormetic responses and subsequently lipidomic analysis was performed on the same treatments. Lipid analysis supported the hypothesis that TNT exposure affected lipid metabolism, and showed that hormetic effects could be related to increases in some polyunsaturated fatty acids and ecosanoids known to be involved in Daphnia growth and reproduction. Chronic toxicity tests was designed to identify the hormetic range of D. magna responses to TNT. Daphnia magna younger than 24 h old and obtained from in-house cultures at the U.S. Army Engineer Research and Development Center, Environmental Laboratory (Vicksburg, MS) were used to initiate the bioassay. The experimental design consisted of one D. magna per test chamber and 10 test chambers per treatment level. In the microarray mass exposure, nominal treatment levels were 0.004, 0.02, 0.12, 0.44, and 1.85 mg/L. Based on the results of the testing in the 50 ml beakers, organisms from three treatments were selected for analysis of gene expression: 0.004, 0.12, and 1.85 mg/L. These treatments were selected to represent a hormetic concentration (0.004 mg/L), a toxic concentration (1.85 mg/L), and an intermediate concentration (0.12 mg/L). A total of 23 arrays used, with 5-6 replicates per each dose (solvent control, 0.004 mg/L, 0.12 mg/L, and 1.85 mg/L).
