Mechanistic evaluation of the reproductive and developmental toxicity of nitrate and nitrite

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Title: Mechanistic evaluation of the reproductive and developmental toxicity of nitrate and nitrite
Author: Hannas, Bethany Reeves
Advisors: Dr. R. Michael Roe, Committee Member
Dr. Gerald A. LeBlanc, Committee Chair
Dr. David Buchwalter, Committee Member
Dr. Jun Tsuji, Committee Member
Abstract: This research investigates the mechanism for reproductive and developmental toxicity associated with the aquatic contaminants nitrate and nitrite, in the crustacean Daphnia magna. Two hypotheses were tested: 1) Nitrate and nitrite are converted to nitric oxide(NO), resulting in disruption of endocrine-related processes; and 2) NO interferes with endocrine signaling by lowering steroid hormone levels or by binding a heme-containing nuclear receptor involved in steroid signaling. In the first study, the developmental and reproductive toxicity of nitrate and nitrite to daphnids was consistent with toxicity elicited by the NO donor, sodium nitroprusside and was ameliorated by the NO-scavenger ß-carotene. Drosophila S2 cells converted nitrate and nitrite to NO in a substrate and cell concentration-dependent manner. Together, these results are consistent with nitrate and nitrite eliciting toxicity via intracellular conversion to NO. Although toxicity was indicative of an anti-ecdysteroid mechanism of action, we did not detect significant, consistent decreases in 20-hydroxyecdysone levels in exposed daphnids. The next study characterized a potential target of NO toxicity in the ecdysteroid signaling pathway. The nuclear receptors E75 (group NR1D) and HR3 (NR1F) were cloned and sequenced from Daphnia magna. E75 possessed amino acid residues in the ligand-binding domain that likely bind heme, a common target of NO binding. HR3 was significantly induced by 20-hydroxyecdysone, whereas E75 was minimally responsive. These results implicate both E75 and HR3 of daphnids as potential targets of the anti-ecdysteroid action of exogenous NO. Finally, we characterized E75 and HR3 regulation of gene transcription, to determine if NO alters this regulatory activity. HR3 activated transcription of a retinoid orphan receptor element (RORE)-driven luciferase reporter. E75 did not activate the reporter, but served to repress HR3 activation. Experiments revealed no evidence that NO interferes with E75 repression of HR3. Therefore, the mechanism by which nitrate and nitrite-derived NO elicits developmental and reproductive toxicity remains unknown. This research highlights the potential threat posed to the reproductive and developmental success of aquatic organisms exposed to nitrate and nitrite. It additionally advances understanding of crustacean endocrinology, while demonstrating the need for further information to identify the mechanism of action for interfering environmental contaminants.
Date: 2009-08-24
Degree: PhD
Discipline: Toxicology

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