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 NOx) 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 (NO3 2) and nitrite (NO2 2) to nitric oxide using amperometric real-time nitric oxide detection. Both NO 3 2 and NO2 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 NO2 2 to nitric oxide. Next, daphnids were continuously exposed to concentrations of the nitric oxide-donor sodium nitroprusside (positive control) and to concentrations of NO3 2 and NO2 2. All three compounds interfered with normal embryo development and reduced daphnid fecundity. Developmental abnormalities wer

Total offspring released from daphnids chronically exposed to increasing concentration of (A) sodium nitroprusside, (B) NaNO₂, or (C) NaNO₃.

<p>Each data point represents the total number of offspring released from a single exposed daphnid over the entire exposure period. Mean ± SD negative control performance is depicted by the solid line bracketed by dotted lines.</p

Concentration-dependent conversion of NO₃⁻ (A) or NO₂⁻ (B) to nitric oxide by <i>Drosophila</i> S2 cells.

<p>Each column represents the mean ± SD of 4–7 experiments with different cell batches.</p

Frequency of neonatal abnormalities among daphnids exposed to increasing concentrations of (A) sodium nitroprusside, (B) NaNO₂, or (C) NaNO₃.

<p>Each data point represents the percentage of abnormal neonates produced by a single maternal daphnid. Mean ± SD negative control performance is depicted by the solid line bracketed by dotted lines.</p

Ecdysteroid levels in daphnids exposed to 1.0 mg N/L sodium nitroprusside (SNP).

<p>Ecdysteroid (pg/individual) values represent the mean ± SD (n = 3–5 treatment groups, with numbers of individuals per group as indicated).</p

NO₂⁻-dependent nitric oxide production at increasing cell densities.

<p>Each bar represents the mean ± SD of 2–3 experiments with different batches of cells.</p

Developmental abnormalities among neonatal daphnids resulting from maternal exposure to NaNO₂.

<p>Images presented are (<b>A</b>) normal control neonate, (<b>B</b>) and (<b>C</b>) neonates derived from maternal exposure to 1.0 and 2.0 mg N/L NaNO₂, respectively.</p

Nitric oxide accumulation in NO₂⁻-exposed perinatal daphnids as indicated by trapping and fluorescent visualization with diacetylaminofluorocene (DAF, 10 µM).

<p>NO₂⁻ exposure concentration (as mg N/l) and duration of exposure are presented to the left of each row of images. DT and RM denote the digestive tract and respiratory membranes, respectively.</p

HESI workshop summary : interpretation of developmental and reproductive toxicity endpoints and the impact on data interpretation of adverse events

Abstract: The Health and Environmental Sciences Institute Developmental and Reproductive Toxicology (HESI-DART) group held a hybrid in-person and virtual workshop in Washington, DC, in 2022. The workshop was entitled, "Interpretation of DART in Regulatory Contexts and Frameworks." There were 154 participants (37 in person and 117 virtual) across 9 countries. The purpose of the workshop was to capture key consensus approaches used to assess DART risks associated with chemical product exposure when a nonclinical finding is identified. The decision-making process for determining whether a DART endpoint is considered adverse is critical because the outcome may have downstream implications (e.g., increased animal usage, modifications to reproductive classification and pregnancy labeling, impact on enrollment in clinical trials and value chains). The workshop included a series of webinar modules to train and engage in discussions with federal and international regulators, clinicians, academic investigators, nongovernmental organizations, contract research organization scientists, and private sector scientists on the best practices and principles of interpreting DART and new approach methodologies in the context of regulatory requirements and processes. Despite the differences in regulatory frameworks between the chemical and pharmaceutical sectors, the same foundational principles for data interpretation should be applied. The discussions led to the categorization of principles, which offer guidance for the systematic interpretation of data. Step 1 entails identifying any hazard by closely analyzing the data at the study endpoint level, while Step 2 involves assessing risk using weight of evidence. These guiding principles were derived from the collective outcomes of the workshop deliberations