Toxicity assessment of 4-amino-2-nitrotoluene.

4-Amino-2-nitrotoluene (4A2NT; CAS 119-32-4) is a degradation product of 2,4-dinitrotoluene. The toxicity data on 4A2NT are limited. Therefore, we collected toxicity data from rats to assess environmental and human health effects from exposures. The approximate lethal dose for both sexes was 5000 mg/kg. A 14-day toxicity study in rats was conducted with 4A2NT in the feed at concentrations of 0, 125, 250, 500, 1000, and 2000 ppm. Based on a 14-day oral dose range toxicity study with 4A2NT in the feed, 2000 ppm was selected as highest concentration for a subsequent 90-day study. An oral 90-day subchronic toxicity study in rats was conducted with concentrations of 0, 500, 1000, or 2000 ppm of 4A2NT in the feed. The calculated consumed doses of 4A2NT in the feed were 0, 27, 52, or 115 mg/kg/d for males and 0, 32, 65, or 138 mg/kg/d for females. A no-observed adverse effect level could not be determined. The lowest observed adverse effect level was 27 mg/kg/d for males and 32 mg/kg/d for female rats based upon decreased body weight gain. The decreased body weight gain in male rats was the most sensitive adverse event observed in this study and was used to derive a benchmark dose (BMD). A BMD of 23.1 mg/kg/d and BMD with 10% effect level of 15.5 mg/kg/d were calculated for male rats, which were used to derive an oral reference dose (RfD). The human RfD of 1.26 µg/kg/d was derived using current United States Environmental Protection Agency guidelines.

Toxicologic characterization of a novel explosive, guanidinium 3,4-dinitropyrazolate (GDNP), in female rats and Ames mutagenicity assay.

Sustainable use of military training ranges requires the development of compounds that have a minimal impact to the environment when used in a weapon system. Guanidinium 3,4-dinitropyrazolate (GDNP) is a novel explosive compound of interest for application in some weapon systems. Little is known of its toxicologic properties. To ensure the health of potentially exposed personnel and the environment, initial toxicity investigations were conducted and the results were compared with another widely used energetic (hexahydro-1,3,5-trinitro-1,3,5-triazine [RDX]). In a microplate Ames assay, GDNP was not cytotoxic to bacterial tester strains at concentrations less than 100 µg/mL. However, GDNP was mutagenic to 4 of 5 bacterial strains with and without S9 metabolic incubation at concentrations as low as 0.7 µg/mL. Unlike RDX, GDNP did not have an affinity for the γ-aminobutyric acid(A) receptor convulsant site and was predicted to not induce seizure. After acute oral dosing in female rats, the median lethal dose in female rats of GDNP in tap water solution was determined to be 720 mg/kg. Daily oral exposure to 500 mg/kg per d of GDNP for 14 days caused weight loss, increased liver and spleen weights, and adverse histopathologic events in kidney and spleen. These adverse events were not observed in animals receiving lower doses of GDNP. In this study, the lowest-observed-adverse-effect-level from oral exposure to GDNP for 14 days was 500 mg/kg per d and the no-observable-adverse-effect-level was 152 mg/kg per d.

Behavioral and physiological effects of RDX on adult zebrafish.

1,3,5-Trinitroperhydro-1,3,5-triazine (RDX) is a nitroamine explosive, with common toxic effects including seizures. Here, we explore the behavioral effects of acute RDX exposure in adult zebrafish Danio rerio, a rapidly developing model in neuroscience and neurotoxicology research. Overall, a 30-min exposure to RDX low dose of 0.1 mM evoked behavioral activation in zebrafish, while a higher dose of 1 mM markedly reduced exploration, increased freezing and evoked seizure-like responses (i.e., bouts of hyperactivity, spasms, and corkscrew swimming). Likewise, whole-body cortisol levels were also significantly elevated in fish exposed to 1 mM (but not 0.1 mM) RDX. In line with clinical and animal data, our study demonstrates the dose-dependent behavioral activation and pro-convulsant effects of RDX in zebrafish-based models.

Assessing the non-cancer risk for RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) using physiologically based pharmacokinetic (PBPK) modeling.

RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) is an explosive used in military applications. It has been detected in ground water surrounding US military installations and at manufacturing facilities. RDX has been shown to produce hepatotoxicity, testicular, and neurological effects in animals, the latter also in humans. The current chronic oral reference dose (RfD) of 0.003 mg/kg/day was derived based on prostate effects in rats. Here, we provide a reevaluation of the risk associated with RDX exposure by examining old and new data and using physiologically based pharmacokinetic (PBPK) modeling approaches. Candidate non-cancer endpoints in rodents were evaluated and the most plausible mode(s) of action were determined. A PBPK model was used to derive appropriate internal doses based on the mode of action, and then a benchmark dose (BMD) and the lower confidence limit on the BMD (BMDL) were determined using these internal doses in animals. Uncertainty factors (UF) were applied to the animal BMDL or no-observed effect level and a human PBPK model was used to determine a human equivalent dose resulting in the candidate RfDs (cRfDs). A proposed chronic RfD of 0.07 mg/kg/day, based on multiple effects observed in rats, was selected from among the cRfDs.

RDX binds to the GABA(A) receptor-convulsant site and blocks GABA(A) receptor-mediated currents in the amygdala: a mechanism for RDX-induced seizures.

BACKGROUND: Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high-energy, trinitrated cyclic compound that has been used worldwide since World War II as an explosive in both military and civilian applications. RDX can be released in the environment by way of waste streams generated during the manufacture, use, and disposal of RDX-containing munitions and can leach into groundwater from unexploded munitions found on training ranges. For > 60 years, it has been known that exposure to high doses of RDX causes generalized seizures, but the mechanism has remained unknown. OBJECTIVE: We investigated the mechanism by which RDX induces seizures. METHODS AND RESULTS: By screening the affinity of RDX for a number of neurotransmitter receptors, we found that RDX binds exclusively to the picrotoxin convulsant site of the γ-aminobutyric acid type A (GABA(A)) ionophore. Whole-cell in vitro recordings in the rat basolateral amygdala (BLA) showed that RDX reduces the frequency and amplitude of spontaneous GABA(A) receptor-mediated inhibitory postsynaptic currents and the amplitude of GABA-evoked postsynaptic currents. In extracellular field recordings from the BLA, RDX induced prolonged, seizure-like neuronal discharges. CONCLUSIONS: These results suggest that binding to the GABA(A) receptor convulsant site is the primary mechanism of seizure induction by RDX and that reduction of GABAergic inhibitory transmission in the amygdala is involved in the generation of RDX-induced seizures. Knowledge of the molecular site and the mechanism of RDX action with respect to seizure induction can guide therapeutic strategies, allow more accurate development of safe thresholds for exposures, and help prevent the development of new explosives or other munitions that could pose similar health risks.

High-affinity near-infrared fluorescent small-molecule contrast agents for in vivo imaging of prostate-specific membrane antigen.

Surgical resection remains a definitive treatment for prostate cancer. Yet, prostate cancer surgery is performed without image guidance for tumor margin, extension beyond the capsule and lymph node positivity, and without verification of other occult metastases in the surgical field. Recently, several imaging systems have been described that exploit near-infrared (NIR) fluorescent light for sensitive, real-time detection of disease pathology intraoperatively. In this study, we describe a high-affinity (9 nM), single nucleophile-containing, small molecule specific for the active site of the enzyme PSMA. We demonstrate production of a tetra-sulfonated heptamethine indocyanine NIR fluorescent derivative of this molecule using a high-yield LC/MS purification strategy. Interestingly, NIR fluorophore conjugation improves affinity over 20-fold, and we provide mechanistic insight into this observation. We describe the preparative production of enzymatically active PSMA using a baculovirus expression system and an adenovirus that co-expresses PSMA and GFP. We demonstrate sensitive and specific in vitro imaging of endogenous and ectopically expressed PSMA in human cells and in vivo imaging of xenograft tumors. We also discuss chemical strategies for improving performance even further. Taken together, this study describes nearly complete preclinical development of an optically based small-molecule contrast agent for image-guided surgery.