Abstract
Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis.
Highlights
Contamination of natural environments by explosives and their degradation products during field use or manufacturing waste streams exposes diverse organisms, including humans, to potential toxicity
The explosive content of the pink water used in this study was determined by high-performance liquid chromatography (HPLC) analysis using a YL-9100 HPLC instrument (Young Lin Instrument Co., Anyang, Korea) with TC-C18 column at 30 ◦C as shown in Materials and Methods (Figure S1)
The major peak in the HPLC profile of pink water was verified as TNT (2,4,6-trinitrotoluene) with a concentration 45.54 ± 0.49 μg/mL; RDX and HMX
Summary
Contamination of natural environments by explosives and their degradation products during field use or manufacturing waste streams exposes diverse organisms, including humans, to potential toxicity. Trinitrotoluene (TNT), a polynitro-organic compound, is one of the main components of explosives and had been detected before in the environment at a concentration of 0.4–21,960 μg/L in ground water, 1.0–3375 μg/L in surface water, 6.7–711,000 mg/kg in sediment, and 0.08–87,000 mg/kg in soil at US military installations, processing facilities and military firing ranges [1]. TNT in soil has been shown to cause lethal and sublethal toxicity in soil invertebrates and freshwater fish and to decrease reproduction rates at concentrations of several hundred mg TNT/kg dry soil [9,11,12,13], as well as in various aquatic species and mammals [1]. It is important that screening tools sensitive enough to detect in vivo biological toxicity of TNT and related compounds at low concentrations are developed
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