Hazard Assessment of High-Nitrogen Compounds: Coupling Transcriptomics with Multi-Cellular Co-Culture Assays Provides Accurate Systemic Toxicity Screening [TNT]

Abstract - Current in vitro hazard assessment techniques lack the systems context necessary to holistically describe in vivo toxicity. To remedy this shortcoming, we have employed the Integrated discrete Multiple Organ Co-Culture (IdMOC) system in conjunction with global-transcriptomic expression assessment to provide a systems context of interactive organ and toxicity pathway responses. Specifically, IdMOC exposures containing hµMan cells representative of five organ types (kidney, liver, lung, vascular endotheliµM and heart muscle) were compared to mono-culture exposures, to evaluate toxic effects in a well-studied legacy munition, 2,4,6-trinitrotoluene (TNT) compared to a structurally similar insensitive munition, 2,4-dinitroanisole (DNAN) having sparsely described toxicity. DNAN toxicity, based on cell viability, was significantly lower than TNT for three cell lines: liver, vascular endotheliµM, and heart. Toxicity pathways enriched in transcriptional analysis of kidney cells exposed to TNT and DNAN through the IdMOC system reflected the literature-based in vivo mammalian toxicity of parent compounds and metabolites where multiple screening values matched chronic dosing levels within 1 order of magnitude. Enriched pathways were primarily unique to each chemical where TNT exposures affected xenobiotic metabolism, oxidative stress, and cell cycle pathways in addition to pathways providing potential screening for hematotoxicity, renal toxicity and urinary cancer. Ten pathway-level responses from IdMOC suggested that DNAN elicited toxicity representative of the primary metabolite, 2,4-dinitrophenol (2,4-DNP). Relative to monoculture, IdMOC results provided a higher nµMber and more robust enrichment of pathways involved in known in vivo toxicity mechanisms for TNT and a profile indicative of in vivo systemic toxicity of DNAN and metabolite 2,4-DNP. HµMan kidney proximal tubule epithelial cells (HK-2) derived from normal tissues (American Type Culture Collection, ATCC®, Manassas, VA) were exposed in mono-culture and co-culture to 2,4,6-tinitrotoluene (TNT). Mono-cultures were conducted in standard 96 well plates whereas co-culutres were conducted in Integrated discrete Multiple Organ Co-Culture (IdMOC) 96 well plates where the kidney cells were co-exposed in a common media with liver (THLE-3), lung (NuLi-1), vascular endotheliµM (eloHAEC), and heart (AC10) cell, all from American Type Culture Collection (ATCC®, Manassas, VA). The mono- and co-culture plates were incubated on shakers at low speed for 24 hours while exposed to TNT at 0 (1% DMSO carrier control), 10µM, and 100µM where all conditions included 4 replicates. In sµMmary, the experiment had a completely randomized design and included a 2 x 3 factorial treatment including 2 culture conditions (mono- and co-cultures) each combined with the 3 TNT exposure levels (0, 10µM, and 100µM) all including x 4 replication. The microarray of one control replicate within the TNT, co-culture exposure treatment was compromised and removed from the data analysis leaving a total of 23 microarray samples.