Effects of Munitions Compounds on Xenobiotic-Activated Nuclear Receptors and Cell Signaling Pathways

David R. Johnson1, ChooYaw Ang2, Tanwir Habib2, Edward J. Perkins1

INTRODUCTION:

• Exposure to certain munitions compounds is known to alter physiological functions in test organisms, however little is known about their molecular and cellular effects.

• Several nuclear receptors are regulated by xenobiotic compounds. These nuclear receptors belong to a class of ligand-activated transcription factors that, when heterodimerized with RXRα and bound to their respective DNA binding elements, activate gene transcription

• Several cell signaling pathways are also affected by xenobiotics, causing dysfunction of normal cellular function and homeostasis

• Whole animal experimentation is time and resource intensive and often generates data that is difficult to interpret

• An initial screening approach would provide detailed mechanistic information that can then be used to guide further in vitro (cell culture) and in vivo (animal) experiments

• The objective of this study was to characterize the effects of new and existing munitions compounds on xenobiotic-activated nuclear receptor and transcription factor pathways in order to better identify potential molecular initiating events that may result in toxicity.

SUMMARY/CONCLUSIONS • TNT and its degradation products 2,4-DNT and 2,6-DNT differentially activated cell signaling pathways, with at

least two pathways commonly shared (AhR and Nrf2/Nrf1). 2,4-Dinitroanisole, a TNT replacement munitions, activated fewer cell signaling pathways than TNT.

• Nitroaromatic munitions compounds appear to regulate nuclear receptor activity more than cyclic nitramine compounds • Most nitroaromatics decrease CAR, PPARα, PPARδ, and LXRα activation. • Select nitroaromatics increase AhR and PPARγ activation.

• DNAN, a TNT replacement compound, also decreases nuclear receptor bioassays like TNT, though the inhibitory effect is less prominent than with TNT. NTO, a RDX replacement compound, increases PPARα and LXRα activation at high concentrations, unlike RDX which does not affect these nuclear receptors.

• These results create a more comprehensive picture of munitions effects within cells and potential down-stream effects within tissues and organisms. Furthermore, rapid nuclear bioassays provide an avenue for material scientist to predict biochemical pathways that may be impacted by newly developed munitions earlier in the development process.

1 Environmental Laboratory, US Army Engineer Research and Development Center, Vicksburg, MS 2 Badger Technical Services, Vicksburg, MS

RESULTS

METHODS: • Chemicals:

•Nitroaromatics: 2,4,6-trinitrotoluene (2,4,6-TNT), 2-amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), 2,4-diamino-6-nitrotoluene (2,4-DANT), 1,3,5-trinitrobenzene (1,3,5-TNB), 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT), 2-methoxy-4-nitrophenol (2-M-4-NP) •Cyclic nitrotriazines: hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), hexahydro-3,5-dinitro-1-nitroso-1,3,5-triazine (MNX), hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) •New munitions: 2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one (NTO), 1,1-diamino-2,2-dinitroethene (FOX-7)

• Cell signaling pathways: Effects of munitions compounds (10 mg/l) on 45 cell signaling pathways were examined using the Cignal 45-pathway reporter array kit (QIAGEN) that reverse-transfected transcription factor-firefly luciferase reporter plasmids into HepaRG liver cells. Cells were then exposed to munitions compounds for 24 h, then examined for signal pathway activation. Data are expressed as relative response ratio (i.e., assay transfection efficiency).

• Nuclear receptor activation bioassays: Chemicals (0, 0.01, 0.1, 1, & 10 mg/l) were incubated with non-human cells containing either the CAR, PPARα, PPARγ, PPARδ, FXR, LXRα, LXRβ, or RXRα reporter assay (Indigo Biosciences, State College, PA) for 24 h according to the manufacturers instructions. AhR reporter assay was conducted using the human CYP1A1 promoter sequence fused to the firefly luciferase gene reporter and transfected into 101L cell line . After 24 h exposure, luminescence was analyzed.

• Cell survival was determined using the live cell multiplex assay (calcein-AM dye) and/or neutral red uptake.

• Statistics: one-way analysis of variance (ANOVA) with Holm-Sidak post hoc test comparison to control samples (* p < 0.05).

Figure 8. LXR activation by munitions compounds

Acknowledgment: This project was funded by the Army’s Environmental Quality & Technology research program. The findings of this presentation are not to be construed as an official Department of the Army position unless so designated by other authorized documents. Permission was granted by the Chief of Engineers to distribute this information.

Abstract 1295, Poster 257

Figure 7. FXR activation by munitions compounds

Figure 1. Transcription factor activation by munitions compounds. Data expressed as relative response ratio (i.e., signal to assay transfection efficiency). N = 6.

Figure 2. AhR activation by munitions compounds

Figure 3. CAR activation by munitions compounds

Figure 4. Cell viability when exposed to munitions compounds

Figure 5. PPAR activation by munitions compounds (A-PPARα, B-PPARγ, C-PPARδ).

Figure 6. Nitroaromatic compounds differentially decrease PPARα activation.