Systems toxicology identifies mechanistic impacts of 2-amino-4,6-dinitrotoluene (2A-DNT) exposure in Northern Bobwhite.

BACKGROUND: A systems toxicology investigation comparing and integrating transcriptomic and proteomic results was conducted to develop holistic effects characterizations for the wildlife bird model, Northern bobwhite (Colinus virginianus) dosed with the explosives degradation product 2-amino-4,6-dinitrotoluene (2A-DNT). A subchronic 60 d toxicology bioassay was leveraged where both sexes were dosed via daily gavage with 0, 3, 14, or 30 mg/kg-d 2A-DNT. Effects on global transcript expression were investigated in liver and kidney tissue using custom microarrays for C. virginianus in both sexes at all doses, while effects on proteome expression were investigated in liver for both sexes and kidney in males, at 30 mg/kg-d. RESULTS: As expected, transcript expression was not directly indicative of protein expression in response to 2A-DNT. However, a high degree of correspondence was observed among gene and protein expression when investigating higher-order functional responses including statistically enriched gene networks and canonical pathways, especially when connected to toxicological outcomes of 2A-DNT exposure. Analysis of networks statistically enriched for both transcripts and proteins demonstrated common responses including inhibition of programmed cell death and arrest of cell cycle in liver tissues at 2A-DNT doses that caused liver necrosis and death in females. Additionally, both transcript and protein expression in liver tissue was indicative of induced phase I and II xenobiotic metabolism potentially as a mechanism to detoxify and excrete 2A-DNT. Nuclear signaling assays, transcript expression and protein expression each implicated peroxisome proliferator-activated receptor (PPAR) nuclear signaling as a primary molecular target in the 2A-DNT exposure with significant downstream enrichment of PPAR-regulated pathways including lipid metabolic pathways and gluconeogenesis suggesting impaired bioenergetic potential. CONCLUSION: Although the differential expression of transcripts and proteins was largely unique, the consensus of functional pathways and gene networks enriched among transcriptomic and proteomic datasets provided the identification of many critical metabolic functions underlying 2A-DNT toxicity as well as impaired PPAR signaling, a key molecular initiating event known to be affected in di- and trinitrotoluene exposures.

Comparison of transcriptional responses in liver tissue and primary hepatocyte cell cultures after exposure to hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine.

BACKGROUND: Cell culture systems are useful in studying toxicological effects of chemicals such as Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), however little is known as to how accurately isolated cells reflect responses of intact organs. In this work, we compare transcriptional responses in livers of Sprague-Dawley rats and primary hepatocyte cells after exposure to RDX to determine how faithfully the in vitro model system reflects in vivo responses. RESULTS: Expression patterns were found to be markedly different between liver tissue and primary cell cultures before exposure to RDX. Liver gene expression was enriched in processes important in toxicology such as metabolism of amino acids, lipids, aromatic compounds, and drugs when compared to cells. Transcriptional responses in cells exposed to 7.5, 15, or 30 mg/L RDX for 24 and 48 hours were different from those of livers isolated from rats 24 hours after exposure to 12, 24, or 48 mg/Kg RDX. Most of the differentially expressed genes identified across conditions and treatments could be attributed to differences between cells and tissue. Some similarity was observed in RDX effects on gene expression between tissue and cells, but also significant differences that appear to reflect the state of the cell or tissue examined. CONCLUSION: Liver tissue and primary cells express different suites of genes that suggest they have fundamental differences in their cell physiology. Expression effects related to RDX exposure in cells reflected a fraction of liver responses indicating that care must be taken in extrapolating from primary cells to whole animal organ toxicity effects.

Validation of a genomics-based hypothetical adverse outcome pathway: 2,4-dinitrotoluene perturbs PPAR signaling thus impairing energy metabolism and exercise endurance.

2,4-dinitrotoluene (2,4-DNT) is a nitroaromatic used in industrial dyes and explosives manufacturing processes that is found as a contaminant in the environment. Previous studies have implicated antagonism of PPARα signaling as a principal process affected by 2,4-DNT. Here, we test the hypothesis that 2,4-DNT-induced perturbations in PPARα signaling and resultant downstream deficits in energy metabolism, especially from lipids, cause organism-level impacts on exercise endurance. PPAR nuclear activation bioassays demonstrated inhibition of PPARα signaling by 2,4-DNT whereas PPARγ signaling increased. PPARα (-/-) and wild-type (WT) female mice were exposed for 14 days to vehicle or 2,4-DNT (134 mg/kg/day) and performed a forced swim to exhaustion 1 day after the last dose. 2,4-DNT significantly decreased body weights and swim times in WTs, but effects were significantly mitigated in PPARα (-/-) mice. 2,4-DNT decreased transcript expression for genes downstream in the PPARα signaling pathway, principally genes involved in fatty acid transport. Results indicate that PPARγ signaling increased resulting in enhanced cycling of lipid and carbohydrate substrates into glycolytic/gluconeogenic pathways favoring energy production versus storage in 2,4-DNT-exposed WT and PPARα (-/-) mice. PPARα (-/-) mice appear to have compensated for the loss of PPARα by shifting energy metabolism to PPARα-independent pathways resulting in lower sensitivity to 2,4-DNT when compared with WT mice. Our results validate 2,4-DNT-induced perturbation of PPARα signaling as the molecular initiating event for impaired energy metabolism, weight loss, and decreased exercise performance.