A comparative study of protein carbonylation and mitochondrial dysfunction using the neurotoxicants 1,3-dinitrobenzene, 3-nitropropionic acid, and 3-chloropropanediol.

This comparative evaluation of neurotoxicants previously identified as models of chemical-induced mitochondrial dysfunction and energy deprivation demonstrated that subtoxic concentrations of 1,3-dinitrobenzene (1,3-DNB), 3-nitropropionic acid (3-NPA), and 3-chloropropanediol (3-CPD) each led to concentration-dependent loss of the mitochondrial membrane potential (ΔΨm) associated with similar patterns of protein carbonylation. Subtoxic concentrations of each neurotoxicant were determined by measuring DI TNC1 cell viability using the MTS cell proliferation assay. Although exposure 1 µM, 10 µM, and 100 µM concentrations of each toxicant did not result in loss of cell viability after 48 h, exposure to each toxicant at these concentrations led to concentration-dependent loss of tetramethyl rhodamine methyl ester (TMRM) fluorescence over the same exposure period. Preincubation with the antioxidant, deferoxamine, was effective in preventing loss of TMRM flurorescence. Through the combined use of two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and Oxyblot analysis, this study demonstrated that exposure to each toxicant resulted in the formation of distinctly similar patterns of protein carbonylation comprised of specific proteins identified with tandem MS/MS. Our results provide insight as to how exposure to different neurotoxicants that enhance oxidative stress may, in fact, lead to mitochondrial injury and subsequent toxicity through selective, yet shared, pathways of protein modification by oxidative carbonylation.

Proteomic identification of carbonylated proteins in 1,3-dinitrobenzene neurotoxicity.

This study demonstrated that 1,3-dinitrobenzene-induced (1,3-DNB) oxidative stress led to the oxidative carbonlyation of specific protein targets in DI TNC1 cells. 1,3-DNB-induced mitochondrial dysfunction, as indicated by loss of tetramethyl rhodamine methyl ester (TMRM) fluorescence, was initially observed at 5h and coincided with peak reactive oxygen species (ROS) production. ROS production was inhibited in cells pre-treated with the mitochondrial permeability transition (MPT) inhibitor, bonkrekic acid (BkA). Pre-incubation with the antioxidant deferoxamine inhibited loss of TMRM fluorescence until 24h after initial exposure to 1,3-DNB. Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and subsequent Oxyblot analysis were used to determine if 1,3-DNB exposure led to the formation of protein carbonyls. Exposing DI TNC1 cells to 1,3-DNB led to marked protein carbonylation 45 min following initial exposure. Pre-treatment with deferoxamine or Trolox reduced the intensity of protein carbonylation in DI TNC1 cells exposed to 1mM 1,3-DNB. Tandem MS/MS performed on protein samples isolated from 1,3-DNB-treated cells revealed that specific proteins within the mitochondria, endoplasmic reticulum (ER), and cytosol are targets of protein carbonylation. The results presented in this study are the first to suggest that the molecular mechanism of 1,3-DNB neurotoxicity may occur through selective carbonylation of protein targets found within specific intracellular compartments of susceptible cells.

The central role of calcium in the effects of cytokines on beta-cell function: implications for type 1 and type 2 diabetes.

The appropriate regulation of intracellular calcium is a requirement for proper cell function and survival. This review focuses on the effects of proinflammatory cytokines on calcium regulation in the insulin-producing pancreatic beta-cell and how normal stimulus-secretion coupling, organelle function, and overall beta-cell viability are impacted. Proinflammatory cytokines are increasingly thought to contribute to beta-cell dysfunction not only in type 1 diabetes (T1D), but also in the progression of type 2 diabetes (T2D). Cytokine-induced disruptions in calcium handling result in reduced insulin release in response to glucose stimulation. Cytokines can alter intracellular calcium levels by depleting calcium from the endoplasmic reticulum (ER) and by increasing calcium influx from the extracellular space. Depleting ER calcium leads to protein misfolding and activation of the ER stress response. Disrupting intracellular calcium may also affect organelles, including the mitochondria and the nucleus. As a chronic condition, cytokine-induced calcium disruptions may lead to beta-cell death in T1D and T2D, although possible protective effects are also discussed. Calcium is thus central to both normal and pathological cell processes. Because the tight regulation of intracellular calcium is crucial to homeostasis, measuring the dynamics of calcium may serve as a good indicator of overall beta-cell function.