Mixed Inhibition of Adenosine Deaminase Activity by 1,3-Dinitrobenzene: A Model for Understanding Cell-Selective Neurotoxicity in Chemically-Induced Energy Deprivation Syndromes in Brain

Abstract

Astrocytes are acutely sensitive to 1,3-dinitrobenzene (1,3-DNB) while adjacent neurons are relatively unaffected, consistent with other chemically-induced energy deprivation syndromes. Previous studies have investigated the role of astrocytes in protecting neurons from hypoxia and chemical injury via adenosine release. Adenosine is considered neuroprotective, but it is rapidly removed by extracellular deaminases such as adenosine deaminase (ADA). The present study tested the hypothesis that ADA is inhibited by 1,3-DNB as a substrate mimic, thereby preventing adenosine catabolism. ADA was inhibited by 1,3-DNB with an IC(50) of 284 μM, Hill slope, n = 4.8 ± 0.4. Native gel electrophoresis showed that 1,3-DNB did not denature ADA. Furthermore, adding Triton X-100 (0.01-0.05%, wt/vol), Nonidet P-40 (0.0015-0.0036%, wt/vol), or bovine serum albumin (0.05 mg/ml or changing [ADA] (0.2 and 2 nM) did not substantially alter the 1,3-DNB IC(50) value. Likewise, dynamic light scattering showed no particle formation over a (1,3-DNB) range of 149-1043 μM. Kinetics revealed mixed inhibition with 1,3-DNB binding to ADA (K(I) = 520 ± 100 μM, n = 1 ± 0.6) and the ADA-adenosine complex (K(IS) = 262 ± 7 μM, n = 6 ± 0.6, indicating positive cooperativity). In accord with the kinetics, docking predicted binding of 1,3-DNB to the active site and three peripheral sites. In addition, exposure of DI TNC-1 astrocytes to 10-500 μM 1,3-DNB produced concentration-dependent increases in extracellular adenosine at 24 h. Overall, the results demonstrate that 1,3-DNB is a mixed inhibitor of ADA and may thus lead to increases in extracellular adenosine. The finding may provide insights to guide future work on chemically-induced energy deprivation.

The Role of Astrocyte Mitochondria in Differential Regional Susceptibility to Environmental Neurotoxicants: Tools for Understanding Neurodegeneration

“…Increased neuronal energy demand may exacerbate astrocytic damage, as observed in the case of DNB exposure (Holton et al, 1997). Involvement of mitochondria in astrocytes is likely related to astrocytic metabolic output in the case of DNB exposure due to the accumulation of astrocyte-derived extracellular adenosine; the ensuing mixed inhibition of extracellular adenosine deaminase by DNB contributes to the accumulation of extracellular adenosine (Wang et al, 2012).…”

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

“…The primary cellular target in the initial phase of 1,3-DNB intoxication are type 1 astrocytes found within the affected brainstem nuclei (Philbert et al, 1987; Romero et al, 1996). Although the molecular mechanism that underlies the selective nature of 1,3-DNB neurotoxicity is still undetermined, research findings support a mechanism that includes, but is not limited to, metabolic perturbation through the inhibition of pyruvate dehydrogenase and adenosine deaminase that could contribute to increased reactive oxygen species (ROS) production, loss of the mitochondrial membrane potential (ΔΨ m ) and induction of the mitochondrial permeability transition (MPT) (Romero et al, 1995; Tjalkens et al, 2000; Miller et al, 2011; Wang et al, 2012). 1,3-DNB-induced oxidative stress and mitochondrial dysfunction has also been linked to the oxidative carbonylation of specific proteins distributed within different intracellular locations (Steiner and Philbert 2011).…”