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

Toxicol Sci. 2012 Feb;125(2):509-21. doi: 10.1093/toxsci/kfr317. Epub 2011 Nov 21.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenosine / metabolism
  • Adenosine Deaminase / chemistry
  • Adenosine Deaminase / metabolism*
  • Adenosine Deaminase Inhibitors / chemistry
  • Adenosine Deaminase Inhibitors / toxicity*
  • Amino Acid Sequence
  • Animals
  • Astrocytes / drug effects*
  • Astrocytes / enzymology
  • Astrocytes / pathology
  • Binding Sites
  • Brain / drug effects*
  • Brain / enzymology
  • Brain / pathology
  • Cattle
  • Cell Line
  • Dinitrobenzenes / chemistry
  • Dinitrobenzenes / toxicity*
  • Dose-Response Relationship, Drug
  • Energy Metabolism / drug effects*
  • Humans
  • Inosine / metabolism
  • Kinetics
  • Mice
  • Models, Molecular
  • Molecular Sequence Data
  • Molecular Structure
  • Neurons / drug effects*
  • Neurons / enzymology
  • Neurons / pathology
  • Neurotoxicity Syndromes / enzymology
  • Neurotoxicity Syndromes / etiology*
  • Neurotoxicity Syndromes / pathology
  • Protein Conformation
  • Protein Denaturation
  • Structure-Activity Relationship
  • Up-Regulation

Substances

  • Adenosine Deaminase Inhibitors
  • Dinitrobenzenes
  • Inosine
  • 3-dinitrobenzene
  • Adenosine Deaminase
  • Adenosine