The Investigation on the Mechanism of Neuroprotection by Adenosine in 1,3-Dinitrobenzene Toxicity.

Abstract

Used as an intermediate in plastic and explosive industries, 1,3-dinitrobenzene (1,3-DNB) causes cell-specific lesions in brain stem. In vivo studies have shown that reduced neuronal activity decreases dependence upon glucose metabolism and reduces neuronal damage in rats. However the molecular mechanism of this neuroprotection has remained elusive. This dissertation hypothesizes that 1,3-DNB is an inhibitor of adenosine deaminase (ADA), and that inhibition of ADA increases local extracellular adenosine levels. Elevated extracellular adenosine provides neuroprotection by binding to the inhibitory adenosine receptor 1 (A1R) to suppress the excitability of neurons. Firstly, using spectrophotometry, we showed that 1,3-DNB inhibited ADA with an IC50 of 284 µM. Computational modeling and kinetics studies suggest mixed inhibition with one and four 1,3-DNB molecules binding to active and peripheral sites of one human ADA molecule, respectively. Second, we examined whether inhibition of ADA increases extracellular adenosine concentration ([Ado ]e) in primary astrocyte-conditioned media using enzyme-based sensors. We found that [Ado]e elevated significantly to 4-6 µM in primary astrocytes, a two or three magnitude increase compared to physiological conditions. Finally we assessed adenosine-mediated neuroprotection in cultures of rat primary neurons. Results show that addition of exogenous adenosine (10 and 100 µM) to 1,3-DNB exposed neurons increased ATP levels by 25% and 50% respectively. 100 µM CPA (an A1R agonist) increased membrane integrity of neurons by approximately 100%. Adenosine 5 µM also significantly delayed onset and reduced the magnitude of cytoplasmic Ca2+ increases by binding to A1R. Both neuroprotection and suppressed excitability are mediated by activation of A1R, as demonstrated by application of AR agonists and antagonists. This work advances understanding of the effects of 1,3-DNB and similar environmental/industrial chemicals on enzymes in the energy metabolism pathway and provides a mechanistic explanation for the observed mixed inhibition of ADA. The role of adenosine-mediated neuroprotection through modulation of cytoplasmic Ca2+ increase elicited by exogenous KCl stimulation is explored using pharmacologic agonists and antagonists of the A1R.