A major complication of HIV-1 infection is the development of HIV-1-associated dementia (HAD). The mechanisms behind HAD are not yet known. Certain studies have indicated that the HIV-1 envelope glycoprotein (gp120) and transregulatory protein (Tat) play a role in the development of HAD. In particular, these proteins affect the integrity of the blood- brain barrier (BBB) by crossing the BBB, altering BBB transporters, and disrupting the BBB. Our studies show that these proteins also induce oxidative stress in RBE4 cells, an in vitro BBB model. We determined that gp120 and Tat induced oxidative stress in RBE4 cells by measuring selected oxidative stress parameters. Subsequently, the levels of glutathione (GSH), the principal intracellular thiol responsible for maintaining the oxidative balance in cells, significantly decreased, indicating that the cells were undergoing oxidative stress. Antioxidants are becoming increasingly popular in oxidative stress-related disorders and hold promise as therapeutic agents. We have also found that N-acetylcysteine amide (NACA), a new thiol antioxidant, significantly increased the levels of GSH in gp120 and Tat-exposed RBE4 cells. Furthermore, many AIDS/HIV-positive patients use addictive drugs, including methamphetamines (METH). Since METH induces oxidative stress as well, drug abusing patients might be at risk of a synergistic effect and increased damage. Our preliminary results showed that METH does indeed work synergistically with gp120 and Tat to induce oxidative stress in RBE4 cells. Since work with RBE4 cells is open to the criticism, discussed under the """"""""Specific Aims"""""""" section, we plan to use a more appropriate in vitro BBB model. Human brain microvascular endothelial cells (HBMVECs) will be used to evaluate the synergistic effect of METH and the two HIV proteins in inducing oxidative stress. Transgenic mice overexpressing gp120 or Tat will serve as our in vivo model. Permeability experiments in BBB models (both in HBMVECs and isolated brain microvascular endothelial cells from the transgenic animals) will also be performed to serve as functional endpoints. Therefore, we propose to determine whether the potent antioxidant NACA protects the BBB from gp120, Tat, or METH (alone and/or in combination).Despite the development and use of effective antiretroviral therapy, HIV-1 associated dementia (HAD) still persists. Certain studies have indicated that the HIV-1 envelope glycoprotein (gp120) and transregulatory protein (Tat) play a role in the development of HAD. One proposed mechanism is that HIV-1 induced neurotoxicity may be due to an increased production of reactive oxygen species (ROS) by HIV-1 proteins (gp120 and Tat). Furthermore, many AIDs/HIV-positive patients use addictive drugs such as methamphetamine (METH). Among these addictive drugs, METH, in particular, induces free radicals in various cells. If neuropathogenesis of HIV infection is found to be caused by a significant increase in oxidative damage by METH and gp120 and/or Tat, antioxidants should be included in the treatment to prevent HIV-induced dementia. Glutathione (GSH) is the principal intracellular thiol responsible for scavenging reactive oxygen species (ROS) and maintaining the oxidative balance in tissues. Cysteine and glutathione delivery compounds protect cells from free radical damage. For example, N-acetylcysteine (NAC) indirectly replenishes GSH through deacetylation to cysteine and prevents oxidative stress. However, NAC has a very low oral bioavailability (30%) and its efficacy is only significant when given intravenously. NAC's failure to provide significant antioxidant effects in vivo might originate from its low solubility and tissue distribution. This inability to provide antioxidant defense may result from NAC's negatively charged carboxyl group. Recently, an amide form of NAC has been synthesized and our laboratory has tested this new compound in various oxidative stress models. Because the carboxylic group in NACA is neutralized, NACA is more lipophilic than NAC and can easily cross the cell membranes. Therefore, we hypothesize that addictive drugs, as exemplified by METH, potentiate the oxidative stress induced by gp120 and Tat at the blood brain barrier (BBB) and that NACA blocks oxidative stress and protects the BBB. ? ? ?

National Institute of Health (NIH)
National Institute on Drug Abuse (NIDA)
Academic Research Enhancement Awards (AREA) (R15)
Project #
Application #
Study Section
NeuroAIDS and other End-Organ Diseases Study Section (NAED)
Program Officer
Lawrence, Diane M
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Missouri University of Science & Technol
Schools of Arts and Sciences
United States
Zip Code
Tobwala, S; Fan, W; Stoeger, T et al. (2013) N-acetylcysteine amide, a thiol antioxidant, prevents bleomycin-induced toxicity in human alveolar basal epithelial cells (A549). Free Radic Res 47:740-9
Tobwala, Shakila; Zhang, Xinsheng; Zheng, Youyou et al. (2013) Disruption of the integrity and function of brain microvascular endothelial cells in culture by exposure to diesel engine exhaust particles. Toxicol Lett 220:1-7
Zhang, X; Tobwala, S; Ercal, N (2012) N-acetylcysteine amide protects against methamphetamine-induced tissue damage in CD-1 mice. Hum Exp Toxicol 31:931-44
Schimel, Andrew M; Abraham, Linu; Cox, Douglas et al. (2011) N-acetylcysteine amide (NACA) prevents retinal degeneration by up-regulating reduced glutathione production and reversing lipid peroxidation. Am J Pathol 178:2032-43
Carey, Joshua W; Pinarci, Eylem Y; Penugonda, Suman et al. (2011) In vivo inhibition of l-buthionine-(S,R)-sulfoximine-induced cataracts by a novel antioxidant, N-acetylcysteine amide. Free Radic Biol Med 50:722-9
Manda, Kalyan Reddy; Banerjee, Atrayee; Banks, William A et al. (2011) Highly active antiretroviral therapy drug combination induces oxidative stress and mitochondrial dysfunction in immortalized human blood-brain barrier endothelial cells. Free Radic Biol Med 50:801-10
Banerjee, Atrayee; Zhang, Xinsheng; Manda, Kalyan Reddy et al. (2010) HIV proteins (gp120 and Tat) and methamphetamine in oxidative stress-induced damage in the brain: potential role of the thiol antioxidant N-acetylcysteine amide. Free Radic Biol Med 48:1388-98
Zhang, Xinsheng; Banerjee, Atrayee; Banks, William A et al. (2009) N-Acetylcysteine amide protects against methamphetamine-induced oxidative stress and neurotoxicity in immortalized human brain endothelial cells. Brain Res 1275:87-95
Ates, Burhan; Ercal, Baris Can; Manda, Kalyan et al. (2009) Determination of glutathione disulfide levels in biological samples using thiol-disulfide exchanging agent, dithiothreitol. Biomed Chromatogr 23:119-23
Ates, Burhan; Abraham, Linu; Ercal, Nuran (2008) Antioxidant and free radical scavenging properties of N-acetylcysteine amide (NACA) and comparison with N-acetylcysteine (NAC). Free Radic Res 42:372-7