HAART drugs induce oxidative stress and mitochondrial dysfunction in blood-brain barrier

The era of highly active antiretroviral therapy (HAART) has controlled AIDS and its related disorders considerably; however, the prevalence of HIV-1-associated neurocognitive disorders (HAND) has been on the rise in the post-HAART era. In view of these developments, we investigated whether a HAART drug combination of 3\u27-Azido-2\u27, 3\u27-deoxythymidine (AZT) and Indinavir (IDV) can alter the functionality of the blood-brain barrier (BBB) endothelial cells, thereby exacerbating the condition. Viability of hCMEC/D3 cells (in vitro model of BBB) that were exposed to the drugs was significantly reduced after a 72 hr treatment, in a dose-dependent manner. Reactive oxygen species (ROS) were highly elevated after the exposure, indicating that mechanisms that induce oxidative stress were involved. Measures of oxidative stress such as glutathione (GSH) and malondialdehyde (MDA) were found to be altered in the treated groups. Loss of mitochondrial membrane potential (Δψm) assessed with fluorescent microscopy and decreased levels of ATP indicated that cytoxicity was mediated through mitochondrial dysfunction. Furthermore, AZT + IDV treatment caused apoptosis in endothelial cells as assessed by the expression of cytochrome c and procaspase-3 proteins. In vivo experiments with HIV-1 transgenic animal treated with AZT+ IDV showed decrease in GSH in the BBB and brain and increase in MDA levels in the BBB. Thiol antioxidant N-acetylcysteine amide (NACA) reversed some of the pro-oxidant effects of AZT+IDV in both invitro and invivo studies. Results from our studies indicate that the AZT + IDV combination can affect the BBB and may play a role in contributing to neurocognitive disorders in HIV-1 infected individuals treated with HAART drugs --Abstract, page iii

Biologically Important Thiols in Aqueous Extracts of Spices and Evaluation of Their in Vitro Antioxidant Properties

The levels of the biologically important thiols in aqueous extracts of different spices were determined using a sensitive high performance liquid chromatography (HPLC) technique. The spices analysed: turmeric, ginger, cardamom, mustard, fenugreek, and coriander showed different levels of thiols. Biologically important thiols or biothiols measured in these spices included glutathione (GSH), cysteine (CYS), N-acetylcysteine (NAC), homocysteine (HCYS), and γ-glutamyl cysteine (GGC). Our results showed that thiol levels varied from 4 to 1089 nM/g weight (dry or wet). Furthermore, none of the biothiols analysed were found in cumin, nutmeg, clove or star anise. We also studied the antioxidant abilities of these aqueous extracts using various in vitro antioxidant methods to correlate between the levels of these thiols and their antioxidant effects. Our results suggested that antioxidant activities may be independent of thiol content and may be, in part the combination of all the phytochemicals present. These results may be useful in explaining the effect of spices on thiol levels in in vitro and in vivo studies

Highly Active Antiretroviral Therapy Drug Combination Induces Oxidative Stress and Mitochondrial Dysfunction in Immortalized Human Blood-brain Barrier Endothelial Cells

The era of highly active antiretroviral therapy (HAART) has controlled AIDS and its related disorders considerably; however, the prevalence of HIV-1-associated neurocognitive disorders has been on the rise in the post-HAART era. in view of these developments, we investigated whether a HAART drug combination of 3′-azido-2′,3′-deoxythymidine (AZT) and indinavir (IDV) can alter the functionality of the blood-brain barrier (BBB) endothelial cells, thereby exacerbating this condition. the viability of hCMEC/D3 cells (in vitro model of BBB) that were exposed to these drugs was significantly reduced after 72 h treatment, in a dose-dependent manner. Reactive oxygen species were highly elevated after the exposure, indicating that mechanisms that induce oxidative stress were involved. Measures of oxidative stress parameters, such as glutathione and malondialdehyde, were altered in the treated groups. Loss of mitochondrial membrane potential, as assessed by fluorescence microscopy and decreased levels of ATP, indicated that cytotoxicity was mediated through mitochondrial dysfunction. Furthermore, AZT + IDV treatment caused apoptosis in endothelial cells, as assessed by the expression of cytochrome c and procaspase-3 proteins. Pretreatment with the thiol antioxidant N-acetylcysteine amide reversed some of the pro-oxidant effects of AZT + IDV. Results from our in vitro studies indicate that the AZT + IDV combination may affect the BBB in HIV-infected individuals treated with HAART drugs

HIV Proteins (gp120 and Tat) and Methamphetamine in Oxidative Stress-induced Damage in the Brain: Potential Role of the Thiol Antioxidant N-acetylcysteine Amide

An increased risk of HIV-1 associated dementia (HAD) has been observed in patients abusing methamphetamine (METH). Since both HIV viral proteins (gp120, Tat) and METH induce oxidative stress, drug abusing patients are at a greater risk of oxidative stress-induced damage. The objective of this study was to determine if N-acetylcysteine amide (NACA) protects the blood brain barrier (BBB) from oxidative stress-induced damage in animals exposed to gp120, Tat and METH. To study this, CD-1 mice pre-treated with NACA/saline, received injections of gp120, Tat, gp120 + Tat or saline for 5 days, followed by three injections of METH/saline on the fifth day, and sacrificed 24 h after the final injection. Various oxidative stress parameters were measured, and animals treated with gp120 + Tat + Meth were found to be the most challenged group, as indicated by their GSH and MDA levels. Treatment with NACA significantly rescued the animals from oxidative stress. Further, NACA-treated animals had significantly higher expression of TJ proteins and BBB permeability as compared to the group treated with gp120 + Tat + METH alone, indicating that NACA can protect the BBB from oxidative stress-induced damage in gp120, Tat and METH exposed animals, and thus could be a viable therapeutic option for patients with HAD

Determination of Glutathione Disulfide Levels in Biological Samples Using Thiol-disulfide Exchanging Agent, Dithiothreitol

A reverse-phase HPLC method incorporating dithiothreitol (DTT) reduction for quantitative determination of oxidized glutathione (GSSG) in biological samples is described here. This method is based on our previous enzymatic reduction technique that uses N-1-(pyrenyl) maleimide (NPM) as a derivatizing agent. In our earlier method, glutathione disulfide (GSSG) was measured by first reducing it to GSH with glutathione reductase (GR) in the presence of NADPH. However, this is a very costly and time-consuming technique. The method described here employs a common and inexpensive thiol-disulfide exchanging agent, DTT, for reduction of GSSG to GSH, followed by derivatization with NPM. The calibration curves are linear over a concentration range of 25-1250 nm (r2 \u3e 0.995). The coefficients of variations for intra-run precision and inter-run precision range from 0.49 to 5.10% with an accuracy range of 1.78-6.15%. The percentage of relative recovery ranges from 97.3 to 103.2%. This new method provides a simple, efficient, and cost-effective way of determining glutathione disulfide levels with a 2.5 nm limit of detection per 5 µL injection volume

N-acetylcysteineamide (NACA) Prevents Inflammation and Oxidative Stress in Animals Exposed to Diesel Engine Exhaust

Diesel exhaust particles (DEPs), a by-product of diesel engine exhaust (DEE), are one of the major components of air borne particulate matter (PM) in the urban environment. DEPs are composed of soot, polycyclic aromatic hydrocarbons (PAHs), redox active semi-quinones, and transition metals, which are known to produce pro-oxidative and pro-inflammatory effects, thereby leading to oxidative stress-induced damage in the lungs. The objective of this study was to determine if N-acetylcysteineamide (NACA), a novel thiol antioxidant, confers protection to animals exposed to DEPs from oxidative stress-induced damage to the lung. To study this, male C57BL/6 mice, pretreated with either NACA (250 mg/kg body weight) or saline, were exposed to DEPs (15 mg/m3) or filtered air (1.5-3 h/day) for nine consecutive days. The animals were sacrificed 24 h after the last exposure. NACA-treated animals exposed to DEP had significant decreases in the number of macrophages and the amount of mucus plug formation in the lungs, as compared to the DEP-only exposed animals. In addition, DEP-exposed animals, pretreated with NACA, also experienced significantly lower oxidative stress than the untreated group, as indicated by the glutathione (GSH), and malondialdehyde (MDA) levels and catalase (CAT) activity. Further, DEP-induced toxicity in the lungs was reversed in NACA-treated animals, as indicated by the lactate dehydrogenase levels. Taken together, these data suggest that the thiol-antioxidant, NACA, can protect the lungs from DEP-induced inflammation and oxidative stress related damage