N-acetylcysteine amide, a thiol antioxidant, prevents bleomycin-induced toxicity in human alveolar basal epithelial cells (A549).

Bleomycin (BLM), a glycopeptide antibiotic from Streptomyces verticillus, is an effective antineoplastic drug. However, its clinical use is restricted due to the wide range of associated toxicities, especially pulmonary toxicity. Oxidative stress has been implicated as an important factor in the development of BLM-induced pulmonary toxicity. Previous studies have indicated disruption of thiol-redox status in lungs (lung epithelial cells) upon BLM treatment. Therefore, this study focused on (1) investigating the oxidative effects of BLM on lung epithelial cells (A549) and (2) elucidating whether a well-known thiol antioxidant, N-acetylcysteine amide (NACA), provides any protection against BLM-induced toxicity. Oxidative stress parameters, such as glutathione (GSH), malondialdehyde (MDA), and antioxidant enzyme activities were altered upon BLM treatment. Loss of mitochondrial membrane potential (ΔΨm), as assessed by fluorescence microscopy, indicated that cytotoxicity is possibly mediated through mitochondrial dysfunction. Pretreatment with NACA reversed the oxidative effects of BLM. NACA decreased the reactive oxygen species (ROS) and MDA levels and restored the intracellular GSH levels. Our data showed that BLM induced A549 cell death by a mechanism involving oxidative stress and mitochondrial dysfunction. NACA had a protective role against BLM-induced toxicity by inhibiting lipid peroxidation, scavenging ROS, and preserving intracellular GSH and ΔΨm. NACA can potentially be developed into a promising adjunctive therapeutic option for patients undergoing chemotherapy with BLM.

N-acetylcysteine amide protects against methamphetamine-induced tissue damage in CD-1 mice.

Methamphetamine (METH), a highly addictive drug used worldwide, induces oxidative stress in various animal organs, especially the brain. This study evaluated oxidative damage caused by METH to tissues in CD-1 mice and identified a therapeutic drug that could protect against METH-induced toxicity. Male CD-1 mice were pretreated with a novel thiol antioxidant, N-acetylcysteine amide (NACA, 250 mg/kg body weight) or saline. Following this, METH (10 mg/kg body weight) or saline intraperitoneal injections were administered every 2 h over an 8-h period. Animals were killed 24 h after the last exposure. NACA-treated animals exposed to METH experienced significantly lower oxidative stress in their kidneys, livers, and brains than the untreated group, as indicated by their levels of glutathione, malondialdehyde, and protein carbonyl and their catalase and glutathione peroxidase activity. This suggests that METH induces oxidative stress in various organs and that a combination of NACA as a neuro- or tissue-protective agent, in conjunction with current treatment, might effectively treat METH abusers.

Pb2+ exposure alters the lens alpha A-crystallin protein profile in vivo and induces cataract formation in lens organ culture.

Epidemiological data supports lead exposure as a risk factor for cataract development. Previous studies which demonstrated oxidative imbalances in the lens following in vivo Pb(2+) exposure support the idea that lead exposure can alter the lens biochemical homeostasis which may ultimately lead to loss of lens clarity with time. alpha-Crystallin, a major lens structural protein and molecular chaperone, undergoes various post-translational modifications upon aging which may contribute to decreased chaperone function and contribute to loss of lens clarity. This study evaluated the impact of 5 weeks of oral Pb(2+) exposure (peripheral Pb(2+) level approximately 30 microg/dL) on the alphaA-crystallin protein profile of the lens from Fisher 344 rats. Decreases in relative protein spot intensity of more acidic forms of alphaA- and betaA(4)-crystallin and of truncated forms of alphaA-crystallin were noted. This data indicates that changes in post-translational processing of crystallins do occur in vivo following short courses of clinically relevant Pb(2+)-exposure. In addition, organ culture of lenses from 4.5-month-old rats in 5 microM Pb(2+) resulted in opacities, demonstrating that lead is toxic to the lens and can induce a loss of lens clarity.

Oxidative effects of lead in young and adult Fisher 344 rats.

Lead poisoning has been extensively studied over the years. Many adverse physiological and behavioral impacts on the human body have been reported due to the entry of this heavy metal. It especially affects the neural development of children. The current study investigates the effect of lead exposure in young (1.5 months) and adult (10 months) male Fisher 344 rats. Five weeks of lead administration resulted in a profound change in the lead levels in the red blood cells (RBCs) of the young lead-exposed group (37.0 +/- 4.47 microg/dl) compared to the control (<1 microg/dl) and adult (27.4 +/- 8.38 microg/dl) lead-exposed groups. Therefore, this study confirms the fact that gastrointestinal absorption of lead in young is greater than that of adults. Furthermore, glutathione and glutathione disulfide (GSSG) levels in RBCs, liver, and brain tissues were measured to determine thiol status; malondialdehyde (MDA) levels of lipid peroxidation and catalase activity were measured to assess changes in oxidative stress parameters. Liver GSSG and MDA levels were significantly higher in the young lead-exposed group than those in the adult lead-exposed group. In RBCs and brains, however, adult lead-exposed animals have shown more elevated MDA levels than young animals exposed to the same lead treatment.

Determination of captopril in biological samples by high-performance liquid chromatography with ThioGlo 3 derivatization.

Captopril, a well-known angiotensin converting enzyme (ACE) inhibitor, is widely used for treatment of arterial hypertension. Recent studies suggest that it may also act as a scavenger of free radicals because of its thiol group. Therefore, the present study describes a rapid, sensitive and relatively simple method for the detection of captopril in biological tissues with reverse-phase HPLC. Captopril was first derivatized with ThioGlo 3 [3H-Naphto[2,1-b]pyran,9-acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)phenyl-3-oxo-)]. It was then detected by fluorescence-HPLC using an Astec C(18) column as the stationary phase and a water:acetonitrile:acetic acid:phosphoric acid mixture (50:50; 1 mL/L acids) as the mobile phase (excitation wavelength, 365 nm; emission wavelength, 445 nm). The calibration curve for captopril was linear over a range of 10-2500 nM and the coefficient of variation acquired for the within- and between-run precision for captopril was 0.5 and 3.8%, respectively. The detection limit of captopril with this method was found to be 200 fmol/20 microL injection volume. Its relative recovery from biological samples was determined to the range from 93.3 to 105.3%. Based on these results, we believe that our method is advantageous for captopril determination.

Antioxidant effect of taurine against lead-induced oxidative stress.

Oxidative stress is proposed as a molecular mechanism in lead toxicity, which suggests that antioxidants might play a role in the treatment of lead poisoning. The present study was designed to investigate whether taurine has a beneficial effect both on Chinese hamster ovary (CHO) cells and on Fisher 344 (F344) rats following lead exposure. Therefore, oxidative stress parameters (glutathione, malondialdehyde levels, catalase, and glucose-6-phosphate dehydrogenase [G6PD] activities) of lead-exposed CHO cells and F344 rats were determined following taurine treatment. Taurine was found to be effective in (1) increasing glutathione levels that had been diminished by lead; (2) reducing malondialdehyde levels, an end-product of lipid peroxidation; (3) decreasing catalase and erythrocyte G6PD activity, which had been increased by lead exposure; and (4) improving cell survival of CHO cells. However, taurine had no effect on blood and tissue lead levels when 1.1 g/kg/day taurine was administered to F344 rats for 7 days, following 5 weeks of lead exposure (2,000 ppm lead acetate). As a result, taurine seems to be capable of fortifying cells against lead-induced oxidative attack without decreasing lead levels. Therefore, administration of taurine, accompanied by a chelating agent, might increase its effectiveness in the treatment of lead poisoning.

Determination of biological thiols by high-performance liquid chromatography following derivatization by ThioGlo maleimide reagents.

The importance of thiols has stimulated the development of a number of methods for determining glutathione and other biologically significant thiols. Methods that are currently available, however have some limitations, such as being time consuming and complex. In the present study, a new high-performance liquid chromatography (HPLC) method for determining biological thiols was developed by using 9-Acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)phenyl)-3-oxo-3H-naphtho[2,1-b]pyran (ThioGlo3) as a derivatizing agent. ThioGlo reacts selectively and rapidly with the thiols to yield fluorescent adducts which can be detected fluorimetrically (lambda(ex) = 365 nm, lambda(em) = 445 nm). The within-run coefficient of variation for glutathione (GSH) by this method ranges from 1.08 to 2.94% whereas the between-run coefficient of variation for GSH is 4.31-8.61%. For GSH, the detection limit is around 50 fmol and the GSH derivatives remain stable for 1 month, if kept at 4 degrees C. Results for GSSG and cysteine are also included. The ThioGlo method is compared to our previous method in which N-(1-pyrenyl)maleimide (NPM) is used to derivatize thiol-containing compounds. The present method offers various advantages over the currently accepted techniques, including speed and sensitivity.

Toxic metals and oxidative stress part I: mechanisms involved in metal-induced oxidative damage.

Toxic metals (lead, cadmium, mercury and arsenic) are widely found in our environment. Humans are exposed to these metals from numerous sources, including contaminated air, water, soil and food. Recent studies indicate that transition metals act as catalysts in the oxidative reactions of biological macromolecules therefore the toxicities associated with these metals might be due to oxidative tissue damage. Redox-active metals, such as iron, copper and chromium, undergo redox cycling whereas redox-inactive metals, such as lead, cadmium, mercury and others deplete cells' major antioxidants, particularly thiol-containing antioxidants and enzymes. Either redox-active or redox-inactive metals may cause an increase in production of reactive oxygen species (ROS) such as hydroxyl radical (HO.), superoxide radical (O2.-) or hydrogen peroxide (H2O2). Enhanced generation of ROS can overwhelm cells' intrinsic antioxidant defenses, and result in a condition known as "oxidative stress". Cells under oxidative stress display various dysfunctions due to lesions caused by ROS to lipids, proteins and DNA. Consequently, it is suggested that metal-induced oxidative stress in cells can be partially responsible for the toxic effects of heavy metals. Several studies are underway to determine the effect of antioxidant supplementation following heavy metal exposure. Data suggest that antioxidants may play an important role in abating some hazards of heavy metals. In order to prove the importance of using antioxidants in heavy metal poisoning, pertinent biochemical mechanisms for metal-induced oxidative stress should be reviewed.

High performance liquid chromatography analysis of D-penicillamine by derivatization with N-(1-pyrenyl)maleimide (NPM).

D-Penicillamine (2-amino-3-mercapto-3-methylbutanoic acid), a well-known heavy metal chelator, is the drug of choice in the treatment of Wilson's disease and is also effective for the treatment of several disorders including rheumatoid arthritis, primary biliary cirrhosis, scleroderma, fibrotic lung diseases and progressive systemic sclerosis. The method proposed incorporates a technique, previously developed in our laboratory, that utilizes the derivatizing agent N-(1-pyrenyl)maleimide (NPM) and reversed-phase high-performance liquid chromatography (HPLC). The coefficients of variation for within-run precision and between-run precision for 500 nM standard D-penicillamine (D-pen) were 2.27% and 2.23%, respectively. Female Sprague-Dawley rats were given 1 g/kg D-pen i.p. and the amounts of D-pen in liver, kidney, brain and plasma were subsequently analyzed. This assay is rapid, sensitive and reproducible for determining D-pen in biological samples.

Can antioxidants be beneficial in the treatment of lead poisoning?

Recent studies have shown that lead causes oxidative stress by inducing the generation of reactive oxygen species, reducing the antioxidant defense system of cells via depleting glutathione, inhibiting sulfhydryl-dependent enzymes, interfering with some essential metals needed for antioxidant enzyme activities, and/or increasing susceptibility of cells to oxidative attack by altering the membrane integrity and fatty acid composition. Consequently, it is plausible that impaired oxidant/antioxidant balance can be partially responsible for the toxic effects of lead. Where enhanced oxidative stress contributes to lead-induced toxicity, restoration of a cell's antioxidant capacity appears to provide a partial remedy. Several studies are underway to determine the effect of antioxidant supplementation following lead exposure. Data suggest that antioxidants may play an important role in abating some hazards of lead. To explain the importance of using antioxidants in treating lead poisoning the following topics are addressed: (i) Oxidative damage caused by lead poisoning; (ii) conventional treatment of lead poisoning and its side effects; and (iii) possible protective effects of antioxidants in lead toxicity.