Rachel Neal

Antioxidant effects of N-acetylcysteine and succimer in red blood cells from lead-exposed rats

This study examined whether lead-induced alterations in selected parameters that are indicative of oxidative stress accompany the toxic effects of lead in red blood cells (RBCs) in vivo. It also explored the possibility that treatment with N-acetylcysteine (NAC) or succimer (meso-2,3-dimercaptosuccinic acid) was capable of reversing parameters indicative of lead-induced oxidative stress. Fisher 344 rats were given 2000 ppm lead acetate in their drinking water for 5 weeks. The lead was then removed and the animals were given NAC (800 mg/kg/day) or succimer (90 mg/kg/day) in their drinking water for 1 week, after which the RBCs were harvested. Animals not given lead and those given lead, but not NAC or succimer, served as negative and positive controls, respectively. At the end of the experiment, blood-lead levels were 35 +/- 4 microg/dl in lead-treated animals, which were reduced to 2.5 +/- 1 microg/dl by treatment with succimer and to 25 +/- 3 microg/dl by treatment with NAC. Lead-exposed animals demonstrated signs of anemia as evidenced by anisocytosis, poikilocytosis, and alterations in hemoglobin, hematocrit, and mean corpuscular volume. Lipid peroxidation, as evidenced by increased malondialdehyde (MDA) content, as well as decreases in reduced glutathione (GSH) and increases in catalase and glucose 6-phosphate dehydrogenase (G6PD) activity were noted in RBCs from lead-treated rats, suggesting that the lead induced oxidative stress. In addition, a significant reduction in blood delta-aminolevulinic acid dehydratase (ALAD) activity suggested that accumulation and autooxidation of delta-aminolevulinic acid might contribute to lead-induced oxidative stress. Treatment with either NAC or succimer reversed lead-induced alterations in MDA and GSH content, but only succimer appeared to partially restore ALAD activity. These results provide in vivo evidence supporting the hypothesis that lead induces oxidative stress in RBCs, which is reversible by treatment with a thiol antioxidant (NAC), as well as a chelating agent (succimer).

ANTIOXIDANT ROLE OF N-ACETYL CYSTEINE ISOMERS FOLLOWING HIGH DOSE IRRADIATION

High dose, acute radiation exposure, as in radiation accidents, induces three clinical syndromes that reflect consequences of oxidative protein, lipid, and DNA damage to tissues such as intestine, lung, and liver. In the present study, we irradiated C57BL/6 mice with 18 Gy whole-body radiation (XRT) and evaluated N-acetyl cysteine (NAC) isomers LNAC and DNAC as potential radioprotectors under conditions that would model the gastrointestinal syndrome. We focused on tissues thought not immediately involved in the gastrointestinal syndrome. Both LNAC and DNAC protected the lung and red blood cells (RBC) from glutathione (GSH) depletion following radiation exposure. However, only LNAC also supplemented the spleen GSH levels following XRT. Protection from increased malondialdehyde (MDA) levels (lung) and increased 8-hydroxy-deoxyguanosine (8-oxo-dG) presence (liver) following XRT was observed with treatment by either isomer of NAC. These results imply that either NAC isomer can act as a radioprotectant against many aspects of oxidative damage; chirality is only important for certain aspects. This pattern would be consistent with direct action of NAC in many radioprotection and repair processes, with a delimited role for NAC in GSH synthesis in some aspects of the problem.

Pro-oxidant effects of δ-aminolevulinic acid (δ-ALA) on Chinese hamster ovary (CHO) cells

Abstract δ-Aminolevulinic Acid (δ-ALA) is a heme precursor accumulated m lead poisoning and acute intermittent porphyria. Although no single mechanism for lead toxicity has yet been defined, recent studies suggest at least some of the lead-induced damage may originate from δ-ALA-induced oxidative stress. The present study was designed to test the hypothesis that δ-ALA accumulation in Chinese hamster ovary (CHO) cells contributes to the cumulative oxidative challenge of lead poisoning as indicated by the oxidative stress parameters glutathione (GSH), glutathione disulfide (GSSG), malondialdehyde equivalents (MDA), and catalase (CAT). It will also examine the possibility that this oxidative challenge can be reversed by treatment with an antioxidant such as N -acetylcysteine (NAC). First in vitro administration of δ -ALA to CHO cells was found to have a concentration-dependent inhibitory effect on colony formation and cell survival. NAC administration was shown to alleviate this inhibition in CHO survival. The oxidative status of CHO cell cultures exposed to increasing concentrations of (5-ALA was then examined. Decreases in GSH levels (P > 0.05) were observed in the δ-ALA-treated cultures as compared to the controls, while GSSG and MDA levels were significantly increased in δ-ALA-treated cells (P

Captopril as an antioxidant in lead-exposed Fischer 344 rats

1 Recent studies suggest that lead induces oxidative stress in various tissues. 2 Captopril ([2S]-1-[3-mercapto-2-methylpropionyl]-L-proline), an angiotensin-converting enzyme inhibitor, is a well-known antihypertensive agent and is also believed to function as an antioxidant. 3 In the present study the antioxidant effect of captopril on lead-induced oxidative stress was studied in Fischer 344 rats. Their liver, brain and kidneys were assayed for glutathione (GSH), glutathione disulfide (GSSG), malondialdehyde concentrations, and catalase activities. The results from animals treated with captopril were compared to results of control and lead-exposed non-treated groups. 4 The captopril-treated samples showed higher GSH:GSSG ratios in the liver, brain and kidneys, as well as slightly decreased malondialdehyde concentrations. The catalase activity was not significantly affected.

EFFECTS OF SOME SULFUR-CONTAINING ANTIOXIDANTS ON LEAD-EXPOSED LENSES

Lead (Pb) is known to negatively affect glutathione (GSH) metabolism in the lens. The present study examined the effects of Captopril, Taurine, and alpha-Lipoic acid on the Pb-induced GSH depletion and lipid peroxide increase in the lenticular system. Captopril administration returned the GSH, cysteine (CYS), and malondialdehyde (MDA) levels to near normal. Following Taurine administration the GSH, CYS and MDA levels were intermediate between the control group and the Pb group levels. Alpha-Lipoic acid administration, however, only increased the CYS levels. No significant changes in oxidized glutathione (GSSG) levels were observed in any treatment group.

RESOLUTION OF CHIRAL THIOL COMPOUNDS DERIVATIZED WITH N-(1-PYRENYL)-MALEIMIDE AND THIOGLO™3

Many chiral thiols such as cysteine, homocysteine, N-acetylcysteine (NAC), and penicillamine are biologically important compounds. Among other roles, they act as antioxidants, therapeutic agents and indicators of disease. When analyzing low levels of these compounds, they are often derivatized in order to increase the sensitivity of the determination. However, it is generally an associated amine functional group that is derivatized. By selectively derivatizing only the thiol moiety with a fluorescent group, one is able to eliminate the large number of amine containing background compounds that are present in biological samples. However, there have been few reports on the enantiomeric resolution of thiol containing amino acids in which the fluorescent tag is linked exclusively through the sulfhydryl group. The first HPLC enantioresolution of N(1-pyrenyl)maleimide (NPM) and ThioGlo™3 derivatized compounds is reported on Teicoplanin and naphthylethyl-carbamate-β-cyclodextrin (NEC-β-CD) chiral stationary phases.

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 Wilsons 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.

Sub-chronic lead exposure alters kidney proteome profiles

The current study examined the impact of sub-chronic lead (Pb)-exposure upon global protein profile in rodent kidney (blood Pb levels ~50 μg/dL; 5 weeks oral Pb-acetate exposure). Utilizing 2D SDS-PAGE for kidney protein separation, greater than 500 protein spots were analyzed by densitometry following background noise removal, spot alignment, and intensity filtering. Approximately 100 protein spots were identified by ESI-MS/MS with mitochondrial, chaperone, antioxidant, and Pb-binding proteins included. Forty-eight protein spots exhibited significant alterations in abundance (18 identified by ESI-MS/MS) including the increased protein abundance of ketohexokinase, enolase, protein disulfide-isomerase, lamda crystallin, lactamase, and glycerol-3-phosphate dehydrogenase. Decreased protein abundances were observed for α-2 microglobulin, glutamate cysteine ligase, prohibitin, homogentisate 1,2-dioxygenase, alpha-ETF, argininosuccinate synthetase and ATP synthase (H+ transporting). These data support the hypothesis that protein profiles in the kidney are altered following sub-chronic physiologically relevant Pb-exposure.