Richard H. Matthews

In vivo indices of oxidative stress in lead-exposed C57BL/6 mice are reduced by treatment with meso-2,3-Dimercaptosuccinic Acid or N-acetylcysteine

Knowledge of leads capacity to disrupt the prooxidant/antioxidant balance within mammalian tissues suggests that definitive therapy for chronic lead poisoning should encompass both chelating and antioxidant actions. The dithiol meso-2,3-Dimercaptosuccinic Acid (DMSA) is the first orally administered metal chelating agent to receive U.S. Food and Drug Administration (FDA) approval for the treatment of childhood plumbism and possesses the potential to function as an antioxidant by removing lead from the site of deleterious oxidation reactions. Five weeks of lead exposure was found to deplete glutathione (GSH) levels, increase oxidized glutathione (GSSG), and promote malondialdehyde (MDA) production in both liver and brain samples taken from C57BL/6 mice. GSH levels increased and GSSG and MDA levels decreased in groups of lead-exposed mice that received 1 mmol/kg DMSA or 5.5 mmol/kg N-acetylcysteine (NAC) for 7 d prior to sacrifice. Treatment with DMSA caused a reduction in blood, liver, and brain lead levels consistent with its function as a chelating agent, while treatment with NAC did not reduce these lead levels. However, NAC did cause a reduction in indices of oxidative stress in both brain and liver samples, which implies that this synthetic thiol-containing antioxidant is capable of abrogating lead-induced oxidative stress in vivo. Overall, these results suggest that lead-induced oxidative stress in vivo can be mitigated by pharmacologic interventions, which encompass both chelating as well as thiol-mediated antioxidant functions.

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.

N-acetylcysteine protects Chinese hamster ovary (CHO) cells from lead-induced oxidative stress

In vitro administration of lead acetate (PbA) to cultures of Chinese hamster ovary (CHO) cells had a concentrationdependent inhibitory effect on colony formation. Colony formation was returned to control levels in lead-treated cultures that were supplemented with 1 mM IV-acetylcysteine (NAC), a well-documented synthetic antioxidant. In order to investigate the nature of NAC’s protective effect, we measured LT-ghrtamyl-Lcysteinylglycine (GSH), oxidized ghrtathione (GSSG), rnalondialdehyde (MDA) and catalase activity both in the presence and absence of NAC in leadexposed CHO cells. Increases in both MDA levels (P < 0.05) and catalase activity (P < 0.05) were observed in cultures that received only PbA, but supplementation with NAC returned these measures to pretreatment levels. The ratio of GSH to GSSG increased in lead-exposed cells incubated in NAG-enhanced media, but declined in cultures treated with PbA only. Our results suggest that NAC can confer protection against lead-induced oxidative stress to CHO cells, possibly through the enhancement of the cell’s own antioxidant defense mechanisms.

Effects of N-acetylcysteine amide (NACA), a thiol antioxidant on radiation-induced cytotoxicity in Chinese hamster ovary cells

Ionizing radiation is known to cause tissue damage in biological systems, mainly due to its ability to produce reactive oxygen species (ROS) in cells. Many thiol antioxidants have been used previously as radioprotectors, but their application has been limited by their toxicity. In this investigation, we have explored the possible radioprotective effects of a newly synthesized thiol antioxidant, N-acetylcysteine amide (NACA), in comparison with N-acetylcysteine (NAC), a commonly used antioxidant. Protective effects of NACA and NAC were assessed using Chinese hamster ovary (CHO) cells, irradiated with 6 gray (Gy) radiation. Oxidative stress parameters, including levels of reduced glutathione (GSH), cysteine, malondialdehyde (MDA), and activities of antioxidant enzymes like glutathione peroxidase, glutathione reductase, and catalase, were measured. Results indicate that NACA was capable of restoring GSH levels in irradiated cells in a dose dependent manner. In addition, NACA prevented radiation-induced loss in cell viability. NACA further restored levels of malondialdehyde, caspase-3 activity, and antioxidant enzyme activities to control levels. Although NAC affected cells in a similar manner to NACA, its effects were not as significant. Further, NAC was also found to be cytotoxic to cells at higher concentrations, whereas NACA was non-toxic at similar concentrations. These results suggest that NACA may be able to attenuate radiation-induced cytotoxicity, possibly by its ability to provide thiols to cells.

High-performance liquid chromatography assay for N-acetylcysteine in biological samples following derivatization with N-(1-pyrenyl)maleimide

N-Acetylcysteine is a thiol antioxidant with expanding clinical importance. A sensitive, rapid method for determining reduced N-acetylcysteine (NAC) concentration in biological samples has been developed which uses a modified reversed-phase high-performance liquid chromatography (HPLC) technique in conjunction with the derivatizing agent N-(1-pyrenyl)maleimide (NPM). The NAC-NPM adduct was analyzed by HPLC with fluorescence detection. The calibration curve for NAC was linear over the range 8-2500 nM and the coefficient of variation obtained for the within-run precision and the between-run precision for 0.5 mM NAC was 1.5% and 2.7%, respectively. Relative recovery of NAC from biological materials ranged between 86% and 96% and the limit of quantitation from biological samples was 32 nM. These results suggest practical advantages relative to other widely-accepted methods of NAC measurement.

Biphasic kinetic plots and specific analogs distinguishing and describing amino acid transport sites in S37 ascites tumor cells

Curve-fitting procedures indicated that exo-2-amino-bicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) modified V and Km for one of two systems serving for histidine transport into the S37 ascites tumor cells. When this system was obliterated by leucine in the medium, BCH had no effect on histidine transport. Curve-fitting procedures similarly suggest N-methyl-alpha-aminoisobutyric acid affected the Km and V values for the other histidine-transporting system and that carboxymethylhistidine (His(Cm)) inhibited both transport systems. His(Cm) further inhibited histidine uptake into leucine-inhibited cells. Km and V values were altered simultaneously in the presence of several inhibitory analogs. Alanine methyl ester markedly inhibited high-concentration histidine uptake, whereas leucine methyl ester markedly inhibited low-concentration histidine uptake. The present results confirm earlier suggestions that our high c system is Christensens A system and our low c system his L system. We also confirm a very high degree of specificity of N-methyl-alpha-aminoisobutyric acid for the A or high c system, and of BCH for the L or low c system. We suggest the utility of combining two approaches to the study of transport system properties; use of specific analogs and modification of biphasic plots. We demonstrate that the carboxyl group is not a prerequisite molecular feature for inhibitory interaction with the A or L system.

In vitro study of the metabolic effects of D-amino acids.

While the L-configuration of amino acids predominates in all known living systems, D-enantiomers of amino acids have been detected with highly sensitive chromatographic techniques in human physiological fluids. In the present study, the survival of Chinese hamster ovary cells (CHO) and HeLa cells was inhibited by exposure to high concentrations of some D- or L-amino acids. Inhibition of colony formation, though, was not necessarily observed to be chiral-dependent. Some L-amino acids (LAAs) were found to be toxic while other D-amino acids (DAAs) were innocuous in both cultures. This is contradictory to the previous observations that DAAs were generally considered to be harmful. Frequently it was implied, although not experimentally proven, that the LAAs were not toxic. One of the metabolites produced by oxidative deamination of D- or LAAs is hydrogen peroxide (H2O2), a reactive oxygen species (ROS) that is decomposed by catalase. Increased intracellular H2O2 can result in peroxidation of lipids. We measured catalase activity and the lipid peroxide levels (LPO) after incubating cells in either D- or LAAs. The amino acids (AAs) that were found to inhibit colony formation were found to be associated with higher levels of catalase activity and LPO. Therefore, we hypothesize that enhanced ROS generation may be, in part, responsible for the observed toxicity of some amino acids.

Characteristics of a transport system serving for the transfer of histidine into S37 ascites tumor cells

Abstract 1. 1. S37 ascites tumor cells accumulate the amino acid histidine by 2 distinguishable processes. One of these has been characterized in an earlier publication ( Biochim. Biophys. Acta , 203 (1970) 457). 2. 2. The second system possesses kinetic parameters such that it could be mistaken for a “non-saturable” uptake process. However, the flux associated with this system may exceed that which might be attributable to “non-saturable” uptake, and competition for uptake by this site is shown to occur. 3. 3. The high c system described in this article resembles the “A” system described by Oxender and Christensen ( J. Biol. Chem. , 238 (1963) 3686), in its specificity. 4. 4. The high c system does not appear to function in exchange of amino acids. 5. 5. Na + stimulates uptake by the high c system, but is not required for transfer of histidine to occur by the high c system. Comparison of uptake in the presence of various alkali metal ions discloses a strong field sequence for interaction of the ions with the transport process. 6. 6. Concentrative uptake by the high c system is dependent upon the availability of metabolic energy. 7. 7. Trans inhibition of the high c system is shown to occur.

Analysis of thiol-containing compounds in biological samples by capillary zone electrophoresis

A new method for the determination of reduced and oxidized glutathione by using capillary zone electrophoresis was developed and compared with the N-(1-pyrenyl)maleimide high-performance liquid chromotography method. The tissue samples were obtained from C57BL/6 mice and homogenized in deionized water. The detection wavelength was set at 214 nm. A 50-cm effective length x 75 microns i.d. uncoated fused silica capillary with a 10 mM phosphate buffer at pH 7.5 was used for the experiment. Samples were introduced by pressure injection for 5s. The voltage for separation was at 20 kV. Between runs, the capillary was regularly washed with 0.1 N NaOH and deionized water, followed by reconditioning with running buffer. The present method was shown to be reproducible and is convenient in that a derivatization step is not required. The method has been used to measure glutathione levels in biological samples and the results show good agreement with those obtained by high-performance liquid chromotography.

Fitting kinetic data for two independent saturable terms by multifit ii, a general purpose curve fitting program in fortran iv☆

Abstract The present report describes multifit ii , a fortran program for fitting data to non-linear equations and its application to biphasic kinetic data. Biphasic plots are encountered, e.g. in cases of enzymatic reactions and in the area of amino acid transport across biological membranes. This program gives a least squares fit of an equation to data, and obviates the need for non-statistical approximations, with their inherent bias. Equations to be fit are written in the form (numerator)/(denominator), where each may contain up to four terms, and each term may be a maximum of fourth order, including up to four independent variables. Data may be fit by the present program in either direct or reciprocal form. The approach taken is an iterative procedure (Newtons method). Scaling and weighting of data are incorporated. Parameters used to generate ideal test data in biphasic kinetic plots were recovered to four significant figures. The utility of the program in fitting real biphasic data obtained from amino acid transport experiments is demonstrated.