Brief Communication

EFFECTS OF SOME SULFUR-CONTAINING ANTIOXIDANTS ONLEAD-EXPOSED LENSES

RACHEL NEAL,* K ATHERINE COOPER,* GARY KELLOGG,† HANDE GURER,* and NURAN ERCAL**Department of Chemistry, University of Missouri—Rolla, Rolla, Missouri 65409 USA and†Springfield Public Health

Department, Springfield, MO, USA

(Received30 April 1998;Revised4 June1998;Accepted28 July 1998)

Abstract—Lead (Pb) is known to negatively affect glutathione (GSH) metabolism in the lens. The present studyexamined the effects of Captopril, Taurine, anda-Lipoic acid on the Pb-induced GSH depletion and lipid peroxideincrease 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 intermediatebetween the control group and the Pb group levels.a-Lipoic acid administration, however, only increased the CYSlevels. No significant changes in oxidized glutathione (GSSG) levels were observed in any treatment group. © 1998Elsevier Science Inc.

Keywords—Lens, Oxidative stress, Lead, Pb, Captopril, Taurine,a-Lipoic acid

INTRODUCTION

Pb is a prevalent environmental toxin which is known tonegatively affect various tissues [1–4]. Pb disturbs len-ticular trace metals, lowers lenticular GSH levels andinhibits retinal and possibly lenticular ion pumps [5,6].Following low level Pb exposure (1,000 ppm in drinkingwater for up to three months) Dwivedi has demonstratedthat GSH metabolizing enzymes, such as glutathionereductase and glutathione-S-transferase, are inhibited byPb exposure [5]. In addition, following the Pb inducedmodification of the lenticular thiol levels, as well as lipidperoxidation status, cataractogenesis has been noted [7].These results implicate oxidative stress as one possiblemechanism for Pb’s lenticular toxicity.

Currently children with blood Pb levels less than 45mg/dL, as in the present study, do not receive chelationtherapy due to the potential side effects and the cost oftreatment [8]. Our laboratory has previously demon-strated that in cases of Pb exposure resulting in blood Pblevels below the medical treatment threshhold of 45mg/dL, antioxidant therapy restores the compromisedliver and brain thiol status [2,9]. The purpose of thepresent study is to provide further evidence supportingantioxidant therapy in a currently untreated segment of

the population. The lens system is ideal for this studysince the oxidative stress induced by chronic Pb intoxi-cation may possibly result in a serious clinical phenom-enon, cataractogenesis.

Captopril, an angiotensin-converting enzyme inhibi-tor and reactive oxygen species (ROS) scavenger, hasbeen shown to protect against oxidative modifications indiquat induced cataract [10]. Taurine, which is present inmillimolar concentrations in the retina and other mem-brane rich tissues, is known to scavenge HOCl andpossibly superoxide and has been demonstrated to pro-tect rod outer segments from structural damage inducedby oxidants [11,12].a-Lipoic acid, a lipophilic antioxi-dant which acts as a cofactor in many enzymatic reac-tions, protects buthionine sulfoxime treated rats fromGSH depletion and cataract formation in the lens [13].

This study tests the hypothesis that the decreasedlenticular redox status initially observed following 1month of Pb poisoning may be reversed by administra-tion of the sulfur containing antioxidants; Captopril, Tau-rine, anda-Lipoic acid. GSH, GSSG, CYS, and MDAlevels were analyzed as selective parameters of the oxi-dative status of these lenses. In addition, the ability ofthese antioxidants to lower the blood Pb levels and raisethe d-aminolevulinic acid dehydratase (ALAD) activityof Pb exposed animals were examined.

Address correspondence to: Nuran Ercal; Tel: (573) 341-6950;E-Mail: nercal@umr.edu.

Free Radical Biology & Medicine, Vol. 26, Nos. 1/2, pp. 239–243, 1999Copyright © 1998 Elsevier Science Inc.Printed in the USA. All rights reserved

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239

MATERIALS AND METHODS

The N-(1-pyrenyl)-maleimide, 1,1,3,3 tetrame-thoxypropane, and 2-vinyl pyridine were purchased fromAldrich (Milwaukee, WI, USA). All other chemicalswere obtained from Sigma (St. Louis, MO, USA).

All experiments were performed with 20 adult maleFisher 344 rats weighing approximately 100–150 gramseach. The animals were housed in stainless steel cages ina temperature controlled room (25°C) with a 12 h light:dark cycle. They were allowed distilled water and stan-dard Purina rat chow ad lib. The control group receiveddistilled water for 6 weeks. The remaining animals wererandomized into the following groups which received2,000 ppm of lead for 5 weeks. During the sixth week,they received: Pb group, distilled water; Captopril group,p.o. 50 mg/kg/day of captopril in distilled water; Taurinegroup, p.o. 1.1 g/kg/day taurine in distilled water;a-Li-poic acid group, i.p. 25 mg/kg/day (vehicle was 1:1ethanol:saline). Following week 6, the animals wereanesthetized with metofane and blood samples were col-lected with lead-free needles via intracardiac puncture.The animals were sacrificed and the lenses were col-lected and maintained at270°C until assayed.

GSH, CYS, and GSSG determinations

The lenses were analyzed by a new method of GSHdetermination which was developed in this laboratory toanalyzeg-glutamyl cycle intermediates [14]. In brief,free sulfhydryl groups were reacted with N-(1-pyrenyl)-maleimide and subsequently separated and detected byreverse phase HPLC with fluorescence detection. GSSGwas derivatized as GSH after initial reduction by gluta-thione reductase and NADPH following free thiol alky-lation by 2-vinylpyridine.

MDA determinations by HPLC

MDA was determined as described by Draper et al. [15].In brief, MDA was derivatized by 2-thiobarbituric acid.Following derivatization, the sample was extracted withbutanol and analyzed by reverse phase HPLC with flu-orescence detection.

Blood Pb determination

Blood Pb levels were determined by graphite furnaceatomic absorption spectroscopy in the CDC certifiedAnalytical Laboratory of the Springfield Public HealthDepartment.

ALAD activity determination

Blood ALAD activity, which is an early subclinicalmarker of Pb intoxication, was determined following the

procedure developed by Berlin and Schaller [16]. Inbrief, the enzyme is incubated with excessd-aminolevu-linic acid. The porphobilinogen formed is mixed withmodified Ehrlich’s reagent and color development ismeasured spectrophotometrically.

Protein determination and statistical analysis

Protein was determined by the Bradford method [17].Tabulated values represent means6SDs of at least fourdata points. One-way analysis of variance (ANOVA) andthe Student-Newman-Keuls multiple comparison testwere used to analyze data from experimental and controlgroups. Values ofp , .05 were considered significant.

RESULTS AND DISCUSSION

Redox disturbances are known to negatively impactbody systems through generation of ROS which modifyproteins, lipids, and DNA [18]. Increases in ROS andinhibition of GSH synthesis have been clearly shown tomodify lenticular proteins, as well as membrane lipids,and lead to cataractogenesis [19,20]. Pb inhibits hemebiosynthesis, P450 activity, and DNA repair mechanisms[21–23]. Pb also causes enhancement of Fe21 toxicityand decreases in the GSH/GSSG ratio [24]. In the lens,Pb inhibits glutathione reductase and glutathione S-trans-ferase, decreases GSH and free protein sulfhydryls, in-creases lipid peroxidation, and leads to cataractogenesis[7]. This study confirms the reported decrease in lentic-ular GSH following increased blood Pb levels and de-creased ALAD activity, a key enzyme in the heme bio-synthesis pathway, which are indicative of Pb exposure[16]. In addition decreased CYS levels and increasedMDA levels support the hypothesis that Pb induces ox-idative stress in the lens. However, GSSG levels did notchange following lead exposure which may be due to anincrease in protein-thiol mixed disulfide formation [25–27]. The results of our study support the hypothesis thatPb exposure compromises the lenticular redox status andleads to the idea that antioxidant supplementation mayreverse the redox disturbance.

Captopril, an angiotensin converting enzyme inhibitorwhich possesses a free sulfhydryl group (Fig. 1), hasbeen proposed as an antioxidant as well as a possibleheavy metal chelator [28,29]. Captopril is absorbedthrough the mucosa resulting in 65% bioavailability andmay be carried as a mixed disulfide [10]. In diquat-induced cataractogenesis, Captopril protected the lensfrom lipid peroxidation, glutathione depletion, and pro-tein sulfhydryl oxidation [10]. In addition to its clearantioxidant potential, captopril has also been demon-strated to chelate Cu11 and Fe31 [30] and to increase

240 R. NEAL et al.

CuZn-superoxide dismutase activity and nonenzymaticantioxidant activity in vivo [31]. Thus, Captopril maymodify the lenticular redox status through either directROS scavenging, stimulation of enzymatic or nonenzy-matic antioxidant activities, or transition metal chelation.In the present study, Captopril treatment following Pbexposure returned GSH and MDA levels to near controlvalues with a slight increase in CYS levels compared tothe Pb group (Tables 1 and 2). However, no significantchange in blood Pb levels was observed following Cap-topril administration. Thus, Captopril administration ap-pears to be a promising antioxidant, but an ineffectivechelator for Pb.

Taurine, a sulfur-containing essential amino acid (Fig.1) which is synthesized in vivo and also is readily ab-sorbed from the diet [32], partially scavenges ROS andprevents changes in membrane permeability following

oxidant injury. H2O2-induced chromosomal aberrationsand sister chromatid exchanges were partially preventedby Taurine administration in vitro [11]. Taurine has beenlinked to membrane stabilization in lymphoblasts leadingto increased viability following iron-ascorbate inducedcell injury [33]. Taurine has also been shown to restorered blood cell membrane Na-K ATPase activity follow-ing ozone exposure [34]. In addition, decreases in Tau-rine have been noted following CYS depletion [35].Therefore Taurine supplementation may have a benefi-cial effect upon the lens through direct ROS scavenging,membrane ATPase stabilization, and lipid peroxidationinhibition. In the present study, Taurine administrationdid result in GSH, CYS (Table 1), and MDA levels(Table 2) that were intermediate to the control and Pbtreated groups. However, the changes were statistically

Table 1. Effects of Various Antioxidants on Lens GSH, GSSG, andCYS Levels

GSH GSSG CYS

Control 9.596 1.23 3.216 0.91 1.046 0.03Pb 4.616 0.59a 2.456 0.94 0.696 0.16a

Pb 1 Captopril 9.496 1.74b 2.906 1.58 0.906 0.09Pb 1 Taurine 8.156 2.66 2.236 1.09 0.866 0.19Pb 1 Lipoic Acid 5.636 1.16a 2.266 0.99 1.006 0.20b

n 5 3–5 animals per group.Units are nmol/mg protein.Glutathione, glutathione disulfide, and cysteine are abbreviated

GSH, GSSG, and CYS respectively.a p , .05 as compared to control group.b p , .05 as compared to Pb group.

Table 2. Effect of Antioxidants on Lenticular MDA Levels, BloodPb Levels and Blood ALAD Levels

Lens MDAlevels

Blood Pblevels

Blood ALADlevels

Control 1.226 0.32 16 0 3.576 0.45Pb 2.646 0.34a 356 4a 1.196 0.32a

Pb 1 Captopril 1.686 0.59b 326 4a NDPb 1 Taurine 1.916 0.56 366 1a 0.686 0.32a

Pb 1 Lipoic Acid 2.276 0.36a 336 3a 1.196 0.55a

n 5 3–5 animals per group.Units are nmol/100 mg protein for MDA,mg/dL for blood Pb, and

U/L for ALAD.Not Determined is abbreviated as ND.Malondialdehyde is abbreviated as MDA.a p , .05 as compared to control group.b p , .05 as compared to Pb group.

Fig. 1. Structures of Captopril, Taurine, anda-Lipoic acid.

241Antioxidants in Pb-exposed lens

nonsignificant. Blood Pb levels and ALAD activity re-mained unchanged, indicating that Taurine does not actas a chelator of Pb.

a-Lipoic acid (Fig. 1) is a well-known disulfide con-taining antioxidant and metal chelator [36,37]. NO pro-duction by lipopolysaccharide exposed macrophages wasinhibited by a-Lipoic acid [38]. In addition,a-Lipoicacid protected cells from single-strand DNA breaks andscavenged singlet oxygen produced following endoper-oxide thermolysis [39].a-Lipoic acid also decreasesCd21 toxicity and forms stable complexes with Mn21,Cu21, and Zn21 [40,41]. However,a-Lipoic acid doesnot seem to protect liver microsomes from Fe-inducedlipid peroxidation [42].a-Lipoic Acid following reduc-tion to dihydrolipoic acid, has been demonstrated toreduce cystine to cysteine which is readily uptaken forGSH synthesis [43]. In the present study,a-Lipoic acidadministration following Pb exposure resulted in nonsig-nificant changes in GSH and MDA levels with a signif-icant increase in CYS levels (Tables 1 and 2). While ourresults confirma-Lipoic Acid’s ability to increase cys-teine levels, the statistically insignificant increase inGSH may result from cysteine utilization for proteinstabilization or from GSH synthesis inhibition. In addi-tion, a-Lipoic acid did not chelate Pb as evidenced by nodecrease in blood Pb levels or increase in ALAD activity.These results indicate thata-Lipoic acid had little ben-eficial effect on the lenticular redox status following Pbexposure.

These results support the hypothesis that Pb inducedmodification of the lenticular redox disturbance may bepartially repaired by antioxidant therapy. It appears thatsulfhydryl containing antioxidants such as Captopril mayreturn the GSH, CYS, and MDA levels to near normal. Infuture studies, the effect of sulfhydryl containing anti-oxidants on the lenticular protein redox status (i.e., pro-tein bound sulfhydryl and activities of glutathione me-tabolizing enzymes) following chronic (at least 3months) Pb exposure, as well as the inhibition of possiblecataractogenesis, should be examined.

Acknowledgements— Dr. Ercal was supported by 1R15ES08016-01from the NIEHS, NIH and the contents of this paper are solely theresponsibility of the authors and do not necessarily represent theofficial views of the NIEHS or NIH. Hande Gurer was supported byThe Scientific and Technical Research Council of Turkey (TUBITAK).

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