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e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 45–54

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ffect of vitamin E and C supplementation onxidative damage and total antioxidant capacityn lead-exposed workers

dela-Leonor Rendón-Ramíreza, María Maldonado-Vegab,artha-Angelica Quintanar-Escorzac, Gerardo Hernándezd,

ertha-Isabel Arévalo-Rivase, Alejandro Zentella-Dehesaf,osé-Víctor Calderón-Salinasa,∗

Biochemistry Department, Centro de Investigación y Estudios Avanzados IPN, México, DF, MexicoCIATEC, León, GTO, MexicoFaculty of Medicine, UJED, Durango, DGO, MexicoSection of Methodology of Science, Centro de Investigación y Estudios Avanzados IPN, México, DF, MexicoHRAEB, León, GTO, MexicoCellular Physiology Institute, UNAM, Mexico

r t i c l e i n f o

rticle history:

eceived 1 January 2013

eceived in revised form

October 2013

ccepted 25 October 2013

vailable online 13 November 2013

eywords:

ntioxidants

a b s t r a c t

The molecular response of the antioxidant system and the effects of antioxidant supplemen-

tation against oxidative insult in lead-exposed workers has not been sufficiently studied. In

this work, antioxidants (vitamin E 400 IU + vitamin C 1 g/daily) were supplemented for one

year to 15 workers exposed to lead (73 �g of lead/dl of blood) and the results were com-

pared with those on 19 non-lead exposed workers (6.7 �g of lead/dl). Lead intoxication was

accompanied by a high oxidative damage and an increment in the erythrocyte antioxidant

response due to increased activity of catalase and superoxide dismutase. Antioxidant sup-

plementations decreased significantly the oxidative damage as well as the total antioxidant

capacity induced by lead intoxication with reduction of the antioxidant enzyme activities.

xidation

lood

uman

ead toxicity

We conclude that antioxidant supplementation is effective in reducing oxidative damage

and induces modifications in the physiopathological status of the antioxidant response in

lead-exposed workers.

© 2013 Elsevier B.V. All rights reserved.

in significant alterations in various organs, the hematolog-

nzymes

. Introduction

ead is one of the metals most commonly used in indus-

ry; its toxicity is a public health problem due mainly to itsnvironmental persistence, inadequate labor security condi-ions and low lead excretion in lead-exposed workers (Nilsson

∗ Corresponding author at: Biochemistry Department, Centro de Investenco, México 07000, DF AP 14-740, Mexico. Tel.: +52 55 57473955.

E-mail address: jcalder@cinvestav.mx (J.-V. Calderón-Salinas).382-6689/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.etap.2013.10.016

et al., 1991; Bleecker et al., 1995; Calderón-Salinas et al., 1996;Ehrlich et al., 1998; Levin and Goldberg, 2000; Patrick, 2006a,b;Quintanar-Escorza et al., 2007). Exposure to lead can result

igación y Estudios Avanzados IPN, México DF. Av. IPN 2508, Zaca-

ical system being an important target (Garcon et al., 2004;Gurer-Orhan et al., 2004). Erythrocytes have high affinity forlead and typically contain the majority of the lead found in

n d p

46 e n v i r o n m e n t a l t o x i c o l o g y a

the blood stream (Leggett, 1993). Several studies suggest thatlead toxicity involves oxidative damage (Ercal et al., 2001;Patrick, 2006a,b; Rendón-Ramírez et al., 2007; Flajs et al., 2009;Hamed et al. 2010; Quintanar-Escorza et al., 2010; Jomova andValko, 2011). Lead intoxication induces inhibition of hem syn-thesis and accumulation of pro-oxidant and autoxidizabilitysubstrates as �-aminolevulinic acid (�-ALA), free hem groupsand free iron ions in erythrocytes (Ercal et al., 2001; Rendón-Ramírez et al., 2007); in turn, accumulation of oxidizablesubstrates can result in superoxide and hydrogen peroxide for-mation (Patrick, 2006b; Flora et al., 2008). Additionally, underlead poisoning, high oxygen concentrations, autoxidizabilityof hemoglobin and vulnerable membrane components to lipidperoxidation make erythrocytes extremely sensitive to oxida-tive damage (Cimen, 2008; Ercal et al., 2001; Rendón-Ramírezet al., 2007; Jomova and Valko, 2011).

Organisms have diverse and highly efficient antioxidantmechanisms, but few and divergent results have been foundin antioxidant responses to lead exposure; differences seem todepend on the experimental model, concentration of lead andexposure time (Kasperczyk et al., 2004a,b; Mishra and Acharva,2004; Haleagrahara et al., 2010; Hamed et al., 2010). Differentinsults induce complex responses in antioxidant enzymaticsystems (Dröge, 2002; Valko et al., 2007; Auten and Davis, 2009),and little has been done to evaluate the effects of exogenousantioxidant treatments in lead intoxicated workers. Non-enzymatic exogenous antioxidants such as vitamins C, E, andB6, taurine, methionine, selenium, zinc, alpha-lipoic acid orN-acetylcysteine have been used as antioxidant treatment inanimal models and shown to lower reactive oxygen species(ROS) that generate cellular damage in different cell types oflead exposed rats (Simon and Hudes, 1999; Flora et al., 2003;Shalan et al., 2005; Patrick, 2006b; Rendón-Ramírez et al., 2007;Wang et al., 2007; Flora et al., 2008). Simultaneous administra-tion of vitamins E – a lipid-soluble non-enzymatic antioxidant-and C – an aqueous antioxidant – is particularly interestingsince it produces a more effective regeneration of the enzy-matic system reducing the oxidation manifestation (Traberand Stevens, 2011). Other studies have demonstrated thesynergistic protective action of vitamins C and E against geno-toxicity, spermatozoa and hippocampus damage (Mishra andAcharva, 2004; Li et al., 2008). In lead-exposed workers, treat-ment with vitamin C, vitamin E, beta-carotene, selenium, zincand chromium prevented the inhibition of �-aminolevulinicacid dehydratase (�-ALAD) activity in blood (Tandon et al.,2001), increased the total antioxidant capacity (TAC) as wellas superoxide dismutase (SOD) and glutathione peroxidase(GPx) activity (Machartová et al., 2000), while abating the toxiceffects associated with inhibition of the calcium pump (Abamet al., 2008). These results suggest investigating the effectsof antioxidant supplements in lead-exposed workers. In thiswork we study the antioxidant benefits of vitamin E and Csupplementation as well its effects on the antioxidant enzy-matic system in these subjects.

2. Materials and methods

A group of lead exposed workers was supplied with vitaminsC and E for one year. The effects on blood lead concentration,

h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 45–54

�-ALAD, SOD, glutathione reductase (GRx) and GPx activities,as well as on lipid peroxidation and erythrocyte TAC was deter-mined. In healthy people, both significant effects and noteffects at all of these vitamins on the antioxidant systems havebeen reported (Traber and Stevens, 2011). To clarify matters, acontrol group of non-lead exposed workers was subjected tothe same treatment for comparison.

The studied group consisted of male workers occupation-ally exposed to lead in a recycling battery factory, particularlyto dust of a mixture of lead oxides; only fifteen workers, withexposure time ranged from 5 to 10 years (mean = 6.8 years),out of twenty subjected to exposure conditions, accepted toparticipate and concluded the treatment for this study. Weremark the difficulty of finding a plant industry willing to allowits employees being studied, much less to be treated in itspremises. Lead exposed workers did not present renal damagealbuminuria or signs or a history of severe neurological dam-age (seizures or severe disturbances of balance), not did theyreceive chelation treatment during this study.

As a control group we used 19 (initially 20, one dropped)male workers clinically healthy and without a history ofoccupational lead exposure. They all showed normal param-eters in blood and urine. None of them presented a historyof, or current, physical symptoms of serious neurological,cardiovascular, renal, hepatic, endocrine, metabolic or gas-trointestinal disease, nor were they subjected to previouspharmacological treatment. Both groups had similar age andsocial position score. Non-lead exposed workers ranged from29 to 42 years old (mean = 34.5), whereas the exposed groupwas from 25 to 41 years old (mean = 32.9). Ranges of social score(Hollingshed, 2011) for non-lead exposed and lead exposedgroups were from 7.0 to 10.5 (mean = 9.2) and from 7.8 to9.6 (mean = 8.6), respectively. All subjects provided written,informed consent, and participation was voluntary. The Med-ical Center-Bajio, IMSS-Mexico, approved the study.

Each subject received 400 IU Alpha Tocopherol Acetate vita-min E (capsules) and 1 g tablet vitamin C per day during themorning for a period of 12 months. These doses were usedin previous studies for different illness associated with oxida-tive damage (Plantinga et al., 2007; Castillo et al., 2011), andno unwanted side effects are mentioned in the literature.We remark that these dosages are within the FDA recom-mended dosage for supplementation (maximum of 1000 mgof vitamin C and 1500 IU for alpha tocopherol). No worker wasreported to have missed his takings, and no worker presentedlateral effects that affected his participation in this study orthat would lead to suspend the use of these antioxidants.Patients were instructed to take them with breakfast at theirworkplace. Also, used dosages are not known to produce pro-oxidant effects, nor did we find such effects in subjects of thenon-exposed group. Besides vitamin supplementation, bettersafety procedures and hygienic and dietetic practices werepromoted for all workers at this site. The use of masks and uni-forms to reduce lead exposure, as well as a diet with highercontent of fiber and reduced calories were recommended tocomply with ethics procedures, even though such practicesmight reduce lead exposure and its effects. Venous blood was

obtained by venous puncture in heparinized tubes. Each sam-ple was centrifuged (700 × g, 10 min) and plasma was collected;white cells were carefully removed by aspiration to avoid loss

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f erythrocytes. To obtain serum, venous blood was collectedn non-heparinized tubes and centrifuged as above.

.1. Blood lead concentration

ead concentration in blood was determined with a Lead Ana-yzer, model 3010B (ESA), as previously described (Quintanar-scorza et al., 2007). Concentration is reported in microgramser deciliter of blood. Lead determinations were run in tripli-ates. The standard curves were calculated with the methodf addition to minimize the matrix effect on the absorptioneak (recovery of 85–113%). Precision of analyses was around7–104% (using ESA Hi and Lo calibrators) and the detectionimit set at 1 �g/dl.

.2. ı-ALAD activity

-ALAD activity in erythrocytes was determined spectropho-ometrically (US/VIS DU 650) (Beckman) and expressed asanomol of porphobilinogen (PBG) per milliliter of erythro-ytes per hour (nmol/ml/h) (Rendón-Ramírez et al., 2007).

.3. Vitamins E and C

erum tocoferol and ascorbate were determined by high-erformance liquid chromatography in a single chromato-raphic run with an internal standard (Merzouk et al., 2003;enton et al., 2003).

.4. Lipid peroxidation

he amount of lipid peroxidation in erythrocytes wasstimated as reported in Quintanar-Escorza et al. (2007)y measuring the thiobarbituric acid-reactive substancesTBARS). The absorbance was measured at 532 nm and 600 nmsing a spectrophotometer US/VIS DU 650 (Beckman). TBARSre expressed as nmol of malondialdehyde equivalents per mlf erythrocyte (Quintanar-Escorza et al., 2010).

.5. Erythrocytes TAC

he antioxidant capacity was measured by the formation ofOS in red blood cell samples. ROS levels were determined byuorescence, as described in Oyama et al. (1994). Briefly, DCF-A was used as a detector of ROS by flow cytometry. This dye

s not fluorescent in reduced state, but it becomes fluorescentnce cellular oxidation and removal of acetate groups by cel-

ular esterases take place (Karlsson et al., 2010). Samples werexcited at 480 nm and the emission detected at 520 nm. Thentensity of fluorescence in presence of fluorophore can be cor-elated with the cellular content of free radicals. Fluorescences measured in arbitrary units per 500,000 cells and expresseds the inverse of these units (1/(FUA/5 × 105 cells).

.6. CAT activity assays

AT activity was determined in erythrocytes by spectropho-ometry and is expressed in U/mg protein. This method

easures the exponential disappearance of H2O2 (3 mM) at40 nm in presence of cellular homogenates (5 Hb/dl) with

a r m a c o l o g y 3 7 ( 2 0 1 4 ) 45–54 47

addition of buffer phosphates pH 7.3. The decay of H2O2

depends on the CAT activity (Aebi, 1984). The hemoglobin con-centration (Hb) was estimated using the reagent described byVan Kampen and Zijlstra (1961).

2.7. SOD activity assays

SOD in erythrocytes inhibits the production of superoxideradicals. The xanthine–xanthine oxidase system, in presenceof oxygen, induces the formation of superoxide radicals; thisradical reduces the nitro blue tetrazolium and gives rise to acolored compound. Since SOD inhibits this reaction, its activ-ity is proportional to the degree of inhibition of nitro bluetetrazolium reduction to superoxide (Sun et al., 1988). Resultsare expressed in units of SOD/g of hemoglobin.

2.8. GRx activity assays

The activity of the enzyme GRx in erythrocytes is based on theformation of reduced glutathione from glutathione disulfidein the presence of the enzyme. Glutathione disulfide formedin one minute reacts with NADPH and the product is mea-sured spectrophotometrically at 340 nm and 37 ◦C, (UV/VISmodelo 30 Beckman-USA). Results of activity of all enzymeswere expressed in U (�M/min)/mg of Hb (Vives-Bauza et al.,2007).

2.9. GPx activity assays

The activity of the enzyme GPx in erythrocytes is based on theformation of glutathione disulfide from reduced glutathione inthe presence of the enzyme. Enzyme activity was measured bymodifying a procedure of Paglia and Valentine (1967). SolutionA consisted of 50 mM potassium phosphate buffer (pH 7), 1 mMEDTA, 1 mM NaN3, 0.2 mM NADPH, 1 IU/ml GSSG-reductase,1 mM GSH, 1.5 mM cumene hydroperoxlde or 0.25 mM H202 ina total volume of 1 ml. Erythrocytes lisated (0.1 ml) was addedto 0.8 ml of Solution A and allowed to incubate 5 min at roomtemperature before initiation of the reaction by the addition of0.1 ml peroxide solution. Absorbance at 340 nm was recordedfor 5 min and the activity was calculated from the slope ofthese lines as �moles NADPH oxidized per minute.

2.10. Statistical analysis

Comparisons between non-lead and lead exposed groups weredone through two-tailed unpaired t-tests. To compare eachgroup with itself before and after the treatment, we used two-tailed paired t-tests; in this case, proportions, instead of meandifferences, were estimated. In all cases the level of signifi-cance was established at P < 0.05. 95% CI were computed toevaluate the biological significance besides the statistical sig-

nificance. For these tests we used GraphPad Prism version 5.03for Windows, GraphPad Software, San Diego California USA,www.graphpad.com. Other, supplementary statistics meth-ods are described where used.

48 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 45–54

Table 1 – Number of individuals presenting clinical symptoms and signs associated to lead intoxication in lead andnon-lead exposed workers.

Non-lead exposed(n = 19)

Lead exposed(n = 15)

% in lead exposed (95%confidence interval)

Dizziness 1 7* 46.6 (21–73)Headache 3 11* 73.3 (45–92)Paresthesia 1 6* 40.0 (16–68)Paresis 0 3* 20.0 (4–48)Abdominal colic 2 5* 33.3 (12–62)Myalgia 2 8* 53.3 (27–79)Motor coordination alteration 1 14* 93.3 (68–100)

∗ Significant difference (P ≤ 0.05) between lead and non-lead exposed workers, according to X2 test. In the third column, percent (lower 95% CI– upper 95% CI) of individuals with a sign or symptom in the lead-exposed group, assuming binomial distribution, ‘exact method’, computedfollowing (Blair and Taylor, 2008).

Table 2 – Biological indices (mean ± SD) of lead intoxication in blood of non-lead exposed and lead exposed workers.

Non-lead exposed (n = 19) Lead exposed (n = 15) Difference betweenmeans ± SD

PbB (�g/dl) 6.7 ± 2.2 73 ± 11 65.8 ± 2.5***

***

�-ALAD activity (nmol PBG/h/ml) 683 ± 61

∗∗∗ p < 0.0001.

3. Results

3.1. Conditions before the treatment

Clinical symptoms and signs associated with chronic leadintoxication as dizziness, headache, paresthesia, paresis,abdominal colic, myalgia and motor coordination alterationwere frequently found in the lead-exposed workers. Percent-age of people with these symptoms was significantly higherthan that of the control group (Table 1). Frequency fromcero to seven symptoms and signs for both lead exposedand non-lead exposed workers follow a binomial distribution

(Fig. 1). For non-lead exposed workers, the probability of pre-senting any sign or symptom ranges from 0.05 to 0.16, whereasfor exposed workers the probability of showing any sign or

Table 3 – Lipid peroxidation and antioxidant defense system stsupplementation. Thiobarbituric acid reactive species concentractivity, SOD activity, GPx activity, GRx activity and serum concexposed and lead exposed workers (mean ± SD).

Non-lead exposed (n = 19)

TBARS (nmol MDA/ml PG) 0.7 ± 0.2

TAC (1/(FUA/5 × 105 cells)) 66 ± 3.7

Enzymatic activity (U/g Hb)CAT 11 ± 1.5

SOD 209 ± 45

GPx 12 ± 1.8

GRx 9.7 ± 2.2

Serum concentration (�mol/l)Vitamin E 24 ± 5.4

Vitamin C 69 ± 9.5

∗ 0.01 < p < 0.05.∗∗ 0.001 < p < 0.01.

∗∗∗ p < 0.001.

138 ± 34 545 ± 17.6

symptom goes from 0.38 to 0.64 ( = 0.05, df = 6, Pearson’s Chi-square test). Blood lead concentration was higher in leadexposed with respect to non-lead exposed workers (P < 0.0001)indicative of a chronic and significant lead exposure. Erythro-cyte �-ALAD activity was significantly lower in lead exposedthan in non-lead-exposed workers (P < 0.0001) also suggestinga severe chronic lead intoxication (Table 2). The oxidative dam-age (lipid peroxidation) and TAC in erythrocytes were higherin lead exposed with respect to non-lead exposed workers(P < 0.0001 and P < 0.0001, respectively) denoting an oxida-tive stress characterized by an increase in the antioxidantresponse, but not enough to contain oxidative insult (Table 3).

A major enzymatic antioxidant response was present in leadexposed workers, as both CAT and SOD activities in eryth-rocytes were much higher in this group than in the controlgroup (P < 0.0001 and P < 0.0001, respectively). However, lower

atus at the beginning of vitamin E/vitamin Cation (TBARS), total antioxidant capacity (TAC), CATentration of vitamins E and C, in blood of non-lead

Lead exposed (n = 15) Difference betweenmeans ± SD

1.3 ± 0.4 0.57 ± 0.01***

110 ± 8.1 44.4 ± 2.08***

37 ± 7.5* 26.0 ± 1.8***

534 ± 55* 324 ± 17.1***

10 ± 2.2 2.0 ± 0.7**

3.1 ± 1.2* 6.6 ± 0.6***

22 ± 4.3 1.1 ± 1.772 ± 7.8 3.7 ± 3.0

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 45–54 49

Fig. 1 – Frequency of individuals with cero to seven clinicalsymptoms and signs associated to lead intoxication innon-lead (A) and lead exposed (B) workers, before (�) anda

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Fig. 2 – Effect of vitamin E/vitamin C supplementation onlipid peroxidation and antioxidant defense system statusin blood of non-lead exposed and lead exposed workers:(A) Lipid peroxidation (TBARS) and (B) total antioxidantcapacity (TAC) before (�) and after (�) supplementation.Values are mean ± SD. ***Significant difference (p < 0.001) for

fter (�) supplementation.

ctivity of GRx in erythrocytes was found in lead exposeds compared to non-lead exposed workers (P < 0.0001), indi-ating a low antioxidant response at the glutathione systemTable 3). Erythrocytes GPx activity (95% CI from 0.6149 to 3.441)nd serum vitamins E and C concentrations were not dif-erent between lead exposed and non-lead exposed workersTable 4).

.2. Effects of vitamins C and E supplementation

dministration of Vitamins E and C for 12 months did notnduce a reduction in blood lead concentration of both workerroups (Table 4); clinical symptoms and signs of lead-exposedorkers were not reduced either (Table 4). Although it isot possible to state an increment in GPx and GRx activities

Table 5), �-ALAD activity increased significantly, twice asuch its initial value, in lead-exposed workers (Table 4). Lipid

eroxidation was reduced to values between 47.1 and 69.4%95% CI) in lead-exposed workers after supplementation withntioxidant vitamins, attaining values non-statistically dif-erent from those of the non-lead exposed workers (P = 0.2119)Fig. 1a). Additionally, TAC was reduced to values between

8.9 to 67.7% (95% CI) in lead-exposed workers after supple-entation with vitamins E and C, restoring the TAC level up

o a similar value of the non-lead exposed workers (P = 0.1298)

paired test before and after supplementation.

(Fig. 1b). CAT and SOD activities decreased to values between33.8% and 48.9% (95% CI), and from 44.2 to 53.2% (95% CI),respectively, after treatment in lead-exposed workers. Despitethis reduction, the activity of CAT was still between 31.8%and 79.3% higher in lead exposed than in non-lead exposedindividuals. SOD activity became after treatment statisticallynon different between lead and no-lead exposed groups(P = 0.0921) (Fig. 2). Finally, although in both groups vitaminsC and E increased significantly their concentrations, non-ledexposed workers were sensibly more affected: between 44and 69% for vitamin C, and between 101 and 153% for vitaminE (95% CI) (Table 5).

4. Discussion

Lead exposed workers presented very high blood lead con-centration and �-ALAD activity severely inhibited, whichindicates that these workers had been extensively exposedto lead. Additionally, they showed clinical symptoms andsigns frequently associated to lead intoxication (Calderón-Salinas et al., 1996; Levin and Goldberg, 2000; Patrick, 2006a;Quintanar-Escorza et al., 2007). They also presented elevatedoxidative damage, similar to that described in other lead expo-sure populations (Gurer-Orhan et al., 2004; Quintanar-Escorza

et al., 2007; Costa et al., 1997; Flajs et al., 2009) (Fig. 3).

The lead-exposed group showed an adaptive responseto oxidative insult – high TAC – due to lead intoxication;

50 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 45–54

Table 4 – Effects of vitamin E/vitamin C supplementation on levels of biological indices of lead intoxication in blood ofnon-lead exposed and lead exposed workers. Blood lead concentration (PbB) and �-aminolevulinic acid dehydrataseactivity (�-ALAD), before and after supplementation (mean ± SD).

Non-lead exposed (n = 19) Lead exposed (n = 15)

Before After CI of ratios (lower 95%CI – upper 95% CI)

Before After CI of ratios (lower 95%CI – upper 95% CI)

PbB (�g/dl) 6.7 ± 2.2 7.1 ± 1.6 0.8–1.0 73 ± 11 70 ± 9.1 0.9–1.0*

�-ALAD activity(nmolPBG/h/ml)

683 ± 61 696 ± 54 1.0–1.04 138 ± 34 315 ± 38 2.0–2.8***

∗ 0.01 < p < 0.05.∗∗∗ p < 0.001.

however, this antioxidant response was insufficient to abatesevere oxidative damage. In some oxidative stress condi-tions (systemic sclerosis and physical fitness, for example)the increment in oxidative damage is accompanied of incre-ments in antioxidant global response (Ogawa et al., 2011;Traustadóttir et al., 2012). However, Haleagrahara et al. (2010)found a significant increment of lipid hydroperoxide and pro-tein carbonyl content, but a significant decrement of totalantioxidants in bone marrow of rats after lead acetate expo-sure. Our results show that high TAC in lead-exposed workersdepended on elevated CAT and SOD activities, since GPxactivity and serum vitamin E and C concentrations were notdifferent –GRx activity was in fact reduced – in lead-exposedworkers. Studies about lead influence on CAT activity haveproduced divergent results in different lead-exposure models.It has been proposed that lead intoxication induces and incre-ment of synthesis of CAT in the erythroblastic system, but aninhibition of its activity has been found instead (Kasperczyket al., 2004a).

In some studies, erythrocytes in lead-exposed workers pre-sented several times higher CAT activity above non-exposedones (Gurer et al., 1999; Gurer and Ercal, 2000); in at leastone study (Sugawara et al., 1991), a decrease in activity of theCAT was observed, while other authors found no influence

of lead exposure on CAT activity (Monteiro et al., 1985). Ourwork found an increase in CAT activity indicating that it is partof the general antioxidant response induced by the oxidative

Table 5 – Effect of vitamin E/vitamin C supplementation on theexposed workers: GPx activity, GRx activity, serum vitamin E colead and non-lead exposed workers. Values are mean ± SD.

Non-lead exposed (n = 19)

Before After CI of ratios (lowerCI – upper 95%

Serum vitamin E(�mol/ml)

24 ± 5.4 52 ± 5.0 2.0–2.5***

Serum vitamin C(�mol/l)

69 ± 9.5 106 ± 9.8 1.4–1.7***

GPx activity (U/gHb)

12 ± 1.8 14 ± 1.3 1.1–1.3**

GRx activity (U/gHb)

9.7 ± 2.2 11 ± 2.1 1.0–1.2

∗ 0.01 < p < 0.05.∗∗ 0.001 < p < 0.01.

∗∗∗ p < 0.001.

insult. With respect to SOD activity in blood of lead intoxicatedpeople, results of clinical research are divergent as well. Com-paring lead exposed populations to non-lead exposed onesdifferent authors have found lower, higher and similar SODactivities (Ribarov and Benov, 1981; Michiels et al., 1994; Yeet al., 1999; Kasperczyk et al., 2004a). In our work, SOD activ-ity in erythrocytes was higher in lead-exposed workers, whichmight be interpreted as a probable response to an increasedproduction of superoxide anion radicals caused by lead expo-sure (Ribarov and Benov, 1981).

Several enzymes in antioxidant defense systems mayprotect against the imbalance between pro-oxidants andantioxidants, however, most of these enzymes containsulfhydryl groups at their active site and might become inac-tive due to a direct binding of lead to the sulfhydryl group(Flora et al., 2008). The effect of the activation induced by theoxidative insult could overcome the inhibitor effect of lead.Augmentation in antioxidant activity of both enzymes, SODand CAT, is frequently found in the presence of different oxida-tive insults, as observed in these lead-exposed workers. Theantioxidant enzymes act in a cooperative or synergistic way toensure global protection, for they frequently act on differentradicals. Efficient protection is attained when there exists anappropriate balance between the activities of these enzymes

(Michiels et al., 1994; Valko et al., 2006).

Vitamins C and E supplementation caused no reductionin the blood lead concentration, in spite of the improvement

antioxidant status in blood of non-lead exposed and leadncentration, serum vitamin C concentration in blood of

Lead exposed (n = 15)

95%CI)

Before After CI of ratios (lower 95%CI – upper 95% CI)

22 ± 4.3 26 ± 5.1 1.1–1.2***

72 ± 7.8 88 ± 7.7 1.1–1.3***

10 ± 2.2 10 ± 2.1 0.9–1.1

3.1 ± 1.2 3.8 ± 1.0 1.0–1.5*

e n v i r o n m e n t a l t o x i c o l o g y a n d p h

Fig. 3 – Effects of vitamin E/vitamin C supplementation onthe antioxidant status in erythrocytes of non-lead exposedand lead exposed workers: (A) CAT activity and (B) SODactivity, before (�) and after (�) supplementation.*a

oatv2abceaoeteCwcot(aimavt

change negligibly after vitamins supplementation in lead-

**Significant difference (p < 0.001) for paired test before andfter supplementation.

f safety and hygienic-dietetic lines for these workers. Someuthors have observed a reduction in blood lead concentra-ion, mostly in animal models, suggesting a chelate effect ofitamin C (Flora and Tandon, 1986; Patrick, 2006b; Flora et al.,008). In contrast, another report stated that rats treated withscorbic acid did not reduce the lead burden in liver, kidney,rain, and blood (Patra et al., 2001). Although it is biologi-ally plausible that vitamin C may affect lead absorption, theffect is more obvious when lead is provided via drinking watert low concentrations combined with high supplementationf this vitamin (Flora et al., 2008). In this study the chelateffect of vitamin C was not observed; however, it is impor-ant to note that lead-exposed workers kept continually beingxposed to lead via respiratory pathway during vitamin E and

supplementation. Supplementation with vitamin E and Cas very effective in reducing lipid peroxidation in erythro-

ytes of lead-exposed workers; simultaneous administrationf vitamin E and C generate a synergistic antioxidant effecthrough the enzymatic regeneration system in a redox cycleTraber and Stevens, 2011). Our results show that the enhancedntioxidant activity was able to compensate the oxidativensult, reducing the oxidative damage and preventing macro-

olecular alterations in lead-exposed workers. Studies in vivo

nd in vitro on the antioxidant effect of vitamin E and/oritamin C in erythrocytes of lead intoxicated rats suggesthat antioxidant supplementation prevents the inhibition of

a r m a c o l o g y 3 7 ( 2 0 1 4 ) 45–54 51

�-aminolevulinic dehydratase activity and lipid peroxidation(Tandon et al., 2001; Patrick, 2006b; Rendón-Ramírez et al.,2007). Vitamin E alone or in combination with CaNa2EDTAwas found to be effective in decreasing the lead-induced lipidperoxide levels of liver and brain in rats (Patra et al., 2001).Although many investigators have shown that lead-inducedoxidative damage and some antioxidant nutrients reducelead toxicity, the mechanisms of dietary supplementation ofantioxidants remain to be further clarified in lead exposedhumans or animals (Hsu and Guo, 2002; Hsu et al., 1988). Inthis work, supplementation with antioxidants produced a sig-nificant increment of �-ALAD activity; the direct effects of leadon the enzyme may explain the non-total recovery of �-ALADactivity. Several models prove that �-ALAD is a protein sus-ceptible to oxidation. Our results suggest that vitamin E andC are effective to prevent protein oxidative damage while pre-serving their activity in lead-exposed workers. Remarkably,the TAC after vitamin E and C supplementation was reducedin lead-exposed workers. This TAC reduction was accompa-nied by lowering SOD and CAT enzymatic activity. One possibleexplanation is that supplemented antioxidants counteract theoxidative insult with the consequent relaxation of the antioxi-dant enzymatic system. This response may be useful in savingenergy and reducing the expenditure of endogen antioxidantsmetabolites. An increment in oxidative damage and antioxi-dant response with high activity of SOD and CAT in red cellswas found in workers exposed to particles derived from a coalelectric-power; after daily supplementation with vitamins C(500 mg) and E (800 mg) during six months, antioxidant enzy-matic activity reached control levels (Possamai et al., 2010).A similar result was obtained in subjects exposed to the air-borne contamination from coal dusts and solid residues ofincineration health services (Wilhelm et al., 2010). In contrast,in animal studies (in vivo and in vitro) lead exposure inducesa reduction in the TAC, and supplementation with exoge-nous antioxidants (epigallocatechin-3-gallate and etlingeraelatior or green tea extracts) increased the enzymatic antioxi-dant activity and, as consequence, the TAC (Yin et al., 2008;Haleagrahara et al., 2010; Hamed et al., 2010). In humanschronically exposed, the organism may be generating a verycomplex adaptive process which causes an energy saving thatreduces enzymatic activities when it uses exogenous antiox-idants; besides, we found (see also Niki and Noguchi, 2004;Choi et al., 2004; Traber and Stevens, 2011) that the antiox-idant used in this work is physiologically integrated in theantioxidant response and have a regeneration enzymatic sys-tem included in the redox cycle. Evidently, the antioxidantenzymatic system contributes primarily to the TAC. On theother hand, vitamins E and C are spent during their scav-enger function, generating oxidized metabolites which areremoved and eliminated (Choi et al., 2004; Niki and Noguchi,2004); consistent with this observation, vitamins E and C con-centrations showed no substantial increment in lead-exposedworkers after vitamins supplementation took place. On theother hand, a quick distribution and redistribution in cellscould cause that concentration of vitamin E and C in serum

exposed workers (Traber and Stevens, 2011). The activity ofGPx and GRx basically changed nothing after vitamins supple-mentation indicating that these antioxidant enzymes do no

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participate in the reduction of the TAC in lead-exposed work-ers.

Different studies have shown that lead can induce oxida-tive stress and damage by producing reactive oxidative speciessuch as superoxide anion (O2

•−), hydrogen peroxide (H2O2),hydroxyl radical (•OH) and singlet oxygen (1O2) (Patrick, 2006a),which might account be responsible of the damage foundin this study. On the other hand, both alpha-tocopherol andvitamin C can remove superoxide anion and others free radi-cals (Traber and Stevens, 2011; Patrick, 2006b), which mightaccounts for the beneficial effects of its supplementation inlead-exposed workers: protecting enzymatic activity, reducingoxidative damage, improving the efficiency of antioxidant sys-tems, allowing the reduction of antioxidant enzymatic activityand, as consequence, the TAC. Treatment with chelates andantioxidant vitamins are currently being carried out in lead-exposed workers with severe neurological damage.

Conflict of interest statement

The authors of this article expressed no conflict of interest.

Acknowledgments

The authors thank Rosas Margarita, Camacho Héctor and RuizAntonio for technical assistance.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.etap.2013.10.016.

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