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 ) 4554

Available online at www.sciencedirect.com

ScienceDirect
journal homepage: www.elsevier.com/locate/etap

Effect of vitamin E and C supplementation on

oxidative damage and total antioxidant capacity
in lead-exposed workers
Adela-Leonor Rendn-Ramrez a , Mara Maldonado-Vega b ,
Martha-Angelica Quintanar-Escorza c , Gerardo Hernndez d ,
Bertha-Isabel Arvalo-Rivas e , Alejandro Zentella-Dehesa f ,
Jos-Vctor Caldern-Salinas a,
a

Biochemistry Department, Centro de Investigacin y Estudios Avanzados IPN, Mxico, DF, Mexico
CIATEC, Len, GTO, Mexico
c Faculty of Medicine, UJED, Durango, DGO, Mexico
d Section of Methodology of Science, Centro de Investigacin y Estudios Avanzados IPN, Mxico, DF, Mexico
e HRAEB, Len, GTO, Mexico
f Cellular Physiology Institute, UNAM, Mexico
b

a r t i c l e

i n f o

a b s t r a c t

Article history:

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

Received 1 January 2013

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

Received in revised form

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

3 October 2013

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

Accepted 25 October 2013

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

Available online 13 November 2013

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

Keywords:

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

Antioxidants

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

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

Oxidation

We conclude that antioxidant supplementation is effective in reducing oxidative damage

Blood

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

Human

lead-exposed workers.

Lead toxicity

2013 Elsevier B.V. All rights reserved.

Enzymes

1.

Introduction

Lead is one of the metals most commonly used in industry; its toxicity is a public health problem due mainly to its
environmental persistence, inadequate labor security conditions and low lead excretion in lead-exposed workers (Nilsson

et al., 1991; Bleecker et al., 1995; Caldern-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
in signicant alterations in various organs, the hematological system being an important target (Garcon et al., 2004;
Gurer-Orhan et al., 2004). Erythrocytes have high afnity for
lead and typically contain the majority of the lead found in

Corresponding author at: Biochemistry Department, Centro de Investigacin y Estudios Avanzados IPN, Mxico DF. Av. IPN 2508, Zacatenco, Mxico 07000, DF AP 14-740, Mexico. Tel.: +52 55 57473955.
E-mail address: jcalder@cinvestav.mx (J.-V. Caldern-Salinas).
1382-6689/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.etap.2013.10.016

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 n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 4554

the blood stream (Leggett, 1993). Several studies suggest that

lead toxicity involves oxidative damage (Ercal et al., 2001;
Patrick, 2006a,b; Rendn-Ramrez et al., 2007; Flajs et al., 2009;
Hamed et al. 2010; Quintanar-Escorza et al., 2010; Jomova and
Valko, 2011). Lead intoxication induces inhibition of hem synthesis and accumulation of pro-oxidant and autoxidizability
substrates as -aminolevulinic acid (-ALA), free hem groups
and free iron ions in erythrocytes (Ercal et al., 2001; RendnRamrez et al., 2007); in turn, accumulation of oxidizable
substrates can result in superoxide and hydrogen peroxide formation (Patrick, 2006b; Flora et al., 2008). Additionally, under
lead poisoning, high oxygen concentrations, autoxidizability
of hemoglobin and vulnerable membrane components to lipid
peroxidation make erythrocytes extremely sensitive to oxidative damage (Cimen, 2008; Ercal et al., 2001; Rendn-Ramrez
et al., 2007; Jomova and Valko, 2011).
Organisms have diverse and highly efcient antioxidant
mechanisms, but few and divergent results have been found
in antioxidant responses to lead exposure; differences seem to
depend on the experimental model, concentration of lead and
exposure time (Kasperczyk et al., 2004a,b; Mishra and Acharva,
2004; Haleagrahara et al., 2010; Hamed et al., 2010). Different
insults induce complex responses in antioxidant enzymatic
systems (Drge, 2002; Valko et al., 2007; Auten and Davis, 2009),
and little has been done to evaluate the effects of exogenous
antioxidant treatments in lead intoxicated workers. Nonenzymatic exogenous antioxidants such as vitamins C, E, and
B6, taurine, methionine, selenium, zinc, alpha-lipoic acid or
N-acetylcysteine have been used as antioxidant treatment in
animal models and shown to lower reactive oxygen species
(ROS) that generate cellular damage in different cell types of
lead exposed rats (Simon and Hudes, 1999; Flora et al., 2003;
Shalan et al., 2005; Patrick, 2006b; Rendn-Ramrez et al., 2007;
Wang et al., 2007; Flora et al., 2008). Simultaneous administration of vitamins E a lipid-soluble non-enzymatic antioxidantand C an aqueous antioxidant is particularly interesting
since it produces a more effective regeneration of the enzymatic system reducing the oxidation manifestation (Traber
and Stevens, 2011). Other studies have demonstrated the
synergistic protective action of vitamins C and E against genotoxicity, spermatozoa and hippocampus damage (Mishra and
Acharva, 2004; Li et al., 2008). In lead-exposed workers, treatment with vitamin C, vitamin E, beta-carotene, selenium, zinc
and chromium prevented the inhibition of -aminolevulinic
acid dehydratase (-ALAD) activity in blood (Tandon et al.,
2001), increased the total antioxidant capacity (TAC) as well
as superoxide dismutase (SOD) and glutathione peroxidase
(GPx) activity (Machartov et al., 2000), while abating the toxic
effects associated with inhibition of the calcium pump (Abam
et al., 2008). These results suggest investigating the effects
of antioxidant supplements in lead-exposed workers. In this
work we study the antioxidant benets of vitamin E and C
supplementation as well its effects on the antioxidant enzymatic system in these subjects.

2.

Materials and methods

A group of lead exposed workers was supplied with vitamins

C and E for one year. The effects on blood lead concentration,

-ALAD, SOD, glutathione reductase (GRx) and GPx activities,

as well as on lipid peroxidation and erythrocyte TAC was determined. In healthy people, both signicant effects and not
effects at all of these vitamins on the antioxidant systems have
been reported (Traber and Stevens, 2011). To clarify matters, a
control group of non-lead exposed workers was subjected to
the same treatment for comparison.
The studied group consisted of male workers occupationally exposed to lead in a recycling battery factory, particularly
to dust of a mixture of lead oxides; only fteen workers, with
exposure time ranged from 5 to 10 years (mean = 6.8 years),
out of twenty subjected to exposure conditions, accepted to
participate and concluded the treatment for this study. We
remark the difculty of nding a plant industry willing to allow
its employees being studied, much less to be treated in its
premises. Lead exposed workers did not present renal damage
albuminuria or signs or a history of severe neurological damage (seizures or severe disturbances of balance), not did they
receive chelation treatment during this study.
As a control group we used 19 (initially 20, one dropped)
male workers clinically healthy and without a history of
occupational lead exposure. They all showed normal parameters in blood and urine. None of them presented a history
of, or current, physical symptoms of serious neurological,
cardiovascular, renal, hepatic, endocrine, metabolic or gastrointestinal disease, nor were they subjected to previous
pharmacological treatment. Both groups had similar age and
social position score. Non-lead exposed workers ranged from
29 to 42 years old (mean = 34.5), whereas the exposed group
was from 25 to 41 years old (mean = 32.9). Ranges of social score
(Hollingshed, 2011) for non-lead exposed and lead exposed
groups were from 7.0 to 10.5 (mean = 9.2) and from 7.8 to
9.6 (mean = 8.6), respectively. All subjects provided written,
informed consent, and participation was voluntary. The Medical Center-Bajio, IMSS-Mexico, approved the study.
Each subject received 400 IU Alpha Tocopherol Acetate vitamin E (capsules) and 1 g tablet vitamin C per day during the
morning for a period of 12 months. These doses were used
in previous studies for different illness associated with oxidative damage (Plantinga et al., 2007; Castillo et al., 2011), and
no unwanted side effects are mentioned in the literature.
We remark that these dosages are within the FDA recommended dosage for supplementation (maximum of 1000 mg
of vitamin C and 1500 IU for alpha tocopherol). No worker was
reported to have missed his takings, and no worker presented
lateral effects that affected his participation in this study or
that would lead to suspend the use of these antioxidants.
Patients were instructed to take them with breakfast at their
workplace. Also, used dosages are not known to produce prooxidant effects, nor did we nd such effects in subjects of the
non-exposed group. Besides vitamin supplementation, better
safety procedures and hygienic and dietetic practices were
promoted for all workers at this site. The use of masks and uniforms to reduce lead exposure, as well as a diet with higher
content of ber and reduced calories were recommended to
comply with ethics procedures, even though such practices
might reduce lead exposure and its effects. Venous blood was
obtained by venous puncture in heparinized tubes. Each sample was centrifuged (700 g, 10 min) and plasma was collected;
white cells were carefully removed by aspiration to avoid loss

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 ) 4554

of erythrocytes. To obtain serum, venous blood was collected

in non-heparinized tubes and centrifuged as above.

2.1.

Blood lead concentration

Lead concentration in blood was determined with a Lead Analyzer, model 3010B (ESA), as previously described (QuintanarEscorza et al., 2007). Concentration is reported in micrograms
per deciliter of blood. Lead determinations were run in triplicates. The standard curves were calculated with the method
of addition to minimize the matrix effect on the absorption
peak (recovery of 85113%). Precision of analyses was around
87104% (using ESA Hi and Lo calibrators) and the detection
limit set at 1 g/dl.

2.2.

-ALAD activity

-ALAD activity in erythrocytes was determined spectrophotometrically (US/VIS DU 650) (Beckman) and expressed as
nanomol of porphobilinogen (PBG) per milliliter of erythrocytes per hour (nmol/ml/h) (Rendn-Ramrez et al., 2007).

2.3.

Vitamins E and C

Serum tocoferol and ascorbate were determined by highperformance liquid chromatography in a single chromatographic run with an internal standard (Merzouk et al., 2003;
Lenton et al., 2003).

2.4.

addition of buffer phosphates pH 7.3. The decay of H2 O2

depends on the CAT activity (Aebi, 1984). The hemoglobin concentration (Hb) was estimated using the reagent described by
Van Kampen and Zijlstra (1961).

2.7.

SOD in erythrocytes inhibits the production of superoxide

radicals. The xanthinexanthine oxidase system, in presence
of oxygen, induces the formation of superoxide radicals; this
radical reduces the nitro blue tetrazolium and gives rise to a
colored compound. Since SOD inhibits this reaction, its activity is proportional to the degree of inhibition of nitro blue
tetrazolium reduction to superoxide (Sun et al., 1988). Results
are expressed in units of SOD/g of hemoglobin.

2.8.

GRx activity assays

The activity of the enzyme GRx in erythrocytes is based on the

formation of reduced glutathione from glutathione disulde
in the presence of the enzyme. Glutathione disulde formed
in one minute reacts with NADPH and the product is measured spectrophotometrically at 340 nm and 37 C, (UV/VIS
modelo 30 Beckman-USA). Results of activity of all enzymes
were expressed in U (M/min)/mg of Hb (Vives-Bauza et al.,
2007).

2.9.

Erythrocytes TAC

The antioxidant capacity was measured by the formation of

ROS in red blood cell samples. ROS levels were determined by
uorescence, as described in Oyama et al. (1994). Briey, DCFDA was used as a detector of ROS by ow cytometry. This dye
is not uorescent in reduced state, but it becomes uorescent
once cellular oxidation and removal of acetate groups by cellular esterases take place (Karlsson et al., 2010). Samples were
excited at 480 nm and the emission detected at 520 nm. The
intensity of uorescence in presence of uorophore can be correlated with the cellular content of free radicals. Fluorescence
is measured in arbitrary units per 500,000 cells and expressed
as the inverse of these units (1/(FUA/5 105 cells).

2.6.

SOD activity assays

Lipid peroxidation

The amount of lipid peroxidation in erythrocytes was

estimated as reported in Quintanar-Escorza et al. (2007)
by measuring the thiobarbituric acid-reactive substances
(TBARS). The absorbance was measured at 532 nm and 600 nm
using a spectrophotometer US/VIS DU 650 (Beckman). TBARS
are expressed as nmol of malondialdehyde equivalents per ml
of erythrocyte (Quintanar-Escorza et al., 2010).

2.5.

47

CAT activity assays

CAT activity was determined in erythrocytes by spectrophotometry and is expressed in U/mg protein. This method
measures the exponential disappearance of H2 O2 (3 mM) at
240 nm in presence of cellular homogenates (5 Hb/dl) with

GPx activity assays

The activity of the enzyme GPx in erythrocytes is based on the

formation of glutathione disulde from reduced glutathione in
the presence of the enzyme. Enzyme activity was measured by
modifying a procedure of Paglia and Valentine (1967). Solution
A consisted of 50 mM potassium phosphate buffer (pH 7), 1 mM
EDTA, 1 mM NaN3, 0.2 mM NADPH, 1 IU/ml GSSG-reductase,
1 mM GSH, 1.5 mM cumene hydroperoxlde or 0.25 mM H2 02 in
a total volume of 1 ml. Erythrocytes lisated (0.1 ml) was added
to 0.8 ml of Solution A and allowed to incubate 5 min at room
temperature before initiation of the reaction by the addition of
0.1 ml peroxide solution. Absorbance at 340 nm was recorded
for 5 min and the activity was calculated from the slope of
these lines as moles NADPH oxidized per minute.

2.10.

Statistical analysis

Comparisons between non-lead and lead exposed groups were

done through two-tailed unpaired t-tests. To compare each
group with itself before and after the treatment, we used twotailed paired t-tests; in this case, proportions, instead of mean
differences, were estimated. In all cases the level of signicance was established at P < 0.05. 95% CI were computed to
evaluate the biological signicance besides the statistical signicance. For these tests we used GraphPad Prism version 5.03
for Windows, GraphPad Software, San Diego California USA,
www.graphpad.com. Other, supplementary statistics methods 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 ) 4554

Table 1 Number of individuals presenting clinical symptoms and signs associated to lead intoxication in lead and
non-lead exposed workers.
Lead exposed
(n = 15)

Non-lead exposed
(n = 19)
Dizziness
Headache
Paresthesia
Paresis
Abdominal colic
Myalgia
Motor coordination alteration

% in lead exposed (95%

condence interval)

7*
11*
6*
3*
5*
8*
14*

1
3
1
0
2
2
1

46.6 (2173)
73.3 (4592)
40.0 (1668)
20.0 (448)
33.3 (1262)
53.3 (2779)
93.3 (68100)

Signicant 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, computed
following (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)
6.7 2.2
683 61

PbB (g/dl)
-ALAD activity (nmol PBG/h/ml)

Lead exposed (n = 15)

Difference between
means SD

73 11
138 34

65.8 2.5***
545 17.6***

p < 0.0001.

3.

Results

3.1.

Conditions before the treatment

Clinical symptoms and signs associated with chronic lead

intoxication as dizziness, headache, paresthesia, paresis,
abdominal colic, myalgia and motor coordination alteration
were frequently found in the lead-exposed workers. Percentage of people with these symptoms was signicantly higher
than that of the control group (Table 1). Frequency from
cero to seven symptoms and signs for both lead exposed
and non-lead exposed workers follow a binomial distribution
(Fig. 1). For non-lead exposed workers, the probability of presenting any sign or symptom ranges from 0.05 to 0.16, whereas
for exposed workers the probability of showing any sign or

symptom goes from 0.38 to 0.64 ( = 0.05, df = 6, Pearsons Chisquare test). Blood lead concentration was higher in lead
exposed with respect to non-lead exposed workers (P < 0.0001)
indicative of a chronic and signicant lead exposure. Erythrocyte -ALAD activity was signicantly lower in lead exposed
than in non-lead-exposed workers (P < 0.0001) also suggesting
a severe chronic lead intoxication (Table 2). The oxidative damage (lipid peroxidation) and TAC in erythrocytes were higher
in lead exposed with respect to non-lead exposed workers
(P < 0.0001 and P < 0.0001, respectively) denoting an oxidative stress characterized by an increase in the antioxidant
response, but not enough to contain oxidative insult (Table 3).
A major enzymatic antioxidant response was present in lead
exposed workers, as both CAT and SOD activities in erythrocytes were much higher in this group than in the control
group (P < 0.0001 and P < 0.0001, respectively). However, lower

Table 3 Lipid peroxidation and antioxidant defense system status at the beginning of vitamin E/vitamin C
supplementation. Thiobarbituric acid reactive species concentration (TBARS), total antioxidant capacity (TAC), CAT
activity, SOD activity, GPx activity, GRx activity and serum concentration of vitamins E and C, in blood of non-lead
exposed and lead exposed workers (mean SD).

TBARS (nmol MDA/ml PG)

TAC (1/(FUA/5 105 cells))
Enzymatic activity (U/g Hb)
CAT
SOD
GPx
GRx
Serum concentration (mol/l)
Vitamin E
Vitamin C

0.01 < p < 0.05.

0.001 < p < 0.01.
p < 0.001.

Non-lead exposed (n = 19)

Lead exposed (n = 15)

Difference between
means SD

0.7 0.2
66 3.7

1.3 0.4
110 8.1

0.57 0.01***
44.4 2.08***

11
209
12
9.7

1.5
45
1.8
2.2

24 5.4
69 9.5

37
534
10
3.1

7.5*
55*
2.2
1.2*

22 4.3
72 7.8

26.0
324
2.0
6.6

1.8***
17.1***
0.7**
0.6***

1.1 1.7
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 ) 4554

Fig. 1 Frequency of individuals with cero to seven clinical

symptoms and signs associated to lead intoxication in
non-lead (A) and lead exposed (B) workers, before () and
after () supplementation.

activity of GRx in erythrocytes was found in lead exposed

as compared to non-lead exposed workers (P < 0.0001), indicating a low antioxidant response at the glutathione system
(Table 3). Erythrocytes GPx activity (95% CI from 0.6149 to 3.441)
and serum vitamins E and C concentrations were not different between lead exposed and non-lead exposed workers
(Table 4).

3.2.

Effects of vitamins C and E supplementation

Administration of Vitamins E and C for 12 months did not

induce a reduction in blood lead concentration of both worker
groups (Table 4); clinical symptoms and signs of lead-exposed
workers were not reduced either (Table 4). Although it is
not possible to state an increment in GPx and GRx activities
(Table 5), -ALAD activity increased signicantly, twice as
much its initial value, in lead-exposed workers (Table 4). Lipid
peroxidation was reduced to values between 47.1 and 69.4%
(95% CI) in lead-exposed workers after supplementation with
antioxidant vitamins, attaining values non-statistically different from those of the non-lead exposed workers (P = 0.2119)
(Fig. 1a). Additionally, TAC was reduced to values between
58.9 to 67.7% (95% CI) in lead-exposed workers after supplementation with vitamins E and C, restoring the TAC level up
to a similar value of the non-lead exposed workers (P = 0.1298)

49

Fig. 2 Effect of vitamin E/vitamin C supplementation on

lipid peroxidation and antioxidant defense system status
in blood of non-lead exposed and lead exposed workers:
(A) Lipid peroxidation (TBARS) and (B) total antioxidant
capacity (TAC) before () and after () supplementation.
Values are mean SD. ***Signicant difference (p < 0.001) for
paired test before and after supplementation.

(Fig. 1b). CAT and SOD activities decreased to values between

33.8% and 48.9% (95% CI), and from 44.2 to 53.2% (95% CI),
respectively, after treatment in lead-exposed workers. Despite
this reduction, the activity of CAT was still between 31.8%
and 79.3% higher in lead exposed than in non-lead exposed
individuals. SOD activity became after treatment statistically
non different between lead and no-lead exposed groups
(P = 0.0921) (Fig. 2). Finally, although in both groups vitamins
C and E increased signicantly their concentrations, non-led
exposed workers were sensibly more affected: between 44
and 69% for vitamin C, and between 101 and 153% for vitamin
E (95% CI) (Table 5).

4.

Discussion

Lead exposed workers presented very high blood lead concentration and -ALAD activity severely inhibited, which
indicates that these workers had been extensively exposed
to lead. Additionally, they showed clinical symptoms and
signs frequently associated to lead intoxication (CaldernSalinas et al., 1996; Levin and Goldberg, 2000; Patrick, 2006a;
Quintanar-Escorza et al., 2007). They also presented elevated
oxidative damage, similar to that described in other lead exposure 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 response
to 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 ) 4554

Table 4 Effects of vitamin E/vitamin C supplementation on levels of biological indices of lead intoxication in blood of
non-lead exposed and lead exposed workers. Blood lead concentration (PbB) and -aminolevulinic acid dehydratase
activity (-ALAD), before and after supplementation (mean SD).
Non-lead exposed (n = 19)

PbB (g/dl)
-ALAD activity
(nmolPBG/h/ml)

Before

After

6.7 2.2
683 61

7.1 1.6
696 54

Lead exposed (n = 15)

CI of ratios (lower 95%

CI upper 95% CI)

Before
73 11
138 34

0.81.0
1.01.04

After

CI of ratios (lower 95%

CI upper 95% CI)

70 9.1
315 38

0.91.0*
2.02.8***

0.01 < p < 0.05.

p < 0.001.

however, this antioxidant response was insufcient to abate

severe oxidative damage. In some oxidative stress conditions (systemic sclerosis and physical tness, for example)
the increment in oxidative damage is accompanied of increments in antioxidant global response (Ogawa et al., 2011;
Traustadttir et al., 2012). However, Haleagrahara et al. (2010)
found a signicant increment of lipid hydroperoxide and protein carbonyl content, but a signicant decrement of total
antioxidants in bone marrow of rats after lead acetate exposure. Our results show that high TAC in lead-exposed workers
depended on elevated CAT and SOD activities, since GPx
activity and serum vitamin E and C concentrations were not
different GRx activity was in fact reduced in lead-exposed
workers. Studies about lead inuence on CAT activity have
produced divergent results in different lead-exposure models.
It has been proposed that lead intoxication induces and increment of synthesis of CAT in the erythroblastic system, but an
inhibition of its activity has been found instead (Kasperczyk
et al., 2004a).
In some studies, erythrocytes in lead-exposed workers presented several times higher CAT activity above non-exposed
ones (Gurer et al., 1999; Gurer and Ercal, 2000); in at least
one study (Sugawara et al., 1991), a decrease in activity of the
CAT was observed, while other authors found no inuence
of lead exposure on CAT activity (Monteiro et al., 1985). Our
work found an increase in CAT activity indicating that it is part
of the general antioxidant response induced by the oxidative

insult. With respect to SOD activity in blood of lead intoxicated

people, results of clinical research are divergent as well. Comparing lead exposed populations to non-lead exposed ones
different authors have found lower, higher and similar SOD
activities (Ribarov and Benov, 1981; Michiels et al., 1994; Ye
et al., 1999; Kasperczyk et al., 2004a). In our work, SOD activity in erythrocytes was higher in lead-exposed workers, which
might be interpreted as a probable response to an increased
production of superoxide anion radicals caused by lead exposure (Ribarov and Benov, 1981).
Several enzymes in antioxidant defense systems may
protect against the imbalance between pro-oxidants and
antioxidants, however, most of these enzymes contain
sulfhydryl groups at their active site and might become inactive due to a direct binding of lead to the sulfhydryl group
(Flora et al., 2008). The effect of the activation induced by the
oxidative insult could overcome the inhibitor effect of lead.
Augmentation in antioxidant activity of both enzymes, SOD
and CAT, is frequently found in the presence of different oxidative insults, as observed in these lead-exposed workers. The
antioxidant enzymes act in a cooperative or synergistic way to
ensure global protection, for they frequently act on different
radicals. Efcient protection is attained when there exists an
appropriate balance between the activities of these enzymes
(Michiels et al., 1994; Valko et al., 2006).
Vitamins C and E supplementation caused no reduction
in the blood lead concentration, in spite of the improvement

Table 5 Effect of vitamin E/vitamin C supplementation on the antioxidant status in blood of non-lead exposed and lead
exposed workers: GPx activity, GRx activity, serum vitamin E concentration, serum vitamin C concentration in blood of
lead and non-lead exposed workers. Values are mean SD.
Non-lead exposed (n = 19)
Before
Serum vitamin E
(mol/ml)
Serum vitamin C
(mol/l)
GPx activity (U/g
Hb)
GRx activity (U/g
Hb)

0.01 < p < 0.05.

0.001 < p < 0.01.
p < 0.001.

After

Lead exposed (n = 15)

CI of ratios (lower 95%

CI upper 95% CI)

Before

After

CI of ratios (lower 95%

CI upper 95% CI)

24 5.4

52 5.0

2.02.5***

22 4.3

26 5.1

1.11.2***

69 9.5

106 9.8

1.41.7***

72 7.8

88 7.7

1.11.3***

12 1.8

14 1.3

1.11.3**

10 2.2

10 2.1

0.91.1

9.7 2.2

11 2.1

1.01.2

3.1 1.2

3.8 1.0

1.01.5*

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 a r m a c o l o g y 3 7 ( 2 0 1 4 ) 4554

Fig. 3 Effects of vitamin E/vitamin C supplementation on

the antioxidant status in erythrocytes of non-lead exposed
and lead exposed workers: (A) CAT activity and (B) SOD
activity, before () and after () supplementation.
***Signicant difference (p < 0.001) for paired test before and
after supplementation.

of safety and hygienic-dietetic lines for these workers. Some

authors have observed a reduction in blood lead concentration, mostly in animal models, suggesting a chelate effect of
vitamin C (Flora and Tandon, 1986; Patrick, 2006b; Flora et al.,
2008). In contrast, another report stated that rats treated with
ascorbic acid did not reduce the lead burden in liver, kidney,
brain, and blood (Patra et al., 2001). Although it is biologically plausible that vitamin C may affect lead absorption, the
effect is more obvious when lead is provided via drinking water
at low concentrations combined with high supplementation
of this vitamin (Flora et al., 2008). In this study the chelate
effect of vitamin C was not observed; however, it is important to note that lead-exposed workers kept continually being
exposed to lead via respiratory pathway during vitamin E and
C supplementation. Supplementation with vitamin E and C
was very effective in reducing lipid peroxidation in erythrocytes of lead-exposed workers; simultaneous administration
of vitamin E and C generate a synergistic antioxidant effect
through the enzymatic regeneration system in a redox cycle
(Traber and Stevens, 2011). Our results show that the enhanced
antioxidant activity was able to compensate the oxidative
insult, reducing the oxidative damage and preventing macromolecular alterations in lead-exposed workers. Studies in vivo
and in vitro on the antioxidant effect of vitamin E and/or
vitamin C in erythrocytes of lead intoxicated rats suggest
that antioxidant supplementation prevents the inhibition of

51

-aminolevulinic dehydratase activity and lipid peroxidation

(Tandon et al., 2001; Patrick, 2006b; Rendn-Ramrez et al.,
2007). Vitamin E alone or in combination with CaNa2 EDTA
was found to be effective in decreasing the lead-induced lipid
peroxide levels of liver and brain in rats (Patra et al., 2001).
Although many investigators have shown that lead-induced
oxidative damage and some antioxidant nutrients reduce
lead toxicity, the mechanisms of dietary supplementation of
antioxidants remain to be further claried in lead exposed
humans or animals (Hsu and Guo, 2002; Hsu et al., 1988). In
this work, supplementation with antioxidants produced a signicant increment of -ALAD activity; the direct effects of lead
on the enzyme may explain the non-total recovery of -ALAD
activity. Several models prove that -ALAD is a protein susceptible to oxidation. Our results suggest that vitamin E and
C are effective to prevent protein oxidative damage while preserving their activity in lead-exposed workers. Remarkably,
the TAC after vitamin E and C supplementation was reduced
in lead-exposed workers. This TAC reduction was accompanied by lowering SOD and CAT enzymatic activity. One possible
explanation is that supplemented antioxidants counteract the
oxidative insult with the consequent relaxation of the antioxidant enzymatic system. This response may be useful in saving
energy and reducing the expenditure of endogen antioxidants
metabolites. An increment in oxidative damage and antioxidant response with high activity of SOD and CAT in red cells
was found in workers exposed to particles derived from a coal
electric-power; after daily supplementation with vitamins C
(500 mg) and E (800 mg) during six months, antioxidant enzymatic activity reached control levels (Possamai et al., 2010).
A similar result was obtained in subjects exposed to the airborne contamination from coal dusts and solid residues of
incineration health services (Wilhelm et al., 2010). In contrast,
in animal studies (in vivo and in vitro) lead exposure induces
a reduction in the TAC, and supplementation with exogenous antioxidants (epigallocatechin-3-gallate and etlingera
elatior or green tea extracts) increased the enzymatic antioxidant activity and, as consequence, the TAC (Yin et al., 2008;
Haleagrahara et al., 2010; Hamed et al., 2010). In humans
chronically exposed, the organism may be generating a very
complex adaptive process which causes an energy saving that
reduces enzymatic activities when it uses exogenous antioxidants; besides, we found (see also Niki and Noguchi, 2004;
Choi et al., 2004; Traber and Stevens, 2011) that the antioxidant used in this work is physiologically integrated in the
antioxidant response and have a regeneration enzymatic system included in the redox cycle. Evidently, the antioxidant
enzymatic system contributes primarily to the TAC. On the
other hand, vitamins E and C are spent during their scavenger function, generating oxidized metabolites which are
removed and eliminated (Choi et al., 2004; Niki and Noguchi,
2004); consistent with this observation, vitamins E and C concentrations showed no substantial increment in lead-exposed
workers after vitamins supplementation took place. On the
other hand, a quick distribution and redistribution in cells
could cause that concentration of vitamin E and C in serum
change negligibly after vitamins supplementation in leadexposed workers (Traber and Stevens, 2011). The activity of
GPx and GRx basically changed nothing after vitamins supplementation indicating that these antioxidant enzymes do no

52

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 ) 4554

participate in the reduction of the TAC in lead-exposed workers.

Different studies have shown that lead can induce oxidative stress and damage by producing reactive oxidative species
such as superoxide anion (O2 ), hydrogen peroxide (H2 O2 ),
hydroxyl radical ( OH) and singlet oxygen (1 O2 ) (Patrick, 2006a),
which might account be responsible of the damage found
in this study. On the other hand, both alpha-tocopherol and
vitamin C can remove superoxide anion and others free radicals (Traber and Stevens, 2011; Patrick, 2006b), which might
accounts for the benecial effects of its supplementation in
lead-exposed workers: protecting enzymatic activity, reducing
oxidative damage, improving the efciency of antioxidant systems, allowing the reduction of antioxidant enzymatic activity
and, as consequence, the TAC. Treatment with chelates and
antioxidant vitamins are currently being carried out in leadexposed workers with severe neurological damage.

Conict of interest statement

The authors of this article expressed no conict of interest.

Acknowledgments
The authors thank Rosas Margarita, Camacho Hctor and Ruiz
Antonio 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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