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Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit
Departamento de Bioqumica, Centro de Investigacin y Estudios Avanzados (CINVESTAV-IPN), Mxico City, Mexico
Departamento de Bioqumica, Facultad de Medicina, Universidad Jurez del Estado de Durango (UJED), Durango, Dgo., Mexico
c
Departamento de Farmacologa, Facultad de Medicina, Universidad Autnoma de Mxico (UNAM), Mxico City, Mexico
b
a r t i c l e
i n f o
Article history:
Received 4 February 2010
Accepted 4 May 2010
Available online 10 May 2010
Keywords:
Trolox
Calcium homeostasis
Human erythrocytes
Oxidative damage
Lead exposure
a b s t r a c t
One important effect of lead toxicity in erythrocytes consists of increasing [Ca2+]i which in turn may
cause alterations in cell shape and volume and it is associated with cellular rigidity, hemolysis, senescence and apoptosis. In this work, we proposed the use of erythrocytes incubated with Pb2+ to assess
association of the mechanisms of lead erythrocyte oxidative damage and calcium homeostasis. Lead incubation produced an increase in [Ca2+]i dose- and time-dependent, which mainly involved Ca2+ entry
mechanism. Additionally, in this in vitro model alterations similar to erythrocytes of lead-exposed workers were produced: Increase in Ca2+ inux, decrease in (Ca2+Mg2+)-ATPase activity and GSH/GSGG ratio;
increase in lipoperoxidation, protein carbonylation and osmotic fragility accompanied of dramatic morphological changes. Co-incubation with trolox, a soluble vitamin-E analog is able to prevent these alterations indicating that lead damage mechanism is strongly associated with oxidative damage with an
intermediate toxic effect via [Ca2+]i increase. Furthermore, erythrocytes oxidation induced with a free
radical generator (APPH) showed effects in [Ca2+]i and oxidative damage similar to those found in erythrocytes incubated with lead. Co-incubation with trolox prevents the oxidative effects induced by AAPH in
erythrocytes. These results suggest that increase of [Ca2+]i depends on the oxidative status of the erythrocytes incubated with lead. We consider that this model contributes in the understanding of the relation
between oxidative damage induced by lead exposure and Ca2+ homeostasis, the consequences related to
these phenomena and the molecular basis of lead toxicity in no excitable cells.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Lead intoxication is one of the leading occupational health
problems. This metal causes a broad range of biochemical, physiological and behavioral dysfunctions (Caldern-Salinas et al., 1996).
The molecular basis of lead toxicity involves: covalent binding to
proteins (Navarro-Moreno et al., 2009; Goering, 1993); oxidative
damage (Gurer-Ohrnan et al., 2004; Rendn-Ramrez et al., 2007)
and interaction with stereospecic sites for divalent cations such
as calcium ion (Ca2+). This latter mechanism effects different biologically signicant calcium-dependent processes, which include:
inter- and intracellular signaling, divalent metal transport, energy
metabolism, enzymatic processes, apoptosis, ionic conduction, cell
adhesion, protein maturation, and genetic regulation (Garza et al.,
2006). To reach its targets, lead ion (Pb2+) is transported into the
erythrocytes through Ca2+ transport systems and competes with
1339
2.1. Subjects
The erythrocytes were obtained from ten healthy male volunteers (2732 years old) with no history of lead exposure. They were
evaluated with clinical analysis of blood and urine, showing normal parameters. Exclusion criteria were the following: History or
current physical ndings of serious cardiovascular, renal, hepatic,
endocrine, metabolic or gastrointestinal disease or previous pharmacological treatment. All subjects provided written, informed
consent and participation was voluntary in accordance with the
Declaration of Helsinki. The study was approved by the Medical
Center-Bajo, IMSS-Mxico.
2.2. Preparation of erythrocytes
Blood was obtained by venous puncture in heparinized tubes.
Each sample was centrifuged at 700g for 10 min and the plasma
and white cells were carefully removed by aspiration to avoid loss
of erythrocytes. The packed cells were washed three more times at
700g for 5 min with isotonic buffer.
2.3. Incubation of erythrocytes to lead
Erythrocytes (2 ml packed cells) were suspended in 10 ml buffer A (300 mOsm, pH 7.4) which contained 144 NaCl; 5 KCl; 10
HEPES; 5 Glucose; 1.8 MgCl2; 1.5 CaCl2; in mmol/l. A solution of
Pb(NO3)2 was added so that nal concentrations Pb2+ were 0.2,
0.4, 2.0 ,4.0 and 6.0 lM and cells were incubated for 0.5120 h
(4 C). Pb2+ and Ca2+ were adjusted in each experiment by titration
of the ion concentration with the selective pH meter PHM95,
ISE25Pb (Radiometer Copenhagen Denmark). Both 0.4 and 4 lM
concentrations are equivalent to 10 and 80 lg/dl respectively of
lead in total blood as reported in lead-exposed workers (Quintanar-Escorza et al., 2007). Samples incubated in the same conditions
without Pb(NO3)2 were used as a control assay. After this process,
the total percent of hemolysis was <5% and similar for all incubations. The packed cells were washed three more times at 700g
for 5 min with isotonic buffer; preparations were maintained at
4 C until use. In some experiments, the erythrocytes were co-incubated with 5 lM trolox for 24 and 120 h at 4 C.
2.4. Calcium uptake assays
Briey, erythrocytes were resuspended at 20% in isotonic buffer.
Incubations were performed at room temperature under constant
stirring. Assays were started by the addition of 1 mmol/l Ca2+ (nal
concentration), containing 45Ca (18.5 lCi/ml) and 10 lmol/l LaCl3.
Lantane was added to prevent 45Ca unspecic binding to the membrane and to inhibit the plasma membrane (Ca2+Mg2+)-ATPase
activity. After 30 min incubation, erythrocytes were separated by
centrifugation in a microfuge TM (Beckman) and washed twice
with isotonic solution. The associated radioactivity was determined by liquid scintillation (Caldern-Salinas et al., 1999a).
2.5. Evaluation of (Ca2+Mg2+) ATPase
The (Ca2+Mg2+)-ATPase activity was assessed in erythrocytes
by measuring the release of inorganic phosphate (Fiske and Subbarow, 1925), using a spectrophotometer US/VIS DU 650 (Beckman).
The (Ca2+Mg2+)-ATPase activity was calculated as the difference
between the amount of phosphate released in the tubes containing
calcium minus the one released in medium without calcium. After
40 min incubation, the activity was stopped by the addition of
(20%) trichloroacetic acid. The results are expressed as nmol of
inorganic phosphate per mg of protein per minutes (nmol Pi/mg
protein/min, after subtraction of a blank run in parallel without
1340
The difference between means was compared using Students ttest, statistical SPSS-w version 14. Differences were considered signicant at P < 0.01.
3. Results
Incubation of freshly drawn erythrocytes with Pb(NO3)2 (0.2,
0.4, 2.0, 4.0, and 6.0 lM) in isotonic solution induced increases in
the [Ca2+]i in a dose-dependent (Fig. 1A) and time-dependent
(12120 h) manner (Fig. 1B). In erythrocytes incubated with high
lead concentrations (4.06.0 lM) the [Ca2+]i for 24 or 120 h, the
[Ca2+]i reached nearly steady values (58 and 120 nM, respectively)
(Fig. 1A); with [Pb2+] 0.4 lM the [Ca2+]i for 48 to 120 h reached
nearly steady values (76 nM) and with 4.0 lM the [Ca2+]i for 48
to 120 h reached nearly steady values (118 nM) (Fig. 1B). In subsequent experiments, we selected incubations with concentrations
0.4 and 4.0 lM of Pb2+ because these values are similar to high
and intermediate lead blood concentration found in lead-exposed
workers (Quintanar-Escorza et al., 2007); the lead-incubation
times selected were 24 and 120 h, which were periods of time
where modications in calcium homeostasis were found. Erythrocytes incubated with lead at short times (<24 h) showed small or
no effects; important hemolysis (>10%) was detected in long periods of incubation (>120 h) and high concentrations (>4.0 lM of
[Pb2+]).
The mechanism by which [Ca2+]i was raised in erythrocytes
incubated with lead was investigated. We found an increase in
Ca2+ inux measured as the uptake of 45Ca2+. Fig. 2A shows that
incubation with 0.4 lM [Pb2+] for 24 h increased Ca2+ uptake 1-fold
as compared with control. After 120 h, the Ca2+ uptake was 3.2-fold
higher. At 4.0 lM [Pb2+] and 24 h of incubation, the calcium uptake
increased 1.7-fold as compared with the control, whereas calcium
uptake after 120 h was 3.8-fold higher (Fig. 2A). As for the Ca2+
1341
160
120 h
120
100
80
24 h
60
40
20
A
*
120
100
*
80
*
60
40
20
0
0
0
0
140
140
24
24
0.4 M Pb 2+
4 M
120
100
0.4 M
80
60
40
20
0
0
12
24
36
48
60
72
84
96
108 120
Time (h)
Fig. 1. (A) Dose-dependent effect of lead on [Ca2+]i in erythrocytes incubated 24 h
(j) or 120 h (h). (B) Time-dependent effect of lead on [Ca2+]i in uo 3-loaded
erythrocyte incubated with 0.4 lM Pb2+ (s) or 4.0 lM Pb2* (d). Results are
presented as mean standard deviation. *P < 0.01 Students t-test (n = 10) between
lead concentration (A) and exposition times (B).
140
120
50
120
4M Pb 2+
40
30
20
10
0
0
24
120
0.4 M Pb2+
24
120
4M Pb 2+
1342
100
Percent hemolysis
80
60
40
Control
24 h
20
120 h
0
0
100
200
300
Osmolarity (mOsm)
Percent hemolysis
100
80
60
Control
40
24 h
120 h
20
0
0
100
200
300
Osmolarity (mOsm)
15
*
12
*
*
6
*
24
0.4 M
120
Pb 2+
24
120
4M Pb 2+
1343
Table 1A
Effects of trolox co-incubation (5 lM) on [Ca2+]i, ATPase(Ca2+/Mg2+) activity and incorporated calcium in erythrocytes incubation without (control) and with lead (0.4 lM).
Results are presented as mean standard deviation.
Calcium parameters
Control
Trolox (5 lM)
[Ca2+]i (nM)
ATPase (Ca2+/Mg2+) activity (nmol Pi/mg/min)
Incorporated calcium (lmol/l)
*
120
24
120
120
24
120
30 2.9
40 3.8
30 2.7
52 2.7*
27 2.1*
61 1.7*
81 2.4*
7 2.8*
127 3.7*
36 1.8
41 3.8
32 2.7
35 2.9
35 2.4
39 3.5
38 2.1
13 1.8*
39 4.1
Table 1B
Effects of trolox co-incubation (5 lM) on [Ca2+]i, ATPase(Ca2+/Mg2+) activity and incorporated calcium in erythrocytes incubated without (control) and with lead (4 lM). Results
are presented as mean standard deviation.
Calcium parameters
Lead (4 lM)
Control
Trolox (5 lM)
[Ca ]i (nM)
ATPase (Ca2+/Mg2+) activity (nmol Pi/mg/min)
Incorporated calcium (lmol/l)
*
24
120
*
30 2.9
40 3.8
30 6.7
65 2.4
18 2.4*
81 2.1*
118 3.1
2 2.7*
145 3.1*
120
24
120
37 1.6
40 3.9
31 3.9
41 3.4
12 3.5*
32 5.7
40 3.1
3 2.7*
38 2.1
Table 2A
Effects of trolox co-incubation (5 lM) on lipid and protein oxidation, [GSH] and [GSSG], GSH/GSSG ratio and osmotic fragility of erythrocytes incubated without (control) and with
lead (0.4 lM). Results are presented as mean standard deviation.
Oxidation and damage parameters
Control
Trolox (5 lM)
120
24
120
120
24
120
0.9 0.3
1.0 0.1
690 42
95 28
7.3 0.1
165 7.0
3.2 0.4*
3.0 0.4*
640 48
143 30
5.0 0.2*
170 9.0
9.0 0.5*
3.6 0.5*
540 43*
246 35*
2.2 0.1*
200 9.0*
0.9 0.5
0.9 0.1
685 44
97 24
7.0 0.3
168 6.0
0.8 0.2
1.6 0.3*
697 47
98 29
7.1 0.1
164 9.0
1.1 0.4
2.0 0.2*
651 45
139 31
4.7 0.2
167 5.0
Table 2B
Effects of trolox co-incubation (5 lM) on lipid and protein oxidation, [GSH] and [GSSG], GSH/GSSG ratio and osmotic fragility of erythrocytes incubated without and with lead
(4 lM). Results are presented as mean standard deviation.
Oxidation and damage parameters
Control
Lead (4 lM)
Trolox (5 lM)
120
24
120
120
24
120
0.9 0.3
1.0 0.1
690 42
95 28
7.3 0.1
165 7.0
9.1 0.3*
4.2 0.5*
544 46*
239 35*
2.3 0.1*
203 7.0*
14.0 0.2*
5.7 0.6*
295 43*
489 28*
0.6 0.1*
247 9.0*
0.9 0.5
0.9 0.1
685 41
97 24
7.0 0.3
168 6.0
4.2 0.8
2.6 0.2*
618 40*
162 25*
3.6 0.1*
185 4.0*
6.3 0.5
2.9 0.3*
539 46*
243 31*
2.4 0.3*
199 6.0*
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Fig. 5. Effect of trolox co-incubation on erythrocytes morphology alteration induced by incubation with lead. (a and b) erythrocytes without lead (control) (24 h); (c and d)
erythrocytes incubated with 0.4 lM Pb2+ (24 h); (e and f) erythrocytes incubated with 4.0 lM Pb2+ (24 h); erythrocytes incubated with 0.4 lM Pb2+ (24 h); (g and h)
erythrocytes without lead (control) (120 h); (i and j) erythrocytes incubated with 0.4 lM Pb2+ (120 h); (k and l) erythrocytes incubated with 4.0 lM Pb2+ (120 h). The pictures
show phase contrast (a, c, e, g, i and k) and Nomarsky (b, d, f, h, j and l) images.
Fig. 6. Effect of trolox co-incubation on erythrocytes morphology alteration induced with 120 h of incubation with lead. (a and b) Erythrocytes without lead (control); (c and
d) erythrocytes incubated with 5.0 lM trolox; (e and f) erythrocytes incubated with 4.0 lM Pb2+; (g and h) erythrocytes co-incubated with 5 lM trolox and 4.0 lM of Pb2+.
The pictures show phase contrast (a, c, e and g) and Nomarsky (b, d, f and h) images.
4. Discussion
In a previous work we provided evidence indicating that in
erythrocytes obtained from lead-exposed workers there is an
increment in [Ca2+]i that is related to an increase in oxidative damage (lipid peroxidation) (Quintanar-Escorza et al., 2007). In order to
deepen molecular mechanisms knowledge between oxidative
stress and calcium homeostasis; in this work, we proposed to use
erythrocytes isolated from healthy donors incubated with Pb2+ as
in vitro model, to assess association of the mechanisms of oxidative
damage and increase in [Ca2+]i and calcium homeostasis in erythrocytes incubated with lead.
Here, we provide strong evidence indicating that the in vitro
model reproduced the effects found in erythrocytes of lead-exposed workers. Indeed, the incubation of erythrocytes with Pb2+ in-
Experimental
conditions
Lipoperoxidation
(nmol MDA/ml)
GSH/
GSSG
ratio
OS50
(mOSm)
[Ca2+]i
(nM)
Control
AAPH (50 lM)
AAPH (50 lM)+
trolox (5 lM)
0.9 0.5
17.1 0.5
2.3 0.9
7.1 0.9
1.8 0.2*
5.7 0.2*
165 7.0
265 9.0*
182 9.0*
31 1.9
114 4.3*
45 2.2*
1345
can be implicated in the Ca2+ inux mechanism present in erythrocytes incubated with Pb2+.
It is important to note that the lack of effect of trolox on the
reduction of (Ca2+Mg2+)-ATPase activity and [ATP] induced by
lead incubation disagrees with evidence indicating that HNE (40H-2,3-trans-nonenal), product of the lipid peroxidation, decreases
the activity of the (Ca2+Mg2+)-ATPase activity in erythrocytes
membranes exposed to lead (Mas-Oliva, 1989; Campagna et al.,
2000). It is possible that in our assays with complete erythrocytes
the oxidative components did not reach the concentration required
to produce damage and that there was a direct covalent inhibitory
effect of Pb2+ on the enzyme.
Trolox co-incubation reduces oxidative damage and results in
an enhancement of the reductor potential of the erythrocytes.
The fact that trolox showed a partial protection to depletation of
[GSH], GSH/GSSG, lipids and proteins oxidative effects induced
by Pb2+ incubation is related with a lowed [Ca2+]i provide evidence
of oxidative damage may be induced increase of [Ca2+]i basically
due to the Ca2+ inux. Correlation between oxidative damage and
alterations in [Ca2+]i homeostasis, possibly due to modication of
the ionic control have been described in lymphocytes of type-2
diabetes patients (Belia et al., 2009).
Oxidation induced by AAPH, independently of other lead toxicity mechanisms, was been able to reduce GSH/GSSG in erythrocytes and induce increments in lipoperoxidation, osmotic
fragility and [Ca2+]i, similar to lead exposition. Trolox protected
partially against oxidation induced by AAPH and the incubation
did not result in [Ca2+]i increment and osmotic fragility and lipoperoxidation. These results suggest that in erythrocytes incubated
with lead, [Ca2+]i depends of the oxidative status of the cell, as has
been suggested in other models (Huang et al., 2009).
It is possible that pathologies that deal with oxidative damage,
also causes increases of [Ca2+]i, or that those pathologies that deal
with increases of [Ca2+]i are related to oxidative damage. In this regard, it is suggestive that erythrocytes affected by age and diseases
such as sickle anemia, hypertension, diabetes, etc., exhibit an
abnormally high [Ca2+]i (Engelmann, 1991; Lidner et al., 1993; Fujita et al., 1999; Shields et al., 2003). Further research is necessary to
conrm these possibilities.
Acknowledgments
The authors thank Ph.D. Mondragn R. for the erythrocyte image and Rosas M., Camacho H. and Montes M. for technical assistance. This work was partially supported by CONACyT fellowship
86896 Grant 21085-5-28.
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