Oxidative damage increases intracellular free calcium [Ca2+]i concentration in human erythrocytes incubated with lead

Toxicology in Vitro 24 (2010) 1338–1346

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Oxidative damage increases intracellular free calcium [Ca2+]i concentration in human erythrocytes incubated with lead M.A. Quintanar-Escorza b, M.T. González-Martínez c, Intriago-Ortega Ma. del Pilar b, J.V. Calderón-Salinas a,* a

Departamento de Bioquímica, Centro de Investigación y Estudios Avanzados (CINVESTAV-IPN), México City, Mexico Departamento de Bioquímica, Facultad de Medicina, Universidad Juárez del Estado de Durango (UJED), Durango, Dgo., Mexico c Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de México (UNAM), México 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+ influx, 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. Ó 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 (Calderón-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; Rendón-Ramírez et al., 2007) and interaction with stereospecific sites for divalent cations such as calcium ion (Ca2+). This latter mechanism effects different biologically significant 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

* Corresponding author. Address: Department of Biochemistry, CINVESTAV-IPN, P.O. Box 14-740, México City 07000, Mexico. Tel.: +52 (55) 5061 3955; fax: +52 (55) 5061 3391. E-mail address: [email protected] (J.V. Calderón-Salinas). 0887-2333/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2010.05.002

this ion (Calderón-Salinas et al., 1999a,b), consequently Ca2+ influx decreases, a phenomenon that may alter calcium homeostasis (Quintanar-Escorza et al., 2007). The Ca2+ is a highly versatile intracellular signal that can regulate many different cellular functions such as contraction, secretion, metabolism, gene expression cell survival and death (Carafoli et al., 2001; Carafoli, 2002; Saris and Carafoli, 2005). The concentration of intracellular free calcium ([Ca2+]i) is determined by a balance between the ion transport systems that introduce Ca2+ into the cytoplasm and the ones that remove it by the combined action of buffers, pumps and exchangers. Human erythrocytes do not contain organelles, and the [Ca2+]i is controlled by the Ca2+ influx rate and the activity of the plasma membrane (Ca2+–Mg2+)-ATPase pump that extrudes Ca2+ against an electrochemical gradient. The erythrocytes affected by age or diseases such as sickle cell anemia, hypertension and diabetes, exhibit abnormally high [Ca2+]i, and appear to have cytoskeleton alterations (Liu et al., 2005). A rise in [Ca2+]i leads to alterations in cell shape and volume and it is associated with cellular rigidity, hemolysis, senescence and apoptosis (Quintanar-Escorza et al., 2007).

M.A. Quintanar-Escorza et al. / Toxicology in Vitro 24 (2010) 1338–1346

Recent studies suggest that there is a cross-talk between Ca2+ and reactive oxygen species (ROS) in different types of cells (Fonfria et al., 2004; Yan et al., 2006). Interactions between Ca2+ and ROS signaling systems can be both stimulatory and inhibitory, depending on the cell environment, the type of targeted macromolecules, species, doses and time exposition of ROS. In senescent erythrocytes, it has been proposed that the oxidative stress contributes to the increased [Ca2+]i (Aiken et al., 1995). The aldehydic lipid peroxidation product 4-hydroxynonenal (HNE) is known to irreversibly inhibit the plasma membrane (Ca2+–Mg2+)-ATPase (McConell et al., 1999). The influence of ZnO nanoparticles on cytotoxicity, oxidative stress and intracellular calcium homeostasis was studied in human bronchial epithelial cells (BEAS-2B). Exposure to ZnO increased [Ca2+]i in a concentration- and time-dependent manner, this effect was partially attenuated by the antioxidant N-acetylcysteine (Huang et al., 2009). Several studies have focused on the possible toxic effects of lead on membrane components and have identified a correlation between these effects and lead-induced oxidative damage. Many authors propose that the formation of free radicals is the most important molecular mechanism of the lead toxicity (Gurer et al., 1998; Rendón-Ramírez et al., 2007). Exposure of rats to lead caused a significant increase in reactive oxygen species, nitric oxide, and [Ca2+]i levels along with altered behavioral abnormalities in locomotor activity, exploratory behavior, learning, and memory that were supported by changes in neurotransmitter levels (Pachauri et al., 2009). In erythrocytes isolated from lead-exposed workers there is a significant increase in intracellular free calcium [Ca2+]i, caused by an enhanced Ca2+ entry mechanism and by (Ca2+– Mg2+)-ATPase inhibition, which is accompanied by lipid peroxidation increase and changes on the cell shape and fragility (Quintanar-Escorza et al., 2007). 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. In the present work, the alterations observed in erythrocytes of lead-exposed workers were reproduced in erythrocytes incubated at different times and concentrations of lead (in vitro): increase in Ca2+ influx, decrease in (Ca2+–Mg2+)-ATPase activity and GSH/ GSGG ratio; increase in lipoperoxidation, protein carbonylation and osmotic fragility accompanied of dramatic morphological changes. This model was used to study if the alterations in Ca2+ homeostasis, cell shape and fragility of lead-exposed erythrocytes were related to oxidative process. Trolox as antioxidant prevents oxidative damage and alteration in Ca2+ homeostasis in erythrocytes incubated with lead. The results obtained suggest that in erythrocytes incubated with lead the increment of [Ca2+]i depends on the oxidative status. Oxidation induced by AAPH (free radicals generator) showed alterations in [Ca2+]i, similar to that observed in erythrocytes incubated with lead; additionally, trolox prevented the oxidative effects induced by AAPH in erythrocytes, the results shown that lead damage mechanism is strongly associated with oxidative damage and an increase in [Ca2+]i as effectors.

2. Materials and methods Fluo-3-AM was obtained from Molecular Probes Inc., 45Ca2+ was obtained from Amersham Co. and the others reagents were analytic grade obtained from Sigma.

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2.1. Subjects The erythrocytes were obtained from ten healthy male volunteers (27–32 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 findings 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-Bajío, IMSS-México. 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 final concentrations Pb2+ were 0.2, 0.4, 2.0 ,4.0 and 6.0 lM and cells were incubated for 0.5–120 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 Briefly, 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+ (final concentration), containing 45Ca (18.5 lCi/ml) and 10 lmol/l LaCl3. Lantane was added to prevent 45Ca unspecific 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 (Calderón-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

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membrane suspension. The protein concentration per unit cell was (4.85 ± 0.38  106 cells/ml = 1 mg of protein) and was similar in all samples (Quintanar-Escorza et al., 2007). 2.6. ATP measurements The ATP concentration was determinate by enzymatic method using an ATP assay Kit Calbiochem. The assay is based on the firefly luciferase-catalyzed oxidation of D-luciferin in the presence of ATP and oxygen, whereby the amount of ATP is quantified by the amount of light (hv) produced. The light can be measured using a luminometer. 2.7. Evaluation of [Ca2+]i Packed erythrocytes (10 ll) were suspended in 10 ml of isotonic buffer (Snitsarev et al., 1996). Erythrocytes were loaded with 1 lmol/l fluo3-AM in the dark, for 45 min at 37 °C. The loaded cells were centrifuged in a conical 15 ml centrifuge tube for 10 min at 500g. The final concentration of packed erythrocytes was 1% in isotonic solution. For fluorescence measurement, 100 ll of cell suspension was added to 2.5 ml of isotonic solution in fluorescence cell kept at 37 °C and under constant magnetic stirring. The [Ca2+]i was measured (within 4 h of blood drawing) by fluorescence using a PTI spectrofluorometer (Photon Technology International, INC). The sample was excited at 500 nm and the emission detected with a cut off filter >515 nm. Calibrations were performed as previously described (Quintanar-Escorza et al., 2007).

2.11. Osmotic fragility erythrocytes The osmotic fragility was measured as the percentage of hemolysis induced by different hypotonic solutions. (OS50) is the osmolarity that produces 50% of hemolysis and was calculated as previously described (Quintanar-Escorza et al., 2007). Hemolysis was estimated by measuring the absorption at 540 nm of hemoglobin released, using a US/Vis DU 650 (Beckman) spectrophotometer. The hemolysis curves are shown in standard format, plotting the percentage of hemolysis as a function of relative tonicity. 2.12. Preparation of erythrocytes for microscopy Erythrocytes were observed in fresh preparations with isotonic solution as previously described in Quintanar-Escorza et al. (2007). The erythrocyte image was obtained by phase contrast microscopy and differential interference contrast (Nomarsky), in a microscopy Axioscope II Mot with digital camera II RC Axiocam and software Axiovision 3.1 (Carl Zeiis). 2.13. Erythrocytes oxidation induced with AAPH incubation Erythrocytes (100 ll) were resuspended in buffer A (0.9 ml). The oxidative damage was induced in erythrocytes by incubation with 150 lM APPH (2,20 -azobis (2-methylpropionamidine) dihydrochloride) for 30 min, at 37 °C, in the dark. In some experiments, the erythrocytes were preincubated with 5 lM trolox for 30 min, at 4 °C before APPH incubation and continued during APPH incubation.

2.8. Lipid peroxidation measurements 2.14. Statistical significance The amount of lipid peroxidation of the 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 using a spectrophotometer US/ VIS DU 650 (Beckman). The TBARS are expressed as nmol of malondialdehyde equivalents per ml of erythrocyte (Jain et al., 1989). 2.9. Protein oxidation measurements The protein carbonyl groups were determined by immunoblotting using a kit (OxyBlotTM; Intergen, Purchase, NY, USA). In brief, red blood cell membrane protein was solubilized with SDS and the protein carbonyl group was labeled with dinitrophenylhydrazone (DNP). The protein samples were then subjected to SDS–polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. After blocking with 5% skim milk, the membrane was incubated with rabbit anti-DNP antibody. After extensive washing with Tris-buffered saline (pH 7.4) with 0.005% Tween-20, the membrane was incubated with horseradish peroxidase conjugated goat anti-rabbit IgG. Immunoreactive proteins were visualized with a chemiluminescence detection system, followed by exposure to Kodak X-ray film. Densitometric analysis was performed using the computational program as reported in Quintanar-Escorza et al. (2007). 2.10. GSH/GSSG measurements The GSH/GSSG ratio was determined by enzymatic method using a GSH/GSSG Ratio Assay kit, Calbiochem. The method is based on the change in color (412 nm) during the reduction of 5,50 -dithiobis-2-nitrobenzoic acid (DTNB) by GSH. GSSG was reduced to GSH by NADPH-glutathione reductase and the amount of GSH was then determined. The calculation of the GSH/GSSG ratio consider stereochemistry of ratio = GSHt  2GSSG/GSSG.

The difference between means was compared using Student’s ttest, statistical SPSS-w version 14. Differences were considered significant 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 (12–120 h) manner (Fig. 1B). In erythrocytes incubated with high lead concentrations (4.0–6.0 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 modifications 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+ influx 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+

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A

160 120 h

120 100 80

24 h

60 40 20

A *

120 100 *

80 *

60 40 20

0 0

1

2

3

4

5

6

Lead concentration (µM)

0 0

B

*

140

Incorporated calcium (µmol/l)

Intracellular free calcium (nM)

140

24

24

0.4 µM Pb 2+

4 µM

120 100

0.4 µM

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 fluo 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 Student’s t-test (n = 10) between lead concentration (A) and exposition times (B).

extruding mechanism, we evaluated the effect of lead incubation on the erythrocyte (Ca2+–Mg2+)-ATPase activity (Fig. 2B). In erythrocytes incubated with 0.4 lM [Pb2+] for 24 and 120 h, the activity was 33% and 83% lower than normal respectively (P < 0.01). In erythrocytes incubated with 4.0 lM [Pb2+] for 24 and 120 h, the activity was of 55% and 95% lower as compared with control respectively (P < 0.01). In erythrocytes incubated with 4.0 lM [Pb2+] for 24 h, [ATP] (4.6 ± 0.2 lmol of ATP/mg of protein) was reduced 8% with respect to control (5.0 ± 0.1 lmol of ATP/mg of protein). Incubation with 4.0 lM [Pb2+] for 120 h, reduced [ATP] (4.4 ± 0.3 lmol of ATP/mg of protein) 12% with respect to control (5.0 ± 0.1 lmol of ATP/mg of protein). Biomarkers of oxidative stress in erythrocytes were measured. The oxidative damage to lipids and proteins appeared to be strongly modified by lead incubation. In erythrocytes incubated with 0.4 lM [Pb2+] for 24 and 120 h, lipoperoxidation was 2.5 and 9-fold higher than control (P < 0.01) (Fig. 4). Lipoperoxidation did not increase in control erythrocytes at the different times of incubation. Likewise, erythrocytes incubated with 4.0 lM [Pb2+] for 24 h showed increased lipid peroxidation 9-fold as compared with control, whereas lipoperoxidation in erythrocytes incubated for 120 h was 15-fold higher (Tables 2A and 2B). The densitometric analysis showed that protein carbonylation (molecular weight between 20 and 100 kDa) in erythrocytes incubated with 0.4 lM [Pb2+] for 24 and 120 h the protein carbonyl-

ATPase (Ca2+ /Mg 2+) activity (nmolPi/mg/min)

140

Intracellular free calcium (nM)

120

50

120 4µM Pb 2+

B

40

30

*

*

20

*

10

*

0 0

24

120

0.4 µM Pb2+

24

120

4µM Pb 2+

Fig. 2. Effects of incubation with lead on erythrocytes calcium transport. (A) Calcium uptake was measured employing 45Ca2+. The erythrocytes were incubated 24 and 120 h before the assay with 0.4 lM Pb2+, 4.0 lM Pb2+ or without lead (control). (B) (Ca2+–Mg2+)-ATPase activity, measured as inorganic phosphate release. The erythrocytes were incubated 24, 120 h before the assay with 0.4 lM Pb2+, 4.0 lM Pb2+ and without lead (control). Results are presented as mean ± standard deviation. *P < 0.01 Student’s t-test (n = 10).

ation was 2-fold and 2.6-fold higher as compared with control (P < 0.01) (Table 2A). In erythrocytes incubated for 24 and 120 h with 4.0 lM [Pb2+], protein carbonylation increased 3.2-fold and 4.7-fold higher, respectively (Table 2B). Glutathione is a powerful intracellular antioxidant, and GSH/ GSSG is a representative marker of the antioxidant capacity of the cells. Erythrocytes incubated with 0.4 lM [Pb2+] for 24 h did not show any significant changes in [GSH] and [GSSG]. The GSH/ GSSG ratio was 32% lower than control (Table 2A). In contrast, in erythrocytes incubated for 120 h, the [GSH] was 22% lower, the [GSSG] was 1.5-fold higher and the GSH/GSSG ratio was 70% lower than control (P < 0.01) (Table 2A). Likewise, in erythrocytes incubated with 4.0 lM [Pb2+] for 24 and 120 h, the [GSH] level was 21% and 57% lower. The [GSSG] was 1.5 and 4-fold higher and the GSH/GSSG ratio was of 69% and 92% lower than control (P < 0.01) (Table 2B). Effects of lead incubation on erythrocyte integrity parameters, such as fragility and morphology were also studied. The fragility was measured as the percentage of hemolysis produced by different hypotonic solutions. Fig. 3A shows that 0.4 lM [Pb2+] for 24 h

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A

100

Percent hemolysis

80

60

40

Control 24 h

20

120 h

0 0

100

200

300

Osmolarity (mOsm)

Percent hemolysis

B

100

80

60 Control

40 24 h 120 h

20

0 0

100

200

300

Osmolarity (mOsm)

Lipid peroxidation (nmol MDA/ ml erythrocytes)

Fig. 3. Effect of incubation with lead on patterns of the hemolysis curves erythrocytes. Erythrocytes were incubated without lead (control) 120 h (d) or 24 h (N) and 120 h (j) with lead 0.4 lM (panel A) or lead 4.0 lM (panel B). Results are presented as mean ± standard deviation. *P < 0.01 Student’s t-test (n = 10) between exposition times.

15 *

12 * *

9

6 *

3

0

0

24

0.4 µM

120

Pb 2+

24

120

4µM Pb 2+

Fig. 4. Effect of incubation with lead on erythrocytes lipoperoxidation. The amount of lipid peroxidation of the erythrocytes was estimated by measuring the thiobarbituric acid-reactive substances (TBARS). The erythrocytes were incubated 24 and 120 h without lead (control) or with 0.4 lM Pb2+ and 4.0 lM Pb2+. Results are presented as mean ± standard deviation. *P < 0.01 Student’s t-test (n = 10).

induced hemolysis as shown by the shift to the right of the curve of osmotic fragility. For erythrocytes incubated with 4.0 lM [Pb2+] for 24 and 120 h, the shift to the right was even larger (Fig. 3B). The osmolarity that produces 50% of hemolysis (OS50) found in erythrocytes incubated with 0.4 and 4.0 lM [Pb2+] for 24 and 120 h increased compared to control, indicating the expected increase in erythrocyte fragility in erythrocytes incubated with lead (Tables 2A and 2B). Thus, lead incubation increased the percentage of hemolysis both in dose and time-dependent manner. In erythrocytes incubated with 0.4 lM [Pb2+] for 24 h OS50 was similar and at 120 h was 0.2-fold higher as compared with control (P < 0.01) (Table 2A). Incubation for 24 h to 4.0 lM [Pb2+] increased OS50 0.2-fold and for 120 h the OS50 was 0.4-fold higher as compared with control. The increase of oxidative damage was accompanied by dramatic morphological alterations, cells without normal biconcave shape, spiculated erythrocytes (echinocytes) were found in a considerable extent in erythrocytes incubated with lead (Fig. 5). Peroxidative damage induced in erythrocytes is encountered by elaborate defense mechanisms including enzymatic and non-enzymatic antioxidants (Rendón-Ramírez et al., 2007). The effect of trolox, a soluble analog of vitamin-E (non-enzymatic antioxidant) was also studied to determine the contribution of oxidative damage on the altered calcium homeostasis and on the cellular morphology induced by lead exposure. Co-incubation with trolox reduced the alterations in calcium transport induced by lead incubation in erythrocytes; this effect depended on time and concentration of incubation with lead. Co-incubation with 5.0 lM trolox prevented the increment [Ca2+]i in erythrocytes incubated with 0.4 lM [Pb2+] or 4.0 lM [Pb2+] for 24 or 120 h (Tables 1A and 1B). The decrement of (Ca2+–Mg2+)-ATPase activity induced by lead was totality prevented by 5.0 lM trolox in erythrocytes incubated with 0.4 lM [Pb2+] for 24 h and only partiality in erythrocytes incubated for 120 h (Table 1A). The trolox co-incubation does not prevent the reduction of (Ca2+–Mg2+)-ATPase activity in erythrocytes incubated with 4.0 lM [Pb2+] for 24 h or 120 h (Table 1B). While co-incubation with 5.0 lM trolox totally prevented the increment of incorporated calcium in erythrocytes incubated with 0.4 lM [Pb2+] or 4.0 lM [Pb2+] for 24 and 120 h (Tables 1A and 1B). Trolox co-incubation totally prevented the lipoperoxidation increase in erythrocytes incubated with 0.4 lM [Pb2+] or 4.0 lM [Pb2+] for 24 and 120 h, (Tables 2A and 2B). The increment in protein carbonylation in erythrocytes incubated with 0.4 lM [Pb2+] for 24 and 120 h was only partially prevented by trolox co-incubation (0.7-fold vs. 2.0-fold and 1.2-fold vs. 2.6-fold higher as compared with control, respectively) (Table 2A). The co-incubation with 4.0 lM [Pb2+] and 5.0 lM trolox for 24 and 120 h showed a reduction in the increment of protein carbonylation (1.8-fold vs. 3.2 and 2.2 vs. 4.7, respectively) (Table 2B). Co-incubation with 5.0 lM trolox totally prevented alterations induced by lead incubation with 0.4 lM [Pb2+] for 24 and 120 h in [GSH], [GSSG] and GSH/GSSG ratio (Tables 2A). The trolox coincubation with 4.0 lM [Pb2+] for 24 and 120 h partially prevented the reduction in [GSH] (10% vs. 21% and 7% vs. 57%, respectively), prevented the increment of [GSSG] (0.6-fold vs. 1.5-fold and 1.5fold vs. 4.1-fold, respectively) and partially prevented the reduction in GSH/GSSG ratio (49% vs. 69% and 66% vs. 92%, respectively) (Table 2B). Trolox co-incubation totally prevents increments of OS50 in erythrocytes incubated with 0.4 lM [Pb2+] and for 24 or 120 h. Whereas, trolox co-incubation prevent only partially (50%) the OS50 increment in erythrocytes incubated with 4.0 lM [Pb2+] and trolox for 24 or 120 h. (Table 2B).

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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

Lead (0.4 lM)

Control

Trolox (5 lM)

Incubation time (h)

[Ca2+]i (nM) ATPase (Ca2+/Mg2+) activity (nmol Pi/mg/min) Incorporated calcium (lmol/l) *

Lead (0.4 lM) + trolox (5 lM)

Incubation time (h)

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

P < 0.01 Student’s t-test (n = 10).

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)

Incubation time (h) 120 2+

[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

Lead (4 lM) + trolox 5 lM

Incubation time (h)

*

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

P < 0.01 Student’s t-test (n = 10).

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

Lead (0.4 lM)

Trolox (5 lM)

Incubation time (h)

Lipoperoxidation (nmol MDA/ml) Protein carbonylation (relative units) [GSH] (lM) [GSSG] (lM) GSH/GSSG ratio OS50 (mOsm) *

Lead (0.4 lM) + trolox (5 lM)

Incubation time (h)

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

P < 0.01 Student’s t-test (n = 10).

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)

Incubation time (h)

Lipoperoxidation (nmol MDA/ml) Protein carbonylation (relative units) [GSH] (lM) [GSSG] (lM) GSH/GSSG ratio OS50 (mOSm) *

Lead (4 lM) + trolox (5 lM)

Incubation time (h)

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*

P < 0.01 Student’s t-test (n = 10).

Additionally, trolox co-incubation prevents morphological alterations induced in erythrocytes incubated with lead at 0.4 lM [Pb2+] and 4.0 lM [Pb2+] for 24 and 120 h (Fig. 6). On the other hand, trolox co-incubation was not able to prevent the reduction of [ATP] in erythrocytes incubated with 4.0 lM [Pb2+] for 24 h (4.5 ± 0.2 vs. 4.6 ± 0.2 lmol of ATP/mg of protein,

respectively) or 120 h (4.3 ± 0.3 vs. 4.4 ± 0.3 lmol of ATP/mg of protein, respectively). Oxidation induced with AAPH (free radical generator) showed similar alterations to that observed in erythrocytes incubated with lead. Erythrocytes incubated 30 min with AAPH increase lipoperoxidation (18-fold), OS50 (0.6-fold), [Ca2+]i (2.7-fold) and reduce

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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.

the GSH/GSSG ratio (25% respect to control). Co-incubation with 5.0 lM trolox partially prevented the alterations induced by APPH incubation; lipoperoxidation increased only 1.5-fold, osmotic fragility 0.1-fold, [Ca2+]i 0.5-fold and GSH/GSSG ratio was 20% lower with respect to the control (P < 0.01) (Table 3).

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-

M.A. Quintanar-Escorza et al. / Toxicology in Vitro 24 (2010) 1338–1346 Table 3 Effects of trolox co-incubation (5 lM) on lipid oxidation, GSH/GSSG ratio, osmotic fragility and [Ca2+]i of erythrocytes incubated with APPH (50 lM). Results are presented as mean ± standard deviation.

*

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*

P < 0.01 Student’s t-test (n = 10).

duced an increase in [Ca2+]i, osmotic fragility and morphological cellular alterations, which were dose- and time-dependent to lead concentration and associated with oxidative damage. It is important to note that the effects of incubation with lead on erythrocytes shown in the in vitro model were similar to those found in erythrocytes of lead-exposed workers; therefore this model may be appropriate to study the molecular basis of lead toxicity of damage present in chronic lead-intoxicated subjects. The erythrocytes incubation with Pb2+ increased significantly [Ca2+]i in a dose- and time-dependent manner. The enhanced [Ca2+]i induced by lead incubation was related to an increase in Ca2+ influx and a decrease in (Ca2+–Mg2+)-ATPase activity (efflux). It should be noted that erythrocytes have not internal stores that might uptake Ca2+, thus both influx and efflux mechanisms are entirely related to these Ca2+ transports. The intracellular signal transduction pathways regulated by reactive ROS remain to be established. Deleterious effects of ROS stem from interactions with various ion transport proteins such as ion channels and pumps, primarily altering Ca2+ homeostasis and inducing cell dysfunction (Belia et al., 2009). One of the most significant phenomena observed during lead poisoning has been attributed to lead-induced oxidative stress, studies in humans and animal models (in vitro and in vivo) suggest that lead-induced oxidative damage contributes to erythrocytes damage in lead exposition. In this regard, the morphological, mechanic and osmotic fragilities induced by lead intoxication in rat erythrocytes were lower in rats treated with vitamin-E (Quintanar-Escorza et al., 2007; Gurer-Ohrnan et al., 2004; Rendón-Ramírez et al., 2007; Ahamed and Siddiqui, 2007). Trolox is a soluble analog to vitamin-E and is one of the most frequently antioxidant used in vitro. Like vitamin-E, trolox is a powerful scavenger of superoxide anions, hydroxyl radical, hydroperoxyl radical and hydrogen peroxide. In order to evaluate the oxidative damage induced in erythrocytes incubated with lead, the protective effect of trolox was evaluated in erythrocytes incubated with AAPH, a water-soluble azo compound which is used extensively as a free radical generator in the study of lipid peroxidation and in the characterization of antioxidants. The fact that trolox prevented the Ca2+ uptake mechanisms induced by high [Pb2+] and a long time incubation, but did not prevent calcium extruding mechanism ((Ca2+–Mg2+)-ATPase activity) indicated that the oxidative damage is rather related to Ca2+ influx mechanisms, and not to efflux processes. Given these effects, it is reasonable to assume that the increase in [Ca2+]i observed in lead-exposed erythrocytes is related to the activation of Ca2+ influx mechanism via lead-induced production of ROS. In this regard, many studies have focused on ROS modulation of VDCC activity. H2O2 has been shown to accelerate the overall channel opening process in neurons, including P/Q type, L type and TRPM2 can be modulated by ROS (Fonfria et al., 2004; Yan et al., 2006). Thus, even though the calcium channels present in erythrocytes have not been identified, here we show evidence suggesting that the redox state

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can be implicated in the Ca2+ influx 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+ influx. Correlation between oxidative damage and alterations in [Ca2+]i homeostasis, possibly due to modification 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 confirm these possibilities. Acknowledgments The authors thank Ph.D. Mondragón 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. References Ahamed, M., Siddiqui, M.K., 2007. Low level lead exposure and oxidative stress: current opinions. Clin. Chim. Acta 383 (1–2), 57–64. Aiken, N.R., Galey, W.R., Satterlee, J.D., 1995. A peroxidative model of human erythrocyte intracellular Ca2+ changes with in vivo cell aging: measurement by 19F-NMR spectroscopy. Biochim. Biophys. Acta 1270 (1), 52–57. Belia, S., Santilli, F., Beccafico, S., De Feudis, L., Morabito, C., Davi, G., Fanò, G., Mariggiò, M.A., 2009. Oxidative-induced membrane damage in diabetes lymphocytes: effects on intracellular Ca(2+) homeostasis. Free Radical Res. 43 (2), 138–148. Calderón-Salinas, J.V., Valdez-Anaya, B., Mazúñiga-Charles, Albores-Medina, A., 1996. Lead exposure in a population of Mexican children. Hum. Exp. Toxicol. 15 (4), 305–311. Calderón-Salinas, J.V., Quintanar-Escorza, M.A., Hernández-Luna, C.E., GonzálezMartínez, M.T., 1999a. Effect of lead on the calcium transport in human erythrocyte. Hum. Exp. Toxicol. 18 (3), 146–153. Calderón-Salinas, J.V., Quintanar-Escorza, M.A., González-Martínez, M.T., Hernández-Luna, C.E., 1999b. Lead and calcium transport in human erythrocyte. Hum. Exp. Toxicol. 18 (5), 327–332. Campagna, D., Huel, G., Hellier, G., Girard, F., Sahuquillo, J., Fagot-Campagna, A., Godin, J., Blot, P., 2000. Negative relationships between erythrocyte Ca-pump activity and lead levels in mothers and newborns. Life Sci. 68 (2), 203–215.

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