The effect of vanadium on platelet function

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 2 ( 2 0 1 1 ) 447–456

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The effect of vanadium on platelet function a,1 ˜ ˜ Díaz b,c,2 , Adriana González-Villalva a,1 , Gabriela Pinón-Zárate , Aurora De la Pena Mirthala Flores-García c,3 , Patricia Bizarro-Nevares a,1 , Erika P. Rendón-Huerta a,4 , Laura Colín-Barenque d,5 , Teresa Imelda Fortoul a,∗ a

Departamento de Biología Celular y Tisular, Facultad de Medicina, Universidad Nacional Autonoma de Mexico (UNAM), CP 04510, Mexico City, Mexico b Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autonoma de Mexico (UNAM), CP 04510, Mexico City, Mexico c Departamento de Biología Molecular, Instituto Nacional de Cardiología “Dr. Ignacio Chavez”, Mexico City, Mexico d Laboratorio de Neuromorfología, FES Iztacala, UNAM CP 54090 Edo. de México, CP 54090, Mexico

a r t i c l e

i n f o

a b s t r a c t

Article history:

Vanadium pentoxide (V2 O5 ) inhalation effect on platelet function in mice was explored,

Received 26 January 2011

as well as the in vitro effect on human platelets. Mouse blood samples were collected

Received in revised form

and processed for aggregometry and flow cytometry to assess the presence of P-selectin

4 August 2011

and monocyte-platelet conjugates. Simultaneously, human platelets were processed for

Accepted 23 August 2011

aggregometry. The mouse results showed platelet aggregation inhibition in platelet-rich-

Available online 30 August 2011

plasma (PRP) at four-week exposure time, and normality returned at eight weeks of exposure, remaining unchanged after the exposure was discontinued after four weeks. This platelet

Keywords:

aggregation inhibition effect was reinforced with the in vitro assay. In addition, P-selectin

Platelet aggregation inhibition

preserved their values during the exposure, until the exposure was discontinued during four

P-selectin

weeks, when this activation marker increased. We conclude that vanadium affects platelet

Vanadium

function, but further studies are required to evaluate its effect on other components of the

Vanadium pentoxide

hemostatic system.

Inhalation

© 2011 Elsevier B.V. All rights reserved.

Abbreviations: ACGIH, American Conference of Governmental Industrial Hygienists; cAMP, cyclic adenosine monophosphate; CD 14, marker for monocytes; CD 41, glycoprotein IIb/IIIa; CD 42, glycoprotein Ib; CD 62p, P-selectin; PPP, platelet poor plasma; PRP, platelet rich plasma; V, vanadium; V2 O5 , vanadium pentoxide. ∗ Corresponding author. Tel.: +52 55 56232182; fax: +52 55 56232399. ˜ E-mail addresses: [email protected] (A. González-Villalva), [email protected] (G. Pinón-Zárate), ˜ Díaz), mirthala fl[email protected] (M. Flores-García), [email protected] (P. [email protected] (A. De la Pena Nevares), [email protected] (E.P. Rendón-Huerta), [email protected] (L. Colín-Barenque), [email protected] (T.I. Fortoul). 1 Tel.: +52 55 56232182; fax: +52 55 56232399. 2 Tel.: +52 55 55232164; fax: +52 55 55730994. 3 Tel.: +52 55 42527370. 4 Tel.: +52 55 56232191. 5 Tel.: +52 55 56232183. 1382-6689/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2011.08.010

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1.

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 2 ( 2 0 1 1 ) 447–456

Introduction

Vanadium (V) is a transition metal and as a pollutant, it has become very important because of the large amounts released to the air from anthropogenic sources as burning of fossil fuels. Venezuelan and Mexican oil has been reported to contain high concentrations of this metal. During the burning of fuel oils, the majority of V released into the atmosphere is as vanadium pentoxide (V2 O5 ), one of the most toxic forms of the metal (Fortoul and Rojas-Lemus, 2007; Rodríguez-Mercado and Altamirano-Lozano, 2006; Nriagu, 1998). Vanadium atmospheric concentrations at rural or urban places ranges from 6 to 1320 ng/m3 (Goc, 2006; WHO, 1998), Occupational exposures may be significant during the cleaning of oil-fired boilers and furnaces, the handling of catalysts, and in the processing of vanadium rich ores. Airborne concentrations of V2 O5 range from 0.7 to 11.7 mg/m3 during smelting and granulation of V2 O5 and as high as 25.1 mg/m3 during the exothermic reaction of V2 O5 with aluminum to make ductile vanadium (WHO, 1998; Ress et al., 2003). Some environmental disasters such the Kuwait oil fires in 1991 may have a contribution in elevating environmental vanadium concentrations; indeed, was the highest measured atmospheric concentration registered in PM10 s was recorded as 1165.8 ng/m3 in Saudi Arabia (Sadiq and Mian, 1994). Vanadium is inhaled through air suspended particles, then it is absorbed and distributed mainly to the bone, liver, kidney and spleen and is excreted via the urine. It has been identified as a source of some occupational health effects such as bronchial asthma and bronchitis named “boilermakers’ bronchitis” (Ress et al., 2003; WHO, 1998). In acute high occupational exposure, patients have a characteristic green tongue (Cooper, 2007; Fortoul and Rojas-Lemus, 2007; Rodríguez-Mercado and Altamirano-Lozano, 2006; Barceloux, 1999; Nriagu, 1998). In experimental models of vanadium toxicity, including our own, hematotoxicity, genotoxicity, neurotoxicity, immunotoxicity and reproductive toxicity have been reported (González-Villalva et al., 2009; Afeseh Ngwa et al., 2009; Colin-Barenque et al., 2008; Avila-Costa et al., 2004, 2005, 2006; Aragón et al., 2005; IPCS, 2000; Altamirano-Lozano et al., 1999, 1993). Vanadium is found in many oxidation states (−1 to +5) and it produces free radicals inducing lipid peroxidation in vitro and in vivo (Scibior et al., 2010; Byczkowski and Kulkarni, 1998; Thompson and McNeill, 1993; Younes and Strubelt, 1991; Younes et al., 1991). Also, V compounds are studied as potential anti-diabetic agents because of their insulin mimetic properties, where they inhibit protein tyrosine phosphatases; there are also many reports suggesting that vanadium effects may diverge from stimulation to cell proliferation or the induction of cell death (Morinville et al., 1998; Mukherjee et al., 2004). In recent years, V has been studied and evaluated for its potential as a carcinogenic and mutagenic element (Altamirano-Lozano et al., 1999; Rojas et al., 1996; Roldan and Altamirano-Lozano, 1990; Rodríguez Mercado et al., 2003; National Toxicology Program, 2002; Assem and Levy, 2009), but also there are vanadium compounds studied as antineoplastic drugs (Bishayee et al., 2010; Faneca et al., 2009; Papaioannou et al., 2004; Evangelou, 2002).

Air pollutants, specially suspended particulate matter, have been related mainly to thromboembolic events (Mills et al., 2009; Kettunen et al., 2007; Nemmar et al., 2003), but also to hemorrhagic disorders (Yamazaki et al., 2007; Villeneuve et al., 2006; Tsai et al., 2003; Halinen et al., 1999). Our group reported thrombocytosis (an increase in platelet number) related to vanadium inhalation, as well as an increase in platelet precursor cells, megakaryocytes. For this reason, it is logical to explore in our model if vanadium can also affect platelet function (González-Villalva et al., 2006; Fortoul et al., 2008, 2009). Platelets are the smallest blood formed structures, which have a critical role in normal homeostasis. When platelets are activated, morphology changes from normal disc-shape to a compact sphere, with large cytoplasmic extensions protruding to facilitate adhesion whilst new glycoproteins are expressed in the external membrane. Platelet-membrane glycoprotein receptors such as CD 41 (IIb/IIIa) or CD 42 (Ib) mediate adhesion to sub-endothelial tissue and subsequent aggregation to form the initial hemostatic plug. P-selectin (CD62p), a glycoprotein located in alfa granules, is translocated to the superficial membrane to facilitate leukocytes’ adhesion (Kaushansky, 2005; Andrews and Berndt, 2004; George, 2000). Measuring platelet-leukocyte conjugates and P-selectin, combined with aggregometry, evaluates platelet function (Tomer, 2004; Villmow et al., 2002). The measure of platelet aggregation is important to assess bleeding problems or thrombosis risk. In fact, there is special interest in assessing platelet function as a marker of hemostatic disorders associated with suspended air pollution or occupational particles toxicity.

2.

Method

2.1.

Mice

Eight-week-old CD-1 male mice weighing 33 ± 35 g were housed in hanging plastic cages kept in an animal facility (with average 21 ◦ C temperature, 57% humidity, controlled lighting 12:12 h light/dark regimen) and fed with PMI rodent laboratory chow and water ad libitum. The experimental protocol was in accordance with the Guide for the care and use of laboratory animals from Institute of Laboratory Animal Resources Commission on Life Sciences National Research Council (2010).

2.2.

Exposure regimens

Inhalation exposures were performed as described by Fortoul et al. (2008). Briefly, exposures were performed with a 0.02 M V2 O5 (99.99% purity, Sigma–Aldrich, St. Louis, MO) suspension. The aerosol inhalation chamber was an acrylic box measuring 45 cm × 21 cm × 35 cm that had a total volume of 3.3 L. A DeVilbiss Ultraneb 99 (Somerset, PA) system was used to nebulize the vanadium solution maintaining a constant flow of 10 L/min; according to the manufacturer, about 80% of the aerosolized particles reaching the mice would be expected

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to have a mass median aerodynamic diameter (MMAD) of 0.5–5 ␮m. Concentrations of V2 O5 in the chamber were quantified as follows: a filter was positioned at the outlet of the ultranebulizer during the whole inhalation time at a flow rate or 10 L/min. After each exposure, the filters were removed and weighed; the element was quantified following the same protocol as with tissue samples. Six filters for each inhalation were evaluated (Fortoul et al., 2002, 2008, 2009). Samples of blood were obtained from mice abdominal aorta and samples of bone marrow were obtained from mice femur and then, were analyzed with a graphite furnace Atomic Absorption Spectrometer (PerkinElmer Mod. 2380). The light source came from a hollow cathode lamp. Accuracy was assured by three random determinations of seven different standard solutions, prepared with the same chemical reactivities used during the metal analysis. For V2 O5 , the wavelength was 318.4 nm; the detection limit was 0.37 ppm and the slit 0.7 nm. Each sample was analyzed in triplicate. In these studies, mice were placed in acrylic box to inhale V2 O5 for 1 h/day, twice a week, for eight weeks. A group of mice were analyzed four weeks after eight weeks of V2 O5 exposure. There were a total of thirty mice in the vanadium exposure group and thirty in the control, which inhaled only the vehiclesaline water. Ten control and ten exposed mice were sacrificed at the four and eight week of inhalation, and at four weeks after cessation of V2 O5 exposure.

2.3. Flow cytometric analysis of platelet-activation markers and platelet–monocyte conjugates According to Villmow et al. (2002), mice blood samples of abdominal aorta were drawn into tubes containing one-tenth volume 3.8% trisodium citrate. The blood was diluted 1:10 with phosphate buffer containing glycine (0.2%, w/v) to quench free aldehyde groups. For detection of platelet-activation markers, diluted whole blood was incubated with phycoerythrin (PE)-labeled anti-CD41 (anti-GPIIb, Beckton and Dickinson) fluorescein isothyocyanate (FITC)-labeled anti-CD14 (Beckton and Dickinson) and biotinylated anti CD62p (Beckton and Dickinson). After 30-min incubation in the dark and at room temperature, the sample was diluted with phosphate buffer, then incubated with Allophycocyanin APC streptavidin to antiCD62p and fixed with paraformaldehyde 0.5%. Prepared samples were analyzed on a FACScalibur, Beckton and Dickinson, at Instituto de Investigaciones Biomédicas, UNAM. Instrument stability was monitored by daily measurement of calibrations beads (Calibrite 3 Beads, Becton Dickinson). Side scatter, forward scatter, and fluorescence signals were measured using the instrument’s logarithmic amplification mode. Data were acquired applying a forward scatter threshold excluding all events smaller than platelets. A minimum of 10,000 platelets was collected per sample. Platelets were defined based on their size and presence of CD41, gated into a FL-4 (APC-channel) histogram, where P-selectin (CD62p) expression on the platelet surface was measured. The percentage of CD62p PE-positive events was determined by setting a threshold according to the isotype control. For visualization of monocyte-platelets conjugates

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monocytes were defined based in their size and presence of CD14 expression, gated into a FL2 (PE-channel) histogram, where CD41 on the platelet surface was measured.

2.4.

Mouse PRP platelet aggregation

Mice blood samples were obtained from abdominal aorta and anticoagulated with sodium citrate, then were centrifuged at 140 × g for 3 min at room temperature (20–24 ◦ C) to obtain the platelet-rich plasmas (PRP), after this step the plasma was withdrawn and poured into another tube. Platelet-poor plasma (PPP) was obtained in a second centrifugation at 400 × g for 15 min. A 250 ␮L of PRP was used for each assay (platelet count was adjusted to 2.5 × 103 /mL) and was incubated for 3 min in an aggregometer (Model 560 CA and accompanying software Model 810 AGGRO/LINK Chronolog, Havertown, PA, USA). The aggregation was induced with 2.5 ␮M ADP (Sigma, Co.) threshold value. The aggregometer operation is based on the amount of light passing through the sample and, therefore, the PPP is used for reference only.

2.5.

Mouse whole blood platelet aggregation

Blood samples from mice were obtained from abdominal aorta and anticoagulated with one-tenth 3.8% sodium citrate. Samples were analyzed using a method that measures electrical impedance (maximal amplitude) between two electrodes immersed in whole blood 5 min after the addition of a platelet agonist (ADP 5 ␮M), using a ChronoLog Aggregometer (ChronoLog 560 model, Havertown, PA, USA). The results are reported as residual platelet aggregation, measured as maximal amplitude of impedance (ohms).

2.6.

Human PRP platelet aggregation assay

Blood was collected by venipuncture employing sodium citrate as anticoagulant from healthy donors at the blood bank of Instituto Nacional de Cardiología “Dr. Ignacio Chávez”. Platelet-rich plasma and platelet-poor plasma was obtained as described above for mice samples. The aggregation was induced with 5 ␮M ADP and it was measured at increasing concentrations of vanadium pentoxide (5 ␮M, 50 ␮M, 500 ␮M and 5000 ␮M).

2.7.

Statistical analysis

Results are expressed as mean ± standard deviation (SD) or standard error of the mean (SEM). ANOVA was performed for statistical evaluation followed by the Tukey post hoc test. Results were compared with controls and statistical significance was considered at p < 0.05.

3.

Results

3.1. V2 O5 concentration in the inhalation chamber and serum The average concentration of V in the chamber was 1436 ␮g/m3 over the exposure period. The average

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Fig. 1 – Vanadium concentration (mean ± standard deviation) in serum and bone marrow (BM) after subchronic exposure to inhaled V2 O5 . None concentration was found in serum or bone marrow of control animals. CSER = Control serum, CBM = Control bone marrow, VSER = Vanadium in serum of exposed animals, VBM = Vanadium in bone marrow of exposed animals.

concentration of V in mice serum after eight weeks of inhalation was 27 ␮g/mL, and the concentration in bone marrow was 22 ␮g/mL, compared with non-detectable concentration in the serum and bone marrow of controls (Fig. 1).

3.2. Effect of inhaled vanadium pentoxide on markers of platelet activation in mice There was no evidence of platelet activation in whole exposure. There was no effect on P-selectin (CD62p) expression during V2 O5 exposure, but the expression was increased at four weeks after the exposure ended (post-exposure time). There was statistical difference between control and postexposure time (p < 0.05). (Figs. 2 and 3). In addition, we did not find changes on platelet–monocyte conjugates measured by flow cytometry (p = 0.488); data not shown.

Fig. 2 – P-selectin (CD62p) mean fluorescence levels ± standard error of the means are displayed. Significant differences compared to control are indicated by asterisks.

3.4. Effect of inhaled vanadium pentoxide on mouse platelet aggregation Vanadium pentoxide inhalation significantly inhibited PRP platelet aggregation at four weeks of exposure (mean 51.207% of inhibition), as in the in vitro assay, but the values returned to normal, at eight weeks of exposure and four weeks after the exposure halted. The percentage of aggregation was higher than control in mice at four weeks after the end of exposure, but this result does not have statistical significance (Table 2).

3.3. Effect of vanadium pentoxide in human platelets in vitro In order to assess if there were a direct effect of V on platelet function, we performed an in vitro assay of human platelets. Vanadium pentoxide inhibited platelet aggregation in a dose–response manner, without incubation and after 10 and 30 min of incubation (Table 1). Maximum effect on platelet aggregation inhibition was at 10 min of incubation with 500 ␮M (53.14%) and 5000 ␮M (87.7%). A representative aggregation trace is presented in Fig. 4. Effective Dose 50 (ED50 ) for platelet aggregation inhibition for V2 O5 without incubation was 724 ␮g; for 10 min of incubation was 400 ␮g and for 30 min of incubation was 387 ␮g.

Fig. 3 – Exemplary histogram presentation showing results of staining for P- selectin (CD62p) at different times of inhaled Vanadium exposure. The completely black pattern curve displays data of controls, the gray pattern curve represents four-week and eight-week exposure (overlapping curves). The black curve at the right, displays data of four weeks after the exposure ended where there was a significant difference compared to control.

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Table 1 – Percentage of platelet aggregation inhibition mean, standard deviation (SD) and standard error of means are displayed for each concentration of V2 O5 without incubation, at 10 min of incubation and at 30 min of incubation. These results were compared to control which was considered 0% inhibition. Percentage of platelet aggregation inhibition (mean)

Standard deviation

Standard error of the means

V2 O5 -w/h incubation 5 ␮M 9.76 50 ␮M 8.3 500 ␮M 22.08 5000 ␮M 66.5

10.28 12.81 14.19 23.63

4.6 5.73 6.34 10.57

V2 O5 10 min incubation 5 ␮M 11.35 50 ␮M 10.2 500 ␮M 53.14 5000 ␮M 87.77

15.07 4.80 13.01 10.12

5.69 1.81 4.91 3.8

V2 O5 30 min incubation 5 ␮M 12.32 50 ␮M 6.8 500 ␮M 47.9 5000 ␮M 71.8

8.44 12.75 8.00 8.80

4.22 6.3 4.00 4.40

A representative trace of inhibition of platelet aggregation by V at week four is shown in Fig. 5. We measured platelet aggregation on mice whole blood at four weeks of exposure of V2 O5 to compare the result with the PRP platelet aggregation and in this case, there was no difference between exposed and control animals. Mean of control platelet aggregation was 9.78  ± 0.94 SEM compared to mean of exposed to four weeks 9.26  ± 0.88 SEM (p = 0.729) (Fig. 6).

4.

Discussion

The U.S. American Conference of Governmental Industrial Hygienists (ACGIH, 2009) established the exposure limit for vanadium respirable dust as V2 O5 is 0.05 mg/m3 . V concentration in inhalation chamber was higher than this limit, but similar to reports of V ranged from 0.2 to 0.5 mg/m3 at workplaces (IARC, 2006; Kiviluoto et al., 1979, 1980). Also, we observed that V concentration increased in blood and bone marrow at subchronic exposure (eighth week), the biological locations where platelets and megakaryocytes, respectively, reside. In our model, we have reported thrombocytosis and a platelet turnover with the presence of giant platelets in

Fig. 4 – A representative trace from the aggregometer showing the inhibition of platelet aggregation with V2 O5 concentration of 5000 ␮M compared to control.

circulating blood. Also, V stimulates the proliferation of their precursors, megakaryocytes, and we described morphological changes in size and granularity of these cells (GonzálezVillalva et al., 2006; Fortoul et al., 2008, 2009). In the present study we evaluated the effect of V on human platelets in vitro, and in our murine model in vivo. Mice provide an excellent model for studying platelets in vivo, although there are some morphological differences that must be considered, there are remarkable similarities between murine and human platelets. Murine platelets are smaller and more numerous but the organelles and glycoprotein subcellular distribution are almost the same on both species (Schmitt et al., 2001). Functionally they are very similar, as shown by Jirouskova et al. (2007) in their review. Also, murine blood

Table 2 – Percentage of platelet aggregation mean and standard deviation are displayed for each time of exposure (control, fourth week, eighth week and four weeks after the exposure ended). Control vs fourth week had significant difference, so it is indicated by asterisk (p < 0.05) and also it reported the percentage of platelet aggregation inhibition. V2 O5 treatment

Percentage of platelet aggregation (mean)

Control Fourth week

100 48.79 *51.207 (percentage of inhibition) 92.727 104.392

Eighth week Postexposure fourth week after treatment

Standard deviation 4.23 12.08

15.3 9.2

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Fig. 5 – Platelet aggregation inhibition at fourth week of V inhalation. This is a representative trace from the aggregometer.

Fig. 6 – Mean and error standard of the means of impedance measured in ohms are displayed. No statistical differences are found between control and exposed animals on whole blood platelet aggregation.

coagulation factors and functions are quite similar to those of humans, so hemostasis is comparable in both species (Emeis et al., 2007; Sachs and Nieswandt, 2007). We found a similar effect on inhibition of platelet aggregation in vitro in human platelet assay and in murine platelets in in vivo model at fourweek time of exposure as we discuss below. Now, we report that there is no evidence of platelet activation during the whole exposure. We investigated this parameter first because of the epidemiological association between air pollution by particulate suspended matter and platelet activation (Nemmar et al., 2010, 2004; Mills et al., 2009; Rudez et al., 2009), and there are no studies about the effect of V occupational exposure on platelet activation. One marker of activation used in this experiment was Pselectin (CD62p) because it is useful to evaluate the risk of thrombosis. P-selectin is normally present in the membrane of alpha granules inside the platelet, but, when platelet is activated, granule contents are released and P-selectin localizes in the platelet membrane (André, 2004). As we have shown, P-selectin remains unchanged during the exposure so it means there is no platelet activation and degranulation does not occur; but at four weeks after the exposure ended, an increased expression of P-selectin (CD62p) was observed. This increase in P-selectin in our model, when the exposure ended, might be a long lasting toxic effect of inhaled V2 O5 as the end result of the slow V elimination (Barceloux, 1999; Nriagu, 1998). This change is consistent with the tendency of hyperaggregation at the same time. The fact that in our model there are no clotting events, besides the thrombocytosis, is supported in part by the absence of platelet activation markers. The finding of platelet–monocyte conjugates without changes is associated to P-selectin because this last molecule plays an important role in generation of these conjugates, so both markers are frequently associated as shown in our model. Besides the finding that there is no platelet activation, we are reporting an inhibition of platelet aggregation in mice at four weeks of exposure to V2 O5, and we also have explored the effect in human platelets in vitro in a dose–response manner. Later, at eight weeks of inhalation and four weeks after the exposure was ceased, platelet aggregation returned to normal values, so we think that platelets might implement a compensatory mechanism to mitigate the damage induced by vanadium that we need to explore in further studies. Another explanation is that mice may develop some tolerance mechanisms due to the repeated exposure. Other researchers have reported such effects in other tissues, as an inflammatory response in the lungs after acute and subacute but not subchronic exposure in monkeys inhaling V2 O5 (Knecht et al., 1992). The effect of V is a direct effect as we can conclude with the in vitro assay. The maximum percentage of inhibition of platelet aggregation (87.77%) was at 10 min of incubation. Also, for longer incubation it needs a lower concentration based on ED50 , 724 ␮g without incubation but 400 ␮g after 10 min incubation. This can be explained because V may enter the platelets by cationic or anionic channels, so at this time V reaches a higher concentration inside them and a greater effect (Goc, 2006; Barceloux, 1999; Nriagu, 1998). Between 10 and 30 min of incubation there was a plateau, ED50 400 ␮g

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and 387 ␮g, respectively, so longer incubations with the same concentration should be performed. For the in vivo mouse model, the explanation is more complex because it is a complete organism and platelets are not independent. There must be many mechanisms involved for V effect that need to be considered. First, is the observation that there is an effect of V on mice platelets because at the fourth week of exposure, we found an inhibition of platelet aggregation on PRP but not on whole blood, so the effect was offset by other components from the hemostatic system. This difference between two aggregation methods could be explained in part because there are no giant platelets on PRP because of centrifugation, but these are present on whole blood. In our model we have found giant platelets and it is reported that this type of platelet is linked with a higher activation (Rinder et al., 1998), so their presence on whole blood could normalize the aggregation. Also, whole blood platelet aggregation is similar to physiological conditions because it is measured in the presence of red and white blood cells, which are known to modulate platelet function (Dyszkiewicz-Korpanty et al., 2005). We have reported leukocytosis with neutrophilia and lymphocytosis in this model after the first week of inhalation (González-Villalva et al., 2009), so the increased cellularity may contribute to normalize the aggregation. Those findings on whole blood are supported by clinical evidence, as there were no bleeding events in the mice. It is possible to suggest that V affects platelet function because it acts on their precursors, megakaryocytes. We have found proliferation of these cells in this model, and V might well also be disrupting the signaling pathways related to megakaryopoyesis and thrombocytosis. In fact we saw changes in shape, size, granularity and ploidy in megakaryocytes and we found changes in platelet morphology also, as previously reported, with the presence of megaplatelets (Fortoul et al., 2009, 2008; González-Villalva et al., 2006). These morphological changes as a result of alterations in megakaryopoiesis may be associated to functional impairment. In fact, there is a myeloproliferative disease, a clonal thrombocytosis called essential thrombocytemia and our model has several criteria for its diagnosis (Schafer, 2006; Tefferi and Gilliland, 2005). In this myeloproliferative disease there is impairment of platelet morphology and function and the consequences varying from hemorrhage to thrombosis (Adams et al., 2009; Elliott and Tefferi, 2004). This possible explanation needs further validation. Although it is difficult to compare our results with others because there are many differences in the compounds used, concentrations or mode of administration, we can discuss some of them taking into account the differences previously mentioned because there are no previous studies on platelets with V2 O5 or with the inhalation route of exposure. Suenaga and Ueki (2004) reported the inhibition of platelet aggregation by orthovanadate, another compound of V; they reported an increase in cAMP which in turn activated Phospholipase A that inhibited Phospholipase C, and as a consequence, calcium release was inhibited and aggregation never occurred. Adenylate cyclase is one of the enzymes activated by V and its activation raises Cyclic AMP (cAMP) (Goc A 2006; Hayashi and Kimurat, 1994; Motoyashiki et al., 1999; Nechay, 1984).

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There are reports of inhibition or enhancing of platelet aggregation related to metal treatments and also it can be explained by changes in the concentration of cAMP (Kumar et al., 2001). An increase in cAMP inhibits calcium release and inhibits aggregation, possibly accounting for our results at four-week exposure on PRP and the in vitro assay of human platelets, because the aggregation trace is typical of calcium release inhibition, but we need to measure calcium concentrations and cAMP to investigate if this mechanism is involved in our model. For other vanadium compounds, there are reports in vitro indicating that pervanadate (vanadyl hydroperoxide) or sodium orthovanadate induced platelet activation and increased platelet aggregation as a consequence of the inhibition of Protein Tyrosine Phosphatases that raises the levels of phosphorylated proteins involved on platelet activation (Pumiglia et al., 1992; McNicol et al., 1993). Vanadium compounds and concentrations are important features to be considered on platelets assays at the time of measuring in vitro effects. In our model we demonstrate that vanadium increases the number of circulating platelets, but also this element exerts an inhibitory effect on platelet function and these qualitative changes of platelets could be associated with mucocutaneous bleeding (Simon et al., 2008; Nurden, 2005). As we debated before in the in vivo model, bleeding problems were not identified in our model, but there are reports of nose bleeding and pulmonary hemorrhage after acute toxicity of intratracheal instillation of vanadium pentoxide powder in rats (Toya et al., 2001), or even after subcutaneous or intraperitoneal administration of vanadium compounds (Evangelou, 2002). Our findings might be of interest at workplace exposure to vanadium, because there are no studies of bleeding disorders related to vanadium occupational exposure. Also, it is important to highlight the association found between air pollution, ambient particulate matter and hemorrhage (Yamazaki et al., 2007; Villeneuve et al., 2006; Tsai et al., 2003; Halinen et al., 1999) or with thrombosis reported in many industrialized cities (Mills et al., 2009; Kettunen et al., 2007; Nemmar et al., 2003). Many of the elements adsorbed on particles are metals and these have been implicated in the adverse effects of air pollution. In fact, platelets are a suitable toxicological model to study the adverse effects of pollutants and particles associated with occupational exposure.

5.

Conclusion

Our findings describe the effect of inhaled vanadium on platelets function in vivo. These results are supported by our previous work, in which we reported morphologic changes as well as an increase in platelets concentrations associated with megakaryocytic proliferation. The effect of V on platelet aggregation was consistent in vitro and in vivo, but it seems to be offset by other components of the hemostatic system, resulting in unseen clinical effects. Further research is needed in order to identify the mechanisms by which V induces this platelet dysfunction. Additional research should be done to dissect the behaviour of other hemostatic components, whilst correlating the toxic vanadium effects on the hemostatic

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system with the increased risk for thrombosis or hemorrhagic events.

Conflict of interest statement The authors declare that they have no conflict of interest.

Ethical statement The experimental protocol was in accordance with the Guide for the care and use of laboratory animals from the Institute of Laboratory Animal Resources Commission on Life Sciences National Research Council (2010).

Acknowledgments First author is a PhD student from the Posgrado en Ciencias Biológicas, UNAM and is supported by CONACyT grant CVU 42414. We thank Carlos Castellanos from the Instituto de Investigaciones Biomédicas, UNAM for his technical assistance. This work was partially supported by grants IN-210409, from DGAPA-UNAM. First author thanks the support given by the Posgrado en Ciencias Biológicas, UNAM.

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