Particulate matter (PM) and nanoparticles (NPs) induce activation and dysfunction of endothelial cells characterized by inhibition of proliferation, increase of adhesion and adhesion molecules expression, increase of ROS production, and death. DHEA has shown anti-inflammatory and antioxidant properties in HUVEC activated with proinflammatory agents. We evaluated if DHEA could protect against some inflammatory events produced by PM10 and TiO2 NPs in HUVEC. Adhesion was evaluated by a coculture with U937 cells, proliferation by crystal violet staining, and oxidative stress through DCFDA and Griess reagent. PM10 and TiO2 NPs induced adhesion and oxidative stress and inhibited proliferation of HUVEC; however, when particles were added in combination with DHEA, the effects previously observed were abolished independently from the tested concentrations and the time of addition of DHEA to the cultures. These results indicate that DHEA exerts significant anti-inflammatory and antioxidative effects on the damage induced by particles in HUVEC, suggesting that DHEA could be useful to counteract the harmful effects and inflammatory diseases induced by PM and NPs.
Particulate matter (PM) is an environmental factor that has been associated with increased cardiovascular morbidity and mortality, particularly mass concentrations of PM with aerodynamic sizes ≤2.5 or ≤10
We have previously shown that PM2.5 and PM10 induce adhesion of U937 cells to human umbilical vein endothelial cells (HUVEC), which was associated with an increase in the expression of adhesion molecules such as E- and P-selectins, ICAM-1, PECAM-1, and VCAM-1 [
In previous works, we have shown that dehydroepiandrosterone (DHEA), an adrenal hormone, has shown anti-inflammatory and antioxidative roles in HUVEC treated with two proinflammatory molecules such as TNF-
In this work, we hypothesized that DHEA could protect HUVEC against inflammatory events induced by PM10 and TiO2 NPs. To test this, we exposed HUVEC to PM10 and TiO2 NPs in combination with DHEA and evaluated the adhesion of monocytic cells, proliferation, and ROS and NO production.
RPMI 1640 and M199 media and trypsin were purchased from GIBCO/BRL (Grand Island, NY, USA), and fetal bovine serum (FBS) was HyClone (Logan, UT, USA). Sterile plastic material for tissue culture was from NUNC and COSTAR. Flow cytometry reagents were purchased from Becton Dickinson, Immunocytometry Systems (San José, CA, USA). TNF-
PM10 were collected from the north zone of Mexico City. Samples were taken three days per week throughout 2007 using a GMW high-volume particle collector (model 1200 VFC HV PM10, Sierra Andersen) to collect particles with mean aerodynamic diameters equal to or smaller than 10
At least 1 mg of particles was weighed and sterilized by autoclave the night before of each experiment. PM10 and TiO2 NPs suspensions in M199 medium, at a concentration of 1 mg/mL, were prepared few minutes before cell exposure. Aliquots were taken from these suspensions and further diluted with culture medium until the required final concentration was obtained. TiO2 NPs used were previously characterized by our work group [
Primary HUVEC cultures were obtained by proteolytic dissociation of the umbilical cord veins from normal deliveries, treated with collagenase type II (0.2 mg/mL), and cultured on gelatin-coated culture dishes in M199 supplemented with 10% FBS, glutamine (2 mM), heparin (1 mg/mL), and endothelial mitogen (20
Human leukemia promonocytic U937 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and L-glutamine (2 mM).
Adhesion was evaluated using U937 cells that were labeled with [3H]-thymidine; 1 × 105 HUVEC were seeded in 24-well tissue-culture plates with 1 mL of supplemented M199 medium and treated with TNF-
Cell number was evaluated by crystal violet staining. HUVEC were cultured on 96-multiwell plates without and with DHEA (1, 10, and 100
The oxidation of 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) into 2,7-dichlorodihydrofluorescein (DCF) was used to assess Ros generation. HUVEC were cultured without or with DHEA (1, 10, and 100
Quantification of nitrite was used as an indirect method to determine the production of NO. Cells were seeded in 96 well plates (NUNC) at a density of 1 × 105 cells/well in M199 (phenol red free) and 10% FBS. Cells were cultured without or with DHEA (1, 10, and 100
All the endpoints were measured at least three times. The results are expressed as mean ± standard deviation. Statistical significance was evaluated using one-way analysis of variance (ANOVA) test using GraphPad Prism, version 2.0 (GraphPad Software, CA, USA), followed by Duncan’s multiple range test (MRT), to assess differences between group means. Differences were considered significant when
Adhesion of U937 cells to HUVEC was evaluated by a coculture assay. DHEA alone did not induce adhesion, whereas the treatment with TiO2 NPs and PM10 induced a 2-fold increase in adhesion, compared to untreated cells; however, this was significantly inhibited until reaching basal levels when HUVEC were exposed to a pretreatment with DHEA (Figure
Effect of DHEA on the adhesion induced by particles. Cells were treated with 1 (D1), 10 (D10), and 100
To examine the possible involvement of DHEA on the inhibition of proliferation induced by TiO2 NPs and PM10, HUVEC were exposed to DHEA alone or in combination with the particles, and proliferation was evaluated by crystal violet. Results showed that DHEA reverted almost completely the inhibition of proliferation induced by TiO2 NPs at any concentration (Figure
Effect of DHEA on the inhibition of proliferation induced by particles. Cells were treated with 1 (D1), 10 (D10), and 100
Oxidative stress was determined indirectly by measuring the H2O2 and nitrite production by H2DCFDA and Griess reagent, respectively. After exposure to TiO2 NPs and PM10 for 24 h, fluorescence from most cells stained with H2DCFDA indicated that intracellular H2O2 had accumulated strongly in HUVEC; however, this was significantly inhibited reaching almost basal levels by pretreatment with DHEA at all concentrations used (Figure
Effect of DHEA on ROS production induced by particles. Cells were treated with 1 (D1), 10 (D10), and 100
Effect of DHEA on NO production induced by particles. Cells were treated with 1 (D1), 10 (D10), and 100
Our previous study showed that exposure of human endothelial cells to TiO2 NPs and PM10 caused cytotoxic damage [
DHEA, at different concentrations, inhibited the adhesion of U937 cells to HUVEC induced by TiO2 NPs and PM10, independently from the time of administration of DHEA to the culture (Figure
In addition, we observed that the antiproliferative effect induced by TiO2 NPs and PM10 on HUVEC was similarly reverted with DHEA (Figure
DHEA, at all tested concentrations, abolished completely the oxidative stress induced by TiO2 NPs and PM10, decreasing the H2O2 and nitrite production (Figures
In other cells, it has been described that DHEA prevented the increased death evoked by glucose deprivation by inhibiting the production of superoxide anion in immunostimulated C6 glioma cells [
As well, in an
In conclusion, our results show that DHEA could be useful as a protective agent in the prevention and treatment of inflammatory and cardiovascular effects induced by urban particulate matter and nanoparticles where endothelial dysfunction is involved.
Dehydroepiandrosterone
Tumor necrosis factor alpha
Human umbilical vein endothelial cells
Reactive oxygen species
2,7-Dichlorodihydrofluorescein diacetate.
The authors thank Dr. Gerardo Tinoco from the Hospital General de Zona de Troncoso 2 A, IMSS, Mexico, for the samples of umbilical cords. E. Huerta-Garcia (doctoral student from the Posgrado en Investigación en Medicina from the Escuela Superior de Medicina, Instituto Politécnico Nacional) was supported by CONACyT scholarship no. 227281. The present study was partially supported by the SEP-CONACyT Grant (106057).