Biochemical and molecular evidences on the protection by magnesium oxide nanoparticles of chlorpyrifos-induced apoptosis in human lymphocytes.

BACKGROUND: Chlorpyrifos (CP) is one of the most widely used organophosphate (OP) insecticides in agricultural and residential pest control with its attendant adverse health effect. In the present study, it is proposed to investigate the possible modulatory role of magnesium oxide nanoparticles (MgO NPs) against CP-induced toxicity in human lymphocytes and determine the mechanisms lying behind this protection by viability and biochemical assays.
MATERIALS AND METHODS: Isolated lymphocytes were exposed to 12 μg/mL CP either alone or in combination with different concentrations of MgO NPs (0.1 μg/mL, 1 μg/mL, 10 μg/mL, and 100 μg/mL). After a 3-day incubation, the viability and oxidative stress markers including cellular mitochondrial activity, caspase-3 and -9 activities, total antioxidant power, lipid peroxidation, and myeloperoxidase (MPO) activity were measured. Also, the levels of tumor necrosis factor-α (TNF-α) as inflammatory index, along with acetylcholinesterase (AChE) activity were measured. Statistical differences were determined using one-way analysis of variance (ANOVA) and Dunnett's multiple comparison tests.
RESULTS: It is indicated that CP-exposed lymphocytes treated with MgO NPs resulted in a substantial reduction in the pace of mortality as well as the stages of oxidative stress in a dose-dependent manner. Also, MgO NPs (100 μg/mL) meaningfully restored CP-induced increase of TNF-α (P < 0.001) and decrease of AChE activity (P < 0.001) and were capable of preventing CP-treated human lymphocytes from apoptosis (P < 0.001).
CONCLUSION: Our results demonstrate that MgO NPs in approximate 100 nm diameter not only make cells resistant to the toxic properties of CP but also attenuate toxic effects of CP, which is demonstrating the potential of MgO NPs to be applied in future immune deficiency therapeutic strategies.

INTRODUCTION

Chlorpyrifos (CP) is one of the most widely used organophosphate (OP) insecticides in agricultural and residential pest control with its attendant adverse health effect. The primary toxicity of CP is attributed to the irreversible inhibition of the enzyme acetylcholinesterase (AChE).[1] Likewise, it has been postulated that CP has multiple effects on the cells including the production of reactive oxygen species (ROS) and initiation of intracellular oxidative stress, thereby disrupting normal cellular development and differentiation.[2] In summation, it has been reported that CP, like other OP compounds, causes immunological abnormalities in animals and man through lymphocytes and other immunocompetent cells,[3] which in the event of chronic exposures result in the incidence of human diseases.[45] Consequently, since the role of CP is on the lift, especially in agriculture, the need to identify factors that have abated the adverse health consequence posed by long- and short-term exposures to the insecticide becomes worthy.

It is always a demand for creating and exploring novel materials for biological applications, particularly in the area of medicine owing to their immense impact on wellness maintenance. Nanomaterials offer promising opportunities for improved and tailored properties for applications in various fields due to their unique physicochemical characterization apparent at the nanoscale such as large surface area, surface reactivity, surface derivatization, and altered electronic properties.[67] Nanomaterials are of special interest not only for basic research but also for their interesting applications in various fields such as pharmaceutical cosmetics, drug delivery, additives to drugs, and food products.[8] In this respect, in the recent years, the efficacy of some antioxidant nanoparticles (NPs) such as nanomagnesium, nanocerium, nanoselenium, and nanoyttrium in disease models of colitis,[9] diabetes,[10] pancreatitis,[111213] diabetic neuropathy,[14] cardiotoxicity,[1516] and spermatotoxicity,[17] have been proven and it has been found that they are useful in reducing oxidative stress.

Magnesium oxide nanoparticle (MgO NP) is one of the metal oxide NPs, which is extensively used in several industries such as pharmaceuticals, cosmetics, food packaging, electrical insulation, desiccant, and medicine.[18] It has the advantage of nontoxic, high thermal stability, biocompatible, low cost, and has considerable potential as an antibacterial agent.[19] A number of studies have also been performed on MgO NPs and have evaluated different mechanistic endpoints. Stoimenov et al. (2002) reported that the nanoscale MgO powder showed a pronounced bacterial action against vegetative cells and spores of bacteria.[20] This antibacterial activity of MgO NPs was reported by other studies.[1921] According to the findings of these studies, the toxicity of NPs toward microorganisms is due to either physical disruption or oxidative stress, which increased as the concentration of MgO increased.[192021] In contrast, Ge et al. (2011) reported low concentration (below 200 μg/mL) of MgO. NPs not only have no cytotoxicity but also promote the human umbilical vein endothelial cells (HUVECs) proliferation.[22] Accordingly, their findings provide elementary cytocompatibility investigation about MgO NPs. This noncytotoxicity of MgO NPs in low concentration was confirmed by other researchers. Lai et al. (2008) showed that the treatment of U87 cells with MgO NPs for 48 h did not significantly reduce their survival until concentrations were higher than 50 μg/mL.[23] In addition, Hasbullah et al. (2013) indicated that MgO NPs have no toxicity on human neuroblastoma SH-SY5Y cell lines.[18] All these researches mentioned above indicated that the beneficial or detrimental biological effects of MgO NPs were dependent on particle composition, particle concentration, particle size, exposure time, and the nature of the biological target.

Given the above evidences, the present endeavor was undertaken to develop a cell culture model using lymphocytes and the immunocompetent cells of the body, and to evaluate compatibility of MgO NPs in low concentration. It is also aimed to investigate the possible modulatory role of MgO NPs against CP-induced oxidative stress and determine the mechanisms lying behind this protection by viability and biochemical assays.

MATERIALS AND METHODS

Chemicals

All the chemicals were purchased from Sigma-Aldrich Chemie (Taufkirchen, Germany) unless otherwise expressed. The MgO NPs suspension was purchased from NanoZino (Tehran, Tehran Province, Iran). The human-specific tumor necrosis factor-α (TNF-α) enzyme-linked immunosorbent assay (ELISA) kit from Bender MedSystems (Vienna, Austria) and ApoFlowEx® fluorescein isothiocyanate (FITC) kit from Exbio (Vestec, Czech Republic) were obtained.

Characterization of magnesium oxide nanoparticle

Ultraviolet visible (UV-Vis) spectra of MgO NPs used in the experiment were recorded (apparatus UV-VIS CECIL BioAquarius 7250, Cambridge, England). The morphology of the NPs was determined using transmission electron microscopic (TEM) technique.

Lymphocyte isolation and care

The study was approved by the institute's review board with code number of 90-04-151-16052. Peripheral blood lymphocytes were isolated from heparinized venous blood, which was obtained from healthy volunteers who were nonsmokers and were not using medicines. Blood was mixed with Ficoll-Paque and centrifuged at 400 g for 30 min. The lymphocytes, from the interface of plasma and Ficoll-Paque, were picked up and washed twice with phosphate buffer, and were then counted based on the trypan blue exclusion method. After washing and weighing, the cells were seeded at a density of 3 × 106 cells/well in the tissue culture medium (RPMI-1640), which consisted of 10% FBS, 2 mM L-glutamine, 100 u/mL penicillin, and 100 μg/mL streptomycin sulfate, and 50 μl/ml LPS for cell growth stimulation. The lymphocyte cultures were grown in a humidified incubator with 5% CO2 at 37° C in 96 microtiter plates.

Experimentation 1: Cytotoxic effect of magnesium oxide nanoparticles on human lymphocytes

Effect of magnesium oxide nanoparticles on human lymphocytes

Before undergoing the following test and seeing the effect of MgO NPs on the prevention of CP-induced toxicity, the human lymphocytes were exposed to various concentrations of MgO NPs. In a former study, Ge et al. (2011) have shown that low concentration (below 200 μg/mL) of MgO NPs showed no cytotoxicity by 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay on HUVECs in vitro.[22] Consequently, in our study human lymphocytes (3 × 106 cells/well) were incubated with the culture medium in combination with different concentrations of MgO NPs (0 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL, 100 μg/mL, and 200 μg/mL) for 72 h at 37° C and 5% CO2 humidified atmosphere. After treatment, the lymphocytes were processed for cytotoxicity by MTT assay.

Lymphocyte viability assay

The assay is based on the reduction of MTT, a yellow tetrazole, to purple insoluble formazon by mitochondrial respiration in viable cells. MTT assay was performed on human lymphocytes cultured after 72 h incubation. Centrifugation was performed and the precipitated lymphocytes were washed twice by phosphate buffer. Then, 50 μl of MTT solution was added and it was reincubated for 4 h at 37° C and 5% CO2 humidified atmosphere. At the end, 150 μl of DMSO solution was added and the absorbance was determined at 570 nm by ELISA reader. The viability of the treatment groups was expressed as the percentage of controls, which assumed 100%.

Experimentation 2: Protective effects of magnesium oxide nanoparticles against chlorpyrifos-induced toxicity

Treatment conditions and experimental groups

According to previous data,[2425] we used 12 μg/mL as the concentration of CP that induced oxidative stress in the lymphocytes. In this regard, cell suspension (3 × 106 cells/well) was incubated with the culture medium in combination with 12 μg/mL CP for 72 h at 37° C and 5% CO2 humidified atmosphere. For protective treatment, optimization of dose was performed by pretreating CP-induced cells with logarithmic concentrations (0.1 μg/mL, 1 μg/mL, 10 μg/mL, and 100 μg/mL) of MgO NPs for 72 h to ascertain the most efficacious dosage. To fulfill this purpose, all the cells were split into half a dozen groups of four per each interval (n = 4). Treatment conditions of experimental groups included:

Con (negative control), lymphocytes in RPMI-1640 medium alone;

CP, lymphocytes in RPMI-1640 medium + CP (12 μg/mL);

MgO NPs I [lymphocytes in RPMI-1640 medium + CP (12 μg/mL) + MgO NPs (0.1 μg/mL)];

MgO NPs II [lymphocytes in RPMI-1640 medium + CP (12 μg/mL) + MgO NPs (1 μg/mL)];

MgO NPs III (lymphocytes in RPMI-1640 medium + CP (12 μg/mL) + MgO NPs (10 μg/mL));

MgO NPs IV [lymphocytes in RPMI-1640 medium + CP (12 μg/mL) + MgO NPs (100 μg/mL)].

Then the lymphocytes were incubated at 37° C and 5% CO2 humidified atmosphere. After a 72-h period, the cell suspensions in all groups were centrifuged. The supernatant solutions were removed for the biochemical assays and the deposited cells were used for viability assays in the succeeding measure.

Viability assays

Mitochondrial activity assay

We used this assay for investigation of the viability of treated lymphocytes as described in the previous section.

Caspase-3 and -9 activities assays

Caspase-3 and -9 activities were measured by colorimetric assays based on the identity of specific amino acid sequences by these caspases. The tetrapeptide substrates were labeled with the chromophore r-nitroaniline (ρNA). ρNA is released from the substrate upon cleavage by caspase and produces a yellow color that is monitored by an ELISA reader at 405 nm. The amount of caspase activity present in the sample is proportional to the quantity of yellow color produced upon cleavage.[26] Briefly, the pretreated lymphocytes were lyzed in the supplied lysis buffer and were incubated on ice for 10 min. The whole cell lysates were incubated in caspase buffer (100 mM HEPES, pH 7.4, 20% glycerol, 0.5 mM EDTA, 5 mM dithiothreitol) containing 100 mM of caspase-3 and -9-specific substrate [N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-ρNA:), N-acetyl-Leu-Glu-His-Asp-p-nitroanilide (Ac-LEHD-ρNA)] for 4 h at 37°C. Then, absorbance was measured at 405 nm. The caspase-3 and -9 activities of the treatment groups were expressed as the percentage of controls, which assumed 100%.

Determination of cell death (apoptosis vs necrosis)

To find out the mode of lymphocyte cell death induced by CP in the presence and absence of MgO NPs, the annexin V-FITC/propidium iodide (PI) staining was performed. The staining of annexin V-FITC and PI indicates the character of death caused by the test compound, i.e., apoptosis or necrosis. The cells (1 × 106) were treated with CP, alone or in combination with several concentrations of MgO NPs for 72 h, washed and stained with annexin V-FITC antibody and PI as per the instructions presented by the producer. The cells were scanned for fluorescence intensity in FL-1 (FITC) and FL-2 (PI) channels. The fraction of cell populations in different quadrants was analyzed using quadrant statistics. Cells in the lower right quadrant represented apoptosis and those in the upper right quadrant represented necrosis or postapoptosis necrosis.[27]

Biochemical assays

Determination of total antioxidant power (TAP)

The method is based on the reduction of Fe3+ tripyridyltriazine (TPTZ) complex (colorless complex) to Fe2+ TPTZ (blue colored complex) formed by the action of the electron donating antioxidants at low pH. The ferric reducing antioxidant power (FRAP) reagent was prepared by mixing 300 mM acetate buffer, 10 mL TPTZ in 40 mM HCl, and 20 mM FeCl3 in the ratio of 10:1:1 at 37° C. Ten μl of H2 O diluted sample was then added to 300 mL freshly prepared reagent warmed at 37° C. An intense blue color complex was formed when Fe3+ TPTZ complex was reduced to Fe2+ form and the absorbance at 593 nm was recorded against a reagent blank after 30 min incubation at 37° C. Data were shown as mmol/μg protein.

Determination of lipid peroxidation (LPO)

To measure LPO, thiobarbituric acid reactive substances (TBARS) were evaluated. TBA reacts with lipid peroxides in the samples producing a measurable pink color that has an optical density at 532 nm read by the ELIZA reader as described in our previous study.[28] The activity was shown as mM.

Determination of myeloperoxidase activity

To assay myeloperoxidase (MPO) activity, we measured it spectrophotometrically as follows: 0.1 mL of supernatant was added to 2.9 mL of 50 mM phosphate buffer containing 0.167 mg/mL O-dianisidine hydrochloride and 0.0005% H2O2. The change in absorbance was recorded by spectrophotometer at 460 nm. MPO activity is set as the absorbance change per minute at 25°C in the final reaction.[29] The MPO activity was shown in unit/mL.

Determination of acetylcholinesterase

AChE activity in lymphocytes was assayed according to the modified Ellman method using acetylthiocholine iodide as the substrate and 5-5-bis dithionitrobenzoic acid (DTNB) as a coloring agent.[30] The action was expressed as unit/mg protein.

Determination of tumor necrosis factor-α

human-specific ELISA kit (Bender MedSystem, Vienna, Austria) was used to quantify TNF-α in the supernatant of lymphocyte culture. To measure the amount of TNF-α, the absorbance of the sample was quantified at 450 nm as the primary wavelength and 620 names as the reference wavelength by the ELIZA reader as described in the kit brochure. Data were shown as ng/mL.

Protein assay

To see the total protein concentration of cells, Bradford reagent was added to dilute samples and the absorbance was measured by the spectrophotometer at 595 nm after 5 min. The bovine serum albumin was used as the criterion.

Statistical analysis of data

The data were first tested for normality (Shapiro-Wilk test) and as this assumption was met, one-way ANOVA and Dunnett's multiple comparison tests were held out by Stats-Direct 3.0.117 to determine the statistical differences while the degree of significance was placed at P < 0.05.[29] Data were presented as mean ± standard error of mean (SEM) of four different experiments, each read in duplicate.

RESULTS

Characterization of magnesium oxide nanoparticles

The optical properties of the MgO NPs were studied in particular by means of the UV-Vis absorption spectra in the wavelength range of 200-700 nm at room temperature. Figure 1 shows the UV-Vis absorption spectrum of the MgO NPs depicting the enhanced absorbance in the low ultraviolet (UV) region. The maximum absorption band of MgO NPs was found at 246.2 nm. The particle size was also examined using transmission electron microscopy (TEM). Figure 2 displays TEM micrographs of MgO NPs, revealing that the particle size is approximately 100 nm.

Figure 1

UV-Vis spectra of MgO NPs used in this study

Figure 2

TEM micrograph of MgO NPs

Lymphocytes viability in the presence of magnesium oxide nanoparticles

As shown in Figure 3, the MTT assay was applied for investigation of the viability of treated lymphocytes after 72 h of exposure. It illustrated that the mitochondrial activity of lymphocytes was about 100% when the MgO NPs suspension concentration was lower than 200 μg/mL. Thus, not only was the range of 0.1-200 μg/mL of MgO NPs safe but also at 100 μg/ml, the viability improved to 25% of the control (P < 0.05).

Figure 3

Effects of various concentrations of MgO NPs in the mitochondrial activity of isolated human lymphocytes; Data are expressed as mean ± SEM; Significantly different from control at *P < 0.05

Protective effects of magnesium oxide nanoparticles against chlorpyrifos-induced toxicity with viability assays

Mitochondrial activity

The results of MTT assay on the cultured lymphocytes post 72 h of different treatments are shown in Table 1. There is a significant decrease in the viability of the CP group as compared with the control (P < 0.01). The groups, which were pretreated with MgO NPs at doses 1 μg/ml, 10 μg/ml, and 100 μg/ml remained more viable in comparison with CP (P < 0.05) but did not prove any different viability when compared with the control. No significant improvement in the lymphocyte viability was observed when the cells pretreated with the group of dose 0.1 μg/mL MgO NPs as compared with CP.

Table 1

Effects of various concentrations of magnesium oxide nanoparticles (MgO NPs) on mitochondrial and caspase-3 and -9 activities of isolated human lymphocytes in the presence of chlorpyrifos (CP)

Caspase-3 and -9 activities

As shown in Table 1, CP-induced apoptosis significantly increased the activities of caspase-3 and-9 when compared with the controls (P < 0.05 and P < 0.001, respectively), whereas pretreatment with different concentrations of MgO NPs in these conditions reduced the activities of caspase-3 and 9 in a dose-dependent fashion. Nevertheless, no obvious change in the activities of caspase-3 and -9 was caused by MgO NPs at 0.1 μg/mL when compared with CP after 3 days but the highest concentration caused a significant (P < 0.001) decrease in caspase-3 and -9 activities in this condition.

Lymphocyte death (apoptosis vs necrosis)

Figure 4 represents typical flow cytometry plots of annexin V and PI staining of different treatments of lymphocyte. After 72 h incubation with CP, there was an increase in the number of lymphocytes that were apoptotic as indicated by an increase in the annexin V-positive and PI-negative cell population as well as an increase in the number of lymphocytes that were necrotic or in the later stages of apoptosis as indicated by the increase in the annexin V-positive and PI-positive cell population as compared with the control group (% of apoptotic cells: 13 vs 2, % of necrotic cells: 27 vs 3). In addition, the treated cells with MgO NPs at dose of 0.1 μg/mL in comparison with CP-treated cells showed no obvious changes in apoptotic and necrotic cell populations (% of apoptotic cells: 13 vs 13, % of necrotic cells: 23 vs 27). In contrast, the groups, which were pretreated with MgO NPs at doses of 1 μg/mL, 10 μg/mL, and 100 μg/mL remained more viable and the number of apoptotic and necrotic cells decreased as compared with the CP (% of apoptotic cells: 9, 5, and 3 vs 13, % of necrotic cells: 6, 3, and 3 vs 23). Interestingly, there were no significant differences in the viable cells in comparison with the control group.

Figure 4

Flow cytometric analyses of apoptosis and necrosis in human lymphocytes induced by CP, alone, or in combination with various concentrations of MgO NPs using Annexin V-FITC and PI double staining; Human lymphocytes (1 × 106/200 μl) were incubated with indicated concentrations of MgO NPs and CP for 72 h and stained with annexin V-FITC/PI as described previously. Quadrant analysis of fluorescence intensity of nongated cells in FL1 (annexin V) vs. FL2 (PI) channels was from 5,000 events. The values shown in the lower left, lower right, upper left, and upper right quadrants of each panel represent the percentage of viable, apoptotic, necrotic, and late apoptotic (postapoptotic necrotic) cells, respectively. The percentages of positive cells were indicated in each panel

Protection effects of magnesium oxide nanoparticles against chlorpyrifos-induced toxicity with biochemical assays

Total antioxidant power

As shown in Figure 5a, there is a decrease in control TAP by administration of CP. In contrast, the groups, which were pretreated with MgO NPs at doses of 0.1 μg/mL, 1 μg/mL, 10 μg/mL, and 100 μg/mL, showed an apparent increase in TAP when compared with the CP group in a dose-dependent manner (P < 0.05, P < 0.01, P < 0.001, and P < 0.001, respectively), which was also significant in comparison with the control group at doses of 10 μg/mL and 100 μg/mL (P < 0.05 and P < 0.001, respectively).

Figure 5

Effects of various concentrations of MgO NPs in TAP values (a), LPO levels (b), MPO activity (c), AChE activity (d), and TNF-α release (e) of isolated human lymphocytes in the presence of CP; Data are expressed as mean ± SEM; Significantly different from control at *P < 0.05, **P < 0.01, ***P < 0.001; Significantly different from CP at #P < 0.05, ##P < 0.01, ###P < 0.001

Lipid peroxidation

There was a significant elevation in LPO (P < 0.01) in CP compared with the control group [shown in Figure 5b]. In addition, the groups which were pretreated with low concentrations of MgO NPs (0.1, 1 and 10 μg/mL) showed no significant reduction in LPO as compared with the CP group and LPO levels of these groups still higher than control group (P < 0.01, P < 0.01 and P < 0.05, respectively). In contrast, high concentration of MgO NPs (100 μg/mL) could decrease LPO as compared with CP, which was significant at dose of 100 μg/mL (P < 0.01).

Myeloperoxidase activity

As depicted in Figure 5c, MPO activity considerably increased in the CP group as compared with the control group (P < 0.05). No significant improvement in the lymphocyte MPO activity was observed when the cells were pretreated with MgO NPs at dose 0.1 μg/mL in comparison with CP. However, treatment with the other concentrations of MgO NPs (1 μg/mL, 10 μg/mL, and 100 μg/mL) decreased MPO activity in a dose-dependent manner. Among these concentrations, 100 μg/mL MgO NPs showed significant effects in comparison with CP (P < 0.05) and reached close to the control values.

Acetylcholinesterase activity

As shown in Figure 5d, AChE activity in the CP group was significantly lower compared with the control group (P < 0.05). The groups, which were pretreated with MgO NPs at doses of 1 μg/mL, 10 μg/mL, and 100 μg/mL, showed an apparent increase in AChE activity as compared with the control and CP groups (P < 0.05 and P < 0.001, respectively). But the group of dose 0.1 μg/mL, MgO NPs showed no difference in AChE activity as compared with CP.

Tumor necrosis factor-α release

As seen in Figure 5e, TNF-α production significantly elevated in the CP group when compared with the control group (P < 0.01). Conversely, administration of MgO NPs in different concentrations decreased TNF-α release in dose-dependent manner as compared with CP, which was significant at doses 1 μg/mL, 10 μg/mL, and 100 μg/mL (P < 0.05, P < 0.05, and P < 0.001, respectively).

DISCUSSION

Previous works, either in vivo or in vitro, have shown that NPs have a wide range of effects on humans. As we know, size, condition, and surface area of NPs,[31] duration of the exposure time[32] and a compactness of them[33] are important in rendering them as toxic property or safety feature. About MgO NPs, in that respect are some articles, which have indicated toxic properties[2234] and also other articles, which have exhibited the positive effects of MgO NPs on different organisms by changing the concentration and size of MgO NPs.[182335] Hence, in this work we prospected increasing maintenance of the human lymphocytes by testing different concentrations of MgO NPs.

Before beginning the task, MgO NPs were investigated by spectroscopic methods for determining the peak concentration. As shown in the Figure 1, peaking absorption of them was approximately 250 nm, which was confirmed range of pure MgO NPs by another study.[36] After that, since the cytotoxic effect of MgO NPs on human lymphocytes in vitro has not been read yet, their toxicity investigated precisely. As shown in the Figure 3, the viability of lymphocytes in exposure to MgO NPs with a scope of 0.1–200 μg/mL for 72 h, based on MTT assay was tested and it showed that MgO NPs are safe and can even increase the viability of the cells. There is a study indicating that concentrations up to 200 μg/mL of MgO NPs have no toxicity on Human umbilical vein endothelial cell (HUVE) cells.[22] The study in which an exposure time of MgO NPs was similar to our approach (72 h) showed similar results with the MTT assay.

Regarding such a straightforward response from the viability test of human lymphocytes, the efficacy of MgO NPs was tested against CP-induced toxicity on lymphocytes.[3738] Interestingly, in the first portion of the study, all results that are channeled out for viability tests of lymphocytes, confirmed each other and showed that MgO NPs could lessen the harmful effects of CP in human lymphocytes. As determined in Table 1, CP significantly decreased the viability of the lymphocytes (P < 0.01) and MgO NPs compensated for that reduction effect. A previous report has indicated that CP has effects on mortality of the cells such as neurons[39] by different mechanisms such as cell signaling and starting cascades, and also changing gene transcription factors, which are activated by reactive oxygen species (ROS).[40] Toxic effect of CP is supported by results of flow cytometry in the present study. As shown in the Figure 4, in the control group, which was not exhibited to any dose of MgO NPs, lymphocytes were distributed mostly in quarter C (83%) and the rate of apoptotic and necrotic cells was really depressed. As we expected, the rates of early and late apoptosis increased significantly by CP (up to 13% and 27%). In contrast, these rates decreased upon treatment with MgO NPs. The best result in the flow cytometric assay was got by a dose of 100 μg/mL MgO NPs, which decreased the percentages of early and late stages of apoptotic cells down to 3% (from 13% and 27% of the CP group). A previous study has reported that magnesium sulfate could decrease apoptosis in nerve cells after cerebral ischemia-reperfusion injury, perhaps via decreasing expression of Bax and caspase-3.[41] In general, there are two mechanisms for justifying the apoptosis; one of the mechanisms for declined apoptotic cells is linked to suppression of caspase enzyme activity,[42] which is depicted in this study by measuring caspase-3 and -9 activities. Given the effects of caspase in Figure 4, we learned that MgO NPs can alleviate caspase activity in CP-induced human lymphocytes. In both caspase-3 and -9, concentration of 100 μg/mL MgO NPs more than other concentrations made a reduction in enzyme activity and was the best effective dose, which correlated well with the flow cytometry results. The other mechanism, which justifies apoptosis and causes internal signal in apoptosis, relates to increasing levels of cytochrome C and apoptosis-inducing factor (AIF) by depressing the mitochondrial function.[43] So because of the positive effects of MgO NPs on the viability of mitochondria, such as decrease in apoptosis, is not surprising.

In the second section of our study, oxidative stress factors were assessed. As shown in the Figure 5, MgO NPs were in a concentration of 100 μg/mL in comparison with the CP group's alleviated levels of LPO and MPO (P < 0.05). Also, the level of TAP, which was decreased by CP upon treatment with MgO NPs increased significantly (P < 0.001). A previous study has shown that CP makes a significant increase in LPO and thus, induces cytotoxicity and apoptosis in the follicular cells.[44] Also, MPO produced a more toxic oxon group by replacing sulfur atom with oxygen in CP; thus, it made more cytotoxicity.[45] Other studies have reported that the metal form of Mg reduces LPO level in the plasma of the rat. The researchers concluded that Mg had an effect on glutathione peroxidase enzyme, which has a central role in LPO.[46] As indicated in another study, Mg in metal form has neuroprotective effect and reduces LPO.[47] Thus, nanoscale and oxide forms of magnesium may have similar properties to the metal form. The present study also showed a substantial elevation in the degree of AChE by MgO NPs compared with the CP group (P < 0.001). It was reported that CP in different tissues such as the kidney, liver, spleen, and brain caused inhibition of AChE activity.[48] Likewise, another study on the blood samples of workers who were in contact with OP pesticide, recommended that measuring AChE activity could be useful for detecting OP poisoning.[49] Because of the freshness of this approach, this is the first time that MgO NPs has been tried out at the level of AChE in lymphocytes but as we know, the reduction of AChE activity is accompanied by oxidative stress;[5051] so increasing of AChE by MgO NPs has a good correlation with the increasing of TAP level.

At the end, due to the important role of lymphocytes in the immune system, the level of TNF-α was investigated. The cytokine TNF-α is one of the best indexes inflammatory processes, which is discharged because of immunologic responses from mononuclear cells in human peripheral blood. TNF-α stimulates production of ROS by inhibiting the action of NF-kB, and by creating oxidative stress resulting in cellular lipid and protein peroxidation, and apoptosis.[52] As shown in Figure 5d, the concentration of 100 μg/mL MgO NPs, which was the best dose in decreasing oxidative stress and apoptosis, lessened the CP-induced increase in TNF-α. This confirms that it can markedly protect lymphocytes against CP toxicity.

Taken together, the present results from MgO NPs on human lymphocytes in terms of increasing the viability and decreasing apoptosis, oxidative stress, and also inflammation show the potential of MgO NPs to be applied in future immune deficiency therapeutic strategies.

CONCLUSION

By this study, the toxic effects of CP as an OP compound, on human lymphocytes was investigated and it was proved that it could increase caspase-3 and-9 activities, which lead to increased apoptosis and decreased viability of the cells. Likewise, it can decrease the antioxidant capacity and make inflammation by increasing TNF-α. Interestingly, our results together with the previous studies, demonstrate that MgO NPs in approximately 100-nm diameter not only make cells resistant to the toxic properties of CP but also attenuate bad effects of CP, which is reported for the first time. Accordingly, the use of MgO NPs in low concentration (100 μg/mL) can play antiapoptotic, antioxidative, and anti-inflammatory roles against toxic properties caused by CP in human lymphocytes.

Financial support and sponsorship

Authors wish the support of Iran National Science Foundation.

Conflicts of interest

There are no conflicts of interest.

AUTHOR'S CONTRIBUTIONS

VH provided assistance in the design of the study, performed some experiments, and participated in manuscript preparation. MNN contributed in the conception and design of the work, provided assistance for all experiments, and participated in manuscript preparation. MR and AM participated in the design and manuscript preparation. MB provided assistance for all experiments. MA conceived the whole project. All the authors have read and approved the content of the manuscript.