Chlorpyrifos and lambda cyhalothrin-induced oxidative stress in human erythrocytes

Abstract

Pesticides are one of the most potentially harmful chemicals introduced into the environment, and their adverse impacts on non-target organisms can be significant. The present study was conducted to shed light on effects of locally used insecticides chlorpyrifos (CPF) and lambda cyhalothrin (LCT) on oxidative stress biomarkers in human erythrocytes. The activity of catalase (CAT), superoxide dismutase (SOD), and protein contents as well as the levels of malondialdehyde (MDA) and osmotic fragility (OF) were measured in human erythrocytes exposed to CPF at concentrations of 0, 100, 500, 1000, and 2000 ppm and LCT at concentrations of 0, 100, 300, 600, and 800 ppm for 1 h and 3 h at 37°C. MDA levels and OF of erythrocytes were significantly higher in erythrocytes incubated with CPF and LCT at increasing concentrations of both insecticides and increased incubation time. However, erythrocyte CAT and SOD activities were decreased at all concentrations of CPF and LCT tested. Protein oxidation products were decreased at lower doses of CPF (100 and 500 ppm); at higher doses (1000 and 2000 ppm), total protein content was increased compared with control. In contrast LCT was associated with decreased in protein contents at all the concentrations. These results clearly demonstrated that CPF and LCT can induce oxidative stress in human erythrocytes (in vitro).

Keywords

Chlorpyrifos lambda cyhalothrin human erythrocytes oxidative stress

Introduction

Pesticides are a class of chemicals, which are broadly classified as organochlorine, organophosphate (OP), carbamates, and pyrethroids. They are widely used to control insect pests of agriculturally important crops. Their excessive use, in the last few decades, has resulted in degradation of the environment including air, water, and soil (Aktar et al., 2009). Also they have negatively affected mankind directly or indirectly. They are known to disturb the biochemical and physiological functions of erythrocytes thereby affecting membrane integrity and inducing the production of reactive oxygen species (ROS) and oxidative tissue damage (Mansour et al., 2009). Pesticides used in the present study belong to two different classes of insecticides, OP (chlorpyrifos (CPF)) and synthetic pyrethroid (lambda cyhalothrin (LCT)). OP insecticides are one of the most widely used insecticides in agriculture and public health accounting for 50% of global insecticidal use (Casida and Quistad, 2004). Because of the extensive use of OP pesticides in agriculture, there is an increased risk of human exposure to these chemicals (Joshi et al., 2007). CPF (O,O-diethyl O-(3,5,6-tricloro-2-pyridinol) phosphorothionate) is a broad spectrum chlorinated OP insecticide utilized extensively in agriculture and residential pest control throughout the world (Cox., 1995; Mehta et al., 2009; Mitra et al., 2008), even though the United States Environmental Protection Agency restricted some of its domestic uses in 2000 based on human health risk (Iyer et al., 2008). CPF is a well-known acetylcholinesterase inhibitor and causes accumulation of acetylcholine resulting in excessive stimulation of post synaptic receptors and consequent signs of toxicity (Al-Badrany and Mohammad, 2007; Zheng et al., 2000). CPF produces moderate acute toxicity in mammals, with rat oral median lethal doses (LD50) of 82–155 mg/kg, and it is classified as moderately hazardous (class II) by the World Health Organization (WHO) (Iyer et al., 2008).

Previous studies reported that OP pesticides (e.g. CPF) can be accumulated and excreted in human milk, and their concentration in milk is proportional to their solubility and lipophilicity. Some estimates for pregnant women and children indicate that CPF exposures exceeded the non observable adverse effect level (NOAEL), even in scenarios of common use (Mansour and Mossa., 2011). CPF treatment in some other studies resulted in increased oxidative stress, as evidenced by enhanced levels of thiobarbituric acid reactive substances (TBARS), accompanied by decreased levels of superoxide scavenging enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in serum, liver, kidney and spleen (Bebe and Panemanogalore, 2003; Verma, 2007; Mansour and Mossa, 2009, 2010).

Synthetic pyrethroids account for more than 30% of insecticides used worldwide in agricultural, domestic and veterinary applications (Naglaa, 2012). These compounds are neurotoxins inducing a transient increase in the sodium permeability of the nerve membrane during excitation (Yavuz et al., 2012), which results in continuous nerve stimulation whereby poisoned organisms lose control of their nervous system and are unable to produce coordinated movement.

LCT, a synthetic type II pyrethroid, is widely used and valued for its broad-spectrum control on a wide range of pests in a variety of applications such as the protection of cotton, cereals, and vegetables as well as in public health application against insect, ticks, and flies, which may act as disease vectors. It is classified as moderately hazardous (class II) by the WHO with rat oral LD50 of 56–79 mg/kg. Residues of LCT have been reported in vegetables and fruits, milk, and blood of dairy cows and also in beef. Consistent with its lipophilic nature, LCT has been found to accumulate in biological membranes leading to oxidative damage by altering antioxidant systems and increasing lipid peroxidation (LPO) in mammals (Naglaa, 2012).

Erythrocytes are particularly sensitive to oxidative damage due to the presence of high polyunsaturated fatty acid content in their membranes and high cellular concentrations of oxygen that make them a convenient cellular model suitable for studies of the toxicity of xenobiotics (Balaji et al., 2014). Pesticide chemicals induce oxidative stress, leading to generation of free radicals (Abdollahi et al., 2004) that can cause cellular damage by reacting with polyunsaturated fatty acids in cellular membranes, nucleotides in DNA, and critical sulfhydryl bonds in proteins. Oxidative stress resulting from increased production of free radicals and ROS and decreases in antioxidant defense leads to damage of biological macromolecules and disruption of normal metabolism and physiology (Mansour and Mossa, 2011). Therefore, the objective of the present study was to investigate the in vitro effect of CPF and LCT on oxidative stress biomarkers in human erythrocytes in time- and dose-dependent manner.

Materials and methods

Preparation and treatment of erythrocytes

Before the start of the experiment, the study was approved by the departmental ethical committee. Blood samples from ten healthy individuals (50% female and 50% male), mean age of 24 ± 3 years with no known hematological/neurological disorders, were collected in heparinized tubes. A full blood count test was performed to check the individuals’ general health before further processing. The samples were analyzed for hematological parameters such as packed cell volume, hemoglobin (Hb), total red blood cells (RBCs), mean cell volume, mean corpuscular Hb, total white blood cells, and total platelets using an automatic hematological assay analyzer (data not shown; BC-3000Plus Mindray Auto Hematology Analyzer).

Samples were then centrifuged at 3000 r/min for 10 min at 4°C for isolation of erythrocytes. The plasma and buffy coat were removed. The erythrocytes were washed three times with cold isotonic saline and centrifuged. After the final wash, the packed erythrocytes were resuspended in phosphate buffer (0.1 mol/L, pH 7.4).

The washed erythrocytes were incubated with various concentrations of CPF (100, 500, 1000, and 2000 ppm) and LCT (100, 300, 600, and 800 ppm) for 1 and 3 h at 37°C. The specific doses of CPF and LCT were selected after screening below, within and above the recommended use of these insecticides in fields and to check the time-dependent effect of the insecticides, erythrocytes were incubated for a minimum 1 and maximum 3 h in diluted solutions containing various doses of both the insecticides. Samples of erythrocytes and phosphate buffer without pesticide stress were used as control.

Pesticide-induced erythrocytes osmotic fragility

Pesticide-induced hemolysis of erythrocyte membrane was done according to Sharma et al. (2009). Osmotic fragility (OF) test was measured by adding 50 µl of washed erythrocyte suspension preincubated with various concentrations (100, 500, 1000, and 2000 ppm) of CPF and LCT (100, 300, 600, and 800 ppm) at 37°C for 1 and 3 h containing 5 ml of 0.1%, 0.5%, and 0.9% saline solutions (pH 7.4). After incubation, the tubes were centrifuged at 5000g for 10 min to pellet the hemolysed cells, and the absorbance of the supernatant was measured at 540 nm. The percent hemolysis for each sample was calculated based on the formula:

Percent hemolysis = \frac{Optical density of test solution}{Optical density of standard solution} \times 100

Determination of MDA contents in erythrocytes

The product of LPO, malondialdehyde (MDA), in control blood samples as well as in pesticide treated blood samples at varying concentrations was determined by the method of Stocks and Dormandy with little modification (1971). Briefly, 0.5 ml of erythrocytes in phosphate buffer (pH 7.4, 0.1 M) was incubated for 1 and 3 h at 37°C at varying concentrations of both the pesticides and centrifuged. To the supernatant, a 2 ml MDA solution was added and then placed in a boiling water bath for 15 min. Contents were cooled in ice water and centrifuged for 15 min at 2500 r/min. The absorbance was taken at 532 nm and the level of LPO was expressed in absorbance units.

Determination of antioxidant enzymes activity

Superoxide dismutase

SOD activity was determined spectrophotometrically using protocol of Beauchamp and Fridovich (1971). The reaction substrate of 250 ml (Nitro blue tetrazolium (NBT): 15.5 mg, riboflavin: 0.02 mg, sodium ethylenediaminetetraacetic acid: 10 mg, and methionine: 485 mg) was prepared. For SOD determination, a reaction of 3 ml contained 2.725 ml reaction substrate, 25 µl of the supernatant, and 25 µl hydrogen peroxide (H₂O₂), and were placed under light at 4000 lux for 20 min. The reduction of NBT by superoxide radicals to blue color formazan was measured at 560 nm.

Catalase

The CAT activity of the hemolysate was assayed by adopting the method of Sinha (1972). Samples treated with various concentrations of CPF and LCT were centrifuged, and the supernatant was used for CAT activity. Briefly, 0.1 ml of erythrocytes suspension and 0.4 ml of H₂O₂ was added to 0.9 ml phosphate. The reaction was stopped after 60 s by adding 2 ml of dichromate acetic acid mixture. The tubes were kept in a boiling water bath for 10 min, cooled, and the color developed was read at 530 nm.

Determination of protein content

Protein content of erythrocytes treated with CPF and LCT was determined using the Bradford assay (1976). Reaction solution containing 0.5 ml Bradford reagent and 2 ml distilled water was prepared, and supernatant (20 µl) was added to the reaction solution with absorbance recorded at 595 nm. All results were expressed as percent of control erythrocytes.

Statistical analysis

The statistical analysis was performed using Statistix 9 data analysis software. Hypothesis testing methods included one-way analysis of variance followed by least significant differences. The difference was considered significant if p value was less than 0.05 (p < 0.05).

Results

Hemolysis studies on erythrocytes

There were significant changes in the degree of erythrocytes OF at 0.1%, 0.5%, and 0.9% sodium chloride (NaCl) concentration (Figure 1). Irrespective of the treatment groups, percentage hemolysis decreased with increasing NaCl concentration. Maximum hemolysis was observed at 0.1% NaCl with increasing concentration of both the pesticides (Figure 1(a) and (c)). Exposure to CPF induced erythrocytes OF to 55.5%, 59.6%, 72.9%, and 86.6% that was significantly greater (p < 0.05) as compared to the control 52.43% at 0.1% NaCl. By incubating the cells for 3 h both the pesticides induced more hemolysis of RBCs compared to 1 h incubated erythrocytes at increasing dose of CPF and LCT at 0.1%, 0.5%, and 0.9% NaCl concentration (Figure 1(b) and (d)). However, minimum hemolysis was observed at physiological saline (0.9% NaCl) that was 8.4% by CPF and 7.4% by LCT at 100 ppm but still that was statistically significant (p < 0.05) compared to the control erythrocytes (4.1%).

LPO in hemolysate

MDA, the end product of oxidative destruction of lipids was significantly increased in human erythrocytes incubated with various concentrations of CPF and LCT in dose- and time-dependent manner as depicted by increase in absorbance level at 532 nm. The results after 3 h exposure to CPF showed significant increase in LPO by 2.2% at 100 ppm, 18.8% at 500 ppm, 25.9% at 1000 ppm, and 16.6% at 2000 ppm as compared to 1-h treatments (Figure 2(a)).

Erythrocytes incubated with LCT also showed significant increase in absorbance revealing the oxidation of membrane lipids induced by LCT at various concentrations. The maximum absorbance was recorded at 800 ppm that was increased by 10.6% after 3 h incubation (Figure 2(b)).

SOD in hemolysate

Incubation of human erythrocytes with CPF and LCT resulted in significant reduction (p < 0.05) in SOD activity in comparison to control erythrocytes. The effects were concentration- and time-dependent. At 100 ppm, SOD activity was decreased to 11.45% by CPF after 1 h incubation while LCT showed decrease in SOD by 7.35% under same concentration and incubation time. The results after 3 h incubation depleted SOD activity further by 5% at 100 ppm, 2.7% at 500 ppm, 14% at 1000 ppm, and 22% at 2000 ppm of CPF (Figure 3(a)). In case of LCT, maximum decrease in SOD activity was recorded at the highest dose (800 ppm) after 3 h incubation that was 28.5% compared to the control (100%) (Figure 3(b)).

CAT in hemolysate

CAT activity was found to be significantly decreased (p < 0.05) in erythrocytes incubated with CPF and LCT in comparison to the control erythrocytes. CPF showed 42.3% decrease in CAT activity at the highest concentration used at 1 h incubation that was further reduced to 7% by incubating erythrocytes for 3 h (Figure 3(c)). The maximum reduction in CAT activity of LCT treatment groups recorded was 47.2% at 800 ppm that was further decreased by 8.7% with increased exposure time to 3 h (Figure 3(d)).

Total protein content

Total protein content decreased in erythrocytes at 100 and 500 ppm of CPF while it increased at higher concentrations of CPF, that is, 1000 and 2000 ppm. Maximum decrease recorded after 3 h incubation was 10.6% at 100 ppm and 19.6% at 500 ppm compared to the control. Higher concentration of CPF, that is, 1000 and 2000 ppm showed significant increase in protein contents of 30.4% and 47.33%, respectively, as compared to the control (Figure 2(c)). In all LCT-treated samples, the protein contents decreased with increased concentration of LCT and incubation time. Maximum recorded decrease was 38.32% at 800 ppm after 3 h incubation (Figure 2(d)).

Correlation between MDA content and various studied parameters

CPF and LCT showed positive correlation between MDA content and OF of erythrocytes in all treatments providing evidence that LPO of membranes can be indirectly measured using OF test. With increase in MDA content, OF of erythrocytes increased with increase in dose of CPF and LCT (Figure 4(a) and (b)).

Negative correlation was found between LPO and antioxidant enzymes CAT and SOD. Both enzymes showed decreased activity with increase in MDA contents in all CPF- and LCT-treated samples (Figure 4(c) and (d)).

Principal component analysis

Principal component analysis (PCA) was applied to the data set to further explore the correlation among the various studied parameters. On the loading plot, the variables T1C, T3C, MIC, M3C, MIL, M3L, O91L, O93C, O11C, O11L, O13C, O13L, O51C, and O53L showed similar behavior (positive correlation) under pesticides stress (Figure 5) while the variables SIC, S3C, S1L, S3L, C1C, C3C, CIL, C3L, TIL, and T3L exhibited negative correlation with group I under CPF and LCT stress.

Cluster analysis

Hierarchical cluster analysis (CA) was performed on the standardized date set by applying Ward’s method. The CA results for the various studied parameters are shown in Figure 6 as dendrogram. In agreement with the PCA, CA identified two clusters of variables. Case 1–4 and case 25–28 are included in group I while the cases 5–24 belong to group II showing similarities with each other.

Discussion

The study of oxidative stress as a possible mechanism of toxicity for various pesticides has become a focus of toxicological research for many investigators, since this biological phenomenon has been involved in the etiology of certain human diseases such as cancer, immunosuppression, and neurodegenerative diseases (Banerjee et al., 2001; Dennog et al., 1999; Koner et al., 1998; Mayne, 2003; Pong, 2003). Oxidative stress in pesticide exposure is evidenced by increased concentration of blood MDA and TBARS, changes in antioxidant status, and altered activities of cellular enzymes (Aly et al., 2010). The present study was done to explore the toxicological changes in human RBCs by measuring the level of LPO, OF, antioxidant enzymes, that is, CAT and SOD, and protein oxidation after pretreatment with various concentrations of the insecticides, CPF, and LCT.

The OF test measures the stability of erythrocytes by testing the cells’ ability to withstand hemolysis in decreasing concentrations of saline solution. Erythrocyte OF can be influenced by various factors that include mechanical damage of the membrane, RBC age, changes in RBC shape, hemoconcentration and acidosis, elevation of body temperature, exercise stress and catecholamine lysolecithin, peroxidation of the erythrocyte membrane, and any factor inhibiting or attenuating glycolysis can cause hemolysis. The insecticides tested in the present study caused increased OF of erythrocytes at concentration limits recommended for agricultural purposes. This demonstrates that insecticide exposure compromises the integrity of the RBC membrane. The change in RBC membrane integrity may have arisen from the increased lipoperoxidative changes as indicated by increased MDA concentration.

These results agreed with those reported in earlier studies that demonstrated increased lipoperoxidative changes and reduction of endogenous antioxidant enzymes in human erythrocytes exposed to CPF (Singh and Bhatia, 2013). Significant increase in erythrocyte OF has been reported following CPF exposure (Ambali et al., 2010, 2011). LPO decreases hydrophobic characteristics of bilayer membrane of erythrocytes changing the affinity and interaction of proteins and lipids impairing the function and homeostasis of erythrocytes (Dargel, 1991). ROS can directly affect the conformation and activities of all sulfhydryl-containing molecules by oxidation of their thiol moiety (Wilcox et al., 2001).

Rodrigues et al. (2011) published a similar trend of 100% erythrocyte hemolysis with the pesticide Roundup at concentration limit recommended for agricultural purpose. Uchendu et al. (2011) reported that LPO can be measured indirectly by erythrocyte OF as a molecular mechanism implicated in CPF poisoning.

The decrease in the SOD and CAT activities in response to CPF insecticide has been reported in previous studies (Aly et al., 2010; Ambali et al., 2011; Tuzmen et al., 2008) and may reflect the level of oxidative damage caused by the pesticide. SOD is involved in dismutation of the oxygen ion (O₂ ⁻) to H₂O₂ and oxygen, and the significant decrease in SOD measured in erythrocytes exposed to CPF may be due to elevated degradation or inactivation of the enzyme. CAT neutralizes H₂O₂ and converts it to H₂O and oxygen (O₂) and the significant decline in the CAT activity observed in erythrocytes exposed to CPF may be due to the reduced conversion of O₂ ⁻ to H₂O₂ by SOD thereby resulting in the accumulation of O₂ ⁻. This accumulated O₂ ⁻ inhibits the activity of CAT. Superoxide radical O₂ ⁻ converts ferroxy state of CAT to ferryl state, which is an inactive form of the enzyme thereby exacerbating the free radical-induced damage to the body tissue (Kono and Fridovich, 1982).

LCT metabolism generates ROS which in turn leads to increased LPO and oxidative stress (Piner and Ünerb, 2012). Therefore, the present study suggests that oxidative damage induced by LCT is attributed to its lipophilic nature as it could penetrate the cell membrane easily and accumulate in biological membranes leading to increased production of ROS. In this aspect, these radicals can destroy proteins, lipids, and DNA by oxidation and attack the cell membrane leading to destabilization and disintegration of cell membrane and decrease its fluidity as a result of LPO and protein oxidation. In accordance with the data obtained from this study, El-Demerdash (2007) and Fetoui et al. (2009) reported that LCT administration resulted in a significant increase in TBARS production.

Decreased activities of antioxidant enzymes were observed in rat liver and in rat kidney by Fetoui et al. (2009, 2010) and in fish liver (Oreochromis niloticus) (Piner and Ünerb, 2012). They indicated the failure of antioxidant defense systems to overcome the increase in ROS induced by LCT exposure. However, data are not available from accidental poisonings, work-related exposures, or other human studies regarding LCT toxicity.

Both principal component analysis (PCA) and CA were used to identify the relationship of studied parameters in a cumulative way. In the present experiment, correlations by PCA revealed that two principal components collectively account for 98.86% of the total variation present in the recorded data. All parameters were mostly present in component 1 explaining strong association with one another. Those variables, which are on positive side and near to 1 and close to one another are having the most relevant anomalies and vice versa. As a whole in component 1, all variables are positively correlated. In component 2, all variables are negatively associated.

In conclusion, data from the present in vitro study demonstrated that insecticides CPF and LCT are capable of inducing hemolysis and LPO in human erythrocytes via oxidative stress mechanisms. The insecticides significantly affected the erythrocytes OF, MDA, erythrocyte proteome, and antioxidant machinery in a time- and dose-dependent manner.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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