Abstract
Pesticides are used in agriculture to protect crops from insects–pests. Most of the field workers of North Indian population are exposed to commonly used insecticides. In the present study, pesticides induced oxidative stress as well as alterations in the level of acetylcholinesterase (AChE) in a total of 70 male healthy agricultural sprayers, exposed to pesticides for about 5 years, were studied and the results were compared with 70 controls. The levels of antioxidant enzymes (superoxide dismutase, CAT, glutathione-S-transferase and glutathione peroxidase), AChE, lipid peroxidation and glutathione (GSH) contents were determined in their blood erythrocytes (red blood cells (RBCs)). The results indicated significant increase in the levels of malondialdehyde as well as the activities of antioxidant enzymes in pesticide-exposed individuals. The levels of GSH, RBC-AChE activity and plasma antioxidant potential were sharply decreased when compared with control subjects. The ferric-reducing ability of plasma (FRAP) assay was carried out to evaluate the antioxidant potential of pesticide in exposed as well as healthy controls. A significant positive correlation was observed between plasma FRAP value and the activity of AChE from RBCs in pesticides sprayers. Furthermore, these results were supported by enhanced messenger RNA expressions of cytochrome P450 isoform 2E1 (CYP2E1) and gutathione-S-transferase isoform pi (GST-pi) in the white blood cells of the randomly selected pesticide-exposed individuals. The decreased GSH level in human red blood cells accompanied by increase in the levels of the activities of antioxidative enzymes and over expressions of CYP2E1 and GST-pi is an indicative of oxidative stress in pesticides-exposed individuals. The reduced activity of AChE indicates possible occurrence of perturbations in blood as well as neurotoxicity in pesticide sprayers.
Introduction
The increased use of pesticides has caused both environmental and public health concerns.
1
Pesticides are widely used in agriculture to improve production, protect stored crops and control disease vectors. Although pesticides usage has benefits, the health risks have been associated to nontarget subjects including humans who are occupationally and/or environmentally exposed to these agrochemicals.
2
These compounds are known to produce toxicity to different systems in the human body resulting in hematological and biochemical perturbations.
3
In north Indian population, the exposure of field workers to the pesticides such as organophosphates (OPs), carbamates (CMs), organochlorines and pyrethriods is very common. Occupational exposure of farmers in particular to these pesticides occurs via dermal absorption and inhalation.
4
Chronic exposure to pesticides is associated with damage to health as these chemicals are reported to induce oxidative stress via production of reactive oxygen species (ROS) such as superoxide anions (O2
For last two decades, the pesticide-induced oxidative stress as a possible mechanism of toxicity has been a focus of toxicological research. 6 Strong association of aerial exposure of these pesticides to genotoxicity and onset of neurological symptoms in the humans have been reported. 7,8 These reports have also indicated that these compounds may cause alterations in the level of acetylcholinesterase (AChE) from red blood cell (RBC) membrane, micronucleus formation, sister chromatid exchanges in mice and human lymphocytes and chromosomal as well as mitotic aberrations in peripheral blood lymphocytes. 9 –11
Erythrocytes (Red blood cells (RBCs)) have been used as a convenient tool to evaluate oxidative stress induced by xenobiotics and prooxidants in terms of alterations in the biochemical constituents of the membrane. 12 The presence of hemoglobin (Hb), oxygen and polyunsaturated fatty acids makes RBCs prone to oxidative stress leading to the osmotic fragility. The RBC membrane integrity is maintained by a balanced action of the prooxidant chemical species and the antioxidant defense system. 13
The evaluation of xenobiotic-induced oxidative stress in terms of the alterations in the levels of the activities of antioxidative enzymes such as cytochrome P450s (CYPs), superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx) as well as the membrane lipid peroxidation (LPO) and fragility has been widely studied in order to ascertain the extent of ROS-mediated toxicity. 14 The ROS have been shown to cause apoptosis as well as altered expression of xenobiotic-metabolizing enzymes. 15,16 In fact to attain a dynamic homeostasis, a balance between the functions of CYPs, phase II and antioxidant enzymes with the ROS is maintained. The major xenobiotic biotransformation reactions of phase I metabolism are catalyzed by CYP isoenzymes by hydroxylation, desaturation, dealkylation, sulfoxidation and nitroreduction. CYPs are involved in metabolic activation and oxidative metabolism of many endogenous and foreign chemicals resulting in toxic metabolites that in turn produce toxicity to vital organs in experimental animals and humans. However, the phase II enzymes such as GST and uridine diphosphate-glucuronyl transferase are mainly involved in early cellular defense against toxicity. The imbalance in it by any means causes excessive production of reactive intermediates and ROS that may cause damage to the macromolecules. 17,18 The impaired dynamic homeostasis due to increased oxidative stress and DNA damage due to increased production of ROS has been shown to be associated with the pesticide-induced health hazards. 8 These notions have been supported by some studies indicating that the prolonged exposure to pesticides alter the expression and activities of oxidative redox indices (SOD, CAT, GPx, GR, etc.). 19,20
The relationship between free radicals and CYPs is well documented. The CYP enzymes, being the most important xenobiotic-metabolizing enzymes, produce ROS during biotransformation of xenobiotics. Cells generate ROS such as superoxide anion (O2
Various efforts have been made in past in order to predict the underlying mechanism of pesticide-induced toxicity using in vitro and animal model systems. These predictions may or may not be directly correlated with humans. Since the consequences of pesticide exposure could vary from one population to another or one ethnic group to another depending upon the environmental conditions, it is inevitable to assess the toxicity induced by pesticides in North Indian suburban/rural population, who have been using different pesticides as spray to protect their crops from the insects–pests. Using blood cells (RBCs and white blood cells (WBCs)) of the pesticide-exposed individuals, we have shown in the present communication that the field occupants and pesticide sprayers from a section of North Indian suburban/rural population exhibit high level of oxidative stress and increased levels of expressions of cytochrome P450 isoform 2E1 (CYP2E1) and gutathione-S-transferase isoform pi (GST-pi). The information from this work may be exploited for proper strategy design toward adequate pesticide formulation and usage as well as better environmental management.
Materials and methods
Chemicals and reagents
Bromophenolblue, 1-chloro-2,4-dinitrobenzene (CDNB), dithiothreitol, ethidium bromide (EtBr), ethoxy resorufin (ERF), ethylene-diamine-tetra-acetic acid (EDTA), glucose-6-phosphate dehydrogenase (GDH), glutathione (GSH; oxidized), GSH (reduced), H2O2, nicotinamide adenine dinucleotide phosphate (NADPH), 4-nitrocatechol, p-nitrophenol, perchloric acid, pyrogallol, resorufin tetrasodium salt, thiobarbituric acid (TBA) and 2,4,6-tri[2-pyridyl]-s-triazine and Tris-base were purchased from Sigma-Aldrich (India). Reverse transcriptase polymerase chain reaction (PCR) kit was purchased from MBI Fermentas (Amherst , New York, USA). Taq polymerase, Taq buffer, deoxyribonucleotide triphosphates (dNTPs), 100 bp ladder, the forward and reverse primers for cytochrome P450 isoform 1A1 (CYP1A1), CYP2E1, GST-pi and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were procured from Bangalore Genei (Bangalore, India). Some common chemicals were procured locally.
Subjects and sample collection
Institutional Ethics Committee clearance was obtained for collecting the human blood samples. Informed consent was obtained from all the participants prior to their inclusion in the study. The blood samples (5 ml) were collected from 70 male occupational pesticide sprayers on different farms and orchards and from 70 healthy males that have no previous or current exposure to pesticides. All the subjects were residents of Allahabad and Lucknow or its adjacent areas in North India. The groups of pesticides most commonly used by order of frequency were organophosphates (OPs), CMs, organochlorines and pyrethroids. The purpose of the study was explained to all the participants and their consents were taken. A detailed questionnaire including demographic characteristics was recorded (Table 1). A complete health assessment of each participant was also performed during the sample collection. It is important to mention that the farmers included in the study usually did not wear gloves or masks or any other protection devices during spraying the pesticides. They were having previous spraying experience of about 3–8 years. Average exposure duration for sprayers and farmers was 3–4 h/week. Mixing of chemicals with bare hands and leakages from the tanks of pesticide during spraying operations were found to be very common for these individuals (Table 1).
Demographic details about the normal as well as occupational pesticide-exposed subjects.
Sample preparation
Venous blood was collected in the heparinized tubes from both normal and occupational sprayers as coded samples from both the control and sprayers. Samples were transported to the analytical toxicology laboratory in ice-cold condition immediately after the collection. The content was centrifuged at 1000g for 10 min for RBC separation. The buffy coat was removed and the remaining RBCs were drawn from the bottom and then the packed RBCs were washed three times with cold phosphate-buffered saline (pH 7.4). After the final wash, the RBCs were lysed by hypotonic shock and different dilutions were used as hemolysate. 21 The analyses of different biochemical indices were carried out in the hemolysate on the same day.
RNA isolation
The total RNA was extracted from 1 ml blood obtained from controls and pesticide-exposed individuals using standard procedure. In brief, 1 ml blood sample was mixed with same volume of TRI BD reagent kit in ice-cold condition. Chloroform was added to each sample (0.2 ml ml−1 of TRI BD reagent used), mixed and kept at room temperature for 10 min. The sample was centrifuged at 12,000g for 15 min at 4°C. The aqueous phase was collected in fresh tube and isopropanol was added to it (0.5 ml ml−1 of TRI BD reagent used). The mixture was mixed gently and kept at room temperature for 10 min. The samples were centrifuged again at 12,000g for 10 min at 4°C. The obtained RNA pellet was washed with 75% ethanol and centrifuged at 7500g for 10 min. The resulting RNA pellet was dried under electric lamp and dissolved in diethylpyrocarbonate (DEPC)-treated water (RNase-free water) and stored at −80°C till further use. The concentration of RNA was determined by reading the absorbance at 260 nm.
cDNA preparation
Complementary DNA (cDNA) synthesis from isolated RNA was performed using oligo dT primers and RevertAid™ minus Mu-LV reverse Transcriptase Kit (Thermo Scientific, Life Science Research, USA) under standard conditions supplied by manufacturer. In brief, 200 ng of total RNA was mixed with 5 µl of oligo dT primer (1 µg µl−1), and the final volume was made up to 11 µl by the addition of RNase-free DEPC-treated water. The content was mixed gently and incubated for 5 min at 65°C. The mixed content was chilled on ice for 1 min and then 4 µl of 5X reaction buffer (supplied with kit), 2 µl of dNTP and 2 µl of RNase-free DEPC-treated water were added. The mixture was incubated at 25°C for 5 min. Finally, reverse transcriptase enzyme (1 µl) was added to the mix and incubated for 60 min at 37°C. The reaction was terminated by heating the content at 70°C for 5 min. cDNA samples prepared were stored at −80°C until further use.
Expression analysis
Forward and reverse primers for CYP1A1, CYP2E1, GST-pi and GAPDH were synthesized as described previously in the literature. 22 –24 Primer sequences and PCR conditions for above mentioned genes are given in Table 2. The PCR products were visualized on 1.2% (w/v) agarose gel in the presence of EtBr (20 µg ml−1) and the density of the bands was analyzed by computerized densitometry system (Alpha Imager System; Alpha Innotech Corporation, San Leandro, CA, USA) and normalized to that of GAPDH.
Primer sequences to amplify the genes involved in the pesticide metabolism.
CYP1A1: cytochrome P450 isoform 1A1; CYP2E1: cytochrome P450 isoform 2E1; GST-pi: gutathione-S-transferase isoform pi; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; FP: forward primer; RP: reverse primer; bp: base pair.
GSH content
RBC GSH was measured by the method of Beutler. 25 This method was based on the ability of the –SH group to reduce 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), which possesses a molar absorption coefficient (∊ = 1.36 × 104 M−1 s−1) and forms a yellow-colored anionic product whose optical density is measured at 412 nm. The concentration of GSH is expressed in milligram per millililer of packed RBCs.
Determination of SOD activity in human RBCs
The activity of human RBC SOD was determined by slight modification of the method described by Marklund and Marklund. 26 In brief, the assay mixture containing hemolysate (50 µl) was incubated with 2.85 ml of 0.05 M Tris–succinate buffer (pH 8.2) at 37°C for 20 min. Reaction was started by adding 100 µl of 8 mM pyrogallol. The increase in absorbance was recorded at 412 nm for 3 min at 30s intervals. An appropriate blank was also run simultaneously. The SOD activity is expressed in unit (50% inhibition of pyrogallol autoxidation per minute) per milligram of Hb.
Determination of CAT activity in human RBCs
The CAT activity in hemolysate of human RBCs was measured spectrophotometrically by monitoring the decrease in H2O2 concentration over increasing reaction time as described by Aebi. 27 The reaction mixture contains hemolysate diluted 1:50 in 50 mM phosphate buffer (pH 7.0) and 10 mM H2O2 in a quartz cuvette. The decomposition rate of the substrate H2O2 at a final concentration of 10 mmol l−1 was monitored at 240 nm and 25°C after adding directly the sample in cuvette. The CAT activity was calculated in units (micromoles of H2O2 decomposed per minute) per milligram of Hb.
Isolation of WBCs from human blood
The isolation of WBCs was carried out according to the method described by Boyum. 28 The whole blood was centrifuged at 250g for 20 min at 20°C to remove platelets and plasma. WBCs were isolated from the buffy coat by dextran sedimentation and further purified with histopaque density gradient centrifugation at 700g for 30 min at 20°C. WBCs were recovered from histopaque 11191/10771 and washed thrice with Hank’s balanced salt solution (pH 7.4, 138 mM sodium chloride (NaCl), 2.7 mM potassium chloride, 8.1 mM disodium hydrogen phosphate and 1.5 mM potassium dihydrogen phosphate) containing 0.6 mM magnesium chloride, 1.0 mM calcium chloride and 10 mM glucose.
CYP1A1 activity assay in WBCs from human blood
The activity of CYP1A1 was assayed according to the method described elsewhere by Pohl and Fouts 29 and Upadhyay et al. 17 The activity of CYP1A1 was monitored in terms of ethoxy resorufin-o-deethylase (EROD) activity spectrofluorimetrically. The incubation mixture (3 ml) containing phosphate buffer, pH 7.4 (100 mM), glucose-6-phosphate (5 mM), GDH (1–2 units), magnesium sulfate (5 mM), BSA (1.6 mg ml−1), ERF (1.5 µM), NADPH (0.6 nM) and varying concentrations of WBC lysate proteins was incubated at 37°C for 20 min in water bath. Methanol of 2.5 ml was added to the incubation mixture to stop the reaction and vortexed for 30s. The incubation mixture was kept on ice for 2 min. Mixture was centrifuged at 825g for 10 min and the supernatant was measured at 550 nm excitation and 585 nm emission wavelengths and the activity was expressed in picomoles of resorufin per minute per milligram of protein.
Measurement of CYP2E1 activity (PNPH assay) in WBCs lysate
CYP2E1 activity was determined according to the method described elsewhere by Koop, 30 with a slight modification. Reaction mixture containing 4-nitrophenol (0.2 mM) mixed with 50 mM Tris-hydrochloric acid (HCl; pH 7.4), 25 mM MgCl2 and varying concentrations of WBCs lysate proteins was incubated at 37°C for 5 min. A 20-μl of 50 mM NADPH was added to initiate the reaction and was further incubated for 10 min. A 500-μl 0.6 N perchloric acid was added to stop the reaction. After centrifugation at 825g for 20 min, supernatant was taken and a 100-μl 10 N sodium hydroxide (NaOH) was added and the absorbance was monitored at 510 nm. CYP2E1 activity was calculated in terms of nanomoles of nitrophenol hydroxylation per minute per milligram of proteins.
GST activity assay
GST activity was measured spectrophotometrically by the method of Habig et al. 31 using CDNB as substrate. In brief, the assay mixture (3 ml) contained 1 mM CDNB in ethanol, 1 mM GSH, 100 mM potassium phosphate buffer (pH 6.5) and sample aliquots. Activity was determined by measuring an increase in the absorbance of the reaction mixture at 340 nm. Enzyme activity was calculated using the extinction coefficient (∊ = 9.6 mM−1 cm−1) and expressed as unites per milligram of Hb.
GPx activity assay
The GPx activity was determined using the method of Paglia and Valentine. 32 The sample was diluted 1:50 in 100 mM phosphate buffer (pH 7.4) containing 1 mM EDTA and added to the assay tube. The final concentrations of the reagents used in the assay were 0.3 mM GSH, 0.3 mM NADPH, 1.1 U ml−1 GR from Saccharomyces cerevisiae (Sigma, St Louis, Missouri, USA) and 44.1 mM phosphate buffer (pH 7.4). After 3 min of incubation at 37°C, the change in absorbance was monitored at 340 nm after the addition of 1.1 mM (final concentration) tert-butyl hydroperoxide. GPx activity was calculated as unit (micromoles of NADPH oxidized per minute) per milligram of Hb.
Quantification of LPO
LPO was measured according to the method of Esterbauer and Cheeseman. 33 Packed RBCs (0.2 ml) were suspended in phosphate buffer (pH 7.4). The lysate (1 ml) was added to 1 ml of 10% trichloroacetic acid and the mixture was centrifuged for 5 min at 3000 r/min. The supernatant (1 ml) was added to 1 ml of 0.67% TBA in 0.05 mol l−1 NaOH and heated for 30 min at 90°C. The reaction mixture was cooled and the absorbance was recorded at 532 nm. The concentration of malondialdehyde (MDA) in RBCs was determined from a standard plot and expressed as nanomoles per milliliter of packed RBCs.
Determination of frap
The ferric-reducing ability of plasma (FRAP) values were determined following the method of Benzie and Strain. 34 Working FRAP reagent was prepared by mixing acetate buffer (300 mM, pH 3.6), 2,4,6-tri[2-pyridyl]-s-triazine (10 mM in 40 mM HCl) solution and FeCl3ċ6H2O (20 mM) solution in 10:1:1 ratio, respectively. FRAP reagent of 3 ml was mixed with 100 μl of plasma; the content was mixed vigorously so that the contents were mixed thoroughly. The absorbance was read at 593 nm at the interval of 30s for 4 min. Aqueous solution of known Fe2+ concentration in the range of 100–1000 μM was used for calibration. Using the regression equation, the FRAP values (micromoles of Fe(II) per milliliter of the plasma) was calculated.
Determination of the activity of AChE (E.C. 3.1.1.7) in RBCs
The membrane bound AChE activity in the human RBCs was analyzed following the methods of Ellman et al. 35 and Beutler. 25 Packed RBCs were suspended in 0.154 M NaCl and to this suspension, β-mercaptoethanol-EDTA stabilizing solution was added and the hemolysate was frozen overnight. The hemolysate was thawed preceding the experimental procedure. The reaction mixture is composed of 50 mM Tris-HCl (pH 7.4) and 5 mM EDTA with 0.5 mM DTNB solution. The reaction was initiated by adding 0.5 mM acetylthiocholine iodide. The increase in optical absorbance was monitored at 412 nm, 28 ± 1°C and 30s intervals for 3 min using a ultraviolet–visible double beam spectrophotometer (Thermo Scientific Chemito Spectroscan UV 2700, Nasik, India) with quartz cuvette (1 cm light path) against a blank. Measurements were made in triplicate for each blood sample. One unit of AChE activity was expressed as nanomoles of substrate hydrolyzed per minute per gram of Hb under experimental conditions using extinction coefficient (∊) 13.6 × 104 M−1 cm−1.
Determination of protein content
Protein concentrations were determined by the Lowry’s method 36 using bovine serum albumin (BSA) as a standard.
Determination of Hb concentration
The conversion of Hb to cyanomethemoglobin by Drabkin reagent was measured against a standard curve using a known procedure. 37 The results are expressed as grams per 100 ml Hb.
Statistical analysis
The samples were coded at the time of preparation and they were decoded before statistical analysis for comparison. All experiments were carried out in duplicate. Student’s t test was used for comparisons between control and exposed groups. The data are expressed as means ± SD, further differences between control and pesticide worker endpoints means were analyzed using the Mann–Whitney nonparametric test and was used to compare the demographic characteristics of studied populations. The level of significance was set at p < 0.05. All analyses were performed with the Prism 5.01 (GraphPad software, Inc., La Jolla, CA, USA) statistical software package.
Results
The mRNA expression pattern in pesticide sprayers
The results of the analysis of messenger RNA (mRNA) expression of CYP1A1 gene as shown in Figure 1(a) indicated that it was not significantly affected in the pesticide-exposed individuals when compared with healthy controls. The expression profile of a housekeeping gene, GAPDH, has been used as a reference. The band density ratio of CYP1A1 to that of GAPDH presented in Figure 1(b) illustrates the similar pattern. In contrast, the levels of mRNA expression of CYP2E1 and GST-pi genes were observed to be increased in the pesticide-exposed individuals when compared with healthy control. The CYP2E1 mRNA expression is found to be significantly increased in comparison with the control (Figure 2(a)). The band density ratio of CYP2E1 to that of GAPDH presented in Figure 2(b) also reflects the similar effect. GST-pi mRNA expression is also more in the WBCs of pesticide-exposed individuals than the normal controls (Figure 3(a)). The band density ratio of GST-pi to that of GAPDH presented in Figure 3(b) also corroborate this increasing expression pattern of GST-pi gene in occupationally pesticide-exposed individuals.
Expression profiles of CYP1A1 gene (a, upper panel) and a housekeeping gene, GAPDH (a, lower panel). The figures shown in (a) represent levels of mRNA expressed from these genes in the randomly selected control and the sprayer subjects. C1–C5 represent control group and E1–E5 represent group of pesticide-exposed individuals. (b) Bar diagram showing band density ratio of CYP1A1 and GAPDH. (c) Bar diagram showing EROD activity as a spectrofluorometric measurement of CYP1A1 activity in the lymphocytes of the control and sprayer subjects. EROD activity was determined as described in Materials and Methods section. GAPDH: glyceraldehyde 3-phosphate dehydrogenase; EROD: ethoxy resorufin-o-deethylase; mRNA: messenger RNA; CYP1A1: cytochrome P450 isoform 1A1.
Expression profiles of CYP2E1 gene (a, upper panel) and a housekeeping gene, GAPDH (a, lower panel). The figures shown in (a) represent levels of mRNA expressed from these genes in the randomly selected control and the sprayer subjects. C1–C5 represent control group and E1–E5 represent group of pesticide-exposed individuals. (b) Bar diagram showing band density ratio of CYP2E1 and GAPDH. (c) Bar diagram showing p-nitrophenol-o-hydroxylation (PNPH) activity as a spectrophotometric measurement of the activity of CYP2E1 in the lymphocytes of the control and sprayer subjects. PNPH activity was determined as described in Materials and Methods section. GAPDH: glyceraldehyde 3-phosphate dehydrogenase; mRNA: messenger RNA; CYP2E1: cytochrome P450 isoform 2E1.
Expression profile of GST-pi gene (a, upper panel) and a housekeeping gene, GAPDH (a, lower panel). The figures shown in (a) represent levels of mRNA expressed from these genes in the randomly selected control and the sprayer subjects. C1–C5 represent control group and E1–E5 represent group of pesticide-exposed individuals. (b) Bar diagram showing band density ratio of GST-pi and GAPDH. GST: glutathione-S-transferase isoform pi; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; mRNA: messenger RNA.
Levels of the activities of CYP1A1 and CYP2E1 in pesticide sprayers
The activities of CYP1A1 and CYP2E1 from the WBCs were assayed by monitoring EROD and extent of p-nitrophenol hydroxylation (PNPH), respectively, as described in Materials and Methods section. The results presented in Figure 1(c) indicated almost no alteration in the activity of CYP1A1 in pesticide sprayers. In contrast, the activity of CYP2E1 exhibited significant increase in the pesticide sprayers (Figure 2(c)) with a good correlation with mRNA expression pattern (Figure 2(b)).
GSH content in pesticide sprayers
The GSH content in the RBCs was found to be drastically decreased to about half in the pesticide-exposed individuals when compared with the healthy controls. The results presented in Table 3 indicated that the decrease was consistent in all the pesticide-exposed individuals and was found statistically significant (p < 0.001).
Effect of pesticides on the levels of antioxidative indices.a
GSH: glutathione; SOD: superoxide dismutase; CAT: catalase; GST: glutathione-S-transferase; GPx: glutathione peroxidase; LPO: lipid peroxidation; CDNB: 1-chloro-2,4-dinitrobenzene; H2O2: hydrogen peroxide; Hb: hemoglobin.
aThe quantitative estimations of the parameters studied were made in the control as well as the pesticide sprayer subjects as described in Materials and Methods section. The results represent the average values after conducting three independent experiments. Values are expressed as means ± SEM.
bMicromoles of GSH-CDNB conjugate formed per minute per milligram of Hb.
cOne unit is equal to 50% inhibition pyrogallol autoxidation per minure per milligram of Hb.
dMicromoles of H2O2 consumed per minute per milligram of Hb.
eMicromoles of GSH utilized per minute per milligram of Hb.
fSignificant changes are expressed as p < 0.001.
gSignificant changes are expressed as p < 0.01.
SOD activity profile in pesticide sprayers
The activity of SOD in the RBCs was found to be markedly increased to almost two folds in the pesticide sprayers when compared with the healthy controls. The data presented in Table 3 indicated that this alteration was consistent in all the exposed subjects and was found statistically significant (p < 0.001).
CAT activity profile in pesticide sprayers
Similar to the trend in the activity of SOD, the CAT activity in the RBCs was also found to be increased by more than 50% in the exposed individuals than healthy controls (Table 3). This increase was recorded to be statistically significant (p < 0.01).
Patterns of the activities of GST and GPx in pesticide sprayers
The data presented in Table 3 indicated that the activities of GST and GPx in the RBCs of pesticide sprayers were sharply increased when compared with those of healthy controls; the fold increase in their activities being about 2 and 1.5, respectively. The analysis of the data suggests that the increase in these indices was consistent and statistically significant (p < 0.001 for GST and p < 0.01 for GPx).
Lipid peroxidation
In order to investigate the level of oxidative damage due to LPO, the assay for measurement of MDA formation in the RBCs of pesticide sprayers was conducted as described in Materials and Methods section. The results presented in Table 3 demonstrated significant (p < 0.001) increase in the level of LPO in the pesticide sprayers when compared with the healthy controls. The increase was consistent in all the representative exposed subjects analyzed in the study.
Level of total antioxidant capacity in pesticides sprayers in terms of frap
The total antioxidant capacity in the pesticides sprayers was measured in terms of FRAP by the method of Benzie and Strain 34 as described in Materials and Methods section. The results of the present study indicated significant (p < 0.001) decline of 51.97% in the blood plasma antioxidation potential of pesticide-exposed individuals than the normal as shown in Figure 4.
Level of antioxidant potential in the RBCs of pesticides sprayers by assaying FRAP; the values are shown as a function of antioxidant potential in pesticides sprayers when compared with control. Student’s t test was used for comparison between control and exposed groups. The results are presented as mean ± SD values derived from 70 control and 70 pesticide-exposed individuals. FRAP values are expressed as micromoles of Fe (II) per milliliter of plasma. FRAP: ferric-reducing ability of plasma; RBC: red blood cell.
Level of AChE activity in the human RBCs of pesticide sprayers
Since the alterations in the AChE activity during oxidative stress have also been reported, the present study therefore is an attempt to assess the status of the activity of AChE in the RBCs isolated from occupationally exposed pesticides sprayers. The data presented in Figure 5 show low level of AChE activity in the pesticides sprayers when compared with that of controls. The pesticides sprayers registered 35% lower level of AChE activity in the RBCs than the normal individuals.
Level of AChE activity in the erythrocytes of pesticides sprayers. AChE activity was assayed using the procedure as described in Materials and Methods section. Student’s t test was used for comparison between control and exposed groups. The results are presented as mean ± SD value derived from 70 control and 70 pesticide-exposed individuals. The unit of AChE activity is expressed as micromoles of acetylthiocholine iodide hydrolyzed per minute per gram of hemoglobin at 37°C. **The level of significance at p < 0.01. AChE: acetylcholinesterase.
Correlation between AChE activity and total antioxidant capacity of plasma (measured as FRAP)
The results shown in Figure 6 represent the correlation between the activity of AChE in the RBCs and total plasma antioxidant capacity, measured in terms of FRAP values. The decrease in AChE correlates significantly (p < 0.001; r 2 = 0.2568) with decrease in the antioxidant capacity of the plasma in the blood of pesticide sprayers. These results indicated that the decline in plasma antioxidant capacity due to increased pesticides-induced oxidative stress might be contributing in significant reduction in the activity of AChE in association with those of pesticides in the pesticide sprayers (Figure 6).
Correlation plot between AChE activity and total antioxidant capacity of plasma (measured as FRAP). AChE activity and FRAP assays were conducted as described in Materials and Methods section. The unit of AChE activity has been expressed as micromoles of acetylthiocholine iodide hydrolyzed per minute per gram of hemoglobin at 37°C. FRAP values are expressed as micromoles of Fe (II) per milliliter of plasma. p < 0.001; r 2 = 0.2568. AChE: acetylcholinesterase; FRAP: ferric-reducing ability of plasma.
Discussion
Pesticides are known to cause free radical-mediated toxicity in organisms via production of ROS. 8 The detrimental effects caused by ROS occur as a consequence of an imbalance between the oxidative and antioxidant indices in an individual due to pesticide-induced toxicity. 10 Inactivation and removal of ROS depend on the reactions involving the antioxidant defense system. 5 The capacity of antioxidant defense is determined by the contributions of certain vitamins, reduced GSH and antioxidant enzymes. 15 The large human population variations may exist regarding antioxidative capacity of each individual, thus affecting individual’s susceptibility against deleterious oxidative reactions. However, very limited information exists concerning the biological variation in antioxidative enzymes in representative population samples.
The pesticides exposures may directly or indirectly modify the antioxidant defense capability of exposed subjects and thus affect their susceptibility to oxidative stress. Oxidative damage, therefore, may be attributed to the consequences of insufficient antioxidant potential. Pesticides are well known to target RBCs by altering their intactness and fluidity of cell membranes. 13 The susceptibility of RBCs and lymphocytes to oxidative stress due to exposure to pesticides is a function of overall balance between the degree of oxidative stress and the antioxidant defense capability. 10,16 Because of the potential deleterious effects of free radicals and hydroperoxides, perturbations that stimulate LPO and weaken antioxidant defense capability may cause an increase in cellular susceptibility to oxidative damage. 12,14
The involvement of oxidative stress in the alteration of membrane integrity has been well documented. The endogenous cellular antioxidants (GSH) and antioxidant enzymes such as SOD, CAT, GST, GPx and so on are involved in the regulation of dynamic homeostasis and are therefore target of various xenobiotics. 21 Efforts made in past have shown positive association of antioxidant defense system with membrane LPO under the influence of pesticide. 38,39 Among various antioxidant mechanisms in the body, SOD, CAT, GPx and GST are thought to be the major enzymes that protect cells from ROS. There is a suggestion that the activity of antioxidant enzymes may play an important role in determining the pesticide-induced toxicity in occupational sprayers. 4,8,16
We assessed the level of endogenous cellular antioxidant (GSH) and membrane LPO. GSH is actively involved in the removal of LPO products. Enhanced susceptibility of cell damage following exposure to toxic chemicals could be related to the efflux of GSH precursors and hence diminished GSH biosynthesis. 40,41 The concentration of GSH is the key determinant of the extent of toxicant-induced cellular injury. On the other hand, increased peroxide level is linked with membrane disruption in various tissue and organs and has been positively correlated with the gravity of the disease and extent of cellular damage. 42
The results from the present study showed a decreased level of GSH in pesticide-exposed individuals when compared with healthy controls, which could cause accumulation of LPO products that in turn may increase oxidative burden to cellular homeostasis resulting in enhanced cellular damage. This observation got support by the examination of membrane LPO (present investigation) that was considerably increased in sprayers when compared with healthy controls.
GST is a phase II enzyme, which is involved in the detoxification of the products of the phase I reactions. An increased expression of GST-pi and the activity of total GST in sprayers as observed in present study could be a compensatory response to overcome the toxic reactions following pesticide exposure. 19 It may be further supported by the fact that GST has high affinity for the LPO products and help reduce the ROS burden. 39,43
Another enzyme, GPx, is the major antioxidant molecule involved in the catalysis of detoxification of endogenous metabolic peroxides and H2O2. Additionally, it is involved in the redox cycling of GSH in the presence of GR and NADPH as a coenzyme. 43,44 The increased activity of GPx in human RBCs (sprayers) as observed in present study could be due to decreased GSH level and/or increased GPx activity. It probably indicates an adaptive measure to tackle any ROS-mediated toxicity due to pesticide accumulation. However, the increased activity of GPx as detected in the present study also could be due to increased production of H2O2 in pesticide-exposed individuals. 45
Furthermore, other antioxidant enzymes such as SOD and CAT along with GSH, GST and GPx are also known to efficiently scavenge toxic-free radicals and are partly responsible for protection against LPO due to acute or chronic pesticide exposure. 46 Increased levels of these enzymes in the present study reflect an activation of the compensatory mechanism for the protection against ROS through the effects of pesticides on progenitor cells.
Alteration in the activity of phase II and antioxidant enzymes prompted us to assess the expression and activity of CYPs that have been reported previously to be involved in free radical generation and oxidative stress. 19,20 CYP2E1 is reported to enhance the toxic effect of many toxicologically important substrates, including ethanol, carbon tetrachloride and acetaminophen by their biotransformation into reactive metabolites. It is directly associated with free radical production such as superoxides and H2O2 in some previous studies. 47 –49 However, CYP1A1 is reported to be potentially involved in the metabolism of various drugs, carcinogens and xenobiotics. 50 CYPs are also involved in organophosphorus activation through oxidative desulfuration of P=S bonds and the increase in their activity could also represent another contributing factor to the imbalance between injuries and defenses. CYP2E1 are reported to activate N-alkyl-nitrosamines, substances also present in tobacco, whereas there are no strong evidences that CYP1A1 is also involved in this process. 51 –53 In the present investigation, the increased activity of CYP2E1 appeared to constitute a risk factor only for sprayers who smoke because its activity increased in sprayers and not in controls, possibly by the formation of carcinogenic DNA adduct with nitrosamines activation products. 11 Moreover, they can also cause type I diabetes mellitus by their cytotoxic effects on pancreatic beta-cells. 2,54
In the present study, the mRNA expressions of CYP1A1, CYP2E1 and GST-pi were monitored in some randomly selected controls and pesticides-exposed subjects. The results displayed no significant alteration in the expression and activity of CYP1A1, which clearly indicated that this CYP isoform was not involved in this process. However, mRNA expressions and activities of CYP2E1 and GST-pi were considerably increased in pesticide-exposed individuals when compared with the healthy controls, which reflected the ROS-mediated peroxidative damage in pesticide sprayers. The role of CYP2E1 and GST in regulating the level of free radical production is well documented in alcoholics. 47 –49 The present study clearly indicates that the occupationally pesticide-exposed population may be under threat of oxidative stress.
OPs and CMs are known pesticides extensively being used in agricultural practices and fields. They are known inhibitors of the activity of AChEs as they modulate the serine residue present at the active site of the enzyme. It results in an excessive accumulation of the neurotransmitter, acetylcholine, at the nerve endings and cause blockade of nerve impulse transmission. The activity of AChE in human RBCs may be considered as a biomarker for evaluating the central cholinergic status. 2 In addition, the alterations in the AChE activity during oxidative stress have also been reported. The results of the present study are in consonance with these reports as it reflected low level of AChE activity in the pesticide sprayers when compared with the healthy controls.
Conclusion
The results of present study are indicative of impairment in the balance between the oxidative and antioxidant indices resulting in altered cellular homeostasis and physiology in the pesticides sprayers. The enzymes such as CYP2E1 and GST-pi were found to be over expressed, whereas CYP1A1 remained uninfluenced. The present findings also emphasize the need to study the expression profiles of these genes in large population of different ethnic groups occupationally exposed to different pesticides and residing in various parts of world in order to ascertain their roles in ROS-mediated toxicity. Since the occupationally exposed subjects involved in this study also used tobacco, smoking and alcohols infrequently, the contribution of these factors to whatsoever extent in eliciting negative impact of the pesticides may not be ruled out. The information obtained from this study may be used to guide public health laws and policies in the work place and residential communities. It may also be exploited for proper strategy design to minimize the risk of pesticide-mediated occupational health hazards to pesticide users as well as toward adequate pesticide formulation and usage for better environmental management.
Footnotes
Funding
Financial support in the form of research fellowship from University Grant Commission (UGC), New Delhi, India, was provided to RKS. This research was supported in part by a grant to NJS from the Research Center, Center for Female Scientific and Medical Colleges, Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia.
Conflict of interest
The authors declared no conflicts of interest.