Volume:09
Issue:02
Year:
2009
Dr Jyotsna A Patil1 MSc PhD (Medical), Dr Arun J Patil1 MSc PhD, Dr Ajit V Sontakke1 MD, Dr Sanjay P Govindwar2 MSc PhD
1 Department of Biochemistry, Krishna Institute of Medical Sciences University, Karad, Maharashtra, Pin-415110
2 Shivaji University Kolhapur, Maharashtra, Pin-416004 India.
Correspondence: Dr Arun J. Patil, Associate Professor, Department of Biochemistry,
Krishna Institute of Medical Sciences University, Karad, District Satara, Maharashtra (India) Pin-415110. Telephone: (R) 91 2164 242321, 91 2164 242312. Email: ajyotsna1@yahoo.co.in
Abstract
This study was undertaken to assess oxidative stress and antioxidant status of sprayers of grape gardens of Western Maharashtra (India). Sixty sprayers of grape gardens (study group) and 30 pesticides-unexposed normal healthy subjects (control group) were taken (age 20 to 45 years) from the Western Maharashtra (India). Demographic, occupational, dietary and clinical data were collected by questionnaire, interview and observation and venous blood samples were collected from both groups.
The serum lipid peroxide level of sprayers of grape gardens (N = 60) was found to be in the range of 2.27 to 6.17 nmol/ml of Mean ± SD, 3.30 ± 0.58 nmol/ml of MDA, whereas that of the pesticides unexposed control group (N = 30) was in the range of 1.68 to 4.50 nmol/ml of Malondialdehyde [MDA] (Mean ± SD, 2.39 ± 0.57 nmol/ml of MDA). This means that the serum lipid peroxide levels of sprayers of grape gardens were significantly increased by 38.07% (P<0.001) as compared to control group. The antioxidant status parameters such as erythrocyte superoxide dismutase, catalase, and plasma ceruloplasmin were significantly decreased by 24.02% (P<0.001), 39.72% (P<0.001), 10.98% (P<0.05) respectively in sprayers of grape gardens as compared to control group. Glutathione Stransferase activity was significantly increased by 80.55% (P<0.01) in sprayers of grape gardens as compared to control group. Serum zinc and copper levels were significantly decreased by 9.06% (P<0.05), 5.37% (P<0.01) respectively in sprayers of grape gardens as compared to control group.
Therefore, this study suggests that exposure to the various pesticides causes an imbalance of prooxidant/antioxidant status in sprayers of grape gardens from Western Maharashtra (India).
Key words:
Catalase (CAT); ceruloplasmin (CP); Cu; environmental health; GST; lipid peroxide (LP);
occupational health; pesticides, sprayers of grape gardens; superoxide dismutase (SOD); Zn.
Introduction
The land used for cash crop like grapes is on the rise particularly in Maharashtra state (India). More pesticides are being used for controlling the various pests in grape gardens and to increase the yield. The environmental pollution and poisoning owing to the widespread use of pesticides during grape cultivation may be disturbing the socio economical status of uneducated farm workers in rural areas (Dave, 1998). Pesticides or their residues are ubiquitous contaminants of our environment and found in air, soil, water, and in human and animal tissue samples from all over the world.
Mainly organochlorines, organophosphorous, carbamates, pyrethroids compounds, and various inorganic compounds are used for controlling the various pests. The common organophosphorous and carbamates pesticides used in grape gardens are Basathrin 25 EC (Cypermethrin 25% EC), Nuvan (Dichlorovas 76% EC), Nuvacron (Monocrotophos 36% EC), Dimethoate 30% EC (CHAMP 30 EC), Phosphamidon 85% SL (Dimecron), Kilex Endosulfan 35% EC, Carbaryl, Cypermethrin 25% EC (JAWAA), Monocrotophos 36% SL, Methomyl.
Pesticides uptake occurs through the skin, eyes, by inhalation, or by ingestion. The fat-soluble pesticides, and to some extent, the water-soluble pesticides are absorbed through intact skin. Sores and abrasions may facilitate uptake through the skin. The vapours of pesticides or aerosol droplets smaller than 5μm in diameters are absorbed effectively through the lungs. Larger inhaled particles or droplets may be swallowed after being cleared from the airways. Ingestion can occur from the consumption of contaminated food or from using contaminated utensils. Contaminated hands may also lead to an intake of pesticides, for example, while pan chewing, tobacco eating, bidi smoking and while spraying, mixing, or handling the pesticides (WHO / UNEP (1990). The dermal exposure is the most important route of uptake of pesticides for exposed workers.
Pesticides are metabolised by oxidation and hydrolysis by esterases and reaction with glutathione, demethylation and glucuronidation may occur. The glutathione transferase reactions produce products that are, in most cases, of low toxicity. Pesticides are mostly eliminated in the urine with lesser amounts in the faeces and expired air.
Headache, fatigue, dizziness, loss of appetite with nausea, abdominal cramps and diarrhoea, blurred vision with watering of eyes, excessive sweating and salivation, bradicardia and twitching of muscles are some of the common signs and symptoms of mild exposures to organophosphate and carbamate insecticides.
Pesticides inhibit a number of enzymes in humans. They affect several physiological systems and processes in the body – the central nervous system (CNS), reproductive, immune, endocrine, cardiovascular and respiratory systems. Not only that, they have an effect on various metabolisms, fluid and electrolyte balance and have carcinogenic potential, particularly in the liver (WHO 1992, 1993).
The adverse effects from exposure to pesticides depends on the dose, the route of exposure, how easily the pesticide is absorbed, and the types of the pesticides, their metabolites, their accumulation and persistence in the body. The toxic effect also depends on the health status of the individual – malnutrition and dehydration are likely to increase sensitivity to pesticides.
The increased formation of reactive oxygen and nitrogen species result in an increase in lipid peroxidation in several tissues mainly the brain, skeletal muscle and red blood cells (RBC) and depleted antioxidant status were reported in several studies of various pesticideexposed populations (Dave 1998, WHO/UNEP 1990). The pesticides may irritate lung macrophages, encouraging them to generate the superoxide radicals and deplete antioxidants status. Therefore, in this study we have planned to see the oxidative stress and antioxidant status of sprayers of grape gardens. To achieve our aim, we have measured serum lipid peroxide, erythrocytes superoxide dismutase, catalase, plasma ceruloplasmin, serum glutathione S-transferase. We also measured serum zinc, and copper levels.
Methods
This study comprises 60 subjects with occupational pesticides exposure i.e. sprayers of grape gardens (study group) and 30 normal healthy subjects, who were not exposed to pesticides (control group). All the study group subjects were in the age range of 20 to 45 years and were taken from Tasgaon Taluka, District Sangli (Western Maharashtra) India. The age-matched normal healthy control subjects working in fields but not performing spraying activities and did not have any kind of pesticide exposure were taken from the same area. Prior to data and biological specimen collection, sprayers of grape gardens were informed on the study objectives and health hazards of pesticides exposure, precautions to reduce pesticides exposure and written consent was obtained from all sprayers and control subjects. Demographic, occupational and clinical data were collected using questionnaires and interviews. All the subjects of the study and control groups belong agricultural families with similar socio-economic status. None of the subjects had a past history of major illness. Dietary intake and food habits of all subjects were normal, which was confirmed periodically by checking their tiffins during their lunch. It was also verified that they had their routine breakfast and dinner. Out of the 60 study group subjects 40% had completed their primary school, 50% had passed high school and 10% attended higher education institutions. Control subjects were selected to provide a similar educational distribution. Subjects who were found to be on drugs for minor illnesses were excluded. Non-smokers, nonalcoholic healthy males, who were occupationally exposed to various pesticides ie sprayers of grape gardens for more than five to 15 years duration of exposure were selected. The study group subjects were engaged continuously four to five hours daily for spraying pesticides in the months of October to January. Blood samples of these subjects were collected in the month of January, because sprayers of grape gardens are most exposed to pesticides at this time of year. The entire experimental protocol was approved by the institutional ethical committee, and utmost care was taken during the experimental procedure according to the Helsinki declaration of 1964. Blood samples were collected by puncturing the anticubital vein into evacuated tubes containing heparin solution as anti-coagulant.
Lipid peroxidation was measured spectrophotometrically by method of Satoh (1978). Serum proteins were precipitated by trichloroacetic acid (TCA) and the mixture was heated for 30 min with thiobarbituric acid in 2M sodium sulfate, in a boiling water bath. The resulting chromogen was extracted with n-butyl alcohol and the absorbance of the organic phase was determined at a wavelength of 530nm. The values were expressed in terms of malondialdehyde (MDA) nmol mL–1 using 1, 1, 3, 3, tetraethoxy propane as the standard.
The activity of erythrocyte superoxide dismutase (SOD) was measured by the method Marklund and Marklund (1988). Superoxide anion is involved in the autooxidation of pyrogallol at alkaline pH 8.5. The superoxide dismutase inhibits the autoxidation of pyrogallol, which can be determined as an increase in absorbance per two minutes at 420 nm. The SOD activity was measured as units mL–1 hemolysate. One unit of superoxide dismutase is defined as the amount of enzyme required to cause 50% inhibition of pyrogllol auto-oxidation.
Erythrocyte catalase was measured by the method of Aebi (1983). Heparinized blood was centrifuged and plasma was removed, and the erythrocytes were washed 2-3 times with 0.9% NaCl and then lysed in 10 volumes of cold deionized water. The whole mixture was centrifuged for 10 min at 3,000 x g. The cell debris was removed and the clear hemolysate was diluted 500 times with phosphate buffer (60 mM) pH 7.4. Catalase decomposes H2O2 to form water and molecular oxygen. In the UV range, H2O2 show a continual increase in the absorption with decreasing wavelength. At 240 nm, H2O2 absorbs maximum light. When H2O2 is decomposed by catalase, then the absorbance decreases. The decreased absorbance was measured at 240nm for every 15 seconds interval up to 1 min and the difference in absorbance ( ∆ A at 240 nm) per unit time is a measure of the catalase activity. The unit of catalase activity was expressed as mM of H2O2 decomposed /mg Hb min–1.
Plasma ceruloplasmin was measured by the method of Herbert and Ravin (1961). Ceruloplasmin oxidises Pphenylenediamine in the presence of oxygen to form a purple-colored oxidised product. The ceruloplasmin concentration was determined from the rate of oxidation of P-phenylenediamine at 37°C at pH 6.0, which has an absorption peak at 530 nm.
Serum Glutathione S-Transferase (GST) was measured by using the Habig et al. (1974) method. The GST activity was determined by measuring the conjugation of 1-chloro-2, 4-dinitrobenzene (CDNB) with reduced glutathione. The conjugation was accompanied by an increase in absorbance at 340nm. The rate of increase is directly proportional to the GST activity in the sample. The serum zinc and copper were measured using a Perkins Elmer model 303 graphite furnace atomic absorption spectrophotometer, which was connected to an Hitachi 165 recorder; values were shown in μg dL–1 (Mert and Henkin, 1971).
Results
See Table 1.0 and Figure 1.0 below.
μmol of conjugate formed/min/mg of protein.Figures indicate Mean ± SD values and those in parenthesis are range of values. * P < 0.05, ** P < 0.01, *** P < 0.001.
Table 1.0 Mean values of lipid peroxide, antioxidants, enzymes and trace elements in sprayers of grape gardens and control groups.
Figure 1.0 Percentage change of mean values of lipid peroxide, antioxidants enzymes and trace elements of sprayers of grape gardens with respect to control group.
Lipid Peroxide (LP), RBC – Superoxide dismutase (SOD), RBC – Catalase (CAT), Plasma Ceruloplasmin (CP), Glutathione S-transferase (GST), Serum Zinc (Zn), Serum Copper (Cu).
Discussion
In this study, we have estimated serum lipid peroxide from 60 sprayers of grape gardens and found statistically significantly increased P < 0.001 (38.07%) as compared to the control group. Increased lipid peroxide level in this study might be caused by various pesticides used in grape gardens. The commonly used pesticides are basathrin, nuvan, nuvacron, dimethoat, phosphamidon, endosulfan, carbaryl, cypermethrin, monocrotophos, and methomyl in Western Maharashtra (India). Most of the sprayers of grape gardens had major complaints of lacrimation, nausea, salivation, snuffling, headache, breathlessness, itching and vomiting.
The role of oxygen free radicals (OFR) has been well established in many chronic disorders, the significance of the implication of OFR in an acute condition like organophosphate (OP) poisoning in sprayers of grape gardens has not been investigated so far. Reactive oxygen species (ROS) are implicated as important pathological mediators in many disorders. Lipid peroxidation constitutes a complex chain reaction of free radicals, which leads to a degradation of polyunsaturated fatty acid in cell membrane (Halliwell and Gutteridge 1986). Present study reveals that the various pesticides increase lipid peroxidation by increasing MDA concentration in plasma of sprayers of grape gardens. Increased plasma MDA concentration in this study definitely accompanied by increased ROS formation. Consequently, enhanced lipid peroxidation, and alteration of antioxidant defence system occurred. Increased lipid peroxide level owing to exposure of OP and carbamate pesticides were reported in several studies (Daves 1998; WHO, 1992, 1993; Patil et al., 2003; Pawar et al., 1978; Prakasam et al., 2001). Moreover, OP, carbamate and endrin increased lipid peroxidation in both liver and kidneys in experimental animals (Pawar et al., 1978). Endosulfan administration also results in a change in membrane permeability of erythrocytes and blood glutathione level (Khanna, et al., 1982).
Pesticides have been shown to initiate peroxidation of lipids in biological membranes (Koryagin et al., 2002; John et al., 2001). In erythrocytes, organophosphate has shown to produce morphological changes that are associated with increased lipid peroxidation (John et al.,2001; Thapar et al., 2002; Altuntas et al., 2002). The effect of lipid peroxidation on membrane lipids, membrane receptors and membrane-bound enzymes can alter the function, structure and fluidity of membranes and may result in altered ion flux (Halliwell et al., 1993). Generation of oxidative stress and consequent lipid peroxidation by pesticides reported in rat and human brain (Verma, and Srivastava et al., 2001; Ranjbar et al., 2002). Increased lipid peroxide levels in sprayers of grape gardens are supported by earlier pesticides exposure studies.
Erythrocytes SOD activity was significantly decreased P <0.001, (24%) in the study group as compared to the control group. Earlier studies in animals have also shown that pesticides such as paraquat, 2,4-D, and endosulfan can inhibit the activity of erythrocyte-SOD and induce oxidative stress in hepatocytes as well as in the central nervous system (Bebe et al., 2003; Yamano et al., 1992;Julka et al., 1992). Moreover, current research indicates that many widely used agricultural chemicals induce oxidative damage in various systems of the body such asin dopaminergic cells of the brain by modulating the antioxidant defence system (Barlow et al., 2005).
The pesticides may irritate lung macrophages, encouraging them to generate the superoxide radical. Pesticides may be more active to an oxygen free radical that re-oxidises to make superoxide, the pesticide may itself be a free radical, or they may deplete antioxidant defences. The overall effect is the production of more free radicals. The superoxide dismutase, glutathione peroxides and glutathione reductase decreased, owing to consumption of enzymes to neutralise free radicals generated by pesticides (Amer et al., 2002).
In the present study, RBC-SOD activity was slightly, though significantly, inhibited in the sprayers of grape gardens. However, it is unclear whether the pesticides or their reactive metabolites suppressed SOD activity. Decreased SOD activity might be caused by the decreased serum zinc and copper levels, since SOD is a Zn-Cu containing enzyme. In this study, we found significantly decreased serum zinc P<0.05, (9%) and copper P<0.01, (5%) in sprayers of grape gardens as compared to the control group. Since SOD is known to be an enzyme induced by its superoxide radical substrate, decreased activities indicates more generation of superoxide radicals in sprayers of grape gardens. Initially, SOD activity may be increased, but owing to prolonged exposure to pesticides, decreased SOD activity was observed in this study.
Erythrocytes catalase activity was significantly decreased P<0.001 (39.72%) in sprayers of grape gardens as compared to control group. Catalase activity in erythrocytes may be explained by their influence on hydrogen peroxide as substrate, which is formed in the process of dismutation of superoxide anion radicals (Shaikh et al., 1999). As catalase is a heme containing enzyme and the fact that pesticides inhibit ALAD activities (Panemangalore and Byers, 1995), which is involved in heme synthesis, it is obvious that pesticides are responsible for the decrease in catalase activity in RBC. Therefore, decreased erythrocytes catalase might be caused by more generation of H2O2 or decreased heme synthesis by the pesticides in this study.
Plasma ceruloplasmin levels were slightly decreased P <0.05, (10.98%) in sprayers of grape gardens as compared to the controls. Ceruloplasmin is a multifunctional enzyme which performs many physiological functions. Ceruloplasmin plays an important regulatory role in iron metabolism whereby it assists the release of iron from cells prior to its uptake by transferrin (Osaki et al., 1971). Ceruloplasmin converts Fe2+ Fe3+ + e- and removes Fe2+ from the blood that may otherwise become involved in the generation of harmful reactive oxygen species. Further, the reduction of Fe3+ to Fe2+ by O2•-would provide a mechanism for the scavenging of O2.The physiological significance of such a mechanism would, of course, be determined by the availability of iron and the relative activities of SOD and ceruloplasmin. SOD is involved first for scavenging the O2, and then ceruloplasmin might be involved. Therefore, decreased plasma ceruloplasmin in sprayers of grape gardens might be caused by more generation O2 by various pesticides.
Serum glutathione-S-transferase activity was significantly increased P< 0.01 (80.5%) in sprayers of grape gardens as compared to the control group. Glutathione-S-transferases are a major family of detoxifying enzymes that catalyze the conjugation of GSH with electophilic centres of lipophilic substrates, thereby increasing its solubility and aiding their excretion from the body (Vontas et al., 2001). Increased GST in this study indicates that the OP and carbamate pesticides are mainly metabolised in the liver and excreted as a conjugate of GSH by the reaction catalysed by GST. A pronounced increase (131%) in the activity of GST was observed in animals chronically exposed to carbofuran (Kaur et al., 2006). Glutathione is a ubiquitous tripeptide that plays a significant role in oxidation-reduction reactions, amino acid transport, detoxification of electrophiles and metals, metabolites of xenobiotics and many carcinogens. Glutathione (GSH) is an endogenous thiol antioxidant that has a multifaceted role in xenobiotic metabolism and is a first line of defence against oxidant-mediated cell injury (Sies, 1999). Studies in animal models suggest that many synthetic organophosphates and organochlorines such as endosulfan and chloryriphos modify the concentrations of GSH (Bebe et al., 2003). The levels of GSH showed a drastic reduction (76%) after acute carbofuran exposure (Cereser et al., 2001). Reduced glutathione is one of the most potent biological molecules that affects the scavenging function in the system. Glutathione together with glutathione dependent systems, glutathione peroxidase (GSH-Px), glutathione-S-transferase, catalase, and superoxide dismutase efficiently scavenge toxic free radicals (Reddy et al., 1984).
Conclusion
This study suggests that pesticides such as basathrin, nuvan, nuvacron, dimethoat, phosphamidon, endosulfan, carbaryl, cypermethrin, monocrotophos, and methomyl cause an imbalance of pro-oxidant/antioxidant status in sprayers of grape gardens from Western Maharashtra (India). This is associated with increased lipid peroxidation with decreased erythrocyte SOD, catalase, plasma ceruloplasmin, zinc, copper and increased glutathione stransferase activities.
The potential risk of pesticides toxicity will persist unless safety measures are taken by the grape gardens owners. The sprayers of grape gardens should use proper protective devices while spraying the pesticides on grape gardens to reduce the pesticides exposure, and regular monitoring is essential to avoid further ill effects through pesticides exposure.
Acknowledgements
We express our deep gratitude to all sprayers of grape gardens and control group subjects who volunteered for this project. We are thankful to grape gardens owners for extending their co-operation.
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