Bifenthrin is a type-I pyrethroid with eight stereoisomers; the cis-isomer being the active moiety. The present study investigated the oxidative stress inducing potential of bifenthrin in eight male buffalo calves. Group I, served as control whereas group II animals were orally administered bifenthrin @ 5.0 mg/kg/day for 21 days. Bifenthrin treated animals showed significantly increased lipid peroxidation, evidenced by increased blood malondialdehyde levels from 17th day onwards, with maximum increase of 54.3%. Blood glutathione levels were decreased significantly (16.5%) by the last day of treatment. There was significant increase in the activity of superoxide dismutase on and after 17th day (96.5%) of administration. Similar alterations were observed in the activity of glutathione peroxidase with maximum incline of 28.8% on 21st day. However, no significant change was evidenced in catalase activity. The present investigation suggests that bifenthrin has a potential to induce oxidative stress on repeated sub-acute exposure in buffalo calves.
Pyrethroids, derivatives of carbamic acid, represent a large variety of compounds which have agricultural and husbandry applications as insecticides, herbicides and fungicides. Synthetic pyrethroid pesticides account for over 30% of the global pesticide use and these are preferentially used in place of organophosphates and organochlorines (El-Tawil and Abdel-Rahman 2001). Bifenthrin is a newly introduced type-I pyrethroid with eight stereoisomers, of which the cis-isomer is the active ingredient (Khan et al., 2013a). It is a synthetic pyrethroid with a broad spectrum insecticidal and acaricidal activity used to control wide range of insect pests in a variety of applications. As a synthetic pyrethroid, bifenthrin is characterized by great photostability, insecticidal activity and low mammalian toxicity and it is widely used for agricultural applications (Domagalski et al., 2010). Pesticide ingestion either by direct or indirect exposure may lead to the generation of reactive oxygen species (ROS), which are detrimental to health. To combat the free radical production, living systems possess an antioxidant defense system, which includes enzyme antioxidants like superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase as well as non-enzymes like glutathione, present endogenously, and vitamins C and E, derived from the diet (Jankov et al., 2001). Exposure to xenobiotics could produce an imbalance between these endogenous antioxidants and exogenous reactive oxygen species (ROS), which can subsequently lead to oxidative damage in organisms (Valavanidis et al., 2006).
Indiscriminate use of insecticides has inadvertently polluted the environment. Exposure to these chemicals is likely to induce certain biological changes in both humans and animals (Dar et al.,2014). In India, buffalo is an important dairy animal which contributes to approximately 70% of its milk production. Although toxicological data on bifenthrin is available in few species, yet there is a paucity of information regarding its toxic effects in bovines. The present investigation was therefore undertaken to evaluate the oxidative stress potential of bifenthrin in buffalo calves following its daily oral administration for 21 days.
Material and Method
The experiment was performed on eight healthy male buffalo calves of 6-12 months age and weighing between 140-160 kg. The animals were acclimatized to the animal shed of department under standarized conditions for 4 weeks prior to the commencement of study. During this period, all animals were subjected to deworming with anthelminthics and regular clinical examination. Animals were fed with green fodder, wheat straw and concentrate as per the standard body requirement with free access to clean drinking water. The experimental protocol followed the ethical guidelines on the proper care and use of animals and had been approved by the Institutional Animal Ethics Committee vide Memo No. 2894-98 dated 28.10.2011.
The animals were randomly divided into two groups of 4 animals each and the baseline values of all parameters were determined. Group I receiving no treatment served as the control. Animals of group II were orally administered with bifenthrin @ 5.0 mg/kg/day for 21 consecutive days. The requisite amount of insecticide was suspended in 50 ml of water and drenched to animals of subsequent group daily for 21 consecutive days. The daily oral dose of bifenthrin was selected on the basis of the recommended concentrations used for crop protection, per acre yield of fodder and average daily consumption of fodder by buffalo calves to resemble the levels of the insecticide to which dairy animals are likely to be exposed under field conditions. All the calves were weighed weekly to make necessary corrections in the dosages of toxicants as per body weight. Blood samples were collected in heparinized vials from the jugular vein of animals on 0, 3, 7, 10, 14, 17, 21 day of treatment and 7th day post treatment to study the extent of lipid peroxidation and various antioxidant parameters. Blood glutathione activity was evaluated by the method described by Beutler et al., 1963. Lipid peroxidation (LPO) was quantified as malondialdehyde (MDA) in 33% packed erythrocyte prepared in PBS buffer according to the method described by Rehman (1984). The activity of catalase (CAT) and superoxide dismutase (SOD) in erythrocyte lysate was determined according to the method described by Aebi (1983) and Marklund and Marklund (1974), respectively. The activity of glutathione peroxidase (GPx) in erythrocyte lysate was assayed by the method of Hafeman et al., 1974. The statistical analysis was performed using SPSS® version 16 and independent student t-test was performed between both the groups in order to test significance.
Results and Discussion
The results on effect of bifenthrin on LPO, GSH, CAT, SOD and GPx in buffalo calves are summarized in Table 1.
Table 1: Effect of subacute toxicity of bifenthrine (@ 5 mg.kg-1. day-1, 21 days) on LPO, GSH, CAT, SOD and GPx in buffalo calves
|Control||6.70 ± 0.46||6.50 ± 0.41||7.01 ± 0.44||7.04 ± 0.38||6.82 ± 0.47||6.50 ± 0.54||6.76 ± 0.47||7.14 ± 0.48|
|Bifenthrine||6.35 ± 0.37||6.63 ± 0.56||7.42 ± 0.33||7.81 ± 0.50||8.09 ± 0.48||9.36 ± 0.32*||9.80 ± 0.44*||8.22 ± 0.36|
|Control||460.9 ± 13.1||466.5±16.4||473.8±12.5||463.7±10.6||461.9±9.20||474.8 ± 9.08||482.1±10.0||471.1 ± 10.89|
|Bifenthrine||453.6 ± 9.76||472.0±11.4||498.7±12.0||505.2±15.9||427.7±8.91||391.8±9.08**||378.9±7.58**||431.4 ± 5.83|
|Control||16.6 ± 0.60||16.0 ± 0.43||16.5 ± 0.20||16.6 ± 0.80||16.7 ± 0.55||16.1 ± 0.50||16.3 ± 0.68||16.2 ± 0.44|
|Bifenthrine||16.5 ± 0.67||16.4 ± 0.93||15.8 ± 0.74||15.0 ± 0.80||15.1 ± 0.54||14.3 ± 0.43||13.9 ± 0.45||15.6 ± 0.29|
|Control||7.67 ± 0.61||7.64 ± 0.70||7.82 ± 0.79||7.51 ± 0.52||7.65 ± 0.70||7.89 ± 0.61||7.95 ± 0.53||7.80 ± 0.59|
|Bifenthrine||6.92 ± 0.51||6.58 ± 0.61||6.01 ± 0.65||7.18 ± 0.60||8.72 ± 0.54||13.6 ± 0.47**||11.72±0.56*||8.74 ± 0.56|
|Control||91.2 ± 2.10||93.5 ± 3.11||94.4 ± 2.83||91.7 ± 2.51||89.1 ± 4.16||88.0 ± 2.56||89.4 ± 2.37||90.4 ± 1.44|
|Bifenthrine||88.5 ± 3.25||83.8 ± 5.06||91.3 ± 3.87||80.8 ± 1.79||75.7 ± 1.56||99.9 ± 1.27||114.0±1.31**||95.5 ± 3.29|
Values are mean ± S.E. of 4 animals
Significant elevation in lipid peroxidation, evidenced by increased blood malondialdehyde levels was observed on 17th day of treatment and thereafter, with maximum increase of 54.3% on 21stday of bifenthrin exposure in buffalo calves. However, there was significant decrease in reduced blood glutathione levels from 17th day onwards with maximum decline of 16.5% on 21st day of treatment.
There was significant increase in the enzymic activity of superoxide dismutase, with maximum incline of 96.5% on the 17th day of bifenthrin exposure in buffalo calves. Similarly, significant elevation in glutathione peroxidase activity, to the tune of 28.8% was observed on 21st day of treatment. However, no significant effect was observed in catalase activity. Oxidative stress occurs due to rupture in the prooxidant-antioxidant balance in favour of the former, leading to characteristic changes in all types of biomolecules and results in tissue damage (Mircescu, 2008). Studies have documented the induction of oxidative stress with bifenthrin in rats (Dar et al., 2014) and goats (Khan et al., 2013a; Khan et al., 2013b). However, no significant data is available on bifenthrin toxicity in bovine species.
Increase in lipid peroxidation on bifenthrin exposure in buffalo calves is in agreement with the previous studies conducted in rats (Dar et al., 2013, Dar et al., 2014) and goats (Khan et al., 2013a, Khan et al., 2013b). In addition, an increase in lipid peroxidation was also observed in rats exposed to cypermethrin (Belma et al., 2001) and deltamethrin (Manna et al., 2005) and in an in vitro study on human erythrocytes (Sadowska-Woda et al., 2010). Malondialdehyde is an important reactive metabolite and an indicator of lipid peroxidation, which represents one of the most frequent reactions caused by free radical attack on biological structures as reflected in elevated MDA levels resulting from disturbance of the oxidant/antioxidant balance in the biological system (Omotuyi et al., 2006) referred to as oxidative stress. Determination of lipid peroxidation indirectly reflects the extent to which cell lipid membranes are attacked by free radicals (Rehman et al., 2006). The increase in extent of lipid peroxidation in the present study could be attributed to the formation of free radicals induced by bifenthrin.
Glutathione is important for the detoxification of toxicants, thus measurement of its activity is considered as a good indicator of antioxidant defense status of an individual. Decreased blood GSH levels as observed in the present study have also been observed in rats (Dar et al., 2014), goats (Khan et al., 2013a, Khan et al., 2013b) and broiler chicks exposed to deltamethrin (Jayasree et al., 2003) and rats exposed to cyfluthrin (Omotuyi et al., 2006). Glutathione is a major endogenous antioxidant that participates in detoxification reactions and counter-balances free radical mediated damage by eliminating the compounds responsible for lipid peroxidation or by increasing the efficiency of NADPH that protects detoxifying enzymes (Machlin and Bandlich, 1987). There is an inverse relationship between oxidative stress and glutathione levels due to increase in its utilization (Dubey et al., 2013). Decline in blood GSH levels in the present investigation, can be justified either due to the inhibited synthesis of GSH or increased utilization of GSH for detoxification of toxicant-induced free radicals following sub acute bifenthrin exposure.
Superoxide dismutase is the first and major line of defense against the action of superoxide radicals and other reactive oxygen species. Superoxide dismutase is a copper-zinc containing enzyme that dismutases superoxide ions produced with consequent formation of H2O2. This H2O2 is then either decomposed by catalase or reduced by GSH dependent mechanism catalyzed by GPx. The enhanced SOD activity in the present investigation might play a protective role in reducing oxidative stress by dismutating bifenthrin induced free radicals. The enhanced dismutation of O2– by SOD cause increase in H2O2 production (Liochev and Fridovich, 2007). Catalase is a heme containing enzyme that catalyzes the reduction of hydrogen peroxide into water and oxygen. This enzyme is important in the removal of hydrogen peroxide generated by SOD (Dubey et al., 2013). Although there was no significant change in catalase, yet non-significant decrease was recorded in its activity in the present study.
Glutathione peroxidase (GPx) is selenium containing tetrameric glycoprotein found in erythrocytes of mammals, which helps in preventing lipid peroxidation of the cell membrane. Glutathione peroxidase reduces lipid hydroperoxides to the corresponding alcohols and free hydrogen peroxide to water (Flohe 1999). Although H2O2 is the common substrate for both GPx as well as catalase, the former one has a much higher affinity for it than later from a kinetic point of view. Thus H2O2 is mostly degraded by glutathione peroxidise, as indicated in the present study. Increase in the GPx activity after bifenthrin administration may act as an adaptive response to minimise free radicals through GSH dependent mechanism as seen by enhanced SOD as well as lipid peroxidation. Significant elevation in LPO and alteration in various antioxidant parameters indicate that bifenthrin at selected dose rate can induce oxidative stress on repeated oral administration in buffalo calves. However, most of the changes were reversible as there was no significant variation between control and treated groups on 7th day of post treatment. The findings of the present investigation strongly suggest that sub-acute exposure to bifenthrin produces significant changes in lipid peroxidation and oxidative profile of buffalo calves thereby indicating its ability to alter antioxidant defense in buffalo calves.
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