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Animal Research International (2005) 2(1): 261 - 266 261SUPEROXIDE DISMUTASE (SOD) ACTIVITY AND SERUM CALCIUM LEVELIN RATS EXPOSED TO A LOCALLY PRODUCED INSECTICIDE “RAMBOINSECT POWDER”OTITOJU Olawale and ONWURAH Ikechukwu Noel EmmanuelPollution Control and Biotechnology Unit, Department of Biochemistry University of Nigeria, Nsukka, EnuguState, NigeriaCorresponding Author: Dr. ONWURAH I. N. E. Pollution Control and Biotechnology Unit, Department ofBiochemistry University of Nigeria, Nsukka, Enugu State, Nigeria. Email: iyknuelo@yahoo.comABSTRACTStudies of superoxide dismutase (SOD) induction in rats exposed to locally producedinsecticide; “Rambo” of which the active chemical compound is permethrin (0.6% w/w) wasperformed. The calcium levels in the blood plasma of the exposed rats were also evaluated.The rats were divi ded into three groups of fi ve rats per cage.Each group of rats was fed with1 %, 5 % or 10 % of the insecticide in their di ets . The control group was fed normal diet. Theeffect of insecticide at various concentrations on superoxide dismutase (SOD) activity in theblood plasma was not significantly different (P > 0.05) in the newly weaned rats (NWR).However, in the middle-aged rats (MAR) and aged rats (AR) groups, the results weresignificantly different (P < 0.05) against the parallel controls. Comparison of the effect of theinsecticide on SOD induction at various concentrations among the groups based on agedifference showed significantly diff erent result (P < 0.05), especially among the groups fedwith 10 % (w/w) of the insecticide in the diet. Serum Ca 2+ level (0.51 ± 0.22mg/ml) increasedf rom newly weaned rats groups to (0.66 ± 0.24mg/ml) in the middle-aged rats and (0.63 ±0.04mg/ml) for the aged rats . The observed Ca 2+ increase was significantly high for rats fed10 % (w/w) concentration of the insecticide in the diet (P < 0.05). This increase tends tosuggest a concentration- dependent effect. The no-observed-effect-concentration (NOEC) wasfound to be 0.006 g permethrin per 100 g of the diet, which is equivalent to 1% of the“Rambo” insecticide per 100g of the feed. Results of this study show that in non-targetorganisms “Rambo” insect powder may induce superoxide dismut ase activity, thus ,suggesting, oxidative-stress related toxicity. The observed increase in calcium ion especiallyat 10 % (w/w) of the insecticide showed that permethrin may induce toxic effects associatedwith cell death via mitochondria uncoupling and loss in ATP metabolism.Keywords: Superoxide dismutase, Calcium ion, Permethrin insecticide, Rambo insect powderINTRODUCTIONIn our enthusiasm for new successes, we haveoften overlooked the fact that we have pollutedour environment and purposely or inadvertentlyhave exposed ourselves and other life forms tohazardous chemicals (e.g. pesticides). Most ofthese chemicals have been implicated as causingcell injury and death especially in non-targetorganisms. (Sotherton, 1991 and Moreby et al.,2001). Toxicity may occur directly as a result of achemical compound being converted to free–radicals or via superoxide anion formation (Bridgeset al., 1983). Free radicals have been implicated inseveral human diseases such as cancer and heartdiseases (Gutheridge, 1994). More so, they causemembrane damage (Onwurah and Eze, 2000), dueto generation of lipid peroxidation product.Detoxification of reactive oxygen species is one ofthe prerequisites of aerobic life, and the multipleline of defence system. The repertoire tocounteract the potentially hazardous reactionsinitiated by oxygen metabolites includes all levelsof protection, prevention, interception and repairs.It comprises non-enzymatic and enzymaticsystems. The enzymes involved in antioxidationare the superoxide dismutase, glutathioneperoxides and catalases (Ledig and Doffoel, 1988).Superoxide dismutase (SOD) catalyses thedestruction (dismutation) of superoxide free –radical ions. These ions are believed to beresponsible for lipid peroxidation and peroxidativehaemolysis of erythrocytes. The action of SODtherefore results in the protection of the biologicalintegrity of cells and tissues against the harmfuleffects of superoxide free radicals (Olusi, 2000).To ameliorate the damage caused by the hydroxylradical formed from superoxide radicals and
OTITOJU Olawale and ONWURAH Ikechukwu Noel Emmanuel 262hydrogen peroxide, organisms have evolvedmechanisms to regulate the concentrations of thetwo reactants. SOD is an important isoenzymefunctioning as superoxide radicals’ scavengers inthe living organisms. Its activity is also induced bydiverse stresses (Bowler et al., 1992), presumably,because of the increase in the concentration ofsuperoxide radicals in cells. SOD is an importantenzyme family in living cells for maintaining normalphysiological conditions and coping with stress.Calcium ions are important and arerequired for many physiological functions.Although, the role of Ca 2+ as a mediator oftoxicant induced cell death has been a subject ofinterest, intracellular Ca 2+ homeostasis is ofimportance to cell viability (Palmeira, 1999). Thecritically important cellular calcium pool forregulation of intracellular events is the cytosolicCa 2+ , and this is control by hormones and growthfactors (Thomas et al., 1984). Loss of the ability torespond to such hormones and growth factors mayresult in cell death (Orrenius et al., 1989). Themechanisms by which Ca 2+ -mobilising hormones,like vasopressin, induce intracellular Ca 2+ transientshave been extensively studied (Kawanishi et al.,1989 and Glennon et al., 1992). Normally,intracellular Ca 2+ homeostasis is maintained by theconcerted operation of cellular transport andcompartmentation systems (Carafoli, 1987).Damages caused by free radicals or superoxideradical on the plasma membrane will lead to a risein cytosolic Ca 2+ concentration, which may causecell injury and finally cell death (Comporti, 1993).Most cells incorporate a variety of veryactive defense and repair systems when exposedto environmental toxicants, and these defensesystems may be overwhelmed during prolongedexposure. The imposed damage may be qualitativeor quantitative in nature. In this study, the effectsof a locally produced insecticide Rambo insectpowder on serum Ca 2+ level and induction of SODwere investigated.MATERIALS AND METHODSTest Sample: The test sample for the experimentwas a locally produced insecticide, Rambo insectpowder that contains 0.60 % (w/w) Permethrin asthe active ingredient. Rambo insect powder isproduced by Gongoni Co. Limited, 89A SharadaIndustrial Estate, Phase 111, Kano, Nigeria.Formulation of Treatment: Differentconcentrations of the insecticide powder in the dietwere prepared by weighing-out a definite amountof growers’ mash (feed) and then mixed with the“Rambo” insect powder. The concentrations of theactive ingredient of the “Rambo” insecticide(permethrin) in the feed were 0.006 g, 0.03 g and0.06 g. This produced either 1 %, 5 % or 10 %(w/w) of the “Rambo” insect powder in the feed.The feed for control contains no “Rambo” powder.All the animals were given sufficient quantity ofwater daily.Procurement and Management ofExperimental Animal: Wilster albino ratsweighing between 120 – 720 g were obtainedfrom the Faculty of Veterinary Medicine, Universityof Nigeria Nsukka (UNN) and maintained on acommercial feed (growers’ mash) for five days inthe animal house of the Department ofBiochemistry, UNN, before the commencement ofthe experiment. The animals were grouped intothree; newly weaned rats (NWR; 2 – 4 weeks,weighing 150 – 185 g), middle aged rats (MAR; 7– 12 weeks, weighing 290 – 335 g), and aged rats(AR; 13 – 16 weeks, weighing 570 – 642 g). Eachgroup was fed with different concentrations of theRambo contaminated diets (1 %, 5 % and 10 %w/w), the control groups were fed with the normaldiet.Protein Determination in the Plasma: Totalprotein concentrations (mg/ml) in the plasma wereanalysed with Follin-Ciocalteau reagent asdescribed by Cunha-Bastos et al. (1999). BovineSerum Albumin (BSA) was used as standardprotein.Superoxide Dismutase Assay: An indirectmethod of inhibiting auto-oxidation of epinephrineto its adrenochrome was used to assay SODactivities in blood plasma (Misra and Fridovich,1971). Auto-oxidation of epinephrine was initiatedby adding 1ml of Fenton reagent prepared asdescribed by Onwurah, (1999) to a mixture ofepinephrine (3 x 10 -4 M), Na 2 CO 3 (10 -3 M), EDTA(10 -4 M), and 1.0ml of deionized water at a finalvolume of 6 ml. The auto-oxidation was read in aspectrophotometer at 480 nm every 30 sec for 5min. The experiment was repeated with 1.0 ml ofthe blood plasma from different blood samplescollected from different groups of animals. A graphof absorbance against time was plotted for each,and the initial rate of auto-oxidation calculated.One unit of SOD activity was defined as theconcentration of the enzyme (mg protein/ml) inthe plasma that caused 50 % reduction in theauto-oxidation of epinephrine (Jewett andRockling, 1993). Superoxide dismutase activity wassubsequently calculated for each sample.Serum Calcium Assay: This was based on themethod of precipitation by chloranilic acid (Cerioti,1974). To 0.5 ml of the serum in a centrifuge tubewas added 0.5 ml of chloranilic acid. This wasmixed thoroughly and centrifuged. The precipitatewas washed in 3 ml of 50 % prapanol–watermixture, and later dissolved in 3.5 ml of citrate
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