Journal of Patna Science College Vol. 1, 1 - 10 [2013] ISSN 2347 - 9604 IMPACT OF TWO PESTICIDES ON SERUM FREE AMINO ACID POOL OF MICE: A COMPARATIVE STUDY USING THIN LAYER CHROMATOGRAPHY. #Bipin Bihari Mishra, *Prakriti verma , #S.R.Padmadeo and #Kumud Ranjan Thakur #Post Graduate Department of Biochemistry Patna University, Patna- 800005 *Post graduate Department of Zoology, Patna University, Patna- 800005 [email protected], [email protected] Abstract : An attempt has been made to identify and quantify the serum free amino acids in normal and pesticide exposed mice. The laboratory mice (Mus musculus) were exposed to Dimethoate (o,o- dimethyl,S-methyl-carbamoyl-methyl phosphorodithioate),an organophosphate commonly known as rogor, with a dose of 20 mg/kg body weight and an organochlorine, Endosulfan (6,7,8,9,10,10hexachloro- 1,5,5a,6,9,9-hexahydro-6,9-Methano-2,4,3- benzodixathiepin-3-oxide) with a dose of 2 mg/kg body weight for 21 days. Every week blood were collected, centrifuged and serum were separated to estimate the free amino acids by Thin layer chromatography. Amino acids were located on the chromatogram with a 0.01% ninhydrin solution in acetone. Each chromatogram reveals 4-5 fractions with a wide range of amino acid such as aspartic acid, glutamic acid, serine, tyrosine, alanine, valine. The quantity and presence of each amino acid depends on the doses and the exposure of pesticides. Other amino acids were present in lower concentration and quantitative estimation was not possible. Finding indicates that the exposure of pesticide brings aiteration of free amino acids content in blood. Key words : Rogor , Endosulfan, amino acid, Thin Layer Chromatography, Mus musculus Introduction : It is well known that pesticides are being widely used in agriculture, which has many harmful effects on living organism. Animals in the natural environment are regularly exposed to low concentration of these xenobiotics, which are sub-lethal. Residual amounts of organochlorines and organophosphate pesticides have been detected in the soil, water reservoirs, vegetables, grains and other food products (John et al., 2003). Man is the ultimate consumer of pesticide residues. These pesticide residues in animal products and other food items ultimately get accumulated in the man especially in the adipose tissue, blood and. lymphoid organs. When fed to man or animals at very low doses daily for months or years, these accumulated pesticides in body, may harm the normal functions causing various diseases in man and animals.The widespread use of pesticides in public health and agriculture has caused severe environmental pollution and health hazards including cases of severe, sub- chronic and chronic human poisoning. Endosulfan (6,7,8,9,10,10 hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-Methano-2,4,3- benzodixathiepin-3-oxide) is one of the organochlorine compound used extensively for the control of agricultural pests. Its metabolites have strong tendencies to get accumulated in different organ and tissue of the body e.g. Adipose tissue and liver (Winter and Street,1992; Thao et al.,1993).Deleterious effect of endosulfan have been studied by many researchers (Sinha et al., 1995;Choudhary et al.,2002) but its effect on serum amino acid pool has not been studied. Similarly, Dimethoate (O,O-dimethyl S– methylcarbomylmethyl phosphodithioate),also known as Rogor , is one of the most important 1 Journal of Patna Science College Vol. 1, 1 - 10 [2013] ISSN 2347 - 9604 organophosphorus insecticides used frequently in agriculture. Many studies have been carried on the toxicity of dimethoate on non-target animal like mice (Betrsian et al., 1995; Kamath et al., 2008). Hassan et al. (1994) have reported several changes in serum parameter and amino acid content in rats after chronic sub lethal dose of dimethoate. In the present investigation, an attempt has been made to examine and compare the effect of Rogor and Endosulfan on serum free amino acid pool at different oral dose. Materials and Methods : Test animal - Mus musculus L.(Swiss albino mice):- For the present investigation adult Swiss albino mice Mus musculus were selected. Thirty female albino mice of same age group and average weight of 23 gm were procured from research laboratory; MAHAVIR CANCER SANSTHAN, Phulwarisharif, Patna. The albino mice were housed in poly propylene cages and maintained in controlled temperature (27degree centigrade), humidity (0.5-10%) and light cycle. They were fed with cereal made bread and gold mohar brand animal feed manufactured by Lipton India limited company Delhi and water ad libitum. All the experimental mice were categorized into following groups:- Group I – Normal, Group II – Endosulfan(E) treatment(E7= treated for 7days,E14 = treated for 14 days and E21= treated for 21 days), and Group III – Rogor (R) treatement (R7= treated for 7days, R14 = treated for 14 days and R21= treated for 21 days). Selection of pesticides : Two pesticides of analytical grade were used, one Endosulfan or “Endocel (EC 35%)” manufactured by “Excel Industries Ltd., Ruvapari Road, Bhawanagar (Gujarat)”, and second Dimethoate or “Rogor (EC 30%)” manufactured by “ANU products, old Faridabad (Haryana)”. The oral LD50 value of endosulfan for mice was calculated by standard interpolation method which was 7.36 mg/kg body weight/day(EXTOXNET, 1996). The oral LD50 value of Rogor for mice was calculated by standard interpolation method which was 160 mg/kg body weight (EXTOXNET, 1996). After calculating the LD50 value of endosulfan and rogor, single sublethal dose of endosulfan (2 mg/kg body weight/day ) and rogor (20 mg/kg body weight/day ) were considered ,their stock solution were prepared in distilled water and administrated orally by gavages method for the interval of 7, 14 & 21 days (3 weeks respectively). The controlled group of mice was given only normal saline. After scheduled interval of exposure of 7th, 14th, and 21th day; the test animal were anesthesized, blood sample were collected in different vials, by puncturing ocular vein with the help of sterile syringe. Blood were extracted and refrigerated at -20oC in sterilized paraffin covered tubes for amino acid and biochemical analysis. Serum was analyzed for quantitative estimation of Total protein respectively. Methods for Amino Acids Estimation : Amino acid profile in the blood serum of Mus musculus were assayed using Thin layer chromatography to arrive at Rf (Retardation factor) values of standard amino acid calculated thus:- R = (Wilson and Walker, 2005) Free amino acid were determined by the method of Moore and Stein (1954) using Thin Layer Chromatographic and quantifie d on Systronic UV-Spe ctrophotometer (UV-U75- Spectrophotometer) at 570 nm. 2 Journal of Patna Science College Vol. 1, 1 - 10 [2013] ISSN 2347 - 9604 Amino acid standard preparation : Stock solution of lysine, glutamine, methionine and other amino acid were prepared by dissolving the proper amount of each amino acid in deionised water. Each stock solution was then used to prepare working solution from which a calibration curve was constructed. Preparation of serum for TLC : A collected blood sample were centrifuged at 3000 rpm for 10 min at 4°C to obtain serum that was then deproteinized with sulphosalicylic acid and centrifuged .The supernatant was stored at low temperature pending analysis of amino acid. Qualitative Analysis of free amino acids of the blood serum of normal and pesticide treated Mus musculus was done using Thin Layer chromatography (TLC). Process : The TLC sheets were activiated by heating in an oven for 30 minute at 1000- 1200 C. A line is drawn three centimeter above the bottom and then 8-10 approximately equal small spots applied at 2cm intervals on silica gel coated 6 of TLC aluminium sheets No-1.05554.0007 purchased from Merck KGa A 64271 Darmstadt, Germany Tel= 49(0) 615172-2440,www.merck.de. Using a micropipette, the prepared test solution and standard is taken and lightly dotted a small amount on each pencil marks. The sheet is then left for sometime or wafted swiftly over a blue flame to speed evaporation. This process is repeated 25 to 30 times, applying at the same area, to build up a concentration. Using n - butanol : acetic acid : water in the ratio of (4 : 1 : 5 ) as elutant, a 0.1% ninhydrin in acetone as spraying reagent. The process has been done after approximate 3-6 hrs. Each amino acid was detected through purple colour spots by heating the sheets at 1100 C for 15 minutes and then the Rf value were calculated. Each Chromatogram reveals 4-5 fractions with a wide range of amino acid such as aspartic acid, glutamic acid, serine, tyrosine, alanine, methionine, valine. The spots were identified by comparing it with the Rf value of standard amino acids. To quantify the amount of amino acid in each spot after chromatography, the sheets were sprayed with ninhydrin to identify the spots. The position of corresponding spot in the sheet were scrapped off and then taken in a test tube adding 5 ml of acetone to it. Then 2 ml. of 1% ninhydrin solution were added. The tube were placed on a water bath for 20 minutes, full colour were developed at the end of this period. The coloured solution was transferred to measuring cylinder (10 ml) made to volume and read on Systronic UV Spectrophotometer (UV-U75-Spectrophotometer) at 570 nm with the help of cuvette.. Reading was compared with reading for known solution of amino acids treated in similar manner. Using lysine (1.13mg./ml) run for comparison.Spectrophotometric reading of amino acid present in each spot of chromatogram were taken to know their quantity. The quantity and presence of each amino acids depends on the doses and the exposure of pesticides. Other amino acids were present in lower concentration and quantitative estimation was not possible.