064 g of 1-methyl-2-phenylindole into 30 ml of acetonitrile to wh

064 g of 1-methyl-2-phenylindole into 30 ml of acetonitrile to which 10 ml of methanol was added to bring the volume to 40 ml. The 37% HCl prepared served as the reagent R2. Preparation of standard The standard (S2) was prepared by dissolving 16.5 ��l of 1, 1, 3, 3-tetramethoxy propane in 10 ml of 20 mM Tris HCl (0.242 g of Tris HCl in 100 ml H2O DW). The solution S2 was diluted to 1:100 in H2O (DW), i.e. 20 ��l of S2 was added to 2 ml of H2O. The final concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 ��M of S2 were prepared according to the method of Siddique et al.[4] The tubes were vortexed after adding 300 ��l of R2 and incubated at 45��C for 40 min. After incubation, the tubes were cooled in ice and centrifuged at 15,000 g for 10 min. The readings were noted at 586 nm, and the standard was prepared. Preparation of larvae homogenate and estimation of lipid peroxidation The larvae (explants) were taken [five larvae per tube; three replicates per treatment in 1.5 ml Tris HCl buffer (ice cold, pH 7.4)] and the homogenate was prepared while keeping the tubes in melting ice. The homogenate was centrifuged at 3000 g for 20 min. 100 ��l of the supernatant, 650 ��l of R1, 100 ��l of distilled water, and 150 ��l of R2 were taken in the microcentrifuge tubes and vortexed. The tubes were incubated at 45��C for 45 min. The tubes were then cooled in melting ice and the readings were noted at 586 nm. Statistical analysis The statistical analysis was done using Statistica Soft Inc, India. The Student’s t-test was applied to observe the significant differences between treatment and untreated groups. Regression analysis was performed using Statistica Soft Inc. RESULTS Figure 1 shows the standard curve for MDA estimation. The regression analysis for the standard shows the ��-coefficient of 0.99958 (P < 0.7689) [Figure 2]. Table 1 and Figure 3 show the mean absorbance value after 24 h of the exposure of CP to larvae. The exposure of 0.0025, 0.025, 0.050, and 0.100 ��l/ml of CP was associated with the mean absorbance values of 0.0440 �� 0.0015, 0.0750 �� 0.0015, 0.0956 �� 0.0012, and 0.1300 �� 0.0011, respectively. The untreated group was associated with the mean absorbance value of 0.0080 �� 0.0005 [Table 1, Figure 3]. Similarly, the exposure of larvae to 0.0025, 0.025, 0.050, and 0.100 ��l/ ml of CP for 48 h was associated with mean value of 0.05600 �� 0.0017, 0.0930 �� 0.0005, 0.1263 �� 0.0020, and 0.1536 �� 0.0046, respectively [Figure 3, Table 1]. The regression analysis was also performed for the treated groups. The value of ��-coefficient (�� = 0.98651; P < 0.135) for 24 h of exposure [Figure 4] clearly shows the concentration effect. The value of ��-coefficient for 48 h (�� = 0.96413; P < 0.02745) [Figure 5] demonstrates the dose as well as the duration effect of CP exposure to third instar larvae of transgenic D. melanogaster (hsp70-lacZ) Bg9.

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