Poly (lactide-co-glycolide), arsenic trioxide, thiobarbituric acid, 5’-5’ dithiobis-2-nitrobenzoic acid, 1-chloro 2, 4-dinitrobenzene and bovine serum albumin were procured from Sigma Chemical Company (USA). Commercial kits for the determination of alanine transaminases, aspartate transaminase, and alkaline phosphatase were procured from Span Diagnostics, Surat (India). Other chemicals and reagents used in this study were of analytical grade and procured from E. Merck (India) and Glaxo (India).
Preparation of arsenic trioxide loaded PLGA nanoparticles
A polymer, PLGA was used to prepare arsenic trioxide nanoparticles with w/o/w double emulsion evaporation technique with minor modifications (Zhao and Zhang 2009). Briefly, the first emulsion was formed between an organic solution of the polymer (100 mg PLGA in 2ml methylene chloride) and an aqueous solution of arsenic trioxide. Then 12ml aqueous solution containing 2.25% of polyvinyl alcohol was added to this primary emulsion and sonicated (Sonics and Materials Inc. USA) to obtain the double emulsion. Afterwards the solvent evaporation was carried out by gentle magnetic stirring at room temperature. The suspension was washed with distilled water and centrifuged three times at 12,000 rpm. Subsequently, the sediments were freeze dried and sterilised. Finally arsenic trioxide PLGA nanoparticles were obtained.
Characterization of As2O3-PLGA nanoparticles, animals and treatments
Physicochemical properties of As2O3-PLGA nanoparticles were verified through transmission electron microscope, scanning electron microscope, Zetasizer and X-Ray diffraction. Due permission from institutional ethical committee was sought before making these experiments. Male albino rats of Wistar strain weighing 250±50g were procured from the animal facility of Jamia Hamdard, New Delhi. They were housed in the animal house of Department of Zoology, Ch. Charan Singh University Meerut (India). All the rats were offered pelleted food (Golden Feeds, New Delhi) and tap water ad libitum and maintained under standard laboratory conditions (room temperature 25±5°C and relative humidity 50±5%). After acclimatization to laboratory conditions for two weeks, the rats were divided into three groups, each containing five rats. Rats of group A were administered a sublethal dose of As2O3-PLGA-NPs dissolved in saline (0.1 mg/100g body weight) by gavage on each alternate day for thirty days. Rats of group B were also given same dose of As2O3 whereas rats of group C were offered saline only to serve as controls.
After scheduled treatments, the rats were starved overnight and sacrificed next morning by light ether anesthesia. Liver was carefully removed and weighed using electronic balance (Sartorius, Germany) and processed for further investigations.
Arsenic accumulation in liver
1g wet liver was digested in 10ml of concentrated nitric acid (A.R. grade) at 80°C for 16 hr. It was diluted to 100ml with double distilled water. 2ml aliquot of the digest was analysed for inorganic arsenic by hydride generation at pH 6.0 using sodium borohydride as the reducing agent. The analyses were performed using atomic absorption spectrophotometer (Electronic Corporation, India). Absorption was recorded at 193.7 nm, using a hollow cathode lamp for arsenic (Electronic Corporation, India).
Small pieces of liver were fixed in 10% neutral formalin at room temperature for 24 hours. They were washed in running tap water overnight. After dehydration, pieces were embedded in paraffin, sectioned (5-6 µ thick) on a rotary microtome and stained with hematoxylin and eosin. The lesions were observed under a light research microscope (Nikon, Japan).
Determination of serum enzymes
Blood was collected from anesthesized rats through cardiac puncture in sterilized tubes. It was allowed to clot at room temperature for about 1 hour. Thereafter, serum was separated by centrifugation and stored at 0°C for further analysis. Serum enzymes aspartate transaminase (AST) (E.C.126.96.36.199), alanine transaminase (ALT) (E.C.188.8.131.52) and alkaline phosphatase (ALP) (E.C.184.108.40.206) were determined according to the method of Rietman and Frankel (1957), using a commercial kit procured from Span Diagnostics (Surat, India).
Lipid peroxidation in the liver of experimental rats was determined by measuring mitochondrial and microsomal malondialdehyde following the method of Jordan and Schenkman (1982). Microsomes were separated using an ultracentrifuge (Sorvel, USA) following the method of Schenkman and Cinti (1978). Thiobarbituric acid reactive substances were measured at 532 nm using a spectrophotometer (Systronics, India). 1’, 1’, 3- tetramethoxypropane (Sigma, USA) was used as the standard.
Glutathione (GSH) was determined in the liver of experimental rats using Ellman’s reagent (5-5’-dithiobis (2-nitrobenzoic acid (Ellman 1959). Sulphosalicylic acid was used for protein precipitation. Absorbance was recorded at 412 nm using a spectrophotometer (Systronics, India).
Glutatione-S-transferase was measured using 1-chloro -2, 4- dinitrobenzene as a substrate following the method of Habig et al. (1974). Enzyme activity was calculated as nm/NADPH/min/mg cytosolic protein. The absorbance was recorded at 340 nm.
CYP450 2E1. (EC 1.14.13)
Microsomes were separated using an ultracentrifuge (Sorvel,USA). Enzyme activity was estimated following the method of Koop (1986). In brief, the reaction mixture consisted of microsomal protein (0.2 mg/ml), 0.1 M potassium phosphate, pH 6.8 and 1 mM p-nitrophenol. Samples were incubated at 37ºC for 3 min prior to the addition of NADPH to start the reaction. After 10 min, the reaction was stopped with 1.5N perchloric acid. Formation of p-nitrocatechol was measured at 510 nm.
Protein content in the liver samples was measured applying the method of Lowry et al (1951). Bovine serum albumin (Sigma, USA) was used as standard.
The data was analysed using SPSS software version 20. P values of less than 0.05 were accepted to be significant.