Rat Hepatic Microsomal Lipid Peroxidation Induced By TNT And Its Metabolites

Introduction2,4,6 -Trinitrotoluene, also known as trinitrotoluene or TNT, is an industrial chemical used as an explosive. It is a flammable solid with the commercial form of yellow monoclinic needles. At room temperature it is highly stable ( detonating only if shocked or heated) , has a low vapor pressure, and is virtually insoluble in water. Chromic human exposure to TNT can result in aplastic anemia, liver damage and cataracts.It is generally been recognized that the one - electron reduction of nitroaromatic compounds by flavin enzyme in the presence of NADPH yields not only its nitro radical but also superoxide anion. The hydrogen peroxide and hydroxyl radical generated from the superoxide anion cause damage to proteins, lipids and nucleic acids. With regard to the biotransformation of TNT, it has been reported that TNT is converted to 4HA and 4A but not a TNT nitro radical. However, it is short of the study on the effect of hepatic toxicity that is induced by TNT and its metabolites. It is reported that NADPH can induce liver microsome lipid peroxidation in vitro. To discover the proposed mechanism, we focus on the reaction of hepatic microsome with TNT and its metabolites using the hepatic microsome model.Materials1. AnimalsFemale Wistar rats, 200 - 250 g, were used in all experiments. Rats were obtained in the Animal Resources Center situated on the campus of the China Medical University ( Shengyang Liaoning Province). The rats had free accessed to food and water with a 12h light -dark cycle.2. ChemicalsNADPH was provided by Biomol Co. TNT was obtained from Fuxin coal mine TNT factory ( Fuxin Liaoning Province) . After recrys-tallized, the purity is more than 99. 9%. 4HA and 4A was obtained from Japanese. All the chemicals were analytical grade.Methods1. Preparation of hepatic microsomeImmediately after sacrifice of the animal, the liver is perfused with ice cold 0. 15mmol/L KC1 through the portal vein, excised, chilled, and homogenized in three volumes of 0. 15mmol/L KC1 Tris - HC1 using a glass homogenizer with a Teflon pestle. The subsequent steps are carried out at 4℃. The 25% homogenate is spun at 10,000 g for 20min and supernatant centrifuged at 105,000 g for 60min in a RC28s ultracentrifuge (SORVALL DUPOND USA) . The pellet is res-perded in 0. 15mmol/L KC1 Tris - HC1 and washed microsomes are obtained by spinning this suspension.2. In vitro lipid peroxidationThe incubation mixture (1 ml) for the estimation of NADPH -induced lipid peroxidation contained 100 ul 10mg/ml microsome (fi-nal concentration 1mg/ml) , 100 ul 5mmol/L NADPH (final concentration 0. 5mmol/L) , 700 ul 5mmol/L MgCl2 in 0. 15mol/L Tris -HC1 buffer (pH7.4, final concentration 0. 1mol/L) , TNT ,4HA and 4A was prepared as a solution from 0. 55 to 140nmol/L (final concentration) in acetone 100 ul or acetone 100ul.3. Estimation of lipid peroxidationAfter incubation at 37C 60 min the malonaldehyde (MDA) formed was estimated immediately by thiobarbituric acid reaction. MDA was detected by UV - visible absorption spectrometry.4. Statistic analysisData were analyzed statistically using AVONA and Dunnett t test.Results1. Differences in content of MDACompared with the control the content of MDA in TNT 0.55 nmol/L group is significantly increased. The content of MDA in TNT 140nmol/L, both 4 HA and 4A 8. 75nmol/ L,35nmol/ L and 140 nmol/ L group is notably lower than that of the control.2. Dose - course of lipid peroxidationIncubation at 37C 60 min, the content of MDA in the group of 0. 55nmol/ L is the highest.3. Time - course of lipid peroxidationIncubation at 37C the content of MDA in the group of 60 min is the highest.4. Addition VitC and GSHAfter adding 10mmol/L VitC (final concentration) or 1mmol/L GSH (final concentration) to TNT ,4HA and 4A, the content of MDAis notably decreased. The incubation mixture is 1ml still.DiscussionThe mechanisms of toxicity of TNT explosives, an important kind of environmental pollutant,...