Inflammatory stress and idiosyncratic drugs hepatotoxicity in rabbit

Back ground: idiosyncratic drug hepatotoxicity is none or time related, unpredictable, occurs infrequently and can be fatal. It was proposed that inflammatory or oxidative stress occurs randomly in patients even after asymptomatic incidence can precipitate drug hepatotoxicity. Aim: To measure hepatotoxicity of diclofenac in rabbit serum following the incidence of inflammatory stress by lipopolysaccharide (LPS) and correlate this to various stress parameters as Malondialdehyed (MDA). Method: 24 rabbits were divided into four groups (6 each) according to type of treatment. Group 1: control (received normal saline). Group 2 received diclofenac sod. (5mg/kg, orally 3times daily for three days). Group 3: received lipopolysaccharide (150μg/kg, i.v, 24 hours before killing. Group 4: received diclofenac sod. + Lipopolysaccharide (5mg/kg orally + 150μg/kg, i.v 24 hours before killing). Then for each animal were measure, liver M D A, liver enzymes. Conclusion: LPS potentiated the hepatotoxic effect of diclofenac sod. The effect is mediated by oxidative and inflammatory reactions as demonstrated by increase in liver tissue M D A. Introduction Drug induce hepatotoxicity is an important human problem.In United Kingdom recent study found that adverse drug reaction responsible for more than 6% of hospital admission and the mortality rate is approximately 2% (1),and it is expected that the real incidence might be greater than estimated by current method (2), (3)). Withdrawal of some efficacious drugs due to idiosyncratic hepatotoxicity leads to deficits in treatment, an example of this is felbamate (4).which was effective in treats sever cases of epilepsy . It’s used markedly reduce because of its association with a plastic anemia and hepatotoxicity in some patients (5).Idiosyncratic adverse drug reaction that target the liver are common cause of acute liver failer in the united states according for more than 10% of cases , the results of 5 years prospective study indicate that many dietary supplement and drugs with different pharmacological targets are associated with idiosyncratic hepatotoxicity.(6).Andrade et al 2005 (7) cited that anti – infective, and musculo skeletal and gastro intestine tract drugs are the main group of drugs, which can cause hepatotoxicity. Material and Methods a. chemicals: Thiobarbituric acid (BDH ,chemical ,Ltd. Pool, England.)Trichloro acetic acid (T C A) from Thomas Baker, Ltd, India .Lipopolysacharide (LPS) from SigmaAldrich, France. b. Drugs: Diclofenac sodium : (100 mg/tab) (triveni chemicals; India); powdered and dissolved in 100 ml distal water to made stock solution contain (1 mg/ml). The drug was given to the rabbit by oral rout in dose of 5 mg/kg three times daily for three days. Lippolysaccharide of E.coli, as powder (vial contain 1o mg) which equivalent to (10.000 μg), dissolved in (100 ml) 0f 0.09% normal saline to make a stock solution has concentration equal to (100μg/kg). The dose used low than that hepatotoxic dose of lippolysacchride of (150 μglkg) AL-Qadisiya Journal of Vet.Med.Sci. Vol./12 No./2 2013 8 c. Animals: the experiment was carried out on 24 local bred sexually mature male rabbits. The range of body weight was (1 1.5 kg), the animal were housed in special room for acclimatization. Rabbits were maintained on free access to food and drinking water. Groups of animals were divided randomly into four groups as following: Group (1) received normal saline (0.9%) Group (2) received diclofenac sod. (5 mg/kg orally, three times daily for three days). Group (3) received lipopolysaccharide (150μg/kg, i.v, 24 hours before killing). Group (4) received diclofenac sod. + Lipopolysaccharide (5mg/kg, orally + 150μg/kg, i.v 24 hours before killing) Prior to the day of experiment each rabbit was kept for more than 10 hours in restraint cage. On the day of experiment the groups of rabbits were allocated randomly to receive either active treatment or physiological saline via pediatric stomach tube advanced through wooden clinical tongue depressor with hole in the center to control jaw movement and prevent the animal from chewing the tube (8). After 3 days of treatment the rabbits were killed by sharp blow on the back of the head, the abdomen was opened and liver removed. The concentrations of the drug used in this study represented the double therapeutic dose of the drug. Preparation of liver homogenate and M D A measurement: A. the liver was removed and cut into small pieces then washed with cold phosphate buffer saline (PBS) and squeezed between filter paper to remove excess phosphate buffer saline. B. five grams of the liver tissue was homogenized by electric homogenizer in (100 ml) of cold phosphate buffer saline until large particles disappeared. C. centrifuge at 2000 rpm for 10 minutes and the supernatant was taken d. (0.1 ml) of this solution add to (1.5 ml) of trichloroacetic acid (TCA). E. after 10 minutes incubation at 37 °C, centrifuge at 3000 rpm for 15 minutes. F. all the supernatant was removed and treat with (1.5ml) of clear solution of 0.067% thiobarbituric acid (TBA) H. incubated in water bath for 30 minutes at 100C. I. absorbance was measured at 532 nm UV wave length. MDA concentration was calculated using the following equation (9) Absorbance MDA = the final result expressed in nmol/g 1.49 x 10-5 Measurement of serum liver enzymes :( ALT, AST): colorimetric method, hazy, to white and umber and adapted for determination of the activity in serum by Reitman (1957).(10) LAlanine + 2-Oxoglutarate-- pyruvate + L-Glutamate. LAspartate +2-Oxoglutarate <--- Oxaloacetate + L –Glutamate. The pyruvate or oxalate react with 2, 4 DNPH to form 2, 4 dinitrophenylhydrazones, which absorbed at 505 nm. We used colorimetric method for determination of alkaline phosphates (ALP) as in the following scheme: Phenyl phosphate---- phenol + phosphate Free phenol liberated by hydrolysis of substrate reacts then with 3 – amino antipyrine in the presence of alkaline from potassium ferric cyanide to form red colored complex which absorbance measured at 510 nm is directly proportional to the (ALP) activity in the serum. AL-Qadisiya Journal of Vet.Med.Sci. Vol./12 No./2 2013 9 Statistical Analysis Analysis was made by using SPSS package version 15. Data were analyzed by one way ANOVA. Paired ttest was used to compare between different concentration and control. The differences are considered to be significant when p<0.05. The correlation between various parameters was evaluated by parsons' correlation analysis.