Journal of Pharmacy Research Vol.4.Issue 4. April 2011 Yohanes Buang et al. / Journal of Pharmacy Research 2011,4(4),951-953 951-953 Research Article ISSN: 0974-6943 Available online through http://jprsolutions.info * Corresponding author. Yohanes Buang 1 Department of Chemistry, Faculty of Science and Engineering, Nusa Cendana University, Jl. Adisucipto Penfui, Kupang-Indonesia 2 Laboratory of Applied Biochemistry, Department of Applied Biological Science,Saga University, Japan 1. INTRODUCTION Peroxisome proliferators are a diverse group of chemicals which includes herbicides, leukotriene antagonists, hypolipidemic drugs, and plasticizers [1-3]. Two major categories of peroxisome proliferator chemicals are fibrate class of the hypolipidemic drugs and phthalate ester plasticiz- ers. One of the phthalate ester plasticizers used in commonly society is di(2-ethylhexyl) phthalate (DEHP). DEHP is widely used in consumer products such as food packaging materials and children’s toys [2-5]. Plastics containing DEHP are also used for tubing and containers for blood transfusions and blood products. Overall, DEHP directly or indirectly interacts with human and animal health cells and affects their peroxisomes. Peroxisomes are single membrane organelles found in virtually all eukaryotic cells [6-8]. Peroxisomes can be derived from the endoplasmic reticulum and replicated by fission [6] and therefore reasonably promotes phospholipids (PL) level, a major component of cell mem- branes, as reported in several studies [2-3]. These proliferated peroxisomes activate nuclear receptor proteins that function as transcription factors regulating the gene expressions [5,9] known peroxisome proliferator activated-receptors (PPARs). Castelein et al. [10] found that PPAR up regulates malic enzyme gene expression which is one of enzymes in the anaplerotic reactions [11]. This enzyme catalyzes conversion of pyruvate to malate and therefore promotes rate of citrate acid cycles to keep energy level needed by the cell. Several authors [4,12-14] reported that PPARα, PPAR expressed in the liver, play essential roles in liver cellular differentiation and development which is associated with an induction of hepatomegaly as a result of an increase in number of liver cells (hyperplasia) and in their sizes (hypertrophy). The hepatomegaly is known as an initial stage of liver cirrhosis and has symptoms of liver failure and serious disorder of the liver structure and the liver cells dysfunction [15-17]. Overall, the hepatomegaly is a crucial health problem in human and animal. Therefore, the screening hepatomegaly associated with dietary manipulation is of interested to study. In present work, the hepatomegaly induced by peroxisome proliferators was studied in Sprague- Dawley (SD) rats as animal model. 2. MATERIALS AND METHODS Animals, diets, and experimental design: All aspects of the experiment were conducted according to guidelines provided by the ethical committee of experimental animal care at Saga University (Saga, Japan). Male SD-rats aged 5 weeks were housed individually in an air- conditional room (24 o C) with a 12-h light/dark cycle. After one week acclimatization, rats were assigned to two groups (five rats each). Basal diets (Control group) were prepared according to recommendations of the American Institute of Nutrition (AIN) and contained (in weight %) 20 of casein, 7 of soybean oil, 1 of vitamin mixture (AIN-93), 3.5 of mineral mixture (AIN-93), 0.25 of choline bitartrate, 0.3 of L-cystine, 0.0014 of ter-butylhydroquinone, 5 of cellulose, 10 of sucrose, 13.2 of α-cornstarch, and β-cornstarch, to make 100. The soybean oil contains palmitic acid 10%, oleic acid 25%, and linoleic acid 55%. The DEHP diet (PPs group) was prepared by supplementation of 1.0% DEHP to the basal diet at the expense of ß-cornstarch. The animals received the diets for 10 days. At the end of the feeding period, rats were killed by decapitation after a 9-h starvation. The livers of rats being anesthetized were excised immedi- ately. Analysis of lipids: Liver lipids were extracted [18] and concentrations of triglyceride (TG), PL, and cholesterol (Chol) were measured by the methods described elsewhere [19-21]. The hepatomegaly induced by peroxisome proliferators is associated with a promotion of anaplerotic reaction Yohanes Buang 1,2 , 1 Department of Chemistry, Faculty of Science and Engineering,Nusa Cendana University, Jl. Adisucipto Penfui, Kupang-Indonesia. 2 Laboratory of Applied Biochemistry, Department of Applied Biological Science,Saga University, Japan. Received on: 04-01-2011; Revised on: 17-02-2011; Accepted on:16-03-2011 ABSTRACT The screening hepatomegaly associated with anaplerotic reaction induced by peroxisome proliferators (1.0% di (2-ethyl hexyl) phthalate) was studied in rats. The liver phospholipid (PL) content of control and PPs group (in mg/g liver) were 36.5±1.0 and 41.7±0.6, respectively. Malic enzyme and carnitine palmitoyl transferase-1 (CPT-1) activities (nmol/min/mg protein) of PPs group were promoted approximately by 4.35- and 2.33-folds, respectively. The increase of liver PL level accompanied with the promotions of malic enzyme and the CPT-1 activities are the key factors of hepatomegaly development accompanied with a promotion of anaplerotic reaction induced by peroxisome proliferators. Key words: peroxisome, DEHP, hepatomegaly, hyperplasia, hypertrophy Preparation of liver subcellular fractions: The mitochondrial and cytosol of liver sub cellular fractions were prepared as described previously [22]. Protein concentration was determined by the method of Lowry et al. [23]. Assays of hepatic enzyme activity: The malic enzyme (EC1.1.1.40) was determined as previ- ously described [24] and the carnitine palmitoyl transferase-1 (CPT-1; EC2.3.1.23) was determined as described by Ikeda et al. [25]. Statistical analyses: Data were analyzed by one-way analysis of variance, and all differences were inspected by Duncan’s new multiple-range test using SPSS statistical software. 3. RESULTS 3. 1 Dietary DEHP promoted liver weight The total food intake for 10 days is shown in Figure 1. The low level of food intake in PPs group was equivalent with the reduction of body weight. However, the weights of liver were significantly higher than that of the control (P<0.05). The liver size of control and PPs groups respectively involved 4.0% and 6.7% of the body weight. The 2.5% body weights and the ratios of liver weights to the 2.5% body weights are reported in Table 1. Figure 1. The metabolic effects of DEHP on growth parameters Rats were fed 1.0% DEHP diet (PPs group) or a diet without DEHP (control group) for 10 days. Values are expressed as mean±SEM of five rats. See Materials and Methods for composition of diets. Clearly define a & b regarding difference of significance at P < 0.05. Table 1. Quantitative rules of hepatomegaly for control (baseline) and treatment groups according to the definition recommended by references 30-31. A. Control Group 1. Body Weight (gram) (a) 206 ± 4 2. 2.5% Body Weight (gram) (b) 5.15 ± 0.1 3. Liver weight (gram) (c) 8.2 ± 0.2 4. Data Ratio: (c/b) 1.59±0.07 B. PPs Group 1. Body Weight (gram) (d) 188 ± 6 2. 2.5% Body Weight (gram) (e) 4.7 ± 0.2 3. Liver weight (gram) (f) 12.6 ± 0.4 4. Data Ratio: (f /c) 1.54±0.09 Values are expressed as mean±SEM of five rats. 3.2 Dietary DEHP induced liver phospholipids level Figure 2 shows the differences in liver lipid contents. The liver PL content of PPs group significantly higher than that of the control group (P<0.05), in which the content (mg/g liver) in control and PPs groups were 36.5±1.0 and 41.7±0.6, respectively. Both liver TG and liver cholesterol levels, however, decreased (P<0.05).