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Journal of Cell and Molecular Biology 2: 19-23, 2003. Haliç University, Printed in Turkey. The short-term effects of single toxic dose of citric acid in mice Tülin Aktaç 1 *, Ayflegül Kabo¤lu 1 , Elvan Bakar 1 and Hamiyet Karakafl 2 1University of Trakya, Faculty of Arts and Sciences, Department of Biology, 22080, Edirne-Turkey; 2University of Trakya, Faculty of Medicine, Department of Biochemistry, 22080, Edirne-Turkey (* author for correspondence) Received 12 April 2002; Accepted 03 July 2002 Abstract The effects of LD25 (480 mg/kg.bw.) dose of citric acid, a food preservative, were investigated on body weight, organ weights (liver, kidney, spleen), creatin kinase (CK), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes in the blood serum, and the liver tissue of mice after 10 days. Citric acid (to experimental groups) and physiological saline (to control groups) were given intraperitoneally. The results of enzyme activities were evaluated using autoanalyzer as IU/L. Even though significant decreases in the body weights were noted when compared to those of the control group (p0.05, kidney: p>0.05, spleen: p>0.05) and serum enzyme levels (CK: p>0.05, LDH,: p>0.05, ALT: p>0.05, AST: p>0.05). Microscopical examination of the liver showed histopathological changes depending on the citric acid. These changes were tissue degeneration, cytoplasmic vacuolisations, nuclear membrane invaginations, picnotic nucleus and necrosis of the hepatocytes. KKeeyy wwoorrddss:: Citric acid, food preservative, enzymes, mouse, liver Farelerde sitrik asidin tek toksik dozunun k›sa süreli etkileri Özet Bir besin koruyucu olan sitrik asidin LD25 (480 mg/kg.va.) dozu farelere intraperitoneal yolla uyguland›. 10 gün sonra hayvanlar›n vücut a¤›rl›klar›, organ a¤›rl›klar› (karaci¤er, böbrek, dalak), kreatin kinaz (CK), laktat dehidrogenaz (LDH), alanin aminotransferaz (ALT) ve aspartat aminotransferaz (AST) enzimlerinin serum düzeyleri ile, karaci¤er dokusu üzerinde sitrik asidin etkileri araflt›r›ld›. Otoanalizörde tayin edilen enzim aktiviteleri U/L olarak de¤erlendirildi. Çal›flmada vücut a¤›rl›klar›nda kontrol grubuna k›yasla anlaml› bir azalma gözlenmesine ra¤men (p0.05 , böbrek: p>0.05, dalak: p>0.05) ve enzim aktivitelerinde (CK: p>0.05, LDH: p>0.05, ALT: p>0.05, AST: p>0.05) anlaml› olmayan bir art›fl gözlendi. Karaci¤erin mikroskopik incelenmesinde doku dejenerasyonu, sitoplazmik vakuolizasyon, nükleer zar çöküntüleri, piknotik nukleuslar ve hepatositlerde nekroz gibi histopatolojik de¤ifliklikler gözlendi. AAnnaahhttaarr ssöözzccüükklleerr:: Sitrik asit, besin koruyucu, enzimler, fare, karaci¤er Introduction Humans are exposed daily to complex mixtures of chemical compounds in their food. One of these substances are antioxidants which are used as food preservatives. However, peroxides of saturated fats and their secondary oxidation products, can be toxic and impair food quality (Würtzen, 1990). Thus despite 19
20 Tülin Aktaç et al. their economic importance, they can have negative effects on living organisms. Xenobiotics entering the organism are held by intestine, kidney and liver cells for detoxification. These cells contain important detoxification enzymes. During the detoxification of xenobiotics, free radicals are produced in oxidation/reduction reactions, and these radicals can have destructive effects on tissues. The toxic effects of many food preservatives on living organisms have been studied by many researchers (Makoveç and Sindelar, 1984; Daniel, 1986; Cabel et al., 1988; Kagan et al., 1990; Jung et al.,1992; Nijhoff and Peters, 1992; Fujitani, 1993; Weemaes et al., 1997; Mc Farlene et al., 1997; Safer and Nughamish, 1999; Kabo¤lu and Aktaç, 2002; Aktaç et al., 2002). Although the citric acid and metal salts (sodium or potassium citrat) are widely used in food industry, there is no report on more detailed effects of citric acid (or its salts) in liver. In addition, soft drinks, cosmetics and drugs, in which citric acid is approved for use, are consumed by most of humans every day. A way of analysing harmfull effects of foreign materials entered to organism is to determine the effects of the chemicals on the enzymes. Enzymes have a very important role in the metabolical process since they are biological catalysts. Thus, their abnormal serum levels indicates various diseases. Among these enzymes are, creatine kinase (CK), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) which are the most important. Therefore, we studied short term treatment of citric acid (10 days) in mice. In these experiments, firstly we tested total body weigths, organ weights (liver, kidney, spleen), and determined the serum levels of creatin kinase (CK), lactate dehydrogenase (LDH), alanin aminotransferase (ALT) and aspartat aminotransferase (AST), and secondly the liver tissue was investigated histopathologically. Material and Methods Male mice (Balb/C albino) weighing 25-30 g were used in our experiments. Five mice were used control group and ten mice were used the citric acid-treated group. Animals were fed by pellet baits and water. LD25 dose (480 mg/kg.bw.) of citric acid (Merck; in physiological saline) were injected intraperitoneally to experiment group mice, and the same amount of physiological saline to control group mice. 10 days after the injection, the mice were killed by cervical dislocation and then the necessary studies were commenced. The livers, kidneys and spleens dissected out, weighed, liver samples were seperated for microscopical examination. Blood samples were also taken for enzyme assays. The serum levels of enzymes were determined using a Merck Mega 600 autoanalyser with the aid of Diasis Kits. Data were analyzed by M.Whitney U test for multiple comparisons for the differences between the control and treated groups. For histological examination, liver samples were fixed with 10% buffered formalin, processed and stained hematoxylin-eosin. Results The effects of citric acid injection on the body weight and liver, kidney, spleen weights was shown Table 1 and 2. Although the liver, spleen and kidney weights were not changed significantly (p>0.05), the body weights were decreased significantly (p0.05) as shown in Table 2. The results of the microscopic investigation showed that liver of mice treated with citric acid has necrotic changes, compare to the control group (Figure 1-6). These changes were slightly degeneration of tissue (Figure 2), cytoplasmic vacuolisation, nuclear membran invaginations (Figure 3, 4) and picnotic nuclei (Figure 5). In addition, we observed degeneration of the blood vessel endothelium (Figure 6). Discussion The effects of xenobiotics in living organisms can investigate in various ways. Among these are, shortterm toxicity tests which are used very commonly. In these methods, many parameter are used to test the effects of xenobiotics. Some of these parameters are body weight, organ weights, blood profile, and histopathological examination. In this study, the shortterm effects of citric acid applied intraperitoneally were investigated. It was reported that the body weight decreases in mouse (Würtzen, 1990), and in rats (Nijhoff and Peters, 1992) by the effects of phenolic antioxidant butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) in chronic studies. In
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