IMMUNOTOXIC ACTION OF POSTORAL ADMINISTRATION OF CARBON TETRACHLORIDE IN MALE ALBINO RAT

Carbon tetrachloride (CCl4) was administered (p.o.) every other day for one month to male Sprague Dawely rats. At the first day after the treatment period, leucocytes (WBCs), and lymphocytes (LYM) numbers, and serum IgM level were significantly higher than those of the control values. At day 15 after CCl4 treatment period WBCs and LYM numbers were below the control levels, whereas, IgM was still above the control level, but lower than the value recorded at day one after CCl4 treatment period. The same dose level and treatment period of CCl4 induced significant increases in serum IL-4 and IL-6 levels that were lasted for 15 days post the administration period. Late administration (i.p.) of DMSO (0.5 ml/100 g b.wt.) at 30 minutes post CCl4 treatment period selectively prevented changes of WBCs and LYM.Tthese results indicate that the toxic effect of CCl4 under the present experimental conditions, could induce changes in both cellular and humoral immune response of rats and these changes could be selectively reversed with time and late administration of DMSO. Nagi A. Ibrahim et al. 24 INTRODUCTION Carbon tetrachloride (CCl4) is a clear, colourless, heavy, and non-flammable liquid (1). The average daily intake of CCl4 for the general population is estimated to be 0.1 μg. Once exposed to this toxic compound by ingestion, inhalation or skin absorption, it is distribution throughout the body, with highest concentrations in the liver, brain, kidney, muscle, fat and blood (1). In spite of the its obvious deleterious effects, CCl4 is still used as a solvent for oils, fats, lacquers, varnishes rubber waxes and resins (2,1). CCl4 is a well known hepatotoxin and nephrotoxin (3,4,5) . It has also been identified as a probable human carcinogen based on evidence of tumors in animals (16). Furthermore, CCl4 has been demonstrated as a potent immunosuppressive agent (7,8,9,10,11) . Several agents have been reported to reduce or erase the toxic effect of CCl4, these include propolis extract (12) , ginkobiloba extract (13,14,15) , black tea extract (15) , and caffeic acid phenthyl ester (4) . The immunoglobulins are proteins with anti-body activity; i.e., they combine specifically with the substance that elicite their formation (immunogen or antigen), and they make up the humoral arm of the immune response. With the possible exception of "natural" antibody, antibodies arise in response to foreign substances introduced into the body. They are therefore products of induced responses. The immunoglobulins, which circulate in body fluids, comprise a heterogeneous family of proteins, they account for approximately 20% of the total plasma proteins (Goodman, 1991). The two hallmarks of immunoglobulins are the specificity of each for one particular antigenic structure and their diversity as a group, which meets the challenge of a vast array of antigenic structures in the environment. In addition to specifically binding antigens, the immunoglobulins express secondary biologic activities, which are important in defense against disease, e.g., transplacental passage, and facilitation of phagocytosis (Goodman, 1991). IgM is secreted by plasma cells (Goldsby et al., 2003). It constitutes approximately 10% of normal immunoglobulins and normally exists as a pentamer with a molecular weight of approximately 900,000(19S) (Goodman, 1991) Immunotoxic Action of Carbon Tetrachloride 25 IgM antibody is prominent in early immune responses to most antigens and predominates in certain antibody responses such as "natural" blood group antibodies. IgM is the major immunoglobulin expressed on the surface of B cells (Goodman, 1991). Cytokines are soluble proteins that affect an array of biological processes including inflammation and immunity. Almost every mammalian cell type has the capability to produce and respond to cytokines (Dinarello, 2007). Interleukin-4 (IL-4) is a cytokine with multiple activities that is mainly produced by the helper CD4 T-lymphocytes of the TH2 subset. It is both an early and late acting growth and differentiation factor for B-lymphocytes which increases cell surface expression of both class II histocompatibility antigens (Noelle et al., 1984; Roehm et al., 1984) and the low affinity receptor for IgE (DeFrance et al., 1987). Interleukin-6 (IL-6) is a poleiotropic cytokine with both pro and anti-inflammatory properties (Barton, 1996). This cytokine is expressed by a variety of cells, including lymphocytes, monocytes, macrophages, neutrophils, endothelial cells, epithelial cells, and fibroblasts (Biffl et al., 1996; van der Boll and van Deventer, 1998). Dimethyl sulfoxide (DMSO), an amphipathic solvent soluble in both aqueous and organic media if often used to dissolve hydrophobic substances employed in biological research (Basile et al., 1996; Szonyi et al. 2001; Harris et al., 2005). In experimental studies, DMSO has been shown to effectively prevent the chemicallyinduced liver or kidney damage (Lind & Grandolfi, 1997, 1999; Gilot et al., 2002; Kishioka et al., 2007). The objective of this study was to further delineate the effects of CCl4 on the immune system in male albino rats and the possible protective action of late administration of DMSO against these effects. MATERIAL AND METHODS Male Sprague-Dawely rats weiging 150-200 g were used in this study. They were obtained from the animal house of the National Research Center, doki, Giza. Rats were housed under standard laboratory conditions. They had free access to a standard food and Nagi A. Ibrahim et al. 26 provided with tap water ad libitum. All animals were acclimatized to these conditions for 10 days before being used. Carbon tetrachloride and dimethyl sulfoxide were applied in the present study as commercial solutions. CCl4 was obtained from Merck (Germany) DMSO City, Egypt was obtained from NOURESH'SHARK Co. 10th of Ramadan The acute post oral LD50 of CCl4 for male Sprague-Dawely rats was determined according to the method of Reed and Meuench (32) and was found to be 0.9 ml/100 g b.wt. , (1.431 g/100 g b.wt.). Animals were randomly allocated into 3 groups (10 rats in each group). Rats of group 1: were not treated and used as controls. Rats of group 2: received postorally CCl4 at the dose level of 1/10 LD50/100 g b.wt. every other day for one month. Rats of group 3: were injected subcutaneously with DMSO at the dose level of 0.05 ml/100 g b.wt 30 minutes after each CCl4 treatment, the desired dose of DMSO was diluted by distilled water to a volume of 0.5 ml for each rat. From each group, 5 rats were deprived of food for overnight before the 1st day and 15 days following termination of each treatment period. These rats were slightly anaesthetized with ether and blood samples were collected through heart puncture into plain tubes and centrifuged at 3000 rpm for 15 minutes. Serum was separated in eppendorfs and kept at -80°C till the immunological analysis were started. Other blood samples were collected into vaccutainer tubes containing EDTA for haematological analysis. Determination of serum immunoglobulin IgM: IgM in serum samples of control and treated rats were assayed by means of immunonephelometry on the BN ProSpec System manufactured by Dade Behring GnbH, Marburg , Germany. Immunotoxic Action of Carbon Tetrachloride 27 Determination of the serum cytokines interleukin-4 (IL-4) and interleukin-6 (IL-6): IL-4 and IL-6 concentrations in serum samples of control and treated rats were measured using AviBion Enzyme-Linked Immunosorbent Assay (ELISA) kits (Orgenium Laboratories, Helsinki, Finland),according to the manufacturer's instructions. Determination of haematological parameters: Total white blood cells (WBCs) number and lymphocyte (Lym) number, were measured following the methods described by Brown (33) using the automated haematology analyzer, Sysmex KX21N manufactured by Sysmex Co. Kobe, Japan. Statistical analysis.