Role of N-Acetyl-L-Cysteine in the Prevention of Hepatotoxicity Induced by Patulin in Male Mice

1 Journal of Pharmaceutical, Chemical and Biological Sciences ISSN: 2348-7658 UGC Approved Journal CODEN: JPCBBG Impact Factor (GIF): 0.701 Impact Factor (SJIF): 3.905 March - May 2018; 6(1):1-10 Published on: March 18, 2018 The work is licensed under Research Article Role of N-acetyl-L-cysteine in the Prevention of Hepatotoxicity Induced by Patulin in Male Mice Mamdouh R. F. El-Sawi, Faried A. E. Hemieda*, Sameh Shabana, Rasha G.S. Khmalj Zoology Department, Faculty of Science, Mansoura University, Mansoura, Egypt *Corresponding Author: Faried A. E. Hemieda, Zoology Department, Faculty of Science, Mansoura University, Mansoura, Egypt Received: 14 January 2018 Revised: 14 February 2018 Accepted: 21 February 2018 ABSTRACT The present study was designed to investigate the possible preventive effect of 7 days pretreatment with N-acetyl-L-cysteine (NAC) on patulin (PAT)-induced hepatotoxicity in male mice. Obtained results showed that intraperitoneal injection (i.p.) of mice with PAT in a single dose (3.75mg/kg) significantly increased hepatic contents of malonic dialdehyde (MDA), accompanied with elevated activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum. On contrary, PAT treatment alone markedly decreased hepatic contents of antioxidant parameters including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rd), glutathione-s-transferase (GST) and reduced glutathione (GSH). Serum concentrations of total protein and albumin were also decreased following administration of PAT. On the other hand, liver contents of p53, caspase-3 and Bax were significantly increased accompanied with markedly decreased hepatic concentration of Bcl-2 after injection of PAT alone, suggesting ability of PAT to induce apoptosis. Treatment with NAC (200mg/kg/day) in mice for 7 days prior exposure to PAT significantly inhibited the above mentioned adverse effects induced by PAT. In conclusion, NAC seems to be effective in the prevention of hepatotoxicity induced by the potent mycotoxin PAT. Keyword: N-acetyl-L-cysteine; patulin; hepatotoxicity; antioxidants; apoptosis INTRODUCTION animal organs including liver, kidney, intestine, Mycotoxins which are produced by fungi can immune system and brain [3,4,5]. There are cause serious diseases and even death in both evidences which indicate that PAT is a humans and animals. PAT, which has a lactone genotoxic and mutagenic [4,6]. However, the structure as shown in Fig. 1, is one of these genotoxicity and cytotoxicity of this mycotoxin mycotoxins that made by certain fungal species are associated with its electrophilic properties of the genera Penicillium, Aspergillus and which facilitate high interaction of it to cellular Bryssochamys [1]. It may grow on a variety of nucleophiles, such as sulfhydryl (thiol) groups. foods including fruits and vegetables, mainly This can cause depletion of cellular sulfhydryl apples and therefore it can cause contamination containing compounds such as GSH for these types of food and their commercial concentration and hence production of oxidative products, such as apple products [2]. stress [7,8]. Furthermore, available data showed Previous studies exhibited that exposure to PAT that administration of PAT in animal was found can induce a number of toxic effects in several to produce apoptosis [9,10]. J Pharm Chem Biol Sci, March - May 2018; 6(1):1-10 Mamdouh et al 2 Fig. 1: Chemical Structure of PAT Fig. 2: Chemical Structure of NAC NAC is considered a powerful Germany. The solid powder of both PAT and antioxidant because it represents a good source NAC was dissolved in distilled water when used of sulfhydryl groups (Fig. 2) which capable of for treatment. scavenging the free radicals, especially radicals of oxygen. Beside this, NAC stimulates GSH Animals and experimental design biosynthesis because it is a precursor of L- Forty healthy male Swiss albino mice weighing cysteine, an essential component of GSH 25-30 g were obtained from Helwan Animal molecule. NAC was therefore recommended as a Farm, Cairo, Egypt. The mice were maintained potential treatment agent for various disorders under controlled environmental circumstances. related to generation of free oxygen radicals [11]. They were fed on commercial rodent pellet diet Published data demonstrated that NAC has a and water was provided ad libitum. Nursing and hepatoprotective effect against several use of the animals were managed under control chemicals-induced toxicity. In both humans and of the Animal Ethics Committee of Mansoura animals, NAC was introduced to treat University, Egypt. After a week of acclimation, hepatotoxicity resulted from exposure to the mice were allocated into four groups, 6 overdoses of acetaminophen [12,13,14]. Also, animals per each, as follows: NAC was found to stabilize the cell membrane Group I: Control mice had no chemical and to decrease the hepatocellular injury as treatment. evidenced by its ability to lower the activity of Group II: Mice were given NAC alone for 7 days hepatic marker enzymes in the plasma (such as in a dose of 200 mg/kg/day [16]. transaminases) and the levels of lipid Group III: Mice were given a single dose of PAT peroxidation, in addition to its improvement of (3.75mg/kg) at the 7th day [17]. the antioxidant system in the liver of rats Group IV: Mice were given both NAC and PAT exposed to CCl4 [15]. In view of these as mentioned above in group (II) and (III) information, it appeared that NAC treatment respectively. may be effective in the protection against PAT- PAT was given to mice by i.p. injection, while induced hepatotoxicity. Interestingly, there are NAC was orally introduced to used animals no clear previous reports on the potential using gastric tube. protective impact of NAC against PAT-induced liver injury, so we considered this point of great Blood and tissue sampling value and worthwhile. Based on this, present After 24 hours of injection of PAT, mice were study was conducted to exploring the efficacy of anesthetized by ether following overnight NAC on the hepatic marker enzymes, non- fasting. Then, the jugular vein of each mouse enzymatic and enzymatic antioxidants, as well was cutted using a sharp razor blade and the as the apoptotic markers in mice exposed to toxic blood flowed was collected into the centrifuge dose of PAT. tube. After blood clotting (10-15 min), the tubes were centrifuged at 3000 rpm for 15 min to MATERIALS AND METHODS separate the blood sera which were kept at - Chemicals 20°C for biochemical analysis. PAT, 4-hydroxy-4H-furo[3,2c] pyran-2(6H), has a On the other hand, the animals were quickly chemical formula C7H604 (Fig. 1) and NAC, N- dissected and the whole liver of each mouse was acetyl-L-cysteine, has a chemical formula removed and cleaned. A known weight of each C5H9NO3SH (Fig. 2). The two chemical liver was homogenized in distilled water at 4°C compounds were obtained from Sigma–Aldrich, J Pharm Chem Biol Sci, March - May 2018; 6(1):1-10 Mamdouh et al 3 forming 10% (w/v) homogenate, and then kept at Maryland, USA, as described by the method of -20°C for the assays. [28,29], respectively. Bax and Bcl-2 concentrations in the liver were determined Biochemical analysis using ELISA kits (MyBioSource, San Dioego, Hepatic contents of MDA, GSH, SOD, CAT and California, USA) on basis of the procedure of GSH-Px were measured by the ELISA technique [30,31], respectively. according to [18, 19, 20, 21, 22] respectively. The ELISA kit was supplied from CUSABIO STATISTICAL ANALYSIS BIOTECH CO., Ltd, Baltimore, Maryland, USA. Obtained results were presented as means ±SE. The activity of GSH-Rd in the liver homogenate Statistically significant differences between was determined according to the method of [23], mean values were evaluated by one way analysis using ELISA kit from Cell Biolabs, Inc, 7758 of variance (ANOVA) using SPSS 19.0 software Arjons Drive, San Diego, CA 92126 USA. considering p ≤ 0.05 as a minimal level of Hepatic GST activity was measured by ELISA significant. kit from Immundiagnostik AG, Stubenwald- Allee 8a, D64625 Bensheim; as described by [24]. RESULTS Serum ALT and AST activities were assayed by Table 1 shows that administration of PAT in a kinetic method according to [25], using kit from single dose (3.75 mg/kg) to male mice produced Spinreact Company, Spain. Total protein content significance increase in the hepatic content of in the serum was estimated colorimetrically lipid peroxidation marker (MDA) and marked using kit from Spinreact Company, Spain decrease in the levels of antioxidant parameters according to the method of [26]. Serum albumin including SOD, CAT, GST, G-Px, G-Rd, and concentration was measured colorimetrically on GSH, as compared to control results. However, 7 basis of the method of [27] using kit from days treatment of mice with NAC prior injection Diamond Company, Egypt. The liver contents of of PAT significantly attenuated the adverse p53 and caspase-3 proteins were quantitatively effects of PAT on the hepatic levels of markers of estimated using ELISA kit provided by lipid peroxidation and antioxidants, in CUSABIO BIOTECH CO., Ltd, Baltimore, comparison with group treated with PAT alone. Table 1: Effect of NAC on PAT-induced oxidative stress in the liver of male mice Parameters Control NAC PAT NAC+PAT MDA (nmol/g) 0.12±0.01 0.12±0.01 0.34±0.01ab 0.21±0.01abc GSH (mg/g) 0.25±0.01 0.28±0.01 0.12±0.01ab 0.19±0.01abc SOD (U/g) 0.29±0.01 0.29±0.01 0.12±0.02ab 0.19±0.01abc CAT (µmol/g) 0.38±0.03 0.44±0.03 0.17±0.01ab 0.26±0.03abc GST (mmol/g) 0.27±0.01 0.29±0.01 0.13±0.01ab 0.18±0.01abc GSH-Px (mµ/mg protein) 0.36±0.02 0.35±0.02 0.11±0.01ab 0.20±0.01abc GSH-Rd (ng/mg protein) 0.28±0.01 0.28±0.02 0.14±0.01ab 0.19±0.01abc Values expressed as mean ± SE from 6 mice in each group.

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