Introduction

Liver diseases are considered one of the most important causes of death in the world(1). Oxidative stresses are known as a mechanism involved in the onset and progress of hepatic damages(2).The increase in the levels of reactive oxygen and nitrogen species can cause hepatocellular injuries (3).

Unsaturated fatty acids are named according to the location of double bond from the carbon of the terminal methyl called Omega carbon. The fatty acids, eicosapentaenoic acid (EPA) and acid and  docosahexaenoic acid (DHA) belong to the Omega-3 fatty acid category. These acids can’t be synthesized by humans and should be included in diet; the main sources of Omega-3 are Fish oil, marine planktons and oceanic fish (4). In a study, Wergedahl et al (2009) showed that combination of fish oil (FO) and fish-protein hydrolysate (FPH) reduce plasma cholesterol levels, which are related to their effects on the reduction of HDL cholesterol level, while the total hepatic cholesterol concentration increased compared to control mice and those receiving FPH and FO alone. The cholesterol reducing effects of combined FPH and FO is related to decreased secretion of low density lipoproteins (LDL) from the liver (5). The studies of Kim Et al. (2013) demonstrated that Omega-3 fatty acid has protective effects against insulin resistance induced by obesity and liver steatosis. Omega-3 hyperlipidemia induced by diet and fatty liver can be improved through induction of cytochrome CYP7A1 expression and the activity of cholesterol catabolism to bile acids (6). In a study by Haast et al. (2015), a direct correlation was found between Omega-3 fatty acid intake and reduced age-related brain deterioration and damage (7). Also, Siegel et al. (2012) showed that Omega-3 fatty acids have beneficial effects on cerebral-cardiovascular diseases (8). Likewise, fish oil Omega-3 emulsion significantly reduces hepatic damage following liver transplantation(9), and has protective effects against liver fibrosis and injuries as well as oxidative stress induced by carbon tetrachloride(10).

Thioacetamide (TAA) is an organic compound containing Thiono–sulfur that is used as a fungicide, an organic solvent and a stabilizer of motor oil. In 1984, Hugh and Nelson first reported that TAA is a hepatotoxic agent. A single dose of this agent can produce lobular necrosis sentry in animals, and chronic induction of thioacetamide can lead to liver cirrhosis and carcinoma (11). The toxic effects of thioacetamide is due to its biological activity exerted through oxidase systems, particularly FAD mono oxygenases and CYP2E1(12).Hence, in conjunction with the measurement of hepatic enzymes, thioacetamide can be useful in pharmaceutical research to induce pathological condition, because many blood factors and enzymes are synthesized in hepatocytes and their measurement is a diagnostic criteria for liver function.

Due to high prevalence of liver disease and the high side effects of current chemotherapies, there is a growing Need for a medicine (s) with minimum complications and high effectiveness. In this regard, the use of fish oil Omega-3 is most promising, since it has antioxidant and anti-inflammatory properties and shows low side effects. Thus, in this research we tried to examine the possible protective effects of this oil on changes in the levels of  alkaline phosphatase (ALP), serum glutamic oxaloacetic Transaminase (SGOT), serum glutamic pyruvic Transaminase (SGPT) and  bilirubin induced by thioacetamide in pretreated rats.

Materials and Methods

Laboratory animals

In This experimental study, 42 adult male Westar rats in a weight range of 200±10g and the age range of 3-2.5 months were used. Animals were randomly divided in 6 Groups of seven, and kept under standard conditions of 20-22 ° c and light cycle of 12 hours light and 12 hours dark. They had easy access to food and water, and all ethical considerations and animal rights were ensured.

Animal treatment : Animals were divided into 6 groups and were treated as follow: the Control which left untreated and subjected to no stresses; the sham 1 which daily received 0.4ml/kg olive oil as a solvent supplement of Fish oil omega-3; the sham 2 which received a single dose of 150 mg/kg thioacetamide; the experimental groups 1, 2 and 3 received a daily dose of 100, 200 and 300 mg/kg Fish oil Omega-3  supplements respectively followed by a single dose of 150 mg/kg thioacetamide. Fish oil omega-3 was administered orally for 3 months, and thioacetamide was injected interperitonaly at  the end of treating period.

48 hours after the last injection,animals were anesthetized with ether, and blood samples were taken from the heart. These samples were kept under laboratory conditions for 20 minutes; then, centrifuged at 5000 RPM for 15 minutes (13, 14).

Serum concentrations of various parameters were measured by appropriate methods: SGPT, SGOT, by DGKC method and ALP by  P-Nitrophenyl phosphate method (15). To measure bilirubin, azo DVD reagents (sulfanilic acid sodium nitrite) were used; they react with bilirubin, producing azo, which is red in alkaline PH. After production, direct bilirubin has a pink color, but after addition of accelerating solution, total bilirubin turns green in alkaline PH (16).

Histological studies

After removal, livers were fixed separately in 10% neutral formalin buffer. Following histological tissue preparation and producing specimen blocks, 4-5 micron thick sections were prepared, stained using hematoxylin-eosin method and studied by light microscope (17).

Statistical analysis

The software program SPSS18 and statistical ANOVA test were used for data analysis. In order to study statistically significant differences among data, the Tukey HSD test was used and significant average difference was sat at . The plasma concentrations of ALP, SGPT , SGOT and bilirubin are presented as average  deviation .

Results and Discussion

The mean serum concentration of SGOT  showed a significant increase in the sham 2 (receiving thioacetamide alone) and experimental groups 2 and 3 (receiving both fish oil omega-3 supplements and thioacetamide) compared with the control and sham 1 while it showed an insignificant reduction in experimental groups 2 and 3 compared with sham 2 (table 1). Similarly, the mean serum level of SGPT increased significantly In the sham 2 and experimental group 2 relative to the control and sham 1 whereas a significant decrease was observed in the experimental group 1 (receiving both 100 mg/kg fish oil omega-3 supplement and thioacetamide) compared with sham 2 (recipient of thioacetamide alone) (p≤.05). Conversely, in respect to mean concentrations of ALP, there were no significant differences among various experimental groups and sham 2, nor was any such differences among sham 2, and control and sham 1 groups (table 1).

Table 1: The effects of different amounts of fish oil omega-3 supplement on various biochemical serum parameters in Male rats poisoned by thioacetamide.

Letter a Represents significant differences between group receiving thioacetamide alone (sham 2), and control and sham 1 groups at the level of P < 0.05; letter b represents significant differences between sham 2 group, and various experimental groups (fish oil omega-3 supplement and thioacetamide) at the level of P <0.05; and letter c represents significant differences among different experimental groups, and control and sham 1 groups at the level of P <0.05.

Histological findings

Histological studies of hepatic samples showed normal hepatocytes with protected cytoplasm and marked nuclei in control and sham 1 groups (figures A and B). In contrast, tissue sections in sham 2 Group (recipient of thioacetamide) in comparison with the control and sham 1 groups indicated hepatocyte necrosis, increase in mitosis, cell death, abnormal mitotic inflammation in portal space and large nuclei (figure C). Although tissue samples of the experimental groups 1 and 2 (receiving 100 and 200 mg/kg fish oil omega-3 supplement and thioacetamide) showed no hepatocyte mitosis, apoptosis and necrosis, they had large nuclei ( figures D and E), whereas the experimental group 3 (recipient of 300 mg/kg fish oil omega-3 supplement and thioacetamide ) showed normal liver tissue ( figure F).

Figure   Click here to View figure

liver injury was also determined by biochemical parameters(plasma SGPT,SGOT,ALP,LDH levels).Many useful medicines such as acetaminophen, and some industrial and environmental toxins can cause severe liver damage through functional interference with reactive free radicals. One of these industrial toxins is thioacetamide. This toxin induces hepatic lobular sentry necrosis,. Liver cirrhosis and hepatocellular carcinoma (HCC) (18).

According to the results of this study, the values of bilirubin,SGPT and SGOT in groups treated with thioacetamide increased significantly compared to the control and sham1 groups.The average concentration of SGPT in the experimental group which received 100mg/kg of fish oil omega-3 supplement and thioacetamide showed a significant decrease compared to the group receiving thioacetamide. The average concentration of billirubin in the experimental groups which received 100mg/kg of fish oil omega-3 supplement and thioacetamide showed a significant decrease compared to the group receiving thioacetamide (P<0.05).This means that the fish oil omega-3 supplement had protective effects on liver cells against damage caused by thioacetamide .The histopathological studies also confirmed these results.

Similarly, Chen Et al. (2012) showed that DHA has beneficial effects on cholestatic liver disease. The beneficial effects of DHA supplement are related to its strong anti-inflammatory and anti-oxidative effects as well as down-regulation of NF-kB, signaling of the  transforming growth factor-beta  and Smad protein through functional interference in the activity of extracellular signal regulating kinase (ERK) (19). More recently, Sherif Et al. (2015) found that cod liver oil can improve damage induced by sodium nitrite through several mechanisms, including blocking of cell death signs, fibrotic mediators and inflammatory cytokines  induced by sodium nitrite (20). Other evidence showed that DHA supplement and fish oil EPA Omega-3 may be the preventive agents in the treatment of liver cirrhosis in mice (21).

According to Li et al. (2014), consumption of diet containing fish oil can reduce systemic inflammation and liver damage induced by infection through up-regulation of the peroxisome proliferator-activated receptor gamma-mediated pathway (PPAR) in septic mice(22).Also, Jangale et al. (2013) determined that fish oil and flax seed oil can alleviate inflammation in diabetic mice induced by streptozotocin-nicotinamide (23). Kim et al. (2013) showed that diet containing Omega-3 can attenuate Hepatic damage caused by ischemia and tissue Reperfusion via reduction of NF-Kb activity (24). At the same time, Popescu et al (2013) indicated that Omega-3 fatty acid along with diet containing natural calorie and diet with natural lipid has protective effects on nonalcoholic fatty liver disease (25). Other studies have shown that fish oil diet prevents hepatocyte cancer in B6C3F1 mice (26). Similarly, Omega-3-rich fish oil improves liver damage caused by LPS through the inhibition of TLR4 signaling pathway and NOD (27).

De Meijer et al. (2009) showed that emulsion based on fish oil prevents parenteral nutrition-associated liver disease (28). Also, Khan et al. (2015) demonstrated that fish and flax seed oil can protect against apoptosis, tissue damage and hepatotoxicity induced by nitric oxide; it can reduce lipid peroxidation and improve body’s antioxidant system (29). Other research showed that the cod liver oil improves hepatic damage induced by sodium nitrite via oxidative stress alleviation, and blocking of MCP-1 and mitochondrial functional response as well as reducing DNA fragmentation (30). It was also found that due to the presence of the antioxidant compounds, Omega-3, lipid emulsion based on fish oil prevents liver diseases associated with intestinal failure (31),since omega-3 fatty acid improves the hepatic inflammatory responses by suppressing inflammatory cytokine production in hepatocytes. In addition, EPA reduces levels of TNF-a and IL-6 in the hepatocytes(32),and DHA improves hepatic injuries induced by valproate through reforming of oxidative stresses and inflammation without having any effect on plasma level of valproate (33). Moreover, it has been shown that addition of fish oil supplement to parental diet reforms the increased levels of hepatic enzymes resulted from liver dysfunction  related to parental nutrition (34). Studies have shown that 10% fish oil and 1 g% artichoke leaf can restore hepatocellular carcinoma in rats (35). Lee et al. (2008) determined that Omega-3 fatty acid can repair hepatocellular damage caused by obstruction of the bile ducts(36); it was also shown that fish oil along with allopurinol And verapamil improve hepatic injuries resulting from ischemia by a significant reduction in oxidative stress and hepatic enzymes (37). In a study by Mardones et al. (2012), it was demonstrated that combination of thyroid hormone and fish oil protocol prevents liver damage resulted from tissue injury and ischemia (38). Furthermore, Omega-3 fatty acid prevents acute liver defects and stimulates liver regeneration following 90% hepatectomy in rats (39). Chiang et al. (2009) showed that fish oil can stimulate anti cell proliferation effect of 1- alpha 25-dihydroxy vitamin D3 on hepatic cancer cells(40); EPA can also improve hepatic toxicity, oxidative stress and inflammation induced by valproate (41) .

In general the results of this study are in line with the results of other studies.It seems that the  oral administration of fish oil omega-3 supplement has  protective effect on thioacetamide induced liver toxicity  by neutralizing free radicals, stimulating the activity of antioxidant enzymes, and reducing the production of  inflammatory cytokinin .As no similar study on the protective effects of fish oil omega-3 supplement on hepatic enzymes and histological changes could be found, it was not possible do a comparative study in this respect. Anyhow, more studies should be conducted to examine the hepatic antioxidant enzymes and molecular changes inducing apoptosis so that the effects of fish oil omega-3 supplement on healing liver toxicity can be determined with higher certainty.

In general, the results of present study showed that Fish oil Omega-3 supplement in rat model with liver dysfunction can cause desirable improvements. Thus, if supported by more experiments, it is possible to add Fish oil Omega-3 supplement to the diet of patients with liver dysfunction.

Acknowledgement

This article is a part of doctoral thesis of. We appreciate the cooperation and contribution of research office of Shiraz Islamic Azad University.

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