Sains Malaysiana 46(9)(2017):

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1 Sains Malaysiana 46(9)(2017): The Effet of Tootrienol-Rih Fration on Oxidative Liver Damage Indued by Fenitrothion (Kesan Fraksi Kaya Tokotrienol ke atas Kerosakan Oksidatif Hepar Diaruh Fenitrotion) PUTRI AYU JAYUSMAN, SITI BALKIS BUDIN*, IZATUS SHIMA TAIB & AHMAD ROHI GHAZALI ABSTRACT Exposure to organophosphate pestiide inluding fenitrothion (FNT) has led to many adverse effets on human health. However, a potent antioxidant like palm oil tootrienol-rih fration (TRF) an redue oxidative damage in various pathologial onditions, ould also redue the adverse effets by FNT. The aim of this study was to evaluate the effet of TRF on oxidative liver damage in FNT indue hepatotoxiity in experimental rats. A total of 40 male Sprague-Dawley rats were randomly divided into four groups of 10, namely ontrol, TRF, FNT and TRF+FNT group. TRF (200 mg/kg body weight) and FNT (20 m/kg body weight) were administered through oral gavage for 28 days. Corn oil whih served as vehile was given orally to the ontrol group. At the end of the study period, liver and blood was taken for oxidative damage and biohemial evaluation and histologial observation. TRF supplementation prevents oxidative liver damage by reduing the hepati malondialdehyde (MDA) and protein arbonyl (PCO) level signifiantly. Besides, TRF also restored the endogenous antioxidants partiularly redued glutathione (GSH), glutathione peroxidase (GPx) and ferri reduing/antioxidant power (FRAP). TRF also prevent liver damage by reduing the liver enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The attenuation of liver damage by TRF was also showed histologially. In onlusion, TRF supplementation showed a potential in preventing oxidative liver damage in FNTtreated rats by reduing the oxidative damage and improving the antioxidant status. Keywords: Antioxidant; oxidative stress; palm oil; vitamin E ABSTRAK Pendedahan kepada organofosfat termasuk fenitrotion (FNT) telah menyebabkan kesan sampingan pada kesihatan manusia. Walau bagaimanapun, antioksidan yang poten seperti fraksi kaya tokotrienol minyak sawit (TRF) berupaya untuk mengurangkan kerosakan oksidatif yang mungkin juga berupaya untuk mengurangkan kesan kerosakan FNT. Kajian ini dilakukan untuk menilai kesan TRF ke atas hepatotoksiti tikus aruhan FNT. Sebanyak 40 ekor tikus jantan Sprague-dawley dibahagikan seara rawak kepada empat kumpulan, sepuluh ekor setiap satu iaitu kumpulan kawalan, TRF, FNT dan TRF+FNT. TRF (200 mg/kg bb) dan FNT (20 mg/kg bb) diberikan seara oral paksa selama 28 hari. Minyak jagung yang merupakan pengangkut telah diberikan seara oral kepada kumpulan kawalan. Pada akhir kajian, hepar dan darah diambil untuk menilai kerosakan oksidatif dan status biokimia serta pemerhatian histologi. Suplementasi TRF melindungi hepar daripada mengalami kerosakan oksidatif dengan menurunkan aras malondialdehid (MDA) dan protein karbonil (PCO) hepar seara signifikan. Tambahan pula, TRF juga mengembalikan aras antioksidan endogenus terutamanya glutation terturun (GSH), glutation peroksidase (GPx) dan pengurangan ferik /kuasa antioksida (FRAP). TRF juga berupaya melindungi hepar daripada mengalami kerosakan dengan merendahkan aras enzim hepar, alanine aminotransferae (ALT) dan aspartate aminotransferase (AST). Pengurangan kerosakan hepar tikus kumpulan TRF+FNT juga telah diperhatikan seara histologi. Kesimpulannya, suplementasi TRF berpotensi memberikan kesan perlindungan pada hepar tikus aruhan FNT dengan mengurangkan kerosakan oksidatif dan meningkatkan status antioksidan. Keywords: Antioksida; minyak sawit; tekanan oksidatif; vitamin E INTRODUCTION Unontrolled appliation of organophosphate (OP) pestiides in agriulture and publi health had inreased the risk of toxiity to non-target organisms (Das & Mukherjee 2000). Human exposure to low-dose pestiide may arise from nutritional (food and drinking water) or environmental soures and may leads to a long-lasting and negative health impat in the long-term (Hernandez et al. 2013). Fenitrothion (O,O-dimethyl O-(4-nitro-m-tolyl)- phosphorotioate) (FNT) is one of the most ommonly used OP pestiides worldwide. It has been lassified as lass II (moderately toxi) ompound by the WHO (2010) and has been ategorised as one of the red list substanes by the European Union (EU) due to its toxiity effets towards the aquati environment (Kozawa et al. 2009). OP pestiides inluding FNT have also been reported to exert their primary toxi effets through the inhibition of holinesterase enzyme (ChE). The irreversible inhibition

2 1604 of ChE leads to aumulation of the neurotransmitter aetylholine and ontinuous stimulation of holinergi reeptors whih then produed the sign and symptoms of toxiity (Hazarika et al. 2003). However, previous researh had reported that the inhibition of ChE enzyme did not fully explain the OP toxiity (Lukaszewiz-Hussain 2010). As a prinipal organ for the drug metabolism and detoxifiation, liver is always prone to xenobioti-indued injury mainly due to metabolite formation (Jaeshke et al. 2002). Liver funtion ould be detrimentally altered, as it is the first organ to enounter the ingested nutrients, drugs and hemials inluding OP pestiides (Al-Attar 2010). FNT at the dose of 20 mg/kg bw was found to ause injury to liver and kidney (Elhalwagy et al. 2008), panreas (Budin et al. 2012) and damaged to sperm and testes (Taib et al. 2013) of experimental rats. Repeated exposure of OP might indue oxidative stress in different tissues through the formation of reative oxygen speies (ROS) as reviewed by Abdollahi et al. (2004). The damage to membrane lipids, protein and DNA was reported to be the endpoint biomarker of oxidative stress-indued toxi effets of pestiide (Tuzmen et al. 2008). In order to redue the toxi effets of OP by targeting the oxidative stress mehanism, many studies had been done to evaluate the protetive effets of various exogenous antioxidants suh as vitamin E (Bhatti et al. 2010; Elhalwagy et al. 2008). Palm oil is one of the major soures of vitamin E that onsist both toopherol and tootrienol isoforms with the ratio of 24: 76 (Aggarwal et al. 2010). Tootrienol is one of the vitamin E isomers whih also possess numerous healthrelated properties that are not shared by other α toopherols (Tiwari et al. 2009). Studies have shown that, tootrienol rih fration (TRF) of palm oil at the dose of 200 mg/kg bw -1 possesses high antioxidant (Paker et al. 2001), antidiabeti (Budin et al. 2009) and anti-hyperholesterolemia (Qureshi et al. 1991) properties. Previous study has reported that TRF has attenuate liver injury indued by FNT in experimental rats (Jayusman et al. 2014). However, the status of oxidative damage was not showed. Thus, the present study was done to determine the ability of TRF in preventing FNT-indued liver oxidative damage in rats by evaluating lipid and protein oxidation and endogenous antioxidants status. MATERIALS AND METHODS CHEMICALS Fenitrothion (Sumithion, 99% purity) was purhased from Supelo Analytial, USA. Palm Oil Tootrienol-rih Fration (Gold Tri. E 70) was purhased from Sime Darby, Malaysia. All other hemials were purhased from Merk, Germany. ANIMALS This study was onduted using male Sprague-Dawley rats (n=40), weighing between 220 and 250 g. The rats were obtained from the Animal Laboratory Unit of Universiti Kebangsaan Malaysia (UKM), Bangi, Malaysia. All rats were alimatized for 1 week under the same laboratory onditions of temperature (25 C to 28 C) with 12 h/12 h light/dark yle and were supplied with standard diet and tap water ad libitum. All the experimental proedures involving the animals were arried out aording to the animal ethis guidelines approved by the UKM Animal Ethi Committee (UKMAEC) with resolution number: FSKB/BIOMED/2010/BALKIS/14-JULY/311-AUGUST JULY EXPERIMENTAL DESIGN Rats were randomly divided into four groups, namely Control, TRF, FNT and TRF+FNT groups. The Control group only reeived orn oil and TRF group reeived TRF (200 mg/ kg bw). TRF and FNT were dissolved in orn oil, whih ats as a vehile. Rats in TRF+FNT group were supplemented with TRF (200 mg/kg bw) 30 min prior to 20 mg/kg bw of FNT both orally (Dirian & Kalender 2012). The FNT dose hosen in this study was based on a previous researh done by Elhalwagy et al. (2008) while TRF dose seletion was based on a study done by Budin et al. (2009). PREPARATION OF SERUM AND TISSUE EXTRACT After the treatment period, rats were anaesthetised and blood samples were olleted by ardia punture to obtain the plasma. The obtained plasma was stored at -40 C for further analysis of oxidative stress and biohemial assay. The rats were sarified and liver obtained. Small slies of the liver were fixed in 10% formalin for histopathologial evaluation. The remaining liver was homogenized with an Ultra Turrax T25 homogenizer in 50 mm ied-old phosphate buffer solution (ph7.4) ontaining 1.15% KCl and 0.1 M EDTA and entrifuged at g for 15 min at 4 C to produe 10% homogenate. Supernatants were olleted, aliquot and stored at -80 C for further analysis of oxidative stress and antioxidant status assay. CHOLINESTERASE AND LIVER ENZYMES ASSAY Cholinesterase enzyme (ChE), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ativities were determined by a semi-automated bioanalyzer BTS-350 (Biosystems S.A., Barelona, Spain). OXIDATIVE STRESS AND ANTIOXIDANT STATUS The homogenised liver and plasma samples were assessed for oxidative stress markers whih were malondialdehyde (MDA) and protein arbonyl using the methods of Stok and Dormandy (1971) and Levine et al. (1990), respetively. The levels of redued glutathione (GSH) and ferri-reduing antioxidant power (FRAP) were assessed aording to the method of Benzie and Strain (1999) and Ellman (1959), respetively. Antioxidant enzymes suh as glutathione S-transferase (GST), atalase (CAT), superoxide dismutase

3 1605 (SOD) and glutathione peroxidase (GPx) were measured in liver homogenate based on method done by Aebi (1984), Beyer and Fridovih (1987), Habig and Jaoby (1974) and Lawrene and Burk (1976), respetively. HISTOPATHOLOGICAL EXAMINATION The fixed liver was proessed in graded series of alohols and embedded in paraffin wax. The paraffin bloks were setioned at 4 μm thik and these setions were stained with haematoxylin and eosin (H&E) for histologial observation under the light mirosope. STATISTICAL ANALYSIS The results were expressed as means ± standard deviation. All data were analysed using the Statistial Pakage for Soial Sienes (SPSS 17.0). The results were analyzed using one way analysis of variane (ANOVA) followed by Tukey s test for multiple omparisons. Statistial signifiant was set at p<0.05. RESULTS EFFECTS OF TRF ON BODY WEIGHT AND LIVER WEIGHT The final body weight and liver (absolute and relative) weights of FNT group were signifiantly redued ompared to ontrol and TRF groups (p<0.05). TRF administration was able to signifiantly inrease the body weight and absolute liver weight in TRF+FNT group ompared to FNT group (p<0.05). However, the relative liver weight was not signifiantly different (Table 1). EFFECTS OF TRF ON CHE AND LIVER ENZYMES ACTIVITIES Table 2 shows a signifiant redution in ChE ativity and elevation of ALT and AST in the plasma of FNT group ompared to ontrol and TRF groups (p<0.05). TRF supplementation signifiantly redued the ALT and AST enzymes ativities in TRF+FNT group ompared to FNT group (p<0.05). However, no signifiant differene was found for ChE in TRF+FNT group ompared to FNT group alone. EFFECTS OF TRF ON OXIDATIVE STRESS MARKERS AND ANTIOXIDANT STATUS Table 3 shows the level of MDA, PC, GSH and FRAP value in the plasma of the experimental groups. In omparison with the ontrol and TRF groups, the levels of plasma MDA and PC were signifiantly elevated in FNT rats (p<0.05). Meanwhile, the level of GSH and FRAP value were signifiantly dereased in FNT group ompared to the ontrol group (p<0.05). TRF supplementation signifiantly dereased PC levels and signifiantly inreased FRAP TABLE 1. Final body weight, absolute and relative liver weight of experimental rats Parameters and treatments Control TRF FNT TRF+FNT Final body weight (g) Absolute liver weight (g) Relative liver weight (%) 354.3± ± ± ± ± ± ±10.63 a,b 6.49±0.67 a,b 2.35±0.18 a,b ±21.89 a,b, 7.6±0.82 a,b, 2.53±0.20 Values are expressed as means ± S.D., for eah group n=6. a Signifiant differene from ontrol group (p<0.05). b Signifiant differene from TRF group (p<0.05). Signifiant differene from FNT group (p<0.05). TRF TRF supplemented group; FNT FNT-indued group; TRF+FNT FNT-indued group supplemented with TRF TABLE 2. Effet of TRF supplementation on plasma ChE and liver enzymes in experimental rats Parameters Control TRF FNT TRF+FNT ChE (U/L) ALT (U/L) AST (U/L) ± ± ± ± ± ± ± a,b ± 4.12 a,b ± a,b ± ± ± Values are expressed as means ± S.D., for eah group n=6. a Signifiant differene from ontrol group (p<0.05). b Signifiant differene from TRF group (p<0.05). Signifiant differene from FNT group (p<0.05). TRF TRF supplemented group; FNT FNT-indued group; TRF+FNT FNT-indued group supplemented with TRF TABLE 3. Effet of TRF supplementation on plasma oxidative stress marker and antioxidant status in experimental rats Parameters Control TRF FNT TRF+FNT MDA equivalent (μmol/mg protein) PCO (nmol/mg protein) GSH (μmol/mg protein) FRAP value (μm) ± ± ± ± ± ± ± ± ± a,b ± a,b 4.85 ± 0.58 a,b ± a,b ± ± ± ± Values are expressed as means ± S.D., for eah group n=6. a Signifiant differene from ontrol group (p<0.05). b Signifiant differene from TRF group (p<0.05). Signifiant differene from FNT group (p<0.05). TRF TRF supplemented group; FNT FNT-indued group; TRF+FNT FNT-indued group supplemented with TRF

4 1606 value in plasma of TRF+FNT group ompared to FNT group (p<0.05). In Table 4, FNT aused a signifiant elevation of hepati MDA and PC levels ompared to ontrol and TRF groups (p<0.05). Conversely, FNT rats supplemented with TRF showed a marked redution in MDA and PC levels ompared to FNT group alone (p<0.05). FNT-intoxiation resulted in a lower GSH level, FRAP value, SOD, GPx, CAT and GST ativities (p<0.05) when ompared to ontrol and TRF groups. Supplementation of TRF to FNT group signifiantly inreased the hepati GSH level, FRAP value and GPx ativity ompared to FNT group (p<0.05). EFFECTS OF TRF ON LIVER HISTOPATHOLOGICAL OBSERVATION By the light mirosopi observation, the ontrol and TRF rats showed a normal histoarhiteture of liver as presented in Figure 1(a) and 1(b). Cords of hepatoytes were radiated from a entral vein towards periphery and separated from eah other by the sinusoid. Centrally loated nuleolus and distint nuleoli were observed at higher magnifiation. Figure 1() and 1(d) shows marked alterations of liver histopathology in FNT-treated rats. Haemorrhage, degeneration of hepati ords and ballooning of hepatoytes with areas of nerosis were observed. In ontrast, TRF supplementation ameliorated the histopathologial hanges in the liver tissue of TRF+FNT rat (Figure 1(e) and 1(f)). Haemorrhage and nerosis were absent and the struture of the hepati lobule was preserved. However, mild pyknoti ells were still present. DISCUSSION Human epidemiologial and animal experimental data indiated that the OP ompounds inluding FNT an be quikly absorbed from the skin and gastrointestinal trat due to its lipid-soluble nature. Beause of its widespread use on fruits, vegetables and stored grains, FNT is the most ommon insetiide residue found in food (Meaklim et al. 2003) whih ould lead to ross-ontamination to nontarget organism. Although FNT was given at 1/30 of the oral LD 50, we observed pathologial hanges in the rat s liver. The present study has showed the benefiial effet of TRF in preventing oxidative liver damage in rats expose to FNT. Inhibition of ChE ativity is an important indiator of OP poisoning. In this study, holinergi risis was observed whereby ChE ativity was found to be inhibited following FNT exposure while, the supplementation of TRF inrease this enzyme ativity marginally. Hene, TRF showed a potential in proteting the inhibition of ChE ativities but it was not a total protetion as desribed in a previous study (Bhatti et al. 2010). Vitamin E is able to protets ChE by preventing the phosphorylation ativity (Verma et al. 2007). Due to the inhibition of ChE, a marked dereased in final body weight was found in FNT group whih was similar to the study using the dimethoate-intoxiated rats (Saafi et al. 2011). Supplementation of TRF inreased the final body weight and absolute liver weight of FNT rats whih might be due to its ability to inrease rat s appetite and the redution of holinergi risis in OP toxiity (ElMazoudy et al. 2011). Besides the inhibition of AChE ativity, oxidative stress has also been lassified as one of the toxiity mehanisms involved in OP repeated exposure (Lukaszewiz-Hussain 2010). The present results showed that FNT indued oxidative damage of liver by inreasing lipid and protein oxidation and altering the enzymati and non-enzymati antioxidants status. Supplementation of TRF showed the ability in ounterating the oxidative liver damage possibly through the inreasing in free radial savenging ativity and improving the antioxidant status whih in line with the previous researh (Bhatti et al. 2010; Elhalwagy et al. 2008). In addition, the reduing of oxidative damage also seen in parameters measured in plasma, whih also support the role of TRF as exogenous antioxidant in biologial system (Budin et al. 2009). TRF possessed its antioxidant ativity through the ability of tootrienol and toopherol to donate phenoli hydrogens (eletrons) to lipid radials (Kamal-Eldin & Appelqvist 1996), suggesting that the antioxidant properties of both vitamin E isoforms in TRF might be responsible for the liver protetion against the oxidative stress. The presene of double bonds in the struture of tootrienol made it highly unsaturated and very effiient TABLE 4. Effet of TRF supplementation on liver oxidative stress marker and antioxidant status in experimental rats Parameters Control TRF FNT TRF+FNT MDA equivalent (μmol/mg protein) PCO (nmol/mg protein) GSH (μmol/mg protein) FRAP value (μm) SOD (unit/mg protein) CAT (unit/mg protein) GPx (nmol/min/mg protein) GST (nmol/min/mg protein) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± a,b ± a,b 4.81 ± 0.45 a,b ± 0.06 a,b ± a,b ± a,b 3.28 ± 0.13 a,b 3.73 ± 0.31 a,b ± ± ± ± ± ± ± ± 0.29 Values are expressed as means ± S.D., for eah group n=6. a Signifiant differene from ontrol group (p<0.05). b Signifiant differene from TRF group (p<0.05). Signifiant differene from FNT group (p<0.05). TRF TRF supplemented group; FNT FNT-indued group; TRF+FNT FNT-indued group supplemented with TRF

5 1607 FIGURE 1. Representative histopathology slides of Haematoxylin and Eosin (H&E) staining in the liver tissue of experimental rats. Figure 1(a) and 1(b) shows normal liver struture of ontrol group and TRF group. (a) Cords of hepatoytes radiated from entral vein are separated by sinusoids ontaining Kuppfer ells ( ) ( 40), (b) In higher magnifiation, roughly uniform nuleus and nuleolus were seen ( 100), () Liver setions of FNT-intoxiated rats showed disruption of hepati ords ( { ), (d) Neroti area (*) and severe haemorrhage (triangle). Ballooning of hepatoytes (star) and pyknoti ells (arrow) were learly seen in higher magnifiation ( 100). Liver setions of TRF+FNT rats showed histopathologial hanges of hepatoytes with attenuated severity ((e) 40 and (f) 100). Some improvements like the absene of severe haemorrhage and nerosis were observed in penetrating the saturated fatty layers of liver and brain tissues (Suzuki et al. 1993). The interation between the radial moleule and vitamin E would therefore stop the propagation of the peroxyl radial hain reation (Paker et al. 2001) and alleviate the oxidative stress response. The inrease in ROS formation would explain a number of deleterious effets suh as membrane damage and loss of ellular integrity (Anderson & Borlak 2007) in the liver. The injury to hepatoytes following OP pestiide intoxiation would ause the release of ytosoli enzymes into blood irulation (Elhalwagy et al. 2008). In the present study, plasma ALT and AST levels were inreased in FNT-intoxiated rats. Redution in the levels of plasma ALT and AST towards the normal values in TRF supplemented rats indiated that TRF protet the strutural integrity of the hepatoyte membrane thus preventing the leakage of its ytosoli enzymes (Bhatti et al. 2010). The mirosopi observations showed that TRF prevent various histopathologial alterations in liver ause by FNT whih inludes vasular ongestion, disruption of hepatoytes and ellular nerosis. The morphologial alterations were onsistent with the liver enzymes levels that have been initially observed. The present findings suggested that TRF ould attenuate the oxidative damage of liver due to the harmful effets of FNT. This finding is onsistent with study done by Bhatti et al. (2010) that shows the supplementation of vitamin E modulate the hepatotoxiity and oxidative stress in experimental rats. CONCLUSION In onlusion, the findings of the urrent study demonstrated that exogenously TRF was apable in proteting the oxidative toxi effets of subhroni FNT exposure. The hepatoprotetive effet of TRF an be orrelated to its antioxidant ativity. In spite of the ameliorative effet of TRF supplementation on some biohemial parameters, it may need more time to onfer full protetion towards the liver and other organs. Further studies are still needed to evaluate the other underlying mehanisms of TRF protetion in FNT-indued liver damage. Moreover, studies an be done to evaluate the reversible effets or the reovery level of the organ and their funtions by stopping the intoxiation with FNT and prolonging the duration of TRF supplementation. ACKNOWLEDGEMENTS The authors would like to express their gratitude to the Ministry of Higher Eduation (MOHE), Malaysia and

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7 1609 Tiwari, V., Kuhad, A., Bishnoi, M. & Chopra, K Chroni treatment with tootrienol, an isoform of vitamin E, prevents intraerebroventriular streptozotoin-indued ognitive impairment and oxidative-nitrosative stress in rats. Pharmaol. Biohem. Be. 93(2): Tuzmen, N., Candan, N., Kaya, E. & Demiryas, N Biohemial effets of hlorpyrifos and deltamethrin on altered antioxidative defense mehanisms and lipid peroxidation in rat liver. Cell Biohemistry and Funtion 26(1): Verma, R.S., Mehta, A. & Srivastava, N In vivo hlorpyrifos indued oxidative stress: Attenuation by antioxidant vitamins. Pesti. Biohem. Phys. 88(2): WHO The WHO Reommended Classifiation of Pestiides by Hazard and Guidelines to Classifiation Geneva: World Health Organization. Programme of Biomedial Siene Shool of Diagnosti and Applied Health Sienes Faulty of Health Sienes Universiti Kebangsaan Malaysia Jalan Raja Muda Abdul Aziz Kuala Lumpur, Federal Territory Malaysia *Corresponding author; Reeived: 24 Marh 2016 Aepted: 6 July 2017