ABSTRACT the various tissues. Key words: Petroleum,
This study was carried out to assess the
antioxidant status of rats fed
diet incorporating catfish contaminated
with crude petroleum oil treated
myrstica extracts. Thirty albino rats of weight 180 to 200 g were used for
the experiment and they were divided into six
groups of five rats each. The
grouping were as follows, group 1: control, group 2: rats were fed crude
petroleum oil contaminated catfish diet (CPO-CCD) only, group 3: CPO-CCD plus
tween 80, group 4, 5, and 6 were given CPO-CCD
and treated with M. myristica water extract (MWE), M.
myristica ethanol extract (MEE) and M. myristica diethyl ether extract
(MDEE). The experiment lasted four weeks. The results showed significant
(p<0.05) decreased in blood reduced glutathione (GSH), blood oxidised glutathione (GSSG), superoxide dismutase (SOD), catalase (CAT) and increase malondialdehyde (MDA) level in the liver, kidney and brain rats fed CPO-CCD only and CPO-CCD + tween 80 when compared to the control. Administration of MWE, MEE and MDEE to the rats fed CPO-CCD significantly (p<0.05) increase the level of blood GSH, blood GSSG, SOD, CAT and decrease MDA level in the liver, kidney and brain when compared with the CPO-CCD only and CPO-CCD + Tween 80. No significant difference was observed in the blood GSH:GSSG ratio and brain GSH level in all the experimental groups. In conclusion, M. myristica extracts exhibited beneficial effect by improvement of the antioxidant status and showed to evade the oxidative insult elicited by the CPO-CCD intoxication and in the various tissues. Key words: Petroleum, Diet, Antioxidants Indices, Monodora Myrstica INTRODUCTION Exposure to crude oil pollution leads to formation of free radicals (Won et al., 2016). Free radical is the most common reactive oxygen species in human (Georgewill and Nwankwoala, 2008). Once polyaromantic hydrocarbons enter the body of a living organism, each are metabolized to form highly reactive molecules such as diol epoxides that are polyromantic hydrocarbons intermediate metabolites that causes oxidative stress (Ekperusi and Aigbodion, 2015; Penning, 2014). Reduced glutathione is a multifunctional intracellular non-enzymatic antioxidant which is well known to be the major thiol-disulphide redox buffer of the cell (Swaran, 2009). Oxidized glutathione is accumulated inside the cells and the ratio of GSH/GSSG is a good measure of oxidative stress of an organisms. Superoxide dismutase is an enzyme that alternately catalyzes the dismutation of the superoxide radical into either ordinary molecular oxygen or hydrogen peroxide (Hayyan et al., 2016). Catalase is also an enzymes. The name catalase was given to the enzyme owing to its catalatic action on the hydrogen peroxide. Antioxidant enzymes can be inactivated by lipid peroxides (Alantary et al., 2014; Hamza and Al-Harbi, 2015). The subchronic exposure of rats to crude oil, decreased tissues catalases activities (Nwaogu et al., 2011). Several studies have shown that, direct exposure to crude oil can disrupt antioxidant status in serum, brain, liver, and kidney (Adedara et al.,2012; Ebokaiwe and Farombi, 2016) Monodora myristica is a spice commonly consumed in the Niger Delta part of Nigeria especially by the Itsekiri, Urhobo and Ndokwa people of Delta State. The seeds of M. myristica have attractive small, possess antioxidant properties and can be used in pharmaceutical industries (Talalaji, 1999). The present study aimed to assess the effect of M. myristica extracts in rats given diet incorporating catfish polluted with crude petroleum oil by evaluating some antioxidant status such as reduced and oxidised GSH, CAT, SOD and lipid peroxidation level. MATERIALS AND METHODS Preparation of the Spice (Monodora myristica) Extracts M. myristica was obtained from Obiaruku main market, Ukwuani Local Government Area (LGA), Delta State and then later identified at the Department of Botany, Delta State University, Abraka, Delta State. The spice was briefly sun-dried to constant weight for two weeks and then crushed into fine particles using blender for at high speed. One hundred grams of the powdered spice was extracted with 500 ml of the respective solvent (hot water (60?C), ethanol (95 % v/v), and diethyl ether, 95 % v/v) and allowed to stand for 48 hrs. The mixture was then filtered using a clean muslin cloth. The filtrate was evaporated to dryness using rotary evaporator attached to a vacuum pump. The extracts were stored in refrigerator (- 4 oC) until required. Crude petroleum oil pollution and diet preparation The crude petroleum oil (CPO) was got from the Nigerian National Petroleum Cooperation (NNPC), refinery, Warri Delta State, Nigeria. Fifty catfish (with length between 20-25 cm and weight between 250-300 g) was got from commercial farm, then acclimatized for 7 days for the experiment. The catfish was divided into two groups; group 1 : control : contains twenty-five catfish which was cultured in plastic aquaria with 30 L borehole water for four weeks. Group 2: also contain twenty-five which was cultured in plastic aquaria with 30 L borehole water and then polluted with crude petroleum oil, 823.3 µl/L as described by Ikeogu et al. (2013) for four weeks. At the end of the experimental period, the catfish was harvested and the used in the preparation of diet for the experimental rats following the method described by Sunmonu and Oloyede (2007). The catfish were oven dried at 40°C and used as a source of protein. The diet for each group were prepared by mixing known quantities of sources of each food class comprising: protein (25 %), corn starch (52 %), groundnut oil (4 %), maize cob (4 %), granulated refined sugar (10 %) and vitamin/mineral mixture (5 %). The food components were mixed together and then made into pellets which was feed rats. Experimental Rats Thirty male albino Wistar rats were used for the study. The rats were allowed to acclimatized for two weeks to suite the laboratory condition. They had free access to water and standard growers mash diet. The rats used for the study were in accordance to the guide for care and use of laboratory animals (NIH, 1985). They were divided into six groups of five rats; group 1: control, group 2: CPO-CCD only, group 3: CPO-CCD plus 1 ml/kg b. wt. of 1 % tween 80, group 4: CPO-CCD plus 200 mg/kg b. wt. of MWE, Group 5: CPO-CCD plus 200 mg/kg b. wt. of MEE and group 6: CPO-CCD plus 200 mg/kg b. wt. of MDEE. Rats in group 1 to 6 received tap water daily throughout the experiment. The administration of the CPO-CCD and extracts orally was allowed for four weeks. Blood Collection and Preparation of Tissue Homogenate The rats were sacrified after 24 hours fast on the last day. The blood was collected by cardiac puncture using hypodermic syringe and needle and then transferred to an anticoagulant tube and organs were harvested. One gram of various tissue (liver, kidney and brain) were homogenized in 10 ml of normal saline and then centrifuged at 2,500 revolution per minutes for 15 minutes to obtained the supernatant which was immediately used for biochemical analysis. BIOCHEMICAL ANALYSIS Estimation of blood GSH/GSSG (Reduced/Oxidized glutathione) ratio. Blood GSH/GSSG was estimated using the enzymatic method described by Tietze (1976). GSSG Sample preparation Thirty microliters (30 ?L) of thiol-scavenging reagent (50 mg 1- methyl pyridinium trifluoromethane sulphonate) to a micro-centrifuge tube and 100 ?L of whole blood was carefully added to the bottom of the centrifuge tube and mixed gently. GSSG sample 130 ?L was incubated at room temperature for 5-10 minutes. Thereafter 270 ?L of ice-cold 5 % MPA was added to the tube and centrifuged at 1000 x g and 4°C for 10 minutes. The supernatant (50 ?L) was added to 700 ?L of assay buffer (phosphate buffer, 2M, pH 8) in a new micro-centrifuge tube, this was placed the on ice until used. GSH Sample preparation Fifty microliter (50 ?L) of whole blood was added to the bottom of a micro-centrifuge tube and then mixed. Thereafter 350 ?L of ice-cold 5% MPA was added to the micro-centrifuge tubes and centrifuged at 1000 x g in 4°C for 10 minutes. Then, 25 ?L of the supernatant was added to 1.5 mL of assay buffer in a new micro-centrifuge tubes and this was placed on ice until required. Procedure Two hundred microliters (200 ?L) of samples and blank were added to 200 ?L of the DTNB solution in respective test-tubes, then 200 ?L of the reductase solution (recombinant glutathione reductase) was added immediately then mixed. These were allowed to incubate at room temperature for 5 minutes, then 200 ?L of 2 mg/mL NADPH solution was added to the test tubes. The absorbance were read and recorded at 412 nm. Standard curve After preparing 1mM stock solution of GSH /GSSG. Different concentrations were prepared and from each of the GSH/GSSG dilution, 200?l was taken and added into 2300 ?l of 0.2 M phosphate buffer pH 7.6 then 500?l of 1mM DTNB was added, these five mixtures were well shaken and incubated for five minutes. After the incubation period, absorbance of each mixture was recorded at 412 nm. 5,5-dithiobis-2-nitrobenzoic acid (DTNB) blank was prepared by adding 500 ?l DTNB to 2500 ?l phosphate buffer pH 7.6. Absorbance of DTNB was also taken at 412 nm. The real absorbance of each mixture was obtained by subtracting absorbance of DTBN blank from absorbance of each of the mixture. The concentration of GSSG is much lower in the reaction mixture compared to GSH,standard calibration curve was plotted separately, 0, 0.50, 0.75, 1.0, and 1.50 ?M GSSG, and 0, 1.0,1.5, 2.0, and 3.0 ?M GSH Concentration of GSH/GSSG ratio (units/ml) = GSH-2GSSG GSSG Estimation of tissue reduced glutathione The reduced glutathione concentration in the liver, kidney and brain were estimated using the method of Ellman (1959). Procedure: To 0.5 ml of tissue homogenate was added 2 ml 10% trichloroacetic acid and centrifuged. One milliliters (1ml) of supernatant was treated with 0.5 ml of Ellman's reagent and 3 ml of phosphate buffer. The colour developed was read at 412 nm. A series of standard were treated in similar manner along with a blank containing 3.5 ml of buffer. Determination of superoxide dismutase activity The activity of SOD in the liver, kidney and brain were assayed using the method of Misra and Fridovich (1972). Procedure: The assay was carried out by adding 0.2 ml of the supernatant to 2.5 ml of 0.05 M carbonate buffer, pH 10.2. The reaction was started by addition of 0.3 ml freshly prepared epinephrine as the substrate to the buffer supernatant mixture and was quickly mixed by inversion. The reference cuvette contained 2.5 ml of the buffer; 0.3 ml of the substrate and 0.2 ml of distilled water. The increase in absorbance at 480 nm due to the adrenochrome formed was monitored every 30 seconds for 120 seconds. Determination of Catalase Activity The method of Kaplan et al. (1972) was adopted for the assay of liver, kidney and brain catalase activity. Procedure: Two milliliter (2 ml) of H2O2 was added to 1ml of sample in the reaction cuvette. Absorbance was read at 360 nm for 70 seconds. The reference cuvette contained 2 ml H2O2 and 1ml of water. The disappearance of hydrogen peroxide was calculated using the Molar extinction co- efficient, ? = 39.4 M-1 cm-1. Determination of Lipid Peroxidation Lipid peroxidation in form of malondialdehyde (MDA) were determined in the liver, kidney and brain by using the method of Buege and Aust (1978). Procedure: One millilitre of the sample was added to 2 ml of TCA-TBA-HCL reagent 0.37% Thioarbituric acid (TBA), 15% Tricarcoxylic acid (TCA) and 0.24 N Hydrochloric acid (HCl) (1:1:1 ratio). The tube was stoppered loosely and immersed in boiling water for 15 minutes and swirled slightly at intervals. The mixture was cooled and centrifuged for 10 minutes at 5000 g. The absorbance was read at 532 nm using the reagent blank. Lipid peroxidation in units/g of wet tissue was calculated with a molar extinction co-efficient of 1.56 x 105M-1 STATISTICAL ANALYSIS The data obtained and results were expressed as mean ±SD. The significant differences between groups were analyzed using one way analysis of variance (ANOVA) and least significant difference (LSD). The SPSS-PC programme package (version 17.0) were used for statistical analysis. A significant threshold of p< 0.05 was regarded statistically significance between the test and control group for the analysis. RESULTS Table 1: Blood GSH, GSSG and GSH:GSSG ratio of rats fed CPO-CCD treated with extracts of M. myristica Groups Blood GSH (units/ml) Blood GSSG (units/ml) Blood GSH:GSSG ratio 1: Control 1.81±0.04 a 0.72±0.03 a 0.99±0.02 a 2: CPO-CCD only 0.42±0.02 b 0.11±0.07 b 0.80±0.05 a 3: CPO-CCD + Tween 80 0.42±0.06 b 0.12±0.05 b 0.83±0.04 a 4: CPO-CCD + MWE 1.09±0.05 a 0.53±0.01 a 1.00±0.01 a 5: CPO-CCD + MEE 1.41±0.32 a 0.56±0.01 a 1.01±0.02 a 6: CPO-CCD + MDEE 1.63±0.10 a 0.61±0.02 a 1.00±0.01 a Values are given in mean ± SD. n=5. Mean values with different superscript letter in the same column differ significantly at p<0.05. Table 2: GSH level in the liver, kidney and brain of rats fed CPO-CCD treated with extracts of M. myristica GSH (units/g wet tissue) Groups Liver Kidney Brain 1: Control 7.19±0.99 a 6.54±0.05 a 3.33±0.47 a 2: CPO-CCD only 3.36±0.94 b 2.24±0.79 b 1.01±0.05 a 3: CPO-CCD + Tween 80 2.34±0.73 b 1.76±0.57 b 2.81±0.08 a 4: CPO-CCD + MWE 3.46±0.54 b 3.31±0.66 b 2.36±0.16 a 5: CPO-CCD + MEE 3.96±0.09 b 4.29±0.19 b 2.20±0.16 a 6: CPO-CCD + MDEE 6.25±0.58 a 5.31±0.29 a, b 2.64±0.08 a Values are given in mean ± SD. n=5. Mean values with different superscript letter in the same column differ significantly at p<0.05. Table 3: Changes in superoxide dismutase activity in the liver, kidney and brain of rats fed CPO-CCD treated with different extracts of M. myristica SOD (units/g wet tissue) Groups Liver Kidney Brain 1: Control 89.41±19.16 a 86.41±14.28 a 73.50±6.52 a 2: CPO-CCD only 57.36±6.12 b 55.07±8.00 b 40.28±3.15 b 3: CPO-CCD + Tween 80 56.07±14.40 b 54.46±3.03 b 40.90±2.67 b 4: CPO-CCD + MWE 66.43±6.98 c 65.29±6.29 c 50.48±4.33 c 5: CPO-CCD + MEE 78.11±6.77 d 74.50±3.38 d 67.32±2.90 d 6: CPO-CCD + MDEE 85.47±3.58 a 82.41±1.42 a 70.23±5.21 a Values are given in mean ± SD. n=5. Mean values with different superscript letter in the same column differ significantly at p<0.05. Table 4: Changes in catalase activity in the liver, kidney and brain of rats fed CPO-CCD treated with extracts of M. myristica. CAT (units/g wet tissue) Groups Liver Kidney Brain 1: Control 74.28±10.19 a 70.18±8.65 a 64.26±5.94 a 2: CPO-CCD only 46.53±12.62 b 40.31±7.62 b 35.71±3.92 b 3: CPO-CCD + Tween 80 46.16±512 b 41.15±2.95 b 36.27±4.37 b 4: CPO-CCD + MWE 55.31±10.19 c 52.28±3.47 c 45.49±3.64 c 5: CPO-CCD + MEE 61.47±11.53 d 60.74±5.42 d 50.39±5.99 d 6: CPO-CCD + MDEE 72.20±5.65 a 68.32±3.57 a 61.17±2.38 a Values are given in mean ± SD. n=5. Mean values with different superscript letter in the same column differ significantly at p<0.05. Figure 1: MDA level in the liver, kidney and brain of rats fed CPO-CCD treated with different extracts of M. myristica. Bars represent mean values from five rats in each group. For each organs, bars with different superscript letter in the same column differ significantly at p<0.05. RESULT AND DISCUSSION Alterations of blood GSH, GSSG and GSH:GSSG ratio of rats fed CPO-CCD treated with different extracts of M. myristica are shown in Table 1. Significant (p<0.05) decrease level of blood GSH and GSSG were observed in rats fed CPO-CCD only and CPO-CCD + tween 80 when compare with control. Treatment of rats fed CPO-CCD with MWE, MEE and MDEE CPO-CCD significantly (p<0.05) increase the level of blood GSH and GSSG. The reduction in GSH and GSSG levels could be a compensatory mechanism by which the rats fed the formulated feed mixed with crude oil contaminated catfish to overcome the effect of the oxidant stress caused by free radicals produced by crude petroleum oil. The is in line with the findings of Shang et al. (2016), indicating that the decreased GSH to GSSG ratio in the blood showed that oxidative stress occurred in the distant organs and systemically upon crude petroleum induced nephrotoxicity. No significant difference were observed in the blood GSH, GSSG and GSH : GSSG ratio levels when rats fed CPO-CCD only were compared rats with fed CPO-CCD + tween 80, these results may be considered as pathological evidences to confirm the nontoxic effect of tween 80 as previously reported (Rowe, 2009). The level of GSH, SOD and CAT activity in the liver, kidney and brain of rats fed CPO-CCD treated with extracts of M. myristica are shown in Table 2, 3 and 4 respectively. Rats fed with CPO-CCD only and CPO-CCD + Tween 80 showed significant (p<0.05) decreased in SOD and CAT activity in the liver, kidney and brain when compared to the control. Administration of MWE, MEE and MDEE to CPO-CCD rats significantly (p<0.05) increased level of SOD and CAT in the liver, kidney and brain when compared with the CPO-CCD only and CPO-CCD + Tween 80 respectively. No significant difference was seen in GSH level in the brain of all the experimental groups. The decrease kidney and liver GSH level in the rats fed CPO-CCD may be due to the decrease in the activity of the hepatic glutamate-cysteine ligase (a key enzyme responsible for glutathione synthesis). The depletion in brain GSH in CPO-CCD induced oxidative stress may leads to increased productions of superoxide, hydroxyl radicals, and H2O2, because there is no known enzymatic defense against hydroxyl radicals (Dringen, 2000), making GSH the only compound capable of scavenging these radicals in the brain. These findings are in line with the study of Aoyama et al. (2008) which states that, when comparing the brain with other organs, the brain is especially vulnerable to oxidative stress. This is because it has lower SOD, CAT, and glutathione peroxidase; GPx activities, while it contains an abundance of lipids with unsaturated fatty acids that are targets of lipid peroxidation (Dringen, 2000). Furthermore, the brain GSH concentration is lower than those of the liver and kidney (Aoyama et al., 2008). The detoxification mechanisms promoted by enhanced glutathione production indicates the protective effects of MDEE, MEE and MWE. This also might be the reason for the restoration of other antioxidant enzymes (SOD and CAT). The marked reduction in SOD activity of rats fed CPO-CCD and the enhanced SOD activity when M. myristica extracts was administered were in agreement with other studies (Nwaogu et al., 2011; Sunmonu and Oloyede, 2007). The inhibition of CAT activity during CPO-CCD induced toxicity may be due to the increased generation of reactive free radicals, which can lead to oxidative stress in the cells. The administration of MDEE, MEE and MWE inversed the catalase activity in the liver, kidney and brain tissues and thus enhance the antioxidant defense against ROS. This findings are in collaboration with Oyinloye et al.(2016) who reported that M. myristica aqueous extract prevent lipid peroxidation and replenish hepatic antioxidant enzymes against cadmium induced liver tissue damage. Figure 1, showed the level of MDA; malondialdehyde (end product of membrane lipid peroxidation) in the tissues (liver, kidney and brain)of rats fed CPO-CCD treated with extracts of M. myristica. MDA level in the respective tissues were significantly (p<0.05) increased in rats fed CPO-CCD only and CPO-CCD + tween 80 when compared with the control rats. However, treatment of rats fed CPO-CCD with the different extracts significantly decreased the level of MDA as compare with that of the control in the respective tissues. The excessive ROS generated during crude petroleum oil toxicity rapidly react with lipid membranes and thus initiates the lipid peroxidation chain reaction, resulting in lipid peroxyl radicals' formation (Ita and Edagha, 2016; Ujowundu et al., 2012; Nwaogu et al., 2011). The elevation of lipid peroxidation caused by rats administered crude petroleum oil has been previously reported (Sunmonu and Oloyede, 2007), which is in line with the results obtained in this study. In the present study, the lower MDA levels in the tissues of rats fed CPO-CCD plus M. myristica extracts, apparently indicating the anti-oxidative protective role of M. myristica extracts against CPO-CCD induced damage on cell membranes. Moreover, MDEE revealed a strong inhibitory ability towards lipid peroxidationas compared with MEE and MWE. CONCLUSION The results of this study showed that M. myristica extracts rescued the CPO-CCD induced tissues damage/lipid peroxidation and improvement of antioxidant status owing to its free radical scavenging properties.