Assessment of selected hepatic serum enzymes in contaminated crude-oil feeds on albino wistar rats

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This study was aimed at investigating the effect of crude oil contaminated feeds on selected serum enzymes and electrolyte levels in Albino wistar rats. Albino wistar rats weighing between 180g to 200g were obtained from Ladoke Akintola University of Technology Ogbomosho, OyoState and used for the study. The animals were grouped into five study groups with six (6) animals in each group. The animals were kept in a wellventilated room comprising control group, crude oil group, garlic treated group, Vitamin C treated group and Vitamin E treated group.

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Nội dung Text: Assessment of selected hepatic serum enzymes in contaminated crude-oil feeds on albino wistar rats

Int.J.Curr.Microbiol.App.Sci (2018) 7(8): 280-290 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 08 (2018) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2018.708.033 Assessment of Selected Hepatic Serum Enzymes in Contaminated Crude-Oil Feeds on Albino Wistar Rats R.U. Ukpanukpong1*, D.I. Basiru1, O.O. Obadare2 and W.A. Omang1 1 Department of Biochemistry, Faculty of Basic Medical Sciences, University of Calabar, Nigeria 2 Department of Biochemistry, College of Natural Sciences, Joseph Ayo Babalola University, Ikeji -Arakeji Osun State, Nigeria *Corresponding author ABSTRACT This study was aimed at investigating the effect of crude oil contaminated feeds on selected serum enzymes and electrolyte levels in Albino wistar rats. Albino wistar rats weighing between 180g to 200g were obtained from Ladoke Akintola University of Keywords Technology Ogbomosho, OyoState and used for the study. The animals were grouped into five study groups with six (6) animals in each group. The animals were kept in a well- Body weight, ventilated room comprising control group, crude oil group, garlic treated group, Vitamin C Serum enzymes, treated group and Vitamin E treated group. The animals were fed with contaminated feeds Electrolyte level 25mg of the antioxidant specified for each group simultaneously for two weeks. However, control group were only fed with the normal while the crude oil group was not treated with Article Info the antioxidant. The levels of LDH and GGT concentrations increased significantly Accepted: (P>0.05) in crude oil only fed group but LDH showed low concentration on treatment with 04 July 2018 vitamin E(P0.05) in the activities of AST, ALT, ALP in groups fed with crude oil contaminated diet when compared to the control and these sharp changes were ameliorated after 28 days of antioxidant treated groups. Introduction total government revenue earning and 25% of the gross domestic product (GDP) (Niger Nigeria is the sixth largest exporter of crude Delta Development Commission, 2006). oil in the world as a result, its air, land and Crude oil and gas pollution is the major water are heavily polluted with crude oil and environmental hazard caused by crude oil and its refined products (Ita and Udofia, 2011). gas exploration, exploitation and production in Crude oil and gas production is the main stay the Niger Delta region of Nigeria and many of the Nigeria economy contributing about parts of the world (Wang et al., 2010). Crude 90% of the nation’s foreign exchange, 80% of oil and gas pollution can occur in form of 280
Int.J.Curr.Microbiol.App.Sci (2018) 7(8): 280-290 spillages due to oil well blowout, corrosion of in organism (Doyle and Lim, 2002). The pipelines, accidental discharges and impact of crude oil spillage and discharge on vandalization. These oil spillages can lead to the ecosystem as a result of oil exploration underground leakages which have impacts on activities is an obvious problem of the environment in the form of underground environmental concern. Similarly, the use of water pollution, soil pollution, health effect crude petroleum as therapeutic and in folkloric and destruction of vegetation (Alam et al., medicine in some parts of mineral oil 2010). producing communities in the Niger-Delta is another deliberate exposure with obvious In Nigeria today, contamination as a result of health implications. Again, the exposure of oil spillage has become one of the severest workers in the oil industries may bring about a forms of environmental pollution. Generally, harvest of ill health, diseases and even death. petroleum and its refined products reaching the terrestrial and aquatic ecosystems arrive as There are accumulating evidences which a consequence of spillage which may result indicate that humans, domestic and wildlife from natural seepages, offshore exploration, species are suffering from adverse health leakages from wells or from oil tankers, consequences from exposure to environmental accident from oil tankers, land based chemicals that interact with the endocrine discharges and sabotage. The toxic effect of system. According to Patrick-Iwuanyanwu et crude oil hydrocarbons in an organism is al., (2011), petroleum has the potential to manifested afterwards to bioaccumulation of elicit multiple types of toxic effects due to its the offending xenobiotics. complicated composition. In experimental animals, petroleum products have been Currently, widespread concern exists demonstrated to have adverse effects regarding the presence of pollutants in the including the generation of free radicals environment which can interfere with normal (Achuba and Osakwe, 2003), increase in endocrine functions in animals, including serum levels of urea and creatinine and humans. Particular attention has been focused significant degenerative changes in the on chemicals that are capable of mimicking or structural integrity of hepatic and renal cells modulating the effects of gonadal hormones, (Patrick-Iwuanyanwu et al., 2011). Acute thereby potentially interfering with the toxicity study of diesel fuel in rats (Rattus reproductive processes and behaviors of rattus) indicated a dose-dependent organisms (Doyle and Lim, 2002). hepatocellular necrosis and adverse alteration in structure and function of renal tubules. Traditional toxicological testing has focused on the ability of substances to cause obvious Acute, chronic and long term effects of harm to adult or developing animals at high chemical compounds (including crude oil) on doses. Some endocrine disruptors are able to living systems could be studied by evaluating affect the hormonal system at very low doses, the biochemical and morphological changes in causing subtle disorders that may not be various organs especially the liver. The basic readily apparent or that may be delayed, even structural component of the liver is the liver onto the next generation. The discovery that cells or hepatocytes. The liver is the principal chemicals could cause harm to animals and organ of metabolism and has a role to play in possibly to humans also in such subtle ways at many body processes most especially low doses has challenged traditional views of detoxification of chemical compounds. toxicity and expanded gaps in toxicity testing Research studies have shown a variety of 281
Int.J.Curr.Microbiol.App.Sci (2018) 7(8): 280-290 adverse effects on the hepatocytes of rats and of paraffin wax, will be viscous and have high catfish following exposure to environmentally cloud and pour points. These paraffins consist toxic compounds (Sunmonu and Oloyede, of isoalkanes and methyl cycloalkanes. Most 2006). In the present study, an attempt is made commonly found naphthenes are five and six to carry out biochemical assessment of serum member rings and occasionally a few rings enzyme and electrolyte in ingested crude oil with seven carbon atoms. Among these, contaminated diets on Wistar Albino Rats. methyl derivatives are the most abundant compounds as compared with the parent Petroleum ("rock oil") is a naturally occurring, bicycle compounds. flammable liquid found in rock formations in the Earth consisting of a complex mixture of Crude oils contains up to 50% of such Oil hydrocarbons of various molecular weights, pollution occurs when oil is introduced into plus other organic compounds. The proportion the environment directly or indirectly by of hydrocarbons in the mixture is highly men’s impacts resulting in unfavorable change variable and ranges from as much as 97% by in such a way that safety and welfare of any weight in the lighter oils to as little as 50% in living organisms is endangered. Crude oil if the heavier oils and bitumens. The spilled into the water spreads over a wide area hydrocarbons in crude oil are mostly alkanes, forming a slick and oil in water immediately cycloalkanes and various aromatic begins to undergo a variety of physical, hydrocarbons while the other organic chemical and biological changes including compounds contain nitrogen, oxygen and evaporation of high volatile fractions, sulfur, and trace amounts of metals such as dissolution of water-soluble fractions, iron, nickel, copper and vanadium. photochemical oxidation, drill, emulsification, microbial degradation and sedimentation The exact molecular composition varies (Colbborn et al., 1993). Crude oil is a complex widely from formation to formation but the mixture of hydrocarbon and organic proportion of chemical elements varies over compounds of Sulphur, Nitrogen, Oxygen and fairly narrow limits as follows. The typical a certain quantity of water which varies in nature of crude oil from different sources is composition from place to place (Ita and different or less identical. The same is true for Udofia, 2011). crude oil also; the individual oil even from the same well at different time of extraction is Crude oil is produced from decay of plants differing in the characteristics in term of and animals over millions of years. It is also chemical composition. Crude oils are complex referred to as mineral oil. Crude oil, which is a but mainly paraffinic, napthenic and aromatic mixture of hydrocarbons and inorganic (Wang et al., 1994). Crude oils contain all compounds, is drilled through rocks. normal alkenes from C1 to C120. However, this percentage rises to 35% in highly Crude oil discharged on the sea surface paraffmic and decreases to zero in highly undergoes physical, chemical and biological biograded oils (Al-Turki, 2010). Methane is alteration. Rapid physical and chemical predominant component of natural gas and processes include spreading and movement by alkanes ranging from pentane to pentadecane wind and currents, injection into the air, are the chief constituents of straight run evaporation of volatile components, dispersion (uncrackcd) gasoline or petrol. Above C17, of small droplets into water, dissolution and the alkanes are solid wax like substances and chemical oxidation. Concurrent with these are crude oils, which contain high concentrations relevant biological processes. They include 282
Int.J.Curr.Microbiol.App.Sci (2018) 7(8): 280-290 degradation by micro-organisms and uptake Drug administration by larger organisms followed by metabolism and storage or discharge (Nelson-Smith, Three drugs: garlic, vitamin C and vitamin E 1972). were used for the study. The drugs were given orally using 2.5mg/kg syringe. Materials and Methods Experimental design Source of animals The animals were grouped into five groups Wistar albino rats, aged between 6 and 8 with each group having six animals including weeks used for this research weighing control. The animals were kept in a well- between 180g and 200g were obtained from ventilated room comprising control group, Ladoke Akintola University of Technology crude oil group, garlic treated group, Vitamin Ogbomosho Oyo State. C treated group and Vitamin E treated group. The animals were fed with contaminated feed The animals were kept in the experimental with 2.7mg/kg of the drug specified for each cage in order to acclimatize to laboratory group simultaneously for 28 days. However, conditions. Regular and adequate feeding control group were only fed with normal meal regimes for one week, i.e. 7 days, before the while the crude oil group was not treated with commencement of the experiment. They were the antioxidant. fed on commercial growers’ mash and water. Blood and serum collection Crude-oil At the end of the experimental period, the The crude oil was obtained from the Shell animals were given chloroform anaesthesia by terminal, well 4 of Shell oil. A class A oil, inhalation to enable unclotted blood to be light and volatile Warri Delta State, Nigeria. collected by cardiac puncture, (Thompson, 1983). With the aid of a 5ml syringe and needle, blood were being collected and Chemicals/Reagents suspended into EDTA plastic bottles. These All the chemicals used in this research were were centrifuged at 1200rpm for 10 minutes to obtained from Famlab Nigeria Limited Lagos obtain serum for determination of enzymes which is of reagent grade obtained from and electrolyte levels. Merck, Germany; BDH chemicals Ltd, Determination of total serum bilirubin level England; May and Baker Ltd, England; Riedel-De-Haen Hannover, Germany and Method Hopkins and Williams Essex, England. Colorimetric method by Jendrassik and Grof Formulation of contaminated diet (1938). 10% crude oil contaminated diet was obtained Principle by thoroughly mixing 45g of feed with 5ml of crude oil. The feed for the control group Direct (conjugated) bilirubin reacts with contained no crude oil (i.e. 0% group). diazotized sulphanilic acid in alkaline medium to form a blue coloured complex. Total 283
Int.J.Curr.Microbiol.App.Sci (2018) 7(8): 280-290 bilirubin was determined in the enzyme Determination of serum Gamma Glutamyl presence. Tranferase (γ-GT) Activity Determination of serum Aspartate Method Aminotransferase (AST) level Colorimetric - Kinetic Method Principle Colorimetric method. Principle At pH 7.8 γ-GT in sample was made to catalyse the transfer of glutamyl terminal GOT residue from γ- glutamyl-p-nitroanalide to the α-oxoglutarate + L-aspartate L- glutamate + oxaloacetate. acceptor, glycyglycine to give yellow coloured γ- glutamyl-glycyglycine. The rate of AST is measured by monitoring formation of the product monitored at 405nm was proportional to the enzyme activity. The concentration of oxaloacetate hydrazine formed with 2,4-dinitrophenylhydrazine. Determination of serum Electrolytes; Na+, K+, Cl - Determination of serum Alanine Aminotransferase (ALT) level Method Method Ion Selective Electrodes (ISE) Colorimetric method. Principle Principle GPT A transducer (or sensor) converts the activity α- oxoglutarate +L- alanine L- of a specific ion dissolved in a solution into an glutamate + pyruvate. electrical potential which was measured by a voltmeter. The voltage was theoretically Alanine aminotransferase was measured by dependent on the logarithm of the ionic Monitoring. activity. The sensing part of the electrode was usually the ion-specific membrane, alone with The concentration of pyruvate hydrazine a reference electrode (Eric et al., 2006). formed with 2, 4- dinitrophenylhydrazine. Statistical analysis Determination of serum Alkaline Phosphatase All the results were expressed as means ± SD Method and all data were analyzed using Analysis of variance (ANOVA). Colorimetric method. Significant differences between the control Principle and treatment groups were determined at ALP (P
Int.J.Curr.Microbiol.App.Sci (2018) 7(8): 280-290 Results and Discussion was significant increase in concentration of ALT and AST level (P>0.05) compared with Results of Lactate dehydrogenase (LDH), control 1 and control 2 (crude oil control) Serum Aspartate Transaminase (AST), respectively. However, ALT in vitamin C Alkaline Phosphatase (ALP), Serum Alanine treated showed significant decrease 94.9±0.04 Transaminase (ALT) and Gamma Glutamate (P
Int.J.Curr.Microbiol.App.Sci (2018) 7(8): 280-290 Figure.1 Effect crude oil contaminated feeds on serum enzymes level after 14 days of administration in Wistar rats 450 400 350 300 LDH 250 GGT ALP 200 ALT 150 AST 100 50 0 control crue oil garlic vitamin C vitamin E Values expressed in Mean ± S.E.M of 8 determinations a= shows significant difference compared with positive control (p< 0.05). b= shows significant difference compared with negative control (p< 0.05) Fig.2 Effect crude oil contaminated feeds on serum enzymes level after 28 days of administration in Wistar rats 350 300 250 LDH 200 GGT ALP 150 ALT 100 AST 50 0 control crude oil garlic vitamin C vitamin E Values expressed in Mean ± S.E.M of 8 determinations a= shows significant difference compared with positive control (p< 0.05). b= shows significant difference compared with negative control (p< 0.05). 286
Int.J.Curr.Microbiol.App.Sci (2018) 7(8): 280-290 Fig.3 Effect crude oil contaminated feeds on electrolyte and bilirubin level after 14 days of administration in Wistar rats 180 160 140 120 Na+ 100 K+ 80 Cl- 60 BIL 40 20 0 control crude oil garlic vitamin C vitamin E Values expressed in Mean ± S.E.M of 8 determinations a= shows significant difference compared with positive control (p< 0.05). b= shows significant difference compared with negative control (p< 0.05). Fig.4 Effect crude oil contaminated feeds on electrolyte and bilirubin level after 28 days of administration in Wistar rats 160 140 120 100 Na+ 80 K+ Cl- 60 BIL 40 20 0 control crude oil garlic vitamin C vitamin E Values expressed in Mean ± S.E.M of 8 determinations a= shows significant difference compared with positive control (p< 0.05). b= shows significant difference compared with negative control (p< 0.05). 287
Int.J.Curr.Microbiol.App.Sci (2018) 7(8): 280-290 Chloride (Cl-) levels was significantly high corroborates with the previous evidence found (P>0.05) in vitamin C and vitamin E treated in (Corpas et al., 2001) reported that serum groups (101.0±0.03 and 97.9±0.03) while it is alkaline phosphatase levels obtained from significantly low (P0.05) in garlic, represent the comparism and the effects of the vitamin C and vitamin E treated groups garlic, vitamin C and vitamin E drugs on (6.9±0.05, 7.9±0.04 and 9.9±0.04). serum enzymes; LDH, GGT, ALP, ALT and AST with positive control and negative This research explored the ameliorative control. The levels of LDH and GGT effects of garlic, vitamin C and vitamin E on concentrations increased significantly serum enzymes and electrolyte of crude oil (P>0.05) with both positive control and crude contaminated feed. Exposure of humans and oil control except in LDH which showed low animals to crude oil contamination is at the concentration on treatment with vitamin E alarming rate as a result of the environmental, (P0.05) increased in the activities clinical diagnosis of heart and liver diseases. of AST, ALT, ALP in rats fed crude oil For example, liver enzymes such as alanine contaminated diet as compared to the control transaminase and aspartate transaminase are agreed with the results of previous studies on usually raised in acute hepatotoxicity but tend crude petroleum and refined petroleum to decrease with prolonged intoxication due to products (Maldonado et al., 2006). The damage to the liver cells. The electrolyte elevated levels of serum marker enzymes balance in the body is greatly affected as a indicate cellular leakage due to damage of the result of the crude oil contaminated feed. structural integrity of the liver (Baranano et There are many enzymes such as al., 2002). In this study, the levels of ALT and phosphatases, dehydrogenases and AST after 28 days increased significantly transferases that are found in the serum which (P>0.05) and the effect of garlic, vitamin C did not originate from the extracellular fluid. and vitamin E were not significant. However, During tissue damage, some of these vitamin C reduces the concentration of ALT biomolecules find their way into the serum, which shows that it has effect on reducing the probably by leakage through disrupted cell toxicity of crude oil contaminated feed. ALP membranes. The increase in serum alkaline generally showed reduction in the phosphatase, glutamaic pyruvic transferase concentration after 28days as a result of the and glutamic oxaloacetic as the concentration effect of garlic, vitamin C and vitamin E of crude oil increased. The mechanisms by drugs therapeutic effect on the crude oil which these enzymes are present in high contaminated feed. The levels of Sodium, concentration in the blood has been attributed Potassium, Chloride ion and bilirubin were to one in which their membrane pockets are significantly low (P
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