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EFFECT OF PRETREATMENT OF CRUDE OIL CONTAMINATED DIET WITH OIL PALM
LEAF ON LIPID PEROXIDATION AND XANTHINE OXIDASE ACTIVITY IN THE KIDNEY OF RAT

Achuba ,F. I .
Department of Biochemistry, Delta State University, PMB 1, Abraka Nigeria. [email protected]

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Abstract

The toxicity of petroleum hydrocarbon across the
living systems is now a common knowledge among the scientific community. What
is lacking is a mini-scale antidote that can be adopted by the inhabitants of
crude oil producing areas of the world. This was the reason for this study. The
study is comprised forty eight female Wister rats divided into six groups of
eight rats each. The rats were fed as flows: Group 1: (Control). Rats in group
2 and 3 were fed with diets treated with 5.0g and 10.0g oil palm leaf
respectively. Rats in group 4 were fed with crude oil contaminated diet (Crude
oil Control). Rats in groups 5 and 6 were fed with contaminated diet mixed with
ground oil palm leaf (5.0 g and 10.0 g respectively). After three and six months
exposure periods respectively, four rats from each group were sacrificed and
the kidney used to prepare supernatant needed for the determinations of lipid
peroxidation and activities of xanthine oxidase,  superoxide
dismutase  and catalase. The
results show that pretreatment of crude oil contaminated diet with oil palm
leaf tend to restore values of lipid peroxidation,
xanthine oxidase activity, superoxide dismutase activity and catalase activity
close to control values. Thus, it is pertinent to state that there exist
potentials in the use oil palm leaf in the treatment of crude oil toxicity. And
indeed setting a fresh agenda for further serious scientific investigations

 

 

 

Keywords: Crude oil, Kidney, Lipid peroxidation, Oil
palm, .Xanthine oxidase,

 

 

 1.0 Introduction

 Humans and animals get exposed to crude oil or
its byproducts when these chemicals are released into the surroundings during
oil exploration activities, equipment failures, corrosion, illegal bunkering,
usage, oil theft and illicit refining 1-3. Crude oil stimulates oxidative
stress in animals 4, 5. Lipid peroxidation, xanthine oxidase superoxide
dismutase (SOD) and catalase activities are part of oxidative stress indices
6. Lipid peroxidation elicits oxidative damage in plants and animals and its
value in conjunction with alterations in the level of antioxidants represent a
measure of oxidative stress. Similarly, the activity of xanthine oxidase is a
defense mechanism as well as measure of oxidative stress in animals 6. Report
has it that the deleterious action of crude oil on the kidney is based on
oxidative stress 7.

 Byproducts of the oil palm tree are important
medicinally. This is because the leaf juice have wound healing property while
the sap is used as laxative 8.This is due to 
 compounds rich in medicinal and antioxidant
properties inherent in oil palm leaf 9, 10. 
The antioxidant action is attributed to the presence of phytochemicals (flavonoid,
tannin and phenols) in the leaves of oil palm tree 11. In fact, oil palm leaf
extract contains more antioxidative phenolic compounds than various green tea
extracts 12. Therefore, oil palm leaf extract is a potential source of
functional food ingredient, based on reports of its health benefit 13 .This
study is aimed at evaluating the protective potentials of oil palm leaf against
crude oil contaminated diet induced nephrotoxicity in rats.

2.0 Materials and methods

The crude oil used for this study was obtained from
Nigeria National Petroleum Corporation (NNPC) Warri, Delta State, Nigeria. The
palm leaf used was obtained from Elaeis guineensis tree in Obiaruku,
Delta state, Nigeria Forty eight (48) female albino wistar rats with weights
ranging from 0.088kg to 0.182 kg obtained from the animal house of Department
of Anatomy, Delta State University, Abraka, Nigeria were used for this study.
The rats were housed in a standard wooden cage made up of wire gauze, net and
solid woods and left to acclimatize for one week on grower’s marsh and tap
water at laboratory temperature of 28o C and
12 hour day/ night regime. After the acclimatization period, the rats
were weighed and grouped.

2.1 Preparation of leaf powder.

The
leaves were isolated from the stock and sun- dried. The dried leaf was then
ground with domestic kitchen blender into a fine powder and stored in a clean
and sealed plastic container

2.2 Treatment of animals

The forty eight (48) female albino wistar rats were
assigned to six (6) groups according to their weights, with eight rats in each
group. Rats in the control, Group 1 were fed with grower’s marsh only. Rats in
Group 2 were fed with grower’s marsh treated with 5g of powdered palm leaf.
Group 3 rats were fed with grower’s marsh treated 10g of powdered palm leaf.
Group 4 rats were fed with grower’s marsh contaminated with crude oil (4ml per
100g of feed).This concentration of crude oil in diet
was established by a pilot study to be tolerated by the rats over a long period.
Rats in Group 5 were fed grower’s marsh contaminated with crude oil (4ml per
100g of feed) plus 5g of powdered palm fronds. While rats in Group 6 were fed
with crude oil contaminated marsh (4ml per 100g of feed) plus 10g of powdered
palm leaves. The rats in each group were allowed access to clean drinking water
while the experiment lasted. The feeds were prepared fresh daily and stale feed
remnants were discarded regularly. This was done every
morning between the hours of 8 am – 9 am and each group provided with 400 g of
the respective diet. The animals in each group were exposed to their
respective diets for three and six months respectively. The National Institute of health guide for the care and use
of laboratory animals (NIH, 1985) was adopted all through the experiment

 

 

2.3 Collection
of samples

After the first exposure period (three months), four rats
from each group were sacrificed and the kidneys were harvested. Five grams (5.0
g) of the kidneys were weighed in chilled conditions and homogenized with 5ml
of normal saline in a mortar. The mixture was diluted
with 45 ml of buffered saline before being centrifuged at 2, 500 rpm and the supernatant was transferred into plastic tubes and
stored at – 4o C in
the refrigerator before used for analysis within forty eight hours. This same
procedure was adopted after six months exposure period.

2.4 Determination of lipid peroxidation and xanthine oxidase activity

The activity of
xanthine oxidase in the kidney of rats was measured using the method of
Bergmeyer et. al. 14, a reaction based on the oxidation of xanthine to uric
acid, a molecule that absorbs light maximally at 290 nm. A unit of activity is
that forming one micromole of uric acid per minute at 25oC. Lipid
peroxidation in the kidney of rats was measured by the thiobarbituric acid
reacting substances TBARS, method of Gutteridge and Wilkins 15.Total
superoxide dismutase activity was assayed using the method of Misra and
Fredorich 16. Catalase was assayed as reported by Rani et al. 17  

2.5Statistical Analysis

Analysis
of variance (ANOVA) and post Hoc Fisher’s test for multiple comparison
was performed using statistical package for social science (SPSS), version 20  to determine statistical significant
differences between means. P values <0.05 were taken as being significantly different   3.0 Results and Discussion The effects of Elaeis guineensis leaf on kidney lipid peroxidation and xanthine oxidase activity against crude oil induced nephrotoxicity in rats after three and six months are shown in tables 1 and 2.  Lipid peroxidation in the kidney of rats exposed to crude oil contaminated diet (group 4) was significantly (P<0.05) higher in comparison with the control (group 1). Rats fed palm leaf pretreated diets (Group 2 and 3) showed significantly lower kidney levels of lipid peroxidation when compared with the control (group 4). Moreover, rats fed crude oil contaminated diets that was pretreated with various amounts of oil palm leaves (Group 5 and 6) exhibited significantly lower kidney lipid peroxidation level when compared with the control (group 1) and  rats fed crude oil contaminated diet alone (group 4) .  Lipid peroxidation, is an index of oxidative stress, induces disturbance of functional loss of biomembranes, that results in inactivation of membrane bound receptors and enzymes 17, 18, 19. The present study shows that the consumption of crude oil contaminated diet increased the level of lipid peroxidation in rats.  This study shows that exposure to hydrocarbons present in crude oil can lead to oxidative damage of the kidney as evident by the rise in renal level of lipid peroxidation. This is based on the premise that metabolism of hydrocarbons present in crude oil generates free radicals 20. This is in consonance with previous studies 5, 6, 7, 21. Oil palm leaf is rich in bioactive phytochemicals such as polyphenols and these polyphenolic compounds are considered to have antioxidant activity that is several folds higher than that of vitamins C and E 22, 23, 24.This may be the basis for the decreased level of lipid peroxidation in the kidney of rats exposed to crude oil that was treated with oil palm leaf The kidney oxidative stress enzyme (xanthine oxidase, Sod and catalase) activities were significantly (P<0.05) lower in rats fed crude oil contaminated diets (group 4) in comparison with all the experimental groups (Tables 1 and 2).Rats fed with oil palm leaf treated crude oil contaminated diet (Groups 5 and 6) have significantly higher xanthine oxidase activities in the kidney when compared with rats fed with crude oil contaminated diet only (group 4). However, rats fed with only oil palm leaf treated diets (Groups 2 and 3) have significantly higher  oxidative stress marker enzyme  activities when compared with rats fed with only crude oil contaminated diet (group 1). Xanthine oxidase is involved in phase one process in the inactivation of xenobiotics in animals 25. The increase in the activity of xanthine oxidase in rats exposed to oil palm leaf treated diet indicates response of the enzyme to enhance the metabolism of endogenous xanthine. This is in a bid to increase the production of uric acid, a potent antioxidant 7, 25, 26. The decrease in activity of xanthine oxidase in rats exposed to crude oil contaminated diet alone shows that the metabolism of crude oil leads to a reduced ability to produce uric acid Nevertheless, the alteration in the activity of oxidative enzymes had been reported as a measure of oxidative stress 27. However, addition of ground oil palm leaf resulted in decrease in toxic effects of crude oil. This is exhibited in the increase in activities of oxidative stress marker enzymes towards control values in rats fed with crude oil contaminated diets that were pretreated with oil palm leaf. This is due to the ability of oil palm leaf to act as an antioxidant, protecting endothelial cells of the kidney against reactive free radicals thereby restoring the level of antioxidant enzymes 11, 13. Substances with antioxidant potentials possess health promoting properties, since they quench free radicals which are involved in many diseases processes 13, 28, 29, 30. Generally, the deleterious action of crude oil on kidney tissue and the protective influence of the oil palm leaf is further highlighted by histological examination of the kidney tissue (Figure 1).Previous study had shown that plant materials with antioxidant properties can attenuate the negative effect of crude oil on experimental animals 31 5.0 Conclusion This study has indicated that the ingestion of crude oil treated diet can result in increase in oxidative stress and consequent kidney damage. However, the crude oil toxicities were reversed by the consumption of diets that were pretreated with oil palm leaf. This study, therefore, shows possible protective role of oil palm leaf against crude oil induced nephrotoxicity.   4.0   References 1  Otitoju O, Onwurah .INE. Preliminary investigation into the possible endocrine disrupting activity of bonny light crude oil contaminated diet on wistar rats. Biokemistri 2007; 19(2):23-28       2   Ovuru SS, Ekweozor IKE. Haematological changes associated with crude oil ingestion in experimental rabbits. Afr. J Biotechnol 2004;3(6):346-348 3 Ogudu AD, Esemuede IH. Crude oil theft and its environmental consequences: The way forward. J Nig Environ Society 2013; 7(4):1-18 4  Achuba FI, Osakwe SA. Petroleum induced free toxicity in African catfish (Clarias   gariepinius). Fish Physiol Biochem 2003 29:97-103 5  Anozie OI, Onwurah IN. Toxic Effects of Bonny Light Crude oil on Rats after Ingestion of contaminated diet. Nig J Biochem Mol Biol 2001; 16:1035-1085 6 Achuba FI.  Petroleum products in soil mediated oxidative stress in cowpea (Vigna ungiculata) and maize (Zea mays) Seedlings. Open J Soil Sci 2014; 4:417-435. 7  Azeez OM, Akhigbe RE, Anigbogu CN.  Oxidative status in rat kidney exposed to petroleum hydrocarbons. J. Nat Sci Biol Med 2013; 4(1):149-154 8 Sasidharan S, Logeswaran S, Latha LY. Wound healing activity of Elaeis guineesis leaf extract ointment. Int J Mol Sci 2012; 13:336-347 9  Chong  KH, Zuraini  Z, Sasidharan S, Devi PVK, Latha LY, Ramanathan S. Antimicrobial activity of Elaeis guineensis leaf. Pharmacology online. 2008;3:379-386 10 Rout SP, Choudary KA, Kar DM, Das L, Jain A. Plants in traditional medicinal system-future source of new drugs. Int J Pharm Pharm Sci 2009'5(4):137-140 11  Phin KC, Syahriel A, Ng, SY. Phytochemical constituents from leaves of Elaeis guineesis and their antioxidant and antimicrobial activities. Int. J Pharm. Pharm Sci 2013; 5(4)137-140 12 Runnie I., Nordin MM, Radzali M, Azizah H, Hapizah N. Antioxidant and hypocholesteromic effects of Elaeis guineensis leaves extract on hypercholesteromic rabbits. ASEAN Food J 2003; 12:137-147 13 Mohamed SK. Oil Palm Leaf: A New Functional Food Ingredient for Health and Disease Prevention. J. Food Process Technol. 2014;5(2):300-306 14  Bergmeyer HV, Gacoehm K, Grassl M. In: Methods of Enzymatic Analysis, HV Bergmeyer    (eds).New York: Academic Press. 1974; 2:  428–429. 15 Guttridge JMC, Wilkins C. Copper dependent hydroxyl radical damage to ascorbic acid formation of thiobarbituric acid reactive products. FEBS Lett. 1982; 137: 327-340.  16   Misra HP, Fridovich I. The role of superoxide ion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 1972 (247): 3170 – 3175. 17    Rani P, Meena UK, Karthikeyan J. Evaluation of antioxidant properties of berries. India J Clin Biochem 2004; 19 (2) 103-110. 18    Halliwell B. Free radicals and antioxidants: a personal view. Nutr Rev1994; 5:253-265. 19    Niki E. Lipid peroxidation products as oxidative stress biomarkers. Biofactors. 2008;34(2):171-180 20    Greenberg ME, Li XM, Giugiu BG, Gu X, Qin J, Salomon RG, Hazen S.The lipid whisker model of the structure of oxidized cell membranes. J Biol Chem 2008; 283:2385-239 21    Achuba FI. Spent engine oil mediated oxidative stress in cowpea (Vigna unguiculata) seedlings. EJEAFChe. 2010; 9(5): 910-917 22    Alisi CS., Ojiako AO., Osuagwu CG, Onyeze GOC (2011) Response pattern of antioxidants in carbon tetrachloride-induced hepatoxicity is tightly logistic in rabbits. Eur J Med Plants. 2011;1:118-129 23    Cowan MM (1999) Plant products as antimicrobial agents. Clin. Microbial. Rev. 1999; 12(4):564-582 24    Lee YL, Jian SY, Lian PY, Mau JL. Antioxidant properties of extract from a white mutant of the mushroom Hypsizigusmarmoreus. J. Food Compos Anal. 2008;21:116-124 25    Jaffri JM, Mohamed S, Ahmad IN, Mustapha NM, Manap YA, Rohimi N. Effects of catechin-rich oil palm leaf extract on normal and hypertensive rats kidney and liver. Food Chem.2011; 128:433–441 26    Ezedom T, Asagba SO. Effect of a controlled food-chain mediated exposure to cadmium and arsenic on oxidative enzymes in the tissues of rat Toxicol Reports 2016 ;(3) :708–715 27    Achuba FI. African land snail Achatina marginatus, as bioindicator of environmental pollution. North- Western J  Zool 2008; 4 (1): 1-5 28    Förstermann U, Xia N,   Li. H. Roles of Vascular Oxidative Stress and Nitric Oxide in the Pathogenesis of Atherosclerosis. Circulation Res.2017; 120:713-735 29    Hybertson BM, Gao, B, Bose, SK., McCord JM. Oxidative Stress in health and disease: The therapeutic potential of Nrf2 activation. Mol Asp Med 2011;32(4):234-246 30    Galli F, Piroddi M., Annetti C, Aisa C,  Floridi E.,  Floridi A (2005) Oxidative stress and reactive oxygen species. Contrib Nephrol 2005; 149: 240-260 31    Achuba FI, Ubogu LA, Ekute BO. Moringa oleifera attenuates crude  oil  contaminated diet induced biochemical effects in wistar albino rats UK J Pharm Biosci 2016;  4(5) 70-77   Table 1.  The effect of  Elaeis guineensis leaf on the level of  oxidative stress indicators in the kidney of rats after three months of exposure to crude oil contaminated diet. Groups Lipid peroxidation    Xanthine oxidase activity (nmol/g  tissue)           (units/g  tissue) SOD activity            Catalase activity  (units/g  tissue)         (nmol/g tissue) Group 1 0.35±  0.05 a                     60.04 ±  4.28 a                                                          26.75 ±  2.21 a                   54.53± 2.55 a Group 2 0.14 ±  0.02 b                    60.83 ±  1.76 a 28.63 ±  3.62 a                   51.33± 3.61 b Group 3 0.10 ±   0.03 b                   69.28 ±  3.34 b                                  29.44 ±  1.47 b                   52.1 2± 1.15 b Group 4 0.76 ±   0.10 c                   42.43 ± 1.78 c 20.10 ± 1.66 c                    46.42± 2.11 c Group 5 0.52  ±  0.01 d                   51.09 ±  2.70 d 22.22 ±  1.80 d                   49.44± 1.52 d Group 6 0.34  ±  0.01 a                   57.05 ±   5.89 a 24.52 ±   1.33 a                  50. 33± 1.66 b   Each value represents mean ± standard deviation. n = 4 in each group. Values not sharing a common superscript letter in the same column differ significantly at (P < 0.05). Group 1: ((Normal Control). Group 2:   feed mixed with 5.0g oil palm leaf. Group 3: feed mixed with 10.0g oil palm leaf. Group 4: Feed mixed with 4ml crude oil (Crude oil Control). Group 5: Contaminated diet mixed with 5.0 g of oil palm leaf. Group 6: contaminated diet mixed with 10.0 g of oil palm leaf.                           Table 2.  The effect of  Elaeis guineensis leaf on the level of  oxidative stress indicators in the kidney of rats after six months of exposure to crude oil contaminated diet Groups Lipid peroxidation    Xanthine oxidase activity (nmol/g  tissue)           (units/g  tissue) SOD activity            Catalase activity  (units/g  tissue)         (nmol/g tissue) Group 1 0.42±  0.08 a                     62.04 ±  3.80 a                                                          28.88 ±  1.11 a                   53.97± 1.45 a Group 2 0.22 ±  0.01 b                    61.41 ±  2.64 a 27.96 ±  3.62 a                   52.36± 2.55 a Group 3 0.11 ±   0.04 b                   68.24 ±  2.22 b                                  29.55 ±  2.81 a                   52.66± 1.22 a Group 4 0.89 ±   0.11 c                   38.43 ± 2.66 c 18.33 ± 1.88c                    43.31± 1.53 c Group 5 0.66  ±  0.12 d                   54.11 ±  3.50 d 23.43 ±  1.92 d                   50.02± 1.68 b Group 6 0.53  ±  0.06 a                   55.44 ±   6.70 a 24.99 ±   1.63 a                  50. 91± 1.74 b   Each value represents mean ± standard deviation. n = 4 in each group. Values not sharing a common superscript letter in the same column differ significantly at (P < 0.05). Group 1: ((Normal Control). Group 2:   feed mixed with 5.0g oil palm leaf. Group 3: feed mixed with 10.0g oil palm leaf. Group 4: Feed mixed with 4ml crude oil (Crude oil Control). Group 5: Contaminated diet mixed with 5.0 g of oil palm leaf. Group 6: contaminated diet mixed with 10.0 g of oil palm leaf.                                                                   Group 1: ((Normal Control). Group 2:   feed mixed with 5.0g oil palm leaf. Group 3: feed mixed with 10.0g oil palm leaf. Group 4: Feed mixed with 4ml crude oil (Crude oil Control). Group 5: Contaminated diet mixed with 5.0 g of oil palm leaf. Group 6: contaminated diet mixed with 10.0 g of oil palm leaf.     Figure 1: Photomicrographs of kidney section of rats fed crude oil contaminated diet and diets pretreated with different amount of ground oil palm leaf

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