Hepatotoxicity and nephrotoxicity of automobile gas oil (diesel oil) and its biochemical effect on hematological and oxidative stress parameters in Wistar male albino rats.

Aims: The study was designed to investigate the hepatotoxicity and nephrotoxicity of automobile gas oil (Diesel oil) and its biochemical effect on hematological and oxidative stress parameters in Wistar male albino rats. Methodology: Preliminary toxicity study to determine the volume of diesel oil that could cause toxicity was carried out using 56 healthy albino rats. Another set of 20 albino rats were grouped into two groups and used for the biochemical analysis. Group I animals were the control group and Group II animals were administered with 1 ml of diesel oil per kg body weight. The hematological Original Research Article Johnson et al.; BJMMR, 6(5): 522-532, 2015; Article no.BJMMR.2015.229 523 parameters were determined using BC-3200 Auto Hematology Analyzer. Liver injuries were measured in the plasma using AST, ALT, ALP, GGT, TP and TB Randox kits respectively. Liver histopathological examination was also determined. The oxidative stress parameters assayed in the liver homogenate were TBARS, SOD, CAT and GSH. Kidney injuries were assayed using urea and creatinine Randox kits. Results: Preliminary toxicity study shows that group II albino rats exhibited changes in behavioural pattern such as salivation, respiratory distress, coma, sedation and death. The LD50 was calculated to be 2.5ml/Kg body weight of diesel oil. The results of this study show that WBC, lymphocyte and granulocyte values were significantly increased (P<0.05) in group II animals compared to group I animals. HGB, RBC and HCT values were significantly reduced (P<0.05) in the group administered with diesel oil compared to the healthy group. All the liver biomarker enzymes: AST, ALT, ALP and GGT were significantly increased (P<0.05) in group II rats compared to group I rats. The plasma TB values increased in group II animals while their plasma TP values significantly reduced (P<0.05) when compared to group I animals. Liver histopathological examination also confirmed that administration of diesel oil caused liver lesions in group II animals. Administration of diesel oil to group II rats caused significant reduction (P<0.05) in the activities of the enzymic antioxidants (SOD and CAT) and non enzymic antioxidant (GSH) values of the liver homogenate. The TBARS values were significantly high in group II rats compared to group I rats. These biochemical parameters (SOD, CAT, GSH and TBARS) indicate oxidative stress in animals administered with diesel oil. Conclusion: The results of this study show that administration of diesel oil is hematotoxic, its affects the liver, kidney and oxidative stress parameters.


INTRODUCTION
Crude oil exploration is the major income of the Nigerian economy and constitutes about 90% foreign exchange earning of the nation. Various environmental pollutants, particularly those associated with crude oil cause many biochemical and toxic effects in terrestrial and marine animals [1][2][3]. Crude petroleum is a mixture of different hydrocarbons and metals [4]. Crude oil is refined into fractions of kerosene, diesel, petrol, heavy gas oils, lubricating oils, as well as residual and heavy fuels, among others. However, kerosene, petrol, and diesel are the most commonly used fractionated crude petroleum products. Crude oil contains significantly high amount of toxic chemicals which can cause a wide range of health effects in people and wildlife, depending on the level of exposure and susceptibility. The chemicals can impair normal growth and development through a variety of mechanisms. They can cause mutations that may lead to cancer and multigenerational birth defects. Exposure of humans and animals to these chemicals is increasing in terms of the environmental level and the different usage of crude oil and its products.
Oxidative stress is the presence of reactive oxygen species (ROS) in excess of the available antioxidant buffering capacity [5]. ROS can damage macro-molecules like: lipids, proteins, and DNA. Oxidative stress is one of the mechanisms for crude oil induced hepatic injury. Metabolism of chemicals or xenobiotic substance takes place in the liver, which accounts for the organ's susceptibility to metabolic induced hepatotoxicity. The crude oil and its products generate free radicals and promote a variety of chemical reactions, such as depletion of reduced glutathiones or inducing lipid peroxidation. Increased levels of lipid peroxidation are indicative of severe liver, kidney and heart damage such as acute myocardial infarction and artherosclerotic plaque stability [6].
This study was designed to investigate the biochemical and toxicological effect of diesel oil on hematological parameters, liver biomarker enzymes, kidney and oxidative stress parameters in Wistar albino rats.

Analysis of the Diesel Oil Used for the Analysis
The diesel oil were bought from Total Nigeria PLC and analysed in SGS Inspection Service Nigeria Limited to ascertain it to be diesel with the SON/DPR Specification using ASTM Methods (American Society for Testing and Material).

Experimental Animals
A total of 76 male wistar albino rats with body weight ranging from 150 to 200g were obtained from Nigeria Institute of Medical Research (NIMR), Lagos, Nigeria. They were acclimatized for one week to Laboratory condition of 23 ±2ºC. They were kept in plastic cages and fed with commercial rat chow and supply with water adlibitum. The rats were used in accordance with NIH Guide for the care and use of laboratory animals; NIH Publication revised (1985) NIPRD Standard Operation Procedures (SOPs).

Preliminary Toxicity Study
Preliminary toxicity study to determine the volume of diesel oil that could cause toxicity was carried out using 56 albino rats. The rats were divided into seven groups of eight rats per group and were administered orally separately with 1, 2, 3 4, 5 and 6 ml/kg body weight of diesel oil respectively. The seventh group was given water ad-libitum. The rats were observed over 72 hour's period for nervousness, salivation, stretching, dullness, in-coordination and death. From the range of doses administered, 1ml/kg body weight was chosen for this study.

Grouping of Experimental Animals
Based on the results of the preliminary toxicity study, another set of 20 healthy male albino rats were grouped into two groups of ten rats per group as shown below: Group I -normal control (healthy group). Group II-animals administered with 1 ml/kg body weight of diesel oil

Collection of Blood Samples
The albino rats were sacrificed after 24 hours fasting on the eighth day. Blood were collected from the albino rats by ocular puncture into EDTA bottles for hematological analysis and the remaining blood were collected in an heparinised bottle and centrifuge at 3000 rpm for 20 minutes using a centrifuge and the plasma stored at 4°C.

Determination of Hematological Parameters
The hemoglobin concentration (HGB), total red blood cell (RBC), white blood cell count (WBC), Hematocrit (HCT), and other hematological parameters were determined in the blood using BC-3200 Auto Hematology Analyzer in University of Lagos Teaching Hospitals (LUTH) in Idi-araba, Lagos, Nigeria.

Determination of Plasma Total Protein (TP) and Total Bilirubin (TB) Values
Plasma bilirubin and total protein were determined using Randox diagnostic kits.

Determination of Kidney Function Test
Plasma urea (UR) and creatinine (CR) values were also determined using Randox diagnostic kits.

Histopathological Studies
The albino rats were sacrificed and their abdomens were cut open to remove the liver. Some of the livers were fixed in Boucin's solution (mixture of 75 ml of saturated picric acid, 25 ml of 40% formaldehyde and 5 ml of glacial acetic acid) for 12 hours, then embeded in paraffin using conventional methods [7]. They were cut into 5 μm thick sections and stained using haematoxylin-eosin dye and finally mounted in di-phenyl xylene. The slides were then examined at a magnification of × 40 under a light microscope for histopathological changes in the liver architecture and their photomicrographs were taken.

Preparation of liver homogenate
The Liver tissues of some of the sacrificed albino rats were excised and the liver samples were cut into small pieces and homogenized in phosphate buffer saline (PBS) to give a 10% (w/v) liver homogenate. The homogenates were then centrifuged at 12,000 rpm for 50 minutes. The supernatant obtained was used for assay of thiobarbituric acid reactive substances (TBARS) content, superoxide dismutase, catalase and reduced glutathione.

Estimation of Lipid peroxidative (LPO) indices
Lipid peroxidation as evidenced by the formation of TBARS was measured in the homogenate by the method of Niechaus and Sameulsson, 1968 [8].

Estimation of superoxide dismutase (SOD)
The liver homogenate was assayed for the presence of SOD by utilizing the technique of magwere et al. [9] with slight modification [9].

Estimation of catalase (CAT)
The liver homogenate was assayed for catalase colorimetrically at 620 nm and expressed as µmoles of H 2 O 2 consumed/min/mg protein as described by sinha, 1972 [10].

Estimation of Reduced glutathione (GSH)
Reduced glutathione (GSH) was determined in the liver homogenate using the method of Ellma, 1959 [11].

Data Analysis
Data analysis was done using the GraphPad prism computer software. Students 't'-test and one-way analysis of variance (ANOVA) were used for comparison. A P-value < 0.05 was considered significant.

Analysis of the Automobile Gas Oil (Diesel Oil)
The Table 1 below shows that the diesel oil used in this research work meet SON/DPR specification.

Preliminary Toxicity Test
Preliminary toxicity test result shows that three animals died after the administration of 2 ml/kg body weight of diesel oil, four died each after the administration of 3 and 4 ml/kg of diesel oil respectively, 6 died after administering 5 ml/kg of diesel and all the eight animals died after administering 6 ml/kg body weight of the diesel oil. In the control group, none of the animals died since they were given only water ad-libitum. The calculated LD 50 is 2.5 ml of diesel oil per kg body weight of the animals.

Effect of Diesel Oil on Hematological Parameters in Male Albino Rats
The different hematological parameters of the experimental groups (Group 1 and II) are shown in Table 2 below. The results of the hematological parameters show that the administration of 1 ml/kg body weight of diesel oil is hematotoxic to the animals in group II.

Effect of Diesel Oil on the Liver and Kidney of Male Albino Rats
The  and increased to 19.8±3.1 mg/dl in the diesel oil infected rats. Mean level of creatinine was 0.4±0.2 mg/dl in the control group animals, the value increased to 0.9±0.7 in group II animals.

Histopathological Examination
The result of the histopathological examination of the liver of the healthy animal and the animal administered with Iml/kg body weight of diesel oil is shown in Figures 1 and 2 below.

DISCUSSION
Crude oil is believed to elicit some therapeutic effects and functions in the treatment of various skin diseases, arthritis, rheumatism, fever and even constitutes an antidote in detoxifying ingested poisons [12]. Majority of the people in Warri and Bonny communities ingest crude oil either directly as curative agents for antipoisoning (snake venom antidotes), anticonvulsion, treatment of skin infection e.g. scabies or indirectly by eating marine animals found in surrounding coastal waters as source of protein [12]. Due to oil spillage, there have been increases in the consumption of these petroleum products. Diesel oil is an essential constituent of human life due to their domestic and industrial use. Table 1 shows that the diesel oil used in this research work met the SON/DPR Specification. Preliminary toxicity study to determine the volume of diesel oil that could cause toxicity shows that the animals exhibited changes in behavioural pattern such as salivation, respiratory distress, coma, sedation and death. The LD 50 was calculated to be 2.5 ml/Kg body weight of diesel oil. Sexena et al. [13], Ohaeri and Eluwa [14] and Patrick-Iwuanyanwu et al. [15] have reported that hematological and biochemical indices are reliable parameter for assessment of the health status of animals. There was a significant increase (P<0.05) in the WBC and lymphocytes number in the animals administered with diesel oil compared to the control group. WBC helps the body to fight infection, defend the body by phagocytosis against invasion by foreign organisms and to produce or at least transport and distribute antibodies in immune response. The observation in this study on WBC is similar to the findings of Momoh and Damazio 2014 where WBC values increase in kerosene administered animals. [16]. Leukocytosis observed in group II rats may be due to luekemia, bone marrow infection and inflammatory disease of the rats administered with diesel oil. Lymphocytosis observed in group II animals is a sign of chronic inflammation, Addison disease and lymphoma of the animals in this group. Table 2 above also shows that granulocytes number significantly increases (P<0.05) in group II animals compared to group I animals, an indication of chronic inflammation, chronic myelomonocytic leukemia, connective tissue infection, lymphoma and myeloproliferative disorders. The primary reason for assessing the RBC is to check the level of anemia and to evaluate normal erythropoiesis. HGB level shows the amount of intracellular iron present, while HCT, indicates the volume of RBC in 100ml of blood and it helps to determine the degree of anemia or polycythaemia [16]. The study shows that there is a significant decrease (P<0.05) in RBC, HGB and HCT in the group administered with diesel oil compared to the normal control group ( Table 2). The significant reduction (P < 0.05) may be attributed to the cytotoxic effects and suppression of the erythropoiesis caused by constituents of the diesel oil. The values of the PLT increased significantly (P<0.05) in group II compared to group I. Changes in the number of platelets (thrombocytes) reveal disturbances in organism hemostasis and indicate hazardous conditions to which the organisms are exposed. The overproduction of platelets (thrombocytosis) is usually a transient event and may be related to stress, inflammation, spleen disorder, reverse effects of thrombopenia, iron deficiency and some types of anemia. Excessive increase in platelets could be related to stimulation of megakaryocytes in the bone marrow [17]. Other hematological parameters (Mid%, MCV, MCH, MCHC, RDW-CV, RDW-SD, MPV, PDW and PCT) show no significant difference (P<0.05) between group I and II animals. It is therefore concluded that diesel oil is highly toxic and are potential damaging agents to the hematopoietic system.
AST is an enzyme found mostly in the liver cell, heart muscle, skeletal muscles and kidneys. Injury to these tissue result in the release of the enzyme in the blood stream. Elevated levels are found in cirrhosis, myocardial infarction and hepatitis [18]. The result from this study showed significant increases in the levels of AST and ALT (Table 3) values in group II rats compared to group I rats. This result is in accordance with the reports of Momoh and Damazio [16] who reported similar increase after administering of kerosine on wistar albino rats. Several studies on seabirds suffering from long term exposures to oil spills show an increase of AST activities compared to birds from non-affected area [19][20]. There were significant increase (P<0.05) in the ALP and GGT values of group II animals compared to the healthy animals. This may imply that damage occur in the liver cells of the rats administered with diesel oil (Table 3), since the activities of these enzymes are reported to be increased in liver damage [21]. The significant increase in these liver marker enzymes (AST, ALT, ALP and GGT) in the plasma is responsible for the hepatotoxicity of the liver in the group administered with diesel oil. Bilirubin is the major breakdown product that results from the destruction of old red blood cells. It is removed from the blood by the liver; hence it is a good indicator of the function of the liver [22]. The total bilirubin values increased significantly (P<0.05) in group II animals compared to group I animals. A rise in plasma level of bilirubin suggests liver cell damage, since liver cells are responsible for removing bilirubin from serum [23]. Ovuru et al. [24] reported an increase in total serum bilirubin concentration in semi adult rabbits exposed to crude oil contaminated diet and attributed this to a metabolic disturbance in the liver arising from defective conjugation and/ or excretion of bilirubin.
The plasma concentrations of urea and creatinine could give an insight into the effect of a compound on the tubular and the glomerular part of the kidney [25]. This study showed significant increased (P<0.05) in urea and creatinine level of group II rats compared to group I rats (Table 3). A rise in serum level of these metabolites suggest the inability of the kidney to excrete these products, which further suggest a decrease in glomerular filtration rate. This affirmation is supported by Counts et al. [26] who reported that chemically induced nephrotocity by halogenated hydrocarbons injure the proximal tubule monolayer, resulting in gaps in epithelial lining, leading to back leak of filtrate and diminishing glomerular filtration.
Histopathological studies also provided important evidence supporting the biochemical analysis and liver antioxidant status In normal control animals, liver sections showed normal hepatic cells with well preserved cytoplasm, prominent nucleus, nucleolus and central vein (Fig. 1). The administration of 1ml/kg body weight of diesel oil induces histopathological damages in the liver with significant degeneration of cells, inflammatory cell infiltration, hepatocyte fibrosis, hepatocyte necrosis and liver lesions were observed in group II animals (Fig. 2).
Oxidative stress is the presence of reactive oxygen species (ROS) in excess of the available antioxidant buffering capacity [5]. ROS can damage molecular targets-lipids, proteins, and DNA, thus altering the structure and function of the cell, tissue, organ, or system [27]. It has been reported that metabolism of aliphatic and aromatic hydrocarbons, the major constituents of petroleum products (e.g. kerosene and diesel) as well as other xenobiotics substances significantly increase the productions of free radical species in various tissues [28][29]. Various authors have earlier showed that petroleum induces the formation of free radicals, which react with some cellular components such as membrane lipids and produce lipid peroxidation products. This predisposes the animals to oxidative stress [30][31]. A progressive significant increase (p<0.05) in the MDA levels in groups II rats compared to group I rats is indicative of increased possibilities of lipid peroxidation and a consequent increase in oxidative stress. In this study there is a significant reduction (P<0.05) in activities of SOD, CAT and reduced glutathione (GSH) in the liver homogenate of group II albino rats compared to group I albino rats (Figs. 3-6). These show hepatic injury from oxidative stress caused by the administration of 1ml/kg body weight of diesel oil. In system, organ and tissue damage, GSH makes up the first line of defence against free radicals resulting from xenobiotic ingestion. The drop in the concentration of liver GSH and corresponding increase in concentration of malondialdehyde indicates hepatocytes damage in the liver homogenate.

CONCLUSION
The results of this work show that administration of diesel oil affects the hematological parameters, causes impairment of the liver and the kidney function and results in oxidative stress in wistar albino rats.

CONSENT
This was not applicable since the study was on animals and not on humans.

ETHICAL APPROVAL
Not applicable.