ALTERATIONS IN THE LEVELS OF SOME SERUM ENZYMES AND LIVER RNA IN GUINEA PIGS TREATED WITH BONNY

LIGHT CRUDE OIL

SOLOMON A NDONIAND

TEBEKEME OKOKO

ABSTRACTThe Bonny light crude oil contains an aromatic and aliphatic fraction in the ratio 1:4 and is the major form prevalent in the Niger Delta region of Nigeria. The intraperitoneal-administration of Bonny light crude to guinea pigs has been associated with alterations in liver chromatin proteins, liver mitochondria and nuclear DNA. This current study was aimed at investigating the changes the intraperitoneal-administration of the Bonny light crude oil could cause to some serum enzymes and RNA in nuclear and mitochondrial fractions of guinea pig liver. The results revealed that the Bonny light crude oil caused marked elevations in the levels of alanine aminotransaminase, aspartate aminotransaminase, and alkaline phosphatase in serum which was dose-dependent suggestive of hepatic damage. The liver nuclear and mitochondrial RNA was also increased in a dose-dependent manner. The findings are ascribed to the interference of the polycyclic hydrocarbons, heavy metals, and other chemical groups in crude oil which interacted with critical molecules on liver and the subsequent upregulation of the transcription of xenobiotic metabolizing enzymes.

Keywords: Bonny light crude oil, alanine aminotransaminase, aspartate aminotransaminase, hepatic, mitochondria, xenobiotic.

IntroductionCrude oils are complex mixtures which originated from both plant and animal sources. Hydrocarbons are the major entities in crude oil together with oxygen, nitrogen, sulphur compounds, and mineral salts mixed with or emulsified in the oil (IARC, 1989). Some of the hydrocarbons present are alkanes, cycloparaffins, aromatics (mono and polyaromatics), and alkane derivatives no wonder it has also been defined as a complex mixture of paraffinic, cycloparaffinic, and aromatic hydrocarbons containing low percentages of sulphur and trace amounts of nitrogen and oxygen derivatives (IARC, 1989).

The type, proportion, and the chemical basis of crude vary with location. The Bonny light crude oil is made up of an aromatic and aliphatic hydrocarbons in the ratio 1:4 and that is why it is termed light crude (Oruambo and Jones, 2007). It is the major type found in the Niger Delta region of Nigeria where spillages occur as a result of broken pipelines and other ways attributed to anthropogenic factors.

Following spillages, plants and lower animals may biocencentrate crude oil which could be biomagnified at higher trophic levels hence it is of utmost importance to study the effects of such exposures. Holmes, et al., (1978) studied the acute effects of petroleum on some birds and

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observed that obvious signs of toxicity were not expressed after the exposure of birds to fairly large doses of petroleum through the oral route. This dose not however rules out the possibility of any systemic effect at the molecular and sub-molecular level. Crude oil also contains 200 ppm nickel and 1200 ppm vanadium and these heavy metals form adducts with protein and nucleic acids (Harnett, et al., 1982). Szaro, et al., (1978) also reported that the administration of crude oil to mice resulted in fatty liver, and increase in hepatic protein. The Bonny light crude oil also causes alterations in liver mitochondrial DNA, chromatin proteins (Oruambo and Jones, 2007) and also causes testicular damage ( ). However reports on alterations in liver RNA are quite limiting. The aim of this current work is to investigate the effect of the Bonny light crude oil on liver mitochondrial RNA, liver nuclear RNA, and some serum enzymes.

Materials and methodAnimalsTwenty guinea pigs weighing 310g – 340g were obtained from the National Institute of Medical Research, Lagos. They were housed in conventional cages and were fed with growers' mash and tap water ad libitum.

Chemicals and reagentsAll chemicals were standard chemicals and solvents of analytical grade and were commercially available thus used without further purification. All buffers were prepared using distilled water. Bonny light crude oil was obtained from the Nigerian National Petroleum Corporation (NNPC) in Port Harcourt.

Treatment of animalsAll experimental procedures for animal studies were approved by the relevant ethical committees. The animals were acclimatized to laboratory conditions for seven days before treatment. Thereafter, the guinea pigs were split into five groups of four each. Guinea pigs in group 1 were intraperitoneally-treated with 1.25 ml/kg b.w. of Bonny light crude oil, animals in group 2 were administered with 2.50 ml/kg b.w., group 3 5.00 ml/kg b.w., group 4 10.00 ml/kg b.w., while the guinea pigs in group 5 were normal (untreated) controls. They were all allowed unlimited access to growers' mash and tap water throughout the study.

Tissue and blood collectionSeven days later, each guinea pig was subjected to light anaesthesia in a chloroform saturated chamber and dissected. The thoracic region was exposed and blood was drawn through direct cardiac puncture and delivered into a sterile sample bottle. Blood was allowed to clot for 10 mins and serum collected after centrifuging at 3500 rpm for 10 mins. The liver was also excised immediately, and washed in ice-cold phosphate buffer (0.25 M, pH 7.5), blotted and a 10 % homogenate prepared in the same buffer. The nuclear and mitochondrial fractions were obtained from the homogenates via centrifugation according to the method of Griffiths, (1995).

Ebokaiwe, et al., 2013; Ebokaiwe, et al., 2015

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Biochemical analysisAlanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined in serum at 37°C according to the method of Reitman and Frankel, (1957). Alkaline phosphatase (ALP) was also determined in serum according to Plummer, (1978). Total RNA in the nuclear and mitochondrial fractions was determined using the orcinol reagent. Briefly, total assay solution was made up of 1.4 ml distilled water, 1.5 ml orcinol reagent (1 g ferric chloride, 2 g orcinol, 200 ml conc. HCl) and 0.1 ml sample in a test tube. This was incubated in boiling water for 20 mins. The test tube was centrifuged at 3000 rpm for 3 mins and the absorbance of the supernatant was read at 660 nm. Total RNA was quantified using a stock RNA standard.

Statistical analysisRepresentative values are expressed as mean ± SEM. Were appropriate, the data were analysed using analysis of variance using Minitab Statistical Software (version 14). A confidence level exhibited at P < 0.05 was considered statistically significant.

Results and DiscussionThe results obtained from the investigation are presented in Tables 1 and 2 as shown below. The crude oil caused almost a 14 % increase in ALT, 8 % increase in AST and a 30 % increase in ALP when administered at a dose of 1.25 ml/kg bw to a 133 % increase in ALT, 121 % increase in AST and 167 % increase in ALP over normal controls when administered at a dose of 10 ml/kg b.w (Table 1). A similar trend was also observed in the mitochondrial and nuclear RNA levels which are shown in Table 2. In all cases, there were significant differences between the groups (P < 0.05).

Table 1. Dose-related changes in the serum levels of alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), and alkaline phosphatase (ALP) activities in guinea pigs caused by Bonny light crude oil.

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Alterations In The Levels Of Some Serum Enzymes And Liver Rna In Guinea Pigs Treated With Bonny Light Crude Oil

Values are presented as means ± S.E.M. of sera from four individual guinea pigs. Means followed by different letters in superscript differ significantly (P < 0.05). (b.w. is body weight)

Table 2. Dose-related changes in nuclear and mitochondrial RNA of livers of guinea pigs caused by Bonny light crude oil.

Values are expressed as means ± S.E.M. of livers from four individual guinea pigs. Means followed by different letters in superscript differ significantly (P < 0.05) (b.w. is body weight; Nu means nuclear, Mito means mitochondrial)

Throughout the experiment, no mortality was recorded and this supports a former report that no obvious signs of toxicity are expressed after the exposure of animals to fairly large doses of petroleum (Holmes, et al., 1978). The Bonny light crude oil caused marked increase in the levels of ALT, AST, and ASP (Table 1) which is an indication of mild liver damage which is dose-dependent. This may be ascribed to several factors. The Bonny light crude oil contains some polyaromatics (20 %) which accumulate in tissues especially the liver after undergoing oxidative metabolism (Kaninsky, et al., 1981; Bergman, 1994; Safe, 1994). In the liver, the metabolites can participate in reactions where nucleic acids serve as nucleophiles and the consequences of carcinogenicity and mutagenicity are manifested subsequently (Beranak, et al., 1980; Lawly, 1984). For instance, the first member of the polyaromatics naphthalene binds covalently to molecules in liver and is an important inhibitor of mitochondrial respiration (Samanta, et al., 2002).

It is quite obvious that the Bonny light crude oil caused RNA induction both in the nucleus and mitochondria (Figure 2). The alteration of the liver mitochondrial RNA due to the Bonny light crude oil and has been partly ascribed to nickel and polyaromatics (Oruambo, et al., 2007). Polyaromatics cause tumours in laboratory animals which could be linked to nucleic acid alterations (Fisher, et al., 2013). Ribonucleic acid is a direct product of transcription hence the dose-dependent increase in the RNA content in the current work may be an evidence to support

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that fact. Polycyclic aromatic hydrocarbons bind to the aryl hydrocarbon receptor in the cytosol which is translocated to the nucleus where it enhances transcription of xenobiotic metabolising enzymes (Fent, 2003) which may sometimes be an initial adaptive response. Similar activity may also occur in the mitochondrion being the power house of the cell and also permeable to adducts of similar characteristics.

ConclusionIt has been revealed that the Bonny Light crude oil cause major alterations in the serum levels of ALT, AST, ALP in guinea pigs which are indices of hepatic damage. The increase in liver nuclear and mitochondrial RNA correlates with the upregulation of xenobiotic metabolising enzymes. Other mechanisms may be involved thus more research is required.

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