INTRODUCTION:

Petrol (or gasoline) is a volatile and inflammable petroleum-derived liquid mixture primarily used for internal combustion of engines. It consists of hydrocarbons (aromatic, saturated and unsaturated) and non-hydrocarbons (N, S, O₂, vanadium and nickel)^1,2. The volatile nature of petrol makes it readily available in the atmosphere any time it is dispensed, especially at petrol filling stations and depots. People are exposed to gasoline fumes during fuelling and refuelling at gas stations, but the gas station attendants are more at risk by virtue of their occupational exposure².

Steinsvåg et al., 2007³ established that people are exposed to known and suspectedcarcinogenic agents, mixtures and exposure circumstances in petroleum industry. Atmospheric concentration of gasoline vapor (approximately 2000 ppm) is not safe when inhaled even for a brief period of time (seconds). During fuelling of vehicles, the concentration of gasoline vapor in the air is between 20 and 200 ppm⁴.This amount is higher when there is a long queue of cars to be fuelled, which is a usual occurrence during fuel scarcity.

Akinosun et al. (2006)⁵, showed that parameters of liver functions are within normal range in Nigerian petrol attendants. This is contrary to the results of liver and renal function tests in Artisans occupationally exposed to petrol in Mechanic Village in Nnewi, Nigeria. The result indicated that occupational exposure to lead in petrol may compromise liver and renal functions⁶. Uboh et al. (2005)⁷, showed that exposure of ungraded concentrations of Nigeria petrol fumes in albino Wistar rats also showed increase in liver function enzymes investigated for two weeks. Further toxicological effect of gasoline was reported by Grebic et al. (2007)⁸.

Apart from gasoline, its constituents are also toxic to the liver especially at high doses. Dose dependent increase in serum liver enzyme activity was observed when female rats were exposed to 0, 1000, 15000 or 2000ppm of p-xylene for four hours. This was considered to be a sign of hepatocellular and hepatobilliary damage⁹. Focal development and growth are believed to be predictive of hepatocarcinogenesis¹⁰. Proliferation of liver endoplasmic reticulum was found in hepatocytes, when rats were exposed to 1000ppm of m-xylene for 6h/day, 5days/week, for 3months. When rats were exposed to a 1:1 combination of m-xylene and toluene (500 plus 500ppm, the changes were a combined effect of each of the solvents¹¹. Long term exposure of a mixture of xylene and ethylbenzene for 8h/day up to 5 months increased the liver weight at a dose of 4000mg/l. at the same dose hypertrophy of the liver centrolobullar zone in the liver including a change in the amount of smooth and rough endoplasmic reticulum. Liver enzymatic activities were increased during the first 6 weeks¹².

In order to replace antiknock leaded derivatives in gasoline, legislations were enacted in the United States and other countries to find safer additives and to reduce CO, O₃, and volatile organic compounds (VOCs) in fuel. It was therefore found that gasoline can be modified to reduce pollution and improve the fuel combustion by adding components containing oxygen. The most widely used oxygenate is Methyl Tertiary-Butyl Ether (MTBE), whereas Ethyl Tertiary-Butyl Ether (ETBE), Tertiary Amyl Ethyl Ether (TAME), methanol and ethanol are possible substitutes^13,14 The resulting fuel is often known as reformulated gasoline (RFG) or oxygenated gasoline. Typically, concentrations up to 15% MTBE by volume (2.7% oxygen by weight) are being used in oxygenated gasoline^15,16.

Methyl tertiary butyl ether has caused the following cancers in rats and mice, kidney, testicular, liver, lymphomas, and leukemias^17,18. This toxicological effect of MTBE was also confirmed by¹⁹ On March 20, 2000, the Clinton-Gore administration announced that it would take action to significantly reduce or eliminate the use of MTBE and increase the use of alternatives such as ethanol²⁰. Finally, in May 2000, legislation was signed by New York’s governor which bans the use, sale, or importation of fuels containing MTBE in the state beginning in 2004. Ethanol costs more to produce, poses challenges to the gasoline distribution system, extends the spread of hydrocarbons through ground water in gasoline plumes, and in the short-term is unlikely to be available in sufficient quantity. Moreover, its metabolite acetaldehyde is a possible carcinogen that undergoes a photochemical reaction in the atmosphere to produce the respiratory irritant peroxylacetate nitrate (PAN). Co-exposure of rats to 300 ppm of xylene and 20% ethanol showed that concurrent ethanol in take increased hepatic and renal microsomal enzyme activities. Stearosis in the liver was more marked in co-exposed animals than in animals exposed ethanol alone²¹.

The literature above shows that addition of oxygenates to gasoline will reduce the gasoline toxicity. Methyl Tertiary-Butyl Ether is toxic and has been banned; we therefore, investigated the effect of oxygenating or reformulating gasoline with methanol and isopropyl ether on liver function. Methanol is less toxic, cheaper and easily produced as alternate fuel. This will go a long way to improve the economy and reduce toxicity of gasoline.

MATERIALS AND METHODS:

Sample Collection and Preparation:

The gasoline sample used in this study was collected from Nigerian National Petroleum Co-operation (NNPC), Nigeria. Three different concentrations – 1216, 1824 and 2432 ppm of gasoline representing GO1, GO2 and GO3 respectively were used. Reformulated or oxygenated gasoline was prepared by mixing 2432ppm of gasoline with 1, 2 and 3 % of a mixture of isopropyl ether and methanol (1:1 ratio), representing RG1, RG2 and RG3 respectively.

Treatment of Animal:

The rats were treated according to the Ethical Guidelines of the Animal Center, University of Port Harcourt, Choba and the experimental protocol was approved by the Animal Studies Committee of University of Port Harcourt, Nigeria.

Seven exposure chambers were used; in which male albino rats were exposed as following:-

Group A - GO1 exposed 1268 ppm of gasoline.

Group B – GO2 exposed 1876 ppm of gasoline

Group C- GO3 exposed 2432 ppm of gasoline.

Group D – RG1 exposed to 2432 ppm of gasoline plus 1% of a mixture of methanol and isopropyl ether (1:1).

Group E – RG2 exposed to 2432 ppm of gasoline plus 2% of a mixture of methanol and isopropyl ether (1:1).

Group F – RG3 exposed to 2432 ppm of gasoline plus 3% of a mixture of methanol and isopropyl ether (1:1).

Group H – CO (Control) not exposed to gasoline but fresh air.

The exposure was done for 8h/day, 5days/week for 3 months. At the end of every month, 3 rats from each group were sacrificed. The blood and the spleen were collected for analysis.

Enzyme Assay:

Biochemical analysis of the serum enzymes for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was by the method of Reitman and Frankel (1957)²².Alkaline phosphatase (ALP) was assayed according to the method of REC (1972)²³. Histopathological analysis was done using the standard stains of haematoxylin and eosin

Statistical analysis:

Statistical analysis was done using the Analysis of Variance (ANOVA). Mean values were considered significant at p<0.05.

RESULTS:

Effects of Various Concentrations of Gasoline on Liver Enzymes:

There was a general increase in serum activities of AST, ALT and ALP after exposure to both gasoline and reformulated gasoline. The levels of both AST and ALP increased as concentration of gasoline increased from 1216 ppm to 1824ppm, and finally to 2432 ppm as shown in figure 1A. The increase was not significant at lower concentration of 1216ppm of gasoline.

Effects of 1, 2 and 3 months of Gasoline exposure on Liver Enzymes:

The levels of the enzymes significantly increased above the normal level (CO) after the first, second and third months for AST and ALT but not for ALP. The level of ALP did not increase above the control except for the first month. Thus there were increases in the release of these enzymes from the liver as time of exposure increased from one month to three months as shown in Figures 1B for AST, ALT and ALP respectively.

Effects of Reformulation of Gasoline on Liver Enzymes:

Reformulation decreased the level of AST and ALT but not that of ALP. Figure 1C shows the levels of AST, ALT, and ALP respectively after exposure to GO3, RG1, RG2 and RG3. The decrease of that of AST was significant. Thus the level of the enzyme increased on exposure to GO3 compared to RG1, RG2 and RG3, though the decrease was not sequential for 1.2 and 3 % of reformulation. ALT level was found to decrease as a result of reformulation but the decrease was highest with 1% rather than 3%. The reformulated gasoline raised the ALP level more than the gasoline alone.

Figure 1. A

Figure 1. B

Figure 1. C

Figures 1.A, 1B and 1C: Effects of (A) Concentration, (B)Time and (C)Reformulation on ALT, AST and ALP in Rat after 3 Months of Gasoline and Reformulated Gasoline Exposure .

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Plate 1 : Normal liver showing undamaged hepatocytes, no signs of congestion of the sinusoids or any form of necrosis.

Plate 2 : GO1 exposed rat Liver showing signs of necrosis, disorganized hepatocytes and sinosoids after 3 months of exposure.

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Plate 3: The liver of rats exposed to G02 showing increased congestion and occlusion of the sinusoidal space with increased sign of necrosis after 3 months of exposure

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Plate 4: The liver of rats exposed to G03 showing vascular congestion, with a diffuse parenchymal inflammatory cell infiltrates and massive necrosis of the liver cell after 3 months of exposure

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Plate 5 The liver of rats exposed to RG1 showing vascular Congestion of the sinuisoids

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Plate 6 : The liver of rats exposed to RG2 showing reduced vascular Congestion of the sinusoids and presence of necrotic cells after 3 months of exposure.

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Plate 7: The liver of rats exposed to RG3 showing highly reduced vascular congestion of the sinusoids and all most absence of necrotic cells after 3 months of exposure.

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Received on 06.06.2010 Modified on 20.07.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 781-784