Beni-Suef University Journal of Basic and Applied Sciences 6 (2017) 253–259

Contents lists available at ScienceDirect

Beni-Suef UniversityJournal of Basic and Applied Sciencesjournal homepage: www.elsevier .com/locate /b jbas

Full Length Article

Hepatotoxicity and genotoxicity of gasoline fumes in albino rats

http://dx.doi.org/10.1016/j.bjbas.2017.04.0072314-8535/� 2017 Beni-Suef University. Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

⇑ Corresponding author.E-mail address: fobillion@yahoo.com (F.O. Owagboriaye).

Folarin O. Owagboriaye a,⇑, Gabriel A. Dedeke b, Joseph S. Ashidi c, A.A. Aladesida b, Wasiu E. Olooto d

aDepartment of Zoology, Faculty of Science, Olabisi Onabanjo University, Ago-Iwoye, Ogun State, NigeriabDepartment of Pure and Applied Zoology, College of Bioscience, Federal University of Agriculture, Abeokuta, Ogun State, NigeriacDepartment of Plant Science, Faculty of Science, Olabisi Onabanjo University, Ago-Iwoye, Ogun State, NigeriadDepartment of Chemical Pathology and Immunology, Faculty of Basic Medical Sciences, Olabisi Onabanjo University, Ago-Iwoye, Ogun State, Nigeria

a r t i c l e i n f o

Article history:Received 24 November 2016Received in revised form 17 April 2017Accepted 21 April 2017Available online 24 April 2017

Keywords:HepatotoxicityGenotoxicityGasoline fumeInhalationAlbino rats

a b s t r a c t

Toxic effects of gasoline fumes have been reported, but evidence of its hepatotoxicity and genotoxicityare rare. Therefore, this study assesses hepatotoxicity and genotoxicity of gasoline fumes on fortyAlbino rats randomly assigned to five experimental treatments (T) with eight rats per treatment (T1,T2, T3, T4 and T5). T1(Control) was housed in a section of experimental animal house free from gasolinefumes while T2, T3, T4 and T5 were exposed to gasoline fumes in exposure chambers for one, three, fiveand nine hours daily respectively for twelve weeks. Serum alanine aminotransferase (ALT), aspartateaminotransferase (AST), alkaline phosphatase (ALP) and histopathological examination of the liver tissueswere used as diagnostic markers to assess liver dysfunction. Genotoxicity test was conducted on the lungtissues using randomly amplified polymorphic DNA fingerprinting polymerase chain reaction (RAPD PCR)technique. Significant increase (p < 0.05) in the level of ALT, AST and ALP for T2, T3, T4 and T5 comparedto T1 were recorded. Photomicrograph examination of the liver sections of T1 showed hepatic tissue withnormal liver cell architecture while that of T2, T3, T4 and T5 revealed degenerative changes in the ultra-structural integrity of the hepatic cells. Genotoxicity test revealed DNA bands at a reducing intensity fromT1 to T5. Dendrogram showed DNA damage in the lungs of T3, T4 and T5 were closely similar and thegenotoxic impact was more in T3. Frequent exposure to gasoline fumes was observed to induce hepatox-icity and genotoxicity, hence impairing the normal liver function and gene structure.� 2017 Beni-Suef University. Production and hosting by Elsevier B.V. This is an open access article under

the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

It has been reported that normal physiological state of anorganism can be altered by substances called xenobiotics, whichcan exist in gaseous, liquid, semi-solid and solid states and caneasily enter into organisms through inhalation, ingestion, dermalcontact and diffusion (Murray, 2003). Gasoline in Nigeria, whichis one of the fractionated products of crude oil, is an example ofa xenobiotic. Gasoline contains very complex and inflammablesubstances which are xenobiotic and endocrine disrupting chemi-cals (EDC). Gasoline and its components are also known to be veryvolatile if left exposed and its fume constitutes chemical pollutantsin the environment (Zahlsen and Tri-Tugaswati, 1993). It com-prises of more than 500 saturated or unsaturated hydrocarbonshaving 3–12 carbons such as n-pentane, n-hexane, toluene andbenzene.

Some adverse effects of xenobiotic have been reported. It hasbeen found to reduce seminal parameters (De Jager, 2006),

impaired semen quality (Aneck-Hahn, 2007), causes male genitalanomalies (Bhatia, 2005) and thus fertility status. It has also beenfound to cause a wide range of biochemical and physiological dys-functions by generating reactive oxygen species and various freeradicals. It also inhibits antioxidant enzymes activities such assuperoxide dismutase (SOD), catalase (CAT) and decreases the levelof glutathione (Rahman and Sultana, 2006).

To satisfy the need of the growing industrial establishmentsover the years in Nigeria, there has been a high increase in the dailydemand for gasoline and other petroleum products (Isa et al.,2013). This increasing daily use has increased the frequency atwhich individuals are exposed to its fume. Some of the uses ofgasoline include fuel for vehicles, cooking and lightning fuel inhomes and outside homes, as chemical feedstock for industries,therapeutic reasons (Huckabay et al., 1995) and as fuel for electric-ity generators at homes, offices and industries. The diverse use ofgasoline has greatly increased the establishment of petrochemicalindustry and petroleum exploration which has been reported asone of the main contributors of environmental and global prob-lems (Carlos and Donna, 2008).

254 F.O. Owagboriaye et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 253–259

Exposures to petroleum products both in and outside petroleumindustries have been reported to have some effects on the users,with those who are occupationally exposed being more likely tobe affected than their counterparts (Rothman et al., 1996). Recentresearch from the national institute for occupational safety andhealth (NIOSH) and the occupational safety and health administra-tion (OSHA) showed that oil and gas workers could be exposed tohydrocarbon gases and vapors while working on or near produc-tion and flowback tanks and the exposure can have immediatehealth effects, including loss of consciousness and death (NIOSH,2016). This means workers can face significant health and safetyrisks when they manually gauge or sample tanks (Esswein et al.,2014; Jordan, 2015). Nine worker fatalities had been recordedamong workers who manually gauged or sampled productiontanks from year 2010 to 2014 in USA (NIOSH, 2015) and exposureto the petroleum vapors are believed to be the primary or contrib-utory factors to the oil and gas extraction workers’ deaths(Harrison et al., 2016). In addition, sudden death due to gasolineinhalation had been reported (Martinez et al., 2012). As reportedin Chi (2015), oil and gas workers exposed to chemicals producedand used in oil and gas industry may suffer occupational diseasesof lungs, skin and other organs at levels depending on the lengthof exposure time and the effects from these occupational healthhazards comprise of dizziness, drowsiness, headaches and nausea(commonly associated with hydrocarbon exposure), dermatitisand irritation and inflammation of respiratory system. However,significant hematopoietic changes were observed among 146 outof 292 workers sampled at filling stations in Baghdad city (Aliand Sahb, 2011). Following a similar trend in Nigeria, Odewabiet al. (2014) observed marked reductions in plasma antioxidantdefense system among gasoline station attendants. Also, Atif andRiffar (2012) had reported hematotoxicity among workers of auto-mobile repair workshops in Pakistan.

As reported by Health Protection Agency (HPA, 2007), the majorroute of exposure is inhalation by workers during production anddistribution of the fuel and general public refueling at the gas sta-tion and consequently brings concentrated hydrocarbons into thelungs and bloodstream. The vaporization or presence of gasolinefume in our environment should therefore necessitate accurateidentification of its potential hazards to human and animal health.

Exposure to gasoline has been reported to cause haematotoxic-ity, nephrotoxicity and alters lipids metabolisms and some bio-chemical activities (Uboh et al., 2008, 2010). HPA (2007) hasreported that exposure to the gasoline vapour may affect the cen-tral nervous system and consequently produce effects such as stag-gered gait, slurred speech, confusion, rapid unconsciousness anddeath due to respiratory failure. Owagboriaye et al. (2016) haverecently reported that inhalation exposure to gasoline fume maybe harmful to the normal body physiology by increasing serumlipid peroxidation, corticosterone and aldosterone level. But littleis known on the hepatotoxic effect of gasoline fume exposure inanimals. Liver is the main organ that chemically alters all com-pound entering the body and this implies that liver could be oneof the major target organ by environmental pollutant, thereby, dis-rupting the metabolic action of this organ.

Genotoxicity is developed to identify the elements or com-pounds presents in the environment having the potential to causemutation by damaging the DNA of animals (Khlood et al., 2011). Ithas been reported that acrylamide, a xenobiotic, can be absorbedinto the circulation and distributed to various organs, and reactswith DNA, neurons, haemoglobin and essential enzymes(Rayburn and Friedman, 2010) causing several toxic effects as ani-mal carcinogen and germ cell mutagen (Ghanayem et al., 2005),but there is paucity of information on the genotoxic effects associ-ated with gasoline fume exposure in animals. It is on this basis thisstudy was therefore designed to clarify the possible hepatotoxic

and genotoxic effects associated with inhalation exposure to gaso-line fumes in albino rat.

2. Materials and methods

2.1. Experimental animal

We used forty apparently healthy adult male albino aged8–9 weeks (220±10 g) obtained from the breeding section of theanimal house of the Department of Zoology, Olabisi OnabanjoUniversity, Ago-Iwoye Nigeria for this study. Prior to thecommencement of the experiment, the rats were acclimatizedunder the laboratory conditions of 25 ± 5 �C and 65 ± 5% RelativeHumidity in the experimental animal house for one week. The ratswere randomized into five experimental treatments (T) with eightrats per treatment. The rats were individually kept in woodencages (65 cm � 35 cm � 50 cm) in a well-ventilated animal houseand were allowed free access to clean drinking water and food.

2.2. Exposure to gasoline fume

The method of exposure earlier described by Uboh et al. (2008)and Owagboriaye et al. (2016) was adopted for this study. The ani-mal cages housing the test groups were placed in an exposurechamber (165 cm � 95 cm � 220 cm). Two calibrated 1000 ml canscontaining 500 ml of gasoline were placed in the chamber one hourprior to the commencement of the exposure to ensure that theexposure chamber was saturated with gasoline fume. The exposedanimals were later placed in the chamber and allowed to inhale thefumes generated from the direct evaporation of liquid gasolinefrom the cans at ambient humidity and temperature. At the endof each daily exposure period, the rats were removed from theexposure chamber. T2, T3, T4 and T5 were exposed to gasolinefume for one, three, five and nine hours respectively at a room tem-perature for a period of twelve weeks while the control treatment,T1 was housed separately in a section of the experimental animalhouse free from gasoline fume and exposed to distilled water. Theexperimental protocol was conducted in accordance with the reg-ulations of the local ethics committee in animal care unit of OlabisiOnabanjo University, Ago-Iwoye, Ogun State, Nigeria. Animalexperiment was performed according to ethical guidelines of ani-mal experimentation (regulation CEE 86/609). The care of the ani-mals was done in accordance with the U.S. public health serviceguidelines (National Research Council, 2011).

2.3. Sample collection

Blood samples were collected from the rats 24 h after the lastexposure by retro orbital sinus with micro haematocrit tube.Serum samples were separated within 1 h after collection of bloodand centrifuged at 3000g for 5 min and stored in a freezer tillassayed. Biochemical analyses on the serum samples were carriedout after the sample collection. The rats were sacrificed and liverexcised through dissection were fixed in 10% neutral-buffered for-malin and processed for histopathological examinations. Lungsamples was also collected and subjected to genotoxicity test.

2.4. Histopathological examinations

The liver specimens were routinely processed and sectioned at4–5 lm thick. The obtained liver sections were stained withHematoxylin-Eosin (H&E) dye before mounting in neutral DPXmedium. Prepared slides were examined at 100 X and 400 Xmagnifications.

Table 1Showing the programs for PCR analysis.

Stages EZ OPA2

1 94 �C, 1:00 min 94 �C, 1:00 min2 54.6 �C, 2:00 min 29 �C, 2:00 min3 72 �C, 2:00 min 72 �C, 2:00 min4 Goto 1, 5� Goto 1, 5�5 94 �C, 0:15 min 94 �C, 0:15 min6 59.6 �C, 0:30 min 34 �C, 0:30 min7 72 �C, 1:00 min 72 �C, 1:00 min8 Goto 5, 45� Goto 5, 45�9 72 �C, 15:00 min 72 �C, 15:00 min10 4 �C, 1 4 �C, 1

F.O. Owagboriaye et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 253–259 255

2.5. Biochemical analysis

Serum samples were analysed for estimation of serum alanineaminotransferases (ALT), aspartate aminotransferases (AST) usingthe standard colorimetric method of Reitman and Frankel (1957).Alkaline phosphatase (ALP) activity was estimated using Randoxcommercial enzyme kit as described by German Society forClinical Chemistry (1972).

2.6. Extraction of DNA from lungs

Extraction of DNA from the animal lung tissues was carried outusing the Qiagen DNA extraction kit which uses the spin columnmethod. 25 mg of lung was cut into small pieces, placed in a micro-centrifuge tube and 180 mL of ATL Buffer and 20 mL of proteinase Kwas added. It was mixed thoroughly by vortexing and incubated at56 �C overnight until the tissue is completely lysed. After completelysis of the tissue, 200 mL of ATL buffer was added, mixed thor-oughly by vortexing then 200 mL of ethanol (96–100%) was added.The whole mixture (including any precipitate) was pipetted intothe DNeasy mini spin column placed in a 2 mL collection tubeand centrifuged at 6000g for 1 min. The flow-through was dis-carded while the spin column was placed in a new 2 mL collectiontube, 500 mL of AW1 buffer was added and centrifuged at 6000g for1 min. The flow-through was discarded while the spin column wasplaced in a new 2 mL collection tube, 500 mL of buffer AW2 wasadded, centrifuged at 20,000g for 3minutes to dry the DNeasymembrane and flow through was discarded. The DNeasy mini spincolumn was then placed in a clean 2 mL microcentrifuge tube and200 mL of buffer AE was pippetted directly onto the DNeasy mem-brane. It was incubated at room temperature for 1 min and thencentrifuged at 6000g for 1 min to elute the DNA. The eluted DNAwas quantified using Fisher Scientific nanodrop spectrophotometerand kept at -20 �C until used (Ausubel et al., 2002).

2.7. Electrophoresis of extracted DNA

100 mL of 1% agarose gel was prepared in 1X TAE (Tris AcetateEDTA) buffer, boiled in a microwave to dissolve the agarose,allowed to cool to about 60 �C and 5 mL of ethidium bromide wasadded and mixed thoroughly. The agarose was then poured intothe gel tray after the comb has been fixed in the tray. The agarosewas allowed to solidify and the comb was removed to create thewells where the DNA was loaded. 2 mL of loading buffer was addedto 18 mL of DNA, this mixture was loaded along with DNA markerinto the wells of the gel. After loading, electrophoresis was carriedout at 70Volts for 45 min in 1X TAE buffer and DNA was viewedusing UVP gel documentation system (Thompson et al., 2012).

2.8. Randomly amplified polymorphic DNA fingerprinting polymerasechain reaction (RAPD PCR)

RAPD PCR was carried out as described by Seufi et al. (2009)using the Qiagen PCR master mix kit. Two operon primers whichhad been previously selected were used for this study because oftheir good RAPD profiles and their good in vivo/in vitro correlation.The primers are:

� EZ: 50-GCATCACAGACCTGTTATTGCCTC-30

� OPA2: 50-TGCCGAGCTG-30

12.5 mL of 2X PCR master mix (PCR reaction buffer, dNTP’s, andamplitaq) and 0.5 mL of primer was pipetted into a DNAase andRNAase free PCR tube. Volume of DNA corresponding to 50 ng ofDNA extract was added and the mixture was made up to 25 mLwith sterilized, filtered distilled water. The samples were placed

in the thermal cycler and PCR was carried out using the programsin Table 1.

2.9. Electrophoresis of PCR products (amplicons)

The amplicons or PCR products were elecrophoresed on 2%agarose gel as described below:

100 mL of 1% agarose gel was prepared in 1X TAE (Tris AcetateEDTA) buffer, boiled in a microwave to dissolve the agarose,allowed to cool to about 60 �C and 5 mL of ethidium bromide wasadded and mixed thoroughly. The agarose was then poured intothe gel tray after the comb has been fixed in the tray. The agarosewas allowed to solidify and the comb was removed to create thewells where the DNA was loaded. 2 mL of loading buffer was addedto 18 mL of DNA extracted and this mixture was loaded along withDNA marker into the wells of the gel. After loading, electrophoresiswas carried out at 70 Volts for 45 min in 1X TAE buffer and DNAwas viewed using UVP gel documentation system (Thompsonet al., 2012).

For genetic distance analysis, PyElph was used to convert thegel images into matrices, which were fed into the clustering pro-gram of Fingerprint Analysis with Missing Data (FAMD version1.31). Based on similarity matrices using the unweighted pairgroup method analysis, FigTree version 1.4.0 was used to generateUPGMA dendrogram.

2.10. Data analysis

Data obtained were subjected to statistical analyses using theStatistical Package for Social Sciences (SPSS) version 20.0. Datawere presented as Mean ± Standard error of mean (SEM). Oneway Analysis of Variance (ANOVA) was conducted to determinesignificant difference between parameters. Post hoc test was doneusing the Student-Newman-Keuls (SNK). P value less than 0.05(P < 0.05) was considered statistically significant.

3. Results

Table 2 shows the level of AST, ALT and ALP in the blood of theexperimental rats exposed to gasoline fume for varying periods oftime. Serum concentrations of AST, ALT and ALP in the experimen-tal rats were significantly different (p < 0.05). T1 recorded the low-est concentrations of AST, ALT and ALP compared to T2, T3, T4 andT5. The T5 rats had significantly higher (p < 0.05) level of AST(415.63 ± 188.23) than the other treatments (126.07 ± 12.25–189.43 ± 2.07). The level of ALT and ALP were also significantlyhigher (p < 0.05) in T5 compared to other treatments.

The liver histopathology of the control and experimental ratsare shown in Plates 3–7. Photomicrograph examination of the liversections of T1 showed a hepatic tissue with normal liver cell archi-tecture. Photomicrograph examination of the liver sections of T2, T3,T4 and T5 revealed increasing level of distorted histoarchitectute,

Table 214986011485245Serum AST, ALT and ALP concentration of the rats exposed togasoline fume.

AST (m/L) ALT (m/L) ALP (m/L)

T1 126.07 ± 12.25b 15.47 ± 1.04c 142.83 ± 12.86b

T2 166.53 ± 3.59b 16.07 ± 1.08c 205.53 ± 8.13a

T3 187.33 ± 7.92b 18.27 ± 1.33bc 205.67 ± 14.84a

T4 189.43 ± 2.07 b 20.63 ± 0.93 b 207.50 ± 5.83a

T5 415.63 ± 188.23a 28.00 ± 3.36 a 222.30 ± 22.84a

**Mean values (±Standard deviation) in the same column having the same super-script are not significantly different (p > 0.05).

256 F.O. Owagboriaye et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 253–259

swelling/degenerated hepatocytes, degenerated endothelium,chromatolytic hepatocytes and pyknotism, patchy inflammationwith remarkable sinusoidal space and large central vein.

The DNA banding profile using EZ and OPA2 primer of the lungsof the rats exposed to varying hours of daily gasoline fume (1 h,3 h, 5 h, 9 h and 0 h [Control]) for 12 weeks are presented in Fig. 1aand bb respectively. DNA banding pattern using EZ primer (50-GCATCACAGACCTGTTATTGCCTC-30) revealed more DNA bands in thecontrol rats than those exposed to the varying hours of gasolinefume (Fig. 1a). The DNA bands ranged from 400 kb and below100 kb which was the lowest band on the marker lane. The numberof DNA bands observed was however lowest in the lungs of ratsexposed to 3 h of gasoline fume daily with bands of 100 kb and300 kb missing. The DNA bands observed in the experimental ratsusing OPA2 primer (50-TGCCGAGCTG-30) ranged from 850 kb to200 kb (Fig. 1b). More number of DNA bands was observed in thelungs of the control rats than those subjected to the varying hoursof gasoline fume exposure. However, a similar DNA banding pat-tern was observed in all the groups of rats subjected to the varying

a

b

Fig. 1. RAPD PCR profile of the lungs of rats exposed to varying hours of gasolinefume for 12 weeks using (a) EZ primer (50-GCATCACAGACCTGTTATTGCCTC-30), and(b) OPA2 primer (50-TGCCGAGCTG-30). 1 = 1 h (T2); 3 = 3 h (T3); 5 = 5 h (T4); 9 = 9 h(T5), 0 = 0 h (T1) and M = Marker.

hours of gasoline fume exposures and the bands were observed toshow a reducing intensity from the control to the rats exposed togasoline fumes.

Coefficient of similarity of the DNA bands of the lungs of ratsdaily exposed to 1 h, 3 h, 5 h, 9 h and 0 h (Control) of gasoline fumeis presented in a dendrogram shown in Fig. 2. Compared to thecontrol, the similarity between the DNA bands of the experimentalrats was followed by rats exposed to 1 h, 5 and 9 h and 3 hrespectively.

4. Discussion

The serum levels of ALT, AST and ALP of rats exposed to gasolinefume for varying periods of time in this study were shown to besignificantly higher and time-dependent, compared to the controlrats. This could be indicative of liver cell damage and injury sincethese enzymes have been shown by Lin et al. (2000) and Charles(2014) to be released greatly into circulation after cellular damage.In this study, the cellular damage to the liver as signified by theincreased serum enzyme activities could be due to the abnormaldynamic properties of cellular membranes following exposure tohydrocarbon components of the gasoline fumes. Masoud et al.(2015) reported increased in activities of the serum enzymesamong gasoline station workers occupationally exposed to ben-zene, toluene and xylenes (BTX) in gasoline as it has also been doc-umented that BTX are the most dangerous elements of gasoline(Adami et al., 2006). This finding confirms Uboh et al. (2009) andMehdi-Araghi and Ahmadi (2013) who had noted increased inthe activities of serum ALT, AST and ALP in rats hourly exposedto gasoline fume due to liver injury.

Histological analysis of the liver tissues of the exposed ratsrevealed that frequent exposure to gasoline fume affects the struc-tural integrity and architecture of the liver cells. This implies thatthe liver is one of the major target organs of gasoline fume-induced injury. Uboh et al. (2009) had earlier reported that thecumulative oxidative damage is likely to be one of the underlyingmechanisms responsible for the hepatotoxic effects of gasolinefumes, as observed in this study.

Genotoxicity is developed to identify the elements or com-pounds presents in the environment having the potential to causemutation by damaging the DNA. RAPD-PCR technique has beenshown to be a powerful tool for gene mapping, population, pedi-gree analysis, phylogenetic studies and strain identification(Grayson et al., 2000). Also, its use in surveying genomic DNA forevidence of various types of damage and mutation suggests thatthey may potentially form the basis of novel genotoxicologicalassays for the detection of DNA damage and mutation (Shimandaand Shima, 1998). Previous study has shown that changes in bandpatterns observed in DNA fingerprint analysis reflect DNA alter-ations from single base changes (point mutations) to complexchromosomal rearrangements (White et al., 1990). DNA adductsand strand breakages with reduced intensity are indicators ofgenotoxic materials (Landis and Yu, 2003). In this study, DNA dam-age induced by gasoline fume was reflected by changes in RAPDprofiles and disappearance of bands which occurred in the profilesgenerated by exposed rats to the gasoline fume. The present datashowed that the RAPD-PCR method is useful for the screeningand characterization of genomic regions that have undergonealterations as the result of gasoline fume exposure. Similar findingshave been reported by Castano and Becerril (2004) and Liu et al.(2005) that used RAPD-PCR to analyze the induced DNA damage.The observed reduction in the intensity of the DNA strand of theexposed rats in this work could be adduced to the impact of thehydrocarbon fractions of the gasoline by inhibiting the polymeriza-tion of the DNA tag polymerase in the PCR reaction, which led to

Fig. 2. UPGMA dendrogram showing clustering of lungs of rats exposed to 1 h, 3 h, 5 h, 9 h and 0 h (Control) of gasoline fume for 12 weeks.

a b

Plate 2. (a) Photomicrograph of liver exposed to gasoline fume for 1 hr (T2). H&E X100: Showing slightly distorted histoarchitechture and dilated central vein (Red Arrow)(b): Photomicrograph of liver exposed to gasoline fume for 1hr (T2). H&E X400: Showing slightly distorted histoarchitechture, swelling/degenerating/hepatocytes andpyknotism (purple arrow) and dilated central vein (Red Arrow).

a b

Plate 1. (a) Photomicrograph of T1. H&E X100: Showing normal histoarchitecture and central vein (Red Arrow). (b): Photomicrograph of T1. H&E X400: Showing normalhepatocytes (yellow arrows); sinusoids (blue arrows) and central vein (Red Arrow).

a b

Plate. 3. (a) Photomicrograph of liver exposed to gasoline fume for 3 h (T3). H&E X100: Showing slightly distorted histoarchitechture, degenerating endothelium and dilatedsinusoids and central vein (Red Arrow) (b): Photomicrograph of liver exposed to gasoline fume for 3 h (T3). H&E X400: Showing extensive distorted histoarchitechture,shrinkage/degenerating/hepatocytes (yellow arrows), pyknotism (purple arrow) and dilated central vein (Red Arrow).

F.O. Owagboriaye et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 253–259 257

a b

Plate. 5. (a) Photomicrograph of liver exposed to gasoline fume for 9 h (T5). H&E X100: Showing extensive distorted histoarchitechture, degenerated endothelium and dilatedsinusoids (blue arrow) and central vein (Red Arrow). (b): Photomicrograph of liver exposed to gasoline fume for 9 h (T5). H&E X100: Showing extensive distortedhistoarchitechture, degenerated endothelium (green arrow); dilated sinusoids (blue arrows) and central vein (Red Arrow); chromatolytic hepatocytes (yellow arrows) andpyknotic cells (purple arrows).

a b

Plate. 4. (a) Photomicrograph of liver exposed to gasoline fume for 5 h (T4). H&E X100: Showing extensive distorted histoarchitechture, degenerated endothelium and dilatedsinusoids and central vein (Red Arrow). (b): Photomicrograph of liver exposed to gasoline fume for 5 h (T4). H&E X100: Showing extensive distorted histoarchitechture,degenerated endothelium (green arrow); dilated sinusoids (blue arrows) and central vein (Red Arrow); chromatolytic hepatocytes (yellow arrows) and pyknotic cells (purplearrows).

258 F.O. Owagboriaye et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 253–259

decrease RAPD band intensity of the exposed rats. Also, band inten-sity decreasing had been previously reported to be as a result of theloss of some alleles (Weinberg, 1991). However, this finding is incontrary to the report by American Petroleum Institute (1980)who had earlier exposed groups of male mice to diesel fuel vapour(6 h/day, 5 days/week, for 8 weeks) and reported no genotoxicityeffects.

5. Conclusion

The results of this work suggested that frequent exposure togasoline fume may induce hepatotoxicity and genotoxicity, henceimpairing the normal liver function and gene structure. Thisassumes significance and public health concern considering theincreasing use of gasoline and consequent exposure to its fumesin Nigeria. Direct exposure to gasoline fume by the populaceshould be discouraged and frequent and timely medical check-upby the petroleum industries workers should therefore be encour-aged to ascertain their health condition. However, more researchesare needed on the genotoxic effect of gasoline fumes on animals.

Conflict of interest

Authors declare no competing interest in this research.

Financial support

This research did not receive any specific grant from fundingagencies in the public, commercial, or not-for-profit sectors.

Roles of author

FOO conceived the experiment. FOO and GAD designed theexperiments. FOO, JSA and WEO performed the experiment. FOOand AAA analyzed and interpreted the data. FOO drafted the manu-script which was critically revised by GAD, JSA, WEO and AAA andgave final approval. All authors agree to be accountable for integ-rity and accuracy in all aspects of the work.

Acknowledgements

Authors appreciate the technical contributions of laboratorytechnologist in the central biotechnology laboratory of the FederalUniversity of Agriculture Abeokuta and Olabisi Onabanjo Univer-sity Ago-Iwoye, Ogun State, Nigeria.

References

Adami, G., Larese, F., Venier, M., Barbieri, P., LoCoco, F., Reisenhofer, E., 2006.Penetration of benzene, toluene and xylenes contained in gasolines throughhuman abdominal skin in vitro. Toxicol. In Vitro. 20, 1321–1330.

Ali, A., Sahb, A., 2011. Hematological assessment of gasoline exposure among petrolfilling workers in Baghdad. J. Fac. Med. 53, 4.

API. American Petroleum Institute., 1980. Mutagenicity evaluation of diesel fuel inthe mouse dominant lethal assay. American Petroleum Institute. API MedicalResearch Publication, Washington (DC), pp. 28–31346.

Aneck-Hahn, N., 2007. Impaired semen quality associated with environmental DDTexposure in young men living in a malaria area in the Limpopo Province. SouthAfrica. J. Androl. 28 (3), 423–434.

Atif, K., Riffar, N.M., 2012. Hematological Evidence of Occupational Exposure toChemicals and Other Factors among Auto-Repair Workers in Rawalpindi.Pakistan. Osong Pub. Health Res. Perspect. 3 (4), 229–238.

F.O. Owagboriaye et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 253–259 259

Ausubel, F.M., Roger, B., Robert, K.E., David, D.M., Seidman, J.G., Smith, J.A., Kevin, S.,2002. Short Protocols in Molecular Biology: A Compendium of Methods fromCurrent Protocols in Molecular Biology. 1, 5th ed. JohnWiley and Sons Inc., NewYork, pp. 10.12–10.18.

Bhatia, R., 2005. Organochlorine pesticides and male genital anomalies in the childhealth and development studies. Environ. Health Persp. 113 (2), 220–224.

Carlos, J.S.P., Donna, M., 2008. Human mercury exposure and adverse health effectsin the Amazon: a review. Cad. Saúde Pública. 24 (4), 503–520. http://dx.doi.org/10.1590/S0102-311X2008001600004.

Castano, A., Becerril, C., 2004. ’In vitro assessment of DNA damage after short- andlong-term exposure to benzo(a)pyrene using RAPD and the RTG-2 fish cell line’.Mut. Res. 552 (1–2), 141–151.

Charles, C.A., 2014. Effect of arsenic trioxide poisoning on hematologicalparameters, liver marker enzymes and kidney of male albino rats. PinnacleBiol. Sci. 2 (2), 262–265.

Chi, N., 2015. 8 Occupational Health Hazards in Oil and Gas Industry That You MustKnow (Part 1). http://oilandgasmanpowerprovider.blogspot.com/2015/10/occupational-health-hazards-in-oil-and-gas-industry.html.

De Jager, C., 2006. Reduced seminal parameters breakthroughs in andrologyassociated with environmental DDT exposure and p, p0-DDE concentration inmen in Chiapas, Mexico: a cross-sectional study. J. Androl. 27 (1), 16–27.

Esswein, E.J., Snawder, J., King, B., Breitenstein, M., Alexander-Scott, M., Kiefer, M.,2014. Evaluation of some potential chemical exposure risks during flowbackoperations in unconventional oil and gas extraction: preliminary results. J.Occup. Environ. Hyg. 11 (10), 174–184.

Ghanayem, B.I., Witt, K.L., El-Hadri, L., Hoffler, U., Kissling, G.E., Shelby, M.D., 2005.Comparison of germ cell mutagenicity in male CYP2EI- null and wild type micetreated with acrylamide: Evidence supporting a glycidamide-mediated effect.Biol. Reprod. 72, 157–163.

Grayson, T.H., Atienzar, F.A., Alexander, S.M., Cooper, L.F., Gilpin, M.L., 2000.Molecular diversity of Renibacterium salmoninarium isolates determined byrandomly amplified polymorphic DNA analysis. Appl. Environ. Microbiol. 66 (1),435–438.

Harrison, R.J., Retzer, K., Kosnett, M.J., 2016. Sudden deaths among oil and gasextraction workers resulting from oxygen deficiency and inhalation ofhydrocarbon gases and vapors — United States, January 2010–March 2015.MMWR Morb. Mortal. Wkly. Rep. 65, 6–9. http://dx.doi.org/10.15585/mmwr.mm6501a2.

HPA. Health Protection Agency., 2007. Petrol: Toxicological overview. Prepared byR.P. Chilcott. CHAPD HQ, United Kingdom. 2nd Version. pp. 1–16.

Huckabay, P., Wendy, D., VanCleave, C., Ostrander, J., 1995. Petroleum sectornotebook paper. Cameron University. J. Appl. Sci. Environ. Manag. 6, 84–86.

Isa, H.A., Hamisu, S., Lamin, H.S., Ya’u, M.Z., Olayande, J.S., 2013. The perspective ofNigeria’s projected demand for petroleum products. J. Petrol. Gas Eng. 4 (7),184–187.

Jordan, T., 2015. Hydrocarbon exposures during tank gauging and samplingoperations. NORA Oil and Gas Sector Council Meeting, Denver, CO: http://www.nationalstepsnetwork.com/docs_tank_gauging/NORA_Oil_and_Gas_CouncilMeeting_March2015.pdf.

Khlood, M. El-Bohi, Gihan, G. Moustafa, Nabela, I. El sharkawi, Laila, M. E. Sabik,2011. Genotoxic effects of acrylamide in adult male albino rats liver. J. Amer.Sci. 7 (1), 1097–1108 (ISSN: 1545-1003). http://www.americanscience.org.

Landis, W.G., Yu, M.H., 2003. Introduction to Environmental Toxicology: impact ofchemicals upon ecological systems. Lewis Publishers CRC Press LLC, New York,USA.

Lin, S.C., Chung, T.H., Ueng, Y.H., Linn, S.H., Hsu, C.L., 2000. The hepatoprotectiveeffects of Solanum alatam moech on acetaminophen-Induced hepatotoxicity inmice. Am. J. Clin. Med. 28, 105–114.

Liu, W., Li, P.J., Qi, X.M., 2005. DNA changes in barley (Hordeum vulgare) seedlingsinduced by cadmium pollution using RAPD analysis. Chemosphere 61 (2), 158–167.

Martinez, M.A., Ballesteros, S., Alcaraz, R., 2012. Reporting a sudden death due toaccidental gasoline inhalation. Forensic Sci. Int. 215 (1–3), 113–120.

Masoud, N., Kiamars, H., Jafar, H., 2015. Early liver and kidney dysfunctionassociated with occupational exposure to sub-threshold limit value levels ofbenzene, toluene, and xylenes in unleaded petrol. Safety Health at Work. 6,312–316.

Mehdi-Araghi, A., Ahmadi, R., 2013. The effects of gasoline vapor on serum alkalinephosphatase in male rats. In: International Conference on Medical Sciences andChemical Engineering (ICMSCE’2013) August 28–29, Penang, Malaysia.

Murray, R.K., 2003. Metabolism of Xenobiotics. In: Janet, F., Jim, R., Janene, M.O.(Eds.). . 26th ed., Harper’s Illustrated Biochemistry 26th ed. The McGraw-HillCompanies, Inc., pp. 626–632.

NRC. National Research Council of the National Academies., 2011. Guide for the careand use of laboratory animals. 8th ed. Committee for the Update of the Guidefor the Care and Use of Laboratory Animals. Institute for Laboratory AnimalResearch. Division on Earth and Life Studies. National Academy Press,Washington DC. www.nap.edu.

NIOSH., 2015. Suspected inhalation fatalities involving workers during manual tankgauging, sampling, and fluid transfer operations on oil and gas well sites, 2010–2014. Cincinnati, OH: U.S. Department of Health and Human Services, Centersfor Disease Control and Prevention, National Institute for Occupational Safetyand Health, http://www.cdc.gov/niosh/topics/fog/data.html.

NIOSH., 2016. NIOSH/OSHA Hazard Alert. Health and Safety Risks for WorkersInvolved in Manual Tank Gauging and Sampling at Oil and Gas Extraction Sites.U.S. Department of Health and Human Services, Centers for Disease Control andPrevention, National Institute for Occupational Safety and Health, DHHS(NIOSH) Publication No. 2016–108.

Odewabi, A.O., Ogundahunsi, O.A., Oyalowo, M., 2014. Effect of Exposure toPetroleum Fumes on Plasma Antioxidant Defense System in PetrolAttendants. British J. Pharmacol. Toxicol. 5 (2), 83–87.

Owagboriaye, F.O., Dedeke, G.A., Aladesida, A.A., Bamidele, J.A., Olooto, W.E., 2016.Assessment of the effect of gasoline fume on stress hormones, antioxidantstatus and lipid peroxidation in albino rat. J. King Saud Univ.-Sci. http://dx.doi.org/10.1016/j.jksus.2016.11.002.

Rahman, S., Sultana, S., 2006. Chemopreventive activity of glycyrrhizin on leadacetate mediated hepatic oxidative stress and its hyperproliferative activity inWistar rats. Chemo. Biol. Intera. 60 (1), 61–69.

Rayburn, J.R., Friedman, M., 2010. I-Cysteine, N-Acetyl-1-cysteine, and Glutathioneprotect Xenopus laevis embryos against Acrylamide-induced Malformationsand Mortality in the frog embryo Teratogenesis Assay. J. Agric. Food Chem. 58(20), 11172–11178.

Recommendations of the German Society for Clinical Chemistry, 1972. Standardmethod for determination of alkaline phosphatase (AP) activity. Z. Klin. Chem.Klin. Biochem. 10, 290.

Reitman, S., Frankel, S.A., 1957. Colorometric method for the determination ofserum glutamic oxaloacetic and glutamic pyruvic transaminase. Am. J. Clin.Pathol. 28, 56–63.

Rothman, N., Li, G.L., Dosemeci, M., Bechtold, W.E., Marti, G.E., Wang, Y.Z., Linet, M.,Xi, L.Q., Lu, W., Smith, M.T., Titenko-Holland, N., Zhang, L.P., Blot, W., Yin, S.N.,Hayes, R.B., 1996. Haematotoxicity among Chinese workers heavily exposed tobenzene. Am. J. Ind. Med. 29, 236–246.

Seufi, A.M., Ibrahim, S.S., Elmaghraby, T.K., Hafez, E.E., 2009. Preventive effect of theflavonoid, quercetin, on hepatic cancer in rats via oxidant/antioxidant activity:molecular and histological evidences. J. Exp. Clin. Canc. Res. 28, 80–88.118611756-9966-28-80.

Shimanda, A., Shima, A., 1998. Combination of genomic DNA fingerprinting intomedaca specific-locus test system for studying environmental germ-linemutagenesis. Mut. Res. 399 (2), 149–165.

Thompson, R., Zoppis, S., McCord, B., 2012. An overview of DNA typing methods forhuman identification: past, present, and future. In: Antonio, Alonso (Ed.), DNAelectrophoresis protocols for forensic genetics. Methods in Molecular Biology.Humana Press Inc, New York, pp. 3–16. http://dx.doi.org/10.1007/978-1-61779-461-1.

Uboh, F.E., Akpanabiatu, M.I., Alozie, Y., 2008. Comparative effect of gasolinevapours on renal functions in male and female albino wistar rats. J. Pharmacol.Toxicol. 3, 478–484.

Uboh, F.E., Ekaidem, I.S., Ebong, P.E., Umoh, I.B., 2009. The Hepatoprotective Effect ofVitamin A against Gasoline Vapor Toxicity in Rats. Gastroenterology Research. 2(3), 162–167.

Uboh, F.E., Akpanabiatu, M.I., Edet, E.E., Ebong, P.E., 2010. Increase activity of serumtotal and prostatic acid phosphatase, alkaline phosphatase, gammaglutamyltransferase and testosterone level in rats exposed to gasoline vapors.J. Med. Medical Sci. 1 (1), 016–020.

Weinberg, R.A., 1991. Tumor suppressor gene. Sci. 254, 1138–1146.White, J.J., Neuwirth, H., Dennis, M.C., Schneider, E.L., 1990. DNA alterations in

prostatic adenocarcinoma and benign prostatic hyperplasia: detection by DNAfingerprint analyses. Mut. Res. 237 (1), 37–43.

Zahlsen, I., Tri-Tugaswati, A., 1993. Review of air pollution and its health impact inIndonesia. Environ. Res. 63, 95–100.