|Year : 2015 | Volume
| Issue : 4 | Page : 868-873
Nuclear anomalies in exfoliated buccal epithelial cells of petrol station attendants in Udaipur, Rajasthan
Rashmi Metgud1, Nidhi Khajuria1, Shrikant Patel1, Sahul Lerra2
1 Department of Oral Pathology and Microbiology, Pacific Dental College and Hospital, Udaipur, Rajasthan, India
2 Consultant, J and K Health Services, Jammu and Kashmir, India
|Date of Web Publication||15-Feb-2016|
Prof and HOD, Department of Oral Pathology and Microbiology, Pacific Dental College and Hospital, Airport Road, Debari, Udaipur - 313 024, Rajasthan
Source of Support: None, Conflict of Interest: None
Aim: The petroleum derivatives consist of a complex mixture of chemical compounds one among which is benzene. Petrol station workers who pump fuel to vehicles absorb the products of fuel fumes and the products of combustion.
Materials and Methods: To study the occupational exposure to petroleum derivatives such as benzene, exfoliated buccal cells from 40 petrol station attendants and 40 age-matched control subjects were examined for micronuclei, binucleation, karyorrhexis and karyolysis frequency by using feulgen and Giemsa stains. Statistical evaluation was performed with ANOVA test.
Results: In the present study, inter-comparison of mean values for micronuclei, binucleation, karyorrhexis and karyolysis using Fuelgen stain between smokers of study and control group, as well as between smokers and non-smokers of study and control group revealed statistically highly significant results with P value 0.00002 and 0.0001 respectively. Whereas inter comparison between non-smokers of study and control group using Feulgen stain and inter comparison between smokers of study and control group, using Giemsa stain revealed statistically significant results with P value 0.0034 and 0.0004, respectively.
Conclusion: This study revealed that the studied individuals belong to a risk group and should periodically undergo biological monitoring and proper care.
Keywords: Exfoliated cells, genotoxicity, micronucleus, nuclear anomalies, occupational exposure, petrol pump workers
|How to cite this article:|
Metgud R, Khajuria N, Patel S, Lerra S. Nuclear anomalies in exfoliated buccal epithelial cells of petrol station attendants in Udaipur, Rajasthan. J Can Res Ther 2015;11:868-73
|How to cite this URL:|
Metgud R, Khajuria N, Patel S, Lerra S. Nuclear anomalies in exfoliated buccal epithelial cells of petrol station attendants in Udaipur, Rajasthan. J Can Res Ther [serial online] 2015 [cited 2022 Jul 4];11:868-73. Available from: https://www.cancerjournal.net/text.asp?2015/11/4/868/146058
| > Introduction|| |
Various environmental and epidemiological studies till now days are being conducted on human population, exposed to various gentoxic agents like atmospheric pollutants. Conclusions derived from these studies suggest increase frequency of DNA and chromosomal damage in human population. Mutagenic and carcniogenic effects of diesel and petroleum vapors have been reported in human and various biological models. 
People residing in industrial areas with event of time become exposed to various types of chemical substances, which potentially could be mutagenic. Once the exposure to the hazardous agents can be identified, measures can be taken to decrease their presence in the environment and to protect the people against its exposure, which can lead to minimize the death rate.  Petroleum products include a large number of organic chemicals constituting of cycloalkanes, straight and branched chain alkenes, which are continued source of pollution in various occupational settings . Higher chain alkanes exhibit co-mutagenic and co-carcinogenic properties. Diesel and gasoline engine exhausts contain a variety of mutagenic and carcinogenic agents (IARC 1989).  Benzene and toluene are major monocyclic hydrocarbons present in refinery oils.
Benzene is said to be carcinogenic in nature. Several studies have shown that benzene can induce various forms of genetic damage including chromosome aberrations, sister chromatid exchanges (SCE), micronuclei formation and DNA damage.  The association between exposure to benzene or benzene-containing mixtures with certain types of leukemia, hematoxicity and other bone marrow disorders has been shown in epidemiological studies in different countries. ,, The current permissible exposure level is 1 part per million (ppm) in air for 8 hours. (EPA 1998) . Short-term exposure to high level of benzene can arouse many symptoms like dizziness, drowsiness and sometimes death. Hughes et al., concluded that cigarette smoke is one of the important indoor sources of benzene. 
In India, petrol-filling workers are employed rather than self-serviced, increasing the opportunity for exposure. Long-term exposure to petrol vapor has shown to affect the different physiological systems in the body.  They are engaged in petrol filling for 8 hours per day and do not wear personal protective equipment and personal hygiene is variable in work place, and the occupational exposure to such derivatives may pose genotoxic risk. Each worker pumps an average 2000 L of petroleum approximately containing 5% benzene in total. Volatile fraction of petrol during refueling of the vehicle is continuously inhaled by the attendants at the petrol station. Such type of occupational exposure can lead to mutogenic risk (Celik et al. 2003).  International Agency for research on Cancer (IARC, 1989) has classified such type of exposure to be carcinogenic to humans. Mark-Vendel et al.(1981)  and Hogstedt et al. (1981)  observed a higher frequency of structural chromosome aberrations in individuals exposed to petroleum vapors.
Exfoliated buccal mucosal cells are capable of metabolizing the carcinogen compounds to reactive chemicals (Salma et al. 1999).  Since the cells express the genotoxic effects they are used to reveal the occupational exposure and compounds effect on micronuclei formation (Heuser et al. 2007).  Micronuclei presence in cells is a reflection of structural and/or numerical chromosomal aberrations arising during mitosis. These cells are easily collected from mouth through non-invasive procedure and can be considered a suitable biomonitoring approach for the detection of increased cancer risk in human population because > 90% of all human cancers are of epithelial origin.  To monitor cytotoxic effects, binucleation, karyolysis and karyorrhexis were also evaluated. This method can be used in the buccal cells without the requirement of cell culture or metaphase preparation. (Torres - Bugarin et al. 2004).  Hence the present study aimed to explore the cytogenetic damage in exfoliated buccal cells obtained from petrol station attendants and control subjects, using micronucleus (MN) assay.
| > Materials and methods|| |
The study was conducted at four different petrol-filling stations located in Udaipur, Rajasthan, India. The study population comprised total 80 healthy subjects devoid of any types of diseases or oral lesions, of whom 40 males were refueling attendants at petrol pump and other 40 males were normal healthy individuals with no occupational exposure to toxic agents Included as as controls. Among 40 males in the exposed group as well as control groups 20 were smokers and 20 non-smokers.
Participants were informed in detail about the planned study and written informed consents were obtained. All subjects were selected based on questionnaire which included questions about age, occupational exposure, smoking habit, use of drugs, alcohol, virus illnesses, recent vaccinations, and radiological exams.
Buccal cell sampling, preparation and staining
Buccal cells originate from a multilayered epithelium that lines the oral cavity. Prior to buccal cell collection the petrol station attendants and control group individuals were advised to rinse their mouth thoroughly with water to remove unwanted debris. Sterile wooden spatula was used to obtain cell samples from buccal mucosa. 
The samples were then applied to clean microscope slides. Smears were air dried and fixed in methanol: Acetic acid (3:1). Slides were stained with Feulgen and Giemsa stains and each slide were evaluated to determine nuclear anomalies. 
Frequencies of micronucleus, binucleation, karyorrhexis and karyolysis in exfoliated 1000 cells were scored per slide. Nuclear abnormalities were classified according to Tolbert et al. (1992). Nuclei less than one-third the diameter of the main nucleus; were determined as micronuclei (MN) [Figure 1] and [Figure 2], cell with two nuclei was considered as binucleate [Figure 3]. Nuclei fragmented into irregular pieces were scored as karyorrhexis. Nuclear dissolution, ghost-like image of the nucleus remains, was evaluated as karyolysis [Figure 2] and [Figure 3]. 
All the data was analyzed by one - way analysis of variance (ANOVA) using SPSS software for differences between groups. Values for P < 0.05 were considered statistically significant. The normality of data was checked before the statistical analysis was carried out.
| > Results|| |
In the present study, inter comparison of mean values for micronuclei, binucleation, karyorrhexis and karyolysis using Fuelgen stain between smokers of study and control groups, as well as b/w smokers and non-smokers of study and control groups revealed statistically highly significant results. Whereas intercomparison of mean values between non smokers of study and control Groups using feulgen stain [Table 1] and between smokers of study and control group using Giemsa stain revealed statistically significant results [Table 2].
|Table 1: Intergroup comparison of mean and standard deviation values for MNC, BNC, KRC, KLC using Fuelgen stain |
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|Table 2: Intergroup comparison of mean and standard deviation values for MNC, BNC, KRC, KLC using Giemsa stain |
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| > Discussion|| |
Petroleum products are a complex mixture of aliphatic and aromatic hydrocarbons with high volatility. Such types of hydrocarbons exhibit co-mutagenic and co-carcinogenic properties.  Benzene is one of such hydrocarbon and is the natural component of petroleum products.  Its content in petrol is in range of 1 − 5%. In India, gasoline contains 3% benzene. The percentage of benzene may be as high as 30% in gasoline in some countries.  Petrol evaporates more in tropical than in temperate countries. Petrol stations are located on streets. Petrol station attendants are workers chronically exposed to low level of benzene during refeulling, inhalation of automobile exhaust for a longer period of time as well as with lifestyle habits like smoking. These exposure may cause cytotoxic and genotoxic effects. 
Cigarette smoking is one of the factors that may influence the rate of cytogenetic damage which include micronuclei formation, binucleation, karyorrhexis and karyolysis.  Suhas et al., 2004 found an increase in micronuclei frequency in beedismokers.  Cigarette smoke is an important source of benzene in indoor air. Hughes et al. 1994 stated that median benzene levels is found to be higher in the homes of smokers (10.5 μg/m 3 ) than those of non-smokers (7 μg/m 3 ).  The mean concentration of benzene in indoor air in homes across Canada was 7.4 μg/m 3 , with a maximum value of 68 μg/m 3 .  Sarto et al. and Piyathilake et al. have reported that smoking increases the micronuclei frequency in buccal cells.  Our finding indicates that smoking significantly increases the frequencies of micronuclei and other nuclear abnormalities in both exposed and control subjects.
Inhaled benzene is absorbed by lung alveoli and mixes with blood, from where about 70% of benzene is directly exhaled by lung but remaining is carried to the liver for detoxification process of benzene and converts it into a number of metabolite like benzene-oxide, phenol and catechol which are considered as toxic substances.  Acute inhalation exposure causes nerve inflammation, central nervous system depression, gastroinstestinal irritation, conculsions and cardiac sensitization. Chronic exposure leads to irreversible injury to the blood - forming organs and anorexia. Benzene can also cause irritation to the skin, eyes and mucous membrane.  Anderson et al. showed that benzene and its metabolites induced DNA damage in human lymphocytes demonstrated by comet assay. An increase of chromosome aberrations in circulating lymphocytes is a risk factor for cancer. Liou et al. demonstrated that people having chromosome type aberrations in their lymphocytes were at higher risk of having cancer. 
Brandt et al. and Zhou et al. (1986) have demonstrated genotoxic effect in workers exposed to low levels of benzene from petrol. , Yager et al., 1990 and Tompa et al., 1994 concluded that occupational exposure to benzene has been associated with chronic myeloid and acute myeloid leukemias and non-Hodgkin's lymphomas. , Various authors like Maltoni et al., 1983 concluded that benzene is a multi-potential carcinogen producing a variety of tumors. Lange et al., 1973 showed that low level of benzene affects human immuno-defence. Hite et al., 1980 demonstrated cytogenetic effects of benzene with micronucleus test on bone marrow from mice. 
Cancer is considered as one of the environmental diseases. In nearly 85% of the cancer, environmental factors and stress are considered as one of the etiological factor.  So it is therefore imperative and important to identify any potential cytogenetic toxicity due to these environmental agents and to assess their biological impact on man.  There are numerous methods to judge the cytogenetic damage which may include sister chromatid exchange, chromosomal aberrations, binucleated cells, presence of micronuclei, telomere lengths, etc.,. The later half of twentieth century has mainly focused on the analyses of chromosomal aberrations in order to judge the genetic damage to a person. ,
Oral mucosa permeability in different regions of the mouth is an important aspect to consider when analyzing the local effects of carcinogenic effects. Non-keratinized tissues, such as the buccal mucosa are shown to be much more permeable than keratinized tissues, such as the palate and gingiva. , Increase in nuclear abnormalities has been observed in buccal cells of women living in a dioxin contaminated area. 
The oral epithelium maintains itself by a system of continuous cell renewal in which new cells produced by mitosis in the basal layer migrate to the surface to replace those that are shed. Thus, the mucosa is composed of progenitor and maturing cell populations.  Micronuclei are chromosomal material that originated from acentric fragments of DNA or complete chromosome that fail in attaching to the mitotic spindle.  Ray et al. stated that once micronuclei are formed, they are not repiarable and stands as an evidence of genetic damage that has occurred in dividing basal cells.  Wu et al. in 2004 concluded in their study that micronuclei can be used as a sensitive cytogenetic biomarker and rapid indicator of genetic damage in the exfoliated cells.  Tolbert et al. concluded that, with the help of micronuclei these genetic changes are detected 1 − 3 weeks earlier.  Moreover, the advantage of this method is that it is non-invasive, relative ease of scoring, limited cost, time efficiency and faster. ,
Analysis of exfoliated cells of buccal mucosa also provides evidence of other nuclear abnormalities such as binucleates, karyorrhexis and karyolysis.  Binucleated cells (BN), are probably indicative of failed cytokinesis following the last nuclear division in the basal cell layer.  Karyorrhexis are indicative of more extensive nuclear chromtin aggregation which leads to disintegration of the nucleus.  Binucleus formation is considered as indicator of cytotoxicity, while karyorrhexis and karyolysis are considered as indicators of apoptosis. It has been postulated that repeated exposure to cytotoxicants can result in chronic cell injury, compensatory cell proliferation, chronic cell injury and ultimately tumor development. 
However, the data on cytogenetic damage in petrol station attendants are rather controversial. Evaluation of sister chromatid exchange (SCE) frequencies in petrol station attendants revealed that there was no significant difference between controls and experimental subjects. , Bukvic et al. 1998 and Pitarque et al. 1996 analysed MN and SCE frequencies in peripheral lymphocytes of petrol station attendants and failed to reveal significant differences for SCE, although they found significant differences for MN. ,
The present study, revealed statistically highly significant results in inter comparison of mean values for micronuclei, binucleation, karyorrhexis and karyolysis using Fuelgen stain and statistically significant results using Giemsa stain between smokers of study and control group. Where as intercomparison between non-smokers of study and control groups revealed statistically significant results using Fuelgen stain. Some nuclear anomalies (binucleates, karyorrhexis, and karyolysis) with non-specific stains are sometimes difficult to interpret and sometimes may be misclassified as micronuclei. Hence nuclear-specific stains were used in the present study and we observed that evaluation using Fuelgen stain revealed better results compared to Giemsa stain.
These findings are in accordance with the results of the studies by Celik et al., Sellappa et al., Rajkokila et al., Gadhia et al. who used different stains as mentioned in [Table 3] and they determined a significant increase in the frequency of nuclear abnormalities in the buccal cells of petrol station workers than the control individuals. ,,,
Our results reveal that petrol station workers are under risk of significant cytogenetic damage. The micronucleus test in exfoliated epithelial cells seems to be a useful biomarker of occupational exposure to genotoxic chemicals. As demonstrated in this study, other nuclear abnormalities, such as binucleates, karyolysis and karyorrhexis, are also useful indices of chemical exposure and toxic response. Therefore, a combination of micronuclei and nuclear abnormalities may increase the sensitivity of the exfoliated epithelial cell technique in assessment of genotoxicty and cytotoxicity.
| > Conclusions|| |
It could be emphasized to educate the working population about the cytogenotoxic effects of petrol exposure and to ensure safe and healthy working atmospheres for the petrol station workers to alleviate the health hazards that they may encounter.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]
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