|Ahead of print publication
Micronucleus assay in the exfoliated cells of buccal mucosa of gasoline station workers in Tehran
Fatemeh Shahsavari1, Sina Mikaeli2, Maedeh Ghorbanpour1
1 Department of Oral and Maxillofacial Pathology, Faculty of Dentistry, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
2 Dentist, Private Practice, Ardabil, Iran
|Date of Submission||27-Jun-2020|
|Date of Decision||14-Aug-2020|
|Date of Acceptance||30-Sep-2020|
|Date of Web Publication||24-Jul-2021|
Department of Oral and Maxillofacial Pathology, Faculty of Dentistry, Tehran Medical Sciences, Islamic Azad University, 9th Neystan St., Pasdaran St., Tehran
Source of Support: None, Conflict of Interest: None
Objectives: One of the most critical landmarks of DNA damage is the micronucleus assay. Enumeration of micronuclei contributes to the early diagnosis of precancerous lesions and cancers; however, there are few studies on the frequency of micronucleus in gasoline station workers. To the best of our knowledge, no study has addressed this issue in Iran. The present study aimed to determine the role of working in the gasoline stations of Tehran city on micronucleus frequency in buccal mucosa.
Materials and Methods: In this historical cohort study, buccal mucosa samples were collected from 110 individuals working at gasoline stations and 100 unemployed persons using wet tongue depressors. After Papanicolaou staining, the percentage of cells containing micronucleus as well as the mean number of micronucleus in the micronucleated cells was reported. Student's t-test, Mann–Whitney test, and regression analyses were used to specify the effect of other variables on the frequency and mean number of micronucleus per cell.
Results: The mean frequency of micronucleus in the case and control group was 29.8 ± 8.2 and 9.3 ± 3.2, respectively, which was statistically significant (P = 0.0001). Furthermore, the mean number of micronucleus in the micronucleated cells of buccal mucosa was significantly higher in individuals who were exposed to gasoline than the control group (P = 0.0001).
Conclusion: The results indicated that exposure to gasoline could increase the frequency of micronucleus. It was also revealed that cigarette and hookah smoking and alcohol consumption, together with working in gasoline stations, increase micronucleus abundance, implying the cumulative carcinogenic effect of these factors.
Keywords: Buccal mucosa, DNA damage, gasoline, genotoxicity, micronucleus
| > Introduction|| |
Cancer is one of the most life-threatening diseases, which is usually diagnosed in advanced stages since there is no early diagnostic marker; thus, the survival rates are reduced despite the best possible treatments. Accordingly, the use of noninvasive diagnostic methods in the early stages of cancer development is of essence. One of the events in the cancer development is damage to DNA of a cell and the entry of this defective cell into the cell cycle. Several methods have been proposed to investigate genetic and chromosomal changes caused by exposure to genotoxic drugs, even though a majority of these methods are expensive and usually evaluate the changes after they occurred.
Micronucleus assay is one of the techniques used to measure DNA changes., Micronucleus is a tiny nuclear object containing whole chromosomes or chromosomal fragments. Such small nuclei might originate from the acentric fragments (chromosomal fragments lacking a centromere) or the chromosomes that cannot migrate with other chromosomes during cell division anaphase. When the acentric components lack a centromere, they do not move toward the nucleus of the daughter cells when the cell is being divided and remains in the cytoplasm as micronucleus. Micronucleus is created by exposure to genotoxic agents in some human cells, including erythrocytes, lymphocytes, and buccal mucosa cells. It is one of the most sensitive DNA damage markers, which has been frequently used to examine the pathological role of occupational and environmental factors in human and animal epidemiological studies., The advantages of this technique compared to the other techniques evaluating the DNA damage are its simplicity, noninvasiveness, and low cost, as it can be used in the early stages of cancer and even before the clinical manifestations to examine nuclear changes in different cells, one of which is human oral buccal mucosa. Buccal mucosal epithelial cells provide an appropriate location for presenting the early genotoxic events caused by inhalation, injection, and exposure to carcinogenic agents., The rapid turnover of epithelial cells, which lasts from 7 to 16 days, allows researchers to study the damages to the epithelial basal cells, which are actually the epithelium-generating layer, after a maximum of 16 days when the target cell is in the exfoliation phase.
Gasoline and its vapor are one of the environmental pollutants, whose long-term exposure has many adverse effects on human health. Previous studies conducted around the world have reported that exposure to gasoline vapor can increase the risk of several types of cancers, such as urinary, skin, laryngeal, and pancreatic cancers and leukemia. BTX (Benzene, toluene, xylene) is a carcinogen substance in gasoline. The gasoline vapor contains these three chemicals, with a higher exposure risk being reported for benzene. The chemical formula of gasoline is a complex combination of hydrocarbons (95% of the gasoline vapor consists of alicylic and aliphatic and less than 2% of gasoline vapor is aromatic). Evidently, some of these compounds are genotoxic, mutagenic, and carcinogenic factors.
Few studies have examined the effects of gasoline and the carcinogenic agents in its compounds, including benzene, on the development of oral cancer and precancerous lesions. Similarly, there is limited research on the effect of gasoline on micronucleus abundance in buccal mucosa.,,,,, Given the long-term exposure for the gas station workers to toxic gasoline vapor and with regard to the lack of information on nuclear changes in these individuals, the present study aimed to investigate the frequency of micronucleus and its relevant factors in buccal mucosa of some individuals working at some gasoline stations in Tehran.
| > Materials and Methods|| |
The present historical cohort study was conducted in Tehran in 2018 on 110 persons working in Tehran gasoline stations, who had at least 1 year of work experience, and 100 healthy unexposed individuals. The participants met the inclusion criteria: not being affected by a systemic disease, no drug use, no recent viral infection, no history of radiotherapy and chemotherapy, and nonemployment in chemical-relevant occupations.
To eliminate other mediating factors affecting the micronucleus frequency, the participants were asked about cigarette smoking, alcohol consumption, and hookah smoking. The participants were asked about the alcohol consumption and hookah smoking, regardless of the amount of consumption. On the other hand, in terms of cigarette smoking, daily consumption was also concerned in both groups to match the group members in this regard. In this study, the term “smoker” refers to those who have smoked regularly for the past 6 months. At the beginning of the study, the subjects were informed of the study objectives and sampling method, and their informed consent was obtained. This study was approved by the Ethics Committee of the University under No.: IR.IAU.DENTAL.REC97/033.
Micronucleus sampling and testing method
Before scraping of buccal mucosa cells, the participants were asked to wash their mouths with water carefully. The buccal mucosa cells were scratched by a tongue depressor, and the cells were spread over small, clean glass slides. The smears prepared on the slides were then fixed using a pathofix spray and then allowed to be dry at room temperature. After staining with Papanicolaou, micronucleus counting was performed by an optical microscope, Nikon - YS100, Japan, at ×400 magnification. In each sample, 500 cells in randomly selected fields were counted, and the frequency of micronucleated cells in the specimens was determined. The result was reported as mean frequency in percentages. The mean number of micronuclei per cells containing micronuclei was also evaluated. For micronucleus counting, the researchers considered the cell with specified margins and nuclei, not overlapping with other cells, and the ones with intact nuclei. Dead or degenerated cells and nuclear bubbles were also excluded from counting. Tolbert's et al. criteria, as described below, were used in such a counting process: (a) Micronucleous is smaller than one third of the nucleus diameter but large enough to characterize the shape and color; (b) it has staining intensity similar to that of the nucleus; (c) it has texture and refraction like the nucleus; (d) it should be on the same plane of focus with the nucleus; (e) it has no overlapping or connection with the nucleus; and (f) it has a smooth and distinct perimeter that represents a membrane. [Figure 1] shows the micronuclei in the exfoliated buccal epithelial cells. It should be noted that the samples were counted by two calibrated oral and maxillofacial pathologists who were blind to the specimens, and the average counts were recorded for each case.
|Figure 1: Micronuclei situated adjacent to the nucleus in the exfoliated buccal cells (a. ×200, b. ×400, Papanicolau staining)|
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After counting the samples, the extracted data were imported to the SPSS software version 24 (IBM, Chicago, United States)and the factors were compared in the two groups using Student's t-test, Mann–Whitney test, and regression analyses.
| > Results|| |
This study encompassed 210 participants (110 persons in the case group and 100 persons in the control group). [Table 1] shows the general characteristics of the subjects in the two groups. There was no significant difference between the number of subjects in the case and the control groups (P = 0.87). The mean age of the participants in the control and case groups was 30.11 ± 4.21 and 29.66 ± 5.24, respectively (P = 0.49), indicating no significant difference in this regard as well.
In this study, all the participants were male, so they were homogenous in the both groups in terms of gender.
For the smokers in the control and case groups, the mean frequency of cigarettes consumed per day was 23 and 22, respectively, and there was no statistically significant difference (P = 0.92) between the groups according to the Chi-square test.
The average experience of working in the gasoline stations was 5.85 ± 1.07 years in the participants of the case group, and no statistically significant difference was found in this regard. In other words, the impact of the intervening factor “the cumulative effect of gasoline exposure” was also removed due to such a homogeneity.
The results revealed that the mean frequency of micronucleus (percentage of cells containing micronucleus to 500 cells under study) was significantly higher in those who were working at the gasoline stations (P = 0.0001), compared to the control group. [Table 2] shows the mean frequency of micronucleus in the buccal mucosal cells for different subgroups of the case and control groups.
|Table 2: Mean frequency of micronucleus in exfoliated buccal mucosa cells (percentage of cells containing MN to 500 cells counted)|
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The mean number of micronucleus in cells containing micronucleus was significantly higher in the participants who were exposed to gasoline (P = 0.0001). [Table 3] presents the mean number of micronuclei per micronucleated cell in the case and control groups.
|Table 3: Mean number of micronuclei per micronucleated cells in the study groups|
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According to the regression analysis results: (a) The variable “group” (working in gasoline station) had a significant, predictable, and positive effect on micronucleus frequency (P = 0.000), which means that nonemployment at the gas station causes a decrease in micronucleus as much as 0.392. (b) The type of consuming materials had a significant, predictable, and positive effect on micronucleus frequency (P = 0.000), suggesting that one unit change in type of consuming materials causes 0.119 units of change in micronucleus rate. (c) Age had no significant or predictable effect on micronucleus frequency (P = 0.24).
| > Discussion|| |
Individuals working at the gas station are workers who are particularly exposed to gasoline vapor through breathing when cars are refilling their fuel tanks. Moreover, they are also exposed to materials released from the car engines. Epidemiological studies on these individuals have revealed the adverse genetic effects of exposure to extremely low levels of benzene produced by gasoline vapor. According to the International Agency for Research on Cancer, exposure to gasoline vapor (especially its known compounds such as benzene) is a carcinogenic substance in humans. Numerous studies have also documented that exposure to gasoline steam can increase the risk of developing several types of cancers such as urinary, skin, laryngeal, and pancreatic cancers and leukemia. Grandjean and Andersen also found a direct relationship between cancer and occupational exposure and claimed that lung cancer was one of the leading causes of death among workers exposed to oil derivatives.
Buccal mucosal epithelial cells are prone to reveal the early genotoxic events that are caused by inhalation, injection, and exposure to carcinogenic agents., Cellular studies on the smears from this area also show some evidence of nuclear abnormalities such as binucleated cells (two nuclei in one cell), micronucleus, karyorrhexis (destructive fragmentation of the nucleus), and karyolysis (dissolution of a cell nucleus). Micronuclei are small independent nuclei next to the cell nucleus and contain chromosome fragments or whole chromosomes. Micronucleus is used as one of the important landmarks to study the genetic changes in human erythrocytes, lymphocytes, and buccal mucosa cells. The most common oral malignancy is squamous cell carcinoma (SCC), which actually originates from the epithelial cells. The prevalence of this type of oral cancer in individuals, especially those exposed to particular risk factors, highlights the necessity of investigating nuclear and cellular changes through using noninvasive and cost-effective methods. This can prevent the progression of malignancies at an early stage before their clinical manifestations.
The present study showed that employment at the gas station had a direct effect on the mean frequency of micronucleus (percentage of cells containing micronucleus to 500 cells under study), indicating a significant difference between the two groups in terms of the mean frequency of micronucleus (P = 0.0001). In this regard, this finding was similar to those of other researchers;,,, however, the frequencies in our study are much higher than those in other similar studies, which might be due to the gasoline quality differences in different countries and other variables related to the working conditions and gas station locations. In the present study, there was an effort, as far as possible, to match other intervening factors known in the formation of micronucleus and cell nuclear changes, even though the impact of environmental variables such as airborne particulate matter and ionizing radiations, as well as the other environmental exposures that are related to individuals' geographical residential place, cannot be ignored as these factors may influence the findings. The differences in the sample size and staining techniques used in different studies may also be another reason for such an inconsistency.
In the present study, the mean frequency of micronucleus specified by the Mann–Whitney test was significantly higher in the smokers of the exposed group (31.23 ± 5.91) than control group (9.5 ± 2.33) (P = 0.0001). This finding is consistent with the findings of other researchers;,,, however, the values in the present study are much higher than those in similar studies. This inconsistency might be due to the same aforementioned reasons. The results of this study and other similar studies indicate that smokers would have a much higher micronucleus rate when exposed to gasoline vapor, which may reflect the synergistic effect of cigarette and gasoline on cytogenetic damage to the epithelial cells.
In this study, the mean frequency of micronucleus in nonsmokers was significantly different between the control and exposed groups as it was significantly higher in the exposed group (P = 0.0001). The findings of other researchers also confirm the case.,, The values obtained in the present study were much higher than the previously reported values. This discrepancy may also be justified according to the reasons outlined above.
In this study, the mean frequency of micronucleus in smokers and alcoholics of the two groups was significantly different as it was much higher in the exposed group (35.52 ± 7.02) than in the control group (10.96 ± 2.85) (P = 0.0001). This finding is similar to the findings of Sellappa's et al. study. In their study, the prevalence of micronucleus in smokers and alcoholics was 13.94 ± 0.09 for the case group and 3.64 ± 0.02 for the control group. Although the values reported in the present study were much higher than those estimated by Sellappa et al., the results of the both studies approve the cumulative effect of cigarette, alcohol, and gasoline on cytogenetic damage to the epithelial cells.
Using the Mann–Whitney test in the present study, the mean frequency of micronucleus in the hookah smokers of the exposed group (31.89 ± 7.55) was significantly higher than the control group (11.07 ± 3.31) (P = 0.0001). The researchers found no similar study addressing the same issue in the literature.
Furthermore, this study showed that both cigarette smoking and alcohol consumption, on the one hand, or hookah smoking, on the other hand, had an evident effect on the frequency of micronucleus in the participants who were not exposed to gasoline, indicating the carcinogenic role of these materials. The study also suggested a significant relationship between type of materials and micronucleus rate (P = 0.0001). This means that micronucleus rate was significantly higher in the case and control groups for those who had a history of smoking alone or with alcohol consumption, as well as a history of hookah smoking, in comparison to the nonsmokers (P = 0.0001). The same finding is also achieved in previous studies.,,, In Kurteshi et al.'s study, the frequency of micronucleus in the control group was higher for those who consumed alcohol (12.65 per 1000 cells) than for smokers (6.54 per 1000 cells). These results may indicate the cumulative effect of cigarettes, alcohol, and hookah accompanied with the exposure to gasoline vapor.
On the other hand, there was no significant relationship between micronucleus rate and age (P = 0.24). This result is consistent with Benites et al.' findings and in contrast with Kurteshi et al.'s (P = 0.001) and Butt et al.'s (P < 0.05)., This disagreement may be due to the fact that the participants in Kurteshi et al.'s study were divided into two groups of older and younger individuals. In Butt et al.'s study, the subjects were also divided into two groups of <25 years and >25 years. This shows that age was considered as a variable in these two studies, whereas the two groups were homogenous in the present study with respect to the mean age to eliminate the effect of this intervening factor.
In this study, the mean number of micronucleus in cells containing micronucleus was also investigated, and the results revealed that it was significantly higher in subjects exposed to gasoline (173.68 ± 42.38) than the control group (132.83 ± 27.27) (P = 0.0001). According to our review of available scientific sources, studies on the micronucleus in gasoline-exposed individuals have only focused on the frequency of this nuclear change in the buccal mucosa,,,,,, and the mean number of micronucleus in the cells containing micronucleus has not been evaluated in previous research. Hence, our study is probably the first case in this regard, so the results of this study could provide the grounds for future research in this field. Furthermore, the high frequency of micronucleus in gas station workers is a warning so that more attention should be paid to the quality of gasoline and the need to purchase gasoline that meets European standards. In addition, the high frequency of micronucleus in control individuals is another warning which is even more serious; hence, the other factors in this area should be identified and eliminated to prevent their harmful consequences. Further studies are recommended to compare micronucleus frequency in gasoline station workers and oral cancer patients.
| > Conclusion|| |
The results indicated that exposure to gasoline could increase the frequency of micronucleus. It was also revealed that cigarette and hookah smoking and alcohol consumption together with working in gasoline stations increase micronucleus abundance, implying the cumulative carcinogenic effect of these factors.
The authors would like to appreciate the efforts made by staffs at Razi Lab, Rasht, Iran.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]