Genotoxicity in Oral Mucosal Epithelial Cells of Petrol Station Attendants: A Micronucleus Study.

INTRODUCTION: Occupational exposure to petrol derivatives possesses an increased risk of various cancers including that of the oral mucosa. Scientific studies have shown the correlation of micronuclei assay (MN) with the cytogenotoxic changes in petrol station attendants. However, very few have reported the use of MN assay as a promising tool for assessing the impact of smoking in these workers. AIM: To explore the cytogenotoxic damage in exfoliated buccal cells obtained from petrol station attendants and control subjects using the MN assay along with additional effects due to smoking.
MATERIALS AND METHODS: The study comprised 60 males who were divided into Group I-IV with each having 15 subjects. These subjects were categorized as exposed smokers, exposed nonsmokers, unexposed smoker group, and unexposed nonsmokers. The MN and additional nuclear abnormalities (karyorrhexis [KH], binucleation [BN], pyknosis [P], and karyolysis [KL]) were calculated in PAP-stained slides.
RESULTS: Statistically higher mean frequencies of overall nuclear anomalies were observed in petrol pump workers in comparison with the control group. Petrol pump smokers carry the highest nuclear anomalies followed by non-exposed smokers than exposed non-smokers and the count was the least among unexposed non-smoker workers. DISCUSSION AND
CONCLUSION: The present study indicated that the petrol pump workers are under higher cytogenotoxic damage. Also, smoking added to the frequency of damage. Thus, MN and other nuclear anomalies are in-vitro reliable biomarker assays available and should be routinely employed as a screening tool in their periodic medical evaluation. Copyright:

INTRODUCTION

Petrol is a complex combination of hydrocarbons; about 95% of compounds in petrol vapor are aliphatic and alicylic compounds and less than 2% are aromatics. Aromatic compounds of petrol are predominantly benzene, toluene, and xylene (BTX). Benzene is the most hazardous component from a toxicological view and has been classified as a human carcinogen by the International Agency for Research and Cancer (IARC, 1989).[1]

Petrochemical workers absorb these petrol fumes either during refueling of vehicles or fuel manipulation; additionally, they inhale volatile vapors emitted by engines during refilling. Thus, they are more prone to cytogenotoxic damage. Occupational exposure to such derivatives possesses an increased risk of cancer of various organs such as the urinary tract, skin, larynx, pancreas, and hematopoietic system. Furthermore, exposure, either by oral ingestion or nasal inhalation of these compounds, has proven carcinogenic effects over the oral mucosa.[2]

Studies have demonstrated increased cytogenetic damage in peripheral blood lymphocytes of petrol pump workers using different genetic endpoints such as sister chromatid exchange (SCE), DNA strand breaks, and micronuclei (MN). In addition, certain other alterations in the morphology of the nucleus including karyorrhexis (KH), karyolysis (KL), pyknosis (P), and binucleus (BN) are also evidence of cytogenetic damage.[3]

MN are cytoplasmic chromatic masses with the appearance of small nuclei that arise from chromosome fragments or intact whole chromosome lagging in the anaphase stage of cell division.[4] MN in exfoliated buccal epithelial cells reflects genotoxic events that occurred in the dividing basal layer. Because more than 90% of all human cancers are of epithelial origin, MN assay is the most suitable biomonitoring approach.[5]

Scientific studies have proven MN as a promising tool for the study of genotoxic and cytogenetic changes in petrol station attendants. A few investigations have reported that cigarette smoking significantly increases the frequencies of micronucleus and other nuclear abnormalities in petrol pump attendants.[6]

In the present study, the assessment of cytogenotoxic damage in oral mucosal epithelial cells of petrol station attendants using the MN assay and other qualitative nuclear changes has been studied based on four-fold objectives. First, we evaluated the micronuclei count in petrol filling station attendants. Second, we evaluated the cytogenetic changes, such as nuclear abnormalities—KH, KL, P, and BN in petrol filling station attendants. Third, we determined the additional effects of smoking in oral mucosal epithelial cells in petrol filling station attendants. Finally, to compare micronuclei count and other nuclear abnormalities in exposed and unexposed groups.

MATERIALS AND METHODS

This prospective study was conducted from July 2019 to December 2019. The study was approved by the Institutional Ethics Committee and all participants were informed about the objectives of the study (Ref No. SRCDSR/ACAD/2018/9313).

A total number of 60 male subjects in the age range of 25 to 45 years were categorized into four groups -[Table 1].

Table 1

Characteristics of groups in the present study

Group	Type of group	Group	Sample size	Source of sample	Age years(mean±SD)	Smoking duration years (mean±SD)	Petrol exposure duration years(mean±SD)
Group I	Exposed	Petrol pump worker smoker	15	Petrol filling station, Delhi	35.67±7.761	8.13±2.20	6.53±2.696
Group II	Exposed	Petrol pump workernon-smoker	15	Petrol filling station, Delhi	32.20±7.664	-	6.27±3.03 **
Group III	Unexposed	Non-workers smokers	15	Routine OPD of college	32.13±7.150	7.40±4.102 **	-
Group IV	Unexposed	Non-workers and non-smokers	15	Routine OPD of college	29.40±4.256 **	-	-

**Non-significant.

Inclusion Criteria:

Petrol pump workers involved in fuelling and working for more than 8 hours for more than 1-year duration for Groups I and II.

Subjects with a smoking history of more than 5 years with 1 packet per day for petrol pump workers (Group I) and healthy individuals (Group III).

Healthy individuals with no smoking habit (Group IV).

Exclusion criteria

Subjects with any oral or systemic problem including any acute illness/endocrine diseases/known immunological diseases for past 1 year/history of viral illness (apart from malaria) or recent vaccination or any medication in past 3 to 4 months.

Subjects with a history of exposure to radiation therapy and exposure to X-rays in the past 6 months/previous work like painting, any other chemical industry, and exposure to petrol fumes and other potential genotoxic substances or with any deleterious habit except tobacco smoking.

Smear preparation and evaluation

In exposed group subjects, samples were collected at the end of the 8-hour work shift and in the unexposed group during their routine dental visit preferably between 10 and 12 AM. Two smears were collected, fixed, and stained using papanicolaou (PAP) stain using the standard protocol and evaluated under the microscope at 40×. Two independent observers did evaluation at different time intervals using the same microscope to eliminate the subjective bias. The intraobserver data were also read at different time intervals to maintain the subjectivity. The criteria given by Tolbert et al (1992)[7] were followed during the counting of micronuclei and other nuclear abnormalities. About 1,000 cells were selected by systematic sampling moving the microscope stage from left to right and then right to left in the “Z” pattern to avoid repetition of cells from different fields and the results were tabulated.

Statistical analysis

The mean ± standard deviation (SD) and percentages were calculated from the results obtained. Non-parametric Mann–Whitney U test was used to compare the two means. Interaction between smoking and exposure was tested with a two-way analysis of variance. The statistical analysis was done using SPSS 21.0 version (Chicago, Inc., USA). A P value of less than 0.05 was considered significant.

RESULTS AND OBSERVATIONS

The demographic characteristics of the groups studied are summarized in Table 1. None of the groups studied showed significant differences in age, duration of smoking habit, and petrol fume exposure [Table 1].

When comparison was carried out between the exposed group (Groups I and II) and the unexposed group (Groups III and IV), a statistically higher difference was obtained in the MN count and overall other nuclear abnormalities in the former group. However, as an individual parameter, a significant result was evident in MN, KH, and BN; however, KL and P failed to reveal a statistical difference [Table 2].

Table 2

Mean MN count and other nuclear abnormalities (KR, KL, P, BN) in t exposed and unexposed groups with their comparisons

Groups	Subjects	Mean±S.D.
		MN Count	KR	KL	P	BN	Total other NA
Exposed (GP-I+GP-II)	30	6.83±1.87	16.73±3.92	19.00±7.23	1.00±0.78	0.97±1.06	37.67±9.36
Unexposed (GP III+GP-IV)	30	3.03±2.28	12.63±4.37	17.27±7.62	0.87±0.81	0.33±0.66	31.10±12.36
P		<0.0001*	0.0001*	0.370**	0.523**	0.008*	0.024*

*Significant and **non-significant

Among all the four groups of all parameters, mean values of MN, KL, and BN were the highest in the exposed smoker's group (Group I) [Figures 1–3]. The frequency of KH and P was the highest in the exposed smoker group (Group II) and unexposed smoker group (Group III), respectively [Table 3 and Figure 4 and 5].

Figure 1

Photomicrograph of a cytological smear from the buccal mucosa showing micronuclei in a study group (PAP staining, 40×)

Figure 3

Photomicrograph of a cytological smear from the buccal mucosa showing binucleation in a study group (PAP staining, 40×)

Table 3

Frequencies of micronuclei and other nuclear abnormalities in all groups

Group	MN (M±SD)	KR (M±SD)	KL (M±S.D)	P (M±S.D)	BN (M±SD)
Group I	7.27±2.187	15.60±2.746	23.73±6.181	0.93±0.704	1.67±0.976
Group II	6.40±1.454	17.87±4.642	14.27±4.698	1.07±0.884	0.27±0.594
Group III	5.07±1.280	15.27±3.348	23.20±4.828	1.53±0.516	0.60±0.828
Group IV	1.00±0.535	10.00±3.684	11.33±4.639	0.20±0.414	0.07±0.258

Figure 4

Photomicrograph of a cytological smear from the buccal mucosa showing karryohexis in a study group (PAP staining, 40×)

Figure 5

Photomicrograph of a cytological smear from the buccal mucosa showing pyknosis in a study group (PAP staining, 40×)

Photomicrograph of a cytological smear from the buccal mucosa showing karryolysis in a study group (PAP staining, 40×)

The intragroup comparison revealed that in the unexposed smoker group (Group III), a higher count of both in total and as an individual parameter when compared with an unexposed nonsmoker group (Group IV). Between the exposed smoker's group (Group I) and the exposed non-smoker group (Group II), a statistically significant difference was obtained in total and as an individual parameter in karyolysis and binucelation [Table 4].

Table 4

Intergroup and intragroup comparisons of MN and other nuclear abnormalities

Comparison	Group	MN	KR	KL	P
Intergroup	I vs. III	0.002	0.768	0.794	0.013 (S)
	II vs. IV	<0.0001	<0.001	0.096	0.002 (S)
Intragroup	I vs. II	0.229	0.115	<0.0001	0.651 (NS)
	III vs. IV	<0.0001	<0.0001	<0.0001	<0.0001 (S)

Intergroup comparison results between the exposed smoker (Group I) and unexposed smoker group (Group III) failed to show an overall significant difference. However, the frequencies of MN, binucleation and pyknosis had a statistically significant difference. On comparison between the exposed non-smoker group (Group II) and unexposed non-smoker group (Group IV), there was a statistically significant difference overall and as an individual parameter except in the frequencies of karyolysis and binucleation [Table 4].

DISCUSSION

According to World Health Organization (WHO), the global cancer burden has risen from 14.1 to 19.3 million new cases and 10.0 million compared with 7.6 million deaths from 2012 to 2020.[8] Various environmental factors and occupational exposure have been associated with cancer as they possess a genotoxic risk. According to the IARC (1989), diesel and gasoline engine exhausts contain a variety of mutagenic and carcinogenic agents.[1] Benzene and toluene are major hydrocarbons present in them. Hughes et al.[9] have concluded that even cigarette smoking is one of the important indoor sources of benzene.

The collection of exfoliated buccal cells is arguably the least invasive method for estimating DNA damage in humans as compared to blood samples or biopsy. Additionally, buccal cells are the first barrier for inhalation or ingestion routes and are capable of metabolizing proximate carcinogens to reactive products. Approximately 90% of human cancers are epithelial in origin. Therefore, exfoliated buccal cells can be considered as a preferred target site for early genotoxic events induced by such carcinogens.[10]

In India, petrol pump workers are exposed to these carcinogens not only through inhalation but also by dermal contact during the fueling and handling process.[11] Thus, taking into account such potential risks involved, the present study was aimed to investigate the genotoxic damage, monitor cytotoxic effects, other nuclear abnormalities in petrol pump workers using the MN assay. The additional toxic effects of smoking were also assessed in petrol pump workers and healthy individuals.

In the present study, a significantly higher MN frequency was noted in petrol pump workers (exposed group) as compared with the healthy group (control group) and these findings were in accordance with previous studies.[3611121314] Also, these were within the baseline range of 0.5 to 11.5 MN per 1,000 cells as stated by Holland et al.[10] The present study and similar other studies reported an increased MN frequency in the exposed group than in the control group and suggested petrol pump workers are at a greater risk of cytogenetic damage.[34612131415161718]

According to Gupta et al.,[19] the origin of MN predominantly is through two mechanisms. The first one is by chromosomal breakage; either whole or fragment–known as clastogenic effect and the second one is aneugenic effect, wherein there is dysfunction of the spindle apparatus. Thus, MN assay is extensively acknowledged as an endpoint for DNA damage estimation. Because buccal exfoliated cells have little less DNA repair capacity as compared to peripheral blood lymphocytes, they can accurately reflect the genetic instability.

Numerous factors, such as age, gender, genotype, oral hygiene, deleterious habit (such as alcohol and smoking), lifestyle, dental health, and occupational exposure can affect the MN frequency. Among petrol pump workers, the MN frequency is influenced by other factors such as the volume of petrol sold, duration of exposure, the average concentration of benzene, toluene, total volatile organic compounds (VOCs), and DNA repair capacity of an individual. Moreover, the MN assay can also alter due to technical reasons such as the timing of cell collection, fixation, staining techniques, number of cells counted, scoring criteria, and other nuclear abnormalities in normal or degenerative cells.[13]

Generally, the age of an individual affects the cell turnover rate and consequently, DNA damage, and repair rate.[13] Thus, to avoid bias, a narrow age range of 25 to 45 years with a mean of 32.35 years was included. Also, only males were included as participants in the study for the homogenization of samples. Besides, in the present study, individuals with more than 5 years of habit history were involved as a participant. Besides, individuals with only a habit history of more than 5 years were involved in the present study as participants as Rajkokila et al.[3] have reported that chronic smokers have a higher degree of nuclear anomalies.

With regard to exposure duration to petrol fumes in the study group, both smokers and nonsmokers had an average range above 6 years of exposure time. According to the toxicological principle of dose–response relationship, the response to a given substance increases as the dose and duration of exposure to the substance increases. Umegbolu et al.[13] stated that more than 2 years' exposure duration could be attributed to greater genotoxic insult. At the beginning of the insult, the body's compensatory mechanisms try to battle with the genotoxins until 2 years, after which they begin to fail as there is a greater accumulation of genotoxins.

Tolbert et al.[7] recommended that at least 1,000 cells should be observed and they have even mentioned the identifying criteria for micronuclei and additional nuclear abnormalities that have to be strictly followed. Another variable factor among the laboratories may lie in the stain employed. The review on MN assays of Holland et al.[10] validated the PAP stain over the Giemsa stain. The reason mentioned by them was that in Giemsa-stained slides, over-analysis was noted due to the inclusion of keratohyalin granules and bacteria leading to false-positive results. Thus, in the present study, PAP-stained slides were analyzed for 1,000 cells using Tolbert et al.[7] criteria.

To investigate cytotoxic effects, other nuclear abnormalities were also evaluated. The exposed group revealed a statistically higher frequency of other nuclear abnormalities in comparison with the non-exposed group. Similar evaluations were noted by a few other investigators also.[261112]

As comprehensively explicated by Tolbert et al.[7] in 1992, both KH and P can be due to apoptosis/necrosis/keratinization of cells. KL is usually a product of cellular necrosis or keratinization thus an indicator of cytotoxicity, which is needed for cancer promotion. BN is considered to be a genotoxicity indicator, a prerequisite for cancer initiation. KH and P are suggestive of genocytotoxicity. Taken as a whole, these results suggest the concept that petroleum is a cytotoxic agent.

It has been postulated by Martins et al.[20] that the repeated exposure to cytotoxic agents results in chronic cellular injury, compensatory cell proliferation, hyperplasia, and ultimately tumor development. A positive correlation between cell proliferation and cancer induction has also been assumed as suggested by Sugano et al.[21] Probably, proliferation increases the risk of mutations within target cells and is also important in selective expansion of (exogenously or endogenously) initiated cells from preneoplastic foci to as far as malignant tumors.[20]

Smoking is an additional factor that is genotoxic to the Nuclear alteration in both control and exposed groups.[20] For this reason, smokers and non-smokers were comparatively investigated here, and in the present study, the higher frequencies were noted in the smoker's group of both exposed and control subjects, which were similar to those reported by previous studies.[3614] It is already an established fact that genotoxic and carcinogenic chemicals released from tobacco can lead to DNA damage and thus higher MN indexes are noted in smoker individuals as compared with nonsmoker healthy individuals.

In addition, in vitro studies on human gingival fibroblasts with exposure to nicotine had seen a statistically significant increase in the micronucleus frequency. However, it is important to keep in mind that in vitro studies do not consider the complex in vivo situation and thus should be interpreted cautiously.[20]

On the basis of current understanding, it is hypothesized that smoking exerts a negative influence on oral mucosa cells by killing them. The main reason for such an assumption is that some tobacco products affect the apoptosis process induced by various stimuli, including ultraviolet light and chemotherapeutic agents in cancer cell lines. However, some authors have argued that nicotine is able to prevent apoptosis in human gingival fibroblasts in vitro and in vivo.[22] This could be the reason why smokers did not present distinct values for cytotoxicity parameters when compared to non-smokers in the exposed group in the present study.

The impact of petrol fumes was analyzed by intergroup comparison between exposed nonsmokers (Group II) and control nonsmokers (Group IV). The significant difference noted was indicative that tobacco with gasoline exposure causes an increase in the rate of frequencies as observed by other researchers due to cytogenetic damage in petrol pump smokers.[615]

The MN can appear in exfoliated buccal cells of healthy individuals after acute exposure to a genotoxic agent within 5 to 7 days or after a delay of up to 21 days. Thus, it can be speculated that exposure to certain genotoxic agents can either promote or inhibit basal cell proliferation and thus confound MN expression.[23] The genomic changes that occur at the onset of exposure to these agents further progress to cytotoxic changes because these mutagenic agents stimulate cellular death to eliminate the genotoxic damage cells.[24]

As stated by Vasquez (2010), cytotoxicity is a confounding factor for mutagenesis, and cytotoxicity is inversely related to genotoxicity.[25]

Thus, the precise association between smoking and petrol needs further elucidation, and not only the MN index but also indices metanuclear changes should be used for complete cytogenotoxicity elucidation in petrol pump workers.

In view of the findings of the study, we strongly recommend the following points:

If possible, a self-service petrol station can be established to avoid unnecessary contact with petrol pump attendants.

Or else provide PPE, face mask shields, gloves, etc., for workers at petrol pump during their shifts.

The use of MN assay should be encouraged for the screening of individuals prior to their employment.

It is necessary to educate petrol pump workers about the hazardous and genotoxic effects not only of petrol fumes but also their age and deleterious habits (alcohol and tobacco usage).

Periodic assessment of their medical health and MN assay should be carried out to assess the risk of cancer development from petrol fumes and/or their habits.

The MN frequencies of petrol pump attendants are more than 1-3 Micronuclei per 1000 cells. They may look for an alternate occupation to prevent further risk of cancer development due to petrol fumes.

Occupations such as firefighting, amalgam filling, shoemaking, and cement factory, also should be avoided by these individuals.

Mass screening is necessary to educate the working population about the genotoxic effect of petrol exposure and ensure a safe and healthy working atmosphere for petrol station workers to alleviate health hazards.

It is important to have necessary guidelines formed by government and private stakeholders to safeguard the health of these petrol pump workers.

CONCLUSION

The current study establishes the fact regarding higher nuclear anomalies in petrol station attendants than in the unexposed group due to genotoxic fumes of petroleum. For these reasons, and with the present study observations, we strongly recommend pre-employment screening for workers at petrol pumps and bio-monitoring of DNA damage at a determined interval using these assays from buccal exfoliated cells of these workers. Nevertheless, future studies, particularly long-term prospective clinical trials, are needed to determine the amount of distortion tolerable biologically and mechanically.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.