Cytogenetic biomonitoring in petrol station attendants: A micronucleus study.

BACKGROUND: Benzene, which is a major organic product, on chronic exposure can result in many malignant disorders, and therefore exposure to gasoline vapors is classified by the International Agency for Research of Cancer as possible carcinogenic to humans. Petrol station attendants are chronically exposed to petroleum derivatives through inhalation of petrol during vehicle refuelling. AIM: This study is aimed to investigate cytogenotoxic damage in exfoliated buccal cells obtained from petrol station workers and control subjects using micronucleus (MN) test.
MATERIALS AND METHODS: This study was carried out on 30 petrol station attendants working at different petrol stations located in Indore. The control group consisted of 30 healthy subjects who were not exposed to benzene. Buccal cell samples were collected at the end of the work shift. Slides were stained and were evaluated to determine the MN frequencies. Exposure monitoring was performed by the detection of phenol excreted in the urine. Urinary phenol measurements were performed following the colorimetric quantitative determination method of Yamaguchi and Hayashi.
RESULTS: Variations in MN frequencies were seen in control and petrol bunk attendants.
CONCLUSION: The MN test in exfoliated epithelial cells seems to be a useful biomarker of occupational exposure to genotoxic chemicals. Phenol is the principal metabolite of benzene. Therefore, phenol concentration in the urine of exposed workers can be used as a biomarker of external exposure.

Introduction

Occupational exposure to benzene in humans has been found to be increasingly associated with acute myeloid leukemia and non-Hodgkin's lymphomas.[1]

Among the individuals occupationally exposed to such mutagenic agents, petrochemical workers and gas station operators are considered particularly because they have to manipulate the fuel and consequently inhale fuel vapors during daily work.[2]

Micronucleus (MN) assay can be applied to measure DNA damage in such human populations. MN are cytoplasmic chromatin masses with the appearance of small nuclei that arise from chromosome fragments or intact whole chromosomes lagging behind in the anaphase stage of cell division. The MN test has been applied for biological monitoring of human populations exposed to mutagenic and carcinogenic agents.[3]

Many studies have been carried out to determine the mutagenic and carcinogenic effects of tobacco since a long time. This study intends to quantify MN in individuals of control group, with no tobacco habit and no pre-existing lesions, and petrol bunk workers with Papanicolaou (Pap) and acridine orange stains and to evaluate its efficacy as a genotoxic biomarker. Another aspect of this study is to evaluate the urinary phenol levels in control group and petrol bunk workers.

Materials and Methods

The study sample consisted of 60 individuals broadly classified into two groups. The control group consisted of 30 individuals in the age group of 20–65, without any clinically observable lesions and without any tobacco (chewing and smoking) habits. The petrol bunk workers group consisted of 30 individuals in age group of 20–65 who were randomly selected from different petrol stations in and around Indore, India. Two smears were obtained from each subject because two staining techniques were used. One smear was immediately stained with acridine orange stain for MN evaluation. The second smear was stained with Pap and evaluated for MN.

Evaluation of micronucleus

The slides were separately evaluated for the presence of MN in acridine orange using fluorescent microscope and Pap-stained slides under light microscope. About 100 cells were counted in acridine orange-stained slides and 100 cells counted in the Pap - stained slides. Scoring criteria for MN according to Tolbert et al.[4] were followed in this study [Figure 1].

Figure 1

Photomicrograph showing micronuclei (a: Acridine orange stain, ×400; b: Pap, ×400)

Urinary phenol estimation

Another aspect of our study was the biochemical analysis in control group and petrol bunk workers. Urinary phenol levels were measured by Yamaguchi and Hayashi method[5] to evaluate the exposure levels between control group and petrol bunk workers. Urine samples from petrol bunk workers were collected at the end of their 8-hour shift.

Results

The mean MN was calculated for each group irrespective of stains. For the control group, the range of MN was 0–20. The mean calculated was 5.02 with a SD of 4.77. For the petrol bunk workers, the range was 0–18. The mean calculated was 6.82 with a SD of 4.77 [Figure 2]. The range of MN in control group when stained with acridine orange stain was 0–6. The mean calculated was 2.40 with a SD of 1.40. The range of MN in control group when stained with Pap stain was 1–20. The mean calculated was 7.63 with a SD of 5.49. The range of MN in petrol bunk workers when stained with acridine orange was 0–13. The mean calculated was 3.83 with a SD of 3.07. The range of MN in petrol bunk workers when stained with Pap was 1–18. The mean calculated was 9.80 with a SD of 4.29 [Figure 3].

Figure 2

Mean micronuclei recorded in groups irrespective of stains used

Figure 3

Mean micronuclei recorded using two stains irrespective of groups

Urinary phenol analysis

In the control group, mean urinary phenol was calculated as 8.40 ± 3.13. In the tobacco users group, the mean urinary phenol calculated was 9.621 ± 2.52 mg/L. In the petrol bunk workers, the mean urinary phenol was 12.2 ± 5.06 mg/L. The range in control group for urinary phenol was 3.57–15.9 mg/L. The range in petrol bunk workers for urinary phenol was 6.0–31.94 mg/L [Figure 4].

Figure 4

Mean urinary phenol levels recorded in two groups

Discussion

Genomic damage is probably the most important fundamental cause of developmental and degenerative disease. It is also well established that genomic damage is produced by environmental exposure to genotoxins, medical procedures (e.g., radiation and chemicals), micronutrient deficiency (e.g., folate), lifestyle factors (e.g., alcohol, smoking, drugs, and stress), and genetic factors such as inherited defects in DNA metabolism and/or repair. It is essential to have reliable and relevant minimally invasive biomarkers to improve the implementation of biomonitoring, diagnostics, and treatment of diseases caused by, or associated with, genetic damage. The MN assay in exfoliated buccal cells is potentially an excellent candidate to serve as such a biomarker.[6-24]

The analysis of MN has gained increasing popularity as an in vitro genotoxicity test and a biomarker assay for human genotoxic exposure and effect.[25] The MN assay can be performed in buccal and other exfoliated cells originating from rapidly dividing epithelial tissue without the need for ex vivo nuclear division, so that the cell cultures required for cytogenetic assays based on analysis of metaphase chromosomes, such as chromosome aberrations and sister chromatid exchanges, are not needed.[26]

The collection of buccal cells has been stated as the least invasive method available for measuring DNA damage in humans, especially in comparison to obtaining blood samples for lymphocyte and erythrocyte assays, or tissue biopsies. The buccal cell MN assay was first proposed in 1983 and continues to gain popularity as a biomarker of genetic damage in numerous applications.[27] Most of the literature that has been published focus primarily on either broad evaluation of noninvasive methods for biomonitoring, the associations of MN or other markers with cancer in various types of exfoliated cells, or are limited to effects of a specific type of exposure, such as smoking and smokeless tobacco or formaldehyde.[28]

In this study, the mean MN value in petrol bunk workers was 9.80 ± 4.29 when stained with Pap and in tobacco users who were nonexposed, the mean MN was 8.57 ± 4.75 and was consistent with the findings of Rajkokila et al.[29] Another study conducted by Benites et al.[30] using Feulgen reaction on gas station workers showed higher MN in exposed group (12.07 ± 4.9) when compared with control group (2.53 ± 2.05). In our study, the mean calculated was 3.83 ± 3.07 in the petrol bunk workers group and was higher when compared with the control group 2.40 ± 1.40.

Our findings are also in agreement with those of HoÈgstedt et al.,[31] who detected a significant increase in the frequency of MN in the lymphocytes of petrol station workers. An increased level of chromosomal deletions in Brazilian station workers was also shown by Santos-Mello and Cavalcante.[32] Oesch et al.[33] detected an increase in DNA strand breaks in mononuclear blood cells of nonsmoker petrol station attendants. Similarly, Andreoli et al.[34] also found that DNA damage in peripheral lymphocytes of petrol station workers increased, using the single-cell gel electrophoresis method.

The turnover rate for the appearance of MN in exfoliated buccal cells in an otherwise normal cell after exposure to an acute genotoxic event, such as ionizing radiation, is estimated to be a minimum of 5–7 days. Interindividual variation during this time course of MN expression, however, has been observed so that peak expression of MN may be delayed up to 21 days.[8935]

Multiple sample times are required to identify optimal timing between 7 and 21 days after exposure because peak expression may vary depending on the effects of the particular DNA damage or chromosomal exposure on basal cell turnover rate. It is possible that certain genotoxic exposures could cause an inhibition or an enhancement of the basal cell proliferation and thus confound the kinetics of MN expression.[26]

Our results reveal that petrol station workers could be under risk of significant cytogenetic damage. The MN test in exfoliated epithelial cells seems to be a useful biomarker of occupational exposure to genotoxic chemicals.

Our study also emphasizes on the fact that DNA-specific stain is more sensitive in evaluating the MN frequencies when compared with DNA nonspecific stain.

Some nuclear anomalies (karyorrhexis, karyolysis, binucleates, and condensed chromatin) with nonspecific stains are sometimes difficult to interpret and may be misclassified as MN. Another possible confounding factor in MN studies is the formation of keratin granules that are found in degenerated cells with nuclear anomalies. These round cytoplasmic bodies, which are formed as a consequence of cell injury, do not contain DNA but may be classified as MN with nonspecific stains.[26]

Phenol is the principal metabolite of benzene. Therefore, phenol concentration in the urine of exposed workers can be used as a biomarker of external exposure. Biological monitoring of petrol station attendants showed substantially higher levels of urinary phenol when workers were compared with subjects with no known exposure to either gasoline or benzene.[36-38] On the other hand, it should also be emphasized that petrol station attendants are not only exposed to hydrocarbons present in petrol vapors but also to the emissions produced by engines during fuel combustion. It was shown that these emissions may also cause cytotoxic and genotoxic effects.[39]

Khoschsorur and Peter[40] reported that urinary phenol concentration in petrol station workers (17.28 mg/g/L creatinine) was higher than in control subjects (4.4 ± 6.6 mg/g/L creatinine). In this study, they also found significantly higher urinary phenol levels in petrol station workers (19.85 mg/g/L creatinine) than in control subjects (7.25 mg/g/L creatinine). Our results are in accordance with the above mentioned studies; urinary phenol in petrol station attendants was found to be increased (12.208 ± 5.061) when compared with control subjects (8.40107 ± 3.13467). However, benzene exposure of petrol station attendants can vary widely due to several factors, such as quantity of fuel pumped, type and number of vehicles filled, protective measures, and total content of benzene in the petroleum. According to the data obtained from the petrol stations around the world, each worker pumps an average 2000 L of petroleum, containing 5% (w/v) benzene, during their 8-h work shift. Therefore, these factors must be taken into account before the petrol station attendants are subjected to biomonitoring in order to evaluate the exact amount of exposure they are subjected to.

Conclusion

This study supports the use of MN as a biomarker for neoplastic progression but only when DNA-specific stains are used. MN can also be used as a biomarker for genotoxic studies for screening huge populations. The use of biochemical analysis along with MN evaluation highlights the exposure of the population to genotoxins and adds to the efficiency of our study.