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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 9  |  Issue : 4  |  Page : 153-157

Micronuclei assay in oral exfoliated buccal cells of pesticides exposed agricultural labourers in Pondicherry population


1 Department of Oral Pathology and Microbiology, Indira Gandhi Institute of Dental Sciences, Sri Balaji Vidyapeeth University, Puducherry, India
2 Department of Anatomy, Mahatma Gandhi Medical College and Research Institute of Dental Sciences, Sri Balaji Vidyapeeth University, Puducherry, India

Date of Web Publication18-Dec-2018

Correspondence Address:
A Aroumougam
Department of Oral Pathology and Microbiology, Indira Gandhi Institute of Dental Sciences, Sri Balaji Vidhyapeeth University, Puducherry
India
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DOI: 10.4103/srmjrds.srmjrds_74_17

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  Abstract 

Background: Occupational exposure is caused by the chemical used in pesticides that are toxic to humans and accounts for significant morbidity and mortality, especially in the developing countries. The biologically active compound present in pesticides is with various degrees of toxicity that leads to DNA damage, which, when left unattended, could lead to the process of carcinogenesis. Therefore, it is important to focus on various biometric procedures in order to evaluate the risk associated with occupational health-related issues. One among the biomonitoring test employed, the micronuclei (Mn) assay on the buccal mucosal cells is used as a part of screening in order to assess the cytotoxic status. This assay has been substantiated in the literature for the last few decades. Aims: To assess the cytotoxic damage in agricultural laborers exposed to pesticides using Mn as a biomarker. Subjects and Methods: The study involved data and sample collections from agricultural laborers (Non exposed and exposed to pesticides) by buccal smears, Papanicolaou staining and comparison of number of Mn scores as per Tolbert's and Thomas criteria, respectively. The parameters before and after exposure were analyzed by paired t-tests and partial correlation. Results: The observation of the study revealed the frequency of Mn was high in exposed individuals irrespective of age and habits when compared to that of the control group. Conclusions: In this study, as the duration of exposure increased, there was an increase in the Mn count. This indicates that there was considerable cytotoxic damage.

Keywords: Buccal cells, cytotoxicity, DNA damage, genotoxicity, micronucleus, pesticides, smokers


How to cite this article:
Aroumougam A, Devy A S, Uma A N, Vezhavendhan N, Vidyalakshmi S, Premlal K R. Micronuclei assay in oral exfoliated buccal cells of pesticides exposed agricultural labourers in Pondicherry population. SRM J Res Dent Sci 2018;9:153-7

How to cite this URL:
Aroumougam A, Devy A S, Uma A N, Vezhavendhan N, Vidyalakshmi S, Premlal K R. Micronuclei assay in oral exfoliated buccal cells of pesticides exposed agricultural labourers in Pondicherry population. SRM J Res Dent Sci [serial online] 2018 [cited 2019 Oct 22];9:153-7. Available from: http://www.srmjrds.in/text.asp?2018/9/4/153/247846


  Introduction Top


A cell is a basic unit of tissue that is organized by a combined interaction of nucleus and cytoplasm which performs the normal physiological functions and maintain the normal homeostasis by undergoing a process of cell division by DNA replication and cell growth in a much Co-ordinated way and controlled by a sequence termed as the cell cycle. These cells are sensitive and reactive when exposed to genotoxic agents such as environmental factors, physical injury, chemical injuries, radiation and genetic factors, and occupational hazards.

Occupational health deals with the health and safety of the individuals in their respective workplace. There are several risk factors that are encountered ranging from accidents, stress-related factors, communicable and noncommunicable diseases to cancer in one's working destination. Such kinds of insults result with different diversities among the individuals and the response to such exogenous exposure results based on the variety of exposure, the rate of metabolites, DNA repair process, and other factors.[1]

One among the occupational exposure is caused by the chemical used in pesticides. They are toxic to humans and accounts for significant morbidity and mortality, especially in the developing countries. The biologically active compound present in the chemicals used in pesticides is with various degrees of toxicity that leads to DNA damage, which when left unattended could lead to the process of carcinogenesis.[2] Therefore, it is important to focus on various biometric procedures in order to evaluate the risk associated with occupational health related issues. One among the bio-monitoring test employed, the micronuclei (Mn) assay on the buccal mucosal cells is used as a part of screening in order to assess the cytotoxic status. This assay has been substantiated in the literature for the last few decades.[3]

Mn are round-to-oval shaped small nuclei that occur whenever a chromosome or a fragment of a chromosome is not incorporated into one of the daughter nuclei during cell division induced by the genotoxic agent.[4] Taking this into account, the study is structured to analyze the cytogenetic damage by Mn assay in farmers exposed to pesticides in Puducherry population. There have been various endeavors to enhance the assessment of exfoliative cytology by extra procedures like an evaluation of thiols, histomorphometry, measurement of atomic DNA, and distinguishing proof of tumor markers such as CK-16, 19, p-53, and viral particles in cytological preparations.[5],[6]

One such strategy is the “Mn test” which decides the chromosomal damage and subsequently the mutagenic impacts on the mucosa.[5] Applying thought, it was reasonable to think about the part of the Mn test connected to exfoliated cells, as a screening technique in evaluating the genomic or cell damage in people who are at high risk for cancer. Therefore, the study is intended to assess the cytotoxic damage in agricultural laborers exposed to pesticides using Mn as a biomarker and to assess the cytogenetic damage in buccal exfoliated epithelial cells in agricultural laborers exposed and nonexposed to pesticides.


  Subjects Top


Sample size

The mean Mn among unexposed to pesticide group of nonsmokers was found to be 1.26 with standard deviation (SD) = 0.54 from reference article.[7] Assuming to detect a minimum of 30% increase of Mn in the exposure to pesticide group of nonsmokers and with α = 0.05 and with β = 10%, the minimum sample size is estimated to be 43 per group after applying the following formula.



Since this study involves two groups, the sample size is 86 in all. The sample size was rounded off to 100.

Sample selection

The study population composed of fifty pesticides exposed agricultural laborers and fifty unexposed controls. The both groups included smokers and alcohol drinking, without tobacco chewing habit, from agricultural laborers working in the rural fields of Puducherry, South India, who had reported to our institution. The respective control groups were matched for age, sex, and socioeconomic status. During sample collection, written consent from the patient was obtained as per the Institutional Ethical Committee (IEC) guidelines, Indira Gandhi Institute of Dental Sciences, Sri Balaji Vidyapeeth, Puducherry, India. A detailed occupational, personal, and medical history was taken. The number of times of exposure to pesticide spraying per day and number of years of exposure was taken into consideration.


  Materials and Methods Top


This was a human prospective comparative study which was conducted over a period of approximately 1½ years. The study protocol has been approved by the Institutional Review Board and IEC.

The study groups and the sample size were divided into two categories as follows: Group 1: agricultural laborers not exposed to pesticides (fifty individuals) and Group 2: agricultural laborers exposed to pesticides (fifty individuals). The individuals who met the following criteria were selected.

Inclusion criteria

Agricultural laborers exposed to pesticides by means of spraying and sprinkling were included in our study.

Exclusion criteria

Agricultural laborers who had the history of genetic disorders, frequent exposure to X-rays, vaccination, medication, lesions in the oral cavity and Non agriculturist were excluded from our study.

The agricultural laborers were asked to gargle the oral cavity for 2–3 times using water before making cytosmear. The cytosmear was obtained from both the unexposed and exposed agricultural laborers. Accordingly, we made smear between 7 and 21 days after pesticide showering. The exfoliated cells were spread on the precoded glass slide and the slides were wet-fixed in 95% alcohol for 30 min followed by Papanicolaou (Pap) technique of Pap stain.[8]

Scoring method

The stained cytosmears were examined under light microscope (UMDOB 3 model, Olympus research and clinical system solutions, Tokyo, Japan) by two observers at ×1000 oil immersion and Mn were scored per 500 epithelial cells in each sample [Figure 1]. The technique, used to count the cells was the zigzag strategy where 1000 cells were screened and identified the number of Mn present. The cells were observed as per Tolbert's criteria[9] for Mn by a senior oral pathologist and a research associate in medical genetics that were blinded from each other. Finally, a data analyst compiled all the readings with an error difference of ±2 cells between the two observers and recorded final positive cells.
Figure 1: High-power view of epithelial cells showing micronuclei

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Scoring criteria

The Mn were analyzed by following Tolbert et al.,[9] have specified the following criteria for scoring:

  1. The Mn should be round and smooth
  2. Diameter of the Mn should be <1/3rd of an associated nucleus
  3. In the field illumination, the Mn should be dark and bright
  4. The staining intensity of the Mn and the nucleus should be similar
  5. The Mn texture should be similar to that of nucleus
  6. It should be in same focal plane as nucleus
  7. No overlap or bridge to the nucleus.


Statistical analysis

Percentages of micronucleated cells are presented as mean, SD, and minimum and maximum values (range). 95% confidence limits were calculated for each group. Groups were analyzed using student t-test and partial correlation. P < 0.05 was considered for statistical significance.


  Results Top


The mean and SD of Mn count for control and exposed groups were shown in [Table 1]. Student's t-test indicated that there was a significant difference (P < 0.001) in Mn counts between the control group and the exposed group. Exposed group have a significant higher mean Mn count than the control. Keeping the number of years exposed as a controlling variable, the partial correlation coefficient between the number of times exposed and the mean Mn count shows a significant value P value (R = 0.509, P < 0.001) [Table 2]. The increase in number of times of the pesticide exposure increases the Mn count.
Table 1: Micronucleus count of the control and the exposed

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Table 2: Partial correlations between the number of times exposed and the mean micronuclei count with the controlling factor to the number of years of exposure

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  Discussion Top


Pesticides are chemicals that are widely used throughout the world. In the past, its use was not managed properly, Hence it has lead to considerable amount of exposure to humans and contamination of natural resources has occurred. Pesticide toxicity has been documented in people working in agricultural fields. The International Agency for Research on Cancer has reported about the mutagenic, carcinogenic, and teratogenic activities of pesticides.[10] In literature negative results have been documented. This may be due to reduced exposure or due to less toxic pesticides. Moreover, the chemicals in pesticides may contribute to the genotoxic effects that can be observed. Hence a specific population in country may be exposed to different types of pesticides. Similarly, they have the potential health risk from exposure to pesticides and should be systematically investigated.[11]

In Tamil Nadu and Puducherry, pesticide consumption was estimated to be 1919 tonnes in 2012–2013 with an upward trend in future consumption leading to widespread exposure. Most of the common pesticides used contain various chemical agents that are highly toxic. Cypermethrin (insecticide), Dichlorvos (insecticide) and Mancozeb (fungicide) are some of the chemicals used in these pesticides which are carcinogenic according to United States Environmental Protection Agency. The genotoxic hazards of the pesticides lead to neuritis, psychiatric manifestations, hepatorenal disorders, neurological and neurodegenerative, immune, metabolic and endocrine disorders as well as mutations leading to cancer.[12] Rafiqkhan found significant increase in Mn induced by pesticides and used Mn as a biomarker.[7] Uma et al. conducted a chromosomal study in leukocyte culture of smokers and Mn assay in exfoliated buccal cells of tobacco related oral squamous cell carcinoma in Puducherry population. They found that a significant rise of chromosomal damage indicating that the Puducherry population might be at a high risk for cancer.[13],[14]

In another study, Kulkarni and Mudur showed high level of Mn in Puducherry population who belong to a high-risk population for oral cancer.[15],[16] This paved the pathway for this study to be conducted in agricultural laborers of Puducherry population, who may or may not be at high risk of cancer. Plenty of various biomonitoring studies concentrating on genomic changes have been done in pesticide-exposed populations from different nations to clarify the hazard-related disorders.[17] Among them, a few studies utilizing the Mn test in peripheral lymphocytes or exfoliated buccal mucosa cells are accessible in the most recent decades. Having considered the severity of these exposures, in this study, we have organized the study applying the Mn test as a biomarker from the exfoliated buccal cells.

As >92% of human carcinomas are caused from the epithelium of the skin, bronchi, alimentary canal and oral cavity, therefore it is insisted in our study that the buccal exfoliated epithelial cells may be used as a tool to measure the genotoxic levels of the pesticide. Moreover, the reason for choosing the buccal exfoliated epithelial cell is that it has the capability of carrying carcinogens which are produced by the reactive products that are ingested or inhaled and is a favorable site for deduction of early genotoxic events.[18] Moreover, the biological samples are easy to collect and are a noninvasive technique.[19] Thus the present study is intended to carry out, to assess cytotoxic damage in agricultural laborers who are exposed and not exposed to pesticides and also to estimate the difference in Mn count among the groups.

In our study, we used Pap technique of Pap stain for identifying Mn. Other stains which can be used to identify Mn can be divided into DNA-specific stain and nonspecific stain. DNA-specific stains are Feulgen, Acridine Orange, DAPI (4′,6-diamidino-2-phenylindole) and propidium iodide. The nonspecific stains are Giemsa, May–Grünwald Giemsa and H and E stain.

In our study, the percentage of micronucleated cells was significant between the control group and exposed group, concerning the mean Mn count which is significantly higher in the exposed group than the control. Many studies showed significant increase in these biomarkers due to pesticide exposure that might lead to Mn formation, chromosomal aberrations, sister chromatid exchange and comet cells, suggesting evidence of the genotoxic effect that is induced by pesticides.[20],[21],[22],[23] On the partial correlation between the number of times exposed and the mean Mn count with the controlling factor to the number of years of exposed, it was found that there was a significant P value (R = 0.509, P value 0.001). From this, we observed that the number of times the pesticide exposures increase the Mn count which is also an added factor. This is also confirmed in another study where it is stated that there was a synergistic effect between the regular usage and occupational exposure.[24] In our study, we also found that the workers were not aware that they are exposed to genotoxic agents or chemicals. Because of this, the rate of such occupational diseases is very high in our country.

The results that was obtained in the present study, significantly showed genetic instability in exfoliated buccal cells due to pesticides exposure in agricultural laborers. We need to educate and create awareness and also to promote occupational safety. Therefore large-scale study is required to explore the effects of DNA-environment interactions. Future studies are aimed with larger samples size, performed longitudinally to assess the visualization of the lesions. Intervention studies may also be considered by utilization of Mn test as a biomarker of genotoxicity in foreseeing the impact of the tumor.


  Conclusions Top


The present study's aim was to assess the cytotoxic damage in agricultural laborers who were exposed to the pesticide by Mn assay using buccal exfoliated cells. In this study, we found that there was a significant increase in a number of Mn as the duration of the exposure increased, this indicates that there was considerable cytotoxic damage thereby it will create awareness among the agriculture laborers of Puducherry to take proper preventive measures.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Hoyos LS, Carvajal S, Solano L, Rodriguez J, Orozco L, López Y, et al. Cytogenetic monitoring of farmers exposed to pesticides in Colombia. Environ Health Perspect 1996;104 Suppl 3:535-8.  Back to cited text no. 1
    
2.
Bolognesi C, Creus A, Ostrosky-Wegman P, Marcos R. Micronuclei and pesticide exposure. Mutagenesis 2011;26:19-26.  Back to cited text no. 2
    
3.
Pradeep MR, Guruprasad Y, Jose M, Saxena K, Deepa K, Prabhu V, et al. Comparative study of genotoxicity in different tobacco related habits using micronucleus assay in exfoliated buccal epithelial cells. J Clin Diagn Res 2014;8:ZC21-4.  Back to cited text no. 3
    
4.
Kamath V, Anigol P, Setlur K. Micronuclei as prognostic indicators in oral cytological smears: A comparison between smokers and non-smokers. Clin Cancer Investig J 2014;4:49-54.  Back to cited text no. 4
    
5.
Burkhardt A. Advanced methods in the evaluation of premalignant lesions and carcinomas of the oral mucosa. J Oral Pathol 1985;14:751-78.  Back to cited text no. 5
    
6.
Ogden GR, Cowpe JG, Wight AJ. Oral exfoliative cytology: Review of methods of assessment. J Oral Pathol Med 1997;26:201-5.  Back to cited text no. 6
    
7.
Rafiqkhan M. Micronucleus assessment as a biomarker and susceptibility to DNA damage in workers occupationally exposed to pesticides. Biomed Res Ther 2014;1:78-84.  Back to cited text no. 7
    
8.
Bancroft JD, Gamble M, editors. Theory and Practice of Histological Technique. 6th ed. China: Elsevier; 2008. p. 127-8.  Back to cited text no. 8
    
9.
Tolbert PE, Shy CM, Allen JW. Micronuclei and other nuclear anomalies in buccal smears: Methods development. Mutat Res 1992;271:69-77.  Back to cited text no. 9
    
10.
International Agency for Research on Cancer. Occupational exposure to pesticides. In: IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man. Vol. 53. Occupational Exposures to Insecticide Application and some Pesticides. Lyon: International Agency for Research on Cancer; 1991. p. 80-5.  Back to cited text no. 10
    
11.
Imamura T, Talcott RE. Mutagenic and alkylating activities of organophosphate impurities of commercial Malathion. Mutat Res 1985;155:1-6.  Back to cited text no. 11
    
12.
Blair A, Freeman LB. Epidemiologic studies in agricultural populations: Observations and future directions. J Agromedicine 2009;14:125-31.  Back to cited text no. 12
    
13.
Uma A, Pajanivel R, Raj S, Lokeshmaran. Smoking-induced satellite associations in a rural population of South India: An in vitro study. Int J Appl Basic Med Res 2011;1:75-9.  Back to cited text no. 13
    
14.
Uma AN, Dhananjay SK, Tirou A, Singh SB, Lokeshmaran A. Comparative cytogenetic study of exfoliative oral mucosal cells in tobacco related potentially malignant disorders in a South Indian Population. J Int Acad Res Multidiscip 2014;2:490-500.  Back to cited text no. 14
    
15.
Kulkarni MR. Head and neck cancer burden in India. Int J Head Neck Surg 2013;4:29-35.  Back to cited text no. 15
    
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Mudur G. India has some of the highest cancer rates in the world. BMJ 2005;330:215.  Back to cited text no. 16
    
17.
Bolognesi C. Genotoxicity of pesticides: A review of human biomonitoring studies. Mutat Res 2003;543:251-72.  Back to cited text no. 17
    
18.
Kashyap B, Reddy PS. Micronuclei assay of exfoliated oral buccal cells: Means to assess the nuclear abnormalities in different diseases. J Cancer Res Ther 2012;8:184-91.  Back to cited text no. 18
    
19.
Benedetti D, Nunes E, Sarmento M, Porto C, Dos Santos CE, Dias JF, et al. Genetic damage in soybean workers exposed to pesticides: Evaluation with the comet and buccal micronucleus cytome assays. Mutat Res 2013;752:28-33.  Back to cited text no. 19
    
20.
Au WW, Sierra-Torres CH, Cajas-Salazar N, Shipp BK, Legator MS. Cytogenetic effects from exposure to mixed pesticides and the influence from genetic susceptibility. Environ Health Perspect 1999;107:501-5.  Back to cited text no. 20
    
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Shaham J, Kaufman Z, Gurvich R, Levi Z. Frequency of sister-chromatid exchange among greenhouse farmers exposed to pesticides. Mutat Res 2001;491:71-80.  Back to cited text no. 21
    
22.
Falck GC, Hirvonen A, Scarpato R, Saarikoski ST, Migliore L, Norppa H, et al. Micronuclei in blood lymphocytes and genetic polymorphism for GSTM1, GSTT1 and NAT2 in pesticide-exposed greenhouse workers. Mutat Res 1999;441:225-37.  Back to cited text no. 22
    
23.
Grover P, Danadevi K, Mahboob M, Rozati R, Banu BS, Rahman MF, et al. Evaluation of genetic damage in workers employed in pesticide production utilizing the comet assay. Mutagenesis 2003;18:201-5.  Back to cited text no. 23
    
24.
Ergene S, Celik A, Cavaş T, Kaya F. Genotoxic biomonitoring study of population residing in pesticide contaminated regions in Göksu Delta: Micronucleus, chromosomal aberrations and sister chromatid exchanges. Environ Int 2007;33:877-85.  Back to cited text no. 24
    


    Figures

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    Tables

  [Table 1], [Table 2]



 

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