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The association of nuclear abnormalities in exfoliated buccal epithelial cells with the health status of different agricultural activities farmers in Peninsular Malaysia

The association of nuclear abnormalities in exfoliated buccal epithelial cells with the health... Background: Pesticide exposure possesses risk of genotoxicity to humans, particularly farmers. Despite accumulating evidences linking genotoxicity to pesticide exposure, epidemiological studies to address pesticide toxicity in occupationally exposed farmers in Malaysia remain underreported. Thus, this study was aimed to determine the presence of nuclear abnormalities through the assessment of micronucleus (MN) and binucleus (BNu) frequencies in exfoliated buccal epithelial cells from farmers who were exposed to pesticides. A cross- sectional study of farmers among different agricultural activities farmers in Bachok and Pasir Puteh, Kelantan, North East of Peninsular Malaysia was done to evaluate the presence of nuclear abnormalities and its correlation with their health status and farming activities. Results: Analysis of buccal cells revealed that the frequency of MN was significantly higher (p < 0.05) in farmers as compared to controls. In contrast, no significant difference (p > 0.05) was observed for BNu frequency in between groups. Correlation analysis showed that apart from a significant (p < 0.05) and positive correlation between the duration of fertilizers exposure and frequencies of MN (r = 0.42, P = 0.001) and BNu (r = 0.37, P = 0.02), no other correlation of various confounding factors on the formation of MN and BNu were observed. Conclusion: In conclusion, pesticide and fertilizers exposure may contribute to the promotion of nuclear anomalies among Malaysian farmers who are engaged in mixed plantation activities. Further assessment of larger populations is important to address and overcome the potential risk of pesticide-induced genotoxicity. Keywords: Pesticides, Farmers, Micronucleus, Binucleus, Buccal cells Background context of occupational exposure, farmers are the most at Worldwide, extensive use of pesticides in the agriculture risk to health related-pesticide toxicity [2]. Long-term ex- sector can produce substantial threats to the environment posure to pesticides has been associated with a number of and human health. Pesticides contain a great variety of human health effects such as hormonal disruption, repro- components that differ in their composition and proper- ductive abnormalities, cancer, neurological disorders and ties, with many that were classified as carcinogenic by the cardiorespiratory symptoms [3–6]. International Agency for Research on Cancer [1]. In the The ability of pesticides to induce DNA damage has been reported in a number of studies [7–10] and a posi- tive correlation between pesticides exposure and the * Correspondence: nfadilah@ukm.edu.my genotoxicity risk has been previously documented [8, Biomedical Science Program, School of Diagnostic and Applied Health 11]. The findings suggested that exposure to pesticide Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia (UKM,), Jalan Raja Muda Abdul Aziz, Kuala Lumpur 50300, Malaysia can be genotoxic and carcinogenic to humans [12] Full list of author information is available at the end of the article © 2016 Abdul Hamid et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abdul Hamid et al. Genes and Environment (2016) 38:7 Page 2 of 9 which impose a need for genotoxicological biomonitor- farmers as registered by the District Farmers Association ing in human populations who are at greater risk of of Bachok and Pasir Puteh. Systematic random sampling pesticide-mediated toxicity. method was employed to recruit farmers and sample Human biological monitoring offers a useful applied size estimation was determined using the method de- approach to evaluate potential genotoxic risk associated scribed in Cochran [29] based on the standard deviation with exposure to various environmental genotoxicants. from a study conducted by Pastor et al. [30]. The inclu- Cytogenetic damage such as formation of micronucleus sion criteria for subjects’ selection were farmers aged be- (MN), chromosomal aberration (CA) and sister chroma- tween 19 to 60 years old who have been working as a tid exchange (SCE) has been widely used as biomarkers farmer for a minimum of 1 year and have never stopped to indicate possible genotoxic risk of a defined exposure working for more than 3 months in a year. The exclu- [13, 14]. Among these, micronucleus testing is the most sion criteria were farmers who have a family history of preferred approach due to it being less laborious, cheap cancer or have been diagnosed for any cancer. A struc- and easy to apply in epidemiological studies [15, 16]. tured questionnaire was used to gather information on Evidences for increase in MN frequency in human popu- socio-demographics, knowledge on safe pesticides hand- lation exposed to pesticide have been reported in several ling practices, pesticides application and the use of per- studies [17–19]. Cytogenetic monitoring is a widely ac- sonal protective equipment (PPE). The study was cepted tool to estimate the genetic damage associated with explained to the potential subjects and informed consent pesticide exposure as is evident from its application in nu- was obtained prior to buccal cell sampling. The ques- merous epidemiological studies conducted to date [20–23]. tionnaire was administered face-to-face. The protocol of MN is known as a product of acentric chromosomal this study was approved by the Universiti Kebangsaan fragments or fragments of the whole chromosomes left Malaysia Ethics Committee (UKMEC) (UKM1.5.3.5/244/ during mitotic cellular division which represent struc- NN-201-2010). This study was conducted as part of a tural or numerical chromosomal aberrations (or both) collaborative research with the Malaysian Agriculture during mitosis [24, 25]. In comparison, binucleated cells and Research Development Institute (MARDI) on the (BNu) is defined as the presence of two nuclei that are health impact associated with pesticides exposure among adherent to each other and can indicate failure in cyto- farmers in Malaysia. kinesis [26]. Together, these biomarkers can allow detec- tion of nuclear anomalies which indicate potential risk Subjects of pesticide-mediated genotoxicity. Sampling of exfoli- The study was carried out on 39 farmers in Bachok (n = ated buccal epithelial cells were preferred due to its less 11) and Pasir Puteh (n = 28), Kelantan. The areas were laborious sample collection technique, a non- invasive selected for its extensive use of pesticides and fertilizers approach and a sensitive method to monitor genetic with mixed plantation activities. The main crops grown damage among human populations [27, 28]. were rice, tobacco and varieties of vegetables such as While pesticide exposure is known for its potential tox- chillies, cucumbers and tapiocas. The control subjects icity effects, epidemiological studies to address pesticide have no record of pesticide exposure and were com- toxicity in occupationally exposed farmers in Malaysia re- posed of 30 office staff from UKM Jalan Raja Muda main underreported. Thus, this study is aimed to deter- Abdul Aziz campus, Federal Territory of Kuala Lumpur, mine the presence of nuclear abnormalities through the Malaysia. assessment of MN and BNu frequencies in exfoliated buc- cal epithelial cells from farmers who were exposed to pes- Buccal cells collection ticides in Bachok and Pasir Puteh, Kelantan, North East of Collection of buccal cells was performed according to Peninsular Malaysia. Influence of various confounding fac- the method as described in [30] with minor modifica- tors which include the use of personal protective equip- tions. Briefly, prior to sample collection, subjects rinsed ment (PPE), type of agriculture activities and demographic their mouth with water for removal of unwanted con- status on the frequencies of MN and BNu were also con- taminants. Buccal cells were obtained by rubbing the sidered. Together, this study can provide information on buccal mucosa layer using a sterile wooden tongue de- the safety aspect of farming activities in Malaysia in order pressor. The buccal cells were washed three times in to promote appropriate pesticide handling practices in 5 ml buffered solution (0.003 M EDTA, 0.01 M Tris– agriculture sectors. HCl, 0.64 M NaCl) at pH 7.0 by centrifugation at 2000 rpm for 10 min each, with a brief vortexing at Methods every interval for dissociation of the cell pellets. After Study design the third wash, cell pellets were added with 5 ml of A cross-sectional study was conducted in the East Coast 0.075 M KCL and 50 μl of 1 % DMSO. The mixtures of Peninsular Malaysia. The sample frame was a list of were incubated for 30 min at room temperature Abdul Hamid et al. Genes and Environment (2016) 38:7 Page 3 of 9 followed by the addition of 1 ml of Carnoy fixative solu- Table 1 General characteristics of studied population tion (methanol and acetic acid at a ratio of 3:1). The Characteristics Farmers Controls mixtures were then centrifuged at 2000 rpm for 10 min. n = 39(%) n = 30(%) Following removal of the supernatant, the cell pellets Age groups were fixed with 10 ml Carnoy fixative solution and 26–35 2(5.1) 6(20.0) stored at -20 °C until further analysis. 36–45 7(17.9) 12(40.0) 46–55 8(20.5) 10(33.3) Evaluation of micronuclei and binucleated buccal cells 56–65 22(56.4) 2(6.7) Fixed cell pellets were washed twice in 10 ml Carnoy fixative solution at 2000 rpm for 10 min. At the second Gender wash, most of the supernatants were discarded except a Male 33(84.6) 21(70.0) leftover solution of approximately 1 ml. 200 μl of cell Female 6(15.4) 9(30.0) suspensions was dropped onto preheated (37 °C) and Education pre-cleaned frosted slides. The slides were allowed to None 2(5.1) 0(0) air-dry for 5–10 min and stained with 0.0025 % acridine Primary school 17(43.6) 2(6.7) orange (AO) for microscopic analysis. Cytological slides were prepared in triplicates for each subject. A total of Secondary school 20(51.3) 19(63.3) 1000 cells were counted for each subject and the fre- University 0(0) 9(30.0) quency of MN and BNu were determined using a fluor- Smoking escence microscope (OLYMPUS XC50) at x200 and Smokers 17(43.6) 9(30.0) x400 magnifications. Non-smokers 22(56.4) 21(70.0) BMI (kg/m ) Stastical analysis Data were analysed using the SPSS statistical software Underweight (<18.5) 4(10.3) 0(0) package version 18.0. The level of significance was set as Normal (18.5–24.9) 13(33.3) 9(30.0) p ≤0.05. Data were tested for normality using the Pre-obese (25.0–29.9) 20(51.3) 15(50.0) Shapiro-Wilk test. Student’s t-test was used for the ana- Obese grade 1 (30.0–34.9) 2(5.1) 3(10.0) lysis of differences between groups. The correlation be- Obese grade 2 (35.0–39.9) 0(0) 3(10.0) tween the two variables was analyzed using Pearson correlation. Results The demographic data of the studied population (Table 1) indicate that the majority of the farmers in Table 2 Commonly reported chronic diseases and health Bachok and Pasir Puteh (56.4 %) were aged between 56 symptoms to 65 years old while in the control group, the majority Farmers Controls of subjects (40.0 %) were aged between 36 to 45 years n = 39(%) n = 30(%) old. Meanwhile, no remarkable differences were ob- Chronic Diseases served for both groups with regards to other general Hypertension 12(30.8) 5(16.7) characteristics, which include educational background, Diabetes Mellitus 2(5.1) 3(10.0) smoking status and body mass index (BMI). The major- Asthma 8(20.5) 2(6.7) ity of farmers (F) and control (C) were pre-obese (F = 51.3 %; C = 50.0 %), completed secondary school (F = Gastric 11(28.2) 3(10.0) 51.3 %; C = 63.3 %) and non-smokers (F = 56.4 %; C = Health-associated symptoms 70.0 %). Sore throat 7(17.9) 3(10.0) With respect to chronic diseases and health-associated Headache 14(35.9) 10(33.3) symptoms (Table 2), farmers had higher percentages of Blurred vision 22(56.4) 10(33.3) hypertension (30.8 %), asthma (20.5 %) and gastric Insomnia 6(15.4) 1(3.3) (28.2 %) as compared to that observed in the control group. Additionally, more than 50 % of farmers claimed Eyes irritation 17(43.6) 7(23.3) to frequently experience blurred vision and numbness. Runny nose 6(15.4) 1(3.3) Other symptoms such as eye irritation, coughing, insom- Numbness 23(59.0) 2(6.7) nia and runny nose were also found to be higher in Cough 17(43.6) 4(13.3) farmers as compared to control. Abdul Hamid et al. Genes and Environment (2016) 38:7 Page 4 of 9 With regards to the application of personal protective Table 4 Agriculture activities among farmers equipments (PPE), a majority of farmers (Table 3) Activities Farmers claimed the use of safety helmets (89.7 %), long-sleeved n = 39(%) shirt (89.7 %), long pants (97.4 %), nose mask (84.6 %) Crops and boots (71.8 %) during pesticide handling. Mean- Vegetables 14(36.0) while, usage of gloves was at a moderate level with only Paddy 10(25.6) 53.8 % who claimed to use it. Eye goggles were found to Tobacco 10(25.6) be the least used PPE with 69.2 % of farmers who Combination of vegetables, paddy and tobacco 5(12.8) claimed that they never used eye goggles while handling pesticides. Farming experiences Additional information with regards to farming activ- Number of years ities were also evaluated. The information includes type 1–10 11(28.2) of crops, farming experiences, duration of pestiside and 11–20 11(28.2) fertilizers exposure and types of pesticide applicators be- 21–30 8(20.5) ing utilized. As shown in Table 4, the main crops grown 31–40 3(7.7) were paddy, vegetables and tobacco. With respect to plantation activities, 36 % of farmers involved in vege- 41–50 6(15.4) table farming while about 25.6 % were paddy and to- Frequencies of pesticides spraying and application of fertilizers bacco farmers. In addition, 12.8 % of farmers involved Frequency per month with mixed farming consisted of paddy, vegetables and Pesticides tobacco. The majority of farmers (28.2 %) have been 1–3 times 14(35.9) farming in between 1 and 10 and 11–20 years. Moreover, >3 times 25(64.1) majority of farmers (64.1 %) had pesticide spraying fre- quencies of more than 3 times in a month with most of Fertilizers them (53.8 %) spent between 1 to 12 h per month for 1–3 times 30(76.9) >3 times 9(23.1) Table 3 Application of personal protective equipment (PPE) Duration of pesticides spraying and application of fertilizers among farmers Spent hours per month PPE n = 39(%) Pesticides Safety helmets 1–12 h 21(53.8) Yes 35(89.7) >12 h 18(46.2) No 4(10.3) Fertilizers Long-sleeve shirts 1–4 h 24(61.5) Yes 35(89.7) >4 h 15(38.5) No 4(10.3) Pesticides application equipments Long Pants Motorized knapsack sprayer 29(74.4) Yes 38(97.4) Mechanical sprayer 5(12.8) No 1(2.6) Other sprayer 5(12.8) Eye goggles Yes 12(30.8) such activities. As for fertilizers exposure, most of No 27(69.2) farmers (61.5 %) spent about 1 to 4 h on fertilizers usage Nose mask per month with majority (76.9 %) exposed to fertilizers Yes 33(84.6) between 1 to 3 times per month. With regards to the No 6(15.4) type of pestiside applicators, farmers mostly used motor- Gloves ized knapsack sprayer (74.4 %) as compared to other Yes 21(53.8) methods. Meanwhile, with regards to common personal habits during and / or after pestiside exposure, majority No 18(46.2) of farmers (>80 %) showed a satisfactory practice on this Boots aspect (Table 5). Yes 28(71.8) The mean ± S.D of micronucleus (MN) and binucleus No 11(28.2) (BNu) per 1000 cells of farmers and controls are as Abdul Hamid et al. Genes and Environment (2016) 38:7 Page 5 of 9 Table 5 Personal habits during and/or after pesticides exposure confounding factors on MN and BNu were observed (Table 7). The frequencies of BNu and MN show a weak Personal habits Farmers positive correlation with the duration of pesticides ex- n = 39(%) posure and the correlation was not significant (p > 0.05). Personal habits during pesticide applications Drinking Discussion Yes 6(15.4) Farmers who are exposed to pesticides are at greater risk No 33(84.6) for cytogenetic damage. To date, a number of studies Eating have been carried out on the health status of pesticide- exposed populations from different countries to eluci- Yes 5(12.8) date the risk associated with pesticide-induced cytogen- No 34(87.2) etic damage [7–10]. However, no definitive conclusions Smoking could yet be established on the association of these fac- Yes 3(7.7) tors. Up to our knowledge, there have been no reports No 36(92.3) in Malaysia concerning the issue, suggesting the need Post-pesticides application practices for such studies to be conducted. In the present study, a preliminary investigation on cytogenetic damage among Change clothes pesticides and fertilizers-exposed farmers from two rural Yes 34(87.2) areas of North East Malaysia, namely Bachok and Pasir No 5(12.8) Puteh was carried out. Cytogenetic damage was deter- Wash hand mined by the frequency of micronucleus (MN) and Yes 37(94.9) binucleus (BN) of isolated buccal epithelial cells. In No 2(5.1) order to identify the confounding factors that may con- tribute to cytogenetic damage, information on demo- presented in Table 6. The frequency of MN was found graphics, pesticide exposure and PPE application were to be significantly higher (p < 0.05) among farmers (6.83 also gathered. ± 6.25) as compared to controls (1.39 ± 0.60). However, The results obtained in this study showed a significant no significant difference was observed for the frequency increase of MN frequencies in farmers when compared of BNu between both groups. to controls. The results obtained for MN is in agreement The intra- (within) groups comparison of MN and with previous studies which observed higher frequency BNu frequencies for age, gender, smoking status, BMI of MN among pesticide-exposed farmers than the non- and duration of employment showed no significant dif- exposed group [31–33]. Although some studies reported ference. In contrast, inter—(between) groups compari- no significant increase in the MN frequencies in son revealed a significantly higher (p < 0.05) frequency of pesticide-exposed farmers [25, 30, 34], the presence of MN among farmers as compared to control at every age MN as indicators for cytogenetic damage has been ac- group. Similarly, a significantly higher (p < 0.05) fre- knowledged as a useful biomonitoring tool in popula- quency of MN was observed among farmers as com- tions occupationally exposed to pesticides [35]. pared to control based on gender and smoking status. In contrast to MN results, BNu frequencies revealed As for BMI classification, a significant difference (p < no significant difference between the two studied groups. 0.05) in the frequency of MN was only seen among nor- The result could be influenced by the presence of un- mal and pre-obese of inter group comparison. A signifi- controlled exogenous factors such as environmental in- cant increase in MN frequencies (p < 0.05) was also fluence that contributed in the evaluation of cytogenetic detected for different duration of employment among damages [36]. Moreover, a lack of comparable data in farmers as compared to control. No significant differ- the literature concerning BNu frequencies as compared ence was seen in the frequency of BNu for inter group to MN indicated the unresolved role of BNu as a marker comparison. for nuclear abnormalities associated with pesticide The correlation between MN and BNu frequencies toxicity. with socio-demographic factors (age, years of employ- Pesticides exposure has been linked to a number of ment, BMI) and duration of exposure to pesticides and human health effects [37, 38]. Thus, an attempt was fertilizers were studied. As shown in Table 7, a signifi- made to identify the possible chronic diseases and symp- cant (p < 0.05) and positive correlation between the dur- toms that are commonly associated with pesticide ex- ation of fertilizers exposure and frequencies of BNu (r = posure among farmers. The number of farmers with 0.37, P = 0.02) and MN were (r = 0.42, P = 0.001) ob- reported hypertension, asthma, gastritis, insomnia, served. However, no other correlation of various runny nose and coughing were higher as compared to Abdul Hamid et al. Genes and Environment (2016) 38:7 Page 6 of 9 Table 6 Frequencies of MN and BNu in farmers and controls according to age, gender, smoking status, BMI and duration of employment. Data are presented as mean ± standard deviation (s.d) Controls (n = 30) Farmers (n = 39) Mean ± S.D Mean ± S.D Groups Frequency of MN (%) Frequency of BNu (%) Frequency of MN (%) Frequency of BNu (%) Age (years) 26–35 1.01 ± 0.32 2.01 ± 0.73 6.65 ± 0.92* 1.50 ± 1.41 36–45 1.47 ± 0.58 1.67 ± 0.94 6.22 ± 1.90* 1.51 ± 0.19 46–55 1.57 ± 0.72 1.49 ± 0.86 8.01 ± 3.09* 1.33 ± 0.45 56–65 1.10 ± 0.14 1.85 ± 0.64 6.61 ± 3.21* 1.63 ± 0.74 Gender Male 1.32 ± 0.55 1.85 ± 0.86 6.75 ± 3.01* 1.49 ± 0.61 Female 1.56 ± 0.70 1.32 ± 0.70 7.23 ± 2.43* 1.80 ± 0.83 Smoking status Smokers 1.25 ± 0.41 1.99 ± 1.08 6.82 ± 2.65* 1.39 ± 0.62 Non-smokers 1.45 ± 0.66 1.56 ± 0.71 6.83 ± 3.15* 1.65 ± 0.65 BMI Underweight - - 6.89 ± 3.04 1.49 ± 0.80 Normal 1.54 ± 0.61 1.59 ± 1.19 7.83 ± 3.19* 1.43 ± 0.77 Pre-obese 1.22 ± 0.47 1.72 ± 0.62 6.22 ± 2.58* 1.58 ± 0.57 Obese grade 1 1.66 ± 0.31 1.30 ± 0.61 6.25 ± 4.74 1.85 ± 0.35 Obese grade 2 1.57 ± 1.25 2.23 ± 0.91 - - Duration of employment (years) 1–10 1.39 ± 0.50 1.74 ± 0.99 7.04 ± 2.39* 1.34 ± 0.61 11–20 1.21 ± 0.64 1.74 ± 0.72 6.14 ± 3.02* 1.54 ± 0.42 21–30 1.67 ± 0.83 1.56 ± 0.93 5.80 ± 2.06* 1.76 ± 0.48 31–40 1.25 ± 0.53 1.63 ± 0.52 7.32 ± 4.20* 1.29 ± 0.88 41–50 - - 8.82 ± 3.71 1.71 ± 1.08 Total 1.39 ± 0.60 1.69 ± 0.84 6.83 ± 2.91* 1.54 ± 0.64 *Farmers versus controls (p < 0.05) the control group. Moreover, more than 50 % of farmers The use of appropriate PPE should give a significant claimed frequent experience of eye irritation, blurred vi- preventive impact in pesticide exposure among farmers. sion and numbness, suggesting a possible association to Significant increase in cytogenetic damage among pesticide exposure. However, due to lack of medical his- farmers with no or little protective clothing during pesti- tory and nutritional status data, direct association of the cide usage has been reported [39–41]. The usage of PPE, manifested symptoms with pesticide exposure could not as observe in this study can be regarded as unsatisfac- be established. Lack of PPE application during pesticide tory. Of the 7 PPE assessed, only 4 of the PPE (safety handling however can be one of the contributing factors helmets, long sleeved shirt, long pants and nose mask) for the reduced quality of health among the farmers. were used by the majority of the farmers. Furthermore, Table 7 Correlation between the frequencies of MN and BNu with the exposure to pesticides and fertilizers Frequency of MN Frequency of BNu (n = 39) (n = 39) Duration of pesticides exposure (hours) 0.15 0.28 (p = 0.37) (p = 0.16) Duration of fertilizers exposure (hours) 0.42 0.37 (p = 0.001)* (p = 0.02)* *p < 0.05 Abdul Hamid et al. Genes and Environment (2016) 38:7 Page 7 of 9 the use of eye goggles, gloves and boots were considered Tobacco smoke is a known genotoxicant and evi- low with a significant number of farmers who claimed dences on its ability to induce DNA damage have been that they never used these PPE throughout their farming comprehensively studied [45]. However, in this study the activities which put them at greater risk of percutaneous influence of smoking on cytogenetic damage showed no exposure to pesticide. In a study conducted by Lander significant correlation as observed in the frequencies of et al. [32], remarkable cytogenetic effects were noted in MN and BNu between smokers and non- smokers workers who did not use gloves, indicating the import- within the pesticide exposed group. Similarly, the effect ance of PPE usage. Although the humid and hot climatic of smoking was not found in MN and BNu frequencies conditions in Malaysia could be the factor that limits the within the control group. The lack of association ob- usage of full protective clothing [42, 43], continued ef- served between the frequencies of studied nuclear anom- fort to increase awareness among farmers on potential alies and tobacco smoke exposure has been reported pesticide hazards and the importance of PPE usage can previously [30, 54]. The effect of tobacco smoke expos- be useful to minimize pesticide related hazards at the ure on genotoxicity is influenced by type of cigarettes workplace. and frequencies of smoking [54] both of which were not The presence of nuclear anomalies can be influenced evaluated in this study. Thus, future studies focusing on by various confounding factors which include the ex- gathering comprehensive information on smoking habits ogenous (alcohol, diet, smoking and pesticide applica- among farmers can provide more definitive correlation tion) and endogenous (age and gender) factors [44–47]. and conclusive evidence. Some studies have reported a positive age effect on MN Duration of farming experience provides information frequencies among workers [33, 48]. However, in this on the degree of exposure to pesticide among farmers study, no significant differences were seen on the fre- [4]. In a review by Bolognesi [14], occupational exposure quencies of MN and BNu for both farmers and controls, to pesticide enhances genotoxic damage in a dose- which is in agreement to previously reported studies [34, dependent manner. In this study, higher frequencies of 49]. MN were observed among farmers with farming experi- Apart from age, the frequency of MN is also influ- ence of 41–50 years as compared to those with shorter enced by gender. Higher MN frequency in women than durations of farming experience. However, the observed in men was commonly observed in populations without difference was not statistically significant. It appears that exposure to any physical or chemical agents, with X duration of farming activities demonstrated no signifi- chromosome micronucleation being shown to contribute cant influence on the frequency of MN and BNu among to the increase of MN frequency in women [44]. In this farmers, which is in agreement with other reported stud- study however, no significant differences for the frequen- ies [55, 56]. To date, studies on the association between cies of MN and BNu between male and female subjects pesticide exposure and frequency of MN and BNu are were observed, though increased MN frequency in fe- inconclusive. Eastmond [57] have indicated that the per- male farmers were observed and is in accordance with a sistence of chromosomal damage is short-lived for acute previous report [49]. exposure while Scarpato et al. [58] noted that damage Level of education may affect the level of knowledge may be reduced during low exposure periods for sea- and awareness on the safety of pesticide handling [50]. A sonal workers. However, increased chromosomal damage previous study reported that the level of education with associated with duration of agricultural employment was at least high school education is required for farmers to reported by a number of studies [17, 58, 59]. The con- show a positive perception on the usefulness of PPE ap- flicting results from studies on pesticide-related cytogen- plication [51]. In a study conducted among Brazilian etic damage were influenced by many factors such as the farmers, the inability to understand the information dis- working environment, the studied populations, the type played on products’ labels led to increased exposure to of exposure to pesticides such as seasonal application of pesticides [52]. A survey conducted on 61 randomly se- pesticides, types of pesticide mixtures and the use of lected fruit-growers in a specific area of Turkey [53] re- PPE [19, 60, 61]. Moreover, Bull et al. [4] has stated in a vealed that pesticide practices were influenced by a review that chromosomal damage itself is regarded as number of characteristics, with those who consider pes- transient and sampling time play an important role on ticides as harmful consisted of farmers who are younger, determining the accuracy of the data. Sampling should better educated and have less experience in fruit- be done within 2 days following acute or cessation of growing. Our findings indicated that almost half of the chronic exposure to overcome false negative results. farmers did not have formal education beyond the sec- However, we have not considered this possibility which ondary education level. Therefore, levels of education could contribute to our non-significant finding. may affect the perception they may have towards the Assessments of correlation between MN and BNu fre- harmfulness of the pesticides. quencies with duration of pesticides and fertilizers Abdul Hamid et al. 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Costa C, Silva S, Coelho P, Roma-Torres J, Teixeira JP, Mayan O. • We provide round the clock customer support Micronucleus analysis in a Portuguese population exposed to pesticides. • Convenient online submission Preliminary survey. Int J Hyg Environ Health. 2007;210:415–8. 46. Fenech M, Neville S, Rinaldi J. Sex is an important variable affecting • Thorough peer review spontaneous micronucleus frequency in cytokinesis-blocked lymphocytes. • Inclusion in PubMed and all major indexing services Mutat Res. 1994;313:203–7. • Maximum visibility for your research 47. Sivayoganathan C, Gnanachandran S, Lewis J, Fernando M. Protective measure use and symptoms among agropesticide applicators in Sri Lanka. Submit your manuscript at Soc Sci Med. 1995;40:431–36. www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genes and Environment Springer Journals

The association of nuclear abnormalities in exfoliated buccal epithelial cells with the health status of different agricultural activities farmers in Peninsular Malaysia

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Springer Journals
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Copyright © 2016 by The Author(s)
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Biomedicine; Human Genetics
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1880-7062
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10.1186/s41021-016-0032-1
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Abstract

Background: Pesticide exposure possesses risk of genotoxicity to humans, particularly farmers. Despite accumulating evidences linking genotoxicity to pesticide exposure, epidemiological studies to address pesticide toxicity in occupationally exposed farmers in Malaysia remain underreported. Thus, this study was aimed to determine the presence of nuclear abnormalities through the assessment of micronucleus (MN) and binucleus (BNu) frequencies in exfoliated buccal epithelial cells from farmers who were exposed to pesticides. A cross- sectional study of farmers among different agricultural activities farmers in Bachok and Pasir Puteh, Kelantan, North East of Peninsular Malaysia was done to evaluate the presence of nuclear abnormalities and its correlation with their health status and farming activities. Results: Analysis of buccal cells revealed that the frequency of MN was significantly higher (p < 0.05) in farmers as compared to controls. In contrast, no significant difference (p > 0.05) was observed for BNu frequency in between groups. Correlation analysis showed that apart from a significant (p < 0.05) and positive correlation between the duration of fertilizers exposure and frequencies of MN (r = 0.42, P = 0.001) and BNu (r = 0.37, P = 0.02), no other correlation of various confounding factors on the formation of MN and BNu were observed. Conclusion: In conclusion, pesticide and fertilizers exposure may contribute to the promotion of nuclear anomalies among Malaysian farmers who are engaged in mixed plantation activities. Further assessment of larger populations is important to address and overcome the potential risk of pesticide-induced genotoxicity. Keywords: Pesticides, Farmers, Micronucleus, Binucleus, Buccal cells Background context of occupational exposure, farmers are the most at Worldwide, extensive use of pesticides in the agriculture risk to health related-pesticide toxicity [2]. Long-term ex- sector can produce substantial threats to the environment posure to pesticides has been associated with a number of and human health. Pesticides contain a great variety of human health effects such as hormonal disruption, repro- components that differ in their composition and proper- ductive abnormalities, cancer, neurological disorders and ties, with many that were classified as carcinogenic by the cardiorespiratory symptoms [3–6]. International Agency for Research on Cancer [1]. In the The ability of pesticides to induce DNA damage has been reported in a number of studies [7–10] and a posi- tive correlation between pesticides exposure and the * Correspondence: nfadilah@ukm.edu.my genotoxicity risk has been previously documented [8, Biomedical Science Program, School of Diagnostic and Applied Health 11]. The findings suggested that exposure to pesticide Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia (UKM,), Jalan Raja Muda Abdul Aziz, Kuala Lumpur 50300, Malaysia can be genotoxic and carcinogenic to humans [12] Full list of author information is available at the end of the article © 2016 Abdul Hamid et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abdul Hamid et al. Genes and Environment (2016) 38:7 Page 2 of 9 which impose a need for genotoxicological biomonitor- farmers as registered by the District Farmers Association ing in human populations who are at greater risk of of Bachok and Pasir Puteh. Systematic random sampling pesticide-mediated toxicity. method was employed to recruit farmers and sample Human biological monitoring offers a useful applied size estimation was determined using the method de- approach to evaluate potential genotoxic risk associated scribed in Cochran [29] based on the standard deviation with exposure to various environmental genotoxicants. from a study conducted by Pastor et al. [30]. The inclu- Cytogenetic damage such as formation of micronucleus sion criteria for subjects’ selection were farmers aged be- (MN), chromosomal aberration (CA) and sister chroma- tween 19 to 60 years old who have been working as a tid exchange (SCE) has been widely used as biomarkers farmer for a minimum of 1 year and have never stopped to indicate possible genotoxic risk of a defined exposure working for more than 3 months in a year. The exclu- [13, 14]. Among these, micronucleus testing is the most sion criteria were farmers who have a family history of preferred approach due to it being less laborious, cheap cancer or have been diagnosed for any cancer. A struc- and easy to apply in epidemiological studies [15, 16]. tured questionnaire was used to gather information on Evidences for increase in MN frequency in human popu- socio-demographics, knowledge on safe pesticides hand- lation exposed to pesticide have been reported in several ling practices, pesticides application and the use of per- studies [17–19]. Cytogenetic monitoring is a widely ac- sonal protective equipment (PPE). The study was cepted tool to estimate the genetic damage associated with explained to the potential subjects and informed consent pesticide exposure as is evident from its application in nu- was obtained prior to buccal cell sampling. The ques- merous epidemiological studies conducted to date [20–23]. tionnaire was administered face-to-face. The protocol of MN is known as a product of acentric chromosomal this study was approved by the Universiti Kebangsaan fragments or fragments of the whole chromosomes left Malaysia Ethics Committee (UKMEC) (UKM1.5.3.5/244/ during mitotic cellular division which represent struc- NN-201-2010). This study was conducted as part of a tural or numerical chromosomal aberrations (or both) collaborative research with the Malaysian Agriculture during mitosis [24, 25]. In comparison, binucleated cells and Research Development Institute (MARDI) on the (BNu) is defined as the presence of two nuclei that are health impact associated with pesticides exposure among adherent to each other and can indicate failure in cyto- farmers in Malaysia. kinesis [26]. Together, these biomarkers can allow detec- tion of nuclear anomalies which indicate potential risk Subjects of pesticide-mediated genotoxicity. Sampling of exfoli- The study was carried out on 39 farmers in Bachok (n = ated buccal epithelial cells were preferred due to its less 11) and Pasir Puteh (n = 28), Kelantan. The areas were laborious sample collection technique, a non- invasive selected for its extensive use of pesticides and fertilizers approach and a sensitive method to monitor genetic with mixed plantation activities. The main crops grown damage among human populations [27, 28]. were rice, tobacco and varieties of vegetables such as While pesticide exposure is known for its potential tox- chillies, cucumbers and tapiocas. The control subjects icity effects, epidemiological studies to address pesticide have no record of pesticide exposure and were com- toxicity in occupationally exposed farmers in Malaysia re- posed of 30 office staff from UKM Jalan Raja Muda main underreported. Thus, this study is aimed to deter- Abdul Aziz campus, Federal Territory of Kuala Lumpur, mine the presence of nuclear abnormalities through the Malaysia. assessment of MN and BNu frequencies in exfoliated buc- cal epithelial cells from farmers who were exposed to pes- Buccal cells collection ticides in Bachok and Pasir Puteh, Kelantan, North East of Collection of buccal cells was performed according to Peninsular Malaysia. Influence of various confounding fac- the method as described in [30] with minor modifica- tors which include the use of personal protective equip- tions. Briefly, prior to sample collection, subjects rinsed ment (PPE), type of agriculture activities and demographic their mouth with water for removal of unwanted con- status on the frequencies of MN and BNu were also con- taminants. Buccal cells were obtained by rubbing the sidered. Together, this study can provide information on buccal mucosa layer using a sterile wooden tongue de- the safety aspect of farming activities in Malaysia in order pressor. The buccal cells were washed three times in to promote appropriate pesticide handling practices in 5 ml buffered solution (0.003 M EDTA, 0.01 M Tris– agriculture sectors. HCl, 0.64 M NaCl) at pH 7.0 by centrifugation at 2000 rpm for 10 min each, with a brief vortexing at Methods every interval for dissociation of the cell pellets. After Study design the third wash, cell pellets were added with 5 ml of A cross-sectional study was conducted in the East Coast 0.075 M KCL and 50 μl of 1 % DMSO. The mixtures of Peninsular Malaysia. The sample frame was a list of were incubated for 30 min at room temperature Abdul Hamid et al. Genes and Environment (2016) 38:7 Page 3 of 9 followed by the addition of 1 ml of Carnoy fixative solu- Table 1 General characteristics of studied population tion (methanol and acetic acid at a ratio of 3:1). The Characteristics Farmers Controls mixtures were then centrifuged at 2000 rpm for 10 min. n = 39(%) n = 30(%) Following removal of the supernatant, the cell pellets Age groups were fixed with 10 ml Carnoy fixative solution and 26–35 2(5.1) 6(20.0) stored at -20 °C until further analysis. 36–45 7(17.9) 12(40.0) 46–55 8(20.5) 10(33.3) Evaluation of micronuclei and binucleated buccal cells 56–65 22(56.4) 2(6.7) Fixed cell pellets were washed twice in 10 ml Carnoy fixative solution at 2000 rpm for 10 min. At the second Gender wash, most of the supernatants were discarded except a Male 33(84.6) 21(70.0) leftover solution of approximately 1 ml. 200 μl of cell Female 6(15.4) 9(30.0) suspensions was dropped onto preheated (37 °C) and Education pre-cleaned frosted slides. The slides were allowed to None 2(5.1) 0(0) air-dry for 5–10 min and stained with 0.0025 % acridine Primary school 17(43.6) 2(6.7) orange (AO) for microscopic analysis. Cytological slides were prepared in triplicates for each subject. A total of Secondary school 20(51.3) 19(63.3) 1000 cells were counted for each subject and the fre- University 0(0) 9(30.0) quency of MN and BNu were determined using a fluor- Smoking escence microscope (OLYMPUS XC50) at x200 and Smokers 17(43.6) 9(30.0) x400 magnifications. Non-smokers 22(56.4) 21(70.0) BMI (kg/m ) Stastical analysis Data were analysed using the SPSS statistical software Underweight (<18.5) 4(10.3) 0(0) package version 18.0. The level of significance was set as Normal (18.5–24.9) 13(33.3) 9(30.0) p ≤0.05. Data were tested for normality using the Pre-obese (25.0–29.9) 20(51.3) 15(50.0) Shapiro-Wilk test. Student’s t-test was used for the ana- Obese grade 1 (30.0–34.9) 2(5.1) 3(10.0) lysis of differences between groups. The correlation be- Obese grade 2 (35.0–39.9) 0(0) 3(10.0) tween the two variables was analyzed using Pearson correlation. Results The demographic data of the studied population (Table 1) indicate that the majority of the farmers in Table 2 Commonly reported chronic diseases and health Bachok and Pasir Puteh (56.4 %) were aged between 56 symptoms to 65 years old while in the control group, the majority Farmers Controls of subjects (40.0 %) were aged between 36 to 45 years n = 39(%) n = 30(%) old. Meanwhile, no remarkable differences were ob- Chronic Diseases served for both groups with regards to other general Hypertension 12(30.8) 5(16.7) characteristics, which include educational background, Diabetes Mellitus 2(5.1) 3(10.0) smoking status and body mass index (BMI). The major- Asthma 8(20.5) 2(6.7) ity of farmers (F) and control (C) were pre-obese (F = 51.3 %; C = 50.0 %), completed secondary school (F = Gastric 11(28.2) 3(10.0) 51.3 %; C = 63.3 %) and non-smokers (F = 56.4 %; C = Health-associated symptoms 70.0 %). Sore throat 7(17.9) 3(10.0) With respect to chronic diseases and health-associated Headache 14(35.9) 10(33.3) symptoms (Table 2), farmers had higher percentages of Blurred vision 22(56.4) 10(33.3) hypertension (30.8 %), asthma (20.5 %) and gastric Insomnia 6(15.4) 1(3.3) (28.2 %) as compared to that observed in the control group. Additionally, more than 50 % of farmers claimed Eyes irritation 17(43.6) 7(23.3) to frequently experience blurred vision and numbness. Runny nose 6(15.4) 1(3.3) Other symptoms such as eye irritation, coughing, insom- Numbness 23(59.0) 2(6.7) nia and runny nose were also found to be higher in Cough 17(43.6) 4(13.3) farmers as compared to control. Abdul Hamid et al. Genes and Environment (2016) 38:7 Page 4 of 9 With regards to the application of personal protective Table 4 Agriculture activities among farmers equipments (PPE), a majority of farmers (Table 3) Activities Farmers claimed the use of safety helmets (89.7 %), long-sleeved n = 39(%) shirt (89.7 %), long pants (97.4 %), nose mask (84.6 %) Crops and boots (71.8 %) during pesticide handling. Mean- Vegetables 14(36.0) while, usage of gloves was at a moderate level with only Paddy 10(25.6) 53.8 % who claimed to use it. Eye goggles were found to Tobacco 10(25.6) be the least used PPE with 69.2 % of farmers who Combination of vegetables, paddy and tobacco 5(12.8) claimed that they never used eye goggles while handling pesticides. Farming experiences Additional information with regards to farming activ- Number of years ities were also evaluated. The information includes type 1–10 11(28.2) of crops, farming experiences, duration of pestiside and 11–20 11(28.2) fertilizers exposure and types of pesticide applicators be- 21–30 8(20.5) ing utilized. As shown in Table 4, the main crops grown 31–40 3(7.7) were paddy, vegetables and tobacco. With respect to plantation activities, 36 % of farmers involved in vege- 41–50 6(15.4) table farming while about 25.6 % were paddy and to- Frequencies of pesticides spraying and application of fertilizers bacco farmers. In addition, 12.8 % of farmers involved Frequency per month with mixed farming consisted of paddy, vegetables and Pesticides tobacco. The majority of farmers (28.2 %) have been 1–3 times 14(35.9) farming in between 1 and 10 and 11–20 years. Moreover, >3 times 25(64.1) majority of farmers (64.1 %) had pesticide spraying fre- quencies of more than 3 times in a month with most of Fertilizers them (53.8 %) spent between 1 to 12 h per month for 1–3 times 30(76.9) >3 times 9(23.1) Table 3 Application of personal protective equipment (PPE) Duration of pesticides spraying and application of fertilizers among farmers Spent hours per month PPE n = 39(%) Pesticides Safety helmets 1–12 h 21(53.8) Yes 35(89.7) >12 h 18(46.2) No 4(10.3) Fertilizers Long-sleeve shirts 1–4 h 24(61.5) Yes 35(89.7) >4 h 15(38.5) No 4(10.3) Pesticides application equipments Long Pants Motorized knapsack sprayer 29(74.4) Yes 38(97.4) Mechanical sprayer 5(12.8) No 1(2.6) Other sprayer 5(12.8) Eye goggles Yes 12(30.8) such activities. As for fertilizers exposure, most of No 27(69.2) farmers (61.5 %) spent about 1 to 4 h on fertilizers usage Nose mask per month with majority (76.9 %) exposed to fertilizers Yes 33(84.6) between 1 to 3 times per month. With regards to the No 6(15.4) type of pestiside applicators, farmers mostly used motor- Gloves ized knapsack sprayer (74.4 %) as compared to other Yes 21(53.8) methods. Meanwhile, with regards to common personal habits during and / or after pestiside exposure, majority No 18(46.2) of farmers (>80 %) showed a satisfactory practice on this Boots aspect (Table 5). Yes 28(71.8) The mean ± S.D of micronucleus (MN) and binucleus No 11(28.2) (BNu) per 1000 cells of farmers and controls are as Abdul Hamid et al. Genes and Environment (2016) 38:7 Page 5 of 9 Table 5 Personal habits during and/or after pesticides exposure confounding factors on MN and BNu were observed (Table 7). The frequencies of BNu and MN show a weak Personal habits Farmers positive correlation with the duration of pesticides ex- n = 39(%) posure and the correlation was not significant (p > 0.05). Personal habits during pesticide applications Drinking Discussion Yes 6(15.4) Farmers who are exposed to pesticides are at greater risk No 33(84.6) for cytogenetic damage. To date, a number of studies Eating have been carried out on the health status of pesticide- exposed populations from different countries to eluci- Yes 5(12.8) date the risk associated with pesticide-induced cytogen- No 34(87.2) etic damage [7–10]. However, no definitive conclusions Smoking could yet be established on the association of these fac- Yes 3(7.7) tors. Up to our knowledge, there have been no reports No 36(92.3) in Malaysia concerning the issue, suggesting the need Post-pesticides application practices for such studies to be conducted. In the present study, a preliminary investigation on cytogenetic damage among Change clothes pesticides and fertilizers-exposed farmers from two rural Yes 34(87.2) areas of North East Malaysia, namely Bachok and Pasir No 5(12.8) Puteh was carried out. Cytogenetic damage was deter- Wash hand mined by the frequency of micronucleus (MN) and Yes 37(94.9) binucleus (BN) of isolated buccal epithelial cells. In No 2(5.1) order to identify the confounding factors that may con- tribute to cytogenetic damage, information on demo- presented in Table 6. The frequency of MN was found graphics, pesticide exposure and PPE application were to be significantly higher (p < 0.05) among farmers (6.83 also gathered. ± 6.25) as compared to controls (1.39 ± 0.60). However, The results obtained in this study showed a significant no significant difference was observed for the frequency increase of MN frequencies in farmers when compared of BNu between both groups. to controls. The results obtained for MN is in agreement The intra- (within) groups comparison of MN and with previous studies which observed higher frequency BNu frequencies for age, gender, smoking status, BMI of MN among pesticide-exposed farmers than the non- and duration of employment showed no significant dif- exposed group [31–33]. Although some studies reported ference. In contrast, inter—(between) groups compari- no significant increase in the MN frequencies in son revealed a significantly higher (p < 0.05) frequency of pesticide-exposed farmers [25, 30, 34], the presence of MN among farmers as compared to control at every age MN as indicators for cytogenetic damage has been ac- group. Similarly, a significantly higher (p < 0.05) fre- knowledged as a useful biomonitoring tool in popula- quency of MN was observed among farmers as com- tions occupationally exposed to pesticides [35]. pared to control based on gender and smoking status. In contrast to MN results, BNu frequencies revealed As for BMI classification, a significant difference (p < no significant difference between the two studied groups. 0.05) in the frequency of MN was only seen among nor- The result could be influenced by the presence of un- mal and pre-obese of inter group comparison. A signifi- controlled exogenous factors such as environmental in- cant increase in MN frequencies (p < 0.05) was also fluence that contributed in the evaluation of cytogenetic detected for different duration of employment among damages [36]. Moreover, a lack of comparable data in farmers as compared to control. No significant differ- the literature concerning BNu frequencies as compared ence was seen in the frequency of BNu for inter group to MN indicated the unresolved role of BNu as a marker comparison. for nuclear abnormalities associated with pesticide The correlation between MN and BNu frequencies toxicity. with socio-demographic factors (age, years of employ- Pesticides exposure has been linked to a number of ment, BMI) and duration of exposure to pesticides and human health effects [37, 38]. Thus, an attempt was fertilizers were studied. As shown in Table 7, a signifi- made to identify the possible chronic diseases and symp- cant (p < 0.05) and positive correlation between the dur- toms that are commonly associated with pesticide ex- ation of fertilizers exposure and frequencies of BNu (r = posure among farmers. The number of farmers with 0.37, P = 0.02) and MN were (r = 0.42, P = 0.001) ob- reported hypertension, asthma, gastritis, insomnia, served. However, no other correlation of various runny nose and coughing were higher as compared to Abdul Hamid et al. Genes and Environment (2016) 38:7 Page 6 of 9 Table 6 Frequencies of MN and BNu in farmers and controls according to age, gender, smoking status, BMI and duration of employment. Data are presented as mean ± standard deviation (s.d) Controls (n = 30) Farmers (n = 39) Mean ± S.D Mean ± S.D Groups Frequency of MN (%) Frequency of BNu (%) Frequency of MN (%) Frequency of BNu (%) Age (years) 26–35 1.01 ± 0.32 2.01 ± 0.73 6.65 ± 0.92* 1.50 ± 1.41 36–45 1.47 ± 0.58 1.67 ± 0.94 6.22 ± 1.90* 1.51 ± 0.19 46–55 1.57 ± 0.72 1.49 ± 0.86 8.01 ± 3.09* 1.33 ± 0.45 56–65 1.10 ± 0.14 1.85 ± 0.64 6.61 ± 3.21* 1.63 ± 0.74 Gender Male 1.32 ± 0.55 1.85 ± 0.86 6.75 ± 3.01* 1.49 ± 0.61 Female 1.56 ± 0.70 1.32 ± 0.70 7.23 ± 2.43* 1.80 ± 0.83 Smoking status Smokers 1.25 ± 0.41 1.99 ± 1.08 6.82 ± 2.65* 1.39 ± 0.62 Non-smokers 1.45 ± 0.66 1.56 ± 0.71 6.83 ± 3.15* 1.65 ± 0.65 BMI Underweight - - 6.89 ± 3.04 1.49 ± 0.80 Normal 1.54 ± 0.61 1.59 ± 1.19 7.83 ± 3.19* 1.43 ± 0.77 Pre-obese 1.22 ± 0.47 1.72 ± 0.62 6.22 ± 2.58* 1.58 ± 0.57 Obese grade 1 1.66 ± 0.31 1.30 ± 0.61 6.25 ± 4.74 1.85 ± 0.35 Obese grade 2 1.57 ± 1.25 2.23 ± 0.91 - - Duration of employment (years) 1–10 1.39 ± 0.50 1.74 ± 0.99 7.04 ± 2.39* 1.34 ± 0.61 11–20 1.21 ± 0.64 1.74 ± 0.72 6.14 ± 3.02* 1.54 ± 0.42 21–30 1.67 ± 0.83 1.56 ± 0.93 5.80 ± 2.06* 1.76 ± 0.48 31–40 1.25 ± 0.53 1.63 ± 0.52 7.32 ± 4.20* 1.29 ± 0.88 41–50 - - 8.82 ± 3.71 1.71 ± 1.08 Total 1.39 ± 0.60 1.69 ± 0.84 6.83 ± 2.91* 1.54 ± 0.64 *Farmers versus controls (p < 0.05) the control group. Moreover, more than 50 % of farmers The use of appropriate PPE should give a significant claimed frequent experience of eye irritation, blurred vi- preventive impact in pesticide exposure among farmers. sion and numbness, suggesting a possible association to Significant increase in cytogenetic damage among pesticide exposure. However, due to lack of medical his- farmers with no or little protective clothing during pesti- tory and nutritional status data, direct association of the cide usage has been reported [39–41]. The usage of PPE, manifested symptoms with pesticide exposure could not as observe in this study can be regarded as unsatisfac- be established. Lack of PPE application during pesticide tory. Of the 7 PPE assessed, only 4 of the PPE (safety handling however can be one of the contributing factors helmets, long sleeved shirt, long pants and nose mask) for the reduced quality of health among the farmers. were used by the majority of the farmers. Furthermore, Table 7 Correlation between the frequencies of MN and BNu with the exposure to pesticides and fertilizers Frequency of MN Frequency of BNu (n = 39) (n = 39) Duration of pesticides exposure (hours) 0.15 0.28 (p = 0.37) (p = 0.16) Duration of fertilizers exposure (hours) 0.42 0.37 (p = 0.001)* (p = 0.02)* *p < 0.05 Abdul Hamid et al. Genes and Environment (2016) 38:7 Page 7 of 9 the use of eye goggles, gloves and boots were considered Tobacco smoke is a known genotoxicant and evi- low with a significant number of farmers who claimed dences on its ability to induce DNA damage have been that they never used these PPE throughout their farming comprehensively studied [45]. However, in this study the activities which put them at greater risk of percutaneous influence of smoking on cytogenetic damage showed no exposure to pesticide. In a study conducted by Lander significant correlation as observed in the frequencies of et al. [32], remarkable cytogenetic effects were noted in MN and BNu between smokers and non- smokers workers who did not use gloves, indicating the import- within the pesticide exposed group. Similarly, the effect ance of PPE usage. Although the humid and hot climatic of smoking was not found in MN and BNu frequencies conditions in Malaysia could be the factor that limits the within the control group. The lack of association ob- usage of full protective clothing [42, 43], continued ef- served between the frequencies of studied nuclear anom- fort to increase awareness among farmers on potential alies and tobacco smoke exposure has been reported pesticide hazards and the importance of PPE usage can previously [30, 54]. The effect of tobacco smoke expos- be useful to minimize pesticide related hazards at the ure on genotoxicity is influenced by type of cigarettes workplace. and frequencies of smoking [54] both of which were not The presence of nuclear anomalies can be influenced evaluated in this study. Thus, future studies focusing on by various confounding factors which include the ex- gathering comprehensive information on smoking habits ogenous (alcohol, diet, smoking and pesticide applica- among farmers can provide more definitive correlation tion) and endogenous (age and gender) factors [44–47]. and conclusive evidence. Some studies have reported a positive age effect on MN Duration of farming experience provides information frequencies among workers [33, 48]. However, in this on the degree of exposure to pesticide among farmers study, no significant differences were seen on the fre- [4]. In a review by Bolognesi [14], occupational exposure quencies of MN and BNu for both farmers and controls, to pesticide enhances genotoxic damage in a dose- which is in agreement to previously reported studies [34, dependent manner. In this study, higher frequencies of 49]. MN were observed among farmers with farming experi- Apart from age, the frequency of MN is also influ- ence of 41–50 years as compared to those with shorter enced by gender. Higher MN frequency in women than durations of farming experience. However, the observed in men was commonly observed in populations without difference was not statistically significant. It appears that exposure to any physical or chemical agents, with X duration of farming activities demonstrated no signifi- chromosome micronucleation being shown to contribute cant influence on the frequency of MN and BNu among to the increase of MN frequency in women [44]. In this farmers, which is in agreement with other reported stud- study however, no significant differences for the frequen- ies [55, 56]. To date, studies on the association between cies of MN and BNu between male and female subjects pesticide exposure and frequency of MN and BNu are were observed, though increased MN frequency in fe- inconclusive. Eastmond [57] have indicated that the per- male farmers were observed and is in accordance with a sistence of chromosomal damage is short-lived for acute previous report [49]. exposure while Scarpato et al. [58] noted that damage Level of education may affect the level of knowledge may be reduced during low exposure periods for sea- and awareness on the safety of pesticide handling [50]. A sonal workers. However, increased chromosomal damage previous study reported that the level of education with associated with duration of agricultural employment was at least high school education is required for farmers to reported by a number of studies [17, 58, 59]. The con- show a positive perception on the usefulness of PPE ap- flicting results from studies on pesticide-related cytogen- plication [51]. In a study conducted among Brazilian etic damage were influenced by many factors such as the farmers, the inability to understand the information dis- working environment, the studied populations, the type played on products’ labels led to increased exposure to of exposure to pesticides such as seasonal application of pesticides [52]. A survey conducted on 61 randomly se- pesticides, types of pesticide mixtures and the use of lected fruit-growers in a specific area of Turkey [53] re- PPE [19, 60, 61]. Moreover, Bull et al. [4] has stated in a vealed that pesticide practices were influenced by a review that chromosomal damage itself is regarded as number of characteristics, with those who consider pes- transient and sampling time play an important role on ticides as harmful consisted of farmers who are younger, determining the accuracy of the data. Sampling should better educated and have less experience in fruit- be done within 2 days following acute or cessation of growing. Our findings indicated that almost half of the chronic exposure to overcome false negative results. farmers did not have formal education beyond the sec- However, we have not considered this possibility which ondary education level. Therefore, levels of education could contribute to our non-significant finding. may affect the perception they may have towards the Assessments of correlation between MN and BNu fre- harmfulness of the pesticides. quencies with duration of pesticides and fertilizers Abdul Hamid et al. 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