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Exploring Cancer Development in Adulthood:Cholinesterase Depression and Genotoxic Effect FromChronic Exposure to Organophosphate PesticidesAmong Rural Farm ChildrenVivien How BS a , Zailina Hashim PhD a , Patimah Ismail PhD a , Salmiah Md Said MD a ,Dzolkhifli Omar PhD b & Shamsul Bahri Mohd Tamrin DVM PhD aa Faculty of Medicine and Health Sciences , Universiti Putra Malaysia , Selangor , Malaysiab Faculty of Agriculture , Universiti Putra Malaysia , Selangor , MalaysiaPublished online: 13 Jan 2014.

To cite this article: Vivien How BS , Zailina Hashim PhD , Patimah Ismail PhD , Salmiah Md Said MD , Dzolkhifli Omar PhD& Shamsul Bahri Mohd Tamrin DVM PhD (2014) Exploring Cancer Development in Adulthood: Cholinesterase Depression andGenotoxic Effect From Chronic Exposure to Organophosphate Pesticides Among Rural Farm Children, Journal of Agromedicine,19:1, 35-43, DOI: 10.1080/1059924X.2013.866917

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Journal of Agromedicine, 19:35–43, 2014Copyright © Taylor & Francis Group, LLCISSN: 1059-924X print/1545-0813 onlineDOI: 10.1080/1059924X.2013.866917

Exploring Cancer Development in Adulthood:Cholinesterase Depression and Genotoxic Effect From

Chronic Exposure to Organophosphate PesticidesAmong Rural Farm Children

Vivien How, BSZailina Hashim, PhDPatimah Ismail, PhD

Salmiah Md Said, MDDzolkhifli Omar, PhD

Shamsul Bahri Mohd Tamrin, DVM, PhD

ABSTRACT. Children are the vulnerable group in the agricultural community due to their earlyexposure to pesticides through the dynamic interplay between genetic predisposition, environment,and host-related factors. This study aims to identify the possible association between the depression inblood cholinesterase level and genotoxic effect among farm children. The results of micronuclei assayand comet assay showed that the reduced blood cholinesterase level from organophosphate pesticideexposure is significantly associated with an increase in chromosome breakage and DNA strand breaks.These genotoxicity end points suggest that farm children’s cells experience early DNA damage that maylead to uncontrolled cell proliferation during their adulthood. Thus, farm children who grow up nearpesticide-treated farmland have a higher probability of developing cancer than children with minimalor zero exposure to pesticides.

KEYWORDS. Children, cholinesterase depression, genotoxicity risk, organophosphate

INTRODUCTION

Organophosphate (OP) pesticides are com-monly used in paddy farmland. During theperiod of rice growth, these pesticides areapplied to fight against various insect pests.1

Nevertheless, studies have shown that only 1%

Vivien How, Zailina Hashim, Patimah Ismail, Salmiah Md Said, and Shamsul Bahri Mohd Tamrin areaffiliated with the Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, Malaysia.

Dzolkhifli Omar is affiliated with the Faculty of Agriculture, Universiti Putra Malaysia, Selangor,Malaysia.

Address correspondence to: Zailina Hashim, PhD, Department of Environmental and Occupational Health,Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia(E-mail: zailina@medic.upm.edu.my).

of the estimated annual pesticide applicationscould reach the target pests.2 Several studieshave suggested that agricultural communitiesliving near farmland are highly exposed to pes-ticides during the interval between applicationand drainage.3 This may be due to the trans-port and fate of emission, drift, deposition,

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36 CHILDHOOD OP EXPOSURE AND CANCER IN ADULTHOOD

sedimentation, leaching, and drainage of pes-ticides from residential settings and sprayingactivity.4,5 Children are particularly vulnerableto pesticides due to their unique age-relatedbehaviors.6,7 Their route and source of exposurediffer from those observed in adults.8 Therefore,children who live in the agricultural communityare at high risk of additional health problemsfrom environmental exposure to pesticides.9

OP pesticides are known to cause depres-sion in blood acetylcholinesterase (AChE). Thishas been widely suggested by occupationalpractitioners as an effective marker to moni-tor OP toxicity level among farm workers.10−12

Monitoring of AChE level has shown that youngchildren from agricultural communities are sus-ceptible to the harmful and toxic effects ofOP exposure.13,14 Studies15,16 have shown thatAChE enzymes are usually low in young infants;therefore, they are more vulnerable to the path-way of OP exposure than farm workers. Sincethere is no other chemical susceptibility toinduce a cholinesterase reduction in the blood,AChE inhibition can be used as a biomarkerof exposure to indicate OP exposure among thestudy population.

Hundekari et al.17 have suggested that bloodAChE inhibition may initiate cellular dysfunc-tion, leading to increased free radical and reac-tive oxygen species (ROS) activities in ery-throcytes. This may cause cellular oxidativestress due to insufficient balance by antioxi-dants, leading to an alteration in cell struc-ture formation.17,18 The elevated ROS leveland decreased regulation of ROS scavenger andantioxidant enzymes are known to cause damageto the nuclei acids, proteins, and lipids. To date,oxidative stress has been considered an effec-tive measure to determine the cumulative OPexposure. As shown in Figure 1, the effectmay exert chromosomal instability, mutation,loss of organelle functions, membrane damage,and other multiple stages of the carcinogenesisprocess.19,20

The effect of children’s exposure to pesticidescan be affected by their pharmacokinetic fac-tors, which are different from those of adults.Children have a lower metabolic capacity thanadults; thus, it is tempting to speculate that acompromise mechanism for an effective bloodflow, oxygen uptake, and release will also beaffected.21,22 As discussed, studies have shown

FIGURE 1. Conceptual model of organophosphate exposure and genotoxicity effect (adaptedfrom Waris and Ahsan44).

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How et al. 37

that depression of blood cholinesterase can beused to indicate OP exposure and increased ROSactivities as a consequence of chronic expo-sure. The question arises as to whether changesin AChE due to OP exposure might contributeto genotoxicity risk among farm children fromtheir early cumulative exposure.

This study aims to fill the knowledge gapwith our hypothesis that there is a relation-ship between blood AChE and the genotoxicityeffect of farm children’s OP exposure in a ruralagricultural environment.

METHODS

This study was approved by the EthicsCommittee of the University Research InvolvingHumans of Universiti Putra Malaysia (UPM/

FPSK/100-9/2-JKEUPM). A cross-sectionalcomparative study was conducted amongfarm children during the vegetative andreproductive phases of paddy farming whereorganophosphate (OP) pesticides are used.

During the screening process, children whoreported with malnutrition, anemia, nephriticproblems, or hepatic diseases were excludedfrom participating in this study, as they tend tohave abnormal levels of blood cholinesterase.Only medically fit children were recruited.Following the screening process, children whofulfilled the inclusion criteria were first askedto obtain consent from their parents; selectionthrough simple random sampling was conductedfrom those who returned with an acknowl-edged consent letter.

A total of 95 children aged 9 to 11 yearswhose schools were located less than 2 km frompaddy farmland, had at least one family memberworking in the farmland, and have stayed in theagricultural village since they were born werechosen. Another 85 school-aged children whoseschool and house were located at least 10 kmfrom the agricultural community were recruitedfor comparison purposes.

In order to minimize physical injury andreduce unwanted invasive techniques, 10 µL ofcapillary blood was collected by finger prick andexfoliated buccal mucosa cells were collected bygently scraping the mucosa of the inner lining of

one or both cheeks. In this study, blood sampleswere used to evaluate OP exposure, and buccalmucosa cells were sampled for genotoxicityassessment.

Biomarker of Exposure (BloodCholinesterase Level)

Monitoring blood cholinesterase level is auseful tool to evaluate the potential OP exposureamong the study population. In this study, ablood cholinesterase test kit (Lovibond, AF267;Tintometer Ltd., UK) was used to monitor theOP exposure level with a few drops of bloodfrom the finger tip. Each sample was analyzedon site as guided by the test kit; readings wereconducted after 4 to 5 minutes based on the col-orimetric principle of the color indicator (bro-mothymol blue solution) used.

Finger-pricked blood was pipetted to around test tube containing 0.5 mL indicatorsolution. Then, 0.5 mL of substrate solution(acetylcholine perchlorate) was added to the testtube. The contents were mixed thoroughly andthen transferred to a 2.5-mm cuvette. Next, thecuvette was placed in the color compartmentto view the indicator color through a prism.Analysis of the results was based on the acid-base blood cholinesterase level (%) obtained(Table 1).

The primary mechanism of OP toxicityis through phosphorylation of the acetyl-cholinesterase enzyme (AChE) at the nerveendings.10,12,23 The colorimetric princi-ple is based on the breakdown process ofacetylcholine (Equation 1). The presence ofacetic acid determines the normal reactionbetween acetylcholine and acetylcholinesterase;

TABLE 1. Analysis of Results of BloodCholinesterase Level (%)

Percentage (%) Indicator

100.0–75.0 Normal62.5–50.0 Overexposure37.5–25.0 Serious over exposure0.0 Very serious and dangerous over

exposure

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38 CHILDHOOD OP EXPOSURE AND CANCER IN ADULTHOOD

otherwise, it performs bases due to theaccumulation of acetylcholine.

Acetylcholine → Acetic acid + Choline (1)

A biomarker of effect is a biological indi-cator of the body’s response to the exposure.The effect of chronic OP exposure is deter-mined by monitoring the genotoxicity conse-quences of subclinical changes. In this study, theend point of genotoxicity effect was determinedby micronuclei (MN) assay and comet assay.Buccal cells were sampled because they providean equivalent sensitive biomarker of genotoxicdamage.24,25

MN assay was conducted based on the stan-dard protocol described previously.26 The endpoint is to measure the cells with the presenceof MN, which is scored based on the cells pre-sented with a main nucleus and smaller nucleicalled micronuclei (MN). The MN are eitherround or oval in shape, and their diameter rangedbetween 1/3 and 1/10 the diameter of the mainnucleus. The level of DNA damage was mea-sured using comet assay following the methoddescribed previously27 and modified based onstandard procedures from the comet assay kit(Trevigen, Gaithersburg, MD, USA). The taillength was measured (µm) to indicate the dis-tance of DNA migration from the body of thenuclear core, and it was used to evaluate theextent of DNA damage.

Statistical Analysis

Univariate analysis was performed to sum-marize the descriptive demographic informationof the study population. An independent t testwas carried out to compare the significant dif-ferences of blood cholinesterase level and geno-toxicity outcome between farm children andthe control group. Simple linear regression wasconducted to evaluate the relationship of the pre-dictive factors of the biomarker of effect (geno-toxicity profile) among the study population.

RESULTS

Table 2 shows that the study population wasequally distributed from 9 to 11 years of age.

TABLE 2. Demographic Characteristics of theStudy Population

Characteristic Farmchildren(N = 95)

Control(N = 85)

Age9 32 (33.68) 26 (30.59)10 31 (32.63) 31 (36.47)11 32 (33.68) 28 (32.94)

GenderFemale 54 (56.84) 43 (50.59)Male 41 (43.16) 42 (49.41)

Body Mass Index (BMI)Underweight 21 (22.11) 8 (9.41)Normal 58 (61.05) 56 (65.88)Overweight 11 (11.58) 14 (16.47)Obese 5 (5.26) 7 (8.24)

Parent smokerNo 46 (48.42) 25 (29.41)Yes 49 (51.58) 60 (70.59)

Other household smokerNo 67 (70.53) 67 (78.83)Yes 28 (29.47) 18 (21.17)

Of the 95 farm children, 43.1% were boysand 56.8% were girls. The body mass index(BMI) was categorized based on the Centers forDisease Control and Prevention (CDC) BMI-for-Age Growth Charts for children. Most ofthe farm children were underweight (22.1%) orof normal weight (61.5%); however, the controlgroup had more children with normal healthyweight (65.8%). Overall, at least 50% of thechildren inhaled environmental tobacco smokefrom their smoking parents or other familymembers.

The level of blood cholinesterase is usedto reflect the cholinesterase depression amongfarm children from their early chronic andlow level of OP exposure. Table 3 high-lights farm children’s overexposure (56.31%)to OP pesticides compared with normal bloodcholinesterase (79.55%) content in the controlgroup. The blood cholinesterase level is deter-mined based on the pH color indicator, whichchanges based on the colorimetric principle forquick field sampling among children.

Two genotoxicity assessments were used todetermine the biomarker of effects from low-level and chronic exposure to OP pesticidesamong the study population. Table 4 shows at

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TABLE 3. Comparison of Blood Cholinesterase Level (%) Among the Study Population

Biomarker of exposure Farm children Control t statistica P valueMean (SD) Mean (SD)

Blood cholinesterase level 56.32 (12.35) 79.55 (13.48) −12.072 <.05∗

aIndependent t test, N = 95 (farm children), N = 85 (control).∗P value is significant at .05 level.

TABLE 4. Comparison of Biomarker of Effect (Genotoxic Risk) Among the Study Population

Biomarker of effect Farm children Control t statistica P valueMean (SD) Mean (SD)

Micronuclei (per 1000 cells) 5.05 (2.45) 2.92 (1.54) 7.09 (160) <.05∗Comet tail length (µm) 8.45 (3.89) 4.38 (1.66) 9.28 (130) <.05∗

aIndependent t test, N = 95 (farm children), N = 85 (control).∗P value is significant at .05 level.

TABLE 5. Predictor Factors of Micronuclei Formation Among the Study Population (N = 180)

Variable SLRa MLRb

b (95% CI) P value b (95% CI) P value

Age (year) 0.105 .623 — —(−0.318, 0.529)

Body mass index (BMI) −0.084 .139 — —(−0.196, 0.028)

Gender −0.629 .070 −0.681 .038(−1.310, 0.052) (−1.325, −0.038)

ETS (parents) 0.801 .023∗ — —(0.109, 1.492)

ETS (other family member) 0.305 .444 — —(−0.480, 1.089)

Blood cholinesterase level −0.044 <.001∗ −0.045 <.001∗(−0.063, −0.026) (−0.063, −0.026)

Note. Gender [1 = boy; 2 = girl]; ETS = environmental tobacco smoke (passive smoking) [1 = Yes, 2 = No].aSimple linear regression; bMultiple linear regression.R2 = .165. The model fits reasonably well. The prediction model of micronuclei formation among study population is:

MN frequency = 8.116 − [0.681 × gender

] − [0.44 × blood cholinesterase level

]

least 1.5- to 2-fold significant increase in geno-toxicity risk among farm children comparedwith the control group. In this context, dif-ferent effects of DNA alteration on a buccalcell are demonstrated by MN assay and cometassay. The MN assay is commonly used todetect fixed mutation that persists in at leastone mitotic cycle through the detection of cen-tromere present, and the comet assay is a

marker for repairable DNA lesions or single-and double-stranded DNA breaks in a singlecell.

Tables 5 and 6 show the simple linearregression used to identify the potential predic-tor factors of genotoxicity effects among thestudy population. There is a significant linearrelationship between micronuclei frequency andcomet tail length of the blood cholinesterase

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40 CHILDHOOD OP EXPOSURE AND CANCER IN ADULTHOOD

TABLE 6. Predictor Factors of Comet Tail Length Among the Study Population (N = 180)

Variable SLRa MLRb

b (95% CI) P value b (95% CI) P value

Age (years) 0.144 .671 — —(−0.523, 0.810)

Body mass index (BMI) 0.037 .681 — —(−0.140, 0.214)

Gender −0.318 .563 — —(−1.399, 0.764)

ETS (parents) 0.717 .200 — —(−0.382, 1.816)

ETS (other family member) −1.101 .078 — —(−2.328, 0.125)

Blood Cholinesterase level −0.087 <.001∗ −0.085 <.001∗(−0.116, −0.059) (−0.115, −0.056)

Note. Gender [1= boy; 2 = girl]; ETS = environmental tobacco smoke (passive smoking) [1 = Yes, 2 = No].aSimple linear regression; bMultiple linear regression.R2 = .185. The model fits reasonably well. The prediction model of comet tail length among study population is:

Comet tail length = 12.548 − [0.085 × blood cholinesterase level

]

level (P < .05). Table 5 shows that the genderand blood cholinesterase level are significantpredictor factors for micronuclei formationamong the study population. Results showthat boys and reduced blood cholinesterasecontributed to 16.5% of micronuclei frequencyvariation. However, 18.5% of the comet taillength variation is explained by the changes inblood cholinesterase level among the study pop-ulation. Overall, a decrease of 1 level of bloodcholinesterase will increase the micronucleiformation by 0.044 and increase the comet taillength by 0.087.

DISCUSSION

By utilizing biomarkers of exposure-effectcontinuum, the study suggests that farm childrenhave markedly reduced blood cholinesterase andincreased risk of genotoxicity effect comparedwith children from the control group. Theseresults are in agreement with other studies.13,17

Farm children who revealed an inhibition ofcholinesterase activity followed by a significantincrease in MN frequency and comet tail lengthclearly highlighted their cumulative OP expo-sure throughout their lifetime. Although paststudies on genotoxicity risk in children from

pesticide exposure are limited, sufficient evi-dence has been gathered to show that farmchildren are vulnerable to developing cancer intheir adulthood.

Previous studies of children’s pharmacoki-netic emphasized their low metabolizing capa-bility in response to toxic chemicals, whichtends to enhance the toxicity and carcinogenicityin their early childhood.13 In humans, oxidativestress is thought to be involved in the devel-opment of cancer from the chronic imbalancebetween the systemic manifestation of ROS andthe biological system’s ability to either detox-ify the reactive intermediates or to repair theresulting damage.28 In order to examine thedegree of cellular damage, genotoxicity effectsare thus used as an indicator for early can-cer risk or DNA damage that is potent toinduce mutated cells and cause cells to becomecancerous.

In the current study, older male farm childrenhad increased genotoxic effects in response toOP pesticide exposure. Past studies on farm chil-dren have shown the unique behavioral patternthat exposes them to multiple routes and sourcesof pesticide contamination,21,29,30 for example,hand-to-mouth hygiene practices, outdoor activ-ities with contaminated sediments and water, orpesticide from the take-home pathway.

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Furthermore, there are contradictory resultsrevealed by children’s BMI association withgenotoxicity effects. This may be due to thedifference in the cellular responses to environ-mental toxicants throughout childhood develop-ment. Past studies have shown that infants whohad prenatal exposure to pesticides are morelikely to have decreased birth weight and length,which may hamper normal development.31,32

However, a study from Gonzalez et al.14 sug-gested that obesity is one of the host factorsthat may affect paraoxonase 1 (PON1) enzymeactivities during pesticide metabolism. The defi-ciency of the PONI enzyme is known to beassociated with an increase in oxidative stressfrom OP pesticide exposure. Although no signif-icant relationship is highlighted in this context,gender, age, and BMI are among the importantdemographic parameters that need to be high-lighted for their influence on the genotoxicityeffect.

Environmental tobacco smoke is known to bea human carcinogen.33,34 It contains toxic, car-cinogenic, and mutagenic chemicals, togetherwith the stable and unstable ROS in the par-ticulate and gas phases that are susceptible toinducing cellular oxidative damage.35 Likewise,early exposure of young children to passivesmoking might cause an additive effect for earlycancerous cell development.36 Under sustainedstress in the exogenous and endogenous environ-ment, the production of ROS is enhanced, whichfurther models the carcinogenic process.18,20,37

Even so, past studies highlighting the genotoxi-city effects caused by parental smoking duringpregnancy or environmental tobacco smoke inrelation to childhood cancer are still controver-sial and inconclusive, as shown in this study.38,39

In view of the different responses shown byMN and comet assays with the depression inblood cholinesterase level, the study suggeststhat a different genotoxicity stress occurs fol-lowing cumulative exposure to genotoxic com-pounds. In fact, cells can initiate a responsesystem by inducing a cell cycle arrest in suf-ficient time for DNA repair under continuousenvironmental stress. If this balance mechanismis disturbed, an insufficient amount of excessiveapoptosis causing cell atrophy might result inuncontrolled cell proliferation, called cancer.40

For instance, some investigation revealed thatfollowing oxidative stress, DNA will tend tobe damaged and cause genomic instability. Thismay help to trigger cancer initiation and pro-gression over time.37,41 Thus, farm children areat disproportionate risk of developing genotox-icity risk from OP pesticide exposure, whichsuggests a relative increase in early cancer-ous cell development. Nevertheless, past studiesshowed that significant damage to cell structureand its functions until it is capable of inducingsomatic mutation neoplastic transformation overtime is expected to remain underappreciated for>15 years.42

Although this research was carefully pre-pared, it is unavoidable that a certain degreeof subjectivity can be found. First, due to thetime limit, this study was conducted on only asmall number of children aged 9 to 11 years.To generalize the results for larger groups, thestudy should have involved more participantsat different age levels. Second, the biomarkerdata used are too limited to draw the conclusionfor genotoxicity outcome. It is recommendedthat the weight-of-evidence approach shouldbe considered, as suggested in “Guidelines forCarcinogen Risk Assessment,”43 as an interpre-tative method of claiming the risk that couldmerge from the chronic use of pesticides.

In conclusion, this study suggests that farmchildren’s early chronic OP exposure causingcholinesterase inhibition is likely to establishgenotoxicity risk that may lead to cancerous celldevelopment if not repaired properly. From thesubtle effects observed in this study, it can stillbe unequivocally stated that a reasonable geno-toxicity risk from OP pesticide exposure duringthe early life of farm children might not causea direct correlation of cancer incidence, but itis sufficient to postulate a high risk of cancerdevelopment in their adulthood.

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