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Cytogenetic damage in peripheral blood lymphocytes of children exposed to pesticides in agricultural areas of the department of Cordoba, Colombia
Accepted Manuscript Title: Cytogenetic damage in peripheral blood lymphocytes of children exposed to pesticides in agricultural areas of the department of Cordoba, Colombia Authors: Javier Alonso Ruiz-Guzm´an, Pamela ´ G´omez-Corrales, Angel Cruz-Esquivel, Jos´e Luis Marrugo-Negrete PII: DOI: Reference:
S1383-5718(16)30447-8 https://doi.org/10.1016/j.mrgentox.2017.10.002 MUTGEN 402848
To appear in:
Mutation Research
Received date: Revised date: Accepted date:
22-12-2016 4-10-2017 9-10-2017
´ Please cite this article as: Javier Alonso Ruiz-Guzm´an, Pamela G´omez-Corrales, Angel Cruz-Esquivel, Jos´e Luis Marrugo-Negrete, Cytogenetic damage in peripheral blood lymphocytes of children exposed to pesticides in agricultural areas of the department of Cordoba, Colombia, Mutation Research/Genetic Toxicology and Environmental Mutagenesis https://doi.org/10.1016/j.mrgentox.2017.10.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cytogenetic damage in peripheral blood lymphocytes of children exposed to pesticides in agricultural areas of the department of Cordoba, Colombia Javier Alonso Ruiz-Guzmána*, Pamela Gómez-Corralesa, Ángel Cruz-Esquivela, José Luis Marrugo-Negretea. a
Water, Applied, and Environmental Chemistry Group, Laboratory of Toxicology
and Environmental Management, Universidad de Córdoba. Montería, Colombia. Postal address: Cra 6 # 76 – 103 Montería, Postal code 354.
* Javier Alonso Ruiz-Guzmán (corresponding author) Water, Applied, and Environmental Chemistry Group, Universidad de Córdoba, Montería, Colombia E-mail: [email protected] Phone: + 57 (4) 7860381 ext 310. Montería, Colombia Fax: + 57 (4) 7860381 ext 310. Montería, Colombia Postal address: Cra 6 # 76 – 103 Montería, Colombia. Postal code 354. Universidad de Córdoba, Toxicology and Environmental Management Laboratory
Pamela Gómez-Corrales [email protected]
Ángel Cruz-Esquivel [email protected]
José Luis Marrugo-Negrete E-mail: [email protected]
Highlights - Pesticides and/or its metabolites were recorded in the children’s urine samples - Frequency of cytogenetic damage was higher in the exposed children that in control group - Results suggest a permanent health risk for pesticide exposure
Abstract Pesticides offer benefits, like optimization of agricultural production and disease control; however, these toxic substances can contaminate the environment and pose risks to human health. The aim of this study was to assess pesticide exposure and frequency of cytogenetic damage in infant populations in agricultural areas of the department of Córdoba, Colombia. Urine and peripheral blood samples were taken from children living in the villages of La Ceibita (municipality of Cereté), Cabuya (municipality of San Carlos), Aguas Negras (municipality of Montería), Pelayito (municipality of San Pelayo), and the city of Monteria (control group). The work evaluated biomarkers of exposure to pesticides (atrazine urinary concentrations (ATZ) and its metabolites) and biomarkers of cytogenetic damage (micronucleus frequency (MN), nuclear buds, and apoptotic cells in peripheral blood lymphocytes). Measurable ATZ concentrations and/or its metabolites were recorded in the Pelayito, Aguas Negras, and Cabuya zones, which had higher MN frequencies, nuclear buds, and apoptotic cells than the control. Infant exposure to one of the more-often used pesticides in the agricultural areas evaluated and an increasing trend in the frequency of markers of cytogenetic damage in the groups of the agricultural areas, as compared to the control group, were evident.
Keywords: micronucleus test, pesticides, atrazine, DNA damage (Source: DeCS)
1. Introduction Pesticide manufacture uses around 1000 active ingredients, sprayed mainly in rural agricultural areas to reduce and/or eliminate populations of insects, fungi, and weeds, among other things, for crop protection [1]. Although these substances offer benefits, such as optimization of agricultural production and disease control, their extensive use tends to pollute the air, water, soil, and food [2], representing risks to human health and the environment [3].
Adverse effects on human health caused by exposure to pesticides include discomfort, permanent damage, and irreversible changes in the nervous, reproductive, and hormonal systems among others. Additionally, DNA alterations that can be transmitted or manifested in subsequent generations, some of which have been classified as carcinogenic and probable or possible carcinogenic for humans, and which may be lethal in cases of severe poisoning [4 – 7]. The association between mutagenic, genotoxic, and carcinogenic activities in humans and exposure to pesticides has been widely recognized [8, 9]. Using biomarkers of cytogenetic damage, such as micronucleus testing (MN), offers evidence of early DNA damage; it has been used to evaluate the effects of these substances on the genetic material of adult and infant populations with occupational or environmental exposure [3, 10 - 12].
These health changes caused by pesticides can occur more easily in infant populations because of their vulnerable stage of development, which makes them more sensitive to the effects of these pollutants [13]. Children may respond differently than adults in the detoxification process, DNA repair, and cell proliferation [14], given that their metabolism is more rapid than that of adults [15]. In this sense, it is more likely that early DNA alterations not adequately repaired in children can lead to a faster development of major alterations in health.
In Colombia, despite being a country with a high use of pesticides for agriculture [16], no studies exist on pesticide exposure in children or evaluations of
cytogenetic alterations in any class of cells in infant populations in agricultural areas potentially exposed to pesticides. However, some studies have evaluated the frequency of chromosome aberrations, MN, deficiencies in lymphocyte DNA repair activity [17], acetylcholinesterase activity [18, 19], and genotoxic risk with a lymphocyte micronucleus assay [20] in adult populations with occupational exposure to pesticides. The aim of this study was to evaluate pesticide exposure and frequency of cytogenetic damage in infant populations in agricultural areas of the department of Córdoba, Colombia. The study used as exposure biomarkers, urinary concentrations of one of the most-often used pesticides in the study areas (atrazine and its metabolites) and as biomarkers of cytogenetic damage, the MN frequency, nuclear buds, and apoptotic cells in peripheral blood lymphocytes.
2. Materials and Methods
2.1. Study area The study was conducted in rural farming areas of the middle basin of the Sinú River in the department of Córdoba, Colombia (Ceibita village - municipality of Cereté: 8°49'35" N - 75°39'03" W, Cabuya - municipality of San Carlos: 8°46'18" N - 75°43'53" W, Aguas Negras - municipality of Montería: 8°48'44.96" N 75°49'1.81" W, Pelayito - municipality of San Pelayo: 8°56'20.8" N - 75°49'5.74" W, and the city of Monteria as the control group: 08°45′27″ N - 75°53′08″ W). The middle basin of the Sinú River is between 10 and 20 m.a.s.l., with an average annual temperature of 29 °C, annual rainfall of 1200 mm, and 85% relative humidity [21]. Traditionally, much of this region has been dedicated to agriculture because of the low concentrations of salts in the Sinú River, a characteristic that provides soil conditions suitable for growing a wide variety of crops (corn, rice, cotton, and cassava, among others), with corn and cotton in the main study areas [22]. Although no official statistics or records are available for the quantities of pesticides used in this region, it is clear that commercial agriculture has evolved and expanded over the decades, which has used and continues to consume large amounts of these substances for crop protection.
2.2. Type of study and target population An exploratory cross-sectional study was conducted in which the frequency of cytogenetic damage markers in peripheral blood lymphocytes (micronucleus assay) and the presence or absence of markers of exposure (metabolites) to pesticides was evaluated in urine samples from children. The study included children between 5 and 15 years of age from both genders (male and female), with a minimum stay in the study area of three years. To reduce the likelihood of bias in the interpretation of the results, children with birth defects, chronic degenerative diseases, requiring permanent medication, who have been exposed to radiation (Xray examinations) within the previous six months and those who that manifested having had the habit of smoking or consuming liquor were excluded from the study.
In each area or study population, an enrollment list (with the parents) of 20 children was made of which a smaller number in each population (Aguas Negras = 17 children, Cabuya = 8 children, Pelayito = 12 children, Ceibita = 13 children, and Control group = 13 children) finally participated in the study, given that on the sampling day some children did not allow to be sampled, or their parents or legal guardians (an adult relative) were unable to attend. All of the participants were selected through non-probability intentional sampling.
2.3. Collection of information A survey was conducted with the parents or legal guardians of children to collect demographic information, regarding exposure characteristics and health status of the children. From this information, the study participants were selected based on the criteria described in the previous section.
2.4. Ethical aspects The Ethics Committee of the Faculty of Health Sciences at Universidad de Córdoba, Colombia approved the study. According to Resolution No. 8430 of 1993 from the Colombian Ministry of Health and Social Protection [23], prior to sampling,
the parents or legal guardians of each child were asked to sign an informed consent form that was explained clearly and completely, indicating the purpose, scope, and limitations of the study and the associated risks. Signing this document meant the parents authorized participation by the children.
2.5. Sampling Urine samples were collected in sterile plastic containers early in the day (between 6:00 am and 7:00 am) by the participants themselves with help from their parents, who were provided instructions for labeling and cold storage. Peripheral blood samples were taken between 7:00 am and 9:00 am through venipuncture on the forearm with heparinized vacutainer tubes (4 mL). Both samples were stored and transported for 1 to 1.5 h in controlled-temperature containers (20 to 22 °C) for the blood samples and plenty of ice for the urine samples, to the Toxicology and Environmental Management Laboratory at Universidad de Córdoba for immediate processing.
2.6. Biomarkers of exposure to pesticides Urinary concentrations of one of the most-often used pesticides in the study areas, atrazine (ATZ) and its metabolites atrazine desisopropyl (ADI) and atrazine desethyl-desisopropyl (ADDI), were used as biomarkers of exposure to pesticides. The analysis was performed with gas chromatography-mass spectrometry (GC-MS Trace 1310 Thermo Scientific gas chromatograph and ISQ Thermo Scientific Mass Spectrometer) after liquid-liquid extraction with ethyl acetate and diethyl ether, and clean-up of the extracts (Florisil and sodium sulfate), under the following chromatography conditions: injection volume = 1 μL, detector temperature = 285 °C, DB5MS capillary column: 30 m x 0.2 mm x ID 0.20 µm and Helium as carrier gas [24]. The limits of detection (LD in ppb) and quantification (LC in ppb) of the method were ATZ: LD = 0.451; LC = 0.455; ADI: LD = 3.194; LC = 3.200; ADDI: LD = 7.123; and LC = 7.130.
2.7. Biomarkers of cytogenetic damage
Frequency of MN, nuclear buds, and apoptotic cells in peripheral blood lymphocytes was used as indicators of cytogenetic damage, assessed with micronucleus assay with block cytokinesis with cytochalasin B, according to the protocol described by [25]. The lymphocytes were separated from the whole blood sample using the Ficoll-1077 technique (Histopaque-1077™), per manufacturer’s instructions. The lymphocyte culture was done in RPMI 160, supplemented with fetal bovine serum, L-Glutamine, antibiotics, and phytohemagglutinin for 72 h at 37 °C and 5% CO2, adding cytochalasin B at 44 h. At the end of cultures, preparations were made on microscope slides with a cytological centrifuge (Cellspin Hanil) at 500 rpm for 5 min, which were dried at room temperature, fixed with 80% cold methanol (v/v), and colored with a 10% Giemsa solution (v/v) in a Sörensen buffer for 15 min. The frequency of DNA damage biomarkers (MN and nuclear buds) was scored in 2000 binucleate cells (1000 duplicated) and the frequency of apoptotic cells was analyzed in 1000 viable cells. Frequency of all alteration was expressed as frequency of alterations for 1000 cells scored and the analyses were performed in an optical microscope (Olympus CX41) at 1000X magnification.
2.8. Statistical analysis Kruskal-Wallis (KW) analysis and Dunn comparison of average-range test were used to assess differences in the frequency of markers of cytogenetic damage (MN, nuclear buds and apoptotic cells) and sociodemographic variables among the study groups. A Spearman correlation analysis was carried out to assess the relationship between markers of cytogenetic damage and concentrations of ATZ and its metabolites (ADI and ADDI) in urine samples, as well as sociodemographic variables that can influence the frequency of these biomarkers, like age, sex, time of residence in the area, distance from home to crops, frequency of pesticide applications, and frequency of pesticide odor detection at home. The analyzes were performed with SPSS Statistics 22 and, in all cases, p <0.05 was established as the criterion for statistical significance.
3. Results
Table 1 presents the sociodemographic characteristics and the use and management of pesticides in the study populations. The distribution by age and sex was similar in the study groups without significant difference among them (age: p = 0.11; sex: p = 0.12), as well as the time of residence in the area (p = 0.30) because most of the children have lived in their populations from birth. All participants attend school and consume potable water from the aqueduct, most without additional pre-treatment prior to consumption, except in the control group where most reported using filters. Most of the children live with parents who do not smoke; most of the children in Pelayito live with parents who work with pesticides, unlike the other groups where the behavior of this variable was inverse and most parents of children in Pelayito have not completed secondary education.
Regarding the management and use of pesticides, except for Pelayito, the parents or guardians of the children reported not storing pesticides inside the house or reusing the packaging for household chores (except four cases in Ceibita for this last variable); all believe that pesticides are dangerous to health and most believe their children are exposed to these substances (except in the control group). With the exception of two cases in Aguas Negras, children do not handle pesticides; however, several children in different study areas perform agricultural tasks, such as fertilizing, harvesting, and cleaning.
Table 2 presents the classification of pesticides commonly used in the study areas, according to their dangerousness, chemical structure, and use, as well as the status of restriction or prohibition in Colombia and the world, and their genotoxic effect in human cells. Mainly, insecticides and herbicides of different types are used and most of these have some sort of restriction in Colombia or other countries.
Table 3 shows the ATZ concentrations and its metabolites (ADI and ADDI) in the urine samples from the different areas evaluated. Among these, Pelayito recorded
the highest percentage of samples with measurable concentrations of the unmodified pesticide (ATZ) and its metabolites, while Aguas Negras only recorded three individuals with measurable concentrations of the ADDI metabolite and only one individual in Cabuya recorded measurable concentrations of the ADI metabolite. No measurable concentrations of any of these compounds was recorded for the samples from Ceibita or the control group.
Table 4 contains the cytogenetic damage markers evaluated. The highest frequencies of MN and apoptotic cells were recorded in Pelayito, Aguas Negras, and Cabuya without significant differences among them (p> 0.05), while the Ceibita and the control groups recorded the lowest rates without significant difference between them (p> 0.05). The frequency of nuclear buds was higher in the Aguas Negras and Cabuya groups, intermediate in Pelayito and Ceibita, and lower in the control group.
No significant correlations (p> 0.05) were noted between the cytogenetic damage markers (MN, nuclear buds, and apoptotic cells) evaluated and the concentrations of ATZ and its metabolites (ADI and ADDI). In addition, no significant differences were recorded in any of the study areas for the sociodemographic variables that can influence these cytogenetic damage markers, such as age, sex, time of residence in the area, distance from home to crops, application frequency of pesticides, and frequency of pesticide odor detection at home. When these sociodemographic variables among the groups of the agricultural areas were compared, significant differences (p <0.05) in the distance from the house to crops, being lower in Pelayito, and frequency of crop spraying, which was higher in Pelayito, were found (Table 5).
4. Discussion
The sociodemographic variables of the study populations show a desirable scenario in terms of education, given that all of the children attend school. This is
especially important in agricultural areas because children who do not attend school may be attracted to the activities in the area, such as agriculture, placing them at risk of greater proximity and exposure to pesticides used in this activity. As for the habits and perception of the health hazards from exposure to pesticides, the apparent parental care of children is notable because, except for the cases in Pelayito and Ceibita, the parents stated that they do not keep pesticides in the house or reuse their packaging in the household. They know that pesticides are dangerous to health and do not allow children to manipulate pesticides (except for two cases in Aguas Negras). However, these few cases of children who handle pesticides (helping in preparations) and several children involved in agricultural tasks (fertilizing, cleaning, and harvesting), as well as the reuse of pesticide containers in household chores, possibly linked to low education levels of the parents (Table 1), suggests the existence of undesirable and avoidable situations that pose risk of exposure to these substances and the consequential adverse effects on health.
In this regard, the presence of ATZ and its metabolites (ADI and ADDI) in urine samples (Table 3) confirm infant exposure, mainly in the participants from Pelayito and Aguas Negras, to one of the pesticides with increased use in the agricultural areas studied. Because this pesticide is removed from the body fairly quickly (24 to 48 h), concentrations in urine only indicate recent exposure [49]; therefore, the absence of measurable concentrations of these markers in the other samples in the agricultural areas may be related to its rapid elimination from the body. The absence of ATZ or its metabolites in a sampling point, as in this study, does not rule out, with sufficient certainty, exposure to it, considering that the populations evaluated are in an environment with potential chronic exposure, given the proximity of housing to the fields where crops are grown throughout the year, with corn being one of the principal crops in which ATZ is one of the most widely used pesticides [50].
This may represent chronic exposure not only to ATZ, but also to the rest of pesticides used on crops, most of which have shown genotoxic effects on different types of human cells [30 - 32, 34, 35, 39, 40, 42 - 44] and have been banned in other countries because of the danger (Table 2). This situation poses a permanent risk of adverse effects on health associated to these substances, which can be caused, for example, by altering processes and genetic instability, evident by evaluating cytogenetic damage biomarkers, such as MN, nuclear buds, and cell apoptosis, among others [3, 25, 51], which have been considered as early biomarkers of cancer risk [52 - 54].
Atrazine is a pesticide known as an endocrine disruptor linked to birth defects in children with prenatal exposure with sufficient evidence of its adverse effects on the health of fetuses, infants, and children [55 - 58]. In contrast, international toxicological evaluations of ATZ have concluded that evidence is insufficient on its carcinogenic effects in humans [55, 59]. In this study, no statistically significant relationships were found between the concentrations of ATZ or its metabolites (ADI and ADDI) and the cytogenetic damage biomarkers evaluated; however, the agricultural areas where measurable concentrations of ATZ and/or its metabolites were recorded had higher frequencies of MN, nuclear buds, and apoptotic cells (Tables 3 and 4). Among these, Pelayito registered the highest percentage of samples with ATZ and its metabolites (ADI and ADDI) and also recorded the shortest distance between homes and crops, highest application frequencies of pesticides, and greater number of children living with parents who work with pesticides (Tables 1, 3, and 5). Similarly, an 8-year follow up study on exposure to organophosphate pesticide in children living in an agricultural area in Washington State reported association between the proximity of homes to the fields and increasing pesticide concentrations in house dust and urinary metabolites [60].
On the other hand, it has been described that variables, like age and gender can influence MN frequency in adult populations, however, in the infant population these variables do not seem to have a definite influence [61], which agrees with the
results of the present study. Exposure to cigarette smoke may also influence the frequency of cytogenetic damage biomarkers. However, the few cases reported in the present study of children living with parents who smoke did not allow the evaluation of possible relationships between these variables.
Few studies have been published on the evaluation of cytogenetic damage in peripheral blood lymphocytes in children living in agricultural areas or exposed to pesticides. Most studies have been conducted with oral epithelium cells because samples are obtained more easily and with non-invasive methods [61]. The results of this research show an increasing trend in the frequency of cytogenetic damage (MN, nuclear buds, and apoptotic cells) in groups from agricultural areas, as compared to the control group. Similar results were described in a study that evaluated cytogenetic damage in children (7 to 11 years of age) in a rural agricultural area with prolonged exposure to pesticides, with a comet assay coupled to the FPG enzyme and testing of cytokinesis-block MN, which reported a significant increase in breaks of single strand of DNA and the frequency of MN in children exposed, as compared to a control group in an area not exposed to pesticides [11]. Another study on lymphocytes in healthy children not exposed to any pollutant, aged between 4 and 14 years in the city of Zagreb, Croatia [62], reported a frequency of MN = 2.32 ± 0.28 and nuclear buds = 1.44 ± 0.19, relatively smaller than those described in this research.
Furthermore, several reviews on cytogenetic alterations in adult population exposed to pesticides have been published. Matheus and Bolaños [10], in a review of 33 publications worldwide (16 on MN in peripheral blood lymphocytes, 13 on oral epithelial cells, and 4 on both techniques), described more frequent cytogenetic abnormalities in individuals exposed to pesticides, as compared to unexposed ones, in 24 of these publications. Another review of 100 publications worldwide described an increase in the frequency of chromosomal aberrations, MN, DNA damage (measured by the comet assay), and/or sister chromatid exchange in peripheral blood lymphocytes or cells of the oral mucosa of individuals with
occupational or environmental exposure to pesticides, in 73 of these publications [12]. In short, most of the studies described above recorded an increasing trend in the frequency of cytogenetic damage in individuals more closely related to pesticides (children or adults), as compared to control groups not exposed, consistent with the results of this study. Recently, a review by Bolognesi and Holland [3], on populations exposed to pesticides in different occupational or environmental scenarios, reported increases in MN frequency associated to pesticide exposure in 36 of 49 studies conducted in different countries in Europe, South America, the USA, Asia, and Australia. Highlighting that increased MN frequency was associated to the extent, duration, and type of exposure and that in occupational scenarios where adequate protection measures were used, no significant increases were noted in MN frequency.
5. Conclusion
Results evidenced infant exposure in the groups from Pelayito, Aguas Negras, and Cabuya to one of the pesticides (ATZ) with greater use in the agricultural areas evaluated and an increasing trend in the frequency of cytogenetic damage markers (MN, nuclear buds, and apoptotic cell) therein, as compared to the unexposed control group. This, coupled with the constant risk from the continuous use of pesticides and the lack of studies on adverse health effects that may be caused by these substances in infant populations in these agricultural areas, suggests the need to continue studies in response to the greater vulnerability of and the special attention needed by infant populations, establishing prevention measures and/or relevant solutions.
Acknowledgements The authors thank Colombia’s Administrative Department of Science, Technology and Innovation, COLCIENCIAS, for financial support [Grant 377-2011, project code 1112-545-31611].
References [1] S. Mostafalou, M. Abdollahi, Pesticides: an update of human exposure and toxicity. Arch Toxicol. 91(2) (2017) 549-599. doi: 10.1007/s00204-016-1849-x. [2] C. Fortes, C. Aprea, Cancer risks from residential exposure to pesticides, Encyclopedia of Environmental Health (2011) 489-497. [3] C. Bolognesi, N. Holland, The use of the lymphocyte cytokinesis-block micronucleus assay for monitoring pesticide-exposed populations, Mutat. Res.: Rev. Mutat. Res. (2016), http://dx.doi.org/10.1016/j.mrrev.2016.04.006. [4] L. Palani, N. Panneerselvam, Toxic effects of pesticides: A review on cytogenetic biomonitoring studies, FU. Med. Biol. 15 (2008) 46-50. [5] C.A. Damalas, I.G. Eleftherohorinos, Pesticide exposure, safety issues, and risk assessment indicators, Int. J. Environ. Res. Public Health 8 (2011) 1402-1419. [6] International Agency for Research on Cancer (IARC), List of classifications by cancer sites with sufficient or limited evidence in humans, Volumes 1 to 118. 2017. http://monographs.iarc.fr/ENG/Classification/Table4.pdf. [7] International Agency for Research on Cancer (IARC), Some organophosphate insecticides and herbicides, Volume 112. 2015. http://monographs.iarc.fr/ENG/ Monographs/vol112/mono112.pdf. [8] C. Bolognesi, Genotoxicity of pesticides: a review of human biomonitoring studies, Mut. Res. 543 (2003) 251–272. [9] C. Infante-Rivard, S. Weichenthal, Pesticides and childhood cancer: An update of Zahm and Ward’s 1998 review, J. Toxicol. Environ. Health, Part B 10 (2007) 8199. [10] T. Matheus, A. Bolaños, Micronúcleos: biomarcador de genotoxicidad en expuestos a plaguicidas, Salus 18 (2014) 18-26. [11] L. Kapka-Skrzypczak, M. Posobkiewic, P. Holownia, J. Niedzwiecka, K. Sawicki, M. Cyranka, M. Kruszewski, Assessing DNA damage in children
environmentally exposed to pesticides through using the comet assay and the micronucleus test, Eur. J. Public Health 23 (2013) 260-260. [12] D. Aiassa, F. Mañas, B. Bosch, N. Gentile, N. Bernardi, N. Gorla, Biomarcadores de daño genético en poblaciones humanas expuestas a plaguicidas, Acta. Biol. Colomb. 17 (2012) 485-510. [13] D. Aiassa, F. Mañas, N. Bernardi, N. Gentile, A. Méndez, D. Roma, N. Gorla, Monitoring genotoxicity in humans exposed to pesticides. Preliminary study in children, Revista Cuestiones de Población y Sociedad 4 (2014) 73-84. [14] V. Garry, Pesticides and children, Toxicol. Applied. Pharmacol 198 (2004) 152-163. [15] M. Neri, M. Ceppi, L.E. Knudsen, D. Franco, R. Barale, R. Puntoni, S. Bonassi, Baseline micronuclei frequency in children: estimates from meta- and pooled analyses, Environ. Health. Perspect. 113 (2005) 226-1229. [16] Superintendencia de Industria y Comercio de Colombia, Estudios económicos sectoriales. Estudio sobre plaguicidas en Colombia. N°7. 2013. http://www.sic.gov.co/drupal/recursosuser/documentos/Estudios-Academicos/Doc umentos-Elaborados-Grupo-Estudios-Economicos/7_Estudio_Sobre_Sector_ Plaguicidas_Colombia_Diciembre2013.pdf. [17] M. Varona, O. Cárdenas, C. Crane, S. Rocha, G. Cuervo, J. Vargas, Cytogenetic alterations in field workers routinely exposed to pesticides in Bogota flower farms, Biomédica 23 (2003) 141-152. [18] M.I. Hanna, M.E. Orozco, Occupational pesticide exposure in cotton growers: valley of the middle Sinu, Económicas CUC 35 (2014) 65-74. [19] M. Varona, R.A. Castro, M.I. Páez, N. Carvajal, E. Barbosa, L.M. León, S.M. Díaz, Impact on health and environment of exposure to pesticides and implementation of best agricultural practices in tomato production, Colombia, 2011, Rev. Chil. Salud Pública 16 (2012) 96-106. [20] C. Bolognesi, G. Carrasquilla, S. Volpi, K.R. Solomon, E.J. Marshall, Biomonitoring of genotoxic risk in agricultural workers from five Colombian regions: association to occupational exposure to glyphosate, J. Toxicol. Environ. Health A 72 (2009) 986-997.
[21] G. Palencia, T. Mercado, E. Combatt, Estudio agroclimático del departamento de Córdoba, Universidad de Córdoba, Montería, 2006. [22] K. Acosta, La economía de las aguas del rio Sinú. Documento de trabajo sobre economía regional, Banco de la Republica, Cartagena, 2013. http://www.banrep.gov.co/docum/Lectura_finanzas/pdf/dtser_194.pdf. [23] Resolución 008430/1993 de 4 de Octubre, por la cual se establecen las normas académicas, técnicas y administrativas para la investigación en salud. (Ministerio de Salud y Protección Social, Bogotá D.C.). [24] The National Institute for Occupational Safety and Health (NIOSH), Manual of analytical methods, fourth ed., Method 8315 Triazine herbicides and their metabolites in urine, 2003. https://www.cdc.gov/niosh/docs/2003154/pdfs/8315.pdf. [25] M. Fenech, Cytokinesis-block micronucleus cytome assay, Nat. Protoc. 2 (2007) 1084-1094. http://www.nature.com/nprot/journal/v2/n5/pdf/nprot.2007.77.pdf. [26] World Health Organization (WHO), The WHO recommended classification of pesticides by hazard and guidelines to classification 2009, WHO, Geneva, 2010. [27] Instituto Colombiano Agropecuario, Restricciones, prohibiciones y suspensión de registros de plaguicidas de uso agrícola en Colombia 2016. http://www.ica.gov.co /getdoc/b2e5ff99-bd80-45e8-aa7ae55f0b5b42dc/PLAGUICIDAS-PROHIBIDOS. aspx. [28] Pesticides Action Network, The list of lists – 3rd edition 2009. http://www.paneurope.info/old/Campaigns/pesticides/documents/cut_off/list%20of%20lists.pdf. [29] Pesticides Action Network, International consolidated list of banned pesticides. 2015. http://pan-international.org/pan-international-consolidated-list-of-bannedpesticides/. [30] D. Li, Q. Huang, M. Lu, L. Zhang, Z. Yang, M. Zong, L. Tao, The organophosphate insecticide chlorpyrifos confers its genotoxic effects by inducing DNA damage and cell apoptosis, Chemosphere. 135 (2015) 387 - 393. [31] S. Sandal, B. Yilmaz, Genotoxic effects of chlorpyrifos, cypermethrin, endosulfan and 2,4-D on human peripheral lymphocytes cultured from smokers
and nonsmokers. Environ. Toxicol. 26(5) (2011) 433 - 442. http://dx.doi.org/.10.1002/ tox.20569. [32] R. Patnaik, R.N. Padhy, Evaluation of geno-toxicity of methyl parathion and chlorpyrifos to human liver carcinoma cell line (HepG2). Environ. Sci. Pollut. Res. 23(9) 2016 8492 - 8499. http://dx.doi.org/.10.1007/s11356-015-5963-8. [33] K.B. Abdelaziz, A.I. El Makawy, A.Z.E. Abd Elsalam, A.M. Darwish, Genotoxicity of chlorpyrifos and the antimutagenic role of lettuce leaves in male mice. Comunicata Scientiae 1(2) (2010) 137-145. [34] F.L. Edwards, C.G. Yedjou, P.B. Tchounwou, Involvement of oxidative stress in methyl parathion and parathion-induced toxicity and genotoxicity to human liver carcinoma (HepG2) cells. Environ. Toxicol. 28 (2013) 342 - 348. [35] G. Argentin, M. Divizia, R. Cicchetti, Oxidative stress, cytotoxicity, and genotoxicity induced by methyl parathion in human gingival fibroblasts: Protective role of Epigallocatechin-3-Gallate, J. Toxicol. Environ. Health A. 78 (2015) 12271240. doi: 10.1080/15287394.2015.1079582. [36] M. Coral, S. Ucman, H. Yildiz, H. Oztas, S. Dalkilic, Potential neoplastic eff ects of parathion-methyl on rat liver, J. Environ. Sci. 21 (2009) 696–699. [37] A.Y. Kocaman, M. Topaktaş, The in vitro genotoxic effects of a commercial formulation of alpha-cypermethrin in human peripheral blood lymphocytes. Environ. Mol. Mutagen. 50(1) (2009) 27-36. Doi: 10.1002/em.20434. [38] A.Y. Kocaman, M. Topaktaş Genotoxic effects of a particular mixture of acetamiprid and α-cypermethrin on chromosome aberration, sister chromatid exchange, and micronucleus formation in human peripheral blood lymphocytes. Environ. Tox. 25(2) (2009) 157-168. Doi: 10.1002/tox.20485. [39] G. Jovtchev, S. Gateva, M. Stergios, S. Kulekova, Cytotoxic and genotoxic effects of paraquat in Hordeum vulgare and human lymphocytes in vitro. Environ. Toxicol. 25(3) (2010) 294 - 303. [40] S.O. Abarikwu, E.O. Farombi, Atrazine induces apoptosis of SH-SY5Y human neuroblastoma cells via the regulation of Bax/Bcl-2 ratio and caspase-3-dependent pathway. Pestic. Biochem. Physio. 118 (2015) 90 - 98.
[41] B.C. Ventura-Camargo, M.A. Marin-Morales, Micronuclei and chromosome aberrations derived from the action of atrazine herbicide in Allium cepa meristematic cells. SDRP-JESES. 1(1) (2016) 1 – 7. [42] C. Bolognesi, G. Carrasquilla, S. Volpi, K.R. Solomon, E.J. Marshall, Biomonitoring of genotoxic risk in agricultural workers from five Colombian regions: association to occupational exposure to glyphosate, J. Toxicol. Environ. Health A 72 (2009) 986 - 997. [43] International Agency for Research on Cancer (IARC), IARC Monographs volume 112: evaluation of five organophosphate insecticides and herbicides. 2015. https://www.iarc.fr/en/media-centre/iarcnews/pdf/MonographVolume112.pdf. [44] C. Monroy, A. Cortés, D. Mercedes, H. Groot, Cytotoxicity and genotoxicity of human cells exposed in vitro to glyphosate, Biomédica 25 (2005) 335-345. [45] C.S. Caramello, M.J. Jorge, N.L. Jorge, L.C. Jorge, Evaluation of herbicide glyphosate effects in the fish Prochilodus lineatus using chromosome aberration test. Rev. Vet. 28(1) (2017) 65-68. [46] B. Holečková, Evaluation of the in vitro effect of glyphosate-based herbicide on bovine lymphocytes using chromosome painting. Bull. Vet. Inst. Pulawy. 50 (2006) 533-536. [47] M.S. Mecchi, Salmonella–Escherichia Coli/mammalian–microsome reverse mutation assay with a confirmatory assay. Unpublished regulatory study. Report Identification Number: CV-2002-186. 2003. [48] B. Kaya, A. Creus, A. Yanikoğlu, O. Cabré, R. Marcos, Use of the Drosophila wing spot test in the genotoxicity testing of different herbicides. Environ. Mol. Mutagen. 36 (2000) 40 - 46. [49] Agency for Toxic Substances and Disease Registry (ATSDR), Atrazina CAS # 1912-24-9. 2003. http://www.atsdr.cdc.gov/toxfaqs/tfacts153.pdf. [50] A.M. Hansen, L.G. Treviño-Quintanilla, H. Márquez-Pacheco, M. VilladaCanela, L.C. González-Márquez, R.A. Guillén-Garcés, A. Hernández-Antonio, Atrazina: un herbicida polémico, Rev. Int. Contam. Ambie. 29 (2013) 65-84. [51] M. Fenech, M. Kirsch-Volders, A.T. Natarajan, J. Surralles, J.W. Crott, J. Parry, H. Norppa, D.A. Eastmond, J.D. Tucker, P. Thomas, Molecular mechanisms
of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells, Mutagenesis 26 (2011) 125-132. [52] E. Murgia, M. Ballardin, S. Bonassi, A.M. Rossi, R. Barale, Validation of micronuclei frequency in peripheral blood lymphocytes as early cancer risk biomarker in a nested case-control study, Mutat. Res. 639 (2008) 27-34. [53] S. Bonassi, A. Znaor, M. Ceppi, C. Lando, W.P. Chang, N. Holland, M. KirschVolders, E. Zeiger, S. Ban, R. Barale, M.P. Bigatti, C. Bolognesi, A. CebulskaWasilewska, E. Fabianova, A. Fucic, L. Hagmar, G. Joksic, A. Martelli, L. Migliore, E. Mirkova, M.R. Scarfi, A. Zijno, H. Norppa, M. Fenech, An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans, Carcinogenesis 28 (2007) 625-631. [54] R.A. El-Zein, M.B. Schabath, C.J. Etzel, M.S. Lopez, J.D. Franklin, M.R. Spitz, Cytokinesis blocked micronucleus assay as a novel biomarker for lung cancer risk, Cancer. Res. 66 (2006) 6449-6456. [55] FAO/WHO, Pesticide residues in food 2007. Joint FAO/WHO meeting on pesticide residues. FAO plant production and protection paper 191. Rome, 2007. ftp://ftp.fao.org/docrep/fao/010/a1556e/a1556e00.pdf. [56] A.J. Agopian, P.J. Lupo, M.A. Canfield, P.H. Langlois, Case-control study of maternal residential atrazine exposure and male genital malformations, Am. J. Med. Genet Part A. 161 (2012a) 977-982. [57] A.J. Agopian, P.H. Langlois, Y. Cai, M.A. Canfield, P.J. Lupo, Maternal residential atrazine exposure and gastroschisis by maternal age, Mater. Child Health J. 17 (2012b) 1768-1775. [58] A.J. Agopian, Y. Cai, P.H. Langlois, M.A. Canfield, P.J. Lupo, Maternal residential atrazine exposure and risk for choanal atresia and stenosis in offspring, J. Pediatr. 162 (2013) 581-586. [59] P. Boffetta, A. Hans-Olov, B. Sir Colin, M. Jack, Atrazine and cancer: a review of the epidemiologic evidence, Eur. J. Cancer Prev. 22 (2013) 169-180. [60] R.A. Fenske, C. Lu, N.J. Simcox, C. Loewenherz, J. Touchstone, T.F. Moate, E.H. Allen, J.C. Kissel, Strategies for assessing children's organophosphorus pesticide exposures in agricultural communities, J. Expo. Anal. Environ. Epidemiol. 10 (2000) 662-671.
[61] N. Holland, A. Fucic, D.F. Merlo, R. Sram, M. Kirsch-Volders, Micronuclei in neonates and children: effects of environmental, genetic, demographic and disease variables, Mutagenesis 26 (2011) 51–56. [62] G. Gajski, M. Geric, V. Orešcanin, V. Garaj-Vrhovac, Cytogenetic status of healthy children assessed with the alkaline comet assay and the cytokinesis-block micronucleus cytome assay, Mutat. Res. 750 (2013) 55-62.
Table 1. Demographic data of study populations.
Variable
Sex Male Female Total Residence time in the zone ≥ 3 years From birth Total Go to school? Yes No Total Live with parents smoking? Yes No Total Parents work with pesticides? Yes No Total Father education level No study Incomplete primary Complete primary Incomplete secondary Complete secondary Technical or Technological University Total Mother education level No study Incomplete primary Complete primary Incomplete secondary Complete secondary Technical or Technological University Total Source of drinking water Tap-drinking Total Make any previous treatment? Yes No Total Save pesticides inside the house? Yes
Study zones Aguas Cabuya Negras N° N° % % Ind Ind
Ceibita
Pelayito
Control
N° Ind
N° Ind
N° Ind
%
%
%
10 7 17
58.8 2 41.2 6 100.0 8
25.0 10 75.0 3 100.0 13
76.9 5 23.1 7 100.0 12
41.7 9 58.3 4 100.0 13
69.2 30.8 100.0
1 16 17
5.9 0 94.1 8 100.0 8
0.0 2 100.0 11 100.0 13
15.4 1 84.6 11 100.0 12
8.3 3 91.7 10 100.0 13
23.1 76.9 100.0
17 0 17
100.0 8 0.0 0 100.0 8
100.0 13 0.0 0 100.0 13
1000 12 0.0 0 100.0 12
100.0 13 0.0 0 100.0 13
100.0 0.0 100.0
1 16 17
5.9 1 94.1 7 100.0 8
12.5 2 87.5 11 100.0 13
15.4 2 84.6 10 100.0 12
16.7 2 83.3 11 100.0 13
15.4 84.6 100.0
5 12 17
29.4 3 70.5 5 100.0 8
37.5 1 62.5 12 100.0 13
7.7 10 92.3 2 100.0 12
83.3 0 16.7 13 100.0 13
0.0 100.0 100.0
1 0 1 11 2 2 0 17
5.9 0.0 5.9 64.7 11.8 11.8 0.0 100.0
0 3 0 1 1 2 1 8
0.0 37.5 0.0 12.5 12.5 25.0 12.5 100.0
0 2 2 3 6 0 0 13
0.0 15.4 15.4 23.1 46.1 0.0 0.0 100.0
1 1 1 4 5 0 0 12
8.3 8.3 8.3 33.3 41.6 0.0 0.0 100.0
1 0 1 2 6 2 1 13
7.7 0.0 7.7 15.4 46.1 15.4 7.7 100.0
0 0 0 7 10 0 0 17
0.0 0.0 0.0 41.2 58.8 0.0 0.0 100.0
0 0 1 4 1 0 2 8
0.0 0.0 12.5 50.0 12.5 0.0 25.0 100.0
2 1 0 8 2 0 0 13
15.4 7.7 0.0 61.5 15.4 0.0 0.0 100.0
0 6 3 3 0 0 0 12
0.0 50.0 25.0 25.0 0.0 0.0 0.0 100.0
0 0 0 0 5 4 4 13
0.0 0.0 0.0 0.0 38.4 30.8 30.8 100.0
17 17
100.0 8 100.0 8
100.0 13 100.0 13
100.0 12 100.0 12
100.0 13 100.0 13
100.0 100.0
3 14 17
17.6 2 82.4 6 100.0 8
25.0 2 75.0 11 100.0 13
15.4 1 84.6 11 100.0 12
8.3 11 91.7 2 100.0 13
84.6 15.4 100.0
0
0.0
0.0
0.0
8.3
0.0
0
0
1
0
No Total Re-used pesticides containers for housework? Yes No Total Considered hazardous to health to pesticides? Yes No Total Think your children are exposed to pesticides? Yes No Total The child has handled pesticides? Yes No Total The child has involved in agricultural work? Yes No Total
17 17
100.0 8 100.0 8
100.0 13 100.0 13
100.0 11 100.0 12
91.7 13 100.0 13
100.0 100.0
0 17 17
0.0 0 100.0 8 100.0 8
0.0 4 100.0 9 100.0 13
30.8 1 69.2 11 100.0 12
8.3 0 91.7 13 100.0 13
0.0 100.0 100.0
17 0 17
100.0 8 0.0 0 100.0 8
100.0 13 0.0 0 100.0 13
100.0 12 0.0 0 100.0 12
100.0 13 0.0 0 100.0 13
100.0 0.0 100.0
15 2 17
88.2 7 11.8 1 100.0 8
87.5 13 12.5 0 100.0 13
100.0 7 0.0 5 100.0 12
58.3 0 41.7 13 100.0 13
0.0 100.0 100.0
2 15 17
11.8 0 88.2 8 100.0 8
0.0 0 100.0 13 100.0 13
0.0 0 100.0 12 100.0 12
0.0 0 100.0 13 100.0 13
0.0 100.0 100.0
3 14 17
17.6 0 82.4 8 100.0 8
0.0 4 100.0 9 100.0 13
30.8 7 69.2 5 100.0 12
58.3 0 41.7 13 100.0 13
0.0 100.0 100.0
Table 2. Pesticides most commonly used in the study area. Restriction status Colombia ** Other countries *** Banned in Saudi Not restricted Arabia Banned in Restricted to European Union technified (EU) and 26 cotton and rice countries overall
Pesticide type
Use
Common name
Active ingredient
HC*
Organophosphorus
Insecticide
Lorsban
Chlorpyrifos
II
Organophosphorus
Insecticide
Methyl parathion
Methyl parathion
Ia
Pyrethroid
Insecticide
Cypermethrin
Cypermethrin
II
Not restricted
Bipyridyl
Triazine
Herbicide
Gramoxone
Paraquat
II
Restricted by air
Herbicide
Atrazine
Atrazine
III
Not restricted
Insecticide
Exalt
Spinetoram
U
Not restricted
Herbicide
Roundup or Glyphosate
Glyphosate
III
Not restricted
Not restricted Use not approved or banned in USA, Sweden and 6 African countries Severe restriction in the UE Not restricted Banned in Sri Lanka
Genetic endpoint in human and other cells DNA damage [30 - 32] and chromosomal mutation [33]. DNA damage [32, 34, 35] and gene mutation [36]. DNA damage [31, 37, 38] and chromosomal mutation [37, 38]. DNA damage and chromosomal mutation [39]. DNA damage [40] and chromosomal mutation [41]. No reported. DNA damage [42 – 44], chromosomal mutation [45, 46] and gene mutation [47, 48].
*: Hazard classification according to WHO [26] (Ia = Extremely hazardous, Ib = Highly hazardous, II = Moderately hazardous, III = Slightly hazardous, U = Unlikely to present acute hazard under normal use). **: Colombian Agricultural Institute [27]. ***: Pesticide Action Network [28, 29].
Table 3. Concentrations of atrazine and its metabolites in urine samples. Pesticides or Zone metabolites*
n
ATZ
Pelayito
ADI
ADDI
N° individuals with measurable concentrations (%)
Concentrations (µg/g creatinine) Mean±SD
Median
12 12 (100.0)
18.6 ± 4.3
18.3
MinimumMaximum 13.0 - 25.5
Pelayito
12 5 (41.7)
3.5 ± 4.6
0.0
0.0 - 14.7
Cabuya
8
--
--
22.2
Pelayito
12 11 (91.7)
1 (12.5)
Aguas Negras 17 3 (17.6)
16.8 ± 10.0 17.1 --
--
0.0 - 43.7 190.6 145.9 127.3
* ATZ = Atrazine, ADI = Desisopropyl-atrazine, ADDI = Atrazine Desethyldesisopropyl.
Table 4. Cytogenetic damage in peripheral blood lymphocytes of children in the study. Variable
Group Aguas Negras Cabuya Pelayito The Ceibita Control
n* 17 8 12 13 13
Mean±SD 5.1 ± 2.7 5.0 ± 3.2 4.7 ± 2.5 1.5 ± 1.7 1.1 ± 1.1
Median 5.0 4.5 5.0 1.0 1.0
Minimum-Maximum** 1 - 11 a 2 - 11 a 0 - 10 a 0-6b 0-4b
Nuclear buds
Aguas Negras Cabuya Pelayito The Ceibita Control
17 8 12 13 13
4.1± 2.2 2.9 ± 1.5 1.6 ± 0.8 1.3 ± 1.3 0.6 ± 0.6
3.0 3.0 1.0 1.0 1.0
2-9a 0 - 5 ab 1 - 3 bc 0 - 5 cd 0-2d
Apoptotic cells
Aguas Negras Cabuya Pelayito The Ceibita Control
17 8 12 13 13
15.9 ± 10.5 30.8 ± 29.5 8.1 ± 5.0 4.5 ± 2.3 2.4 ± 2.9
15.0 21.5 8.5 4.0 1.0
3 - 40 a 0 - 76 a 0 - 17 ab 2 - 9 bc 0-9c
MN
*: n = Number of individuals sampled. **: At each variable, letters “a” and "c” correspond to the highest and lowest values, respectively; and groups sharing at least one letter are not significantly different (Dunn test, p <0.05).
Table 5. Variable determinants of pesticide exposure in the study zones. Variable*
Zone
n**
Mean±SD
Median
DHC (m)
Aguas Negras Cabuya Pelayito The Ceibita
17 8 12 13
42.2 ± 61.4 49.9 ± 101.7 4.8 ± 5.9 26.9 ± 24.6
20 10 1 20
MinimumMaximum*** 2 - 200 a 5 - 300 a 1 - 20 b 10 - 80 a
FSP (times/year)
Aguas Negras Cabuya Pelayito The Ceibita
17 8 12 13
58.1 ± 39.1 44.3 ± 32.3 105.0 ± 24.1 14.5 ± 3.5
64 32 108 12
6 - 96 b 22 - 96 b 36 - 144 a 12 - 20 c
FPO (times/year)
Aguas Negras Cabuya Pelayito The Ceibita
17 8 12 13
28.2 ± 40.6 72.3 ± 94.2 78.0 ± 61.1 80.0 ± 95.2
0 27 108 52
0 – 96 a 0 – 224 a 0 – 180 a 0 – 240 a
*: DHC = Distance from house to crops, FSP = Frequency of spraying pesticides on crops, FPO = Frequency of pesticide odor detection at home. **: n = Number of individuals sampled. ***: At each variable, letters “a” and "c” correspond to the highest and lowest values, respectively; and groups sharing at least one letter are not significantly different (Dunn test, p <0.05).
S1383-5718(16)30447-8 https://doi.org/10.1016/j.mrgentox.2017.10.002 MUTGEN 402848
To appear in:
Mutation Research
Received date: Revised date: Accepted date:
22-12-2016 4-10-2017 9-10-2017
´ Please cite this article as: Javier Alonso Ruiz-Guzm´an, Pamela G´omez-Corrales, Angel Cruz-Esquivel, Jos´e Luis Marrugo-Negrete, Cytogenetic damage in peripheral blood lymphocytes of children exposed to pesticides in agricultural areas of the department of Cordoba, Colombia, Mutation Research/Genetic Toxicology and Environmental Mutagenesis https://doi.org/10.1016/j.mrgentox.2017.10.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cytogenetic damage in peripheral blood lymphocytes of children exposed to pesticides in agricultural areas of the department of Cordoba, Colombia Javier Alonso Ruiz-Guzmána*, Pamela Gómez-Corralesa, Ángel Cruz-Esquivela, José Luis Marrugo-Negretea. a
Water, Applied, and Environmental Chemistry Group, Laboratory of Toxicology
and Environmental Management, Universidad de Córdoba. Montería, Colombia. Postal address: Cra 6 # 76 – 103 Montería, Postal code 354.
* Javier Alonso Ruiz-Guzmán (corresponding author) Water, Applied, and Environmental Chemistry Group, Universidad de Córdoba, Montería, Colombia E-mail: [email protected] Phone: + 57 (4) 7860381 ext 310. Montería, Colombia Fax: + 57 (4) 7860381 ext 310. Montería, Colombia Postal address: Cra 6 # 76 – 103 Montería, Colombia. Postal code 354. Universidad de Córdoba, Toxicology and Environmental Management Laboratory
Pamela Gómez-Corrales [email protected]
Ángel Cruz-Esquivel [email protected]
José Luis Marrugo-Negrete E-mail: [email protected]
Highlights - Pesticides and/or its metabolites were recorded in the children’s urine samples - Frequency of cytogenetic damage was higher in the exposed children that in control group - Results suggest a permanent health risk for pesticide exposure
Abstract Pesticides offer benefits, like optimization of agricultural production and disease control; however, these toxic substances can contaminate the environment and pose risks to human health. The aim of this study was to assess pesticide exposure and frequency of cytogenetic damage in infant populations in agricultural areas of the department of Córdoba, Colombia. Urine and peripheral blood samples were taken from children living in the villages of La Ceibita (municipality of Cereté), Cabuya (municipality of San Carlos), Aguas Negras (municipality of Montería), Pelayito (municipality of San Pelayo), and the city of Monteria (control group). The work evaluated biomarkers of exposure to pesticides (atrazine urinary concentrations (ATZ) and its metabolites) and biomarkers of cytogenetic damage (micronucleus frequency (MN), nuclear buds, and apoptotic cells in peripheral blood lymphocytes). Measurable ATZ concentrations and/or its metabolites were recorded in the Pelayito, Aguas Negras, and Cabuya zones, which had higher MN frequencies, nuclear buds, and apoptotic cells than the control. Infant exposure to one of the more-often used pesticides in the agricultural areas evaluated and an increasing trend in the frequency of markers of cytogenetic damage in the groups of the agricultural areas, as compared to the control group, were evident.
Keywords: micronucleus test, pesticides, atrazine, DNA damage (Source: DeCS)
1. Introduction Pesticide manufacture uses around 1000 active ingredients, sprayed mainly in rural agricultural areas to reduce and/or eliminate populations of insects, fungi, and weeds, among other things, for crop protection [1]. Although these substances offer benefits, such as optimization of agricultural production and disease control, their extensive use tends to pollute the air, water, soil, and food [2], representing risks to human health and the environment [3].
Adverse effects on human health caused by exposure to pesticides include discomfort, permanent damage, and irreversible changes in the nervous, reproductive, and hormonal systems among others. Additionally, DNA alterations that can be transmitted or manifested in subsequent generations, some of which have been classified as carcinogenic and probable or possible carcinogenic for humans, and which may be lethal in cases of severe poisoning [4 – 7]. The association between mutagenic, genotoxic, and carcinogenic activities in humans and exposure to pesticides has been widely recognized [8, 9]. Using biomarkers of cytogenetic damage, such as micronucleus testing (MN), offers evidence of early DNA damage; it has been used to evaluate the effects of these substances on the genetic material of adult and infant populations with occupational or environmental exposure [3, 10 - 12].
These health changes caused by pesticides can occur more easily in infant populations because of their vulnerable stage of development, which makes them more sensitive to the effects of these pollutants [13]. Children may respond differently than adults in the detoxification process, DNA repair, and cell proliferation [14], given that their metabolism is more rapid than that of adults [15]. In this sense, it is more likely that early DNA alterations not adequately repaired in children can lead to a faster development of major alterations in health.
In Colombia, despite being a country with a high use of pesticides for agriculture [16], no studies exist on pesticide exposure in children or evaluations of
cytogenetic alterations in any class of cells in infant populations in agricultural areas potentially exposed to pesticides. However, some studies have evaluated the frequency of chromosome aberrations, MN, deficiencies in lymphocyte DNA repair activity [17], acetylcholinesterase activity [18, 19], and genotoxic risk with a lymphocyte micronucleus assay [20] in adult populations with occupational exposure to pesticides. The aim of this study was to evaluate pesticide exposure and frequency of cytogenetic damage in infant populations in agricultural areas of the department of Córdoba, Colombia. The study used as exposure biomarkers, urinary concentrations of one of the most-often used pesticides in the study areas (atrazine and its metabolites) and as biomarkers of cytogenetic damage, the MN frequency, nuclear buds, and apoptotic cells in peripheral blood lymphocytes.
2. Materials and Methods
2.1. Study area The study was conducted in rural farming areas of the middle basin of the Sinú River in the department of Córdoba, Colombia (Ceibita village - municipality of Cereté: 8°49'35" N - 75°39'03" W, Cabuya - municipality of San Carlos: 8°46'18" N - 75°43'53" W, Aguas Negras - municipality of Montería: 8°48'44.96" N 75°49'1.81" W, Pelayito - municipality of San Pelayo: 8°56'20.8" N - 75°49'5.74" W, and the city of Monteria as the control group: 08°45′27″ N - 75°53′08″ W). The middle basin of the Sinú River is between 10 and 20 m.a.s.l., with an average annual temperature of 29 °C, annual rainfall of 1200 mm, and 85% relative humidity [21]. Traditionally, much of this region has been dedicated to agriculture because of the low concentrations of salts in the Sinú River, a characteristic that provides soil conditions suitable for growing a wide variety of crops (corn, rice, cotton, and cassava, among others), with corn and cotton in the main study areas [22]. Although no official statistics or records are available for the quantities of pesticides used in this region, it is clear that commercial agriculture has evolved and expanded over the decades, which has used and continues to consume large amounts of these substances for crop protection.
2.2. Type of study and target population An exploratory cross-sectional study was conducted in which the frequency of cytogenetic damage markers in peripheral blood lymphocytes (micronucleus assay) and the presence or absence of markers of exposure (metabolites) to pesticides was evaluated in urine samples from children. The study included children between 5 and 15 years of age from both genders (male and female), with a minimum stay in the study area of three years. To reduce the likelihood of bias in the interpretation of the results, children with birth defects, chronic degenerative diseases, requiring permanent medication, who have been exposed to radiation (Xray examinations) within the previous six months and those who that manifested having had the habit of smoking or consuming liquor were excluded from the study.
In each area or study population, an enrollment list (with the parents) of 20 children was made of which a smaller number in each population (Aguas Negras = 17 children, Cabuya = 8 children, Pelayito = 12 children, Ceibita = 13 children, and Control group = 13 children) finally participated in the study, given that on the sampling day some children did not allow to be sampled, or their parents or legal guardians (an adult relative) were unable to attend. All of the participants were selected through non-probability intentional sampling.
2.3. Collection of information A survey was conducted with the parents or legal guardians of children to collect demographic information, regarding exposure characteristics and health status of the children. From this information, the study participants were selected based on the criteria described in the previous section.
2.4. Ethical aspects The Ethics Committee of the Faculty of Health Sciences at Universidad de Córdoba, Colombia approved the study. According to Resolution No. 8430 of 1993 from the Colombian Ministry of Health and Social Protection [23], prior to sampling,
the parents or legal guardians of each child were asked to sign an informed consent form that was explained clearly and completely, indicating the purpose, scope, and limitations of the study and the associated risks. Signing this document meant the parents authorized participation by the children.
2.5. Sampling Urine samples were collected in sterile plastic containers early in the day (between 6:00 am and 7:00 am) by the participants themselves with help from their parents, who were provided instructions for labeling and cold storage. Peripheral blood samples were taken between 7:00 am and 9:00 am through venipuncture on the forearm with heparinized vacutainer tubes (4 mL). Both samples were stored and transported for 1 to 1.5 h in controlled-temperature containers (20 to 22 °C) for the blood samples and plenty of ice for the urine samples, to the Toxicology and Environmental Management Laboratory at Universidad de Córdoba for immediate processing.
2.6. Biomarkers of exposure to pesticides Urinary concentrations of one of the most-often used pesticides in the study areas, atrazine (ATZ) and its metabolites atrazine desisopropyl (ADI) and atrazine desethyl-desisopropyl (ADDI), were used as biomarkers of exposure to pesticides. The analysis was performed with gas chromatography-mass spectrometry (GC-MS Trace 1310 Thermo Scientific gas chromatograph and ISQ Thermo Scientific Mass Spectrometer) after liquid-liquid extraction with ethyl acetate and diethyl ether, and clean-up of the extracts (Florisil and sodium sulfate), under the following chromatography conditions: injection volume = 1 μL, detector temperature = 285 °C, DB5MS capillary column: 30 m x 0.2 mm x ID 0.20 µm and Helium as carrier gas [24]. The limits of detection (LD in ppb) and quantification (LC in ppb) of the method were ATZ: LD = 0.451; LC = 0.455; ADI: LD = 3.194; LC = 3.200; ADDI: LD = 7.123; and LC = 7.130.
2.7. Biomarkers of cytogenetic damage
Frequency of MN, nuclear buds, and apoptotic cells in peripheral blood lymphocytes was used as indicators of cytogenetic damage, assessed with micronucleus assay with block cytokinesis with cytochalasin B, according to the protocol described by [25]. The lymphocytes were separated from the whole blood sample using the Ficoll-1077 technique (Histopaque-1077™), per manufacturer’s instructions. The lymphocyte culture was done in RPMI 160, supplemented with fetal bovine serum, L-Glutamine, antibiotics, and phytohemagglutinin for 72 h at 37 °C and 5% CO2, adding cytochalasin B at 44 h. At the end of cultures, preparations were made on microscope slides with a cytological centrifuge (Cellspin Hanil) at 500 rpm for 5 min, which were dried at room temperature, fixed with 80% cold methanol (v/v), and colored with a 10% Giemsa solution (v/v) in a Sörensen buffer for 15 min. The frequency of DNA damage biomarkers (MN and nuclear buds) was scored in 2000 binucleate cells (1000 duplicated) and the frequency of apoptotic cells was analyzed in 1000 viable cells. Frequency of all alteration was expressed as frequency of alterations for 1000 cells scored and the analyses were performed in an optical microscope (Olympus CX41) at 1000X magnification.
2.8. Statistical analysis Kruskal-Wallis (KW) analysis and Dunn comparison of average-range test were used to assess differences in the frequency of markers of cytogenetic damage (MN, nuclear buds and apoptotic cells) and sociodemographic variables among the study groups. A Spearman correlation analysis was carried out to assess the relationship between markers of cytogenetic damage and concentrations of ATZ and its metabolites (ADI and ADDI) in urine samples, as well as sociodemographic variables that can influence the frequency of these biomarkers, like age, sex, time of residence in the area, distance from home to crops, frequency of pesticide applications, and frequency of pesticide odor detection at home. The analyzes were performed with SPSS Statistics 22 and, in all cases, p <0.05 was established as the criterion for statistical significance.
3. Results
Table 1 presents the sociodemographic characteristics and the use and management of pesticides in the study populations. The distribution by age and sex was similar in the study groups without significant difference among them (age: p = 0.11; sex: p = 0.12), as well as the time of residence in the area (p = 0.30) because most of the children have lived in their populations from birth. All participants attend school and consume potable water from the aqueduct, most without additional pre-treatment prior to consumption, except in the control group where most reported using filters. Most of the children live with parents who do not smoke; most of the children in Pelayito live with parents who work with pesticides, unlike the other groups where the behavior of this variable was inverse and most parents of children in Pelayito have not completed secondary education.
Regarding the management and use of pesticides, except for Pelayito, the parents or guardians of the children reported not storing pesticides inside the house or reusing the packaging for household chores (except four cases in Ceibita for this last variable); all believe that pesticides are dangerous to health and most believe their children are exposed to these substances (except in the control group). With the exception of two cases in Aguas Negras, children do not handle pesticides; however, several children in different study areas perform agricultural tasks, such as fertilizing, harvesting, and cleaning.
Table 2 presents the classification of pesticides commonly used in the study areas, according to their dangerousness, chemical structure, and use, as well as the status of restriction or prohibition in Colombia and the world, and their genotoxic effect in human cells. Mainly, insecticides and herbicides of different types are used and most of these have some sort of restriction in Colombia or other countries.
Table 3 shows the ATZ concentrations and its metabolites (ADI and ADDI) in the urine samples from the different areas evaluated. Among these, Pelayito recorded
the highest percentage of samples with measurable concentrations of the unmodified pesticide (ATZ) and its metabolites, while Aguas Negras only recorded three individuals with measurable concentrations of the ADDI metabolite and only one individual in Cabuya recorded measurable concentrations of the ADI metabolite. No measurable concentrations of any of these compounds was recorded for the samples from Ceibita or the control group.
Table 4 contains the cytogenetic damage markers evaluated. The highest frequencies of MN and apoptotic cells were recorded in Pelayito, Aguas Negras, and Cabuya without significant differences among them (p> 0.05), while the Ceibita and the control groups recorded the lowest rates without significant difference between them (p> 0.05). The frequency of nuclear buds was higher in the Aguas Negras and Cabuya groups, intermediate in Pelayito and Ceibita, and lower in the control group.
No significant correlations (p> 0.05) were noted between the cytogenetic damage markers (MN, nuclear buds, and apoptotic cells) evaluated and the concentrations of ATZ and its metabolites (ADI and ADDI). In addition, no significant differences were recorded in any of the study areas for the sociodemographic variables that can influence these cytogenetic damage markers, such as age, sex, time of residence in the area, distance from home to crops, application frequency of pesticides, and frequency of pesticide odor detection at home. When these sociodemographic variables among the groups of the agricultural areas were compared, significant differences (p <0.05) in the distance from the house to crops, being lower in Pelayito, and frequency of crop spraying, which was higher in Pelayito, were found (Table 5).
4. Discussion
The sociodemographic variables of the study populations show a desirable scenario in terms of education, given that all of the children attend school. This is
especially important in agricultural areas because children who do not attend school may be attracted to the activities in the area, such as agriculture, placing them at risk of greater proximity and exposure to pesticides used in this activity. As for the habits and perception of the health hazards from exposure to pesticides, the apparent parental care of children is notable because, except for the cases in Pelayito and Ceibita, the parents stated that they do not keep pesticides in the house or reuse their packaging in the household. They know that pesticides are dangerous to health and do not allow children to manipulate pesticides (except for two cases in Aguas Negras). However, these few cases of children who handle pesticides (helping in preparations) and several children involved in agricultural tasks (fertilizing, cleaning, and harvesting), as well as the reuse of pesticide containers in household chores, possibly linked to low education levels of the parents (Table 1), suggests the existence of undesirable and avoidable situations that pose risk of exposure to these substances and the consequential adverse effects on health.
In this regard, the presence of ATZ and its metabolites (ADI and ADDI) in urine samples (Table 3) confirm infant exposure, mainly in the participants from Pelayito and Aguas Negras, to one of the pesticides with increased use in the agricultural areas studied. Because this pesticide is removed from the body fairly quickly (24 to 48 h), concentrations in urine only indicate recent exposure [49]; therefore, the absence of measurable concentrations of these markers in the other samples in the agricultural areas may be related to its rapid elimination from the body. The absence of ATZ or its metabolites in a sampling point, as in this study, does not rule out, with sufficient certainty, exposure to it, considering that the populations evaluated are in an environment with potential chronic exposure, given the proximity of housing to the fields where crops are grown throughout the year, with corn being one of the principal crops in which ATZ is one of the most widely used pesticides [50].
This may represent chronic exposure not only to ATZ, but also to the rest of pesticides used on crops, most of which have shown genotoxic effects on different types of human cells [30 - 32, 34, 35, 39, 40, 42 - 44] and have been banned in other countries because of the danger (Table 2). This situation poses a permanent risk of adverse effects on health associated to these substances, which can be caused, for example, by altering processes and genetic instability, evident by evaluating cytogenetic damage biomarkers, such as MN, nuclear buds, and cell apoptosis, among others [3, 25, 51], which have been considered as early biomarkers of cancer risk [52 - 54].
Atrazine is a pesticide known as an endocrine disruptor linked to birth defects in children with prenatal exposure with sufficient evidence of its adverse effects on the health of fetuses, infants, and children [55 - 58]. In contrast, international toxicological evaluations of ATZ have concluded that evidence is insufficient on its carcinogenic effects in humans [55, 59]. In this study, no statistically significant relationships were found between the concentrations of ATZ or its metabolites (ADI and ADDI) and the cytogenetic damage biomarkers evaluated; however, the agricultural areas where measurable concentrations of ATZ and/or its metabolites were recorded had higher frequencies of MN, nuclear buds, and apoptotic cells (Tables 3 and 4). Among these, Pelayito registered the highest percentage of samples with ATZ and its metabolites (ADI and ADDI) and also recorded the shortest distance between homes and crops, highest application frequencies of pesticides, and greater number of children living with parents who work with pesticides (Tables 1, 3, and 5). Similarly, an 8-year follow up study on exposure to organophosphate pesticide in children living in an agricultural area in Washington State reported association between the proximity of homes to the fields and increasing pesticide concentrations in house dust and urinary metabolites [60].
On the other hand, it has been described that variables, like age and gender can influence MN frequency in adult populations, however, in the infant population these variables do not seem to have a definite influence [61], which agrees with the
results of the present study. Exposure to cigarette smoke may also influence the frequency of cytogenetic damage biomarkers. However, the few cases reported in the present study of children living with parents who smoke did not allow the evaluation of possible relationships between these variables.
Few studies have been published on the evaluation of cytogenetic damage in peripheral blood lymphocytes in children living in agricultural areas or exposed to pesticides. Most studies have been conducted with oral epithelium cells because samples are obtained more easily and with non-invasive methods [61]. The results of this research show an increasing trend in the frequency of cytogenetic damage (MN, nuclear buds, and apoptotic cells) in groups from agricultural areas, as compared to the control group. Similar results were described in a study that evaluated cytogenetic damage in children (7 to 11 years of age) in a rural agricultural area with prolonged exposure to pesticides, with a comet assay coupled to the FPG enzyme and testing of cytokinesis-block MN, which reported a significant increase in breaks of single strand of DNA and the frequency of MN in children exposed, as compared to a control group in an area not exposed to pesticides [11]. Another study on lymphocytes in healthy children not exposed to any pollutant, aged between 4 and 14 years in the city of Zagreb, Croatia [62], reported a frequency of MN = 2.32 ± 0.28 and nuclear buds = 1.44 ± 0.19, relatively smaller than those described in this research.
Furthermore, several reviews on cytogenetic alterations in adult population exposed to pesticides have been published. Matheus and Bolaños [10], in a review of 33 publications worldwide (16 on MN in peripheral blood lymphocytes, 13 on oral epithelial cells, and 4 on both techniques), described more frequent cytogenetic abnormalities in individuals exposed to pesticides, as compared to unexposed ones, in 24 of these publications. Another review of 100 publications worldwide described an increase in the frequency of chromosomal aberrations, MN, DNA damage (measured by the comet assay), and/or sister chromatid exchange in peripheral blood lymphocytes or cells of the oral mucosa of individuals with
occupational or environmental exposure to pesticides, in 73 of these publications [12]. In short, most of the studies described above recorded an increasing trend in the frequency of cytogenetic damage in individuals more closely related to pesticides (children or adults), as compared to control groups not exposed, consistent with the results of this study. Recently, a review by Bolognesi and Holland [3], on populations exposed to pesticides in different occupational or environmental scenarios, reported increases in MN frequency associated to pesticide exposure in 36 of 49 studies conducted in different countries in Europe, South America, the USA, Asia, and Australia. Highlighting that increased MN frequency was associated to the extent, duration, and type of exposure and that in occupational scenarios where adequate protection measures were used, no significant increases were noted in MN frequency.
5. Conclusion
Results evidenced infant exposure in the groups from Pelayito, Aguas Negras, and Cabuya to one of the pesticides (ATZ) with greater use in the agricultural areas evaluated and an increasing trend in the frequency of cytogenetic damage markers (MN, nuclear buds, and apoptotic cell) therein, as compared to the unexposed control group. This, coupled with the constant risk from the continuous use of pesticides and the lack of studies on adverse health effects that may be caused by these substances in infant populations in these agricultural areas, suggests the need to continue studies in response to the greater vulnerability of and the special attention needed by infant populations, establishing prevention measures and/or relevant solutions.
Acknowledgements The authors thank Colombia’s Administrative Department of Science, Technology and Innovation, COLCIENCIAS, for financial support [Grant 377-2011, project code 1112-545-31611].
References [1] S. Mostafalou, M. Abdollahi, Pesticides: an update of human exposure and toxicity. Arch Toxicol. 91(2) (2017) 549-599. doi: 10.1007/s00204-016-1849-x. [2] C. Fortes, C. Aprea, Cancer risks from residential exposure to pesticides, Encyclopedia of Environmental Health (2011) 489-497. [3] C. Bolognesi, N. Holland, The use of the lymphocyte cytokinesis-block micronucleus assay for monitoring pesticide-exposed populations, Mutat. Res.: Rev. Mutat. Res. (2016), http://dx.doi.org/10.1016/j.mrrev.2016.04.006. [4] L. Palani, N. Panneerselvam, Toxic effects of pesticides: A review on cytogenetic biomonitoring studies, FU. Med. Biol. 15 (2008) 46-50. [5] C.A. Damalas, I.G. Eleftherohorinos, Pesticide exposure, safety issues, and risk assessment indicators, Int. J. Environ. Res. Public Health 8 (2011) 1402-1419. [6] International Agency for Research on Cancer (IARC), List of classifications by cancer sites with sufficient or limited evidence in humans, Volumes 1 to 118. 2017. http://monographs.iarc.fr/ENG/Classification/Table4.pdf. [7] International Agency for Research on Cancer (IARC), Some organophosphate insecticides and herbicides, Volume 112. 2015. http://monographs.iarc.fr/ENG/ Monographs/vol112/mono112.pdf. [8] C. Bolognesi, Genotoxicity of pesticides: a review of human biomonitoring studies, Mut. Res. 543 (2003) 251–272. [9] C. Infante-Rivard, S. Weichenthal, Pesticides and childhood cancer: An update of Zahm and Ward’s 1998 review, J. Toxicol. Environ. Health, Part B 10 (2007) 8199. [10] T. Matheus, A. Bolaños, Micronúcleos: biomarcador de genotoxicidad en expuestos a plaguicidas, Salus 18 (2014) 18-26. [11] L. Kapka-Skrzypczak, M. Posobkiewic, P. Holownia, J. Niedzwiecka, K. Sawicki, M. Cyranka, M. Kruszewski, Assessing DNA damage in children
environmentally exposed to pesticides through using the comet assay and the micronucleus test, Eur. J. Public Health 23 (2013) 260-260. [12] D. Aiassa, F. Mañas, B. Bosch, N. Gentile, N. Bernardi, N. Gorla, Biomarcadores de daño genético en poblaciones humanas expuestas a plaguicidas, Acta. Biol. Colomb. 17 (2012) 485-510. [13] D. Aiassa, F. Mañas, N. Bernardi, N. Gentile, A. Méndez, D. Roma, N. Gorla, Monitoring genotoxicity in humans exposed to pesticides. Preliminary study in children, Revista Cuestiones de Población y Sociedad 4 (2014) 73-84. [14] V. Garry, Pesticides and children, Toxicol. Applied. Pharmacol 198 (2004) 152-163. [15] M. Neri, M. Ceppi, L.E. Knudsen, D. Franco, R. Barale, R. Puntoni, S. Bonassi, Baseline micronuclei frequency in children: estimates from meta- and pooled analyses, Environ. Health. Perspect. 113 (2005) 226-1229. [16] Superintendencia de Industria y Comercio de Colombia, Estudios económicos sectoriales. Estudio sobre plaguicidas en Colombia. N°7. 2013. http://www.sic.gov.co/drupal/recursosuser/documentos/Estudios-Academicos/Doc umentos-Elaborados-Grupo-Estudios-Economicos/7_Estudio_Sobre_Sector_ Plaguicidas_Colombia_Diciembre2013.pdf. [17] M. Varona, O. Cárdenas, C. Crane, S. Rocha, G. Cuervo, J. Vargas, Cytogenetic alterations in field workers routinely exposed to pesticides in Bogota flower farms, Biomédica 23 (2003) 141-152. [18] M.I. Hanna, M.E. Orozco, Occupational pesticide exposure in cotton growers: valley of the middle Sinu, Económicas CUC 35 (2014) 65-74. [19] M. Varona, R.A. Castro, M.I. Páez, N. Carvajal, E. Barbosa, L.M. León, S.M. Díaz, Impact on health and environment of exposure to pesticides and implementation of best agricultural practices in tomato production, Colombia, 2011, Rev. Chil. Salud Pública 16 (2012) 96-106. [20] C. Bolognesi, G. Carrasquilla, S. Volpi, K.R. Solomon, E.J. Marshall, Biomonitoring of genotoxic risk in agricultural workers from five Colombian regions: association to occupational exposure to glyphosate, J. Toxicol. Environ. Health A 72 (2009) 986-997.
[21] G. Palencia, T. Mercado, E. Combatt, Estudio agroclimático del departamento de Córdoba, Universidad de Córdoba, Montería, 2006. [22] K. Acosta, La economía de las aguas del rio Sinú. Documento de trabajo sobre economía regional, Banco de la Republica, Cartagena, 2013. http://www.banrep.gov.co/docum/Lectura_finanzas/pdf/dtser_194.pdf. [23] Resolución 008430/1993 de 4 de Octubre, por la cual se establecen las normas académicas, técnicas y administrativas para la investigación en salud. (Ministerio de Salud y Protección Social, Bogotá D.C.). [24] The National Institute for Occupational Safety and Health (NIOSH), Manual of analytical methods, fourth ed., Method 8315 Triazine herbicides and their metabolites in urine, 2003. https://www.cdc.gov/niosh/docs/2003154/pdfs/8315.pdf. [25] M. Fenech, Cytokinesis-block micronucleus cytome assay, Nat. Protoc. 2 (2007) 1084-1094. http://www.nature.com/nprot/journal/v2/n5/pdf/nprot.2007.77.pdf. [26] World Health Organization (WHO), The WHO recommended classification of pesticides by hazard and guidelines to classification 2009, WHO, Geneva, 2010. [27] Instituto Colombiano Agropecuario, Restricciones, prohibiciones y suspensión de registros de plaguicidas de uso agrícola en Colombia 2016. http://www.ica.gov.co /getdoc/b2e5ff99-bd80-45e8-aa7ae55f0b5b42dc/PLAGUICIDAS-PROHIBIDOS. aspx. [28] Pesticides Action Network, The list of lists – 3rd edition 2009. http://www.paneurope.info/old/Campaigns/pesticides/documents/cut_off/list%20of%20lists.pdf. [29] Pesticides Action Network, International consolidated list of banned pesticides. 2015. http://pan-international.org/pan-international-consolidated-list-of-bannedpesticides/. [30] D. Li, Q. Huang, M. Lu, L. Zhang, Z. Yang, M. Zong, L. Tao, The organophosphate insecticide chlorpyrifos confers its genotoxic effects by inducing DNA damage and cell apoptosis, Chemosphere. 135 (2015) 387 - 393. [31] S. Sandal, B. Yilmaz, Genotoxic effects of chlorpyrifos, cypermethrin, endosulfan and 2,4-D on human peripheral lymphocytes cultured from smokers
and nonsmokers. Environ. Toxicol. 26(5) (2011) 433 - 442. http://dx.doi.org/.10.1002/ tox.20569. [32] R. Patnaik, R.N. Padhy, Evaluation of geno-toxicity of methyl parathion and chlorpyrifos to human liver carcinoma cell line (HepG2). Environ. Sci. Pollut. Res. 23(9) 2016 8492 - 8499. http://dx.doi.org/.10.1007/s11356-015-5963-8. [33] K.B. Abdelaziz, A.I. El Makawy, A.Z.E. Abd Elsalam, A.M. Darwish, Genotoxicity of chlorpyrifos and the antimutagenic role of lettuce leaves in male mice. Comunicata Scientiae 1(2) (2010) 137-145. [34] F.L. Edwards, C.G. Yedjou, P.B. Tchounwou, Involvement of oxidative stress in methyl parathion and parathion-induced toxicity and genotoxicity to human liver carcinoma (HepG2) cells. Environ. Toxicol. 28 (2013) 342 - 348. [35] G. Argentin, M. Divizia, R. Cicchetti, Oxidative stress, cytotoxicity, and genotoxicity induced by methyl parathion in human gingival fibroblasts: Protective role of Epigallocatechin-3-Gallate, J. Toxicol. Environ. Health A. 78 (2015) 12271240. doi: 10.1080/15287394.2015.1079582. [36] M. Coral, S. Ucman, H. Yildiz, H. Oztas, S. Dalkilic, Potential neoplastic eff ects of parathion-methyl on rat liver, J. Environ. Sci. 21 (2009) 696–699. [37] A.Y. Kocaman, M. Topaktaş, The in vitro genotoxic effects of a commercial formulation of alpha-cypermethrin in human peripheral blood lymphocytes. Environ. Mol. Mutagen. 50(1) (2009) 27-36. Doi: 10.1002/em.20434. [38] A.Y. Kocaman, M. Topaktaş Genotoxic effects of a particular mixture of acetamiprid and α-cypermethrin on chromosome aberration, sister chromatid exchange, and micronucleus formation in human peripheral blood lymphocytes. Environ. Tox. 25(2) (2009) 157-168. Doi: 10.1002/tox.20485. [39] G. Jovtchev, S. Gateva, M. Stergios, S. Kulekova, Cytotoxic and genotoxic effects of paraquat in Hordeum vulgare and human lymphocytes in vitro. Environ. Toxicol. 25(3) (2010) 294 - 303. [40] S.O. Abarikwu, E.O. Farombi, Atrazine induces apoptosis of SH-SY5Y human neuroblastoma cells via the regulation of Bax/Bcl-2 ratio and caspase-3-dependent pathway. Pestic. Biochem. Physio. 118 (2015) 90 - 98.
[41] B.C. Ventura-Camargo, M.A. Marin-Morales, Micronuclei and chromosome aberrations derived from the action of atrazine herbicide in Allium cepa meristematic cells. SDRP-JESES. 1(1) (2016) 1 – 7. [42] C. Bolognesi, G. Carrasquilla, S. Volpi, K.R. Solomon, E.J. Marshall, Biomonitoring of genotoxic risk in agricultural workers from five Colombian regions: association to occupational exposure to glyphosate, J. Toxicol. Environ. Health A 72 (2009) 986 - 997. [43] International Agency for Research on Cancer (IARC), IARC Monographs volume 112: evaluation of five organophosphate insecticides and herbicides. 2015. https://www.iarc.fr/en/media-centre/iarcnews/pdf/MonographVolume112.pdf. [44] C. Monroy, A. Cortés, D. Mercedes, H. Groot, Cytotoxicity and genotoxicity of human cells exposed in vitro to glyphosate, Biomédica 25 (2005) 335-345. [45] C.S. Caramello, M.J. Jorge, N.L. Jorge, L.C. Jorge, Evaluation of herbicide glyphosate effects in the fish Prochilodus lineatus using chromosome aberration test. Rev. Vet. 28(1) (2017) 65-68. [46] B. Holečková, Evaluation of the in vitro effect of glyphosate-based herbicide on bovine lymphocytes using chromosome painting. Bull. Vet. Inst. Pulawy. 50 (2006) 533-536. [47] M.S. Mecchi, Salmonella–Escherichia Coli/mammalian–microsome reverse mutation assay with a confirmatory assay. Unpublished regulatory study. Report Identification Number: CV-2002-186. 2003. [48] B. Kaya, A. Creus, A. Yanikoğlu, O. Cabré, R. Marcos, Use of the Drosophila wing spot test in the genotoxicity testing of different herbicides. Environ. Mol. Mutagen. 36 (2000) 40 - 46. [49] Agency for Toxic Substances and Disease Registry (ATSDR), Atrazina CAS # 1912-24-9. 2003. http://www.atsdr.cdc.gov/toxfaqs/tfacts153.pdf. [50] A.M. Hansen, L.G. Treviño-Quintanilla, H. Márquez-Pacheco, M. VilladaCanela, L.C. González-Márquez, R.A. Guillén-Garcés, A. Hernández-Antonio, Atrazina: un herbicida polémico, Rev. Int. Contam. Ambie. 29 (2013) 65-84. [51] M. Fenech, M. Kirsch-Volders, A.T. Natarajan, J. Surralles, J.W. Crott, J. Parry, H. Norppa, D.A. Eastmond, J.D. Tucker, P. Thomas, Molecular mechanisms
of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells, Mutagenesis 26 (2011) 125-132. [52] E. Murgia, M. Ballardin, S. Bonassi, A.M. Rossi, R. Barale, Validation of micronuclei frequency in peripheral blood lymphocytes as early cancer risk biomarker in a nested case-control study, Mutat. Res. 639 (2008) 27-34. [53] S. Bonassi, A. Znaor, M. Ceppi, C. Lando, W.P. Chang, N. Holland, M. KirschVolders, E. Zeiger, S. Ban, R. Barale, M.P. Bigatti, C. Bolognesi, A. CebulskaWasilewska, E. Fabianova, A. Fucic, L. Hagmar, G. Joksic, A. Martelli, L. Migliore, E. Mirkova, M.R. Scarfi, A. Zijno, H. Norppa, M. Fenech, An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans, Carcinogenesis 28 (2007) 625-631. [54] R.A. El-Zein, M.B. Schabath, C.J. Etzel, M.S. Lopez, J.D. Franklin, M.R. Spitz, Cytokinesis blocked micronucleus assay as a novel biomarker for lung cancer risk, Cancer. Res. 66 (2006) 6449-6456. [55] FAO/WHO, Pesticide residues in food 2007. Joint FAO/WHO meeting on pesticide residues. FAO plant production and protection paper 191. Rome, 2007. ftp://ftp.fao.org/docrep/fao/010/a1556e/a1556e00.pdf. [56] A.J. Agopian, P.J. Lupo, M.A. Canfield, P.H. Langlois, Case-control study of maternal residential atrazine exposure and male genital malformations, Am. J. Med. Genet Part A. 161 (2012a) 977-982. [57] A.J. Agopian, P.H. Langlois, Y. Cai, M.A. Canfield, P.J. Lupo, Maternal residential atrazine exposure and gastroschisis by maternal age, Mater. Child Health J. 17 (2012b) 1768-1775. [58] A.J. Agopian, Y. Cai, P.H. Langlois, M.A. Canfield, P.J. Lupo, Maternal residential atrazine exposure and risk for choanal atresia and stenosis in offspring, J. Pediatr. 162 (2013) 581-586. [59] P. Boffetta, A. Hans-Olov, B. Sir Colin, M. Jack, Atrazine and cancer: a review of the epidemiologic evidence, Eur. J. Cancer Prev. 22 (2013) 169-180. [60] R.A. Fenske, C. Lu, N.J. Simcox, C. Loewenherz, J. Touchstone, T.F. Moate, E.H. Allen, J.C. Kissel, Strategies for assessing children's organophosphorus pesticide exposures in agricultural communities, J. Expo. Anal. Environ. Epidemiol. 10 (2000) 662-671.
[61] N. Holland, A. Fucic, D.F. Merlo, R. Sram, M. Kirsch-Volders, Micronuclei in neonates and children: effects of environmental, genetic, demographic and disease variables, Mutagenesis 26 (2011) 51–56. [62] G. Gajski, M. Geric, V. Orešcanin, V. Garaj-Vrhovac, Cytogenetic status of healthy children assessed with the alkaline comet assay and the cytokinesis-block micronucleus cytome assay, Mutat. Res. 750 (2013) 55-62.
Table 1. Demographic data of study populations.
Variable
Sex Male Female Total Residence time in the zone ≥ 3 years From birth Total Go to school? Yes No Total Live with parents smoking? Yes No Total Parents work with pesticides? Yes No Total Father education level No study Incomplete primary Complete primary Incomplete secondary Complete secondary Technical or Technological University Total Mother education level No study Incomplete primary Complete primary Incomplete secondary Complete secondary Technical or Technological University Total Source of drinking water Tap-drinking Total Make any previous treatment? Yes No Total Save pesticides inside the house? Yes
Study zones Aguas Cabuya Negras N° N° % % Ind Ind
Ceibita
Pelayito
Control
N° Ind
N° Ind
N° Ind
%
%
%
10 7 17
58.8 2 41.2 6 100.0 8
25.0 10 75.0 3 100.0 13
76.9 5 23.1 7 100.0 12
41.7 9 58.3 4 100.0 13
69.2 30.8 100.0
1 16 17
5.9 0 94.1 8 100.0 8
0.0 2 100.0 11 100.0 13
15.4 1 84.6 11 100.0 12
8.3 3 91.7 10 100.0 13
23.1 76.9 100.0
17 0 17
100.0 8 0.0 0 100.0 8
100.0 13 0.0 0 100.0 13
1000 12 0.0 0 100.0 12
100.0 13 0.0 0 100.0 13
100.0 0.0 100.0
1 16 17
5.9 1 94.1 7 100.0 8
12.5 2 87.5 11 100.0 13
15.4 2 84.6 10 100.0 12
16.7 2 83.3 11 100.0 13
15.4 84.6 100.0
5 12 17
29.4 3 70.5 5 100.0 8
37.5 1 62.5 12 100.0 13
7.7 10 92.3 2 100.0 12
83.3 0 16.7 13 100.0 13
0.0 100.0 100.0
1 0 1 11 2 2 0 17
5.9 0.0 5.9 64.7 11.8 11.8 0.0 100.0
0 3 0 1 1 2 1 8
0.0 37.5 0.0 12.5 12.5 25.0 12.5 100.0
0 2 2 3 6 0 0 13
0.0 15.4 15.4 23.1 46.1 0.0 0.0 100.0
1 1 1 4 5 0 0 12
8.3 8.3 8.3 33.3 41.6 0.0 0.0 100.0
1 0 1 2 6 2 1 13
7.7 0.0 7.7 15.4 46.1 15.4 7.7 100.0
0 0 0 7 10 0 0 17
0.0 0.0 0.0 41.2 58.8 0.0 0.0 100.0
0 0 1 4 1 0 2 8
0.0 0.0 12.5 50.0 12.5 0.0 25.0 100.0
2 1 0 8 2 0 0 13
15.4 7.7 0.0 61.5 15.4 0.0 0.0 100.0
0 6 3 3 0 0 0 12
0.0 50.0 25.0 25.0 0.0 0.0 0.0 100.0
0 0 0 0 5 4 4 13
0.0 0.0 0.0 0.0 38.4 30.8 30.8 100.0
17 17
100.0 8 100.0 8
100.0 13 100.0 13
100.0 12 100.0 12
100.0 13 100.0 13
100.0 100.0
3 14 17
17.6 2 82.4 6 100.0 8
25.0 2 75.0 11 100.0 13
15.4 1 84.6 11 100.0 12
8.3 11 91.7 2 100.0 13
84.6 15.4 100.0
0
0.0
0.0
0.0
8.3
0.0
0
0
1
0
No Total Re-used pesticides containers for housework? Yes No Total Considered hazardous to health to pesticides? Yes No Total Think your children are exposed to pesticides? Yes No Total The child has handled pesticides? Yes No Total The child has involved in agricultural work? Yes No Total
17 17
100.0 8 100.0 8
100.0 13 100.0 13
100.0 11 100.0 12
91.7 13 100.0 13
100.0 100.0
0 17 17
0.0 0 100.0 8 100.0 8
0.0 4 100.0 9 100.0 13
30.8 1 69.2 11 100.0 12
8.3 0 91.7 13 100.0 13
0.0 100.0 100.0
17 0 17
100.0 8 0.0 0 100.0 8
100.0 13 0.0 0 100.0 13
100.0 12 0.0 0 100.0 12
100.0 13 0.0 0 100.0 13
100.0 0.0 100.0
15 2 17
88.2 7 11.8 1 100.0 8
87.5 13 12.5 0 100.0 13
100.0 7 0.0 5 100.0 12
58.3 0 41.7 13 100.0 13
0.0 100.0 100.0
2 15 17
11.8 0 88.2 8 100.0 8
0.0 0 100.0 13 100.0 13
0.0 0 100.0 12 100.0 12
0.0 0 100.0 13 100.0 13
0.0 100.0 100.0
3 14 17
17.6 0 82.4 8 100.0 8
0.0 4 100.0 9 100.0 13
30.8 7 69.2 5 100.0 12
58.3 0 41.7 13 100.0 13
0.0 100.0 100.0
Table 2. Pesticides most commonly used in the study area. Restriction status Colombia ** Other countries *** Banned in Saudi Not restricted Arabia Banned in Restricted to European Union technified (EU) and 26 cotton and rice countries overall
Pesticide type
Use
Common name
Active ingredient
HC*
Organophosphorus
Insecticide
Lorsban
Chlorpyrifos
II
Organophosphorus
Insecticide
Methyl parathion
Methyl parathion
Ia
Pyrethroid
Insecticide
Cypermethrin
Cypermethrin
II
Not restricted
Bipyridyl
Triazine
Herbicide
Gramoxone
Paraquat
II
Restricted by air
Herbicide
Atrazine
Atrazine
III
Not restricted
Insecticide
Exalt
Spinetoram
U
Not restricted
Herbicide
Roundup or Glyphosate
Glyphosate
III
Not restricted
Not restricted Use not approved or banned in USA, Sweden and 6 African countries Severe restriction in the UE Not restricted Banned in Sri Lanka
Genetic endpoint in human and other cells DNA damage [30 - 32] and chromosomal mutation [33]. DNA damage [32, 34, 35] and gene mutation [36]. DNA damage [31, 37, 38] and chromosomal mutation [37, 38]. DNA damage and chromosomal mutation [39]. DNA damage [40] and chromosomal mutation [41]. No reported. DNA damage [42 – 44], chromosomal mutation [45, 46] and gene mutation [47, 48].
*: Hazard classification according to WHO [26] (Ia = Extremely hazardous, Ib = Highly hazardous, II = Moderately hazardous, III = Slightly hazardous, U = Unlikely to present acute hazard under normal use). **: Colombian Agricultural Institute [27]. ***: Pesticide Action Network [28, 29].
Table 3. Concentrations of atrazine and its metabolites in urine samples. Pesticides or Zone metabolites*
n
ATZ
Pelayito
ADI
ADDI
N° individuals with measurable concentrations (%)
Concentrations (µg/g creatinine) Mean±SD
Median
12 12 (100.0)
18.6 ± 4.3
18.3
MinimumMaximum 13.0 - 25.5
Pelayito
12 5 (41.7)
3.5 ± 4.6
0.0
0.0 - 14.7
Cabuya
8
--
--
22.2
Pelayito
12 11 (91.7)
1 (12.5)
Aguas Negras 17 3 (17.6)
16.8 ± 10.0 17.1 --
--
0.0 - 43.7 190.6 145.9 127.3
* ATZ = Atrazine, ADI = Desisopropyl-atrazine, ADDI = Atrazine Desethyldesisopropyl.
Table 4. Cytogenetic damage in peripheral blood lymphocytes of children in the study. Variable
Group Aguas Negras Cabuya Pelayito The Ceibita Control
n* 17 8 12 13 13
Mean±SD 5.1 ± 2.7 5.0 ± 3.2 4.7 ± 2.5 1.5 ± 1.7 1.1 ± 1.1
Median 5.0 4.5 5.0 1.0 1.0
Minimum-Maximum** 1 - 11 a 2 - 11 a 0 - 10 a 0-6b 0-4b
Nuclear buds
Aguas Negras Cabuya Pelayito The Ceibita Control
17 8 12 13 13
4.1± 2.2 2.9 ± 1.5 1.6 ± 0.8 1.3 ± 1.3 0.6 ± 0.6
3.0 3.0 1.0 1.0 1.0
2-9a 0 - 5 ab 1 - 3 bc 0 - 5 cd 0-2d
Apoptotic cells
Aguas Negras Cabuya Pelayito The Ceibita Control
17 8 12 13 13
15.9 ± 10.5 30.8 ± 29.5 8.1 ± 5.0 4.5 ± 2.3 2.4 ± 2.9
15.0 21.5 8.5 4.0 1.0
3 - 40 a 0 - 76 a 0 - 17 ab 2 - 9 bc 0-9c
MN
*: n = Number of individuals sampled. **: At each variable, letters “a” and "c” correspond to the highest and lowest values, respectively; and groups sharing at least one letter are not significantly different (Dunn test, p <0.05).
Table 5. Variable determinants of pesticide exposure in the study zones. Variable*
Zone
n**
Mean±SD
Median
DHC (m)
Aguas Negras Cabuya Pelayito The Ceibita
17 8 12 13
42.2 ± 61.4 49.9 ± 101.7 4.8 ± 5.9 26.9 ± 24.6
20 10 1 20
MinimumMaximum*** 2 - 200 a 5 - 300 a 1 - 20 b 10 - 80 a
FSP (times/year)
Aguas Negras Cabuya Pelayito The Ceibita
17 8 12 13
58.1 ± 39.1 44.3 ± 32.3 105.0 ± 24.1 14.5 ± 3.5
64 32 108 12
6 - 96 b 22 - 96 b 36 - 144 a 12 - 20 c
FPO (times/year)
Aguas Negras Cabuya Pelayito The Ceibita
17 8 12 13
28.2 ± 40.6 72.3 ± 94.2 78.0 ± 61.1 80.0 ± 95.2
0 27 108 52
0 – 96 a 0 – 224 a 0 – 180 a 0 – 240 a
*: DHC = Distance from house to crops, FSP = Frequency of spraying pesticides on crops, FPO = Frequency of pesticide odor detection at home. **: n = Number of individuals sampled. ***: At each variable, letters “a” and "c” correspond to the highest and lowest values, respectively; and groups sharing at least one letter are not significantly different (Dunn test, p <0.05).