Feminization of the agricultural labor is common in Mexico; these women and their families are vulnerable to several health risks including genotoxicity. Previous papers have presented contradictory information with respect to indirect exposure to pesticides and DNA damage. We aimed to evaluate the genotoxic effect in buccal mucosa from female farmers and children, working in the agricultural valley of Maneadero, Baja California. Frequencies of micronucleated cells (MNc) and nuclear abnormalities (NA) in 2000 cells were obtained from the buccal mucosa of the study population (
Health risk associated with different labors is related to contact with corrosive, infectious, carcinogen, cytotoxic, mutagenic, or genotoxic agents. Research around genetic toxicology and risk assessment or workplace exposure is important since exposure to several hazardous agents is common and could aid in health issues; agricultural activity is often associated with exposure to high volumes of pesticides, mainly organochlorines, organophosphorus, carbamates, pyrethroids, and various inorganic compounds, which are used to control pests in the agriculture zone of Baja California, Mexico [
Women that work as farmers in the agricultural valley in Maneadero, Baja California, usually are coming from the south of the country, most of them from indigenous ancestors with scarce education, and most of them are not provided with medical insurance or social security, even though they are continuously exposed to pesticides [
In Maneadero valley children are indirectly exposed to pesticides through contact with their farmer mothers and the environment, since most of them live less than 500 m from the agricultural areas. Children are especially vulnerable to adverse effects from pesticides, from conception through birth, due to their constant growth and excessive proliferation rate. Furthermore, early in life exposure to pesticides may be particularly detrimental given that children do not have adult level of enzymes to detoxify until after age of 7 [
Several studies have evaluated pesticides effects in offspring from farmers and described the presence of neurological damage, respiratory affection, birth defects, diabetes, cardiovascular diseases, hormonal problems, and genotoxicity [
Nuclear abnormalities (NA) are additional biomarkers that can be recognized through the performance of the MN test; these abnormalities can occur during cell differentiation, indicating DNA damage, cytotoxicity, or cell death when observed in high frequencies [
There has been a clear association between pesticide exposure and genotoxic damage previously; nonetheless most studies did not evaluate genotoxicity through the MN test; we believe that since this is a more practical methodology especially for children, our results can be replicated to search for genotoxicity in farmers and their children working in different countries, without the requirement of expensive, more elaborated, and invasive techniques [
The purpose of the present study was to evaluate the genotoxic effect through the MN test and nuclear abnormalities (NA) in buccal mucosal cells of farmers who work at the agricultural valley of Maneadero and children living in close proximity to the fields.
This study was approved by the research and bioethics committee of Universidad Autonoma de Baja California with registry number 5-031-074-07-001. The study complied with Mexican Research Regulations and the Declaration of Helsinki. Each participant gave their informed consent before sampling.
Study subjects were selected in an intentional nonprobabilistic way, and only after acceptance via informed consent they were included in the study; cases were all encountered, working in the agricultural fields of Maneadero, Baja California, being constantly exposed to several groups of pesticides. Controls were unexposed women selected from a distant population (16 km) in Ensenada, Baja California, who were healthy housewives or black coated workers. We divided the study population into four groups: (1) farmers, (2) unexposed, (3) farmers’ children, and (4) unexposed children. Study subjects were interviewed to obtain personal and clinical information according to predesigned questioner, including the following: age, alimentary habits, smoking, alcohol intake, and time of exposure to pesticides. Subjects with history of cancer, radiotherapy, current infections, and dental procedures within the previous month were excluded from the study.
Each participant rinsed their mouth with water to keep it clean and exfoliated buccal mucosal cells were collected through a smear of the inside lining of the subjects’ oral mucosa of both cheeks. The smears were directly transferred to slides previously coded and dried at room temperature. Afterwards, these slides were fixed with 80% ethanol for 48 h and stained with acetoorcein for 2 h and fast green for 10 minutes as previously described [
The cells in the oral mucosa samples were analyzed using a Carl Zeiss IVFL Axiostar Plus microscope with the objective 100x/1.25. The MNC score was quantified manually and blindly by the same observer, for a total of 2000 cells in order to identify both normal cells and abnormal MN or other NA, using the HUMNxl scoring criteria [
For descriptive purposes about age and exposure years results are expressed as mean (standard deviation); indigenous language, education, daily income, time of exposition, smoking, drinking, and diet habits are expressed as percentages; MNc and NA counts are expressed as mean (standard deviation) and reported as the number of occurrences per 2000 cells. Except for age, the distribution of the variables was not able to pass normality test (Shapiro-Wilks test
Maneadero valley is located 10 km at the south of Ensenada with parcel extension of 4,200 hectares in which 40% of the obtained products are produced the entire year and 60% are seasonal. Main commercial activity in this region is agriculture and products obtained from this region include tomatoes, potatoes, zucchinis, cilantro, lettuce, onions, grapes, strawberry, olive, asparagus, green tomatillo, barley, alfalfa, rye, and flowers. Different pesticides are used in this zone and are described in Table
Main pesticides used in Baja California, Mexico.
Mechanism of action | Uses | Chemical group | Concentration %, EPA classification |
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Organophosphate | |||
Inhibits acetylcholinesterase and is a DNA alkylating agent, classified as carcinogenic, mutagenic, and teratogenic | Insecticide | Diazinon | 25–90, not likely (IV) |
Azinphos-methyl | 35, not likely (I) | ||
Malathion | 90, evidence (IV) | ||
Dimethoate | 40, C (II) | ||
Methamidophos | 39.6–48.3, E (IB) | ||
Herbicide | Bensulide | 12.5, not likely (III) | |
Glyphosate | 48, E (IV) | ||
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Carbamate | |||
Rapid onset; inhibits acetylcholinesterase and other enzymes | Insecticide | Methomyl | 90, E (IB) |
Insecticide | Oxamyl | 24–42, E (IA) | |
Fungicide | Mancozeb | 56.4–80, B2 (III) | |
Fungicide | Maneb | 75–80, B2 (III) | |
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Organochlorines | |||
GABA receptor antagonist inhibits Ca2+, Mg2+ channels. | Insecticide | Endosulfan | 25–48, not likely (I) |
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Pyrethroid | |||
Affects Na+ channels | Insecticide | Permethrin | 34–48, C (IB) |
Bifenthrin | 10, C (II) | ||
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Biperidiles | |||
Interferes in electrons transference and inhibits the reduction of NADP to NADPH during photosynthesis, with superoxide radical formation | Herbicide | Paraquat | 24, C (IA) |
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Others | |||
Mechanism of action not clearly stablished | Fungicide | Copper oxychloride | 85.0, D (III) |
Fungicide | Chlorothalonil | 54, likely (IV) |
In the present study a total of 144 women and their offspring were included; mean age of farmers (
Social and demographic characteristics of farmers from the Maneadero valley and unexposed women.
Farmers ( |
Unexposed ( |
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% | % | ||
Indigenous language | Yes | 27 | 24 |
Not | 73 | 76 | |
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Education | None | 58 | 5 |
Basic | 23 | 19 | |
Middle | 4 | 14 | |
Higher | 15 | 62 | |
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Daily income | MXP | $115.5 | $189.5 |
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Time of residence in sampled locations | 1 to 5 years | 30 | 36 |
6 to 10 years | 5 | 9 | |
10 or more years | 61 | 55 | |
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Years of exposition to pesticides | 1 to 5 years | 51 | NA |
6 to 10 years | 22 | ||
10 to 25 years | 27 | ||
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Smoke | Yes | 6 | 0 |
Not | 94 | 100 | |
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Alcohol | Yes | 6 | 9 |
Not | 94 | 91 | |
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Fruit intake (per week) | 3.00 ± 2.27 | 5.09 ± 1.30 | |
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Vegetable intake (per week) | 2.63 ± 2.67 | 5.18 ± 1.94 | |
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Additional supplements (vitamins) | Yes | 24 | 0 |
Not | 15 | 100 | |
Unanswered | 3 | — |
MXP: Mexican pesos; NA: nonapplicable.
We compared frequencies of MNc and NA between farmers and unexposed women and found significant differences in MNc (
Frequencies of micronuclei and nuclear abnormalities in farmers from the Maneadero valley and children.
Women ( |
Children ( |
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Farmers ( |
Unexposed ( |
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Farmers |
Unexposed ( |
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Age | 35.5 ± 12.4 | 27.7 ± 9.4 | 0.0047 | 7.8 ± 3.2 | 10.3 ± 3.4 | 0.0111 |
MNc | 4.5 ± 5.5 | 0.7 ± 0.9 | <0.0001 | 2.5 ± 2.5 | 0.1 ± 0.2 | <0.0001 |
LN | 2.5 ± 3.7 | 2.6 ± 2.5 | 0.3512 | 3.7 ± 5.3 | 0.7 ± 2.3 | <0.0001 |
BN | 3.1 ± 2.9 | 2.6 ± 2.1 | 0.7317 | 2.2 ± 2.0 | 2.3 ± 2.0 | 0.2833 |
KR | 2.1 ± 4.4 | 1.0 ± 2.3 | 0.2480 | 3.0 ± 5.1 | 1.0 ± 1.2 | 0.9863 |
CC | 2.0 ± 3.4 | 0.8 ± 1.7 | 0.0376 | 2.4 ± 4.2 | 0.1 ± 0.3 | <0.0001 |
PN | 0.3 ± 0.6 | 2.0 ± 2.3 | <0.0001 | 0.3 ± 0.5 | 1.4 ± 1.8 | 0.0043 |
KL | 6.4 ± 12.1 | 1.2 ± 1.6 | 0.0558 | 4.4 ± 6.4 | 4.3 ± 3.1 | 0.0617 |
MNc = micronucleated cells; LN = lobulated nucleus; BN = binucleated cells; PN = pyknotic cells; CC = condensed chromatin; KR = karyorrhexis; KL = karyolysis. Results are presented as mean ± SD from number of occurrences per 2000 counted cells.
Frequencies of micronucleated cells and nuclear abnormalities in farmers and unexposed women. F = farmers; U = unexposed; MNc = micronucleated cells; LN = cells with lobulated nucleus; BN = binucleated cells; KR = karyorrhexis; CC = cells with condensed chromatin; PN = pyknotic cells; and KL = karyolysis.
The presence of MNc in the exposed group reflects both genotoxic damage and cell death. With respect to NA, some authors suggest that the presence of binucleated cells (BN) is indicative of genotoxic damage, while pyknosis, condensed chromatin, karyorrhexis, and karyolysis are originated after cytotoxic damage [
Even though the clinical significance of NA presence has not been fully elucidated, an honest attempt has been made by previous researchers to try to understand their biological meaning; all of these NA have been associated with the presence of diverse chronic and degenerative diseases, as well as with exposition to hazardous chemicals. In general, the presence of BN is a reflection of failure of cytokinesis due to either defects in formation of the microfilament ring or cell cycle arrest due to malsegregation of chromosomes or telomere dysfunction [
Previous studies in Mexico evaluating genotoxic damage after pesticide exposure were mostly performed in lymphocytes, and significant increased frequencies of MNc were found in exposed subjects [
During the present study we were interested in a small subgroup of farmers that were found to have >5 MNc (14 out of 37 women); this subgroup could be at risk of developing health related issues. Because of this, we searched for other factors that could be responsible for higher MNc frequencies, such as alcohol consumption and tobacco use, living in close proximity to the fields (<500 mts2), time of exposition to pesticides (>5 years), and increased age (>35 years); we found that none of these factors, previously associated with increased MNc frequency, was significantly associated with higher frequencies of MNc; results from this analysis are represented in Table
Risk factors that could increase MNc frequency in farmers (
Variable | Samples | >5 MNc | <5 MNc | OR (CI 95%) |
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Alcohol consumption and tobacco use | 5 (14) | 3 (8) | 2 (5) | 5.36 (0.74–38.70) | 0.1102 |
Living in close proximity to fields (<400 m2) | 21 (57) | 7 (19) | 14 (38) | 2.17 (0.46–10.20) | 0.4613 |
Time of exposition to pesticides (>5 years) | 18 (49) | 5 (14) | 13 (34) | 1.07 (0.25–4.59) | 1.000 |
Age (>35 years of age) | 11 (30) | 5 (14) | 6 (16) | 3.50 (0.75–16.27) | 0.1249 |
MNc: micronucleated cells, CI: confidence interval. Fisher’s exact test (two-sided); a
There are only a few studies that assess the distances where people involved in farming are living; one of these studies was carried out by Lee and colleagues [
When we evaluated frequencies of MNc in association with time of exposure to pesticides, we found that there was a slight increase in MNc frequencies after acute exposition <5 years (
With respect to children, the highest frequencies of MNc were found in farmers’ children (
Micronuclei and nuclear abnormalities frequencies in indirectly exposed children and unexposed children. MNc = micronucleated cells; LN = cells with lobulated nucleus; BN = binucleated cells; KR = karyorrhexis; CC = cells with condensed chromatin; PN = pyknotic cells; and KL = karyolysis.
The induction of MNc formation after indirect exposure to pesticides might be due to the fact that farmers’ homes are located in close proximity to the fields and additionally by contact with clothes or personal objects impregnated with pesticides. Children are especially susceptible to genotoxic damage due to differences in the mutagens metabolism, distribution, and excretion, when compared to adults [
Our findings suggest that in farmers’ children there is a chromosomal instability represented by increased frequencies of MNc and NL; additionally they could suffer from cytotoxicity, since CC represents cells that are no longer transcriptionally active and undergoing apoptosis. The presence of PN represents an irreversible condensation of chromatin; this NA is often found with CC and KR although their biological significance is not clear [
In order to clearly identify the genotoxic damage that these children are exposed to, future research is needed, where information about the degree of exposition is registered, including lifestyle, nutrition, and immune function in order to be able to elucidate mechanisms for micronuclei generation during childhood and adult life.
Farmers are at risk of developing genotoxic damage secondary to pesticide exposition; more importantly their children who are living in close proximity to the fields demonstrate this genotoxic damage. Children can be easily monitored for genotoxic damage through the MN test; it would be of extreme importance to develop more studies with prospective design in order to obtain information about relative risks and even more important to design strategies directed towards the prevention of future genotoxic damage of the identified population with higher MNc and NA frequencies, given the fact that they are susceptible to health related issues.
The authors declare that there is no conflict of interests regarding the publication of this paper.