Mutagenesis, Vol. 17, No. 1, 79-82,
January 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
A follow-up study on micronucleus frequency in Spanish agricultural workers exposed to pesticides
Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain and 1 Delegación Provincial de la Consejería de Salud de Almería, Carretera de Ronda 101, Almería, Spain
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
|---|
To determine whether occupational exposure to a complex mixture of pesticides results in a significant increase in the level of cytogenetic damage, a follow-up study was planned on 39 greenhouse workers from Almería (southeastern Spain). Taking into account that pesticide exposure can be season-related, two blood samples were taken from each individual at different times: one in a period of high exposure (sample A, spring–summer) and the other in a period of lower exposure (sample B, autumn–winter). Using the cytokinesis block micronucleus technique the frequency of binucleated cells with micronuclei (BNMN) and the cytokinesis blocked proliferation index (CBPI) were determined in peripheral blood lymphocytes. The results obtained indicate that there were no statistically significant differences in BNMN frequencies between the two sampling periods nor between exposed and controls. ANCOVA analysis of repeated measures revealed that the age of the individuals showed a direct relation with BNMN in the first study period. With regard to CBPI, a significant and season-related effect was found.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Undesirable effects of pesticides have been reported in farmers, including genotoxic effects, such as cancer and other genetic diseases (IARC, 1991
; Dich et al., 1997), including leukemia (Blair and Zahm, 1995), bladder cancer (Viel and Chalier, 1995), non-Hodgkin's lymphoma (Waddell et al., 2001) and pancreatic cancer (Ji et al., 2001), among others.
Biomonitoring studies using somatic cells have been extensively used to evaluate the possible genotoxic risk of a defined exposure and some indicators, such as chromosome aberrations, have been shown to be a relevant biomarker for further cancer incidence (Hagmar et al., 1994, 1998). In addition, the use of appropriate biomarkers in these biomonitoring studies can provide useful tools to elucidate the mechanisms of action of the exposure. In this context, different studies have been conducted in human populations occupationally exposed to pesticides, although the results obtained are not conclusive and conflicting (Scarpato et al., 1996a; Venegas et al., 1998; Gómez-Arroyo et al., 2000; Gregorio d'Arce and Colus, 2000; Lucero et al., 2000). One of the possible causes of these divergent data, among others, could be seasonal variations in the levels of pesticide exposure, as shown by Carbonell et al. (1995). Thus, depending on the occupational levels of exposure at the time of sampling, the detected effect can be more or less noteworthy. The best way to detect the effects of seasonal variation is to use a follow-up study approach, which allows a comparison of genetic damage levels over time (Steenland et al., 1985; Carbonell et al., 1995; Scarpato et al., 1996b; Lander et al., 2000). This approach also permits one of the most problematical aspects of human biomonitoring studies, selection of the matched control group, to be overcome. Nevertheless, and in spite of the advantages of this type of approach (Gutiérrez et al., 1999), follow-up studies are rarely conducted, mainly due to the difficulty of obtaining repeated blood samples from the same individual over time.
Therefore, to investigate a potential seasonal effect of pesticide exposure, a follow-up biomonitoring study was carried out by analysing the variation in frequency of micronuclei in peripheral blood lymphocytes in a group of agricultural workers from Almería (southeastern Spain). This population was previously studied (Lucero et al., 2000), without any indication of cytogenetic differences between the exposed and control groups. Nevertheless, and due to the intensive greenhouse agricultural activity in the region, we considered that it would be advisable to carry out a more complete study of this exposed population with a follow-up design and in this manner rule out a season-related effect of pesticide exposure.
It must be pointed out that both peripheral blood cells and micronuclei are widely used for this type of study (Carrano and Natarajan, 1988; Surrallés et al., 1992; Fenech, 1993; Gutiérrez et al., 1997). In particular, the simplicity of scoring micronuclei as well as the ability to detect exposure to both clastogenic and aneugenic agents make micronuclei a good biomarker in human biomonitoring studies (Kirsch-Volders et al., 1997).
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Study population
The study was performed in a group of 39 healthy men working in greenhouses in the province of Almería (Spain), exposed to different mixtures of pesticides, principally carbamates, organophosphates and pyrethroids. For more detailed information see Lucero et al. (2000). In addition, a control group constituting 22 healthy men from the same area, working as clerks, without previous occupational exposure to pesticides or any particular environmental agent, was also studied.
Blood samples were obtained at two different times: in a period of high exposure (March–April, sample A) and in a period of lower exposure (November–December, sample B), after a break for the summer holidays followed by a pause of 3 weeks in the application of pesticides.
Prior to the study all the individuals gave informed consent and blood samples were collected and further manipulated in accordance with ethical standards. All participants completed a questionnaire. See Lucero et al. (2000) for more details.
Lymphocyte cultures and MN analysis
Blood was obtained from each subject by venipuncture using heparinized vacutainers and immediately sent to Barcelona for the establishment of lymphocyte cultures. Lymphocyte cultures were set up by adding 0.5 ml of whole blood to 4.5 ml of RPMI 1640 medium supplemented with 15% heat-inactivated fetal calf serum, 1% antibiotics (penicillin and streptomycin) and 1% L-glutamine (all provided by Gibco Life Technologies, Paisley, UK). Lymphocytes were stimulated by addition of 1% phytohaemagglutinin (Gibco) and incubated for 72 h at 37°C.
A cytochalasin B (Cyt-B) (Sigma, St Louis, MO) solution was prepared in dimethylsulphoxide at a final concentration of 6 µg/ml (Surrallés et al., 1994) and added to the cultures after 44 h incubation to arrest cytokinesis. At 72 h incubation the cultures were harvested by centrifugation at 800 r.p.m. for 8 min. Next, in order to eliminate red cells and to preserve cytoplasm, the cell pellet was treated with a hypotonic solution (2–3 min in 0.075 M KCl at 4°C). Cells were then centrifuged and a methanol/acetic acid (3:1 v/v) solution was gently added. This fixation step was repeated twice and the resulting cells were resuspended in a small volume of fixative solution and dropped onto clean slides. Finally, they were stained with 10% Giemsa (Merck, Darmstadt, Germany) in phosphate buffer, pH 6.8, for 10 min.
To determine the frequency of binucleated cells with micronuclei (BNMN) and the total number of micronuclei, a total of 1000 binucleated cells with well preserved cytoplasm (500 per replicate) were scored for each subject. In addition, 500 lymphocytes were scored to evaluate the percentage of cells with one to four nuclei and the cytokinesis block proliferation index (CBPI) was calculated (Surrallés et al., 1995). Microscopic scoring was performed on coded slides and, to minimize variability, the same expert performed all the microscopic analyses.
Statistical methods
To analyse possible differences between the control and exposed groups, with regard to age and lifestyle habits such as smoking and coffee and alcohol consumption, a Mann–Whitney U-test was performed.
An analysis of covariance (ANCOVA) of repeated measures was carried out to detect differences over time (BNMN and CBPI), as well as to evaluate the effects of covariates (age, alcohol and coffee). BNMN data were transformed to square roots in order to homogenize the variances and make them independent of the means (Draper and Smith, 1981).
A logistic binary regression analysis was used to evaluate differences in the incidence of miscarriages, considering the possible effects of age, alcohol, coffee and exposure to pesticides. Owing to the lack of normality of some haematological and biochemical data, logarithmic transformation was used. Differences between groups were evaluated by the t-test for independent samples, while the t-test for dependent samples was used to compare the variation in the frequency of each selected variable during and after the period of highest exposure. In addition, the 
2 test was used to compare the frequencies between the two groups for other variables, such as hygiene, illnesses, intoxication symptoms, etc.
The statistical software used for the data analyses was STATISTICA (StatSoft, Tulsa, OK) and SPSS version 10.0 (SPSS Inc., Chicago, IL).
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Table I
shows the main characteristics (age, lifestyle and exposure) for both groups (exposed and control). The groups differed significantly only with respect to average age (P = 0.041, U-test) and coffee consumption (P = 0.004, U-test), which were higher in the control group. Another aspect to note in this population is that the average period working in agriculture was 
8 years, which corresponds with a great increase in the number of greenhouses in Almería in the last 10–15 years.
|
Table II
shows the mean values (± SE) for the cytogenetic variables evaluated in both groups and samples. Although the MN values are higher in control sample B than in control sample A, the difference was not statistically significant. Comparing the results reported here with other data from our laboratory corresponding to biomonitoring studies of agricultural workers from Poland and Greece ( Pastoret al., 2001a, b), it appears that the MN values found in the population from Almería are lower in both control and exposed individuals.
|
± SE) of the cytogenetic parameters analysed in the groups studied
In the case of BNMN levels, ANCOVA showed that neither exposure nor the differences with season had any effect on BNMN (Table III
). Of the covariates introduced in the analysis only the age of individuals in sample A had a significant influence on micronucleus frequency (Table IV), in agreement with other authors (Migliore et al., 1991), whilst a lack of effect has also been indicated (da Silva et al., 1997). With regard to CBPI, the ANCOVA analysis revealed a significant effect only with season (Table III). Thus, in both groups the CBPI value was slightly higher in sample B when compared with sample A, suggesting enhanced proliferation of lymphocytes in vitro during the period autumn–winter. Sample B was drawn after a period of low application of pesticides in the winter. Therefore, the increase in proliferation index may reflect a decrease in the cytotoxic effects of pesticides in agricultural workers. Nevertheless, taking into account that an increased level of CBPI was also observed in the control group, the possibility of seasonal fluctuations that induced variations in CBPI over time cannot be ruled out.
|
|
Thus, the results described above indicate that in the group of agricultural workers no significant induction of cytogenetic damage was observed at the two sampling times under these particular conditions of exposure. Our findings agree with other follow-up studies on exposure to pesticides showing no significant changes in micronuclei, chromosomal aberrations (CA) and sister chromatid exchanges (SCE) (Steenland et al., 1985
; Scarpato et al., 1996b). However, in other studies pesticide sprayers sampled at two different times were found to show an increase in the level of CA after the pesticide spraying season (Carbonell et al., 1995; Lander et al., 2000).
Up to now, many biomonitoring studies have been performed in agricultural workers from different regions and under a variety of exposure conditions. In this context, it is not surprising that the results obtained have shown high variability. The usual explanation for this has been, mainly, different levels of exposure (Scarpato et al., 1996a). Nevertheless, the agricultural workers in this study were selected because of the particular characteristics of their working area, where agricultural activity is intensive and exclusively inside greenhouses, leading to a high level of exposure. In addition, the climatic conditions of the area allow three or four crops per year, which implies continual use and application of pesticides throughout the year. The continued exposure to pesticides would also suggest that this kind of exposure could induce an adaptive response. This possible adaptive response would induce an increase in apoptosis of the damaged cells in vitro or a delay in nuclear division, allowing repair of the damage, preventing the detection of exposure effects as BNMN cells (Kirsch-Volders and Fenech, 2001).
On the other hand, another possible explanation for the lack of genotoxic damage could be the protective measures taken by the workers. It is assumed that all the workers who participated in this study were aware that they carried out a job with a potential risk. In accordance with the information obtained from the questionnaires, 93% of them normally used some protective measures. Nevertheless, given the temperature conditions inside the greenhouses, these protective measures may not be followed all year round.
Concerning the adverse health problems associated with pesticide exposure (Weisenburger, 1993), it is interesting to note that from the answers to the questionnaire the percentage of miscarriages in the exposed group (50%) was higher than in the controls (20%), although no significant differences were found in the logistic regression analysis, neither with respect to exposure (P = 0.093) nor for the rest of the variables included as covariates (age, P = 0.186; alcohol, P = 0.522; coffee, P = 0.261). Reproductive dysfunctions, such as spontaneous abortions, infant prematurity, congenital malformations and reduced fertility, have been associated with pesticide exposure in previous investigations (Czeizel et al., 1993; Garry et al., 1996); thus, the high percentage of miscarriages found in the exposed group could be a consequence of pesticide exposure.
In addition, the values obtained for the biochemical and haematological parameters revealed that there were no statistically significant differences between the exposed group and the control group for the parameters analysed (data not shown). Although the plasma cholinesterase (PChE) levels in greenhouse workers were lower in the period of major exposure (sample A) with respect to the period of minor exposure (sample B), both values (12 059.83 ± 369.13 versus 10 501.51 ± 348.28) are within the normal range (<13 200 U/l). Nevertheless, this difference could be attributed to the fact that in the March–April period higher exposure to pesticides produced a reduction in the concentration of PChE. In this context it should be noted that several pesticides, such as organophosphates and carbamates (most used by these workers), are reported to be the major cause of a depression of serum cholinesterases (Yeary et al., 1993) and, as consequence, this is used as a good biomarker of exposure.
In addition, the 2 analysis did not reveal any significant differences between groups for the list of symptoms with high probability of being induced by pesticide exposure (asthenia, dermatitis, cramps, paresthesia, etc.). However, a higher percentage of asthenia (34.5%) and conjunctivitis (13.8%) was observed in the agricultural workers when compared with the controls (20% asthenia and 6.6% conjunctivitis).
|
Acknowledgments |
|---|
We would like to thank G.Umbert and A.Corral for their expert technical assistance in the preparation and scoring of samples, M.McCarthy for her secretarial skills and the Servei d'Estadística (UAB) for statistical advice. This investigation was supported in part by a grant from the European Union (CT96-0300, INCO-Copernicus), awarded to Dr S.M.Piperakis (National Centre for Scientific Research `Demokritos', Athens, Greece).
|
Notes |
|---|
2 Present address: DNA Repair Group, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon Cedex 08, France
3 To whom correspondence should be addressed. Email: richand.marcos{at}uab.es
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
-
Blair,A. and Zahm,S.H. (1995) Agricultural exposures and cancer. Environ. Health Perspect., 103, 205–208.
Carbonell,E., Valbuena,A., Xamena,N., Creus,A. and Marcos,R. (1995) Temporary variations in chromosomal aberrations in a group of agricultural workers exposed to pesticides. Mutat. Res., 344, 127–134.[Web of Science][Medline]
Carrano,A.V. and Natarajan,A.T. (1988) Considerations for population monitoring using cytogenetic techniques. ICPEMC Publication no. 14. Mutat. Res., 204, 379–406.[Web of Science][Medline]
Czeizel,A.E., Elek,C., Gundy,S., Métneki,J., Nemes,E., Reis,A., Sperling,K., Tímár,L., Tusnády,G. and Virágh,Z. (1993) Environmental trichlorfon and cluster congenital abnormalities. Lancet, 341, 539–542.[Web of Science][Medline]
da Silva,L.G., Rocha Lieber,S., Artur Ruiz,M. and Antonio de Souza,C. (1997) Micronucleus monitoring to assess human occupational exposure to organochlorides. Environ. Mol. Mutagen., 29, 46–52.[Web of Science][Medline]
Dich,J., Zahm,S.H., Hanberg,A. and Adami,H.O. (1997) Pesticides and cancer. Cancer Causes Control, 8, 420–443.[Web of Science][Medline]
Draper,N.R. and Smith,H. (1981) Applied Regression Analysis, 2nd Edn. Wiley, New York, NY.
Fenech,M. (1993) The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations. Mutat. Res., 285, 35–44.[Web of Science][Medline]
Garry,V.F., Schreinachers,D., Harkins,M.E. and Griffith,J. (1996) Pesticide appliers, biocides and birth defects in rural Minnesota. Environ. Health. Perspect., 104, 394–399.[Web of Science][Medline]
Gómez-Arroyo,S., Díaz-Sánchez,Y., Meneses-Pérez,M.A., Villalobos-Pietrini,R. and De León-Rodríguez,J. (2000) Cytogenetic biomonitoring in a Mexican floriculture worker group exposed to pesticides. Mutat. Res., 466, 117–124.[Web of Science][Medline]
Gregorio d'Arce,L.P. and Colus,I.M. (2000) Cytogenetic and molecular biomonitoring of agricultural workers exposed to pesticides in Brazil. Teratog. Carcinog. Mutagen., 20, 161–170.[Web of Science][Medline]
Gutiérrez,S., Carbonell,E., Galofré,P., Creus,A. and Marcos,R. (1997) Micronuclei induction by 131I exposure: study in hyperthyroidism patients. Mutat. Res., 373, 39–45.[Web of Science][Medline]
Gutiérrez,S., Carbonell,E., Galofré,P., Creus,A. and Marcos,R. (1999) Cytogenetic damage after 131-iodine treatment for hyperthyroidism and thyroid cancer. Eur. J. Nucl. Med., 26, 1589–1596.[Web of Science][Medline]
Hagmar,L., Brøgger,A., Hansteen,I.-L., Heim,S., Högstedt,B., Knudsen,L., Lambert,B., Linnainmaa,K., Mitelman,F., Nordenson,I., Reuterwall,C., Salomaa,C., Skerfving,S. and Sorsa,M. (1994) Cancer risk in humans predicted by increased levels of chromosomal aberrations in lymphocytes: Nordic Study Group on the Health Risk of Chromosome Damage. Cancer Res., 54, 2919–2922.
Hagmar,L., Bonassi,S., Stromberg,U., Brøgger,A., Knudsen,L., Norppa,H. and Reuterwall,C. (1998) Chromosomal aberrations in lymphocytes predict human cancer: a report from the European Study Group on Cytogenetic Biomarkers and Health (ESCH). Cancer Res., 64, 159–163.
IARC (1991) Occupational Exposures in Insecticide Application and Some Pesticides, IARC Monographs no. 53. IARC, Lyon.
Ji,B.T., Silverman,D.T., Stewart,P.A., Blair,A., Swanson,G.M., Baris,D., Greenberg,R.D., Hayes,R., Brown,L.M., Lillemoe,K.D., Schoenberg,J.B., Pottern,L.M., Schwartz,A.G. and Hoover,R.N. (2001) Occupational exposure to pesticides and pancreatic cancer. Am. J. Ind. Med., 40, 225–226.
Kirsch-Volders,M. and Fenech,M. (2001) Inclusion of micronuclei in non-divided mononuclear lymphocytes and necrosis/apoptosis may provide a more comprehensive cytokinesis block micronucleus assay for biomonitoring purposes. Mutagenesis, 16, 51–58.
Kirsch-Volders,M., Elhajouji,A., Cundari,E. and Van Hummelen,P. (1997) The in vitro micronucleus test: a multi-endpoint assay to detect simultaneously mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction. Mutat. Res., 392, 19–30.[Web of Science][Medline]
Lander,B.F., Knudsen,L.E., Gamborg,M.O., Jarventaus,H. and Norppa,H. (2000) Chromosome aberrations in pesticide-exposed greenhouse workers. Scand. J. Work Environ. Health, 26, 436–442.[Web of Science][Medline]
Lucero,L., Pastor,S., Suárez,S., Durbán,R., Gómez,C., Parrón,T., Creus,A. and Marcos,R. (2000) Cytogenetic biomonitoring of Spanish greenhouse workers exposed to pesticides: micronuclei analysis in peripheral blood lymphocytes and buccal epithelial cells. Mutat. Res., 464, 255–262.[Web of Science][Medline]
Migliore,L., Parrini,M., Sbrana,I., Biagini,C., Battaglia,A. and Loprieno,N. (1991). Micronucleated lympocytes in people occupationally exposed to potential environmental contaminants: the age effect. Mutat. Res., 256, 13–20.[Web of Science][Medline]
Pastor,S., Gutiérrez,S., Creus,A., Cebulska-Wasilewska,A. and Marcos,R. (2001a) Micronuclei in peripheral blood lymphocytes and buccal epithelial cells of Polish farmers exposed to pesticides. Mutat. Res., 495,147–156.
Pastor,S., Gutiérrez,S., Creus,A., Xamena,N., Piperakis,S. and Marcos,R. (2001b) Cytogenetic analysis of Greek farmers by using the micronucleus assay in peripheral lymphocytes and in buccal cells. Mutagenesis, 16, 539–545.
Scarpato,R., Migliore,L., Angotzi,G., Fedi,A., Migli,L. and Loprieno,N. (1996a) Cytogenetic monitoring of a group of Italian floriculturists: no evidence of DNA damage related to pesticide exposure. Mutat. Res., 367, 73–82.[Web of Science][Medline]
Scarpato,R., Migliore,L., Hirvonen,A., Falck,G. and Norppa,H. (1996b) Cytogenetic monitoring of occupational exposure to pesticides: characterization of GSTM1, GSTT1 and NAT2 genotypes. Environ. Mol. Mutagen., 27, 263–269.[Web of Science][Medline]
Steenland,K.M.S., Carrano,A., Clapp,D., Ratcliffe,J., Ashworth,L. and Meinhardt,T. (1985) Cytogenetic studies in humans after short-term exposure to ethylene dibromide. J. Occup. Med., 37, 729–732.
Surrallés,J., Carbonell,E., Marcos,R., Degrassi,F., Antoccia,F. and Tanzarella,C. (1992) A collaborative study on the improvement of the micronucleus test in cultured human lymphocytes. Mutagenesis, 7, 407–410.
Surrallés,J., Antoccia,A., Creus,A., Degrassi,F., Peris,F., Tanzarella,C., Xamena,N. and Marcos,R. (1994) The effects of cytochalasin-B concentration on the frequency of micronuclei induced by four standard mutagens. Results from two laboratories. Mutagenesis, 9, 347–353.
Surrallés,J., Xamena,N., Creus, A, Catalán,J., Norppa,H. and Marcos,R. (1995) Induction of micronuclei by five pyrethroid insecticides in whole blood and isolated human lymphocyte cultures. Mutat. Res., 341, 169–184.[Web of Science][Medline]
Venegas,W., Zapata,Y., Carbonell,E. and Marcos,R. (1998) Micronuclei analysis in lymphocytes of pesticide sprayers from Concepción (Chile). Teratog. Carcinog. Mutagen., 18, 123–129.[Web of Science][Medline]
Viel,J.F. and Chalier,B. (1995) Bladder cancer among French farmers: does exposure to pesticides in vineyards play a part? Occup. Environ. Med., 52, 587–592.
Waddell,B.L., Zahm,S.H., Baris,D., Weisenburger,D.D., Holmes,F., Burmeister,L.F., Cantor,K.P. and Blair,A. (2001) Agricultural use of organophosphate pesticides and the risk of non-Hodgkin's lymphoma among male farmers (United States). Cancer Causes Control, 12, 509–517.[Web of Science][Medline]
Weisenburger,D.D. (1993) Human health effects of agrichemical use. Hum. Pathol., 24, 571–576.[Web of Science][Medline]
Yeary,R.A., Eaton,J., Gilmore,E., North,B. and Singell,J. (1993) A multiyear study of blood cholinesterase activity in urban pesticide application. J. Toxicol. Environ. Health., 39, 11–25.[Web of Science][Medline]
Received on June 18, 2001; accepted on September 26, 2001.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Bull, K. Fletcher, A.R. Boobis, and J.M. Battershill Evidence for genotoxicity of pesticides in pesticide applicators: a review Mutagenesis, March 1, 2006; 21(2): 93 - 103. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||