Immunomodulatory effects of the fungicide Mancozeb in agricultural workers
Introduction
Ethylenebisdithiocarbamates (EBDCs) are widely used in agriculture as fungicides, mainly on fruit, vineyard, and potato crops. Their extensive world-wide consumption can be ascribed to their low acute toxicity and their short environmental persistence.
In the last decades, evidences have been emerging supporting an immunomodulatory effect of these compounds and other dithiocarbamates (Lombardi et al., 1991, Padget et al., 1992, Pruett et al., 1992). One compound of this group, the sodium diethyldithiocarbamate (Imuthiol), was shown to be a potent in vivo immunomodulator, influencing maturation and activation of T cells, NK cell activity, IgG secretion, and prolonging immunological memory (Renoux and Renoux, 1980, Renoux et al., 1988). Based on these properties, Imuthiol has been experimented as immunostimulator for the therapy of different clinical immunodeficiency conditions, including HIV infections; however, the results of double-blind placebo-controlled trials failed to show any beneficial effects (Hersh et al., 1991, Vanham et al., 1993). In workers occupationally exposed to an EBDC compound, the ethylenebisdithiocarbamate of manganese and zinc (Mancozeb), an increase in serum IgG, IgE, and α2-macroglobulin was observed (Jablonicka et al., 1989). Studies carried out by our research group demonstrated, in a group of workers engaged in Mancozeb production, an increase in T-cell proliferative responses, suggesting an immunostimulator effect in conditions of low-level, prolonged occupational exposure (Colosio et al., 1996). On the other hand, ethylenebisdithiocarbamate of zinc did not show any immunoenhancing effect in response to T-cell mitogens, since only a cytocidal effect on spleen lymphocytes was shown (Renoux et al., 1988), whereas sodium methyldithiocarbamate has also been reported to cause a significant immunosuppression in mice following in vivo exposure (Padget et al., 1992, Pruett et al., 1992). At present, given the number of conflicting reports, there is insufficient evidence either in humans or animals to confirm, or to rule out, the hypothesis that EBDC compounds can act as immunomodulators in occupational or environmental exposures.
This study has been carried out with the aim of evaluating the immune system of a group of agricultural workers occupationally exposed to Mancozeb.
Section snippets
Population under study
The study was carried out in Northern Italy, in a rural area named “Oltrepò Pavese”, on 13 vineyard workers (12 males and 1 female; mean age: 41, range: 25–54) and 13 matched healthy controls (12 males and 1 female; mean age: 40, range: 25–50). Agricultural workers were engaged in pesticide application on vineyard. In the period of the investigation (May–June), workers applied by tractor, three to four times, a Mancozeb-based plant-protection product, with a daily use of 5–10 kg of active
Personal data collection
Both exposed and controls were asked to fill in a questionnaire aimed at collecting data on health status, smoking habits, alcohol intake, and working conditions. Information on the possible presence of confounding factors, such as pharmacological treatment, surgery, and vaccination, was also collected.
Urinary ETU
Mancozeb exposure was assessed through the determination of the urine concentration of a major Mancozeb metabolite, ethylenethiourea (ETU). Urinary ETU was analyzed by high performance liquid chromatography (HPLC) with UV diode array detection, as previously reported (Colosio et al., 2002). Detection limit of the assay was 0.5 μg/g creatinine.
Blood analysis
The following parameters were measured: complete and differential blood cell count, serum immunoglobulins (IgA, IgE, IgG, IgM), complement fractions (C3 and C4), anti-nuclear, anti-mitochondrial, and anti-smooth muscle autoantibodies (ANA, AMA, ASMA), and lymphocyte subpopulations CD3 (total T-Lymphocytes), CD4 (T-helper), CD8 (T-suppressor-cytotoxic); CD19 (B-cells), CD25 (activated T-cells), and CD56/CD16 (NK cells). The immune function study was completed with functional assays as described
Serum immunoglobulins
Sera were obtained by centrifugation of peripheral blood and kept frozen at −20 °C until analysis. IgA, IgE, IgG, and IgM were determined by nephelometry (Behring nephelometer analyzer, Behringwerke Marburg, Germany). Results are expressed as ng/dl.
Lymphocyte subpopulations
Peripheral heparinized blood (50 μl) was stained with appropriate concentration of fluorescein- or phycoerythrin-conjugated monoclonal antibodies against CD3 (CD3 isotype mouse IgG1, clone SK7, FITC), CD4 (CD4 isotype mouse IgG1, clone SK3, phycoerythrin), CD8 (CD8 isotype mouse IgG1, clone SK1, phycoerythrin), CD19 (CD19 isotype mouse IgG1, clone SJ25C1, phycoerythrin), CD25 (clone 2A3, isotype mouse IgG1, conjugated PE, phycoerythrin), and CD56/CD16 (CD16/CD56 isotype mouse IgG1, clone B73.1,
Proliferative responses
Functional assays were performed using whole blood added of sodium citrate as anticoagulants. Sodium citrate was chosen instead of heparin or EDTA as anticoagulant, since functional assays were performed using the whole blood assay and heparin may be contaminated with endotoxin, while EDTA interferes with cell activation. Blood was diluted 1:10 with cell culture medium RPMI 1640 (Sigma, St Louis, USA) containing 2 mM l-glutamine, 0.1 mg/ml streptomycin, 100 IU/ml penicillin. Microcultures for
Cytokine production
For the evaluation of cytokine production, macro-cultures were set up in open 12 × 75 polypropylene reaction tubes (Falcon, Oxnard, CA, USA) containing 1 ml of 1:10 diluted whole blood, in medium alone or in the presence of the following stimuli: lipopolysaccharide (LPS) at final concentration of 1 μg/ml, PHA 1.2 μg/ml, and PWM 1 μg/ml. For TNF-α release, cells were incubated for 24 h, while for IL-4 and γ-IFN for 72 h at 37 °C in a humidified 5% CO2 incubator. Cell-free supernatants obtained
Natural killer cytotoxic activity
Lytic activity of NK cells against K562 target cells was assessed by evaluating lactate dehydrogenase (LDH) release according to Korzeniewski and Callewaert (1983) and Morales and Ottenhof (1983) with slight modification. The myeloid leukemia cell line K562 was grown in RPMI 1640 medium supplemented with 10% fetal calf serum (Sigma), containing 2 mM l-glutamine, 0.1 mg/ml streptomycin, 100 IU/ml penicillin. For use as targets, cells were resuspended at 4 × 106 cells/ml and serially diluted to
Statistical analysis
Statistical analysis was carried out using the statistical package SPSS 10.0 for Windows. Because of the small sample size and the skewness of some data-sets, the test of significance was based on non-parametric techniques namely Mann–Whitney test for comparing two independent samples and Wilcoxon's test for paired data before and after exposure. Spearman's correlation coefficient was applied to measure the degree of correlation between dose (urine ETU concentration) and selected immune
Urinary ETU
Pesticide application led to a significant increase of urine ETU concentration in post-exposure (median 2.5 μg/g creatinine) in comparison with pre-exposure (median <0.5 μg/g creatinine P = 0.008) and with controls samples (median <0.5 μg/g creatinine; P = 0.0001). Prior to exposure, levels of urine ETU in workers were comparable to controls (Fig. 1).
Immunoglobulin levels and blood cells parameters
Blood cells counts (Table 1) showed a statistically significant decrease in the percentage of eosinophil in post-exposure samples compared either
Discussion
We investigated the effects of the fungicide Mancozeb on the immune system in a group of occupationally exposed agricultural workers. The levels of urinary excretion of ETU, the major metabolite of Mancozeb, confirmed that absorption of the fungicide took place during application. The investigation of serum, cellular, and functional immune parameters suggested that Mancozeb exerts a slight immunomodulatory effect characterized by (i) an increase in total leukocyte counts; (ii) an increase in
Acknowledgments
This work was partially supported by the Center for Toxicological Risk Assessment of the University of Milan and by the Italian National Institute of Occupational Safety and Prevention (ISPESL). Contract B66/DML/00.
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