Neutrophil function in workers exposed to organophosphate and carbamate insecticides

Neutrophil function in workers exposed to organophosphate and carbamate insecticides

International Journal of Immunopharmacology 21 (1999) 263±270 Neutrophil function in workers exposed to organophosphate and carbamate insecticides M...

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International Journal of Immunopharmacology 21 (1999) 263±270

Neutrophil function in workers exposed to organophosphate and carbamate insecticides M.L.S. Queiroz*, M.D. Fernandes, M.C. Valadares Department of Pharmacology/Hemocenter, Faculty of Medical Sciences, UNICAMP PO Box 6111, CEP 113084-100, Campinas, SP, Brazil Received 26 August 1998; accepted 31 December 1998

Abstract Neutrophil function in 40 workers occupationally exposed to carbamate and organophophate insecticides were examined and compared to those of non-exposed individuals. Phagocytosis and intracellular killing of Candida albicans and Candida pseudotropicalis by neutrophils were studied. Two species of Candida were used since in individuals with myeloperoxidase de®ciency neutrophils are unable to kill Candida albicans, while Candida pseudotropicalis can be e€ectively lysed. Phagocytosis of both antigens was normal in all the workers studied. On the other hand, there was a considerable reduction in the ability of neutrophils from exposed workers to kill Candida albicans, whereas Candida pseudotropicalis was e€ectively lysed. This ®nding indicates some interference with the myeloperoxidase activity in the exposed population. The levels of cholinesterase activity in all workers were normal. These results demonstrate that exposure to carbamates and organophophates insecticides may lead to changes in neutrophil function even in workers presenting no impairment in the cholinesterase activity. # 1999 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. Keywords: Anti-ChE insecticides; Immunology; Engulfment and killing capabilities; Neutrophils; Myeloperoxidase

1. Introduction In the past decade, the agricultural use of insecticides in combination, such as carbamates and organophosphates, has become increasingly popular. Exposure to cholinesterase-inhibiting agents is considered a major health problem for the 2.5 to 5 million farmworkers in the United States. Clinical manifestations of such exposure are extremely diverse primarily resulting from * Corresponding author. 0192-0561/99/$20.00 # 1999 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 2 - 0 5 6 1 ( 9 9 ) 0 0 0 0 6 - 5

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the inhibition of cholinesterase (ChE) activity, including several types of cancer, genotoxic e€ect, teratogenic e€ects, sterility, spontaneous abortion and immunosuppression (Sullivan, 1989; Wills, 1972; Midtling, Barnett, & Coye, 1985; Wilk, 1986; Kahn, 1976; Ciesielski, Loomis, Mims, & Auer, 1994; Lander & Ronne, 1995). Several studies on the immunotoxic e€ects of ChE-inhibiting compounds in experimental animals have demonstrated decreased numbers of cells in the spleen and thymus (Ladics, Smith, Heaps, & Loveless, 1994). Other authors observed an inhibition of chemotaxis in rabbit neutrophils and inhibition of Interleukin-2 in the mouse (Ward, 1968; Casale, Vennerstrom, Bavari, & Wang, 1993). Dose-dependent decreases in serum levels of SRBC-speci®c IgM antibody has been reported (Ladics et al., 1994; InstitoÂris, Siroki, & DeÂsi, 1995). The evaluation of immunotoxic e€ects of ChE-inhibiting insecticides in humans is limited to few studies. Lee, Moscati, and Park (1979) were the ®rst to draw attention to the possible e€ects of organophosphates on human leukocyte function. These workers demonstrated that leukocyte proliferation to phytohemagglutin in vitro was decreased in the presence of these compounds. Subsequently, Hermanowicz and Kossman (1984) showed a marked impairment in neutrophil chemotaxis and neutrophil adhesion in workers exposed to organophosphate. Newcombe and Esa (1992) reported a decreased number of both monocyte expressing C3b receptors and number of receptors in each cell after exposure of organophosphate. In addition, a reduction in the natural killer cell activity was observed in these workers. In these studies, ChE activity was suppressed in the majority of the groups examined. Recently, Klucinski et al. (1996) observed that workers exposed to combinated insecticides, including ChE-inhibiting, had increased serum IgM, IgG and IgA concentration, changes in NBT-dye reduction, reduced CD4/CD8 ratio and low myeloperoxidase levels. Based on the above reports, we designed the present study to investigate the possible changes in the phagocytosis and bactericidal capabilities of neutrophils isolated from farmworkers exposed simultaneously to organophosphates and carbamates insecticides who presented no changes in ChE activity.

Table 1 Symptoms presented by Anti-ChE insecticides exposed workers and controls Frequency (%) Symptoms CNSa Muscarinic e€ects Nicotinic e€ects a

CNSÐCentral Nervous System.

Dizziness Headache Memory loss Visual disturbance Increased sweating Abdominal pain Muscular weakness Myalgias



12.5% 42.5% 2.5% 10.0% 7.5% 7.5% 20.0% 15.0%

± 5.0% ± ± ± ± 2.5% ±

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2. Experimental procedures 2.1. Population The study consisted of 40 male farmworkers who manipulated Anti-ChE insecticides, organophosphate (marathon, formation, dichorvos) and carbamates (diacarp, cartap, tamaron, diazinon, folidol, triatox, dicarzol). The workers were attended in the ambulatory of toxicology of the Medical Faculty in this University. The exposed group consisted in syntomatic and assyntomatic individuals employed in ¯oricultural producing, fruit-bearing, cotton and co€ee plantations. The mean exposure period to insecticides was 83 months (range: 02±480) and the mean age of the workers was 37 years (range: 19±62). Each worker was examined in a standard fashion by a physician and a complete occupational history was noted. Table 1 presents the occurrence of symptoms possibly related to insecticides exposure. Only 16% of the workers used the Individual Equipment of ProtectionÐE.P.I. (face masks, synthetic gloves and protective clothes such as rubber aprons or whole-body clothes). Control group consisted of 40 male subjects of comparable age and race with no history of organophosphate and carbamate insecticides exposure who were chosen from blood donors arriving at the University hospital blood bank. A similar protocol was applied to the non-exposed individuals. In both study groups, drinking and smoking habits did not di€er signi®cantly. Blood samples from each exposed worker and control subject were collected in vacutainers for a laboratory check of activity of cholinesterase in red blood cells and for the study of engulfment and killing capabilities of neutrophils. The hematological parameters were assessed by a cell Counter Haematological Roche Cobas Helios (RAB 006B). 2.2. Preparation of yeast cells Candida pseudotropicalis and Candida albicans were cultured for 8 h at 378C on Sabouraud glucose-agar slants and were harvested, washed in Hank's balanced salt solution (HBSS) and counted. To opsonize the yeast cells, 100 ml of pooled non-inactived normal human AB serum was added to 5  104 yeast cells. After 30 min, TC 199 medium (GIBCO, Grand Island, NY) was added to give a ®nal concentration of 5  104 cells/ml. 2.3. Isolation of polymorphonuclear cells and study of their phagocytic and lytic functions Blood obtained by venipuncture was immediately placed on a clean glass slide, the edges of which were sealed. No anticoagulant was used. After 2 h of incubation in a humid chamber at 378C, the blood clot was removed and the slides were washed with warm TC 199 medium. The polymorphonuclear (PMN) cells adhering to the glass slides were incubated with 1 ml of the 5  106 cells/ml suspension of opsonized yeast cells. After a 30 min incubation, the yeast cells remaining in suspension were removed by gently washing the slides three times with warm TC 199 medium. The slides were then air dried and stained with Giemsa prior to evaluating Candida phagocytosis and killing by the adherent cells. Live yeast cells were distinguished from dead yeast cells by their blue staining Giemsa, while the latter cells remained unstained and


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appeared as `ghost' images within the phagocyte. Phagocytic activity was expressed as the number of bacteria phagocytized per 100 PMN cells. Lytic activity was expressed as the percentage of ghost images (dead yeast cells) among the phagocytized Candida cells. 2.4. Analytical methods Cholinesterase activity in blood samples from exposed workers and non-exposed individuals were measured by two methods, tintometric method (Edson, 1958) and espectrophotometric method (Sittert, 1987). For the tintometric method, the sample was incubated with acetylcoline and the indicator used was bromothymol blue. Changes in colour, re¯ecting acid produced by hydrolysis of acetylcoline, were measured by comparison with coloured glass standard. The time required to reach 100% of acetylcholinesterase activity is established with the nonexposed, and them compared to the exposed. In the espectrophotometric method, the cholinesterase activity was measured by DTNB-method modi®ed according to technique described by Ellman, Courtney, Andres, and Featherstone (1961) measuring the increase in absorbance at 405 nm due to the reaction of thiocoline formed by the enzymatic hydrolysis of

Fig. 1. Phagocytosis of Candida albicans (n=39) and Candida pseudotropicalis (n=36) by polymorphonuclear leukocytes isolated from workers exposed to Anti-ChE insecticides [E,*] and non-exposed controls [NE,w] n=30. Horizontal lines indicate the geometric means.

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butyrylthiocoline with dithiobis nitrobenzoic acid (DTNB) forming the yellow nitrobenzoate anion. 2.5. Statistical analysis Statistical comparisons of the results from exposed and non-exposed individuals were performed using the unpaired Student's t-test. Associations between variables were evaluated based on the Pearson correlation coecient (Beiguelman, 1991). 3. Results Cholinesterase activity in red blood cells was used as an indicator of exposure. Biological screening of ChE activity was performed by tintometric method (Edson, 1958). For determination of both plasma and erythrocyte ChE activity, the modi®ed Ellman espectrophotometric method was used (Sittert, 1987), since it provides excellent precision and

Fig. 2. Lysis of Candida albicans (n=39) and Candida pseudotropicalis (n=36) by polymorphonuclear leukocytes isolated from workers exposed to Anti-ChE insecticides [E,*] and from non-exposed persons [NE,w] n=30. Horizontal lines indicate the geometric means. P < 0.001 compared to non-exposed individuals.


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accuracy (Coye, Lowe, & Maddy, 1986). The 40 workers studied presented undetectable impairment in the ChE activity in blood cells. The individual values for neutrophil phagocytosis and killing of C. albicans. (n=39) and C. pseudotropicalis (n=36) are presented in Figs. 1 and 2, respectively. No changes were observed in the phagocytosis of either antigen as determined by the number of cells containing organisms. However, lysis of C. albicans was signi®cantly decreased (P < 0.001ÐStudent's t-test) in the exposed population when compared to the non-exposed individuals. Conversely, the lysis of C. pseudotropicalis by neutrophils was not altered. We did not observe any alterations in the hematological parameters studied (Table 2). There was no correlation between all the parameters measured. 4. Discussion The present study con®rms and extends previous observations of the ability of insecticides to induce injures on the immune system. We investigated phagocytosis and lysis of C. albicans and C. pseudotropicalis by neutrophils obtained from workers exposed to carbamate and organophosphate insecticides. Two species of Candida were used because in the individuals with myeloperoxidase (MPO) de®ciency neutrophils are unable to kill C. albicans (Lehrer & Cline, 1969a,b), whereas C. pseudotropicalis can be e€ectively lysed (Lehrer, 1975), indicating the presence of di€erent lytic mechanisms depending on the microorganism (Lehrer, 1972). The results demonstrated that phagocytosis of both Candida species by neutrophils was normal. However, lytic activity of C. albicans, but not of C. pseudotropicalis, was impaired. This defect was observed in exposed workers with normal ChE activity. In addition, out of 40 workers studied, 29 presented the classical symptoms of Anti-ChE insecticides intoxication (Table 1) (Aaron & Howland, 1994). The results provide evidence that neutrophil function may be altered in workers chronically exposed to Anti-ChE insecticides. The neutrophil is packed with granules whose contents are essential to the killing and degradation of micoorganisms. These granules contain proteases, hydrolytic enzymes, defensins, collegenase, lysozyme, apolactoferrim, C5 splitting enzyme and MPO. The encounter with micoorganisms or other external stimuli triggers a cascade of events a€ecting neutrophil adhesion, migration, engulfment and bactericidal function (Bogomolski-Yahalom & Matzner, 1995). Table 2 Hematological parameters in Anti-ChE insecticides exposed (n=40) workers and controls (n=40) Haematological parameters




5.0020.27a 45.2222.61 87.7524.84 7.6121.68

4.6420.33 41.6822.20 89.8723.91 7.3821.09

RBC (106/mm3) Hct (%) MCV (mm3) WBC (103/mm3) a b

These values indicate the geometric means. RBCÐRed Blood Cells; HctÐHematocrit; MCVÐMean Corpuscular volume; WBCÐWhite Blood Cells.

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Data in the literature demonstrate that workers exposed to carbamates had a reduction in the phagocytic parameters. These authors suggested that this e€ect is probably a consequence of the suppressive activity of these compounds on cell oxidases and/or other systems such as cythochrome P-450, NADPH, ¯avoprotein, cytoyochrome b5 (Tsvetkova, Andonova, Zvetkova, & Blagoeva, 1992). Experimental studies seem to indicate that this e€ect is dosedependent (Ladics et al., 1994). Our observation of an impaired ability to kill C. albicans indicates that Anti-ChE insecticides exposure may also lead to a de®ciency in MPO. This enzyme in the presence of hydrogen peroxide and halide produces hypoclorous acid and chlorine. Both of these products can be instrumental in destroying target cells. However, an increased incidence of infection was not found in the Anti-ChE exposed workers. In this regard, most of the patients with hereditary MPO-de®cient do not have serious bacterial infections, probably due to compensatory function by other microbicidal mechanisms. Nevertheless, in these patients, Candida infections are frequent and bacterial killing is delayed (Bogomolski-Yahalom & Matzner, 1995). Moreover, the respiratory burst is mildly prolonged. Work in progress in our laboratory seem to indicate an impaired respiratory burst of neutrophils from workers exposed to Anti-ChE insecticides. We therefore demonstrate that, in spite of the normal ChE activity, there was a decrease in the lytic activity of MPO dependents-antigens in organophaphates and carbamates insecticides exposed workers. Acknowledgements This work was supported by grants from the Fundac° aÄo de Amparo aÁ Pesquisa do Estado de SaÄo Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientõ ®co e TecnoloÂgico (CNPq). References Aaron, C. K., & Howland, M. A. (1994). Insecticides: organophosphates and carbamates. In: L. R. Goldfrank, Goldfrank's Toxicologic Emergencies, (5th ed.) (pp. 1105±1116). New York: Appleton & Lange. Beiguelman, B. (1991). Curso praÂtico de bioestatõ stica. In: Revista Brasileira de GeneÂtica, (2nd ed.). S: RibeiraÄo PretoaÄo Paulo. Bogomolski-Yahalom, V., & Matzner, Y. (1995). Disorders of neutrophil function. Blood Reviews, 9, 183±190. Casale, G. P., Vennerstrom, J. L., Bavari, S., & Wang, T. L. (1993). Inhibition of interleukin 2 driven proliferation of mouse CTLL2 cells, by selected carbamate and organophosphate insecticides and congeners of carbaryl. Immunopharmacol. Immunotoxicol., 15, 199±215. Ciesielski, S., Loomis, D. P., Mims, S. R., & Auer, A. (1994). Pesticide exposures, cholinesterase depression, and symptoms among North Carolina migrant farmworkers. Am. J. Public Health, 84, 446±451. Coye, M. J., Lowe, J. A., & Maddy, K. T. (1986). Biological monitoring of agricultural workers exposed to pesticides: I. Cholinesterase activity determinations. J. Occup. Med., 28, 619±627. Edson, E. F. (1958). Blood tests for users of O.P. insecticides. World Crops, 10, 49±51. Ellman, G. L., Courtney, K. D., Andres, V., & Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol., 7, 88±95. Hermanowicz, A., & Kossman, S. (1984). Neutrophil function and infectious disease in workers occupationally


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