Determination of the immunotoxic potential of pesticides on functional activity of sheep leukocytes in vitro

Toxicology 188 (2003) 73 /81 www.elsevier.com/locate/toxicol

Determination of the immunotoxic potential of pesticides on functional activity of sheep leukocytes in vitro Juraj Pistl *, Nata´lia Kovalkovicˇova´, Vanda Holovska´, Jaroslav Lega´th, Ivan Mikula Department of Microbiology and Immunology, University of Veterinary Medicine, 041 81 Kosˇice, Slovakia Received 8 August 2002; accepted 27 January 2003

Abstract The effect of eight pesticides with different chemical structure (atrazine, bentazone, chloridazone, dichlofluanid, endosulfan, MCPA, simazine, triallate) on sheep peripheral blood phagocytes and lymphocytes was examined under in vitro conditions by iodo-nitro-tetrazolium reductase test and leukocyte migration-inhibition assay. The pesticides, dissolved in DMSO, were tested at the concentrations of 10
1 /10
6 M. The significant suppression of metabolic activity of phagocytic cells was registered after exposure to dichlofluanid (10
1 /10
3 M), endosulfan, simazine and triallate (10
1 M). The significant cytotoxic effect (the decrease of spontaneous migration of leukocytes) was registered for bentazone, dichlofluanid, endosulfan and MCPA (10
1 M); chloridazone (10
1 M /10
2 M) and triallate (10
1 / 10
5 M). The significant immunotoxic effect (the decrease of lymphocyte activation with PHA) was observed for atrazine (10
1 /10
2 M); bentazone (10
2 /10
4 M); dichlofluanid, endosulfan (10
2 /10
3 M); MCPA (10
2 /10
6 M) and simazine (10
1 /10
4 M). Three of the pesticides tested suppressed both, the metabolic activity of phagocytes and mitogenic activation of lymphocytes (dichlofluanid, endosulfan and simazine). Triallate suppressed the metabolic activity of phagocytes and showed a strong cytotoxic effect. Pesticides atrazine, bentazone and MCPA influenced the mitogenic activation of lymphocytes and chloridazone showed a significant cytotoxic effect. The different chemical structure of pesticides influenced the metabolic activity of phagocytic cells as well as mitogenic activation of lymphocytes to various intensity. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Sheep; Pesticides; Metabolic activity of phagocytes; Lymphocyte activation

1. Introduction Abbreviations: DMSO, dimethylsulfoxide; IMA, index of metabolic activity; INT, iodo-nitro-tetrazolium reductase test; LMIA, leukocyte migration-inhibition assay; MA, metabolic activity; MI, migration index of leukocytes; PHA, phytohemagglutinine. * Corresponding author. E-mail address: [email protected] (J. Pistl).

Pesticides together with heavy metals from emissions are dominant compounds of the chemical load on the environment of man and animals (Kacˇma´r et al., 1999; Kore´nekova´ et al., 2000). Besides their beneficial effect on agricultural

0300-483X/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0300-483X(03)00046-5

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J. Pistl et al. / Toxicology 188 (2003) 73 /81

practice and prevention of man and animals from the vectors of diseases, their extensive use raises many problems due to their toxicity for non-target organisms, persistence, and combined effects with other agrochemicals and the environmental factors (Kocˇisˇova´ and Toporcˇa´k, 2000). About 2/3 /4/5 of farm animals are exposed to harmful effect of pesticides (Bo¨hmer et al., 1991). The effect of persistent exposure to pesticide chemicals on the integrity of the immune system has recently drawn considerable interest as an additional indicator of potential problems. Immunotoxicants are factors of the external environment which cause significant changes (modulation) in the immune mechanisms in humans and animals (Dietert et al., 1996). The initial references to the susceptibility of the immune system and its potential use for detection of subclinical toxic states were published in the seventies and early eighties (Vos and Van Genderen, 1973; Loose et al., 1978; Faith et al., 1980). The main attention began to focus on the immune system as an important object of immunotoxic action of pesticides (Descotes, 1988). The immune system is more sensitive and reacts more rapidly than other organ systems to the effect of pesticides, even in concentrations of these chemicals lower than those necessary for acute systemic toxicoses (Black et al., 1992; Raszyk et al., 1997). Several methods were developed to determine the immunosuppressive or immunostimulative action of xenobiotics using the following: dose and time-dependence of response, toxicokinetic, determination of direct and indirect effects on the immune system as well as biological relevance of in vitro tests to the effects of agents in vivo (Kacˇma´r et al., 1999). Long-term animal studies determining the toxicological risk of these compounds are time consuming and expensive to perform; ethical factors also play an important role. Nowadays, there is a tendency to reduce the number of test animals and minimize their suffering in the experiments which calls for simple, reproducible and reliable in vitro test systems for the quick screening of the immunotoxic potency of pesticides. The aim of this study was to determine the action of several pesticides of different chemical structure with regard to biological effects on the

function of phagocytes and lymphocytes isolated from the peripheral blood of sheep under in vitro conditions.

2. Methods 2.1. The pesticides tested Pesticides */atrazine, bentazone, chloridazone, dichlofluanid, endosulfan, MCPA, simazine and triallate were dissolved in dimethylsulfoxide (DMSO, Lachema, Brno, Czech Republic), of which the final concentration in the maintenance medium was 1%. The basic molar concentrations of pesticide, freshly prepared before each experiment, were 10
1 /10
6 M. These pesticide concentrations were added to leukocyte suspensions in 1% volumes; i.e. the actual molar concentrations were 100 fold lower than the basic one. The characteristics of pesticides are summarized in Table 1 (The Pesticide Manual, 1994). 2.2. Sampling procedure Blood samples were obtained from 5 clinically healthy 1-year-old Merino sheep reared at University facilities. They were withdrawn from a jugular vein into 1.5% EDTA. Leukocytes were isolated from sheep peripheral blood by the method of osmotic shock of erythrocytes according to Karlson and Kaneko (1973). Immunological assays. For the analysis of immunological parameters the assays were chosen to evaluate the functional activity of both cell types */phagocytes and lymphocytes. 2.3. Iodo-nitro-tetrazolium reductase test (INT) Quantitative evaluation of tetrazolium reductase activity of phagocytes was carried out according to the method of Lokaj and Oburkova (1975) to determine the metabolic activity (MA) of phagocytes during phagocytosis. The suspension of leukocytes (1 /107 per ml) was prepared in a maintenance medium RPMI 1640 with 25 mM HEPES, 0.3 mg per ml L-glutamine (Gibco, Germany), supplemented with 100 mg per ml

Table 1 The characteristics and features of pesticides tested (The Pesticide Manual, 1994) Chemical family

Molecular formula

Molecular weight

CAS register number

Producer purity

Use

Toxicity in animals per oral LD50 (mg per kg body weight)

Atrazine

Triazines

C8H14ClN5

215.7

1912-24-9

Herbicide

Bentazone

Benzothiadiazoles

C10H12N2O3S

240.3

25057-89-0

Chloridazone

Pyridazinones

C10H8ClN3O

221.6

1698-60-8

Herbicide

Rat 1869 /3080, mouse 1750 /3992, rabbit 750 Rat
/1000, dog
/500, rabbit 750, cat 500 Sheep 160

333.2

1085-98-9

Fungicide

Rat
/5000

406.93

115-29-7

Sigma-Aldrich, USA; 99.2% Sigma-Aldrich, USA; 99.9% Sigma-Aldrich, USA; 99% Supelco, Bellefonte, USA; 98% Supelco, Bellefonte, USA; 99% Sigma-Aldrich, USA; 99.1% Supelco, Bellefonte, USA; 99% Supelco, Bellefonte, USA; 99%

Insecticide acaricide

Rat 70, dog 77

Herbicide

Rat 900 /1160, mouse 550

Herbicide

Sheep 500, rat, mouse, rabbit
/5000

Herbicide

Rat 1100

Dichlofluanid Sulphamides Endosulfan

Organochlorines

C9H11Cl2FN2O2S2 C9H6Cl6O3S

MCPA

Chlorophenoxys

C9H9ClO3

200.6

94-74-6

Simazine

Triazines

C7H12ClN5

201.66

122-34-9

Triallate

Thiocarbamates

C10H16Cl3NOS 304.7

2303-17-5

Herbicide

J. Pistl et al. / Toxicology 188 (2003) 73 /81

Pesticide

75

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J. Pistl et al. / Toxicology 188 (2003) 73 /81

streptomycin, 100 IU per ml penicillin (Gibco, Germany) and 10% foetal calf serum (Gibco, Germany). Leukocyte suspension was then divided into two parts. One portion of leukocytes was incubated at 37 8C for 45 min with starch (1% suspension of Amylum oryzae in phosphate-buffered saline) alone or with 1% DMSO (dissolvent of pesticides) and with different concentrations of the pesticides tested (10
1 M /10
6 M); the second one was incubated under the same conditions, but without starch. All suspensions of cells contained 0.1% INT (3/-4-iodophenyl /2-/4-nitrophenyl/-5-phenyl/-tetrazolium chloride, Lachema, Brno, Czech Republic) and were tested in triplicate. Immediately after a 45-min incubation, the cell lysis by methanol was performed and the content of formasan in the supernatant (red colored product after INT reduction) was determined spectrophotometrically at 485 nm. The results were described in the form of an index of metabolic activity (IMA) based on the ratio of mean optical density (OD485) of leukocyte suspensions with starch (actually phagocytic cells*/an increased metabolism) and pesticide to the leukocyte suspensions treated with pesticide but without the starch (phagocytes without stress */a basic metabolism). 2.4. Leukocyte migration-inhibition assay (LMIA) The LMIA analyzing the reactive capacity of lymphocytes to mitogenic activation was carried out according to Bendixen et al. (1976). Leukocyte suspensions (2 /108 per ml) were prepared in a culture medium RPMI 1640 with 25 mM HEPES, 0.3 mg per ml L-glutamine (Gibco, Germany), supplemented with 100 mg per ml streptomycin, 100 IU per ml penicillin (Gibco, Germany), and 10% foetal calf serum (Gibco, Germany). Cell suspensions (50 ml) were placed to glass capillaries (Skla´rny Kavalier, Czech Republic). One end of the capillaries was carefully closed by heat. After centrifugation for 2 min at 400 g, the capillaries were cut at the border of sediment and supernatant, placed into the wells of plastic plates (24well tissue culture plate, Sarstedt, USA) and incubated at 37 8C for 18 h in 5% CO2 atmosphere with 1 ml RPMI 1640 medium containing different

concentrations of pesticides (10
1 /10
6 M) dissolved in DMSO with and without 10 mg per ml phytohemagglutinine (PHA, Sigma, Germany). All tests were made in triplicate for each animal. T-lymphocytes activated with PHA produce higher levels of lymphokines including the migration inhibition factor (MIF). MIF causes inhibition of leukocyte migration (monocytes, polymorphonuclears). The intensity of this inhibition depends on the amount of MIF produced by the activated lymphocytes. The areas of leukocyte migration were measured by a planimeter after 17.5 fold magnification of migration areas. Results were expressed as a migration index (MI), represented as a ratio of mean leukocyte migration areas with PHA and areas of leukocyte migration without PHA. In the case of routine interpretation of LMIA, MIB/0.9 signalizes a positive reaction /activation of Tlymphocytes with PHA and more intensive activation of lymphocytes by mitogen is characterized by lower MI value. The cytotoxic effect of different concentrations of pesticides was determined by the ratio of spontaneous leukocyte migration areas from control cell suspensions (without pesticides and mitogen, containing only 1% DMSO) and the areas of leukocyte migration from suspensions treated with pesticides, without PHA. A cytotoxic effect has been postulated if the mean leukocyte migration area from suspensions treated with pesticide (without PHA) was significantly smaller than the area of spontaneous leukocyte migration from cell suspension which served as a control (without pesticide and PHA, containing only 1% DMSO). If the cytotoxic effect was not observed, the MI was calculated as a ratio of mean leukocyte migration area with pesticide and PHA and the area of leukocyte migration with pesticide and without PHA (MIpesticide). The immunotoxic effect was determined by comparing two migration indices: MIpesticide to MI of the control cell suspension (MIcontrol) calculated as a ratio of mean leukocyte migration area with PHA (without pesticide) and the area of leukocyte migration without PHA (without pesticide). An immunotoxic effect has been postulated

J. Pistl et al. / Toxicology 188 (2003) 73 /81

if the mean values of MIpesticide and MIcontrol were significantly different. The immunotoxic effect was characterized as a decreased ability of lymphocytes to respond to the mitogenic stimuli of PHA (PHA, polyclonal activator of T lymphocytes). 2.5. Statistical analysis The evaluation of IMA and MI values of the exposed cells with regard to the control was performed by the Student’s t -test.

3. Results Four of the pesticides tested (dichlofluanid, endosulfan, simazine and triallate, Table 2) suppressed significantly the metabolic activity of phagocytic cells isolated from the peripheral blood of sheep. Dichlofluanid caused significant decrease IMA at the concentration of 10
1 M (P B/0.001) / 10
3 M (P B/0.05). Endosulfan, simazine and triallate decreased IMA significantly at the concentration of 10
1 M (P B/0.05, Table 2). The results of LMIA are summarized in Table 3. A significant cytotoxic effect (a decrease in spontaneous migration of leukocytes) was registered with bentazone (10
1 M, P B/0.01); chloridazone (10
1 M, P B/0.001; 10
2 M, P B/0.05); dichlofluanid and endosulfan (10
1 M, P B/0.001); MCPA (10
1 M, P B/0.05) and triallate (10
1 M, P B/0.001; 10
2 /10
5 M, P B/0.05). No

77

cytotoxic effect was recorded with atrazine and simazine (Table 3). A significant immunotoxic effect (the decrease of lymphocyte activation with PHA) was observed with atrazine (10
1 /10
2 M, P B/0.001); bentazone (10
2 /10
3 M, P B/0.001; 10
4 M, P B/ 0.05); dichlofluanid (10
2 M, P B/0.001; 10
3 M, P B/0.05); endosulfan (10
2 /10
3 M, P B/ 0.01); MCPA (10
2 /10
6 M, P B/0.001; at 10
7 M, non-significant difference compared with the control) and simazine (10
1 /10
4 M, P B/0.001, Table 3). No immunotoxic effect was recorded with chloridazone and triallate (Table 3). Three of the pesticides tested suppressed both the metabolic activity of phagocytes and the mitogenic activation of lymphocytes (dichlofluanid, endosulfan and simazine). Triallate suppressed the metabolic activity of phagocytes too but with regard to LMIA showed a strong cytotoxic effect. Pesticides atrazine, bentazone and MCPA influenced the mitogenic activation of lymphocytes and chloridazone showed a significant cytotoxic effect in LMIA.

4. Discussion The susceptibility of the immune system to xenogenous substances, which can result in immunosuppression, depends on both the properties of the respective chemical and the complex nature of the immune system, e.g. antigen recognition and processing, cellular interactions */cooperation,

Table 2 The mean values (9/S.D.) of the indices of metabolic activity of sheep peripheral phagocytes in the presence of different concentrations of pesticides tested (***P B/0.001; *P B/0.05) concentration pesticide Atrazine Bentazone Chloridazone Dichlofluanid Endosulfan MCPA Simazine Triallate

Control

2.769/1.08

DMSO

2.589/0.94

10
1 M

10
2 M

10
3 M

10
4 M

10
5 M

10
6 M

2.189/0.74 2.019/0.61 2.289/0.95 0.919/0.14*** 1.349/0.16* 2.219/1.26 1.489/0.47* 1.489/0.44*

2.039/1.36 1.999/0.38 2.019/0.87 1.439/0.52* 1.99/0.38 2.309/0.70 2.069/0.34 1.879/1.81

2.069/1.37 2.029/0.15 2.689/0.99 1.299/0.24* 2.589/0.82 2.289/0.82 1.979/0.27 2.429/1.13

2.049/1.71 2.319/0.69 2.389/0.99 2.159/0.37 3.09/0.96 2.259/0.70 1.909/0.69 2.069/1.35

2.179/1.9 2.359/0.27 2.279/0.84 2.199/0.17 3.109/0.72 2.189/0.21 1.879/0.96 2.819/1.27

1.959/0.9 2.299/0.31 2.209/1.73 2.179/0.14 3.159/0.80 2.349/0.43 1.869/0.34 2.729/1.29

9/S.D., standard deviation; DMSO, solvent control.

J. Pistl et al. / Toxicology 188 (2003) 73 /81

78

Table 3 Determination of cytotoxic effect in relation to spontaneous migration and immunotoxic effect in relation to migration indices of sheep leukocytes exposed to tested pesticides in LMIA Pesticide Control DMSO Atrazine

Bentazone

Chloridazone

Dichlofluanid

Endosulfan

MCPA

Simazine

Triallate

Concentration (M )

Area of spontaneous leukocyte migration (cm2)a9/S.D.

MI9/S.D.

10
1 10
2 10
3 10
4 10
5 10
6 10
1 10
2 10
3 10
4 10
5 10
6 10
1 10
2 10
3 10
4 10
5 10
6 10
1 10
2 10
3 10
4 10
5 10
6 10
1 10
2 10
3 10
4 10
5 10
6 10
1 10
2 10
3 10
4 10
5 10
6 10
7 10
1 10
2 10
3 10
4 10
5 10
6 10
1 10
2 10
3 10
4

27.569/8.90 26.619/9.94 27.759/4.11 28.259/5.73 23.339/6.44 21.809/2.41 19.509/4.38 24.339/4.03 13.679/2.07** 24.009/5.48 27.009/1.55 25.679/1.37 28.509/0.58 25.679/2.58 5.679/2.25*** 17.509/2.74* 26.009/3.22 20.009/4.37 19.209/8.44 22.679/1.51 2.339/1.86*** 23.509/6.72 21.009/4.73 22.209/6.83 27.839/7.25 22.339/8.09 5.279/1.17*** 18.379/3.16 20.679/3.34 23.509/3.42 20.279/4.28 22.089/5.28 16.009/3.10** 25.339/5.24 23.339/5.16 18.679/3.72 19.679/1.37 22.339/6.09 24.509/0.55 25.839/0.75 26.679/2.34 24.009/0.63 27.679/1.03 23.679/4.23 25.339/3.39 2.339/1.37*** 15.679/1.86* 15.679/2.88* 15.429/1.43*

0.449/0.17 0.479/0.14 0.859/0.14*** 0.719/0.17*** 0.569/0.51 0.529/0.40 0.559/0.22 0.539/0.38 0.719/0.24*** 0.769/0.11*** 0.599/0.10* 0.439/0.11 0.469/0.04

Effect

IT IT

CT IT IT IT

CT CT 0.439/0.33 0.459/0.03 0.419/0.10 0.399/0.11 0.669/0.21*** 0.589/0.23* 0.439/0.21 0.609/0.52 0.539/0.20 0.669/0.26** 0.659/0.29** 0.579/0.14 0.489/0.33 0.429/0.44 0.909/0.17*** 0.969/0.20*** 1.199/0.57*** 0.879/0.25*** 0.689/0.36*** 0.319/0.04 0.889/0.06*** 0.699/0.22*** 0.639/0.21*** 0.639/0.05*** 0.479/0.14 0.439/0.14

CT IT IT

CT IT IT

CT IT IT IT IT IT / IT IT IT IT

CT CT CT CT

J. Pistl et al. / Toxicology 188 (2003) 73 /81

79

Table 3 (Continued ) Pesticide

Concentration (M )

Area of spontaneous leukocyte migration (cm2)a9/S.D.

MI9/S.D.

10
5 10
6

15.679/1.37* 23.339/2.88

0.409/0.13

Effect CT

***P B/0.001; **P B/0.01; *P B/0.05; MI, migration index of leukocytes; CT, cytotoxic effect; IT, immunotoxic effect; S.D., standard deviation; DMSO, solvent control; LMIA, leukocyte migration inhibition assay. a Magnification 17.5/.

regulation and amplification, activation and differentiation of cells, and production of mediators by various cell types (Luster et al., 1993). Free living, grazing animals are among the animal groups immediately exposed to and most affected by the harmful influence of pesticides. We have used sheep as model animals to donate peripheral blood leukocytes as they are close to wild ruminants and are a better indicator of environmental exposure than rodents, which are kept strictly under laboratory conditions. The in vitro methods are utilizable for screening of immunotoxicity of xenobiotics if the effect of these substances is implemented through a direct action of the respective chemical on the immune cells and their membrane receptors. On the other hand, these methods cannot detect indirect, secondary effects of chemicals on important internal organs such as liver (hepatotoxicity */production of hepatoproteins with immunomodulative properties) and the neuroendocrine system (neuroendocrine toxicity */chemically induced changes in hormones or neuroactive substances with immunomodulative properties; Dietert et al., 1996). From a wide range of immunological methods recommended for immunotoxicological investigations (Luster et al., 1993; Vandebriel et al., 1995; Dietert et al., 1996) we selected the INT because it is an objective, well reproducible, and fast assay for the measurement of functional activity of phagocytes. The LMIA was selected for the evaluation of functional activity of lymphocytes on the basis of polyclonal activation. This test was preferred to the more sophisticated lymphoprolipherative test because it enables to differentiate the cytotoxic and immunotoxic effects of chemicals in vitro. On the other hand, there is a good response to the antigenic or mitogenic activation in the

lymphoprolipherative test but the cytotoxicity cannot be differentiated sufficiently. Presented results clearly indicate the different sensitivity of phagocyte and lymphocyte functions dependent on the different chemical composition and activities of pesticides. The phagocytic cells showed higher, dose-dependent resistance to the chemicals tested than lymphocytes. Four the pesticides tested, atrazine, bentazone, MCPA and chloridazone, did not influence the metabolic activity of phagocytic cells. However, the mitogenic activation of lymphocytes was inhibited by the first three of them. Chloridazone showed cytotoxic activity with regard to LMIA */the motility of phagocytic cells was decreased but their metabolic activity in the INT test was not influenced. This suggests that the mechanism of action of pesticides on the same cell type differs. Our results obtained in vitro are very close to those recorded under in vivo conditions with the pesticides tested. An exposure of mice to atrazine (1/2/1.64 LD50) had no marked effect on stimulation of lymphocytes by mitogens and impairment of the production of some cytokines (Fournier et al., 1992; Hooghe et al., 2000). In vitro results agree with those of 3-month subchronic intoxication of sheep with herbicide Bentazone TP (1/10 and 1/20 LD50) which revealed a decrease in the response of lymphocytes to mitogenic activation from the eighth week of the experiment but no effect of this pesticide on phagocytic activity (Mikula et al., 1992). Acute intoxication with herbicide chloridazone after its peroral administration at a single dose of 80 mg per kg body weight (1/2 LD50) caused a significant increase in metabolic activity of peripheral blood phagocytes after 12 h; however, PHA activation of lymphocytes was significantly

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J. Pistl et al. / Toxicology 188 (2003) 73 /81

decreased in the lymphocyte migration-inhibition test (Pistl et al., 2002). Banerjee and Hussain (1986, 1987) evaluated the effect of subchronic and chronic doses of endosulfan in albino rats immunized with tetanus toxoid. Both cellular and humoral immunoresponses were decreased in dose/time dependent patterns. Kurkure et al. (1993) studied the immunosuppressive effects of endosulfan in white Leghorn chicks after dietary exposure. Birds vaccinated with Newcastle disease virus had decreased antibody titer against this virus. The immunosuppressive effect of sublethal and subchronic concentrations of endosulfan was also detected in Swiss albino mice (Bhatia et al., 1998). Khurana et al. (1998) described significant depression in the functional activity of macrophages, assessed by nitroblue tetrazolium reduction test, in broiler chicks supplied feed with 30 ppm of endosulfan. There is no information regarding immunosuppressive effect of dichlofluanid. But after treatment of wood furniture with this fungicide contact skin allergic reactions were described in humans (Hansson and Wallengren, 1995). The effects of herbicide MCPA on leukocytes were evaluated by cytokine production in vitro. MCPA stimulated the production of tumor necrosis factor-alpha (Hooghe et al., 2000). Agricultural exposure to MCPA may exert short term immunosuppressive effects in farmers (Faustini et al., 1996). Long-term contact with low doses of simazine (1/1000 LD50) may induce changes in the immune system because of the disorders in the T-lymphocyte development after 7-month per oral exposure of rats (Barshtein et al., 1991). Immunodeficiency and pathological changes in the thymus after exposure to low doses of simazine were also described (Palij et al., 1991). Simazine showed concentration-related effects on interferon / gamma and tumor necrosis factor/alpha production (Hooghe et al., 2000). There is little information about the effects of triallate on the immune system. Low repeated doses of the herbicide induced generalized suppression of non-specific immunobiological reactivity of organism connected with the decrease in phagocy-

tic activity (De Bruin, 1980). A decrease in phagocytosis of peritoneal macrophages and Tlymphocyte blastogenesis in the presence of phytohemagglutinin in male rats after subchronic per oral exposure to triallate (5 mg/kg twice per week) was recorded (Blakley et al., 1998). It can be concluded that the in vitro methods of the determination of metabolic activity of phagocytes and activation of lymphocytes with mitogen enable quantitative and qualitative evaluation of the direct dose-dependent inhibitory effect of some pesticides on the functions of sheep peripheral blood phagocytes and lymphocytes. The methods show that the tested pesticides with different chemical structure inhibited different cell types and functions to various intensity. We consider these in vitro experiments the basic screening examination of cytotoxic and immunotoxic effects of pesticides.

Acknowledgements This work was supported by The Ministry of Education and Science of the Slovak Republic (Grant Vega No.1/8115/01) and National Reference Laboratory for Pesticides, University of Veterinary Medicine, Kosˇice.

References Barshtein, Y.A., Palij, G.K., Persidskij, J.V., Benjaminov, V.O., Pusˇkar, M.S., 1991. The immunomorphological analysis of long-term intoxication by low doses of herbicide simazine. Biull. Eksp. Biol. Med. 112 (12), 657 /659 (In Russian). Black, R.D., Scott, D., Oehme, F.W., 1992. Immunotoxicity in the bovine animal: a review. Vet. Hum. Toxicol. 34, 438 / 442. Blakley, B.R., Yole, M.J., Brousseau, P., Boermas, H., Fournier, M., 1998. Effect of 2,4 dichlorophenoxyacetic acid, frifluralin and triallate herbicides on immune function. Vet. Hum. Toxicol. 40, 5 /10. Banerjee, B.D., Hussain, Q.Z., 1986. Effect of sub-chronic endosulfan exposure on humoral and cell-mediated immune responses in albino rats. Arch. Toxicol. 59, 279 /284. Banerjee, B.D., Hussain, Q.Z., 1987. Effects of endosulfan on humoral and cell mediated immune responses in rats. Bull. Environ. Contam. Toxicol. 38, 435 /441. Bhatia, A., Thind, H., Kaur, J., 1998. Effect of endosulfan on numerical values and functions of mice cells involved in

J. Pistl et al. / Toxicology 188 (2003) 73 /81 immune response. J. Ecotoxicol. Environ. Monit. 8, 257 / 261. Bendixen, G., Bentzen, K., Clausen, J.E., et al., 1976. Inhibition of human leucocyte migration. In Natvig, J.B., Permann, P., Wigzel, H. (Eds.) Lymphocytes. Isolation, Fractionation and Characterization., Scand. I. Immunol., Suppl. 5, Universitetsforlaget, Nyegaard, Oslo, pp. 244 /267. Bo¨hmer, D., Ujha´zy, E., Tomo, I., .Sˇkorvaga, P., Balonova´, T., 1991. Experimental possibilities for detection of embryotoxicity risk of xenobiotics. Agroche´mia 31, 55 /57. De Bruin, A., 1980. Biochemical Toxicology of Environmental Agents. Elsevier, Amsterdam, p. 1543. Descotes, J., 1988. Immunotoxicity of Pesticides. Immunotoxicology of Drugs and Chemicals. Elsevier, Amsterdam, pp. 347 /363. Dietert, R.R., Golemboski, K.A., Kwak, H., Ha, R., Miller, T.L., Davison, T.F., 1996. Environment */immunity interactions. In: Davison, T.F., Morris, T.R., Payne, L.N. (Eds.), Poultry Immunology. Carfax Publishing, Abingdon, UK, pp. 343 /356. Faith, R.E., Luster, M.L., Vos, J.G., 1980. Effects on immunocompetence by chemicals of environmental concern. In: Hodgson, E., Bend, J.R., Philpot, R.M. (Eds.), Reviews in Biochemical Toxicology 2. Elsevier, Holland, pp. 173 / 211. Faustini, A., Settimi, L., Pacifici, R., Frano, V., Zuccaro, P., Forastiere, F., 1996. Immunological changes among farmers exposed to phenoxy herbicides: preliminary observations. Occup. Environ. Med. 53, 583 /585. Fournier, M., Friborg, J., Girard, D., Mansour, S., Krzystyniak, K., 1992. Limited immunotoxic potential of technical formulation of the herbicide atrazine (Aatrex) in mice. Toxicol. Lett. 60, 263 /274. Hansson, C., Wallengren, J., 1995. Allergic contact dermatitis from dichlofluanid. Contact Dermatitis 32, 116. Hooghe, R.J., Devos, S., Hooghe /Peters, E.L., 2000. Effects of selected herbicides on cytokine production in vitro. Life Sci. 66, 2519 /2525. Kacˇma´r, P., Pistl, J., Mikula, I., 1999. Immunotoxicology and veterinary medicine. Acta veterinaria 68, 57 /79. Karlson, G.P., Kaneko, J.P., 1973. Isolation of leucocytes from bovine peripheral blood. Proc. Soc. Exp. Biol. Med. 142, 853 /856. Kocˇisˇova´, A., Toporcˇa´k, J., 2000. Protection of environment by use of biorational pesticides. Dezinfekce, Dezinsekce, Deratizace 4, 108 /109 (In Slovak).

81

Kore´nekova´, B., Skalicka´, M., Nad’, P., 2000. Nickel accumulation by cattle from industrial polluted area. Biologia Bratislava 55 (Suppl. 8), 59 /62. Khurana, S.K., Chauhan, R.S., Mahipal, S.K., 1998. Immunotoxic effects of cypermethrin and endosulfan on macrophage functions of broiler chicks. Ind. J. Anim. Sci. 68, 105 /106. Kurkure, N.V., Bhandarkar, A.G., Joshi, M.V., et al., 1993. Immunosuppressive and histotoxic effects of endosulfan in chicks. Ind. J. Anim. Sci. 63, 1258 /1260. Loose, L.D., Pittman, K.A., Benitz, K.F., Silkworth, J.B., Mueller, W., Coulston, F., 1978. Environmental chemicalinduced immune dysfunction. Ecotoxicol. Environ. Saf. 2, 173 /198. Lokaj, V., Oburkova, P., 1975. Determination of tetrazoliumreductase activity of leukocytes. Imunol. Zprav. 6, 42 /44 (In Czech). Luster, M.I., Portier, C., Pait, D.G., Rosenthal, G.J., Germolec, D.R., Corsini, E., Blaylock, B.L., Pollock, P., Kouchi, Y., Craig, W., White, K.L., Munson, A.E., Comment, C.E., 1993. Risk assessment in immunotoxicology. II. Relationships between immune and host resistance tests. Fundam. Appl. Toxicol. 21, 71 /82. Mikula, I., Pistl, J., Kacˇma´r, P., 1992. Immune response of organism at subchronic intoxication with herbicide bentazon TP. Vet. Hum. Toxicol. 34, 507 /509. Palij, G.K., Barsˇtejn, J.A., Persibskij, J.V., Benjamikov, V.O., Pusˇkar, M.S., Jakubovskij, M.M., 1991. Immunomorphological characterisation of staphylococcal infection on the basis of long-term effect of low doses of simazine. Mikrobiolog. zˇurnal 53 (2), 62 /68 (In Russian). The Pesticide Manual, 1994. Cambridge, 1994, pp. 1341. Pistl, J., Kovalkovicova´, N., Reichel, P., Mlynarcˇ´ıkova´, H., Lega´th, J., Holovska´, V., Kova´cˇ, G., 2002. Immunological and haematological parameters in sheep after acute intoxication by herbicide chloridazone. In: Industrial Toxicology’02, Slovenska´ Lupca, 15 /17 May 2002, pp. 82 /86. Raszyk, J., Toman, M., Gajdu´sˇkova´, V., Nezveda, K., Ulrich, R., Jarosˇova´, A., Docekalova´, H., Salava, J., Pala´c, J., 1997. Effects of environmental pollutants on the porcine and bovine immune systems. Vet. Med. Czech. 42, 313 /317. Vandebriel, R.J., Garsen, J., Van Loveren, H., 1995. Methods in immunotoxicology. Methods Neurosci. 24, 151 /169. Vos, J.G., Van Genderen, H., 1973. Toxicological aspects of immunosuppression. In: Deichmann, W.B. (Ed.), Pesticides and the Environment: A continuing Controversy. Intercontinental Medical Book Corporation, New York, pp. 527 / 545.