Asian J Androl 2005; 7 (1): 97-101

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^.Original Article^.

Effect of malathion on the male reproductive organs of earthworms, Eisenia foetida

Omar Espinoza-Navarro¹, Eduardo Bustos-Obregn²

¹Department of Biology and Health, Faculty of Sciences, University of Tarapac, Arica, Chile
²Laboratory of Biology of Reproduction, ICBM, Faculty of Medicine, University of Chile, Santiago, Chile

Abstract

Aim: To observe the cytotoxic effect of the organophosphate insecticide malathion in the reproductive tissues of the earthworms, Eisenia foetida. Methods: Worms were nourished in soil treated with malathion at single sub-lethal doses of 0, 80, 150, 300 and 600 mg.kg^-1 soil. (LD₅₀ = 880 mg.kg^-1 soil) and evaluated on days 1, 5, 15 and 30 after exposure. The body weights were recorded and male reproductive organs evaluated. Results: Malathion-treated animals showed a significant reduction in body weight in a dose-dependent manner. Malathion treatment modified the disposition of spermatozoa in the basal epithelium of the spermatheca. The Br-deoxyuridine test showed a significant rise in cells in phase S on days 5 and 15. Also, a higher percentage of spermatogonia with fragmented DNA were observed by means of the TdT-mediated dUTP nick-end labeling (TUNEL) technique in the spermatheca of treated animals. Conclusion: Treatment with malathion decreased the body weight and the spermatic viability in spermatheca, altering the cell proliferation and modifying the DNA structure of spermatogonia. (Asian J Androl 2005 Mar; 7: 97-101)

Keywords: Eisenia foetida; malathion; reproductive organs; apoptosis

1 Introduction

Organophosphorates are frequently used in pesticides due to their low half life. Malathion (5-1, 2-di-etoxycarbonyl ethyl O, O dimethyl phosphorodithioate) is considered one of the most innocuous organophos-phorates for humans. This fact has led to an indiscriminate use of this pesticide [1]. Malathion-induced morphologic alterations have been found in the mouse testes[2]. In birds, malathion induces germ cell degeneration possibly by altering cholinergic functions [3].

Routine evaluation of the effect of xenobiotics on human health has required the use of mammals as biological models [4]. Because of restriction on experimental subjects, there has been an increasing interest in alternative models. Eisenia foetida (Annelida, Oligochaeta) is a good candidate as a complementary model for in situ and laboratory studies [5]. Worms make direct contact with the ground and absorb pesticides both from the skin and the digestive system, thus the pollutants are absorbed 5 to 10 times [6].

The present work evalu ates the changes produced by the insecticide on male reproductive organs.

2 Materials and methods

2.1 Animals

Earthworms E. foetida (agricultural "Los Nogales", Paine, Chile) were kept in plastic boxes with soil-like substrate (size of particle: smaller than 2 mm) and dampened with water (pH 6.5 with a final humidity of 50 % at 21-22 ºC). There were forty individuals in each treatment group.

2.2 Pesticide and treatment

A commercial solution of malathion (57 %, Labora-torios Farmagro S.A., Lima, Per) was used. Groups of 40 worms were treated in duplicated assay (n=80). Malathion (LD₅₀ = 880 mg.kg^-1 soil) was applied at a single dose of 80, 150, 300 and 600 mg.kg^-1 soil dissolved in 10 mL of distilled water. Control group was given only distilled water. The effects were evaluated on days 1, 5, 15 and 30.

2.3 Observations

Body weights were recorded at the beginning and the end of each treatment day. Male gonad segments were fixed in Bouin's solution for 12 hours and embedded in paraffin. Longitudinal sections (5 µm) were processed for histology and immunocytochemistry.

For the determination of cell proliferation, the BrdU staining kit was used (Zimed, San Francisco, California, USA) based on the streptavidin-biotin and diaminoben-cidine system. Nuclei were stained brown when there was BrdU incorporation. Cells that did not incorporate BrdU showed blue staining by hematoxyline. DNA fragmentation was detected using the TdT-mediated dUTP nick-end labeling (TUNEL) method. The marked cells (dark brown) had their DNA fragmented. A negative reaction was recognized by the nuclei turning blue, which indicates intact DNA.

The changes on male gonad segments were observed according to the method used by Sobarzo and Bustos-Obregn [7]. Four longitudinal sections for each animal were observed under light microscopy at 400. To evaluate the percentage of cell proliferation and DNA fragmentation, 300 cells from each slide were examined.

2.4 Statistical analysis

Comparison of results was performed using the non-parametric ANOVA test (Kruskal-Wallis) and Dunn's multiple comparisons posttest. INSTAT software (Graphpad software, USA) was used to analyze the data. P < 0.05 was considered significant.

3 Results

3.1 Body weights

A significant reduction in the body weight of the animals treated with malathion was observed each day of treatment with 80, 150, 300 and 600 mg.kg^-1 (Figure 1).

3.2 Immunohistochemical studies

An evaluation of the effects of malathion on E. foetida spermatheca is shown in Figure 2. Slides of control individuals showed an ordered and proliferative basal epithelium. Spermatozoa were distributed occupying all the internal lumen of the structure (Figure 2A). Immunohistochemistry of the spermatheca of treated worms showed vacuolization of the basal epithelium and a very anomalous disposition of spermatozoa, which were concentrated in the center of the structure without contacting the basal epithelium (Figure 2B).

3.3 Cell proliferation in seminal vesicles

Spermatogonial morules of control worms showed a negative reaction for cell proliferation (Figure 3A). In contrast, malathion-treated worms presented positive signs of cell proliferation (Figure 3B). Figure 4 shows the percentage of positive BrdU cells in the seminal vesicles of worms treated with malathion. It could be observed that cell proliferation was altered (cf.the controls), increasing statistically at 5, 15 and 30 days with 150 mg.kg^-1. The higher dose of malathion increased the BrdU signal on days 1 and 5. The percentage of cell proliferation in the control group was about 15 %.

3.4 DNA fragmentation in spermatogonia

Figure 5 shows the cytotoxic damage in worms treated with 600 mg.kg^-1 on days 5 and 15. A significant increase in cells with fragmented DNA was seen. On days 1 and 30, the percentage of DNA fragmentation did not show significant differences between the control group and the treated group. The percentage of TUNEL positive cells in germinal cells of control worms was 11 %. Figure 6 shows a positive TUNEL reaction in E. Foetida on day 5 with 600 mg.kg^-1 of malathion. Morules with fragmentation of DNA had a brown nuclei and morules without fragmentation of DNA had a blue nuclei.

4 Discussion

Currently, there is much concern about the adverse effects of environmental chemical agents in biological systems [8]. Malathion is widely used in agriculture, houses and gardens and is lethal to many living systems. However, little is known about the chronic and sub-lethal effects it has on reproduction. The present results show a significant loss of weight in earthworms exposed to malathion (Figure 1), with a toxic dose-dependent effect, which supports the results of other authors [9, 10]. Leland et al. [11] detected the morphologic changes and symptomatic effects characteristic of acetylcholinesterase inhibition (including weight lost, reduced burying ability and curling) in earthworms exposed to soil polluted by organophosphates. This loss of corporal mass has a multiplicative effect in E. foetida, as it also alters its feeding ability, and therefore, its reproductive performance [12, 13]. In the spermatheca of animals treated with malathion (Figure 2) vacuolization of the basal epithelium and absence of integrated spermatozoa to the microvilli were seen. Bustos-Obregn and Goicochea [5] observed a similar phenomenon with parathion, another organophosphorate pesticide, which indicates that these kinds of pesticides may affect the spermatheca content, eliciting phagocytosis of spermatozoa or modifying the anchorage sites between spermatozoa and microvilli in the basal epithelium. This would explain the central disposition of spermatozoa in the spermatheca. These findings are worthy of further investigations. BrdU incorporation and TUNEL studies showed that malathion affected the cell proliferation and DNA fragmentation. These results were clearly observed on days 5 and 15. Blasiak et al. reported that malathion has an acute cytotoxic effect leading to cellular death in human lymphocytes, without causing damage to the DNA; but its active metabolites, malaoxon and isomalathion act on DNA, breaking its chains [14]. Bustos-Obregn and Gonzlez-Hormazabal observed that malathion interfered with mouse testicular function, altering the ADN structure of spermatogonia and maturing spermatids [15]. Thirty days after treatment, cell proliferation was observed, but no significant differences in DNA fragmentation were seen. Studies in mice have reported that malathion was a potent cell cycle inhibitor that induces DNA damage and capable of interfering with DNA replication; this suggests that malathion is a genotoxic agent and may be regarded as a potential germ cell mutagen [16].

These effects tend to disappear, possibly because of the detoxifying action of E. foetida through peritoneal yellow cells, also known as chloragogenous cells, that are found throughout the intestine. These cells play a detoxifying function, similar to hepatocytes in vertebrates and they would allow total biodegradation of malathion and its metabolites [17].

In conclusion, malathion is a cytotoxic agent that interferes with the reproductive function of E. foetida, decreasing significantly the body weight dose-dependently. This pesticide also impedes the appropriate anchorage of the sperms on the microvilli of the basal epithelium of spermatheca, possibly altering their viability. In addition malathion alter the cell proliferation and affect the DNA structure of spermatogonia.

Acknowledgment

The work was partially financed by the University of Tarapac (Arica, Chile) research project 4760 and Banco Santander Central Hispano, Madrid, Spain.

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