Cytotoxic effect of endosulfan on rat Sertoli-germ cell coculture

Reproductive Toxicology 13 (1999) 291–294

Cytotoxic effect of endosulfan on rat Sertoli-germ cell coculture Neelima Sinha, Neeta Adhikari, Ram Narayan, Daya K. Saxena* Industrial Toxicology Research Centre, Incharge–Embryotoxicology Division, Mahatma Gandhi Marg, PO Box 80, Lucknow–226 001, India Received 19 January 1999; Revision received 24 April 1999; Accepted 25 April 1999.

Abstract Endosulfan induced testicular impairment has been reported in vivo in rats. The present study was conducted to evaluate the cytotoxic effects of endosulfan in vitro employing rat testicular cells in culture, that is Sertoli– germ cell coculture. Cytotoxic changes induced by endosulfan (0, 2, 20, 40, and 80 ␮M) in mixed cultures of Sertoli and germ cells were seen after 24 and 48 h of treatment. Endosulfan led to an increase in germ cell detachment from the Sertoli cell monolayer in a dose dependent manner. A loss in the viability of detached cells was observed in all treated groups. Substantial leakage of the cytosolic enzyme lactate dehydrogenase was observed in the medium after 24 and 48 h endosulfan treatment at concentrations of 20 to 80 ␮M. The extent of toxicity was greater after 48 h of treatment. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Sertoli– germ cell coculture; Endosulfan; Cytotoxity; Rat

1.

Introduction

Endosulfan (6,7,8,9,10-hexachloro 1,5,5a,9,6,9,9a-hexahydro 6 –9 methano-2,3,4 benzodioxa-thiepin-3-oxide) is an organochlorine insecticide that is extensively used to control pests in vegetables, cotton, and fruits [1]. The accumulation of endosulfan has been reported in various crops in India and other developing countries [2,3]. Endosulfan-induced testicular impairment in vivo was reported previously from this lab [4,5] and elsewhere [6,7]. Our studies revealed that endosulfan led to a decline in daily sperm production along with increased sperm abnormalities and altered activities of testicular marker enzymes in both mature and immature rats. In addition, vacuolation of Sertoli cells was observed histologically (Sinha, unpublished). Testicular cells, especially Sertoli– germ cell coculture, have been used by many workers to investigate the mechanisms by which recognised toxicants exert their effect [8,9]. Metabolic cooperation exists between Sertoli cells and germ cells and any disturbance in this interaction may lead to testicular dysfunction [10]. In the present work we observed whether endosulfan evokes cytotoxic effects on Sertoli– germ cell coculture, which is an initial requirement * Corresponding author. Tel.: ⫹91-522-211547 (ext. 218, 219). Fax: ⫹91-0522-228227. E-mail address: [email protected] (D. K. Saxena)

for the evaluation of further effects on Sertoli– germ cell interaction.

2.

Materials and methods

2.1. Materials Collagenase (type I), DNAse I, and Eagle’s minimum essential medium (EMEM) were procurred from Sigma, St. Louis MO. Hank’s balanced salt solution (HBSS), fetal calf serum (FCS), and trypsin were purchased from Hi-media Laboratory Pvt. Ltd., Mumbai India. Tissue culture dishes were obtained from Tarson India. Technical Grade endosulfan (95.32% purity) was a gift from M/S Bharat Pulverising Mills Pvt. Ltd., Mumbai India. 2.2. Animals Weaned male albino rats of the Druckray strain were procured from the Industrial Toxicology Research Centre animal breeding colony and housed in polycarbonate cages in an air conditioned room with 12-h light/dark cycles and 60 to 70% humidity. Animals were acclimatized for a week prior to experimentation and fed a pelleted diet (Ashirwad, Ropar, Punjab, India) and water ad libitum. Preparation of Sertoli– germ cell coculture: Sertoli– germ

0890-6238/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 0 - 6 2 3 8 ( 9 9 ) 0 0 0 2 0 - 9

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cell coculture was prepared from 28 to 30 d old rats according to the method of Ku and Chapin [11] with slight modifications. In brief, testes from 10 to 14 rats were collected under aseptic condition in sterile HBSS, decapsulated, minced, and allowed to settle in culture tubes. HBSS was decanted and the tubules transferred to fresh HBSS solution containing 0.4% trypsin and 10 ␮g DNAse I/mL, placed in a water bath at 32°C for 15 min. The flask was manually shaken at intervals to facilitate separation of the tissue into cord like individual tubules. The tubules were then aspirated, centrifuged at 80 ⫻ g, and transferred to a flask containing collagenase (0.1%) in HBSS, placed in a water bath at 32°C for 40 to 45 min with manual shaking for further breaking up of the tubules. The tubules were again aspirated, centrifuged, and washed with EMEM (containing 100 ␮g penicillin/mL, 100 ␮g streptomycin/mL), and 10% FCS. The supernatant was discarded and the pellet was aspirated, resuspended in EMEM, and centrifuged at 40 ⫻ g for 3 to 4 min. This step was carried out 3 or 4 times until the supernatant was clear. The pellet served as the Sertoli– germ cell coculture stock. Aliquots (3.5 mL) of this suspension were plated in 35-mm tissue culture dishes and incubated at 32°C in a humidified atmosphere with 5% CO2/95% air. The density of the plated cells was 11 ⫻ 106 cells/dish. 2.3. Treatment of cultures After incubation of cells for 24 h in EMEM containing 10% (v/v) FCS, the medium was replaced with serum free medium (3.5 mL/dish) containing endosulfan (2, 20, 40, and 80 ␮M) dissolved in dimethyl sulphoxide (DMSO; final DMSO concentration 0.4% v/v). Control cultures received medium containing 0.4% (v/v) DMSO only. The cultures were incubated for an additional 48 h (a total of 72 h in culture). The medium was replaced after every 24 h with medium containing same quantity of the test material. The 20 ␮M concentration corresponded to endosulfan testicular tissue levels in vivo at the effect dose of 10 mg/kg/d in our previous work [5]. 2.4. Assessment of germ cell exfoliation Each time the medium was collected it was centrifuged at 80 ⫻ g and the cells pelleted. The cell pellet was resuspended in an appropriate volume of serum free medium and germ cells detached from the Sertoli– germ cell monolayer were counted using a haemocytometer. 2.5. Viability of detached cells The viability of detached cells was studied by staining with trypan blue (1:1) and counting cells in a haemocytometer. Viable cells were recorded as the percentage of cells excluding trypan blue.

Table 1 Effect of endosulfan on germ cell detachment in rat testicular cell cultures No. of germ cell detached (as % of control) Endosulfan concentration (␮M) 2 20 40 80

Treatment time (h) 24

48

7.30 142.92a,b 233.33a,b,c 470.31a,b,c,d

9.70a 182.04a,b 279.61a,b,c a,b,c,d 541.74

Values are mean for three culture dishes. The control cell counts were 0.219 ⫾ 0.01 and 0.206 ⫾ 0.01 ⫻ 105/dish after 24 and 48 h, respectively. P ⬍ 0.05 by Student’s t-test compared to acontrol, b2 ␮M endosulfan, c 20 ␮M endosulfan, d40 ␮M endosulfan.

2.6. Measurement of lactate dehydrogenase (LDH) in the medium The leakage of the cytosolic enzyme LDH into the medium after endosulfan treatment was performed according to the method of Kornberg [12]. Media from treated and control plates were collected and centrifuged to pellet the cells. The clear medium was used for measurement of enzyme. Protein in the sample also was measured [13]. 2.7. Statistical analysis Data were analysed with Student’s t-test. The significance level was ascertained at P ⬍ 0.05.

3.

Results

The Sertoli– germ cell cocultures from 28-d-old rats generally contains Sertoli cells, primary spermatocytes, at various stages of meiotic division, and few spermatogonia as already reported by Gray and Beamand [14]. In the present study, the Sertoli cell monolayer was obtained after 20 to 24 h of initial plating with attached germ cells. The cells were 92 to 95% viable as assessed by the trypan blue exclusion test. The germ cells progressively detached from the monolayer into the culture medium with time. The percent detachment increased with the addition of endosulfan to the medium. The Sertoli cell monolayer, however, remained intact throughout the course of the experiment. 3.1. Germ cell exfoliation Exfoliation of germ cells into the medium after endosulfan exposure was expressed as percent (increase) of control. Endosulfan produced an increase in germ cell detachment in a dose dependent manner after 24 h of exposure (Table 1). No significant changes were observed in the group exposed to 2 ␮M. At 48 h, a similar trend in germ cell detachment

N. Sinha et al. / Reproductive Toxicology 13 (1999) 291–294 Table 2 Effect of endosulfan on the viability of detached cells in rat Sertoli– germ cell cocultures

Table 3 Effect of endosulfan on LDH leakage in the medium of Sertoli– germ cell cocultures

Percent viability of detached cells Endosulfan concentration (␮M) Control 2 20 40 80

48

74.41 67.45 50.41a,b 39.86a,b,c 30.85a,b,c,d

69.58 60.86a 42.31a,b,* 31.27a,b,c,* 26.96a,b,c,d,*

Values are mean percent of three culture dishes. P ⬍ 0.05 by Student’s t-test compared to acontrol, b2 ␮M endosulfan, c 20 ␮M endosulfan, d40 ␮M endosulfan, *corresponding 24-h value.

was observed with even the lowest dose showing a significant increase in germ cell detachment. The number of germ cells detached after 48 h was similar to that observed after 24 h. 3.2. Viability of detached cells The viability of exfoliated germ cells was 74.41 and 69.58% in the control group after 24 and 48 h of treatment, respectively. The viability of detached cells decreased progressively (67 to 31% after 24 h and 61 to 27% after 48 h) with the increase in the concentration of endosulfan. A significant loss in viability was recorded even in the lowest concentration after 48 h of treatment. The decrease in viability of detached cells was both concentration and duration dependent, the loss being maximum after 48 h of treatment (Table 2). 3.3. Leakage of LDH The leakage of LDH into the medium was calculated as nmol NADH oxidised/min/mg protein. LDH leakage in the control cells both after 24 and 48 h intervals was constant. Exposure to endosulfan resulted in substantial leakage of LDH into the medium at the doses of 20, 40, and 80 ␮M (Table 3). The leakage was both concentration and duration dependent.

4.

LDH leakage (nmol NADH oxidised/min/ mg protein)

Treatment time (h) 24

Discussion

Xenobiotics can disturb testicular function by interfering with several pathways operating within the testis. Testicular lesions may be characterised by early detachment of spermatocytes and spermatids from the germinal epithelium leading to a disruption in the normal interaction between Sertoli cells and germ cells that are responsible for maintaining the integrity of the epithelium [15]. In the present study, endosulfan exposure resulted in the detachment of germ cells in the culture medium. The percent cell detach-

293

Endosulfan concentration (␮M) Control 2 20 40 80

Treatment time (h) 24

48

4.23 ⫾ 0.20 3.95 ⫾ 0.16 17.09 ⫾ 0.61a,b 20.08 ⫾ 0.91a,b,c 25.91 ⫾ 0.98a,b,c,d

4.68 ⫾ 0.22 4.94 ⫾ 0.20* 19.36 ⫾ 0.35a,b,* 23.35 ⫾ 0.34a,b,c,* 29.86 ⫾ 0.63a,b,c,d,*

Values are mean ⫾ SE of three culture dishes per concentration. P ⬍ 0.05 by Student’s t-test compared to acontrol, b2 ␮M endosulfan, c 20 ␮M endosulfan, d40 ␮M endosulfan, *Corresponding 24-h value.

ment increased with endosulfan concentration both after 24 and 48 h of treatment. This shedding of germ cells in vitro is a possible reason for the low sperm production observed in our earlier in vivo studies on mature and immature rats following endosulfan exposure [4,5]. Similarly, Foster et al. [16] showed that a correlation existed between germ cell detachment in vitro and testicular damage in vivo after treatment with various phthalate esters. Williams and Foster [17] using in vitro methods demonstrated that 1,3-DNB (dinitrobenzene) had a very prominent effect on the detachment of germ cells from the Sertoli cell layer. Similarly, the percent viability of detached cells decreased with increasing endosulfan concentration, and was both dose and duration dependent. This effect appears probably to be due to direct cytotoxic action of endosulfan on the germ cells. However, the role of Sertoli cells in this process cannot be ruled out. Our result is in contrast with that reported by Gray and Beamand [14], who have found no differences in the viability of cells in control and mono-(2ethylhexyl)phthalate (MEHP) treated cultures. They attributed their result to the lack of direct action of MEHP on germ cells. The cytotoxicity of any compound/xenobiotic can be characterised by the use of the viability stains such as trypan blue [18] and more sensitive parameter like leakage of the cytosolic enzyme lactate dehydrogenase [19]. LDH leakage into the medium occurs with even minor damage to the cytoplasmic membrane [20]. The present study confirmed that endosulfan leads to cytoplasmic membrane damage at all the tested concentrations above 2.0 ␮M. From the present study, it is clear that endosulfan leads to cytotoxicity in Sertoli– germ cell coculture, which might disturb the normal interaction between Sertoli and germ cells, thereby leading to testicular dysfunction. Acknowledgements We thank the Director of Industrial Toxicology Research Centre for his keen interest and encouragement in this study.

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The authors, NS and NA, are thankful to Council of Scientific and Industrial Research, New Delhi, for providing financial assistance as Senior Research Associateships. We thank M/S Bharat Pulverising Mills Pvt. Ltd., Mumbai for supply of technical grade endosulfan as a gift. ITRC Communication Number 2042.

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