ISI Impact Factor (2004): 1.096

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Prof. Yi-Fei WANG,

Joint action of phoxim and fenvalerate on reproduction in male rats

Asian J Androl  2004 Dec; 6: 337-341         

Keywords: joint action; phoxim; fenvalerate; sperm; reproductive function

Abstract

Aim: To evaluate the joint action of phoxim and fenvalerate on the reproductive function in male Sprague-Dawley rats. Methods: The 2×2 factorial analysis experiment was used in the study. The pesticides were orally given at daily doses of phoxim (Pho) 8.2 mg/kg, fenvalerate (Fen) 3.3 mg/kg and Pho 8.2+Fen 3.3 mg/kg (Pho:Fen = 5:2), 5 days a week for 60 days. Sperm motility was measured with computer-assisted sperm motility analysis (CASA) and daily sperm production estimated. Immunoenzymatic method and electron microscopy were used to evaluate the serum testosterone and the testicular morphology, respectively. Results: There were significant decreases in sperm motility parameters in the treated animals, including straight line velocity (VSL), beat cross frequency (BCF), linearity (LIN) and straightness (STR). After treated with Fen, significant decreases in VSL, LIN and STR were demonstrated. Significant decreases of daily sperm production were seen in animals treated with Pho and Pho+Fen in comparison with the controls. Serum testosterone levels were not significantly changed in the treated groups. Factorial ANOVA showed that no significant interactions were noted between Pho and Fen in sperm motility, sperm production and serum testosterone. Both the single and mixed pesticides caused various degrees of testicular lesions, involving vacuolation of endoplasmic reticulum and necrosis of Sertoli cells. Conclusion: The pesticides may cause sperm motility changes and testicular lesions in male rats. The action of Pho and Fen may be additive.

1 Introduction

It has been shown that endocrine disrupting chemicals (EDCs) are associated with alteration of sexual differentiation and increase incidence in testicular cancer, hypospadias and cryptorchidism [1]. Some pesticides are well known EDCs [2, 3].

Due to the development of insect resistance to a single pesticide, the present trend is to develop mixed pesticides. Since organochlorine insecticides have been banned or restricted since the 1960s, organophosphate and pyrethroid are used as the main pesticides. It has been reported that single pesticides impairs male reproductive function [4-7]. Phoxim (Pho), one of the organophosphorus pesticides, has been shown to decrease the testicular weight and sperm motility, associated with an increase in the percentage of dead and morphologically abnormal spermatozoa. Pho also causes decreases in testosterone levels and testicular lesions [8]. Fenvalerate (Fen), a synthetic pyrethroid, induces significant reduction in testis weight, epididymal sperm count, sperm motility and marker testicular enzymes for testosterone biosynthesis [9].

In the present study, the 2×2 factorial analysis experiment, a classical method for assessment of combined action, has been used to evaluate the joint action of Pho and Fen on reproductive function in male rats.

2 Materials and methods

Pho (purity 91.4 %) and Fen (purity 91.8 %) were obtained from the Liyang Chemical Company (Jiangsu, China) and the Baofeng Chemical Company (Jiangsu, China), respectively. The desired doses were prepared in corn oil. Osmium trichloride, epoxy resin EPON-Araldite 812, uranyl acetate and lead citrate used in electron microscopy were purchased from the Electron Microscopy Sciences (PA, USA).

Male adult Sprague-Dawley rats, weighing 230-290 g and about 3 months of age, were housed under standardized conditions (25 ℃±2 ℃, 12 h light/12 h darkness, humidity 50 % - 55 %) with free access to pelleted food and tap water. The rats were randomly chosen and divided into 1 control and 3 treatment groups (n=8 each). The pesticides were orally given at daily doses of Pho 8.2 mg/kg (1/180 LD50), Fen 3.3 mg/kg (1/180 LD50) and Pho + Fen 8.2 + 3.3 mg/kg (Pho:Fen = 5:2), five days a week for 60 days to cover a complete spermatogenic cycle. The same volume of oil was given to control animals.

Sperm were collected and prepared as described [10,11]. The animals were sacrificed by the end of administration and cauda epididymides were removed and placed in 3 mL M199 medium supplied with 0.4 % bovine serum albumin (BSA) in a 35-mm glass petri dish at 37 ℃. After the fat was trimmed, they were transferred to another petri dish with 3 mL of the same medium. Three to four deep cuts were made along the proximal and distal cauda of each epididymis. After diffusing for five min at 37 ℃, the tissue was removed, the sperm suspension collected and kept at 37 ℃. Sperm suspension was diluted with fresh M199 medium to give an approximate concentration of 6.0× 10⁵ sperm/mL. Aliquots of 10 µL of the diluted sperm suspension were loaded at the counting chambers with a depth of 100 µm. The slides were viewed using a Nikon Labophot-2 microscope equipped with a negative-phase contrast objective and a video camera connected to a video-cassette recorder (Nikon, Japan). Sperm motility was analyzed using the HST computer-assisted sperm motility analysis system (CASA, Hobson Tracking Systems Ltd., Sheffield, England) operating with a HST-7 V1B software. The following sperm motility parameters were determined: curvilinear velocity (VCL, µm/s), average path velocity (VAP, µm/s), straight line velocity (VSL, µm/s), beat cross frequency (BCF, Hz), straightness (STR, %) and linearity (LIN, %).

Daily sperm production in the rats was determined as described previously [12]. Frozen testes were thawed on ice, decapsulated and homogenized in 50 mL icecold 0.9 % NaCl solution containing 0.01 % Triton X-100 using a tissue mixer. The homogenate was settled for 1 min and then mixed. The number of sperm head was counted in 4 chambers of a hemocytometer. The number of spermatozoa produced per gram testicular tissue per day was calculated according to the formula: A×B×C×D/E/F. A= average count of sperm heads in the 4 chambers, B= square factor (5.0), C= hemocytometer factor (10⁴), D= dilution factor (50), E= testis weight (g) and F= the time (days) during spermatogenesis that these cell are resistant to homogenization (6.1 days).

Serum testosterone was determined by the immuno-enzymatic method (IEMA) according to the instruction of the kit (BIOZYME Co., Switzerland). The inter- and intra- assay coefficients of variation are <15 % and <10 %, respectively.

After the animals were killed, the right testis was excised and cut in halves. One-half of it was fixed in 5 % glutaraldehyde. After washing in PBS for 4 times, the prefixed tissue was postfixed in 1 % osmium trichloride for 2 hours and was embedded in epoxy resin EPON-Araldite 812. Ultra-thin sections were obtained with an ultramicrotom, doubled stained with uranyl acetate and lead citrate and observed under a transmission electron microscope (Hitachi H-300, Japan).

The data were expressed as mean±SD and were analyzed using factorial design ANOVA. All the statistical analyses were performed with SAS software. The significance level was set at 0.05. The 2×2 factorial analysis experiment was used. Factorial design ANOVA was employed to clarify the interaction between Pho and Fen. If P>0.05, the interaction was considered as additive and when P<0.05, synergistic or antagonistic.

When rats were treated with Pho and Pho+Fen, significant decreases in sperm motility parameters of VSL, BCF, LIN and STR were seen in comparison with the controls (P<0.01). No significant changes were found in VCL and VAP. Fen treatment caused significant decreases in VSL (P<0.05), LIN and STR (P<0.01). There was no significant difference in motility parameters of VCL, VAP and BCF between the control group and the treated. Factorial analysis showed that no significant interaction was noted between Pho and Fen (Table 1).

Table 1. Effects of Pho and Fen on rat sperm motility. means±SD. ^aP>0.05, factorial analysis of interaction between Pho and Fen, ^bP<0.05, ^cP<0.01, compared with controls.

Significant decreases of daily sperm production were seen in animals treated with Pho and Pho+Fen (P<0.05). No obvious decrease was found in the group treated with Fen. There was no significant interaction between Pho and Fen (Table 2).

Table 2. Effects of Pho and Fen on rat daily sperm production and serum testosterone. means±SD. ^aP>0.05, factorial analysis of interaction between Pho and Fen, ^bP<0.05, compared with controls.

Serum testosterone levels were similar in both the controls and the groups treated with Pho, Fen or Pho+Fen. No significant interaction between Pho and Fen was found (Table 2).

As shown in Figure 1, in the group treated with Pho+Fen, degenerative changes were observed with vacuolation and necrosis of endoplasmic reticulum in Sertoli cell. Necrotic spermatids were occasionally seen at the surface of the germinal epithelium. Various degrees of testicular degenerative changes were also seen in the single pesticide treated groups (data not shown).

4 Discussion

The reported effects of pesticides on reproductive system have been mostly limited to a single pesticide and in the present study the combined effects of two pesticides were investigated.

Pho and Fen exerted different effects on daily sperm production. Pho decreased it, while Fen did not. Both Pho and Fen did not change serum testosterone level. Factorial ANOVA showed that no significant interactions were noted between the two groups in sperm motility, sperm production and serum testosterone. Both single and mixed pesticides caused various degrees of testicular lesions. The joint action of the two pesticides was considered additive. One did not enhance the toxicity of the other at the dose levels employed.

Organophosphate and pyrethroid exerted toxic effects through different mechanisms. Organophosphorus pesticides inhibited acetylcholinesterase and caused acetylcholine accumulation, which resulted in hyperexcitability of the nervous system. Pyrethroid pesticides acted as a neuropoison interfering the ionic conductance by prolonging the sodium current [13,14]. Limited toxicity studies showed that some organophosphorus pesticides might enhance the toxicity of pyrethroid. Changes in the metabolism pathway might be important in modifying the pesticide toxicity [15]. It was well established that pyrethroid pesticides are subjected to hydrolysis by plasma and tissue esterases. Organophosphorus pesticides could increase the toxicity of pyrethroid by inhibiting esterase activity [16]. The addictive effect of these two pesticides shown in this study might be due to the use of low doses. The mechanism required further study.

In this study, testicular lesions including vacuolation of endoplasmic reticulum and necrosis of Sertoli cell were recorded. The decreases of daily sperm production and sperm motility were consistent with the histological findings in Pho and Pho+Fen treated groups. Results from present study suggested that Pho and Pho+Fen affected sperm production and motility indirectly through the effects on Sertoli cells. The decrease in daily sperm production is the result of a concomitant necrosis of Sertoli cells. The dose of Fen used in this study may not impair Sertoli cells to the extent of decreasing sperm production, so only changes in sperm motility were observed. No obvious changes of serum testosterone were found in single and mixed pesticide groups, suggesting that these two pesticides might exert their effects on the endocrine system through other mechanisms.

In addition to testicular factors, epididymal dysfunction may be involved in abnormal sperm motility. Some chemicals such as chlorohydrin and methylchloride can be classified as epididymal toxicants, either having direct or indirect effects on the epididymal epithelium [17]. It is possible that the pesticides affected the sperm motion parameters by effects on the epididymis.

In conclusion, the joint action of mixed Pho and Fen on reproductive function in male rats may be considered additive. Single pesticide alone or mixed pesticides may cause sperm motility changes and testicular lesion with various degrees.

The authors gratefully acknowledge the financial assistance from the Preliminary Study of an Important Project in the National basic Research, MOST (No. 2001 CCA 04900) and the Greatest Project in the National Basic Research (No 2002 CB512908). We also thank Dr. Hebron C. Chang for his suggestions on discussion.

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Correspondence to: Dr. Xin-Ru Wang, Institute of Toxicology, Nanjing Medical University, 140, Hanzhong Road, Nanjing 210029, China.
Tel: +86-25-8686 2939, Fax: +86-25-8652 7613
Email: xrwang@njmu.edu.cn
Received 2004-01-29 Accepted 2004-10-08