Histopathological, biochemical and molecular changes of reproductive function after malathion exposure of prepubertal male mice

Abstract

We aimed in the present work to evaluate the implication of oxidative stress in the toxicological effects of subchronic malathion exposure on reproductive function in mice. In this respect, we used prepubertal male mice separated into two groups: a control and a malathion treated group. Animals were treated by gavage (per orally, p.o.) with malathion at 200 mg kg⁻¹, body weight (b.w.) during thirty days. We found that malathion treatment leads to the alteration of semen parameters such as a decrease of testosterone level and acetylcholinesterase activity, an induction of apoptosis and necrosis in spermatozoa as well as a decrease of reproductive performance of male mice. The histopathological examination showed a marked change in the testis tissue. Malathion intoxication was by an increase of malondialdehyde (MDA) level, a decrease of sulfhydril groups (–SH) content, as well as a depletion of antioxidant enzyme activities such as catalase (CAT), total superoxide dismutase (SOD), Cu/Zn–SOD and Mn–SOD in testis and epididymis. More importantly, malathion treatment clearly induced a decrease in mRNA expression of COX isoenzyme in cauda and epididymis as well as GPx-4 in testis and GPx-5 in epididymis. These data suggest that a marked deregulation of reproductive function in prepubertal male mice exposed to malathion might be partly due to pro-oxidant properties of the examined compound.

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1. Introduction

Organophosphate insecticides (OPs) constitute one of the most widely used classes of pesticides being employed for both agricultural and pest control. Malathion is one of the most important OPs extensively used against several pests. Malathion induces several neurological and endocrine alterations in humans and different wildlife species.¹ However, subchronic exposure to malathion induces a mild cognitive dysfunction (including problems in identifying words, colors or numbers and inability to speak fluently) and hormonal imbalances leading to infertility, breast pain, menstrual disturbances, adrenal gland exhaustions and early menopause. There is now considerable evidence that male reproductive function is in decline in the wild life populations.² The disturbance of the reproductive function may be due to the disturbed testicular apoptosis and altered hepatic biotransformation of steroids. In rat model, malathion exposure induces a reduction of reproductive organs weights and spermatozoa parameters with an increase in sperm death and abnormalities.³ In the same context, Pina-Guzmán et al.⁴ demonstrated that OPs affects the late step of spermatogenic maturation in mice leading to DNA damage and reduces chromatin in spermatogonia and spermatids. Furthermore, malathion is considered as an endocrine disruptor due to its estrogenic and anti-androgenic activities,⁵ via stimulation of estrogen (ER) and androgen (AR) receptors expression.^6,7

It was also reported that malathion induced toxicity through the inhibition of acetylcholinesterase (AchE) and subsequent activation of cholinergic receptors.^8,9 However, other reports demonstrated that malathion exposure induces oxidative stress in many tissues such as brain,¹⁰ liver,¹¹ kidney¹¹ and heart.¹² Recently, we have showed that malathion exposure during lactation induced cholinesterase inhibition in plasma, erythrocyte and brain of rats pups¹⁰ as well as oxidative stress and alteration of biochemical markers in liver and kidney.¹¹

Hence, the present study aimed to investigate the impact of subchronic exposure to malathion on the reproductive function of prepubertal male mice as well as the implication of oxidative stress in such effect.

2. Materials and methods

2.1. Chemicals

Malathion (98% purity) (fyfanon 50 EC 500 g l⁻¹), acetylthiocholine iodide, 5.5′-dithiobis-2-nitrobenzoic acid (DTNB), triton X-100, eosin stain, and RPMI (Roswell Park Memorial Institute), were purchased from SIGMA and Invitrogen.

2.2. Animals and dosage exposures

Male and female mice were purchased from Pasteur Institute (Tunis, Tunisia) and used in accordance with the local ethic committee of Tunis University for use and care of animals in conformity with the NIH recommendations. They were housed in standard cages (40 × 28 × 16 cm) and provided with food (standard pellet diet-Badr, Utique-TN) and water ad libitum and maintained in animal house at controlled temperature (22 ± 2 °C) with a 12 hours light-dark cycle and the relative humidity was about 50–60%. Primiparous females were placed three per cage with one male breeder and vaginal smear examined daily in the evening. The day 0 of pregnancy was confirmed by the presence of a vaginal smear of both vaginal cells typical of the oestrous stage and spermatozoids. At the weaning age (21 days), after the lactational period of their offspring (prepubertal male mice) were separated and then randomly divided into two groups of 16 animals each: control and malathion treated group. Mice in the treated group received by gavage malathion in corn oil at (200 mg kg⁻¹, b.w.) during 30 days. Control group received equal amount of corn oil for the same period.

2.3. Body and reproductive organ weights

Initial (weight of start point) and final (weight of end point) body weights were recorded. Male mice from each group were killed by decapitation. The reproductive organs were stripped from fatty tissues and blood vessels, blotted, and their absolute weights were determined using precision balance Denver S-603 series. Clinical signs of body and reproductive organs were evaluated for toxicological criteria. To normalize the data for statistical analysis, organ weights were expressed per 100 gram body weight.

2.4. Plasma acetylcholinestrase activity (AChE)

The acetylcholinesterase activity was determined in plasma by the method of Ellman et al.¹³ using acetylthiocholine iodide as a substrate. The rate of hydrolysis acetylthiocholine was measured at 405 nm by the reaction of thiocholine with dithiobisnitrobenzoic acid (DTNB) to give the yellow 5-thio-2-nitrobenzoate anion. The enzyme activity was expressed as nmol of substrate hydrolyzed/min/mg protein.

2.5. Evaluation of reproductive hormone in plasma

Immediately, when animals reached the mating period, peripheral blood was collected. The plasma was separated and used for measurement of steroid hormone. The concentration of testosterone was measured by commercial enzyme-linked immune-absorbent assays (ELISA) purchased from demeditec (Ref, DEV 9911).

2.6. Evaluation of sperm characteristics

2.6.1. Sperm collection. After the test period, the laparotomy was conducted for all exposed and control male mice after body and organs weights measurement. Tesis and epididymis were carefully excised. The sperm count was assessed from right cauda epididymis while sperm motility and morphology were analyzed from the left one. Epididymis was excised and minced in 1 ml of RPMI to obtain spermatozoa suspension. The other testis and epididymis were frozen in −20 °C until use.

2.6.2. Sperm count. The cauda epididymal sperm count was performed according to the method of Vega et al.¹⁴ Epididymal sperm counts were expressed as number of spermatozoa per epididymis. To minimize the error, the count was repeated three times for each sample.

2.6.3. Sperm motility. Ten μl of sperm suspension was layered onto a warmed microscope slide. Sperm motility was assessed by counting all progressive motile, non-progressive motile and immotile spermatozoa. The number of motile spermatozoa in each field was divided by the total number, and the average of the fields was assayed. The percentage of motile spermatozoa was thereafter determined.¹⁵

2.6.4. Sperm viability. Sperm viability was assessed using the eosin stain.¹⁶ The staining was performed with one drop of freshly collected semen (10 μl) and two drops of solution (20 μl) of eosin. The live was unstained (intact) and dead (purple to red-stained head with damaged membranes). The dye exclusion was evaluated in 100 spermatozoa. Sperm viability was defined as the percentage of dead sperm cells. Viability was evaluated according to WHO guidelines.¹⁷

2.6.5. Sperm morphology. A drop of sperm suspension was smeared on a slide and air-dried and made permanent. The smeared slide was stained with 1% eosin. Morphological sperm defects were evaluated and examined on optical microscope.¹⁸ At least one hundred spermatozoa from different fields in each slide were examined and classified for criteria of morphological abnormalities (head, tail and tail-head) according to Filler.¹⁹ Abnormal sperm cells were counted and the percentage was calculated.

2.6.6. Assessment of sperm production. Sperm content per gram was determined using the method previously described by Vega et al.¹⁴ and Narayana et al.²⁰ with slight modifications. Briefly, after thawing at temperature (25–27 °C), the whole epididymal and the testicular tissues were homogenized for 5 min in 5 ml of physiological saline (0.9% NaCl) containing 0.05% (v/v) Triton X-100 using a manual homogenizer. The homogenates were diluted with 1.5 ml of saline solution; spermatozoa and spermatids were counted. Three counts per sample were averaged.²¹ These count values were used to obtain the total number of spermatids per testis and sperm per epididymis; which was then divided by the testis or epididymis weight to determine the number per gram of testis or epididymis.

2.7. Measurement of semen DNA fragmentation by flow cytometry

Flow cytometry analysis was performed as described by Perticarari et al.²² Each 30 days, the epididymal sperms of malathion and control mice were tested. Briefly, 100 μl of epididymal semen were stained for 20 minutes in the dark at room temperature using 2 ml of 10 mM solution of Syto 16 green fluorescent nucleic acid stain (molecular) and 10 μl of 7-amino-actinomycin-D (7-AAD) (Invitrogen). After the incubation period, 1 ml of PBS solution was added and the sample was analyzed by flow cytometry. The nucleic acid stain Syto 16 has been demonstrated to be able to distinguish apoptotic from non-apoptotic cells in several apoptosis models, including cell lines and stem cells and, recently, also in clinical samples.^22–24 Necrotic cells can be identified by using 7-AAD, a ready-to-use solution for the exclusion of nonviable cells in flow cytometric analysis. 7-AAD penetrates only dead cells.

2.8. Lipid peroxidation

Lipid peroxidation (LPO) was detected by the determination of MDA production determined by the method of Begue and Aust.²⁵ Briefly, homogenates of testis and epididymis were centrifuged at 1000g for 10 min at 4 °C to sediment cell debris. Supernatants were suspended in PBS, pH = 7.4, mixed with BHT-TCA solution (1‰ BHT dissolved in 20% TCA w/v) centrifuged at 1000g for 35 min and finally mixed with 0.5 N HCl and 120 mM TBA (thiobarbituric acid) in 26 mM Tris and heated in water bath at 80 °C for 10 min. After cooling, the absorbance of the resulting chromophore was measured at 532 nm using a UV-visible spectrophotometer (Beckman DU 640B). MDA levels were determined by using an extinction coefficient for MDA–TBA complex of 1.56 × 10⁵ M⁻¹ cm⁻¹.

2.9. Thiol groups measurement

Total concentration of thiol groups (–SH) was performed according to Hu and dillard method.²⁶ Briefly, aliquots from testis or epididymis tissues were mixed with 100 μl of 10% SDS and 800 μl of 10 mM phosphate buffer (pH 8) and the absorbance was measured at 412 nm (A0). Then, 100 μl of DTNB were added and incubated at 37 °C during 60 min. After incubation, the absorbance of the sample was measured at 412 nm (A1). The thiol groups concentration was calculated from A1 − A0 subtraction using a molar extinction coefficient of 13.6 × 103 M⁻¹ × cm⁻¹. Results were expressed as nmol of thiol groups per mg of protein.

2.10. Antioxidant activities assays

SOD activity was determined by using modified epinephrine assay of Misra and Fridovich.²⁷ At alkaline pH, superoxide anion O₂˙⁻ causes the autoxidation of epinephrine to adenochrome, while competing with this reaction, SOD decreased the adenochrome formation. One unit of SOD is defined as the amount of the extract that inhibits the rate of adenochrome formation by 50%. Enzyme extract was added in 2 ml reaction mixture containing 10 μl of bovine catalase (0.4 U μl⁻¹), 20 μl epinephrine (5 mg ml⁻¹) and 62.5 mM sodium carbonate/bicarbonate buffer pH 10.2. Changes in absorbance were recorded at 480 nm. Characterization of SOD isoforms was performed using KCN (2 mM), which inhibits Cu/Zn–SOD or H₂O₂ (5 mM), affecting both Cu/Zn–SOD and Fe–SOD whereas Mn–SOD was insensitive to both inhibitors.²⁸

CAT activity was assayed by measuring the initial rate of H₂O₂ disappearance at 240 nm.²⁹ The reaction mixture contained 33 mM H₂O₂ in 50 mM phosphate buffer pH 7.0 and CAT activity calculated using the extinction coefficient of 40 mM⁻¹ cm⁻¹ for H₂O₂.

2.11. Total RNA isolation and RT-PCR analysis

Total RNA was prepared using Trizol reagent according to the manufacturer's instructions. Total RNA (1 μg) was reverse transcribed using MMLV reverse transcriptase (Invitrogen, Tunis, Tunisia) by incubation at 25 °C for 10 min, at 42 °C for 60 min and at 99 °C for 5 min. The synthesized cDNA was amplified using Taq DNA polymerase (Invitrogen, Tunis, Tunisia) and the following specific primers:

GAPDH. F: 5′-GTGGATATTGTTGCCATCA-3′

R: 5′-ACTCATACAGCACCTCAG-3′

S-16. F: 5′-TCCGCTTGCACTGGGCTTCAAGTCTT-3′

R: 5′-GCCAAACTTCTTCTTGGATTCGCAGCG-3′

COX-1. R: 5′-TGGAGAAGTGCCAGCCCAACTCCC-3′

F: 5′-GGGGCAGGTCTTGGTGTTGAGGCA-3

COX-2. F: 5′-TCCAAATGAGATTGTGGGAAAATTGCT-3′

R: 5′-AGATCATCTCTGCCTGAGTATCTT-3′

GPx-4. F: 5′-AGTACAGGGGTTTCGTGTGC-3′

R: 5′-CGGCAGGTCCTTCTCTATCA-3′

PCR conditions were 30 cycles of 94 °C for 30 s, 59 °C for 30 s and 72 °C for 30 s, followed by 5 min incubation at 72 °C. PCR products were run on 1.5% agarose gel and then stained with ethidium bromide.

2.12. Histopathological examination

Immediately after sacrifice, small pieces of testis and epididymis were harvested and washed with ice-cold saline. Tissue fragments were then fixed in a 10% neutral buffered formalin solution, embedded in paraffin and used for histopathological examination. 5 μm thick sections were cut, deparaffinized, hydrated and stained with hematoxylin and eosin (HE). The testis and epididymis sections were examined in control and malathion treatment.

2.13. Protein determination

Protein concentration was determined according to Bradford method³⁰ using bovine serum albumin (BSA) as standard.

2.14. Statistical analysis

Data were analyzed by unpaired Student's t-test or one-way analysis of variance (ANOVA) and were expressed as means ± standard error of the mean (SEM). Data are representative of ten independent experiments. All statistical tests were two-tailed, and a p value of 0.05 or less was considered significant.

3. Results

3.1. Mortality and macroscopic symptoms of malathion toxicity

In the present study, no mortality was noted after suchronic exposure of prepubertal male mice to malathion (200 mg kg⁻¹, b.w., p.o.) during 30 days. However, cholinergic signs of adverse toxicological effects were observed such as sluggishness, muscular tremors, irregular movements, and abdominal tremble. The progression of these signs proceeds to the last week of treatment. Concerning the testis as well as the reproductive accessories, no macroscopic alterations were found until the end of the experiment.

3.2. Body and reproductive organs weights

Data from Table 1 showed that subchronic malathion (200 mg kg⁻¹, b.w., p.o.) exposure during 30 days induced a significant decrease of body weight gain by 29%. Malathion treatment also increased testis, epididymis, prostate and seminal vesicles relative weights respectively by 18%, 25%, 19% and 19%.

Table 1 Effects of malathion exposure (200 mg kg⁻¹ b.w., p.o.) of prepubertal male mice during 30 days on body and reproductive organs weights (n = 16). *: p < 0.05 versus control group

	Control	Malathion	% of increase or decrease
Gain body weight during 30 days (%)	273.14 ± 6.51	194.27 ± 8.12*	29
Relative testis weight (g/100 g b.w.)	0.52 ± 0.012	0.63 ± 0.06*	18
Relative epididymis weight (g/100 g b.w.)	0.09 ± 0.001	0.12 ± 0.02*	25
Relative prostate weight (g/100 g b.w.)	0.13 ± 0.004	0.16 ± 0.002*	19
Relative seminal vesicles weight (g/100 g b.w.)	0.39 ± 0.01	0.48 ± 0.05*	19

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3.3. Evaluation of biochemical findings

After 30 days of malathion treatment, a significant inhibition of plasma AChE activity (52%) compared to control group was observed (Fig. 1). Plasma testosterone levels were also drastically reduced by 58% in the animals treated with malathion at 200 mg kg⁻¹ b.w. during 30 days (Fig. 2).

Fig. 1 Effects of malathion exposure (200 mg kg⁻¹ b.w., p.o.) of prepubertal male mice during 30 days on plasma acetylcholinesterase activity (n = 16). *: p < 0.05 versus control group.

Fig. 2 Effects of malathion exposure (200 mg kg⁻¹ b.w., p.o.) of prepubertal male mice during 30 days on plasma testosterone concentration (n = 16). *: p < 0.05 versus control group.

3.4. Evaluation of reproductive performance quality

3.4.1. Semen analysis. Malathion exposure of male mice at 200 mg kg⁻¹, b.w., during 30 days induced a significant decrease of epididymal spermatozoa count and testicular spermatids enumeration as well as a reduction of sperm motility, while the number of dead sperm increased significantly (Table 2). Morphological abnormalities of spermatozoa were categorized by head or tail (Table 2 and Fig. 3).

Table 2 Effects of malathion exposure (200 mg kg⁻¹ b.w., p.o.) of prepubertal male mice during 30 days on reproductive performance quality (n = 16). *: p < 0.05 versus control group

	Control	Malathion
Sperm concentration (10^6 ml^−1)	7 ± 2	2.7 ± 1*
Motility (%)	69 ± 7	40 ± 5*
Viability (%)	79 ± 4	22 ± 6*
Morphology, normal forms (%)	86.1 ± 4.52	46 ± 3*
Abnormal head (%)	5.8 ± 0.96	14 ± 2.1*
Abnormal tail (%)	8.1 ± 1.41	39 ± 4.2*
Sperm count 10^6 g^−1 epididymis	132 ± 16.4	96 ± 1.98*
Spermatid count10^6 g^−1 of testis	200 ± 29.3	118 ± 5.89*

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Fig. 3 Patterns of sperm abnormalities after malathion exposure (200 mg kg⁻¹ b.w., p.o.) of prepubertal male mice during 30 days. (A) Normal sperm with acrosome (arrow), (B–D) clumped head with normal tail (*), (F) unstained megacephaly head and normal tail with cytoplasm droplet (*), (E–G) normal head with looped tail (*).

3.4.2. DNA fragmentation. The mean percentage of living, apoptotic, and necrotic spermatozoa of control and malathion treated groups are shown in Fig. 4. Flow cytometry technique demonstrated that malathion exposure significantly increased the percentage of spermatozoa with fragmented DNA (apoptotic spermatozoa: Syto 16) compared to control group (Fig. 4A and B). Furthermore, the mean percentage of necrotic spermatozoa (7-AAD) also increased in malathion treated group when compared to control mice (Fig. 4C).

Fig. 4 Flow cytometry analysis of malathion effect on viability, apoptosis and necrosis in spermatozoa cells. Prepubertal male mice were treated with malathion (200 mg kg⁻¹ b.w., p.o.) during 30 days. Epididymal spermatozoa were subjected to FACS analysis in order to detect Syto 16 (A) and 7-AAD (B) expression. A representative for histogram for viability, apoptosis and necrosis is shown in (C). Data are expressed as mean ± SEM (n = 16). *: p < 0.05 versus control group.

3.5. Oxidative stress status

The implication of oxidative stress in the malathion induced reprotoxicity in mice was also studied and the results are shown in Fig. 5. We firstly found that malathion exposure induced a significant increase of MDA level, as index of LPO, a decrease of –SH groups as well as a depletion of CAT activity in both testis and epididymis tissues (Fig. 5A–C). We also showed in the present work that malathion exposure significantly reduced the total SOD, Cu/Zn–SOD, Mn–SOD but not Fe–SOD activities in testis (Fig. 5D). Concerning the epididymis tissue, the depletion of SOD activity was only significant for total-SOD and Mn–SOD (Fig. 5E).

Fig. 5 Effects of malathion exposure (200 mg kg⁻¹ b.w., p.o.) of prepubertal male mice during 30 days. on MDA level (A), –SH groups content (B) as well as CAT (C), total SOD, Cu/Zn–SOD, Mn–SOD and Fe–SOD activities (D and E) in testis and epididymis tissues (n = 16). *: p < 0.05 versus control group.

3.6. Expression of COX isozymes, GPx-4 and GPx-5 in reproductive organs

We further looked at the effect of malathion (200 mg kg⁻¹, b.w.) exposure during 30 days on COX isoenzymes and GPx-4 in testis as well as GPx-5 in cauda epididymis of male mice. Cauda epididymis from male mice was found to express both COX-1 and COX-2 isozymes. The expression levels were normalized by S-16 expression detected in the same PCR reaction, and these values were compared with those of the control group (Fig. 6). As expected, there was no change in the expression of S-16 in control and treated groups. However, malathion exposure caused a decrease in both COX iso-enzymes COX-1 and COX-2 when compared to control group (Fig. 6). The same results were found for GPx-4 in epididymis and GPx-5 in testis (Fig. 7).

Fig. 6 RT-PCR analysis of malathion effect on COX-1 and COX-2 expression in cauda epididymis of male mice. Prepubertal male mice were treated with malathion (200 mg kg⁻¹ b.w., p.o.) during 30 days. Cauda epididymis homogenates were subjected to RT-PCR analysis to detect COX-1 (A) and COX-2 (B). The relative expression of COX isoforms was quantified by densitometry and normalized to S-16 expression. A representative histogram of COX-1 and COX-2 is shown in (C). Data are expressed as mean ± SEM (n = 8). *: p < 0.05 versus control group.

Fig. 7 RT-PCR analysis of malathion effect on GPx-4 and GPx-5 expression respectively in testis and epididymis of male mice. Prepubertal male mice were treated with malathion (200 mg kg⁻¹ b.w., p.o.) during 30 days. Testis and epididymis homogenates were respectively subjected to RT-PCR analysis to detect GPx-4 (A) and GPx-5 (B). The relative expression of GPx isoforms was quantified by densitometry and normalized to GAPDH expression. A representative histogram of COX-1 and COX-2 is shown in (C). Data are expressed as mean ± SEM (n = 8). *: p < 0.05 versus control group.

3.7. Testis and epididymis histopathology

The histological study revealed that testis section of control mice showed a normal spermatogenic and Sertoli cells in the seminiferous tubules (Fig. 8A and B). Leydig cells were found in the interstitial connective tissue between the seminiferous tubules, and appeared to be uniform in size and shape. They were lined with regularly arranged rows of spermatogenic cells at different stages of maturation (Fig. 8B). In contrast, malathion exposure showed a necrosis in some seminiferous tubules as well as a clear depletion of spermatogenic cells number. We also observed an edema in the interstitial tissue of malathion treated group (Fig. 8C and D).

Fig. 8 Testis histology showing the effect of malathion exposure (200 mg kg⁻¹ b.w., p.o.) during 30 days. Normal architecture in control group (A) (×100) and (B) (×400). Histological changes in malathion treated group (C) (×100) and (D) (×400). N: necrosis in seminiferous tubules; E: edema in interstitial tissue and *: decrease of spermatozoa density.

4. Discussion

The present study aimed to evaluate the putative implication of oxidative stress in the subchronic effects of malathion on the reproductive function in male mice.

We firstly showed a significant loss of body weight gain in the malathion-treated mice as well as an increase of testis, epididymis, prostate and seminal vesicles relative weights. These results corroborate those of other authors who demonstrated morphologic changes and symptomatic effects, characteristic of acetylcholinesterase inhibition including accumulation of acetylcholine and subsequent activation of cholinergic, muscarinic, and nicotinic receptors and neurological deficits in male mice exposed to malathion.³¹ This loss of corporal mass has consequently numerous toxic effects in male mice, as their feeding ability, and therefore, their reproductive performances.³²

Male mice exposure to malathion during 30 days showed a significant decrease of epididymal spermatozoa count, testicular spermatids enumeration and sperm motility as well as a clear increase of viability. In addition, flow cytometry analysis demonstrated a significant increase of apoptotic (Syto 16) and necrotic (7-AAD) spermatozoa percentages in the malathion treated group when compared to control mice. In the same context, several studies attempted to make a link between apoptosis and the ejaculated human sperm and male infertility as well as sperm parameter defects.³³ However, the influence of apoptosis on sperm function and quality is controversial.^34–37 Apoptotic cells can be identified because they show a decreased Syto 16 fluorescence, probably because of changes in their DNA structure.³⁸ The combination of Syto 16 with a vital stain offers a sensitive, simple, inexpensive live-cell method for the discrimination of live, apoptotic and necrotic cells.^39,40 However, viable cells can be identified because they show an intact Syto 16 fluorescence. It has been also demonstrated that malathion exposure altered the DNA structure of spermatogonia and spermatids.^3,41 However, studies on mice have reported that malathion was a potent cell cycle inhibitor that induces DNA damage and is capable of interfering with DNA replication; this suggests that malathion is a genotoxic agent and may be regarded as a potential germ cell mutagen.⁴² Many studies have suggested an alteration of the mechanism of steroidogenic hormones production. Indeed, it is probable that the production of gonadotropins has been affected by malathion in male mice by the disruption of the hypothalamic pituitary gonadal axis.⁴³ However, other laboratories indicated that subchronic treatment with OPs caused alterations in hormone levels.^44,45 This decrease in testosterone level may be caused by the inability of the neuroendocrine cells of hypothalamic–pituitary axis to respond to the feedback.

In agreement with previous results, the current study showed that malathion exposure impaired motility and viability in mature spermatozoa collected after 30 days. It is likely that these effects of malathion and other OPs might be due, at least in part, to their ability to cross the blood–testis barrier.⁴⁶ This in turn may cause the degeneration of the spermatogenic and Leydig cells, which disrupts spermatogenesis and reduces sperm counts. Supporting this idea, we found in the present work that subchronic malathion exposure induced necrosis in some seminiferous tubules as well as edema in the interstitial tissue. The sperm themselves may also be damaged by the oxidative effects of OPs, which affect the activities of mitochondrial enzymes and the structure of the microtubules in the sperm. This in turn reduces their motility.

We also showed in the present study that subchronic exposure of male mice to malathion during 30 days induced oxidative stress status in testis and epididymis as assessed by increased LPO, decreased –SH groups level as well as a depletion of antioxidant enzyme activities such as CAT, total SOD, Cu/Zn–SOD and Mn–SOD but not Fe–SOD activity. Our data fully corroborated other investigations which evidenced that organophosphorus administration to male mice provoked an alteration in antioxidant enzymes activities in testis and epididymis tissues.⁵ However, it has been reported that toxic effects of malathion as well as other OPs are mainly related to their ability to cross the blood–testis barrier,⁴⁶ leading to oxidative stress and lipid peroxidation and consequently damages in the biological membranes of reproductive organs.⁴⁷ Thus, malathion exposure may cause the degeneration of the spermatogenic and Leydig cells, which disrupts spermatogenesis and reduces sperm counts. In addition, the sperm may also be indirectly damaged by the reactive oxygen species (ROS), as superoxide anion, hydroxyl radical and hydrogen peroxide induced by malathion exposure, which affect the activity of mitochondrial enzymes as well as the structure of the microtubulus in the sperm.⁴⁸ Consequently, ROS may contribute to infertility caused by defective sperm function.⁴⁸

More importantly, we showed that malathion administration caused a significant decrease in both COX isozymes COX-1 and COX-2 when compared to control group. On the other hand, The COX-1 isozyme is expressed by basal cells of epididymal epithelium, whereas COX-2 is detected in principal cells of intact epididymis under conditions not known to be associated with inflammation.⁴⁹ It is well known that COX-1 plays an important role in the regulation of electrolytes and water secretion by the epididymis, while the function of COX-2 remains obscure until now.⁴⁹ Recently, COX-2 has been shown to be the predominant COX isoforms in the epithelium of the distal vas deferens, where the constitutive expression of COX-2 is several times greater than in any other organs.⁵⁰ Evidence is accumulating that COX-2 plays a role in the regulation of physiological processes other than mediating inflammation.⁵¹ The observed decrease of mRNA COX isoforms after malathion exposure might be due in part to the decrease of testosterone secretion.⁵² On other hand an important decrease was shown for GPx-4 mRNA expression in testis and GPx-5 mRNA expression in epididymis after malathion exposure. This decrease can be correlated by the inhibition of enzyme activity after excessive free radical production. However, GPx-5 is strongly expressed under androgenic control in the caput epididymidis.⁵³ Other selenium-containing GPxs (GPx-1, GPx-3, and GPx-4) were found to be expressed by the mammalian epididymis but at much lower levels.^54,55 The GPx-5 gene has long been reported to be a highly tissue-restricted gene expressed in all the mammals that have been tested so far, under androgenic control only within the adult epididymis.⁵⁶ However, GPx-5 could work both in vitro and in vivo, as would be expected for an antioxidant scavenger.^57,58

5. Conclusion

We clearly demonstrated in the present work that malathion exposure of prepubertal male mice during 30 days exerts endocrine disruption and cytotoxic impacts in the reproductive function. These effects are in part due to its prooxidant properties.

Declaration of interest

The authors alone are responsible for the content of this paper.

Abbreviations

MDA	Malondialdehyde
AChE	Acetyl cholinesterase
GPx	Glutathione peroxidase
SOD	Superoxide dismutase
CAT	Catalase
OPs	Organophosphorus

Acknowledgements

Financial support of the Tunisian Ministry of Higher Education and Scientific Research is gratefully acknowledged. Financial disclosures: none declared.

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