Malathion induced testicular toxicity and oxidative damage in male mice: the protective effect of curcumin

The widespread use of the organophosphorus (OP) pesticide (malathion) in agricultural and household applications and the high rates of food contamination can lead to exposure of humans to high levels of this toxic chemical (Babu et al. 2006; dos Santos et al. 2011; Salama et al. 2015; Ibrahim et al. 2011).

Exposure to malathion [O, O-dimethyl-S-(1,2-dicarbethoxyethyl) phosphorodithioate] causes distinct cytopathological and histopathological changes in the male mammal’s reproductive system. Uzunhisarcikli et al. reported that the duration and concentration of exposure to malathion have the major role of these changes that include higher percentage of sperm abnormality, defects in spermatogenesis, and lowered sperm count (Mahgoub and El-Medany 2001; Bustos-Obregón and González-Hormazabal 2003; Uzunhisarcikli et al. 2007). Geng et al. found in addition marked loss of the testis weights, final body weight, and sperm mobility (Geng et al. 2015).

The ability of organophosphorus compounds including malathion to cross the blood–testis barrier accounts for the damage that affect the testis (Nahid et al. 2016). The testicular biological membranes are damaged by malathion through oxidative stress mechanism and lipid peroxidation induction (Geng et al. 2015).

Leydig and spermatogenic cell degeneration might occur in this condition as a result of the oxidative damage, which decreases the sperm counts and deactivates spermatogenesis (Uzunhisarcikli et al. 2007; Penna-Videau et al. 2012). Penna-Videau and his colleagues reported that steroidogenesis is impaired by malathion exposure and this in turn stimulates the proliferation of the seminiferous epithelium beside the apoptosis that affect germ cells (Penna-Videau et al. 2012). Moreover, reduced chromatin and damaged DNA can be caused by malathion exposure that affect the maturation of spermatogenic cells in mice in its late stages (Ojha and Srivastava 2014; Moore et al. 2011). The structure of the microtubules and mitochondrial enzymes of the sperm could be affected and injured by the oxidative damage induced by OPs that affects and destroys the sperm. Latchoumycandane et al. reported previously that the functions of the sperm and their mobility are reduced, causing infertility because of reactive oxygen species generation (Latchoumycandane et al. 2002).

Previous researchers have identified the mechanism of testicular damage induced by malathion exposure in rats and humans: first, because of the lipophilic feature of malathion which in turn facilitates its prompt tissue diffusion. This diffusion causes accumulation of acetylcholine in the target organ causing all the manifestations of OP poisoning (Bajgar 2005). Induction of the oxidative stress damage with the generation of reactive oxygen species (ROS) and the loss of balance between the oxidant/antioxidant systems are another way of organ damage by malathion (Bayrami et al. 2012).

Oxidative stress might be relieved by using antioxidant therapy. Recently, there is a major inclination toward the utilization of natural products as antioxidant agents. This pattern requires the assessment of the pharmaceutical properties of these natural products (Sepand et al. 2013; Kim et al. 2018; Huang et al. 2018; Ibrahim et al. 2018). Curcumin (diferuloylmethane), a polyphenol, is the active component of the condiment turmeric (Curcuma longa) and has been used for treatment purposes for hundreds of decades (Gupta et al. 2012; Joint 2003; Bulku et al. 2012). It acts as a free radical scavenger and antioxidant (Ak and Gülçin 2008) inhibiting lipid peroxidation and oxidative DNA damage and enhances the activities of antioxidant enzymes such as glutathione-S-transferase (Piper et al. 1998). Its free radical scavenging antioxidant property helps it in reducing genotoxic damage caused by organophosphorus exposure (Kumar et al. 2014).

Curcumin has been used in clinical trials as anticancer agents with its activity resulting from multiple biochemical mechanisms. Studies have shown that this compound can inhibit the malignant proliferation of tumor cells and enhance chemosensitivity of liver cancer, gastric cancer, and colon cancer (Kunati et al. 2018; Gall Troselj and Kujundzic 2014).

The protective effect of curcumin has been investigated against the effect of many OP compounds on several organs (Hassani et al. 2015). This study was designed to evaluate the protective activity of curcumin on malathion-induced testicular toxicity in male mice by assessment of histopathological changes of the testis, oxidative markers, plasma testosterone level, and plasma acetylcholinesterase activity.

No animal mortalities were found throughout the experimental period.

Effect of malathion and curcumin on the histological structure of the testis

Sections examined from cases of control group (Fig. 1a, b) and group treated with curcumin alone (Fig. 1c, d) revealed normal testicular tissue with seminiferous tubules showing full maturation to spermatids and mature spermatozoa in the lumen. Sections from the group treated with malathion (Fig. 2a–c) showed hypospermatogenesis, maturation arrest at stages of primary and secondary spermatocytes focally in 100% of cases, foci of germ cell aplasia (Sertoli cell only) in about 40% of cases, sloughing and disorganization, foci of intratubular necrosis and inflammatory infiltrate, interstitial edema and Leydig cell hyperplasia. Sections from the group treated by both malathion and curcumin (Fig. 3a, b) showed normal spermatogenesis in 50% of cases, mixed hypospermatogenesis and maturation arrest in 50% of cases, no germ cell aplasia, sloughing and disorganization (50%), no necrosis or inflammatory infiltrate, mild interstitial edema in 25% of cases, and no Leydig cell hyperplasia.

Immunohistochemistry

Bcl-2 was expressed in the testis of mice from all groups but with different intensity and distribution. In the control, curcumin, and malathion + curcumin groups, there was moderate cytoplasmic Bcl-2 expression. Expression was prominent mainly in spermatocytes and spermatids. Leydig cells also showed some immunoreactivity. Bcl-2 expression was weak in the malathion-treated group. There was no expression in spermatogonia (Fig. 4). As regards percent area of expression of Bcl-2 immunoreaction of the testis, a statistically significant difference was observed between malathion-treated group and both control and curcumin groups (p < 0.05). There was a statistically significant difference in the expression of Bcl-2 immunoreaction in the malathion + curcumin group compared with the malathion group (p < 0.05) as presented in Table 4.

Parameters	G1 (control)	G2 (curcumin)	G3 (malathion)	G4 (malathion + curcumin)
Bcl-2	45.148 ± 3.02	42.366 ± 5.55	17.259 ± 2.67 ab	43.714 ± 2.83 c
Caspase3	34.416 ± 3.79	35.620 ± 3.65	34.914 ± 2.03	34.485 ± 3.72

Data are mean ± SD

G1 control, G2 curcumin, G3 malathion, G4 malathion + curcumin

^aCompared to the control (G1) group—significant at p < 0.05

^bCompared to curcumin (G2) group—significant at p < 0.05

^cCompared to malathion (G3) group—significant at p < 0.05

Caspase3 was moderately expressed in the testis of mice from all groups. The expression was mainly cytoplasmic. It presents in all cells from spermatogonia to spermatids. There was no difference in expression among groups (Fig. 5). As regards percent area of expression of Caspase3 immunoreaction of the testis, no statistically significant difference was observed among groups (p > 0.05) as presented in Table 4.

Insecticides have been widely used for decades, and human exposure to these insecticides always causes public health concern. Malathion is an organophosphate insecticide widely used in agriculture, residential areas, public recreation areas, and pest control programs, and it is also used for the treatment of the head and body lice (Stone et al. 2014). The compounds of organophosphorus are broadly utilized as acaricides and insecticides in agriculture. They are likewise often utilized in drug and industry. Residues of organophosphate (OP) insecticides have been recognized in the food products, grains, vegetables, water bodies, and soil (Poet et al. 2004). In addition, because of the extensive accessibility of organophosphorus pesticides, toxicity is widespread (Garcia et al. 2003). Previous researches have reported that the male reproductive system have been affected by some OP insecticides (Farag et al. 2000). Malathion is an organophosphorus insecticide that affects the reproductive system. Humans, birds, and other animals are in contact to increased levels of this insecticide (malathion), due to its common utilization and the rising rates of food contamination (Babu et al. 2006).

In this study, we introduced malathion at a dose of 1/50 of the oral LD50 and observed the pathological changes in mice testes. None of the mice died during the interval of experiment.

Malathion caused changes in the mouse testis, notably by a decrease in their weight. The same finding was demonstrated in a research done by Geng et al. and Slimen et al. (2014; 2015). The reported data presented by Lafuente et al. (2008) dedicated that oral exposure to malathion caused adverse effects on the reproductive system of male mice following intake of 27 mg/kg per day for 1 month, including lowered weight of the body and testes.

LH and FSH activities depend on both the quantity of these hormones and availability of their specific receptors in the testis. It was clarified that there is an adverse effect on testicular function on exposure to environmental pollutants mediated by lowering LH secretion by pituitary and steroidogenesis by Leydig cell (Geng et al. 2015; Magnusson and Ljungvall 2014). By the end of the month of this study, the levels of LH and testosterone in the group treated by malathion were significantly lower than their levels in mice in the group taken as control and the FSH is higher in the malathion-treated group. Thus, malathion administration suppressed LH and testosterone release and increased the level of FSH. This is in line with the finding of Mahgoub and El-Medany (2001) who found elevated FSH in the insecticide-treated group. However, decreased levels of LH, FSH, and testosterone have been reported by other authors (Maitra and Mitra 2008; Elbetieha and Da’as 2003). This may be made clear by the supposed antagonism of malathion to androgen receptor. It is known that antagonists to androgen receptor can change the glycosylation of gonadotrophins, and this leads to the inhibition of their levels (Sikka and Naz 1999). Some researchers considered a change in the mechanism of production of steroid hormones. It is probable that production of gonadotropins was affected by malathion in male mice due to the interruption of the hypothalamic–pituitary–testicular axis (Roohbakhsh et al. 2011).

Gonadotrophins and testosterone are key hormones, which regulate spermatogenesis. LH is secreted by the pituitary gland then induces Leydig cells to secrete testosterone. Many OPs were tested for their effect on plasma levels of testosterone (Blanco-Muñoz et al. 2010; Satar et al. 2004). In our experiment, introducing malathion for 4 weeks was associated with a reduction in plasma levels of testosterone. There may be pathological alterations in the interstitial cells of Leydig, which may be the reason for that effect. However, levels of plasma testosterone showed no significant effect under organophosphorus insecticide exposure as mentioned by Okamura et al. (2005). Some researchers described the adverse effect of malathion on the fertility through its reduction effect on levels of plasma testosterone (Contreras and Bustos-Obregón 1999). Studies have shown that many organophosphate pesticides including malathion can interfere with hormone levels through activating estrogen, androgen, and other hormone receptors (McKinlay et al. 2008; Mnif et al. 2011).

The current study depicted the low level of acetylcholinesterase in malathion-treated group. These results are similar to the previous researchers who stated that pseudocholinesterase and acetylcholinesterase activity have been inhibited by the organophosphorus pesticides in the body tissues (John et al. 2001; Kalender et al. 2006; Rubin et al. 2002). These changes depend on concentration and intensify with longer exposure. The effects of malathion and other OPs may be at least partially because of they are able to cross the blood–testis barrier and enhance oxidative stress and peroxidation of lipids causing damage to the testes (Uzunhisarcikli et al. 2007). Malathion does not directly inhibit action of the acetylcholinesterase before it is biotransformed. The toxicity to mammals of organophosphorus pesticides as malathion depends on mixed function oxidase (MFO) induced activation to its analogous oxygen correspondent, which directly inhibit acetylcholinesterase (Pope 1999). Therefore, biological effects of malathion can be due to electrophilic attack on the cells with concomitant production of reactive oxygen species (Johnson et al. 2000). Therefore, the harmful effects of malathion on reproductive system of male mice may be due to its electrophilic attack on their cells.

Erythrocyte membranes damage through a mechanism of oxidative stress had been reported in previous researches after malathion administration orally for more than 6 weeks at a dose of 0.13 mg/kg (John et al. 2001). In addition, alterations in CAT and SOD activity in the different regions of the brain and oxidative damage had been induced by malathion exposure (Fortunato et al. 2006a, b). Our results clearly verified other researches, which told that giving organophosphorus insecticides to male mice changed the activity of antioxidant enzymes in tissues of reproductive organs in the form of decreased GSH level with suppressed SOD, CAT, and GPx activity.

A major underlying mechanism of OP toxicity is oxidative stress that results from the generation of many free radicals, and correspondent depletion of antioxidant enzymes. The results are oxidation of DNA, protein, and lipids (Koltuksuz et al. 2000). Moreover, OPs themselves can directly attack cellular membranes and form lipid peroxidation products or enter the cytoplasm and generate free oxygen radicals. The result is increased oxidative stress, leading to a more depletion of antioxidant enzymes in the cell and disruption of the oxidant–antioxidant scale favoring the oxidant limb (Pearson and Patel 2016).

Insecticides can cause variable histopathological and cytopathological lesions in the testes of male mice (Mahgoub and El-Medany 2001; Uzunhisarcikli et al. 2007). These changes include defective spermatogenesis (Farag et al. 2000; Khan et al. 2001; Uzunhisarcikli et al. 2007).

The organophosphorus insecticides including malathion may lead to degeneration of the germ and Leydig cells. This disrupts spermatogenesis and reduces sperm numbers (Uzunhisarcikli et al. 2007). This coincides with our results that showed marked hypospermatogenesis and even germ cell aplasia in the group treated by malathion.

Uzun et al. (2009) declared malathion-caused necrosis in the seminiferous tubules and edema in interstitial tissue. This agrees with our results. The same researchers also mentioned that destruction of DNA, proteins, and lipids through a mechanism of oxidative stress had been promoted by the deleterious effects of OPs (malathion) within the testis. This same happened when curcumin was used in our experiment to reduce the oxidative injuries. Oldereid et al. (2001) stated also that the spermatogenic inhibition induced by cisplatin may be due to the formation of free radicals in the testicular tissue. Spermatogenesis improved among curcumin-treated mice. This may be associated with the antioxidant and free radical scavenger properties of the curcumin. This coincides with our hypothesis about curcumin.

Oldereid et al. (2001) had reported that all male germ cell levels in humans may undergo spontaneous apoptosis. Bcl-2 family proteins have been suggested to play a particular role in the process of maturation and differentiation occurs in spermatogenesis of human, as they have been found to be distributed within distinguished germ cell levels. In the current study, expression was more prominent in spermatocytes and spermatids. However, Tanaka et al. (2002) showed that Bcl-2, BAX, caspase1, and caspase3 are expressed in germ cells of the human testes, especially in basal cells such as spermatogonia, with expression only rarely in spermatids.

Previous studies have reported that utilization of caspase3 apoptosis played a vital role in the process of human testicular spermatogenesis, and the increase in apoptosis has been mentioned to happen in the process of hypospermatogenesis, maturation arrest, and syndrome of Sertoli cell only (Almeida et al. 2013).

Geng et al. (2015) concluded that malathion effects on the testicular spermatogenic function of male rats and its working mechanism may involve cell apoptosis induced by the downregulation of Bcl-2 and upregulation of Bax. This coincides with our results that showed weak Bcl-2 in the group that was exposed to malathion. This was mentioned previously by Khorsandi et al. (2013) in their study.

Our research clarified that curcumin administration minimized malathion-induced testicular toxicity. Therefore, we can assume that curcumin could protect against oxidative stress caused by free radical. It does so by scavenging free radicals and minimizing lipid peroxidation and decreasing antioxidant consumption. Majority of antioxidants have either a functional phenolic or a β-diketone group. Curcumin has several functional groups, including the β-diketone group, carbon–carbon double bonds, and phenyl rings having variable content of methoxy and hydroxyl substituents (Priyadarsini et al. 2003; Wright 2002). However, curcumin was said to be incapable to prevent MDA production (Reyes-Gordillo et al. 2007). After absorption from the intestine, curcumin is bio-transformed to dihydrocurcumin, tetrahydrocurcumin, and hexahydrocurcumin. These compounds are conjugated to glucuronide, and then, they are more polar and better absorbed than curcumin. Therefore, the pharmacological actions of curcumin are largely induced by curcumin’s hydro soluble metabolites (Maheshwari et al. 2006). The methoxy and phenolic groups on benzene rings of curcumin are vital structures which contribute to antioxidant capabilities of curcumin (Samuhasaneeto et al. 2009).

Curcumin has been popular in the past 20 years due to its multiple biological and pharmacological effects, which can modify multiple cell-signaling pathways. Its action is mediated by direct or indirect interaction with multiple molecular targets (Aggarwal and Sung 2009). Its metabolites, curcumin glucuronide (COG) and curcumin sulfate (COS), may retain some of the pharmacological activities of curcumin and responsible for its biological effects (Prasad et al. 2014).

Our finding that giving curcumin to malathion-exposed mice mollified the toxic effects of malathion on the testis supports the idea that malathion causes their harmful effects by causing destructive oxidation of lipids, proteins, and DNA of testicular tissue.

This study showed that curcumin ameliorates malathion-induced testicular damage. The curcumin effect is mediated through the enhancement of antioxidant enzymes and suppression of oxidative stress markers. Other in vitro studies are needed to define the pathways involved in curcumin actions while competing malathion-induced testicular damage.