Protective Effect of N-Acetyl Cysteine on Chlorpyrifos-Induced Testicular Toxicity in Mice

As reported by the World Health Organization (WHO), unsuccessful pregnancy has been globally increased. Researchers found that 48.5 million couples worldwide were unable to have a child after five years of unprotected regular sexual intercourse (1).

Almost all people working on agricultural fields are exposed to various toxins that may cause reproductive toxicity. Pesticides are widely used for eliminating pests to protect corps and fruits. Organophosphate pesticides are regarded as dangerous types of pesticides for the environment as they can affect humans and animals health (2). Chlorpyrifos (O,O-Diethyl O-3,5,6-trichloropyridin- 2-yl phosphorothioate), is an organophosphate pesticide which can cause adverse effects on the reproductive system of both males and females (3). For instance, seminiferous tubules were significantly degenerated in chlorpyrifos (CPF)-treated mice (4). In addition, sexual hormones disturbance and also defects in sperm production have been reported following CPF exposure (5). CPF was also shown to increase DNA impairment (6, 7) and induce harmful effects in different organs such as the thyroid (8) and lung (9). CPF permanently binds acetylcholinesterase and inhibits deactivation of acetylcholine in the synapses. So, acetylcholine signaling may last longer. This process is irreversible unless new acetylcholinesterase enzymes are synthesized. It has been reported that CPF also induces oxidative stress (8, 10).

Acetylcysteine, also known as N-acetyl cysteine (NAC) is widely used in management of acetaminophen overdose, cystic fibrosis and also chronic obstructive pulmonary disease. NAC may be useful in toxins treatments, since it can escalate glutathione levels and prevent further injuries caused by lipid peroxidation (11). Furthermore, a significant improvement of sperm motility and morphology were observed by NAC treatment in varicocele and also other models induced by synthetic drugs such as paracetamol (12, 13). Moreover, NAC may protect male genital system against strong toxins such as arsenic trioxide in (14). It has been observed that NAC has more marked effects compared to vitamin C in improvement of sperm parameters (15). Therefore, this study was conducted to investigate the protective effect of NAC on histopathology of testis and sperm parameters in CPF-treated mice.

In CPF-exposed mice, a considerable reduction in sperm parameters was found. Meanwhile, NAC could not significantly improve sperm motility, morphology and count. NAC in combination with CPF 5 mg/kg.b.w. considerably prevented further damages to sperm motility and morphology; However, NAC could not improve sperm counts caused by CPF-induced toxicity. In a similar study on CPF reproductive toxicity, a significant decrease in sperm motility and counts was observed by CPF gavage at 20 mg/kg.b.w (18).

In addition, CPF considerably decreased the level of antioxidant enzymes and glutathione in plasma. Nigella sativa oil can act like NAC as a potent protective agent which statistically improved sperm parameters, antioxidant enzymes activity and testosterone level (18). According to our results, adverse effects of CPF is likely irreversible and has negative effects on genitalia systems.

For decades, CPF devastating effects on spermatogenesis process was unclear. Other scientists investigated various pesticides at different doses for their negative effects on spermatogenesis process (19). Organophosphates or any other chemicals, such as different toxins, that have adverse effects on tissues and cells may be inadvertently absorbed through the skin or digestion system. In order to confront these harmful effects, consumption of antioxidant substances like syrup of Malva sylvestris (20) along with hydroalcoholic extract of Fumaria parviflora (21) or products such as propolfenol (22) is highly recommended to protect against damages induced by toxins, particularly against those cause in the genital system. However, some antioxidant materials such as catechin and quercetin did not have significant influences in this regard (9). Nevertheless, natural nutrients such as ginger and cinnamon could be effective on male genital dysfunction due to their anti-oxidant efficacy (23).

Vitamin C and E have been widely used in previous studies and were introduced as beneficial protective materials to compensate damages induced by organophosphates such as malathion, a broad spectrum organophosphate pesticide that could decrease sperm parameters and induce histopathological alteration (24). Although vitamin C and E are potent protective materials against various toxins, a lower dose of intraperitoneal NAC possibly has a more marked impact on sperm parameters based on our findings but use of food or fruits overfilled with these vitamins is suggested for people who are daily exposed to pesticides (25). Another study showed that vitamin C only resulted in a significant improvement of sperm motility (26).

The present study indicates that NAC at 35 mg/kg.b.w somehow significantly increased seminiferous tubules area, diagonal diameter, and spermatogonia, spermatocytes and spermatids counts. Meanwhile CPF 0.5 mg/ kg.b.w could not considerably reduce seminiferous tubules area and diagonal diameter. Furthermore, CPF 5 mg/kg.b.w significantly diminished seminiferous tubules.

Based on these findings, NAC in combination with CPF ameliorates the pesticide’s adverse effects on testis. It seems that intraperitoneal injection of NAC, even at a low dose has a more marked effect on sperm parameters than gavage administration (15). It has been proven that NAC can affect lipid peroxidation (LPO) (27). Therefore, NAC might decrease ROS elevation caused by CPF. However, in the present study, NAC exact effects on sexual hormones or anti-oxidant enzymes such as superoxide dismutase, catalase, or glutathione in treated groups, were not evaluated. But considering significant reductions in testis germinal cells, oxidative stress level was probably elevated by CPF and NAC ameliorated the adverse effect of CPF on the testis.

According to our results, seminiferous tubules area and diagonal diameters were not affected by CPF. It suggests that resting times at the end of each week and also seven days after the last injection of CPF might provide a chance for the immune system to recover and regenerate genital and possibly other tissues. Therefore we did not expect NAC to protect these two unaffected variables. Meanwhile, we assume that CPF at the dose of 0.5 mg/kg.b.w could not significantly diminish seminiferous tubules area following four-week administration. Maybe by longer treatment periods, CPF could induce more destructive effects at the dose of 0.5 mg/kg.b.w It is clear that NAC is able to confront negative effects of CPF toxicity in male genital system but what if we could use NAC at doses higher than 35 mg/kg.b.w? In this case, we probably observe NAC protective effects against CPF typical tissue toxicity. Further in vivo studies using intraperitoneal injections, are highly recommended to affirm our data.

Both low and high doses of CPF can decrease sperm parameters. Also, this pesticide at 5 mg/kg.b.w dose significantly diminishes the length and diagonal diameter of seminiferous tubules. NAC significantly improved CPF adverse effects on sperm parameters and spermatogenesis cells except spermatogonia. However, this antioxidant could not statistically ameliorate the histopathological alterations of seminiferous area induced by CPF.