INTRODUCTION

The general population is exposed to metals at low concentrations voluntarily (food supplements) or involuntarily through ingesting contaminated food or water and contact with contaminated soil, dust and air¹. Lead is a contaminant widely distributed among heavy metals in all environmental and biological systems². The detection and prevention of lead toxicity is an important public health problem³. Although the population's exposure to lead has decreased in the last three decades with the introduction of low-lead paints, lead-free petroleum, and the ban on lead solder in food cans⁴, subclinical exposure remains in other sources such as lead pipes, toys, insecticides, oil refineries, construction material, firearm bullets, x-rays, and industrial by-products³. We must add the high exposure of the populations that live near the mines, tailings, and deposits of this metal, such as in Oroya and Callao, where high levels of lead in the blood of the inhabitants have been reported, especially children⁵and even in newborns⁶, so its potential risk to human health is still a subject of much debate.

Lead toxicity has been related to different pathologies in humans⁷, and its relationship with the observed damage to reproductive function in humans and rodents^8,9is suggested. Most reports indicate that workers exposed to pb+2 exhibit decreased sperm density and a high rate of teratozoospermia¹⁰. Lead occurs in nature as an oxide or salt; thus, lead acetate (AP) has been shown to produce infertility in mice^11,12; evaluation from the first to fifth-week post-injection of ap at a single dose of 100 mg/kg body weight (BW), increased oxidative stress and sperm morphological abnormalities, while decreased tail epididymal sperm count¹³.

In turn, lead nitrate (PB(NO3)2) (NP) has a multitude of uses and is a product of economic importance¹⁴, it is embryotoxic in rats¹⁵but not teratogenic¹⁶and its genotoxic effects are still highly controversial^17,18. It has been reported that 44.8 mg/kg/pc of NP induces DNA damage in blood cells even after seven days of exposure¹⁴. The mechanism by which lead exerts toxic effects on the reproductive system has not yet been fully elucidated. Still, it has been reported to induce oxidative stress in different tissues with the consequent generation of reactive oxygen species (ROS)^13,19. Damage from ROS leads to infertility by two mechanisms: damage to the sperm membrane that decreases sperm motility and its ability to fuse with the oocyte, and damage to sperm DNA that alters the paternal genomic contribution to the embryo. Sperm DNA fragmentation has been related to many reproductive aspects²⁰and is predictive of fertilization and pregnancy rates²¹demonstrating its evaluation's importance. Currently, there are no reports on the effect of NP on the integrity of sperm DNA. Considering that exposure to lead is a serious public health problem, this work attempts to clarify whether a single dose of NP (50mg/kg/pc ) affects male reproductive characteristics and sperm DNA integrity assessed seven days post-injection.

METHODS

RESULTS

Significant differences were observed between the final and initial body weights during the experiment, being lower and negative in the NP group (p < 0.05) (table 1). The consequences of the reproductive organs: testicles, epididymis, prostate, seminal vesicle (table 1), physiological values of mobility (figure 1 ) and sperm concentration (figure 2).

Figure 1: Sperm motility in the treatment group PB(NO3)2 (50mg/kg/pc) vs control (H2O). 

Figure 2: Sperm concentration in the treatment group PB(NO3)2 (50mg/kg/pc) vs control (H2O). 

Figure 3: Sperm DNA fragmentation in the PB(NO3)2 (50mg/kg/pc) vs control (H2O). 

The sperm count evaluated with tunel between both groups was not altered after treatment with NP (p>0.05) (figura 3); the positive control in this test was 99%, and the negative control 0%. The DNA fragmentations did not show significant differences (p > 0.05).

DISCUSSION

The present work investigates the effects of a single dose of NP in mouse spermatozoa. We must note that a systemic toxic effect of NP was evidenced by decreasing the body weight of the treated mice (table 1, p < 0.05). However, the weights of the testicles, epididymis, prostate, and seminal vesicle, which are androgen-dependent target organs²⁵, did not decrease, so NP would not be exerting a toxic or anti-androgenic effect on the male reproductive system(table 1, p > 0,05).

NP would not have a localized effect on sperm physiology because no significant differences were observed in sperm motility and concentration between the two groups (figures 1andfigure 2, p > 0.05); sperm transit and maturation in the epididymis would not be affected by NP. In this research, within the spermatogenic cycle of the mouse, we evaluated the spermatozoa deposited in the epididymis²², these spermatozoa have already exchanged their histones for protamines, which allows them to strongly condense the paternal genome within the nucleus²⁶. While the histones of a somatic cell compact the DNA forming nucleosomes, protamines, proteins unique to spermatozoa, wind and condense DNA into much larger subunits known as toroids²⁶. In this way, sperm chromatin experiences strong compaction, 6 times more than in a somatic cell²⁷protecting the integrity of sperm DNA^25,27,28. However, the multiple cysteine residues that surround the spermatic nucleus are oxidized to form disulfide bridges between protamines and stabilize chromatin during the final stages of sperm maturation²⁸, achieving complete protection of sperm DNA only in the epididymis itself. Devi¹⁴ observed DNA damage in blood cells in mice evaluated seven days after administration of a dose of NP (44.8 mg/kg/pc), a similar concentration used in this work (50 mg/kg/pc). Therefore, we expected that NP causes damage to the DNA of the spermatozoa; however, our results show the opposite (figure 3, p > 0,05), which demonstrates the importance of protamination as a sperm protection mechanism against PN²⁹.

At the same time, we believe that the epididymis, having many antioxidant enzymes³⁰, could adequately counteract the well-documented effect of lead on the induction of ROS in different tissues³¹. Furthermore, it has been shown that the effects of lead on the reproductive system depend on the exposure time, returning to normal values after a prolonged exposure time³², which suggests that an animal exposed to this metal is capable of adapting to its toxic effects; in the present work, by administering a single dose of NP, we ruled out the possibility of not observing damage to the reproductive system, sperm physiology, and sperm DNA integrity due to a prolonged exposure time to NP.

As limitations of the study we had the logistical nature in the institution where the work was carried out; mainly due to power cuts during the weekends that harmed the dosage and the continuity of the monitoring of the animals. In addition, the impossibility of entering the investigation pavilion on sundays due to lack of surveillance personnel. All this caused delays and repetition of the experimental design.

CONCLUSIONS

We conclude that a single dose of lead nitrate 50mg/kg/pc does not alter reproductive parameters or damage sperm DNA in mice.