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 (
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
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 (
Chlorpyrifos (99%) and NAC (99%) technical grade were purchased from Sigma-Aldrich (St. Louis, MO, USA) [lot No. LC13116V and 616-91-1, respectively]. Also dimethyl sulfoxide (DMSO) was provided from Sigma too (St. Louis, MO, USA) [Lot No. 67-68-5].
Forty-two healthy adult BALB/c mice (6-8 weeks old) were obtained from the Animal Research Unit, Babol Medical University, Babol. Animal care and handling was done based on Animal Research Unit and following approval of Ethics Committee (MUBABOL.HRI. REC.1395.73). The animals were habituated to laboratory conditions for 1 week before initiation of the experiment. Mice were maintained on 12 hours light-dark cycle at 21-24°C with 50-60% humidity. Mice had free access to normal diet and water, ad libitum. The animals were divided into seven groups: group I (control group) received normal saline, group II (sham group) received DMSO (0.75% solution), group III received NAC 35 mg/kg.b.w, group IV (high CPF) received CPF 5 mg/kg. b.w, group V (low CPF) received CPF 0.5 mg/kg.b.w, and group VI and VII received CPF at low (0.5 mg/kg.b.w) and high (5 mg/ kg.b.w) doses, respectively along with NAC on a daily basis. In groups VI and VII, NAC was given intraperitoneally from five days before the experimental timeline, in order to acclimate mice with this antioxidant. All groups were treated intraperitoneally except the control group. Treatment was conducted for 4 weeks and injections in all groups were administrated on five consecutive days each week. One week after the last injection, mice were sacrificed using anesthetics to evaluate sperm parameters and testis histopathological alterations.
Here, 15 µL DMSO was added to 1985 µL distilled water to prepare 2 ml DMSO solution to be administered to the sham group. Also, 1 mL DMSO was added to CPF powder vial (1 mg) in order to prepare CPF stoke solution (1mg/1mL). Afterward, 15 µL CPF was added to 135 µL distilled water and after pipetting, the whole solution was added to 1850 µL distilled water to prepare 2 ml High CPF (5 mg/kg.b.w) solution. Eventually, 200 µL of high CPF solution was added to 1800 µL distilled water to prepare low CPF (0.5 mg/kg.b.w) solution. NAC was dissolved in water at 35 mg/kg.b.w. It should be noted that fresh CPF solutions were daily prepared.
Seven days after the last day of treatment, mice were
anaesthetized via an inhalation induction chamber and
sacrificed. Right testis of each animal was excised and
put in 10% formalin solution for histopathological evaluations.
Afterward, the caudal of left epididymis of each
animal was excised and put in petri dish containing 3 mL
Ham’s F10 (St. Louis, MO, USA) [Lot No. 87120401].
According to diffusion method (
From semen samples prepared by diffusion method,
almost 50 µL semen from each mouse was smeared by
a pipette on a slide. Afterwards, maximum 100 sperms
were observed on right upper quarter of each slide to
examine sperm count, motility and morphology. Sperm
normality percentage for each mouse was easily calculated
using a counter by knowing about mice sperm abnormalities
Testis specimens were kept in 10% neutral buffered
formalin. For testis histopathological evaluations, 5 µm
sections were prepared from each testis, stained with
haematoxylin and eosin (H&E) and observed under a
light microscope. Images were captured by Olympus
optical microscope equipped with a Canon HD camera
at magnifications ×4, ×10 and also ×40 at four random
points. Afterwards, data were evaluated on a proper personal
computer using Motic software instruction (
Data were presented as mean ± standard error (SE). Statistical analysis was performed in SPSS (version 22, SPSS Inc., Chicago, IL) using one-way analysis of variance (ANOVA) followed by Tukey as the post hoc test.
Effect of NAC on sperm parameters in CPF-induced mice
|Control||DMSO||NAC||Low CPF||High CPF||Low CPF+NAC||High CPF+NAC|
|Sperm motility (%)||75.83 ± 0.83||71.66 ± 1.66||75.83 ± 0.83||45 ± 2.23a||33.33 ± 4.77a||75 ± 0.1b||57 ± 4.03b|
|Sperm normality (%)||66 ± 2.73||62.50 ± .17||63.66 ± 1.02||54 ± 1.98a||30.66 ± 0.7a||59.16 ± 1.60b||62.83 ± 1.85b|
|Sperm count (sperm cell concentration/ml)||87.5 ± 3.09||90 ± 4.47||93.33 ± 4.21||55 ± 2.23a||65.83 ± 5.23a||82.5 ± 3.59b||70 ± 2.58|
The data are presented as mean ± SE (n=6). Sperm count is expressed as number×106 per caudal epididymis. a; Indicates a significant difference as compared to control group (P<0.05), b; Indicates a significant difference as compared to CPF group (P<0.05), NAC; N-Acetyl Cysteine, CPF; Chlorpyrifos, and DMSO; Dimethyl sulfoxide,
Male mice that received CPF (0.5 and 5 mg/kg.b.w/day) for 35 days showed signs of toxicity such as salivation, diarrhea and tremor. No death was recorded throughout the study period.
According to our data, no significant differences were
found in sperm characteristics between DMSO and control
Average count of spermatogonia, spermatocytes
and spermatid in DMSO group slightly decreased but
it was not significant; however, a significant increase
was observed in NAC group (P<0.001,
Based on data given in Figures 2 and 3, there was no significant increase in mean seminiferous tubules area and diameter in DMSO group compared to control (P>0.05); but, NAC showed a significant increase in both variables (P<0.001). While high CPF treatment significantly diminished seminiferous tubules, low CPF treatment (0.05 mg/kg.b.w) caused no considerably damage in seminiferous tubules shape. NAC could not ameliorate the effects caused by high CPF (P>0.05).
Bars presents mean ± SE of spermatogonia, spermatocytes and spermatid cells counts in different groups. *; Indicates a significant difference in spermatogonia counts as compared to control group (P<0.05), **; Indicates a significant difference in spermatocytes counts as compared to control group (P<0.05), ***; Indicates a significant difference in spermatids counts as compared to control group (P<0.05), NAC; N-Acetyl Cysteine, CPF; Chlorpyrifos, and DMSO; Dimethyl sulfoxide.
Bars presents mean ± SE of seminiferous tubules area in different groups. *; Indicates a significant difference as compared to control group (P<0.05), NAC; N-Acetyl Cysteine, CPF; Chlorpyrifos, and DMSO; Dimethyl sulfoxide,
Bars presents mean ± SE of seminiferous tubules diagonal length in different groups. *; Indicates a significant difference as compared to control group (P<0.05), NAC; N-Acetyl Cysteine, CPF; Chlorpyrifos, and DMSO; Dimethyl sulfoxide.
Histopathological difference is shown between experimental groups. It is demonstrated a massive destruction in CPF groups.
However NAC considerably improved histopathology of testis.
Based on data given in Figure 4, there was no significant increase in mean seminiferous tubules area and diameter in DMSO group compared to control (P>0.05); but, NAC showed a significant increase in both variables (P<0.001). While high CPF treatment significantly diminished seminiferous tubules, low CPF treatment (0.05 mg/kg.b.w) caused no considerably damage in seminiferous tubules shape. NAC could not ameliorate the effects caused by high CPF (P>0.05).
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 (
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 (
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 (
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
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 (
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.