Reproductive effects of subchronic exposure to acetamiprid in male rats

Acetamiprid, a selective agonist of nicotinic acetylcholine recetors, is one of the most widely used neonicotinoids. There is limited data about toxicity of acetamiprid on male reproductive system. Therefore, the study aimed to investigate the reproductive toxic potential of acetamiprid in male rats orally treated with acetamiprid with low (12.5 mg/kg) medium (25 mg/kg) or high dose (35 mg/kg) for 90 days. According to our results, sperm concentration and plasma testosterone levels decreased in dose dependent manner. Gonadotropin-releasing hormone (GnRH), follicle-stimulating hormeone (FSH), luteinizing hormone (LH) levels increased at low and medium dose groups and acetamiprid caused lipid peroxidation and glutathione (GSH) depletion in the testes. Histologic examinations revealed that acetamiprid induced apoptosis in medium and high dose groups and proliferation index dramatically decreased in high dose group. In conclusion, acetamiprid caused toxicity on male reproductive system in the high dose. The mechanism of the toxic effect may be associated with oxidative stress, hormonal disruptions and apoptosis.

medium-and high-dose groups, respectively, compared to the beginning of the experiment. Similarly, there was no significant change in the testicular weights (p > 0.05) as compared to the control group ( Table 1).

Effect of acetamiprid on sperm count and morphology. Sperm count and morphology evaluations
were performed according to the World Health Organization (WHO) guideline 15 . The number of sperm was significantly decreased in the medium-and high-dose groups (p ≤ 0.004) as compared to the control group (Table 2). However normal sperm count and abnormal sperm count did not show any significant difference among the treatment groups. In the abnormal sperm morphology evaluations, significantly increase in the flattened headed sperm was observed in the high dose treatment group (p = 0.046).
Effect of acetamiprid on hormone levels and oxidative stress parameters. The plasma testosterone (≥ 23%) and cholesterol (≥ 12%) levels decreased in a dose dependent manner. The decrement of cholesterol level was found statistically significant in medium and high dose groups while the testosterone concentration decreased nonsignificantly. The plasma luteinizing hormone (LH), gonadotropin-releasing hormone (GnRH) and inhibin B (INHB) hormone levels increased significantly in the low and medium dose groups (≥ 2%, p < 0.05) as compared to the control group. The follicle-stimulating hormone (FSH) level also increased significantly in all the treatment groups (≥ 1.57 fold, p < 0.0005). However, it was observed that the plasma INHB levels declined 11% at the high dose of acetamiprid group.
The antioxidative parameters (Reduced glutathione (GSH) and total antioxidant status (TAS)) was found to decrease significantly in plasma and testes tissues in a dose-dependent manner (p < 0.05). Additionally, the oxidative parameters (Malondialdehyde (MDA) and Total oxidant status (TOS) of plasma and testes tissue levels   Table 2. Evaluations of sperm morphology and counts in the Sprague Dawley rats treated orally with acetamiprid. Data were shown as mean ± standard error of the means (SEM). *p < 0.05, **p < 0.005, ***p < 0.0005.

Effects of acetamiprid on histopathology of testes.
In the control group, normal testicular morphology with regular spermatogenic cells and seminiferous tubules with regular basement membrane was observed.
In the low dose group (score 7.6) vacuole formation was observed in the germinal epithelium of some tubules. Regular basement membrane was observed in this group. In the medium-dose group (score 7.9), immature cells were observed in many tubule lumens. Decrease number of spermatogenic germ cells, damaged spermatogenic cells with vacuole formation and luminal immature cell rashes, were observed in a large number of seminiferous tubules in the high-dose group (score 8.1). Irregular and undulating basement membrane was observed in medium-dose and high-dose groups (Figs. 1 and 2). Seminiferous tubule score was decreased significantly all the treatment groups (p ≤ 0.01) as compared to the control group (Table 4). Proliferative cell nucleus antigen (PCNA)-positive cells were observed as dark brown in the seminiferous tubules of all groups. were observed (Fig. 3). Proliferation index was significantly decreased in all the treatment groups (p ≤ 0.01) as compared to the control group (Table 4).
Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL)-positive cells in the seminiferous tubules of all groups were observed as dark brown (Fig. 4). The apoptotic index was significantly increased in the medium (p < 0.1), and high-dose groups (p ≤ 0.01) compared to the control group (Table 4).

Discussion
To identify the toxic effects of acetamiprid which is one of the widely used neonicotinoid insecticide on the reproductive system, sperm morphology, oxidative stress parameters, apoptosis, changes in the body and testicular weights were evaluated. In the present study, body weight decreased minimally in all acetamiprid-treated groups at the last day of experiment comparing to the beginning of the experiment except for control group and this may be associated with acetamiprid treatment and there was no significant change in testicular weights among the treatment groups. Zhang et al. 7 also reported acetamiprid reduced body, and testis weights, in Kunming male mice treated with 30 mg/kg acetamiprid orally during 35 days. Similarly, Devan et al. 8 observed a decrease in testicular absolute organ weight, and an increase in absolute organ weights, which of these changes are statistically non-significant in Wistar rats treated with orally acetamiprid at doses of 27.5, 55 and 110 mg/kg body weight. In the same study, NOAEL value was found to be ≤ 55 mg/kg.
It has been demonstrated that acetamiprid caused to change in serum testosterone levels and seminiferous tubule diameter and thickness in albino mice exposed to 0.16 mg/ml, and 0.22 mg/ml, by adding acetamiprid to drinking water for four weeks 16 . According to a study which was conducted by Rasgele 17 , acetamiprid did not increase the sperm abnormalities at the doses of 0.62, 1.25, and 2.50 μg/mL after 24 and 48 hours on in Mus musculus mice. A study conducted by Kong et al. 18 demonstrated that acetamiprid increases LH levels and decreases testosterone levels and sperm count in Sprague Dawley rat treated with 10 mg/kg and 30 mg/kg dose. Zhang et al. 7 also showed 30 mg/kg acetamiprid treatment decreased intact acrosome rate of sperms and testosterone level in Kunming male mice. In a study conducted by Mosbah et al. 19 , acetamiprid has been reported to increase in body weight and decline in testis weights, sperm count, plasma testosterone level, sperm motility in Wistar rats dosed by 27 mg/kg acetamiprid by gavage (  we also found that acetamiprid induced sperm abnormalities and decreases sperm concentration and plasma testosterone levels in a dose dependent manner. Since testosterone which is main circulating androgen is synthesized from cholesterol 20 , the decrease of the plasma testosterone may result from decrease of plasma cholesterol level Eacker et al. 21 , and it was found that plasma cholesterol level declines significantly in a dose dependent manner. Besides, acetamiprid may induce oxidative stress in Leydig cells and can play a role in decreasing testosterone secretion 19,22 .
In addition to testosterone, GnRH, FSH, LH, and INHB hormones have important roles in spermatogenesis. The balance of these hormones is necessary for the proper functioning of the testes 22 . Thus, plasma levels of the hormones were also evaluated. Acetamiprid was found to disrupt the hormonal balance in the present study. It has been reported that increase in serum FSH and LH levels, and decrease in serum testosterone and INHB levels has been detected in patients who have low sperm concentration, and this can be used as biomarker for the diagnosis of the infertility in the clinic 23,24 . In the current study, we demonstrated that acetamiprid caused to increase FSH and LH plasma levels in all treatment groups. Although sperm count diminished in a dose dependent manner, the rate of the increase of these hormones was found to be lower in the high dose group. Interestingly, the plasma INHB level increased at the low and medium dose group whereas it decreased at the high dose group.
It has been stated that acetamiprid leads to oxidative stress in many organs such as liver, kidney, brain of different species 8,25 . Oxidative stress has been shown to cause damage in DNA, proteins and lipids of sperm and also induce apoptosis. As it is known, these mechanisms might play a role in decrease of sperm count and impairment of sperm function 26 . It is known that oxidative stress is one of the common reason of testicular dysfunction which leads to infertility 27 . Reactive oxygen species (ROS) production in the testis leads to oxidative damage and destroys steriodogenesis and spermatogenesis 28 . Some studies showed acetamiprid induced lipid peroxidation and reduced antioxidant enzymatic activity in the testes 12,18 . In the current study, acetamiprid led to lipid peroxidation, GSH depletion, increase in TOS, and decrease in TAS in testis homogenates in a dose dependent manner. These results were also confirmed in the plasma. However, these parameters were more affected in testis homogenate than plasma. This can be associated with high potential of ROS formation in the cells that use molecular oxygen in steroid biosynthesis in testes 29 . Oxidative stress also affects the regulation of proliferation, apoptosis and transcription in the testis 30 . Thus, acetamiprid-induced oxidative stress might result in reduced sperm count, increased sperm abnormalities and apoptosis induction in the study. Moreover, it has been stated testosterone might have antioxidant and antiapoptotic function in testes and protect sperm from DNA damage 31,32 and www.nature.com/scientificreports www.nature.com/scientificreports/ decreased testosterone level may augment acetamiprid-induced oxidative stress in the present study. Besides, it has been stated INHB is released more in oxidative stress 32 . However, INHB level decreased nonsignificantly at high dose treatment group which we observed extensive oxidative stress compared to control group. This might be associated with dramatically increased apoptosis in testes at the high dose group.
As it is well known, the alterations of apoptosis have been involved in pathology of the many diseases such as cancer and neurodegenerative diseases 33 . It has been also showed that cell injury in testis may result in apoptosis 34 . In the present study, TUNEL cell count and apoptotic index were used to evaluate apoptotic cells. The number of apoptotic cell-containing seminiferous tubules was higher in the exposure groups than in the control group. The apoptotic index levels were 10.6, 26.3 and 57.5, respectively, in the low-, medium-and high-dose groups. Increased apoptosis may be a consequence of acetamiprid-induced oxidative stress. Histopathological examination revealed apoptotic cells in the tubules of all exposed groups, mostly in spermatogonia and primary spermatocytes. The number of apoptotic cells increased in a dose-dependent manner. According to the results of seminiferous tubule scoring and histopathological examination, the basement membranes of the seminiferous tubules were found to be regular in the control and low-dose exposure groups. In the medium-and high-dose exposure groups, the presence of basal membranes showing irregular and undulate structure in many seminiferous tubules was detected. In the high-dose group, a decrease in spermatogenic germ cell counts, spermatogenic cells with vacuole formation and luminal immature cell rashes were observed in most seminiferous tubules.
Spermatogenesis perturbation and weak sperm were seen in some seminiferous tubules of the Wistar rats treated with 27 mg/kg oral acetamiprid for 45 consecutive days. The presence of pelleted cell particles in the lumen, tubulous atrophy, disorganisation and degenerative direction of the seminal epithelium were observed 35 .    Table 4. Histopathological scoring, proliferation index and apoptotic index in the testes of Sprague Dawley rats treated with oral acetamiprid. *p < 0.05, # p < 0.1, ## p < 0.01.
According to a study, degeneration of spermatogonia and edematous changes in seminiferous tubules and occlusion of blood vessels in the interstitial space have been reported in Swiss albino male mice treated with 2.3 mg/kg and 4.6 mg/kg acetamiprid for 30, 60 and 90 days 36 . In the present study, the proliferation index decreased in a dose-dependent manner, particularly in the high-dose exposure group by PCNA immunohistochemical technique.
In conclusion, acetamiprid was found to have potentially toxic to the male reproductive system on Sprague Dawley rats at the dose of 35 mg/kg. Underlying mechanisms of the toxic effects can be associated with oxidative stress and apoptosis in the testes. This data emphasizes the importance of taking precautions against environmental and occupational exposure to agricultural drugs. However, further mechanistic studies are needed to understand the molecular mechanisms of the acetamiprid induced reproductive toxicity.

Material and Method
Animals and their synchronization. A total of 48 (8-10 weeks old) male Sprague-Dawley rats with body weights of 300-350 g were obtained from Istanbul University Aziz Sancar Institute of Experimental Medicine and were divided into 4 groups, control group (n = 10), low dose group (n = 11), medium dose group (n = 12) and high dose group (n = 12). Rats were housed in polystyene standard cages with 4-5 animals in each, maintained at 21-23 °C and humidity (55 ± 5%) at Istanbul University Faculty of Pharmacy Animal Facility Unit (EDEHAB). Rats synchronized for a 12 h light/12 h dark cycle (LD 12:12). Standard pellet chow and tap water provided ad libitum throughout the experiment. Acetamiprid administration was performed during light span and commenced at 10:00 AM equal to 3 hours after light onset i.e. HALO-3 to prevent time dependent differences. The study was approved by Istanbul University Local Ethics Committee of Experimental Animals (IUHADYEK; 2016/35) and all experiments were performed in accordance with Istanbul University Animal Experiments Local Ethics Committee Guideline 37,38 . Chemicals and reagents. Acetamiprid (97% of purity) was kindly gifted from Hektaş Ticaret T.A.Ş.
(Istanbul, Turkey) and freshly suspended in an aqueous solution of 0.5% methylcellulose (MC) on each study day. Acetamiprid was orally administered to the rats in a fixed fluid volume (4 mL/kg body weight) 37 .
Sperm morphology was evaluated using phosphate-buffered saline (PBS 10×), Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM-F12), and fetal bovine serum (FBS) (Multicell Wisent, Quebec, Canada). For histological evaluation of testes, polylysine coated microscope slides and covers (Thermo Scientific, Munich, Germany) and microtome blades (Cologne, Germany), analytical grade chemicals (Sigma Aldrich, Munich, Germany and Merck Millipore, Munich, Germany) were used, while plastic wares from Eppendorf (Nijmegen, Netherlands) and Nest Biotechnology (Munich, Germany).  39 . According to the European Food Safety Authority (EFSA) 2016 data, reproductive NOAEL was 51 mg/kg 40 . The NOAEL of acetamiprid determined was 38.7 mg/kg mg/kg by two generation study in rats 41 . In our preliminary study, oral dose of 50 mg/kg of acetamiprid resulted in 25% of animal death. Accordingly, the acetamiprid dose groups were divided as highest dose group (35 mg/kg/ day), medium dose group (25 mg/kg/day), and the low dose group (12.5 mg/kg/day). Acetamiprid was administered to rats during 90 days, by using oral gavage used for the rats. The control group was given only vehicle i.e., 0.5% MC.
At the end of the 90 th day, the rats were exposed to inhalation anaesthesia for very short time. Then, they were sacrificed by removing large volume of blood from the orbital veins and immediately incised from abdominal to breast area. The blood samples were collected in EDTA tubes for hormone and biochemical analysis. The testes and epididymis were dissected, and sperms were collected from the epididymis. The left testis was homogenized in a sufficient amount of PBS (1×), and stored at −80 °C until biochemical analysis. The right testis was fixed in a sufficient amount of 10% neutral buffered formalin, and stored at room temperature until histological examinations.
Body weight and testis weights. Body weights of all animals were measured (Sartorius, Mettler H20, Germany) three times a week and at the end of the experiment. Mortality and clinical signs e.g. posture, locomotor activity, were controlled every day. Testicles were removed on sacrifice and weighed (Precisa XB220A, Switzerland) for the toxicological evaluation (U.S. Enviromental Protection Agency 1996). The testes were also evaluated macroscopically to reveal any enlargement, shrinkage, gaps due to tissue loss, tissue softening, tissue foreign coloring, and altered content.
The epididymis was cleaned from peripheral structures carefully, and weights were recorded. The somatic index was calculated by employing the following formula:  www.nature.com/scientificreports www.nature.com/scientificreports/ suspension was centrifuged at 2,230 rpm for 3 min. The supernatant was centrifuged again and then was added to trypan blue (1:1, v/v). The mixture was spread on the Thoma slide. The sperms were counted under a Leica microscope (Leica Microsystems Co., Wetzlar, Germany) at 20X magnification.
Sperm morphology. To assess sperm morphology, one drop of the epididymis suspension was spread on the slides and allowed to dry at room temperature. The morphological examinations of spreading samples were completed within one week. The samples were prepared in accordance with the Diff-Quick staining protocol (ADR, Istanbul, Turkey), and were carried out with 3 spreads for each sample and 200 sperm were counted for each preparation. The sperms were classified as normal, headless, detached head, flattened head, pinhead, bent neck, bent tailed, coiled tail and multiple abnormalities 42,43 . Hormone and biochemical analysis in plasma and testicular tissue. The blood samples were centrifuged at 3000 rpm for 20 min −4 °C (Hettich Universal 32 R Germany). The plasma was separated and kept in −20 °C refrigerator (Arcelik 2041, Turkey). The quantitative determination of plasma cholesterol, testosterone, LH, FSH, GnRH and INHB was carried out in plasma by commercial kits according to the manufacturer's instructions (Elabscience, Wuhan, China, and SunRed, Shanghai, China).
The testes tissues were homogenized in PBS (1:5, w/v) and kept at −80 °C (Daihan-Scientific Wisecry, South Korea). Biochemical parameters GSH, MDA, TAS and TOS indicating oxidative damage were measured by enzyme-linked immunosorbent assay (ELISA) commercial kits according to the manufacturer's instructions (Elabscience, Wuhan, China, and SunRed, Shanghai, China). The optical density (OD) was measured at 450 nm, with a reference wavelength of 620 to 630 nm using an Epoch microplate spectrophotometer (BioTek Inc., Bad Friedrichshall, Germany).
Histological examination of testicular tissue. For the histological examination, the right testis was fixed in 10% neutral buffered formalin, dehydrated in a series of graded alcohol and embedded in paraffin 44,45 . Hematoxylin and eosin (H&E) staining was applied to the middle part of testis taken from paraffin blocks at a thickness of 4 μm for histopathologic analysis, and periodic acid Schiff (PAS) staining was performed to evaluate changes of seminiferous tubule basement membrane. The evaluation was performed on each of the 30 seminiferous tubules with a 20X microscopic magnification. The first evaluated seminiferous tubule was randomly selected, and the section was shifted clockwise to evaluate the others. Histopathologic scoring was assessed by the modified Johnson scoring method (Table 5) 46 .
PCNA immunohistochemistry. The PCNA immunohistochemistry were done by using commercial kit from Invitrogen (Carlsbad, California, USA). For PCNA immunohistochemistry, half of the right testes were fixed for 72 h in 10% neutral buffered formalin. The paraffin sections with a thickness of 4 μm, which were collected on the positively charged slides, were done according to previous study 47 . Randomly selected, twenty seminiferous tubules were examined in the stained sections. The cells with brown nuclear staining were accepted PCNA positive and all of the stained and nonstained germ cells were counted. The ratio of PCNA positive cells to the total number of germ cells, "PCNA index, " was calculated for each seminiferous tubule. The average PCNA index in each case was obtained by dividing the sum of all PCNA indices by the number of seminiferous tubules in which the calculation was carried out. Detection of apoptosis by TUNEL assay. TUNEL assay were done by using ApopTag Plus Peroxidase In Situ Apoptosis Kit (Millipore, Massachusetts-USA) according to manufacturer's protocol with slight modifications. Twenty seminiferous tubules were examined in the stained sections. The seminiferous tubules containing 3 or more apoptotic cells were counted as TUNEL. The apoptotic index was calculated by the following formula: