Selenium reduces acrylamide‐induced testicular toxicity in rats by regulating HSD17B1, StAR, and CYP17A1 expression, oxidative stress, inflammation, apoptosis, autophagy, and DNA damage

This study investigated the effects of Selenium (Se) on testis toxicity induced by Acrylamide (ACR) in rats. In our study, 50 male adult rats were used, and the rats were divided into five groups; control, ACR, Se0.5 + ACR, Se1 + ACR, and Se1. Se and ACR treatments were applied for 10 days. On the 11th day of the experimental study, intracardiac blood samples from the rats were taken under anesthesia and euthanized. Sperm motility and morphology were evaluated. Dihydrotestosterone, FSH, and LH levels in sera were analyzed with commercial ELISA kits. MDA, GSH, TNF‐α, IL‐6, and IL‐1β levels and SOD, GPx, and CAT, activities were measured to detect the level of oxidative stress and inflammation in rat testis tissues. Expression analysis of HSD17B1, StAR, CYP17A1, MAPk14, and P‐53 as target mRNA levels were performed with Real Time‐PCR System technology for each cDNA sample synthesized from rat testis RNA. Testicular tissues were evaluated by histopathological, immunohistochemical, and immunofluorescent examinations. Serum dihydrotestosterone and FSH levels decreased significantly in the ACR group compared to the control group, while LH levels increased and a high dose of Se prevented these changes caused by ACR. A high dose of Se prevented these changes caused by ACR. ACR‐induced testicular oxidative stress, inflammation, apoptosis, changes in the expression of reproductive enzymes, some changes in sperm motility and morphology, DNA, and tissue damage, and Se administration prevented these pathologies caused by ACR. As a result of this study, it was determined that Se prevents oxidative stress, inflammation, apoptosis, autophagy, and DNA damage in testicular toxicity induced by ACR in rats.


| INTRODUCTION
Acrylamide (ACR), which is not a natural compound, is a highly toxic compound with high chemical activity and a molecular weight of 71.08 g/mol, and it can be synthesized chemically.ACR has uses many fields as industry, printing, textile, water treatment systems, cosmetics, and research laboratories. 1,2It is not naturally present in foods, but ACR occurs when carbohydrate and protein-rich foods such as meat, bread, French fries, chips, cereals, and roasted coffee are exposed to temperatures above 120 C. 3,4 ACR, which has a wide range of uses in daily life and can be exposed to food continuously, is known as a class 2A carcinogen for humans. 5It has been reported that ACR, which is easily soluble in water, can pass from the mother to the fetus through the placenta and may cause permanent damage to the fetus. 6The organism's remainder (about 15%) is converted to oxidized glycidamide by an enzyme reaction involving cytochrome P450 2E1.A large amount (about 85%) of ACR is conjugated with glutathione. 7ACR mutagenicity in humans and mice is due to the ability of glycidamide to react with DNA.In addition, both ACR and glycidamide can react rapidly with hemoglobin and essential enzymes. 8As a result of chronic exposure to ACR, experimental animals develop genotoxicity 9,10 hepatotoxicity, 11 neurotoxicity, 12 nephrotoxicity, 13 and testicular toxicity. 14In different studies, it has been determined that ACR causes sperm anomalies, a decrease in sperm count, testicular damage, decreased glutathione levels in the sperm nucleus and tail, an increase in lipid peroxidation, and infertility due to DNA damage. 15,16These effects of ACR arise due to the disruption of the oxidant and antioxidant balance and the resulting oxidative stress.In addition, it has been reported that the administration of ACR in experimental animals causes atrophy of testes and seminal vesicles and decreases mating ability. 17Some studies have shown that antioxidant compounds can alter the detrimental effects of ACR on various tissues, 18,19 and one of these compounds is Selenium (Se). 13 is a trace element and has crucial roles in diverse biological and physiological processes in humans and animals, and its antioxidant, antiinflammatory, and anti-cancer effects. 20,21In addition, Se is required for normal testicular development and male fertility. 22The effects of Se on toxicity induced by ACR in the brain, 23,24 and kidney 13 have been reported by studies.Various studies have made it clear that Se is essential for spermatogenesis and male fertility, possibly due to its vital role in the modulation of antioxidant defense mechanisms and other basic biological pathways. 25In addition, in some studies, it has been reported that Se application is protective against testicular damage in testicular damage caused by various agents. 26,27Despite all this information, the effects of Se against ACR toxicity in the testes of rats remain unknown.In line with the literature information, this study aimed to determine the effects of Se on testicular toxicity induced by ACR in rats and the pathways through which these effects occur.

| Animals
Fifty adult male Sprague-Dawley rats (12 weeks old, 200-220 g) were used, and rats were produced from the Atatürk University Medical Experimental Application and Research Center.The rats were housed in a room with a controlled temperature (24 ± 1 C) with 45 ± 5% humidity with a 12 h light/12 h dark cycle with free access to standard rat food and water.Rats were acclimatized to the laboratory environment for at least 7 days before experiments.

| Experimental design
The rats were weighed before the experiment began and randomly divided into five groups.The rats were weighed before each injection, and Se and ACR were made.Experimental groups were formed as follows: Control group: Rats received a daily dose of saline intragastric (i.g.) for 10 consecutive days.
Se1 group: Rats received a daily dose of Se (1 mg/kg, i.g.) for 10 consecutive days. 13enty-four hours after the final administration (11th day), the rats were weighed, and intra-cardiac blood samples were taken from the rats under sevoflurane anesthesia (Sevorane 100% liquid, Abbott Laboratories, _ Istanbul, Turkey).After the serums in the blood samples were separated, they were stored in a deep freezer at À80 C until the analysis.After they were decapitated, the testis tissues of rats were excised and weighed.The right testis of each rat was fixed in 10% formaldehyde for histopathological, immunohistochemical, and immunofluorescence examination.The left testis of each rat was immediately harvested and then stored in a À80 C freezer for use in ELISA and Real-Time PCR analysis.

| Sperm analysis
After the euthanasia of the rats, the testicles were weighed, and the mean testicular weights (MTW) were calculated for each rat.For each randomly selected animal, one of the cauda epididymides was dissected in 5 mL of physiological saline in a petri dish and kept on a preheated table (35 C) for 5 min to induce sperm cell migration.The resulting liquid was accepted as a semen sample.Sperm motility was examined microscopically according to the visual estimation method.Briefly, approximately 20 μL of semen was dropped onto an object slide.The semen sample was then covered with a coverslip.The slide was placed on the heated stage with a light microscope (Zeiss Primo Star, Carl Zeiss, Oberkochen, Germany).Sperm motility was determined by visual estimation for each rat.At least two different areas were evaluated, and the mean motility score was recorded as the final motility score. 30 determine sperm density, semen samples were diluted 1/100 with physiological saline in a centrifuge tube.Tubes were vortexed at 2500 rpm for 15 s.Approximately 20 μL of semen was transferred to the Thoma counting chamber.Spermatozoa were counted in two chambers under a light microscope at 400Â magnification.The arithmetic means a score of the two chambers was calculated as the final value.The value obtained by multiplying this score by 5 Â 106 was expressed as the epididymal sperm density and spermatozoa/mL.
For the determination of sperm morphology, approximately 10 μL of semen was dropped on a slide, and 10 μL of eosin dye (5%) was added to it and mixed with the coverslip.The smear slide was prepared and then quickly dried (at 50-55 C) in front of the heater.All slides were evaluated under a light microscope at Â400 magnification.
Abnormalities of sperm cells were calculated on each slide according to the morphological structures of the cells (head, middle, and tail of sperm cells).A total of 200 sperm were counted on each slide, and abnormal sperm rates were calculated according to these results.

| Determination of serum dihydrotestosterone, FSH, and LH levels
After 30 min of taking, the blood was centrifuged at 3500-4000 rpm for 10 min at 4 C, and the serum was separated.The serum dihydrotestosterone, FSH, and LH levels were analyzed using commercial ELISA kits (Sunred, Shanghai, China) according to the manufacturer's instructions.

| Preparation of the tissues for biochemical analysis
Testis tissues stored in the freezer (À80 C) were taken out and weighed on the analysis day.Phosphate buffer was added to obtain a 10% homogenate, and it was homogenized in a MagNA Lyser (Roche Diagnostics Corporation) for 30 s at a speed setting of 6000.Homogenates were centrifuged for 20 min at 10 000 rpm at +4 C, and supernatants were used for assaying the biochemical analyses.

| Measurement of lipid peroxidation and antioxidant enzyme activities and levels
Lipid peroxidation in the testicular tissues were analyzed using commercial ELISA kits.The levels of MDA in testis supernatants were quantified using commercial ELISA kits (Sunred, Shanghai, China) according to the manufacturer's instructions.The SOD, GPx, and CAT activities and GSH levels were analyzed using commercial ELISA kits (Sunred, Shanghai, China) according to the manufacturer's instructions.

| Determination of inflammation parameters
To determine inflammation in testicular tissues obtained from experimental groups, TNF-α, IL-1β, and IL-6 levels were measured using commercial ELISA kits (Sunred, Shanghai, China).

| RNA isolation and cDNA synthesis
The testis tissues were stabilized in RNA Stabilization Reagent (RNAlater, Qiagen), then homogenized with Tissue Lyser II (Qiagen) device.Total RNA was isolated from homogenized testis tissues following the RNeasy Mini Kit (Qiagen) instructions.cDNA was synthesized by reverse transcription of complementary DNA from isolated RNA samples according to the instructions of the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA).As previously described, the purity and concentration of isolated RNA and cDNA were determined with a nucleic acid measurement device (Nanodrop Take3 plate, Biotek). 31The sequences of PCR primer pairs used for each gene are shown in Table 1.

| Relative analysis of gene expressions
Expression analysis of HSD17B1, StAR, CYP17A1, MAPk14, and P-53 as target genes were conducted with StepOne Plus Real-Time PCR System technology (Applied Biosystems) for each cDNA sample synthesized from rat testis RNA.The expression analysis of β-actin was used as an endogenous reference gene.Quantitative real-time PCR was conducted according to the One-Step TaqMan Gene Expression Assays Probe-based technology (Primer Design Ltd., Southampton, UK), as previously described. 32The data obtained were expressed as a fold-change in expression using the 2 ÀΔΔCt method compared to the healthy group. 33

| Histopathological examination
Tissue samples taken for histopathological examination were fixed in 10% formalin solution for 48 h.As a result of routine tissue follow-up procedures, paraffin blocks were embedded and were taken in sections of 4 μm thickness from each block.The preparations prepared for histopathological examination were stained with hematoxylineosin (HE) and examined with a light microscope (Leica DM 1000, Germany).Sections were evaluated as absent (À), mild (+), moderate (++), and severe (+++) according to their histopathological findings.

| Immunohistochemical examination
All sections taken on the adhesive (poly-L-Lysin) slides for immunoperoxidase examination were passed through the xylol and alcohol series.After washing the sections with distilled water, they were kept in 3% H 2 O 2 for 10 min endogenous peroxidase inactivation was achieved.To reveal the antigen in the tissues, it was treated with antigen retrieval solution for 2 Â 5 min at 500 W in a microwave oven and then left to cool.Tissues were incubated with the autophagic regulator Beclin-1 (Cat no: sc-48 341, dilution ratio: 1/100 Santa Cruz, USA) for 30 min at 37 C. Followed up according to the immunohistochemistry kit procedure (Abcam HRP/DAB Detection IHC kit).3-3 0 Diaminobenzidine (DAB) was used as chromogen.Ground staining was done with hematoxylin.

| Double immunofluorescence examination
After 4 μm thick sections were taken on adhesive slides, deparaffinization, and dehydration processes were performed and washed with PBS.To mask the antigen in the nucleus, it was boiled in the microwave four times at 5 min intervals and left to cool at room temperature.After cooling, it was washed with PBS and left for 10 min by dripping the protein block.Primary antibodies (mouse-monoclonal 8-OHdG; sc-66 036 Santa Cruz, USA) and rapid-polyclonal H2A.X I 0856 protein tech, dilution ratio: 1/100 USA) were prepared in the appropriate dilution, dropped, and kept at the appropriate temperature and time depending on the conditions of use and washed with PBS.Immunofluorescence antibody was used as a secondary antibody (mouse-FITC Cat No: ab6717, Diluent Ratio: 1/1000 and rabbit-Texas-red sc-3917 Diluent Ratio: 1/100) and kept in the dark for 45 min and washed with distilled water.Then, secondarily, DAPI with DNA marker mounting medium (Cat no: D1306 diluent ratio: 1/100, Thermo, USA) is dripped and left in the dark for 5 min, then the tissues will be washed with distilled water and covered with a coverslip.The processed tissues will be examined under a fluorescent microscope (Zeiss AXIO).

| Statistical analysis
At the end of the studies, one-way ANOVA was used to analyze more than two independent groups in the SPSS 20.00 statistical data program.Then, the quantitative values were evaluated by applying the Tukey test.All values were expressed as mean ± standard error (±SEM), and p < .05 was considered significant.SPSS 20.00 program was used for statistical analysis in histopathological examinations, and the data were evaluated with p < .05considered significant.Duncan's test was used for comparison between groups.The non-parametric Kruskal-Wallis test was used to detect group interaction, and the Mann-Whitney test was used to determine the differences between groups.
To determine the intensity of positive staining from the pictures obtained as a result of immunohistochemical and immunofluorescent staining, five random areas were selected from each image and evaluated in the ZEISS Zen Imaging Software program.Data were statistically defined as mean and standard deviation (mean ± SD) for % area.One-way ANOVA followed by Tukey's test was performed to compare positive immunoreactive cells and immunopositive stained areas with healthy controls.As a result of the test, a p < .05 was considered significant, and the data were presented as mean ± SD.

| Effects of Se on body weight and testicular weight in ACR-induced testicular toxication
At the beginning of the experiments, the body weights of the rats did not differ between the experimental groups.At the end of the experimental study, the body weights of the rats were weighed, and it was T A B L E 1 Primers used in real-time PCR experiments.

Gene name
Primer sequence Accession number determined that there were lost body weights in the ACR, Se0.5 + ACR, and Se1 + ACR groups.At the end of the experiment, the body weights of the rats in the control and Se1 groups were significantly higher than those in the ACR-applied groups (p < .05).Testicular weights were lower in the other groups compared to the control, but it was not statistically significant ( p > .05,Table 2).

| Sperm analysis results
The spermatological parameters obtained in the study are presented in + ACR and Se1 + ACR groups were lower than control ( p < .001p< .05)When the serum LH levels were examined, it was observed that there was a significant increase in the ACR group compared to the other groups ( p < .05).On the other hand, a significant decrease was observed in the Se0.5 + ACR and Se1 + ACR groups compared to the ACR group (Figure 1).

| Effects of Se and ACR on HSD17B1, StAR, and CYP17A1 expression
The effects of Se on HSD17B1, StAR, and CYP17A1 mRNA expression levels in ACR-induced testicular toxicity were analyzed using Real-Time PCR (Figure 2).HSD17B1, StAR, and CYP17A1 mRNA expression were decreased in the ACR group compared to the control group (p < .05).Both doses of Se significantly increased HSD17B1, StAR, and CYP17A1 mRNA expression compared to the ACR group ( p < .05).

| Effects of Se and ACR on MAPk14 and P-53 mRNA expression
The MAPk14 and P-53 mRNA expression levels in testis tissues were analyzed using real-time PCR (Figure 3).

| Effects of Se and ACR on testicular inflammation
TNF-α, IL-1β, and IL-6 levels were significantly increased ( p < .001) in the ACR group compared to the control (Figure 6A-C).Both doses of Se prevented the ACR-induced increase in TNF-α but were still higher than in the control (Figure 6A).The IL-1β level was lower in the Se0.5 + ACR group than in the ACR group, but it was not statistically significant.A high dose of Se prevented the increase in IL-1β but was higher than the control (Figure 6B).Both doses of Se significantly inhibited the ACR-induced increase in IL-6 level.IL-6 was higher than the control in the treatment groups (Figure 6C).

| Histopathological findings
When the testicular tissues of the rats in the experimental groups were examined histopathologically, it was determined that the testicular tissues of the rats in the control and Se1 groups had a normal histological appearance (Figure 7).In the testicular tissues of the rats in the ACR group, severe edema in the interstitial spaces, severe hyperemia in the vessels, severe degeneration in the spermatocytes in the seminiferous tubule walls, and severe thinning in the wall thickness due to necrosis, marginal hyperchromasia in the nuclei of the degenerated spermatocytes, and multinucleated giant cells in their lumens was observed (Figure 7).Moderate edema and hyperemia in the intertubular spaces in testicular tissues of the Se0.5 + ACR group, moderate degeneration, and mild necrosis in spermatocytes were detected (Figure 7).In the testes of the Se1 + ACR group, mild degeneration was observed in the tubular wall, spermatocytes, and hyperemia in the interstitial vessels (Figure 7).There was a statistically significant difference when compared with the ACR group.Histopathological findings are summarized in Table 4.

| Immunohistochemical findings
When testicular tissues of rats in the control and Se1 groups were examined immunohistochemically, Beclin-1 expression was evaluated as negative (Figure 7).In the ACR group, severe cytoplasmic Beclin-1 expression was observed in spermatocytes in the seminiferous tubular walls (Figure 7).Moderate intracytoplasmic Beclin-1 expression was detected in the spermatocytes of the Se0.5 + ACR group (Figure 7).
Mild intracytoplasmic Beclin-1 expression was observed in the tubular wall and spermatocytes in the Se1 + ACR group (Figure 7).There was a statistically significant difference ( p < .05)when compared with the ACR group.Immunohistochemical findings are summarized in Table 5.

| Double immunofluorescence findings
When the testicular tissues of the rats in the experimental groups were examined by immunofluorescence method, 8-OHdG and H2A.X expressions in the control and Se1 groups were evaluated as negative (Figure 8).In the ACR group, severe 8-OHdG, and H2A.X expressions were detected in spermatocytes (Figure 8).In the Se0.5 + ACR group, moderate levels of 8-OHdG and H2A.X expressions were detected in spermatocytes in the seminiferous tubule walls (Figure 8).In the Se1 + ACR group, mild 8-OhDG, and H2A.X expressions were observed in the tubular wall and spermatocytes (Figure 8).There was a statistically significant difference ( p < .05)when compared with the ACR group.
Immunofluorescence findings are summarized in Table 5.

| DISCUSSION
ACR is used as an industrial chemical in many areas such as polymers, cosmetics, paper, textiles, wastewater treatment, and the production of laboratory gels. 34However, ACR has been found to occur in some foods subjected to heat treatment. 3As a result of studies with experimental animals, it has been shown that different doses of ACR cause neurotoxicity, genotoxicity, reproductive toxicity, and embryonic toxicity. 35The testicular damage and functional changes induced by ACR in the reproductive system are fundamental problems for male reproductive health.In recent years, male infertility cases have become a global health problem.In recent years, compounds with antioxidant and anti-inflammatory effects have become widespread to prevent the damage caused by ACR on the testis. 36In this study, which we presented in line with this information, the protective effects of Se application on testis toxicity induced by ACR in rats were investigated.
Studies in rats have shown that ACR causes to decrease in sperm count and viability and increases abnormal sperm morphology. 37In our study, similar to the findings obtained in this study, it was observed that ACR application caused a decrease in sperm count and viability.In contrast, it caused an increase in abnormal sperm morphol- various animal models have determined that the LH-testosterone signaling pathway has a critical role in the initiation and maintenance of spermatogenesis. 39Our study established that ACR application significantly changed serum dihydrotestosterone, FSH, and LH levels compared to healthy rats.In a study, some natural compounds inhibit ACR-induced reproductive hormone disorders. 40We determined that our findings are compatible with the literature and that Se inhibits ACR-induced reproductive hormone disorders.
StAR is a crucial regulatory enzyme in the first step of the steroidogenic pathway and ensures cholesterol transport from the outer to the inner mitochondrial membrane. 41The luteinizing hormone receptor (LHR)-mediated steroidogenic pathway in Leydig cells regulates StAR expression and activation. 42In our study, it was observed that ACR application significantly inhibited StAR mRNA expression in testicular tissue and ACR suppressed StAR-mediated testosterone synthesis.On the other hand, StAR mRNA expression increased in the groups treated with Se compared to the ACR group.
catalyzes the formation of pregnenolone from mitochondrial cholesterol, and pregnenolone is converted to testosterone by metabolic reactions under the influence of 3β-HSD and 17β-HSD.
3β-HSD plays a crucial role in changing the 3β-hydroxyl from pregnenolone to the keto group, which initiates testosterone biosynthesis. 43The decrease in the expression of these enzymes causes a decrease in the synthesis of the testosterone hormone. 44Oxidative stress is one of the most critical factors that can reduce the expression of these enzymes. 45Our study determined that ACR application significantly reduced CYP17A1 and 17β-HSD mRNA expression in testis tissue.This is evidence that ACR negatively affects CYP17A1 and 17β-HSD-mediated testosterone synthesis.On the other hand, it was determined that Se significantly inhibited the ACR-induced decrease in CYP17A1 and 17β-HSD mRNA expressions in testicular tissues, thus suppressing the decrease in ACR-induced testosterone synthesis.These results suggest that this effect of Se is especially shown by reducing oxidative stress.
During vital activities in cells, chemical compounds known as oxidants are regularly released, and antioxidant systems control the production of oxidants.Disruption of the balance between oxidant and antioxidant systems is defined as oxidative stress. 46Oxidative stress is critical in the pathogenesis of many diseases and toxicities. 47In experimental studies of the testicular damage model induced by ACR in rats, it was determined that ACR significantly increased the MDA levels in testicular tissue and decreased GSH levels and GPx, SOD, and CAT activities. 48Consistent with the literature, our study determined that ACR application significantly increased the MDA levels in renal oxidative stress caused by ACR. 13 In a study, Se application prevented increased MDA levels and decreased SOD, GPx, and CAT activities in testicular toxicity induced by bisphenol in experimental animals. 49Similar to the literature, our study determined that the Se application showed antioxidant activity by suppressing the testicular oxidative stress induced by ACR.
TNF-α, IL-1β, and IL-6, known as pro-inflammatory cytokines, cause the acute responses of inflammation by inducing the expression of adhesion molecules in endothelial cells, stimulating inflammatory cells, and causing circulating leukocytes to adhere to the endothelium in the inflammatory process. 50A study determined a significant increase in TNF-α and IL-1β levels in ACR-induced neurotoxicity in mice compared to the control and ACR-induced inflammation. 51The increase in ROS levels in ACR toxicity stimulates an increase in pro-inflammatory cytokine levels.Our previous study determined that ACR stimulated renal inflammation in rats and caused a significant increase in TNF-α, IL-1β, and IL-6 levels compared to the control.Se significantly prevented ACR-induced inflammation. 13Ghorbel et al. 52 reported a significant increase in liver TNF-α, IL-1β, and IL-6 levels in rats treated with ACR compared to the control.Our study determined that ACR caused testicular inflammation by significantly increasing TNF-α, IL-1β, and IL-6 levels, and our findings were consistent with the literature.Many studies have determined that Se application provides an anti-inflammatory effect in rats with experimental testicular damage. 53Our results were consistent with the literature that ACR stimulated inflammation by increasing pro-inflammatory cytokines in the rat testis, and Se application provided an anti-inflammatory effect.
Hammad et al. 54 stated that due to increasing doses of ACR caused severe disorders in tissues.Fiedan et al. 55 observed that ACR application caused congestion, interstitial edema, and degeneration in spermatogenic cells in seminiferous tubules, giant spermatid cells, necrosis, and calcification in testes of rats.In another study, multinucleated giant cells, vacuolization, and apoptotic cell formation were determined in the seminiferous tubules in rat testes exposed to different doses of ACR. 56In addition, it has been reported that ACR reduced testosterone levels produced by the suppression of the expression of fundamental transcription factors and genes that play a role in the steroidogenic pathway in Leydig cells. 57Totani et al. 58 that consuming oil containing 7% ACR decreased the spermatozoid density with cells spilled into the tubules of seminiferous contortus lumen and newly formed giant cell formations in the testicular tissues of rats.Our study determined that ACR application caused severe degeneration, necrosis, and vascular hyperemia in in testis tissue, and Se significantly reduced these ACR-induced changes.
8-OHdG and H2A.X are essential markers of DNA damage after oxidative stress.It is often used as a biomarker in oxidative DNA damage. 59Studies have shown that ACR application significantly increases 8-OHdG expressions in tissues and various body fluids. 60Studies also show that H2A.X expression increases due to oxidative stress. 61Our results were consistent with the literature.We determined that ACR significantly increased testicular 8-OHdG and H2A.X expression, and both doses of Se prevented the increase in both 8-OHdG and H2A.X expression.This situation suggested that ACR application caused DNA damage in testis tissue mediated by oxidative stress, and Se prevented this damage.
As stated in previous studies, p53 is a good marker of nuclear apoptosis. 62,63Again, with some studies, DNA damage develops in the testicular tissue originating from ACR, and as a result, the expression of p53, a marker of apoptosis, increases. 64The findings of our study are similar to these studies.We determined that ACR application increased p53 mRNA expression in testis tissue.On the other

3. 6 |
Effects of Se and ACR on MDA and GSH levels in testicular tissuesMDA level significantly increased in the ACR group compared to the control group ( p < .0001).Both doses of Se significantly prevented ACR-induced lipid peroxidation ( p < .05).Se administration alone did not significantly increase the MDA level (Figure4A).GSH levels were significantly decreased in the ACR and Se0.5 + ACR groups compared to the control ( p < .05).The high dose of Se significantly prevented ACR-induced GSH reduction (Figure4B).
ogy.Considering the groups treated with Se, it was determined that Se increased sperm count and viability and decreased the abnormal sperm rate.These findings show that Se positively affects sperm count and morphology by suppressing ACR-induced oxidative stress and inflammation in testicles with its antioxidant and antiinflammatory activity.Dihydrotestosterone, FSH, and LH hormones have essential effects on testicular physiology.FSH plays a central role in regulating and promoting spermatogenesis in Sertoli cells.The primary function of LH is to stimulate and regulate testosterone synthesis from interstitial Leydig cells in the testis.Testosterone and FSH regulate spermatogenesis in the seminiferous tubules. 38As a result of studies from F I G U R E 6 Effects of Se and ACR on TNF-α (A), IL-1β (B), and IL-6 (C) levels in testicular tissues of rats.Statistical significance (control vs. others: ***p < .001;**p < .01;*p < .05;ACR vs. others: ###p < .001;##p < 0.01; #p < .05;Se0.5 + ACR vs. Se1 + ACR: ɣ: p < .05)was analyzed Tukey's multiple comparisons tests followed one-way ANOVA.

7
testicular tissue and decreased GSH levels and GPx, SOD, and CAT activities.Our previous study reported that ACR-induced renal oxidative stress stimulated lipid peroxidation and decreased SOD, GSH, GPx, and CAT activities.At the same time, Se application prevented T A B L E 4 Scoring of histopathological findings in testicular sockets.Testicular tissue, edema (stars) in the intertubular spaces, degeneration of spermatocytes (thin arrows), necrosis (thick arrows), spermatic giant cells (arrowheads), H&E, cytoplasmic Beclin-1 expression in spermatocytes (arrowheads), IHC-P, bar: 50 μm.

Table 3 .
According to our results, no difference was found between the motility values of the control and Se1 groups.The lowest (p < .05)motilityrateamong the groups was detected in the ACR and Se0.5 + ACR groups.However, the motility value increased significantly (p < .05) in the Se1 + ACR group compared to the ACR group.While the abnormal sperm rate was similar in the control and Se1 groups, it was highest in the ACR group (p < .05).However, a significant improvement was observed depending on the dose in the groups treated with Se (p < .05).Dihydrotestosterone, FSH, and LH levels were evaluated in serum samples obtained from experimental groups.There was a significant decrease in dihydrotestosterone level in the ACR group compared to the other groups (p < .05),and the dihydrotestosterone level in the Se1 + ACR group was lower than in the control group (p < .01).The FSH level in the ACR group was lower than the control ( p < .001),Se1 + ACR (p < .05),and Se1 (p < .01) group.The FSH level in Se0.5 Similarities were found in the rates of dead sperm in the control and Se1 groups.ACR administration significantly increased the dead sperm rate compared to the control group, while this value decreased significantly in the Se1 + ACR group compared to the ACR group.There was no statistical difference in the Se0.5 + ACR group compared to the ACR and Se1 + ACR groups, but the dead sperm rate increased significantly (p < .05)compared to the control and Se1 groups.Semen density was similar in the control and Se1 groups and decreased significantly (p < .05) in the ACR group compared to the control, Se0.5 + ACR, and Se1 groups.3.3 | Effects of Se and ACR on serum dihydrotestosterone, FSH, and LH levels Body and testes weights of the experimental groups' rats at the experimental study's beginning and end.Sperm motility, abnormal sperm rate, dead sperm ratio, and density parameters in the experimental groups.