Carvacrol reduces abnormal and dead sperm counts by attenuating sodium arsenite‐induced oxidative stress, inflammation, apoptosis, and autophagy in the testicular tissues of rats

Arsenic (As) is a highly toxic metalloid. Carvacrol (CAR) is the active ingredient of Lamiaceae plants and has various biological and pharmacological properties. The present study investigated the protective effects of carvacrol (CAR) against testicular toxicity induced by sodium arsenite (SA). Rats were given SA (10 mg/kg) and/or CAR (25 or 50 mg/kg) for 14 days. Semen analyzes showed that CAR increased sperm motility and decreased the percentage of abnormal and dead sperm. It was determined that the oxidative stress induced by SA decreased with the increase of Nrf‐2 and HO‐1 expressions, SOD, CAT, GPx, and GSH levels, and MDA levels decreased after CAR treatment. It was observed that autophagy and inflammation triggered by SA in testicular tissue were alleviated by suppressing the expressions of LC3A, LC3B, MAPK‐14, NF‐κB, TNF‐α, IL‐1β, iNOS, and COX‐2 biomarkers in rats given CAR. Also, CAR treatment suppressed SA‐induced apoptosis by inhibiting Bax and Caspase‐3 expressions in testicles and up‐regulating Bcl‐2 expression. Histopathological analyzes showed that rats given SA had deterioration in tubule structure and spermatogenesis cell line, especially a serious loss of spermatogonia cells, atrophy of seminiferous tubules, and deterioration of germinal epithelium. In the group given CAR, the germinal epithelium and connective tissue were in normal morphological structure and an increase in seminiferous tubule diameters was observed. As a result, it was determined that oxidative stress, inflammation, autophagy, and apoptosis induced by SA were suppressed by CAR, thus protecting the testicular tissue from damage and increasing semen quality.

production of agricultural pesticides, the production of wood preservatives, glass production, and other mining industry applications, as well as medical applications such as the treatment of leukemia. 4,5 In addition, the most important source of As exposure is the use of drinking water containing inorganic As, 2 and it has been reported that approximately 150 million people worldwide are affected by Ascontaminated water. 3 Acute and chronic exposure to As which has various symptoms such as nausea, vomiting, abdominal pain, encephalopathy, and neuropathy causes toxicity in many tissues due to its water solubility and reaction with endogenous thiol groups and impairs the functions of these tissues. 4,6 Due to its toxic effects and environmental distribution, As has been evaluated by the World Health Organization (WHO) as one of the 10 chemicals that threaten public health. 1 In the literature, there are various studies on As toxicity and preservatives that can be used against As toxicity. 6,7 In studies on animal models, it has been shown that As crosses the blood-testis barrier and concentrates in these tissues, thus affecting reproductive capacity by causing a decrease in testicular weights, inhibition of antioxidant enzymes and spermatogenesis, and a decrease in steroidogenesis. 4,6 Oxidative stress occurs when the amount of free oxygen radicals is higher than normal levels and the antioxidant defense mechanism is suppressed, 8 and this is considered as an important mechanism in the emergence of the toxic effects of sodium arsenite (SA), an electrophilic agent. 4,9 SA modulates protein metabolism by directly binding to sulfhydryl groups in proteins or indirectly increasing ROS production, thus affecting the vital functions of cells. 9 ROS resulting from As exposure cause inflammation in tissues. It also triggers apoptosis by preventing cell growth and cell division. 6 On the other hand, previous studies have reported that the use of naturally occurring substances with antioxidant and anti-inflammatory properties can both avoid the side effects that may occur from the use of traditional chelating agents and provide protection against tissue damage that may occur due to As toxicity. 6,10 Carvacrol (CAR) (5-isopropyl-2-methylphenol), the predominant monoterpenic phenol, is the active ingredient of Lamiaceae plants including Origanum, Thymus and Corydothymus species. [11][12][13] It is known that this compound, which is generally used as a food additive and flavoring agent 14 has pharmacological properties such as antioxidant, anti-inflammatory, anti-apoptotic, anti-cancer, antifungal, insecticidal and antibacterial. [14][15][16][17] It is thought that the hydroxyl group in the aromatic ring gives CAR antioxidant properties. It also shows antiinflammatory effect by suppressing cyclooxygenase-2 and proinflammatory cytokines. 18 Last but not least, prior research suggests that CAR provides protection against reproductive damage due to various reasons (eg, diabetes-related damage or toxicity from chemotherapeutic agents). 13,15 In the present study, it was investigated whether CAR has a protective effect against the toxic effects of SA on testicular tissue. For this purpose, in addition to semen analysis, oxidative stress, inflammation, apoptosis and autophagy markers in testicular tissue were analyzed using biochemical and molecular methods, and the histological structure of testicular tissue was examined.

| Ethics committee approval and animals
The ethics committee approval was given by Atatürk University Animal Experiments Ethics Committee (Approval No. 2021/10/249). The male Sprague Dawley rats aged 10-12 weeks were weighed before starting experiment, and those weighing 250-300 g participated in the experiment. The environment in which they were kept had 24 ± 1 C and 45 ± 5% humidity. In addition, the animals were subjected to a 12-h dark and 12-h light cycle during the experiment.

| Experimental groups and selection of doses
Before the study, rats were divided into 5 groups (control, CAR, SA, SA + CAR 25 Group, SA + CAR 50 Group). There were 7 animals in each group. The doses of SA and CAR were determined by reference to previous studies. 18,19 According to WHO, it has been reported that exceeding the limit of 10 μg/L in drinking water may adversely affect human health. 20 In the study of Baltaci, Uygur, Caglar, Aktas, Aydin, Ozen, 19 it was confirmed that this dose of SA inhibited antioxidant enzymes in the testicles, caused lipid peroxidation, increased the number of tunnel positive apoptotic cells and caused histopathological damage in the testicles. In line with this information, 10 mg/kg dose of SA was chosen in the current study. On the other hand, it has been previously stated that 25 and 50 mg/kg doses of CAR attenuate cadmium-induced oxidative stress, inflammation and apoptosis in the liver and kidney. 18 In another study, it was reported that these two doses of CAR corrected lipid peroxidation and increased the activities of antioxidant enzymes in benzo(a)pyrene-induced lung toxicity. 21 However, it is not known how much of these doses can be met by diet.
The animals in the control group were given orally saline and corn oil for 14 days. CAR dissolved in corn oil at a dose of 50 mg/kg was administered orally to animals in the CAR group for 14 days. SA dissolved in physiological saline at a dose of 10 mg/kg was administered orally to animals in the SA group for 14 days. Animals in the SA + CAR 25 Group were given oral SA dissolved in saline at a dose of 10 mg/kg for 14 days, and CAR dissolved in corn oil at a dose of 25 mg/kg was administered orally 30 min later. Animals in the SA + CAR 50 Group were given oral SA dissolved in saline at a dose of 10 mg/kg for 14 days, and CAR dissolved in corn oil at a dose of 50 mg/kg was administered orally 30 min later.
At the end of the administration period, the animals were placed under mild sevoflurane anesthesia and as soon as the testicular tissues were removed, they were weighed and sperm analysis was performed.
After the sperm analysis, testicular tissues were powdered in liquid nitrogen in a homogenizer and stored at À80 C until biochemical and molecular analysis. After the sperm analysis, one of the testicular tissues were pulverized in liquid nitrogen in a homogenizer and stored at À80 C until biochemical and molecular analysis. Other testis tissue was used for histopathological analysis. SOD, CAT and GPx activities in the obtained supernatants were analyzed using methods reported by Sun, Chang, Pan, 23 Aebi, 24 and Lawrence, Burk. 25 Also, the total protein levels of the homegenates were analyzed using the method of Lowry, Rosebrough, Farr, Randall. 26 Table 1. The β actin gene was used to normalize the data.

| Spermatological analysis
Cauda epididymis taken from rats was prepared by the method used by Kandemir, Caglayan, Aksu, Yildirim, Kucukler, Gur, Eser. 29 Cauda epididymis was trimmed in a petri dish by dilution with Tris solution. It was waited for the spermatozoa to pass into the liquid. The obtained semen fluid was used in sperm analysis. To determine sperm motility, a heating plate was placed on a light microscope (Primo Star; Carl Zeiss), a slide was placed on it, 20 μL of sperm fluid was added and covered with a coverslip. At 400Â magnification, three different image areas were selected and the motility value prediction score was scored.
Sperm density was determined by the method described by Aksu, Akman, Özkaraca, Ömür, Ucar. 30 990 μL of eosin solution was added to 10 μL of semen sample in an eppendorf tube vortexed at 1000 g for 15 s and 10 μL of the mixture was transferred to a thoma slide.
After the passive movement of the fluid stopped, it was examined with the help of a light microscope at 400Â magnification and the spermatozoa were counted. Sperm density was expressed as the result of multiplying the obtained number by 5 Â 10 6 .
Abnormal sperm rate was determined by the method described by Sönmez, Türk, Yüce. 31 The rate of dead spermatozoa was determined by the method described by Türk, Ateşşahin, Sönmez, Çeribaşi, Yüce. 32 20 μL of the semen fluid obtained from the cauda epididymis was dropped onto the slide, and 20 μL of eosin dye was added and mixed. The mixture was covered with a coverslip and examined under a light microscope at 400Â magnification. The rate of sperm with abnormal was determined by counting 200 spermatozoa from each sample. The slides prepared to find the abnormal sperm count were used to determine the rate of dead-viable sperm. The semen whose heads were painted were considered dead. A total of 200 spermatozoa were counted for each sample and expressed as %.

| Histopathological analysis
Testicular tissues were fixed in a 10% neutral buffered formalin solution. After 24 h, the tissues were dehydrated by passing them through increasing grade alcohol to follow up. After passing through xylene to make the tissues transparent, they were treated with paraffin and then paraffin blocks were prepared. Sections of 5 μm were taken from paraffin blocks with the help of a microton. Sections were stained with Hematoxylin-Eosin (HE) following the procedures for histological evaluation and photographed under a microscope. To measure the diameter of the seminiferous tubule and lumen, 10 tubules were randomly selected for each animal and measurements were made in accordance with the literature. 33 To measure the diameter of the seminiferous tubule and lumen, 10 round-shaped tubules were randomly selected from each animal. Tubular diameter was averaged for each animal. In the same section, the lumens of the same tubules were measured and averaged. For measurements, the total and lumen diameter of each tubule were measured in μm at 10Â magnification by drawing parallel to its width. ImageJ 1.51v 9 program was used for metric analysis.

| Statistical analysis
Statistical analysis of the data obtained from the study was carried out with the IBM SPSS program using the One-way analysis of variance (ANOVA) test and Tukey's multiple comparison test.

| Carvacrol activates Nrf-2 and HO-1 genes suppressed by sodium arsenite in testicular tissue
The data obtained show that there is a serious decrease in Nrf2 and HO-1 gene expressions after sodium arsenide administration (p < .001). On the other hand, CAR treatment significantly increased Nrf2 and HO-1 expressions in testicular tissues compared to SA group. In addition, while it was observed that a high dose of CAR increased Nrf2 expression more, there was no significant difference between doses on HO-1 expression. The results are given in Figure 1 3.2 | Carvacrol alleviates the oxidative stress triggered by sodium arsenite in testicular tissue SA significantly inhibited SOD, CAT, and GPx activities in the testicular tissues of rats compared to the control group. Additionally, there was a significant decrease in GSH stores compared to the control T A B L E 1 Primer sequences.

| Carvacrol interrupts the inflammatory pathway triggered by sodium arsenite in testicular tissue
According to the data presented in Figure 2, it was observed that NF-κB and IL-1β relative protein levels are presented in Figure 3.
The results showed that there was a significant increase in NF-κB and IL-1β levels in testicular tissue with the effect of SA. After CAR administration, it was observed that these increases related to SA were inhibited.

| Carvacrol reduces the apoptotic effect of sodium arsenite in testicular tissue
Data on proapoptotic Bax and Caspase-3 gene expressions and antiapoptotic Bcl-2 gene expressions in testis tissue are summarized in

| Carvacrol reduces the autophagic effect of sodium arsenide in testicular tissue
Data for LC3A and LC3B mRNA transcript levels reflecting the autophagic state of testicular tissue are summarized in Figure 5. obvious that high-dose CAR administration on autophagy is more effective than low-dose (LC3A; p < .01, LC3B; p < .001).

| Testicular weights and sperm analysis results after carvacrol and sodium arsenite administrations
Testicular weight and sperm analysis results of rats after CAR and SA treatments are presented in Table 3  F I G U R E 4 Bax, Bcl-2 and Caspase-3 mRNA transcript levels in testicular tissues of rats treated with sodium arsenide and carvacrol. Statistical significance (letters indicate the difference between groups; p < .05) was analyzed using One Way ANOVA.

| Histopathology results
Testicular tissue seminiferous tubules of the control and carvacrol groups showed normal histological structure and regular cell arrangement. Germinal epithelium and interstitial area morphology were smooth in these groups ( Figure 6A.a, B

| DISCUSSION
In the present study, the protective effects of CAR on testicular toxicity caused by arsenic, which influence millions of people around the world, were examined and it was seen that CAR could be a promising compound against SA-induced testicular toxicity.
Oxidative stress significantly affects fertility. 29 Previous studies have reported that arsenic impairs spermatogenesis by inducing oxidative stress, resulting in a decrease in sperm quality and quantity. 6 SOD, CAT and GPx enzymes are at the base of the cellular antioxidant defense line. 34,35 SOD is responsible for scavenging superoxide radicals, and catalase is responsible for the decomposition of H 2 O 2 into water and molecular oxygen. [36][37][38] While GSH helps maintain the redox state in cells, it also plays a role in removing metals from cells. 9 Mice treated with arsenic have been reported to have a significant reduction in GSH levels. 9 In another study, it was reported that LPO, which is an important indicator of oxidative stress in the testicles, increased after SA administration, while a decrease in GSH, SOD, CAT and GPx levels occurred. 6 In this study, it was observed that GSH stores were significantly reduced in the testes of rats given SA, probably to compensate for excessive ROS production. it was thought that SA inhibited SOD, CAT and GPx enzymes and this occured either by directly acting on sulfhydryl groups or by excessive increase in ROS production, as mentioned before. naringin treatment, Nrf-2 and HO-1 expressions were triggered in rats. 40 Similarly, in the presented study, it was observed that the Nrf-2 and HO-1 mRNA transcript levels decreased in the testicular tissues of rats, possibly due to high oxidative stress, in the administration of SA, and that CAR upregulated Nrf-2 and HO-1 expressions by attenuating oxidative stress with its ROS scavenging feature.
Increasing evidence shows that in addition to oxidative stress, inflammation, which is triggered significantly by oxidative stress 43 and caused by the increase of pro-inflammatory cytokines has an important role in tissue damage induced by arsenic. 5 NF-κB is an important factor regulating the transcription of the pro-inflammatory cytokines, and together they play a key role in spermatogenesis, testicular steroidogenesis and semen maturation. 44 MAPK14 is involved in the regulation of the inflammatory response by activating NF-κB in various cell types. 11,45,46 In a previous study, it was reported that administration of SA to mice increased IL-1β, IL-6 and TNF-α levels and Nos2 was up-regulated. In this report, it was also reported that reproductive dysfunction develops with testicular atrophy as a result of excessive NO production and increased pro-inflammatory cytokines. 6 Another study has similarly confirmed that arsenic exposure increases the production of pro-inflammatory cytokines and this plays a role in the pathogenesis of testicular damage. 10 50 In the study, it is thought that SA triggers caspase-3 expression by causing cytochrome c release from mitochondria. It is thought that this may protect against male infertility by reducing the testicular toxicity induced by SA.
Based on the knowledge that arsenite can induce oxidative stress and oxidative stress can induce autophagy, 51 which is the cell's selfeating process, the effects of CAR against SA toxicity were also investigated with autophagic markers. The data obtained showed that the expressions of LC3A and LC3B, 52 which are involved in autophagosome formation, increased significantly after SA administration, triggering autophagy in testicular tissue. Although autophagy is an essential event required by cells in normal physiological processes, it causes tissue damage when it occurs at a high level. 53,54 This situation has also been reported in many previous studies, which is consistent with our results. 51,55 In the study, it was also observed that CAR down-regulated LC3A and LC3B expressions and protected testicular tissue from SA-induced autophagy.
It is known that there is a close relationship between arsenic exposure and male reproductive dysfunction. 3 There was no significant difference between testicular weights and sperm densities in the study. However, it was determined that sperm motility decreased significantly after SA administration, and the percentages of dead and abnormal sperm were significantly increased compared to other groups. In addition, it was observed that CAR administration brought these values closers to the control group levels. In a previous study, it was reported that a significant decrease in sperm counts occurred in rats given arsenite. 2 In another study, it was reported that arsenic administration caused a decrease in testicular weights and sperm motility, and this occurred as a result of increased lipid peroxidation. 5 Baltaci, Uygur, Caglar, Aktas, Aydin, Ozen 19 reported that SA and quercetin administrations did not cause any change in testicular weights in accordance with the present study.

| CONCLUSION
Data from the study showed that SA inhibits antioxidant enzymes and depletes GSH stores, thereby causing oxidative stress by causing lipid peroxidation. As seen in the findings obtained, this situation is thought to trigger a series of reactions such as inflammation, autophagy and apoptosis in the testicular tissue. Moreover, after SA application, it was determined that histological irregularities occurred in the testicular tissue and sperm quality decreased. However, due to the antioxidant property of CAR, it was determined that free radicals were swept away, GSH stores were renewed and antioxidant enzyme activities were recovered. It was concluded that with the relief of oxidative stress, inflammation, autophagy and apoptosis were suppressed, histological irregularities returned to normal and sperm quality improved.