Acutely applied melatonin prevents CCl4-induced testicular lesions in rats: the involvement of the oxidative capacity and arginine metabolism

Carbon tetrachloride (CCl 4 ) represents an organic chemical that causes reactive oxygen species derived organ disturbances including male infertility. Melatonin (MLT) is a neurohormone with strong antioxidant capacity, involved in numerous physiological processes. In this study we evaluated the capability of MLT, administered in a single dose of 50 mg/kg, to preserve the testicular tissue function after an acute administration of CCl 4 to rats. The disturbance in testicular tissue and the effects of MLT after CCl 4 exposure were estimated using biochemical parameters that enabled us to determine the tissue (anti)oxidant status and the intensity of arginine/nitric oxide metabolism. Also, the serum levels of testosterone and the histopathological analysis of tissue gave us a better insight into the occurring changes. A significant diminution in tissue antioxidant defences, arginase activity and serum testosterone levels, followed by the increased production of nitric oxide and extensive lipid and protein oxidative damage, was observed in the CCl 4 -treated group. The application of MLT after the CCl 4 caused changes, clearly visible at both biochemical and histological level, which could be interpreted mainly as a consequence of general antioxidant system stimulation and a radical scavenger. On the other hand, the application of MLT exerted a limited action on the nitric oxide signalling pathway.


INTRODUCTION
Although there are numerous legal regulations related to the carbon tetrachloride (CCl 4 ) management, this organic industrial compound still remains a major hazard risk to the ones exposed (Sherry et al., 2018). This industrial pollutant, which can enter the organism through various routes, is known to cause multi-organ damage by altering cell/tissue function and structure (Sönmez et al., 2014). When CCl 4 comes in contact with the organism it is rapidly metabolized in all tissues that possess cytochrome P-450 enzymes (Slater, Cheeseman, Ingold, 1985). The products of this metabolism are highly reactive trichloromethyl (CCl 3 • ) and trichloromethyl peroxy (CCl 3 O 2 • ) radicals that are the main culprits of cell macromolecule damage (Radulović et al., 2013;Wu, Zhang, Song, 2018). Among the cell components most vulnerable to the damaging potential of these generated free radicals are polyunsaturated fatty acids, which represent major building molecules of cell and organelle membranes (Manibusan, Odin, Eastmond, 2007).
A decline in male fertility can be the consequence of the exposure to numerous exogenous agents such as smoking, alcoholism, drugs (prescribed and non-prescribed) and even some toxic chemicals (e.g. CCl 4 ) (Türket al., 2016). Cell membrane fluidity, depending on the amounts of polyunsaturated fatty acids, is suggested to be needed for the adequate functioning of spermatozoa and germ cells. Having this mind, one can say that these cells are highly susceptible to the reactive oxygen species (ROS) damaging potential (Khan, 2012;Türk et al., 2016). Since the organs that are part of the male reproductive system express cytochrome P-450, it is not unexpected that they could suffer from exposure to CCl 4 either (Türk et al., 2016). Apart from ROS, the formed nitric oxide (NO), from arginine in the presence of inducible nitric oxide synthase (iNOS), is known to react with O 2 •thus generating highly reactive peroxynitrites that could damage biomolecules to a similar manner as ROS (Abdel Moneim, 2016).
Melatonin (MLT) is a neurohormone produced primarily by the pineal gland cells, however, contemporary studies suggest that it might be synthesized by numerous other cells (Sokolović et al., 2018). The main role of MLT is passing the information about the daily cycle of light and darkness to the body, additionally, it regulates core temperature, sleep-wake rhythms, immune system function, tissue antioxidative defences, hemostasis and glucose levels (Claustrat, Brun, Chazot, 2005). Several studies dealt with MLT's potential in preventing testicular tissue damage in different animal models (Parlaktas et al., 2008;Mirhoseini et al., 2014;Sokolović et al., 2015;El-Shafaei et al., 2018), and there is only one report dealing with the effects of pre-treatment with MLT on acute testis damage induced by CCl 4 (Wang et al., 2018).
The aim of our study was to evaluate whether the exogenous MLT administered after CCl 4 would prevent rat testicular tissue damage by determining the changes in serum and tissue biochemical parameters, as well as in tissue morphology.

Chemicals, drugs, reagents, and instruments
All solvents (analytical grade) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA), while commercial ELISA kits were obtained from AbCam (Cambridge, UK). Melatonin (>95%) was dissolved in absolute ethanol and further diluted in a sterile saline solution (0.85% NaCl) prior to the administration at a dose of 50 mg/kg. The dose, treatment regimen and route of application (intraperitoneal injection; i.p.) were chosen based on the previous publications (Sokolović et al., 2018;Ničković et al., 2018). In these publications the efficacy of MLT in preventing brain ischemic and traumatic injury, liver and pancreatic damage was proven (Ničković et al., 2018 and references cited within). Spectrophotometric analyses were performed using V-1800 Shimadzu spectrophotometer and/or microplate reader (Multiscan Ascent; Labsystems, Helsinki, Finland).

Animals and housing
Healthy male Wistar rats (250-300 g, n=24) were obtained from the Vivarium of the Institute of Biomedical Research, Medical Faculty, Niš, Serbia. The animals were maintained under standard husbandry conditions in plastic cages with a temperature of 23 ± 2 °C, relative humidity 55±10% and 12/12 h light/dark cycle. All animals were fed with standard laboratory food pellets, and water was provided ad libitum. The experiments were performed in accordance with the Declaration of Helsinki and European Community guidelines for the ethical handling of laboratory animals (EU Directive of 2010; 2010/63/ EU). The experimental procedures were commenced after being approved by the Animal Ethics Committee of the Republic of Serbia. The ethical approval number is 323-07-01762/2019-05, and the approval was obtained on 1 st February, 2019.

Experimental design
Before the experiment, animals were divided into four equal experimental groups (6 animals per group) and further housed in large spacious cages throughout the experiment. The acute testicular injury was induced by injecting a single dose of CCl 4 (50%, v/v). The groups of animals were treated as follows: Group I (Vehicle control): Animals treated with vehicle (0.85% NaCl saline solution containing 8% ethyl alcohol) once 24 hours before the end of the experiment in a volume of 1 ml/kg. Group II (MLT control): Animals treated once with MLT (50 mg/kg) in a volume of 1 ml/kg. Group III (CCl 4 control): Animals were given a single CCl 4 dose (1 ml/kg). Group IV (CCl 4 + MLT): Animals treated with MLT (50 mg/kg) once 1 h after a single CCl 4 dose. All animals were sacrificed by an overdose of ketamine (Ketamidor, 10%) 24 hours after the last treatment and a blood sample was withdrawn by a cardiac puncture using a vacutainer system. One testis was collected for homogenization and further biochemical analyses (stored at -80 °C), while the second one was fixed in 10% formalin and processed following routine pathohistological techniques.

Serum isolation and determination of testosterone levels
After blood collection, the tubes were left to clot at room temperature and were centrifuged at 3000 rpm for 10 min in order to obtain serum which was used for testosterone levels determination. Serum testosterone levels were determined using a standard Rat Testosterone ELISA kit (Cusabio Biotech. Co., LTD, Wuhan, Hubei, China), where the detection limits were from 0.13 ng/ mL to 25.6 ng/mL, with an assay sensitivity of 0.06 ng/ml. The procedure was performed following the manufacturer's instructions.

Tissue homogenization and biochemical parameter determination
Testicular tissue was homogenized in an ice-cold distilled water (10%, w/v) and the obtained homogenate was centrifuged at 12,000 rpm, for 10 min at 4°C in order to obtain a clear supernatant which was further used for the determination of tissue biochemical parameters. The protein content in supernatants was determined by using Lowry's method (Lowry et al., 1951) and the amount of proteins was calculated based on the bovine serum albumin standard curve.

Determination of enzymatic antioxidants
Superoxide dismutase activity in testicular tissue homogenates was determined using colorimetric SOD determination kit (Abcam653454) following the manufacturer's instructions. The assay is based on the reaction of water-soluble tetrazolium salt (WST) as a reagent, which in the presence of superoxide anion results in the decrease of WST formation that can be detected at 450 nm. The obtained results are presented as the percent inhibition of the superoxide anions compared to the control group.
Glutathione peroxidase (GPx) activity was determined in rat testes homogenates using a standard spectrophotometric method that is described elsewhere (Sokolović et al., 2018). The method is based on the reaction between the GSH, which remains after the enzymatic reaction that utilizes GSH and H 2 O 2 , and DTNB reagent. The enzyme activity was expressed as U/mg of proteins.
T h e c a t a l a s e a c t iv it y w a s m e a s u r e d spectrophotometrically, after the reaction of the enzyme with H 2 O 2 as a substrate and ammonium molybdate, at 405 nm. The reaction mixture consisted of tissue homogenate and reaction buffer and was initiated by H 2 O 2 (Ničković et al., 2018). After incubation at room temperature for 1 min, the reaction was stopped by adding ammonium molybdate solution. The activity was expressed as CAT U/g of testicular tissue proteins.

Determination of total tissue antioxidant capacity
Testes tissue antioxidant capacity (TAC) was determined by using a colorimetric assay kit (Abcam65329). The reaction was based on the conversion of Cu 2+ ions to Cu + ions. The degree of conversion was measured calorimetrically at 570 nm. The obtained results are expressed as a change in % activity from the one obtained in testicular tissue of the untreated animals.

Determination of non-enzymatic antioxidant capacities
The amount of GSH in testes tissue homogenates was determined using standard Ellman method, which is based on the reaction of non-protein thiols with DTNB reagent. The absorbance of the reaction mixture was measured at 412 nm, where the amounts of GSH were determined using a standard curve constructed with different concentrations of GSH. The obtained results are expressed as μmol of GSH/mg of proteins.

Determination of G6PD activity
The activity of G6PD was determined using a commercial colorimetric kit (Abcam, USA; ab102529). The reaction was based on a decrease in NADH concentration in the presence of the G6PD substrate. The enzyme activity was calculated based on the standard curve values obtained using a standard enzyme solution. The results are expressed as mU/g of tissue proteins.

Determination of biochemical parameters related to arginine metabolism
The arginase activity was determined using a colorimetric method which is based on a reaction between ornithine and ninhydrin. The coloured reaction absorbance was measured at 515 nm and the obtained values were corrected with those obtained from the control (Porembska, Kedra, 1975). The results are expressed as nmol/mg of proteins.
The quantitative determination of rat testis tissue iNOS was performed using CUSBIO (CSB-08325r) sandwich enzyme immunoassay kit following the manufacturer's instructions. The obtained values are expressed as U/mg of tissue proteins.
The concentration of nitrates/nitrites present in testicular tissue homogenates was determined in a mixture consisting of tissue homogenate and Griess reagent (Radulović et al., 2015). The absorbance of the mixture was measured at 540 nm using a microplate reader and the final concentrations were calculated using a standard curve of sodium nitrate. The final concentration of nitrates/nitrites in the sample was expressed as nmol/ mg of testicular tissue proteins.
The amount of citrulline in testis tissue homogenate was determined according to a standard method that is based on a reaction with diacetyl monoxime and thiosemicarbazide (Boyde, Rahmatullah, 1980). The absorbance of the developed colour was measured at 530 nm and the results were presented as nmol/mg of proteins.

Lipid and protein damage determination
The amount of lipid peroxidation was measured using a spectrophotometric in which the malondialdehyde (MDA) reacts with thiobarbituric acid, under increased heat, and forms a coloured product that can be measured at 532 nm (Sokolović et al., 2018). The concentration of MDA in testicular tissue was calculated using a standard curve and the results are expressed as nmol of MDA per mg of tissue proteins.
Protein carbonyl content was used for the quantification of oxidatively modified proteins. Their content was determined spectrophotometrically using 2,4-dinitrophenylhydrazine as a colour reagent (Levine et al., 1994). Reactive carbonyl derivatives were assessed using a molar extinction coefficient for 2,4-dinitrophenylhydrazine at 370 nm (22×10 3 L/mol/ cm) and the obtained results are expressed as μmol/g of testicular tissue proteins.

Pathological analysis
The fixed testis was processed using routine histological techniques that include tissue dehydrated through graded concentrations of ethyl alcohol and further embedment in paraffin wax. Tissue was cut into 4µm thin slices and stained with Mayer's haematoxylin and eosin (HE). The changes in the structure of the testicular tissue were scored following a previously described Johnsen scoring system (Johnsen, 1970). From each animal, a randomly selected testis was fixated and examined. The score was based on the analysis of at least 20 tubules from each tissue section and the results are given as a mean value for the selected group. In each of the seminiferous tubule, the level of sperm maturation and the germ cell appearance and their presence was examined in order to obtain a score value for a single tubule.

Statistical analysis
The obtained results are presented as mean values ± standard deviations (SD). Statistically significant differences were determined by One-Way Analysis of variance (ANOVA), followed by Tukey's post hoc test for multiple comparisons (GraphPad Prism, ver. 5.03; San Diego, CA). Probability values (p) less than, or equal to, 0.05 were considered to be statistically significant.

RESULTS AND DISCUSSION
The disturbance in testicular tissue antioxidant/ oxidant capacities is known to lead to male infertility, since a decline in antioxidant capacities leads to poor sperm quality and function (Wright, Milne, Leeson, 2014). By applying MLT to animals exposed to toxicant inducing testes tissue oxidative damage, this study was designed to determine whether this neurohormone could be used for potential treatment of infertility or at least in part to prevent testicular tissue damage. The application of CCl 4 has been previously proven to decrease male rat reproductive organ weight (testes, epididymis, and accessory sex organs) (Sönmez et al., 2014), which was corroborated by the results of this study as well. The obtained relative for testis weight indicates that the exposure to CCl 4 statistically significantly decreased tissue weight, compared to the control group. The application of MLT after CCl 4 did not cause a significant amelioration in testis tissue weight, however, the value was not significantly different from the one obtained for the control group either (data not shown).
Different factors, such as testosterone levels, are previously found to be related to a decrease in male reproductive organ weight (Sönmez et al., 2014), where the disturbance in androgenesis causes negative changes to testicular tissue mass (Klinefelter and Hess, 1998). The serum testosterone levels in rats exposed to CCl 4 were significantly decreased compared to both untreated animals (group I) and to those treated with MLT only (group II) (Figure 1). The acute treatment with MLT after exposure to CCl 4 (group IV) was not able to significantly prevent a decrease in the serum testosterone levels, but the testosterone levels in this group were not different from the ones measured in the serum of animals from the control group (Figure 1). Such results related to a decrease in the serum testosterone levels in the rats treated with CCl 4 are in agreement with a slight decrease in testes tissue mass ( Figure 1). Here again, the application of MLT did not prevent, at least not significantly, a decrease in testosterone concentrations, although the effect is notable since the values are not different from the ones found in the control group of animals. These results are in line with the described physiological functions of MLT. Melatonin directly inhibits, at the level of hypothalamus and hypophysis, the release of gonadotropin-releasing hormone and luteinizing hormone (Li, Zhou, 2015). Thus, it is unlikely that MLT can increase testosterone levels by acting at the hypothalamo-hypophyseal axis. The maintenance of testosterone levels is probably related to the MLT ability to preserve Leydig cells viability/ function that was challenged by the CCl 4 metabolites and/or excessively produced ROS.
Cell antioxidant potential is tightly regulated by both enzymatic and non-enzymatic antioxidant systems and any disturbance in these precisely regulated systems, the one that could be seen after CCl 4 application, leads to the disturbance in normal cell function (Bin-Jaliah, Sakr, 2018). In the present study, we determined the activity of different enzymes (SOD, CAT, GPx, and G6PD) and non-enzymatic antioxidant (GSH), whose function is related to the elimination of harmful ROS, and found them to be statistically significantly decreased, compared to the control group, after the exposure to CCl 4 (group III) ( Table I). The two most prominent enzymes involved in the removal of harmful ROS are CAT and SOD. These two enzymes are found to be significantly decreased in the rats exposed to CCl 4 , possibly due to the significant enzyme activation in order to eliminate ROS or due to the enzyme activity inhibition in the presence of ROS (Ighodaroa, Akinloye, 2018). When MLT was applied to the healthy animals, group II, it did not cause any significant changes in the enzymatic and non-enzymatic antioxidant capacities of rat testicles compared to group I ( Table I). The application of MLT after exposure to CCl 4 led to the preservation/generation of both enzymatic and non-enzymatic antioxidant defences in testicular tissue, however, in the case of SOD, CAT and GPx activity, and in the case of TAC concentrations the effect of MLT was found to be significant ( Table I). The observed activity of MLT might be related to the ability of this neurohormone to increase the expression of these enzymes, as well as to its ability to scavenge free radicals (Sokolović et al., 2018). Values are given as means values ± SD, n=6. One-way ANOVA followed by Tukey's test was used to compare the groups. * p<0.001; ** p<0.01; *** p<0.05 vs. Control (group I), # p<0.001; ### p<0.05 vs. CCl 4 treated animals (group III).

IV (CCl 4 + MLT)
Johnsen's testicular score 10 10 8.5 9 The enzymes that utilize GSH as a substrate for toxic products elimination, such as GPx, can be considered useful parameters in biomonitoring tissue oxidative capacities (Song et al., 2013). The activity of GPx, as well as the concentrations of GSH, in testicular tissue of rats exposed to CCl 4 , were found to be decreased (Table  I), probably due to their excessive "consumption" in order to eliminate toxic ROS. Although MLT was able to partially preserve tissue GPx activity, the concentration of GSH still remained decreased compared to the untreated control (Table I). Additionally, the enzyme whose activity was found to be decreased by the exposure to CCl 4 is G6PD (Table I). The G6PD deficiency is connected to the increased sensibility of cells towards different ROS, as well as to the action of NO and peroxynitrite (Ho, Cheng, Chiu, 2007). The application of MLT was previously reported to increase the activity of G6PD in both isolated erythrocytes (in vitro) and in rats a few hours (1-4 h) after its intraperitoneal injection (Ho, Cheng, Chiu, 2007). In a similar manner, although not absolute, we found that the application of MLT prevented a decline in testicular G6PD activity induced by CCl 4 application. Thus, this kind of activity could be brought in connection with MLT possible therapeutic potential for G6PD deficiency anemias (Ciftçi, Bilici, Küfrevioğlu, 2001) and yet again to its ability to increase tissue antioxidant capacities by increasing the intracellular (cytosolic) pool of NADPH (Ho, Cheng, Chiu, 2007). Determination of TAC reflects all substances (small molecules and proteins) that are involved in the protection of tissue from excessive ROS production (Sokolović et al., 2018). As expected, a decrease in TAC was found in the animals exposed only to CCl 4 , probably due to the significant reaction of antioxidant with ROS of different origin. When applied on its own, MLT did not alter TAC, which is not completely in accordance with some previous reports where MLT caused an increase in plasma TAC (Grzelak et al., 2005). However, when MLT was given to animals after CCl 4, the concentration of TAC was increased (Table I). Such discrepancy might be related to MLT ability to affect TAC only when there is significant ROS production, but not under normal (physiological) ROS production.
As explained in the introductory part of this study, an increase in ROS causes polyunsaturated fatty acids damage, thus initiating a chain reaction which potentially could alter spermatozoa cell membrane fluidity (Khan, 2012;Türk et al., 2016). Oxidatively modified lipids, estimated through the levels of MDA, were found statistically significantly increased in the animals exposed to CCl 4 , compared to the untreated ones ( Figure  2A). The treatment with MLT was able to completely prevent an increase in MDA that follow CCl 4 application, maintaining the levels of MDA at the concentrations similar to those found in healthy animals ( Figure 2A). An increase in MDA content was found to negatively correlate with sperm count, motility and morphology in men (Benedetti et al., 2012). Although this is the first study evaluating MLT's potential in preventing testicular tissue damage induced by CCl 4, it does not come as a surprise that MLT prevented an increase in MDA content. These results could partially be expected since some of the previous studies provided us with evidence that this neurohormone inhibits testicular tissue MDA formation induced by different toxic substances (Parlaktas et al., 2008;Mirhoseini et al., 2014;Sokolović et al., 2015;El-Shafaei et al., 2018). Apart from lipids, generated ROS could oxidatively modify amino acids i.e. proteins, where a protein carbonylation is considered an early biomarker of oxidative tissue damage (Ong et al., 2002). The amount of carbonylated protein content was significantly increased in the groups of animals that received CCl 4 , compared to the untreated ones ( Figure 2B). A single dose of MLT significantly prevented an increase in carbonylated protein content compared to the CCl 4 treated group, however, the level of the measured parameter still remained higher than in the control group of animals ( Figure 2B). Such protein modification is found to affect protease activity, thus causing a decrease in their activity and consequently their removal (Kumar et al., 2007). The administration of MLT failed to completely prevent the formation of this biomarker to the same extent as it did in the case of MDA ( Figure 2B). Previous studies also proved MLT's potential in preventing protein carbonylation process in testicular tissue of rats subjected to ischemia and reperfusion injury (Kurcer et al., 2008) or exposed to different toxic substances (Parlaktas et al., 2008), and radiation (Sokolović et al., 2015).
The pathohistological analysis did not reveal any lesions in testicular tissue of the control (group I) and the melatonin-treated (group II) group ( Figure  3A and B). In the groups that received CCl 4 , with or without MLT, microscopic analysis revealed occasional loss of germ cells compared to the untreated animals ( Figure 3C and D). These oxidative lipid/protein changes in testicular tissue partially overlap with a mild reduction in germ cell number (Figure 2 and  3). This reduction should not be regarded as mild since a short exposure to CCl 4 (only 24 hours) can be regarded as an acute one, and the extensive changes are expected after chronic exposure (Türk et al., 2016;Wang et al., 2018). The single previous study proved that MLT in a dose of 10 mg/kg applied after CCl 4 can prevent germ cell damage and can cause a reduction in testicular cells depolarization. The authors suggested that the mechanism of MLT action potentially involves regulation of tissue antioxidative capacities and downregulation of telomerase and caspase-3 (Wang et al., 2018). The role of NO in the pathogenesis of the CCl 4 mediated testicular tissue damage has been previously evaluated, and a significant increase in NO concentrations and iNOS activity was detected in the animals treated with this toxic chemical (Abdel Moneim, 2016), which is the case in the present study as well (Table I). Also, one may say that the exposure to this toxicant caused a shift in arginine metabolism in testicular tissue, where this metabolism is changed from arginase (significantly decreased) to iNOS (significantly increased) ( Table I). The iNOS activity and NO do not primarily originate from inflammatory cells, as is the case in some other tissues (e.g. liver) since no significant increase in inflammatory cells has been observed on the examined tissue sections. Probably the source of the investigated arginine/nitric oxide metabolism parameters are Sertoli, Leydig and germ cells whose function has been previously reported to be dependent on this signalling pathway (Auharek et al., 2012). Since the NO is found to be involved in testosterone production/secretion regulation (Jarazo et al., 2015), it is interesting that MLT maintained testosterone levels, but did not affect (decreased) NO production (Table I). This can be probably again explained by some other mechanism of action of MLT that affects cell function directly, rather than through other mediators. Besides the cell macromolecules damage induced by peroxynitrates, the generated NO is found to promote α-fodrin proteolysis in germ cells, consequently causing cell necrosis (Shiraishi, Naito, Yoshida, 2001). The findings of the present study are in line with such mechanism since the rare loss of germ cells was seen and the Johnsen's testicular score was almost identical in the two groups exposed to CCl 4 (Table II), and the levels of NO were not different either (Table I).
In conclusion, melatonin was found to be effective in reducing carbon tetrachloride induced rat testicular toxicity by increasing tissue's enzymatic and nonenzymatic capacities and by preventing lipid and protein oxidative damage. However, it was found MLT had very little impact on the investigated arginine/nitric oxide metabolism parameters and on the limited changes in the microscopic testicular tissue induced by CCl 4 . Our results suggest that MLT acts rather as a general antioxidant system stimulator and a radical scavenger than as a modulator of the nitric oxide signalling pathway.