Ameliorative effect of morin on dutasteride- tamsulosin-induced testicular oxidative stress in rat

Abstract

Objectives: Dutasteride-Tamsulosin (DUT-TAM), a drug of choice for the treatment of prostate enlargement (Benign Prostatic Hyperplasia, BPH) has been implicated in testic- ular toxicity. This study investigated the protective effect of morin, a plant-derived flavonoid on DUT-TAM-induced testicular toxicity in Wistar rat.
Methods: Twenty-four male Wistar rats (110–140 g) were randomly divided into four treatment groups (n=6). Group A animals served as the control and were administered olive oil, Group B animals were administered 5.4 mg/kg b.w. of dutasteride + 3.4 mg/kg b.w of tamsulosin., Group C animals were administered 100 mg/kg b.w. of morin, while Group D animals were administered DUT-TAM (5.4 mg/kg b.w. of dutasteride + 3.4 mg/kg b.w. of tamsulosin) and morin (100 mg/kg b.w.). The administration lasted for two weeks. Results: DUT-TAM-induced abnormal sperm morphology (31.8%), significantly reduced (p<0.05) sperm count, sperm motility, live-dead sperm ratio, testicular superoxide dis- mutase (SOD), catalase, glutathione-S-transferase (GST) and acid phosphatase (ACP) activities, as well as the levels of ascorbic acid and reduced glutathione (GSH) which were ameliorated by co-treatment with morin. Also, DUT-TAM- induced increase in testicular malondialdehyde level and the activities of alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT) and lactate dehydrogenase (LDH) were significantly reversed (p<0.05) by co-treatment with morin.
Conclusions: These results indicated the protective ability of morin against Dutasteride-Tamsulosin-induced testic- ular toxicity and oxidative stress.

Keywords: dutasteride; oxidative stress; prostate enlarge- ment; tamsulosin; testis.

Introduction

Antinflammatory drugs used as remedy for the symptoms associated with prostate enlargement (Benign Prostatic Hy- perplasia, BPH) are known to elicit deleterious side effects [1–3]. Dutasteride-Tamsulosin (DUT-TAM), one of the drugs of choice in the treatment of BHP, is a combination of 2 drugs, dutasteride and tamsulosin [4, 5]. Dutasteride, (5α, 17β)-N {2, 5, bis (trifluoromethyl) phenyl}-3-oxo-4-azaandrost-1-ene- 17-carboxamide) (Figure 1a), is a 5α-reductase inhibitor (5ARI), while Tamsulosin, (R)-5[2-[[2-(2-Ethoxyphenoxy) ethyl]amino]-propyl]-2-methoxybenzene-sulfonamide mon- ohydrochloride (Figure 1b), is an α1-adrenergic blocking agent (α-blocker) [6]. Dutasteride-Tamsulosin is associated with some adverse effects, including impotence, reduced libido and ejaculatory dysfunction [7]. While the side effects of tamsulosin include asthenia, dizziness, headache and postural hypotension [8], that of dutas- teride include increased blood glucose, glycated haemo- globin, dislipidemia, and increased activities of plasma transferases [9].
Compelling experimental evidences have suggested that many factors, such as dietary elements, inflammatory mediators and oxidative stress affect the incidence of BPH [10]. Also, the effect of BPH and its treatment with DUT-TAM on the antioxidant system of the testes might have delete- rious side effects on this organ in spite of its impressive antioxidant defense capacity in counteracting the destruc- tive tendencies of free radicals which may negatively affect its functions, including spermatogenesis [3, 11]. However, it is believed that supplementation with antioxidants may help in preventing tissues from drug-induced oxidative stress, as well as restoration of systemic antioxidant status to improve the clinical outcomes of patients with BPH [10].
Morin, or 3,5,7,2′,4′-pentahydroxy flavone (Figure 1c), is one of the polyphenol identified in Prunus dulcis (almonds), Ficus carica (Figs), Psidium guajava (Guava) and other members of the Moraceae plant family [12]. The antiapoptotic, antiinflammatory, antioxidant, hepatoprotective and neuro- protective activities of morin have been demonstrated by various studies [12, 13]. For example, morin augments anti- oxidative protection against liver carcinogenesis induced by N-nitrosodiethylamine [14]. This flavone also offers pro- tection, in vitro against H2O2-induced toxicity by curbing generation of reactive oxygen species via antioxidative mechanisms, including induction of catalase activity [15]. The effectiveness of morin in boosting cell viability and decreasing the reactive oxygen species level, as well as pro- tecting mitochondrial integrity and inhibiting DNA fragmen- tation of primary rat hepatocytes following exposure to high glucose concentration has also been reported [16].
The protective effects of administration of antioxidants along with several drugs as a strategy in preventing tissues from drug-induced oxidative stress is well-known [3, 17]. However, the protective capacity of morin against DUT-TAM- induced testicular damage and testicular oxidative stress has not yet been studied. This study therefore assumed and hypothesized that morin will protect the testis against DUT-TAM-induced oxidative damage. The objective of this study therefore was to evaluate the ameliorative effect of morin on DUT-TAM-induced testicular toxicity and oxidative stress.

Materials and methods

Chemicals and reagents

Dutasteride, tamsulosin hydrochloride (Duodart®) is a product of Catalent Germany Schorndorf GmbH, Schorndorf Germany. Gluta- thione (GSH), 1-chloro-2, 4-dinitrobenzene (CDNB), 5′,5′-din- trobenzoic acid (DTNB), thiobarbituric acid (TBA), and epinephrine were purchased from Sigma Chemical Company (London, UK). Morin is a product of Sigma-Aldrich chemical company, USA. Acid phos- phatase (ACP), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT) kits were products of Agape Diagnostics®, Switzerland GmbH. All other chemicals and reagents were purchased from British Drug House, Poole London.

Experimental animals

Twenty four male Wistar rats, of 125 g average weight (about 42 days old), used for this study were sourced from the College of Health Sciences, Ladoke Akintola University Osogbo, Osun State Nigeria. The animals were kept in the Animal Facility at the Chemical Sciences Department, Ajayi Crowther University Oyo, Oyo State, Nigeria. The animals were kept in well-aerated cages and allowed free access to animal chow (Vital feed®) and clean water ad libitum. The 24 animals were randomised into 4 groups of 6 animals per group. Both morin and DUT-TAM were prepared by dissolving them in olive oil prior to administration to the animals based on their corresponding body weight. DUT-TAM in doses of 5.4 mg/kg and 3.4 mg/kg body weight equivalent to 270 and 170 mg, respectively of the adult human dose calculated as described by Paget and Barnes [18], while the dose of morin used is the recommended dose from literature [19, 20] The grouping of the animals is as shown in Table 1. 1 mL each of the prepared drug (DUT-TAM) and morin were administered by oral gavage using oral intubator, once daily for 2 weeks. The animals were euthanized by slight pentobarbital overdose 24 h following the last administration.

Care of animals

The animals were given humane treatment as approved by the Ethical Review Committee of the Ajayi Crowther University, Oyo, Oyo State Nigeria (FNS/ERC/2018/0055) on December 23, 2018, in line with the Guide for the Care and Use of Laboratory Animals as outlined by the National Academy of Science and published by the National Institutes of Health [21].

Collection of tissue samples homogenate

place of test samples. Protein concentration was estimated by extrapolation from the curve prepared using known concentrations of bovine serum albumin.

Determination of superoxide dismutase activity

The activity of testicular superoxide dismutase (SOD) was estimated as described by Mishra and Fridovich [26]. Briefly, 0.2 mL of the diluted sample (sample: distilled water; 1:9), mixed with 2.5 mL of carbonate buffer (0.05 M, pH 10.2) was equilibrated in the spectrophotometer. Thereafter, 0.3 mL of freshly prepared epinephrine (0.3 mM) was added to the mixture and promptly mixed by inversion, while the blank contained 0.2 mL of distilled water in place of sample. The absorbance was monitored at 30 s interval for 2.5 min at 480 nm. The activity of SOD in the testes was calculated using the formular:
The testes were removed from the animals, rinsed in ice-cold 1.15% KCL, blotted and weighed for sub-cellular fraction preparation. For every two testes removed from the animals, one was fixed in 10% formalin solution for subsequent histopathology. The testes were weighed, macerated and homogenised in 4 volume of refrigerated 0.1 M phosphate buffer of pH 7.4, and then centrifuged for 15 min at 13,000 g using a temperature-controlled centrifuge (CENCOM Analytika, Ath- ens, Greece). The supernatant, termed post mitochondria fraction (PMF) was obtained and stored frozen for subsequent analysis.

Testicular and epididymal sperm number, progressive sperm motility assay and volume

Sperm from the minced testis and epididymis were obtained by filtra- tion, using a nylon mesh. The numbers of spermatozoa were estimated by counting, with the aid of a Neubauer hemacytometer [22]. Evaluation of sperm motility was done using ×400 magnification immediately they were isolated from the cauda, using the entire field per sample. The average was taken as the final motility, expressed in percentage [23].

Evaluation of sperm morphological and live-dead ratio

Sperm smears were prepared on glass slide, fixed in ethanol (95%), and stained with Eosin (1%) and Nigrosin (5%) stains. From each of the rats, a minimum of 100 sperms were examined for deformity, using the method described in Wyrobeck and Bruce [24].

Determination of catalase activity

The activity of testicular catalase was determined as described by Sinha [27]. Briefly, a reaction mixture of 2 mL of hydrogen peroxide (800 μ moles), 2.5 mL of 0.01 M phosphate buffer (pH 7.0) and 0.5 mL of diluted sample (1:50) was rapidly constituted at 25 °C, 1 mL of reaction mixture was withdrawn and promptly added to 2 mL dichromate/acetic acid solution at an intervals of 1 min to measure the residual H2O2 in the solution. The chromic acetate generated was quantified at 570 nm and the residual H2O2 estimated extrap- olating from the standard curve for hydrogen peroxide. The activity of catalase was expressed as micromole of H2O2 used up/min/mg protein.

Determination of glutathione-S-transferase activity

Glutathione-S-transferase, GST activity was estimated as described by Habig et al. [28]. Briefly, assay mixture contained 30 μL of 0.1 M reduced glutathione, 2.79 mL 0.1 M phosphate buffer (pH 6.5), 150 μL CDNB (3.37 mg/mL) and 30 μL tissue sample. Absorbance was read at 340 nm against the blank after 60 s. The activity of GST in the sample was estimated using the relation.

Estimation of ascorbic acid level

The concentration of testicular ascorbic acid in samples was estimated as described by Jagota and Dani [29]. Briefly, 0.5 mL of homogenate and 0.8 mL of 10% TCA combined, mixed, and left on ice for 5 min and thereafter subjected to centrifugation (3000×g for 5 min). 1 mL of supernatant was added to 0.2 mL of diluted Folin’s solution (Folin’s solution: ddH2O, 1:10) and stirred strongly. The absorbance of the blue color developed was read after 10 min at 760 nm. The ascorbic acid concentration (μg/mL) was obtained by extrapolating from the ascorbic acid standard curve prepared using different concentrations of ascorbic acid using a procedure similar to the one described above.

Determination of reduced glutathione content

The testicular reduced glutathione (GSH) in the samples was quanti- fied by the method described by Jollow et al. [30]. Briefly, the reaction mixture contained 1.8 mL of distilled water, 0.2 mL of sample and 3 mL of sulphosalicylic acid (4%). The reaction mixture was filtered after 150 s, the resulting filtrate (1 mL) and 4 mL phosphate buffer (0.1 M) were mixed, followed by the addition of 0.5 mL Ellmans’ reagent. The blank was prepared as appropriate, and the absorbance (412 nm) was read, and the concentration of GSH in tissues determined by extrap- olation from the GHS standard curve.

Assessment of lipid peroxidation

The level of testicular lipid peroxidation was estimated by quantifica- tion of thiobarbituric acid reacting substances (TBARS) in the homog- enate sample as described by Varshney and Kale [31]. Briefly, 3.2 mL Tris-KCl buffer, 1.0 mL of 30% TCA, 0.8 mL of the test sample, 1.0 mL of 0.75% thiobarbituric acid were all mixed together. The temperature of the mixture was raised to 95 °C and maintained at same for 1 h in a water bath. The temperature of the mixture brought to 25 °C and then centri- fuged at 4000 g for 15 min. The absorbance of the clear supernatant was read at 532 nm, using distilled water as blank. The lipid peroxidation (nmole/mg protein) was estimated using the following equation: MDA = Abs × Volume of reaction mixture E532 × Volume of sample × mg protein where: MDA is malondialdehyde, E532=1.56×105 M−1 Cm−1, the molar extinction coefficient for MDA=

Assays for testicular marker enzymes

The activities of testicular γ-glutamyl transferase (GGT), lactate dehy- drogenase (LDH), acid phosphatase and alkaline phosphates (ACP and ALP) were estimated according to the manufacturer’s instruction (Agape
Diagnostic kits). Estimation of the activity of GGT was done following the method described by [32] which was based on the quantitation of 5 amino-2-nitrobenzoate (at 405 nm), which was generated when GGT acts on L-γ-glutamyl-3-carboxy-4-nitroanilide. ALP and ACP activities were estimated according to the method of Tietz [33]. The hydrolysis of p-nitrophenyl phosphate produces the yellowish p-nitrophenol, the resulting colour intensity, which is proportional to enzyme activity, can then be monitored at 405 nm. Estimation of LDH activity was according to the method of Cabaud and Wroblewski [34]. LDH catalyses the reductive conversion of pyruvate by NADH to lactate and NAD+; the catalytic concentration of LDH is determined by measuring rate of decline in NADH activity at 340 nm.

Histopathology of the testis

Histopathology of the testes was done using the method of Krause [35]. Briefly, small sections of the testes were cut out and fixed in 10% formalin for a day (24 h). The tissues were dehydrated through ascending grades of ethanol to absolute ethanol and fixed in paraffin wax. 5 μm of the sample was carefully cut, using a microtome; the cut sample was stained in aqueous dyes, mounted on a glass using Bal- sam mounting medium was put on the tissue section and a thin glass cover slip was placed on the mounting medium, observed using ×400 magnification of the microscope, photomicrographs were then ob- tained in a bright field.

Statistical analysis

Data was expressed as Mean ± SD. The data were analyzed using one way analyses of variance (ANOVA), comparison between treatments and control was by Duncan Multiple Range, using SigmaPlot® soft- ware. Differences were considered as significant at p-values less than 0.05 (p<0.05).

Results

Protective effect of morin on dutasteride- tamsulosin-induced alterations in sperm parameters in rats

Table 2 shows the potential protective effect of morin on DUT-TAM-induced alterations in the sperm parameters of rat. DUT-TAM significantly reduced (p<0.05) the percentage of sperm motility, live-dead sperm ratio as well as sperm count by 31.6, 11.3 and 36.2% respectively when compared with the control. However, co-administration of morin with DUT-TAM significantly reversed (p<0.05) the observed reduction in these sperm parameters relative to the DUT-TAM treated group. Table 3 presents the protective effect of morin on DUT-TAM-induced changes in the sperm morphology of rat. DUT-TAM significantly increased (p<0.05) the % of abnormal sperm morphology by 31.8% when compared with the con- trol. However, the co-administration of DUT-TAM and morin significantly reduced (p<0.05) the abnormality relative to the DUT-TAM treated group.
The protective effect of morin on DUT-TAM-induced changes in testicular ascorbic acid and GSH is shown in Figure 2. Ascorbic acid and GSH levels were significantly decreased (p<0.05) in DUT-TAM-treated group by 46.2 and 42.2% respectively when compared with the control. The DUT-TAM - (5.4 mg/kg b.w dutasteride + 3.4 mg/kg b.w of tamsulosin); Morin=(100 mg/kg b.w). The results are expressed as Mean ± SD for six rats in each group. *Significantly different (p<0.05) from the control. aSignificantly different (p<0.05) from dutasteride/tamsulosin group. Values in parentheses represent percentage (%) decrease/ increase.

Protective effect of morin on dutasteride- tamsulosin-induced alterations in the activities of testicular SOD, catalase and GST in rats

The protection provided by administration of morin against DUT-TAM-induced alterations in testicular SOD, catalase and GST are presented in Table 4. The activities of these enzymes were reduced by 51.9, 40.6 and 41.3% respectively in the DUT-TAM-treated rats relative to control. However, administration of morin significantly protects against decreased in SOD, catalase and GST activities relative to the DUT-TAM treated group. co-administration of DUT-TAM and morin significantly attenuated (p<0.05) the decreased in ascorbic acid and GSH levels relative to the DUT-TAM group. Figure 3 shows protection provided by administration of morin on DUT-TAM-induced alterations in testicular lipid peroxida- tion in rats. Testicular lipid peroxidation was significantly increased (p<0.05) by 49.8% by DUT-TAM-treatment when compared with the control. The combined treatment of DUT-TAM and morin was seen to significantly attenuate the increase in testicular malondialdehyde when compared with the DUT-TAM group.

Effect of morin on dutasteride-tamsulosin- induced changes in the activities of testicular GGT, ALP, ACP and LDH in rats

Figure 4 shows the protection afforded by administration of morin on DUT-TAM-induced alterations in testicular activities of GGT and ALP in rats. Testicular GGT and ALP activities significantly increased (p<0.05) in DUT-TAM group by 56.1 and 27.6% respectively relative to the con- trol. However, the combined administration of DUT-TAM and morin attenuated this increase relative to the DUT-TAM group. The protective effect of morin on DUT-TAM-induced changes on testicular ACP and LDH activities is shown in Figure 5. The ACP activity was significantly decreased (p<0.05) in DUT-TAM treated group by 35.7% when compared to the control. In contrast, LDH activity was significantly increased (p<0.05) by 35.6% when compared to the control. Co-administration of DUT-TAM and morin was seen to significantly ameliorate the decrease in ACP activity relative to the DUT-TAM group. Also, the co-treatment of DUT-TAM and morin was seen to attenuate the increase in the LDH activity relative to the DUT-TAM group.

Effect of morin on dutasteride-tamsulosin treatment on the histology of the testes

The Photomicrograph of testicular tissue is shown in Figure 6. In this photomicrograph, the DUT-TAM-treated group has some visible damage to the testicular morphology. The control group shows testicular tissue with normal architecture, and the tubules are well disposed with active spermatogenesis. However, the co- treatment of DUT-TAM and morin was seen to reverse the damages to the testicular tissues relative to the DUT-TAM group.

Discussion

This study investigated the protective role of orally administered morin in DUT-TAM-induced testicular toxicity in rat. Dutasteride-Tamsulosin is used in the treatment of the symptoms of prostate enlargement/BPH; but it may provoke oxidative stress or diminish systemic antioxidant capacity in subjects. The spermatogenesis function of testis can be impaired by oxidative stress, even though, it has an efficient antioxidant defence systems; thus, the resultant oxidative stress occasioned by the drug can lead to poor sperm quality thereby resulting in male infertility [36, 37].
Morin, a bi-flavonoid is a potent antioxidant derived basically plants products [38]. It has the ability to scavenge free radicals, thereby preventing cellular oxidative damage [39]. Considering the numerous antioxidative properties of morin, we hypothesized that morin could inhibits the DUT-TAM-induced dependent damages in the testis of rat.
In this study, results indicated that sperm count, motility as well as live-dead ration were significantly reduced by DUT-TAM administration. Decreased sperm count and motility is usually associated with onset of testicular tissue damage, attributable to oxidative damage. Consequently, a confirmed indicator of elevated ROS level in the testes is a significant decrease in sperm motility [40]. Elevated ROS levels modulate enzymatic content of the sperm, leads to increased lipid peroxidation, and consequently hinders membrane fluidity and sperm motility [41]. Interestingly, administration of morin led to significantly improvement in the number, the viability and the motility of sperm cells. Previous studies have shown the protective effect of morin on silver nanoparticles-induced [42] and titanium dioxide nanoparticles-induced [43, 44] sperm abnormalities as revealed by reversal of silver nanoparticles and titanium nanoparticle-induced reduced sperm motility, viability percent, sperm concentration, and a higher abnormality ratio. Thus, results from this study indicated augmentation of tissue antioxidative protective mechanism by morin, which in turn ameliorated DUT-TAM-elicited damages in the testis and enhanced the number of sperm, sperm live-dead ratio and motility.
Observation from this study also showed that there was increased number of abnormal sperm cells in DUT-TAM- treated rats, indicating that the toxic effect of DUT-TAM might be through its impact on the physiology of Sertoli cells, as a result of DUT-TAM-induced oxidative damage. Also, significant decrease in the activities of the antioxidant enzymes, including, catalase, GST and SOD, were observed following treatment of animals with DUT-TAM. The antiox- idant enzymes, SOD and catalase are known as major components of the innate antioxidant protective system against ROS [45]. Superoxide dismutase and catalase take care of superoxide radical and hydrogen peroxide respec- tively, both working together to convert these toxic ROS to innocuous products, such as water and oxygen. The observed decrease in the activities of these antioxidant en- zymes by DUT-TAM might have exposed the testes to oxidative assault [36]. Furthermore, GST, an important enzyme in the elimination of drugs and toxicant, is also involved in peroxidase activity. The peroxidase activity of GST relies on availability of GSH, therefore, systemic bal- ance of these enzyme are important to mop-up ROS, including, superoxide anion and peroxide generated in the testes [46]. Reports have shown that morin protects cells from hydrogen peroxide-induced damage by inhibiting ROS generation and by inducing catalase activation [15]. Co- administration of morin therefore protects the testes against oxidative stress by enhancing the effectiveness of the anti- oxidant enzymes.
Ascorbic acid is an important antioxidant component of the cell, and it is involved in prompt scavenging ROS and other reactive species [3]. It is also essential in the replen- ishment of vitamin E, an important component in the proper functioning of the cell membrane [47]. The ascorbic acid in the testes is kept in a reduced state by GSH-dependent dehydroascorbate reductase. The level of GSH is an indicator of the oxidative stress status of the cell [48]. Thus, changes in GSH concentration may affect the general redox status of the testis [49]. The decrease in the antioxidant status of the rat implies an increased vulnerability of the testicular compo- nents to free radicals produced by the drug. The results from this study showed that DUT-TAM caused a significant reduction in the testicular GSH and ascorbic acid. Membrane lipids are prime targets for the damaging effects of free radicals [50]. Thus, uncontrolled oxidative stress may result in membrane lipid peroxidation and, ultimately, testicular damage and loss of testicular functions [3]. In this study, the observed elevation in the level of TBARS, a measure MDA in the testis of animals treated with DUT-TAM implied increased lipid peroxidation as a result of oxidative stress precipitated by the drug. Co-treatment with morin protected the testis via attenuation of lipid peroxidation and reduced production of free radical derivatives as evident from the decrease level of testis TBARS. Therefore, morin offered protection against oxidative stress by scavenging free radicals.
The role of morin as an in vivo anti-lipoperoxidative agents have been confirmed in several studies [42–44, 51, 52]. The activities of marker enzymes in the testes, including, ACP, ALP, LDH and GGT are accepted as indices of sper- matogenesis. Alkaline phosphatase and LDH perform vital roles in spermatogenesis, sperm preservation and viability [53]. The observed elevation in the activity of testicular ALP and LDH indicates possible degeneration of the testes [54]. Lactate dehydrogenase is directly related with spermato- genesis and testicular development, thus, the increased activity of the enzyme following the administration of DUT-TAM could be due to systemic attempt to boost sper- matogenesis and testicular development after oxidative onslaught occasioned by the drug [47]. Gamma-glutamyl transferase is a marker enzyme of Sertoli cell function of testis. The noticeable increase in the activity of testicular GGT in DUT-TAM-treated animals could be associated with the relative activity of GGT in the testicles and epididymis, or an alteration in the role of the enzyme in germ cells development and sperm maturation [55]. In the spermatogenic cells, there is an increase in the specific activity of ACP as the germ cells differentiate from spermatogonia into spermatocytes and spermatids [56]. In this study, a significant decrease in the activity of ACP following treatment with DUT-TAM as observed is a characteristics of testicular atrophy associated with damage to germ cells by many xenobiotics in the liter- ature [57]. However, co-treatment of DUT-TAM along with morin, effectively ameliorated these observed effects in the activities of these enzymes.

Conclusion

From this study, it may be concluded that DUT-TAM im- pairs testicular antioxidant system and causes degenera- tive changes in the germ cells. However, morin, a potent antioxidant, positively modulates the effect of the drug on the antioxidant status by ameliorating the oxidative dam- age and effectively protects against DUT-TAM-induced testicular oxidative stress; this may be due to its intrinsic antioxidant properties. These results demonstrate the cytoprotective ability of morin against DUT-TAM-induced testicular toxicity.

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