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Improved Histoarchitectural Changes with Angiotensin Receptor Blockers in Early Testicular and Cauda Toxicity in Rats/Mejora de los Cambios en la Histoarquitectura con Bloqueadores de Receptores de Angiotensina en la Toxicidad Precoz Testicular y de Cauda en Ratas.

INTRODUCTION

Male hypogonadism is a common problem in patients with liver cirrhosis which partially improves after liver transplantation (Foresta et al., 2008). The underlying mechanism of such problem is complex and not well explained. Liver is involved in metabolism of sex hormones, thus pronounced changes in such metabolism, free serum testosterone level, and sex hormone-binding globulin level in blood could be possible causes (Nitsche et al., 2014). Thioacetamide-induced liver damage was associated with testicular toxicity (Kang et al., 2006). In testicular toxicity, use of histopathological measures is more sensitive parameter of testicular damage than use of testis weight and sperm counts and thus can detect early toxicity.

Proliferating cell nuclear antigen (PCNA) is a nuclear matrix protein necessary for several cell cycle pathways. Significant decreases in PCNA immunohistochemistry measured on formalin-fixed paraffin-embedded rat testes have been used to identify and quantify of the proliferation-related toxicity. Thus PCNA assay is potentially useful in vivo biomarker for detecting early testicular toxicity and for follow-up of compounds with low testicular toxicity (D'Andrea et al., 2008). The block of effects of angiotensin II whether by angiotensin converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs) have been found to protect the testicular tissue against multiple insults (Alves-Pereira et al., 2014). Both captopril and telmisartan reversed the cadmium-induced testicular damage with nonsignificant differences in-between. They increased serum testosterone level and the testicular reduced glutathione and decreased testicular MDA level and expression of testicular caspase-3 (Fouad & Jresat, 2013). ACEIs and ARBS are used to treat cardiovascular diseases like hypertension and heart failure, to prevent renal failure in cases of hypertension and/or diabetes, and to decrease the risk of stroke. Generally ARBs are more tolerated than ACEIs.

Taken together, this study was designed to evaluate the morphological changes in testicular histoarchitecture before and after treatment with angiotensin receptor blockers in early testicular toxicity induced by low dose thioacetamide in rats.

MATERIAL AND METHOD

Animals and Drugs. The study was approved by the Institutional Research Ethics Committee. It agreed with the International guidelines for use of Laboratory animals. Sprague-Dawley male rats (200-250 g) were housed in cages at 22 [degrees]C room temperature with food and water ad libitum. Drugs and chemicals were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA).

Induction of low-grade testicular toxicity. Low grade testicular toxicity was induced in rats by injection of thioacetamide (TAA, 50 mg/kg) intraperitoneally once daily for two weeks. This small dose was used before, to induce low-grade hepatotoxicity. The rats were randomly divided into four groups (n = 8): negative control group (distilled water), TAA (positive control) group, candesartan cilexetil group (0.2 mg/kg/day) (Gaur & Kumar, 2011), and losartan potassium group (7.5 mg/kg/day) (Croquet et al., 2002). Treatments were given orally for three weeks. At the end of treatment duration, blood was collected through the retroorbital plexus, centrifuged at 4000 rpm for 5 min, and serum was separated and kept at -80[degrees]C for biochemical measurements. Rats were sacrificed by cervical dislocation and the male system was removed as one block for preparation of the testicular homogenate, and histopathological and immunohistochemical examination. For histopathological and immunohistochemical examination the testis and cauda were fixed in 10 % neutral buffered formalin then processed for paraffin sections. Slides were stained by hematoxylin and eosin for general structure, periodic acid Schiff (PAS) for basement membrane, Masson trichrome for collagen, and immunohistochemically for caspase-3 and proliferating cell nuclear antigen (PCNA). Photographs were taken for all groups and compared (Suvarna et al., 2013).

Assay of serum testosterone. The serum testosterone level was measured by an ELISA kit (Sigma, MO, US) as per instructed by the manufacturer.

Measurement of testicular MDA and GSH. The testes were homogenized in ice-cold medium of 50 mM Tris-HC1 (pH 7.4), the homogenate was centrifuged at 1000 xg for 10 min at 4 [degrees]C, the supernatant was used for measurement of MDA & GSH. For estimation of MDA, the testis homogenate was incubated in 10 % trichloroacetic acid and 0.67 % thiobarbituric acid (1 mL of each) for 30 min (Ohkawa et al., 1979). The GSH was determined by the reduction of Elman's reagent (Ellman, 1959).

Immunohistochemistry of testis and cauda

Caspase-3. The testicular tissue sections were dewaxed and rehydrated. Endogenous peroxidase was blocked using 0.3 % [H.sub.2][O.sub.2] in methanol for 15 min. Then sections were incubated with primary diluted polyclonal antibodies for caspase-3 (Thermo Fisher Scientific Co., USA) for 30 min. and subsequently with biotinylated polyvalent secondary antibody (Thermo Fisher Scientific Co., USA) for 30 min. Metal Enhanced DAB Substrate Working Solution was added to the tissue followed by counterstaining with hematoxylin stain (Kim et al., 2001; Elgawish & Abdelrazek, 2014).

Proliferative cell nuclear antigen (PCNA). After deparaffinization and rehydration steps, the testicular tissue sections were incubated in methanol containing 0.3 % H202 and normal goat serum for 15 minutes to decrease non-specific peroxidase reactions. Then, they were incubated for 30 minutes with a primary antibody against PCNA (Santa Cruz Biotech., Texas, US) and subsequently with goat anti-rat IgG as a secondary antibody (Sigma, MO, USA). After that, tissue sections were incubated with the peroxidase/anti-peroxidase complex for 90 min. Diaminobenzidine was used as chromogen followed by counterstaining with hemotoxylin and examination by a light microscope (Sternberger, 1986; Altay et al., 2003; Dkhil et al.,2016).

Statistical analysis. Data was expressed as means [+ or -] SEM and analyzed using SPSS version 18. One-way ANOVA followed by Tukey's multiple comparison post-hoc test was used to assess differences between groups. P < 0.05 was considered to be statistically significant.

RESULTS

Serum testosterone and Testicular MDA and GSH. TAA decreased serum testosterone level, increased testicular MDA level while decreased testicular GSH level compared with the normal control group. Both candesartan and losartan significantly reversed these change with non-significant difference in-between (Table I).

The histoarchitectural study. Both candesartan and losartan protected against TAA-induced testicular and cauda toxicity as shown using the following stains: Hematoxylin and eosin (Figs. 1 and 2), Masson trichrome (Fig. 3), PAS (Fig. 4), Caspase-3 (Fig. 5), and PCNA (Fig. 6).

DISCUSSION

Liver fibrosis could decrease male fertility (Foresta et al.). The increased markers of lipid peroxidation and the decreased natural antioxidants in the testicular tissue supported the postulation of a direct toxic effect of TAA on testicular tissue via stimulating oxidative mechanisms (Kang et al.). The effect of TAA may be directly on the spermatogenic cells or via damage of their supporting Sertoli cells (Lenzi et al., 2002; Cheng, 2014). In the present study, the decrease in Leydig cells population could be explained in view of reported testosterone changes in patients with liver fibrosis (Nitsche et al.). It was found that exposure to flutamide during intrauterine life resulted in a chronic apoptotic germ cell death in the adult rat testicular tissue correlated with an increase in the expression and activation in germ cells of caspases-3 and -6 which are two important components in the apoptotic machinery (Omezzine et al., 2003). In rats, lead acetate significantly increased level of testicular caspase-3 expression. PCNA immunohistochemistry showed three differences between the testes of control and hypothyroid rats indicating its usefulness as a marker to differentiate between the two regarding germ cell kinetics and spermatogenesis (Tousson et al., 2011).

PAS was used in the present study to demonstrate any changes in the tubule basement membrane and the result showed that it was thickened in TAA group which may have a role in impaired spermatogenesis and germ cell degeneration. Thickening of basement membrane and excessive deposition of extracellular matrix proteins are characteristics of fibrotic disorders of multiple organs including liver and kidney (Rosenbloom et al., 2010). In rats, PAS staining was used as a histomorphological method to detect disorders in seminiferous tubular histoarchitecture (Ogedengbe et al., 2016). In the present study, both candesartan and lorsartan modulated the TAA-induced testicular toxicity and all histological and immunohistochemical alteration in both testicular tissue and cauda epididymis tubules with non-significant differences in-between. Candesartan relieved the cisplatin-induced testicular damage by modulating the expression patterns of the testicular nephrin-podocin complex (Enatsu et al., 2015). In rats, losartan partially reversed the angiotensin II type 1 receptor dependent increase in interstitial fibrosis and impairment of spermatogenesis that occurred post-vasectomy due to oxidative stress (Shiraishi et al., 2003). Losartan protected against testicular germ cell apoptosis caused by an experimental varicocele (Bolat et al., 2016). Limitations of the current study include lack of quantitative histological analysis and use of only two ARBs members.

In conclusion, treatment with ARBs (candesartan and losartan) significantly reversed thioacetamide-induced low-grade testicular and cauda toxicity in rats. This could be potentially useful for early treatment of male patients with occupational toxicant-induced reproductive dysfunction especially if they are using ARBs for other comorbidities.

ACKNOWLEDGEMENTS. Funding: This project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah under grant no. J-735-247-38. The authors, therefore, acknowledge with thanks DSR for technical and financial support. The participation of the medical students Yasser A. Alkhairy, Abdulrahman M. Khafagy, Hassan Gabbani, Mohammed Abdulhamid Alfuraydi, Hesham Flemban, and Tarek Abou-Taleb is gratefully acknowledged.

FADDLADDEEN, K. A.; MURAD, H. A. M. & ALI, S. S.Mejora de los cambios en la histoarquitectura con bloqueadores de receptores de angiotensina en la toxicidad precoz testicular y de cauda en ratas. Int. J. Morphol., 37(2):515-521, 2019.

RESUMEN: La disfuncion reproductiva es una complicacion por muchas enfermedades y toxinas. Su diagnostico y tratamiento tempranos son inmensamente importantes. Aqui se evaluaron los cambios morfologicos en la histoarquitectura en la toxicidad precoz testicular y cauda antes y despues del tratamiento con bloqueadores de receptores de angiotensina. Se indujo dano testicular de bajo grado usando tioacetamida (TAA, 50 mg / kg / dia) por via intraperitoneal durante dos semanas en ratas. Las ratas se dividieron aleatoriamente en cuatro grupos (n = 8) tratados diariamente por via oral durante tres semanas de la siguiente manera: control normal (agua destilada), TAA (control positivo), TAA + candesartan (0,2 mg / kg) y TAA + losartan (7,5 mg / kg). Se midieron la testosterona serica, el malondialdehido testicular y el glutation. Los cambios en la histoarquitectura de los testiculos y la epidermis de la cauda se evaluaron mediante Hematoxilina y Eosina para determinar la estructura general., con tricromicro de Masson para el colageno, acido periodico de Schiff para la membrana basal y la caspasa-3 y el antigeno nuclear de celulas proliferantes (PCNA) para analisis inmunohistoquimico. Las ratas TAA mostraron disminucion de la testosterona serica y glutation testicular, aumentos en el malondialdehido testicular, cambios degenerativos y apoptosis en celulas germinales, engrosamiento de la lamina basal tubular y aumentos en la expresion de la caspasa 3, y disminucion en la expresion de PCNA. Los ARB (candesartan y losartan) revirtieron significativamente estos cambios con diferencias no significativas en el medio. El tratamiento con BRA (candesartan y losartan) revirtio significativamente la toxicidad testicular y cauda inducida por TAA en ratas. Esto podria ser potencialmente util para el tratamiento temprano de pacientes con disfuncion reproductiva inducida por toxicos ocupacionales, especialmente si estan usando BRA para otras comorbilidades.

PALABRAS CLAVE: Caspasa-3; Masson; Antigeno nuclear de celulas en proliferacion; Bloqueadores de receptores de angiotensina.

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Khadija Abduljalil Faddladdeen (1); Hussam Aly Murad (2) & Soad Shaker Ali (3)

(1) Department of Biology, Faculty of Science, King Abdulaziz University (KAU), Jeddah, Saudi Arabia (SA).

(2) Department of Pharmacology, Faculty of Medicine, Rabigh, KAU, Jeddah, Saudi Arabia.

(3) Department of Anatomy and Histology, Faculty of Medicine, KAU, Jeddah, Saudi Arabia.

FADDLADDEEN, K. A.; MURAD, H. A. M. & ALI, S. S. Improved histoarchitectural changes with angiotensin receptor blockers in early testicular and cauda toxicity in rats. Int. J. Morphol., 37(2):515-521, 2019.

Corresponding author:

Prof. Hussam Aly Sayed Murad

Department of Pharmacology

Faculty of Medicine Rabigh

King Abdulaziz University

Jeddah

SAUDI ARABIA

E mail: hamurad@kau.edu.sa

HussamMurad@med.asu.edu.eg

muradha2000@yahoo.com

Received: 16-10-2018

Accepted: 14-01-2019
Table I. Effects of treatment with ARBs on parameters of
thioacetamide-induced testicular toxicity.

Group            Serum                   Testicular GSH
                 testosterone
                 (mmol/L)                ([micro]g/g testis)

Normal control   3.26 [+ or -] 0.14      57.63 [+ or -] 0.90
Thioacetamide    1.71 [+ or -] 0.11      38.59 [+ or -] 1.55
TAA+Candesartan  2.82 [+ or -] 0.18 (*)  55.31 [+ or -] 1.33 (*)
TAA+Losartan     2.90 [+ or -] 0.14 (*)  53.75 [+ or -] 1.09 (*)

Group            Testicular MDA
                 (mmol/mg protein)

Normal control   16.91 [+ or -] 0.45
Thioacetamide    29.61 [+ or -] 0.63
TAA+Candesartan  17.65 [+ or -] 0.32 (*)
TAA+Losartan     18.14 [+ or -] 0.38 (*)

Data was expressed as mean [+ or -] SEM. (*): P < 0.05 vs. thioactamide
(TAA) group.
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Author:Faddladdeen, Khadija Abduljalil; Murad, Hussam Aly; Ali, Soad Shaker
Publication:International Journal of Morphology
Date:Jun 1, 2019
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