Erectile dysfunction drugs altered the activities of antioxidant enzymes, oxidative stress and the protein expressions of some cytochrome P450 isozymes involved in the steroidogenesis of steroid hormones

Abstract

Objectives: Infertility is a global health problem with about 15 percent of couples involved. About half of the cases of infertility are related to male-related factors. A major cause of infertility in men is oxidative stress, which refers to an imbalance between levels of reactive oxygen species (ROS) and antioxidants. Erectile dysfunction drugs (EDD), known as phosphodiesterase inhibitors (PDEIs), have been used for the treatment of ED. It has been shown that oxidative stress plays an important role in the progression of erectile dysfunction. Oxidative stress can be alleviated or decreased by non-antioxidants and antioxidant enzymes. The present study was undertaken to determine if these compounds could have a role in the incidence of infertility, especially after long-term use. Therefore, the present study aims to investigate the effect of EDD on the activities of antioxidant enzymes, free radical levels as well as the protein expression of different cytochrome P450 isozymes involved in the steroidogenesis of different hormones. In addition, the activity of both 17β-hydroxysteroid dehydrogenase and 17-ketosteroid reductase were assayed. The architectures of both livers and testes cells were investigated under the influence of EDD.

Methods: A daily dose of Sildenafil (1.48 mg/kg), Tadalafil (0.285 mg/kg) and Vardenafil (0.285 mg/kg) were administered orally to male rabbits for 12 week. Western immunoblotting, ELISA, spectrophotometric and histopathological techniques were used in this study.

Results: The present study showed that Sildenafil, Vardenafil, and Tadalafil treatments significantly decreased the levels of glutathione and free radicals in both livers and testes of rabbits. Also, Vardenafil and Sildenafil induced the activity of superoxide dismutase and catalase whereas, glutathione S-transferase, glutathione reductase, and glutathione peroxidase activities inhibited in livers of rabbits. The protein expression of cytochrome P450 isozymes (CYP 11A1, 21A2, and 19C) which are involved in the steroidogenesis was markedly changed in both livers and testes of rabbits after their treatments for 12 weeks. After the treatment of rabbits with these medication, the protein expression of CYP11A1 was slightly down-regulated in both livers and testes except Sildenafil up-regulated such protein expression. In addition, the protein expressions of CYP11A1 and CYP 19C in both livers and testes were down-regulated after treatment of rabbits with Sildenafil, Vardenafil, and Tadalafil for 12 weeks. Also, these drugs inhibited the activity of both 17β-hydroxysteroid dehydrogenase and 17-ketosteroid reductase in testes of rabbits. Moreover, Sildenafil, Vardenafil, and Tadalafil-treated rabbits showed a decrease in spermatocytes and the number of sperms in the testes.

Conclusions: It is concluded that ED drugs induced the activities of both SOD and catalase which consequently decreased MDA level. Decrement in MDA levels and oxidative stress could therefore sustain the erection for a long period of time. On the other hand, it is not advised to use these drugs for a long-term since the protein expressions of CYP isozymes involved in steroidogenesis as well as the numbers of spermatocytes in testes were decreased.

Fig 1. Influence of Sildenafil, Tadalafil, and Vardenafil on the protein expression of CYP11A1 (A), CYP21A2 (B), and CYP19 (C) after treatment of male rabbits with three times a week for 12 weeks.

Lanes 1, 2, 3, and 4 (testes and liver) represent control, Sildenafil, Tadalafil, and Vardenafil, respectively.

Fig 2

(A) Photomicrograph of T.S in the liver of control rat showing normal liver architecture, normal hepatocytes (H), central vein (C.V), and sinusoidal space(S) (H&E, X 400). (B): Photomicrograph of T.S in the liver of Sildenafil- treated group showing: Loss of the normal hepatocytic structure, fatty degeneration with pyknotic nuclei (black square & black arrow), dilation of central vein (C.V) & blood sinusoids (S) with congestion (black dotted arrow. (H&E, X400). (C): Photomicrograph of T.S in the liver of Tadalafil group treated rats showing: Loss of the normal hepatocytic architecture, vacuoles, degeneration of hepatocytes with pyknotic nuclei (black square& black arrow), fibrosis and congestion around the portal area and (black circle), dilation and congestion of portal vein (blue dotted arrow), congestion in hepatocytes and blood sinusoids (black dotted arrow), binucleated hepatocyte (green dotted arrow). (H&E, X400). (D): Photomicrograph of T.S in the liver of Vardenafil-treated group showing: Hydropic swelling ballooning hepatocytes (blue dotted arrow), fat vacuoles (green circle). (H&E, X400).

Fig 3

(A): Photomicrograph of a T.S in rabbit testis of the control group showing the normal morphological architecture of seminiferous tubules (S) and Leydig cell (L), the different stages of spermatogenesis; spermatogonia (black dotted arrow), primary spermatocyte (yellow dotted arrow), secondary spermatocyte (blue dotted arrow), spermatids (green arrow) and lumen filled with spermatozoa (H&E, X 400). (B): Photomicrograph of a T.S in rabbit testis of Sildenafil -treated group showing severe degeneration and disorganization seminiferous tubules (black square); sloughing germinal epithelia of seminiferous tubules into the lumen with the absence of spermatogenic stages, exfoliation of germ cells with the absence of spermatozoa and presence of cellular debris and immature germ cells (black square), vacuolations between germ cells (black arrow). Atrophy in Leydig cell (L), hemorrhage in seminiferous tubules (yellow circle), and enlargement space between seminiferous tubules (green dotted arrow) (H&E, X 400). (C): Photomicrograph of a T.S in rabbit testis of Tadalafil -treated group showing seminiferous tubules lost its shape and appeared with irregular outline and disorganization in seminiferous tubules (black square); sloughing germinal epithelia of seminiferous tubules into the lumen with the absence of spermatogenic stages, exfoliation of germ cells with an absence of spermatozoa and presence of cellular debris and immature germ cells (black square), vacuolations between germ cells (black arrow). Atrophy in Leydig cell (L), hemorrhage in seminiferous tubule (yellow circle), and enlargement space between seminiferous tubules (green dotted arrow) (H&E, X 400). (D): Photomicrograph of a T.S in rabbit testis of Vardenafil -treated group showing marked degeneration and disorganization in seminiferous tubules (black square); sloughing germinal epithelia of seminiferous tubules into the lumen with the absence of spermatogenic stages, exfoliation of germ cells with fewer & deformed spermatozoa (blue circle), vacuolations between germ cells (black arrow). Atrophy in Leydig cell (L) and enlargement space between seminiferous tubules (green dotted arrow) (H&E, X 400).