Therapeutic effect of sodium alginate on bleomycin, etoposide and cisplatin (BEP)-induced reproductive toxicity by inhibiting nitro-oxidative stress, inflammation and apoptosis

Abstract

Impaired spermatogenesis and male infertility are common consequences of chemotherapy drugs used in patients with testicular cancer. The present study investigated the effects of sodium alginate (NaAL) on testicular toxicity caused by bleomycin, etoposide, and cisplatin (BEP). Rats in group 1 received normal saline, while groups 2 and 3 were treated with 25 and 50 mg/kg of NaAL, respectively. Group 4 was treated with a 21-day cycle of BEP (0.5 mg/kg bleomycin, 5 mg/kg etoposide, and 1 mg/kg cisplatin), and groups 5 and 6 received BEP regimen plus 25 and 50 mg/kg of NaAL, respectively. Then, sperm parameters, testosterone levels, testicular histopathology and stereological parameters, testicular levels of malondialdehyde (MDA), nitric oxide (NO), and total antioxidant capacity (TAC), and the expression of apoptosis-associated genes including Bcl2, Bax, Caspase3, p53, and TNF-α were evaluated. Our findings revealed that NaAL improved sperm parameters, testosterone levels, histopathology, and stereology parameters in BEP-administrated rats. NaAL also improved testis antioxidant status by enhancing TAC and ameliorating MDA and NO. Further, modifications to the expression of Bcl2, Bax, Caspase3, p53, and TNF-α suggested that NaAL alleviated BEP-induced apoptosis and inflammation. Collectively, NaAL protects rats' testes against BEP-evoked toxicity damage through the modulation of nitro-oxidative stress, apoptosis, and inflammation.

Figure 1

The schematic of experimental treatment plan. BEP chemotherapy regimen was administrated intraperitoneally (IP) for a 21-day period. Sodium alginate was injected (IP) (25 and 50 mg/kg/d) one week before and after BEP administration; and, on days 2, 9 and 16 of BEP therapy, as well. Unt: untreated, E: etoposide, P: cisplatin, B: bleomycin, Na: sodium alginate, W: week.

Figure 2

A test system composed of two elements (an unbiased counting frame and a basic tile with a Merz grid inside it was superposed on the image. If the sperm’s head was lay inside the frame and did not touch the forbidden (continued) lines, it was sampled (here 2 sperms). The sperm’s tail length was estimated using the following formula: ΣL = (π/2)·(1/asf)·(a/l)·ΣI, where, ‘‘a/l’’ was the Merz grid constant, ‘‘asf’’ was the area of the basic tile divided by the area of the counting frame, and ‘‘ΣI’’ was the summation of the intersections of the tails with the semicircles (arrowheads; here 4). The total estimated length was divided by the number of counted sperm’s to achieve tail length per sperm.

Figure 3

The effect of one cycle of BEP regimen with or without sodium alginate on weight gain (%) and testis weight (g). Cntrl: Control; NaAL 25 and NaAL 50: rats injected with 25 or 50 mg/kg of sodium alginate; BEP + NaAL 25 and BEP + NaAL 50: rats treated with BEP regimen plus 25 or 50 mg/kg of melatonin respectively. ^aP < 0.05 versus Control group; ^bP < 0.01 versus Control group; ^cP < 0.05 versus BEP group; ^dP < 0.01 versus BEP group.

Figure 4

Light microscope micrograph of the testis in the controls and experimental groups. (A) normal structure of the germinal epithelium and interstitial tissue are seen in the control group. (B) and (C) testicular tissue in the control groups treated with 20 and 50 mg/kg of NaAL, respectively, show normal architecture of the testis. (D) testicular tissue in the BEP-treated rats showing destructive changes such as atrophic (yellow stars), necrotic (res stars) tubules, and degeneration of interstitial connective tissue (E,F) testis structure in the BEP-treated rats received 20 and 50 mg/kg of NaAL, respectively, showing improvement of testicular tissue toward normal structure (H&E staining, 100×, scale bar = 100 µm).

Figure 5

The effect of one cycle of BEP regimen with or without sodium alginate on testosterone level in the controls and experimental groups (n = 5). Cntrl: Control; NaAL 25 and NaAL 50: rats injected with 25 or 50 mg/kg of sodium alginate; BEP + NaAL 25 and BEP + NaAL 50: rats treated with BEP regimen plus 25 or 50 mg/kg of melatonin respectively. ^aP < 0.05 versus Control group; ^bP < 0.01 versus Control group; ^cP < 0.05 versus BEP group; ^dP < 0.01 versus BEP group.

Figure 6

The effect of one cycle of BEP regimen with or without sodium alginate on sperm count, sperm viability, sperm motility, progressive motility, and sperm morphology in controls and experimental groups (n = 5). Cntrl: Control; NaAL 25 and NaAL 50: rats injected with 25 or 50 mg/kg of sodium alginate; BEP + NaAL 25 and BEP + NaAL 50: rats treated with BEP regimen plus 25 or 50 mg/kg of melatonin respectively. ^aP < 0.05 versus Control group; ^bP < 0.01 versus Control group; ^cP < 0.05 versus BEP group; ^dP < 0.01 versus BEP group.

Figure 7

Different abnormalities observed in experimental rats after one cycle of BEP regimen and one week recovery period. Papanicolaou test, scale bar = 5 µm.

Figure 8

The effect of one cycle of BEP regimen with or without sodium alginate on testicular levels of malondialdehyde (MDA), nitric oxide (NO), and total antioxidant capacity (TAC) in the controls and experimental groups (n = 5). Cntrl: Control; NaAL 25 and NaAL 50: rats injected with 25 or 50 mg/kg of sodium alginate; BEP + NaAL 25 and BEP + NaAL 50: rats treated with BEP regimen plus 25 or 50 mg/kg of melatonin respectively. ^aP < 0.05 versus Control group; ^bP < 0.01 versus Control group; ^cP < 0.05 versus BEP group; ^dP < 0.01 versus BEP group.

Figure 9

The effect of one cycle of BEP regimen with or without sodium alginate on Bax, Bcl-2, Caspase-3 (C3), and p53 genes expression and also Bax/Bcl-2 ratio of testis in controls and experimental groups. Cntrl: Control; NaAL 25 and NaAL 50: rats injected with 25 or 50 mg/kg of sodium alginate; BEP + NaAL 25 and BEP + NaAL 50: rats treated with BEP regimen plus 25 or 50 mg/kg of melatonin respectively. ^aP < 0.05 versus Control group; ^bP < 0.01 versus Control group; ^cP < 0.05 versus BEP group; ^dP < 0.01 versus BEP group.

Figure 10

The effect of one cycle of BEP regimen with and without sodium alginate on TNF-α, p53 and Bcl-2 expression in the testis. Intense labeling of p53 and TNF-α are seen in spermatogonia and primary spermatocyte of the BEP-treated group, while the expression in other groups that received NaAL is down-regulated. The expression of Bcl-2 in the control and NaAL-treated groups is remarkably positive, yet in the BEP-treated group, the testicular cell demntrates a negative immunostaining reaction. Yellow arrowheads: positive immunostaining cells, Red arrowheads: negative immunostaining cells (Scale bar = 50 µm, × 400).