Protective Role of Spirulina platensis Against Bifenthrin-Induced Reprotoxicity in Adult Male Mice by Reversing Expression of Altered Histological, Biochemical, and Molecular Markers Including MicroRNAs

Abstract

: The potential reprotoxicity of bifenthrin remains unclear if only the common clinical indicators of reproductive disease are examined. The present study aimed to investigate the efficacy of Spirulina platensis, a microalga rich in antioxidant compounds, against bifenthrin-induced testicular oxidative damage in male mice. At the first, we demonstrate that administration of bifenthrin resulted in a decline of testosterone level and in deterioration of sperm quality that was correlated with significant transcription changes of some specific mRNA and microRNA involved in cholesterol transport, testosterone synthesis, and spermatogenesis. At the biochemical level, we found that oxidative stress was obvious in the bifenthrin group, as evidenced by increase in malondialdehyde (MDA), protein carbonyls (PCO), reactive oxygen species (ROS), and nitrite oxide (NO) that was correlated with activation of genes related to mitochondrial apoptotic signal pathways. We then brought, for the first time to our knowledge, solid and complete experimental evidences that administration of mice with Spirulina extract was sufficient to protect against deleterious effects BF in testicular tissues by abrogating the change in antioxidant enzyme activities; the increase in MDA, PCO, and NO concentrations; and the altered expression level of miRNA and mRNA involved in spermatogenesis. We finally demonstrate that Spirulina restores the production of testosterone in mice as well as epididymal sperm viability and motility. These results suggest a potential antitoxic activity of Tunisian Spirulina deserving further attention.

Keywords: Reprotoxicity; Spirulina antioxidants; apoptosis; microRNA; oxidative stress; pesticides.

Figure 1

Observation under an optical microscope of the eosin-stained spermatozoa of mice (100 × magnification), (A) control (normal morphology of the spermatozoa); (B) mid-piece abnormality of the spermatozoa; (C) head abnormality of the spermatozoa; (D) tail abnormality of the spermatozoa.

Figure 2

(A) Testicular sections of control mice, which show normal spermatogenesis (400× haematoxylin and eosin (H&E)): note the normal cell arrangement in the seminiferous tubules. The interstitial spaces also appear normal. (B) Testicular sections of mice treated with 5 mg per kg b.w. per day of BF (400 × H&E): note the atrophic seminiferous tubules with a large proportion of tubules showing signs of degeneration and disorganization. Sloughing of germ cells into tubular lumen. (C) Testicular sections of mice treated with 5 mg per kg per day of BF and 500 mg per kg per day of SP (400 × H&E): note the increase of germ cells in the seminiferous tubules. The interstitial spaces appear normal. (D) Testicular sections of mice treated with 500 mg per kg per day of SP (400 × H&E): note the normal cell arrangement in the seminiferous tubules. The interstitial spaces also appear normal. Ti, interstitium; Sg, spermatogonia; Spzs, spermatozoa. (E) Effect of different treatments on testosterone level. Values are expressed as means SD of eight mice in each group. All groups vs. control group: ^A p < 0.05; ^B p < 0.01; ^C p < 0.001. All groups vs. BF group: ^D p < 0.05; ^E p < 0.01, ^F p < 0.001.

Figure 3

Effect of different treatments on ROS (A), MDA (B), PCO (C), NO (D), levels, antioxidant enzyme activities (SOD (E), CAT (F), and GPx (G)), GSH concentration (H) and DNA fragmentation (I) in testes of controls and mice treated with bifenthrin (BF), Spirulina (SP), or their combination (SP + BF). Values are expressed as means SD of eight mice in each group. All groups vs. control group: ^A p < 0.05; ^B p < 0.01; ^C p < 0.001. All groups vs. BF group: ^D p < 0.05; ^E p < 0.01, ^F p < 0.001.

Figure 4

Effects of different treatments on mRNA levels of genes related to cholesterol transport and testosterone synthesis including SRB1 (A), LDL-R (B), PBR (C), StAR (D), P450scc (E), P450-17α (F), 3β-HSD (G) and 17β-HSD (H) in testes of mice. Values are presented as means ± SE. All groups vs. control group: ^A p < 0.05; ^B p < 0.01; ^C p < 0.001. All groups vs. BF group: ^D p < 0.05; ^E p < 0.01, ^F p < 0.001.

Figure 5

Assessment of apoptosis-related genes in mice testes. The quantifications of apoptosis related genes including Bcl-2 (A), Bax (B), Bid (C), Apaf-1 (D), Cytochrome c (E), p53 (F), TNF (G), Fas lg (H), Faim (I) and caspases 3 (J), 9 (K), 8 (L) was performed by real-time quantitative polymerase chain reaction (RT-PCR). All samples were run in triplicate and results are presented as mean ± SEM. All groups vs. control group: ^A p < 0.05; ^B p < 0.01; ^C p < 0.001. All groups vs. BF group: ^D p < 0.05; ^E p < 0.01, ^F p < 0.001.

Figure 6

Effects of different treatments on spermatogenesis and apoptosis related miRNA including miR-17 (A), miR-34c (B), miR-34b (C), miR-449a (D), miR-449c (E), miR-122 (F), miR-146b (G) and miR-509 (H). Values are presented as means ± SEM. All groups vs. control group: ^A p < 0.05; ^B p < 0.01; ^C p < 0.001. All groups vs. BF group: ^D p < 0.05; ^E p < 0.01, ^F p < 0.001.