Table 2.
Androgenic/antiandrogenic effects of MCs and consequences on male reproductive toxicity.
| Toxin | Experimental Model | Assays Performed | Concentration Ranges and Exposure Conditions | Main Results | References |
|---|---|---|---|---|---|
| Pure MC-LR standard | Male SD rats | Sperm analysis. Determination of T, LH, and FSH serum levels. |
5, 10, and 15 µg/kg b.w. for 28 d | Decrease of epididymal sperm concentration and motility. Slight testicular atrophy, slight deformation of androgonial and spermatogenic cells, and reduced numbers of Sertoli cells and mature sperm. Decreased T, LH, and FSH serum levels. | [71] |
| Leydig cells | Cell viability by MTT. Measurement of apoptosis. Determination of T concentration and ROS production. Detection of LPO and SOD activity. |
0.5, 5, 50, and 500 nM for 12, 24, and 48 h | Reduced cell viability. Decrease of T content. Increase of ROS and LPO, reduced cytosolic SOD activity. |
||
| Pure MC-LR standard | Male SPF mice | Sperm morphology. Determination of T, LH, and FSH serum levels. Histopathological evaluation of testis. TUNEL assay for detection of apoptotic cells in testis. |
1, 3.2, and 10 µg/L for 3 and 6 months | Sperm motility decreased with 3.2 and 10 µg/L. Serum levels of T decreased, whereas FSH and LH levels increased. At the highest MC-LR level: testicular atrophy and derangement of spermatogenic cells. Six-month results showed a significant increase of apoptotic cells. |
[83] |
| Pure MC-LR standard | Male BALB/c mice | Expression in hypothalamus, pituitary, and testis of gnrh, lh and fsh genes by RT-PCR. Determination of T, LH, and FSH serum levels. |
3.75, 7.5, 15, and 30 µg/kg b.w. for 1, 4, or 7 d | Expression of gnrh gene was downregulated. fshβ and lhβ genes were upregulated at 1 and 4 days; however, its expression was downregulated at 14 days. Serum levels of T, LH, and FSH increased at 1 and 4 days, and decreased at 14 days. |
[45] |
| Leydig cells | Determination of MC-LR uptake. Cell viability by CCK-8 test. T concentrations. |
1, 10, 100, 250, 500, 750, and 1000 nM for 2 h | MC-LR was not able to enter Leydig cells. There were no statistically significant differences in cell viability and T concentration. |
||
| Pure MC-LR standard | Male Wistar rats | Histology of testis. Mitochondrial swelling and DNA damage. Determination of T, LH, and FSH serum levels. Determination of ROS production. Expression in testis of different cytoskeletal and mitochondrial genes by RT-PCR. |
1 and 10 µg/kg for 50 d | Testis index decreased (10 µg/kg group). Blockage in seminiferous tubules was observed and spermatogonia showed apoptotic characters and cytoplasmic shrinkage. DNA fragmentation was observed. Serum levels of LH and FSH increased, while T levels decreased. ROS production was increased. The expression of all mitochondrial genes was increased, whereas some cytoskeletal genes decreased (β-actin, β-tubulin, Radixin) and some increased (vimentin, ezrin, moesin). | [84] |
| Pure MC-LR standard | Male C57BL/6 Mice | Determination of T, FSH, and LH serum levels. Evaluation of epididymal sperm. Expression of gnrh, lh and fsh genes in hypothalamus, pituitary, and testis by RT-PCR. |
3.75, 7.5, 15, 30 µg/kg b.w. for 1, 4, 7, and 14 d | Significant reduction in epididymal sperm production. T and LH production was inhibited. Reduction of lh and gnrh genes expression. |
[72] |
| Leydig cells | Cell viability and detection of T levels. | 1, 10, 100, 250, 500, 750, and 1000 nmol/L for 48 h | No significant differences on cell viability and T production | ||
| Pure MC-LR standard | Male SD rats | Detection of GnRH, FH, LSH, and T serum levels. TUNEL assay for detection of apoptotic hypothalamic cells. Hypothalamus gnrh1, lhβ, and fshβ detection by RT-PCR. Western blot and immunofluorescence for MC-LR detection in GnRH neurons. |
30 µg/Kg body weight for 1, 3, 5, 7, and 14 d | Serum levels of GnRH, LH, FSH, and T showed a similar pattern of early-stage increase and late-stage decline. Remarkable apoptotic hypothalamic cells after 7 and 14 days of exposure. MC-LR could be observed in GnRH neurons. |
[46] |
| GT1-7 cells | Immunocytochemistry for MC-LR detection. Cell viability, LDH rate, and GnRH concentration. Measurement of the concentrations of cAMP, Ca2+, and AC activity by ELISA. |
1, 10, 100, 1000, and 10,000 nM for 48 h | MC-LR could be taken up by GT1-7 cells. Cell viability decreased and LDH release increased. A concentration-dependent increase of cAMP, Ca2+, and AC activity. |
||
| Pure MC-LR standard | Male BALB/c mice | Determination of macrophages enrichment in testes by flow cytometry. Expression in testis of Hsd3β, Hsd17β, StAR, TNF-α, IL-6 and MCP-1 genes by qRT-PCR. Western blot, coimmunoprecipitation, and ELISA immunohistochemical analysis. Analysis of testicular tissues by immunofluorescence. |
1, 10, 20, and 30 µg/L for 180 d | At 20 and 30 µg/L, the number of Leydig cells, mRNA levels of StAR, Hsd3β, and Hsd17β decreased. Protein levels of Hsd3β decreased at 10 µg/L. Immunohistochemical staining assay demonstrated an increase of macrophages in testes and flow cytometry identified that MC-LR could modulate the number of testicular macrophages. Levels of TNF-α, IL-6, and MCP-1 increased. Leydig cells apoptosis was drastically increased when co-cultured with macrophages in the presence of MC-LR. mRNA levels of TNF-α increased in a time-dependent manner in macrophages, peaking at 3 h post exposure. GAS6 mRNA levels increased at 24 h, and GAS6 cytokine levels declined in medium at 6 h. After addition of MC-LR into the co-culture system of macrophages and LCs, no marked increase of GAS6 in the cytoplasm of LCs. |
[73] |
| RAW264.7 cells | Determination of macrophage polarization by flow cytometry. | 500 nM for 24 h | Apoptosis in LCs increased in a time-dependent manner when LCs were cultured with MC-LR. Moreover, TNF-α levels in macrophages were significantly elevated at mRNA and protein levels. Phosphorylation levels of NF-κB were upregulated in macrophages. | ||
| Pure MC-LR standard | Male BALB/c mice | MC-LR uptake in GnRH neurons by immunofluorescence. GnRH and T levels in serum by Western blot. |
20 µg/Kg body weight for 7 d | GnRH mRNA in hypothalamus and the content in serum decreased. Immunofluorescence staining in hypothalamus tissue sections showed the presence of MC-LR in the cytoplasm of GnRH neurons. | [74] |
| GT1-7 cells | GnRH synthesis and secretion by RT-PCR. Intracellular levels of cAMP and Ca2+ by ELISA. Determination of Ca2+ and cAMP by ELISA. Measurement of soluble GnRH by HPLC-MS/MS. |
10, 50, 100, 500, and 1000 nM for 1, 3, 6, 12, 24, and 48 h | MC-LR can be transported into GT1-7 cells. Declination of gnrh mRNA levels, and increase followed by a decrease of GnRH releasing. Intracellular levels of cAMP and Ca2+ increased. PKC kinase activity enhanced with the time of MC-LR exposure, meanwhile, a time- and concentration-dependent increasing trend of phosphorylation levels of PKC and NF-κB was observed. PKA kinase activity enhanced with the time of MC-LR-exposure, and Prkacb (PKA catalytic subunits) and the phosphorylation of Creb increased with the time and concentration of MC-LR. The toxin enhanced the phosphorylation of downstream proteins in the MAPK pathway, such as the phosphorylation of Erk and p38. mRNA levels of Oct-1, Dlx-2, Otx-2 decreased, and c-Fos and c-Jun increased gradually. Their protein expressions showed similar trends. |
||
| Pure MC-LR standard | Male ICR mice | Determination of GnRH concentration in the brain by immunofluorescence. Determination of GnRH serum levels by HPLC-MS. Determination of T serum levels by ELISA. |
1, 7.5, 15, and 30 µg/L for 180 d | GnRH and T serum levels decreased. | [75] |
| GT1-7 cells | Cell viability. Determination of GnRH levels. Determination of cAMP levels by ELISA. Determination of PKA kinase activity. |
1 mg/mL for 0.25, 0.5, 1, 3, and 6 h | Cell viability and GnRH levels decreased. cAMP levels increased. Upregulation of PKA activity. |
||
| Pure MC-LR standard | Male ICR mice | Sperm count. T measurement by radioimmunoassay. Immunohistochemistry of testis. Immunoblotting of testis. Detection of MC-LR. |
Daily i.p. injection of 20 µg/kg for 35 d | Serum and testicular T levels decreased. MC-LR located in both Leydig cells and seminiferous tubules. Testicular steroidogenic proteins (StAR, CYP11A1, and CYP17A1) downregulated. |
[76] |
| TM3 cell line | Cell viability. Detection of MC-LR. ROS detection. |
0.05–20 µM MC-LR for 6–24 h | MC-LR was increased in a concentration-dependent manner in MC-LR-treated TM3 cells. Concentrations of MC-LR used did not affect viability. p-GCN2 and p-eIF2α increased at 24 h. Intracellular ROS began to increase at 6 h and continued to rise at 12 h and 24 h. Pre-treatment with PBN alleviated MC-LR-induced activation of GCN2/eIF2α signaling in the cells. |
||
| Pure MC-LR standard | Male BALB/C mice | Immunohistochemistry of the brain. Apoptosis detection by TUNEL. Serum levels of T and GnRH by ELISA. Electron microscopy of the brain. |
1, 7.5, 15, and 30 μg/L 180 d | Serum T and GnRH declined. GnRH levels were downregulated in the brain. Remarkable apoptotic cells in hypothalamus. Hypothalamus tissues displayed increased expression of active-caspase3. Hypothalamus neuron cells showed distinct ultrastructure changes. | [77] |
| GT1-7 cell line | Cell viability. Analysis of cell cycle. Cell apoptosis by flow cytometry. ROS content detection. Measurement of intracellular Ca2+ mRNA expression of gnrh by qRT-PCR. |
50, 500, and 1000 nM | GnRH protein released into the supernatant decreased. mRNA levels of gnrh were remarkably reduced. MC-LR induced cell cycle dysregulation, apoptosis, and inhibited cell growth. Generation of ROS increased. MC-LR was able to cause the release of intracellular Ca2+. Pre-treatment with an endoplasmic reticulum stress inhibitor, such as 4-phynyl butyric acid, reduced apoptosis rate in the cells. The pre-treatment with the autophagy inhibitor 3-MA could increase the apoptosis caused by the toxin. |
||
| MC-LR from a purified extract from Microcystis aeruginosa and MC-LR standard | Male Nile tilapia | Determination of E2 and T serum levels. Expression in brain, gonads and liver of different genes in HPGL axis by RT-PCR. |
9.6 µg/L for 28 d | Serum E2 and T levels increased. Transcripts of 13 genes (GHRH, PACAP, etc.) were altered. |
[78] |
| Pure MC-LR standard | Male Rana nigromaculata | Determination of sperm motility and deformity. Testes histopathology by electron microscopy. Quantification of E2 and T serum levels. Analysis of SF-1 and P450 aromatase protein expression by commercial kit. |
0.1, 1, and 10 µg/L for 7 and 14 d | The number of sperm cells and sperm motility decreased in all groups. The sperm abnormality rate was significantly increased. Frogs manifested ultrastructural changes in a concentration-dependent manner. Testosterone content was significantly decreased in all groups, while estradiol content was significantly increased. The protein content of p450 aromatase and SF-1 significantly increased. | [79] |
| Pure MC-LR standard | Male frogs (Rana nigromaculata) | MC-LR concentration in testes by HPLC-MS. Determination of E2, T, LH, and FSH serum levels. Evaluation of cAMP content in testes by ELISA. Expression in brain and testis of genes implicated on the HPG axis by qRT-PCR. |
0.1, 1, and 10 µg/L for 7 and 14 d | MC-LR in the testes increased in a concentration- and time-dependent manner. Serum levels of T decreased, while E2 and FSH increased. cAMP content decreased at 7 d but significantly increased at 14 d. mRNA levels of StAR, CYP11A1, CYP17A1, and CYP19A1 were upregulated, whereas HSD3B2 and HSD17B3 levels were downregulated. mRNA levels of AR and ESR1 were upregulated. | [80] |
| Pure MC-LR standard | Macrobrachium rosenbergii | Immunolocalization of MC-LR in testis. Histopathological examinations. Determination of T serum levels. TUNEL assay for determination of testicular germ cells apoptosis. Expression in testis and eyestalk of gonadal-development-related genes (hsp70, sox9) by RT-PCR. |
0.5 and 5 µg/L for 1, 2 or 3 weeks | MC-LR was found in testis, spermatocytes, and Leydig cells. T serum levels decreased. Inhibitory effect on testis development, and testicular germ cells apoptosis. Hsp70 was upregulated. | [81] |
| Pure MC-LR standard | Male parental and F1 embryo Macrobrachium nipponense | Serum concentrations of T and E2. Transmission electron microscopy of testis. Determination of sperm quality. Measurement of testicular antioxidant ability. Detection of apoptotic cells by TUNEL assay. F1 mRNA expression of different genes associated with apoptosis, immune function, and antioxidant capacity by qRT-PCR and protein expression by Western blot. |
0.5 and 5 µg/L for 28 d | T serum levels decreased, but elevated serum E2 levels were found. After 4 weeks, the testes of male prawns showed disturbed development. Increased percentages of spermatogonia (SPG) and spermatocytes (SPC). ROS levels of testis in F0 generation prawns increased, with increased testicular SOD and CAT activities. In contrast, activities of GPx1 and GST decreased. MDO contents increased but GSH content decreased. Treatment with increasing MC-LR concentrations resulted in increased levels of the toxin in the testis of F0 and F1 embryos. Sperm DNA damage and apoptotic cells. Malformation rates in F1 larvae from MC-LR-treated F0 males significantly increased. Lower mRNA levels of Mn-SOD, GPx1, and CAT. Transcript abundances of apoptosis-related genes (Caspase-3 (Casp3), caspase-9 (Casp9)) were higher. Protein levels of Casp3 and p53 increased in F1. |
[82] |
| Pure MC-LR standard | GT1-7 cells | Cell viability by CCK-8 test. MC-LR uptake by Western blot. Expression of gnrh and OATPs genes by RT-PCR. Determination of OATPs proteins expression by Western blot. Measurement of soluble GnRH by HPLC-MS/MS. |
10, 20, 50, 100, 200, 300, 400, 500, 750, 1000, and 50,000 nM for 48 h | Cell viability gradually decreased. MC-LR can be transported into GT1-7 cells. Gnrh transcription levels decreased with increasing MC-LR concentrations. Oatp1a4, Oatp1a5, Oatp5a1, and Oatp2b1 genes were expressed in GT1-7 cells. Only Oatp1a5 protein was expressed. |
[89] |
| OATP1a5-KO GT1-7 cells | Expression of OATPs genes by RT-PCR. Determination of OATPs proteins expression by Western blot. Cell viability by CCK-8 test. |
500 nM for 48 h | Pattern of Oatp1a4, Oatp5a1, and Oatp2b1 mRNA expression in the Oatp1a5-KO GT1-7 cells was the same as in the intact cells. Like the intact cells, Oatp1a5-KO cells did not express Oatp1a4, Oatp5a1, or Oatp2b1 at the protein level, evidenced by Western blot assay. Cell viability still unchanged. |
AC: adenylate cyclase; cAMP: cyclic adenosine monophosphate; CAT: catalase; c-KIT: stem cell factor receptor; cDNA: deoxyribonucleic acid; E2: 17β-estradiol; eIF2α: eukaryotic translation initiation factor 2α; FSH: follicle-stimulating hormone; GAS6: growth arrest-specific 6; GCN2: general control nonderepressible 2; GPx1: glutathione peroxidase 1; GnRH: gonadotropin-releasing hormone; GSH: glutathione; GSI: gonadosomatic index; hpf: hours post-fertilization; HPG: hypothalamic-pituitary-gonadal; HPGL: hypothalamic-pituitary-gonadal-liver; HPI: hypothalamic-pituitary-interrenal axis; HPLC-MS/MS: High-performance liquid chromatography–tandem mass spectrometry; hsp: heat shock proteins; IL-6: interleukine 6; i.p.: intraperitoneal injection; Kg b.w.: Kg per body weight; LC-MS: Liquid Chromatography- mass spectrometry; LDH: lactate dehydrogenase; LH: luteinizing hormone; LPO: lipid peroxidation; 3-MA: 3-methyladenine; MAPK: mitogen-activated protein kinases; MCP-1: monocyte Chemoattractant Protein-1; MDO: malondialdehyde; Mn-SOD: manganese suporoxide dismutase; MTT: 3-(4;5-dimethylthiazol-2-yl)-2;5-diphenyltetrazolium bromide; OATP: organic anion transporting polypeptide; Oct-1: organic cation transporter 1; PBN: N-Tert-Butyl-α-Phenylnitrone; pepck: phosphoenolpyruvate carboxykinase; PI: propidium iodide; PKA/C: protein kinase A/C; pomc: pro-opiomelanocortin; PP1/2A: protein phosphatases 1 and 2A; ROS: reactive oxygen species; RT-PCR: real time polymerase chain reaction; SF-1: steroidogenic factor 1; SOD: superoxide dismutase; SOX9: SRY-Box Transcription Factor 9; SD: Sprague-Dawley; SPF: specific pathogen free; StAR: steroidogenic acute regulatory protein; T: testosterone; TNF-α: tumor necrosis factor-α.