Astaxanthin Relieves Busulfan-Induced Oxidative Apoptosis in Cultured Human Spermatogonial Stem Cells by Activating the Nrf-2/HO-1 pathway.

Many child cancer patients endure anticancer therapy containing alkylating agents before sexual maturity. Busulfan (BU), as an alkylating agent, is a chemotherapy drug, causing DNA damage and cytotoxicity in germ cells. In the present study, we aimed to investigate the protective effect of astaxanthin (AST), as a potent antioxidant and powerful reactive oxygen species (ROS) scavenger, on BU-induced toxicity in human spermatogonial stem cells. For this purpose, testes were obtained from four brain-dead donors. After tissue enzymatic digestions, testicular cells were cultured for 3 weeks for spermatogonial stem cell (SSC) isolation and purification. K562 cell line was cultured to survey the effect of AST on cancer treatment. The cultured SSCs and K562 cell line were finally treated with AST (10µM), BU (0.1nM), and AST+BU. The expression of NRF-2, HO-1, SOD2, SOD3, TP53, and apoptotic genes, including CASP9, CASP3, BCL2, and BAX, were assayed using real-time PCR. Moreover, ROS level in different groups and malondialdehyde level and total antioxidant capacity in cell contraction of SSCs were measured using ELISA. Data showed that AST significantly upregulated the expression of NRF-2 gene (P<0.001) and protein (P<0.005) and also significantly decreased the production of BU-induced ROS (P<0.001). AST activated the NRF-2/HO-1 pathway that could remarkably restrain BU-induced apoptosis in SSCs. Interestingly, AST upregulated the expression level of apoptosis genes in the K562 cell line. The results of this study indicated that AST reduces the side effects of BU on SSCs without interference with its chemotherapy effect on cancerous cells through modulation of the NRF-2/HO-1 and mitochondria-mediated apoptosis pathways.

Busulfan-mediated oxidative stress and genotoxicity decrease in sperm of Satureja Khuzestanica essential oil-administered mice.

Here, we studied the protective effects of Satureja Khuzestanica essential oil (SKEO) as a potent anti-oxidant, against damage caused by chemotherapy with busulfan in testis and epididymal sperm of adult mice. The NMRI adult mice were assigned: G1: control, G2: was treated with busulfan (4 days, 3.2 mg/kg), G3: was treated with busulfan (4 days, 3.2 mg/kg) and SKEO (28 days, 225 mg/kg) at the same time, and G4: was pre-treated with SKEO (7 days, 225 mg/kg) and subsequently co-treated with busulfan (4 days, 3.2 mg/kg) and SKEO (28 days, 225 mg/kg). Apoptosis and Bcl-2 family gene expression were evaluated in sperm by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and Real-Time PCR, respectively. The level of oxidative stress was studied in sperm and testis by Superoxide Dismutase (SOD) and Glutathione Peroxidase (GPx) assays. Lactate Dehydrogenase (LDH), and Thiobarbituric assays were used for analyzing cytotoxicity and lipid peroxidation in testis and sperm of mice, (TBA) respectively. The results showed a significant decrease in the percentage of apoptotic sperm in G4 versus G2 and G3 (p < 0.05). SKEO pre-treatment potentially increased Bcl-2 expression and decreased BAX expression in sperm of G4 compared with G2 and G3. The activities of SOD and GPx were increased, also, LDH and TBA decreased significantly in testis and sperm of G4 compared with G2 and G3 (p < 0.05). SKEO pre-treatment had a notable role in reducing oxidative stress, apoptosis, cytotoxicity, and genotoxicity in sperm of busulfan-treated mice. In addition, cytotoxicity and oxidative stress were decreased significantly in testes of this group. Thereby, SKEO may inhibit busulfan-mediated apoptosis in sperm via decreasing oxidative stress and regulating Bcl-2 family genes expression. In conclusion, the beneficial properties of SKEO pre-treatment and co-treatment by its herbal potent anti-oxidants may reduce adverse effects of chemotherapy in the reproductive system in a rodent system. ABBREVIATIONS: SKEO: Satureja Khuzestanica essential oil; SOD: superoxide dismutase; GPx: glutathione peroxidase; LDH: lactate dehydrogenase; GC-MS: gas chromatography/mass spectrometry; TdT: terminal deoxynucleotidyl transferase; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; ROS: reactive oxygen species.

Paracrine effects of human amniotic epithelial cells protect against chemotherapy-induced ovarian damage.

BACKGROUND: Human amniotic epithelial cells (hAECs) are attractive candidates for regenerative medical therapy, with the potential to replace deficient cells and improve functional recovery after injury. Previous studies have demonstrated that transplantation of hAECs effectively alleviate chemotherapy-induced ovarian damage via inhibiting granulose cells apoptosis in animal models of premature ovarian failure/insufficiency (POF/POI). However, the underlying molecular mechanism accounting for hAECs-mediated ovarian function recovery is not fully understood. METHODS: To investigate whether hAECs-secreting cytokines act as molecular basis to attenuate chemotherapy-induced ovarian injury, hAECs or hAEC-conditioned medium (hAEC-CM) was injected into the unilateral ovary of POF/POI mouse. Follicle development was evaluated by H&E staining at 1, 2 months after hAECs or hAEC-CM treatment. In addition, we performed a cytokine array containing 507 human cytokines on hAECs-derived serum-free conditioned medium. Finally, we further investigated whether hAECs could affect chemotherapy-induced apoptosis in primary human granulosa-lutein (hGL) cells and the tube formation of human umbilical vein endothelial cells (hUVECs) via a co-culture system in vitro. RESULTS: We observed the existence of healthy and mature follicles in ovaries treated with hAECs or hAEC-CM, whereas seriously fibrosis and many atretic follicles were found in the contralateral untreated ovaries of the same mouse. To distinguish cytokines involved in the process of hAECs-restored ovarian function, hAEC-CM was analyzed with a human cytokines array. Results revealed that 109 cytokines in hAEC-CM might participate in a variety of biological processes including apoptosis, angiogenesis, cell cycle and immune response. In vitro experiments, hAECs significantly inhibited chemotherapy-induced apoptosis and activated TGF-ß/Smad signaling pathway within primary granulosa-lutein cells in paracrine manner. Furthermore, hAEC-CM was shown to promote angiogenesis in the injured ovaries and enhance the tube formation of human umbilical vein endothelial cells (hUVECs) in co-culture system. CONCLUSIONS: These findings demonstrated that paracrine might be a key pathway in the process of hAECs-mediating ovarian function recovery in animal models of premature ovarian failure/insufficiency (POF/POI).

The anticlastogenic potential of fatty acid methyl esters.

To test for possible anticlastogenic effects of fatty acids, the methyl esters of fatty acids--short-chain to long-chain--were examined on busulfan in Chinese hamster bone-marrow cells using the chromosome aberration test. When the experimental animals were treated with fatty acid esters and the mutagen, the chromosome-breaking actions of busulfan were not modulated by the short-chain fatty acids, but the fatty acids from lauric acid (C12) up to nonadecanoic acid (C19) reduced the rate of aberrant metaphases from 9.4 to about 3% at doses of 100 mg/kg and less. Other chemical properties of the fatty acids (saturated or not, number of double bonds, even- or odd-numbered) had no influence on the anticlastogenic effects. The only exceptions to this rule were arachidonic acid, which had no effect, and gamma-linolenic acid, which had no consistent effect on the action of busulfan.

Anticlastogenic effect of beta-carotene in Chinese hamsters. Time and dose response studies with different mutagens.

beta-Carotene exhibited dose-dependent anticlastogenic effects on aberrations induced by the direct-acting mutagens thio-TEPA, methyl methanesulfonate and busulfan in the in vivo chromosome aberration test (bone marrow cells, Chinese hamsters). No effect was seen when retinol was used. Apparent differences in the action of beta-carotene on aberrations induced by the three applied mutagens may be due to differences in the solubility of the compounds and to the different routes of administration.

Combinations of cytotoxic agents that have less than expected toxicity on normal tissues in mice.

A pretreatment dose of cyclophosphamide reduced lethality caused by high doses of busulphan or cyclophosphamide. In the case of cyclophosphamide given prior to busulphan, increased survival could be attributed to greater regeneration of haemopoietic stem cells in animals that received the combined dose compared with those that received busulphan alone. The mechanism by which cyclophosphamide pretreatment increased the animals' tolerance to a large dose of cyclophosphamide has not yet been elucidated. However, the urothelium in mice given the combined treatment was much less damaged than the urothelium in mice given the large dose alone, and its a known that bladder damage is a major feature of toxicity in patients given high-dose cyclophosphamide. This sparing combination exerted its expected toxicity on Lewis lung tumours, however, and so provided a useful differential effect against tumour tissue.