Wen-peng Zhao

Fluoride-induced oxidative stress and apoptosis are involved in the reducing of oocytes development potential in mice

The present study was conducted to investigate the mechanisms of excessive-fluoride-induced reduction of oocyte development potential in mice. The development morphology of oocyte and the changes of pathomorphology in ovary were observed. The protein expression levels of apoptosis factors, including Bax, Bcl-2, casepase-3, casepase-9 and cytochrome c, and the mRNA expression levels of antioxidant enzymes, including SOD1, GSH-Px1, CAT and inducible nitric oxide synthase were measured by Western blot and real-time PCR, respectively. DNA damage in the ovary was analysed by single cell gel electrophoresis and TUNEL staining. Results indicated that the structure and function of ovarian cells were seriously damaged, followed, the development potential of oocyte was reduced by excessive fluoride. The expression levels of apoptosis factors were up-regulated and antioxidant enzymes were significantly down-regulated. Meanwhile, the contents of ROS, MDA, NO and iNOS were significantly increased. Whereas, the activities of SOD1, GSH-Px1 and CAT was significantly decreased compared with the control group. Simultaneously, the results of DNA analysis indicated that the tail length and tailing ratio of ovarian cells were significantly increased in the fluoride group. In summary, the results provided compelling evidence that excessive fluoride intake can reduce the development potential of oocyte by inducing oxidative stress and apoptosis in the ovary of female mice.

Ca 2+ metabolic disorder and abnormal expression of cardiac troponin involved in fluoride-induced cardiomyocyte damage

Our previous study indicated that excessive fluoride (F) induces ATP5J and ATP5H proactive expression by interfering cardiomyocyte mitochondrial dysfunction in mice. This study aimed to investigate underlying mechanisms of F¯ induced damage to cardiomyocytes. A total of 100 mg/L F¯ was added to distilled water to treat Kunming mice for 70 days. Pathological and morphological changes in myocardial tissues were observed under transmission electron microscope and light microscope. Content of ATP and ATP enzyme distributed in cardiomyocytes were determined by fluorescence and ATP enzyme staining. Expression levels of troponin (Tn) C, TnI, TnT and tropomyosin (TPM) were measured by immunofluorescence, western blot, and real-time polymerase chain reaction. Contents of Ca2+ in blood, myocardial cells and faeces were also detected by confocal microscopy and ethylenediaminetetraacetic acid. Using 100 mg/L F¯ resulted in nuclaer enrichment, the myocardial fibre breakage and mitochondrial lysis. Following mitochondrial structure damage, contents of ATP and ATP enzyme significantly decreased in the fluoride group. Expression levels of TnT and TnI were significantly down-regulated, whereas that of TPM was up-regulated. Content of Ca2+ in cardiomyocytes of fluoride group visibly increased. Interestingly, contents of Ca2+ in blood and faeces decreased. These findings reveal that excessive F ingestion induces Ca2+ metabolic disorder, and an abnormal expression of cardiac Tn are involved in F-induced cardiomyocyte damage.

JNK/STAT signalling pathway is involved in fluoride-induced follicular developmental dysplasia in female mice

Excessive fluoride (F) intake decreases the development of potential oocytes by inducing oxidative stress and apoptosis in female mice in our previous study. This study aims to investigate the underlying mechanisms of F-induced follicular developmental dysplasia. Pathomorphological changes in the ovary tissues were observed under light and transmission electron microscopes. DNA damage and proliferation in granulosa cells were analysed by TUNEL staining and BrdU measurement. The protein expression of cell proliferation related regulatory factors including JNK, STAT3, STAT5, CDK2, CDK4, PCNA and Ki67 in the ovary tissues was measured by immunohistochemistry and Western blot analyses. Results indicated that the structure of granulosa cells in the ovary was seriously damaged by excessive F, evident by the swollen endoplasmic reticulum, mitochondria with vacuoles and nucleus shrinkage. F treatment also considerably enhanced the apoptosis and inhibited the proliferation of granulosa cells. The number of granulosa cells around the oocyte decreased after F treatment. The expression levels of STAT3, CDK2, CDK4 and Ki67 in the ovary tissues were up-regulated, and STAT5 and PCNA did not change significantly after F treatment, whereas JNK expression was down-regulated with increasing F dose. In summary, changes in the expression levels of JNK, STAT3, STAT5, CDK2, CDK4, PCNA and Ki67 in the JNK/STAT signalling pathway are involved in F-induced follicular dysplasia in the ovary.

PI3K/AKT signaling pathway involvement in fluoride-induced apoptosis in C2C12 cells

To investigate the mechanisms of fluoride-induced apoptosis, a fluoride-induced C2C12 skeletal muscle cell (C2C12 cell) model was established in this study, and the viability of the C2C12 cells was measured using an MTT assay. Cell morphological changes were observed via haematoxylin and eosin staining and transmission electron microscopy. Apoptosis was monitored through Hoechst staining. The mRNA and protein expression of PI3K, PDK1, AKT1, BAD, Bcl-2, Bax and caspase-9 were detected through real-time PCR and western blotting, respectively. The results showed that the survival rates of C2C12 cells decreased gradually with an increasing fluoride doses. The C2C12 cell structure was seriously damaged by fluoride, presenting with pyknosis, mitochondrial ridge disruption and swollen endoplasmic reticulum. Furthermore, the expression of mRNA in PI3K, BAD, Bcl-2, Bax and caspase-9 were significantly increased in the fluoride group (P < 0.01), while the expression of PDK1 was markedly decreased (P < 0.01). The expression of protein in BAD, Bcl-2 and Bax were significantly increased in the fluoride group (P < 0.01), while the expression of PDK1 and P-AKT1 was markedly decreased (P < 0.01). In conclusion, fluoride-induced apoptosis in C2C12 cells is related to the PI3K/AKT signaling pathway.

Mitochondrial respiratory chain complex abnormal expressions and fusion disorder are involved in fluoride-induced mitochondrial dysfunction in ovarian granulosa cells

Excessive fluoride intake has a strong female reproductive toxicity, which can result in follicular developmental dysplasia and decrease oocytes developmental potential. The underlying mechanisms of fluoride-induced mitochondrial dysfunction in ovarian granulosa cells remain largely unknown. In this study, the ultrastructure changes of mitochondria and DNA damage in ovarian granulosa cells were observed under transmission electron microscope and TUNEL staining. Then, the ATP content and ROS level in granulosa cells were measured. The expression of mitochondrial fusion proteins and mitochondrial respiratory chain complexes, including OPA1 and Mfn1, and NDUFV2, SDHA and CYC1, in the ovarian tissues were measured by immunohistochemistry, Western blot and Quantitative real-time PCR analyses. The expression of ATP5j and ATP5h in the ovarian tissues was also measured. Results show that fluoride treatment considerably damages mitochondrial ultrastructure and enhances the apoptosis of granulosa cells. The ATP content greatly decreased, whereas the ROS level increased after fluoride treatment. The expression level of Mfn1 in the ovarian tissue was up-regulated, whereas OPA1 expression had no significant change. The expression levels of NDUFV2, SDHA and CYC1 were considerably up-regulated, and the expression of ATP5j and ATP5h were down-regulated after fluoride treatment. In summary, the damage in the mitochondrial ultrastructure, ATP content decrease, ROS level increase and the abnormal expression of OPA1, Mfn1, NDUFV2, SDHA, CYC1, ATP5j and ATP5h in ovary tissue are closely associated with fluoride-induced mitochondrial dysfunction, which might be responsible for the follicular developmental dysplasia and the potential decrease in oocyte development induced by fluoride in female mice.