Manganese-induced apoptosis in rat myocytes

• Published in: Journal of biochemical and molecular toxicology 2007-07, Vol.21 (3), p.94-100
• Main Authors: Yang, Huijuan, Sun, Yiguo, Zheng, Xiaoxiang
• Format: Article
• Language: English
• Subjects: Animals; Animals, Newborn; Apoptosis; Apoptosis - drug effects; Cell Nucleus Shape - drug effects; Cells, Cultured; Cytotoxicity; Gene Expression Regulation; Manganese; Manganese - toxicity; Muscle Cells - drug effects; Muscle Cells - metabolism; Myocytes; Proto-Oncogene Proteins c-bcl-2 - genetics; Rat; Rats; Rats, Sprague-Dawley; RNA, Messenger - genetics; Tumor Suppressor Protein p53 - genetics
• ISSN: 1095-6670; 1099-0461
• DOI: 10.1002/jbt.20172

Manganese can be toxic to the heart, causing dysfunction following long exposure. In our experiments, we examined the cytotoxicity of manganese in neonatal rat ventricular myocytes (NRVM) by MTT assays in vitro. Results showed that after incubation in the different concentrations of manganese for 24 h, apparent cytotoxicity was observed. At 500, 1000, and 1500 2 μM of manganese, the percentage of cell viability dropped to 82% ± 6.13, 78% ± 5.28, and 66% ± 4.22, respectively. When cells were treated for 48 h, all concentrations tested exerted toxic effect; especially from 500 to 1500 μM the cell viability dropped from 67% ± 4.84 to 37% ± 3.25. Apoptosis in NRVM was then examined by flow cytometry. Results showed that the percentage of apoptotic cells treated with 500 μM of manganese for 24 h increased from 4% ± 0.84 to 7% ± 1.16. After 48 h of incubation, this percentage increased to 11% ± 0.91. There was no significant difference between control groups (0 μM manganese) after 24 and 48 h incubation. The morphological changes of NRVM nuclei were visualized with the fluorescent DNA‐binding dye Hoechst33342 after incubation in 500 μM of manganese for 48 h. Compared with normal nuclei, apoptotic nuclei showed the typical features of fragmentation and condensation. To investigate whether there are any apoptotic gene expression changes during apoptosis, we examined the expression level of Bcl‐2, Bax, and P53 mRNAs after treatment with 500 μM of manganese for 48 h. The Bcl‐2 mRNA expression decreased while the expression of Bax as well as P53 mRNAs increased. These results suggested that manganese cytotoxicity on NRVM could induce apoptosis in NRVM cells. The apoptosis process might involve, and be promoted by, the changes of the expression levels of P53, Bcl‐2, and Bax proteins. © 2007 Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:94–100, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20172