Title page for ETD etd-08052008-151905


Document Type Master's Dissertation
Author Potgieter, Marnie
URN etd-08052008-151905
Document Title Cellular effects of Coenzyme Q10 and Triton X on primary chicken embryo heart and muscle cell cultures
Degree MSc
Department Anatomy
Supervisor
Advisor Name Title
Dr M Bester Co-Supervisor
Prof E Erasmus Supervisor
Keywords
  • Triton X
  • cellular effects
  • cell cultures
  • Coenzyme Q10
Date 2008-04-23
Availability unrestricted
Abstract
Coenzyme Q10 is a lipid-soluble coenzyme, synthesized in mammalian tissue to support energy production, and also act as an antioxidant. Certain medication, stress and age may deplete the body’s endogenous Coenzyme Q10 store. Numerous disease conditions have been shown to benefit from Coenzyme Q10 supplementation. It is a lipid-soluble component of virtually all cell membranes, and is located in the hydrophobic domain of the phospholipid bilayer of cellular membranes. It is also the only known lipid-soluble antioxidant that animal cells can synthesize de novo, and for which there exist enzymatic mechanisms which can regenerate it from its oxidized product formed in the course of its antioxidant function. The aim of this study was to investigate the cellular effects of Coenzyme Q10 and Triton X-100 on primary chicken embryo heart and muscle cell cultures. Triton X-100, a well known membrane disrupter, extensively used by cell biologists for that purpose, was used to investigate whether Coenzyme Q10 might offer protection to cell membranes exposed to disruption. Due to the correlation found between the chemical structures of nonylphenol and Triton X-100, it was decided to determine whether Triton X-100 possess estrogenic properties. Using the Recombinant Yeast Screen Assay for estrogenic activity, it was found that Triton X-100 induced weak estrogenic activity.

The primary heart and skeletal muscle cell cultures were established by harvesting skeletal muscle tissue and hearts from 13 day old chicken embryos. After establishment of the cell cultures, the concentrations of Coenzyme Q10 and Triton X-100 were tested for cytotoxicity using the MTT, NR, and CV assays, in the form of a combined colorimetric cytotoxicity assay. The MTT assay revealed an increase in cell viability in both cell cultures upon exposure to Triton X-100 and Coenzyme Q10, alone, and in combination. Triton X-100 and Coenzyme Q10, alone, and in combination, caused a decrease in lysosomal membrane integrity, as measured by the NR assay, and both substances, alone, and in combination, had no effect on cellular proteins, as measured by the CV assay.

Scanning electron microscopy (SEM) was done to determine the cellular effect of heart and skeletal muscle cell cultures on the external surface, more specifically the membranes, of cells in culture. Triton X-100 in the concentrations used in the study, caused membrane disruption, ranging from complete membrane lyses at the highest concentrations to membrane ruptures and apoptotic blebbing in lower concentrations. SEM revealed that no adverse effects were caused by Coenzyme Q10 on the membrane structure, in dissimilarity, cell differentiation and proliferation, including myoblast formation were seen in the presence of all the concentrations of Coenzyme Q10. Numerous ion channels were observed on cellular surfaces exposed to Coenzyme Q10. Upon exposure to 0.005% Triton X-100, after pre-treatment with Coenzyme Q10, SEM revealed a “membrane patch” formation on membranes disrupted by Triton X-100. Damage to cell membranes in the presence of Triton X-100, were less severe when cells were pre-treated with Coenzyme Q10. Confocal microscopy was utilized to investigate intracellular occurrences in the presence of Triton X-100 and Coenzyme Q10. Using Mito Tracker Red to stain active respiring mitochondria and DAPI to stain nuclei, confocal microscopy confirmed the observations made by SEM, that Coenzyme Q10 enhance cell proliferation and differentiation, and that the adverse effects to cells exposed to Triton X-100 are less severe after pre-treatment with Coenzyme Q10. ROS generation was detected, using dichlorodihydrofluorescein diacetate, in cultures exposed to Triton X-100, and none in the presence of Coenzyme Q10. In the presence of Triton X-100, after pre-treatment with Coenzyme Q10, ROS generation was remarkably lower.

The study provided apparent evidence that Coenzyme Q10 offer protection to cardiac and skeletal muscle cells in culture after exposure to relatively low concentrations of the membrane disrupter Triton X-100. Coenzyme Q10 also promotes the process of proliferation and differentiation in primary chicken embryonic cultures of heart and skeletal muscle cells.

© University of Pretoria 2007

E963 /ag

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