ToxSci Advance Access published online on April 15, 2003
Toxicological Sciences, doi:10.1093/toxsci/kfg084
Toxicological Sciences © Society of Toxicology 2003; all rights reserved
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1 Oklahoma State University, Stillwater, OK 74078; GlaxoSmithKline Inc., 5 Moore Drive, Research Triangle Park, NC 27709
* To whom correspondence should be addressed. E-mail: Sabu.k.kuruvilla{at}gsk.com.
Uncouplers of oxidative phosphorylation have relevance to bioenergetics and obesity. The mechanisms of action of chemical uncouplers of oxidative phosphorylation on biological systems were evaluated using differential gene expression. The transcriptional response in human rhabdomyosarcoma cell line, RD, was elucidated following treatment with FCCP, a classical uncoupling agent. Changes in mitochondrial membrane potential were used as the biological dosimeter. There was an increase in membrane depolarization with increasing concentrations of FCCP. The concentration at 75% uncoupling (20 µM) was chosen to study gene expression changes, using cDNA based large-scale differential gene expression (LSDGE) platforms. At the above concentration, subtle light microscopic and clear gene expression changes were observed at 1, 2 and 10 h. Statistically significant transcriptional changes were largely associated with protein synthesis, cell cycle regulation, cytoskeletal proteins, energy metabolism, apoptosis and inflammatory mediators. Bromodeoxyuridine (BrdU) and Propidium iodide (PI) assays revealed cell cycle arrest to occur in the G1 and S phases. There was a significant initial decrease in the intracellular ATP concentrations. The following 7 genes were selected as potential molecular markers for chemical uncouplers: seryl-tRNA synthetase (Ser-tRS), glutamine-hydrolyzing asparagine synthetase (Glut-HAS), mitochondrial bifunctional methylenetetrahydrofolate dehydrogenase (Mit BMD), mitochondrial heat shock 10-kDa protein (Mit HSP 10), proliferating cyclic nuclear antigen (PCNA), cytoplasmic beta-actin (Act B) and growth arrest & DNA damage-inducible protein 153 (GADD153). Transcriptional changes of all 7 genes were later confirmed with RT-PCR. These results suggest that gene expression changes may provide a sensitive indicator of uncoupling in response to chemical exposure.
© 2003 Society of Toxicology
Molecular and Genetic Toxicology
Effects of Minimally Toxic Levels of Carbonyl Cyanide P-(Trifluoromethoxy) Phenylhydrazone [FCCP], Elucidated through Differential Gene Expression with Biochemical and Morphological Correlations
2 GlaxoSmithKline Inc., 5 Moore Drive, Research Triangle Park, NC 27709
3 Oklahoma State University, Stillwater, OK 74078
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