Journal Article

Effects of Minimally Toxic Levels of Carbonyl Cyanide P-(Trifluoromethoxy) Phenylhydrazone (FCCP), Elucidated through Differential Gene Expression with Biochemical and Morphological Correlations

Sabu Kuruvilla, Charles W. Qualls, Ronald D. Tyler, Sam M. Witherspoon, Gina R. Benavides, Lawrence W. Yoon, Karen Dold, Roger H. Brown, Subbiah Sangiah and Kevin T. Morgan

in Toxicological Sciences

Volume 73, issue 2, pages 348-361
Published in print June 2003 | ISSN: 1096-6080
Published online June 2003 | e-ISSN: 1096-0929 | DOI: http://dx.doi.org/10.1093/toxsci/kfg084
Effects of Minimally Toxic Levels of Carbonyl Cyanide P-(Trifluoromethoxy) Phenylhydrazone (FCCP), Elucidated through Differential Gene Expression with Biochemical and Morphological Correlations

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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 carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (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 adenosine triphosphate (ATP) concentrations. The following seven 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 and DNA damage-inducible protein 153 (GADD153). Transcriptional changes of all seven genes were later confirmed with reverse transcription-polymerase chain reaction (RT-PCR). These results suggest that gene expression changes may provide a sensitive indicator of uncoupling in response to chemical exposure.

Keywords: FCCP; uncoupling agent; gene expression; molecular marker; cell cycle; protein synthesis

Journal Article.  9892 words.  Illustrated.

Subjects: Medical Toxicology ; Toxicology (Non-medical)

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