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 microM) 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.