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

Abstract

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.

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

“…9, D and E, and supplemental Tables 1 and 2). Previous comprehensive gene studies using microarrays in rhabdomyosarcoma cells exposed to FCCP showed that the uncoupler mimics the hypoxic environment, where a sudden drop in ATP production as a result of loss of OxPhos inhibits protein synthesis and induces G 1 -S cell cycle arrest and DNA damage and ultimately apoptosis (57). Here, such global effects were also evident in HepG2 as SR4 severely impacted a number of metabolic processes vital to cell growth and proliferation.…”

“…SCARA3 (also referred to as the cellular stress response (CSR) gene) has been shown to deplete ROS and thus play an important role in protecting cells from oxidative stress (61). FCCP did not produce any increase in ROS, and based on recent microarray gene analysis, no associations between FCCP and any components the ETC as well as ROS scavenger genes were identified (57). Although speculative at this time, the activity of SR4 on the ETC components could be one of its unique characteristics that differentiate it from other uncouplers, such as FCCP, allowing it to have increased cytotoxicity in cancer cells.…”

Comparison of the binding behavior of FCCP with HSA and HTF as determined by spectroscopic and molecular modeling techniques

“…Carbonylcyanide p ‐(trifluoromethoxy) phenylhydrazone (FCCP) (molecular structure, Scheme ; formula, C 10 H 5 F 3 N 4 O; molecular weight, 254.17 gr/mol) is a potent mitochondrial uncoupler that completely destroys oxidative metabolism and ATP synthesis . In the presence of FCCP, cellular respiration proceeds until all of the available oxygen is reduced and immediate oxidation of substrates proceeds with little or no phosphorylation of ADP, in other words, FCCP separates oxidation from phosphorylation .…”

2,4‐Dinitrophenol induces apoptosis in As4.1 juxtaglomerular cells through rapid depletion of GSH

“…The proton gradient and membrane potential are the proton motive force that is used to drive ATP synthesis. Coupling of electron transport through the respiratory chain and ATP generation can be disrupted by uncoupling agents such as cyanide m-chloropheylhydrazone, caybonyl cyanide p-(trifluoromethoxy) phenylhydroazone, and 2,4-dinitrophenol (Kuruvilla et al, 2003;Futakawa et al, 2006). Mitochondria are also one of the most important sources of ROS production at mitochondrial respiratory chain level (Nohl et al, 2005).…”