Francisco Dieguez

Project title: Role of Nuclear Factor-kappa B in the Molecular Toxicology of Mercury in Kidney and Brain Cells

| Project type: Thesis/Dissertation
Completed in: 2003 | Faculty advisor: James S. Woods


Nuclear Factor kappa B (NF-κB) is a thiol-dependen transcriptional factor that promotes cell survival and protects cells from apoptotic stimuli. Hg2+, one of the strongest thiol-binding agents known, impairs LPS-mediated NF-κB activation and transcriptional activity in normal rat kidney epithelial (NRK52E) cells at low concentrations (<15 µM) by binding to specific reduced thiol moieties in the NF-κB activation pathway.The inhibitory effects of Hg2+ were observed in both LPS- and TNFα-stimulated NRK52E cells. Since NF-κB prevents apoptosis in many models, we hypothesized that attenuation of NF-κB activation by Hg2+ may increase the sensitivity of kidney cells to apoptotic agents to which kidney cells are otherwise resistant. In untreated cells, fewer than 10% were apoptotic when evaluated by DNA fragmentation (TUNEL) analysis. However, when tumer necrosis factor (TNF) was given following Hg2+ treatment (0.5 to 5 µM for 30 min), the proportion of cells undergoing apoptosis increased by 2 to 6 fold over that seen following TNF alone. Kidney cells pretreated with specific NF-κB inhibitors (Bay11-7082 and SN50) prior to TNF showed a significant increase in apoptosis. These findings are consistent with the view that Hg2+ enhances the sensitivity of kidney cells to apoptotic stimuli as a consequence of inhibition of NF-κB activity. Additional studies show that rats pretreated in vivo with Hg2+ (0.75 mg/kg) for 18 hours followed by LPS (10 mg/kg) have impaired NF-κB activation and an increased cytochrome-C release in kidney cortex. Since apoptosis is known to play a key role in the pathogenesis of renal failure, inhibition of NF-κB activity may define a molecular mechanism by which Hg2+ toxicity is initiated in kidney cells. We further show that the specific effects of Hg2+ on NF-κB activation are not restricted to kidney cells or to inorganic mercury per se. Preliminary studies with a brain cell model revealed that methyl mercury (CH3Hg2+) impairs TNF-mediated NF-κB activation in primary rat astrocytes at doses similar to those observed with Hg2+ in kidney cells. The inhibitory effects of mercury compounds on NF-κB activation may underlie a novel mechanism by which mercury could elicit toxic effects to various organs.