Impaired mitochondrial function and oxidative stress in rat cortical neurons: implications for gadolinium-induced neurotoxicity.

Gadolinium (Gd), a rare-earth lanthanides metal, is widely utilized for various industrial and medical purposes, particularly in brain magnetic resonance imaging. However, its potential effects on the impairment of the central nervous system remain uncertain, especially with regard to the mitochondria, the potential primary target in metal-induced neural injury. This study investigates the effects of gadolinium on mitochondrial energy metabolism, ROS accumulation, and cell death toward cortical neurons. Results show that the metabolic activity of the mitochondria significantly decreased as early as 3h after exposure of cells to gadolinium chloride. Subsequently, significant elevation of intracellular ROS, decrease in ATP synthesis, depolarization of mitochondrial membrane potential, release of cytochrome c and activation of caspase-3 were observed. Following these changes, increased release of LDH into culture medium and DNA fragmentation were detected. Inhibition of both cytochrome c release and caspase-3 activation could significantly reduce Gd-induced neuron cell death. All these results suggest that gadolinium cause neuron cell apoptosis primarily by inhibiting mitochondrial function and inducing oxidative stress. The present work provides new insight into the toxicological mechanism of gadolinium in neurons.

Gadolinium chloride-induced hepatocyte proliferation is prevented by antibodies to tumor necrosis factor alpha.

Gadolinium chloride (GdCl(3)) destroys large Kupffer cells and has been used extensively in mechanistic studies in a number of disease and toxicity processes; however, it cannot be used to study hepatocyte turnover since it increases cell proliferation itself. The mechanism by which GdCl(3) activates cell turnover in liver is unknown, but several possibilities exist. Here it was demonstrated that a direct mitogenic action on hepatocytes is unlikely since GdCl(3) did not stimulate the growth of primary rat hepatocyte in vitro. Therefore, it was hypothesized that GdCl(3) acts indirectly through mitogenic cytokines of nonparenchymal cell origin. Antibodies to tumor necrosis factor alpha (TNFalpha) were used to evaluate if TNFalpha is causally responsible for GdCl(3)-induced cell proliferation. GdCl(3) treatment of rats in vivo increased hepatocyte replication 5-fold in 24 h and 3-fold in 48 h. Pretreatment with specific anti-TNFalpha antibodies completely prevented these effects. However, when antibody treatment was delayed until 24 h after GdCl(3), increased cell proliferation was not prevented, suggesting that TNFalpha production during the first 24 h after treatment is responsible for activation of a signaling cascade involving other mitogens that sustain hepatocyte replication at 48 h. Twenty-four hours after treatment with GdCl(3), TNFalpha mRNA transcripts were increased 2-fold over control, an effect that was prevented by pretreatment with anti-TNFalpha antibody. NFkappaB, which is known to be involved in TNFalpha transcription, was activated by GdCl(3) about 4.5-fold over control 8 h after treatment in vivo, an increase not observed when antibodies to TNFalpha were present. When GdCl(3) was added to macrophages in culture, TNFalpha was nearly doubled 4 h after treatment. Additionally, conditioned media harvested from macrophages treated with GdCl(3) for 2 to 8 h stimulated the growth of HepG2 cells in culture about 2-fold, while antibodies to TNFalpha completely prevented this effect. Taken together, these data are consistent with the hypothesis that TNFalpha released from Kupffer cells at early time points prior to their destruction is causally responsible for triggering a cascade of events responsible for GdCl(3)-induced cell proliferation.

Mechanism of rise and decay of thapsigargin-evoked calcium signals in MDCK cells.

We studied the effect of thapsigargin on intracellular calcium levels ([Ca2+]i) measured by fura-2 fluorimetry in Madin Darby canine kidney (MDCK) cells. Thapsigargin elevated [Ca2+]i dose dependently with an EC50 of approximately 0.15 microM. The Ca2+ signal consisted of a slow rise, a gradual decay and a plateau. Depletion of the endoplasmic reticulum Ca2+ store with thapsigargin for 7 min abolished the [Ca2+]i increases evoked by bradykinin. Removal of extracellular Ca2+ reduced the thapsigargin response by approximately 50%. The Ca2+ signal was initiated by Ca2+ release from the internal store followed by capacitative Ca2+ entry (CCE). The thapsigargin-evoked CCE was abolished by La3 and Gd3+, and was partly inhibited by SKF 96365 and econazole. After depletion of the internal Ca2+ store for 30 min with another inhibitor of the internal Ca2+ pump, cyclopiazonic acid, thapsigargin failed to increase [Ca2+]i, thus suggesting that the thapsigargin-evoked Ca2+ influx was solely due to CCE. We investigated the mechanism of decay of the thapsigargin response. Pretreatment with La3+ (or Gd3+) or alkalization of extracellular medium to pH 8 significantly potentiated the Ca2+ signal; whereas pretreatment with carbonylcyanide m-chlorophynylhydrozone (CCCP) or removal of extracellular Na+ had no effect. Collectively, our results imply that thapsigargin increased [Ca2+]i in MDCK cells by depleting the internal Ca2+ store followed by CCE, with both pathways contributing equally. The decay of the thapsigargin response might be significantly governed by efflux via the plasmalemmal Ca2+ pump.