Copper induced apoptosis in Caco-2 and Hep-G2 cells: Expression of caspases 3, 8 and 9, AIF and p53.

Copper (Cu) is an essential trace metal needed to ensure cell function. However, when present at high concentrations it becomes toxic to organisms. Cell death, induced by toxic levels of copper, was previously observed in in vitro studies. However, there is no consensus about the cell death pathway induced by Cu and it is still not known whether this occurs as a result of the direct action of the metal or by indirect effects. In the present work, we intend to identify the influence of different Cu concentrations in the induction of apoptosis and to explore the potential signaling pathways, using two different in vitro cell culture models (Caco-2 and Hep-G2). Cells were exposed, during 6, 12, 24 and 48h, to Cu concentrations corresponding to IC50 and 1/8 of IC50, according to the viability assays. Then, considering the different apoptosis pathways, the expression of caspases 3, 8 and 9, apoptosis inducing factor (AIF) and p53 genes was analyzed by quantitative real time PCR. The results suggested that different Cu concentrations could trigger different apoptotic pathways, at different times of exposure. In both cell lines, apoptosis seems to be initiated by caspase independent pathway and intrinsic pathway, followed by extrinsic pathway. In conclusion, this study demonstrates that Cu induces the activation of apoptosis through caspase dependent and independent pathways, also suggesting that apoptosis activation mechanism is dependent on the concentration, time of exposure to Cu and cell type.

Neuropeptide Y stimulates autophagy in hypothalamic neurons.

Aging is characterized by autophagy impairment that contributes to age-related disease aggravation. Moreover, it was described that the hypothalamus is a critical brain area for whole-body aging development and has impact on lifespan. Neuropeptide Y (NPY) is one of the major neuropeptides present in the hypothalamus, and it has been shown that, in aged animals, the hypothalamic NPY levels decrease. Because caloric restriction (CR) delays aging, at least in part, by stimulating autophagy, and also increases hypothalamic NPY levels, we hypothesized that NPY could have a relevant role on autophagy modulation in the hypothalamus. Therefore, the aim of this study was to investigate the role of NPY on autophagy in the hypothalamus. Using both hypothalamic neuronal in vitro models and mice overexpressing NPY in the hypothalamus, we observed that NPY stimulates autophagy in the hypothalamus. Mechanistically, in rodent hypothalamic neurons, NPY increases autophagy through the activation of NPY Y1 and Y5 receptors, and this effect is tightly associated with the concerted activation of PI3K, MEK/ERK, and PKA signaling pathways. Modulation of hypothalamic NPY levels may be considered a potential strategy to produce protective effects against hypothalamic impairments associated with age and to delay aging.

Unaltered hepatic oxidative phosphorylation and mitochondrial permeability transition in wistar rats treated with nimesulide: Relevance for nimesulide toxicity characterization.

Nonsteroidal anti-inflammatory drugs have been associated with hepatotoxicity in susceptible patients. One such example is nimesulide, a preferential cyclooxygenase 2-inhibitor, widely used for the treatment of inflammation and pain. It was suggested that nimesulide could exert its hepatotoxicity by altering hepatic mitochondrial function, which was demonstrated in vitro. The objective of this study was to verify whether liver mitochondria isolated from rats treated with doses of nimesulide well above therapeutic levels possessed decreased calcium tolerance and oxidative phosphorylation, which indicates in vivo nimesulide mitochondrial toxicity. Male and female rats received nimesulide or its vehicle twice daily, for 5 days, and were killed on the seventh day for the isolation of liver mitochondria. Mitochondrial respiration, transmembrane electric potential, and calcium tolerance were characterized in all experimental groups. Nimesulide had no effect on liver mitochondrial function. Indexes of mitochondrial integrity, calcium loading capacity, and oxidative phosphorylation efficiency were unchanged between liver mitochondria from treated and control animals. In the animals tested, no evidence of degraded mitochondrial function due to nimesulide administration could be found. The results corroborate the notion that despite recognized in vitro mitochondrial toxicity, nimesulide does not cause detectable mitochondrial dysfunction in Wistar rats, even when administered in much higher concentrations than those known to have anti-inflammatory effects.