Calcium-induced cytotoxicity in hepatocytes after exposure to extracellular ATP is dependent on inorganic phosphate. Effects on mitochondrial calcium

In isolated mitochondria extensive uptake of Ca2+ in the presence of an "inducing agent," e.g. inorganic phosphate (Pi), causes permeabilization of the mitochondrial inner membrane and a collapse of the mitochondrial membrane potential. In this study we tested whether the effect of phosphate occurs in intact hepatocytes. Rat hepatocytes were incubated with ATP to induce a sustained increase in intracellular Ca2+ ([Ca2+]i), dissipation of the mitochondrial membrane potential, and cell death (Zoeteweij, J. P., van de Water, B., de Bont, H. J. G. M., Mulder, G. J., and Nagelkerke, J. F. (1992) Biochem. J. 288, 207-213). Omission of Pi from the incubation medium delayed the loss of viability. The nonhydrolyzable ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S) had similar effects on [Ca2+]i and viability, but now omission of extracellular Pi completely protected against cytotoxicity. Exposure to ATP or ATP gamma S induced a large cellular uptake of Pi. With the use of video-microscopy a significant increase in mitochondrial free calcium was observed before the onset of cell death. Accumulation of mitochondrial calcium was reduced when extracellular Pi was omitted. These results suggest that, after induction of high [Ca2+]i by ATP in hepatocytes, 1) mitochondria accumulate calcium which is associated with cell toxicity and 2) intracellular Pi increases which stimulates mitochondrial calcium uptake. These observations support a calcium-dependent mitochondrial dysfunction, induced by phosphate, as a valid model for ATP-induced cytotoxicity in hepatocytes.Toxicolog

Role of mitochondrial Ca2+ in the oxidative stress-induced dissipation of the mitochondrial membrane potential. Studies in isolated proximal tubular cells using the nephrotoxin 1,2-dichlorovinyl-L-cysteine

The relationship between mitochondrial Ca2+, oxidative stress, and a dissipation of the mitochondrial membrane potential (delta psi) was investigated in proximal tubular kidney cells. Freshly isolated proximal tubular cells from rat kidney were exposed to the nephrotoxin 1,2-dichlorovinyl-L-cysteine (DCVC). DCVC stimulated the formation of hydroperoxides as determined by flow cytometry using the hydroperoxide-sensitive compound dichlorofluorescein. This was prevented by the antioxidant diphenylphenylenediamine (DPPD) and the iron chelator desferrioxamine. Studies in individual cells with video-intensified fluorescence microscopy showed that a DCVC-induced increase in the intracellular free calcium concentration ([Ca2+]i) was accompanied by an increase in the mitochondrial free calcium concentration ([Ca2+]m). The latter increase was selectively prevented by an inhibitor of the mitochondrial calcium uniporter, ruthenium red (RR). Chelation of cellular Ca2+ with EGTA acetoxymethyl ester (EGTA/AM) completely prevented the formation of hydroperoxides, whereas inhibition of the uptake of Ca2+ by the mitochondria with RR reduced it. This indicates that the increase in [Ca2+]m is important for the induction of oxidative stress by DCVC. DPPD and desferrioxamine did not protect against a DCVC-induced increase in [Ca2+]i and [Ca2+]m, indicating that oxidative stress is the consequence rather than the cause of the cellular calcium perturbations. DCVC decreased delta psi and caused cell death; both effects were clearly delayed by EGTA/AM and RR, although they could not prevent a decrease in delta psi. The latter decrease was completely prevented by inhibition of the beta-lyase-mediated metabolism of DCVC with aminooxyacetic acid. Like EGTA/AM, inhibition of oxidative stress with DPPD and desferrioxamine delayed the decrease in delta psi. This strongly suggests that the decrease in delta psi caused by metabolites of DCVC directly is potentiated by Ca(2+)-dependent DCVC-induced hydroperoxide formation. The importance of both hydroperoxide formation and mitochondrial damage in DCVC-induced cell killing is discussed.Toxicolog

Mutual insurance as an elusive concept in traditional rural communities

During the last two decades, economists have paid increasing attention to the role of informal risk-sharing arrangements as a privileged way through which traditional rural communities can achieve a significant degree of protection against income fluctuations and other hazards beyond their control. This article however argues that when they enter into such arrangements members of these communities are guided by a principle of balanced reciprocity (they expect a return from any contribution or payment they make) rather than by a true logic of mutual insurance. More precisely, they do not conceive of insurance as a game where there are winners and losers and where income is redistributed between lucky and unlucky individuals. None the less, traditional agrarian societies have proven able to develop a restricted range of sustainable forms of mutual insurance that avoid the aforementioned problem.

Mitochondria and Cell Death. Mechanistic Aspects and Methodological Issues.

Mitochondria are involved in cell death for reasons that go beyond ATP supply. A recent advance has been the discovery that mitochondria contain and release proteins that are involved in the apoptotic cascade, like cytochrome c and apoptosis inducing factor. The involvement of mitochondria in cell death, and its being cause or consequence, remain issues that are extremely complex to address in situ. The response of mitochondria may critically depend on the type of stimulus, on its intensity, and on the specific mitochondrial function that has been primarily perturbed. On the other hand, the outcome also depends on the integration of mitochondrial responses that cannot be dissected easily. Here, we try to identify the mechanistic aspects of mitochondrial involvement in cell death as can be derived from our current understanding of mitochondrial physiology, with special emphasis on the permeability transition and its consequences (like onset of swelling, cytochrome c release and respiratory inhibition); and to critically evaluate methods that are widely used to monitor mitochondrial function in situ