Ada Serroni

The killing of cultured hepatocytes by N-acetyl-p-benzoquinone imine (NAPQI) as a model of the cytotoxicity of acetaminophen

The killing of isolated hepatocytes by N-acetyl-p-benzoquinone imine (NAPQI), the major metabolite of the oxidation of the hepatotoxin acetaminophen, has been studied previously as a model of liver cell injury by the parent compound. Such studies assume that the toxicity of acetaminophen is mediated by NAPQI and that treatment with exogenous NAPQI reproduces the action of the endogenously produced product. The present study tested these assumptions by comparing under identical conditions the toxicity of acetaminophen and NAPQI. The killing of hepatocytes by acetaminophen was mediated by oxidative injury. Thus, it depended on a cellular source of ferric iron; was potentiated by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase; and was sensitive to antioxidants. By contrast, the cytotoxicity of NAPQI was not prevented by chelation of ferric iron; was unaffected by BCNU; and was insensitive to antioxidants. Thus, the killing of cultured hepatocytes by NAPQI occurs by a mechanism different from that of acetaminophen. The killing by NAPQI was preceded by a collapse of the mitochondrial membrane potential and a depletion of ATP. Monensin potentiated the cell killing, and extracellular acidosis prevented it. These manipulations are characteristic of the toxicity of mitochondrial poisons, and are without effect on the depletion of ATP and the loss of mitochondrial energization. Thus, mitochondrial de-energization by a mechanism unrelated to oxidative stress is a likely basis of the cell killing by NAPQI. It is concluded that treatment of cultured hepatocytes with NAPQI does not model the cytotoxicity of acetaminophen in these cells.

Peroxidation-dependent and peroxidation-independent mechanisms by which acetaminophen kills cultured rat hepatocytes

Acetaminophen killed cultured hepatocytes prepared from male rats induced with 3-methylcholanthrene by two distinct mechanisms. With 0.5 to 5 mM acetaminophen, cell killing within 4 h depended on the inhibition of glutathione reductase by 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) and was accompanied by the peroxidation of cellular lipids as assessed by the accumulation of malondialdehyde. The antioxidant diphenylphenylenediamine (DPPD) prevented both the peroxidation of lipids and the death of the cells. By contrast, DPPD had no effect on the metabolism of acetaminophen as assessed by the extent of the covalent binding of [3H]acetaminophen; by the rate and extent of the depletion of glutathione; and by the accumulation of acetaminophen metabolites in the culture medium. It is concluded that the peroxidation of the phospholipids of cellular membranes is the mechanism whereby 0.5 to 5 mM acetaminophen lethally injures cultured hepatocytes. With 10-20 mM acetaminophen, cell killing at 4 h still depended on BCNU. However, the amount of malondialdehyde in the cultures progressively decreased in parallel with the decreasing ability of DPPD to protect the cells. With 20 mM acetaminophen, there was no evidence of lipid peroxidation, and DPPD had no protective effect. Thus, a second mechanism of lethal cell injury with 10-20 mM acetaminophen is independent of lipid peroxidation and insensitive to antioxidants.

Independent antioxidant action of vitamins E and C in cultured rat hepatocytes intoxicated with allyl alcohol

The relationship between the metabolism of alpha-tocopherol (vitamin E) and ascorbate (vitamin C) was examined in cultured hepatocytes intoxicated with allyl alcohol. Alcohol dehydrogenase rapidly metabolizes allyl alcohol to the potent electrophile acrolein. Acrolein depletes the glutathione (GSH) content of the hepatocytes, thereby sensitizing the cells to the constitutive flux of activated oxygen species. Supplementation of the medium with 1 microM alpha-tocopherol phosphate (alpha-TP) prevents the 85% decline in cellular vitamin E seen after 16-18 hr in culture. In cells supplemented with alpha-TP, allyl alcohol produced a concentration-dependent decline in the cellular content of alpha-tocopherol, and these cells were more resistant to cell killing than hepatocytes not supplemented with alpha-TP. alpha-TP concentrations that raised the cellular alpha-tocopherol above the physiological level completely protected hepatocytes against the killing by allyl alcohol. In cells with physiological alpha-tocopherol, vitamin E declined within 30 min of exposure to allyl alcohol. This decrease paralleled the peroxidation of lipids, but preceded the decrease in cellular ascorbate. Under these conditions, a decline in ascorbate correlated with the loss of cell viability. Cells supplemented with at least 3 mM ascorbate prevented the decline in alpha-tocopherol. However, ascorbate acts as an independent antioxidant at these concentrations. In the absence of killing by allyl alcohol, the loss of cellular ascorbate did not depend on the presence or absence of cellular alpha-tocopherol. These data indicate that vitamins E and C act as separate antioxidants and that ascorbate does not regenerate the tocopheroxyl radical in cultured rat hepatocytes.

Re-evaluation of the distinction between type I and type II cells : The necessary role of the mitochondria in both the extrinsic and intrinsic signaling pathways upon fas receptor activation

Cyclosporin A (CyA) and bongkrekic acid (BK) prevented Fas‐induced apoptosis in two type I cell lines (H9 and SKW6.4) and two type II cell lines (Jurkat and CEM). CyA and BK inhibited the release of cytochrome c in all four cell lines. In type I cells and in CEM cells, CyA and BK did not prevent the translocation of Bax to the mitochondria. In these same cells, full‐length Bid decreased in the mitochondria and cytosol. The cleavage product of Bid, tBid, appeared in the cytosol and to a lesser extent in the mitochondria. In Jurkat cells, Bid also decreased in the cytosol, but increased in the mitochondria. Similar to the other cells, tBid appeared in the mitochondria and cytosol. In the type I H9 and SKW6.4 cells and type II Jurkat cells, the caspase‐8 inhibitor Z‐Ile‐Glu(OMe)‐Thr‐Asp(OMe)‐CH2F (IETD) prevented the cell killing. In the type I cells, IETD prevented the translocation of Bax, the degradation of Bid and the accumulation of tBid. By contrast, IETD only marginally protected the type II CEM cells. In these cells in the presence of IETD, Bax translocated to the mitochondria, in the absence of any degradation of Bid or accumulation of tBid. In the type I H9 cells, IETD produced a depletion of ATP, an effect that did not occur in the type II CEM cells. It is concluded that in type I cells the extrinsic signaling pathway is mitochondrial dependent to the same extent as is the intrinsic pathway in type II cells. J. Cell. Physiol. 208: 556–565, 2006.

The inhibition of lipid peroxidation by disulfiram prevents the killing of cultured hepatocytes by allyl alcohol, tert-butyl hydroperoxide, hydrogen peroxide and diethyl maleate

Disulfiram is a potent antioxidant that prevented the peroxidation of microsomal phospholipids induced by ADP/Fe3+ at concentrations as low as 1 microM. However, disulfiram had a biphasic action when used to assess the role of lipid peroxidation in the killing of cultured hepatocytes by an acute oxidative stress. At a relatively low concentration (10 microM), the antioxidant activity of disulfiram predominated, and there was protection against the killing of the hepatocytes by allyl alcohol, tert-butyl hydroperoxide, hydrogen peroxide, and diethyl maleate. As the concentration of disulfiram was increased above 10 microM, the extent of protection progressively decreased. Thus, with higher concentrations of disulfiram, there was a second action whose consequence is to obscure the protective effect of the lower doses. With the agents studied, this additional and as yet undefined action of disulfiram leads to the killing of the hepatocytes by a mechanism that is unrelated to the peroxidation of lipids. This biphasic action of disulfiram must be appreciated in any attempt to use this compound to assess the role of lipid peroxidation in toxic cell injury.