Apoptotic injury in cultured human hepatocytes induced by HMG-CoA reductase inhibitors
Introduction
3-Hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase inhibitors such as statins are the most widely used cholesterol-lowering agents for prevention of obstructive cardiovascular events [1], [2], [3]. However, severe adverse events including myopathy, rhabdomyolysis and hepatotoxicity associated with lipophilic statins sometimes limit the lipid-lowering therapy with these agents [4], [5]. Among them, hepatotoxicity characterized by the elevation of plasma transaminases is most common. In particular, the incidence is heightened, when statin is used in combination with fibrate compounds [6], [7], although such combination therapy is necessary for the severe refractory hyperlipidemic patients [8]. It has been reported that the frequency of the elevation of transaminases is 0.5–2.0% during the monotherapy with statin [9], [10] but the rate is markedly elevated to 1.3–10% by the combination therapy with statin and fibrate [6], [7], [11]. However, the precise mechanisms underlying the statin-induced hepatotoxicity remain to be clarified.
It has been reported that a number of lipophilic statins cause apoptosis in a variety of cells, including striated muscle cells [12], cardiac myocytes [13], vascular smooth muscle cells [14], [15], [16], and endothelial cells [17], [18], by decreasing the amounts of farnesyl pyrophosphate and geranylgeranyl pyrophosphate due to the inhibition of HMG-CoA reductase. However, the hydrophilic pravastatin does not possess the toxic action on these cells, since unlike lipophilic statin, the hydrophilic statin can hardly penetrate into cells other than hepatocytes where the hydrophilic statin is transported through the tissue-specific organic anion transporter such as a human liver-specific transporter [19].
The aim of the present study was to compare the toxic action in cultured human hepatocytes among lipophilic and hydrophilic statins and to determine the cellular mechanisms underlying the statin-induced hepatotoxicity. For this purpose, the effects of various statins with different lipophilicity on the viability and nuclear morphology were examined in cultured human hepatocytes. The role for caspases in statin-induced hepatocyte injury was subsequently determined.
Section snippets
Chemicals
Atorvastatin, cerivastatin and pravastatin were gifts from Sankyo Co. Ltd. The following chemicals and reagents were obtained from commercial sources: fluvastatin, lovastatin, simvastatin, (±)mevalonolacton, and caspase inhibitors such as zLHED-fmk, zDEVD-fmk and zIETD-fmk were obtained from Calbiochem. Fenofibrate and geranylgeraniol were purchased from Sigma-Aldrich. Fetal bovine serum (FBS) was from JRH Biosciences. Caspase substrates such as Ac-DEVD-AMC for caspase-3, Ac-IETD-AMC for
Effect of various statins on the viability of Chang liver cells
Lipophilic statins, including simvastatin (log P=4.4), lovastatin (log P=3.91), cerivastatin (log P=2.32), fluvastatin (log P=1.73) and atorvastatin (log P=1.59), decreased the viability of cultured human hepatocytes, whereas a hydrophilic statin pravastatin (log P=−0.47) had no influence on the cell viability (Fig. 1). The hepatotoxic effect of lipophilic statins was augmented by a low concentration (10 μM) of fenofibrate (Fig. 2), although fenofibrate alone reduced concentration-dependently the
Discussion
In the present study, a variety of lipophilic statins but not the hydrophilic statin reduced the viability of cultured human hepatocytes as assessed by WST-8 assay. WST-8 is taken up into living cells and reduced to the water-soluble formazan by the mitochondrial NADH enzymes, thus is widely used for the cell viability assay [29]. Although the WST-8 assay also reflects cell proliferation, the rate of proliferation of the cultured hepatocytes used in the present study was extremely low and no
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