Elsevier

Biochemical Pharmacology

Volume 67, Issue 12, 15 June 2004, Pages 2175-2186
Biochemical Pharmacology

Apoptotic injury in cultured human hepatocytes induced by HMG-CoA reductase inhibitors

https://doi.org/10.1016/j.bcp.2004.02.037Get rights and content

Abstract

Hepatotoxicity is the major complaint during therapy with lipid-lowering agents such as statins, although the cellular mechanisms underlying the statin-induced liver injury are not fully understood. Using cultured human hepatocytes, we investigated the effects of lipophilic as well as hydrophilic statins on the cell viability. Lipophilic statins, including simvastatin, lovastatin, cerivastatin, fluvastatin and atorvastatin, reduced the viability of hepatocytes as assessed by the mitochondrial enzyme activity to reduce WST-8, however, a hydrophilic pravastatin did not cause cell injury. The simvastatin-induced loss of cell viability was attenuated by mevalonate or geranylgeranyl pyrophosphate. Simvastatin-induced DNA fragmentation and increased the number of cells stained with annexin V and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, both of which were reversed by caspase inhibitors such as zDEVD-fmk, zLEHD-fmk and zIETD-fmk. Consistent with these data, the activities of caspase-3, caspase-9 and caspase-8 were elevated by simvastatin. Simvastatin reduced the protein content and mRNA expression for bcl-2 without affecting bax mRNA expression. On the other hand, both lipophilic and hydrophilic statins significantly reduced the content of endogenous cholesterol. These findings suggest that lipophilic statins cause an apoptotic injury in human hepatocytes by stimulating caspase-3 subsequent to the activation of caspase-9 and caspase-8, in which the inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase may be involved.

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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