Mitigation of statins-induced cytotoxicity and mitochondrial dysfunction by L-carnitine in freshly-isolated rat hepatocytes

N Abdoli, Y Azarmi, M A Eghbal, N Abdoli, Y Azarmi, M A Eghbal

Abstract

Statins are widely used as anti hyperlipidemic agents. Hepatotoxicity is one of their adverse effects appearing in some patients. No protective agents have yet been developed to treat statins-induced hepatotoxicity. Different investigations have suggested L-carnitine as a hepatoprotective agent against drugs-induced toxicity. This study was designed to evaluate the effect of L-carnitine on the cytotoxic effects of statins on the freshly-isolated rat hepatocytes. Hepatocytes were isolated from male Sprague-Dawley rats by collagenase enzyme perfusion via portal vein. Cells were treated with the different concentrations of statins (simvastatin, lovastatin and atorvastatin), alone or in combination with L-carnitine. Cell death, reactive oxygen species (ROS) formation, lipid peroxidation, and mitochondrial depolarization were assessed as toxicity markers. Furthermore, the effects of statins on cellular reduced and oxidized glutathione reservoirs were evaluated. In accordance with previous studies, an elevation in ROS formation, cellular oxidized glutathione and lipid peroxidation were observed after statins administration. Moreover, a decrease in cellular reduced glutathione level and cellular mitochondrial membrane potential collapse occurred. L-carnitine co-administration decreased the intensity of aforementioned toxicity markers produced by statins treatment. This study suggests the protective role of L-carnitine against statins-induced cellular damage probably through its anti oxidative and reactive radical scavenging properties as well as its effects on sub cellular components such as mitochondria. The mechanism of L-carnitine protection may be related to its capacity to facilitate fatty acid entry into mitochondria; possibly adenosine tri-phosphate or the reducing equivalents are increased, and the toxic effects of statins toward mitochondria are encountered.

Keywords: Hepatotoxicity; Isolated rat hepatocytes; L-carnitine; Mitochondria; Statins.

Figures

Fig. 1
Fig. 1
Reactive oxygen species formation after statin and L-carnitine administration to isolated rat hepatocytes. A; atorvastatin, B; simvastatin, C; lovastatin. The fluorescent activity of dichlorofluorescin, which is directly linked to the amount of reactive oxygen species, was measured at different time points. Data are given as mean ± SEM for three experiments. ***; Significant as compared to control group (P<0.001). **; Significant as compared to statins-treated group (P<0.01). *; Significant as compared to statins-treated group (P<0.05). The sole administration of L-carnitine did not cause any toxicity toward rat hepatocytes at mentioned concentration.
Fig. 2
Fig. 2
Lipid peroxidation after statins administration to isolated rat hepatocytes. A; atorvastatin, B; simvastatin, C; lovastatin. Thiobarbituric acid reactive substances test was assessed in different time schedules to investigate statins-induced cytotoxicity in experimental groups. Carnitine (100 μM) caused no significant lipid peroxidation when administered alone. Data are given as mean ± SEM for three experiments. **; Significant as compared to control group (P<0.01). **; Significant as compared to statins-treated group (P<0.01). The sole administration of carnitine did not cause any toxicity toward rat hepatocytes at mentioned concentration.
Fig. 3
Fig. 3
Hepatocytes reduced glutathione (GSH) levels after statins administration. A; atorvastatin, B; simvastatin, C; lovastatin. Data are given as mean ± SEM for three experiments. The Ellman reagent (DTNB) test was employed to assess hepatocytes glutathione content. **; Significant as compared to control group (P<0.01). ***; Significant as compared to control group (P<0.001). *; Significant as compared to statins-treated group (P<0.05). The sole administration of carnitine did not cause any toxicity toward rat hepatocytes at mentioned concentration.
Fig. 4
Fig. 4
Hepatocytes oxidized glutathione levels after statins administration. A; atorvastatin, B; simvastatin, C; lovastatin. Data are given as mean ± SEM for three experiments. ***; Significant as compared to control group (P<0.001). *; Significant as compared to statins-treated group (P<0.05). **; Significant as compared to statins-treated group (P<0.01). The sole administration of carnitine did not cause any toxicity toward rat hepatocytes at mentioned concentration.
Fig. 5
Fig. 5
Statins-induced collapse in cellular mitochondrial potential (ΔΨm) and the role of L-carnitine administration. A; atorvastatin, B; simvastatin, C; lovastatin. Rhodamine 123 test was employed to assess the mitochondrial membrane potential. ***; Indicates P<0.001 versus control group. **; Indicates P<0.01 versus drug-treated groups. *; Indicates P<0.05 versus drug-treated groups.

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