Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism
Induction of megamitochondria by some chemicals inducing oxidative stress in primary cultured rat hepatocytes
Introduction
There is a vast literature describing megamitochondria (MG) formation both in physiological and pathological conditions 1, 2, 3, 4, 5. Formation of MG can be seen in any source of tissues, such as skeletal muscles and adrenal glands, but the liver may be the one which is most frequently associated with the phenomenon [1].
We have been studying the mechanism of the formation of MG induced by various chemicals, such as ethanol and hydrazine, using rats as experimental animals 3, 4. MG are induced in the liver of animals given a hydrazine-diet for 6–7 days, and animals become very weak losing appetite during the course of the experiment. Malnutritional states, such as essential fatty acid deficiency [6]and riboflavin deficiency [7], also induce MG in the liver. Therefore, it is difficult to analyze factor(s) which induces MG as long as in vivo experimental models are employed. Thus, in the present study we have attempted to induce MG in culture hepatocytes using various chemicals.
Recently, we have succeeded in suppressing the formation of MG in the liver induced by chloramphenicol (CF) [5]and hydrazine [8]using 4-hydroxy-2,2,6,6-tetramethyl-1-piperidine-1-oxyl (4-OH-TEMPO), and have found at the same time that 4-OH-TEMPO lowers enhanced lipid peroxidation induced by CF and hydrazine. These data have led us to speculate that free radicals are intimately related to the mechanism of the formation of MG 5, 8.
In the present study, we have tested hydrazine, bromobenzene and hydrogen peroxide, inducers of free radicals. If hydrogen peroxide is capable of inducing MG, this would be a direct evidence to support the idea that free radicals are intimately related to the phenomenon. Furthermore, we have also tested in the present study erythromycin, a specific inhibitor of mitochondrial protein synthesis, and cycloheximide, a specific inhibitor of cytoplasmic protein synthesis, to clarify the correlation between the inhibition of protein synthesis and the mechanism of the formation of MG.
Section snippets
Isolation and culture of hepatocytes
Hepatocytes were isolated by a collagenase perfusion technique [9]from phenobarbital-pretreated or control male Wistar rats weighing 100 to 120 g. Phenobarbital was given to the animal for 3 days by intraperitoneal (8 mg/100 g body weight/day). Cells with a viability of more than 90% determined by Trypan blue exclusion were used in the present study. Hepatocytes with seeding density of 6×104 cells cm−2 were cultivated on type I collagen coated dishes at 37°C in an atmosphere of 5% CO2, 95% air
Effects of various chemicals on the size and distribution of mitochondria in cultured hepatocytes
Mitochondria of hepatocytes cultured for up to 22 h in the presence of 2 mM hydrazine became moderately enlarged (Fig. 1B) compared with those of the control (Fig. 1A) cultured in the absence of hydrazine when they were examined by confocal laser microscope. On the other hand, mitochondria of hepatocytes isolated from rats pretreated with phenobarbital showed distinct morphological changes when they were cultured in the presence of hydrazine: Mitochondria of hepatocytes cultured in the presence
Discussion
We have succeeded in the present study in inducing MG in cultured hepatocytes by hydrazine and hydrogen peroxide, inducers of free radicals, and erythromycin, a specific inhibitor of mitochondrial protein synthesis. These chemicals have been found at the same time to enhance lipid peroxidation remarkably. Namely, free radicals generated by different mechanisms or via different sources seem to be involved in the induction of MG. In the light of results obtained in the present study, it seems
Acknowledgements
The authors wish to thank Dr. M. Wozniak, Department of Biochemistry, Medical University of Gdansk, Poland, for valuable discussion. This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan (08670169).
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