Induction of megamitochondria by some chemicals inducing oxidative stress in primary cultured rat hepatocytes

doi:10.1016/S0005-2760(97)00140-9

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism

Volume 1349, Issue 3, 30 November 1997, Pages 242-250

Biochimica et Biophy...

https://doi.org/10.1016/S0005-2760(97)00140-9 Get rights and content

Abstract

Effects of hydrazine, hydrogen peroxide and bromobenzene, inducers of free radicals, and those of erythromycin and cycloheximide, inhibitors of protein synthesis on structural changes of mitochondria in primary monolayer culture of rat hepatocytes were examined using laser confocal microscope and electron microscope. After 22 h of incubation of hepatocytes with 0.2 mM hydrogen peroxide or 10 μg ml⁻¹ of erythromycin, mitochondria became extremely enlarged. Mitochondria of hepatocytes isolated from control rats became slightly to moderately enlarged in the presence of 2 mM hydrazine, while those of hepatocytes isolated from phenobarbital-pretreated animals became extremely enlarged in the presence of 2 mM hydrazine. Cycloheximide (0.5–10.0 μg ml⁻¹) and bromobenzene (0.1–1.0 mM) failed to induce structural changes of mitochondria. The level of cytochrome P-450 in freshly prepared hepatocytes from phenobarbital-treated rats was 2.5 times higher than that from the control rats, and remained about three times higher than the latter after 22 h of incubation with 2 mM hydrazine. The level of malondialdehyde was invariably elevated when megamitochondria were induced. These results may suggest that oxidative stress is intimately related to the mechanism of the formation of megamitochondria and that the inhibition of cytoplasmic protein synthesis seems not to contribute the phenomenon. However, the detailed mechanism by which free radicals may induce megamitochondria remains to be elucidated.

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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Induction of megamitochondria by some chemicals inducing oxidative stress in primary cultured rat hepatocytes

Abstract

Introduction

Section snippets

Isolation and culture of hepatocytes

Effects of various chemicals on the size and distribution of mitochondria in cultured hepatocytes

Discussion

Acknowledgements

Toxicol. Appl. Pharmacol.

Methods. Biol.

Methods Enzymol.

J. Biol Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochem. Pharm.

Arch. Biochem. Biophys.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

Arch. Biochem. Biophys.

Free Rad. Biol. Med.

Anat. Rec.