Tatsuo Matsuhashi

Suppression of the formation of megamitochondria by scavengers for free radicals.

In the present study we have attempted to suppress the formation of megamitochondria by scavengers for free radicals since conditions for the formation of megamitochondria are often intimately related to the generation of free radicals. We employed three different experimental conditions to induce megamitochondria in the liver: ethanol, hydrazine and chloramphenicol (CP). Scavengers for free radicals tested were: alpha-tocopherol, coenzyme Q10(CoQ10) and 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl(4-OH-TEMPO). Allopurinol (AP), a xanthine oxidase inhibitor, was also tested. Results obtained were as follows. (1) Changes observed in the liver of animals treated with ethanol, hydrazine or CP were: formation of megamitochondria; decreases in the body weight and the weight of the liver; remarkable increases in the level of lipid peroxidation; increases in the activity of xanthine oxidase. (2) 4-OH-TEMPO was most effective in improving these changes. A mechanism of the formation of megamitochondria is proposed stressing the role of free radicals in the mechanism.

Complete Suppression of Ethanol-Induced Formation of Megamitochondria By 4-Hydroxy-2,2,6,6-Tetramethyl-Piperidine-1-Oxyl (4-OH-Tempo)

An attempt has been made to suppress the ethanol-induced formation of megamitochondria (MG) in the rat liver by 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (4-OH-TEMPO), a free radical scavenger, and by allopurinol (AP), a xanthine oxidase inhibitor. Changes observed in the liver of animals given ethanol (EtOH) for 1 month were remarkable decreases both in the body weight gains during the course of the experiment and in the liver weight at the time of sacrifice compared to those of the control; remarkable increases in the level of thiobarbituric acid reactive substances and lipid soluble fluorophores both in microsomes and mitochondria; decreases in the content of cytochrome a+a3 and b and lowered phosphorylating ability of mitochondria; and formation of MG in the liver. A combined treatment of animals with EtOH plus 4-OH-TEMPO completely suppressed the formation of MG in the liver induced by EtOH and distinctly improved the changes caused by EtOH, as specified above, while AP partly suppressed the MG formation. Results described herein provide additional insight into chronic hepatotoxicity of EtOH besides that previously reported. A novelty of the present work is that we were able for the first time to demonstrate reversibility of EtOH-mediated ultrastructural changes of the liver by a simple administration of aminoxyl-type free radical scavenger, 4-OH-TEMPO. Our results suggest that free radicals may be involved in the mechanism of the formation of MG induced by EtOH.

ROLE OF FREE RADICALS IN THE MECHANISM OF THE HYDRAZINE-INDUCED FORMATION OF MEGAMITOCHONDRIA

The effect of 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl(4-OH-TEMPO), a scavenger for free radicals, and 4-hydroxypyrazolo [3,4-d(pyrimidine)allopurinol], a xanthine oxidase inhibitor, on the hydrazine-induced changes of mitochondrial ultrastructure and those in the antioxidant system of the liver were investigated using rats as experimental animals. Animals were placed on a powdered diet containing 0.5% hydrazine for 7 d in the presence and absence of a combined treatment with 4-OH-TEMPO or allopurinol. Results obtained were as follows. 4-OH-TEMPO completely prevented the hydrazine-induced formation of megamitochondria in the liver, while it was partly prevented by allopurinol. The following changes observed in hydrazine-treated animals were improved almost completely by 4-OH-TEMPO:decreases in the body weight and liver weight; lowered rates of ADP-stimulated respiration and coupling efficiency of hepatic mitochondria; remarkable elevation of the level of lipid peroxidation. Improving effects of allopurinol were incomplete. The present results suggest that free radicals may play a key role in the mechanism of the hydrazine-induced formation of megamitochondria and that a part of free radicals generated during the hydrazine intoxication is ascribed to the degradation of purine nucleotides via xanthine oxidase. A general mechanism of the megamitochondria formation induced in various pathological conditions besides the case of hydrazine are discussed.

Mechanism of the formation of megamitochondria in the mouse liver induced by chloramphenicol

Correlation between chloramphenicol-induced formation of megamitochondria in the mouse liver and oxidative stress was studied by lipid peroxidation analysis and electron microscopic technique. Chloramphenicol suppressed increases in the body weight and liver weight of experimental animals and at the same time induced a remarkable increase in lipid peroxidation in the liver during the formation of megamitochondria. A spin trapping agent, 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl, abolished all these changes induced by chloramphenicol. Namely, both the body weight and liver weight of chloramphenicol-treated animals stayed at the same levels as those of the control, and the formation of megamitochondria was completely suppressed. Allopurinol, a xanthine oxidase (EC 1.2.3.2) inhibitor, partly inhibited the changes induced by chloramphenicol, as described above. These results suggest that chloramphenicol-induced formation of megamitochondria is not simply ascribed to the suppression of the dividing process of mitochondria due to lowered protein synthesis in mitochondria but is intimately related to oxidative stress. Furthermore, the results obtained with allopurinol may indicate that enhanced levels of lipid peroxidation observed in chloramphenicol-treated animals are partly due to enhanced rate of the degradation of purine nucleotides catalyzed by xanthine oxidase.

Suppression of the hydrazine-induced formation of megamitochondria in the rat liver by coenzyme Q10

The effects of coenzyme Q10 (CoQ10) on the hydrazine-induced changes in the structure of mitochondria and those in antioxidant systems of the liver were investigated using rats as experimental animals. Animals were placed on a powdered diet containing 1.0% hydrazine for 7-8 days in the presence or absence of the combined treatment with CoQ10. Results obtained were as follows: (a) treatment of animals with CoQ10 prevented the hydrazine-induced formation of megamitochondria in the liver; (b) changes observed in the liver of the hydrazine-treated animals in comparison to the control were increases in the contents of α-tocopherol and CoQ analogs, increases in the levels of lipid peroxidation, decreases in the level of reduced glutathione with increases in that of oxidized glutathione, and increases in the ratio of unsaturated to saturated fatty acids in phospholipid domains of mitochondrial membranes; and (c) administration of CoQ10 to hydrazine-treated animals suppressed enhanced lipid peroxidation and improved lowered adenosine diphosphate/O ratios of mitochondria. The present data suggest that CoQ10 suppresses the hydrazine-induced formation of megamitochondria by scavenging free radicals generated from hydrazine and its metabolites.

Studies on urea synthesis in the liver of rats treated chronically with ethanol using perfused livers, isolated hepatocytes, and mitochondria

Changes in urea synthesis in the liver of rats treated with 32% ethanol in the drinking water for up to 6 months were studied using perfused livers, isolated hepatocytes, and mitochondria. Results obtained from ethanol-treated rats are summarized as follows: (1) the mitochondria of the hepatocytes of rats treated with ethanol for 2 months or longer became enlarged to various degrees, (2) the levels of ammonia in the serum remained within a normal range, while those in liver tissue were elevated compared with the control, (3) urea synthesis from ammonia in perfused livers was decreased markedly, while that from citrulline remained in the normal range, (4) the activities of carbamyl phosphate synthetase (CPS; EC 2.7.2.5) and ornithine transcarbamylase (OTC; EC 2.1.3.3) in mitochondria were unchanged compared with those of the control, and (5) the levels of ATP in liver tissue and the ability of mitochondria to synthesize ATP were decreased markedly compared with the control. Both the level of ATP in the hepatocytes and the synthesis of urea from ammonia by perfused livers of rats treated with ethanol were resistant to externally added ethanol, while those of control animals were severely affected. These results suggest that the intracellular level of ATP is intimately related to urea synthesis in both control and ethanol-treated animals, and lowered levels of ATP may be a key factor in the suppression of urea synthesis in ethanol-treated animals.

Reduction of 2,4-dinitrotoluene by Wistar rat liver microsomal and cytosol fractions.

2,4-Dinitrotoluene (2,4-DNT) is an important industrial nitroaromatic compound. 2,4-Diaminotoluene (2,4-DAT), one of the urinary metabolites of 2,4-DNT, is carcinogenic when fed to rats. The objectives of these studies were to determine whether 2,4-DAT was formed from 2,4-DNT in rat liver and to clarify the nature of enzymes responsible for reduction of 2,4-DNT to 2,4-DAT. Data obtained from thin-layer and high-pressure liquid chromatography indicated that metabolites produced by microsomal preparations were 2-amino-4-nitrotoluene (2A4NT) and its isomer (4A2NT). This microsomal activity is probably mediated by cytochrome P-450 because the reduction is blocked by carbon monoxide and primary amines [aniline, n-octylamine, and 2,4-dichloro-6-phenylphenoxyethylamine (DPEA)]. In contrast, 2,4-DNT was metabolized via 2A4NT and 4A2NT to 2,4-DAT by cytosolic preparations. The greatest part of the reduction activity was due to cytosolic xanthine oxidase because the reduction was blocked by allopurinol. The results of this investigation suggest that reduction of 2,4-DNT to 2,4-DAT by cytosolic xanthine oxidase may play a role in 2,4-DNT hepatocarcinogenicity.

Potentiating effects of organomercuries on clastogen-induced chromosome aberrations in cultured Chinese hamster cells

Mercury compounds are among the most serious environmental pollutants. In this communication, the potentiating effects of organic and inorganic mercuries on clastogen-induced chromosome aberrations were studied in Chinese hamster CHO K1 cells. Post-treatment with monoalkylated mercuries--methyl mercuric chloride (MeHgCl) and ethyl mercuric chloride (EtHgCl)--increased the number of breakage- and exchange-type aberrations induced by 4-nitroquinoline 1-oxide (4NQO) and methyl methanesulfonate. With the DNA crosslinking agents mitomycin C (MMC) and cisplatin, MeHgCl enhanced both types of aberrations while EtHgCl enhanced breakage-type aberrations only. Since these monoalkylated mercuries did not show clastogenic effects by themselves under the present experimental conditions, the increases in chromosome aberrations were not additive. Dialkylated mercuries (dimethyl mercury and diethyl mercury) and inorganic mercuries (HgCl and HgCl2) did not show any potentiating effects. When MMC- or 4NQO-treated cells were post-treated with MeHgCl during the G1 phase, both breakage- and exchange-type aberrations were enhanced. Treatment with EtHgCl during the G1 phase also enhanced both types of aberrations induced by 4NQO. With MMC, however, G1 treatment with EtHgCl did not show any potentiating effect. MeHgCl and EtHgCl treatments during the G2 phase enhanced breakage-type aberrations only. Based on these results, the following possible mechanisms for potentiation of clastogenicity by monoalkylated mercuries were suggested; (1) they interfere with repair of base lesions induced by 4NQO and MMS during the pre-replicational stage, thereby increasing unrepaired DNA lesions which convert into DNA double-strand breaks in S phase, (2) MeHgCl (but not EtHgCl) also inhibits repair of crosslinking lesions during the pre-replicational stage, and (3) their G2 effects enhance breakage-type aberrations only.