Effect of thyroid state on H2O2 production by rat liver mitochondria

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Abstract

It has been suggested that activation of mitochondrial respiration by thyroid hormone results in oxidative tissue injury secondary to increased reactive oxygen species production. In order to throw light on this subject, the effects of thyroid state on O2 consumption and H2O2 release by rat liver mitochondria were investigated. Hypothyroidism decreased the rates of O2 consumption and H2O2 release by succinate or pyruvate/malate-supplemented mitochondria during both State 4 and State 3 respiration, whereas hyperthyroidism increased such rates. Conversely, with both substrates and during either respiration phase, the percentage of O2 released as H2O2 was not significantly affected by thyroid state. On the other hand, the capacity of mitochondria to remove H2O2 increased by about 17% in hyperthyroid rats and decreased by about 35% in hypothyroid ones. This result indicates that the ratio between H2O2 production and release and so the percentage of O2 turned into H2O2 instead of being reduced to water increase in the transition from hypothyroid to hyperthyroid state. In light of previous observations that mitochondrial content of cytochromes and ubiquinone also increases in such a transition, the modifications of H2O2 production appear to be due to a modulation by thyroid hormone of the mitochondrial content of the autoxidisable electron carriers. This view is supported by measurements of H2O2 release in the presence of respiratory inhibitors, which show that the thyroid state-linked changes in H2O2 production occur at H2O2 generator sites of both Complex I and Complex III.

Introduction

It is well known that the main biological process leading to reactive oxygen species (ROS) generation is electron transport within inner mitochondrial membranes (Chance et al., 1979). One-electron autoxidation of mitochondrial carriers appears to lead to production of superoxide anion radical (Oradical dot2) (Turrens and Boveris, 1980). Then superoxide dismutase in the mitochondrial matrix converts superoxide to H2O2 (Loschen et al., 1974), which can be turned into the highly reactive radical dotOH radical via the Fenton reaction. ROS can attack various cellular substances including membrane polyunsaturated fatty acids, which are subjected to a chain of peroxidative reactions (Kehrer, 1993, Halliwell and Gutteridge, 1998). To neutralise the oxidative effects of ROS, biological systems are endowed with a great number of antioxidant substances (Yu, 1994). When ROS generation exceeds the antioxidant capacity of cells, potentially harmful oxidative stress develops (Sies, 1991). This phenomenon has been related to many disorders (Freeman and Crapo, 1982, Halliwell and Gutteridge, 1990, Kehrer, 1993), and it has also been suggested that activation of mitochondrial respiration by thyroid hormone results in oxidative tissue injury secondary to increased ROS production (Asayama and Kato, 1990). It is well established that experimental hyperthyroidism is associated with increased hepatic levels of lipid peroxidation indicators including: (i) thiobarbituric acid reactive products both in microsomal fraction (Fernández et al., 1985) and homogenate (Fernández et al., 1985, Venditti et al., 1997); (ii) hydroperoxides in microsomal fraction (Landriscina et al., 1988), homogenate (Venditti et al., 1997, Venditti et al., 1999) and mitochondria (Venditti et al., 1999). Extensive information on the effects of the hypothyroid state on liver peroxidative processes is lacking. However, it has been reported that the levels of lipid peroxidation products in the liver from hypothyroid rats (Venditti et al., 1997) and mice (Guerrero et al., 1999) do not differ from euthyroid values. It is possible that opposite changes in rate of mitochondrial ROS production are in part responsible for the pattern shown in hyperthyroid and hypothyroid liver. Previous reports indicate that ROS production by liver mitochondria is higher in hyperthyroidism (Swaroop and Ramasarma, 1985, Fernández and Videla, 1993) and lower in hypothyroidism (Swaroop and Ramasarma, 1985). However, neither the mechanisms nor the sites responsible for the different rate of ROS production have been studied. To throw light on this subject, we investigated the effects of thyroid state on rates of both oxygen consumption and H2O2 release in intact mitochondria from rat liver using Complex I- and Complex II-linked substrates and inhibitors specific for different segments of the respiratory chain. In order to obtain information on H2O2 production by mitochondria, we also determined their capacity to remove the peroxide.

Section snippets

Animals

Male Wistar rats (60 days old) were used in the experiments. From day 45, animals were randomly assigned to one of three groups: euthyroid, hypothyroid, and hyperthyroid. Hypothyroidism was induced by administering methimazole (MMI) in drinking water (0.02% w/v) for 15 days. Hyperthyroidism was elicited by daily i.p. injections of triiodothyronine (T3) (10 μg/100 g body weight) for 10 days in MMI-treated rats. All animals were kept under the same environmental conditions, i.e. at a room

Determination of thyroid state

The thyroid state of different groups of animals was characterised by the physiological data reported in Table 1. The values indicate the effectiveness of MMI and T3 treatment administration to achieve hypothyroid and hyperthyroid state, respectively.

Cytochrome oxidase activity

In liver homogenates and mitochondria, COX activity was decreased by the MMI treatment, but was increased by T3 treatment (Table 2) in agreement with previous observations that liver oxidative capacity (expressed as COX activity per mg of protein) (

Discussion

Until now information regarding the effects of thyroid hormone on the rates of H2O2 release by liver intact mitochondria has come from only one study showing that hyperthyroidism increases and hypothyroidism decreases the rate of H2O2 release during succinate-supported State 4 respiration (Swaroop and Ramasarma, 1985). The present study shows that such thyroid state-linked changes also take place with Complex I-linked substrates and during State 3 respiration. Because in the cells mitochondria

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