Comparative study of the effects of benzoquinones on mitochondria isolated from rat liver and insect flight muscle

Abstract

This study was intended to investigate the effects of four biologically active benzoquinones namely: maesanin, maesaquinone, juglone and embelin on mitochondria isolated from rat liver and insect flight muscle. These benzoquinones occur naturally in the plant family myrsinaceae. Five species of myrsinaceae have been identified in Kenya and are widely distributed. Several studies have shown that most of the benzoquinones have pharmacological potential and can also protect stored grains against insect pests. There is, however, no report relating the interaction of benzoquinones with insect energy metabolism. Three insect species were used in this study i.e. Schistocerca gregaria (Lipid and carbohydrate metab oltsmg), Periplaneta americana (carbohydrate me1.abolising) and Glos~ina morsitans (amino acid metabolising) . The effects of benzoquinones on energy metabolism was determined by measuring the rate of oxygen consumption, oxidative phosphorylation and ATPase activity. Mitochondria from the insect thoracic muscle and rat liver mitochondria were both prepared by differential centrifugation of the homogenates from the respective tissues. The rate of oxygen consumption was recorded polarographically by an oxygen electrode at 250C in a closed magnetically stirred chamber. ATPase activity was measured enzymatically by pyridine nucleotide coupled assays using Perkin Elmer spectrophotometer. In the absence of benzoquinones (controls) the respiratory control ratios were found to be above 3 for the four types of mitochondria studied, which was an indication that the mitochondria were well coupled and could effectively phosphorylate ADP to ATP. When either maesaquinone or embelin were added in the reaction chamber after the substrate, the-rate of oxygen consumption was increased. Subsequent additions ...of ADP and CCCP after the e- ,'" benzoquinones increased the rate of oxygen consumption in rat liver and Glossina mitochondria. These observations indicated that the rrutochondrta were partially uncoupled. Embelin had similar effect on Schistocerca mitochondria. However, when either ADP or CCCP were added after the benzoquinones (maesaquinone and embelin), there was no further stimulation in the rate of oxygen consumption by Periplaneta mitochondria. A similar effect was observed with maesaquinone on Schistocerca mitochondria. From these observations, it was suggested that the mitochondria were completely uncoupled. Juglone was found to completely uncouple the mitochondria from rat liver and flight muscle of Schistocerca and Periplaneta while it inhibited oxygen consumption by mitochondria isolated from Glossina. The uncoupling (partial or complete) was depicted by low respiratory control ratios (about 1.00) and ADP/0 ratios. When the effect of maesanin was "investigated, it was found that addition of this benzoquinone after-the substrates (malate plus glutamate or succinate) inhibited both state 4 and state 3 rates of oxygen consumption. This effect was observed on the four types of mitochondria used. From this observation it was suggested that maesanin possibly inhibited the electron flow along the electron transport chain. Further experiments with the known inhibitors of electron transport chain i.e. rotenone for Site I, antimycin for Site II and KCN for Site III indicated that maesanin inhibited the electron transport chain before cytochrome C. This was found to be true for the three types of insects and rat liver mitochondria. The ATPase activity was studied on both directions i.e. toward ATP formation (ATPase phosphorylating) and toward ATP hydrolysis (ATPase dephosphorylating). Effects of benzoquinones on ATPase (phosphorylating) indicated that the enzyme activity was inhibited by maesanin in all the four types of mitochondria. On the other hand, maesaquinone and embelin were found to stimulate the activity of this enzyme though in Glossina the stimulation was not significant. Juglone also had a similar effect in all the four cases though it lowered the activity in Glossina mitochondria. " The ATPase activity in the dtreetton of ATP hydrolysis of mitochondria from the four sources was stimulated by maesaquinone and embelin except that in the Glossina mitochondria the stimulation was not significant. However, juglone inhibited the activity and maesanin had rio significant effect. These observations indicate that these benzoquinones interfere with energy generation. The fact that benzoquinones either uncouples the mitochondria or inhibits the electron transport chain and interferes with ATPase activity suggest that they interrupt energy metabolism in the mitochondria of the three types of insects and rat liver. It is yet to be investigated whether in vivo the mechanism of toxicity is the same in insect and rat.