No abstract

Ethidium bromide (2,7-diamino-9-phenylphenanthridinium-1 0-ethyl bromide) inhibits mitochondria1 DNA (1), RNA (2), and protein syntheses (3, 4). Ethidium bromide can intercalate with the nucleic acids (5) and it is commonly thought that inhibition of these processes occurs by this mechanism rather than by inhibiting ATP generation. Ethidium bromide can combine with mitochondrial membranes (6) producing a color shift in the ethidium spectrum which indicates interaction with a low dielectric site on the membrane. This interaction is enhanced by the presence of ATP but not ADP nor AMP (7). Degradation of mitochondrial DNA may imply that energy processes are inhibited by ethidium bromide and ethidium bromide was considered an uncoupler of oxidative phosphorylation (8).Since an intact energy transducing membrane is required to show temperature discontinuities in Arrhenius plots for state 3 and state 4 respirations (9), we have studied the interaction of ethidium bromide on respiratory control in intact mitochondria. This communication presents evidence that ethidium bromide, at mutagenic concentrations of 1 pg/106 liver cells, is a phosphorylation inhibitor and not an uncoupler of mitochondrial respiratory control processes. Exhaustive washing of mitochondria did not remove inhibition caused by ethidium bromide nor did it effect more than 27% removal of bound dye.Materials and Methods. Hepatic mitochondria were prepared from Sprague-Dawley rats as described previously (10). Respiratory rates were determined polarographically (10) and the conventions of Chance and Williams (11) were used to identify respiratory states and in computing respiratory control ratios (RCR). High amplitude volume changes of the organelles (swelling and contraction cycles) and mito-chondrial ATPase (ATP phosphohydrolase, EC 3.6.1.4) activity were monitored and assayed as already described (1 2). Uncoupler stimulated ATPase and energized contraction of the organelles were both inhibited completely by oligomycin (3 and 6 pg/mg mitochondrial protein, respectively). Mitochondrial protein was solubilized with 1 % deoxycholate and estimated by a biuret method (13). Sucrose, EDTA, and sodium deoxycholate were from Fisher Scientific Co. ; mannitol was from Pfanstiehl Laboratories; ethidiilm bromide, ADP, ATP, substrates, and other biochemicals were from Sigma Chemical Co. Ethidium bromide solutions were protected from light at all times.Results. Ethidium bromide depressed Pi acceptor control of respiration in mitochondria oxidizing either glutamate or SUCcinate (Fig. 1). The effect was concentration dependent and resulted from selective inhibition of respiration in the active state (state 3). This inhibition was specifically responsible for complete depression of the RCR to unity; at no time were the organelles released from control by uncoupling, i.e., the resting state respiration (state 4) was unaltered by ethidium bromide throughout its effective concentration range. As in the case of depression of the RCR by a large number of lipophilic organic c...