Naoki YAMANAKA, Toshio IMANARI,* Zenzo TAMURA,*

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1 J. Biochem., 73, (1973) Uncoupling of Oxidative Phosphorylation of Rat Liver Mitochondria by Chinoform Naoki YAMANAKA, Toshio IMANARI,* Zenzo TAMURA,* and Kunio YAGI Institute of Biochemistry, Faculty of Medicine, University of Nagoya, Nagoya and *Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo Received for publication, October 7, Chinoform (5-chloro-7-iodo-8-quinolinol) uncoupled the oxidative phosphorylation of rat liver mitochondria in the presence of Mg ions. 2. Release of the controlled respiration was induced by chinoform both in the presence and absence of inorganic phosphate, and was insensitive to oligomycin. ATPase activity of intact mitochondria was induced by chinoform in the presence of Mg ions, and was inhibited by oligomycin. These data indicate that chinoform affects the "non-phosphorylated high-energy intermediate." 3. Prior addition of Mg ions was essential for the uncoupling action of chinoform. Chinoform-Mg chelate uncoupled oxidative phosphorylation in the absence of Mg ions. Mg ions could be replaced by other cations such as Fe ions. 4. Chinoform glucuronide (5-chloro-7-iodo-8-quinolyl-ƒÀ-D-glucuronic acid) had no uncoupling action. Chinoform, 5-chloro-7-iodo-8-quinolinol, has been widely used in the treatment of intestinal disorders caused by virulent microorganisms. Recently in Japan, however, administration of this drug at high dosage has been implicated in the etiology of a neuropathy called SMON,** often characterized by the appearance of a green substance in fur on the tongue, urine and faeces of the patients (1-3). The green substance was found to be an iron chelate of chinoform, and excretion of free chinoform in the urine was also observed (4, 5). This led us to reexamine in detail the toxicity of this ** This name is an abbreviation of "subacute myelo - optico neuropathy." drug. By analogy with the fact that 2, 4-dinitrophenol (DNP), a typical uncoupler of oxidative phosphorylation, is known to be a neurotoxin, one of our approaches towards understanding of the relationship between SMON and chinoform was to examine the effect of this drug on mitochondrial oxidative phosphorylation. Results reported in this communication indicate that metal chelates of chinoform uncouple oxidative phosphorylation. MATERIALS AND METHODS Reagents-Chinoform was obtained commercially and used after recrystallization from ethanol. Chinoform glucuronide, 5-chloro-7- Vol.73, No.5,

2 994 N. YAMANAKA, T. IMANARI, Z. TAMURA, and K. YAGI iodo-8-quinolyl-ƒà-d-glucuronic acid, was synthesized by the method of Matsunaga and Tamura (6). Chinoform-Mg chelate was prepared as follows: a solution of 120 mg of chinoform in 10 ml of dioxane was added dropwise to 50 mg of MgSO4 E7H2O and 2 g of NH4Cl in 80 ml of a mixture of dioxane and water (1: 1, v/v) with vigorous shaking. The solution was made slightly alkaline with 10% ammonium hydroxide and stood for 30 min at room temperature. It was then mixed with 20 ml of water and stood for 3 hr. The chinoform-mg chelate was precipitated and was collected by filtration, washed with water, dried at 105 Ž and recrystallized from methanol. Hexokinase [EC ] (16 units/mg), ADP and oligomycin were purchased from Sigma Chemical Co. Oligomycin and DNP were dissolved in ethanol at concentrations of 100ƒÊg/ml and 10 mm, respectively. Preparation of Rat Liver Mitochondria- Rat liver mitochondria were isolated by the method of Hogeboom (7), using medium containing 0.21 M mannitol, 0.07 M sucrose, and 0.1 mm EDTA (8). Three additional washings were performed to reduce the contents of light mitochondria and other contaminants. Mitochondrial protein was determined by the biuret method (9). Measurements of Oxygen Uptake and Inorganic Phosphate-Oxygen uptake at 20 Ž was measured with a Beckman Oxygen Sensor. The standard reaction mixture (5.0 ml) contained 0.3 M mannitol, 10 mm KCl, 10 mm inorganic phosphate, 5 mm Tris-HCl (ph 7.4), 2.5 mm Mg ions, and 0.25 mm EDTA. The reaction was started by adding 12 mg mitochondrial protein and 50 ƒêmoles of succinate. Solution of 0.1 M chinoform, chinoform glucuronide or chinoform-mg chelate in dimethyl sulfoxide, was added to the reaction mixture as indicated in the figures. Dimethyl sulfoxide at the concentrations employed had no effect on oxygen uptake. States 3 and 4 of the released respiration are due to the definition of Chance and Williams (10). The reaction medium for measurement of the P/0 ratio contained 0.3 M mannitol, 10 mm KCl, 1.0 mm inorganic phosphate, 5 mm Tris-HCl (ph 7.4), 2.5 mm Mg ions, 0.25 mm EDTA, 0.2 mm ADP, 10 mm glucose, and 0.15 mg of hexokinase. Inorganic phosphate was measured by the method of Berenblum and Chain (11) as modified by Martin and Doty (12). Measurement of Induction of ATPase Activity-The reaction medium (1.0 ml) contained 0.3 M mannitol, 5 mm Tris-HCl (ph 7.4), 10 mm ATP, and 6 mg of mitochondrial protein. Induction of ATPase by the test substance was measured by determining the liberated inorganic phosphate after incubation of the reaction mixture with the test substance at 20 Ž for 3 min. Inorganic phosphate was measured as described above. RESULTS Effect of Chinoform on Oxidative Phosphorylation in Rat Liver Mitochondria-The controlled respiration in the standard reaction mixture was released by addition of 2ƒÊmoles of chinoform (final concentration, 0.4 mm) (Fig. 1). In the presence of chinoform, esterification of inorganic phosphate was inhibited, resulting in decrease of the P/O ratio from 2 to 0, as shown in Table I. These results indicate that chinoform acts as an uncoupler. To examine the site of action of chinoform in this uncoupling, the effect of oligomycin on the uncoupling action of chinoform was investigated. As shown in Fig. 2, the release of respiration by chinoform was not inhibited by Fig. 1. Effect of chinoform on oxygen uptake by rat liver mitochondria. The medium (5.0 ml) contained 0.3 M mannitol, 10 mm KCl, 10 mm inorganic phosphate, 5 mm Tris-HCl (ph 7.4), 2.5 mm Mg ions, and 0.25 mm EDTA. J. Biochem.

UNCOUPLING EFFECT OF CHINOFORM 995 TABLE I. Effect of chinoform on the P/O ratio of mitochondria. The reaction medium (5.0 ml) contained 0.3 M mannitol, 10 mm KCl, 1.

3 UNCOUPLING EFFECT OF CHINOFORM 995 TABLE I. Effect of chinoform on the P/O ratio of mitochondria. The reaction medium (5.0 ml) contained 0.3 M mannitol, 10 mm KCl, 1.0 mm inorganic phosphate, 5 mm Tris-HCl (ph 7.4), 2.5 mm Mg ions, 0.25 mm EDTA, 0.2 mm ADP, 10 mm glucose, and 0.15 mg of hexokinase. Oxygen uptake was measured with a Beckman Oxygen Sensor and decrease in inorganic phosphate was estimated as described in " MATERIALS AND METHODS." TABLE II. Effect of chinoform on ATPase activity of mitochondria. were added sequentially in the order shown from left to right. ATPase activity in intact mitochondria was estimated by the amount of inorganic phosphate liberated. The reaction medium (1.0 ml) contained 0.3 M mannitol, 5 mm Tris-HCl (ph 7.4), 10 mm ATP, and 6 mg mitochondrial protein. ions (2.5 mm) were added after addition of chinoform. The reaction was continued for 3 min at 20 Ž after adding the compounds shown. Inorganic phosphate liberated was measured as described in " MATE- RIALS AND METHODS." Fig. 2. Effect of oligomycin on the induction of respiration by chinoform. The medium (5.0 ml) was the same as for Fig. 1. oligomycin. The effect of chinoform on ATPase activity was also examined. As shown in Table II, ATPase activity was induced by addition of chinoform, as by addition of DNP. However, this induction of ATPase by chinoform was completely inhibited by oligomycin. It was found that inorganic phosphate was not essential for the release of the controlled respiration by chinoform (see Fig. 4). for Mg Ions for the Uncoupling Effect of Chinoform \Chinoform did not release the controlled respiration in the absence of Mg ions. Moreover addition of chinoform first and then Mg ions did not release the respiration, but further addition of ADP produced the state transition. DNP added in this period released the respiration (Fig. 3). This was verified by the data on the effect of chinoform on the P/O ratio (Table I) and on ATPase activity (Table II). Figure 4 shows the effect of EDTA on the release of the respiration by chinoform in the presence of Mg ions. EDTA diminishes the effect of chinoform, probably due to formation of an EDTA-Mg chelate. The requirement for Mg ions was examined further. When chinoform dissolved in dimethyl sulfoxide was mixed with the standard reaction mixture containing Mg ions, formation of chinoform-mg chelate could be demonstrated by absorption measurement (4). No chinoform-mg chelate was detected when chinoform solution was mixed with the standard reaction mixture without Mg ions. Instead, chinoform was precipitated as a fine sediment and the latter did not seem to react appreciably with Mg ions. Thus the prior existence of Mg ions seems necessary for formation of the chinoform-mg chelate under the present experimental conditions. Conditions which do not favor formation of chinoform-mg chelate seem to prevent chinoform from acting as an ucoupler of oxidative phosphorylation. However, chinoform-mg chelate caused uncoupling in the absence of Mg ions Vol. 73, No. 5, 1973

4 996 N. YAMANAKA, T. IMANARI, Z. TAMURA, and K. YAGI Fig. 3. Requirement of the presence of Mg ions for release of respiration by chinoform. The medium (5.0 ml) contained 0.3 M mannitol, 10 mm KCl, 10 mm inorganic phosphate, and 5 mm Tris-HCl (ph 7.4). Fig. 5. Effect of chinoform-mg chelate on oxygen uptake of rat liver mitochondria. The medium (5.0 ml) contained 0.3 M mannitol, 10 mm KCl, and 10 mm Tris-HCl (ph 7.4). Fig. 4. Effect of EDTA on induction of respiration by chinoform. The medium (5.0 ml) contained 0.3 M mannitol, 10 mm KCl, and 10 mm Tris-HCl (ph 7.4). (Fig. 5), though the extent of uncoupling was somewhat less than that observed in medium containing Mg ions when chinoform was added. of Mg Ions by Other Cations an iron chelate of chinoform was found in the urine of SMON patients, the effect of iron on the uncoupling effect of chinoform was then examined. Prior addition of Fe3+ (FeCl3) was found to induce the uncoupling effect of chinoform (Fig. 6). Similar results were obtained when Fe2+(FeCl2) or Mn ions were used instead of Fe3+. These results suggest that cations which form chelation compounds with chinoform are all effective in inducing the uncoupling effect of chinoform. Effect of Chinoform Glucuronide-Chino- J. Biochem.

5 UNCOUPLING EFFECT OF CHINOFORM 997 form glucuronide (0.4 and 0.8 mm) did not enhance the rate of succinate oxidation in the presence of Mg ions (Fig. 7). Further addition of ADP to this reaction mixture released the controlled respiration and the respiration rate of state 4 was reestablished when ADP added had all been phosphorylated to ATP. Then, the uncoupling effect of DNP was observed. Similar results were obtained with chinoform glucuronide in the absence of Mg ions. These results show that chinoform glucuronide, which is synthesized from chinoform in living body- (13), has no effect on oxidative phosphorylation in mitochondria. DISCUSSION Fig. 6. Effect of Fe3+ on the induction of respiration by chinoform. The medium (5.0 ml) contained 0.3 M mannitol, 10 mm KCl, 10 mm Tris-HCl (ph 7.4), and 0.8 mm Fe3+ (FeCl3). Fig. 7. Effect of chinoform glucuronide on oxygen uptake of rat liver mitochondria. The medium (5.0 ml) was the same as for Fig. 1. The present data clearly indicate that chinoform uncouples oxidative phosphorylation. Chinoform probably attacks the "non-phosphorylated high-energy intermediate" (10) produced during the oxidative phosphorylation for the following reasons. The uncoupling action of chinoform is not inhibited by oligomycin, ATPase activity of mitochondria is induced by chinoform and inorganic phosphate is not essential for the release of the controlled respiration by chinoform. Under the present experimental conditions, the prior addition of Mg ions was essential for the occurrence of the uncoupling effect of chinoform. This seems to be due to formation of chinoform-mg chelate, since chinoform-mg chelate was found to release the controlled respiration in the absence of Mg ions, though the effect was smaller than that obtained by sequential addition of Mg ions and chinoform. It was further confirmed that the chinoform- Mg chelate was formed on sequential addition of Mg ions and chinoform to the medium, but not on their addition in the reverse order. It was also found that for the uncoupling action of chinoform Mg ions can be replaced for other cations, such as Fe ions. From these results we conclude that the requirement of cation for manifestation of the uncoupling effect of chinoform can be ascribed, at least partly, to formation of a chinoformcation chelate. The induction of the uncoupling effect of chinoform by formation of a chelate may be due to an increase in hydrophobicity, since Hemker (14) and Terada and Muraoka (15) reported that introduction of a hydrophobic group(s) into an uncoupler increased the uncoupling effect. If we assume that the toxicity of chinoform in provoking SMON has some relation to the uncoupling effect reported here, these cations must be considered to be involved in the toxicity. Excretion of chinoform-fe chelate by patients with SMON (4, 5) supports this possibility, even though it does not exclude the possible toxicity of free chinoform. Vol. 73, No, 5, 1973

6 998 N. YAMANAKA, T. IMANARI, Z. TAMURA, and K. YAGI The glucuronide of chinoform has no uncoupling effect. This result is in accord with the view that chinoform can be detoxified in the liver through formation of its glucuronide. REFERENCES 1. T. Takasu, A. Igata, and Y. Toyokura, Igaku no Ayumi, 72, 539 (1970). 2. A. Igata, T. Takasu, and Y. Toyokura, Igaku no Ayumi, 72, 639 (1970). 3. A. Igata, H. Hasebe, and T. Tsuji, Nihon Iji Shinpo, 2421, 25 (1970). 4. M. Yoshioka and Z. Tamura, Igaku no Ayumi, 74, 320 (1970). 5. T. Imanari and Z. Tamura, Igaku no Ayumi, 75, 547 (1970). 6. I. Matsunaga and Z. Tamura, Chem. Pharm. Bull., 19, 1056 (1971). 7. G.H. Hogeboom, "Methods in Enzymology," ed. by S.P. Colowick and N.O. Kaplan, Academic Press, New York, Vol.1, p.16 (1955). 8. B. Chance and B. Hagihara, Biochem. Biophys.. Res. Commun., 3, 1 (1960). 9. A.G. Gornall, C.S. Bardawill, and M.M. David, J. Biol. Chem., 177, 751 (1949). 10, B. Chance and G.R. Williams, Adv. Enzymol. 17, 65 (1956). 11. J. Berenblum and E. Chain, Biochem. J., 32, 295 (1938). 12. J.B. Martin and D.M. Doty, Anal. Chem., 21,. 965 (1949). 13. K. Liewendahl, V. Kivikangas, and B.A. Lamberg, Nucl. Med. (Stuttg.), 6, 32 (1967). 14. H.C. Hemker, Biochim. Biophys. Acta, 63, 46. (1962). 15. H. Terada and S. Muraoka, Mol. Pharmacol., 8, 95 (1972). J. Biochem.

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