APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1990, p. 2114-2119
Vol. 56, No. 7
0099-2240/90/072114-06$02.00/0 Copyright ©D 1990, American Society for Microbiology
Effects of Cyclohexane, an Industrial Solvent, on the Yeast Saccharomyces cerevisiae and on Isolated Yeast Mitochondria SALVADOR URIBE,* PABLO RANGEL, GLORIA ESPINOLA, AND GABRIELA AGUIRRE Instituto de Fisiologia Celular, Universidad Nacional Aut6noma de Mexico, Apartado Postal 70-600, 04510 Mexico D.F., Mexico Received 12 February 1990/Accepted 22 April 1990
Little information on the effects of cyclohexane at the cellular or subcellular level is available. In Saccharomyces cerevisiae, cyclohexane inhibited respiration and diverse energy-dependent processes. In mitochondria isolated from S. cerevisiae, oxygen uptake and ATP synthesis were inhibited, although ATPase activity was not affected. Cyclohexane effects were similar to those reported for beta-pinene and limonene, suggesting that the cyclohexane ring in these monoterpenes may be a determinant for their biological activities. MATERIAL AND METHODS Materials. All reagents were of the highest purity available commercially. Bovine serum albumin, NADP, ADP, car-
Cyclohexane is a nonsubstituted alicyclic hydrocarbon used as an industrial solvent. Cyclohexane is regarded as relatively nontoxic, and it has been increasingly used as a substitute for benzene (9). Among unicellular organisms, only one bacterium, tentatively identified as a Nocardia sp., has been described to grow by using cyclohexane as the sole carbon source. This organism was isolated from estuarine mud beds (16). Apparently, there are no studies on the biological effects of cyclohexane on any microorganism, and no studies have been conducted on the interaction of cyclohexane with organelles from eucaryotic cells. In mammals, acute hexacarbon intoxication has narcotizing effects (15, 18). In rats, intravenously injected 1.1 mM cyclohexane led to vestibuloocular reflex excitation, probably because of inhibition of cerebellar modulation (18). Chronically exposed humans develop a syndrome known as glue sniffer's polyneuropathy and reversible renal tubule damage. Addiction to glue sniffing can result in a massive accumulation of cyclohexane and other solvents in many tissues (4, 6). After prolonged exposure to high concentrations of cyclohexane, degenerative changes in the liver and kidneys develop (4). The cyclic nonsubstituted monoterpenes beta-pinene, alpha-pinene, and limonene contain a cyclohexane ring in their molecules. These monoterpenes inhibit oxygen consumption and uncouple oxidative phosphorylation in mitochondria isolated from rat liver (20), yeast cells (21, 22), and mung bean (3). In yeast mitochondria, the respiratory chain inhibition was located at the level of NADH and succinate oxidases (22). In rat liver mitochondria, cyclohexane uncoupled oxygen consumption, although at higher concentrations than the monoterpenes. Benzene, toluene, and phenol did not elicit the effects observed with cyclohexane or the monoterpenes (20). The possibility that the cyclohexane ring in the monoterpene structure might be a factor in the biological activities of beta-pinene and limonene was explored by analyzing the effects of cyclohexane on the same yeast cells and on isolated yeast mitochondria. The data may be useful to predict the effects of cyclohexane on soil yeast cells and even in other eucaryotic cells.
*
bonyl cyanide m-chlorophenylhydrazone (CCCP), ATP, MES (morpholineethanesulfonic acid), N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), safranin 0, 3,3'-dipropylthiacarbocyanine, oligomycin, mannitol, imidazole, trizma base, and succinic and ascorbic acids were from Sigma Chemical Co., and cyclohexane, dimethylformamide, trichloroacetic acid, triethanolamine, H3PO4, and KCI were obtained from J. T. Baker. Yeast cells were from a commercial strain of Saccharomyces cerevisiae (La Azteca S.A.). Methods. (i) Yeast cell culture. Yeast cells (30 g) were incubated for 8 h at 30°C in a 2-liter Erlenmeyer flask containing 1 liter of culture medium (2) with aeration (3 liters per min) through an aquarium glass sprayer. After 8 h, the cells were centrifuged at 3,000 rpm for 10 min in a tabletop centrifuge, washed, suspended in 500 ml of water, and aerated for 16 more hours. The cells were centrifuged at 3,000 rpm for 10 min in a tabletop centrifuge, washed, and resuspended to 50% (wet weight/vol). The buffer used in this last resuspension was different depending on whether experiments with intact cells (20 mM MES-triethanolamine, pH 6.0) or mitochondria (see below) were to be performed. In experiments with intact cells, a final concentration of 8.3 mg of yeast (wet weight) per ml of medium (or 25 mg [wet weight] per ml) was used. (ii) Isolation of yeast mitochondria. After the 24-h incubation described above, 40 g (wet weight) of yeast cells was washed and suspended in 40 ml of medium consisting of 0.6 M mannitol, 10 mM imidazole, and 0.1% bovine serum albumin adjusted to pH 6.8 with HCl. The cell suspension was mixed with 50% (vol/vol) 0.45-mm glass beads in a Braun homogenizer metal flask and subjected to homogenization at 4°C for 15 s. After cell disruption, the suspension was treated with DNase for 15 min at room temperature and then the mitochondria were isolated by differential centrifugation as described previously (7). (iii) Mitochondrial protein concentration. Protein concentration was determined by the biuret method (5). Unless otherwise indicated, a concentration of 0.5 mg of mitochondrial protein per ml of medium was used in experiments. (iv) Oxygen consumption. Oxygen was measured with a Clark electrode in a water-jacketed closed chamber at 30°C connected to an oxygen meter (YSI 5300) and a chart recorder. Oxygen concentration in the buffer at the level of
Corresponding author. 2114
VOL. 56, 1990
EFFECTS OF CYCLOHEXANE ON YEAST CELLS Yeast
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containing 20 mM MES-triethanolamine, pH 6.0. Final volume, 3 ml. Numbers next to traces are cyclohexane concentrations (mM). The initial substrate concentration was either 166 mM ethanol or 10 mM glucose.
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(7). (v) Potassium uptake. Potassium concentrations were determined with a monovalent cation electrode (model 476220; Corning Glass Works) attached to an expanded-scale pH meter and a chart recorder. The K+ sensitivity was calibrated after each experiment by the addition of samples of known K+ concentration. (vi) Yeast membrane potentials. Membrane potentials in the cell and in situ mitochondria were measured by using the fluorescent potential indicator 3,3'-dipropylthiacarbocyanine [DiSC3(3)], as described before (16), in a Mark I Farrand spectrofluorometer at excitation-emission wavelengths of 540 to 590 nm (8). (vii) Mitochondrial transmembrane potentials. Mitochondrial transmembrane potentials were measured in a DW2-C SLM Aminco spectrophotometer in dual mode with wavelengths at 511 to 533 nm by using safranin 0 as described by Akerman and Wikstrom (1). (viii) ATP synthesis by mitochondria. The synthesis of ATP was measured as described by Trautschold et al. (19) following the ATP-dependent reduction of NADP in a DW2-C SLM Aminco spectrophotometer in split mode at a wavelength of 340 nm. (ix) Mitochondrial ATPase activity. Mitochondrial ATPase activity was monitored by measuring Pi liberated from ATP. P1 was determined as described by Sumner (17). Cyclohexane-dimethylformamide solutions. Solutions of 0.2 M cyclohexane in dimethylformamide were made and kept in a capped test tube in ice until used. Cyclohexane-dimethylformamide was added to the incubation medium, making a water-in-oil suspension just before the addition of mitochon-
oxygen per g
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dria or cells (21). Controls using dimethylformamide at the highest concentrations used were included in each experiment with cells or mitochondria, and no effects were detected (data not shown).
RESULTS Oxygen consumption by intact yeast cells was measured by using either glucose or ethanol as substrates in the presence of increasing concentrations of cyclohexane (0.2 to 5.0 mM) (Fig. 1). The effects of cyclohexane on oxygen consumption by yeast cells were similar with either ethanol (Fig. 1, left) or glucose (Fig. 1, right): at 0.2 and 0.5 mM cyclohexane, no effects were detected. The addition of 1 to 5 mM cyclohexane resulted in increasing inhibition of oxygen consumption. At 5 mM cyclohexane, oxygen consumption by yeast cells was inhibited approximately 90% after 1 min (Fig. 1). Potassium uptake by yeast cells can be energized either by the glycolytic pathway, using glucose as a substrate, or through oxidative phosphorylation, using ethanol as a substrate (8). The effects of cyclohexane were different depending on whether glucose or ethanol was used as a substrate. With ethanol, K+ uptake was inhibited by concentrations of cyclohexane similar to those that affected oxygen consumption (Fig. 2, left). At 0.2 and 0.5 mM cyclohexane, almost no effects were detected, and then at 1 mM cyclohexane, a mild inhibition of K+ uptake was detected. At 2 and 5 mM, cyclohexane inhibited completely the uptake of K+ by yeast cells. When glucose was used as a substrate instead of ethanol, the inhibition of K+ uptake by cyclohexane was much milder; even at 5 mM cyclohexane, inhibition was only partial (Fig. 2, right).
APPL. ENVIRON. MICROBIOL.
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FIG. 3. Effects of increasing concentrations of cyclohexane on the membrane potentials of yeast cells by using either ethanol (A) or glucose (B) as a substrate. Incubation mixture was as described in the legend to Fig. 1, except the final volume was 2 ml and 0.5 ,uM DiSC3(3) was added. Numbers below traces are cyclohexane concentrations (mM). Yeast (Y), DiSC3(3) (D), and 6 ,uM CCCP were added.
The effects of cyclohexane on oxygen consumption suggested an interaction of cyclohexane with mitochondria. This was analyzed by measuring the transmembrane potential of in situ mitochondria by using DiSC3(3) (8). Increasing concentrations of cyclohexane were tested by using either ethanol (Fig. 3A) or glucose (Fig. 3B) as a substrate. When DiSC3(3) was added to yeast cells in the absence of cyclohexane (Fig. 3A and B, traces 1), an initial small increase in fluorescence was observed which seems to be a mixture between the increase in fluorescence due to the uptake of DiSC3(3) by the cell and the quenching due to dye concentration by the mitochondria. Then, when an uncoupler was added, fluorescence increased, probably because of the exit of the dye from the mitochondria into the cytoplasm (8). In the presence of 0.2 mM (data not shown), 0.5 mM (Fig. 3A and B, traces 2), and 1 mM (Fig. 3A and B, traces 3) cyclohexane, no effects on the transmembrane potentials were detected, compared with the controls. In the presence of ethanol, the addition of 2.0 mM cyclohexane resulted in less fluorescence increase and the addition of CCCP did not result in any further increase in fluorescence. When glucose was used as a substrate, the addition of 2.0 mM cyclohexane resulted in an increase in fluorescence higher than that of the control (Fig. 3B, trace 4). Then, the addition of CCCP resulted in a further increase which was not as large as that in the control. The effects of 5.0 mM cyclohexane on yeast cells were the same regardless of the substrate (Fig. 3A and B, traces 5). At 5 mM, cyclohexane inhibited the fluorescence increase both before and after the addition of CCCP, even in the presence of glucose, suggesting that glycolysis was inhibited. The results with yeast cells suggested that mitochondria were affected by the lower concentrations of cyclohexane used. In order to analyze further the effects of cyclohexane, mitochondria were isolated and oxygen consumption was measured by using substrates for different respiratory chain oxidases, i.e., ethanol, succinate, or ascorbate-TMPD. The effects of cyclohexane on oxygen consumption by mitochondria in state 4, state 3, and the uncoupled state were tested. In the presence of ethanol, the rate of oxygen consump-
tion in state 4 remained unaffected with up to 0.5 mM
cyclohexane (Fig. 4A); 1 mM cyclohexane inhibited state 4 respiration approximately 40%, and .2 mM produced almost complete inhibition of state 4 oxygen uptake. The effects on state 3 respiration appeared at lower solvent concentrations (Fig. 4A): 0.2 mM cyclohexane inhibited state 3 by 30%, and 0.5 mM cyclohexane produced 50% inhibition. At .1.0 mM cyclohexane, no transition from state 4 to state 3 occurred upon the addition of ADP (Fig. 4A). In the uncoupled state, cyclohexane had the following effects: from 0.2 to 1 mM cyclohexane, additions of 6 ,uM CCCP produced a slightly higher stimulation of respiration than in the control; however, 2 and 5 mM cyclohexane eliminated the mitochondrial response to the uncoupler (Fig. 4A). When succinate was used, cyclohexane also affected consumption (Fig. 4B). State 4 respiration remained unaffected at 0.2 and 0.5 mM cyclohexane. At 1.0 mM, the addition of cyclohexane resulted in 50% inhibition of the respiratory rate. At 2.0 and 5.0 mM, cyclohexane led to 80% inhibition of the respiratory chain. The transition from state 4 to state 3 was slightly diminished with additions of 0.2 and 0.5 mM cyclohexane. At .1.0 mM cyclohexane, the respiratory control disappeared. In the uncoupled state, the response to CCCP was not inhibited completely even at 5.0 mM cyclohexane. When ascorbate was used as a substrate, 0.2 to 1.0 mM cyclohexane produced an acceleration of mitochondrial respiration in either state 4 or the uncoupled state (Fig. 4C). At 2 mM cyclohexane, a slight decrease in oxygen consumption was observed; however, even at 5 mM cyclohexane, the inhibition of state 4 was not higher than 30% of the control (Fig. 4C). By using ascorbate, the transition from state 4 to state 3 was very slight, and thus no attempts were made to analyze the effects of cyclohexane on state 3. In situ, the mitochondrial membrane potential was decreased at very low cyclohexane concentrations. This was further studied by measuring the transmembrane potential in isolated mitochondria (Table 1). The mitochondrial potential supported by either ethanol or succinate was affected similarly. The addition of concentrations of 0 to 1.0 mM cyclooxygen
VOL. 56, 1990
EFFECTS OF CYCLOHEXANE ON YEAST
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FIG. 4. Effects of increasing concentrations of cyclohexane on the oxygen consumption rate in isolated yeast mitochondria. Reaction mixture: 0.6 M mannitol-10 mM H3PO4-20 mM KCl-0.1% bovine serum albumin-triethanolamine (pH 6.5)-166 mM ethanol or 30 mM succinate- or 6 mM ascorbate-100 ,ug of TMPD. Mitochondrial concentration was 0.5 mg of prtoein per ml, temperature was 30°C, and final volume was 3 ml. (A) Ethanol, (B) succinate, (C) ascorbate-TMPD. State 3 was started by adding 240 nmol of ADP. The uncoupled state was initiated by the addition of 6 ,uM CCCP. Symbols: 0, state 4; 0, state 3; O, uncoupled state. In panel C, no effects of ADP addition were detected; therefore, state 3 was not plotted.
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Cyclohexane concentration (mM) hexane did not affect the transmembrane potential. At 2.0 mM, cyclohexane produced a small decrease, and the addition of 5.0 mM cyclohexane inhibited the transmembrane potential by approximately 50% (Table 1). Cyclohexane inhibited oxidative phosphorylation by isolated mitochondria (Table 2). ATP synthesis in the control was 40.5 nmol of ATP per min per mg of protein. At 0.2 mM cyclohexane, no effects were detected. At 0.5 mM cyclohexane, ATP synthesis decreased to 28 nmol of ATP per min per mg of protein. At 1 mM cyclohexane, the synthesis rate was 16 nmol of ATP per min per mg of protein, and 2 and 5 mM cyclohexane inhibited ATP synthesis to the same level as that seen in the presence of oligomycin (Table 2). The effects on ATP synthesis could be due to a direct interaction of cyclohexane with the F1F0 ATPase. This was tested by measuring the hydrolytic component of the reaction in a hypotonic medium at pH 8.5 (7) (Table 2). At 0.2, 0.5, 1, and 2 mM cyclohexane, ATPase was accelerated slightly. At 5.0 mM, cyclohexane produced a small inhibition. In this hypotonic system, CCCP did not affect ATPase activity. Oligomycin inhibited ATPase activity by 90%. DISCUSSION In yeast cells, mitochondrial functions were affected at lower cyclohexane concentrations than glycolysis. This was observed in the intact cells for which the rate of oxygen consumption was inhibited by cyclohexane to the same extent regardless of whether the respiratory chain was
energized directly by ethanol or through the glycolytic pathway. In addition, the ethanol- or glucose-supported mitochondrial potential measured in situ collapsed at cyclohexane concentrations that did not affect the glucose-mediated plasma membrane potential (8). Another indication of the effects of low concentrations of cyclohexane on mitochondria was that in the presence of ethanol, potassium uptake was inhibited at lower concentrations than it was in the presence of glucose. When ethanol was used, the effects of 5 mM cyclohexane were similar to those of the uncoupler CCCP (Fig. 3A, compare traces 5 and 6); however, when the substrate used was glucose, the effects of 5 mM cyclohexane (Fig. 3B, trace 5) were different from the effects of the uncoupler (Fig. 3B, trace 6) but similar to the effects 5 mM cyclohexane and the uncoupler on the ethanol-supported yeast. These results suggest that at the higher concentrations used, cyclohexane inhibited glycolysis-supported dye uptake as well as the TABLE 1. Effects of increasing concentrations of cyclohexane on mitochondrial membrane potential by using ethanol or succinate as a substratea Cyclohexane concn (mM)
0.0 0.2 0.5 1.0 2.0 5.0
Membrane potential (mV) by using: Ethanol Succinate
180 186 194 183 163 98
0 5 11 8 10 6
180 190 199 171 157 82
0 10 11 10 9 4
a Experimental conditions were as described in the legend to Fig. 4. Safranin-0 (10 pLM) was added to each cuvette, and the change in absorbance at 511 to 533 nm was monitored by using a double-beam spectrophotometer (SLM Aminco) in dual mode.
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APPL. ENVIRON. MICROBIOL.
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TABLE 2. Effects of different concentrations of cyclohexane on mitochondrial ATP synthesis and ATPase activitya Cyclohexane (nmol/mg of protein Cyclohexane concn (mM)
0 0.2 0.5 1.0 2.0 5.0
CCCP (6 p.M) Oligomycin (10 p.g/mg of protein)
per min) from: ATP synthesis ATP hydrolysis
40.5 42.0 28.0 16.0
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0
188 214 270 307 271
0 0
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0
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The higher concentrations of cyclohexane tested seemed to inhibit glycolysis, as suggested by the results from the whole cell which included decreased glucose-supported K+ uptake or DiSC3(3) fluorescence increase. This would be in agreement with reports suggesting that the in vivo effects of cyclohexane might be due to glycolysis inhibition (4). Cyclohexane inhibited the metabolism of yeast cells and isolated yeast mitochondria. The widespread use of cyclohexane in industry and in addictive inhalable substances (4, 6, 9, 15, 18) indicates the need to study the toxic effects and detoxification mechanisms of cyclohexane in organisms at all levels of the phylogenetic scale.
a Experimental conditions were as described in the legend to Fig. 4. The ATP synthesis assay was an enzyme-linked assay using hexokinase and glucose-6-phosphate; the reduction of NADP+ at 340 nm was monitored in an Aminco DW-2 spectrophotometer in split mode.
LITERATURE CITED 1. Akerman, K. E. O., and M. K. F. Wikstrom. 1976. Safranine as a probe of the mitochondrial membrane potential. FEBS Lett. 68:191-197. 2. De Kloet, S. R., R. K. A. Van Wormeskerken, and V. V. Konigsberger. 1961. Studies of protein synthesis by protoplast of Saccharomyces carlsbergensis. Biochim. Biophys. Acta 47:
respiratory chain. This would be in agreement with others who have reported that cyclohexane inhibits glycolysis (4). In isolated yeast mitochondria, cyclohexane inhibited the respiratory chain at the level of succinate oxidation, affecting both ethanol and succinate uptake. The ascorbateTMPD-supported oxygen uptake was only slightly affected by the cyclohexane concentrations used. Some authors have proposed an inhibition of the respiratory chain to explain the toxic effects of cyclohexane vapor inhalation in rats (18). Beta-pinene and limonene inhibit oxygen uptake at the level of succinate and NADH oxidation in mitochondria from diverse sources (3, 20-22). Both monoterpenes have a cyclohexane ring in their molecules. Cyclohexane inhibited the respiratory chain at the same site where monoterpene inhibition has been reported, although at higher concentrations than the monoterpenes (3, 20-22). The effects of the monoterpenes and of cyclohexane on the respiratory chain of yeast or rat liver mitochondria are not produced by other hydrophobic solvents such as benzene, toluene, or phenol (20), suggesting that the respiratory chain inhibitory effects may be specific for the cyclohexane structure and not just due to a hydrophobicity effect. There is a wide variety of carbon sources that yeast cells can grow on, including different hydrocarbons such as n-alkanes (12); however, cyclohexane seems to be a difficult substrate to use by most organisms. This would explain its occurrence in the molecules of diverse allelopathic compounds (11). Cyclohexane inhibited oxidative phosphorylation without affecting ATP hydrolysis by the F1Fo ATPase; therefore, a direct effect on the enzyme was ruled out. Inhibition of ATP synthesis was observed at cyclohexane concentrations that had no apparent effects on the transmembrane potential (0.5 and 1 mM cyclohexane). That is, even in the absence of effects on the membrane potential, the synthesis of ATP was inhibited. There are several reports indicating that a variety of substances such as anesthetics, diverse uncouplers, and the ionophore gramicidin A inhibits oxidative phosphorylation without having a major effect on the membrane potential (10, 13, 14). These results have led to proposals for the existence of a proton source other than the extramitochondrial pool which is also available for ATP synthesis. This second proton pool cannot be measured because of its intramembrane location, but it may be affected by these different compounds. As a result, ATP synthesis could be inhibited without collapsing the bulk proton gradient (10, 13, 14).
138-143. 3. Douce, R., M. Neuburger, R. Bigny, and G. Pauly. 1978. Effects of P-pinene on the oxidative properties of purified intact plant mitochondria, p. 207-214. In G. Ducet and R. Lance (ed.), Plant mitochondria. Elsevier/North-Holland Publishing Co., Amsterdam. 4. Dreisbach, R. H., and W. 0. Robertson. 1987. Handbook of poisoning, p. 190. Simon & Schuster, East Norwalk, Conn. 5. Layne, N. 1957. Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymol. 3:447-454. 6. Mutti, A., S. Lucertini, M. Falzoi, A. Cavatorta, and I. Franchini. 1981. Organic solvents and chronic glomerulonephritis: a cross sectional study with negative findings for aliphatic and alicyclic C5-C7 hydrocarbons. J. Appl. Toxicol. 1:224-226. 7. Pena, A., M. Z. Pina, E. Escamilla, and E. Pina. 1977. A novel method for the rapid preparation of coupled yeast mitochondria. FEBS Lett. 80:209-213. 8. Pefia, A., S. Uribe, J. P. Pardo, and M. Borbolla. 1984. The use of cyanine dye in measuring membrane potential in yeast. Arch. Biochem. Biophys. 231:217-225. 9. Perbellini, L., and F. Brugnore. 1980. Lung uptake and metabolism of cyclohexane in shoe factory workers. Int. Arch. Occup. Health 45:261-269. 10. Pick, U., M. Weiss, and H. Rottenberg. 1987. Anomalous uncoupling of photophosphorylation by palmitic acid and by gramicidin-D. Biochemistry 26:8295-8302. 11. Rice, E. L. 1979. Allelopathy, an update. Bot. Rev. 45:15-109. 12. Rose, A. H. 1987. Responses to the chemical environment, p. 5-40. In A. H. Rose and J. S. Harrison (ed.), The yeasts, 2nd ed., vol. 2. Yeasts and the environment. Academic Press, London. 13. Rottenberg, H. 1985. Proton coupled energy conversion. Chemiosmotic intramembrane coupling. Mod. Cell Biol. 4:47-83. 14. Rottenberg, H., and K. Hashimoto. 1986. Fatty acid uncoupling of oxidative phosphorylation in rat liver mitochondria. Biochemistry 25:1747-1755. 15. Savolaien, H., and P. Pfaffli. 1980. Burden and dose-related neurochemical effects of intermittent cyclohexane vapour inhalation in rats. Toxicol. Lett. 7:17-22. 16. Stirling, L. A., and R. J. Watkinson. 1977. Microbial metabolism of alicyclic hydrocarbons: isolation and properties of a cyclohexane-degrading bacterium. J. Gen. Microbiol. 99:119-125. 17. Sumner, J. B. 1946. A method for the colorimetric determination of phosphorus. Science 100:413-414. 18. Tham, R., I. Bunnfors, B. Eriksson, B. Larsby, S. Lindgren, and L. M. Odkvist. 1984. Vestibulo-ocular disturbances in rats exposed to organic solvents. Acta Pharmacol. Toxicol. 54: 58-63. 19. Trautschold, I., W. Lamprecht, and G. Scheitzer. 1985. UVmethod with hexokinase and glucose-6-phosphate dehydrogenase, p. 346-357. In H. U. Bergmeyer (ed.), Methods
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of enzymatic analysis, vol 7: metabolites 3-tri- and dicarboxylic acids, purines, pyrimidines, and derivatives, coenzymes, inorganic compounds. VCH Verlagesellschaft, New York. 20. Uribe, S., R. Alvarez, and A. Pena. 1984. Effects of beta-pinene a non substituted monoterpene on rat liver mitochondria. Pes-
tic. Biochem. Physiol. 22:43-50. 21. Uribe, S., and A. Pena. 1990. Toxicity of beta-pinene and limonene emulsions on yeast. Dependence on droplet size. J. Chem. Ecol. 16:1399-1408. 22. Uribe, S., J. Ramirez, and A. Pena. 1985. Effects of P-pinene on yeast membrane functions. J. Bacteriol. 161:1195-1200.
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Vol. 56, No. 7
0099-2240/90/072114-06$02.00/0 Copyright ©D 1990, American Society for Microbiology
Effects of Cyclohexane, an Industrial Solvent, on the Yeast Saccharomyces cerevisiae and on Isolated Yeast Mitochondria SALVADOR URIBE,* PABLO RANGEL, GLORIA ESPINOLA, AND GABRIELA AGUIRRE Instituto de Fisiologia Celular, Universidad Nacional Aut6noma de Mexico, Apartado Postal 70-600, 04510 Mexico D.F., Mexico Received 12 February 1990/Accepted 22 April 1990
Little information on the effects of cyclohexane at the cellular or subcellular level is available. In Saccharomyces cerevisiae, cyclohexane inhibited respiration and diverse energy-dependent processes. In mitochondria isolated from S. cerevisiae, oxygen uptake and ATP synthesis were inhibited, although ATPase activity was not affected. Cyclohexane effects were similar to those reported for beta-pinene and limonene, suggesting that the cyclohexane ring in these monoterpenes may be a determinant for their biological activities. MATERIAL AND METHODS Materials. All reagents were of the highest purity available commercially. Bovine serum albumin, NADP, ADP, car-
Cyclohexane is a nonsubstituted alicyclic hydrocarbon used as an industrial solvent. Cyclohexane is regarded as relatively nontoxic, and it has been increasingly used as a substitute for benzene (9). Among unicellular organisms, only one bacterium, tentatively identified as a Nocardia sp., has been described to grow by using cyclohexane as the sole carbon source. This organism was isolated from estuarine mud beds (16). Apparently, there are no studies on the biological effects of cyclohexane on any microorganism, and no studies have been conducted on the interaction of cyclohexane with organelles from eucaryotic cells. In mammals, acute hexacarbon intoxication has narcotizing effects (15, 18). In rats, intravenously injected 1.1 mM cyclohexane led to vestibuloocular reflex excitation, probably because of inhibition of cerebellar modulation (18). Chronically exposed humans develop a syndrome known as glue sniffer's polyneuropathy and reversible renal tubule damage. Addiction to glue sniffing can result in a massive accumulation of cyclohexane and other solvents in many tissues (4, 6). After prolonged exposure to high concentrations of cyclohexane, degenerative changes in the liver and kidneys develop (4). The cyclic nonsubstituted monoterpenes beta-pinene, alpha-pinene, and limonene contain a cyclohexane ring in their molecules. These monoterpenes inhibit oxygen consumption and uncouple oxidative phosphorylation in mitochondria isolated from rat liver (20), yeast cells (21, 22), and mung bean (3). In yeast mitochondria, the respiratory chain inhibition was located at the level of NADH and succinate oxidases (22). In rat liver mitochondria, cyclohexane uncoupled oxygen consumption, although at higher concentrations than the monoterpenes. Benzene, toluene, and phenol did not elicit the effects observed with cyclohexane or the monoterpenes (20). The possibility that the cyclohexane ring in the monoterpene structure might be a factor in the biological activities of beta-pinene and limonene was explored by analyzing the effects of cyclohexane on the same yeast cells and on isolated yeast mitochondria. The data may be useful to predict the effects of cyclohexane on soil yeast cells and even in other eucaryotic cells.
*
bonyl cyanide m-chlorophenylhydrazone (CCCP), ATP, MES (morpholineethanesulfonic acid), N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), safranin 0, 3,3'-dipropylthiacarbocyanine, oligomycin, mannitol, imidazole, trizma base, and succinic and ascorbic acids were from Sigma Chemical Co., and cyclohexane, dimethylformamide, trichloroacetic acid, triethanolamine, H3PO4, and KCI were obtained from J. T. Baker. Yeast cells were from a commercial strain of Saccharomyces cerevisiae (La Azteca S.A.). Methods. (i) Yeast cell culture. Yeast cells (30 g) were incubated for 8 h at 30°C in a 2-liter Erlenmeyer flask containing 1 liter of culture medium (2) with aeration (3 liters per min) through an aquarium glass sprayer. After 8 h, the cells were centrifuged at 3,000 rpm for 10 min in a tabletop centrifuge, washed, suspended in 500 ml of water, and aerated for 16 more hours. The cells were centrifuged at 3,000 rpm for 10 min in a tabletop centrifuge, washed, and resuspended to 50% (wet weight/vol). The buffer used in this last resuspension was different depending on whether experiments with intact cells (20 mM MES-triethanolamine, pH 6.0) or mitochondria (see below) were to be performed. In experiments with intact cells, a final concentration of 8.3 mg of yeast (wet weight) per ml of medium (or 25 mg [wet weight] per ml) was used. (ii) Isolation of yeast mitochondria. After the 24-h incubation described above, 40 g (wet weight) of yeast cells was washed and suspended in 40 ml of medium consisting of 0.6 M mannitol, 10 mM imidazole, and 0.1% bovine serum albumin adjusted to pH 6.8 with HCl. The cell suspension was mixed with 50% (vol/vol) 0.45-mm glass beads in a Braun homogenizer metal flask and subjected to homogenization at 4°C for 15 s. After cell disruption, the suspension was treated with DNase for 15 min at room temperature and then the mitochondria were isolated by differential centrifugation as described previously (7). (iii) Mitochondrial protein concentration. Protein concentration was determined by the biuret method (5). Unless otherwise indicated, a concentration of 0.5 mg of mitochondrial protein per ml of medium was used in experiments. (iv) Oxygen consumption. Oxygen was measured with a Clark electrode in a water-jacketed closed chamber at 30°C connected to an oxygen meter (YSI 5300) and a chart recorder. Oxygen concentration in the buffer at the level of
Corresponding author. 2114
VOL. 56, 1990
EFFECTS OF CYCLOHEXANE ON YEAST CELLS Yeast
Yeast
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Time (min) FIG. 1. Effects of increasing concentrations of cyclohexane
on
oxygen consumption by yeast cells with either ethanol or glucose as a substrate. Experimental conditions: 25 mg (wet weight) of yeast cells was added to the incubation mixture (room temperature)
containing 20 mM MES-triethanolamine, pH 6.0. Final volume, 3 ml. Numbers next to traces are cyclohexane concentrations (mM). The initial substrate concentration was either 166 mM ethanol or 10 mM glucose.
Mexico City, Mexico, is 400 nanogram-atoms
per g.
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Time (min) FIG. 2. Effects of increasing concentrations of cyclohexane on K+ transport by yeast cells with either ethanol or glucose as a substrate. Experimental conditions are as described in the legend to Fig. 1, except the final volume was 8 ml.
The
oxymeter was adjusted to 100% before the addition of the biological sample, equivalent to 1,200 nanogram-atoms of
(7). (v) Potassium uptake. Potassium concentrations were determined with a monovalent cation electrode (model 476220; Corning Glass Works) attached to an expanded-scale pH meter and a chart recorder. The K+ sensitivity was calibrated after each experiment by the addition of samples of known K+ concentration. (vi) Yeast membrane potentials. Membrane potentials in the cell and in situ mitochondria were measured by using the fluorescent potential indicator 3,3'-dipropylthiacarbocyanine [DiSC3(3)], as described before (16), in a Mark I Farrand spectrofluorometer at excitation-emission wavelengths of 540 to 590 nm (8). (vii) Mitochondrial transmembrane potentials. Mitochondrial transmembrane potentials were measured in a DW2-C SLM Aminco spectrophotometer in dual mode with wavelengths at 511 to 533 nm by using safranin 0 as described by Akerman and Wikstrom (1). (viii) ATP synthesis by mitochondria. The synthesis of ATP was measured as described by Trautschold et al. (19) following the ATP-dependent reduction of NADP in a DW2-C SLM Aminco spectrophotometer in split mode at a wavelength of 340 nm. (ix) Mitochondrial ATPase activity. Mitochondrial ATPase activity was monitored by measuring Pi liberated from ATP. P1 was determined as described by Sumner (17). Cyclohexane-dimethylformamide solutions. Solutions of 0.2 M cyclohexane in dimethylformamide were made and kept in a capped test tube in ice until used. Cyclohexane-dimethylformamide was added to the incubation medium, making a water-in-oil suspension just before the addition of mitochon-
oxygen per g
I
0
dria or cells (21). Controls using dimethylformamide at the highest concentrations used were included in each experiment with cells or mitochondria, and no effects were detected (data not shown).
RESULTS Oxygen consumption by intact yeast cells was measured by using either glucose or ethanol as substrates in the presence of increasing concentrations of cyclohexane (0.2 to 5.0 mM) (Fig. 1). The effects of cyclohexane on oxygen consumption by yeast cells were similar with either ethanol (Fig. 1, left) or glucose (Fig. 1, right): at 0.2 and 0.5 mM cyclohexane, no effects were detected. The addition of 1 to 5 mM cyclohexane resulted in increasing inhibition of oxygen consumption. At 5 mM cyclohexane, oxygen consumption by yeast cells was inhibited approximately 90% after 1 min (Fig. 1). Potassium uptake by yeast cells can be energized either by the glycolytic pathway, using glucose as a substrate, or through oxidative phosphorylation, using ethanol as a substrate (8). The effects of cyclohexane were different depending on whether glucose or ethanol was used as a substrate. With ethanol, K+ uptake was inhibited by concentrations of cyclohexane similar to those that affected oxygen consumption (Fig. 2, left). At 0.2 and 0.5 mM cyclohexane, almost no effects were detected, and then at 1 mM cyclohexane, a mild inhibition of K+ uptake was detected. At 2 and 5 mM, cyclohexane inhibited completely the uptake of K+ by yeast cells. When glucose was used as a substrate instead of ethanol, the inhibition of K+ uptake by cyclohexane was much milder; even at 5 mM cyclohexane, inhibition was only partial (Fig. 2, right).
APPL. ENVIRON. MICROBIOL.
URIBE ET AL.
2116
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FIG. 3. Effects of increasing concentrations of cyclohexane on the membrane potentials of yeast cells by using either ethanol (A) or glucose (B) as a substrate. Incubation mixture was as described in the legend to Fig. 1, except the final volume was 2 ml and 0.5 ,uM DiSC3(3) was added. Numbers below traces are cyclohexane concentrations (mM). Yeast (Y), DiSC3(3) (D), and 6 ,uM CCCP were added.
The effects of cyclohexane on oxygen consumption suggested an interaction of cyclohexane with mitochondria. This was analyzed by measuring the transmembrane potential of in situ mitochondria by using DiSC3(3) (8). Increasing concentrations of cyclohexane were tested by using either ethanol (Fig. 3A) or glucose (Fig. 3B) as a substrate. When DiSC3(3) was added to yeast cells in the absence of cyclohexane (Fig. 3A and B, traces 1), an initial small increase in fluorescence was observed which seems to be a mixture between the increase in fluorescence due to the uptake of DiSC3(3) by the cell and the quenching due to dye concentration by the mitochondria. Then, when an uncoupler was added, fluorescence increased, probably because of the exit of the dye from the mitochondria into the cytoplasm (8). In the presence of 0.2 mM (data not shown), 0.5 mM (Fig. 3A and B, traces 2), and 1 mM (Fig. 3A and B, traces 3) cyclohexane, no effects on the transmembrane potentials were detected, compared with the controls. In the presence of ethanol, the addition of 2.0 mM cyclohexane resulted in less fluorescence increase and the addition of CCCP did not result in any further increase in fluorescence. When glucose was used as a substrate, the addition of 2.0 mM cyclohexane resulted in an increase in fluorescence higher than that of the control (Fig. 3B, trace 4). Then, the addition of CCCP resulted in a further increase which was not as large as that in the control. The effects of 5.0 mM cyclohexane on yeast cells were the same regardless of the substrate (Fig. 3A and B, traces 5). At 5 mM, cyclohexane inhibited the fluorescence increase both before and after the addition of CCCP, even in the presence of glucose, suggesting that glycolysis was inhibited. The results with yeast cells suggested that mitochondria were affected by the lower concentrations of cyclohexane used. In order to analyze further the effects of cyclohexane, mitochondria were isolated and oxygen consumption was measured by using substrates for different respiratory chain oxidases, i.e., ethanol, succinate, or ascorbate-TMPD. The effects of cyclohexane on oxygen consumption by mitochondria in state 4, state 3, and the uncoupled state were tested. In the presence of ethanol, the rate of oxygen consump-
tion in state 4 remained unaffected with up to 0.5 mM
cyclohexane (Fig. 4A); 1 mM cyclohexane inhibited state 4 respiration approximately 40%, and .2 mM produced almost complete inhibition of state 4 oxygen uptake. The effects on state 3 respiration appeared at lower solvent concentrations (Fig. 4A): 0.2 mM cyclohexane inhibited state 3 by 30%, and 0.5 mM cyclohexane produced 50% inhibition. At .1.0 mM cyclohexane, no transition from state 4 to state 3 occurred upon the addition of ADP (Fig. 4A). In the uncoupled state, cyclohexane had the following effects: from 0.2 to 1 mM cyclohexane, additions of 6 ,uM CCCP produced a slightly higher stimulation of respiration than in the control; however, 2 and 5 mM cyclohexane eliminated the mitochondrial response to the uncoupler (Fig. 4A). When succinate was used, cyclohexane also affected consumption (Fig. 4B). State 4 respiration remained unaffected at 0.2 and 0.5 mM cyclohexane. At 1.0 mM, the addition of cyclohexane resulted in 50% inhibition of the respiratory rate. At 2.0 and 5.0 mM, cyclohexane led to 80% inhibition of the respiratory chain. The transition from state 4 to state 3 was slightly diminished with additions of 0.2 and 0.5 mM cyclohexane. At .1.0 mM cyclohexane, the respiratory control disappeared. In the uncoupled state, the response to CCCP was not inhibited completely even at 5.0 mM cyclohexane. When ascorbate was used as a substrate, 0.2 to 1.0 mM cyclohexane produced an acceleration of mitochondrial respiration in either state 4 or the uncoupled state (Fig. 4C). At 2 mM cyclohexane, a slight decrease in oxygen consumption was observed; however, even at 5 mM cyclohexane, the inhibition of state 4 was not higher than 30% of the control (Fig. 4C). By using ascorbate, the transition from state 4 to state 3 was very slight, and thus no attempts were made to analyze the effects of cyclohexane on state 3. In situ, the mitochondrial membrane potential was decreased at very low cyclohexane concentrations. This was further studied by measuring the transmembrane potential in isolated mitochondria (Table 1). The mitochondrial potential supported by either ethanol or succinate was affected similarly. The addition of concentrations of 0 to 1.0 mM cyclooxygen
VOL. 56, 1990
EFFECTS OF CYCLOHEXANE ON YEAST
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FIG. 4. Effects of increasing concentrations of cyclohexane on the oxygen consumption rate in isolated yeast mitochondria. Reaction mixture: 0.6 M mannitol-10 mM H3PO4-20 mM KCl-0.1% bovine serum albumin-triethanolamine (pH 6.5)-166 mM ethanol or 30 mM succinate- or 6 mM ascorbate-100 ,ug of TMPD. Mitochondrial concentration was 0.5 mg of prtoein per ml, temperature was 30°C, and final volume was 3 ml. (A) Ethanol, (B) succinate, (C) ascorbate-TMPD. State 3 was started by adding 240 nmol of ADP. The uncoupled state was initiated by the addition of 6 ,uM CCCP. Symbols: 0, state 4; 0, state 3; O, uncoupled state. In panel C, no effects of ADP addition were detected; therefore, state 3 was not plotted.
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Cyclohexane concentration (mM) hexane did not affect the transmembrane potential. At 2.0 mM, cyclohexane produced a small decrease, and the addition of 5.0 mM cyclohexane inhibited the transmembrane potential by approximately 50% (Table 1). Cyclohexane inhibited oxidative phosphorylation by isolated mitochondria (Table 2). ATP synthesis in the control was 40.5 nmol of ATP per min per mg of protein. At 0.2 mM cyclohexane, no effects were detected. At 0.5 mM cyclohexane, ATP synthesis decreased to 28 nmol of ATP per min per mg of protein. At 1 mM cyclohexane, the synthesis rate was 16 nmol of ATP per min per mg of protein, and 2 and 5 mM cyclohexane inhibited ATP synthesis to the same level as that seen in the presence of oligomycin (Table 2). The effects on ATP synthesis could be due to a direct interaction of cyclohexane with the F1F0 ATPase. This was tested by measuring the hydrolytic component of the reaction in a hypotonic medium at pH 8.5 (7) (Table 2). At 0.2, 0.5, 1, and 2 mM cyclohexane, ATPase was accelerated slightly. At 5.0 mM, cyclohexane produced a small inhibition. In this hypotonic system, CCCP did not affect ATPase activity. Oligomycin inhibited ATPase activity by 90%. DISCUSSION In yeast cells, mitochondrial functions were affected at lower cyclohexane concentrations than glycolysis. This was observed in the intact cells for which the rate of oxygen consumption was inhibited by cyclohexane to the same extent regardless of whether the respiratory chain was
energized directly by ethanol or through the glycolytic pathway. In addition, the ethanol- or glucose-supported mitochondrial potential measured in situ collapsed at cyclohexane concentrations that did not affect the glucose-mediated plasma membrane potential (8). Another indication of the effects of low concentrations of cyclohexane on mitochondria was that in the presence of ethanol, potassium uptake was inhibited at lower concentrations than it was in the presence of glucose. When ethanol was used, the effects of 5 mM cyclohexane were similar to those of the uncoupler CCCP (Fig. 3A, compare traces 5 and 6); however, when the substrate used was glucose, the effects of 5 mM cyclohexane (Fig. 3B, trace 5) were different from the effects of the uncoupler (Fig. 3B, trace 6) but similar to the effects 5 mM cyclohexane and the uncoupler on the ethanol-supported yeast. These results suggest that at the higher concentrations used, cyclohexane inhibited glycolysis-supported dye uptake as well as the TABLE 1. Effects of increasing concentrations of cyclohexane on mitochondrial membrane potential by using ethanol or succinate as a substratea Cyclohexane concn (mM)
0.0 0.2 0.5 1.0 2.0 5.0
Membrane potential (mV) by using: Ethanol Succinate
180 186 194 183 163 98
0 5 11 8 10 6
180 190 199 171 157 82
0 10 11 10 9 4
a Experimental conditions were as described in the legend to Fig. 4. Safranin-0 (10 pLM) was added to each cuvette, and the change in absorbance at 511 to 533 nm was monitored by using a double-beam spectrophotometer (SLM Aminco) in dual mode.
2118
APPL. ENVIRON. MICROBIOL.
URIBE ET AL.
TABLE 2. Effects of different concentrations of cyclohexane on mitochondrial ATP synthesis and ATPase activitya Cyclohexane (nmol/mg of protein Cyclohexane concn (mM)
0 0.2 0.5 1.0 2.0 5.0
CCCP (6 p.M) Oligomycin (10 p.g/mg of protein)
per min) from: ATP synthesis ATP hydrolysis
40.5 42.0 28.0 16.0
1.5 3.0 1.4 1.2
0
188 214 270 307 271
0 0
168 ± 6.0 190 ± 2.0
0
18 ± 4.0
± ± ± ±
± ± ± ± ±
1.0 0.5 2.0 5.0 6.0
The higher concentrations of cyclohexane tested seemed to inhibit glycolysis, as suggested by the results from the whole cell which included decreased glucose-supported K+ uptake or DiSC3(3) fluorescence increase. This would be in agreement with reports suggesting that the in vivo effects of cyclohexane might be due to glycolysis inhibition (4). Cyclohexane inhibited the metabolism of yeast cells and isolated yeast mitochondria. The widespread use of cyclohexane in industry and in addictive inhalable substances (4, 6, 9, 15, 18) indicates the need to study the toxic effects and detoxification mechanisms of cyclohexane in organisms at all levels of the phylogenetic scale.
a Experimental conditions were as described in the legend to Fig. 4. The ATP synthesis assay was an enzyme-linked assay using hexokinase and glucose-6-phosphate; the reduction of NADP+ at 340 nm was monitored in an Aminco DW-2 spectrophotometer in split mode.
LITERATURE CITED 1. Akerman, K. E. O., and M. K. F. Wikstrom. 1976. Safranine as a probe of the mitochondrial membrane potential. FEBS Lett. 68:191-197. 2. De Kloet, S. R., R. K. A. Van Wormeskerken, and V. V. Konigsberger. 1961. Studies of protein synthesis by protoplast of Saccharomyces carlsbergensis. Biochim. Biophys. Acta 47:
respiratory chain. This would be in agreement with others who have reported that cyclohexane inhibits glycolysis (4). In isolated yeast mitochondria, cyclohexane inhibited the respiratory chain at the level of succinate oxidation, affecting both ethanol and succinate uptake. The ascorbateTMPD-supported oxygen uptake was only slightly affected by the cyclohexane concentrations used. Some authors have proposed an inhibition of the respiratory chain to explain the toxic effects of cyclohexane vapor inhalation in rats (18). Beta-pinene and limonene inhibit oxygen uptake at the level of succinate and NADH oxidation in mitochondria from diverse sources (3, 20-22). Both monoterpenes have a cyclohexane ring in their molecules. Cyclohexane inhibited the respiratory chain at the same site where monoterpene inhibition has been reported, although at higher concentrations than the monoterpenes (3, 20-22). The effects of the monoterpenes and of cyclohexane on the respiratory chain of yeast or rat liver mitochondria are not produced by other hydrophobic solvents such as benzene, toluene, or phenol (20), suggesting that the respiratory chain inhibitory effects may be specific for the cyclohexane structure and not just due to a hydrophobicity effect. There is a wide variety of carbon sources that yeast cells can grow on, including different hydrocarbons such as n-alkanes (12); however, cyclohexane seems to be a difficult substrate to use by most organisms. This would explain its occurrence in the molecules of diverse allelopathic compounds (11). Cyclohexane inhibited oxidative phosphorylation without affecting ATP hydrolysis by the F1Fo ATPase; therefore, a direct effect on the enzyme was ruled out. Inhibition of ATP synthesis was observed at cyclohexane concentrations that had no apparent effects on the transmembrane potential (0.5 and 1 mM cyclohexane). That is, even in the absence of effects on the membrane potential, the synthesis of ATP was inhibited. There are several reports indicating that a variety of substances such as anesthetics, diverse uncouplers, and the ionophore gramicidin A inhibits oxidative phosphorylation without having a major effect on the membrane potential (10, 13, 14). These results have led to proposals for the existence of a proton source other than the extramitochondrial pool which is also available for ATP synthesis. This second proton pool cannot be measured because of its intramembrane location, but it may be affected by these different compounds. As a result, ATP synthesis could be inhibited without collapsing the bulk proton gradient (10, 13, 14).
138-143. 3. Douce, R., M. Neuburger, R. Bigny, and G. Pauly. 1978. Effects of P-pinene on the oxidative properties of purified intact plant mitochondria, p. 207-214. In G. Ducet and R. Lance (ed.), Plant mitochondria. Elsevier/North-Holland Publishing Co., Amsterdam. 4. Dreisbach, R. H., and W. 0. Robertson. 1987. Handbook of poisoning, p. 190. Simon & Schuster, East Norwalk, Conn. 5. Layne, N. 1957. Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymol. 3:447-454. 6. Mutti, A., S. Lucertini, M. Falzoi, A. Cavatorta, and I. Franchini. 1981. Organic solvents and chronic glomerulonephritis: a cross sectional study with negative findings for aliphatic and alicyclic C5-C7 hydrocarbons. J. Appl. Toxicol. 1:224-226. 7. Pena, A., M. Z. Pina, E. Escamilla, and E. Pina. 1977. A novel method for the rapid preparation of coupled yeast mitochondria. FEBS Lett. 80:209-213. 8. Pefia, A., S. Uribe, J. P. Pardo, and M. Borbolla. 1984. The use of cyanine dye in measuring membrane potential in yeast. Arch. Biochem. Biophys. 231:217-225. 9. Perbellini, L., and F. Brugnore. 1980. Lung uptake and metabolism of cyclohexane in shoe factory workers. Int. Arch. Occup. Health 45:261-269. 10. Pick, U., M. Weiss, and H. Rottenberg. 1987. Anomalous uncoupling of photophosphorylation by palmitic acid and by gramicidin-D. Biochemistry 26:8295-8302. 11. Rice, E. L. 1979. Allelopathy, an update. Bot. Rev. 45:15-109. 12. Rose, A. H. 1987. Responses to the chemical environment, p. 5-40. In A. H. Rose and J. S. Harrison (ed.), The yeasts, 2nd ed., vol. 2. Yeasts and the environment. Academic Press, London. 13. Rottenberg, H. 1985. Proton coupled energy conversion. Chemiosmotic intramembrane coupling. Mod. Cell Biol. 4:47-83. 14. Rottenberg, H., and K. Hashimoto. 1986. Fatty acid uncoupling of oxidative phosphorylation in rat liver mitochondria. Biochemistry 25:1747-1755. 15. Savolaien, H., and P. Pfaffli. 1980. Burden and dose-related neurochemical effects of intermittent cyclohexane vapour inhalation in rats. Toxicol. Lett. 7:17-22. 16. Stirling, L. A., and R. J. Watkinson. 1977. Microbial metabolism of alicyclic hydrocarbons: isolation and properties of a cyclohexane-degrading bacterium. J. Gen. Microbiol. 99:119-125. 17. Sumner, J. B. 1946. A method for the colorimetric determination of phosphorus. Science 100:413-414. 18. Tham, R., I. Bunnfors, B. Eriksson, B. Larsby, S. Lindgren, and L. M. Odkvist. 1984. Vestibulo-ocular disturbances in rats exposed to organic solvents. Acta Pharmacol. Toxicol. 54: 58-63. 19. Trautschold, I., W. Lamprecht, and G. Scheitzer. 1985. UVmethod with hexokinase and glucose-6-phosphate dehydrogenase, p. 346-357. In H. U. Bergmeyer (ed.), Methods
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of enzymatic analysis, vol 7: metabolites 3-tri- and dicarboxylic acids, purines, pyrimidines, and derivatives, coenzymes, inorganic compounds. VCH Verlagesellschaft, New York. 20. Uribe, S., R. Alvarez, and A. Pena. 1984. Effects of beta-pinene a non substituted monoterpene on rat liver mitochondria. Pes-
tic. Biochem. Physiol. 22:43-50. 21. Uribe, S., and A. Pena. 1990. Toxicity of beta-pinene and limonene emulsions on yeast. Dependence on droplet size. J. Chem. Ecol. 16:1399-1408. 22. Uribe, S., J. Ramirez, and A. Pena. 1985. Effects of P-pinene on yeast membrane functions. J. Bacteriol. 161:1195-1200.
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