Sonochemistry of Quinones in Argon-Saturated Aqueous Solutions

850

Chem. Res. Toxicol. 1999, 12, 850-854

Sonochemistry of Quinones in Argon-Saturated Aqueous Solutions: Enhanced Cytochrome c Reduction Raphael C. Lawson, Amaris Ferrer, Wilmarie Flores, and Antonio E. Alegrı´a* Department of Chemistry, CUH Station, University of Puerto Rico at Humacao, Humacao, Puerto Rico 00791 Received April 15, 1999

Sonolysis of argon-saturated aqueous quinone solutions resulted in an enhancement in ferricytochrome c (Cyt c) reduction. Upon addition of superoxide dismutase, Cyt c reduction was partially inhibited, thus implying a role of superoxide ion in this reduction process. Neither quinone hydrophobicity nor reduction potential exclusively controls the Cyt c reduction enhancement, although a preference for hydrophobicity versus reduction potential is noted.

Introduction Quinonoid compounds are widely distributed in nature. Both the naturally occurring and the synthetic ones play important roles in biological systems. For instance, electron transport in mitochondria is known to be mediated by ubiquinone (1), whereas 2-methyl-1,4-naphthoquinone (2-MNQ)1 and some anthracyclines are wellknown for their antitumor activities (2). The biological functions and activity of quinones are believed to be mediated by their corresponding semiquinones (3). These free radical intermediates can be formed by either chemical or enzymatic one-electron reduction of the parent quinones. The reduction potential of the quinone/ semiquinone couples is an important property which controls the reactivity of semiquinones (4). Semiquinones formed in biochemical systems often undergo a sequence of reactions commonly termed redox cycling. One product of such a reaction is the superoxide anion radical (O2•-). Further reaction of the superoxide with hydrogen peroxide produces the damaging hydroxyl radical (OH•) through the iron-catalyzed Haber-Weiss reaction (5).The antitumor effect of some quinone-containing drugs is known to be enhanced upon exposure to ultrasound (6). A synergistic effect of cell killing for hematoporphyrin combined with ultrasound has been ascribed to the formation of the superoxide anion radical because cell damage was suppressed by superoxide dismutase (SOD) (7). Even when tumor temperatures are maintained at normal body temperatures during in vivo ultrasound treatments with anti-cancer quinone-containing drugs, marked enhancements in the drug cytotoxicity result. Thus, nonthermal mechanisms, involving acoustically induced cavitation, may be considered responsible (8, 9). The chemical effects of ultrasound are attributed to acoustic cavitation, a term often used to describe the formation of small gas bubbles in a liquid, their continuous absorption of energy from the ultrasonic source with * To whom correspondence should be addressed. Phone: (787) 8523222. Fax: (787) 850-9422. E-mail: [email protected]. 1 Abbreviations: Cyt c, horse heart ferricytochrome c; UBQ-0, 2,3dimethoxy-5-methyl-1,4-benzoquinone; 2-MNQ, 2-methyl-1,4-naphthoquinone; SOD, superoxide dismutase; 2,6-DMBQ, 2,6-dimethyl-pbenzoquinone; E17, one-electron redox potential at pH 7; E1/2, half-wave reduction potential.

concomitant changes in size, and eventual violent implosion. Temperatures as high as 5000 °C and pressures as high as hundreds of atmospheres are generated during the violent implosion of the gas bubbles (10). Such extreme conditions are responsible for thermal dissociation of water vapor into hydroxyl radicals and hydrogen atoms (11, 12). Riesz and Kondo have observed the formation of ubiquinol upon irradiating argon-saturated aqueous ubiquinone solutions with a 50 kHz ultrasonic generator (13). Evidence was found which suggests that superoxide radicals, generated from the thermal dissociation of water vapor, are responsible for the reduction of ubiquinone to ubiquinol. However, the possible role of hydrogen atoms as quinone reducing agents was not discarded (13). Quinones are known to increase catalytically the rate of oxygen consumption in the presence of reducing agents (14), and this effect has been found to be dependent on the quinone one-electron reduction potential (14, 15). Since oxygen molecules are produced during the sonication of argon-saturated water (16), the rate of superoxide production in these solutions in the presence of quinone is expected to increase upon sonication. In view of the fact that the superoxide anion radical is known to reduce ferricytochrome c (Cyt c) (17), an enhancement in the reduction of Cyt c might be expected for argon-saturated quinone solutions spiked with Cyt c and irradiated with ultrasound. There is evidence to suggest that a gradient of hydrophobicity exists at the interfacial region of a cavitation bubble which decreases toward the bulk of the solution (18, 19). Thus, the enhancement in Cyt c reduction might depend on the hydrophobicity of the quinones as well as on their one-electron reduction potentials at pH 7, E17. It is of interest to determine in this work whether such an enhancement can occur during the sonication of argon-saturated aqueous solutions containing quinone and Cyt c and, if possible, the relative importance of quinone hydrophobicity and redox potential on the observed enhancement.

Materials and Methods Chemicals. The quinones (Figure 1) selected for these measurements comprise large ranges of redox potentials (E17 ) -80 to -203 mV) and hydrophobicities (octanol/buffer parti-

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Sonochemistry of Quinones

Figure 1. Quinones being studied. tion constants ranging from 8 to 224). Ubiquinone (UBQ-0), 2-methyl-1,4-naphthoquinone (2-MNQ), tetramethyl-p-benzoquinone, ferricytochrome c (Cyt c) from horse heart, superoxide dismutase (SOD) from bovine kidney (EC 1.15.1.1), sodium phosphate, ascorbic acid, and octanol were purchased from Sigma Chemical Co. (St. Louis, MO). The quinones, 1,4naphthoquinone (1,4-NQ), 2,6-dimethyl-p-benzoquinone (2,6DMBQ), p-benzoquinone, and methyl-p-benzoquinone, were obtained from J. T. Baker Chemical Co. (Waterbury, CT), Alfa Aesar Chemical Co. (Ward Hill, MA), Pfaltz & Bauer (Waterbury, CT), and Aldrich Chemical Co. (Milwaukee, WI), respectively. All quinones were purified by sublimation under vacuum conditions. All other chemicals were of the highest purity commercially available and used without further purification. Stock solutions of quinone, Cyt c, phosphate buffer, and SOD were prepared using argon-saturated water. SOD was assayed according to the procedure described previously (20). Denatured SOD was prepared by autoclaving at 120 °C for 15 min. Sonochemical Measurements. The procedure used for sonicating aqueous samples is analogous to that described by Miyoshi et al. (21). A solution (1.5 mL) containing 0.5 mM quinone, 20 µM Cyt c and 20 mM phosphate buffer (pH 7.4) was placed in a disposable Pyrex tube (Corning catalog no. 980013, Corning Inc.) and the tube clamped in the center of an ultrasonic cleaning bath (Bransonic 1200) operating at a frequency of 50 kHz. The water inside the bath and the meniscus of the aqueous sample to be sonicated were maintained at the same level to ensure sufficient reproducibility in the standing wave field (13). The temperature of the coupling water was 26 °C. Since a major factor that limits the capability of most ultrasonic cleaning baths is their low acoustic intensities (22), partially degassing the coupling water, by exposure to vacuum, prior to being used produced a considerable increase in the acoustic intensity of our ultrasonic generator. This is evident from Fricke dosimeter analysis (see below). The oxygen content of this partially degassed coupling water, at the ultrasound bath, was adjusted using a Clarke type oxygen electrode to give a constant absorbance reading after Fricke dosimetry. Each sample was purged with argon which was passed through an oxygen-removing cartridge (Johnson Matthey Inc., Ward Hill, MA) and a water bubbler to prevent contamination with traces of oxygen and the loss of sample through evaporation, respectively. The gas was allowed to enter the sample solution through

Chem. Res. Toxicol., Vol. 12, No. 9, 1999 851 Teflon tubing at a flow rate of ∼40 mL/min for 5 min before sonication and during sonication. For comparison, an argonsaturated Fricke dosimeter solution was sonicated under identical experimental conditions using air-saturated and partially degassed coupling water. After sonication for 2 min using partially degassed water, the change in absorbance at 302 nm for 1.5 mL of Fricke dosimeter was 0.17 ( 0.01 (SD, n ) 4). When 1.5 mL of argon-saturated Fricke dosimeter solution was sonicated for 10 min using air-saturated bath water, the absorbance reading that was obtained was only 0.12 ( 0.01 (SD, n ) 4). The Fricke dosimeter solution was prepared according to the procedure described in the literature (23). Spectrophotometric Measurements. The reduction of Cyt c was monitored at 550 nm using a Beckman DU-7400 spectrophotometer. Absorbance readings were obtained immediately following every 2 min of sonication using a 1 cm path length quartz cell. Quinones with low solubility in water were prepared by centrifuging suspensions in water and determining the concentration of quinone in the supernatant using UV-vis spectroscopy. Dissolved Oxygen Monitoring. The dissolved oxygen content of the degassed water was measured amperometrically with a Clark type oxygen electrode (YSI model 5300, Yellow Springs Instruments, Yellow Springs, OH). Calibration was carried out with air ([O2] ) 240 µM)- and N2-saturated water. Octanol/Buffer Partition Coefficients. Octanol/phosphate buffer (pH 7.4) partition coefficients were determined using a procedure similar to that described previously (24). Ferricytochrome c Reduction Rate Measurements in the Absence of Ultrasound. These were measured spectrophotometrically at 550 nm immediately after addition of the concentrated argon-saturated ascorbate solution to an airsaturated phosphate buffer (pH 7.4) solution containing Cyt c and quinone. The concentrations of reagents immediately after mixing were 20 µM Cyt c, 0.1 mM quinone, and 0.5 mM ascorbate. Ultrasound-Induced Destruction of Quinones. Quinone decomposition percent after ultrasound irradiation for 6 min was determined from the relative decrease in the chromatographic peak area as determined by HPLC. A Waters analytical HPLC system equipped with a 600 solvent delivery pump, a UV/vis tunable detector, and a µBondapack C18 (3.9 mm × 300 mm) column was used. A mobile phase of MeOH/H2O with a convenient ratio of solvents was used at a flow rate of 1 mL/ min. Half-Wave Redox Potential Measurements. Half-wave potentials (E1/2) were obtained using differential pulse polarography (DPP). A PAR model 264A polarographic analyzer and a PAR model 303A static mercury electrode (Princeton Applied Research) were used to record the polarograms. Quinones were dissolved in 0.1 M phosphate buffer (pH 7.4) to give a final concentration of 0.1 mM. Each solution was purged with nitrogen for 8 min prior to being analyzed. A pulse amplitude of 50 mV, a scan rate of 2 mV/s, and a drop time of 1.0 s were used to acquire the polarograms under an atmosphere of nitrogen. A Ag/AgCl electrode was used as the reference electrode and a platinum wire as the auxiliary electrode.

Results Sonication of argon-saturated aqueous Cyt c solutions containing 0.5 mM MNQ in 20 mM phosphate buffer (pH 7.4) resulted in an enhancement in Cyt c reduction, as is evidenced by the growth of reduced Cyt c absorption at 550 nm (Figure 2). A similar behavior was detected for all the quinones being studied here (Figure 3). Only a limited extent of Cyt c reduction was detected upon sonicating identical samples in the absence of quinones (Figure 3). Figure 4 illustrates the effect of sonication time on the absorbance at 550 nm of solutions containing NQ sonicated both in the absence and in the presence of

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Figure 4. Effect of SOD addition (112 units/mL) on the extent of ferricytochrome c reduction during ultrasound irradiation of argon-saturated solutions containing 20 µM Cyt c, 0.50 mM NQ, and 20 mM phosphate buffer (pH 7.4). Absorbance is corrected for initial absorbance. ∆A ) Aw/sonication - Awo/sonication. Figure 2. Change in the absorption spectrum of a 20 µM ferricytochrome c argon-saturated aqueous solution containing 0.5 mM MNQ and 20 mM phosphate buffer (pH 7.4) before sonication (s), after sonication for 2 min (- - -), and after sonication for 6 min (- - -).

Table 1. Redox Potentials and Octanol/Water Partition Coefficients of Quinones and Their Corresponding Cytochrome c Reduction Efficiencies in the Presence of Ascorbate or under Ultrasound Irradiation

quinone

E17 partition (mV)a constant

MNQ -203 224 ( 5 UBQ-0 -130 8.0 ( 0.6 NQ -100 75.0 ( 0.1 DMBQ -80 19 ( 1 MNQ (purged with O2)

∆A after sonication for 6 minb 0.242 ( 0.023 0.144 ( 0.034 0.153 ( 0.019 0.192 ( 0.012 0.08 ( 0.01

∆Ac per % SOD min inhibitiond 0.25 0.35 0.55 0.75

31 57 76 39

a Obtained from ref 26 with the exception being that of UBQ-0 which was estimated from Figure 5. b Argon-saturated sample containing 0.5 mM quinone and 20 µM Cyt c in 20 mM phosphate buffer (pH 7.4). c SOD activity of 112 units/mL; % inhibition ) 100(∆Awo/SOD - ∆Aw/SOD)/∆Awo/SOD. d Initial Cyt c reduction rates in air-saturated samples containing 0.1 mM quinone, 20 µM Cyt c, and 0.5 mM ascorbate in 20 mM phosphate (pH 7.4).

Figure 3. Ferricytochrome c reduction enhancements during sonication of argon-saturated solutions containing 20 µM Cyt c and 20 mM phosphate buffer (pH 7.4). The quinone concentration was 0.50 mM. ∆A ) Aw/sonication - Awo/sonication.

SOD, indicating the production of superoxide ions in these sonolyses. Also shown in Figure 4 are data for NQ samples not exposed to ultrasound. When quinone solutions saturated with oxygen were sonicated under identical experimental conditions, the change in absorbance at 550 nm was very small (Table 1). This observation is due to the lower maximum temperature expected for imploding gas bubbles when O2, a diatomic gas, is used as opposed to Ar (25). Therefore, lower sonochemical yields are expected when O2 is used as opposed to Ar. Comparing the absorbance readings for argon-saturated quinone solutions sonicated for 6 min clearly shows a substantial increase in the extent of Cyt c reduction compared to that of samples sonicated in the absence of quinones (Table 1 and Figure 3). It is evident from the observations described above that ultrasound irradiation of the quinone solutions resulted in a dramatic increase in the extent of Cyt c reduction. Addition of 112 units/ mL SOD to the quinone solutions resulted in an incomplete inhibition of Cyt c reduction (Table 1).

Since the one-electron reduction potential at pH 7 is an important thermodynamic property that affects the electron-transfer properties of the quinones, we found looking for a correlation between this property and the enhanced Cyt c reduction observed under the influence of ultrasound interesting. To the best of our knowledge, the E17 value of UBQ-0 has not been reported. We therefore resorted to electrochemical methods to estimate this value. Using differential pulse polarography (DPP), we determined the E1/2 values of a series of p-benzoquinone derivatives. These quinones were chosen so that these were water-soluble, and their E1/2 values correlate with the Hammet σ substituent effects (27). The halfwave potentials were determined in phosphate buffer (pH 7.4). Consequently, the electrode reaction is Q + 2e- + 2H+ f QH2, a well-behaved electrochemical system between pH 7 and 9 (28). A linear correlation (correlation coefficient of 0.99) was obtained between E1/2 and E17 (Figure 5). Such a good correlation was not obtained if quinones other than p-benzoquinones were used. From the equation of this linear relation, the E17 value of UBQ-0 was estimated to be -130 mV. The one-electron reduction potentials and the initial rates of Cyt c reduction are compared in Table 1. Since a hydrophobicity gradient exists at the interfacial region of the cavitation bubble (18, 29), quinones with

Sonochemistry of Quinones

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c most probably via the scavenging of superoxide radicals. Thus, the differences in the extent of Cyt c reduction cannot be ascribed to differences in the rates of sonochemical quinone destruction. On the basis of the mechanism formulated by Riesz et al. (13, 29), the following reactions can be written to account for the enhanced Cyt c reduction observed in this study: )))

•

Figure 5. Linear relationship used in the estimation of E17 for UBQ-0 (point marked with an arrow). E1/2 values were determined in this work, and E17 values were taken from ref 26. Points in this graph correspond to p-benzoquinone (*), methyl-p-benzoquinone (1), DMBQ ([), and tetramethyl-pbenzoquinone (×). A value of -130 mV for the E17 of UBQ-0 was interpolated from this plot.

Figure 6. Percentage of quinone present in the sonicated sample as a function of sonication time in the presence (black symbols) and in the absence (white symbols, only shown for UBQ-0) of Cyt c. The sample composition is as stated in the legend of Figure 3.

larger hydrophobicities should accumulate in this region more extensively, and as indicated in the Introduction, this could probably have an effect on the extent of Cyt c reduction. Therefore, we examined the octanol/buffer partition coefficients of all the quinones being studied, to detemine if this property correlates with the extent of Cyt c reduction. Partition coefficients are listed in Table 1. To determine if the extent of sonochemical destruction of quinones accounts for the differences in the extents of Cyt c reduction observed, we measured the percent of available quinone after sonolysis for 6 min (Figure 6).

Discussion We have shown in this work that ultrasound irradiation of argon-saturated quinone solutions enhances the reduction of Cyt c (Figure 3 and Table 1). As shown in Figure 6, more than 88% of the initial DMBQ, UBQ-0, and NQ and more than 80% of the initial MNQ were still available after sonication for 6 min. Exclusion of Cyt c from UBQ-0-containing samples strongly accelerated UBQ-0 destruction, indicating the protective role of Cyt

H2O 98 H• + •OH

(1)

OH + •OH f H2O + •O•

(2)

2•O• f O2

(3)

H• + O2 f HO2•

(4)

HO2• f H+ + O2•-

(5)

Q + H• f Q•- + H+

(6)

•-

Q + O2

hQ

•-

+ O2

(7)

O2•- + Cyt(III)c f O2 + Cyt(II)c

(8)

Q•- + Cyt(III)c f Q + Cyt(II)c

(9)

The fact that quinones enhance Cyt c reduction implies that reactions 6-9 accelerate the shuttling of electrons from the reducing agent, which could be H or O2•-, to Cyt c as compared to the absence of quinones. Due to its charged nature (30), Cyt c is expected to be located in the bulk solution, where scavenging of hydrogen atoms or superoxide ions is limited as a result of the distance from the cavitation bubble. Therefore, partitioning of quinones into the interphase of the cavitation bubble is expected to facilitate the scavenging of these reducing equivalents where they are produced, followed by their transportation to Cyt c in the bulk solution. Quinones with the largest hydrophobicity should have also the largest probability of partitioning into the inner side of the cavitation bubble and thus will be more accessible to reducing equivalents produced in the gas phase of the bubble. Therefore, if only hydrophobicity were important in this process, the most hydrophobic quinones should shuttle electrons more readily to oxygen or Cyt c. However, quinones with more positive E17 values are expected to reduce oxygen or Cyt c faster than those with more negative E17 values. In fact, initial Cyt c reduction rates in air-saturated samples containing quinone, Cyt c, ascorbate, and phosphate buffer increase with an increase in quinone redox potential (Table 1). Although the extent of quinone-enhanced ascorbate reduction of Cyt c in the absence of ultrasound increases with increasing quinone E17 values, the extent of quinoneenhanced sonochemical reduction of Cyt c correlates better with quinone hydrophobicity than with E17 values (linear correlation factor of 0.67 for hydrophobicity vs 0.50 for E17). Thus, hydrophobic quinones could be sonochemically reduced in a preferred manner compared to hydrophilic quinones. This lack of correlation between solute kinetic properties in homogeneous solutions with the behavior of these solutes in aqueous solutions exposed to ultrasound has been reported previously for sonolysis of volatile compounds in argon-saturated aqueous solutions. The efficiency of hydroxyl radical scavenging by such solutes was found to correlate with the hydrophobicity of the solutes but not with their specific reactivity

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toward •OH, as measured in homogeneous solutions (18). The importance of the equilibrium interfacial concentration of the solute, i.e., the gas-water surface excess, has been previously recognized in controlling the solute role in sonochemical reactions (31, 32). Since SOD partially inhibits Cyt c reduction in the presence of quinones, both reactions 8 and 9 should be responsible for Cyt c reduction. However, a decrease in the extent of Cyt c reduction upon addition of SOD could also be the result of an SOD-induced shift to the left in the equilibrium position of reaction 7 due to the withdrawal of O2•-. The presence of quinones accelerates the rate of Cyt c reduction by the direct semiquinone reduction of Cyt c (reaction 9) and/or oxygen (opposite of reaction 7). As observed previously by other authors (15, 33, 34), a smaller extent of SOD inhibition is expected for quinones with more positive E17 values, because the equilibrium constant for reaction 7 should be larger and thus more semiquinone ions will be available for direct Cyt c reduction after the SOD-induced shift in equilibrium 7. However, we observed no such trend in our SODinhibited cytochrome c reduction data. This lack of a correlation could be ascribed to the existence of a heterogeneous environment during sonication as a result of the formation of cavitation bubbles. For instance, redox potentials of the quinones might be affected by their partitioning into the hydrophobic layer of the cavitation bubble.

Acknowledgment. We express appreciation to NIGMS/MBRS for Grant S06-GM08216 which provided financial support.

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