J. Phys. Chem. B 1999, 103, 3963-3964
3963
Mechanism for the Photochemical Production of Superoxide by Quinacrine Susan E. Forest, Michael J. Stimson, and John D. Simon* Department of Chemistry, P. M. Gross Chemistry Laboratory, Duke UniVersity, Durham, North Carolina 27708 ReceiVed: January 22, 1999; In Final Form: March 9, 1999
Transient absorption spectroscopy has been used to elucidate the UV-A induced photodynamics of quinacrine. Following excitation of quinacrine in a pH 7.2 buffer solution at 395 nm, the excited molecule ejects an electron into the surrounding aqueous solution. The quantum efficiency for solvated electron formation at this excitation wavelength is determined to be in the range of 0.20-0.40. These results provide a simple mechanism that accounts for the generation of superoxide (O2-) following UV excitation of quinacrine in aqueous solution.
Introduction Quinacrine is predominantly used as an antimalarial drug and an effective agent for the treatment for lupus erythematous and giardiasis.1,2 However, use of quinacrine can result in adverse side effects that can be ocular, neuromuscular, hematologic, and cutaneous in nature.1-3 Cutaneous reactions that produce discolorations, lichenoid eruptions, and exfoliative erythroderma3 are among the most severe. The mechanism(s) by which quinacrine causes these adverse physiological effects is not clear. An early photosensitization study found that quinacrine does not have significant net photochemical activity in aqueous solution4 while another source concludes that it acts as a superoxide antioxidant.2 However, recent EPR and laser flash photolysis experiments have determined that photoexcited quinacrine is a producer of superoxide and hydroperoxy adducts and that a quinacrine photoproduct produces singlet oxygen.5 Superoxide and hydroperoxy adducts are known to interfere with normal cell processes, so these reaction channels could be responsible for the adverse effects of quinacrine. In fact, many of the adverse cutaneous and ocular reactions associated with the use of quinacrine, which absorbs over the range of λ ) 300-500 nm (see Figure 1), are thought to be light induced.4-6 However, how photoexcitation of quinacrine generates these reactive oxygen species is not known. Herein, we will focus on a mechanism for the photochemical production of superoxide by quinacrine.
Figure 1. Absorption spectrum of quinacrine in a pH 7.2 buffer solution.
A solution with a concentration of 0.94 mM was made from dissolving quinacrine in a potassium phosphate buffer (pH 7.2). In order to prevent photodamage and to guarantee that only fresh quinacrine molecules were being excited, a room-temperature circulating system was used to flow the sample. This was important since photoexcitation was thought to produce species that might absorb the excitation light. Experiments were carried out in a 2 mm path length cuvette.
Experimental Section
Results and Discussion
Transient absorption experiments were performed on quinacrine in a room-temperature pH 7.2 buffer solution. A homebuilt Ti:sapphire oscillator and regenerative amplifier with a compressor/stretcher produced femtosecond pulses centered at 790 nm. The sample was excited by the second harmonic of the 395 nm laser light, which was created by a BBO crystal. The residual of the fundamental was focused into a piece of fused silica to generate a white light continuum. Various 10 nm interference filters were used to select probe wavelengths. We obtained continuum absorption spectra only in the region of 550-900 nm. This range was limited in wavelength (550900 nm) and time resolution (750 fs) by our methods for probe light generation and detection. However, individual wavelength studies allowed us to observe the faster dynamics. Further details on the experimental setup have been described elsewhere.7
The transient absorption spectrum measured 100 ps after 395 nm excitation for the region of 600-900 nm is shown in Figure 2. This spectrum resembles the spectrum of the solvated electron, e-(H2O).8 We carried out the same experiment on a sample of potassium iodide in water, a system known to produce solvated electrons. The spectrum observed in that case was identical to that observed following the 395 nm excitation of quinacrine, confirming that solvated electrons are produced. Individual wavelength femtosecond dynamic studies on the quinacrine solution in the region from 700 to 800 nm reveal that the solvated electron is formed within the instrument response time of the apparatus. Studies of the buffer solution under identical focusing conditions rule out the possibility that the electron results from solvent ionization. Furthermore, from a powerdependence study of the transient signal (the pump power was
10.1021/jp9902809 CCC: $18.00 © 1999 American Chemical Society Published on Web 04/22/1999
3964 J. Phys. Chem. B, Vol. 103, No. 19, 1999
Forest et al.
Figure 2. Transient absorption spectrum of a quinacrine solution recorded 100 ps following excitation at 395 nm (dotted). This spectrum is identical to that of solvated electrons (dashed).8 Scatter of laser light makes it difficult to obtain an accurate absorbance in the vicinity of 800 nm.
varied from 100 nJ to 1 µJ), we find that the yield of solvated electron is linear with excitation energy. Thus, we conclude that the photoionization is a one-photon process. Light at 395 nm is characteristic of the low-energy region of the UVA range of the spectrum. The observation of the formation of e-(H2O) at this excitation wavelength suggests that this process may play an important role in the adverse photobiology of quinacrine. If we assume that the excited region of the sample is cylindrical in shape, the quantum efficiency of this process (Φ) can be determined from eq 1,
Φ)
[e-(H2O)] [Q*]
)
Ae-/e-l np(1 - 10-Aq)/2πr2l
)
2πr2Aenp(1 - 10-Aq)e(1)
where Ae- and e- are the absorbance and extinction coefficient of the solvated electron, respectively. Aq is the absorbance of quinacrine at the excitation wavelength and the parameters np, r, and l are the moles of photons in the excitation pulse, the radius, and length of the excitation cylinder, respectively. The absorbance of the solvated electron is determined from the experimental data and e- at l ) 750 nm is 18 000 M-1 cm-1.8 The values of Aq and np are easily measured, and l ) 0.2 cm, the path length of the sample cell. The value of r, which corresponds to the beam waist in the sample, is difficult to determine with high accuracy. However, we can use eq 1 to plot the dependence of Φ on the value of r. Figure 3 shows such a plot for the experimental conditions used in this study. We estimate the beam waist to be between 250 and 350 µm, which corresponds to a photoionization quantum efficiency between 0.20 and 0.40. The results discussed above provide a mechanism for the photochemical formation of superoxide (O2-) following UV-A irradiation of quinacrine in aqueous solutions. It has been demonstrated that e-(H2O) reacts with ground state oxygen molecules to form superoxide.9 Our results show that the photochemistry of quinacrine can provide the needed solvated electron following one-photon absorption in the UVA. This active oxygen species has been implicated in cellular damage and deleterious effects to tissues.9 The highly reactive superoxide can be oxidized back to oxygen or it can be reduced further to form hydrogen peroxide
Figure 3. Pictorial representation of the quantum efficiency of solvated electron formation as a function of the radius of the excitation beam. The shaded region represents a beam radius of 250-350 µm (characteristic of the experimental conditions used in this study), and corresponds to a quantum efficiency of 0.2-0.4.
(H2O2). During normal biological processes, superoxide and hydrogen peroxide are formed in small quantities and there are natural defense mechanisms, such as superoxide dismutase, which effectively remove superoxide and other active oxygen species. However, under certain conditions, such as intake of drugs, UV radiation, and/or metabolic dysfunction, these reactive oxygen species can be generated in sufficient quantity to exceed normal defense capabilities of the body. This can result in deleterious effects on tissues.9 Because quinacrine has been demonstrated to bind and accumulate in melanin-containing tissues,6 overloading these cells with superoxide produced from the UVA photoexcitation of quinacrine may be relevant to ocular and cutaneous (melanin-containing) tissue damage. We are currently studying the photophysics and photoreactivity of the quinacrine-melanin complex. Acknowledgment. This work was supported by the National Institute of General Medical Sciences and the MFEL Program administered by the Office of Naval Research. References and Notes (1) Webster Jr., L. T. The Pharmacological Basis of Therapeutics, 8th ed.; Pergamon: New York, 1990; Chapter 42, p 1005. (2) Wallace, D. J. Sem. Arthritis Rheum. 1989, 18, 282. (3) Tanenbaum, L.; Tuffanelli, D. L. Arch. Dermatol. 1980, 116, 587. (4) Moore, D. E.; Hemmens, V. J. Photochem. Photobiol. 1982, 36, 71. (5) Motten, A. G.; Martinez, L. J.; Holt, N.; Sik, R. H.; Reszka, K.; Chignell, C. F.; Tønnesen, H. H.; Roberts, J. E. Photochem. Photobiol. 1999, 69, 282. (6) Kristensen, S.; Orsteen, A.-L.; Sande, S. A.; Tønnesen, H. H. J. Photochem. Photobiol. B: Biol. 1994, 26, 87. (7) Chang, Y. J.; Simon, J. D. J. Phys. Chem. 1996, 100, 6406. (8) Migus, A.; Gauduel, Y.; Martin, J. L.; Antonetti, A. Phys. ReV. Lett. 1987, 58, 1559. Kimura, Y.; Alfano, J. C.; Walhout, P. K.; Barbara, P. F. J. Phys. Chem. 1994, 98, 3450. (9) Ranadive, N. S.; Menon, I. A. Pathol. Immunopathol. Res. 1986, 5, 118.
