0031-865392$05.oO+0.00 Pergamon Press plc
Photochemistry and Photobiology Vol. 55, No. 3, pp. 359-366, 1992 Printed in Great Britain
PHOTOSENSITIZATION BY ANTICANCER AGENTS-10. ortho-SEMIQUINONE AND SUPEROXIDE RADICALS PRODUCED DURING ANTHRAPYRAZOLE-SENSITIZED OXIDATION OF CATECHOLS KRZYSZTOFRESZKA'*,J. WILLIAM L O W Nand ~ COLINF. CHIGNELL' 'Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA and *Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2 (Received 30 May 1991; accepted 21 August 1991) Abstract-Photosensitized oxidation of catechol, 3,4-dihydroxybenzoic acid (DHBA), 3,4-dihydroxydihydrocinnamic acid (DHCA), and 3,4-dihydroxy-phenylalanine(DOPA) by novel anticancer agents, anthrapyrazoles (AP), has been studied employing EPR and the spin trapping technique. The formation of o-semiquinone radicals, the one-electron oxidation products of the catechols, stabilized in the form of zinc ion complexes, has been demonstrated. Rate constants for the disproportionation of the semiquinone radical/Zn*+ complexes in (DMSO)/acetate buffer (pH 4.5,1:l voYvol; 100 mM Zn2+) mixture have been determined to be 0.35 X lo4, 14 x 104,8.8 X lo4 and 3 X lo4 M-I S K Ifor catechol, DHBA, DHCA and DOPA respectively. The presence of oxygen enhanced rather than inhibited the photogeneration of the o-semiquinone radicals and facilitated their EPR detection. The EPR spectrum of the superoxide radical adduct with the spin trap 5,5-dimethyl-l-pyrroline-N-oxide was observed for the first time during photosensitized oxidation of the catechols in acidic aqueous solutions and in DMSOlacetate buffer mixture.
INTRODUCTION
Anthrapyrazoles (AP)t have been developed as alternatives to the widely prescribed but highly cardiotoxic anticancer agent, doxorubicin. The main rationale behind their synthesis was that appropriate modification of the anthracenedione chromophore might diminish cardiotoxicity by reducing the potential to form semiquinone free radicals (Leopold et al., 1985; Showalter et al., 1986). This was achieved by incorporating another ring (D in Fig. 1) into the chromophore and modification of the central quinone moiety to a quasi iminoquinone (Fig. 1). A noteworthy feature of some anthrapyrazoles is their powerful photosensitizing properties. Deshydroxy derivatives (without OH groups in ring A), such as AP in Fig. 1, are particularly photoactive in the generation of singlet oxygen upon visible light illumination in ethanolic solutions (Reszka et af., 1986a). One of these deshydroxy derivatives, AP1, readily oxidized several biochemical electron donors, such as ascorbic acid, 3,4-dihydroxyphenylalanine (DOPA) and NADH (Reszka et al., 1986b,
I
R9
Ri
AP1
(CH2),NH(CH2),0H
R,
AP2
(CH2),NH(CH2),0H
CH3
AP3
H
(CH2),NH(CH 2),0H
Figure 1. Structures of anthrapyrazoles studied in this work.
c). The same agent, APl, was able to induce single strand breaks in DNA, but only in the presence of light and a reducing cofactor (Hartley et al., 1988). Both EPR and spin trapping studies showed that the photooxidation reactions involve formation of free radical species including hydroxyl radicals. Superoxide radical was observed only when 'To whom correspondence should be addressed. NAD(P)H was used as a substrate. Hydrogen pert A bbreviations: AP, anthrapyrazole; DETAPAC, diethylenetriamine-pentaacetic acid; DHBA, 3,4-dihydroxy- oxide accumulated during DOPA photooxidation benzoic acid; DHCA, 3,4-dihydroxy-dihydrocinnamic (Reszka et al., 1986b) as shown by the effect of acid; DMPO, 5,5-dimethyl-l-pyrroline-N-oxide;catalase on oxygen consumption. It was then postuDMSO, dimethyl sulfoxide; DOPA, 3,4-dihydroxy- lated that radical forms of the sensitizer and of phenylalanine; EPR, electron paramagnetic resonance; hf, hyperfine; NADH, reduced diphosphopyridine the substrate are obligatory intermediates in this nucleotide; SOD, superoxide dismutase; TEMPO, 2,2, reaction, and that reoxidation of the semireduced sensitizer may produce superoxide radicals. How6,6-teramethyl-l-piperidinyloxy. 359
KRZYSZTOF RESZKAer al.
360
ever, no spectroscopic evidence was provided at that time to support this hypothesis. The primary objective of this study then was to provide spectroscopic evidence for the anthrapyrazole-photosensitized oxidation of catechols to osemiquinone radicals. This was achieved by using EPR spectroscopy in conjunction with the metal ion spin stabilization technique (Felix and Sealy, 1981; Plancherel and von Zelewsky , 1982; Kalyanaraman et a f . , 1985). Additional goals were to examine the detectability of the o-semiquinone radicals in aerated solutions and to seek evidence for the formation of superoxide radicals during photosensitized oxidation of catechols. MATERIALS AND METHODS
Anthrapyrazoles APl, AP2, and AP3 (FIg. 1) were kindly supplied by Dr. H. D. H. Showalter (WarnerlLambert. Park Davis. MI) and used as received. Zinc acetate, diethylenetriamine-pentaacetic acid N-oxide (DMPO), (DETAPAC), 5,5-dimethyl-l-pyrroline deuterium oxide and 2.2.6,6,-teramethyl-l-piperidinyloxy (TEMPO), catechol and its derivatives 3.4dihydroxyphenylalanine (DOPA), 3.4-dihydroxybenzoic acid (DHBA). 3,4-dihydroxyhydrocinnamic acid (DHCA). were obtained from Aldrich Chemical Company (Milwaukee. WI). Superoxide dismutase (EC 1.15.1.1) from bovine liver was purchased from Sigma Chemical Company (St. Louis. MO). DMSO was from Fisher Scientific. DMPO was distilled in vucuo and stored at -22°C. Stock solutions of AP (5 mM), and the catechols (0.1 M and 0.01 M) were prepared in deionized water. Zinc acetate stock solutions (0.9 M ) were prepared in sodium acetatelacetic acid buffer, pH 4.5 (the required pH was achieved by a drop-wise addition of concentrated acetic acid). The acetate buffer in deuterium oxide was prepared assuming pD = pH + 0.5. Electron paramagnetic resonance (EPR) measurements were performed on a Varian E-Line Century Series EPR Spectrometer operating at 9.5 GHz. Samples in a flat quartz EPR cell (0.3 mm thickness) were illuminated directly inside the microwave cavity of the spectrometer using a 1 kW Xe arc lamp and a Schoeffel grating monochromator. Excitation wavelength corresponded to the absorption maximum appropriate for a given anthrapyrazole in the visible range and optical density at A,,, was always below 0.15 in the 0.3 mm EPR flat cell. Anthrapyrazoles were the only light-absorbing species in our systems. Kinetic measurements were conducted monitoring growth and decay of the central, most intense line of the EPR spectrum of catechol-derived radical, or the most intense low-field line of DHCA-, DHBA- or DOPAderived radicals. The concentration of the photochemically-induced radicals was assessed by comparison of a second integral of their EPR spectra with that of a standard solution of TEMPO recorded under similar conditions. Ultravioletivisible absorption spectra were measured in a 0.2 cm quartz cell using a Hewlett-Packard diode array spectrophotometer Model HP 8451A. Unless othenvise stated all experiments were performed in a mixture of DMSO and 0.1 M acetic acidlsodium acetate buffer, pH 4.5 (measured pH of this mixture was 6.0). to which appropriate amounts of the stock solutions of the AP, catechols and Zn2+ were added. The final DMSO/H20 ratio was I : 1 volivol. Where necessary, the samples in the EPR cell were deaerated with a stream of nitrogen gas for 5 min.
RESULTS
Absorption spectra
Structures of the anthrapyrazoles used in this study (AP1, AP2, AP3) are shown in Fig. 1. They share the same chromophore skeleton and differ only in the nature of their R1 and R2 substituents. Preliminary measurements showed that in aqueous buffer, pH 4.5, the anthrapyrazoles (0.2 mM) tend to aggregate, and for that reason most experiments were conducted in DMSO/acetate buffer, pH 4.5 (1:l vol/vol) mixture. In this mixture the longwave absorption bands of AP are blue-shifted (2-4 nm) and are more intense (50-60°/0) compared with the spectra measured in aqueous solutions. The most intense visible bands for AP1, AP2, and AP3 are observed at 480,488 and 474 nm, respectively. Zinc ions (0.2 M ) did not affect the absorption spectra of the anthrapyrazoles. Electron paramagnetic resonance measurements Direct detection of o-semiquinone radicals. For our EPR study of the formation of semiquinone radicals the following catechol derivatives (RH,) were selected: catechol (X = H), DOPA (X = CH2CHNH2COZH), DHCA (X = CH*CH2CO*H), and DHBA (X = C02H). When AP2 (0.2 mM) was illuminated in the presence of catechol (1 mM) and Zn2+ (100 mM) in aerated DMSO/buffer mixture, the EPR spectrum shown in Fig. 2(B) was observed. Control experiments showed that light and all the above reactants were necessary to produce this spectrum. The EPR signal was assigned to catechol-derived o-semiquinone radical complexed with Zn2+ ion (R7/Zn2+). When a deaerated sample was illuminated, a similar EPR spectrum was observed, but its amplitude was approximately 6 times lower than that obtained in the presence of air. This observation indicates that AP2 can oxidize catechol in Type I reaction. Subsequent aeration and exposure of the same solution produced an EPR spectrum close to the original one shown in Fig. 2(B). Thus oxygen markedly enhances the production of radicals and in this way facilitates their detection. When DOPA, DHBA, or DHCA were used instead of catechol, the EPR spectra shown in Figs. 3(A), (B) and (C) respectively were recorded. Similar spectra, although with much lower intensity. were recorded in aerated aqueous buffer without DMSO (not shown). Hyperfine (hf) couplings determined in these two solvents are given in Table 1. The values for the aqueous sample are in good agreement with those reported by other researchers for the same species (Felix and Sealy, 1981; Kalyanaraman et al., 1985). Kinetic measurements of the photoinduced EPR signals were performed next. Illumination of an aerated DMSOhuffer solution of catechol, AP and
Photosensitization by anticancer agents
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A
Figure 2. EPR spectrum from system containing AP2 (0.2 mM), catechol(1 mM) and ZnZ+(100 mM) in aerated DMSO/acetic buffer, pH 4.5. (A) before, and (B) during illumination at 488 nm. Instrumental settings: microwave power 5 mW, modulation amplitude 0.165 G, time constant 0.25 s, scan rate 8 min, and gain 2 x 104 (A) and 10 x 103 (B).
Zn2+ caused an immediate generation of the signal which reached a steady-state level in approximately 2 min from the beginning of exposure (Fig. 4). Figure 5 shows similar kinetics for DHCA. The steady state amplitude depended on the concentration of the sensitizer, catechol and zinc ions as shown in Fig. 6(A), (B) and (C) respectively. The dependence on [Zn”] is particularly notable because it affected both the steady state concentration of radicals, the duration of the steady state, and kinetics of the signal decay in dark. Thus, a strong signal from RT/Zn2+ could be observed at zinc ion concentrations as low as 1.3 or 2.5 mM [Figs. 4(B) and 5(B)], which is significantly lower than the routinely used concentration of 0.1-0.2 M (Kalyanaraman ef al., 1985; Felix and Sealy, 1981). When catechol (1 mM) was used as an oxidizable substrate the steady state signal began to decrease after ca 10 min exposure at [Zn”] = 10 mM, but this period was reduced to less than 2 min at [Zn”]
RH2
Re-
Figure 3. EPR spectra of o-semiquinone/Znz+complexes derived from: (A) DOPA (4 mM), [Zn*+] = 100 mM; (B) DHBA (2 mM), [ZnZ+]= 100 mM; (C) DHCA (4 m M ) , [Znz+] = 2.5 mM. Radicals were generated illuminating AP2 (0.2 mM), used as a sensitizer and the appropriate catechol derivative in aerated DMSO/acetic buffer mixture. Instrumental settings: microwave power 5 mW, modulation amplitude 0.33 G, time constant 0.25 s, scan rate 4 and 8 min, gain 1.6, 2.0 and 1.25 x lo‘ for (A), (B) and (C) respectively.
> 200 mM (not shown). This may indicate faster depletion of reactants (catechol and oxygen) at higher zinc concentration; however, it is not clear why and how zinc ions might accelerate this process. DHCA, DHBA, and DOPA behaved similarly to catechol both at the high and low zinc ion concentrations (not shown). After cessation of illumination the photoinduced signal decayed with kinetics which depended upon [Znz+] (Figs. 4 and 5). Dark decay at [Zn2+] = 100 mM obeyed second order kinetics (Fig. 7) which indicates that the radicals are depleted in a bimolecular process, most likely by disproportionation. The rate constant of radical termination for catechol was determined to be 2k = 3.5 x lo3 M-’s-’. For semiquinones from DOPA, DHCA and DHBA the
R’-IZnZ+
KRZYSZTOFRESZKAel a / .
362
1-
"i-
t
Figure 4. Kinetics of the generation and the dark decay of the EPR signal of R ' / Z n 2 + . ( A ) [ A m ] = 0.2 mM, [catechol] = 1.0 mM, [Znz'] = 100 mM; (B) [Am] = 0.2 mM. [catechol] = 2 mM, [Zn2+] = 2.5 mM; instrumental settings: same as in Fig. 2 but scan rate 16 min, gain 1.25 and 2 x 1oJ for ( A ) and (B) respectively. Arrows indicate when the light was turned ON ( t ) and OFF ( 1). Table 1 . Hyperfine couplings of photochemically-induced EPR spcctra from Zn"-complexed o-semiquinone radicals in DMSOlacetic buffer. pH 4.5 ( 1:l vol/vol) Hyperfine coupling (G) Compound
a,
Catechol
(1.58 0.5
DOPA
nrt 0.316 0.25
DHBA
0.55 0.47
DHCA
-
a,
a,
3.83 3.83 3.9 3.9 3.85 3.9§ 3.83 3.45 3.45 4.09 4.02
a,,
a@
Ref.
0.58 - This work 0.5 0.70 6.29$ This work 0.706 5.82$§ This work 0.87 2.65 3.11 1.01 - This work 0.85 P 0.75 3.92 This work (2H) 0.62 4.02 7 (2H)
'Kalyanaraman er a / ., 1985. tnr-no! rcsolvcd $This is a sum of hf couplings on two nonequivalent p hydrogens i n thc 5ldc chain of DOPA. Poor resolution is caused hy sclcctivcly broadened lines 2 due to a hindered rotation of the side chain and an oxygen broadening effect. However, computer simulation using this # value produced a spectrum which tits the 8 most intense lines of the experimental spectrum [Fig. 3(A)]. §In l(XJ"&aqueous buffer, pH 4.5. FFclix and Scalv. 1981.
ON
Figure 5. Kinetics of the generation and the dark decay of EPR signal of R'/Zn2'. ( A ) [ A P l ] = 0.2 mM, [ D H C A ] = 4.0 mM, [Znz+] = 100 mM; (B) [Zn2+] = 1.3 m M in aerated DMSO/acetic buffer p H 4.5. Instrumental settings: same as in Fig. 2 but modulation amplitude 0.33 G, scan rate 4 min, and gain 8 and 25 X 10' for ( A ) and (B) respectively.
2k values are 3 x lo4 M - ' s-I , 8.8 x lo4 M - l s - l , and 14 x 10' M-' s-I respectively. The rate constant for similar complex from DOPA in aqueous buffer was reported to be 2k = 1.1 x lo4 M-' s - I (Kalyanaraman et al., 1985). Dark decay of the EPR signals observed at low [Znz+] (1-50 mM) showed two components [Figs. 4(B) and 5(B)]. The first one was a rapid decrease of the amplitude immediately after cessation of illumination, followed by the second much slower kinetics. To establish whether there are other spectroscopic differences between radicals observed at low and high Zn2' concentrations we used catechol and DHCA because they produced particularly conspicuous pattern of kinetic dependence on [Zn2+]. Both the g-factors and the hf splitting were similar regardless of the zinc concentration. For RT/Zn2+ from catechol the g value was 2.0040 and for DHCA it was 2.0038 (vs Fremy's salt of g = 2.0055). These g values are different from those of uncomplexed radicals for which g = 2.0046 and 2.0047 were reported (Kalyanaraman et al., 1985). The initial fast kinetics cannot be attributed to the fast decay of the uncomplexed radical anions. R', (2k = 107-108 M - I S - 1 ) (Kalyanaraman ef af.,1985), since in the absence of zinc the EPR signals were not observed. Thus besides the kinetic differences the radicals observed at low and high zinc concen-
Photosensitization by anticancer agents
--
i-
-
363
I O -
08-
.
N
;0 6 -
I
0 0 00
[ API
]lmM)
40
80 2 120
160
200 L L 240 - & 280 - d
TIME ( s e c l
Figure 7. Reciprocal of [R:/Zn*'] from catechol vs time for the dark decay [from Fig. 4(A)]. Data were used to calculate the termination rate constant.
500
a 4
1
d
-_.....
catechol (10 mM), DETAPAC (0.5 mM), and DMPO (80 mM) in aerated acetate buffer pH 4.5. The spectrum has hyperfine couplings aN = 14.45 G, aH = 11.45 G, and aH = 1.25 G and was assigned to the DMP0/02H adduct. These couplings are close to values reported previously for the same adduct produced enzymatically in acetate buffer, pH 4.6: aN = 14.4 G, = 11.4 G, &$ = 1.3 G (Mottley and Mason, 1986). In the presence of superoxide dismutase (SOD) (100 p,g/mL) the intensity of the spectrum was diminished by ca 46% (data not shown) vs the control sample upon similar light exposure, suggesting the intermediacy of the superoxide radical in the adduct formation. Figure 8(A) shows the control run in dark, before exposure. EPR spectra similar to those in Fig. 8(B) were observed when DOPA, DHCA or DHBA were used instead of catechol. This observation confirms the production of OYO2H during the APphotosensitized oxidation of catechols and this is the first time that the EPR spectrum of the DMPO/02H adduct has been detected during catechol(amine) photooxidation. Figure 8(D) shows spectrum observed when AP2 (0.2 mM), catechol (10 mM), DETAPAC (0.5 mM) and DMPO (80 mM) were illuminated in aerated DMSOhuffer pH 4.5. It contains components from two DMPO adducts: one is a peroxy radical (I) and another, much weaker, a carboncentered (11) radical. By analogy with what was observed in aqueous buffer, the adduct I can be assigned to DMP0/02H, although its hf couplings, aN = 13.8 G, a& = 11.14 G, and a;l = 1.37 G, are slightly different from those found in aqueous solution (vide supra). This can be explained by lower polarity of the sample due to the presence of DMSO, which is known to affect hf couplings (Harbour and Hair, 1978). In fact the values of the above couplings are between those established for the DMP0/02H adduct in aqueous solution and in DMSO, as expected. When SOD (100 &mL) was
1 . -.
L
Figure 6 . Steady state EPR amplitude of the photochemically generated R'/ZnZ+ from catechol vs [APl], at 1.0 mM catechol, and 50 mM ZnZ+(A); vs [catechol], at 0.2 mM AP2, and 100 mM Zn2+ (B); vs [Znz+]. at 0.2 mM AP2, and 1.0 m M catechol (C) in DMSOlacetic buffer pH 4.5.
trations seem to be similar. In all these experiments AP2 could be replaced by AP1 or AP3, producing similar results. This confirms that anthrapyrazoles of structures shown in Fig. 1 are good photosensitizing agents. Spin trapping. It was anticipated that the photosensitized oxidation of catechols may lead to superoxide formation and therefore we employed the spin trap DMPO to detect this radical. Figure 8(B) shows the EPR spectrum observed upon illumination of a sample consisting of AF'2 (0.2 mM),
364
KRZYSZTOF RESZKAef a / .
Figure R . EPR spectra from systems containing AP2 (0.2 mM). catechol (10 mM). DETAPAC (0.5 mM), and DMPO (80 mM) in acetic buffer pH 4.5 (A and B ) and in DMSOiacetic buffer pH 1.5 mixture (1:l volivol) (C and D). ( A ) and ( C ) before. and (B) and (D) during illumination. Instrumental settings: microwave power 20 mW. modulation amplitude 0.53 G; time constant 0.25 s. scan rate 1min; gain 2.5 x 10' ( A . B and C), 1.6 x 10' (D).
introduced into this system the intensity of the signal decreased b) ca 20% (data not shown), in agreement with the involvement of the superoxide radical in the adduct formation. The relatively small inhibitory effect of SOD may be due to decreased enzyme activity at acidic pH (4.5)and/or the presence of DMSO at high concentration in our samples. Alternatively. the spectrum in Fig. 8(D) may be assigned to the DMPO adduct of a methyl peroxyl radical formed in the presence of DMSO (vide infra). This adduct has hyperfine couplings similar to those of the DMPO/OzH radical but in contrast to the latter is not SOD-sensitive (Kalyanaraman ef al., 1983). (We wish to mention that although the formation of the superoxide radical in our system is highly likely and seems to be supported by the SOD effect, SOD may not be reliable in a system in which singlet oxygen is generated (vide infra) because the enzyme quenches singlet oxygen (Lion er al., 1980) with high rate constant (2.73 x loy M-' s-l ) in phosphate buffer pH 7.1 (Suzuki et al., 1990)). Adduct I1 was identified as DMPO/CH3 radical based on its hf couplings. a N = 15.8 G. aH = 22.4 G. which are close to those reported for this species in aqueous DMSO, ax = 16.10 G, aB = 23.00 G (Mossoba and Gutierrez, 1985). Because both hydrogen peroxide and consequently hydroxyl radicals can be produced during AP-photosensitized oxidation of catechols (Reszka et al., 1986a). the methyl radical may have its origin in the reaction between OH and DMSO (Lagercrantz and Forshult, 1969). If some methyl radicals react with oxygen first and then add to DMPO, they will give rise to the DMPOiOOMe adduct. Next *e examined the effect of D 2 0 on the
athrapyrazole-sensitized oxidation of catechols. An earlier study showed that anthrapyrazoles generate singlet oxygen in ethanolic solutions (Reszka er al., 1986a) and that certain catechol(amine)s can be oxidized by singlet oxygen (de Mol el al., 1979; Reszka and Sealy, 1984). It was therefore anticipated that if singlet oxygen contributes to catechol oxidation in our system, its longer lifetime in D20 (55.0 vs 4.2 ps in water, Rodgers, 1983) should result in more superoxide radicals and consequently a more intense EPR spectrum of the corresponding DMPO adduct. It was found that in D20-acetate buffer pD 4.5 the EPR signal of DMPO/02D was approximately 90% larger in comparison with the control ( H 2 0 ) sample. For a sample prepared in DMSOlacetate buffer ( D 2 0 ) pD 4.5 the enhancement factor was ca 50%. This stimulating effect of D 2 0 indicates that singlet oxygen is produced upon illumination of anthrapyrazoles in both aqueous solutions and in DMSO/buffer mixtures and that '02 is partially involved in catechol oxidation. DISCUSSION
In this work we have demonstrated that osemiquinones are produced during anthrapyrazolephotosensitized oxidation of catechols and that this process is accompanied by the formation of superoxide radicals. To facilitate the detection of the semiquinone radicals the metal ion spin stabilization technique has been employed. Radical complexes with the Zn2+ ion proved to be the most stable and for this reason this metal ion was used in the present study (Felix and Sealy, 1981; Kalyanaraman et al., 1985). The photophysical properties of anthrapyrazoles are still not well characterized; however, the observed photoreactivity of these agents in aerated and oxygen-free solutions as well as the D 2 0 effect indicate that they may function as Type 1 and Type I1 photosensitizers. In the particular case of the oxidation of catechols the possible events are illustrated in Schemes 1 and 2 for these two types of processes respectively. It is likely that, in the systems studied in this work, the two mechanisms operate simultaneously although the importance of the Type I1 process may be further suppressed by the decreasing oxygen concentration in the course of the reaction. Pathway c in Scheme 1 regenerates the original state of the sensitizer and prevents back electron transfer. It is likely that it is this reaction and/or singlet oxygen formation (pathway b, Scheme 2) which is responsible for the enhanced production of the radicals observed in the presence of oxygen. In an anaerobic environment the two processes essential for efficient radical formation, will be either totally blocked (Type I1 process), or significantly slowed down (Type I process), resulting in lower steady state concentrations of the radical-metal ion
Photosensitization by anticancer agents
365
0;
+ RH2 -+
R: t H202
(2)
k = 5.6 x lo4 M - I s - ' 202H -+ H202 + 0 k
=
=
(3)
2
(4)
7.6 x lo5 M - l s - I
0 5 + OzH + H t - + H 2 0 2 + 0 k
2
8.5 x lo7 A 4 - l s - I
The stronger EPR signal of the DMPOIperoxyl radical in DMSO/buffer mixture may be due to several factors: the higher content of A P monomers, which may possess superior sensitizing properties over dye aggregates which are more abundant in aqueous solutions; the longer lifetime of the superoxide radical in the presence of DMSO; and the likely overlapping of two similar signals from D M P 0 / 0 2 H and DMPO/OOMe adducts. Accumulation of H202 during AP-sensitized oxidation of DOPA has been demonstrated (Reszka et al., 1986b) and it is certain that photosensitized oxidation of other catechol(amine)s will produce the same species. Neither H 2 0 z nor oxygen react efficiently with o-semiquinone radicals (Kalyanaraman el al., 1984; Pate1 and Willson, 1973). The observation that the o-semiquinone radicals accumulate readily in aerated solutions indicates that their oxidation by dissolved O2 is inefficient and cannot be the source of the superoxide radicals detected in our systems. It is more likely that the 0 3 / 0 2 H 'radicals are produced via path c in Scheme 1 andlor 2.
Scheme 1
Scheme 2
complexes and consequently a weaker EPR signal. No EPR signal from the semireduced sensitizing compound ( A V was observed. This is in agreement with the observation of Graham er d.(1987), who found that the radiolytically-produced radical anion of a structurally-related anthrapyrazole CONCLUSIONS (analog of AP1 with O H at position 7 of the A ring, In this work we have demonstrated that photoFig. 1) decays by disproportionation with the rate oxidation of catechols sensitized by anthrapyrazoles constant of 5 x lo8 M-' s-l, or reacts with oxygen may involve Type I and Type I1 processes and with a rate constant of 8 x loRM-I s-l. The rate proceeds through a one-electron oxidation step, to constants for the reaction between radical anions of form the corresponding o-semiquinone radicals. In the anthrapyrazoles studied in this work and O2 illuminated aerated solutions of the anthrapyrazoles should be equally high. Both these reactions preand catechols superoxide radical is formed as demclude direct EPR observation of the sensitizeronstrated employing DMPO as the spin trapping derived radicals. agent. Since catechol(amine)s are natural comUsing DMPO we demonstrated the formation of ponents of cells it seems likely that biological superoxide radical during photosensitized oxidation activity of the deshydroxy anthrapyrazole anticancer of catechols at pH 4.5. At this pH a significant agents could be enhanced by photoactivation. amount of the superoxide ions exists in the protonated form (pK, (0,H) = 4.88, Behar et d . ,1970) Acknowledgement-This research was supported in part which adds much faster to DMPO ( k ( D M P 0 t 0 2 H ) by a grant to J . W. L. from the National Cancer Institute of Canada. = 6.6 x lo3 M-' s-', Eq. 1) than its conjugated base, 0; (k(DMPO+Oq = 10M-' s-l) REFERENCES (Finkelstein et al., 1980). Therefore this reaction can successfully compete with the scavenging of the Behar, D., G. Czapski. J. Rabani, L. M. Dorfman and H. A. Schwartz (1970) The acid dissociation constant superoxide by catechols, Eq. 2 (Bors et al., 1978), and kinetics of the perhydroxyl radical. J . Phys. Chem. and dismutation processes (Eqs. 3 and 4). 02H t DMPO + DMPO/OzH
(1)
14. 3209-3213. Bors. W., C. Michel, M. Saran and E. Lengfelder (1978)
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