Luminescence investigations of redox cycling of adriamycin

Luminescence investigations of redox cycling of adriamycin

Chemosphere 44 (2001) 83±90 Luminescence investigations of redox cycling of adriamycin Irena Kruk a,* , Teresa Michalska a, J ozef Køadny b, Lidia...

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Chemosphere 44 (2001) 83±90

Luminescence investigations of redox cycling of adriamycin Irena Kruk


, Teresa Michalska a, J ozef Køadny b, Lidia Kubera-Nowakowska




Institute of Physics, Technical University of Szczecin, Al. Piast ow 48/49, 70-310 Szczecin, Poland Clinic of General and Vascular Surgery, Pomeranian Medical Academy, ul. Powsta nc ow Wielkopolskich 72, 70-111 Szczecin, Poland Received 31 March 2000; received in revised form 5 January 2001; accepted 12 February 2001

Abstract The light emission from the adriamycin ‡ Co2‡ ‡ H2 O2 system has been studied. Chemiluminescence, ¯uorescence and absorption spectra were measured. The ¯uorescence spectra were time-dependent exhibiting maxima at 555, 590 and 645 nm. The chemiluminescence spectra consist of four bands with maxima at around 460±500, 550±580, 640 and 700 nm. Free radical reaction inhibitors, 1 O2 -quenchers and catalase inhibited the light emission indicating that hydroxyl radical, superoxide anion radical and singlet oxygen are generated during the redox cycling of adriamycin. Chemiluminescence studies revealed that adriamycin undergoes chemiexcitation under our experimental conditions. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Adriamycin; Luminescence; Oxygen free radicals; Singlet oxygen

1. Introduction Adriamycin (doxorubicin), a quinone-containing anthracycline antibiotic, is one of the most clinically used antitumor agents. The quinone±semiquinone redox cycle of the drug is considered to be important for its antineoplastic action in cancer therapy (Iwamoto et al., 1974; Vichi et al., 1995). Unfortunately, the clinical usage of the drug is limited by its neurotoxicity, hepatoxicity or nephrotoxicity (Lenaz and Page, 1976; Zidenberg-Cherr and Keen, 1986; Goeptar et al., 1995). The adriamycin side-e€ects limit a long-term maintenance treatment. One of theories of the drug induction of cellular alteration is oxygen free radical generation and release of catecholamines (Goodman and Hochstein, 1977; Kalyanaraman et al., 1980; Doroshow, 1995; Malisza and Hasino€, 1995).


Corresponding author. Tel.: +48-91-4342-113; fax: +48-9143-42113. E-mail address: [email protected] (I. Kruk).

In previous papers (Michalska et al., 1996; Bounias et al., 1997) we have reported that adriamycin is readily reduced to the semiquinone form which can activate molecular oxygen producing oxygen radicals (superoxide anion radical, O2 , hydroxyl radical, HO ) and hydrogen peroxide (H2 O2 ) as well as increases generation of hydroxyl radicals from the Co2‡ ‡ H2 O2 system. In this paper, we report the ®rst spectroscopic evidences that the redox cycling of adriamycin in darkness is associated with generation of electronically excited molecules which can cause harmful damage by transferring their energy to organelles ``photochemistry in darkness''.

2. Materials and methods Adriamycin hydrochloride was obtained from Farmitalia Carlo Erba. Catalase from bovine liver with about 3000 U mg 1 protein activity was from I.B and BAM Wroclaw (Poland), compounds used as hydroxyl radical scavengers were obtained from Merck (Darmstadt, Germany) as were singlet oxygen scavengers. Other reagents were analytical grade from POCH (Gliwice,

0045-6535/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 1 ) 0 0 0 5 5 - 8


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Poland), and were used without further puri®cation. The solutions were prepared in redistilled water immediately prior to use and were protected from air in darkness. Chemiluminescence intensity was measured with an M12FQC51 photomultiplier with S-20 cathode sensitive in the range 200±800 nm, operating jointly with Zeiss K200 recorder (Germany). The method has been described previously (Aboul-Enein et al., 1998). Fluorescence spectra were measured at 298 K with a spectro¯uorometer consisting of monochromators SPM-2, M12FQC51 photomultiplier operating jointly with a computer. For spectrophotometric studies a UV/VIS spectrophotometer M-40 from Zeiss, Jena (Germany) was used. Chemiluminescence spectra were performed with a set of cut-o€ ®lters (GOST 9411±66) and an EMI9203B photomultiplier. The method has been described previously (Kruk et al., 1989). All results are representative for at least three measurements and are reported with mean values  standard deviation. 3. Results and discussion We have previously reported (Michalska et al., 1996) that adriamycin greatly increased the HO-radical generation from the following reaction:

Co2‡ ‡ H2 O2 ! Co3‡ ‡ HO ‡ HO


and the yield of the HO production increased with increasing pH. The data obtained showed that observed increase in the radical generation could be achieved by redox cycling mechanism generating O2 and semiquinone free radical of adriamycin (SQ ), which reduces Co3‡ to Co2‡ ion. This reaction is accompanied by chemiluminescence (CL). The quantum yield of the CL is strongly dependent on the pH value (Fig. 1), adriamycin and H2 O2 concentration (Fig. 2). The kinetics of the light emission manifest di€erent behaviour depending on the pH value and H2 O2 concentration. The stimulatory action of HO concentration re¯ects a sharp rise of intensity (about 30 s at pH 10.8) followed by a rapid decay. The water solution of adriamycin alone exhibits absorption in the UV region with the maxima at 220, 240 and 262 nm and a broad band with maximum centered at 480±500 nm corresponding to quinone in the visible region (Fig. 3(A)). Fig. 3(B) shows the change of the absorption spectra of adriamycin in the visible region with the reaction time shortly after its addition to the Co2‡ ‡ H2 O2 system. As can be seen from the ®gure the mixing of the reagents is accompanied with the formation of a new product absorbing at around 600 nm, i.e.,

Fig. 1. The e€ect of pH on the chemiluminescence intensity of the 0.1 mM adriamycin ‡ 0:5 mM CoCl2 ‡ 10 mM H2 O2 system measured at 310 K (0.15 M phosphate bu€er, pH 7.4 and pH 8; 0.1 M carbonate bu€er; pH 9.2 and pH 10.8). Reactions were initiated by the addition of H2 O2 . A representative study of three experiments is shown.

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Fig. 2. E€ects of H2 O2 and adriamycin concentration on time course of the chemiluminescence from the system of adriamycin ‡ CoCl2 ‡ H2 O2 measured in carbonate bu€er, pH 10.8. Curves: 1 ± 1 mM H2 O2 ; 2 ± 5 mM H2 O2 ; 3 ± 10 mM H2 O2 . Adriamycin concentration in these experiments was 0.1 mM. Curve 4 ± as curve 1 but without adriamycin; curve 5 ± as curve 1 but for decreased concentration of adriamycin (0.05 mM). Concentration of CoCl2 was 0.5 mM, pH 10.8, temperature 310 K.

Fig. 3. Part A. UV and visible spectrum of adriamycin in water. Part B. Absorption spectral change of the 0.1 mM adriamycin ‡ 0:5 mM CoCl2 ‡ 10 mM H2 O2 system. Conditions: 0.15 M phosphate bu€er, pH 8; temperature 301 K.


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adriamycin±Co3‡ complex (Michalska et al., 1996) which self-reduces the cobalt moiety to form a Co2‡ ion and semiquinone form of the drug. The rate of the decrease in the 480±500 nm band increased as the alkalinity of the solution and the H2 O2 concentration were increased (data not shown). This phenomenon can be explained in terms of the quinone disappearance. The decrease of quinone is a very important condition for the appearance of the CL (Kruk et al., 1989). To determine the origin of the excited species causing the observed CL, measurements of the spectra of the ¯uorescence and CL were performed. The ¯uorescence emission spectra were measured at various pH values (Fig. 4). The spectra cover the range from 540 to 750 nm and show maxima at about 555, 590 and 645 nm. The ¯uorescence bands at 555 and 590 nm were also reported by Carmichael et al. (1983) for air-free and oxygen-saturated solutions of adriamycin during irradiation with visible light. The authors observed a decrease in the both maxima intensity in the presence of air and oxygen. In the presence of oxygen the authors observed generation of O2 and HO radicals. The ¯uorescence band at 645 nm additionally observed in our experiments may arise from the adriamycin±Co2‡ complex. Fig. 5 shows the ¯uorescence emission spectra of the adriamycin ‡ H2 O2 ‡ Co2‡ system at pH 8 for various time of the reaction course. As can be seen from the ®gure the emission slowly decreases with an increase in the reaction time, but no new band is observed.

The CL spectra of the above system measured at two di€erent values of pH (8.0 and 10.8) are shown in Fig. 6. The spectra are spread through the full visible region and have four emission maxima at around 460±500, 550±580, 640 and 700 nm. The ¯uorescence bands match the 530±700 nm CL region, however, distinct shift in the maxima position occurs. The position of the bands with maxima at 640 and 700 nm may correspond to the simultaneous transition in the singlet oxygen …1 O2 † dimols h i   2O2 1 Dg ! 2O2 3 Rg with vibrational quantum number of (1.0) for 580 nm, (0.0) for 640 nm and (0.1) for 700 nm, whereas the emission at about 500 nm is due to the h i h i 3 ! 2O R 2O2 1 Dg ; 1 R‡ 2 g g transition (Khan and Kasha, 1970; Khan, 1987). The emission at 460 nm may result from the excited form of a semiquinone (Stau€ and Bartolomes, 1970). Since the ¯uorescence band of adriamycin overlaps the emission resulting from dimols of 1 O2 , the CL spectrum may also be interpreted in terms of the activated CL: h i 1 2O2 1 Dg ; 1 R‡ g ‡ adriamycin h i ! 2O2 3 Rg ‡ 1 adriamycin

Fig. 4. The e€ect of pH on the ¯uorescence emission spectra of the adriamycin ‡ CoCl2 ‡ H2 O2 system detected 5 min after the reaction start …kexc ˆ 480 nm†. Remaining conditions as in Fig. 3.

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Fig. 5. Change in the ¯uorescence emission spectra of the adriamycin ‡ CoCl2 ‡ H2 O2 system …kexc ˆ 480 nm†. Remaining conditions as in Fig. 3. 1

adriamycin ! 1 adriamycin ‡ hm

If 1 O2 is generated, then the addition of 1 O2 -quenchers and free radical scavengers to the reaction mixture, such as HO and O2 should lead to a decrease in the CL intensity. The e€ect of 1 O2 -quenchers was studied quantitatively (Table 1). The quenchers reduce the light emission according to the Stern±Volmer equation (Stern and Volmer, 1919). The calculated rate constants do not di€er signi®cantly from those reported by other authors (Bellus, 1979; Foote, 1979). It is worth mentioning that methionine and histidine beside being the good scavengers of 1 O2 are capable of scavenging of HO radicals with a high eciency, whereas NaN3 also reacts with O2 . All free radical scavengers tested inhibit the light emission (Fig. 7). As can be seen the CL from the adriamycin ‡ Co2‡ ‡ H2 O2 system is e€ectively inhibited by SOD (O2 and 1 O2 scavenger), deoxyribose, cysteine, thiourea, glutathione, mannitol (HO scavenging abilities) and catalase (decomposition of H2 O2 ). The strong quenching e€ect exerted by catalase indicates that H2 O2 participates directly in the generation of the CL emitters. Also an addition of ascorbic acid to the reaction mixture causes a strong decrease of the light intensity. This compound can reduce the drug on the twoelectron way as well as to remove O2 from the reaction at the reaction rate 2:7  105 M 1 s 1 (Halliwell and Gutteridge, 1989). Results described here, together with our previous experiments (Michalska et al., 1996; Bounias et al.,

1997) indicate that the CL observed in the redox-cycling metabolism of a quinone-containing drug, adriamycin (Q), can result from the following reactions: Q ‡ HO

! SQ  ‡ HO


SQ  ‡ O2 ! Q ‡ O2


First, in alkaline solution adriamycin undergoes univalent reduction to the semiquinone anion (SQ  ), then the semiquinone transfers its electron to molecular oxygen with concomitant generation of O2 . This suggestion ®nds con®rmation in a study of photoirradiation of the drug performed by Carmichael et al. (1983). The workers observed a direct electron transfer from the electronically excited molecule of adriamycin or its anion radical to molecular oxygen with formation of O2 and HO in the presence of oxygen. Superoxide anion radical could also be generated in a redox cycling reaction between Co2‡ and Co3‡ during formation of paramagnetic complex with molecular oxygen (Matsuura and Nishinaga, 1984) Co2‡ ‡ O2 ! Co2‡    O2 3‡

! Co




Co3‡    O2 …4†

Semiquinones are able of reducing Co3‡ to Co2‡ ion, thus to supply the Co2‡ ion for reaction (1) SQ  ‡ Co3‡ ! Q ‡ Co2‡



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Fig. 6. Chemiluminescence spectrum for the 0.1 mM adriamycin ‡ 0:05 mM CoCl2 ‡ 10 mM H2 O2 system measured in carbonate bu€er at pH 10.8 (blackened area) and in phosphate bu€er, pH 8 (bright area). The size of the bars approximates the standard deviation. Temperature 291 K. Table 1 The e€ect of 1 O2 quenchers on the chemiluminescence from the 0.1 mM adriamycin ‡ 0:5 mM CoCl2 ‡ 5 mM H2 O2 systema Quenchers

Quenching constant kq  10 7 …M 1 s 1 †

Hydroquinone Histidine Methionine Sodium azide 5,5-dimethylcyclohexandione-1,3

4:1  0:3 2:2  0:8 6:2  1:7 2:0  0:4 6:7  0:9

SQ  ‡ H2 O2

Co ions


Q ‡ HO ‡ HO


It should be noted that reactions generating O2 are the key processes limiting the generation of excited states because the addition of SOD reduced the light emission by 70% (Fig. 7). Superoxide radicals may be involved in several reactions with concomitant formation of 1 O2 : O2 ‡ O2 ! …O4 † O2


! … O 2 †2 ‡

…7† O22 

Reactions were performed at pH 8 in 150 mM phosphate bu€er. Temperature 310 K. Quenching constants (kq ) are the total bimolecular rate constant for deactivation of 1 O2 .

…O4 † ‡

The semiquinone of adriamycin could be involved in the reaction generating the HO radical (Gutteridge, 1984):

The latter reaction is known as Haber±Weiss reaction and could be catalyzed by Co ions (Haber and Weiss, 1934).


O2 ‡ H2 O2 ! 1 O2 ‡ HO ‡ HO

…8† …9†

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Fig. 7. Quenching e€ect exerted by O2 ; HO scavengers and catalase on the chemiluminescence intensity of the 0.1 mM adriamycin ‡ 0:5 mM CoCl2 ‡ 5 mM H2 O2 system measured in 0.15 M phosphate bu€er, pH 8. Inhibitors were added to the reaction mixture 10 min after the start of the reaction. Concentration of inhibitors were: 5 mM deoxyribose; 10 mM mannitol, thiourea and ascorbic acid; 2.5 mM cysteine; 5 mM glutathione; 100 lg=ml catalase and SOD.

Singlet oxygen may also be generated in the reaction of scavenging O2 by HO , which occurs at a rate controlled by di€usion (Arneson, 1970): HO ‡ O2 ! 1 O2 ‡ HO


Pairs of 1 O2 molecules in the 1 Dg ; 1 R‡ g states can combine their electronic excitation giving the dimols emission at 480, 585, 640 and 700 nm. The base catalyzed disproportionation of H2 O2 is also accompanied by the 1 O2 generation (Smith and Kulig, 1976): H2 O2 ‡ HO H2 O2 ‡ HOO

! HOO ‡ H2 O ! H2 O ‡ HO ‡ 1 O2

…11† …12†

The results presented in this paper show that an understanding of the photochemistry in darkness of adriamycin is important because of its known toxicity and eventually possible future application of a combination therapy with scavengers of oxygen free radicals and/or quenchers of singlet oxygen.

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