A Variety of Compounds Have Been Demonstrated to Be Active

2478 Vol. 35 (1987)

Chem. Pharm. Bull. 35( 6 )2478-2483(1987).

Singlet Oxygen-Producing Activity and Photodynamic Biological Effects of Acridine Compounds

YOSHIHISAIWAMOTO,* HISASHI YOSHIOKAand YASUTAKEYANAGIHARA

ShizuokaCollege of Pharmacy, 2-2-1 Oshika, Shizuoka-shi422, Japan

(ReceivedNovember 10, 1986)

The singlet oxygen-producing activities and photodynamic biological activities of acridine compounds were compared. Singlet oxygen was trapped by 2,2,6,6-tetramethyl-4-piperidone (TEMP), and 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TEMPO) produced was detected by electron spin resonance (ESR) spectrometry. TEMPO production was inhibited by NaN3 and enhanced in D2O, confirming it to be an adequate ESR spectroscopic indicator for singlet oxygen. Comparison of TEMPO-producing activities revealed that acriflavine and proflavine, which are the most potent photosensitizers both in cell inactivation and petite induction of yeast, produced the most intense and long-lived ESR signals. However there was no clear difference of TEMPO-producing activity between biologically ineffective acridines such as acridine and quinacrine and effective ones such as acridine yellow and 6,9-diamino-2-ethoxyacridine. These results suggested that the differences observed in the photodynamic biological effects among the acridine compounds depend mainly on the differences of drug distribution and only partly on the differences of singlet oxygen-producing activity of the dye molecule itself. Keywords- singlet oxygen production; photodynamic action; acridine compound; ESR detection; 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TEMPO); yeast; Saccharomyces cerevisiae; cell inactivation; petite induction

A variety of compounds have been demonstrated to be active photo-sensitizers in different organisms.1) In previous studies,1a,2) acriflavine (AF) was identified as an active photo-sensitizer in yeast. Irradiation of AF-pretreated cells with common fluorescent lamps brought about cell inactivation, petite induction and an increase in the reversion rate from tryptophan auxotrophy to prototrophy.2b) These biological effects were suggested to be induced by the type II photodynamic action mediated by singlet oxygen.2) Singlet oxygen production has been detected indirectly in terms of nitroxide radical production by the electron spin resonance (ESR) method.3) Using this method, the production of the nitroxide radical in photo-irradiated AF solution was demonstrated.4) Comparison of the photodynamic activities of acridine compounds5) revealed that proflavine (PF), acridine yellow (AY), 3,6-diamino-2-ethoxyacridine (DAEA) and acridine orange (AO), in addition to AF, were highly toxic and mutagenic, but acridine (A), quinacrine (Q) and 9-aminoacridine (AA) were not. These findings led us to compare the singlet oxygen-producing activities and the photodynamic biological activities among acridine compounds. The results presented here indicate that nitroxide radical production in photo-irradiated dye solution is closely related to singlet oxygen production. All acridines used here could produce nitroxide radical by photodynamic action. AF or PF, which induce intense and various photodynamic biological effects, produced the most intense and long-lived ESR signals of nitroxide radical.

Materialsand Methods Detectionof SingletOxygen- Detailsof the methodwere given in the previousreport.4) No. 6 2479

Preparation of Reaction Mixture All the solution were prepared in glass using redistilled water. The components of the standard reaction mixture were 2 x 10 -2 M 2,2,6,6-tetramethyl-4-piperidone (TEMP), 10 -5 M dye solution and 1/15 M phosphate buffer (pH 9.2). In order to determine the inhibitory effect of sodium azide, 50 mm NaN3 was added. An anoxic sample was prepared as follows. A reaction mixture containing 2 x 10 -2 M TEMP, 10-5 M AF and 1/15 M phosphate buffer (pH 9.2) was frozen by the use of liquid nitrogen in a quartz tube with a capillary side branch. Then the tube was evacuated, thawed and frozen repeatedly in order to remove molecular oxygen thoroughly without changing the volume of the contents. Finally the neck of the evacuated tube was heat- sealed. A part of the contents (about 50 pp was decanted into the capillary branch and the neck of the capillary was heat-sealed. A D20 sample was prepared as follows. First, 0.5 ml of 2/15 M phosphate buffer (pH 9.2) and a mixture of 0.1 ml of 0.2 M TEMP and 0.1 ml of 10 M AF were lyophilized separately in order to avoid the decomposition of AF or TEMP by the concentrated alkali salts. Then the former residue was dissolved in 1 ml of deuterium oxide (D20) to make a 1/15 M phosphate buffer (pH 9.2), and the latter residue was dissolved in it. Irradiation of Samples Samples were irradiated with an ultraviolet (UV) lamp (Toshiba SHL-100 UV-2 type 100 W) from 3 cm distance. Measurement of ESR Spectra Spectra were recorded on a JEOL JES-3BS X spectrometer (X band) with 100 kHz field modulation (0.8 G). Relative intensity of 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TEMPO) signals was expressed as the mean + S.D. (mm) of the three peak-to-peak heights as described in the previous report') Photodynamic Action of Acridine Compounds on Yeast A haploid yeast, Saccharomyces cerevisiae DP1 1B/517 (a, his,, trp,, p+ , w+ , CR), was grown for 18 h in YPD medium at 30 •Ž with shaking. Cells were washed with 1/15 M phosphate buffer (pH 7.0), sonicated to separate clumping cells and resuspended at a cell density of 108/m1 in the buffer. Cells (l06/ml) were incubated with 1-100 /LM acridine compounds at 30 •Ž for 60 min in the dark. Aliquots of sensitized cell suspension were taken into Thunberg tubes and irradiated with fluorescent lamps (National 15 W x 4, 7 cm distance), while the remaining cell suspensions were kept in the dark as a control. Cells sensitized with AF, PF, AY, DAEA or AO were irradiated for 30 min, and those with A, Q or AA for 60 min. Cell suspensions, irradiated or unirradiated, were spread on plates of YPD medium after suitable dilution. After 3 d of incubation at

30 •Ž in the dark, the frequencies (%) of petite colonies were determined by the tetrazolium-overlay method') as described in the previous report') Survivors were calculated from the number of colonies relative to the dark control. Chemicals Acriflavine was purchased from Aldrich. It is a 70 : 30 mixture of 3,6-diamino-10- methylacridinium chloride (euflavine) and 3,6-diaminoacridine (proflavine). Acridine orange and deuterium oxide were purchased from Merck, acridine hydrochloride and acridine yellow from Wako, quinacrine dihydrochloride and 9-aminoacridine hydrochloride from Nakarai, 6,9-diamino-2-ethoxyacridine lactate from Sigma, proflavine hemi- sulfate from Tokyo Kasei and 2,2,6,6-tetramethyl-4-piperidone from Aldrich. Authentic TEMPO was synthesized as described elsewhere2)

Results

TEMPO Production in AF Solution by Photo-Irradiation and Inhibition by the Addition of NaN3

In order to confirm that the TEMPO production specifically depends on singlet oxygen,

TEMPO production was compared under aerobic and anoxic conditions and in the presence

of NaN3. Each sample was taken into a capillary tube as described in Materials and Methods.

The ESR spectrum of each sample was measured after photo-irradiation as described in the

Fig. 1. Time Course of TEMPO Production by

Photo-Sensitization of Acriflavine

Standard, D20, anoxic and NaN3 samples were

prepared as described in Materials and Methods. Each capillary was irradiated with a UV lamp from 3 cm distance. The ESR results are expressed as the mean •} S.D. (mm) of the peak-to-peak height. Stan- dard sample, •›-•›; D20 sample, •œ-•œ, anoxic sample, •¢-•¢; NaN3 sample, •£•£. 2480 Vol. 35 (1987) previous report') Typical triplet signals of nitroxide radical (TEMPO) were detected in the irradiated aerobic sample. Relative intensity of TEMPO signals increased with increasing irradiation time but sharply decreased after 8 min, as shown in Fig. 1. However, TEMPO signals were not detected in the anoxic sample. Further, 50 mm NaN3 completely inhibited the TEMPO production. Enhancement of TEMPO Production in D2O TEMPO production by the photodynamic action of AF was determined in D20 as described in Materials and Methods. The intensity of signals of TEMPO radical was higher and more long-lived in D2O than in H20 as shown in Fig. 1. These results clearly indicated that TEMPO production is closely related to singlet oxygen production. Comparison of the TEMPO Production Among Acridine Compounds Time courses of the TEMPO production by 8 acridine compounds (Fig. 2) were compared. As shown in Fig. 3, all acridine compounds used here induced TEMPO production by photo-activation. AF and PF produced the most intense and long-lived ESR signals. The

proflavine acridine yellow 6,9-diamino-2-ethoxyacridine euflavine

acridine orange acridine quinacrine 9-aminoacridine

Fig. 2. Chemical Structures of the Acridine Compounds

Fig. 3. Time Course of TEMPO Production by Photo-Sensitization of Acridine Compounds

Samples containing 2 x 10-2 M TEMP and 10 M acridine compound in 1/15 M phosphate buffer (pH 9.2) were irradiated with a UV lamp. Acriflavine (AF), •›-•›; proflavine (PF), •œ-•œ; acridine yellow (AY), •¢-•¢; 6,9-diamino-2-ethoxyacridine (DAEA), A-A; acridine orange (AO), V-V; acridine (A), V-V; quinacrine (Q), 0-0; 9- aminoacridine (AA), No. 6 2481

Fig. 4. Cell Inactivation by Photodynamic Ac- Fig. 5. Petite Induction by Photodynamic Ac- tion of Acridine Compounds at Various Con- tion of Acridine Compounds at Various Con- centrations centrations Yeast cells were incubated with acridine com- Yeast cells were treated as described in the legend pounds in the dark and irradiated under fluorescent to Fig. 4. The frequencies () of petite colonies were lamps. The values of cell survival (") were calculated determined by the tetrazolium overlay method as from the number of colonies relative to that of the described in Materials and Methods. Symbols are the dark control as described in Materials and Methods. same as in Fig. 3. Symbols are the same as in Fig. 3.

ESR signals produced by AO or AA were of average intensity, but were long-lived. Cell Inactivation by Photodynamic Action of Acridine Compounds As shown in Fig. 4, all acridine compounds induced photodynamic cell inactivation more or less. However, they could be divided into two groups in terms of their efficacy in cell inactivation. AF, PF, AY, DAEA and AO induced extensive cell inactivation, which depended on dye concentration, within 30 min of photo-irradiation. However, the activities of A, Q and AA were very low and were detected only at high concentration (100 ,um) and after prolonged irradiation (60 min). The cell inactivation activities of 8 acridines were ranked as follows; AO > PF > AF = AY > DAEA »A = Q = AA. Cell inactivation was not observed in any dark control. Petite Induction by Photodynamic Action of Acridine Compounds The difference between active acridines and inactive ones was clear-cut, as shown in Fig. 5. AF, PF, AY and DAEA induced petites remarkably after 30 min of photo-irradiation, while, A, Q, and AA did not even after 60 min of irradiation. Petite induction by PF was maximum at 1 pM and decreased with increasing concentration. Such a decrease of the induction rate at 10 ,um was also observed in AF or DAEA photo-sensitization. Only the petite induction by AY was higher at 10 ,um than at 1 pM. At high concentration, the dye molecules present in the solution, cytoplasm or mitochondrial matrix, or bound to membranes, may absorb light, so that less energy is available to activate the dye molecules.21,5) None of the acridine compounds induced detectable petites without irradiation under such resting conditions.

Discussion The ESR method for the detection of singlet oxygen is based on the assumption that 2482 Vol. 35 (1987)

TEMP is specifically oxidized by singlet oxygen to TEMPO, which is a stable nitroxide radical whose characteristic signals are detectable by ESR measurement. In this work, TEMPO production was confirmed to be an adequate ESR spectroscopic

indicator of singlet oxygen production. Namely, TEMPO production was completely inhibited in the presence of NaN3, which is an efficient quencher of singlet oxygen (Fig. 1).8)

Furthermore, TEMPO production was remarkably enhanced in D2O (Fig. 1), in which the half-life of singlet oxygen is extended.9) The signal intensity of TEMPO is not necessarily proportional to the amount of singlet oxygen. However, if singlet oxygen is not produced, TEMPO will not be produced under our experimental conditions. The greater the amount of singlet oxygen produced, the greater the

amount of TEMPO, and larger TEMPO signals should be produced. Therefore we may be able to judge the singlet oxygen-producing activity from the signal intensity of TEMPO, at least semi-quantitatively. Using this method, the singlet oxygen-producing activities of 8 acridine compounds were compared (Fig. 3). All the acridine compounds were proved to have singlet oxygen-producing ability AF and PF, which are the most potent photo-sensitizers in both cell inactivation and

petite induction (Figs. 4 and 5) produced the most intense TEMPO signals. A and Q, which had been believed to be ineffective in terms of cell-inactivating activity,5) produced as much singlet oxygen as AY and DAEA, which effectively induce both cell inactivation and petite mutation. Reexamination of the photodynamic cell-inactivating activities of A, Q and AA

(Figs. 4 and 5) revealed that those have the ability to inactivate yeast cells, but only at high concentration (100 ƒÊM) and after prolonged irradiation (60 min). These results suggest that all acridine compounds used here have similar abilities to produce singlet oxygen but the distribution of each sensitizer in the cell and the interaction with each target may be different and be important determinants of the photodynamic biological activities of each photo-sensitizer. AF, PF, DAEA and AY, which have amino substituents at position 3 and/or 6, may be efficiently incorporated by yeast cells and intercalated into mitochondrial deoxyribonucleic acid (DNA). Photo-irradiation may activate these dyes and produce singlet oxygens which inactivate yeast cells and induce petite mutation. On the other hand A, Q and AA, which do not have an amino substituent at position 3 or 6, may not interact with cell components or be incorporated into mitochondrial DNA efficiently. AO which has dimethylamino substituents at the 3 and 6 positions is a well known photosensitizer1a,8b,10) and was reported to be an effective dye for phototherapy for cancer.11) In this work it inactivated yeast cells effectively (Fig. 4) but did not induce petite mutations

(Fig. 5). The TEMPO-producing activity of AO was average, but the life time of TEMPO was long. These results suggested that AO is a potent singlet oxygen-producer and interacts with cell components, but is not accumulated in yeast mitochondria. We may be able to clarify the drug distribution within the cell on the basis of the specific biological effects of each photo- sensitizer.

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