International Journal of Radiation Biology
ISSN: 0955-3002 (Print) 1362-3095 (Online) Journal homepage: http://www.tandfonline.com/loi/irab20
DNA Damage Induced by Hypocrellin-A Photosensitization Eh-Hua Cao, Shu-Min Xin & Long-Sheng Cheng To cite this article: Eh-Hua Cao, Shu-Min Xin & Long-Sheng Cheng (1992) DNA Damage Induced by Hypocrellin-A Photosensitization, International Journal of Radiation Biology, 61:2, 213-219, DOI: 10.1080/09553009214550841 To link to this article: http://dx.doi.org/10.1080/09553009214550841
Published online: 03 Jul 2009.
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Date: 16 March 2016, At: 05:01
INT . J . RADIAT . BIOL .,
1992,
VOL.
61,
NO .
2, 2 1 3-219
DNA damage induced by hypocrellin-A photosensitization EH-HUA CAO*t, SHU-MIN XINt and LONG-SHENG CHENGt (Received 7 November 1991 ; second revision received 11 June 1991 ; accepted 24 June 1991)
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Abstract. Hypocrellin-A (HC-A) isolated from Hypocrellia bambusae Sacc., is a new and effective photosensitizer . Illumination of sarcoma 180 cells with visible light in the presence of HC-A leads to a decrease in cell viability and 3 H-TdR incorporation, causes DNA strand breakage, and results in the selective destruction of guanine moieties in DNA . HC-A photosensitization causes an increase in the 0260/0280 ratio in the circular dichroism spectra of DNA in vitro . Of the four usual 2'-deoxynucleotides illuminated in the presence of HC-A only 2'-deoxyguanylic acid was destroyed .
1. Introduction Hypocrella bambusae Sacc . is used as a traditional medicine in China . Its major effective constituent is hypocrellin-A (HC-A, Figure 1), a derivative of 3,10-dihydroxy-4,9-perylenequinone, the molecular formula being C30H25010 (Chen et al. 1981) . HC-A can be metabolized rapidly in vivo (Fu et al . 1988) . So we presume that HC-A should have no long term damaging effects and thus be harmless after suitable administration . It is used successfully as a new drug in clinical phototherapy for the treatment of skin diseases, in particular keloid and white lesions of the vulva (Wan and Chen 1980) . Photosensitization of HC-A was shown to be lethal to sarcoma 180 (S 180) cells growing in mice (Dong et al. 1987) . HC-A is an effective photosensitizer (Wan and Chen 1980, An et al. 1985, Ma et al. 1989) . It can produce '0 2 , 02 , HC-A-, H202 and even abstract hydrogen to form free radicals, under some conditions active oxygen formation may involve unstable non-oxygen free radials (Diwu 1988, Zhang et al. 1989) . Some investigations have focused on membrane damage (Zheng and Cheng 1986, Cheng et al . 1988, 1989) . There is evidence suggesting that DNA as well as protein is involved in the effect of the drug on cells . HC-A photosensitization induced 0 . 47 times as many strand breaks as were produced by y rays at the D 10 dose in HeLa cells (Cao and Cheng 1987, 1988) . In order to further investigate the mechanisms of HC-A photosensitization on biological sys*Author for correspondence . t Institute of Biophysics, Academia Sinica, Beijing, 100080, People's Republic of China .
tems, some of its effects on DNA in cells, on DNA isolated from cells and on nucleotides are described in this paper . 2. Materials and methods 2.1 .
HC-A and other chemicals
HC-A was obtained from the Institute of Microbiology, Yunan Province, People's Republic of China . It was isolated from the crude product of Hypocrella bambusae Sacc. using thin layer chromatography separation followed by acetone purification . Deoxyribonucleotides including 2'-deoxyguanylic-
0 200
nm
Figure 1 . Absorption spectra of HC-A (5 x 10 -s M) . HC-A was illuminated for ( ) 0min ; ( ) 5 min; (----) 10 min ; (- - - •- •) 20 min; (- - - ) 30 min and • ( ---) - 60 min .
0020-7616/92 $3 .00 © 1992 Taylor & Francis Ltd
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En-hua Cao et al .
acid (dGMP) 2'-deosyadenylic acid (dAMP), 2'deoxycytidylic acid (dCMP) and 2'deosythymidylic-acid (dTMP) were purchased from Boehringer Co. Dimethyl sulphoxide (Aldrich Chem . Co .) was used as solvent to prepare 2 x 10 -3 M HC-A solution (dimethyl sulphoxide : water= 1 :1) . S 180 cell DNA was prepared from S180 cells by extraction with phenol according to the methods described by Marmur (1961), the isolated DNA was treated with proteinase K and RNAase, the content of protein was < 1 % (Lowry et al. 1951) . A stock solution of DNA was prepared in 0 . 15 M sodium chloride and 0. 15 M sodium citrate/10(SSC/10) .
even more effective-thus a high pressure sodium lamp is a convenient light source) was focused on the sample by a convex lens with an irradiation intensity of 1300 mW/cm 2 measured by a BTY-8204 photometer (from the Beijing Institute of Solar Energy) . A cell viability of 0 . 1 (D 10) was obtained with an irradiation time of 90 s . When a cell suspension was irradiation with cobalt-60 y rays the D10 absorbed dose was 4 .8 Gy (the dose rate was 3 .2 Gy/min) . The irradiation time was thus 90 s, the same as for illumination by visible light . The illumination unit was placed in a dark room . For irradiation of DNA solutions the temperature was maintained at 25°C for 30 min . DNA was then precipitated with ethanol, the precipitate was extensively washed with ethanol and the DNA redissolved in SSC/ 10 .
2.2. Cell cultures and analyses of single strand break (SSB) and base composition sodium Downloaded by [] at 05:01 16 March 2016
2 .4 .
S180 tumour cells were prepared as previously described (Cao and Chang 1988) . A suspension of S180 cells (10 6 cells/ml phosphate buffered saline (PBS)) was divided into two parts : one underwent no treatment and served as a control, the other was treated with HC-A (final concentration 5 x 10 -5 M) and incubated at 37°C for 2 h . SSB in DNA of S180 cells was determined by hydroxylapatite batch assay (DNA grade, Bio-Gel HTP, Bio Rad) as previously described (Kanter and Schwartz 1979, Cao and Cheng 1988) . The trypan blue exclusion test for cell viability was carried out according to the method of Phillips (1973) . Clonogenic efficiency was not measured since S 180 cells were anchorage- independent . Determination of 3H-TdR incorporation was carried out as described previously (Dong et al. 1987), cells were incubated for 2 h in DMEN (Dulbecco's modification of Eagle's medium) containing 15% bovine serum and 5 x 10 -5 M of HC-A, then irradiated with visible light . Cells were incubated for 4 h after the addition of 3 H-TdR (7 .4 x 104 Bq/ml) . Base composition analyses on formic acid hydrolysates (88%, at 175°C for 30 min) were determined by paper chromatography using an isopropanol : water : hydrochloric acid mixture as the solvent and eluted as described previously (Wyatt and Cohen 1953) . 2 .3 . Illumination procedure The cell suspension with or without HC-A were illuminated in an ice bath with a 500 W xenon lamp, equipped with a water filter to remove IR and UV light . The light (300-700 nm-for clinical use wavelengths of 490-580 nm are employed and maybe
Physico-chemical analyses
The circular dichroism (CD) spectra were described previously (Maiti and Nandi 1987) . The UV absorbtion spectra were measured with a Hitachi U-3200 spectrophometer, the fluorescence spectra were determined with a Hitachi 850 fluorescence spectrophotometer . 2.5 . Active oxygen quenching Photosensitized oxidations of dGMP were carried out in SSC/10, pH 7 .0. The concentrations of the active oxygen quenchers sodium azide, 1,4,diazabicyclo(2,2,2)octane (DABCO), sodium formate and superoxide dismutase (SOD) were 40 mm, 1 .0 mM, 100 mm and 2400U, respectively . Solutions were stirred for 5 min and irradiated in a darkened room, then the absorbance at 260 nm was measured . 3 . Results 3.1 . Photolysis of HC-A HC-A has a maximal absorbance in SSC/ 10 solution at 475 nm with two other small bands at 545 and 589 nm in the visible region, and an absorption band at 270 nm in the UV region . The absorbance in the visible region decreased with increasing illumination time. In the UV region (Figure 1) the absorbance increased with a red shift . HC-A has fluorescence emission at 605 nm when the excitation wavelength is 465 nm . The fluorescence spectrum of HC-A in the presence of DNA showed no changes in peak position, but its fluorescence intensity only
Photosensitization of DNA
increased slightly and this was not dependent on DNA concentration .
3 .2 . Inhibition of cell growth
Figure 2 shows viability curves for cells exposed to light and HC-A or to y rays . According to these curves, light exposure of 90 s, and a y ray dose of 4 . 8 Gy both correspond to a reduction in the surviving fraction from 1 to 0 . 1 . When S180 cells were exposed to HC-A followed by illumination, a characteristic dose-dependent inhibition of 3 H-TdR incorporation was observed (Figure 2) .
21 5
marked difference between control cells and cells treated by HC-A plus light : 52 . 4±3 . 3% of dsDNA (in air), 43 . 7±4. 7% (in oxygen) and 73 . 2+2 . 8% (in nitrogen) . SSBs induced by HC-A plus light increased with an increase in illumination time . The number of SSBs was 2 . 3 x 10` 0 breaks per Da DNA when survival was 10% . The SSB induced by HC-A plus light was about half that produced by y irradiation when the two methods of treatment were compared at the same survival level (Figure 3) .
3 .4 . Selective destruction of guanine residues in DNA S 180 cells were irradiated by visible light for 30 min in ice in the presence of HC-A . Then DNA
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3 .3 . SSBs in DNA of cells
The percentage of double strand DNA (dsDNA) was 85 . 6+3-9% for control cells, 80-5+5-3% for cells treated by HC-A without light, and 83-8+4-6% for cells exposed to light for 10 min . Thus no significant differences were observed from untreated control cells . However, there was a
r-Ray dose 0
rad 600
300
k~
=
100
1
3 .5 . Changes of DNA structure
Light treatment of DNA solution containing HC-A resulted in a shift of an absorption maximum
D10
-
was isolated from cells and a determination of base composition was carried out . HC-A photosensitization resulted in the selective destruction of guanine residues in DNA . This was four to seven times higher than for adenine, thymine and cytosine (Figure 4) . This effect which was dependent upon HC-A concentration was not seen when DNA was incubated with HC-A in the dark or when it was illuminated without HC-A .
may\
-r-Ray
Di0
10
a
H C _A+Light
z
0 v 0 O
100
C
0
v >`
g
3
o
a
H-Td R
5
CD H N
0 V C
Gamma Ray
j -
s -- HC-A+L1ght
-41
20
B 0
05
1
2
min
Light exposure time
Figure 2 . Viability and H-TdR incorporation of cells incubated for 2 h with HC-A (5 x 10 M) . A . Viability exposed to (----) light alone; ( •- ---) y rays irradiation alone; (--- .-) light and HC-A . B. H-TdR incorporation . The data points represent the mean of three or more separate experiments .
-5
3
1 0
15
Dose /D10 Figure 3 . Comparison of efficiency of y rays and HC-A plus light in producing DNA SSBs . The scales on the abscissa are adjusted according to the viability curves; i .e . a light exposure of 90 s and a y ray dose of 4 . 8 Gy both correspond to one irradiation dose required to reduce the viable fraction from 1 .0 to 0 . 1 Dlo for the two modes of treatment . The data points represent the mean of three or more separate experiments .
216
En-hua Cao
et al . the shape of the positive band of light-actived DNA with a blue shift of the maximum . It can be seen that the shape of the CD spectra at pH 3-0 changes significantly with various treatment . The ellipticity ratios of 0 260 /8280 was 0-78 for unilluminated DNA, but 0-80 and 0-84 for illuminated DNA for 30 min and 60 min, respectively . The shape of the CD spectrum of each DNA at pH 3-0 remained unaltered for an observation period of 1 h, which indicates clearly that the slow depurination of DNA at pH 3-0 does not significatly affect the observed ellipticity values . Therefore, the increase in the ratios of 0260/0280 represents the loss of guanine-cytosine content of DNA (Maiti and Nandi 1987) . These physio-chemical effects were both a function of the duration of illumination and of HC-A concentration .
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HC-A ug/ml
3 .6 . Deoxyribonucleotide changes analysis
Figure 4 . Base composition of DNA from S180 cells illuminated in the presence of HC-A . S180 cells were illuminated for 30min (450Jcm -2 ) with 7-5, 15, 30 and 60 µg mg -1 of HC-A . The concentration of DNA isolated from S 180 cells was 0 . 6 mg ml -1 . The data points represent the mean of three or more separate experiments.
from 257-2 to 258-5 nm . The CD spectrum of illuminated DNA in the UV region indicated that the 0 value at 275 nm and -0 value at 220-250 nm was decreased by increasing the illumination time in SSC/10, pH 7.0 (Figure 5) . When compared to unirradiated DNA some changes can be noticed in
Exposure of the four usual 2'-deoxynuclectides to HC-A in the presence of light revealed that only dGMP was modified . This effect depended upon the duration of the illumination (Figure 6) . Illumination led to the destruction of dGMP only in the presence of HC-A and oxygen . Photosensitization of dGMP was suppressed in the presence of nitrogen, however significantly increased in D2 0The addition of the '0 2 quencher DABCO (Foote 1979), the -OH scavenger sodium formate and the 02 scavenger SOD could partly eliminate the HCA-photosensitized photooxidation (Figure 7) . Sodium azide could partly inhibit the photooxidation of dGMP at the low concentration (10-40 mm) and completely at concentrations higher than 80 mm .
4
4 . DiScnssion 2 TO P_
0
_2
-6
v 220 250 280
A
B 310
220 250 280
310
nm Figure 5 . CD spectra of DNA (100µgm1 -1 ) . A . In citratephosphate buffer, pH 7 .0 . B . pH 3 .0 . (-) Unilluminated DNA ; ( ) DNA illuminated for 60 min ; (----) and DNA illuminated for 30 and 60min in the presence of HC-A(5x10 -5 M), respectively.
HC-A photosensitization was found to decrease the viability of 5180 cells and inhibit DNA synthesis . In previous work (Cao and Cheng 1988) we reported that the colony-forming ability of HeLa cells treated with HC-A plus light decreased . It suggested a possible role for DNA damage in cell killing . SSBs were observed in illuminated S 180 cells with HC-A. This is the result of a photochemical reaction, since no increase of SSBs appears if the samples are kept in the dark . This photosensitizing effect is highly oxygen dependent . But in nitrogen the observed SSB might involve non-oxygen free radicals . There are two kinds of sites in DNA where
217
Photosensitization of DNA
adenine, thymine and cytosine . However, the yield of damaged thymine is the highest in aqueous DNA solution following y ray irradiation. It is suggested that HC-A photosensitization resulted in the selective destruction of guanine residue in DNA . There might be cases where the altered base is so labile that it might be released from the DNA strand . Apurinic endonucleases, present in cells may subsequently lead to the generation of SSB in DNA . The observed spectral shifts of treated DNA are consistent with the destruction of the guanine residues in DNA (Gutter et al. 1977), which, of the four bases, has the lowest one-electron potential (Jovanvic and Simic 1986) . The increase of the 0260/0280 ratio seen in the CD spectra after illumination may reflect the decrease of guanine-cytosine content caused by the destruction of guanine residues in DNA (Maiti and Nandi 1987) . Thus, these physicochemical changes are also consistent with damage to guanine in the DNA . The degradation of DNA in vitro caused by HC-A plus light is similar to other photosensitizers (hematoporphyrin, methylene blue, acridine orange and riboflavine), which have been shown to also selecti-
1 .0
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0 .5
0
260
300
30
n m
Figure 6 . Difference absorption spectra of deoxyguanylic acid illuminated in the presence of HC-A. Deoxyguanylic acid(l x 10 -4 m) was illuminated for ( ) 0min ; ( ) 5 min; (---) 10 min ; (•-) 20 min ; (- - -) 30 min; (- - ) 60 min in the presence of HC-A (5 x 10 -5 m) .
radiation damage might manifest itself, the bases and the sugar moiety. Damage to the latter may result in strand breaks and alkali-labile sites which are connected into strand breaks when the damaged DNA is treated with alkali . It is possible that the observed SSBs might be correlated with this kind of damage . For the effect of HC-A plus light, indirect damage such as lipid oxidation and radical formation may give rise to the DNA strand breakage observed (Piette 1990) . In order to determine the chemical nature of the base damage, which might be different from those produced by ionizing radiation, we determined the types of base damage in DNA by chemical means . The above mentioned results showed that illumination of S180 cells with HC-A caused the degradation of the guanine moieties of DNA at a rate four to seven times higher than for
min
Time Figure 7 . Effect of quenchers on HC-A-photosensitized photodegradation of dGMP in SSC/10 buffer (a) without and with (f') HGA in air ; (f') HC-A in nitrogen ; (f3 ) He-A in D2 0 (HC-A=5 x 10 -5 m) ; (b) NaN3 ; (c) DABCO ; (d) SOD ; (e) sodium formate. A0 , absorbance at 260 nm before irradiation, A, absorbance at 260 nm after irradiation. The data points represent the mean of three or more separate experiments .
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218
En-hua Cao
vely destroy the guanine residues (Simon and Vunakis 1962, Sastry and Gordon 1966, Speck et al. 1975, Gutter et al. 1977) . In view of the photodamage mechanism involving guanine, we have determined the effects of HC-A photosensitization on deoxynucleotides . Out of the four usual 2'-deoxyribonucleotides illuminated in the presence of HC-A only dGMP was destroyed . We have observed that the destruction of dGMP only took place in the presence of HC-A plus light and oxygen, the effect was enhanced by D 2 0. Kinetic studies on the HC-A sensitized photooxidation of dGMP in SSC/ 10 neutral buffer in the presence of DABCO and NaN 3 showed that photooxidations were partly eliminated . The yield of singlet oxygen from HC-A is as high as 0=0 . 24 (An et al . 1985) . Results shown above suggest that singlet oxygen formed by energy transfer from excited triplet HC-A to ground state oxygen appears to be a major factor for the oxidation of guanine . On the other hand, a high concentration of NaN 3 also quenches the -OH free radical (Halliwell 1985) . Thus, the effects of sodium formate, SOD and NaN 3 may be related to other forms of active oxygen or free radicals (Zhang et al . 1989, Ma et al . 1989) . In the experiments, because HC-A was initially dissolved in DMSO, the concentration of DMSO in irradiated sample was about 0 . 15 mm . Thus, a small amount of -OH free radicals formed by HC-A photosensitization might be scavenged by DMSO in the reaction system .
Acknowledgement We thank the National Natural Science Foundation of China for support of this work .
References AN, J .-Y ., ZHANG, M .-H ., Wu, G .-Y ., Liu, J .-Y., MA, G .-Z . and JIANG, L . J ., 1985, Photochemistry of hypocrellin A . Scientia Sinica (Science in China), Series B, 975-982 . CAO, E .-H . and CHENG, L .-S ., 1987, Comparison of DNA single
strand breakages and repair in HeLa cells by treatment with hypocrellin-A and light or y-ray radiation research . Proceedings of the eighth International Congress of Radiation Research, 1, 177 . CAO, E .-H. and CHENG, L .-S .,
1988, DNA single-strand breakage and its rejoining in HeLa cells caused by hypocrellin A photosensitization . Acta Biologiae Experimentalis Sinica, 21, 79-85 .
et al . E ., PUFF, des hypocrellins and seines photooxidationsproduktes peroxyhypocrellin . Liebigs Annals Chemistry, 1880-1885 . CHENG, L .-S ., WANG, J . Z . and Fu, S . M ., 1988, Effect of hypocrellin A sensitization on the lateral mobility of cell membrane proteins . Journal of Photochemistry and Photobiology (B : Biology) 2, 385-398 . CHENG, L .-S ., TANG, X . and ZHENG, J.-H ., 1989, ESR study of hypocrellin A induced photodamage on erythrocyte membranes . Chinese Journal of Biochemistry Biophysics, 21, 129-135 . Diwu, Z . J ., 1988, Mechanism of hypocrellin A sensitized photooxidation. PhD dissertation . Institute of Photographic Chemistry. Beijing, People's Republic of China . DONG, C .-Y ., JAI, H .-T., ZHANG, L . and MA, C .-M ., 1987, The inhibitory effect of the new photosensitizer hypocrellin A on experimental tumors . Chinese Biochemistry Journal, 3, 468-472 . FOOTE, C . S ., 1979, Quenching of singlet oxygen . In Organic CHEN, W.-S., CHENG, T .-T., WAN, X .-Y., FRIEDRICHS, H . and BREEITMAIER, E ., 1981, Die struktur
Chemistry, A Series of Monographs, Vol 40, Singled Oxygen,
edited by H . H . Wasserman and R . W . Murray (New York : Academic Press), pp . 139-167 . Fu, N .-W ., CHU, Y .-X ., YAN, L .-X., Yu, B. F., TANG, J . and AN, J .-Y ., 1988, Tissue distribution in r-bearing rats and tumor uptake of Tc-hypocrellin A . Second Chinese National Symposium on Photobiology, pp 31-32 . GUTTER, B ., SPECK, W. and ROSENKRANZ, H ., 1977, Photodynamic modification of DNA by Hematoporphyrin. Biochimica et Biophysica Acta, 475, 307-314. HALLIWELL, B ., 1985, Free Radicals in Biology and Medicine (New York : Oxford University Press), p 50 . JOVANVIC, S . V . and SIMIC, M . G ., 1986, One-electron redox potentials of purines and pyrimidines . Journal of Physical Chemistry, 90(5), 974-978 . KANTER, P . M. and SCHWARTZ, H . S ., 1979, A hydroxylapatite batch assay for quantition of cellular DNA damage . Analytical Biochemistry, 97, 77-78 . LOWRY, O . H ., ROSENBROUGH, N . J ., FARR, A. L . and RANDALL, R . J ., 1951, Protein measurement the folin phenol reagent, Journal of Biological Chemistry, 193, 265-270 . MA, J .-S ., YAN, F ., WANG, C .-Q. and AN, J . Y ., 1989, Hypocrellin A sensitized photooxidation of bilirubin, Photochemisty and Photobiology, 50, 827-830 . MAITI, M ., and NANDI, R ., 1987, Spectropolarimetric determination of the guanine-cytosine content of DNA, Analytical Biochemistry, 164, 68-71 . MARMUR, J ., 1961, A procedure for the isolation of deoxyribonucleic acid from microorganisms . Journal of Molecular Biology, 3, 205-218 . PHILLIPS, H . J ., 1973, Dye exclusion testgs for cell viability . In Tissue Culture, Methods and Applications, edited by P. F. Kruse and M . K . Patterson (New York : Academic Press), pp 406-408 . PIETTE, J ., 1990 . Mutagenic and genotoxic properties of singlet oxygen, Journal of Photochemistry and Photobiology (B : Biology), 4, 335-342 . SASTRY, K . S . and GORDON, M . P ., 1966, The photosensitized degradation of guanosine by acridine orange . Biochimica et Biophysica Acta, 129, 42-48 . SIMON, M . I . and VUNAKIS, VAN, H ., 1962, The photodynamic reaction of methylene blue with deoxyribonucleic acid . Journal of Molecular Biology, 4, 430 443 . SPECK, W . T., CHEN, C . C . and ROSENKRANZ, H . S ., 1975, In vitro studies of effects of light and riboflavine on DNA and HeLa cells. Pediatric Research, 9, 150-153 .
Photosensitization of DNA WAN, X .-Y. and CHEN, Y .-T ., 1980, A new photochemothera-
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peutic agent-hypocrellin Kexue Tongbao (Science Communication) 24, 1148-1149 . WATT, G . R . and COHEN, S . S ., 1953, The bases of the nucleic acids of some bacterial and animal viruses the occurrence of 5-hydroxymethylcytosine, Biochemistry Journal, 55, 774-782 .
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ZANG, Z .-Y ., ZHANG, L .-Y., Xu, G .-Y ., TAO, N .-B . and WANG, D.-H ., 1989, Characteristics of the pri reaction of hypocrellin A photosensitization Scientia Sinica (Science in China) Series B, 1063-1071 . ZHENG, J .-H . and CHENG, L .-S ., 1986, The mechanism of
hypocrellin A photosensitization in erythrocyte membranes. Acta Biophysics Sinica, 2, 312-318 .
ISSN: 0955-3002 (Print) 1362-3095 (Online) Journal homepage: http://www.tandfonline.com/loi/irab20
DNA Damage Induced by Hypocrellin-A Photosensitization Eh-Hua Cao, Shu-Min Xin & Long-Sheng Cheng To cite this article: Eh-Hua Cao, Shu-Min Xin & Long-Sheng Cheng (1992) DNA Damage Induced by Hypocrellin-A Photosensitization, International Journal of Radiation Biology, 61:2, 213-219, DOI: 10.1080/09553009214550841 To link to this article: http://dx.doi.org/10.1080/09553009214550841
Published online: 03 Jul 2009.
Submit your article to this journal
Article views: 3
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=irab20 Download by: [137.189.171.235]
Date: 16 March 2016, At: 05:01
INT . J . RADIAT . BIOL .,
1992,
VOL.
61,
NO .
2, 2 1 3-219
DNA damage induced by hypocrellin-A photosensitization EH-HUA CAO*t, SHU-MIN XINt and LONG-SHENG CHENGt (Received 7 November 1991 ; second revision received 11 June 1991 ; accepted 24 June 1991)
Downloaded by [] at 05:01 16 March 2016
Abstract. Hypocrellin-A (HC-A) isolated from Hypocrellia bambusae Sacc., is a new and effective photosensitizer . Illumination of sarcoma 180 cells with visible light in the presence of HC-A leads to a decrease in cell viability and 3 H-TdR incorporation, causes DNA strand breakage, and results in the selective destruction of guanine moieties in DNA . HC-A photosensitization causes an increase in the 0260/0280 ratio in the circular dichroism spectra of DNA in vitro . Of the four usual 2'-deoxynucleotides illuminated in the presence of HC-A only 2'-deoxyguanylic acid was destroyed .
1. Introduction Hypocrella bambusae Sacc . is used as a traditional medicine in China . Its major effective constituent is hypocrellin-A (HC-A, Figure 1), a derivative of 3,10-dihydroxy-4,9-perylenequinone, the molecular formula being C30H25010 (Chen et al. 1981) . HC-A can be metabolized rapidly in vivo (Fu et al . 1988) . So we presume that HC-A should have no long term damaging effects and thus be harmless after suitable administration . It is used successfully as a new drug in clinical phototherapy for the treatment of skin diseases, in particular keloid and white lesions of the vulva (Wan and Chen 1980) . Photosensitization of HC-A was shown to be lethal to sarcoma 180 (S 180) cells growing in mice (Dong et al. 1987) . HC-A is an effective photosensitizer (Wan and Chen 1980, An et al. 1985, Ma et al. 1989) . It can produce '0 2 , 02 , HC-A-, H202 and even abstract hydrogen to form free radicals, under some conditions active oxygen formation may involve unstable non-oxygen free radials (Diwu 1988, Zhang et al. 1989) . Some investigations have focused on membrane damage (Zheng and Cheng 1986, Cheng et al . 1988, 1989) . There is evidence suggesting that DNA as well as protein is involved in the effect of the drug on cells . HC-A photosensitization induced 0 . 47 times as many strand breaks as were produced by y rays at the D 10 dose in HeLa cells (Cao and Cheng 1987, 1988) . In order to further investigate the mechanisms of HC-A photosensitization on biological sys*Author for correspondence . t Institute of Biophysics, Academia Sinica, Beijing, 100080, People's Republic of China .
tems, some of its effects on DNA in cells, on DNA isolated from cells and on nucleotides are described in this paper . 2. Materials and methods 2.1 .
HC-A and other chemicals
HC-A was obtained from the Institute of Microbiology, Yunan Province, People's Republic of China . It was isolated from the crude product of Hypocrella bambusae Sacc. using thin layer chromatography separation followed by acetone purification . Deoxyribonucleotides including 2'-deoxyguanylic-
0 200
nm
Figure 1 . Absorption spectra of HC-A (5 x 10 -s M) . HC-A was illuminated for ( ) 0min ; ( ) 5 min; (----) 10 min ; (- - - •- •) 20 min; (- - - ) 30 min and • ( ---) - 60 min .
0020-7616/92 $3 .00 © 1992 Taylor & Francis Ltd
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En-hua Cao et al .
acid (dGMP) 2'-deosyadenylic acid (dAMP), 2'deoxycytidylic acid (dCMP) and 2'deosythymidylic-acid (dTMP) were purchased from Boehringer Co. Dimethyl sulphoxide (Aldrich Chem . Co .) was used as solvent to prepare 2 x 10 -3 M HC-A solution (dimethyl sulphoxide : water= 1 :1) . S 180 cell DNA was prepared from S180 cells by extraction with phenol according to the methods described by Marmur (1961), the isolated DNA was treated with proteinase K and RNAase, the content of protein was < 1 % (Lowry et al. 1951) . A stock solution of DNA was prepared in 0 . 15 M sodium chloride and 0. 15 M sodium citrate/10(SSC/10) .
even more effective-thus a high pressure sodium lamp is a convenient light source) was focused on the sample by a convex lens with an irradiation intensity of 1300 mW/cm 2 measured by a BTY-8204 photometer (from the Beijing Institute of Solar Energy) . A cell viability of 0 . 1 (D 10) was obtained with an irradiation time of 90 s . When a cell suspension was irradiation with cobalt-60 y rays the D10 absorbed dose was 4 .8 Gy (the dose rate was 3 .2 Gy/min) . The irradiation time was thus 90 s, the same as for illumination by visible light . The illumination unit was placed in a dark room . For irradiation of DNA solutions the temperature was maintained at 25°C for 30 min . DNA was then precipitated with ethanol, the precipitate was extensively washed with ethanol and the DNA redissolved in SSC/ 10 .
2.2. Cell cultures and analyses of single strand break (SSB) and base composition sodium Downloaded by [] at 05:01 16 March 2016
2 .4 .
S180 tumour cells were prepared as previously described (Cao and Chang 1988) . A suspension of S180 cells (10 6 cells/ml phosphate buffered saline (PBS)) was divided into two parts : one underwent no treatment and served as a control, the other was treated with HC-A (final concentration 5 x 10 -5 M) and incubated at 37°C for 2 h . SSB in DNA of S180 cells was determined by hydroxylapatite batch assay (DNA grade, Bio-Gel HTP, Bio Rad) as previously described (Kanter and Schwartz 1979, Cao and Cheng 1988) . The trypan blue exclusion test for cell viability was carried out according to the method of Phillips (1973) . Clonogenic efficiency was not measured since S 180 cells were anchorage- independent . Determination of 3H-TdR incorporation was carried out as described previously (Dong et al. 1987), cells were incubated for 2 h in DMEN (Dulbecco's modification of Eagle's medium) containing 15% bovine serum and 5 x 10 -5 M of HC-A, then irradiated with visible light . Cells were incubated for 4 h after the addition of 3 H-TdR (7 .4 x 104 Bq/ml) . Base composition analyses on formic acid hydrolysates (88%, at 175°C for 30 min) were determined by paper chromatography using an isopropanol : water : hydrochloric acid mixture as the solvent and eluted as described previously (Wyatt and Cohen 1953) . 2 .3 . Illumination procedure The cell suspension with or without HC-A were illuminated in an ice bath with a 500 W xenon lamp, equipped with a water filter to remove IR and UV light . The light (300-700 nm-for clinical use wavelengths of 490-580 nm are employed and maybe
Physico-chemical analyses
The circular dichroism (CD) spectra were described previously (Maiti and Nandi 1987) . The UV absorbtion spectra were measured with a Hitachi U-3200 spectrophometer, the fluorescence spectra were determined with a Hitachi 850 fluorescence spectrophotometer . 2.5 . Active oxygen quenching Photosensitized oxidations of dGMP were carried out in SSC/10, pH 7 .0. The concentrations of the active oxygen quenchers sodium azide, 1,4,diazabicyclo(2,2,2)octane (DABCO), sodium formate and superoxide dismutase (SOD) were 40 mm, 1 .0 mM, 100 mm and 2400U, respectively . Solutions were stirred for 5 min and irradiated in a darkened room, then the absorbance at 260 nm was measured . 3 . Results 3.1 . Photolysis of HC-A HC-A has a maximal absorbance in SSC/ 10 solution at 475 nm with two other small bands at 545 and 589 nm in the visible region, and an absorption band at 270 nm in the UV region . The absorbance in the visible region decreased with increasing illumination time. In the UV region (Figure 1) the absorbance increased with a red shift . HC-A has fluorescence emission at 605 nm when the excitation wavelength is 465 nm . The fluorescence spectrum of HC-A in the presence of DNA showed no changes in peak position, but its fluorescence intensity only
Photosensitization of DNA
increased slightly and this was not dependent on DNA concentration .
3 .2 . Inhibition of cell growth
Figure 2 shows viability curves for cells exposed to light and HC-A or to y rays . According to these curves, light exposure of 90 s, and a y ray dose of 4 . 8 Gy both correspond to a reduction in the surviving fraction from 1 to 0 . 1 . When S180 cells were exposed to HC-A followed by illumination, a characteristic dose-dependent inhibition of 3 H-TdR incorporation was observed (Figure 2) .
21 5
marked difference between control cells and cells treated by HC-A plus light : 52 . 4±3 . 3% of dsDNA (in air), 43 . 7±4. 7% (in oxygen) and 73 . 2+2 . 8% (in nitrogen) . SSBs induced by HC-A plus light increased with an increase in illumination time . The number of SSBs was 2 . 3 x 10` 0 breaks per Da DNA when survival was 10% . The SSB induced by HC-A plus light was about half that produced by y irradiation when the two methods of treatment were compared at the same survival level (Figure 3) .
3 .4 . Selective destruction of guanine residues in DNA S 180 cells were irradiated by visible light for 30 min in ice in the presence of HC-A . Then DNA
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3 .3 . SSBs in DNA of cells
The percentage of double strand DNA (dsDNA) was 85 . 6+3-9% for control cells, 80-5+5-3% for cells treated by HC-A without light, and 83-8+4-6% for cells exposed to light for 10 min . Thus no significant differences were observed from untreated control cells . However, there was a
r-Ray dose 0
rad 600
300
k~
=
100
1
3 .5 . Changes of DNA structure
Light treatment of DNA solution containing HC-A resulted in a shift of an absorption maximum
D10
-
was isolated from cells and a determination of base composition was carried out . HC-A photosensitization resulted in the selective destruction of guanine residues in DNA . This was four to seven times higher than for adenine, thymine and cytosine (Figure 4) . This effect which was dependent upon HC-A concentration was not seen when DNA was incubated with HC-A in the dark or when it was illuminated without HC-A .
may\
-r-Ray
Di0
10
a
H C _A+Light
z
0 v 0 O
100
C
0
v >`
g
3
o
a
H-Td R
5
CD H N
0 V C
Gamma Ray
j -
s -- HC-A+L1ght
-41
20
B 0
05
1
2
min
Light exposure time
Figure 2 . Viability and H-TdR incorporation of cells incubated for 2 h with HC-A (5 x 10 M) . A . Viability exposed to (----) light alone; ( •- ---) y rays irradiation alone; (--- .-) light and HC-A . B. H-TdR incorporation . The data points represent the mean of three or more separate experiments .
-5
3
1 0
15
Dose /D10 Figure 3 . Comparison of efficiency of y rays and HC-A plus light in producing DNA SSBs . The scales on the abscissa are adjusted according to the viability curves; i .e . a light exposure of 90 s and a y ray dose of 4 . 8 Gy both correspond to one irradiation dose required to reduce the viable fraction from 1 .0 to 0 . 1 Dlo for the two modes of treatment . The data points represent the mean of three or more separate experiments .
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En-hua Cao
et al . the shape of the positive band of light-actived DNA with a blue shift of the maximum . It can be seen that the shape of the CD spectra at pH 3-0 changes significantly with various treatment . The ellipticity ratios of 0 260 /8280 was 0-78 for unilluminated DNA, but 0-80 and 0-84 for illuminated DNA for 30 min and 60 min, respectively . The shape of the CD spectrum of each DNA at pH 3-0 remained unaltered for an observation period of 1 h, which indicates clearly that the slow depurination of DNA at pH 3-0 does not significatly affect the observed ellipticity values . Therefore, the increase in the ratios of 0260/0280 represents the loss of guanine-cytosine content of DNA (Maiti and Nandi 1987) . These physio-chemical effects were both a function of the duration of illumination and of HC-A concentration .
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HC-A ug/ml
3 .6 . Deoxyribonucleotide changes analysis
Figure 4 . Base composition of DNA from S180 cells illuminated in the presence of HC-A . S180 cells were illuminated for 30min (450Jcm -2 ) with 7-5, 15, 30 and 60 µg mg -1 of HC-A . The concentration of DNA isolated from S 180 cells was 0 . 6 mg ml -1 . The data points represent the mean of three or more separate experiments.
from 257-2 to 258-5 nm . The CD spectrum of illuminated DNA in the UV region indicated that the 0 value at 275 nm and -0 value at 220-250 nm was decreased by increasing the illumination time in SSC/10, pH 7.0 (Figure 5) . When compared to unirradiated DNA some changes can be noticed in
Exposure of the four usual 2'-deoxynuclectides to HC-A in the presence of light revealed that only dGMP was modified . This effect depended upon the duration of the illumination (Figure 6) . Illumination led to the destruction of dGMP only in the presence of HC-A and oxygen . Photosensitization of dGMP was suppressed in the presence of nitrogen, however significantly increased in D2 0The addition of the '0 2 quencher DABCO (Foote 1979), the -OH scavenger sodium formate and the 02 scavenger SOD could partly eliminate the HCA-photosensitized photooxidation (Figure 7) . Sodium azide could partly inhibit the photooxidation of dGMP at the low concentration (10-40 mm) and completely at concentrations higher than 80 mm .
4
4 . DiScnssion 2 TO P_
0
_2
-6
v 220 250 280
A
B 310
220 250 280
310
nm Figure 5 . CD spectra of DNA (100µgm1 -1 ) . A . In citratephosphate buffer, pH 7 .0 . B . pH 3 .0 . (-) Unilluminated DNA ; ( ) DNA illuminated for 60 min ; (----) and DNA illuminated for 30 and 60min in the presence of HC-A(5x10 -5 M), respectively.
HC-A photosensitization was found to decrease the viability of 5180 cells and inhibit DNA synthesis . In previous work (Cao and Cheng 1988) we reported that the colony-forming ability of HeLa cells treated with HC-A plus light decreased . It suggested a possible role for DNA damage in cell killing . SSBs were observed in illuminated S 180 cells with HC-A. This is the result of a photochemical reaction, since no increase of SSBs appears if the samples are kept in the dark . This photosensitizing effect is highly oxygen dependent . But in nitrogen the observed SSB might involve non-oxygen free radicals . There are two kinds of sites in DNA where
217
Photosensitization of DNA
adenine, thymine and cytosine . However, the yield of damaged thymine is the highest in aqueous DNA solution following y ray irradiation. It is suggested that HC-A photosensitization resulted in the selective destruction of guanine residue in DNA . There might be cases where the altered base is so labile that it might be released from the DNA strand . Apurinic endonucleases, present in cells may subsequently lead to the generation of SSB in DNA . The observed spectral shifts of treated DNA are consistent with the destruction of the guanine residues in DNA (Gutter et al. 1977), which, of the four bases, has the lowest one-electron potential (Jovanvic and Simic 1986) . The increase of the 0260/0280 ratio seen in the CD spectra after illumination may reflect the decrease of guanine-cytosine content caused by the destruction of guanine residues in DNA (Maiti and Nandi 1987) . Thus, these physicochemical changes are also consistent with damage to guanine in the DNA . The degradation of DNA in vitro caused by HC-A plus light is similar to other photosensitizers (hematoporphyrin, methylene blue, acridine orange and riboflavine), which have been shown to also selecti-
1 .0
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0 .5
0
260
300
30
n m
Figure 6 . Difference absorption spectra of deoxyguanylic acid illuminated in the presence of HC-A. Deoxyguanylic acid(l x 10 -4 m) was illuminated for ( ) 0min ; ( ) 5 min; (---) 10 min ; (•-) 20 min ; (- - -) 30 min; (- - ) 60 min in the presence of HC-A (5 x 10 -5 m) .
radiation damage might manifest itself, the bases and the sugar moiety. Damage to the latter may result in strand breaks and alkali-labile sites which are connected into strand breaks when the damaged DNA is treated with alkali . It is possible that the observed SSBs might be correlated with this kind of damage . For the effect of HC-A plus light, indirect damage such as lipid oxidation and radical formation may give rise to the DNA strand breakage observed (Piette 1990) . In order to determine the chemical nature of the base damage, which might be different from those produced by ionizing radiation, we determined the types of base damage in DNA by chemical means . The above mentioned results showed that illumination of S180 cells with HC-A caused the degradation of the guanine moieties of DNA at a rate four to seven times higher than for
min
Time Figure 7 . Effect of quenchers on HC-A-photosensitized photodegradation of dGMP in SSC/10 buffer (a) without and with (f') HGA in air ; (f') HC-A in nitrogen ; (f3 ) He-A in D2 0 (HC-A=5 x 10 -5 m) ; (b) NaN3 ; (c) DABCO ; (d) SOD ; (e) sodium formate. A0 , absorbance at 260 nm before irradiation, A, absorbance at 260 nm after irradiation. The data points represent the mean of three or more separate experiments .
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En-hua Cao
vely destroy the guanine residues (Simon and Vunakis 1962, Sastry and Gordon 1966, Speck et al. 1975, Gutter et al. 1977) . In view of the photodamage mechanism involving guanine, we have determined the effects of HC-A photosensitization on deoxynucleotides . Out of the four usual 2'-deoxyribonucleotides illuminated in the presence of HC-A only dGMP was destroyed . We have observed that the destruction of dGMP only took place in the presence of HC-A plus light and oxygen, the effect was enhanced by D 2 0. Kinetic studies on the HC-A sensitized photooxidation of dGMP in SSC/ 10 neutral buffer in the presence of DABCO and NaN 3 showed that photooxidations were partly eliminated . The yield of singlet oxygen from HC-A is as high as 0=0 . 24 (An et al . 1985) . Results shown above suggest that singlet oxygen formed by energy transfer from excited triplet HC-A to ground state oxygen appears to be a major factor for the oxidation of guanine . On the other hand, a high concentration of NaN 3 also quenches the -OH free radical (Halliwell 1985) . Thus, the effects of sodium formate, SOD and NaN 3 may be related to other forms of active oxygen or free radicals (Zhang et al . 1989, Ma et al . 1989) . In the experiments, because HC-A was initially dissolved in DMSO, the concentration of DMSO in irradiated sample was about 0 . 15 mm . Thus, a small amount of -OH free radicals formed by HC-A photosensitization might be scavenged by DMSO in the reaction system .
Acknowledgement We thank the National Natural Science Foundation of China for support of this work .
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