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Effect of Copper Phthalocyanine Derivatives on Sister Chromatid Exchanges in BrdU-substituted Chromosomes of Allium cepa
J.PlantPhysiol. Vol. 132.pp. 557-560(1988)
Effect of Copper Phthalocyanine Derivatives on Sister Chromatid Exchanges in BrdU-substituted Chromosomes of A Ilium cepa A. ]UARRANZ, M. J.
HAZEN,
and J. C.
STOCKERT
Departamento de Biologla, Facultad de Ciencias, Universidad Autonoma de Madrid, 28049-Madrid, Spain Received September 22, 1987 . Accepted September 29, 1987
Summary The effect of some copper phthalocyanine derivatives on sister chromatid exchanges (SCEs) in meristematic cells of Allium cepa roots was studied. A significant increase in SCEs frequencies was observed when BrdU-substituted chromosomes were subjected to the cationic phthalocyanines Alcian blue 8GS, Astra blue and Cuprolinic blue for 3 hours at the end of the G 1 period of the second division cycle. Among the anionic phthalocyanine compounds, only Luxol fast blue MBSN increases the SCEs over the control baseline.
Key words: Allium cepa chromosomes, meristematic cells, sister chromatid exchanges, phthalocyanine derivatives. Abbreviation list: BrdU, 5-bromo-2'-deoxyuridine; dT, unlabelled thymidine; FdU, 5-fluorodeoxyuridine; SCEs, sister chromatid exchanges; FPG, Fluorescence plus Giemsa; SE, standard error.
Introduction DNA damage processes in living cells can be detected, among other cytogenetic techniques, by using the sister chromatid exchanges (SCEs) test (Latt 1974, Perry and Evans 1975). SCEs represent the interchange of DNA replication products of apparently homologous loci, the exchange process probably involving breakage and reunion of DNA strands. It is known that certain physical and chemical agents, particularly DNA intercalating compounds (Kato 1973, Latt and Loveday 1978, Armas-Portela et al. 1985), influence the occurrence of SCEs (Raj and Heddle 1980, Latt et al. 1981). Copper phthalocyanines have been used for many years as histological stains due to their specificity for binding to certain tissue components (Gurr 1971, Lillie 1977). For example, Alcian blue 8GS and Astra blue are currently being applied for the histochemical demonstration of acid glycosaminoglycans (Bloom and Kelly 1960, Scott 1972, 1980); Luxol fast blue MBSN and Methazol fast blue 2G are used for the selective staining of myelin sheaths (Thompson 1966, Lillie 1977), and the phthalocyanine-like dye Cuprolinic blue has interesting applications in the detection of RNA and acid © 1988 by Gustav FlScher Verlag, Stuttgart
proteoglycans (Tas et al. 1983, Van Kuppevelt et al. 1984, Juarranz et al. 1986). Apart from the histochemical use of these compounds other phthalocyanine derivatives were found to be selectively retained in several kinds of tumors after treatments in vivo (Rousseau et al. 1983, 1985, Chan et al. 1986). Some of these agents have been reported as effective photosensitizers for cell killing and tissue damage (Ben-Hur and Rosenthal 1985 a, b, c, Chan et al. 1986) and they were proposed as substitutes for porphyrins in the photodynamic therapy of tumors (Spikes 1986). Since SCEs are very sensitive indicators of the effect of mutagenic and carcinogenic agents on eukaryotic chromosomes (Wolff 1977, Latt et al. 1979, Latt et al. 1981), we have studied the frequency of SCEs in plant chromosomes when subjected to some cationic and anionic copper phthalocyanine derivatives.
Material and methods Roots of Allium cepa bulbs were grown in tap water renewed every 24h and aerated continuously at a rate of 10-20cm3/min. Bulbs were maintained in the dark and at a constant temperature of
558
A. jUARRANZ, M. J.
HAZEN,
and J. C. STOCKERT
Table 1: Phthalocyanine and phthalocyanine-like derivatives used in this work [for the chemical structure of these dyes see Gurr (1971), Lillie (1977), Scott (1972, 1973, 1980)]. Chemical structure of the chromophore, substituents (R) and counterions (X). Phthalocyanine ring
C.I. 74.360 74.180 74.240
Cuprolinic blue
Dye
Source
R
Luxol fast blue MBSN Methazol fast blue 2G Sirius light turquoise blue GL Alcian blue 8GS Astra blue l ) Cuprolinic blue
(Serva) (Gurr) (Bayer) (Serva) (Merck) (BDH)
?R: 4R: 2R: 4R: 3R:
X
-SO) -S03-SO) -CH2SC[N(CH3)z]t -S02NH(CH2hNH(CH3)t
X X
=
X X X
=
X
= = = =
diarylguanidinium +
C6H13NHt
Na+ ClCH 3COOCH30SO)
I) Refered to as Astra blue 6GLL by Scott (1980).
25±0.5°C. In order to analyse the frequency of SCEs, roots were treated with 1O- 4 M 5-bromo, 2' -deoxyuridine (BrdU) and 1O- 7 M 5-fluor, 2'-deoxyuridine (FdU) for a complete cell division cycle (20 h), followed by another cycle in the presence of 10 - 4 M thymidine (dT). Finally, the roots were treated with 0.05 % colchicine for 3 h and fixed overnight in ethanol-acetic acid (3 : 1) at 4°C. The phthalocyanine derivatives used were Alcian blue 8GS, Astra blue, Luxol fast blue MBSN, Methazol fast blue 2G, Sirius light turquoise blue GL, and the phthalocyanine-like dye, Cuprolinic blue. The source and chemical characteristics of these compounds are given in Table 1. Solutions for treatments were prepared in tap water from a 11 mg% solution in distilled water, except for Luxol fast blue MBSN and Methazol fast blue 2G, which were prepared in 1 % ethanol or acetic acid respectively, as these two compounds hardly dissolve in water. The treatment with phthalocyanine solutions (1.1JLg/ml, 2.2JLg/ml and 5.5JLg/ml) was performed in the presence of dT for 3 h at the end of the G I period of the second division cycle. To visualize the occurrence of SCEs, the fluorescenceplus-Giemsa (FPG) chromosome technique was performed according to the modification of Hazen et al. (1985).
Results T able2 summarizes the frequencies of seEs obtained for all the experimental treatments. When the bromo-substitution of DNA was followed by a treatment with cationic phthalocyanine derivatives, the SeEs frequencies significantly increased (p < 0.01, Student's t test) over the control baseline levels (Fig. 1). The increase in the seEs rates in-
Table 2: Frequency of SCEs in controls and after treatments with several cationic and anionic phthalocyanine dyes. Treatments on BrdUsubstituted chromosomes
Dose (JLg/ml)
Control Cationic dyes Alcian blue 8GS
Astra blue Cuprolinic blue
Anionic dyes Luxol fast blue MBSN
Methazol fast blue 2G
2.79±0.19 1.1 2.2 5.5 1.1
5.5 1.1 2.2 5.5 1.1 2.2 5.5 1.1
2.2 5.5 Sirius light turquoise blue GL
a), b) SCEs/chromosome
1.1
2.2 5.5
3.87±0.20 4.70±0.20 5.02±0.20 5.55±0.20 5.81±0.20 3.88±0.15 4.67±O.29 5.84±O.20 2.89±0.21 2.93±O.20 4.20±O.20 3.12±O.21 3.05±0.20 3.30±0.20 2.93±O.19 2.94±O.19 2.95±O.19
a) mean ± S.E. b) 300 metaphase chromosomes were studied in each case.
Copper phthalocyanine derivatives effect on sister chromatids
559
It is well known that most intercalating agents introduce changes in the DNA structure, which could be expected to result in breaks and/or exchanges of DNA strands, thus being SCEs inductors (Wolff 1977, Kato 1977, Latt et al. 1981, Cortes and Hazen 1984, Villanueva et al. 1986). In this context, it seems that SCEs induced by cationic phthalocyanines could be related to a possible intercalative binding mode to DNA. Luxol fast blue MBSN was the only anionic derivative able to increase the SCEs frequency over the baseline level. In this respect it is tempting to assume that the diarylguanidinium counterion of this derivative could be responsible for the increase of SCEs, because all the anionic compounds used have essentially the same phthalocyanine ring system and substituents. Diarylguanidinium is a cationic aromatic compound which seems able to interact with DNA, and further studies are required to clarify its influence on SCEs. Acknowledgements Fig. 1: Alhum cepa metaphase chromosomes showing the SCEs induced by a treatment with Cuprolinic blue (5.5 Itgl ml). duced by these dyes was found to be concentration-dependent. The SCEs rates for BrdU-substituted chromosomes subjected to the anionic phthalocyanines Methazol fast blue 2G and Sirius light turquoise blue GL were not statistically different from control levels. On the contrary, Luxol fast blue MBSN induced an increase in the SCEs frequency when used at the highest concentration. Discussion Phthalocyanines have been reported as rather non toxic compounds for mammalian organisms and for several protozoans, crustaceous and fishes (for review see Spikes 1986). Nevertheless, as far as we know, there is no previous information on the uptake and genotoxicity of phthalocyanines in living plant cells. Our results show that the three cationic agents, as well as the anionic Luxol fast blue MBSN, produce a DNA damage in vivo which is reflected by an increase of the SCEs frequency in plant cell chromosomes. Intercalation is a binding mode firstly introduced by Lerman (1971) to explain the formation of stable complexes between DNA and planar aromatic dyes. Although planar compounds of small size (e.g. acridines) can easily intercalate between base pairs of DNA, this process could be more difficult for bigger molecules with voluminous side chains such as certain phthalocyanine dyes (Scott 1972). However, strong hydrophobic, face-to-face interactions could be expected to occur between the planar phthalocyanine chromophore of Alcian blue 8GS and the bases of polynucleotides (Stockert and Juarranz 1980, Stockert et al. 1982), the intercalating capacity being confirmed for Cuprolinic blue (Dougherty and Pilbrow 1984, Dougherty et al. 1985). This allows us to assume that the cationic phthalocyanines could bind to DNA in vivo by intercalative mechanisms.
This work was supported by a grant (2320-83) from the Comisi6n Asesora de Investigaci6n Ciendfica y Tecnica, Spain.
References ARMAS-PORTELA, R., M. J. HAZEN, and J. C. STOCKERT: Increase in sister chromatid exchanges in BrdU-substituted chromosomes of Alhum cepa induced by the combined effect of pyronin Y and green light. Mutat. Res. 158,77 -80 (1985). BEN-HuR, E. and I. ROSENTHAL: Photosensitized inactivation of chinese hamster cells by phthalocyanines. Photochem. Photobio!. 42,129-133 (1985 a). - - The phthalocyanines: a new class of mammalian cell photosensitizers with a potential for cancer phototherapy. Int. J. Radiat. Bio!. 47, 145-147 (1985 b). - - Factors affecting the photokilling of cultured chinese hamster cells by phthalocyanines. Radiat. Res. 103,403-409 (1985 c). BLOOM, G. and J. W. KELLY: The copper phthalocyanin dye «Astrablau» and its staining properties, especially the staining of mast cells. Histochemie 2,48-57 (1960). CHAN, W. J., R. SVENSEN, D. PHILLIPS, and 1. R. HART: Cell uptake, distribution and response to alluminium chloro suphonated phthalocyanine, a potential anti-tumour photosensitizer. Br. J. Cancer 53, 255-264 (1986). CORTES, F. and M. J. HAZEN: Chromosomal aberrations and SCEs in Allium cepa root-tip cells treated with caffeine and pyronin y. Mutat. Res. 127, 119-122 (1984). DOUGHERTY, G. and J. R. PILBROW: Physico-chemical probes of intercalation. Int. Rev. Biochem. 16, 1179-1192 (1984). DOUGHERTY, G., J. R. PILBROW, A. SKOROBOGATY, and T. D. SMITH: Electron spin resonance spectroscopic and spectrophotometric investigation of the binding of tetracationic porphyrins and pophyrazines with calf thymus DNA. J. Chern. Soc. Faraday Trans. 2, 1739-1759 (1985). GURR, E.: Synthetic Dyes in Biology Medicine and Chemistry, Academic Press, London (1971). HAZEN, M.J., A. VILLANUEVA, A. JUARRANZ, M. CANETE, and J. C. STOCKERT: Photosensitizing dyes and fluorochromes as substitutes for 33258 Hoechst in the fluorescence-plus-Giemsa (FPG) chromosome technique. Histochemistry 83, 241-244 (1985).
560
A. jUARRANZ, M. J. HAzEN, and J. C. STOCKERT
]UARRANZ, A., J. M. FERRER, A. TATO, M. CAN-ETE, and J. C. STOCKERT: Metachromatic staining and electron dense reaction of glycosaminoglycans by means of Cuprolinic Blue. Histochem. J. 19, 1-6(1987). KATO, H.: Induction of sister chromatid exchanges by UV light and its inhibition by caffeine. Exp. Cell Res. 82, 383-390 (1973). - Spontaneous and induced sister chromatid exchanges as revealed by the BrdU-labelling method. Int. Rev. Cyto!' 33, 55-97 (1977). LATT, S. A.: Sister chromatid exchanges, indices of human chromosome damage and repair: a detection by fluoroscence and induction by mitomycin C. Proc. Nat!' Acad. Sci. USA 71, 3162-3166 (1974). LATT, S. A. and K. S. LOVEDAY: Characterization of sister chromatid exchange induction by 8-methoxy psoralen plus near UV light. Cytogenet. Cell Genet. 21, 184-200 (1978). LATT, S. A., R. R. SCHRECK, K. S. LOVEDAY, arid C. F. SHULER: In vitro and in vivo analysis of sister chromatid exchange. Pharmaco!' Rev. 30,501-535 (1979). LATT, S. A.,]. ALLEN, S. E. BLOOM, A. KARRANO, E. FALKE, D. KRAM, E. SCHNEIDER, R. SCHRECK, R. TICE, B. WHITFIELD, and S. WOLFF: Sister chromatid exchanges: a report of the gene-tox program. Mutat. Res. 81, 17 -62 (1981). LERMAN, L. S.: Structural considerations in the interactions of DNA and acridines. J. Mo!. Bio!. 3, 18 -30 (1971). LILLIE, R. D.: H.]. Conn's Biological Stains, 9th edition. Williams & Wilkins, Baltimore (1977). PERRY, P. and H. J. EVANS: Cytological detection of mutagen-carcinogen exposure by sister chromatid exchange. Nature 258, 121-124 (1975). RAJ, A. S. and J. A. HEDDLE: Simultaneous detection of chromosomal aberrations and SCE. Experience with DNA intercalating agents. Mutat. Res. 78, 253 - 260 (1980). ROUSSEAU, ]. H., D. AUTENRIETH, and J. E. VAN LIER: Synthesis, tissue distribution and tumor uptake of 99Tc-tetra-sulfo-phthalocyanine. Int. J. App!. Radiat. Isot. 34, 571-579 (1983).
ROUSSEAU, ]. H., H. Au, G. LAMOUREUX, E. LEBEL, and J. E. VAN LIER: Synthesis, tissue distribution and tumor uptake of 99mTcand 67Ga-tetrasulfophthalocyanine. Int. J. App!. Radiat. Isot. 36, 709-716 (1985). SCOTT, J. E.: Histochemistry of Alcian blue. III. The molecular biological basis of staining by Alcian blue 8GX and analogous phthalocyanins. Histochemie 32, 191-212 (1972). - Affinity, competition and specific interaction in the biochemistry and histochemistry of polyelectrolytes. Biochem. Soc. Trans. 1,787-806 (1973). - The molecular biology of histochemical staining by cationic phthalocyanin dyes: the design of replacements for Alcian blue. J. Microscopy 119, 373-381 (1980). SPIKES, ]. D.: Phthalocyanines as photosensitizers in biological systems and for the photodynamic therapy of tumors. Photochern. Photobio!' 43, 691-699 (1986). STOCKERT,]. C. and A. jUARRANZ: Temperature dependent staining reaction of chromatin by Alcian blue. Z. Naturforsch. 35 c, 1092-1093 (1980). STOCKERT, J. c., O. D. COLMAN, and A. ]UARRANZ: Chromatin staining reactions by oxidized p-phenylenediamine and Alcian blue. Acta Histochem. 25 (supp!.), 129-136 (1982). TAS,]., D. MENDELSON, and c.J. F. VAN NOORDEN: Cuprolinic blue: a specific dye for single-stranded RNA in the presence of magnesium chloride. 1. Fundamental aspects. Histochem. J. 15, 801-814 (1983). THOMPSON, E. B.: Selected Histochemical and Histopathological Methods, Ch. C. Thomas Publishers, Springfield, lllinois (1966). VAN KUPPEVELT, T. H. M. S. M., F. P. M. CREMERS, J. G. W. DOMEN, and C. N. A. KUYPER: Staining of proteoglycans in mouse lung alveoli. II. Characterization of the Cuprolinic blue-positive anionic sites. Histochem. J. 16, 671-686 (1984). VILLANUEVA, A., M.]. HAZEN, and J. C. STOCKERT: Photodynamic effect of the porphyrin derivative meso-tetra (4-N-methyl-pyridyl) porphine on sister chromatid exchanges in meristematic cells. Experientia 42, 1269-1271 (1986). WOLFF, S.: Sister chromatid exchanges. Ann. Rev. Genet. 11, 183-201 (1977).
Effect of Copper Phthalocyanine Derivatives on Sister Chromatid Exchanges in BrdU-substituted Chromosomes of A Ilium cepa A. ]UARRANZ, M. J.
HAZEN,
and J. C.
STOCKERT
Departamento de Biologla, Facultad de Ciencias, Universidad Autonoma de Madrid, 28049-Madrid, Spain Received September 22, 1987 . Accepted September 29, 1987
Summary The effect of some copper phthalocyanine derivatives on sister chromatid exchanges (SCEs) in meristematic cells of Allium cepa roots was studied. A significant increase in SCEs frequencies was observed when BrdU-substituted chromosomes were subjected to the cationic phthalocyanines Alcian blue 8GS, Astra blue and Cuprolinic blue for 3 hours at the end of the G 1 period of the second division cycle. Among the anionic phthalocyanine compounds, only Luxol fast blue MBSN increases the SCEs over the control baseline.
Key words: Allium cepa chromosomes, meristematic cells, sister chromatid exchanges, phthalocyanine derivatives. Abbreviation list: BrdU, 5-bromo-2'-deoxyuridine; dT, unlabelled thymidine; FdU, 5-fluorodeoxyuridine; SCEs, sister chromatid exchanges; FPG, Fluorescence plus Giemsa; SE, standard error.
Introduction DNA damage processes in living cells can be detected, among other cytogenetic techniques, by using the sister chromatid exchanges (SCEs) test (Latt 1974, Perry and Evans 1975). SCEs represent the interchange of DNA replication products of apparently homologous loci, the exchange process probably involving breakage and reunion of DNA strands. It is known that certain physical and chemical agents, particularly DNA intercalating compounds (Kato 1973, Latt and Loveday 1978, Armas-Portela et al. 1985), influence the occurrence of SCEs (Raj and Heddle 1980, Latt et al. 1981). Copper phthalocyanines have been used for many years as histological stains due to their specificity for binding to certain tissue components (Gurr 1971, Lillie 1977). For example, Alcian blue 8GS and Astra blue are currently being applied for the histochemical demonstration of acid glycosaminoglycans (Bloom and Kelly 1960, Scott 1972, 1980); Luxol fast blue MBSN and Methazol fast blue 2G are used for the selective staining of myelin sheaths (Thompson 1966, Lillie 1977), and the phthalocyanine-like dye Cuprolinic blue has interesting applications in the detection of RNA and acid © 1988 by Gustav FlScher Verlag, Stuttgart
proteoglycans (Tas et al. 1983, Van Kuppevelt et al. 1984, Juarranz et al. 1986). Apart from the histochemical use of these compounds other phthalocyanine derivatives were found to be selectively retained in several kinds of tumors after treatments in vivo (Rousseau et al. 1983, 1985, Chan et al. 1986). Some of these agents have been reported as effective photosensitizers for cell killing and tissue damage (Ben-Hur and Rosenthal 1985 a, b, c, Chan et al. 1986) and they were proposed as substitutes for porphyrins in the photodynamic therapy of tumors (Spikes 1986). Since SCEs are very sensitive indicators of the effect of mutagenic and carcinogenic agents on eukaryotic chromosomes (Wolff 1977, Latt et al. 1979, Latt et al. 1981), we have studied the frequency of SCEs in plant chromosomes when subjected to some cationic and anionic copper phthalocyanine derivatives.
Material and methods Roots of Allium cepa bulbs were grown in tap water renewed every 24h and aerated continuously at a rate of 10-20cm3/min. Bulbs were maintained in the dark and at a constant temperature of
558
A. jUARRANZ, M. J.
HAZEN,
and J. C. STOCKERT
Table 1: Phthalocyanine and phthalocyanine-like derivatives used in this work [for the chemical structure of these dyes see Gurr (1971), Lillie (1977), Scott (1972, 1973, 1980)]. Chemical structure of the chromophore, substituents (R) and counterions (X). Phthalocyanine ring
C.I. 74.360 74.180 74.240
Cuprolinic blue
Dye
Source
R
Luxol fast blue MBSN Methazol fast blue 2G Sirius light turquoise blue GL Alcian blue 8GS Astra blue l ) Cuprolinic blue
(Serva) (Gurr) (Bayer) (Serva) (Merck) (BDH)
?R: 4R: 2R: 4R: 3R:
X
-SO) -S03-SO) -CH2SC[N(CH3)z]t -S02NH(CH2hNH(CH3)t
X X
=
X X X
=
X
= = = =
diarylguanidinium +
C6H13NHt
Na+ ClCH 3COOCH30SO)
I) Refered to as Astra blue 6GLL by Scott (1980).
25±0.5°C. In order to analyse the frequency of SCEs, roots were treated with 1O- 4 M 5-bromo, 2' -deoxyuridine (BrdU) and 1O- 7 M 5-fluor, 2'-deoxyuridine (FdU) for a complete cell division cycle (20 h), followed by another cycle in the presence of 10 - 4 M thymidine (dT). Finally, the roots were treated with 0.05 % colchicine for 3 h and fixed overnight in ethanol-acetic acid (3 : 1) at 4°C. The phthalocyanine derivatives used were Alcian blue 8GS, Astra blue, Luxol fast blue MBSN, Methazol fast blue 2G, Sirius light turquoise blue GL, and the phthalocyanine-like dye, Cuprolinic blue. The source and chemical characteristics of these compounds are given in Table 1. Solutions for treatments were prepared in tap water from a 11 mg% solution in distilled water, except for Luxol fast blue MBSN and Methazol fast blue 2G, which were prepared in 1 % ethanol or acetic acid respectively, as these two compounds hardly dissolve in water. The treatment with phthalocyanine solutions (1.1JLg/ml, 2.2JLg/ml and 5.5JLg/ml) was performed in the presence of dT for 3 h at the end of the G I period of the second division cycle. To visualize the occurrence of SCEs, the fluorescenceplus-Giemsa (FPG) chromosome technique was performed according to the modification of Hazen et al. (1985).
Results T able2 summarizes the frequencies of seEs obtained for all the experimental treatments. When the bromo-substitution of DNA was followed by a treatment with cationic phthalocyanine derivatives, the SeEs frequencies significantly increased (p < 0.01, Student's t test) over the control baseline levels (Fig. 1). The increase in the seEs rates in-
Table 2: Frequency of SCEs in controls and after treatments with several cationic and anionic phthalocyanine dyes. Treatments on BrdUsubstituted chromosomes
Dose (JLg/ml)
Control Cationic dyes Alcian blue 8GS
Astra blue Cuprolinic blue
Anionic dyes Luxol fast blue MBSN
Methazol fast blue 2G
2.79±0.19 1.1 2.2 5.5 1.1
5.5 1.1 2.2 5.5 1.1 2.2 5.5 1.1
2.2 5.5 Sirius light turquoise blue GL
a), b) SCEs/chromosome
1.1
2.2 5.5
3.87±0.20 4.70±0.20 5.02±0.20 5.55±0.20 5.81±0.20 3.88±0.15 4.67±O.29 5.84±O.20 2.89±0.21 2.93±O.20 4.20±O.20 3.12±O.21 3.05±0.20 3.30±0.20 2.93±O.19 2.94±O.19 2.95±O.19
a) mean ± S.E. b) 300 metaphase chromosomes were studied in each case.
Copper phthalocyanine derivatives effect on sister chromatids
559
It is well known that most intercalating agents introduce changes in the DNA structure, which could be expected to result in breaks and/or exchanges of DNA strands, thus being SCEs inductors (Wolff 1977, Kato 1977, Latt et al. 1981, Cortes and Hazen 1984, Villanueva et al. 1986). In this context, it seems that SCEs induced by cationic phthalocyanines could be related to a possible intercalative binding mode to DNA. Luxol fast blue MBSN was the only anionic derivative able to increase the SCEs frequency over the baseline level. In this respect it is tempting to assume that the diarylguanidinium counterion of this derivative could be responsible for the increase of SCEs, because all the anionic compounds used have essentially the same phthalocyanine ring system and substituents. Diarylguanidinium is a cationic aromatic compound which seems able to interact with DNA, and further studies are required to clarify its influence on SCEs. Acknowledgements Fig. 1: Alhum cepa metaphase chromosomes showing the SCEs induced by a treatment with Cuprolinic blue (5.5 Itgl ml). duced by these dyes was found to be concentration-dependent. The SCEs rates for BrdU-substituted chromosomes subjected to the anionic phthalocyanines Methazol fast blue 2G and Sirius light turquoise blue GL were not statistically different from control levels. On the contrary, Luxol fast blue MBSN induced an increase in the SCEs frequency when used at the highest concentration. Discussion Phthalocyanines have been reported as rather non toxic compounds for mammalian organisms and for several protozoans, crustaceous and fishes (for review see Spikes 1986). Nevertheless, as far as we know, there is no previous information on the uptake and genotoxicity of phthalocyanines in living plant cells. Our results show that the three cationic agents, as well as the anionic Luxol fast blue MBSN, produce a DNA damage in vivo which is reflected by an increase of the SCEs frequency in plant cell chromosomes. Intercalation is a binding mode firstly introduced by Lerman (1971) to explain the formation of stable complexes between DNA and planar aromatic dyes. Although planar compounds of small size (e.g. acridines) can easily intercalate between base pairs of DNA, this process could be more difficult for bigger molecules with voluminous side chains such as certain phthalocyanine dyes (Scott 1972). However, strong hydrophobic, face-to-face interactions could be expected to occur between the planar phthalocyanine chromophore of Alcian blue 8GS and the bases of polynucleotides (Stockert and Juarranz 1980, Stockert et al. 1982), the intercalating capacity being confirmed for Cuprolinic blue (Dougherty and Pilbrow 1984, Dougherty et al. 1985). This allows us to assume that the cationic phthalocyanines could bind to DNA in vivo by intercalative mechanisms.
This work was supported by a grant (2320-83) from the Comisi6n Asesora de Investigaci6n Ciendfica y Tecnica, Spain.
References ARMAS-PORTELA, R., M. J. HAZEN, and J. C. STOCKERT: Increase in sister chromatid exchanges in BrdU-substituted chromosomes of Alhum cepa induced by the combined effect of pyronin Y and green light. Mutat. Res. 158,77 -80 (1985). BEN-HuR, E. and I. ROSENTHAL: Photosensitized inactivation of chinese hamster cells by phthalocyanines. Photochem. Photobio!. 42,129-133 (1985 a). - - The phthalocyanines: a new class of mammalian cell photosensitizers with a potential for cancer phototherapy. Int. J. Radiat. Bio!. 47, 145-147 (1985 b). - - Factors affecting the photokilling of cultured chinese hamster cells by phthalocyanines. Radiat. Res. 103,403-409 (1985 c). BLOOM, G. and J. W. KELLY: The copper phthalocyanin dye «Astrablau» and its staining properties, especially the staining of mast cells. Histochemie 2,48-57 (1960). CHAN, W. J., R. SVENSEN, D. PHILLIPS, and 1. R. HART: Cell uptake, distribution and response to alluminium chloro suphonated phthalocyanine, a potential anti-tumour photosensitizer. Br. J. Cancer 53, 255-264 (1986). CORTES, F. and M. J. HAZEN: Chromosomal aberrations and SCEs in Allium cepa root-tip cells treated with caffeine and pyronin y. Mutat. Res. 127, 119-122 (1984). DOUGHERTY, G. and J. R. PILBROW: Physico-chemical probes of intercalation. Int. Rev. Biochem. 16, 1179-1192 (1984). DOUGHERTY, G., J. R. PILBROW, A. SKOROBOGATY, and T. D. SMITH: Electron spin resonance spectroscopic and spectrophotometric investigation of the binding of tetracationic porphyrins and pophyrazines with calf thymus DNA. J. Chern. Soc. Faraday Trans. 2, 1739-1759 (1985). GURR, E.: Synthetic Dyes in Biology Medicine and Chemistry, Academic Press, London (1971). HAZEN, M.J., A. VILLANUEVA, A. JUARRANZ, M. CANETE, and J. C. STOCKERT: Photosensitizing dyes and fluorochromes as substitutes for 33258 Hoechst in the fluorescence-plus-Giemsa (FPG) chromosome technique. Histochemistry 83, 241-244 (1985).
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A. jUARRANZ, M. J. HAzEN, and J. C. STOCKERT
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