Chem. Res. Toxicol. 2005, 18, 1545-1552
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The Mechanism of Guanine Specific Photooxidation in the Presence of Berberine and Palmatine: Activation of Photosensitized Singlet Oxygen Generation through DNA-Binding Interaction Kazutaka Hirakawa,*,† Shosuke Kawanishi,‡ and Toru Hirano§ Division of Applied Science and Basic Engineering, Faculty of Engineering, Shizuoka University, Johoku 3-5-1, Hamamatsu, Shizuoka 432-8561, Japan, Department of Environmental and Molecular Medicine, Mie University School of Medicine, Edobashi 2-174, Tsu, Mie 514-8507, Japan, and Photon Medical Research Center, Hamamatsu University School of Medicine, Handayama 1-20-1, Hamamatsu, Shizuoka 431-3192, Japan Received June 28, 2005
The mechanism of DNA damage by photoexcited alkaloids, berberine and palmatine, was examined using 32P-labeled DNA fragments obtained from human genes. Berberine and palmatine easily bind to DNA, leading to the formation of strong fluorescent complexes. The binding constants of berberine and palmatine to DNA, estimated from an analysis of their fluorescence enhancements, indicate the formation of stable complexes. Photoexcited berberine and palmatine caused DNA cleavage, specifically at almost all guanine residues, under the aerobic condition after Escherichia coli formamidopyrimidine-DNA glycosylase or piperidine treatment, suggesting the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo), an oxidized product of 2′-deoxyguanosine, and further oxidized products. The formation of 8-oxodGuo was confirmed by HPLC measurement. The quantum yield of 8-oxodGuo formation by berberine was almost the same as that induced by palmatine. Berberine and palmatine did not cause DNA photodamage under anaerobic conditions. Scavengers of singlet oxygen (1O2), such as sodium azide and methional, inhibited DNA damage. These findings suggest that photoexcited berberine and palmatine give rise to 8-oxodGuo through 1O2 generation. The photosensitized 1O2 generation from these alkaloids was examined using near-infrared luminescence measurements. Emission at ca. 1270 nm was observed during photoexcitation of the DNA-alkaloid complexes. This emission was quenched by sodium azide, a scavenger of 1O . In the absence of DNA, berberine and palmatine could not show the emission. This 2 spectroscopic study has shown that photoexcited alkaloids can generate 1O2 only when the DNA-alkaloid complexes are formed. In conclusion, berberine and palmatine easily bind to DNA and induce guanine specific photooxidation via 1O2 formation. The present study suggests that berberine and palmatine can act as functional photosensitizers enabling a switch in phototoxicity via 1O2 formation by the interaction with DNA.
Introduction The damage of DNA induced by photosensitizers through the generation of reactive oxygen species (ROS)1 and electron transfer has been extensively investigated (1-12). These mechanisms contribute to photocarcinogenesis and phototoxicity (9). Furthermore, photosensitized DNA damage has received attention from a therapeutic viewpoint such as photodynamic therapy (PDT) (13, 14). Critical sites of action of reactive species in PDT * To whom correspondence should be addressed. Tel: +81-53-4781287. Fax: +81-53-478-1287. E-mail:
[email protected]. † Shizuoka University. ‡ Mie University School of Medicine. § Hamamatsu University School of Medicine. 1 Abbreviations: ROS, reactive oxygen species; PDT, photodynamic therapy; 1O2, singlet oxygen; HPLC-ECD, electron chemical detector coupled to high-performance liquid chromatography; 8-oxodGuo, 8-oxo7,8-dihydro-2′-deoxyguanosine; dGuo, 2′-deoxyguanosine; DTPA, diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid; SOD, superoxide dismutase; Fpg, E. coli formamidopyrimidine-DNA glycosylase; •OH, hydroxyl radical.
include mitochondria and lipid membrane, and DNA is also a potential target of PDT. We have previously reported the mechanism and sequence specificity of photosensitized DNA damage through electron transfer (type I mechanism) and singlet oxygen (1O2) generation (type II mechanism) (9-12). Consecutive guanine residues, such as underlined G of 5′-GG or 5′-GGG, act as the hole trap (15-18) and are selectively damaged through the type I mechanism, whereas the type II mechanism causes the oxidation of every guanine residue (1, 9). If the DNA-damaging activity of a photosensitizer can be controlled, such photoinduced DNA damage offers a promising tool for PDT. The interaction between DNA and photosensitizers should play an important role in the photosensitized reaction. Therefore, the environmental effect of the DNA strand should be one of the key factors to control the activity of photosensitizers. Berberine is the main alkaloid constituent of Goldenseal (Hydrasis canadensis L.) and has been
10.1021/tx0501740 CCC: $30.25 © 2005 American Chemical Society Published on Web 09/29/2005
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Chem. Res. Toxicol., Vol. 18, No. 10, 2005
Figure 1. Structures of berberine and palmatine.
employed to treat skin diseases, including psoriasis (19) and eye infections (20). The phototoxicity and DNAphotodamaging abilities of berberine to HaCaT keratinocytes have been reported (21). The use of berberine as a fluorescent strain for cells (22), dermosomes (23), and energized mitochondria (24) indicates that there is a strong interaction with cellular components such as DNA. Recently, an NMR study demonstrated that berberine binds to a minor groove of DNA (25). The complex formation between DNA and photosensitizers is useful to design an experimental system to clarify the environmental effects of a DNA strand on a photosensitized reaction. In addition, the clarification of the mechanism of DNA damage by these alkaloids is important to design a photosensitizer, which damages DNA as the specific target of PDT. In this study, DNA damage by photoirradiated berberine and its analogue, palmatine (Figure 1), was examined using 32P-5′-end-labeled DNA fragments obtained from a human gene. Using an electrochemical detector coupled to high-performance liquid chromatography (HPLC-ECD), we also measured the content of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) (26), an oxidized product of 2′-deoxyguanosine (dGuo), formed by photoirradiated berberine and palmatine. The interaction between DNA and berberine or palmatine was examined using fluorescence measurements and molecular mechanics calculations. The activities of photosensitized 1O2 generation by these alkaloids were examined using nearinfrared luminescence measurements.
Experimental Procedures Materials. Restriction enzymes (HindIII and MroI) and T4 polynucleotide kinase were purchased from New England Biolabs (Beverly, MA). Restriction enzymes (EcoRI and XbaI) and calf intestine phosphatase were from Boehringer Mannheim GmbH (Mannheim, Germany). [γ-32P]ATP (222 TBq mmol-1) was from New England Nuclear (Boston, MA). Diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid (DTPA) was from Dojin Chemicals Co. (Kumamoto, Japan). Calf thymus DNA, catalase (45000 units mg-1 from bovine liver), and superoxide dismutase (SOD) (3000 units mg-1 from bovine erythrocytes) were from Sigma Chemical Co. (St. Louis, MO). Nuclease P1 was from Yamasa Shoyu Co. (Chiba, Japan). Berberine chloride and piperidine were from Wako Chemicals Co. (Osaka, Japan). Palmatine chloride was from Aldrich Chemicals Co. (Milwaukee, WI). Berberine chloride and palmatine chloride were purified by silica gel column chromatography with water-ethanol (1/5, v/v) as an eluent. Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) was from Trevigen Co. (Gaithersburg, MD). D2O was from Kanto Chemical Co. (Tokyo, Japan).
Hirakawa et. al. Measurement of Fluorescence Spectra of Berberine and Palmatine. Fluorescence spectra of berberine and palmatine were measured with a RF-5300PC spectrophotometer (Shimadzu, Kyoto, Japan) on 365 nm excitation. The samples contained 50 µM berberine or palmatine with or without calf thymus DNA in 10 mM sodium phosphate buffer (pH 7.8). Preparation of 32P-5′-End-Labeled DNA Fragments. DNA fragments were obtained from the human p16 (27) tumor suppressor gene and the c-Ha-ras-1 protooncogene (28). A singly 32P-5′-end-labeled double-stranded 158 bp p16 fragment (MroI 6173-EcoRI* 6330) was prepared from pGEM-T Easy Vector (Promega Corp., Madison, WI) as described previously (29). A singly labeled 261 bp c-Ha-ras-1 fragment (AvaI* 1645-XbaI 1905) was prepared from plasmid pbcNI, which carries a 6.6 kb BamHI chromosomal DNA segment containing the human c-Ha-ras-1 protooncogene, according to the method described previously (30, 31). Nucleotide numbering starts with the BamHI site (28). The asterisk indicates 32P-labeling. Detection of DNA Damage Induced by Photoexcited Berberine or Palmatine. The standard reaction mixture in a microtube (1.5 mL Eppendorf) contained 32P-labeled DNA fragment (
