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Copper-mediated DNA damage caused by purpurin, a natural anthraquinone

Copper-mediated DNA damage caused by purpurin, a natural anthraquinone Background: Purpurin (1,2,4-trihydroxy-9,10-anthraquinone), a natural red anthraquinone pigment, has historically been used as a textile dye. However, purpurin induced urinary bladder tumors in rats, and displayed a mutagenic activity in assay using bacteria and mammalian cells. Many carcinogenic dyes are known to induce bladder cancers via DNA adduct formation, but carcinogenic mechanisms of purpurin remain unknown. In this study, to clarify the mechanism underlying carcinogenicity of purpurin, copper-mediated DNA damage induced by purpurin was exam- ined using P-labeled DNA fragments of human genes relevant to cancer. Furthermore, we also measured 8-oxo-7,8- dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in calf thymus DNA. Results: Purpurin plus Cu(II) cleaved P-labeled DNA fragments only under piperidine treatment, indicating that purpurin caused base modification, but not breakage of the DNA backbone. In the absence of Cu(II), purpurin did not induce DNA cleavage even with piperidine treatment. Purpurin plus Cu(II) caused piperidine-labile sites predomi- nantly at G and some T residues. Bathocuproine, a Cu(I) chelator, completely prevented the occurrence of piperidine- labile sites, indicating a critical role of Cu(I) in piperidine-labile sites induced by purpurin plus Cu(II). On the other hand, methional, a scavenger of a variety of reactive oxygen species (ROS) and catalase showed limited inhibitory effects on the induction of piperidine-labile sites, suggesting that ROS could not be major mediators of the purpurin- induced DNA damage. Considering reported DNA adduct formation by quinone metabolites of several carcinogenic agents, quinone form of purpurin, which is possibly generated via purpurin autoxidation accompanied by Cu(I)/Cu(II) redox cycle, might lead to DNA adducts and piperidine-labile sites. In addition, we measured contents of 8-oxodG. Purpurin moderately but significantly increased 8-oxodG in calf thymus DNA in the presence of Cu(II). The 8-oxodG formation was inhibited by catalase, methional and bathocuproine, suggesting that Cu(I)-hydroperoxide, which was generated via Cu(I) and H O , caused oxidative DNA base damage. 2 2 Conclusions: We demonstrated that purpurin induces DNA base damage possibly mediated by Cu(I)/Cu(II) redox cycle both with and without ROS generation, which are likely to play an important role in its carcinogenicity. Keywords: Purpurin, Copper, DNA damage, Reactive oxygen species Introduction Anthraquinones are a class of organic compounds abundant in the universe of natural substances and are found in various plant parts such as roots, rhizomes, fruits, and flowers [1 ]. Purpurin (1,2,4-trihydroxy- *Correspondence: s-oikawa@med.mie-u.ac.jp 9,10-anthraquinone) is a natural red anthraquinone Department of Environmental and Molecular medicine, Mie University pigment mostly isolated from madder root (Rubia tinc- Graduate School of Medicine, Edobashi 2-174, Tsu, Mie 514-8507, Japan Full list of author information is available at the end of the article torum) [2]. Historically, purpurin has been used not © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kobayashi et al. Genes and Environment (2022) 44:15 Page 2 of 8 only as a textile dye but also as an ingredient of herbal Preparation of  P‑5 ′‑end‑labeled DNA fragments medicine and a food coloring agent. Purpurin has been DNA fragments were obtained from the human p16 reported to show beneficial biological activities such as tumor suppressor gene [8] and the c-Ha-ras-1 protoon- antioxidant, antimutagenic, antimicrobial, neuropro- cogene [9]. A fragment containing exon 2 of the human tective, antiadipogenic, and anticancer activities [2]. p16 tumor suppressor gene was obtained as described On the other hand, purpurin was reported to induce previously [10]. The 5′ end-labeled 460-base pair frag - marked hyperplasia of pelvic epithelium and urinary ment (EcoRI* 9481-EcoRI* 9940) containing exon 2 was bladder tumors (papilloma and carcinoma) in rats [3]. also further digested with BssHII to obtain the singly Purpurin had also a mutagenic activity in Salmonella labeled 309-base pair fragment (EcoRI* 9481-BssHII without mammalian microsomal activation [4, 5] and 9789) and the 147-base pair fragment (BssHII 9794- was active in various mutagenesis assay systems using EcoRI* 9940). DNA fragments were also prepared from mammalian cells including V79-HGPRT assay, DNA- plasmid pbcNI, which carries a 6.6-kb BamHI chromo- repair assay in primary rat hepatocytes, and transfor- somal DNA segment containing the human c-Ha-ras-1 mation of C3H/M2 mouse fibroblasts [5 ]. Working protooncogene. The singly labeled 98-base pair fragment exposed to dyes has been identified as one of the (AvaI* 2247-PstI 2344) and 337-base pair fragment (PstI major risk factors for occupational bladder cancer [6, 2345-AvaI* 2681) were obtained as previously described 7]. Many carcinogenic dyes induce carcinogenesis via [11]. For reference, nucleotide numbering starts with the DNA adduct formation, but carcinogenic mechanisms BamHI site [9]. An asterisk indicates P-labeling. of purpurin remain unknown. In this study, to clarify the mechanism underlying the Detection of damage to isolated P‑labeled DNA carcinogenicity of purpurin, we investigated purpurin- fragments induced DNA damage in the presence of copper ions. Reaction mixtures containing P-labeled DNA frag- We analyzed DNA damage using P-5′-end-labeled ment, 20 μM/base calf thymus DNA, purpurin and 20 μM DNA fragments obtained from the c-Ha-ras-1 pro- CuCl in 200 μl of sodium phosphate buffer (pH 7.8) con- tooncogene and the p16 tumor suppressor gene. We taining 5 μM DTPA were incubated for 1 h at 37 °C under also measured the contents of 8-oxo-7,8-dihydro-2′- light shielding. To examine the effects of reactive oxygen deoxyguanosine (8-oxodG), a relevant indicator of oxi- species (ROS) scavengers and bathocuproine, these rea- dative DNA damage, in calf thymus DNA using a high gents were added before the addition of purpurin. The performance liquid chromatograph equipped with an DNA fragments were heated in 1 M piperidine for 20 min electrochemical detector (HPLC-ECD). at 90 °C, followed by electrophoresis on an 8% poly- acrylamide/8 M urea gel as previously described [11]. An autoradiogram was obtained by exposing an X-ray film (FUJIFILM Corp., Tokyo, Japan) to the gel. Band inten- Materials and methods sity was quantified using ImageJ software (National Insti - Materials tutes of Health). EcoRI, AvaI, PstI and T polynucleotide kinase were The preferred cleavage sites were determined by purchased from New England Biolabs Ltd. (Ipswich, directly comparing the positions of the oligonucleotides MA, USA). BssHII was purchased from Takara Bio with those produced by the chemical reactions of the Inc. (Shiga, Japan). [γ- P] ATP (222 TBq/mmol) Maxam-Gilbert procedure [12], using a DNA-sequencing was purchased from Perkin Elmer, Inc. (Waltham, system (LKB 2010 Macrophor, LKB Pharmacia Biotech- MA, USA). Purpurin, catalase (30,000 units/mg from nology Inc., Uppsala, Sweden). The autoradiogram was bovine liver) and 3-(methylthio) propionaldehyde obtained by exposing an imaging plate (BAS-MS2040, (methional) were purchased from Sigma-Aldrich Co., FUJIFILM Corp.) to the gel. The relative amounts of oli - LLC. (St Louis, MO, USA). Copper chloride dihydrate gonucleotides from the treated DNA fragments were (CuCl ·2H O), ethanol, mannitol and sodium formate 2 2 measured using a laser scanner (Typhoon FLA-9500, GE were purchased from Nacalai Tesque (Kyoto, Japan). Healthcare, Buckinghamshire, England) and analyzed Diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid with ImageQuant TL software (GE Healthcare). (DTPA) and bathocuproine disulfonic acid were pur- chased from Dojindo Laboratories (Kumamoto, Japan). Analysis of 8‑oxodG formation in calf thymus DNA Nuclease P (500 units/vial) and piperidine were pur- Measurement of 8-oxodG in calf thymus DNA was chased from FUJIFILM Wako Pure Chemical Co., Ltd. performed as described previously [13]. The reaction (Osaka, Japan). Calf intestinal phosphatase (500 units/ mixtures, containing 100 μM/base calf thymus DNA, vial) was purchased from Roche Diagnostics GmbH purpurin and 20 μM CuCl in 400 μl of 4 mM sodium (Mannheim, Germany). 2 Koba yashi et al. Genes and Environment (2022) 44:15 Page 3 of 8 phosphate buffer (pH 7.8) containing 5 μM DTPA, were cleaves DNA at sugars with modified bases, it is reason - incubated for 1 h at 37 °C under light shielding. To exam- able to conclude that purpurin caused base modification ine the effects of ROS scavengers and bathocuproine, but not breakage of deoxyribose phosphate backbone in these reagents were added before the addition of purpu- the presence of Cu(II). rin. After ethanol precipitation, the DNA was digested into nucleosides with nuclease P and calf intestine phos- Eec ff ts of scavengers and bathocuproine on DNA damage phatase. The amount of 8-oxodG was measured using an induced by purpurin in the presence of Cu(II) HPLC-ECD as described previously [14]. Effects of the scavengers and bathocuproine on the dam - age of P-labeled DNA fragments by purpurin plus Statistical analysis Cu(II) were examined. Bathocuproine, a Cu(I)-specific Results were presented as means ± standard deviation chelator [15], prevented purpurin-induced DNA damage (SD). Differences were analyzed by student t-test. P val - in P-labeled DNA fragments in the presence of Cu(II) ues less than 0.05 were considered to be statistically (Fig.  2 and Fig. S1B). Furthermore, methional, a scaven- significant. ger of a variety of ROS [16], partially inhibited the DNA damage. Catalase also showed a slight inhibitory effect on Results the DNA damage. On the other hand, the DNA damage Damage of  P‑labeled DNA fragments by purpurin was not prevented by typical free hydroxyl radical (•OH) in the presence of Cu(II) scavengers such as ethanol, mannitol and sodium for- Figure  1 shows the autoradiogram of DNA fragments mate. These results suggest that ROS could not be major incubated with purpurin plus Cu(II) and followed by mediators of the purpurin-induced DNA damage. treatment with or without piperidine. Oligonucleotides were detected on the autoradiogram as a result of the Site specificity of DNA damage by purpurin in the presence DNA cleavage. In the presence of Cu(II), purpurin caused of Cu(II) DNA cleavage with piperidine treatment, although no The patterns of DNA damage induced by purpurin in the or little DNA cleavage was observed without piperi- presence of Cu(II) were determined by DNA sequencing dine treatment (Fig.  1 and Fig. S1A). Meanwhile, in the using the Maxam-Gilbert procedure [12]. The relative absence of Cu(II), purpurin did not induce DNA cleavage intensity of DNA damage obtained by scanning autora- even with piperidine treatment (data not shown). DNA diogram with a laser scanner is shown in Fig.  3. Purpu- cleavage induced by purpurin plus Cu(II) was detected rin cleaved DNA frequently at guanine (G) and at some at 5 μM of purpurin, and the degree of the DNA cleav- thymine (T) residues in DNA fragments obtained from age was increased at 10, 20, 50 and 100 μM. As piperidine the human c-Ha-ras-1 protooncogene with piperidine Fig. 1 Autoradiogram of P-5′-end-labeled DNA fragments incubated with purpurin in the presence of Cu(II). Reaction mixtures contained the P-5′-end-labeled 147-bp fragment, 20 μM/base calf thymus DNA, the indicated concentrations of purpurin and 20 μM CuCl in 200 μl of 4 mM sodium phosphate buffer (pH 7.8) containing 5 μM DTPA. After incubation at 37 °C for 1 h under light shielding, the DNA fragments were treated with or without hot piperidine and electrophoresed on a polyacrylamide gel Kobayashi et al. Genes and Environment (2022) 44:15 Page 4 of 8 as ethanol, mannitol and sodium formate did not. Batho- cuproine showed an inhibitory effect on the 8-oxodG for - mation. These results suggest the involvement of H O , 2 2 ROS other than •OH and Cu(I) in the 8-oxodG formation caused by purpurin plus Cu(II). Discussion The present study demonstrated that purpurin caused copper-mediated DNA damage in P-labeled DNA fragments. Cleaved oligonucleotides were observed only under piperidine treatment, indicating that pur- purin caused base modification, but not breakage of the deoxyribose-phosphate backbone, in the presence of Cu(II). Bathocuproine completely prevented the piper- idine-labile sites induced by purpurin plus Cu(II), while methional and catalase also displayed limited inhibitory effects on the DNA damage (Fig.  2). Site specificity of the purpurin-induced piperidine-labile sites (predominantly at G) is inconsistent with our previous studies indicating that many ROS-generating agents plus Cu(II) frequently cleaved DNA at cytosine (C) and T under piperidine treatment [17–19]. These results suggest that mecha - nisms other than ROS generation could mainly con- tribute to purpurin-induced DNA damage. Some DNA Fig. 2 Eec ff ts of ROS scavengers and bathocuproine on adducts formed in DNA base has been reported to lead purpurin-induced DNA damage in P-5′-end-labeled DNA fragments. to DNA strand break under piperidine treatment [20– Reaction mixtures contained the P-5′-end-labeled 147-bp fragment, 20 μM/base calf thymus DNA, 50 μM purpurin, each scavenger or 22]. Many quinones are reported to form DNA adduct bathocuproine and 20 μM CuCl in 200 μl of 4 mM sodium phosphate 2 through covalently binding to nucleobases of DNA [23]. buffer (pH 7.8) containing 5 μM DTPA. After incubation at 37 °C for Notably, o-quinone metabolites of carcinogens such as 1 h under light shielding, the DNA fragments were treated with estrogen and bisphenol A contribute to their carcino- piperidine and electrophoresed on a polyacrylamide gel. The genic mechanism via DNA adduct formation [24, 25]. concentrations of each scavenger and bathocuproine were as follows: 0.2 M ethanol, 0.1 M mannitol, 0.1 M sodium formate, 0.6 M u Th s, we considered that the o-quinone form of purpu - methional, 50 U catalase, 50 μM bathocuproine rin, which is generated via purpurin autoxidation accom- panied by Cu(I)/Cu(II) redox cycle, could lead to DNA adducts and piperidine-labile sites (Fig.  5). Although a recent study has reported that purpurin can react with treatment (Fig.  3A). In DNA fragments obtained from Cu(II) generating a metal complex (2:1) under certain the p16 tumor suppressor genes, DNA cleavages at T as conditions [26], the complex was not detected by ultravi- well as G residues were induced by purpurin under piper- olet-visible spectroscopy under our conditions (data not idine treatment (Fig.  3B). Thus, purpurin caused piperi - shown). dine-labile sites predominantly at G and some T residues. Interestingly, the present study indicated that purpurin plus Cu(II) also induced oxidative damage. We examined Formation of 8‑oxodG in calf thymus DNA by purpurin the ability of purpurin to induce 8-oxodG formation. in the presence of Cu(II) 8-OxodG causes DNA misreplication resulting in muta- To investigate oxidative DNA damage, we measured the tion or cancer [27, 28] and is one of the oxidative DNA contents of 8-oxodG, an indicator of oxidative base dam- products generated by the reaction with ROS [29]. Pur- age, in calf thymus DNA treated with purpurin in the purin modestly but significantly increased formation of presence of Cu(II). The formation of 8-oxodG was sig - 8-oxodG in calf thymus DNA in the presence of Cu(II). nificantly increased by purpurin treatment in a concen - To clarify the mechanism of purpurin-induced 8-oxodG tration-dependent manner (Fig.  4A). Figure  4B shows formation, we investigated effects of ROS scavengers and the effects of ROS scavengers and bathocuproine on the bathocuproine. Catalase and bathocuproine exhibited Cu(II)-mediated 8-oxodG formation induced by purpu- inhibitory effects on the increase of 8-oxodG, although rin. Catalase and methional significantly inhibited the not complete. Methional also showed similar results, 8-oxodG formation, while typical •OH scavengers such Koba yashi et al. Genes and Environment (2022) 44:15 Page 5 of 8 32 32 Fig. 3 Site specificity of purpurin-induced DNA damage in P-5′-end-labeled DNA fragments. Reaction mixtures contained the P-5′-end-labeled (A) the 337-bp fragment or (B) the 309-bp fragment, 20 μM/base calf thymus DNA, 50 μM purpurin and 20 μM CuCl in 200 μl of 10 mM sodium phosphate buffer (pH 7.8) containing 5 μM DTPA. After incubation at 37 °C for 1 h under light shielding, the DNA fragments were treated with piperidine and electrophoresed on a polyacrylamide gel. DNA bases in bold represent preferentially cleaved bases Fig. 4 Formation of 8-oxodG in calf thymus DNA by purpurin in the presence of Cu(II). (A) Reaction mixtures contained 100 μM/base calf thymus DNA, the indicated concentrations of purpurin and 20 μM CuCl in 400 μl of 4 mM sodium phosphate buffer (pH 7.8) containing 5 μM DTPA. (B) Reaction mixtures contained 100 μM/base calf thymus DNA, 100 μM purpurin, 20 μM CuCl and each scavenger or bathocuproine in 400 μl of 4 mM sodium phosphate buffer (pH 7.8) containing 5 μM DTPA. The concentrations of each scavenger and bathocuproine were as follows: 0.2 M ethanol, 0.1 M mannitol, 0.1 M sodium formate, 0.6 M methional, 50 U catalase, 50 μM bathocuproine. Reaction mixtures were incubated at 37 °C for 1 h under light shielding. After ethanol precipitation, the DNA was digested to nucleosides with nuclease P and calf intestine phosphatase, then analyzed with an HPLC-ECD system. * p < 0.05 vs 0 μM. # p < 0.05 vs purpurin plus Cu(II). Significance was analyzed using Student t-test Kobayashi et al. Genes and Environment (2022) 44:15 Page 6 of 8 Fig. 5 A possible mechanism of DNA base damage induced by purpurin and Cu(II) while •OH scavengers did not inhibit the 8-oxodG forma- Future research should focus on the association between tion. These results suggest that Cu(I)/Cu(II) redox cycle copper and purpurin for understanding of mechanism of and concomitant H O production possibly via autoxida- purpurin carcinogenicity. 2 2 tion of purpurin lead to generate Cu(I)-hydroperoxide, which induces oxidative DNA base damage including 8-oxodG formation (Fig. 5). Conclusions A higher level of serum copper was observed in blad- We demonstrated that purpurin plus Cu(II) induces Cu(I)/ der cancer patients than in healthy controls [30]. A recent Cu(II) redox cycle-mediated DNA base damage, possibly case-controlled clinical study reported that plasma level through both ROS-independent and -dependent mecha- of copper was significantly and positively associated with nisms, which are likely to contribute to its carcinogenicity. bladder cancer development, suggesting that the increase Abbreviations in plasma copper may be one of the risk factors for blad- 8-oxodG: 8-oxo-7,8-dihydro-2′-deoxyguanosine; DTPA: Diethylenetriamine- der cancer [31]. Many studies have reported that copper N,N,N′,N″,N″-pentaacetic acid; HPLC-ECD: A high performance liquid chromato- graph equipped with an electrochemical detector; ROS: Reactive oxygen species. plays a role in promoting carcinogenesis though induc- ing oxidative stress and resultant DNA damage [32–34]. Supplementary Information Copper ions have been known to be particularly adept The online version contains supplementary material available at https:// doi. at helping other agents to damage DNA and to enhance org/ 10. 1186/ s41021- 022- 00245-2. mutagenic activities via production of ROS [35, 36]. Our previous studies also demonstrated that copper mediated Additional file 1. the various carcinogens-induced ROS production and oxidative DNA damage [19, 37, 38]. However, the present Acknowledgements study suggests that purpurin plus Cu(II) leads to DNA Not applicable. damage through not only ROS generation but also pos- Authors’ contributions sible DNA adduct formation mediated by Cu(I)/Cu(II) SO and HK conceived and designed the study. HK, SO and YM wrote paper. HK, redox cycle (Fig. 5). Therefore, both oxidative DNA dam - SO, YM, RI, YH and Shinya K performed experiment and data analysis. SO, HK, Sho- age and DNA adduct could be involved in the carcino- suke K and MM performed data curation. Shosuke K and MM reviewed and criti- cally edited the paper. All authors have read and approved the final manuscript. genic mechanism of purpurin. Several previous studies reported that purpurin is also Funding capable of binding DNA based on voltammetric, elec- This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 19H03885, 18 K19673. trochemical and spectroscopic analysis [39–41]. These findings suggest that purpurin, copper and DNA might Availability of data and materials coexist and interact with each other and lead to DNA Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. damage via ROS generation and DNA adduct formation. Koba yashi et al. Genes and Environment (2022) 44:15 Page 7 of 8 producing oxygen radicals and evidence for its repair. Carcinogenesis. 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Das P, Guina PS, Mandal PC, Paul M, Paul S, Das S. Cyclic voltammetric studies of 1,2,4-trihydroxy-9,10-anthraquinone, its interaction with calf thymus DNA and anti-leukemic activity on MOLT-4 cell lines: a comparison with anthracycline anticancer drugs. J Phys Org Chem. 2011;24(9):774–85. https:// doi. org/ 10. 1002/ poc. 1827. 41. Wang QX, Gao F, Yuan XL, Li WQ, Liu AF, Jiao K. Electrochemical studies on the binding of a carcinogenic anthraquinone dye, Purpurin (CI 58 205) with DNA. Dyes Pigments. 2010;84(3):213–7. https:// doi. org/ 10. 1016/j. dyepig. 2009. 09. 004. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? 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Copper-mediated DNA damage caused by purpurin, a natural anthraquinone

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Abstract

Background: Purpurin (1,2,4-trihydroxy-9,10-anthraquinone), a natural red anthraquinone pigment, has historically been used as a textile dye. However, purpurin induced urinary bladder tumors in rats, and displayed a mutagenic activity in assay using bacteria and mammalian cells. Many carcinogenic dyes are known to induce bladder cancers via DNA adduct formation, but carcinogenic mechanisms of purpurin remain unknown. In this study, to clarify the mechanism underlying carcinogenicity of purpurin, copper-mediated DNA damage induced by purpurin was exam- ined using P-labeled DNA fragments of human genes relevant to cancer. Furthermore, we also measured 8-oxo-7,8- dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in calf thymus DNA. Results: Purpurin plus Cu(II) cleaved P-labeled DNA fragments only under piperidine treatment, indicating that purpurin caused base modification, but not breakage of the DNA backbone. In the absence of Cu(II), purpurin did not induce DNA cleavage even with piperidine treatment. Purpurin plus Cu(II) caused piperidine-labile sites predomi- nantly at G and some T residues. Bathocuproine, a Cu(I) chelator, completely prevented the occurrence of piperidine- labile sites, indicating a critical role of Cu(I) in piperidine-labile sites induced by purpurin plus Cu(II). On the other hand, methional, a scavenger of a variety of reactive oxygen species (ROS) and catalase showed limited inhibitory effects on the induction of piperidine-labile sites, suggesting that ROS could not be major mediators of the purpurin- induced DNA damage. Considering reported DNA adduct formation by quinone metabolites of several carcinogenic agents, quinone form of purpurin, which is possibly generated via purpurin autoxidation accompanied by Cu(I)/Cu(II) redox cycle, might lead to DNA adducts and piperidine-labile sites. In addition, we measured contents of 8-oxodG. Purpurin moderately but significantly increased 8-oxodG in calf thymus DNA in the presence of Cu(II). The 8-oxodG formation was inhibited by catalase, methional and bathocuproine, suggesting that Cu(I)-hydroperoxide, which was generated via Cu(I) and H O , caused oxidative DNA base damage. 2 2 Conclusions: We demonstrated that purpurin induces DNA base damage possibly mediated by Cu(I)/Cu(II) redox cycle both with and without ROS generation, which are likely to play an important role in its carcinogenicity. Keywords: Purpurin, Copper, DNA damage, Reactive oxygen species Introduction Anthraquinones are a class of organic compounds abundant in the universe of natural substances and are found in various plant parts such as roots, rhizomes, fruits, and flowers [1 ]. Purpurin (1,2,4-trihydroxy- *Correspondence: s-oikawa@med.mie-u.ac.jp 9,10-anthraquinone) is a natural red anthraquinone Department of Environmental and Molecular medicine, Mie University pigment mostly isolated from madder root (Rubia tinc- Graduate School of Medicine, Edobashi 2-174, Tsu, Mie 514-8507, Japan Full list of author information is available at the end of the article torum) [2]. Historically, purpurin has been used not © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kobayashi et al. Genes and Environment (2022) 44:15 Page 2 of 8 only as a textile dye but also as an ingredient of herbal Preparation of  P‑5 ′‑end‑labeled DNA fragments medicine and a food coloring agent. Purpurin has been DNA fragments were obtained from the human p16 reported to show beneficial biological activities such as tumor suppressor gene [8] and the c-Ha-ras-1 protoon- antioxidant, antimutagenic, antimicrobial, neuropro- cogene [9]. A fragment containing exon 2 of the human tective, antiadipogenic, and anticancer activities [2]. p16 tumor suppressor gene was obtained as described On the other hand, purpurin was reported to induce previously [10]. The 5′ end-labeled 460-base pair frag - marked hyperplasia of pelvic epithelium and urinary ment (EcoRI* 9481-EcoRI* 9940) containing exon 2 was bladder tumors (papilloma and carcinoma) in rats [3]. also further digested with BssHII to obtain the singly Purpurin had also a mutagenic activity in Salmonella labeled 309-base pair fragment (EcoRI* 9481-BssHII without mammalian microsomal activation [4, 5] and 9789) and the 147-base pair fragment (BssHII 9794- was active in various mutagenesis assay systems using EcoRI* 9940). DNA fragments were also prepared from mammalian cells including V79-HGPRT assay, DNA- plasmid pbcNI, which carries a 6.6-kb BamHI chromo- repair assay in primary rat hepatocytes, and transfor- somal DNA segment containing the human c-Ha-ras-1 mation of C3H/M2 mouse fibroblasts [5 ]. Working protooncogene. The singly labeled 98-base pair fragment exposed to dyes has been identified as one of the (AvaI* 2247-PstI 2344) and 337-base pair fragment (PstI major risk factors for occupational bladder cancer [6, 2345-AvaI* 2681) were obtained as previously described 7]. Many carcinogenic dyes induce carcinogenesis via [11]. For reference, nucleotide numbering starts with the DNA adduct formation, but carcinogenic mechanisms BamHI site [9]. An asterisk indicates P-labeling. of purpurin remain unknown. In this study, to clarify the mechanism underlying the Detection of damage to isolated P‑labeled DNA carcinogenicity of purpurin, we investigated purpurin- fragments induced DNA damage in the presence of copper ions. Reaction mixtures containing P-labeled DNA frag- We analyzed DNA damage using P-5′-end-labeled ment, 20 μM/base calf thymus DNA, purpurin and 20 μM DNA fragments obtained from the c-Ha-ras-1 pro- CuCl in 200 μl of sodium phosphate buffer (pH 7.8) con- tooncogene and the p16 tumor suppressor gene. We taining 5 μM DTPA were incubated for 1 h at 37 °C under also measured the contents of 8-oxo-7,8-dihydro-2′- light shielding. To examine the effects of reactive oxygen deoxyguanosine (8-oxodG), a relevant indicator of oxi- species (ROS) scavengers and bathocuproine, these rea- dative DNA damage, in calf thymus DNA using a high gents were added before the addition of purpurin. The performance liquid chromatograph equipped with an DNA fragments were heated in 1 M piperidine for 20 min electrochemical detector (HPLC-ECD). at 90 °C, followed by electrophoresis on an 8% poly- acrylamide/8 M urea gel as previously described [11]. An autoradiogram was obtained by exposing an X-ray film (FUJIFILM Corp., Tokyo, Japan) to the gel. Band inten- Materials and methods sity was quantified using ImageJ software (National Insti - Materials tutes of Health). EcoRI, AvaI, PstI and T polynucleotide kinase were The preferred cleavage sites were determined by purchased from New England Biolabs Ltd. (Ipswich, directly comparing the positions of the oligonucleotides MA, USA). BssHII was purchased from Takara Bio with those produced by the chemical reactions of the Inc. (Shiga, Japan). [γ- P] ATP (222 TBq/mmol) Maxam-Gilbert procedure [12], using a DNA-sequencing was purchased from Perkin Elmer, Inc. (Waltham, system (LKB 2010 Macrophor, LKB Pharmacia Biotech- MA, USA). Purpurin, catalase (30,000 units/mg from nology Inc., Uppsala, Sweden). The autoradiogram was bovine liver) and 3-(methylthio) propionaldehyde obtained by exposing an imaging plate (BAS-MS2040, (methional) were purchased from Sigma-Aldrich Co., FUJIFILM Corp.) to the gel. The relative amounts of oli - LLC. (St Louis, MO, USA). Copper chloride dihydrate gonucleotides from the treated DNA fragments were (CuCl ·2H O), ethanol, mannitol and sodium formate 2 2 measured using a laser scanner (Typhoon FLA-9500, GE were purchased from Nacalai Tesque (Kyoto, Japan). Healthcare, Buckinghamshire, England) and analyzed Diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid with ImageQuant TL software (GE Healthcare). (DTPA) and bathocuproine disulfonic acid were pur- chased from Dojindo Laboratories (Kumamoto, Japan). Analysis of 8‑oxodG formation in calf thymus DNA Nuclease P (500 units/vial) and piperidine were pur- Measurement of 8-oxodG in calf thymus DNA was chased from FUJIFILM Wako Pure Chemical Co., Ltd. performed as described previously [13]. The reaction (Osaka, Japan). Calf intestinal phosphatase (500 units/ mixtures, containing 100 μM/base calf thymus DNA, vial) was purchased from Roche Diagnostics GmbH purpurin and 20 μM CuCl in 400 μl of 4 mM sodium (Mannheim, Germany). 2 Koba yashi et al. Genes and Environment (2022) 44:15 Page 3 of 8 phosphate buffer (pH 7.8) containing 5 μM DTPA, were cleaves DNA at sugars with modified bases, it is reason - incubated for 1 h at 37 °C under light shielding. To exam- able to conclude that purpurin caused base modification ine the effects of ROS scavengers and bathocuproine, but not breakage of deoxyribose phosphate backbone in these reagents were added before the addition of purpu- the presence of Cu(II). rin. After ethanol precipitation, the DNA was digested into nucleosides with nuclease P and calf intestine phos- Eec ff ts of scavengers and bathocuproine on DNA damage phatase. The amount of 8-oxodG was measured using an induced by purpurin in the presence of Cu(II) HPLC-ECD as described previously [14]. Effects of the scavengers and bathocuproine on the dam - age of P-labeled DNA fragments by purpurin plus Statistical analysis Cu(II) were examined. Bathocuproine, a Cu(I)-specific Results were presented as means ± standard deviation chelator [15], prevented purpurin-induced DNA damage (SD). Differences were analyzed by student t-test. P val - in P-labeled DNA fragments in the presence of Cu(II) ues less than 0.05 were considered to be statistically (Fig.  2 and Fig. S1B). Furthermore, methional, a scaven- significant. ger of a variety of ROS [16], partially inhibited the DNA damage. Catalase also showed a slight inhibitory effect on Results the DNA damage. On the other hand, the DNA damage Damage of  P‑labeled DNA fragments by purpurin was not prevented by typical free hydroxyl radical (•OH) in the presence of Cu(II) scavengers such as ethanol, mannitol and sodium for- Figure  1 shows the autoradiogram of DNA fragments mate. These results suggest that ROS could not be major incubated with purpurin plus Cu(II) and followed by mediators of the purpurin-induced DNA damage. treatment with or without piperidine. Oligonucleotides were detected on the autoradiogram as a result of the Site specificity of DNA damage by purpurin in the presence DNA cleavage. In the presence of Cu(II), purpurin caused of Cu(II) DNA cleavage with piperidine treatment, although no The patterns of DNA damage induced by purpurin in the or little DNA cleavage was observed without piperi- presence of Cu(II) were determined by DNA sequencing dine treatment (Fig.  1 and Fig. S1A). Meanwhile, in the using the Maxam-Gilbert procedure [12]. The relative absence of Cu(II), purpurin did not induce DNA cleavage intensity of DNA damage obtained by scanning autora- even with piperidine treatment (data not shown). DNA diogram with a laser scanner is shown in Fig.  3. Purpu- cleavage induced by purpurin plus Cu(II) was detected rin cleaved DNA frequently at guanine (G) and at some at 5 μM of purpurin, and the degree of the DNA cleav- thymine (T) residues in DNA fragments obtained from age was increased at 10, 20, 50 and 100 μM. As piperidine the human c-Ha-ras-1 protooncogene with piperidine Fig. 1 Autoradiogram of P-5′-end-labeled DNA fragments incubated with purpurin in the presence of Cu(II). Reaction mixtures contained the P-5′-end-labeled 147-bp fragment, 20 μM/base calf thymus DNA, the indicated concentrations of purpurin and 20 μM CuCl in 200 μl of 4 mM sodium phosphate buffer (pH 7.8) containing 5 μM DTPA. After incubation at 37 °C for 1 h under light shielding, the DNA fragments were treated with or without hot piperidine and electrophoresed on a polyacrylamide gel Kobayashi et al. Genes and Environment (2022) 44:15 Page 4 of 8 as ethanol, mannitol and sodium formate did not. Batho- cuproine showed an inhibitory effect on the 8-oxodG for - mation. These results suggest the involvement of H O , 2 2 ROS other than •OH and Cu(I) in the 8-oxodG formation caused by purpurin plus Cu(II). Discussion The present study demonstrated that purpurin caused copper-mediated DNA damage in P-labeled DNA fragments. Cleaved oligonucleotides were observed only under piperidine treatment, indicating that pur- purin caused base modification, but not breakage of the deoxyribose-phosphate backbone, in the presence of Cu(II). Bathocuproine completely prevented the piper- idine-labile sites induced by purpurin plus Cu(II), while methional and catalase also displayed limited inhibitory effects on the DNA damage (Fig.  2). Site specificity of the purpurin-induced piperidine-labile sites (predominantly at G) is inconsistent with our previous studies indicating that many ROS-generating agents plus Cu(II) frequently cleaved DNA at cytosine (C) and T under piperidine treatment [17–19]. These results suggest that mecha - nisms other than ROS generation could mainly con- tribute to purpurin-induced DNA damage. Some DNA Fig. 2 Eec ff ts of ROS scavengers and bathocuproine on adducts formed in DNA base has been reported to lead purpurin-induced DNA damage in P-5′-end-labeled DNA fragments. to DNA strand break under piperidine treatment [20– Reaction mixtures contained the P-5′-end-labeled 147-bp fragment, 20 μM/base calf thymus DNA, 50 μM purpurin, each scavenger or 22]. Many quinones are reported to form DNA adduct bathocuproine and 20 μM CuCl in 200 μl of 4 mM sodium phosphate 2 through covalently binding to nucleobases of DNA [23]. buffer (pH 7.8) containing 5 μM DTPA. After incubation at 37 °C for Notably, o-quinone metabolites of carcinogens such as 1 h under light shielding, the DNA fragments were treated with estrogen and bisphenol A contribute to their carcino- piperidine and electrophoresed on a polyacrylamide gel. The genic mechanism via DNA adduct formation [24, 25]. concentrations of each scavenger and bathocuproine were as follows: 0.2 M ethanol, 0.1 M mannitol, 0.1 M sodium formate, 0.6 M u Th s, we considered that the o-quinone form of purpu - methional, 50 U catalase, 50 μM bathocuproine rin, which is generated via purpurin autoxidation accom- panied by Cu(I)/Cu(II) redox cycle, could lead to DNA adducts and piperidine-labile sites (Fig.  5). Although a recent study has reported that purpurin can react with treatment (Fig.  3A). In DNA fragments obtained from Cu(II) generating a metal complex (2:1) under certain the p16 tumor suppressor genes, DNA cleavages at T as conditions [26], the complex was not detected by ultravi- well as G residues were induced by purpurin under piper- olet-visible spectroscopy under our conditions (data not idine treatment (Fig.  3B). Thus, purpurin caused piperi - shown). dine-labile sites predominantly at G and some T residues. Interestingly, the present study indicated that purpurin plus Cu(II) also induced oxidative damage. We examined Formation of 8‑oxodG in calf thymus DNA by purpurin the ability of purpurin to induce 8-oxodG formation. in the presence of Cu(II) 8-OxodG causes DNA misreplication resulting in muta- To investigate oxidative DNA damage, we measured the tion or cancer [27, 28] and is one of the oxidative DNA contents of 8-oxodG, an indicator of oxidative base dam- products generated by the reaction with ROS [29]. Pur- age, in calf thymus DNA treated with purpurin in the purin modestly but significantly increased formation of presence of Cu(II). The formation of 8-oxodG was sig - 8-oxodG in calf thymus DNA in the presence of Cu(II). nificantly increased by purpurin treatment in a concen - To clarify the mechanism of purpurin-induced 8-oxodG tration-dependent manner (Fig.  4A). Figure  4B shows formation, we investigated effects of ROS scavengers and the effects of ROS scavengers and bathocuproine on the bathocuproine. Catalase and bathocuproine exhibited Cu(II)-mediated 8-oxodG formation induced by purpu- inhibitory effects on the increase of 8-oxodG, although rin. Catalase and methional significantly inhibited the not complete. Methional also showed similar results, 8-oxodG formation, while typical •OH scavengers such Koba yashi et al. Genes and Environment (2022) 44:15 Page 5 of 8 32 32 Fig. 3 Site specificity of purpurin-induced DNA damage in P-5′-end-labeled DNA fragments. Reaction mixtures contained the P-5′-end-labeled (A) the 337-bp fragment or (B) the 309-bp fragment, 20 μM/base calf thymus DNA, 50 μM purpurin and 20 μM CuCl in 200 μl of 10 mM sodium phosphate buffer (pH 7.8) containing 5 μM DTPA. After incubation at 37 °C for 1 h under light shielding, the DNA fragments were treated with piperidine and electrophoresed on a polyacrylamide gel. DNA bases in bold represent preferentially cleaved bases Fig. 4 Formation of 8-oxodG in calf thymus DNA by purpurin in the presence of Cu(II). (A) Reaction mixtures contained 100 μM/base calf thymus DNA, the indicated concentrations of purpurin and 20 μM CuCl in 400 μl of 4 mM sodium phosphate buffer (pH 7.8) containing 5 μM DTPA. (B) Reaction mixtures contained 100 μM/base calf thymus DNA, 100 μM purpurin, 20 μM CuCl and each scavenger or bathocuproine in 400 μl of 4 mM sodium phosphate buffer (pH 7.8) containing 5 μM DTPA. The concentrations of each scavenger and bathocuproine were as follows: 0.2 M ethanol, 0.1 M mannitol, 0.1 M sodium formate, 0.6 M methional, 50 U catalase, 50 μM bathocuproine. Reaction mixtures were incubated at 37 °C for 1 h under light shielding. After ethanol precipitation, the DNA was digested to nucleosides with nuclease P and calf intestine phosphatase, then analyzed with an HPLC-ECD system. * p < 0.05 vs 0 μM. # p < 0.05 vs purpurin plus Cu(II). Significance was analyzed using Student t-test Kobayashi et al. Genes and Environment (2022) 44:15 Page 6 of 8 Fig. 5 A possible mechanism of DNA base damage induced by purpurin and Cu(II) while •OH scavengers did not inhibit the 8-oxodG forma- Future research should focus on the association between tion. These results suggest that Cu(I)/Cu(II) redox cycle copper and purpurin for understanding of mechanism of and concomitant H O production possibly via autoxida- purpurin carcinogenicity. 2 2 tion of purpurin lead to generate Cu(I)-hydroperoxide, which induces oxidative DNA base damage including 8-oxodG formation (Fig. 5). Conclusions A higher level of serum copper was observed in blad- We demonstrated that purpurin plus Cu(II) induces Cu(I)/ der cancer patients than in healthy controls [30]. A recent Cu(II) redox cycle-mediated DNA base damage, possibly case-controlled clinical study reported that plasma level through both ROS-independent and -dependent mecha- of copper was significantly and positively associated with nisms, which are likely to contribute to its carcinogenicity. bladder cancer development, suggesting that the increase Abbreviations in plasma copper may be one of the risk factors for blad- 8-oxodG: 8-oxo-7,8-dihydro-2′-deoxyguanosine; DTPA: Diethylenetriamine- der cancer [31]. Many studies have reported that copper N,N,N′,N″,N″-pentaacetic acid; HPLC-ECD: A high performance liquid chromato- graph equipped with an electrochemical detector; ROS: Reactive oxygen species. plays a role in promoting carcinogenesis though induc- ing oxidative stress and resultant DNA damage [32–34]. Supplementary Information Copper ions have been known to be particularly adept The online version contains supplementary material available at https:// doi. at helping other agents to damage DNA and to enhance org/ 10. 1186/ s41021- 022- 00245-2. mutagenic activities via production of ROS [35, 36]. Our previous studies also demonstrated that copper mediated Additional file 1. the various carcinogens-induced ROS production and oxidative DNA damage [19, 37, 38]. However, the present Acknowledgements study suggests that purpurin plus Cu(II) leads to DNA Not applicable. damage through not only ROS generation but also pos- Authors’ contributions sible DNA adduct formation mediated by Cu(I)/Cu(II) SO and HK conceived and designed the study. HK, SO and YM wrote paper. HK, redox cycle (Fig. 5). Therefore, both oxidative DNA dam - SO, YM, RI, YH and Shinya K performed experiment and data analysis. SO, HK, Sho- age and DNA adduct could be involved in the carcino- suke K and MM performed data curation. Shosuke K and MM reviewed and criti- cally edited the paper. All authors have read and approved the final manuscript. genic mechanism of purpurin. Several previous studies reported that purpurin is also Funding capable of binding DNA based on voltammetric, elec- This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 19H03885, 18 K19673. trochemical and spectroscopic analysis [39–41]. These findings suggest that purpurin, copper and DNA might Availability of data and materials coexist and interact with each other and lead to DNA Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. damage via ROS generation and DNA adduct formation. Koba yashi et al. Genes and Environment (2022) 44:15 Page 7 of 8 producing oxygen radicals and evidence for its repair. Carcinogenesis. 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Journal

Genes and EnvironmentSpringer Journals

Published: May 9, 2022

Keywords: Purpurin; Copper; DNA damage; Reactive oxygen species

References