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 examined using 32P-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 32P-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 predominantly 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 H2O2, caused oxidative
DNA base damage.
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.