Formation of genotoxic metabolites from anthraquinone glycosides, present in Rubia tinctorum L.

Rubia tinctorum L., a medicinal plant used for the treatment of kidney and bladder stones, contains a characteristic spectrum of 9,10-anthraquinone derivatives, which are substituted in only one of the aromatic benzo rings. The majority of the anthraquinones present in the plant itself or in plant extracts are glycosides. We investigated the metabolism of two such glycosides, alizarinprimeveroside (AlP) and lucidinprimeveroside (LuP). AlP given orally to rats was metabolized to alizarin (Al) and 1-hydroxyanthraquinone (1-HA). The reductive cleavage of AlP was also observed after treatment of this compound with rat liver enzymes (S9) and NADPH. 1-HA has been reported to induce unscheduled DNA synthesis (UDS) in primary rat hepatocytes (PRH) and intestinal and liver tumors in rats after chronic treatment. The in vitro genotoxicity of 1-HA was confirmed by our present investigations. We also observed that the glycoside AlP was active at inducing UDS in PRH, but the compound was inactive in the Salmonella/microsome assay. Oral administration of LuP to rats resulted in the excretion of lucidin and rubiadin. When LuP was treated with rat liver extract and NADPH, the compound was reduced to rubiadinprimeveroside (RuP), which was hydrolyzed to rubiadin. We have recently shown that lucidin is highly genotoxic in a battery of short-term tests. We now report that rubiadin is also highly genotoxic in Salmonella typhimurium. However, in contrast to lucidin, it requires metabolic activation. In the UDS assay in PRH, rubiadin was even more potent than lucidin and equal to the positive control DMBA. In addition, the glycoside LuP is active in the Salmonella/microsome assay as well as in the UDS assay. The present work demonstrates that the uptake of the anthraquinone glycosides AlP and LuP leads to the rodent carcinogen 1-HA, and to the highly genotoxic compounds lucidin and rubiadin. This extends our previous studies and supports our suggestion that the therapeutic use of Rubia tinctorum may involve a carcinogenic risk.

Evaluation of DNA-binding activity of hydroxyanthraquinones occurring in Rubia tinctorum L.

The naturally-occurring anthraquinones (AQs), alizarin (1,2-dihydroxyanthraquinone) and lucidin (1,3-dihydroxy-2-hydroxymethylanthraquinone), were incubated with DNA in the presence of S9 mix. The isolated DNA was analysed by 32P-postlabelling for the presence of aromatic adducts. Only lucidin formed up to five different DNA adducts in the range from 0.995 to 3.05 adducts/10(8) nucleotides. Lucidin was also incubated with polynucleotides poly[d(A-T)] and polydG*polydC in the presence of S9 mix. Analysis of polydG*polydC revealed a similar adduct pattern to that obtained with lucidin-modified DNA. Alizarin, lucidin, a glycoside mixture containing alizarinprimeveroside and lucidinprimeveroside, and Rubia Teep (a herbal drug made from Rubia tinctorum containing lucidin) were incubated with primary rat hepatocytes for 24 h and the isolated DNA was analysed by 32P-postlabelling. Lucidin, the glycoside mixture and Rubia Teep gave rise to DNA adducts, but alizarin did not. Male Parkes mice were treated orally for 4 days with alizarin (10 mg/d), lucidin (2 mg/d), the glycoside mixture (20 mg/d) or Rubia Teep (1/2 tablet/d) and DNA was isolated from liver, kidney, duodenum and colon. Analysis by 32P-postlabelling revealed that lucidin, the glycoside mixture and Rubia Teep, but not alizarin, formed DNA adducts in all the tissues examined but that the adduct patterns were organ-specific.