Possible contribution of rubiadin, a metabolite of madder color, to renal carcinogenesis in rats.

Madder color (MC) has been shown to exert carcinogenic potential in the rat kidney in association with degeneration, karyomegaly, increased cell proliferation of renal tubule cells and increased renal 8-OHdG levels. To clarify the causal relationship of components and metabolites of MC to renal carcinogenesis, male F344 rats were fed lucidin-3-O-primeveroside (LuP) or alizarin (Alz), and the genotoxic LuP metabolites lucidin (Luc) or rubiadin (Rub) for up to 26 weeks. After one week and four weeks, Luc did not induce any renal changes. In contrast, after one week, cortical tubule degeneration was apparent in the Alz and LuP groups, and cytoplasmic swelling with basophilic change and karyomegaly in the outer medulla was observed only in the Rub group. LuP and Rub increased the proliferative activity of tubule cells in the outer medulla, and Alz and LuP increased renal 8-OHdG levels. After 26 weeks, Rub but not Alz induced atypical tubules, a putative preneoplastic lesion, and karyomegaly in the outer medulla. These results indicate that Rub may be a potent carcinogenic metabolite of MC, targeting proximal tubule cells in the outer medulla, although oxidative stress increased by Alz or LuP might also be involved in renal carcinogenesis by MC.

Induction of kidney and liver cancers by the natural food additive madder color in a two-year rat carcinogenicity study.

Madder color (MC) extracted from the roots of Rubia tinctorum (madder root) has been used as a food coloring in Japan. Our previous studies revealed MC to have obvious subchronic and chronic toxicity and potent carcinogenicity targeting rat liver and kidney. In the present two-year carcinogenicity study, conducted to further elucidate the long-term effects of MC and its target organs, male and female F344 rats were fed diet containing 0%, 2.5%, and 5.0% MC for 104 weeks. Body weights were significantly decreased in treated groups of both sexes throughout the feeding period. However, survival rates at week 104 were higher in treated groups of both sexes than in controls. Relative weights of the kidneys and liver were significantly increased in treated groups of both sexes. Histopathologically, karyomegaly and atypical tubules/hyperplasias, as well as renal cell adenomas and carcinomas were significantly increased in treated groups of both sexes with dose-dependence. Moreover, the incidence of hepatocellular adenomas and/or carcinomas was increased significantly with a dose-relation in treated groups of both sexes. These data provide clear evidence that MC exerts unequivocal carcinogenicity against renal tubule cells and hepatocytes in rats.

One-year chronic toxicity of madder color in F344 rats--induction of preneoplastic/neoplastic lesions in the kidney and liver.

To evaluate chronic toxicity of madder color (MC), a natural food colorant extracted from the roots of Rubia tinctorum L., F344 rats were fed diet containing 0%, 0.2%, 1.0% or 5.0% MC for 53 weeks. Hematological changes including anemia and serum biochemical alterations indicating hepatotoxicity were demonstrated at 5.0% in both sexes. Relative weights of the liver were significantly increased from 1.0% in both sexes, and those of the kidney were significantly increased from 1.0% in males and from 0.2% in females. Histopathologically, atypical renal tubule hyperplasias were increased at 1.0% or higher in both sexes in association with increase of cell proliferative activity in the tubules. A renal cell adenoma was observed in a male rat receiving 5.0% MC. In addition, glutathione S-transferase placental form-positive liver cell foci were significantly increased at 5.0% in both sexes. These results indicate that MC has chronic toxicity targeting kidney, liver and blood cells. Moreover, the results strongly suggest that MC may have the carcinogenic potential in the kidney and the liver.

Promotion potential of madder color in a medium-term multi-organ carcinogenesis bioassay model in F344 rats.

A medium-term multi-organ carcinogenesis bioassay in rats was conducted to assess any possible tumor promoting effects of madder color extracted from the root of madder. Male F344 rats were divided into 5 groups of 20 each. All rats of groups 1 to 4 were given DMD treatment, consisted of multicarcinogens, N-nitrosodiethylamine (DEN), N-methyl-N-nitrosourea (MNU), and N-bis (2-hydroxypropyl) nitrosamine (DHPN), for 4 wk, while group 5 served as untreated control without carcinogens. The animals were then administered a basal diet containing madder color at doses of 5.0% (group 1), 2.5% (group 2 with 0.75% additional dextrin), or 0 (groups 3 with 1.5% additional dextrin, 4 without dextrin and 5) for the following 28 wk (total 32 wk). The total amount of dextrin in groups 1 to 3 diets was adjusted to 1.5% by extra dextrin because madder color powder contained dextrin. Key organs were observed histopathologically and glutathione S-transferase placental form (GST-P) positive foci of the liver were quantified. In the liver, 5.0% and 2.5% treated groups showed statistically significant dose-related increases in both number and area of GST-P positive foci, number: 2.81 +/- 0.90 and 1.96 +/- 0.93 (groups 1 and 2), area: 0.99 +/- 2.49 and 0.37 +/- 0.77, as compared with control, number: 0.87 +/- 0.72, area: 0.06 +/- 0.06 (group 3). In the kidneys, incidences (and numbers) of adenoma treated with 5.0% and 2.5%, 47.4% (0.20 +/- 0.24), and 47.4% (0.13 +/- 0.15) (groups 1 and 2) were significantly increased compared to control, 0% (0) (group 3). In conclusion, madder color demonstrated significant tumor promoting effects in the liver and kidneys in the DMD model.

A 13-week subchronic toxicity study of madder color in F344 rats.

A 13-week repeated oral dose toxicity study of madder color (MC), a natural food colorant extracted from the roots of Rubia tinctorum L., was performed using F344 rats. Five groups of animals, each consisting of 10 males and 10 females, were fed diet containing 0, 0.6, 1.2, 2.5 or 5.0% MC for 13 weeks. During the experiment, lower body weight was evident from the 2.5% dose. Hematologically, fluctuation in red blood cell (RBC) parameters suggestive of weak anemia (females), and slight increases of platelet counts (both sexes) and white blood cell (WBC) counts (males) were observed at higher doses. Serum biochemically, slight fluctuations were observed in many parameters, including increased total protein (TP), conjugated bilirubin, Ca, and inorganic phosphate, and decrease of the albumin/globulin (A/G) ratio in both sexes, with dose-dependence for TP and A/G from 0.6% in females. Histopathological changes were mainly observed in the renal proximal tubules, such as microvesicular vacuolar degeneration in the cortex and karyomegaly in the outer medulla involving both sexes, lesions being evident even with 0.6%. In the outer medulla, elevation of cell proliferation activity as assessed with proliferating cell nuclear antigen was observed in males from 2.5%. Severity of focal necrosis of hepatocytes was increased only in females at 5.0%, while the increased relative liver weight as with the increased conjugated bilirubin was evident in both sexes from 1.2%. The results thus suggest that MC exerts mild toxicity, targeting liver, kidneys, and possibly RBCs and WBCs, some renal changes being evident from 0.6% in diet, that is attributable to be the lowest-observed adverse effect level (305.8-309.2mg/kg body weight/day).

Cytotoxic anthraquinones from the stems of Rubia wallichiana Decne.

From the stems of Rubia wallichiana DECNE, thirty-four structurally related compounds were isolated and identified. Three of them, namely rubiawallin-A (1), -B (2), and -C (3), constitute the first report of their occurrence from the natural source. Their structures were determined by comprehensive analyses of their 1D and 2D NMR, and electron impact (EI) mass spectral data. Furthermore, an in vitro screening of cytotoxicity of the isolated compounds was also evaluated. Among the testing compounds, 1-hydroxy-2-hydroxymethyl-3-methoxyanthraquinone (4) demonstrated most effective cytotoxicity towards Hepa-3B and Colo-205 cells.