Lack of tumor-promoting effects of flavonoids: studies on rat liver preneoplastic foci and on in vivo and in vitro gap junctional intercellular communication.

Possible tumor-promoting activity of four flavonoids, quercetin (QC), tangeretin (TG), flavone (FO), and flavanone (FN), was examined in a rat liver short-term carcinogenesis assay as well as with in vivo and in vitro assays of inhibition of gap junctional intercellular communication (GJIC). Rat hepatocarcinogenesis was induced by aflatoxin B1 treatment followed by a selection phase (2-acetylaminofluorene treatment and partial hepatectomy), then treatment with or without test chemicals (in vivo studies of antipromotion were not performed). Using glutathione S-transferase placental form (GST-P)-positive foci, we compared the effects of flavonoids (at 1,000 ppm in the diet) with the effects of phenobarbital (PB) on the occurrence of liver preneoplastic lesions. In addition, we studied the effects of flavonoids on GJIC in the livers derived from these experiments and in two types of cultured cells. No significant difference in the number and area of GST-P-positive foci was found after one or three months of treatment between any flavonoid group and control group. In the positive control group, PB markedly increased the numbers and areas of preneoplastic lesions at three months. Whereas PB also decreased by 60% the average size of lucifer yellow dye spread in slices of liver parenchyma free of preneoplastic lesions among the different flavonoids, only TG decreased the dye transfer in vivo: by 30% at one month and 50% at three months. With the dye transfer assay applied to a rat liver epithelial cell line (REL) and the Chinese hamster V79 metabolic cooperation assay, none of the tested flavonoids (< or = 25 microM) inhibited GJIC. Conversely, protective properties were seen for some of the compounds in antipromotion in vitro studies, because TG and FN enhanced the dye transfer in REL cells and FO, TG, and QC partly prevented the inhibition of metabolic cooperation by 12-O-tetradecanoylphorbol-13-acetate. Thus, taken together, our results suggest that QC, FO, and FN do not show tumor-promoting activity. Concerning TG, some discrepancies in the in vivo data are observed. Some of them (GJIC inhibition in liver slices) are probably more relevant to promotion of hepatocarcinogenesis.

Studies on the modulating effects of retinoic acid and retinol acetate using dye transfer and metabolic cooperation assays.

The effects of retinoic acid and retinol acetate on gap junctional communication were examined in two in vitro tests. Rat liver epithelial cell line IAR 203 was used for dye transfer assays, and hamster lung fibroblast V79 cells were used for metabolic cooperation assays. A reversible dose-dependent inhibition of dye transfer was detected after a 1-hr treatment with retinoic acid or retinol acetate at concentrations ranging from 10 to 50 microM. On the other hand, enhancement of dye transfer was observed after a 24-hr treatment with retinoic acid at 0.1 microM. A dose-dependent inhibition of metabolic cooperation was obtained with retinoic acid at noncytotoxic concentrations ranging from 5 to 50 microM. Retinoids and TPA (1 ng/ml) acted synergistically in their inhibition of cell communication. Thus, the assays appear to be complementary: the dye transfer assay was useful in studying the time course and the reversibility of the inhibition or enhancement of dye transfer, whereas the metabolic cooperation assay was effective in quantifying the inhibitory effect of TPA or retinoids and interactions between them.

Biological activity of a fluorescent 12-O-tetradecanoyl-phorbol-13-acetate derivative.

A newly synthesized fluorescent TPA derivative, O-(N-dansylamino-3-tetradecanoyl)-12,O-acetyl-13-phorbol (Dansyl-TPA), inhibited metabolic cooperation of Chinese hamster V79 cells in culture and also the specific binding of [3H]phorbol dibutyrate to V79 cells. Dansyl-TPA should be a valuable tool for studying the cellular targets of TPA.