Protective and detrimental effects of kaempferol in rat H4IIE cells: implication of oxidative stress and apoptosis.

Flavonoids are ubiquitous substances in fruits and vegetables. Among them, the flavonol kaempferol contributes up to 30% of total dietary flavonoid intake. Flavonoids are assumed to exert beneficial effects on human health, e.g., anticancer properties. For this reason, they are used in food supplements at high doses. The aim of this project was to determine the effects of kaempferol on oxidative stress and apoptosis in H4IIE rat hepatoma cells over a broad concentration range. Kaempferol is rapidly taken up and glucuronidated by H4IIE cells. The results demonstrate that kaempferol protects against H2O2-induced cellular damage at concentrations which lead to cell death and DNA strand breaks in the absence of H2O2-mediated oxidative stress. Preincubation with 50 microM kaempferol exerts protection against the loss of cell viability induced by 500 microM H2O2 (2 h) while the same concentration of kaempferol reduces cell viability by 50% in the absence of H2O2 (24 h). Preincubation with 50 microM kaempferol ameliorates the strong DNA damage induced by 500 microM H2O2 while 50 microM kaempferol leads to a significant increase of DNA breakage in the absence of H2O2. Preincubation with 50 microM kaempferol reduces H2O2-mediated caspase-3 activity by 40% (4 h) while the same concentration of kaempferol leads to the formation of a DNA ladder in the absence of H2O2 (24 h). It is concluded that the intake of high dose kaempferol in food supplements may not be advisable because in our cellular model protective kaempferol concentrations can also induce DNA damage and apoptosis by themselves.

Low concentrations of flavonoids are protective in rat H4IIE cells whereas high concentrations cause DNA damage and apoptosis.

Dietary flavonoids possess a wide spectrum of biochemical and pharmacological actions and are assumed to protect human health. These actions, however, can be antagonistic, and some health claims are mutually exclusive. The antiapoptotic actions of flavonoids may protect against neurodegenerative diseases, whereas their proapoptotic actions could be used for cancer chemotherapy. This study was undertaken to determine whether a cytoprotective dose range of flavonoids could be differentiated from a cytotoxic dose range. Seven structurally related flavonoids were tested for their ability to protect H4IIE rat hepatoma cells against H(2)O(2)-induced damage on the one hand and to induce cellular damage on their own on the other hand. All flavonoids proved to be good antioxidants in a cell-free assay. However, their pharmacologic activity did not correlate with in vitro antioxidant potential but rather with cellular uptake. For quercetin and fisetin, which were readily taken up into the cells, protective effects against H(2)O(2)-induced cytotoxicity, DNA strand breaks, and apoptosis were detected at concentrations as low as 10-25 micromol/L. On the other hand, these flavonoids induced cytotoxicity, DNA strand breaks, oligonucleosomal DNA fragmentation, and caspase activation at concentrations between 50 and 250 micromol/L. Published data on quercetin pharmacokinetics in humans suggest that a dietary supplement of 1-2 g of quercetin may result in plasma concentrations between 10 and 50 micromol/L. Our data suggest that cytoprotective concentrations of some flavonoids are lower by a factor of 5-10 than their DNA-damaging and proapoptotic concentrations.

Polyphenols from plants used in traditional Indonesian medicine (Jamu): uptake and antioxidative effects in rat H4IIE hepatoma cells.

Phytochemical investigation of plants used in traditional Indonesian medicine (Jamu) yielded lignans (pinoresinol, 9 alpha-hydroxypinoresinol and salicifoliol), flavonoids (3-O-beta-(D)-glucopyranosyl-(1-->6)-beta-(D)-glucopyranosylkaempferol, luteolin and apigenin) and coumarins (coumarin, 8-hydroxycoumarin and 5-hydroxycoumarin). The beneficial effects of the respective plants for human health are thought to be associated with antioxidative activity. In the present study, the antioxidative capacity of the isolated compounds was determined in an in-vitro assay. Luteolin and kaempferol (cleavage product of 3-O-beta-(D)-glucopyranosyl-(1-->6)-beta-(D)-glucopyranosylkaempferol, which is thought to be formed in the intestine) showed strong antioxidant activity; pinoresinol and 9 alpha-hydroxypinoresinol showed only minor antioxidative effects. The coumarins, as well as apigenin and 3-O-beta-(D)-glucopyranosyl-(1-->6)-beta-(D)-glucopyranosylkaempferol were inactive. The antioxidative effects of luteolin, kaempferol and pinoresinol were further investigated in H4IIE rat hepatoma cells. A strong protective effect of kaempferol and luteolin was found against H2O2-mediated intracellular reactive oxygen species formation measured using the dichlorofluorescein assay and H2O2-mediated DNA strand breaks. Pinoresinol did not have a protective effect against H2O2-mediated DNA-damage, but in the dichlorofluorescein assay, an antioxidative effect was detectable. During studies with H4IIE cells, kaempferol, luteolin and pinoresinol were taken up by the cells within 60 min. The flavonoids were found to be relatively toxic at higher concentrations, while pinoresinol was less cytotoxic. In conclusion, kaempferol and luteolin, at low concentrations (< or = 50 microM), protect H4IIE cells against oxidative stress but are cytotoxic at higher concentrations; the biological effects of pinoresinol are less prominent in comparison. These results are important for the identification of pharmacologically active substances from traditional Indonesian medicinal plants.