I

Figure 5 DNA lesions, as measured by the mean number (± SD) of sister chromatid exchanges (SCE) per 25 metaphase (SCE; O), and induction of apoptosis, as measured by the expression of mitochondrial Apo2.7 molecules (Apo2.7+; •) in a representative experiment. To measure SCE, PHA-activated peripheral blood mononuclear cells were incubated with quercetin and syringin at 0, 0.1, 1, 10 and 100 ptM for 72 h, while the investigation of apoptosis was performed on isolated lymphocytes after a 24 h incubation period as described (Büssing et al., 1996a). " p<0.05 (Wilcoxon's signed rank test)

Figure 5 DNA lesions, as measured by the mean number (± SD) of sister chromatid exchanges (SCE) per 25 metaphase (SCE; O), and induction of apoptosis, as measured by the expression of mitochondrial Apo2.7 molecules (Apo2.7+; •) in a representative experiment. To measure SCE, PHA-activated peripheral blood mononuclear cells were incubated with quercetin and syringin at 0, 0.1, 1, 10 and 100 ptM for 72 h, while the investigation of apoptosis was performed on isolated lymphocytes after a 24 h incubation period as described (Büssing et al., 1996a). " p<0.05 (Wilcoxon's signed rank test)

Co-carcinogenicity of the flavonoids, however, is still controversial. The putative genotoxic metabolites of quercetin vary for different genetic end-points considered, and the fate of flavonoids might partly account for the conflicting data about their genotoxicity in vivo and carcinogenic activity (Rueff et al., 1986). Oliveira et al. (1997) addressed the question of exposure to low levels present in the diet and approved an adaptive response by low doses of quercetin to challenging doses of quercetin, hydrogen peroxide and mitomycin C, using induction of chromosomal aberrations in V79 cells as the end point. Moreover, oral uptake of quercetin at concentrations that were about 103 times greater than the estimated average human intake of total flavonoids did not show mutagenicity/carcinogenicity in mice, as measured by micronucleus test and Ames Salmonella tester strain TA 98 (Aeschenbacher et al., 1982). Calomme et al. (1996) stated that quercetin should not merely be regarded as a genotoxic risk factor in the human diet, since its mutagenicity may be inhibited by accompanying compounds including other flavonoids, and since quercetin itself exhibited an anitmutagenic effect against 2-aminofluorene in the Ames test (Calomme et al., 1996), and produced anti-genotoxic effects in combination with food mutagens such as 3-amino-1-metyl-5H-pyrido (4,3-b)indole (Trp) and 2-amino-3-methylimidazo (4,5-f)quinoline (IQ) effects in human lymphocytes and sperm (Anderson et al., 1997).

The mutagenic or anti-mutagenic effects of quercetin are further dependent on the mutagen tested and its activation (Ogawa et al., 1985). In fact, quercetin enhanced the mutagenicity of tricyclic aromatic amines (aminofluorene, aminoanthracene, aminophenanthrene) and their acetamides, whereas the mutagenicity of aniline derivatives, biphenyl derivates, and bi- and tetra-cyclic amino derivatives are depressed (Ogawa et al., 1985, 1987a, b, c). Here, quercetin may promote N-hydroxylation and deacetylation in the microsomes, and inhibits deacetylation in the cytosol. The mutagenicity-modulation of heterocyclic amines (Trp-P-1, Trp-P-2, Glu-P-1, Glu-P-2) by quercetin was liable to be affected by the content of S9 in a mammalian metabolic activation system (Ogawa et al., 1987b). Mutagenicity and mutagenicity-enhancing effects of flavonoids, as measured in the Ames test, seem to depend on the number of hydroxyl groups substituted at the 3', 4' and 5' position of the B ring, and the presence of a free hydroxy or methoxy group in the 7 position of the A ring, while the presence of a hydroxyl group at the 2' position in the B ring of the flavonoid molecule markedly decreases its mutagenic activity (Ogawa et al., 1987a; Czeczot et al., 1990).

An inhibition of quercetin's mutagenic activity was observed by the addition of metal salts (MnCl2, CuCl2, FeSO4, and FeCl3), probably by facilitating catalytic oxidation of the flavonoid, while ascorbate, superoxide dismutase, NADH and NADPH enhanced the mutagenic activity of quercetin (Hatcher and Bryan, 1985). Addition of liver homogenate (S9 mix) may enhance the mutagenic activity of quercetin by scavenging superoxide radicals, thus inhibiting its autoxidation, and possibly by reducing quinone oxidation products of quercetin (Hatcher and Bryan, 1985). However, other reports point out that in mammalian cells quercetin is unable to induce SCE and point mutations, and that a putative clastogenic effect of the flavonoid is abolished by the addition of liver homogenate (van der Hoeven et al., 1984).

Modulation of the detoxifying systems by flavonoids and their metabolites may be one of the key factors to explain the conflicting findings. In fact, polyphenolic flavonoids such as quercetin, myriacetin and kaempferol were observed to decrease the content of nuclear antioxidant defense glutathione (GSH) and glutathione S-transferase (Sahu and Gray, 1996), and thus can lead to oxidative DNA damage, which may be responsible for their mutagenic effects. However, depletion of reduced GSH by quercetin occurred prior to death of lymphocytes, Caco-2, HepG2, and HeLa cells (Duthie et al., 1997), indicating that oxidative stress by itself may induce apoptosis, or that oxidative DNA damages induces arrest of the cell cycle via accumulation of the tumour-suppressor protein p53. Indeed, cell death induced by quercetin was observed in G1 and S phase of the cell cycle of leukemic cell lines (Yoshida et al., 1992; Wei et al., 1994; Larocca et al., 1996), an effect associated with a suppression of growth-related genes histone H4, cyclin A and B, and p34cdcc2 (Yoshida et al., 1992). In the non-tumour cell line C3H10T1/2CL8 induction of apoptosis and induction of the p53 protein occurred out of the G2/M phase of the cell cycle (Plaumann et al., 1996). The G2/M arrest seems to be p53-dependent as it did not occur in p53 knockout fibroblasts. Also in MDA-MB468 human breast cancer cells, the block of cell cycle at the G2/M phase was associated with a prevention of the accumulation of newly synthesized p53 protein (Avila et al., 1994).

The exact underlying mechanisms leading to apoptosis are unclear. The initial interaction of quercetin with DNA may have a stabilizing effect on its secondary structure, but prolonged treatment leads to an extensive disruption of the double helix (Alvi et al., 1986). Quercetin may also block signal transduction pathways by inhibiting protein tyrosine kinases and serine/threonine protein kinases (Hagiwara et al., 1988; Ferriola et al., 1989; Kang and Liang, 1997), 1-phosphatidylinositol 4-kinase and 1-phosphatidylinositol 4-phosphate 5-kinase resulting in a reduction of inositol-1,4,5-trisphosphate (Nishioka et al., 1989; Kang and Liang, 1997) which should decrease the release of Ca2+ from intracellular sources. An early down-regulation of the c-myc and Ki-ras oncogenes (Csokay et al., 1997; Weber et al., 1997) is suggested to be part of the antiproliferative action of quercetin in K562 human leukemia (Csokay et al., 1997). Larocca et al. (1996) postulated that quercetin exerts its growth inhibitory action by interaction with type II estrogen binding sites and subsequent induction of transforming growth factor (TGF)-ß1 expression and secretion.

Findings of Yokoo and Kitamura (1997) elucidated a novel action of quercetin as an apoptosis inhibitor. Pretreatment with quercetin protected mesangial cells from hydrogen peroxide (H2O2)-induced apoptosis. A similar effect was observed in other cell types including LLC-PK1 epithelial cells and NRK49F fibroblasts. This cytoprotective effect was found to be mediated via suppression of the tyrosine kinase-c-Jun/activator protein-1 (AP-1) pathway triggered by oxidant stress. However, quercetin did not inhibit caspase-3 activation and Apo2.7 expression in ML I-treated human lymphocytes (Büssing, unpublished results).

Although the bioavailability of quercetin and its derivatives from Viscum album is unclear, one may not ignore the fact, that flavonoids from VA-E applied repeatedly to tumour patients may accumulate in the blood. However, Lorch was unable to detect quercetin in mistletoe grown on appletree, pine tree or fir tree: VA-E from fir tree contain predominantly homoeriodyctiol-glycoside, and in mistletoe from pine 5,7-dimethoxy-4-hydroxyflavanon (personal communication). Both flavonoids were found in about 3 mg/g dried plant material. However, whole plant extracts from Viscum album were recognised not to induce mutagenic and/or genotoxic effects (see Stein, this book).

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