A study enrolling Turkish medicinal herbs found that fruits of Viscum album (bought from a herbalist market) significantly increased the number of DNA strand breaks as measured by COMET assay (alkaline single cell gel electrophoresis), while the extract, however, did not produce a positive response in the microsomal activation assay (Ames test) (Basaran et al., 1996). These conflicting results were not approved by other groups with commercially available aqueous VA-E extracts from fresh plant material (Büssing et al., 1994, 1995a, c, 1996a; Mengs et al., 1997; Mengs, 1998), and with crude, heated or ethanolic extracts from Korean mistletoe (Ham et al., 1998). Moreover, VA-E contain DNA stabilization properties for peripheral blood mononuclear cells. This effect has been demonstrated with the significant reduction of spontaneous and cyclophosphamide (CP)-induced sister chromatid exchange (SCE)-inducing DNA lesions of human peripheral blood mononuclear cells with the whole plant extract Helixor A (Büssing et al., 1994, 1995c, 1996a). In rapidly proliferating amniotic fluid cells, the fermented VA-E Iscador P significantly reduced the SCE level only at very high concentrations (Büssing et al., 1995b). Concomitantly, the drug extract Helixor A also protected CP-mediated depression of activation-associated surface molecules, specifically interleukin-2 receptor a chains (CD25) and transferrin receptors (CD71) on T cells (Büssing et al., 1995c). However, purified components, such as the ML and viscotoxins, did not prevent the activation marker depression on T cells mediated by CP (Büssing et al., 1995c), indicating that the whole plant extract is effective, not the purified compounds. In leukemic Jurkat T cells, however, the simultaneous addition of CP and VA-E resulted in a more severe decline of cell numbers than CP alone (Büssing et al., 1996a). In mice infected with mamma carcinoma cells and treated with CP and VA-E (Isorel), the number of lung metastases was severely reduced compared to animals treated with CP or VA-E alone (Büssing et al., 1996b). Thus, in the murine model and in cultured leukemic cells, VA-E produced no protective effects.
Using a fermented VA-E (Iscador M), Kovacs and co-workers (1991) observed an improved incorporation of [3H]-thymidin in the DNA of UV-damaged lymphocytes from breast cancer patients after subcutaneous application. Based on sedimentation of DNA strand breaks on an alkaline sucrose gradient, VA-E treatment further increased DNA repair of radiation and CP-induced damage of lymphocytes in breast cancer patients (Kovacs et al., 1995, 1996). Murine models illustrated the protective effects of VA-E for CP-treated and y-radiated mice with increased survival and reduced leukocytopenia (Kuttan and Kuttan, 1993b; Narimanov et al., 1992). Recently published studies have shown that administration of a fermented VA-E (Iscador M) significantly reduced sarcomas induced in the C57BL/6 mice by 20-methylcholanthrene administration (Kuttan et al., 1996, 1997). Only one animal among 15 developed sarcoma within an observation period of 160 days in contrast to all control-mice that developed sarcoma within 80 days.
Irregardless of the mechanism of action, the results indicate that VA-E reduces DNA damages induced by carcinogens and probably inhibits tumour development. Whether these effects are of benefit for the patients especially during chemotherapy, is under investigation in an ongoing controlled and randomized clinical study.
Apart from this, cellular abnormalities such as uni-, bi- and multipolar anaphases, resulting in tetra- and aneuploidy, asynchronic bimitoses, and micronuclei were detected in the root tips of onions (Allium cepa) after treatment with the drug extract produced under the conditions described by Winterfield and co-workers (Acatrinei, 1966, 1967, 1969). These effects were thought to be related to a destruction of the spindle apparatus by the mistletoe extract. The findings are in agreement with those of our group (Büssing et al., 1998b), as we observed increased frequencies of telomeric associations and C-anaphases in lymphocytes treated with aqueous drug extracts from mistletoe from fir trees and by ML III at 10 ng/ml. Clearly, a correlation exists with chromosomal affections and apoptotic changes induced by VA-E (Table 2). Using a whole plant extract from VA-E (Lektinol) Mengs et al. (1997) did not observed gene mutations and chromosomal aberrations, as measured in bacteria and mammalian cells.
Using non-toxic concentrations of ML I, ML II and ML III (0.001, 0.014, 0.14 ng/ ml), the number of micronuclei in the human lung carcinoma cell line Calu-1 did not change (Köteles et al., 1998), while ML I at >74 ng/ml induced these cytogenetic damages. After irradiation of Calu-1 cells, the number of micronuclei decreased within 1 h in response to ML I and ML II at 0.001 and 0.014 ng/ml, while ML III, however, showed a slight increase of these cytogenetic damages. Further, intraperitoneal treatment of irradiated rabbits (1 Gy) with ML I at 1 ng/kg BW irradition revealed a strong decrease of micronuclei within 24 h (Köteles et al., 1998). As the induction of micronuclei was associated with a decrease of viability in lymphocytes, lymphoblasts and the Calu-1 cells, one may suggest that radiation-damaged cells are more sensitive to the ML-mediated apoptosis. Again, a correlation between cytogenetic damage and cell death was evident. Whether this deletion of abberation-carrying cells may have clinical relevance in the treatment of irradiated cancer patients has to be clarified in clinical studies.
MECHANISM OF ACTION: BIOLOGICALLY ACTIVE COMPONENTS
Viscum album contains a variety of biologically active components (see both, Becker and Pfüller, this book). While the cytotoxic properties of VA-E were linked to the ML and to the viscotoxins, the immunomodulating properties were attributed not only to the ML but also to alkaloids, polysaccharides and oligosaccharides (i.e. rhamnogalacturonan), viscotoxins, a 5 kDa peptide, the vesicles from Viscum album, and an undefined non-lectin antigen present in a fermented extract from pine trees. The compounds that exert the protective effects of the plant extracts remain to be characterized. The role of flavonoids and phenolic acids are conflicting, as some of them may act as anti-carcinogens or inhibit the growth of tumour cells, whereas others act as co-carcinogens, are mutagenic or able to induce DNA damage.
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