Accidental cell death and generation of reactive oxygen intermediates in human lymphocytes induced by thionins from Viscum album L.

Abstract

The cytotoxic mechanisms of thionins from Viscum album L., the viscotoxins, were investigated in human granulocytes and lymphocytes. The time course of viscotoxin effects indicate accidental cell death, i.e. membrane permeabilization, degradation of cytoplasm and chromatin, swelling of mitochondria with loss of their cristae, and generation of reactive oxygen intermediates within 1±2 h, followed by secondary apoptosis-associated events. The viscotoxin homologue purothionin from whole-wheat flour and viscotoxin B, however, did not induce cell death in cultured lymphocytes. Cytotoxicity of cationic and amphipathic viscotoxin was prevented only by cleavage of its disulphide bridges.Keywords: thionins; viscotoxins; reactive oxygen intermediates; apoptosis.The viscotoxins are toxic proteins from Viscum album L., isolated first by Winterfeld and co-workers [1] and by Samuelsson and co-workers [2,3]. They are related to the family of a-and b-thionins, a group of highly basic cysteine-rich polypeptides found in the endosperms of several Gramineae, which consist of 45±47 amino acids, and have three or four disulfide bridges (reviewed in [4]). Several thionins have been sequenced and they appear to be highly homologous at the amino-acid level [4,5]. The cysteine residues, which form three disulfide bridges, the thyrosine residue at position 13, and in addition, the distribution of hydrophobic and hydrophilic residues are highly conserved [4±7]. Because of their positive charge, thionins interact with the negatively charged phospholipids making up the membranes, resulting in either pore formation or a specific interaction with a specific lipid domain [4]. The positively charged viscotoxins were found to form complexes with the negatively charged DNA, resulting in protection of the double helix against thermal denaturation [8], but, leaving the DNA conformation unaffected.Thionins are toxic to laboratory animals, insect larvae, bacteria, yeasts and mammalian cells (reviewed in [4,9]). However, there are only few reports concerning the cytotoxicity of viscotoxin to cultured cell lines. Konopa et al. [10] reported that the isolated polypeptides exhibited cytotoxic activity against the human tumour cell lines KB and HeLa in a concentration range (ED 50 ) of 0.2±1.7 mg´mL 21 . Surprisingly, Yoshida sarcoma cells were found to be more sensitive to viscotoxin than the leukaemia cell line . However, the mechanisms of viscotoxin-mediated cytotoxicity are unclear.To analyse their cytotoxic properties, we cultured peripheral blood cells with thionins from mistletoe (viscotoxin) and from whole-wheat flour (purothionin), and apoptosis-inducing proteins [12] such as mistletoe lectin (ML I) and the RNA synthesis-inhibiting Streptomyces toxin actinomycin D. The galactose-binding ML I belongs to the family of ribosomeinhibiting proteins with a lectin B chain linked by hydrophobic bonds and disulfide bridges to the catalytic A chain, which is a highly specific N-glycosidase and cleaves the N-glycosidic bond of at least one particular adenin...

Transgenic tomato plants expressing an Arabidopsis thionin (Thi2.1) driven by fruit-inactive promoter battle against phytopathogenic attack

“…However, due to the general consensus that thionins are toxic to biological systems (Bussing et al 1999), the production of transgenic tomato using thionin genes needs to be done carefully. Thus, the commonly used constitutive promoter, CaMV35S, could not be used in the present study.…”

“…However, their use has been confined to homologous or model systems, not extended to crop improvement. This may be attributable to the fact that thionins, in general, are considered toxic to animals, as demonstrated in laboratory animals and mammalian cells in vitro (Bohlmann 1994;Bussing et al 1999). This prompted us to investigate the use of thionins to genetically engineer disease resistance in crops, by adopting a safe and careful engineering strategy.…”

Immunomodulatory Effects of <i>Viscum album</i> Extracts on Natural Killer Cells: Review of Clinical Trials

“…They revealed DNA stabilizing properties [9][10][11] as well as cytotoxic and growth-inhibitory effects [12][13][14]. The cytotoxic effects of VA extracts are predominantly induced by ML via apoptosis [15][16][17], while viscotoxins mediate necrotic cell death [16,18].…”

Small, novel proteins from the mistletoe Phoradendron tomentosum exhibit highly selective cytotoxicity to human breast cancer cells

“…Furthermore, high expression levels of viscotoxin A3 complementary DNA (cDNA) in transgenic Arabidopsis thaliana have been shown to enhance resistance against the pathogen Plasmidiophora brassicae [24]. Viscotoxins are toxic to mammals [25] and other biological systems [26,27]. The toxicity of viscotoxins and that of other thionins is associated with increased permeability of plasma membrane, leading to depolarization and Ca-influx, and, potentially, to cell death.…”