DNA-protective activities of hyperforin and aristoforin.

The aim of this study was to explain the molecular mechanisms of action of hyperforin, a phluoroglucinol derivative found in Hypericum perforatum L. and its more stable derivative aristoforin. DNA-topology assay revealed partial DNA-protective activities of hyperforin and aristoforin against Fe(2+)-induced DNA breaks. In order to assess molecular mechanisms underlying DNA-protective activity, the potential antioxidant activity of hyperforin and aristoforin was investigated using DPPH and OH scavenging assays, reducing power assay and Fe(2+)-chelating assay. We also studied interaction of hyperforin and aristoforin with DNA using established protocols for fluorescence titration. The ability of the studied compounds to relax topoisomerase I with electrophoretic techniques was investigated. The reduction in the fluorescence of hyperforin indicated an interaction between hyperforin and DNA with a binding constant of 0.2×10(8)M(-1). We suggest that a mechanism of hyperforin/aristoforin DNA-protective abilities is based on free radicals (mainly OH) scavenging activity.

Gentiana asclepiadea and Armoracia rusticana can modulate the adaptive response induced by zeocin in human lymphocytes.

Zeocin is a member of bleomycin/phleomycin family of antibiotics isolated from Streptomyces verticullus. This unique radiomimetic antibiotic is known to bind to DNA and induce oxidative stress in different organisms producing predominantly single- and double- strand breaks, as well as a DNA base loss resulting in apurinic/apyrimidinic (AP) sites. The aim of this study was to induce an adaptive response (AR) by zeocin in freshly isolated human lymphocytes from blood and to observe whether plant extracts could modulate this response. The AR was evaluated by the comet assay. The optimal conditions for the AR induction and modulation were determined as: 2 h-intertreatment time (in PBS, at 4°C) given after a priming dose (50 µg/ml) of zeocin treatment. Genotoxic impact of zeocin to lymphocytes was modulated by plant extracts isolated from Gentiana asclepiadea (methanolic and aqueous haulm extracts, 0.25 mg/ml) and Armoracia rusticana (methanolic root extract, 0.025 mg/ml). These extracts enhanced the AR and also decreased DNA damage caused by zeocin (after 0, 1 and 4 h-recovery time after the test dose of zeocin application) to more than 50%. These results support important position of plants containing many biologically active compounds in the field of pharmacology and medicine.

Biological activity of plant extract isolated from Papaver rhoeas on human lymfoblastoid cell line.

Varied medicinal plants are known as a source of natural phytochemicals with antioxidant activities that can protect organisms from oxidative stress and from various chronic diseases. Papaver rhoeas has a long history of medicinal usage, especially for ailments in adults and children. The possible cytotoxicity, genotoxicity and potential antioxidant effect of plant extract isolated from flowers of Papaver rhoeas was investigated in human lymfoblastoid cell line (TK6). Antioxidant activity of this extract was determined using the DPPH assay. The plant extract exhibited dose dependent free radical scavenging ability. The growth activity assay was used for determination of cytotoxicity. To assess potential genotoxicity the comet assay was used. The lower extract concentrations (0.25 and 0.5 mg/ml) neither exerted cytotoxic, nor genotoxic effects in TK6 cells but they stimulated cell proliferation. The concentration 25 mg/ml scavenged almost 85% of DPPH free radical. On the other hand, this concentration had strong cytotoxic and genotoxic effect on TK6 cells. The balance between beneficial and harmful effects should be always considered when choosing the effective dose.

Growth inhibitory effect of ethyl acetate-soluble fraction of Cynara cardunculus L. in leukemia cells involves cell cycle arrest, cytochrome c release and activation of caspases.

An extract of artichoke Cynara cardunculus L. (CCE) has been shown to exhibit antioxidant and antigenotoxic properties. In this study, the ability of CCE to inhibit the growth of L1210 and HL-60 leukemia cells was studied. Treatment of leukemia cells with a variety of concentrations of CCE (500-2500 microg/microL) for 24 h resulted in dose-dependent inhibition of leukemia cell growth. The cell growth inhibition was accompanied by G(0)/G(1) cell cycle arrest and by a loss of cells in S phase. Futhermore, apoptosis detected as a sub-G(0) cell population and apoptotic DNA fragmentation was observed. More detailed analyses of apoptosis induced by CCE in HL-60 cells revealed that apoptosis progressed through the caspase-9/-3 pathway, as release of cytochrome c, caspase-9/-3 activations and specific proteolytic cleavage of poly(ADP-ribose) polymerase. Taken together, the results suggest that CCE exerts an antiproliferative activity on leukemia cells and induces apoptosis of these cells through a mitochondrial/caspase dependent pathway.

Antigenotoxic effect of extract from Cynara cardunculus L.

The extract of artichoke Cynara cardunculus L. (CCE) was investigated for its potential antigenotoxic and antioxidant effects using four experimental model systems. In the Saccharomyces cerevisiae mutagenicity/antimutagenicity assay, CCE significantly reduced the frequency of 4-nitroquinoline-N-oxide-induced revertants at the ilv1 locus and mitotic gene convertants at the trp5 locus in the diploid Saccharomyces cerevisiae tester strain D7. In the simultaneous toxicity and clastogenicity/anticlastogenicity assay, it exerted an anticlastogenic effect against N-nitroso-N'-methylurea-induced clastogenicity in the plant species Vicia sativa L. On the contrary, despite CCE not being mutagenic itself, in the preincubation Ames assay with metabolic activation, it significantly increased the mutagenic effect of 2-aminofluorene in the bacterial strain Salmonella typhimurium TA98. In the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay, CCE exhibited considerable antioxidant activity. The SC50 value representing 0.0054% CCE corresponds to an antioxidant activity of 216.8 microm ascorbic acid which was used as a reference compound. Although the mechanism of CCE action still remains to be elucidated, different possible mechanisms are probably involved in the CCE antigenotoxic effects. It could be concluded that CCE is of particular interest as a suitable candidate for an effective chemopreventive agent.

Genetic risk assessment of acid waste water containing heavy metals.

The mutagenic/cancerogenic potential of acid-mine water from the Slovak mining area Rudnany containing a high load of toxic metals was evaluated after its application to three model test organisms (bacteria Salmonella typhimurium, yeast Saccharomyces cerevisiae and plant Vicia sativa L.). The results obtained from the modified preincubation Ames assay proved that 1000-fold diluted waste water exhibited mutagenic effect in three (TA97, TA98, TA102) of four bacterial strains. In the test on yeast the toxicity and genotoxicity increased as a function of the concentration. At the highest concentration used (0.06%) the frequency of revertants increased 6 times and convertants increased 4.5 times above the control level. In the simultaneous phytotoxicity and clastogenicity assay, concentration dependent toxicity and statistically significant clastogenicity was proved. We can conclude that heavy metals might be responsible for the genotoxic/cancerogenic potential of the test water. However, we do not entirely exclude the possibility that its genotoxicity might be promoted by its high acidity.

The comparative genotoxicological study of new local anesthetics, 3-(2-alkoxyphenylcarbamoyloxy)quinuclidium chlorides, on Salmonella typhimurium, Saccharromyces cerevisiae, Vicia faba, Hordeum vulgare and Drosophila melanogaster.

Potential gentoxicity of five new local anesthetics, derivatives of phenylcarbamic acid differing in the length of the alkyl chain of the alkoxy substituent, was studied on five test systems. There was a direct relationship with increased toxic effect in bacteria and yeast as a function of the elongation of the alkyl chain of the alkoxy substituents of the phenylcarbamic acid esters. On the other hand, no structure-toxicity relationship was found after application of 3-(2-alkoxyphenylcarbamoyloxy)-quinuclidium chlorides on plants and Drosophila. All anesthetics were nonmutagenic to Salmonella typhimurium strains TA97, TA98, TA100, and TA102 in the absence and in the presence of S9 mix. Pentyloxy and heptyloxy derivatives increased rates of genetic changes in Saccharomyces cerevisiae, mainly revertants at the isoleucine locus. Pentyloxy and hexyloxy derivatives increased the frequency of chromosome aberrations in Vicia faba root-tip meristems. No chlorophyll mutations were detected after treatment of Hordeum vulgare with pentyloxy, hexyloxy and heptyloxy derivatives. No sex-linked recessive lethals were scored in Drosphila melanogaster males. The rates of aneuploids induced in their germ cells were significantly increased after treatment with butoxy and octyloxy derivatives. However, the local toxic and genotoxic effects of test anesthetics on the microorganisms of the anesthetized tissues may be of some importance. In particular, the genotoxic effect exhibited in fungi by the heptyloxy derivative, a potent local anesthetic, was remarkable.

Genotoxicity evaluation of Rastim 30 DKV, the plant growth regulator, on five test systems.

The possible mutagenic activity of Rastim 30 DKV, a new plant growth regulator, was studied on five model test systems. It did not increase the frequency of His+ revertants in Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1538 in the absence and the presence of S9 mix. It slightly increased rates of genetic changes in Saccharomyces cerevisiae, mainly convertants at the tryptophan locus. No clastogenic effect was observed after Vicia faba root-tip meristem treatment, and at the lowest concentration used its mitotic activity was significantly increased. No chlorophyll mutants after the treatment of two cultivars of barley were observed. Though no sex-linked recessive lethals were scored in Drosophila melanogaster males, the rates of aneuploids induced in their germ cells were significantly increased.