Antimutagenic, Antirecombinogenic, and Antitumor Effect of Amygdalin in a Yeast Cell-Based Test and Mammalian Cell Lines.

Amygdalin is a major component of the seeds of Rosaceae family of plants such as apricots, peaches, cherry, nectarines, apples, plums, and so on, as well as almonds. It is used in alternative medicine for cancer prevention, alleviation of fever, cough suppression, and quenching thirst. The aim of the present study is to determine the mutagenic and recombinogenic effects of amygdalin in a test system Saccharomyces cerevisiae and to evaluate its potential antitumor effect in a yeast cell-based test and colon cancer cell lines. Results obtained show that concentrations 25, 50, and 100 µg/mL did not have any cytotoxic, mutagenic, and carcinogenic effect in yeast cell-based tests. Pretreatment with amygdalin at concentration 100 µg/mL leads to around twofold of the cell survival and decrease of reverse mutation frequency, induced by the alkylating agent methyl methanesulfonate. The frequency of gene conversion and mitotic crossing-over is around threefold lower. The anticarcinogenic potential of amygdalin at the same concentration is presented as around fourfold reduction of Ty1 retrotransposition induced by hexavalent chromium. In summary, data presented in this study provide evidence concerning the inability of amygdalin itself to provoke events related to the initial steps of tumorigenesis. In addition, the observed antimutagenic/antirecombinogenic effect could be activation of error-free and error-prone recombination events. Based on the high selectivity toward normal or tumor cell lines, it could be speculated that amygdalin has higher cytotoxic effect in cell lines with higher proliferative and metabolic activity, which are the majority of fast developing tumors.

Antioxidant, antimutagenic, and anticarcinogenic effects of Papaver rhoeas L. extract on Saccharomyces cerevisiae.

The aim of this work was to analyze the antioxidant and antimutagenic/anticarcinogenic capacity of Papaver rhoeas L. water extract against standard mutagen/carcinogen methyl methanesulfonate (MMS) and radiomimetic zeocin (Zeo) on a test system Saccharomyces cerevisiae. The following assays were used: 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, quantitative determination of superoxide anion (antireactive oxygen species [antiROS test]), DNA topology assay, D7ts1 test--for antimutagenic--and Ty1 transposition test--for anticarcinogenic effects. Strong pro-oxidative capacity of Zeo was shown to correlate with its well-expressed mutagenic and carcinogenic properties. The mutagenic and carcinogenic effects of MMS were also confirmed. Our data concerning the antioxidant activity of P. rhoeas L. extract revealed that concentration corresponding to IC(50) in the DPPH assay possessed the highest antioxidant activity in the antiROS biological assay. It was also observed that a concentration with 50% scavenging activity expressed the most pronounced antimutagenic properties decreasing Zeo-induced gene conversion twofold, reverse mutation fivefold, and total aberrations fourfold. The same concentration possessed well-expressed anticarcinogenic properties measured as reduction of MMS-induced Ty1 transposition rate fivefold and fourfold when Zeo was used as an inductor. Based on the well-expressed antioxidant, antimutagenic, and anticarcinogenic properties obtained in this work, the P. rhoeas L. extract could be recommended for further investigations and possible use as a food additive.

New cell-based assay indicates dependence of antioxidant biological activity on the origin of reactive oxygen species.

The mobility of the Ty1 transposon in Saccharomyces cerevisiae was found to vary proportionally with the level of ROS generated in cells, which provides the possibility to determine antioxidant activity by changes in a cellular process instead of using chemical reactions. The study of propolis, royal jelly, and honey with the newly developed Ty1antiROS test reveals an inverse exponential dependence of antioxidant activity on increased concentrations. This dependence can be transformed to proportional by changing the source of ROS: instead of cell-produced to applied as hydrogen peroxide. The different test responses are not due to excess of added hydrogen peroxide, as evidenced by the exponential dependence found by usage of yap1Δ tester cells accumulating cell-generated ROS. Results indicate that the activity of antioxidants to oxidative radicals depends on the origin of ROS, and this activity is elevated for cell-generated ROS compared to ROS added as reagents in the assay.

The positive response of Ty1 retrotransposition test to carcinogens is due to increased levels of reactive oxygen species generated by the genotoxins.

In previous laboratory and environmental studies, the Ty1 short-term test showed positive responses (i.e. induced mobility of the Ty1 retrotransposon) to carcinogenic genotoxins. Here, we provide evidence for a causal relationship between increased level of reactive oxygen species and induction the mobility of the Ty1 retrotransposon. Results obtained in concentration and time-dependent experiments after treatment, the tester cells with carcinogenic genotoxins [benzo(a)pyrene, benzo(a)anthracene, ethylmethanesulfonate, formamide], free bile acids (chenodeoxycholic, lithocholic acids) and metals (arsenic, hexavelant chromium, lead) showed a simultaneous increase in both cellular level of the superoxide anions and Ty1 retrotransposition rates. Treatment with the noncarcinogenic genotoxins [benzo(e)pyrene, benzo(b)anthracen, anthracene], conjugated bile acids (taurodeoxycholic, glycodeoxycholic acids) and metals (zinc, trivalent chromium) did not change significantly superoxide anions level and Ty1 retrotransposition rate. The induction by carcinogens of the Ty1 mobility seems to depend on the accumulation of superoxide anions, since the addition of the scavenger N-acetylcysteine resulted in loss of both increased amount of superoxide anions and induced Ty1 retrotransposition. Increased hydrogen peroxide levels are also involved in the induction of Ty1 retrotransposition rates in response to treatment with carcinogenic genotoxins, as evidenced by disruption of YAP1 gene in the tester cells. It is concluded that the carcinogen-induced high level of reactive oxygen species play a primary and key role in determination the selective response of Ty1 test to carcinogenic genotoxins.

The role of reactive oxygen species in the induction of Ty1 retrotransposition in Saccharomyces cerevisiae.

Here we provide evidence for a dependence between the increased production of reactive oxygen species and the activation of Ty1 retrotransposition. We have found that the strong activator of Ty1 mobility, methylmethane sulphonate, can not induce Ty1 retrotransposition in cells with compromised mitochondrial oxidative phosphorylation (rho(-); sco1Delta), which is the major source for production of reactive oxygen species (ROS) in Saccharomyces cerevisiae. The quantitative estimation of superoxide anions in living cells showed that rho(+) cells exposed to methylmethane sulphonate increase Ty1 retrotransposition and superoxide levels. The increase of superoxide anions by the superoxide generator menadione is accompanied by induction of Ty1 mobility without any treatment with a DNA-damaging agent. Higher frequencies of retrotransposition were found in rho(+) and rho(-) cells treated with exogenously added hydrogen peroxide or in cells with disrupted YAP1 gene characterized by increased intracellular levels of hydrogen peroxide. These data indicate that increased levels of ROS may have an independent and key role in the induction of Ty1 retrotransposition.

Ty1 transposition induced by carcinogens in Saccharomyces cerevisiae yeast depends on mitochondrial function.

The transposition of the Ty mobile genetic element of Saccharomyces cerevisiae is induced by carcinogens. While the molecular background of spontaneous Ty1 transposition is well understood, the detailed mechanism of carcinogen induced Ty1 transposition is not clear. We found that mitochondrial functions participate in the Ty induced transposition induced by carcinogens. Contrary to the parental rho(+) cells rho(-) mutants (spontaneous or induced by ethidium bromide) do not increase the rate of Ty1 transposition upon treatment with carcinogens. Preliminary results strongly suggest that the absence of oxidative phosphorylation in rho(-) mutants is the reason for the inhibited Ty transposition. The lack of carcinogen induced Ty1 transposition in rho(-) cells is not specific for a particular carcinogen and represents a general feature of different carcinogenic substances inducing rho(-). It is concluded that carcinogen induced Ty1 transposition depends on the functional state of mitochondria and cannot take place in cells with compromised mitochondrial function (rho(-)).

The Ty1 transposition assay: a new short-term test for detection of carcinogens.

An assay based on induction by carcinogens of Ty1 transposition in Saccharomyces cerevisiae is proposed. A tester strain was developed that contains a marked Ty1 element, which allows following the transposition in the genome as a whole and a mutation, which increases cellular permeability. Hypersensitivity to chemical agents, higher cell wall porosity and transformability with plasmid DNA evidenced an enhanced cellular permeability of the tester cells. The increased permeability resulted in higher sensitivity to carcinogens. The treatment with different laboratory carcinogens induced Ty1 transposition rates in the tester strain by a factor of 10 to 20, compared to the controls. The induction is not stress-generated by the cytotoxicity of carcinogens, since treatment with NaN3 at concentrations killing 50% of the cells did not increase the transposition rate. The increase of Ty1 transposition in tester cells is specific for active carcinogens and a positive response with procarcinogens was obtained only in presence of S9 mix. The Ty1 transposition test responded positively to a number of Ames-test or DEL-test negative carcinogens. The positive response of Ty1 test was statistically significant and verified in kinetics and concentration-dependent experiments. It is concluded that the Ty1 transposition test can be used, in addition to the Ames assay, as a short-term test for detection of carcinogens.