DNA repair of hydrogen peroxide-induced damage in human lymphocytes in the presence of four antimutagens. A study with alkaline single cell gel electrophoresis (comet assay).

We assessed four antimutagenic compounds' influences on DNA repair in human lymphocytes exposed in vitro to hydrogen peroxide (20 microM, 5 min, at 4 degrees C). DNA damage and repair were estimated by means of alkaline single cell gel electrophoresis (comet assay). It was noticed that the enhancement of DNA repair was relatively strongest when fluphenazine was present in the cell culture medium. In the cases of anthocyanins and alkylresorcinols, the effects were almost 6-9 times weaker than that of FPh. The effect of todralazine on DNA repair was relatively weakest. Further study should be done on fluphenazine as a potential DNA repair-enhancing compound.

Impact of four antimutagens on apoptosis in genotoxically damaged lymphocytes in vitro.

An antimutagenic activity of fluphenazine, todralazine, anthocyanins and alkylresorcinols was established in a battery of short-term cytogenetic tests. One of the possible mechanisms of their antimutagenic action could be an increase in apoptotic elimination of heavily-damaged cells from a culture. In this paper we provide data on quantitative estimation of the antimutagens' impact on apoptosis in lymphocyte cultures exposed in the G(0)-phase to genotoxic agents: hydrogen peroxide (0.2mM, 20 min.) or benzo[a]pyrene (40 microM, 90 min.), and then cultured for 36 hrs in the presence of a lectin (PHA-M, 1% v/v) and each of the tested antimutagens. Apoptosis was estimated by means of microscopic examination of cell smears stained with a mixture of fluorochromes (ethidium bromide/acridine orange) as well as of the results of DNA separation with the field inversion gel electrophoresis. By microscopic examination we assessed that the frequencies of cells exhibiting morphological features of apoptosis considerably increased in the cultures containing the antimutagens. The FIGE separation of DNA from those cultures proved that the DNA content in the 30-50 kb domain was markedly elevated, as compared with the control cultures that did not contain antimutagens. It was established in the regression analysis that the apoptosis-enhancing effect significantly depended on the concentration of each tested antimutagen in a culture medium. However, marked differences of apoptosis-enhancing potency were noticed among the four antimutagens. The multicriterial analysis proved that the apoptosis-enhancing effects of fluphenazine and also, to a smaller extent, by alkylresorcinols, were many times stronger than those of anthocyanins and of todralazine. The results suggest that the enhancement of apoptosis by fluphenazine and by alkylresorcinols can explain a major part of their antimutagenic activity, whereas in the case of anthocyanins and of todralazine other mechanisms of antimutagenic action should be sought for.

A comparison of the methods applied to detect apoptosis in genotoxically-damaged lymphocytes cultured in the presence of four antimutagens.

The sensitivity of the available methods of apoptosis detection in lymphocyte cultures was tested. Cells were preincubated with genotoxic agents: hydrogen peroxide (0.2 mM; 20 min.) and benzo[a]pyrene (40 microM; 90min.), and then cultured for 36h in the presence of a lectin (PHA-M; 1% v/v) and one of the following potentially antimutagenic agents: alkylresorcinols, anthocyanins, todralazine and fluphenazine. It was established that staining with a mixture of fluorochromes (ethidium bromide and acridine orange) provided the highest amount of detected apoptotic cells, and the best repeatability of the results in subsequent experiments. Calculation of the Spearman's rank correlation coefficients proved that there was a high correlation between the results obtained by the ethidium bromide/acridine orange method and those obtained by identifying genomic DNA fragmentation by means of FIGE-electrophoresis. Therefore, these two methods were chosen for further studies of the tested antimutagens' impact on apoptosis in genotoxically-damaged lymphocytes.

Antimutagenic activity of fluphenazine in short-term tests.

Fluphenazine, an antipsychotic drug that belongs to the phenothiazine family, reduced the genotoxicity of direct- and indirect-acting mutagens in the Ames test, both in the presence and in the absence of promutagen-activating S9 fraction. In short-term tests on human lymphocytes, the inhibitory effect of fluphenazine on the genotoxicity of standard mutagens was strongest in the cytokinesis-blocked micronucleus assay and in the thioguanine resistance test, and weakest in the sister chromatid exchange test. Fluphenazine also considerably reduced the level of free radicals estimated in in vitro samples of human granulocytes. The results suggest that, in the range of the tested concentrations, fluphenazine could be considered for use to prevent the genotoxicity of daunorubicin, methyl methanesulfonate, benzo[a]pyrene, and mitomycin C. Reduction in the level of free radicals appears to be an important mechanism of the antimutagenic action of fluphenazine.

Evaluation of antimutagenic effect of todralazine in cultured lymphocytes.

Todralazine, an antihypertensive drug from the hydrazinophthalazine group, significantly decreased the activities of benzo[a]pyrene and mitomycin C in three short-term genotoxicity tests in human lymphocyte cultures. The thioguanine resistance test, the cytokinesis-blocked micronucleus assay and the sister chromatid exchange test were used to demonstrate the antimutagenicity of todralazine. Todralazine lowered the level of free radicals generated by human granulocytes in vitro in the presence of benzo[a] pyrene and also in the presence of the granulocyte activator and tumor promoter phorbol myristate acetate. These results, together with our previous data obtained in the standard bacterial Ames test, strongly suggest that todralazine is a good antimutagen in vitro and deserves further research on its inhibitory action on mutagenesis and carcinogenesis.

Antimutagenic activity of anthocyanins isolated from Aronia melanocarpa fruits.

Anthocyanins belong to the flavonoid family and are ubiquitous in plants, especially in flower petals and fruit peels. We established that anthocyanins isolated from fruits of Aronia melanocarpa markedly inhibited the mutagenic activity of benzo(a)pyrene and 2-amino fluorene in the Ames test. In the Sister Chromatid Exchanges (SCEs) test with human blood-derived lymphocytes cultured in vitro, a significant decrease of SCEs frequency induced by benzo(a)pyrene was observed in the presence of anthocyanins. In the case of mitomycin C the effect of anthocyanins on SCEs frequency was smaller but still noticeable. Anthocyanins markedly inhibited the generation and release of superoxide radicals by human granulocytes. The results suggest that the antimutagenic influence of anthocyanins is exerted mainly by their free-radicals scavenging action as well as by the inhibition of enzymes activating promutagens and converting mutagens to the DNA-reacting derivatives. These preliminary data seem to be important in the aspect of a possible antimutagenic and anticarcinogenic potency of anthocyanins commonly present in fruits and vegetables.

Antimutagenic activity of alkylresorcinols from cereal grains.

Alkylresorcinols, natural amphiphilic compounds commonly found in cereal grains, markedly decreased mutagenic activity of four standard mutagens examined in the Ames test. The effect was the strongest in the case of indirect-acting mutagens, benzo[a]pyrene and 2-aminofluorene. In the case of direct-acting mutagens, daunorubicin and methyl methanesulfonate, the diminution of the mutagenic activity by the alkylresorcinols was smaller but still noticeable. In the Sister Chromatid Exchanges test (SCEs) with cultured in vitro human blood-derived lymphocytes, a significant decrease of SCEs frequency induced by benzo[a]pyrene was observed in the presence of alkylresorcinols. These preliminary results seem to be important in the aspect of possible antimutagenic and anticarcinogenic potency of alkylresorcinols found in cereal grains.