Study of the genotoxic potential of iron ions by the SOS chromotest.

Modulation of iron ions induced SOS response by scavengers of reactive oxygen species was studied in SOS chromotest in three strains of Escherichia coli (PQ37, PQ300 and OG400). Iron ions in the absence of reductants induced SOS response generated by reactive oxygen species in a dose dependent manner and this was inhibited in the presence of catalase.

Influence of ascorbic acid on cytotoxic activity of copper and iron ions in vitro.

We investigated survival of two kinds of human embryonic cells (CLV102, Lu106) and human melanoma cells (Mel8) exposed to exogenous iron and copper ions in the absence or in the presence of ascorbic acid, catalase and superoxide dismutase. Iron ions produced cytotoxicity towards both kinds of cells dependent on its concentration. Catalase suppressed the cytotoxicity induced by iron ions in Lu106 cells. whereas in CLV102 and Mel8 cells, was ineffective. By contrast, superoxide dismutase abolished the cytotoxicity of iron ions towards CLV102 cells, whereas in Lu106 and Mel8 cells, was ineffective. The mixture of iron ions with ascorbic acid was less cytotoxic than iron ions themselves or ascorbic acid itself, only in CLV102 and Lu106 cells. Ascorbic acid enhanced drastically cytotoxic effect of copper ions in all kinds of cells.

Adaptive response towards adriamycin in vitro: circumvention with verapamil.

In an attempt to identify mechanisms of adaptive response to adriamycin (ADR), we have earlier isolated ADR-resistant cell lines CHO/R and ME18/R by short-term pulse exposures of parent cell lines to this drug, followed by single-cell cloning. The results presented in this study have shown that the development of resistance to ADR was accompanied by cross-resistance to vinblastine and methotrexate. The resistance of tested cell lines towards ADR was substantially reversed by verapamil (VPL) at non-toxic concentrations. VPL abolished also the capability of these cell lines to express adaptive response after treatment of the cells with a conditioning dose of ADR. From the results of our study, we conclude that similar characteristics play a role in the mechanism of the phenomenon of adaptive response as in the mechanism of pleiotropic multidrug resistance.

Capability of adriamycin and busulfan to induce adaptive response in vitro.

The capability to induce an adaptive response by low doses of busulfan (BS) or adriamycin (ADR) was studied in two kinds of mammalian cells with acquired or inherent resistance to ADR (ME18/R and V3) cultured in vitro. The results indicate the presence of an adaptive response to ADR in both kinds of used cells pretreated with a low priming dose of ADR. In the same kind of cells no adaptive response to BS after priming with a low dose of this drug was found.

Studies on the nature of cytotoxic effects induced by dihydralazine.

The effects of scavengers on the cytotoxicity of dihydralazine were evaluated. Pretreatment of two kinds of mammalian cells cultured in vitro with catalase or sodium dismutase abolished partially or almost totally the effects of the drug. Hydroxyl radicals scavengers dimethylsulphoxide and D-mannitol minimized the cytotoxic response of both kinds of cells.

Studies on adaptation to adriamycin in cells pretreated with hydrogen peroxide.

Various investigations have reported the occurrence in bacterial and mammalian cells of an adaptive response to the toxic effects of oxidants or agents that cause oxidation via redox reactions. In our previous study, it was shown that several cell lines pretreated with a low dose of hydrogen peroxide (H2O2) exhibited an adaptive response to subsequent high doses of adriamycin (ADR), whereas other cell lines did not. Based on the observation that the cell lines utilized differed in their sensitivity towards adriamycin, we undertook the present investigation with the goal of evaluating possible relationships between the levels of antioxidant enzymes and sensitivity towards adriamycin. Another aim was to determine relationships between the inducibility of these enzymes and the occurrence of adaptation. We utilized African Green monkey kidney (V3), human embryo (CLV98), human melanoma (ME18), and Chinese hamster ovary (CHO) cell lines and experimentally developed adriamycin-resistant human melanoma (ME18/RN) and Chinese hamster ovary (CHO/RN) cell sublines. Cytotoxicity was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and trypan blue exclusion. The levels of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) were determined in the same kind of experiment as that revealing the occurrence of adaptation. The rank order established for catalase activities was similar to that for sensitivity towards adriamycin. Aberrant increases in the tested enzymes were demonstrated in experimental groups of all kinds of cells. We conclude that in our cell systems catalase is a major determinant of adriamycin resistance. Whether the occurrence of the adaptive response under study is dependent on the contribution of catalase, itself dependent on the degree of resistance to the drug, is discussed.

Studies on the nature of genotoxic and cytotoxic effects induced by hydralazine.

The nature of genotoxic and cytotoxic effects induced by hydralazine was analyzed taking into account possible protection of cells by catalase, superoxide dismutase and dimethyl sulfoxide. For the experiments designed to evaluate the influence of scavengers on the genotoxicity expressed as the SOS induction factor the E. coli PQ37 strain was used. The cytotoxic effects were investigated in V3 cells cultured in vitro. The genotoxicity and cytotoxicity of hydralazine were suppressed by catalase in a dose-dependent manner but they were enhanced by superoxide dismutase. No protective effect of dimethyl sulfoxide was observed. Our results indicate that H2O2 plays an essential role in the genotoxicity and cytotoxicity of hydralazine.

Protective role of reactive oxygen species scavengers against toxicity of 3-chloropyridine and 2-chloropyridine N-oxide.

The nature of biological effects of substituted pyridines and their N-oxides is a matter for discussion. Our previous study demostrated that 3-chloropyridine is cytotoxic and clastogenic. No cytotoxic activity was observed with 2-chloropyridine tested in the same dose range. In this study experiments were performed to assess cytotoxicity and clastogenicity of 2-chloropyridine N-oxide and 3-chloropyridine N-oxide. In the dose range from 800 to 3200 mug 2-chloropyridine/ml, N-oxide showed a dose-dependent cytotoxicity and clastogenicity. In the same dose range, 3-chloropyridine N-oxide was found to be non-cytotoxic and non-clastogenic. The nature of the effects induced by 3-chloropyridine and 2-chloropyridine N-oxide was analysed on the basis of possible protection by scavengers of reactive oxygen species. The cytotoxicity of both compounds was effectively suppressed by catalase and hydroxyl radicals scavengers. Pretreatment of V(3) cells with dimethyl sulfoxide or catalase provided protection against the ability of both compounds to induce chromosomal aberrations. From the data of this study we conclude that cytotoxicity and clastogenicity of 3-chloropyridine and 2-chloropyridine N-oxide are linked to the generation of reactive oxygen species.

Role of pyridine N-oxide in the cytotoxicity and genotoxicity of chloropyridines.

During metabolic processes, substituted pyridines may undergo N-oxidation and decomposition. It is a matter of discussion whether these processes influence the reactivity of this class of compounds. To elucidate this problem, the cytotoxicity and clastogenicity of two selected Chloropyridines on cultured V(3) cells was assessed in the absence and in the presence of pyridine N-oxide. For this purpose two cytotoxicity assays in parallel with chromosomal analysis were performed. In the dose range from 400 to 3200 mug/ml, 3-chloropyridine showed a dose-dependent cytotoxicity and clastogenicity towards V(3) cells, whereas in the same dose range 2-chloropyridine was found to be non-cytotoxic and non-clastogenic. Pyridine N-oxide (200 mug/ml) showed protective effects against 3-chloropyridine-induced cytotoxicity and clastogenicity. Under analogous experimental conditions cytotoxic and clastogenic effects were induced by 2-chloropyridine. From these studies it appears that the expression of toxicity by halogenated pyridines differing in the position of the halogen moiety may be influenced in different ways by the potential metabolite.

Transforming activities of methotrexate, hydroxyurea and 5-fluorouracil in different cell systems.

The transforming ability of methotrexate, hydroxyurea and 5-fluorouracil was studied in mass cultures of embryo cells from BN/a, DBA/2 and Swiss mice. As estimated by virological methods the cells from BN/a mice were infected persistently with ectromelia virus. In parallel a quantitative transformation assay with a cloned BALB/3T3 cell line was employed. Oncogenic transformation could be induced repeatedly in mass cultures from BN/a mice after treatment with every of chemotherapeutic agents studied. Neither of the drugs induced transformation in the cultures deriving from two other strains of mice. In the cloned BALB/3T3 cell line from the three drugs tested only methotrexate induced the appearance of transformed foci, however, the yield of transformants was very low. It has been concluded that under the employed experimental conditions the transforming ability of hydroxyurea and 5-fluorouracil could be expressed exclusively in the cells infected with ectromelia virus. The transforming ability of methotrexate seems to depend either on the presence of viral infection or on the properties of cells enabling additional events necessary for transformation.

Methionine regulation of N-5-methyltetrahydrofolate: homocysteine methyltransferase and its influence on the growth and protein synthesis in normal, neoplastic, and transformed cells in culture.

Normal human embryonic fibroblasts (CLV-58) and normal embryonic rat fibroblasts (REF) revealed equal growth in media containing 0.2 mM DL-homocysteine thiolactone (HOM) or in methionine (METH) enriched with 1.5 microM cyanocobalamin and 0.1 mM folic acid, whereas L 5178Y lymphoblasts and transformed rat fibroblasts (REF-S) showed more or less acute need for exogenous METH. In normal cells general protein synthesis remained unimpaired during 24 hours after substitution of METH by HOM. In L 5178Y and REF-S cells, however, protein synthesis was significantly inhibited. Measurements of N-5-methyltetrahydrofolate:homocysteine methyltransferase revealed a two- to threefold increase of enzyme activity in normal cells grown on HOM and in L 5178Y and REF-S cells grown on HOM with added METH. HeLa cells showed variability in growth pattern, in general protein synthesis response, and in inducibility of methyltransferase upon substitution of METH by HOM.