Assessment of the cytotoxicity of aluminium oxide nanoparticles on selected mammalian cells.

The rapid development of nanotechnology raises both enthusiasm and anxiety among researchers, which is related to the safety use of the manufactured materials. Thus, the aim of this study was to investigate the effect of aluminium oxide nanoparticles on the viability of selected mammalian cells in vitro. The aluminium oxide nanoparticles were characterised using SEM and BET analyses. Based on Zeta (ζ) potential measurements and particle size distribution, the tested suspensions of aluminium oxide nanoparticles in water and nutrient solutions with or without FBS were classified as unstable. Cell viability, the degree of apoptosis induction and nanoparticles internalization into the cells were assessed after 24 h of cell exposure to Al2O3 nanoparticles. Our results confirm the ability of aluminium oxide nanoparticles to penetrate through the membranes of L929 and BJ cells. Despite this, there was no significant increase in apoptosis or decrease in cell viability observed, suggesting that aluminium oxide nanoparticles in the tested range of concentrations has no cytotoxic effects on the selected mammalian cells.

In vitro angiomodulatory activity of sera from type 2 diabetic patients with background retinopathy.

Diabetic retinopathy is the leading cause of adult vision loss and blindness. The most important contributors to the development of diabetic retinopathy are hyperglycemia and hypoxemia that lead to increased vasopermeability, endothelial cell proliferation, and pathological neovascularization. In our previous studies, close relationship between proangiogenic activity of sera from type 2 diabetes mellitus patients (DM2) with background retinopathy, assessed in the in vivo serum-induced mouse cutaneous test (SIA), and VEGF and IL-18 serum concentration were observed. Moreover, it was clearly shown that IGF-1 might play an important role in the negative regulation of neoangiogenesis induced by DM2 patients' sera by diminishing the VEGF stimulatory effect. To confirm the observed phenomenon we evaluated the effect of DM2 patients' sera on the in vitro proliferative activity of human endothelial cells, which is critical for the sprouting and generation of new blood capillaries. Endothelial proliferative activity was significantly higher in the presence of sera from DM2 patients than from healthy controls (P<0.001), as estimated by the MTT test. Moreover, the examined sera from DM2 patients were characterized by increased IL-18 (P<0.05), diminished IGF-1 (P<0.02), and unchanged VEGF levels compared with those in controls. In conclusion, the present study showed a strong stimulatory effect of DM2 patients' sera on the proliferation of endothelial cells, which, along with the findings of our previous studies, proves that the described phenomenon is universal and valid for both animal and human endothelium.

Influence of vitamin D3 metabolites on cell proliferation and cytotoxicity of adriamycin in human normal and neoplastic cells.

The common features of biological activity displayed by vitamin D family members and adriamycin suggest the possibility of synergistic effects of the combination of these compounds. Until now, the mechanisms responsible mainly for adriamycin cytotoxic action have not been indicated. Therefore, observation of the possible common cell targets for adriamycin and vitamin D metabolites could shed more light on the mechanisms of cytotoxic activity of adriamycin. In the present study, the influence of calcidiol (25-hydroxyvitamin D(3)) and calcitriol (1alpha,25-dihydroxyvitamin D(3)) on the proliferation and cytotoxicity of adriamycin was studied. The following cell lines were tested: normal human fibroblasts-CRL 1502, human melanoma cells-ME18 and its subline-ME18/R, resistant to adriamycin. As was shown, 72 h of incubation with calcidiol or calcitriol, both at 10 microM, inhibited growth (to approx. 60%) only of the ME18 cells. Dose and time dependence of this effect has been confirmed. Antiproliferative events did not correlate with an increase of adriamycin cytotoxicity. It was noted that calcidiol and calcitriol had no significant influence on the adriamycin IC(50) values in any cell lines tested. These results point to the divergent mechanisms of action of adriamycin and vitamin D(3) metabolites.

Activation of programmed cell death (apoptosis) by adriamycin in human neoplastic cells.

We have studied the occurrence of the apoptosis phenomenon in the action of adriamycin (ADR) on human melanoma cells sensitive (ME18) and resistant (ME18/R) to ADR. The study has shown that the intensity of apoptotic morphological changes noted in melanoma cells depended on the duration of the ADR treatment. We have not observed any positive correlation between the induction of apoptosis and sensitivity to ADR. We have used a fluorescence microscope and flow cytometer to evaluate apoptotic events in cells treated with ADR.

Elaboration of HPLC method for biotin determination in multiple vitamin drugs and comparison with microbiological method.

We were searching for optimal analytical conditions for the biotin determination by the HPLC method with detection at the wavelength 200 nm in multiple vitamin drugs. Statistical parameters of the HPLC method and the microbiological method of the biotin determination, were compared.

Tofisopam--evaluation of mutagenic and genotoxic properties.

The mutagenic properties of tofisopam, the member of the 2,3-benzodiazepine family, were evaluated on the basis of Ames test with Salmonella typhimurium TA1537, TA97, TA98, TA100 and TA102 strains. The genotoxic properties of tofisopam were estimated on L929 cell line with the cytokinesis-block technique. Under the experimental conditions, no mutagenic activity of tofisopam in tester bacteria strains was found, and no genotoxic activity was observed.

The use of HPLC method for determination of the folic acid in multi-component vitamin preparations.

In the present study we tried to use HPLC method to determine the folic acid content in multivitamin and multimineral pharmaceutical preparations. The reverse phases technique was applied and measurements were made at the wavelength 282 nm. The elaborated method, because of its sufficient precision, accuracy as well as short time retention of folic acid may be used as an alternative method to microbiological.

The influence of low H2O2 concentrations on DNA content distribution in cell cycle and expression of cyclins in human cells.

The previous study has shown several cell lines pretreated with a low dose of hydrogen peroxide to exhibit an adaptive response to subsequent high doses of adriamycin. We undertook the present investigation to examine whether a relationship exists between the hydrogen peroxide pretreatment and perturbation in the phases of the cell cycle observed as the changes in DNA content distribution. We utilized human embryo cells, human melanoma cells and human melanoma adriamycin resistant subline, experimental developed. The data obtained in the present study suggest that hydrogen peroxide at low concentration can modify the normal rate of cell cycle progression.

The in vitro study of influence of antioxidant enzymes level and repair capacity on cytotoxic bleomycin activity in human and mouse cells.

Bleomycin, an antitumour antibiotic was used to study the possible relationship between DNA single strand breaks repair capacity, antioxidant enzymes level and cytotoxic activity of the drug in mouse cells: AKR and BALB/c and in human cells: CRL 2088 and CRL 1307 (xeroderma pigmentosum). The BALB/c and CRL 1307 cells were used because of having defects in DNA repair capacity. A positive correlation was shown to exist between IC50 values and repair ability which suggested that DNA single strand breaks could be responsible for cytotoxic effects of bleomycin in human and mouse cells. Also antioxidant enzymes level have occurred as, at least partly, participating in bleomycin cytotoxic efficiency. About 10-fold higher resistance of AKR cells to bleomycin in comparison with the other cells, as appeared here, did not exhibit the straight correlation with antioxidant status of the cells. It prompts participation of the other mechanism in bleomycin cytotoxic action than that based on free radical generation. Also drug distribution and metabolism should be considered as a possible factor needed in bleomycin efficacy evaluation.

Simultaneous determination of vitamins A, D3 and E in multiple pharmaceutical preparations by HPLC method.

This study makes possible the use of reversed phase HPLC with detection at 265 nm for the simultaneous quantitative determination of vitamin A, D3 and E in multiple pharmaceutical preparations. We have also proposed a very simple method of isolation of these vitamins from multivitamin preparations. On the basis of statistical analysis of the data, we can note that conditions for HPLC method used in our work gave us precise and accurate results.

Studies on the mechanisms of an H2O2 adaptive dose on DNA damage in human neoplastic cells treated with adriamycin.

The possible influence of a hydrogen peroxide adaptive dose on DNA damage induced by adriamycin treatment in human melanoma cells, sensitive (ME18) and resistant (ME18/R) to adriamycin was investigated. In our earlier work, it was shown that the human melanoma resistant subline exhibited, in contrast to the parental cell line, the capacity to evoke an adaptive response provoked by low doses of hydrogen peroxide. This was observed as a diminished cytotoxic effect of adriamycin used at a toxic dose. The current work showed that an adaptive dose of hydrogen peroxide (2 microM) reduced DNA--single strand breaks, generated by a challenging dose of adriamycin in both, sensitive and resistant human melanoma cells. For better understanding of the adaptive response mechanism, exogenous free radical scavengers were used. In this study, it was shown that superoxide dismutase used at a concentration of 200 u/ml and mannitol at concentration of 100 mM modulated adriamycin--generated DNA--single strand breaks in parental melanoma cells. None of the exogenous scavengers, used in this study, influenced the cytotoxic effects of adriamycin either in sensitive or in resistant melanoma cells.

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.

DNA repair of adriamycin induced damage in mouse and human cells differing in catalase activity.

Catalase is known to counteract cytotoxic effect of adriamycin, used as an anti-neoplastic drug. In cells with low catalase activity no repair of adriamycin induced lesions was observed up to 48 h post treatment. In cells with high catalase activity after 48 h the repair was either complete or partial depending on the human or mouse cell type used.

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.

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.

DNA damage and repair in normal and neoplastic cells treated with adriamycin.

Adriamycin (ADR), a common antineoplastic drug, was used to study DNA repair synthesis, cell cytotoxicity and DNA single strand breaks in normal human fibroblasts--CLV98 and human melanoma cells--ME18. No repair synthesis was observed in ME18 and CLV98 cells exposed to adriamycin in concentrations up to 10(-5) M. ME18 cells were less sensitive to ADR treatment than CLV98 cells. Adriamycin-induced DNA single strand breaks (at ADR concentration: 1 microgram/ml) were incompletely repaired in ME18 cells and unrepaired in CLV98 cells within 24 h after drug removal. Within 48 h strand breaks were completely repaired in both kinds of cells. No repair of single strand breaks was observed in ME18 and CLV98 cells after drug treatment in the concentration of 5 micrograms/ml.

Evaluation of potential carcinogenic properties of Tolpa Peat Preparation in vitro.

For the evaluation of the potential carcinogenic properties of TPP the Syrian hamster embryo cell transformation test was employed. TPP did not induce morphological cell transformation in these cells.