Herbicide acetochlor interferes with proliferation activity of MCF-7 cells enhanced by estradiol.

It is well documented that pesticides used in agricultural processes may have detrimental effects upon human health. Moreover, many of these compounds have been indicated as potential endocrine and reproductive disruptors. In the present study, the ability of herbicide acetochlor to affect a growth of estrogen-sensitive MCF-7 mammary epithelial carcinoma cells was studied using E-screen test. Acetochlor alone did not affect proliferation of MCF-7 cells. Significant inhibition of estradiol-induced (10-14-10-8 M) MCF-7 cell growth by the action of acetochlor (10-5 M) and interaction between these chemicals were observed. Estradiol-stimulated MCF-7 cell proliferation was decreased by 41% of positive control (17ß-estradiol, 10-8 M, 100%) in the presence of acetochlor (10-5 M). Our results demonstrate that acetochlor might interfere with estradiol signaling conducted to altered proliferation activity of MCF-7 cells and might support endocrine disruptive effects of acetochlor.

Estrogenic/antiestrogenic activity of homoisoflavonoids from bulbs of Muscari racemosum (L.) Miller.

The homoisoflavonoid fraction obtained from the Muscari racemosum bulb ether extract (MRBEE) exhibited estrogenic activity by inducing proliferation of MCF7 cells in a dose-dependent manner. In the presence of estradiol MRBEE exhibited a dose-dependent antiestrogenic activity.

Biological activity of some 4-anilinoquinazolines: cytotoxic, genotoxic and antiprotease effects, induction of necrosis and changes of actin cytoskeleton.

Fourteen substituted 4-anilinoquinazolines have been tested for cytotoxic effect and structure activity relationships. The most active derivatives were substituted by chlorine or bromine group in the aromatic ring, in the pyrimidine ring by morpholine group and in the aniline skeleton by nitro group in position 4 or 2. Derivatives 6-bromo-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline, 6-bromo-2-morpholin-1-yl)-4-anilinoquinazoline, 2-(morpholin-1-yl)-4-(4'-bromoanilino)-quinazoline and 6-chloro-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline inhibited growth of tumor cell lines HeLa, B16 and L1210. Mutagenic data provided by Ames test showed, that the compounds 6-bromo-2-morpholin-1-yl)-4-anilinoquinazoline and 2-(morpholin-1-yl)- 4-(4'-bromoanilino)quinazoline did not exhibit the mutagenic effect, whereas the compounds 6-bromo-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline and 6-chloro-2-(morpholin-1-yl)-4-(4'-nitroanilino) quinazoline increased slightly the number of revertants of the strain TA 98 without metabolic activation. Concentration 26 micromol/L of 6-bromo-2-(morpholin-1-yl)-4-anilinoquinazoline induced necrosis of tumor cells B16. Concentration 5.2 micromol/l induced a significant increase of filamentous actin in the transformed HepG2 cells. Derivatives 6-bromo-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline, 6-bromo-2-morpholin-1-yl)-4-anilinoquinazoline, 2-(morpholin-1-yl)-4-(4'-bromoanilino)quinazoline and 6-chloro-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline exhibited antiprotease effect on plasmine. This results could be relevant for the anticancer properties of these compounds.

Comparison of three in vitro assays at evaluation of IC50 of acetylsalicylic acid, ferrous sulfate, amitriptyline, methanol, isopropanol and ethylene glycol in human cancer cells HeLa.

Evaluation of the 50% inhibitory concentration (IC50) of acetylsalicylic acid, ferrous sulfate, amitriptyline, methanol, isopropanol and ethylene glycol was done on human cancer cells cultured in in vitro conditions. Three different in vitro assays were used in this study: the plating efficiency test, the microprotein test and the neutral red uptake test. Obtained results were evaluated by statistical methods. All used methods seem to be useful for screening a cytotoxic potential of the tested chemicals. The knowledge of cytotoxic effects of frequently used chemicals on mammalian cells is important not only for necessary in vitro genotoxicity and carcinogenicity studies but also for assessing the toxicity of chemicals to find out possible hazards to the human health. Results presented in this paper underline the usefulness of the wider methodological approach for the comparison of the different endpoints as well as a necessity for selection of a battery of in vitro cytotoxicity tests allowing to estimate the possible harmful effects of xenobiotics.

Interaction of acetochlor with estrogen receptor in the rat uterus. Acetochlor--possible endocrine modulator?

The aim of the present study was to investigate the ability of the chloroacetanilide herbicide acetochlor to interact with the endocrine system. The modulation of the binding of [3H]estradiol-17beta to protamine sulphate-precipitated uterine nuclear and cytoplasmic estrogen receptors was analysed for this purpose. Two different stages of reproductive life cycle of female rats, virgin and uniparous, were used. Our results demonstrate that acetochlor interacts in a specific manner with high-affinity limited-capacity uterine estrogen receptors. A significant difference (p < 0.001) in estrogen receptor density was observed between two control groups: uniparous rats (Bmax = 43.634 +/- 9.516) and virgin rats (Bmax = 154.375 +/- 21.462), suggesting an intrinsic difference in the nuclear estrogen receptor levels between female rats in different reproductive life cycle stages. Consequently, a different response to acetochlor treatment was noted. Exposure to acetochlor significantly (p < 0.001) increased estrogen receptor binding activity in the nuclear fraction of uniparous female rats (Bmax = 123.324 +/- 5.666) in comparison to control (Bmax = 43.634 +/- 9.516). In exposed virgin female rats, no significant difference was detected when compared to the corresponding control group. These results should prompt us to more thoroughly evaluate potential hazards of acetochlor to human and wildlife endocrine systems.

Stobadine inhibits lysosomal enzyme release in vivo and in vitro.

This study investigated the ability of stobadine, an effective cardioprotective drug with antiarrhythmic, antihypoxic and oxygen free radical scavenging properties, to protect cells against cyclophosphamide-induced toxic and cytotoxic damage in vivo and in vitro. Cyclophosphamide-induced toxic damage in female ICR mice was accompanied by marked increase in the activity of lysosomal enzymes in the spleen and kidney. Administration of stobadine prior to cyclophosphamide inhibited these biochemical changes. The in vivo protective effect of stobadine was comparable with its in vitro effect established in HeLa cells.

Comparison of isoproterenol-induced changes in lysosomal enzyme activity in vivo and in vitro.

Isoproterenol was used as a drug which, when administered in high doses, is able to induce lysosomal enzyme activity changes in in vivo conditions. We correlated lysosomal enzyme activity in the absence and presence of isoproterenol, obtained in whole animals and in HeLa and HepG2 cells in tissue culture. In vivo experiments: male Wistar rats (270-300 g) were treated subcutaneously with isoproterenol in various doses. Effect of isoproterenol on lysosomal enzyme activity was assayed in the heart after differential centrifugation. In vitro experiments: Isoproterenol in concentrations 0.1-100 microg/ml was added to HeLa and HepG2 cells and the activity of lysosomal enzyme was measured in the cell homogenate. In the sedimentable and nonsedimentable fractions of the rat myocardium, the isoproterenol-induced changes in the activity of lysosomal enzyme were time-and dose-dependent. In HeLa cells, isoproterenol administration caused a dose-dependent increase of lysosomal enzyme activity, while in HepG2 cells the activity remained unchanged. Thus the isoproterenol-induced changes in lysosomal enzyme activity in the rat myocardium were comparable with the results found in vitro in HeLa cells.

Detection of apoptotic changes in HeLa cells after treatment with paracetamol and sodium fluoride.

Apoptosis is genetically programmed cell death, an irreversible process of cell senescence with characteristic features (cell shrinkage, chromatin condensation, DNA fragmentation, apoptotic bodies) different from necrosis. Several effective in vitro methods for qualitative and quantitative detection of apoptotic events have been developed. Chromatin degradation, reductions in cell volume and other apoptosis-associated changes in cell morphology and physiology can be quickly analysed by multiparametric flow cytometry (FC) using small numbers of intact cells. One further method used for morphological determination of apoptotic changes is the fluorescent microscopic technique (FM) based on labeling of cells with fluorochromes acridine orange and ethidium bromide. In our experiments FC was used for determination of DNA changes in HeLa cells based on staining of DNA by fluorochrome propidium iodide (PI). Among the tested chemicals (paracetamol and sodium fluoride) apoptotic process could only be detected in paracetamol-treated cells. Apoptosis was induced mainly in cells treated with paracetamol (concentration range 4-5 mg/ml) for 8 h and following incubation for 18 h in fresh medium without paracetamol. The results obtained by the FM method correlated with the results obtained by FC.

Dual effect of pseudorabies virus growth factor (PRGF) displayed on actin cytoskeleton.

Pseudorabies virus growth factor (PRGF) was shown to possess transforming activity as well as transformation repressing activity in in vitro systems. In order to better understand these phenomena we studied actin cytoskeleton and its alterations induced by PRGF using normal human fibroblasts VH-10 and transformed cell line HeLa. For specific detection of filamentous actin cells were stained with phalloidin conjugated with fluorescein isothiocyanate (FITC)-phalloidin. PRGF was applied to VH-10 cells for various length of time from 10 min up to 48 h. The effect was very fast and changes in actin filament composition could be detected already after 10 min. In comparison to untreated cells the staining of treated cells was more diffuse and a number of actin microfilaments in individual stress fibers became reduced. After 30 min thick short actin bundles appeared in the perinuclear region. A 24-h exposure resulted in a large reduction of actin bundles. After additional 24 h a partial restoration of actin cytoskeleton in cells was observed. In transformed HeLa cells PRGF induced opposite process than in normal cells: the number of filamentous actin structures increased. We hypothesise that PRGF may act as a transcription-like factor and may initiate changes in gene expression which consequently result in actin cytoskeleton alterations.

Apoptosis Versus Cytotoxicity in HeLa Cells Exposed to Paracetamol.

Apoptosis is a programmed form of cell death which occurs in response to specific stimuli. It is distinguished from necrotic or accidental cell death by unique events, including the degradation of chromatin and a loss of cellular volume. In contrast to necrotic cell death, cell membrane integrity and mitochondrial function are thought to be maintained until the apoptotic process is well advanced. One of the novel assays for detecting apoptosis is flow cytometry. In our experiments, we used a flow cytometric assay to detect DNA changes in a human cell line (HeLa) exposed to paracetamol, by measuring propidium iodide binding. We were able to detect the apoptotic process in cells exposed to paracetamol. Apoptosis did not correlate with cytotoxicity, and was only found in samples exposed to 4-5mg/ml paracetamol for 8 hours in minimum essential medium and incubated in fresh medium without paracetamol for 14-19 hours. The greatest effect was noted 18 hours after paracetamol exposure. These results were confirmed by studying cell morphology and chromatin condensation by fluorescent microscopy with the fluorochromes acridine orange and ethidium bromide. Our results support the hypothesis that, in cultured cells, apoptosis is induced by a relatively narrow range of chemical concentrations which are known to inhibit the cell cycle, and that apoptosis and inhibition of cell proliferation coincide to some degree.

Cu-complex counteracts the effect of paraquat on polymerized actin.

The copper complex tetraanhydroaminobenzaldehyde is a synthetic compound with structural (16 atoms in a macrocyclic ring, four donor nitrogens associated with Cu(2+)) and functional similarity to the enzyme superoxide dismutase. Its ability to catalyse superoxide anion to molecular oxygen and hydrogen peroxide makes this complex interesting since it may be used to avoid or at least reduce different kinds of injuries induced by oxidative stress. To confirm this hypothesis at the cell level we used human hepatoma cell line HepG2 and a widespread herbicide, paraquat, known to induce production of free oxygen radicals through its reduction/oxidation as the model system. One of target sites described for paraquat is actin cytoskeleton. This phenomenon was used to detect and to characterize the possible ability of the Cu-complex to influence the effect of paraquat on actin structures. We investigated the Cu-complex in three different conditions: (1) the simultaneous effect of paraquat and the Cu-complex; (2) pretreatment with the Cu-complex following paraquat exposure; (3) exposure to paraquat following the treatment with Cu-complex. Phalloidin, conjugated with fluorescein isothiocyanate, known to bind specifically to filamentous actin, was used to stain filamentous actin structures. Flow cytometry showed that the Cu-complex and paraquat interacted with the actin cytoskeleton in a competitive-like manner.

Macrocyclic Cu(II)tetraanhydroaminobenzaldehyde complex--antiproliferative activity in vitro and in vivo.

The macrocyclic Cu(II)-tetraanhydroaminobenzaldehyde complex Cu(TAAB)C12 induces various cytotoxic effects in the dependence on a cell line, concentration and time of exposition. The highest complex concentration of 914 nmol/l induces degeneration of certain part of the HeLa cells population after 24 hours of cultivation. The concentration of 183 nmol/l causes a delayed cytotoxic effect on HeLa and HepG2 cells. After 24 hours of culturing 15.4-28.4% of cell population proliferated but after 48 and 72 hours 2.0-42.3% of the cell population degenerated. The cytotoxic effect on V79 cells is directly dependent on the actual concentration and time of the complex influence. The cytotoxic concentrations of Cu(TAAB)Cl2 induce an integrity damage of cytoplasmatic membrane and two phase unbalanced growth. Cu(TAAB)Cl2 possesses an antileukemic activity which at the dose of 10 mg/kg of body weight is not accompanied by side toxic effects on mice.

Requirement of amino-terminal modification for striated muscle alpha-tropomyosin function.

Striated muscle alpha-tropomyosin expressed in Escherichia coli is unacetylated, polymerizes poorly, and binds weakly to F-actin (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). To define the structural requirements of NH2-terminal modification for striated tropomyosin function, an acetylated recombinant tropomyosin and an unacetylated short fusion recombinant tropomyosin were compared. An acetylated recombinant chicken striated muscle alpha-tropomyosin was expressed in insect Sf9 cells using the baculovirus expression vector system. The purified tropomyosin (approximately 15 mg/liter of insect cell suspension) polymerized, comigrated with chicken striated alpha-tropomyosin purified from muscle on two-dimensional polyacrylamide gels, was blocked at the NH2 terminus, and had the same actin affinity as muscle tropomyosin. These results conclusively show the importance of NH2-terminal acetylation for striated tropomyosin function. To learn if a short fusion peptide would substitute for amino-terminal acetylation, tropomyosin with AlaSer-Arg on the NH2 terminus was constructed and expressed in E. coli as an unacetylated protein. This f3-tropomyosin bound to actin with a 10-fold higher affinity than striated muscle alpha-TM and, unlike muscle tropomyosin, exhibited a shear-dependent viscosity. The altered function of f3-tropomyosin shows that the naturally occurring acetylated NH2 terminus is required for full, normal function. It is proposed that a major requirement for cooperative binding of striated muscle tropomyosin to actin is modification of the alpha-amino group of methionine to be an amide, as when acetylated or in a peptide bond in a fusion protein, to make the extreme NH2 terminus more hydrophobic. The results are discussed in terms of known coiled coil structure.

Immunofluorescent detection of actin cytoskeleton in spontaneously transformed and B77-supertransformed cells using antibody to tropomyosin.

Differences in immunofluorescence of actin cytoskeleton between normal rat fibroblasts and two transformed cell lines SAMIV and SAMB77 were detected by antiserum to tropomyosin. In both transformed cell lines reduction in number and shortening of microfilament bundles (stress fibers) were observed. In some transformed cells ruffling membranes (peripheral ruffles) were seen. Differences were found in actin cytoskeleton among individual cells of spontaneous transformants (SAMIV) and supertransformants (SAMB77).

BrdU-induced changes in the provirus of PR-RSV in mammalian cells.

Previously we demonstrated differences in the organization and methylation pattern of integrated Rous sarcoma proviral sequences in helper-dependent virogenic rat TWERC cells. In the present study we attempted to induce changes in the integrated viral genome of TWERC cells using 5-bromodeoxyuridine (BrdU). Four clones (A, B, C, and F) derived from the parental cell line were treated for 10 months with different concentrations of BrdU. Restriction enzyme analysis of the parental cell line and its clones showed that the cells contained in their genomic DNA two copies of deleted provirus. In TWERC cells, the PR-RSV provirus lost the whole env gene and part of the 3' end of the pol gene. The proviral sequences in DNA from the TWERC-derived clones were found to be hypermethylated. A slightly different situation was seen in clone C where demethylation of the provirus in the 3' region and in the 5' end of the genome was found. The level of mRNA expression both in the parental cells and in the clones correlated with the methylation pattern of the PR-RSV provirus. Clone C was less methylated and expressed more virus-specific RNA. The possible role of BrdU in these events is discussed.

Using tropomyosin polymorphic phenomenon for detection of differences between rat spontaneous tumor cells and their in vitro oncovirus supertransformats.

Biochemical and immunological comparative studies of rat tumor cells of spontaneous origin and in vitro supertransformed cell populations have been done. We have focused on characterization of differences in tropomyosin molecular forms in the individual cell populations. Our experiments have shown that differences of tropomyosin forms exist not only between spontaneous transformants and supertransformants but also between supertransformants and normal rat fibroblasts. It means that superinfection of spontaneous transformants by avian sarcoma virus B77 have induced changes in tropomyosin synthesis but a pattern of tropomyosin forms in super-transformants has not been equal or similar to that of normal fibroblasts.

Quantitative changes of tropomyosin synthesis in RSV-transformed mammalian cells in comparison with normal fibroblasts.

Protein profiles of three tumor mammalian cell lines and three control cell lines were analyzed by two-dimensional gel electrophoresis. Comparison of the cells labeled metabolically with 35S-methionine has revealed obvious differences in an area of molecular weights 34 000--36 000 daltons and pH 4.5-5.0. Two spots have been proposed to be alpha- and beta-tropomyosin subunits. Their synthesis decreases after cell transformation.