The cytotoxic effect of heavy metals on an L-cell culture

The dependence of some parameters of L-cells culture viability on different concentrations of heavy metals was studied. Considerable cytotoxic effect of low concentrations of nickel (0.025 mcg/ml) and lead (0.05 mcg/ml) was shown. Copper and chrome at concentrations of 0.25-0.5 mcg/ml promote cells proliferation between third and fifth days of cultivation. Nickel at concentration 0.025 mcg/ml and lead at all investigated concentrations synchronize cells division in culture. Increasing of giant polynucleas cells level in culture was characteristic for investigated metals. The maximum levels of this type cells were caused by the action of nickel, chrome and copper.     

Conditions of the reversibility of metaphase arrest and induction of multi-polar mitoses after treatment with nocodazole

Nocodazole at a concentration 0.02 mcg/ml or higher arrests PE cells (pig kidney embryo cells) in K-metaphase. Accumulation of mitotic cells by incubation with 0.02 or 0.6 mcg/ml nocodazole occurs linearly and at the same rate during 12-16 hours. After nocodazole is removed, the mitotic index is resumed to the normal rate. The maximum time of the reversible mitotic arrest in PE cells in 16 hours. After the incubation of cells with 0.2 mcg/ml nocodazole, the time of the reversible mitotic arrest is 12 hours. After the incubation of cells with 0.02 or 0.2 mcg/ml nocodazole, no multipolar mitoses are observed. After the 4 hours incubation with 0.6 mcg/ml nocodazole, multipolar mitotic figures are observed 1.5-2.5 hours after drug removal. It is concluded that the induction of multipolar divisions requires no prolonged mitotic arrest, but it may be caused by a complete depolymerization of spindle microtubules.     

The effect of fractionated exposure to unequal concentrations of chemical mutagens

The effect of unequally fractionated concentrations of dipin and thiophosphamid on chromosomes of human lymphocytes was investigated at Go phase. There were used five low concentrations of mutagens 2, 0.2, 2.10(-2), 2.10(-3), 2.10(-4) mcg/ml and one high concentration 20 mcg/ml by which cells have been treated. Decrease of chromosome breaks and exchanges were observed, but the level of the aberration cell was stable. The "protective" levels for dipin were in concentrations of 0.2, 2.10(-2), 2.10(-3) mcg/ml. Only one "protective" concentration was 2.10(-2) mcg/ml.     

Cellular behavior and centriole distribution during multipolar mitosis induced by nocodazole action

After recovery from the nocodazole blockade, mitoses in PE cells proceed differently depending on the time of treatment and on the drug concentration. Cells, treated with 0.02 mcg/ml for 3 hours or less, have a recovery period of 1-1.5 hours, however cells, treated with 0.02 mcg/ml for more than 3 hours or with 0.2 mcg/ml at a time, have a recovery period of 3-4 hours. In both the cases anaphase and cytokinesis proceed normally. The 0.6 mcg/ml nocodazole concentration results in the appearance of only multipolar mitoses during recovery. The minimal-time multipolarity induction is 1 hour. Cytokinesis is disturbed in 60% of multipolar mitoses: two of the three daughter cells are fused to form a binucleated cell. A complete disruption of the mitotic apparatus causes one of the diplosomes to dissociate. In the first minutes of recovery, the other diplosome dissociates too. In tripolar telophase centrioles distributed among the spindle poles according to the 2 : 2 : 0 pattern, as a rule. Thus, the deranging of the mitotic spindle is a necessary and sufficient condition for the induction of multipolar mitoses in tissue culture cells. This derangement accompanies the dissociation of diplosomes, but single daughter centrioles do not form a spindle pole.     

Cytotoxic and cytostatic effect of avermectines on tumor cells in vitro]

Effect of natural avermectin complex (aversectin C) and separate avermectins A1, A2, B1 and B2 in the cell culture of murine myeloma Ns/o, Erlich carcinoma ascites and human larynx carcinoma Hep-2 was investigated. It was shown that aversectin C within the concentrations of 0.1 to 1.0 mcg/ml inhibited proliferation of tumor cells and induced their death. Proliferation inhibition was due to the delay of the cells cycle start (lag-phase prolongation) and blocking of mitotic cycle. Ns/o cells death had apoptosis signs: chromatin condensation and fragmentation, DNA fragmentation. It was demonstrated that only avermectin A1 has cytotoxic activity within the concentrations used, avermectins A2 and B2 had cytostatic activity, avermectin B1 showed no activity under the experimental conditions.     

Study of antiviral activity of different drugs against bovine herpes virus and pestivirus]

In vitro antiviral activity of 11 different drugs against the viruses of infectious bovine rhionotracheitis (IBR) and bovine viral diarrhea (BVD) was studied. The ID50 of the drugs were determined in monolayers of cell cultures MDBK and KCT: 20 mcg/ml for anandin, 25 mcg/ml for polyprenole, 50 mcg/ml for bromuridin, methisazone, aciclovir, gossypole, ribavirin and liposomal ribavirin, 100 mcg/ml for eracond, and 200 mcg/ml for phosprenil and argovit. Phosprenil was the only drug that showed virucidal activity against the IBR virus. All the drugs inhibited reproduction of the IBR virus in sensitive cell culture MDBK: 100,000-fold inhibition by bromuridin, aciclovir, ribavirin and methisazone, 1000-10000-fold inhibition by liposomal ribavirin, gossypole, anandin, polyprenole and phosprenil, 100-fold inhibition by eracond and argovit. As for the BVD virus, bromuridin, phosprenil, polyprenole, methisazone, aciclovir, gossypole, argovit, ribavirin and liposomal ribavirin also showed their activity in cell culture KCT (100-10,000-fold inhibition). The other drugs were ineffective.     

2,4-Dichlorophenoxyacetic acid causes chromatin and chromosome abnormalities in plant cells and mutation in cultured mammalian cells

The cytotoxic and mutagenic effects of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) on shallot root tip cells and on V79 Chinese hamster fibroblast cells were examined and compared. In shallot root tips 2,4-D caused changes in mitotic activity, as well as changes in chromosome and chromatin structure, and also changes during the cell cycle. 2,4-D also showed mutagenic and cytotoxic effects on V79 cells in culture in concentrations higher than 10 micrograms/ml. The results in both systems (plant and mammalian cells) were in agreement showing mutagenic activity of 2,4-D in the concentration range higher than usually used in establishing plant tissue culture (greater than 5 micrograms/ml).     

In vitro antiviral activity of fullerene amino acid derivatives in cytomegalovirus infection]

The in vitro effect of 5 water soluble fullerene C60 amino acid derivatives (FAD) on the development of cytomegalovirus infection was studied in the schemes of the therapeutic, prophylactic and virucidal action. The following compounds as FAD were used: fullerene conjugated with Na salt of gamma-aminobutyric acid (C60-ABA-Na), 2 derivatives based on Na salts of fullerene-gamma-aminobutyric acid and fullerene-omega-caproic acid (C60-ABA-OH-Na and C60-ACA-OH-Na respectively) and 2 derivatives based on methyl ethers of the above mentioned fullerene amino acids (C60-ABA-OH-CH3 and C60-ACA-OH-CH3). All the FAD were able to inhibit the development of the virus cytopathogenic action in the cell culture. However, the compounds had different antiviral properties. C60-ABA-OH-Na, C60-ABA-CH3 and C60-ACA-CH3 showed marked antiviral activity in the prophylactic scheme. 50-Percent inhibition of the virus cytopathogenic action (ID50) was observed when concentrations of the compounds were 0.31, 5 and 25 mcg/ml respectively. C60-ACA-OH-Na inhibited the development of cytomegalovirus infection in the cell culture only in the scheme of the therapeutic action (ID50 4 mcg/ml). C60-ABA-Na had the highest antiviral effect. In a concentration of 0.22 mcg/ml it inhibited the cytomegalovirus plague-forming capacity by 50% in both the prophylactic and the virucidal schemes. The chemotherapeutic index of the compound was within the limits of 2500 to 5450.     

The trypanocidal action of fluorouracil on Crithidia oncopelti in the presence of lipid metabolic modifiers]

Effect of fluorouracil in combination with ascorbate and L-valin, modifiers of lipid metabolism was studied in cultured protozoa Crithidia oncopelti. The inhibitory effect of preparation in a concentration of 100 mcg/ml gradually decreased in the course of cultivation. Its readdition to a 96-hour culture did not increase its effect on protozoa cells. Combined addition of fluorouracil and modifiers (100 mcg/ml each) resulted in insignificant decrease of the cell accumulation in the culture as compared with the effect of fluorouracil alone. When fluorouracil and modifiers were readded to the 96-hour culture, the trypanostatic effect of preparation was 2.5 times enhanced. This enhancement was confirmed by destructive alterations in cell morphology and by the culture lysis by 192 h of protozoa cultivation.     

Isolation of canine rotavirus and study of its immunobiological properties]

Canine rotavirus was isolated in MA104 roller culture of rhesus macaque cells. Two passages in gnotobiotic puppies and two in colostrum-free puppies resulted in isolation of strain P of canine rotavirus. After 20 passages in MA104 culture the virus was adapted to MDCK culture. Optimal conditions for accumulation of canine rotavirus and its antigen (9.01 g TCD50/ml) in MDCK culture are trypsin pretreatment of the virus inoculate in the final concentration of 50 mcg/ml for 30 min at 37 degrees C, presence of trypsin (10 mcg/ml) in the maintenance medium, multiplicity of infection 0.1 TCD50/ml, and incubation in roller culture at 37 degrees C during 24-30 h. After 60 passages in cell culture, canine rotavirus completely lost its virulence for colostrum-free puppies but retained antigenic activity and induced manifest seroconversion in infected.     

The effect of estrogen on the incorporation of 3H-thymidine by PHA-stimulated human peripheral blood lymphocytes

The effect of estrogen on the incorporation of tritiated thymidine by phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes was evaluated in 15 adult males. Varying concentrations of diethylstilbestrol diphosphate (DEP-S) were added to peripheral blood lymphocytes with and without PHA to study the effects of estrogen on blastogenesis. Maximum suppression of blastogenesis occurred after the addition of 500 mcg/ml culture of DEP-S. The absence and presence of DEP-S 500 mcg/ml culture resulted in a 52% reduction in lymphocytic reactivity (p.002). It was concluded that this reduction or suppression of lymphocytic blastogenesis in the presence of estrogen suggests that the palliative effects of estrogenic therapy in treating patients with hormone-dependent tumors may be countered by its adverse effect on the host's immunologic responsiveness to malignancy.     

Experimental study of Ingavirin antiviral activity against human adenovirus

Antiviral properties of Ingavirin were investigated in the Hep-2 cell culture with respect to the human respiratory tract virus (type 5 adenovirus). In concentrations of Ingavirin of 1000, 100 and 10 mcg/ml the generated posterity showed lower infective capacity (by 250, 100 and 10 times respectively). The electron microscopy of the infected cells confirmed the Ingavirin ability to disturb the adenovirus normal morphogenesis.     

Effects of exposure time using preliminary concentrations on the mutagenic effect of the basic action of one-center and dual-center mutagens

The effect of exposition with pretreatment for thiophosphamide and dipin of human lymphocytes at Go phase was investigated. There were used 5 low concentrations of mutagens: 2, 0, 2; 2.10(-2); 2.10(-3), 2.10(-4) mcg/ml with different exposure: 1/4 hr, 1/2 hr, 1 hr and 4 hr and high concentration of 20 mcg/ml by which cell have been treated. There was discovered the dependence of the "protective" concentration on the exposition: the increase of exposition of pretreatment induced the decrease of "protective" concentration and vice versa.     

Investigation of remaxol efficacy in complex therapy of experimental generalized tuberculosis on mice

Efficacy of remaxol in complex chemotherapy of generalized drug resistant tuberculosis was studied on mice. Mycobacterium tuberculosis 5419 SPBNIIF isolated from a patient with freshey diagnosticated pulmonary tuberculosis resistant to isoniazid (10 mcg/ml), rifampicin (40 mcg/ml), streptomycin (10 mcg/ml) and ethionamide (30 mcg/ml) was used in the experiments. The main polychemotherapy included 4 antituberculosis drugs in the highest therapeutic doses: isoniazid, amikacin, ethambutol and tavanic, the treatment course was 8 weeks. Remaxol was administered in a dose of 25 ml/kg intraperitoneally 5 times a week (14 injections). Significant activating effect of remaxol on the tension of the lung tissue local immunity was revealed by changes in the granuloma cell composition (from mainly epitheliod to mainly lymphoid) and by more frequent large lymphohistiocytic infiltrates. The use of remaxol also greatly increased the absorptive and digestive activity of the peritoneal macrophages phagocytosis, inhibited in the process of the experimental tuberculosis development.     

Transforming activities of sodium fluoride in cultured Syrian hamster embryo and BALB/3T3 cells

The transforming activity of sodium fluoride was studied in the SHE and the BALBl3T3 cell culture systems. Initiating and promoting activities were then investigated by means of the orthogonal methodology. Sodium fluoride was found to induce morphological transformation of SHE cells seeded on a feeder layer of X-irradiated cells at high concentrations (75–125 g/ ml). When the cells were seeded in the absence of a feeder-layer, the transformation frequencies increased in a dose-dependent manner with the concentrations of sodium fluoride ranging from 0 to the highly toxic concentration of 200 g/ml. In the BALBl3T3 cell system, sodium fluoride was negative in the standard Kakunaga procedure, while through the experiment designed by table L₈ (2⁷) of the orthogonal method, an initiating-like effect and a weak promoting activity were detected within the concentrations ranging from a 25 g/ ml to a 50 g/ ml concentration which is highly toxic for BALBl3T3 cells. From these results, it is suggested that, besides a genetic mode of action, sodium fluoride could possibly act through a non-genotoxic mechanism.Abbreviations CE cloning efficiency - NaF sodium fluoride - SHE Syrian hamster embryo - TF transformation frequency     

Mechanistic investigations of low dose exposures to the genotoxic compounds bisphenol-A and rotenone

A complete hazard and risk assessment of any known genotoxin requires the evaluation of the mutagenic, clastogenic and aneugenic potential of the compound. In the case of aneugenic chemicals, mechanism of action (MOA) and quantitative responses may be investigated by studying their effects upon the fidelity of functioning of components of the cell cycle. These present studies have demonstrated that the plastics component bisphenol-A (BPA) and the natural pesticide rotenone induce micronuclei and modify the functioning of the microtubule organising centres (MTOCs) of the mitotic spindles of cultured mammalian cells in a dose-dependent manner. BPA and rotenone were used as model compounds in an investigation of dose response relationships for the hazard/risk assessment of aneugens. Thresholds of action for the induction of aneuploidy have been predicted for spindle poisons on the basis of the multiple targets, which may need disabling before a quantitative response can be detected. The cytokinesis blocked micronucleus assay (CBMA) methodology was utilised in the human lymphoblastoid cell lines AHH-1, MCL-5 and Chinese hamster V79 cell lines. A no observable effect level (NOEL) at 10.8 microg/ml BPA was observed for MN induction. Rotenone showed a small increase in MN induction with the first significant effect at 0.25 ng/ml in V79 cells but there was no significant effect in the metabolically competent cell line, MCL-5. For a mechanistic evaluation of the aneugenic effects of BPA and rotenone, fluorescently labelled antibodies were used to visualise microtubules (alpha-tubulin) and MTOCs (gamma-tubulin). The NOELs for tripolar mitotic spindle induction in V79 cells were 7 microg/ml for BPA and 80 pg/ml for rotenone (concentrations which produced similar changes to mitotic index (M.I.)). Interestingly there was close proximity to the NOEL of 10.8 microg/ml BPA for micronucleus (MN) induction in the human lymphoblastoid AHH-1 cell. Multiple MTOCs can therefore be predicted as a possible mechanism for MN induction. The similarity in concentration inducing tripolar mitosis, M.I. and MN changes suggests immunofluorescence analysis to be a useful dose setting assay with emphasis on the mechanism.     

A trophic effect of epidermal growth factor (EGF) on rat colonic mucosa in organ culture

The development of an organ-culture system for rat colonic mucosa has enabled a direct assessment of the effect of epidermal growth factor (EGF) on cell division. An augmented mitotic index (AIm) has been employed to identify changes in cell proliferation. Explants of colonic mucosa from four animals were maintained in a medium containing serum for five days. On the fifth day of culture, half of the explants received fresh medium containing EGF (40 ng/ml) and the remainder (controls) fresh medium only. At 6, 12, 24 and 48 hr thereafter groups of both experimental and control explants received the metaphase-arresting drug vincristine (4 micrograms/ml) for 3 hr prior to fixation. The proportions of vincristine-arrested metaphases within the explants were determined. Analysis of the data indicates that when serum is present exogenous EGF exerts a trophic effect which increases with time (P less than 0.001). In a second experiment colonic explants from four animals were maintained for five days in a serum-free medium and were then divided into groups, each of which received one of a range of concentrations of EGF. The AIm was determined for each group after 36 hr. It was found that increasing concentrations of EGF produce a small but significant increase in cell proliferation (P less than 0.01). This effect, however, was less pronounced than that seen when serum was present. These results suggest that EGF has a trophic action on the colon and interacts with additional factors found in serum.     

A trophic effect of epidermal growth factor (EGF) on rat colonic mucosa in organ culture

Abstract. The development of an organ-culture system for rat colonic mucosa has enabled a direct assessment of the effect of epidermal growth factor (EGF) on cell division. An augmented mitotic index (AI_m) has been employed to identify changes in cell proliferation.
Explants of colonic mucosa from four animals were maintained in a medium containing serum for five days. On the fifth day of culture, half of the explants received fresh medium containing EGF (40 ng/ml) and the remainder (controls) fresh medium only. At 6,12,24 and 48 hr thereafter groups of both experimental and control explants received the metaphase-arresting drug vincristine (4 μ g/ml) for 3 hr prior to fixation. The proportions of vincristine-arrested metaphases within the explants were determined. Analysis of the data indicates that when serum is present exogenous EGF exerts a trophic effect which increases with time ( P < 0.001).
In a second experiment colonic explants from four animals were maintained for five days in a serum-free medium and were then divided into groups, each of which received one of a range of concentrations of EGF. The AI_m was determined for each group after 36 hr. It was found that increasing concentrations of EGF produce a small but significant increase in cell proliferation ( P < 0.01). This effect, however, was less pronounced than that seen when serum was present.
These results suggest that EGF has a trophic action on the colon and interacts with additional factors found in serum.     

Mutagenic activity of fluorides in mouse lymphoma cells

The L5178Y mouse lymphoma cell forward-mutation assay was used to test for the mutagenic activity of sodium and potassium fluoride at the thymidine kinase locus. Mutants were detected by colony formation in soft agar in the presence of trifluorothymidine. Mutagenic and toxic responses were observed in the concentration range of 300-600 micrograms/ml with both sodium and potassium fluoride. Approximately 3-fold increases in mutant frequency were observed for concentrations in the 500-700 micrograms/ml range that reduced the relative total growth to approximately 10% in the absence or presence of a rat-liver S9 activation system. A sample of 30% sodium fluoride-70% sodium bifluoride (NaHF2) induced a similar mutagenic response but was more toxic with respect to the fluoride concentration. A specificity for fluoride ions in causing mutagenesis was indicated by the fast that much higher concentrations of sodium or potassium chloride were necessary to cause toxicity and increases in the mutant frequency. The possible involvement of chromosomal changes was signaled by the predominant increase in the small colony class of mutants.     

Cytogenetic effect of malathion in in vitro culture of human peripheral blood

Chromosomal aberrations, sister-chromatid exchanges and mitotic indices were observed in human peripheral leukocytes, treated with four different concentrations of malathion (0.02, 0.2, 2 and 20 μg/ml), an organophosphate pesticide, added to the culture medium at 0, 24 and 48 h after culture initiation. These cultures showed a dose-dependent increase in the frequency of chromosomal aberration as well as sister-chromatid exchanges. There was a significant decreases in mitotic index at all concentrations.