Clastogenicity of 1-nitropyrene, dinitropyrenes, fluorene and mononitrofluorenes in cultured Chinese hamster cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on 1-nitropyrene (NP), 3 dinitropyrenes (DNPs), fluorene and 4 mononitrofluorenes with and without metabolic activation (rat S9 mix). The 3 DNPs (1,3-, 1,6- and 1,8-DNP) induced chromosomal aberrations in the absence of S9 mix. The frequencies of cells with aberrations after treatment for 48 h were 43% at 2 micrograms/ml of 1,3-DNP, 55% at 0.1 microgram/ml of 1,6-DNP and 45% at 0.025 microgram/ml of 1,8-DNP, indicating the order of clastogenic potency as 1,8- greater than 1,6- greater than 1,3-DNP. On the other hand, 1-NP, which is known to be a direct-acting mutagen in bacteria, was negative in the chromosomal aberration test without S9 mix, but clearly positive with S9 mix. This effect was dependent on the concentration of the S9 fraction in the reaction mixture. High-pressure liquid chromatography analysis showed that 1-NP was converted by S9 mix to several metabolites, including 1-aminopyrene (AP). The clastogenic activity of 1-AP, however, was equivocal without S9 mix, suggesting that active clastogens other than 1-AP exist. Fluorene induced chromosomal aberrations only in the presence of S9 mix (61.8% at 25 micrograms/ml). 1-, 2-, 3- and 4-nitrofluorene (NF) were more clastogenic in the presence of S9 mix than in the absence of S9 mix, suggesting that NFs were converted to more active clastogens by S9 mix.     

Evaluation of magnolia bark extract in chromosomal aberration assays

Magnolia bark extract (MBE) has been used historically in traditional Chinese and Japanese medicines, and more recently as a component of dietary supplements and cosmetic products. The genotoxic potential of MBE was studied in two in vitro chromosomal aberration assays. In Chinese hamster ovary (CHO) cells, exposure for 3 h to MBE at concentrations of 0-30 microg/ml in the absence of a metabolic activation system (S9) and 0-7 microg/ml with S9 did not induce chromosomal aberrations, whereas higher concentrations were cytotoxic and did not allow for analysis of aberrations. Extended exposure for 18 h without metabolic activation at concentrations up to 15 microg/ml also resulted in a negative response. In V79 cells derived from Chinese hamster lung tissue, treatment for 6h with concentrations up to 52 and 59 microg/ml in the absence and presence of S9, respectively, did not increase the incidence of chromosomal aberrations compared to negative controls. Furthermore, MBE exposure for 24 h without metabolic activation did not induce aberrations. The results of these studies demonstrate that MBE is not genotoxic under the conditions of the in vitro chromosomal aberration assays in CHO and V79 cells, and support the safety of MBE.     

SCE induction and cell-cycle delay by toxaphene

Toxaphene is genotoxic in mammalian cell systems and also inhibits cell replication. It was therefore used to investigate possible masking of SCE induction due to cell-cycle delay. In this study, toxaphene-treated Chinese hamster lung (Don) cells exhibited a dose-dependent decrease in cell-cycle progression compared with untreated cells. At high, nontoxic toxaphene levels (15 micrograms/ml), cell cycling also slowed as the toxaphene treatment time was increased. Toxaphene induced significantly higher numbers of SCEs in treated cells, demonstrating a dose- and treatment time-relationship. Slopes of dose-response curves were 0.29, 0.43 and 0.77 SCE/micrograms toxaphene for 20.5 h, 24.5 h and 28.5 h incubation, respectively. There were no changes in SCE values in control cultures even when slower dividing cells were sampled e.g. at longer incubation times. Thus, higher SCE values in Chinese hamster cells were not associated per se with slower or more delayed cells. The results demonstrate that longer toxaphene treatment times were not necessary for obtaining sufficient harlequin-stained cells for SCE analysis, but that higher numbers of SCEs occurred in slower dividing cells, following prolonged incubation of cultures treated with toxaphene.     

Genetic toxicology evaluation of commercial beers, III. SCE, chromosome aberrations, and forward mutation (HGPRT) of commercial beer products in CHO cells.

Concentrated organic residues extracted from 5 blended aliquots of commercial beers were evaluated for their ability to induce sister chromatid exchange (SCE), chromosomal aberrations and forward mutation in Chinese hamster ovary (CHO) cells. Each extract was prepared by blending 4 commercial beers of similar ingredients and brewing method, passing the beer pool over XAD-2 resin, extracting the resin and concentrating the extract. Studies were performed both with and without metabolic activation using variable amounts of reconstituted residues from 225-fold concentrates of the blended samples. CHO cultures were treated with 0.75 microliters/ml through 10.0 microliters/ml of the concentrates in the SCE assays, 1.0 microliters/ml through 10.0 microliters/ml of the extracts in the aberration assays and 2.5 microliters/ml up to 20 microliters/ml for forward mutation assays. In preliminary screening for SCE as an indicator of potential DNA damage, a significant increase was observed for 3 of 5 concentrated samples; however, no increase in SCE was induced by any of the 5 samples when S9 was added as a source of exogenous metabolic activation. More definitive tests for induction of genetic events, i.e., chromosome aberrations and forward HGPRT mutations, were negative for all 5 extracts whether or not S9 mix was present. Since SCE were not induced in tests with metabolic activation and since there was no concordant aberration or point mutation induction, the preliminary indication of potential DNA damage shown by elevated SCE under conditions without metabolic activation appears to have little biological significance.     

Chromosomal aberrations and sister-chromatid exchanges in Chinese hamster cells treated in vitro with hexavalent chromium compounds.

Chinese hamster cells (CHO line) were treated in vitro for 30--39 h with hexavalent chromium compounds (K2Cr2O7 and Na2CrO7), at concentrations ranging from 0.1 to 1.0 microgram of Cr6+ per ml, in medium containing BUdr. Chromosomal aberrations and sister-chromatid exchanges were scored on BUdr-labelled 2nd division metaphases, collected at the end of treatment and stained with Giemsa. Treatment with mitomycin C 0.009--0.030 microgram/ml) was carried out as a control for the responsiveness of the cell system to chromosomal damage. Both chromium compounds induced marked mitotic delays. Chromosomal aberrations were increased about 10-fold by exposure to Cr6+ (1.0 microgram/ml). The principal aberrations observed were single chromatid gaps, breaks and interchanges, whose frequencies increased proportionally to the concentration of chromium. Dicentric chromosomes, isochromatid breaks, chromosome and chromatid rings were also induced. The frequenyc of sister-chromatid exchanges was hardly doubled 30 h after exposure to Cr6+ at 0.3 microgram/ml, whereas it was trebled 39 h after treatment, in the cells whose division cycle had been slowed down by chromium.     

Clastogenicity of low pH to various cultured mammalian cells.

It has been reported that low pH itself can be clastogenic to Chinese hamster ovary cells or mouse lymphoma L5178Y cells. On the other hand, there was no indication that low pH is clastogenic to rat or human lymphocytes. Therefore, in order to evaluate the generality of clastogenicity of low pH conditions, chromosomal aberration tests were carried out on Chinese hamster cell line cells (CHO-K1, CHL, Don and V79 379A) and human cells (HeLa and peripheral lymphocytes used as whole-blood cultures). The cytotoxicity of low pH to each cell line was also evaluated by counting surviving cells. The treatment medium used was Eagle's MEM containing 15 mM MES or Bis-Tris as an organic buffer to maintain the acidity of the medium for the 6-h or 24-h treatment period, and pH adjustment was done with NaOH or HCl. Chromosomal aberrations were induced at pH 6.5 or below in CHO or CHL cells, and the maximum frequency was 24.7% at pH 6.0 or 34% at pH 6.3, respectively. About 5-10% of Don or HeLa cells had aberrations over the range of pH 6.6-6.0 or pH 6.6-6.3, respectively. In V79 379A cells or human lymphocytes, however, aberrant cells amounted to about 8% at near pH 6.0, where cell survival was low (less than 20%). About 90% of aberrations induced in each cell line examined were chromatid-type gaps and breaks. When CHO or CHL cells were treated with acidic medium for 6 h plus 18 h recovery in fresh medium, about 20% of cells had aberrations including chromatid exchanges at pH 5.5 or pH 5.7, respectively. These results indicate that clastogenicity of low pH is a general finding, although the extent of it varies with cell type, and that the clastogenicity is associated with varying extents of cytotoxicity. The mechanisms of clastogenesis at low pH are not known, but might involve inhibition of DNA or protein synthesis or DNA-repair enzymes.     

On the genotoxicity of the pesticides Endodan and Kilacar in 6 different test systems

Two pesticides, the fungicide Endodan (ethylene thiuram monosulphide) and the insecticide-acaricide Kilacar (bis(parachlorophenyl)cyclopropyl methanol), produced or used in the neighbouring countries of Bulgaria and Greece were investigated in a coordinated research programme for their genotoxic effects in a variety of test systems. This included the Ames test, Aspergillus nidulans for mitotic segregation, in vitro human lymphocyte cell cultures for SCE and chromosomal aberrations, in vivo bone marrow cells in hamsters and rats and the dominant lethal test in rats. The genotoxicity of Endodan was found to range from negative to slightly positive in different test systems. At concentrations of 7.5 and 12.0 micrograms/plate together with S9 mix it induced base-pair substitutions in the TA100 strain of Salmonella typhimurium at a rather low level. At a dose of 93 mg/kg b.w. it also caused chromosomal aberrations in acutely treated hamster bone marrow cells. A significant increase of SCE was also found in human lymphocyte cultures at a concentration of 20.0 micrograms/ml. Endodan was found to be negative in A. nidulans for somatic segregation, lymphocyte cultures for chromosomal aberrations and mitotic activity and in rats for dominant lethals and chromosomal aberrations. Kilacar was found to be a weak mutagen in the TA97 strain of S. typhimurium at concentrations of 2.5 and 5.0 micrograms/plate together with S9 mix. At concentrations of 1.0, 1.5 and 2 micrograms/ml Kilacar increased the number of mitotic segregants in A. nidulans by 160%, 220% and 156% respectively over the control. In Syrian hamster bone marrow cells after acute administration at concentrations of 0, 40, 80 and 160 mg/kg, the MI was 5.50, 4.30, 3.10 and 1.30 respectively, and an increase in chromosomal aberrations of about 300% over the control was observed with a concentration of 80 mg/kg. In human lymphocytes no significant changes were observed in either MI or SCE. In the dominant lethal test after chronic treatment of male rats at doses of 5.1, 10.2 and 102.0 mg/kg b.w. no significant mutagenic effect was found although a decrease was shown in the percentage of females with implants mated with treated males in the first week.     

Cytogenetic effects induced by cytochalasin B in Chinese hamster ovary cells

We determined the kinetics of the induction of chromosomal aberrations and micronuclei (MN) by mitomycin C (MMC, 0.1 µg/ml) in Chinese hamster ovary (CHO) cells treated with cytochalasin B (Cyt-B, 3 µg/ml). In cells treated with Cyt-B as well as with Cyt-B plus MMC the highest yield of binucleated cells was obtained 24 h after treatment. After 40 h of treatment with Cyt-B the frequency of MN in binucleated cells was significantly higher than that observed at previous times in the same cultures as well as in controls. In cultures treated with MMC the frequency of MN increased with time, reaching the highest value at 24 h. The frequency of chromosomal aberrations was also significantly higher in cells treated both with Cyt-B and Cyt-B plus MMC than in controls and exceeded that of MN in parallel cultures. These data confirm the capacity of MMC to induce chromosomal alterations in mammalian cells; in particular they indicate that Cyt-B is able to induce cytogenetic effects in CHO cells. Using immunofluorescence microscopy, after reaction with CREST antikinetochore antibodies, we found that in cells treated with Cyt-B or Cyt-B plus MMC the frequency of MN without kinetochore was, respectively, about 70 and 85%, indicating that under our experimental conditions MN originate mainly from acentric chromatid fragments. Present data suggest that the method based on the blockage of cytokinesis by Cyt-B normally used in the MN assay should be reconsidered.     

The induction of chromosomal aberrations and SCEs by visible light in combination with dyes. II. Cell cycle dependence, and the effect of hydroxyl radical scavengers during light exposure in cultures of Chinese hamster ovary cells sensitized with acridine orange

Chinese hamster ovary (CHO) cells were synchronized by mitotic shake-off, treated with the fluorochrome acridine orange (AO; 0.5 micrograms/ml), washed free of excess dye and subsequently exposed to visible light (2 X 40 W/8 Wm-2). The light exposure was performed on cells in the G1, G1/S, S or G2 phase of the cell cycle. AO + light induced high frequencies of aberration in the S phase and even higher in the G1 phase. The aberrations observed were all of the chromatid type. The chromosome-type aberrations (dicentrics, rings) obtained when cells in the G1 phase were exposed to X-rays were not found after corresponding treatments with AO + light. With the exception of an increased frequency of gaps, no chromosomal aberrations were induced in G2-phase cells. Sister-chromatid exchanges were efficiently produced by the photodynamic system in the G1, G1/S and S phase of the cell cycle. In other experiments, AO-treated unsynchronized CHO cells were exposed to light in the presence of the hydroxyl radical scavengers mannitol (100 mM) and 5-dimethyl thiourea (100 mM). In parallel experiments these scavengers were found to reduce markedly the chromosome breaking effects by X-rays but had no influence on the photodynamic induction of chromosomal alterations. The results presented show that the visible light-induced chromosomal alterations in CHO cells sensitized with the fluorochrome AO are obtained by an S-dependent mechanism. Furthermore, the results indicate that the hydroxyl free radical does not play a major role in the production of chromosomal alterations by AO + light.     

Use of the cytokinesis-block method for the analysis of micronuclei in V79 Chinese hamster lung cells: results with mitomycin C and cyclophosphamide

The cytochalasin B (CYB)-blocked binucleated cell assay has been explored to analyze micronuclei and cell cycle kinetics using 2 known mutagenic carcinogens in V79 Chinese hamster lung cells. To determine the optimum time to obtain the maximum number of binucleated cells for micronucleus analysis, duplicate cultures of exponentially growing cells were treated with 3 micrograms/ml CYB for varying durations (8-48 h). A peak appearance of binucleated cells at 16 h in the presence of CYB suggested this as an optimum time for micronucleus analysis in binucleated V79 cells. To evaluate the capacity for induction of micronuclei in V79 cells, 2 mutagenic carcinogens, mitomycin C (0.125-1.0 micrograms/ml) and cyclophosphamide (2-12 micrograms/ml) were tested in duplicate cultures. Mitomycin C, a direct-acting alkylating agent, caused approximately an 18-fold increase in micronucleus frequency over controls at the highest concentration tested (1.0 micrograms/ml), and this increase occurred in a dose-related manner (r = 0.92). The concentrations of mitomycin C tested also caused a significant dose-related cell cycle delay, thus suggesting cytotoxicity to V79 cells. Cyclophosphamide, an indirect-acting alkylating agent, requiring the presence of S9 mix, caused approximately a 17-fold increase in micronucleus frequency over controls at the highest tested concentration (12 micrograms/ml), with a clear dose response (r = 0.99). The various concentrations of cyclophosphamide also caused cytotoxicity in a dose-related fashion. Thus, this study demonstrates the usefulness of the cytokinesis-block method in V79 cells as a possible screen to analyze micronucleus induction and cytotoxicity. Because this approach is much less labor intensive than conducting a structural chromosomal analysis, this assay has great potential both as an initial screen for clastogenic activity and as a tool for investigating the underlying mechanisms for clastogenicity.     

Chromosomal aberrations induced in vitro by 3,7- and 3,9-dinitrofluoranthene

The chromosomal aberration test using a Chinese hamster cell line (CHL) was carried out with 3,7- and 3,9-dinitrofluoranthene (DNF) with and without exogenous metabolic activation (rat liver S9 mix). The highest dose tested was limited to 20 μg/ml because of the compounds' insolubility in dimethyl sulfoxide. Both DNFs induced chromosomal aberrations in the absence of S9 mix; the frequency was not very high. Results were reproducible, but without clear dose-response relationships. Neither DNF induced chromosomal aberrations in the presence of S9 mix. Both DNFs did not induce polyploid cells under any conditions.     

Genotoxic effects of o-phenylphenol metabolites in CHO-K1 cells

The effects of microsomal activation and/or deactivation on the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) in cultured Chinese hamster ovary cells (CHO-K1 cells) by o-phenylphenol (OPP) were studied, and concurrently the metabolites were determined. After a 3-h incubation in the presence of 15% S9 mix (45 microliters/ml of S9), OPP (25-150 micrograms/ml) dose-independent SCEs and chromosomal aberrations were induced, while the amount of phenylhydroquinone (PHQ) metabolite produced from OPP did not increase linearly in the higher doses. The maximum induction of chromosomal aberrations was 18% at the 150 micrograms/ml dose, and of SCEs 13.8/cell at 75 micrograms/ml. The corresponding control values were 3% and 5.8/cell. The lowest dose required to induce SCEs in the presence of S9 mix was 25 micrograms/ml. Changing the percent of S9 mix (0-50%) while holding the OPP dose constant (100 micrograms/ml) produced a correlation between SCEs and the production of PHQ. PHQ caused cytogenetic effects both with and without S9 mix, however, in the absence of S9 mix it was more lethal and was oxidized to phenylbenzoquinone (PBQ). These results suggest that the enhanced cytogenetic effects of OPP by the addition of S9 mix correlated with the amount of PHQ produced or with the further oxides of PHQ such as phenylsemiquinone and/or PBQ which are capable of being produced from PHQ spontaneously or by the mixed-function oxidase system.     

Streptonigrin induces delayed chromosomal instability involving interstitial telomeric sequences in Chinese hamster ovary cells

We analyzed the induction of chromosomal aberrations in Chinese hamster ovary (CHO) cells exposed to the radiomimetic compound streptonigrin (SN), in order to determine whether interstitial telomeric sequences (ITSs) are involved in the long-term clastogenic effect of this antibiotic. CHO cells were treated with a single concentration of SN (100ng/ml), and the frequency of unstable chromosomal aberrations was determined at three times after treatment (18h, and 6 and 15 days) by using PNA-FISH with a pan-telomeric probe. Cytogenetic analysis revealed a higher frequency of aberrations at 18h and 6 days after treatment in SN-exposed cultures vs. untreated cultures. The percentage of damaged cells and the yield of SN-induced aberrations at 6 days after treatment increased on average twofold compared with the ones at 18h after treatment. Moreover, a significant decrease in the frequency of aberrations was observed in SN-exposed cells at 15 days after treatment, resulting in a frequency of aberrations significantly lower than the frequency of aberrations observed in the corresponding control cultures. These data indicate that SN induces delayed chromosomal instability in CHO cells, and that the in vitro clastogenic effect of this compound persists for at least 6 days but less than 15 days after treatment. In addition, we found that SN induces delayed ITSs instability, cytogenetically detectable as additional FISH signals and centromeric breaks involving dissociation of the telomeric signal 6 days after treatment. We propose that the delayed effect of SN on ITSs results from breakage of heterochromatic centromeric ITSs blocks and further insertion of these sequences at the sites of mono- or isochromatid breaks occurring at G2 or G1-S phases of the cell cycle, respectively, since most of the additional FISH signals were present as single or double dots, and located at interstitial sites of the involved chromosomes.     

Modes of genotoxicity of a macromolecular antibiotic, SN-07, a novel type of interstrand DNA cross-linker

The modes of genotoxicity of a novel macromolecular antitumor antibiotic (SN-07) were examined using both prokaryotic and eukaryotic cells in vitro. The antibiotic induced a frameshift-type reverse mutation in Ames Salmonella typhimurium TA98 at 1.6-400 ng/plate with and without S9 mix. SN-07 also induced chromosomal aberrations and a forward mutation (6-TGr) in Chinese hamster V79 cells after 1 h treatment at 12.5-100 ng/ml without metabolic activation. The alkaline elution technique revealed that SN-07 induced interstrand DNA cross-linking dose-dependently after treatment with 2.5-10 micrograms/ml for 1 h followed by elution at pH 12.1, but it did not induce the dose-dependent cross-linking after the same treatment followed by elution at pH 12.6. It was also found that SN-07 induced single-strand DNA breaks (pH 12.1) and alkali-labile (pH 12.6) sites after treatment with 0.1-10 micrograms/ml for 1 h followed by 24-h post-incubation.     

Chromosomal aberration tests on 29 chemicals combined with S9 mix in vitro.

A metabolic activation system with rat-liver microsome fraction plus cofactors (S9 mix) was applied to chromosomal aberration tests in vitro for the screening of chemical mutagens or carcinogens in the environment. Dialkylnitrosamines only induced chromosomal aberrations in Chinese hamster cells (CHL) when treated with S9 mix. The incidence of chromosomal aberrations in CHL varied with experimental conditions, e.g. incubation time, recovery time, components of S9 mic and inducers used for preparation of S9. For dimethylnitrosamine (DMN), the maximal incidence was obtained when the cells were incubated with S9 mix for 3 h and harvested 24 h after treatment. Therefore, this system (3 h incubation and 24 h recovery) was routinely applied to further screening of other chemicals with S9 prepared from PCB-pretreated rats. 10 carcinogens (e.g. 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, quinoline, etc.) out of 16 induced aberrations when they were treated with S9 mix, whereas the remaining 6 carcinogens (e.g., 3-methyl-cholanthrene, 4-o-tolylazo-o-toluidine, etc.) induced few or no aberrations even after activation. Two insecticides, allethrin and diazinon, were strongly positive at relatively low doses only when they were activated with the S9 mix. Medical drugs, such as ethenzamide, methyl p-hydroxybenzoate and nitrofurazone, and a food additive, sodium hypochlorite, were positive on activation. Chemicals used for industry, such as styrene monomer and tris-dichloropropylphosphate, were also positive in our activation system.     

Butachlor is cytotoxic and clastogenic and induces apoptosis in mammalian cells

The ability of butachlor to induce cytotoxicity, clastogenicity and DNA damage was assessed using Chinese hamster ovary cells (CHO), Swiss mouse embryo fibroblasts (MEF) and human peripheral blood lymphocytes. A dose and time dependent loss of viability was evident upon treatment of CHO cells with butachlor. Cell killing to an extent of 50% was observed when cells were treated with 16.2 micrograms/ml of butachlor for 24 hr or with 11.5 micrograms/ml for 48 hr. The herbicide induced micronuclei significantly in cultured lymphocytes at 24 and 48 hr of treatment suggesting that it is clastogenic. To understand the mechanism of cell death caused by butachlor, its effect on DNA strand breaks was studied in MEF. A concomitant decrease in cell viability was observed with increase in DNA strand breaks. Agarose gel electrophoresis of DNA from herbicide treated CHO cells and cytochemical staining indicate the induction of apoptosis by butachlor.     

Genetic toxicity of the benzene metabolite trans, trans-muconaldehyde in mammalian and bacterial cells

Previous studies in our laboratory identified trans,trans-muconaldehyde (MUC), a six-carbon diene dialdehyde, as a microsomal metabolite of benzene. This ring-opened metabolite of benzene was also shown to be hematotoxic in mice in a manner similar to benzene. To further explore the role of MUC in relation to benzene toxicity, a number of test systems were utilized to determine its genotoxic potential. In B6C3F1 mice, MUC induced a highly significant increase in sister-chromatid exchange (SCE), the lowest effective dose being 3 mg/kg, but failed to induce any micronuclei (MN). In Chinese hamster ovary (CHO) cells, MUC at concentrations up to 0.8 micrograms/ml was negative in the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) assay. Dose-related increases in the percentage of cells with MN were observed in CHO cells treated with 0.4-0.8 micrograms/ml MUC. MUC did not-cause unscheduled DNA synthesis in rat primary hepatocytes. Treatment of Salmonella typhimurium TA97 with MUC induced a low level of mutations at concentrations ranging from 10 to 70 micrograms/ml with or without S9 activation. MUC was inactive in strains TA1535, TA100, TA1538 and TA98. In CHO cells and rat primary hepatocytes, MUC was cytotoxic at 0.4 and 4.0 micrograms/ml, respectively. Concentrations of 100 micrograms/plate MUC were toxic for bacterial cells. The present findings indicate that MUC is nonmutagenic or minimally mutagenic in bacterial and mammalian in vitro systems. In mammalian cells, MUC is highly cytotoxic and genotoxic.     

Evaluation of anti-coccidial drugs' inhibition of Neospora caninum development in cell cultures

Eight anti-coccidial drugs were examined for their efficacies in preventing development of Neospora caninum in bovine monocyte cell cultures. Lasalocid sodium (0.05 microgram/ml), monensin sodium (0.05 microgram/ml), piritrexim (0.01 microgram/ml), pyrimethamine (0.05 microgram/ml), and trimethoprim (5.0 micrograms/ml) were effective in preventing development of intracellular N. caninum tachyzoites (P less than 0.05). No differences (P greater than 0.05) in mean numbers of infected cells compared to controls were observed in cultures treated with amprolium hydrochloride (10.0 micrograms/ml), sulfadiazine (200.0 micrograms/ml), and sulfamethoxazole (200.0 micrograms/ml).     

Cytological effects of kepone on Chinese hamster cells

Cytological and cytotoxic effects of kepone on Chinese hamster cells (M3-1) were investigated. Cells treated with 2 micrograms, 4 micrograms, and 6 micrograms/ml of kepone did not show any morphological abnormalities. However, cytological observations showed that chromosome breaks, chromatid breaks, dicentric chromosomes, and chromosome interchanges were produced by these treatments. Cytotoxic studies revealed dose response and time response reactions to kepone. Cell toxicity was greater at the 30 micrograms/ml concentration, producing 100 percent cell death within 24 hours.     

Dependence of mammalian DNA replication on DNA supercoiling. I. Effects of ethidium bromide on DNA synthesis in permeable Chinese hamster ovary cells.

Chinese hamster ovary cells labelled with [14C]thymidine were made permeable, incubated with various concentrations of the intercalating dye ethidium bromide, and centrifuged through neutral sucrose gradients. The gradient profiles of these cells were qualitatively similar to those obtained by centrifuging DNA from untreated, lysed permeable cells through gradients containing ethidium bromide. The sedimentation distance of DNA had a biphasic dependence on the concentration of ethidium bromide, suggesting that the dye altered the amount of DNA supercoiling in situ. The effect of ethidium bromide intercalation on incorporation of [3H]dTMP into acid-precipitable material in an in vitro DNA synthesis mixture was measured. The incorporation of [3H]dTMP was unaffected by less than 1 microgram/ml of ethidium bromide, enhanced up to two-fold by 1--10 microgram/ml, and inhibited by concentrations greater than 10 micrograms/ml. Alkaline sucrose gradient analysis revealed a higher percentage of small DNA fragments (6--20 S) in the cells treated with 2 micrograms/ml ethidium bromide than in control cells. These fragments attained parental size within the same time as the fragments in control cells. In cells treated with 2 micrograms/ml ethidium bromide, a significant fraction of newly synthesized DNA resulted from new starts, whereas in untreated cells practically none of the newly synthesized DNA resulted from new starts. These results suggest that relaxation of DNA supercoiled structures ahead of the replication fork generates spurious initiations of DNA synthesis and that in intact cells the rate of chain elongation is limited by supercoiled regions ahead of the growing point.