Activity of 1,1,1- and 1,1,3-trichloroacetones in a chromosomal aberration assay in CHO cells and the micronucleus and spermhead abnormality assays in mice

1,1,1- and 1,1,3-trichloroacetones (TCA) result from the disinfection of municipal water supplies with chlorine, and are direct-acting mutagens in the Ames/Salmonella assay. The objective of this study was to further investigate the genotoxicity of these compounds in mammalian cells using an in vitro chromosomal aberration assay in Chinese hamster ovary (CHO) cells and the micronucleus and spermhead abnormality assays in mice. Both compounds induced significant increases in structural chromosomal aberrations in CHO cells in the presence and in the absence of rat S9 metabolic activation (MA). 1,1,3-TCA was more cytotoxic to CHO cells but 1,1,1-TCA resulted in a higher proportion of cells with aberrations. The clastogenic activities of both compounds were reduced in assays conducted with MA. Neither compound resulted in the induction of a significant increase in micronucleated polychromatic erythrocytes from bone marrow of Swiss-Webster mice when administered by oral gavage; nor were effects seen on the incidence of sperm with head-shape abnormalities, testis weight, or epididymal sperm concentration in B6C3F1 mice 21 or 35 days after treatment. These data indicate that the drinking water contaminants 1,1,1- and 1,1,3-TCA are clastogenic in vitro, but are not clastogenic to bone marrow cells in vivo, and do not adversely affect several indicators of testicular function in mice.     

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.     

Cloning and characterization of small circular DNA from Chinese hamster ovary cells.

Small polydisperse circular (spc) DNA was isolated and cloned, using BglII from Chinese hamster ovary (CHO) cells. The properties of 47 clones containing at least 43 different BglII fragments are reported. The majority of the clones probably contain entire sequences from individual spcDNA molecules. Most of the clones were homologous to sequences in CHO cell chromosomal DNA, and many were also homologous to mouse LMTK- cell chromosomal sequences. The majority of homologous CHO cell chromosomal sequences were repetitive, although a few may be single copy. Only a small fraction of cloned spcDNA molecules were present in every cell; most occurred less frequently than once in 15 cells. Localization studies indicated that at least a portion of spcDNA is associated with the nucleus in CHO cells.     

High chromosomal sensitivity of Chinese hamster xrs 5 cells to restriction endonuclease induced DNA double-strand breaks

The cytogenetic effects of restriction endonucleases (RE) and X-rays were examined in the radiosensitive mutant Chinese hamster cell line xrs 5 and its normal parental line CHO K1. Cells were permeabilized with Sendai virus and exposed to Pvu II and Eco RV which induce blunt-ended double-strand breaks (dsb) in the DNA of cells, or Bam H1 and Eco R1 which induce cohesive-ended dsb with a four-base overlap. Treated cells were then assayed for the presence of metaphase chromosomal aberrations by sampling at multiple fixation times and in experiments where cells were exposed to graded series of RE concentrations. Exposure to X-rays or RE causing blunt-ended dsb was found to be between two and three times more effective in xrs 5 than in CHO K1 cells. We interpret this higher chromosomal sensitivity of xrs 5 cells as reflecting the reported defect in dsb repair in xrs 5. Both xrs 5 and CHO K1 cells yielded less aberrations after exposure to Bam H1 or Eco R1 than after exposure to Pvu II or Eco RV, confirming our previous results and demonstrating that cohesive-ended dsb are less damaging than blunt-ended dsb. Multiple fixation time experiments showed that the higher sensitivity of xrs 5 was evident at several different sampling times after treatment. Similarly the low yield of aberrations after exposure of cells to Bam H1 was evident at all sampling times. Overdispersion of chromosomal aberrations was observed in samples exposed to RE. This is thought to be due to a non-uniform permeabilization of the cell population to RE. Our results indicate that RE-induced dsb are handled by cells in a similar way to those arising during X-ray exposure.     

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.     

DNA damage in Chinese hamster cells repeatedly exposed to low doses of X-rays

In a recent paper we reported the results of an experiment carried out by analysing chromosomal damage in Chinese hamster (CHO) cells exposed to low doses of X-rays. The present investigation was undertaken in order to validate those results using a different approach, the single cell gel electrophoresis assay (comet assay) immediately after irradiation. Cells were cultured during 14 cycles, irradiation treatment was performed once per cycle when the cells were at 90-95% of confluence. Doses of 2.5, 5.0 and 10.0 mSv were used. Sequential irradiation of CHO cells induced a decrease of cells without migration and an increase of cells showing DNA damage with the three doses employed. Significant increases of low-level damaged cells (p < 0.001) were found for the 14 exposures when compared to controls except for the first irradiations with 2.5 and 10 mSv, respectively. No significant increase of the frequency of cells with severe damage was observed in any case. These findings could be explained by assuming a complex interactive process of cell recovery, DNA damage and repair together with the induction of genomic instability, the incidence of bystander effects as well as some kind of radioadaptative response of the cells. If these phenomena are limited to the cell line employed deserves further investigation.     

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.     

Chromosomal aberrations induced by the restriction endonuclease Alu I in Chinese hamster ovary cells: influence of duration of treatment and potentiation by cytosine arabinoside

Induction of chromosomal aberrations by the restriction endonuclease Alu I in Chinese hamster ovary cells (CHO) has been studied. Treatment of cell pellets with Alu I for a time as short as 1 min was found to induce significant increase in the frequency of chromosomal aberrations. Alu I was found to be effective both in trypsinized cells as well as in cells which were collected with a rubber policeman, indicating that trypsinization of cells is not a prerequisite for the entry of the enzyme into the cells. Treatment of cells with Alu I in the presence of 1-beta-D-arabinosylcytosine (ara C) led to an increase in the induced frequency of aberrations, most probably due to the inhibition of ligation of DNA-strand breaks by ara C.     

Enhancement of cytogenetic and cytotoxic effects on multidrug-resistant (MDR) cells by a calcium antagonist (verapamil)

The cytogenetic effects of a calcium antagonist, verapamil, on anticancer antibiotic-induced chromosomal damage and cytotoxicity were studied in multidrug-resistant (MDR) Chinese hamster ovary (CHO) cells in vitro. Nine colchicine-resistant (CHr) sublines were obtained by stepwise culturing with increasing concentrations of colchicine. Compared with the parent CHO cells, CHr sublines exhibited an approximately 2.6- to 120-fold higher resistance to colchicine. CHr sublines were cross-resistant to mitomycin C (MMC), actinomycin D (ACD), daunomycin (DM), bleomycin (BLM) and adriamycin (ADM). These anticancer antibiotics are known to induce chromosomal aberrations in various cell types. However, one MDR subline, CHr-500, showed resistance to induction of chromosomal aberrations by MMC. In CHr-500 cells, verapamil at a non-toxic concentration of 10 micrograms/ml enhanced the MMC-induced chromosomal damage and cytotoxicity to the levels seen in the sensitive parent cells. The increase in chromosomal damage in the presence of verapamil was correlated with the increase in cytotoxicity.     

Adenosine deaminase gene amplification in deoxycoformycin-resistant mammalian cells

Deoxycoformycin (dCF)-resistant mutants of rat hepatoma, mouse LMTK-, and Chinese hamster ovary (CHO) cells have been isolated and shown to overproduce adenosine deaminase (ADA). The overproduction of ADA was found to be due to ADA-gene amplification in rat and mouse cells but not in CHO cells. Deoxycoformycin-resistant rat hepatoma cells have large HSRs (homogeneously staining regions), mouse cells carry DMs (Double minutes), and CHO cells do not appear to have any gross chromosomal anomalies. When dCF-resistant rat hepatoma and mouse cells are selected by increasing the concentration of the inhibitor in small increments, there is a good correlation between the increase in ADA gene copy number and the increase in the level of expression of ADA, suggesting that all of the amplified genes are equally active in the expression of ADA.     

Bleomycin induces delayed instability of interstitial telomeric sequences in Chinese hamster ovary cells

We analyzed the behavior of interstitial telomeric sequences (ITSs) in the progeny of Chinese Hamster Ovary (CHO) cells exposed to the radiomimetic compound bleomycin (BLM) in order to determine if ITSs play some role in the long-term clastogenic effect of this antibiotic. To this end, CHO cells were treated with a single concentration of BLM (2.5μg/ml), and the frequency of unstable chromosomal aberrations was determined at several times after treatment (18h, and 6, 15 and 34/36 days) by using PNA-FISH with a pan-telomeric probe [(TTAGGG)n repeats]. Cytogenetic analysis revealed a higher frequency of aberrations at 18h and 6 days after treatment in BLM-exposed cultures vs. untreated cultures, although the yield of BLM-induced aberrations decreased on average five times 6 days after treatment compared with the one induced 18h after treatment. Moreover, no significant differences in the frequency of aberrations were observed between untreated and BLM-exposed cells at 15 or 34/36 days after treatment. These data indicate that, in terms of unstable aberrations, the in vitro clastogenic effect of BLM on CHO cells persists for at least 6 days but less than 15 days after exposure. In addition, we found that BLM induces ITSs instability, cytogenetically detectable as acentric fragments (18h after treatment) or additional (new) FISH signals (6 days after treatment). We propose that the delayed effect of BLM on ITSs mainly results from breakage of heterochromatic ITSs blocks and further insertion of these sequences at the sites of monochromatid breaks occurring at G2 phase of the cell cycle, since most of the additional FISH signals were present as single dots and located at interstitial sites of the involved chromosomes.     

Chromosomal damage induced by maleic hydrazide in mammalian cells in vitro and in vivo

The induction of sister-chromatid exchanges (SCE) and chromosomal aberrations (Ch.Ab.) by the herbicide maleic hydrazide (MH) has been investigated in Chinese hamster ovary (CHO) cells grown in vitro and in bone marrow cells of mice treated in vivo. MH induces SCE and Ch.Ab. in CHO cells without metabolic activation; however, no induction of SCE was found in the in vivo experiments.     

The effect of 1,10-phenanthroline on the chromosome damage and sister-chromatid exchanges induced by streptozotocin in mammalian and insect cells

The effect of the metal chelating agent 1,10-Phenanthroline (PNT) on the streptozotocin (STZ)-induced chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) and mosquito (Aedes albopictus) cells was investigated. Treatment of CHO and mosquito cells with STZ produced a significant and dose-response increase in the yield of CAs as well as SCEs (p<0.05). The addition of PNT prevented the induction of CAs by STZ in both types of cells, causing a significant decrease in the frequency of STZ-induced CAs (46.5-72.5%) (p<0.05). This fact indicates that intracellular transition metals are implicated in STZ-induced CAs and that the Fenton reaction (Fe(2+)+H(2)O(2)-->OH degrees +OH(-)+ Fe(3+)) is partly responsible for the production of CAs by this compound. On the other hand, the addition of PNT to CHO and mosquito cell cultures did not prevent the induction of SCEs by STZ. Therefore, it is valid to assume that the induction of CAs and SCEs by STZ occurs by different mechanisms.     

Comparative genotoxic effects of the cooked-food-related mutagens Trp-P-2 and IQ in bacteria and cultured mammalian cells

As part of a major study to evaluate the mutagenicity of chemicals produced during the cooking of foods, we examined the responses of bacteria and cultured Chinese hamster cells to the compounds Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) and IQ (2-amino-3-methylimidazo[4,5-f]quinoline), constituents identified in cooked beef and fish. In the Ames/Salmonella tester strain TA1538, both compounds were confirmed to be extremely potent mutagens that were active at levels below 1 ng/plate in the presence of hamster-liver S9 microsomal fraction. 50-fold higher doses of both compounds were required for mutagenicity in the uvr+ tester strain TA1978. Trp-P-2 also behaved as a strong mutagen in CHO cells using the standard exogenous activation with hamster-liver S9 fraction. At concentrations below 1 microgram/ml it produced dose-dependent increases in cell killing, mutations at the hprt and aprt loci, sister-chromatid exchanges, and chromosomal aberrations. An excision-repair-deficient strain was about 2-fold more sensitive than the normal CHO cells with respect to these genotoxic effects of Trp-P-2. IQ had unexpectedly weak activity for all genetic endpoints in the CHO cells, and it produced clear-cut responses only in the repair-deficient cells and only above a concentration of 10 micrograms/ml. The toxicity that was observed with IQ was not affected by the repair capacity of the cells and was not associated with chromosomal aberrations, indicating that damage to cellular structures other than nuclear DNA was likely the predominant pathway for cell killing. Because the culture conditions normally used for CHO cell exposure were shown to be competent in producing bacterial mutagenicity with IQ, it was concluded that the active metabolite of IQ was present in the medium but was somehow ineffective in reaching the DNA of CHO cells and/or reacting with it. These results suggest that the relative mutagenic potency of compounds in Salmonella may bear no direct relationship to relative mutagenicity in CHO cells, emphasizing precaution in attempting to extrapolate microbial data to mammalian somatic cells. This study illustrates the use and merits of a multi-endpoint assay for genetic damage in CHO cells, the utility of using CHO cells that are defective in excision repair of DNA, and the importance of comparative testing between bacterial and mammalian systems.     

Exogenous catalase introduced in CHO cells by electroporation does not protect against chromosome damage induced by ionizing radiation.

The relative importance of hydrogen peroxide generated as a consequence of irradiation with X-rays for the production of chromosomal aberrations has been studied in cultured CHO cells. Catalase introduced into cells by electroporation protected DNA from strand breakage induced by hydrogen peroxide given 4h later, and the yield of chromosome aberrations was also reduced. Nevertheless, when the cells were irradiated after treatment with catalase following a similar protocol and the yield of chromosomal aberrations analyzed at metaphase, no protective effect was observed as compared with cells treated with X-rays alone. These observations seem to support the hypothesis that hydroxyl radicals generated from hydrogen peroxide are not a major factor responsible for chromosome damage induced by ionizing radiation.     

Genome sequence comparison between Chinese hamster ovary (CHO) DG44 cells and mouse using end sequences of CHO BAC clones based on BAC-FISH results

Chinese hamster ovary (CHO) cells have frequently been used in biotechnology as a mammalian host cell platform for expressing genes of interest. Previously, we constructed a detailed physical chromosomal map of the CHO DG44 cell line by fluorescence in situ hybridization (FISH) imaging using 303 bacterial artificial chromosome (BAC) clones as hybridization probes (BAC-FISH). BAC-FISH results revealed that the two longest chromosomes were completely paired. However, other chromosomes featured partial deletions or rearrangements. In this study, we determined the end sequences of 303 BAC clones (BAC end sequences), which were used for BAC-FISH probes. Among 606 BAC-end sequences (BESs) (forward and reverse ends), 558 could be determined. We performed a comparison between all determined BESs and mouse genome sequences using NCBI BLAST. Among these 558 BESs, 465 showed high homology to mouse chromosomal sequences. We analyzed the locations of these BACs in chromosomes of the CHO DG44 cell line using a physical chromosomal map. From the obtained results, we investigated the regional similarities among CHO chromosomes (A–T) and mouse chromosomes (1–19 and sex) about 217 BESs (46.7% of 465 high homologous BESs). Twenty-three specific narrow regions in 13 chromosomes of the CHO DG44 cell line showed high homology to mouse chromosomes, but most of other regions did not show significant correlations with the mouse genome. These results contribute to accurate alignments of chromosomes of Chinese hamster and its genome sequence, analysis of chromosomal instability in CHO cells, and the development of target locations for gene and/or genome editing techniques.     

Cytological characterization of repair-deficient CHO cell line 43-3B. I. Induction of chromosomal aberrations and sister-chromatid exchanges by UV and its modulation with 3-aminobenzamide

The induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) by short-wave ultraviolet (UV) and X-irradiation was studied in Chinese hamster ovary (CHO) wild-type (WT) cells and one of its UV-hypersensitive mutants, 43-3B. The results indicate that CHO 43-3B show high levels of spontaneously occurring chromosomal aberrations and SCEs; these levels are, respectively, approximately 4 and 1.7 times those found in WT CHO. Treatment with UV produced a considerable delay in the cell-cycle progression of the mutant cells compared to the WT cells. Doses of UV that had no effect on WT cells, significantly induced chromosomal alterations in the mutant in a dose-dependent manner. An approximately 5-fold increase in the induced frequencies of SCEs was obtained in 43-3B cells after UV treatment. No synergistic effect was observed with UV irradiation and the inhibitor of poly(ADP-ribose) synthetase, 3-aminobenzamide (3AB), in either cell type. The frequency of SCEs in the mutant cell lines was lower than would be expected if the effects of UV and the inhibitor were additive. X-Ray alone in G1 and in combination with 3AB in G2 did not induce increased frequencies of chromosomal aberrations in mutant cells in comparison to the WT cells.     

Multiple mechanisms generate extrachromosomal circular DNA in Chinese hamster ovary cells.

Seven cloned small circular DNA molecules from CHO cells were sequenced and examined for the presence of homologies to each other and to a number of other functional sequences present in transposable elements, retroviruses, mammalian repeat sequences, and introns. The sequences of the CHO cell circular DNA molecules did not reveal common structural features that could explain their presence in the circular DNA population. A gene bank was constructed for CHO chromosomal DNA and sequences homologous to two of the seven small circular DNA molecules were isolated and sequenced. The nucleotide sequences present at the junction of circular and chromosomal DNA suggest that a recombination process involving homologous pairing may have been involved in the generation of one, but not the other, of the two circular DNA molecules.     

Effect of follicle-stimulating hormone (FSH) on radiation-induced chromosomal aberrations in mouse germ cells and Chinese hamster cells in vitro

Earlier observations on the induction by X-rays of reciprocal translocations in stem-cell spermatogonia of the rhesus monkey have established a correlation between the level of follicle-stimulating hormone (FSH) in blood at the moment of irradiation and the final recovery of translocations (van Buul, 1980). In the present study, FSH treatment of mice did not induce chromosomal aberrations in bone-marrow cells or stem-cell spermatogonia, nor did it change the radiosensitivity of stem-cell spermatogonia for the induction of chromosomal translocations. Experiments in vitro with Chinese hamster ovary cells (CHO), however, showed a clear radiosensitizing effect of FSH on the induction of structural chromosomal aberrations.     

Cleistanthin B causes G1 arrest and induces apoptosis in mammalian cells

Cleistanthin B is a potential anticancer agent isolated from the tropical plant Cleistanthus collinus. We have previously shown that cleistanthin B is clastogenic and induces micronuclei formation and chromosomal aberrations. We now show that this compound inhibits DNA synthesis in Chinese hamster ovary (CHO) cells and induces apoptosis in cervical carcinoma (SiHa) cells. Flow cytometric analysis of cleistanthin treated CHO cells revealed that they were blocked in G1. Cervical carcinoma (SiHa) cells exposed to cleistanthin B shrank, rounded up and had condensed chromatin and fragmented nuclei. DNA isolated from cleistanthin treated cells exhibited the characteristic apoptotic ladder when electrophoresed in agarose gels. These results were confirmed by flow cytometry. Etoposide, a structurally similar compound also induced apoptosis in these cells although with a difference. Etoposide induced apoptosis after permitting cells to enter into S phase, while cleistanthin B stopped entry of cells into S phase and subsequently drove them to apoptosis.