Methyl mercury uptake and associations with the induction of chromosomal aberrations in Chinese hamster ovary (CHO) cells

In order to evaluate possible health effects of environmental exposure of humans towards methyl mercury species, relevant exposure experiments using methyl mercury chloride in aqueous solution and Chinese hamster ovary (CHO) cells were performed. The solution was monitored for the presence of monomethyl, dimethyl and elemental mercury by several analytical techniques including chromatographic as well as atomic absorption and mass spectrometric methods. Methyl mercury induces structural chromosomal aberrations (CA) and sister chromatid exchanges (SCE) in CHO cells. At a concentration of methyl mercury in the culture medium of 1.0 x 10(-6) M where the frequencies of CA and SCE are significantly elevated, the intracellular concentration was 1.99 x 10(-16) mol/cell. Possible biochemical processes leading to the cytogenetic effects are discussed together with toxicological consequences, when humans (e.g. workers at waste deposits) are exposed to environmental concentrations of methyl mercury.     

In vivo induction of sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) by lynestrenol

Genotoxicity study of synthetic progestin lynestrenol, was carried out on mouse bone marrow cells using sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) as parameters. Lynestrenol was studied at three different doses (6.87, 13.75 and 27.50 mg/kg body wt.). SCE and CA increased significantly as compared to normal control when treated with lynestrenol at 13.75 and 27.50 mg/kg body wt. The present results suggest that lynestrenol has both a genotoxic and cytotoxic effects in mouse bone marrow cells.     

Search for DNA interchange corresponding to sister chromatid exchanges in Chinese hamster ovary cells.

Chinese hamster ovary cells (CHO) grown for one cycle in bromodeoxyuridine (BrdU) contain a small amount (0.5%) of unusually dense double stranded DNA. This dense DNA has been previously interpreted as being bifilarly substituted with BrdU and hence evidence that sister chromatid exchange (SCE) formation proceeds via the Holliday model of recombination. However, the amount of this dense DNA is 100 times greater than that expected based on the SCE frequency in similarly cultured CHO cells, and it is not increased by treating the cells with mitomycin C. Moreover, contrary to expectations for bifilary substituted DNA, the amount of this dense DNA is not reduced by growing BrdU-labeled cells for a second cycle in TdR. Finally, DNA isolated from CHO cells contains a minor band (0.5%) with a density 0.025 gm/cc greater than that of the main band, whether or not BrdU has been incorporated. These results call into question the identification of this unusually dense DNA as bifilarly substituted and hence its previously postulated relationship to SCE formation.     

Restriction endonuclease Bam H I induces chromosomal aberrations in Chinese hamster ovary (CHO) cells

Treatment of Chinese hamster ovary (CHO) cells with the restriction endonuclease Bam H I (recognition site: G/GATCC) leads to high frequencies of chromosomal aberrations. Experiments with bromodeoxyuridine-labelled chromosomes show that the aberrations occur nearly exclusively in first post-treatment metaphases. The results are interpreted to mean that only some of the cells take up the enzyme and that these cells are the ones showing the aberrations. Cells which do not take up the enzyme show up as differentially stained metaphases and have no aberrations. Why some cells take up the restriction enzyme and others not is not known, possibly this is dependent on the physiological condition of the cells.     

Analysis of bromodeoxyuridine-induced single and twin sister chromatid exchanges in tetraploid Chinese hamster ovary cells

Culture of cells in high exogenous levels (>10^–4 M) of bromodeoxyuridine (BrdUrd) or thymidine will increase the baseline sister chromatid exchange (SCE) frequency. The effect is thought to be related to the balance of the DNA precursors thymidine and deoxycytidine. Exogenous addition of deoxycytidine will reverse this effect. Single and twin SCEs were analysed in Colcemid-induced tetraploid Chinese hamster ovary cells exposed to different concentrations of BrdUrd to determine at what stage SCEs are induced by high levels of BrdUrd. In cells exposed to low concentrations of BrdUrd (10^–5 M), equal numbers of SCEs were induced in each of the two cell cycles. With increasing concentrations of BrdUrd (10^–4 to 2×10^–4 M), SCE frequency increased in both cell cycles, but far more SCEs were induced in the second cell cycle. Deoxycytidine (2×10^–4 M) reduced the frequency of SCEs primarily by reducing the frequency of SCEs induced in the second cell cycle. Treatment with 3-aminobenzamide (3AB), a potent inhibitor of poly(ADP-ribose) polymerase, produced effects similar to exposure to high levels of BrdUrd including inducing SCEs in the second replication cycle. This suggests a similar mechanism of action. Deoxycytidine had no effect on 3AB-induced SCEs, however, and there was no interaction between 3AB and high exogenous levels of BrdUrd in SCE induction. Thus these two agents probably act through different mechanisms.     

No induction of chromosomal aberrations in Chinese hamster ovary cells by chrysophanol

Chrysophanol is an anthraquinone which occurs in several herbal drugs, e.g. senna, a commonly used laxative. As there are only limited data on its clastogenic potential we have investigated its capability to cause chromosomal aberrations in the Chinese hamster ovary cell assay. There were no significant increases in chromosomal aberrations when chrysophanol was tested up to its limit of solubility with or without metabolic activation. We conclude that chrysophanol had no clastogenic activity under the conditions described.     

Comparative effectiveness in the induction of sister chromatid exchanges and chromosome aberrations

The dose curves for 5 chemicals were studied to compare the efficiency of induction of SCEs and chromosomal aberrations by "polycentric" mutagens. The number of SCEs was found to increase linearly with the dose while that of chromosomal aberrations--nonlinearly. The efficiency of SCEs induction by these mutagens was found to be 25-50 times as high as in the induction of chromosomal aberrations. Division of alkylating mutagens into "monocentric" and "polycentric" is shown to be useful. It reflects their different efficiency in damaging one or simultaneously two DNA strands. The correlation between SCEs and formation of aberrations of the chromatid type is stated.     

Effects of various cyclophosphamide concentrations in vivo on sister chromatid exchanges (SCE) and chromosome aberrations of Chinese hamster bone marrow cells

Summary The in vivo SCE formation and the induction of chromosome aberrations in the bone marrow of Chinese hamsters (Cricetulus griseus) were studied after various concentrations of cyclophosphamide, and the sensitivity of the two test methods was compared. The administration of 1.0, 5.0, 13.3, 25.0, and 40.0 mg/kg body weight induced a dose-dependent increase in SCE. The frequency of chromosome aberration, however, was not increased significantly with doses of 1.0 and 5.0 mg/kg body weight. Only with doses of more than 13.3 mg is a significant induction of chromosome aberrations seen. Therefore the SCE test system seems to be 10 times more sensitive than the induction of chromosome aberrations in the same cell type.This work is a part of the M.D. thesis of G. Roszinsky-Köcher, to whom offprint requests should be sent     

Influence of Neurospora endonuclease on trenimon-induced structural chromosomal aberrations and sister-chromatid exchanges in human peripheral lymphocytes and Chinese hamster ovary cells

Human peripheral lymphocytes and Chinese hamster ovary cells were treated in the G1 phase of the cell cycle with the trifunctional alkylating agent trenimon (TRN) and post-treated with a single-strand specific endonuclease from Neurospora crassa (NE). TRN induces chromosomal aberrations of the chromatid type (CA) and sister-chromatid exchanges (SCE). NE post-treatment leads to an elevation of the frequencies of CA but not of SCEs. This indicates that TRN induced CA are the result of DNA double-strand breaks and that the SCEs originate from other types of lesions, most probably base damage.     

Mechanisms for sister chromatid exchanges and their relation to the production of chromosomal aberrations

By taking advantage of the fact that fluorescent light (FL) induces strand breaks only in bromodeoxyuridine(BrdU)-substituted DNA, and that those breaks eventually lead to the formation of sister chromatid exchanges (SCEs), the response of SCEs to FL was studied carefully in Chinese hamster chromosomes in which, out of four DNA strands, BrdU-substitution had occurred either in one or three strands. The FL-induced SCE frequency did not differ greatly between these two types of chromosomes. However, when they were submitted to caffeine treatment, a drastic increase in the frequency was detected in the trifilarly-substituted chromosomes while a significant decrease occurred in the unifilarly-substituted chromosomes. Based on these results, a working hypothesis was developed that the SCE can arise by at least two different mechanisms, one operating at replicating points probably utilizing the machinery of DNA replication, and the other acting only in the post-replicational DNA portion, probably in a similar fashion as assumed in a general model of crossing over in the eukaryote. These dual mechanisms may account for the discrepancy encountered in the explanations of the induction of SCEs by various exogenous agents as well as spontaneous SCEs. The present study also showed that some, but clearly not all, of chromatid deletions are the outcome of the failure to complete SCEs arising through these mechanisms.     

SCE induction in Chinese hamster ovary cells (CHO) exposed to G agents

Cultured Chinese hamster ovary (CHO) cells were exposed to two neurotoxic organophosphates, either satin (GBI, GBII) at 1.4 x 10⁻³ M or soman (GD) at 1.1 and 2.2 x 10^t-3 M for 1 h, grown and their metaphase chromosomes scored for sister-chromatid exchanges (SCE). No cytotoxicity was seen with either agent at any dose level tested. Since histograms of SCE per cell showed that they were non-symmetrically arrayed around the mean, the number of SCEs were analyzed by using the nonparametric tests, Mann-Whitney and Kruskall-Wallis. Agents GBI and GBII did not show any significant increase in SCE over baseline. On the other hand, GD demonstrated a statistically significant increase in SCE with and without metabolic activation. Ethyl methanesulfonate (EMS) alone at 5 x 10⁻³ M and cyclophosphamide (CP) at 10⁻⁴ M in the presence of rat microsomes (S9) induced a 3- and 8-fold increase in SCE per cell, respectively.     

Induction of chromosomal aberrations in Chinese hamster ovary cells by triethyllead acetate.

Organolead compounds enter the environment primarily through the combustion of leaded gasoline and industrial discharge. Lead and lead-containing compounds have been shown to induce a broad spectrum of toxic effects, including hematopoietic, renal, neurologic, and carcinogenic effects. In this study, the mutagenic activity of triethyllead acetate (Et3PbAc) was determined by measuring the induction of chromosomal aberrations in Chinese hamster ovary cells. The results indicate that Et3PbAc is very cytotoxic and a potent clastogen. In preliminary cytotoxicity studies used to determine appropriate test concentrations for chromosomal aberration analysis, the LC50 of Et3PbAc was approximately 10 microM in the absence of metabolic activation, and 80 microM in the presence of metabolic activation. The maximal response was greater with metabolic activation than without. However, a much higher dose was required to elicit a significant response in the presence of metabolic activation than in its absence.     

The effect of continuous gamma irradiation on formation of sister chromatid exchanges (SCEs) and chromosomal aberrations in meristematic cells ofVicia faba

Germinated seeds ofVicia faba were continuously irradiated at low dose rate of gamma rays (0.05 Gy h-¹) up to a total accumulated dose of 2 Gy. The FPG (fluorescence plus Giemsa) technique of differential chromatid staining was used to monitor the frequency of sister chromatid exchanges (SCEs) in irradiated root tip meristem cells. The results of the experiments have demonstrated that SCE frequency is raised by continuous gamma irradiation only in plant cells containing BrdU in the chromosomal DNA. No effect concerning SCE formation was recorded at continuous irradiation of meristematic cells of Vicia faba with native, i. e. BrdU-nonsubstituted, DNA. In contrast to SCEs, a significant increase was found in the yield of chromosomal aberrations in all variants of irradiation.     

Sister chromatid exchanges in human cells and Chinese hamster cells: Evidence that the rate of sister chromatid exchanges is a function of ploidy

The frequency of sister chromatid exchanges (SCE) in the chromosomes of the diploidy and polyploidy of Chinese hamster cells and human cells has been studied using BUdR-DAPI (bromodeoxyuridine, 4′-6-diamidino-2-phenylindol) fluorescence. The rate of SCEs per cell under constant control conditions is in proportion to the ploidy levels. In addition, the frequency of SCEs observed in a given human chromosome (nos. 1) is also directly proportional to the number of such chromosomes presented in the cells. The mean of SCEs in human chromosome numbers 1 is very similar (0.46–0.48) for diploid, triploid, and tetraploid cells. The results suggest that the rate of SCEs is a function of cellular ploidy levels.     

Role of homologous recombination in the alpha-particle-induced bystander effect for sister chromatid exchanges and chromosomal aberrations

The bystander effect for sister chromatid exchanges (SCEs) and chromosomal aberrations was examined in hamster cell lines deficient in either DNA-PKcs (V3 cells, deficient in nonhomologous end joining, NHEJ) or RAD51C (irs3 cells, deficient in homologous recombination, HR). Cells synchronized in G0/G1 phase were irradiated with very low fluences of alpha particles such that < 1% of the nuclei were traversed by an alpha particle. Wild-type cells showed a prominent bystander response for SCE induction; an even greater effect was observed in V3 cells. On the other hand, no significant induction of SCE was observed in the irs3 RAD51C-deficient bystander cells irradiated at various stages in the cell cycle. Whereas a marked bystander effect for chromosomal aberrations occurred in V3 cells, the induction of chromosomal aberrations in irs3 bystander cells was minimal and similar to that of wild-type cells. Based on these findings, we hypothesize that HR is essential for the induction of SCE in bystander cells; however, HR is unable to repair the DNA damage induced in NHEJ-deficient bystander cells that leads to either SCE or chromosomal aberrations.     

Genotoxicity assessment in iron deficiency anemia patients using sister chromatid exchanges and chromosomal aberrations assays

Previous studies have shown that iron deficiency anemia is associated with oxidative stress produced by a decrease in antioxidant enzyme activities and/or a high level of oxidants. Because oxidative stress induces DNA damage, we investigated genotoxicity in lymphocytes from patients with iron deficiency anemia (IDA) using chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) assays. Eighteen IDA subjects and a similar number of age-matched healthy controls were included in the study. The results demonstrated that IDA was associated with a slight increase in the frequency of spontaneous CAs and a decrease in the frequency of SCEs (P < 0.05). In addition, the level of SCEs was positively correlated with both the ferritin concentration (r = 0.485, P < 0.05) and hemoglobin content (r = 0.514, P < 0.05) in subjects. Moreover, vitamin E treatment reduced the frequency of SCEs in IDA patients and control subjects by the same percentage (～30%) without affecting the magnitude of the difference in the levels of SCEs between the two groups. In conclusion, our results demonstrated that IDA has a differential effect on the frequency of spontaneous CAs and SCEs.     

Effect of sodium butyrate on autophagy and apoptosis in Chinese hamster ovary cells

Sodium butyrate (NaBu), which is widely used in recombinant Chinese hamster ovary cell (rCHO) cultures for high-level expression of therapeutic proteins, is known to induce apoptosis in a dose-dependent manner. Lately, the significance of autophagy has increased in the field of CHO cell culture due to the fact that autophagy is related to the programmed cell death mechanism. To determine the effect of NaBu on autophagy as well as apoptosis of rCHO cells, rCHO cells producing erythropoietin were subjected to NaBu treatment. NaBu treatment up to 5 mM increased cleaved forms of PARP, caspase-3, and Annexin V positive population, confirming the previous results that NaBu induces apoptosis. Concurrently, NaBu treatment increased the level of accumulation of the autophagic marker, LC3-II, independently of nutrient depletion, suggesting that NaBu induces autophagy. To elucidate the potential role of autophagy induced by NaBu, a representative autophagy inducer (rapamycin) or an inhibitor (bafilomycin A1) was added to cultures together with NaBu. It was found that autophagy had the potential role of a positive cell survival mechanism under NaBu treatment. Furthermore, gradual reduction in mitochondrial membrane potential/mass and recruitment of a mitophagy protein, Parkin, to the mitochondria were observed under NaBu treatment, suggesting that this positive function of autophagy might be mediated by the autophagic removal of damaged mitochondria. Taken together, autophagy was observed in rCHO cell culture under NaBu treatments and the results obtained here support the positive effects of autophagy induced by NaBu treatments.