Mutual interactions among ingredients of betel quid in inducing genotoxicity on Chinese hamster ovary cells

The purpose of this study is to explore the mutual interactions among the chemical ingredients of betel quid including arecoline, sodium fluoride, catechin and glycyrrhizin in producing genotoxicity on Chinese hamster ovary (CHO) cells using the micronucleus method. Our results show that arecoline at a rather low concentration of 0.2–2 μM which could be in the oral cavity during betel quid chewing and NaF (0.8–2.4 mM) significantly elevated the number of micronucleated cells in a concentration-dependent manner. In addition, significant prolongation of cell cycles was observed by treatment with arecoline (≥2.0 μM) or NaF (2.4 mM) in CHO cells. Both catechin and glycyrrhizin could antagonize not only the increased micronucleated cells induced by arecoline and NaF but also the prolonged cell cycle induced by arecoline in CHO cells. This finding implies that the adjuvant ingredients, catechu and liquorice root extract provide not only a flavor but also an antagonist against the genotoxicity of arecoline and fluoride containing betel quid.     

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 lack of genotoxicity of sodium fluoride in a battery of cellular tests

In a comprehensive assessment of genotoxicity, sodium fluoride was evaluated in a battery of cellular tests providing different genetic end points and biotransformation capabilities. The tests included the following: rat hepatocyte primary culture/DNA repair assay, Salmonella typhimurium histidine locus reversion assay, adult rat liver epithelial cell/hypoxanthine guanine phosphoribosyl transferase mutation assay, and sister chromatid exchange in two target cell types, human peripheral blood lymphocytes and Chinese hamster ovary cells. Negative findings were made in all assays, indicating that sodium fluoride is not genotoxic in these assays.Abbreviations ARL adult rat liver epithelial cell - CHO Chinese hamster ovary cell - HGPRT hypoxanthineguanine phosphoribosyl transferase - HPBL human peripheral blood lymphocyte - HPC hepatocyte primary culture - SCE sister chromatid exchange     

Genotoxic evaluation of chronic fluoride exposure: sister-chromatid exchange study

As part of a continuing investigation, this study was conducted to examine the genotoxic effects of chronic exposure to sodium fluoride (NaF) in drinking water on the frequency of sister-chromatid exchange (SCE) in the bone-marrow cells of male Chinese hamsters. Animals at about 3 weeks of age were randomly assigned to 6 groups, each with at least 3 hamsters, and were maintained on a low fluoride diet (less than 0.2 ppm F) throughout the experiment. At 4 weeks of age, the animals in groups I-V began to receive drinking water containing fluoride at concentrations of 0, 1, 10, 50 and 75 ppm, respectively. Group VI was treated with cyclophosphamide and served as the positive control. The animals were sacrificed at 24 weeks of age by cervical dislocation. The humeri and plasma were analyzed for fluoride content, which was found to increase with the increase in fluoride concentration in drinking water. Slides of chromosomes from bone-marrow cells were prepared and blindly examined for the frequency of SCE. The mean scores of SCE for the hamsters receiving drinking water containing F concentrations up to 75 ppm for 21 weeks ranged from 4.28 to 6.28 per cell, and were not significantly different from those of the negative controls (4.60-5.44/cell). The results indicated that chronic fluoride exposure had no effect on the frequency of SCE in Chinese hamster bone-marrow cells under the conditions of the present investigation.     

Regulation of CTP:phosphocholine cytidylyltransferase activity and subcellular location by phosphorylation in Chinese hamster ovary cells. The effect of phospholipase C treatment.

The phosphorylation state of cytidylyltransferase in Chinese hamster ovary (CHO) cells was correlated with its subcellular distribution and activity in vivo. Western blot analysis of soluble and particulate fractions from control and phospholipase C-treated cells revealed slower migrating forms of cytidylyltransferase present only in the soluble fraction of control cells. These were abolished by incubating the soluble fraction at 37 degrees C in the presence of 5 mM Mg2+ but persisted if 135 mM NaF was present in the incubation. CHO cells were labeled with 32Pi, and cytidylyltransferase was immunoprecipitated from soluble and particulate fractions from control and phospholipase C-treated cells. The slower migrating forms of cytidylyltransferase, present in the soluble fraction of control cells, were phosphorylated at multiple sites. Although an equivalent amount of cytidylyltransferase was immunoprecipitated from the particulate fraction of phospholipase C-treated cells, it contained little 32P. Pretreatment of the CHO cells with okadaic acid, an inhibitor of type 1 and 2A phosphatases, prevented the stimulation of cytidylyltransferase in vivo by phospholipase C. These results demonstrate that dephosphorylation of soluble cytidylyltransferase is required for the phospholipase C-mediated translocation of cytidylyltransferase in CHO cells.     

Effect of exogenous thymidine on sister-chromatid exchange frequency in Chinese hamster ovary cells with bromodeoxyuridine- and chlorodeoxyuridine-substituted chromosomes

There are conflicting reports on the effect of exogenous thymidine (dThd) on the frequency of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Thymidine has been reported either to increase or to have no effect on SCE frequency under similar experimental conditions. To resolve this controversy, we have carried out a series of experiments to examine the effect of dThd on CHO cells cultured with 5-bromodeoxyuridine (BrdUrd). In addition, we have examined the effect of dThd on CHO cells cultured with 5-chlorodeoxyuridine (CldUrd), a much more potent inducer of SCEs than BrdUrd. The addition of 100 microM dThd to the culture medium caused a consistent decrease in the yield of SCEs in cells grown in BrdUrd for two cell cycles. The decrease was even greater when cells were grown in dThd and CldUrd. Analysis of twin and single SCEs indicated that dThd must be present during the first cell cycle to reduce the frequency of SCEs. Because excess dThd is thought to have an effect when DNA replicates on a template substituted with a halogenated nucleoside, dThd at concentrations from 100 microM to 9 mM was added to cultures for the second cell cycle after a first cell cycle in BrdUrd. In this experiment, the presence of dThd increased SCE frequency in a dose-dependent manner. The results suggest that if dThd competes with halogenated nucleosides and thus decreases their incorporation into DNA, SCEs are suppressed in the subsequent cell cycle, whereas if excess dThd creates a dNTP pool imbalance, SCEs can be increased.     

Assessment of genotoxic potential of ethylenediamine: in vitro and in vivo studies

Ethylenediamine (EDA) was evaluated for potential genotoxic activity using a battery in vitro and in vivo mammalian tests. The tests employed were the Chinese hamster ovary (CHO) gene mutation assay, the sister-chromatid exchange (SCE) test with CHO cells, unscheduled DNA synthesis (UDS) assays with primary rat hepatocytes and a dominant lethal study with Fischer 344 rats. EDA did not produce a positive, dose-related, mutagenic effect in either the CHO mutation assay or in the SCE test when evaluated both with and without the addition of a rat-liver S9 activation system. With hepatocytes, no positive effects of EDA upon UDS values were noted in 2 separate studies using either a scintillation counting procedure or an autoradiographic method to determine UDS activity. In a dominant lethal study, male rats fed for 23 weeks with dietary levels of EDA X 2HCl of 0, 0.05, 0.15 or 0.50 g/kg/day, and mated with 1 virgin female/week for 3 consecutive weeks, showed no dose-related or statistically significant effects upon fertility, total number of implantations/female, or the number of living and dead implants per female; marked effects upon the incidence of dominant lethal mutations were noted in the positive control group injected intraperitoneally with one dose of 0.25 mg/kg triethylenemelamine. We conclude that EDA was not genotoxic in the in vitro and in vivo mammalian test systems employed.     

Cytotoxicity and genotoxicity testing of sodium fluoride on Chinese hamster V79 cells and human EUE cells.

The cytotoxic effects of sodium fluoride (NaF) on hamster V79 cells and human EUE cells were studied by measuring the cloning efficiency and DNA, RNA and protein synthesis in cells cultured in the presence of NaF. Potential mutagenicity of NaF was followed on the basis of induced 6-thioguanine-resistant mutants in treated Chinese hamster V79 cells. The results showed that the addition of 10-150 micrograms of NaF per ml of culture medium induced 10-75% cytotoxic effect on hamster V79 cells but had no toxic effect on human EUE cells. NaF was cytotoxic to human EUE cells at considerably higher concentrations (200-600 micrograms/ml). Growth of both cell types with 100 and 200 micrograms of NaF per ml caused inhibition of 14C-thymidine, 14C-uridine and 14C-L-leucine incorporation. This means that NaF inhibits macromolecular synthesis whereby damaging effects were less drastic in human EUE cells. The results of detailed mutagenicity testing on hamster V79 cells showed that NaF did not show any mutagenic effect after long-term (24-h) incubation of hamster cells in the presence of 10-400 micrograms of NaF per ml of culture medium.     

Assignment of a human DNA-repair gene associated with sister-chromatid exchange to chromosome 19

The Chinese hamster ovary (CHO) cell mutant, EM9, is defective in rejoining strand breaks, hypersensitive to chlorodeoxyuridine (CldUrd), and has a high frequency of sister-chromatid exchange (SCE). Somatic cell hybrids constructed from fusion of EM9 cells with normal human lymphocytes and fibroblasts, and selected in CldUrd, extensively segregate human chromosomes but preferentially retain markers of human chromosome 19. The SCE frequency in the hybrid clones is low as in normal CHO cells, but in CldUrd-sensitive subclones, which lose the human chromosome 19 markers, SCE frequencies return to mutant levels. We therefore assign a human gene designated repair complementing defective repair in Chinese-hamster (RCC) to chromosome 19. Since this is the second (of two) human genes complementing repair-deficiency mutations in CHO cells assigned to the 19, the assignment and organization of DNA-repair genes is discussed in the light of hemizygosity in CHO cells and the evolutionary conservation of mammalian linkage groups.     

Induction of sister-chromatid exchanges in human lymphocytes and Chinese hamster cells exposed to aflatoxin B1 and N-methyl-N-nitrosourea.

The effect of a 1-h exposure to aflatoxin B1 (AFB1) in inducing sister-chromatid exchange in Chinese hamster ovary (CHO) cells and human lymphocytes in the presence or absence of mixed function oxidase ("S9 mix") was compared. CHO cells were also exposed to a graded series of doses of N-methyl-N-nitrosourea, a powerful inducer of SCE whose action was independent of the presence or absence of S9 mix. CHO and human cells showed a close correlation in response to SCE induction by AFB1 and in both cell systems the additon of mixed function oxidases in the S9 mix resulted in a marked enhancement of action of AFB1.     

Suppression of the frequencies of sister chromatid exchanges in Bloom's syndrome fibroblasts by co-cultivation with Chinese hamster cells

Summary The effect of co-cultivation of Bloom's syndrome fibroblasts with Chinese hamster ovary cells (CHO) on the incidence of sister chromatid exchanges (SCEs) was studied. The results show that suppression of the frequency of SCEs in Bloom's syndrome cells occurs only if cell to cell contact is present with CHO cells, without any effect on the SCE frequency in the latter.It is suggested that possible genetic heterogeneity between different Bloom's syndrome patients can be studied using the method of co-cultivation.     

Vitamin C is positive in the DNA synthesis inhibition and sister-chromatid exchange tests

Ascorbate caused a dose-dependent increase in sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells and in human lymphocytes. Moreover, in the DNA synthesis inhibition test with HeLa cells, ascorbate gave results typical of DNA-damaging chemicals. Catalase reduced SCE induction by ascorbate, prevented its cytotoxicity in CHO cells, and prevented its effect on HeLa DNA synthesis. Ascorbate reduced induction of SCE in CHO cells by N-methylN′-nitrosoguanidine (MNNG) by direct inactivation of MNNG.     

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.     

Combinatorial treatment with lithium chloride enhances recombinant antibody production in transiently transfected CHO and HEK293E cells

Lithium chloride (LiCl), which induces cell cycle arrest at G2/M phase, is known as a specific production rate (q _p)-enhancing additive in recombinant Chinese hamster ovary (CHO) cell culture. To determine the potential of LiCl as a chemical additive that enhances transient gene expression (TGE), LiCl was added to the CHO-NK and human embryonic kidney 293E (HEK293E) cell cultures before and/or after transfection with polyethylenimine as a transfection reagent. The effect of this addition on transfection efficiency (pre-treatment) and q _p enhancement during TGE (post-treatment) was examined. For the TGE of monoclonal antibody (mAb) in CHO-NK cells, pretreatment alone with 10 mM LiCl and post-treatment alone with 5 mM LiCl resulted in 1.2- and 3.4-fold increase of maximum mAb concentration (MMC), respectively, compared with the TGE without LiCl treatment. Furthermore, combinatorial treatment with LiCl (10 mM for pre-treatment and 5 mM for post-treatment) synergistically increased the TGE of mAb (5.3-fold increase in MMC). Likewise, combinatorial treatment with LiCl (10 mM for pre-treatment and 15 mM for post-treatment) in HEK293E cells synergistically increased the TGE of mAb (4.9-fold increase in MMC). Taken together, the data obtained here demonstrate that combinatorial treatment with LiCl is a useful means to improve TGE in CHO as well as HEK293 cells.     

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.     

Hydroxyurea retards the progression of G2 cells

Continuous treatment of monolayer cultures of Chinese hamster ovary (CHO) cells with 1.0 mM HU reduced the rate at which G2 cells entered mitosis. Although all cells in G2+M at the start of treatment eventually entered mitosis, early G2 cells, which were exposed to HU the longest, entered mitosis 45 min later than untreated G2 cells. This perturbation of G2 progression should be considered when employing HU as a synchronizing agent in mammalian cell populations.     

Effect of tetrandrine on micronucleus formation and sister-chromatid exchange in both in vitro and in vivo assays

The genotoxicity of tetrandrine, a drug potentially useful for the treatment of silicosis, was studied using the micronucleus and the sister-chromatid exchange (SCE) assay systems. Cultured Chinese hamster lung (V79) cells were used for the in vitro micronucleus and sister-chromatid exchange studies. Mouse bone marrow was used for the in vivo micronucleus assay and mouse spleen cells for the in vivo/in vitro sister-chromatid exchange analysis. The results show that SCE levels in V79 and in spleen cells were significantly elevated by treatment with tetrandrine at doses above 0.08 mg/ml and 100 mg/kg bw, respectively. Increased tetradrine-induced SCE in vitro was metabolic activation dependent. Tetrandrine failed to induce micronuclei at any of the doses tested. A decrease of replicative index with an increase in the concentration of tetrandrine was found both in vitro and in vivo. These results indicate that tetrandrine is a weak indirect-acting genotoxicant.     

The use of sister-chromatid exchange in Chinese hamster primary lung cell cultures to measure genotoxicity

Primary cell cultures derived from Chinese hamster lung (CHL) were established, and their response for the induction of sister-chromatid exchange (SCE) by direct- and indirect-acting mutagens was characterized. An increase in SCE frequency was induced in CHL cells by 3-methylcholanthrene (MCA), benzo[a]pyrene (BaP), and 2-aminoanthracene (2AA). The SCE frequency increased slightly after exposure to cyclophosphamide, but did not respond to the hepatocarcinogen dimethylnitrosamine (DMN). A slight increase in SCE frequency by DMN was observed in the CHL system with use of Aroclor-1254-induced rat liver homogenate fraction (S9). This response to DMN in CHL cells was lower than that seen when CHO cells were the target in the presence of S9. At low (1) and high (20) passages, the CHL cells responded with a similar dose-related increase in SCE frequency to direct- (ethyl methanesulfonate, EMS) and indirect- (MCA) acting mutagens. This response indicates that even after prolonged culturing in vitro, the cells retained the ability to metabolically activate xenobiotic promutagens. The induction of SCE by MCA occurred at concentrations that also induced macromolecular binding. SCE induction was also examined in primary lung cell cultures from animals exposed by nose-only inhalation to MCA aerosol. A significant increase in SCE frequency above controls was observed in cells from animals after a single exposure to MCA. No detectable increase in SCE frequency was observed after repeated inhalation exposures. Because CHL cells are of lung origin and showed metabolic activity, the CHL system appears to be appropriate for study of the genotoxic potential of inhaled compounds.     

Sodium fluoride-induced chromosome aberrations in different stages of the cell cycle: a proposed mechanism

In an attempt to clarify the controversy about sodium fluoride (NaF) clastogenicity, the induction of chromosome aberrations in Chinese hamster ovary cells (CHO) by NaF was investigated. Following a protocol used for screening chemicals for clastogenic activity, significant increases of aberrant cells were observed when cells were exposed to NaF for 4 h and harvested 8 h later. Cell-cycle kinetic studies demonstrated most cells were exposed in G2 of the cell cycle. Smaller increases in aberrant cells were observed when cells were harvested 20 h later (most cells were exposed in G1/S). The sensitivity of G2 cells to NaF was investigated further, along with the induction of aberrations at low doses. The results indicated that G2 cells are sensitive to NaF and the percent of aberrant cells increased with dose and length of exposure. With a 3-h exposure until harvest, no statistically significant increase in aberrant cells was observed at doses below 10 micrograms/ml NaF. These data are consistent with a threshold for NaF-induced clastogenicity around 10 micrograms/ml, as has been proposed previously (Scott and Roberts, 1987). It thus may be predicted that clastogenic effects would not occur in humans exposed to the levels of fluoride that are present in drinking water or dentifrices. An understanding of the mechanism of NaF-induced clastogenicity would help to clarify this point. It has previously been reported that NaF inhibits DNA synthesis/repair. The types of aberrations, mostly deletions and gaps, the induction of endoreduplicated cells, the cell-cycle delay and the sensitivity of G2 cells to NaF observed are similar to that reported in the literature for DNA synthesis/repair inhibitors like aphidicolin (APC). Similarities in the induction of aberrations by NaF and APC were confirmed in experiments with G2 cells. Based on these results and those previously reported for NaF and APC, it is proposed that NaF-induced aberrations may occur by an indirect mechanism involving the inhibition of DNA synthesis/repair.     

Induction of sister-chromatid exchanges by pesticides in primary rat tracheal epithelial cells and Chinese hamster ovary cells

Possible induction of sister-chromatid exchanges by butachlor, paraquat, phorate and monocrotophos was examined in primary rat tracheal epithelial (RTE) and Chinese hamster ovary (CHO) cells. At dose levels that killed less than 50% of the cell population, monocrotophos induced SCEs positively in CHO and RTE cells, while paraquat was positive only in RTE cells. In two trials of the same experiment, paraquat and butachlor in CHO cells, and phorate in either RTE or CHO cells failed to induce a significant number of SCEs at any dose level within the ranges assayed. On the other hand, in RTE cells, butachlor induced a significant number of SCEs at a dose level of 5 micrograms/ml in one trial, but was insignificant in another. The inductions in these assays were, however, dose-dependent. The addition of S9 mixture did not alter the results of SCE induction by these 4 pesticides in CHO cells. RTE cells were more vulnerable to paraquat in cytotoxicity and SCE assays than CHO cells. Cytotoxicities were ranked as butachlor greater than phorate greater than paraquat greater than monocrotophos to CHO cells and paraquat greater than butachlor greater than phorate greater than monocrotophos to RTE cells. Significant cell cycle delays were only found in the treatments with the highest dose levels of butachlor, paraquat and phorate in CHO cells. In addition, this is the first report on SCE induction in RTE cells.