Gene mutation and sister chromatid exchange in Chinese hamster cells

The mutation in hypoxanthine phosphoribosyl transferase gene and the induction of sister chromatid exchange (SCE) were comparatively studied treating Chinese hamster ovary cells with the mutagens ethylmethanesulphonate. N-methyl-N'-nitro-N-nitrosoguanidine, Mitomycin C and X-ray. All the agents exerted strong mutagenic effects and showed a dose-dependent relationship for the induction of SCEs.     

The mutagenic potential of high flash aromatic naphtha

Catalytic reforming is a refining process that converts naphthenes to aromatics by dehydrogenation to make higher octane gasoline blending components. A portion of this wide boiling range hydrocarbon stream can be separated by distillation and used for other purposes. One such application is a mixture of predominantly 9-carbon aromatic molecules (C9 aromatics, primarily isomers of ethyltoluene and trimethylbenzene), which is removed and used as a solvent — high-flash aromatic naphtha. A program was initiated to assess the toxicological properties of high-flash aromatic naphtha since there may be human exposure through inhalation or external body contact. The current study was conducted partly to assess the potential for mutagenic activity and also to assist in an assessment of carcinogenic potential. The specific tests utilized included the Salmonella/mammalian microsome mutagenicity assay, the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) forward mutation assay in CHO cells, in vitro chromosome aberration and sister chromatid exchange (SCE) assays in CHO cells, and an in vivo chromosome aberration assay in rat bone marrow.There was no evidence that high-flash aromatic naphtha was either a gene or chromosomal mutagen. Thus it is unlikely to be a genotoxic carcinogen.Abbreviations Brdu 5-Bromo-2-deoxyuridine - C9 Aromatic species with 9 carbons (i.e., ethyl toluene and trimethyl benzene isomers) - CE Cloning efficiency - CHO Chinese hamster embryo - CP Cyclophosphamide - DMSO Dimethyl sulfoxide - HGPRT Hypoxanthine-guanine phosphoribosyl transferase - HVAC Heating, Ventilation, Air Conditioning - 3MC 3 Methylcholanthrene - MMC Mitomycin C - MMS Methyl methanesulfonate - S9 S9 Mammalian microsomal enzyme activation mixture - SCE Sister chromatid exchange     

In vivo mutagenicity studies with iodinated glycerol azeo

Previous studies have shown that iodinated glycerol azeo is positive in a number of in vitro mutagenicity assays including the Ames assay (TA100; TA1535), mouse lymphoma assay, Chinese hamster ovary (cytogenetic) assay and in one in vivo study, the sex-linked-recessive-lethal assay in Drosophila. Prior studies have also shown that the drug is negative in the mouse micronucleus assay. We now report that the drug is also negative for mutagenic activity in a number of other in vivo tests. Single intraperitoneal doses of 25, 125 and 250 mg/kg were without effect in the rat bone marrow chromosomal aberration assay. Single oral doses of 30, 75, 150 and 300 were negative in the rat hepatocyte DNA-repair assay. Single intraperitoneal doses of 30 and 100 mg/kg were without effect in the sister chromatid exchange (SCE) assay in the mouse. Statistically significant effects were seen at 200 and 300 mg/kg in the initial SCE assay and at 300 and 350 mg/kg in the confirmatory SCE assay. The rationale for considering the SCE results to be anomalous and thus not relative to the overall safety evaluation of the drug is presented.     

Mutagenic and genotoxic evaluation of bisphenol F diglycidyl ether (BFDGE) in prokaryotic and eukaryotic systems

The epoxy resin bisphenol F diglycidyl ether (BFDGE), was examined for its mutagenicity in prokaryotic assays (Salmonella typhimurium His(-) and Escherichia coli Trp(-) tests) and its genotoxicity in eukaryotic systems (sister chromatid exchange (SCE) and micronucleus tests in human lymphocytes), in the presence or absence of an exogenous metabolizing system (S9 from rat liver). In the prokaryotic tests, the concentrations of BFDGE ranged between 100 and 5000 micro g per plate, and in the eukaryotic assays from 12.5 to 62.5 micro g/ml. The compound is able to induce mutagenic effects in bacterial strains TA100, TA1535, WP2uvrA and IC3327, as revealed by the increase observed in the number of induced revertants. With respect to the genotoxicity assays, BFDGE induces an increase in the frequency of sister chromatid exchanges and micronuclei in human peripheral blood lymphocytes.     

Toxicity of 6-thioguanine and 8-azaguanine to non-dividing liver cell cultures

8-azaguanine and 6-thioguanine were both toxic to non-dividing liver cells in primary cultures. In addition, these agents were toxic to an established line of liver-derived epithelial cells brought to growth arrest by serum deprivation. These observations demonstrate that the toxicity of 8-azaguanine and 6-thioguanine can occur at least in part through mechanisms that do not involve effects on DNA synthesis or incorporation of the analogs into DNA.Abbreviations AG 8-azaguanine - ARL adult rat liver epithelial cell line - HGPRT hypoxanthine-guanine phosphoribosyl transferase - WME Williams Medium E     

Comparison of the Ames assay and the induction of sister chromatid exchanges: Results with ten pharmaceuticals and five selected agents

Seven antischistosomal drugs, two antimalarial drugs, and one antiamoebic drug were tested in all five Ames strains for induction of mutation, as well as for induction of cytotoxicity, inhibition of cellular progression, and the induction of sister chromatid exchanges in two cultured mammalian cell lines. We found that two agents shown to be negative in the Ames test were positive for sister chromatid exchange induction. Based on qualitative and quantitative evaluation, we find that all but three of the pharmaceuticals should be considered to be potential human carcinogens.Abbreviations AA 2-aminoanthracene - 9AACC 9-aminoacridine - AM amoscanate - BrdUU bromodeoxyuridine - CA chloroquine diphosphate - CHO Chinese hamster ovary - CQ chloroquine - DAPI 46-diamidino-2-phenylindole - DHY dehydroemetine - DMSO dimethyl sulfoxide - EB ethidium bromide - FCS fetal calf serum - FN 4-fluoro-3-nitrophenyl azide - HY hycanthone - ICP inhibiting cell progression - LU lucanthone - MEM minimal essential medium - 2NF 2-nitrofurantoin - 4NPD 4-nitro-O-phenylenediamine - NZ niridazole - OL oltipraz - OX oxaminiquine - PBS phosphate buffered saline - PQ primaquine - PZ praziquantel - SA sodium azide - SCE sister chromatid exchange     

Bromodeoxyuridine tablet methodology for in vivo studies of DNA synthesis.

5-Bromodeoxyuridine (BrdU) tablets with different physical characteristics are useful in a wide variety of studies requiring detection of DNA replication in vivo. These tablets can effect a high substitution of BrdU in DNA, thereby permitting sister chromatid differentiation in chromosomes stained with 33258 Hoechst alone or in conjunction with Giemsa. Baseline and cyclophosphamide-induced in vivo sister chromatid exchange frequencies in mouse spleen, marrow, and thymus were measured and found to be significantly greater than those in spermatogonia. Sister chromatid exchange analysis was also extended to mouse liver and to Chinese hamster and Armenian hamster marrow cells. Sister chromatid differentiation was observed in Armenian hamster meiotic tissue, and evidence for interhomolog chromatid exchange obtained.     

Three-Way Differentiation of Chinese Hamster Ovary Chromosomes by Immunoperoxidase Technique Using a Monoclonal Anti-Bromodeoxyuridine Antibody

A new permanent staining procedure for sister chromatid differentiation (SCD) in cultured Chinese hamster ovary cells has been established by combining the three-way differentiation in third mitosis (M3) chromosomes and the immunoperoxidase reaction developed with 3,3'-diaminobenzidine using a monoclonal anti-bromodeoxyuridine (BrdU) antibody. This procedure allows SCD at very low BrdU concentrations, and the evaluation of the sister chromatid exchange frequencies on a per cell-cycle basis.     

Sister chromatid exchange frequency,cellular replication and relative cloning efficiency in human teratocarcinoma-derived cells

To determine the relationships between the induction of specific biological responses and exposure to DNA-damaging agents, human teratocarcinoma-derived cells were exposed to either ethyl methanesulfonate or to methyl methanesulfonate, and sister chromatid exchange, cellular proliferation and relative cloning ability measured. SCE increased while cellular proliferation and relative cloning ability each decreased in a concentration-dependent manner. Methyl methanesulfonate was consistently more efficient in inducing biological responses than was ethyl methanesulfonate. When the individual responses were compared, the decrease in cellular proliferation paralleled the reduction in cloning efficiency. A strong correlation was also observed between the reduction in relative cloning ability and sister chromatid exchange frequency. Because these relationships are similar to those previously described in other mammalian cell lines, the observations in our study suggest that the P3 cell line is an appropriate choice for modeling effects of toxicant exposure in human cells.Abbreviations AGT average generation time - BUdR 5-bromodeoxyuridine - CHO Chinese hamster ovary - EMS ethyl methanesulfonate - ENU N-ethyl-N-nitrosourea - MMS methyl methanesulfonate - MNU N-methyl-Nnitrosourea - SCE sister chromatid exchange     

Effects of copper ions released from metallic copper on CHO-K1 cells

The aim of this study was to investigate the cytotoxic and genotoxic effect of copper extracts obtained from metallic copper in Chinese hamster ovary (CHO-K1) cell line using neutral red (NR), sister chromatid exchange (SCE), chromosomal aberrations (CA) and cell-cycle kinetics tests. Cells were cultured in Ham-F10 with different copper-containing extracts obtained after the immersion of copper disks for 1, 2, 3, 9, 12, 24, 48 and 72 h in culture medium. Results from cytotoxicity assay showed an inverted U-shape response evidenced in changes in lysosomal activity and mitotic index. The analysis of CA revealed an increase of abnormal metaphases for copper concentration (cCu) in the 5.67-7.42 mg/L dose-range (p<0.001). In addition, SCE frequencies were higher for treated cells when compared with controls in the 1.56-7.42 mg/L concentration range (p<0.001). The absence of metaphases indicated cytotoxicity for cCu≥10.85 mg/L. Results show that cells close to copper-containing materials releasing copper ions are susceptible to cytotoxic and genotoxic effects.     

Differences between rat liver epithelial and fibroblast cells in metabolism of purines

Epithelial and fibroblast cells from adult rat liver were found to differ markedly in their metabolism of the purine hypoxanthine. Both cell types took up hypoxanthine and possessed hypoxanthine-guanine phosphoribosyl transferase for phosphoribosylating the purine. However, in the transferase assay, lysates from epithelial cells converted hypoxanthine predominantly to inosine monophosphate, with small amounts of the nucleoside inosine as product, whereas fibroblast cell lysates converted hypoxanthine predominantly to inosine. The inosine appeared not to be produced by direct ribosylation of the base, since fibroblast cell lysates had less purine nucleoside phosphorylase activity than epithelial cell lysates. Rather, the inosine produced by fibroblast lysates appeared to be derived from inosine monophosphate through catabolism of the mononucleotide by 5' nucleotidase. An inhibitor of 5' nucleotidase, thymidine triphosphate, reduced the amount of inosine formed.     

Characterization of sister chromatid exchange induction by 8-methoxypsoralen plus near UV light

The combination of 8-methoxypsoralen and near UV light is highly effective in inducing sister chromatid exchanges (SCEs) in Chinese hamster ovary cells. Appreciable increases in SCEs can be effected by treatments compatible with cell survival, and effects of a single dose of alkylation persist over multiple generations. Both the frequency and location of SCEs induced at different times within the DNA synthesis period varies in a manner indicating that exchange induction is restricted to regions which replicated during or after DNA damage.     

Genotoxicity and carcinogenicity testing of 1,2-dibromopropane and 1,1,3-tribromopropane in comparison to 1,2-dibromo-3-chloropropane

The activities of 1,2-dibromopropane (DBP) and 1,1,3-tribromopropane (TBP) were studied in seven genotoxicity assays, (i) SOS-induction inE. coli, (ii) DNA repair in primary rat hepatocyte culture, (iii) theSalmonella/microsome assay, (iv) a host-mediated assay usingSalmonella, (v) the somatic mutation and recombination assay inDrosophila melanogaster, (vi) HGPRT-mutagenesis assay in ARL 18 cells, and (vii) micronucleus formation assay in mouse polychromatophylic erythrocytes (PCE), forestomach (FS), glandular stomach (GS), duodenum (D), jejunum (J), cecum (C) and liver (L). The halopropanes were also tested for tumor formation in the fishDanio rerio. DBP was active in assays (ii), (v), (vii FS) and (vii L). TBP was positive in assays (ii) and (iii), strongly positive in (vii L) and borderline positive in (iv). However, neither DBP nor TBP induced tumors in fish, in contrast to the carcinogenic 1,2-dibromo-3-chloropropane. The genotoxicity and potential carcinogenicity of DBP and TBP in mammals is discussed.Abbreviations 2-AA 2-aminoanthracene - DBCP 1,2-dibromo-3-chloropropane - DBP 1,2-dibromopropane - HGPRT hypoxanthine-guanine phosphoribosyl transferase - i.p. intraperitoneal(ly) - NQO 4-nitro-quinoline-1-oxide - PCE polychromatic erythrocytes - TBP 1,1,3-tribromopropane - WME Williams' medium E     

In vitro metabolic activation of ethidium bromide and other phenanthridinium compounds: mutagenic activity in Salmonella typhimurium.

The induction of mutation by a variety of mutagens has been measured utilizing the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells (CHO/HGPRT) system). These mutagens include physical agents such as UV light and X-rays, and chemicals such as alkylating agents, ICR-191, and metallic compounds. This system can also be modified for study of the mutagenicity of promutagens such as dimethylnitrosamine (DMN) which require biotransformation for mutagenic action, either through the addition of a rat liver microsomal activation preparation or through a host-mediated activation step using Balb/c athymic mice.     

Mutagenicity of an antitumor protein, neocarzinostatin, in Escherichia coli.

An assay is described for the measurement of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells utilizing resistance to 6-thioguanine (TG). Optimal selection conditions are defined for such parameters as phenotypic expression time prior to selection, and TG concentration and cell density which permits maximum mutant recovery. The nature of the TG-resistant mutants is characterized by several physiological and biochemical methods. The data demonstrate that more than 98% of the mutant clones isolated by this selection procedure contain altered HGPRTase activity. The CHO/HGPRT system thus shows the specificity necessary for a specific gene locus mutational assay.     

Genetic toxicity evaluation of McN-5195: A novel analgesic

McN-5195, (±)trans-3-(2-bromophenyl)octahydroindolizine, a novel analgesic, was tested for genotoxic potential in a battery of tests with endpoints of mutagenicity, chromosomal alterations and DNA damage/ repair. McN-5195 was not mutagenic when tested in the Ames test using strains TA98, TA100, TA1535, TA1537 and TA 1538, in the absence of metabolic activation and in the presence of Aroclor 1254-induced rat or hamster S-9. Negative results were also obtained in the mouse lymphoma assay in the absence of activation, but reproducible mutagenic responses were seen in this mammalian cell assay in the presence of rat S-9 at high levels of induced toxicity (reduced cell growth). Testing of the enantiomers of McN-5195 in this assay supported these findings. A predominance of small mutant colonies in the mouse lymphoma assay suggested a potential chromosomal effect of McN-5195. This was confirmed with positive findings in an in vitro cytogenetics assay using CHO cells, again at toxic exposure levels and only in the presence of S-9. McN-5195 did not induce DNA repair in the primary rat hepatocyte/DNA repair assay, nor did it induce alterations in vivo of chromosome structure or number when tested in a rat bone marrow cytogenetics assay. The findings from this battery of tests indicate that McN-5195 has modest genotoxic activity when tested in the presence of rat liver S-9 in in vitro systems sensitive to cytogenetic change. The absence of genotoxicity in vitro in Salmonella and intact liver cells and in vivo in rat bone marrow suggests that McN-5195 is unlikely to present a genotoxic risk to whole animals.Abbreviations 2-AA 2-anthramine - 9-AA 9-aminoacridine HCI - 2-AAF 2-acetylaminofluorene - AO acridine orange - CHO Chinese hamster ovary - CP cyclophosphamide - EMS ethylmethane sulfonate - ³H-dThd methyl-³H-thymidine - LDH lactate dehydrogenase - 3-MCA 3-methylcholanthrene - McN-5195 (±)-trans-3-(2-bromophenyl) octahydroindolizine - McN-5195-11 hydrochloride salt of McN-5195 - Na azide sodium azide - RCG relative clonal growth - RSG relative suspension growth - RTG relative total growth - SMF spontaneous mutation frequency - TEM triethylenemelamine - TFT trifluorothymidine     

A review and analysis of the Chinese hamster ovary/hypoxanthine guanine phosphoribosyl transferase assay to determine the mutagenicity of chemical agents. A report of phase III of the U.S. Environmental Protection Agency Gene-Tox Program

Published literature on the Chinese hamster ovary cell/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) assay from mid-1979 through June 1986 was reviewed and evaluated. Data from the papers considered acceptable include test results on 121 chemicals belonging to 25 chemical classes. A total of 87 chemicals were evaluated positive, 3 negative, and 31 inconclusive. Mutagenicity data on 49 of the 121 chemicals evaluated could also be compared with in vivo animal carcinogenicity data. 40 of the 43 reported animal carcinogens were considered mutagenic. Caprolactam, the only definitive noncarcinogen in the group of 49, was not mutagenic. The CHO/HGPRT assay was concluded to be an appropriate assay system for use in the screening of chemicals for genotoxicity.     

DNA-mediated transfer of a human DNA repair gene that controls sister chromatid exchange.

The Chinese hamster cell line mutant EM9, which has a reduced ability to repair DNA strand breaks, is noted for its highly elevated frequency of sister chromatid exchange, a property shared with cells from individuals with Bloom's syndrome. The defect in EM9 cells was corrected by fusion hybridization with normal human fibroblasts and by transfection with DNA from hybrid cells. The transformants showed normalization of sister chromatid exchange frequency but incomplete correction of the repair defect in terms of chromosomal aberrations produced by 5-bromo-2'-deoxyuridine.     

Genotoxic effects of fluoride evaluated by sister-chromatid exchange

The purpose of this investigation was to study the genotoxic potential of fluoride (in the form of sodium fluoride, NaF) using in vitro and in vivo sister-chromatid exchange (SCE) assays with Chinese hamster cells. The NaF concentrations used in cultures of Chinese hamster ovary (CHO) cells ranged from 0 to 6.3 mM, both with and without S9 activation. Fluoride analysis of the culture medium demonstrated that it contained little indigenous fluoride, and the concentration of added fluoride was not affected by the components of the medium or the S9 mix. The CHO cells cultured in 6.3 mM NaF almost vanished, and at the concentration of 5.3 mM NaF in cultures without S9 microsome, only M1 cells were observed. In in vivo studies, Chinese hamsters were intubated with NaF dosages of 0, 0.1, 1.0, 10, 60 and 130 mg/kg, and the bone marrow (CHBM) cells were examined for SCE frequencies. Bone fluoride data showed that the intubated NaF was effectively absorbed. Death occurred in 3 of the 8 animals given 130 mg NaF/kg. The results indicated that NaF, in dosages up to 5.3 mM in CHO cell cultures and 130 mg/kg in in vivo CHBM cells, did not significantly increase the SCE frequencies over those observed in the negative (distilled water) controls. However, examination of the cell cycle revealed an inhibitory effect of NaF on cell proliferation with doses of NaF at or greater than 1.0 mM in cultured CHO cells and at or greater than 60 mg NaF/kg in in vivo CHMB cells. The results of the present study indicated an inhibition of the cell cycle and death of the cells with increasing concentrations of fluoride but not effect of fluoride on SCE frequency in CHO and CHBM cells.     

Genetic damage is not produced by normal cryopreservation procedures involving either glycerol or dimethyl sulfoxide: a cautionary note, however, on possible effects of dimethyl sulfoxide

Cryopreservation of chinese hamster ovary cells in tissue culture with either glycerol or dimethyl sulfoxide did not result in chromosome damage as measured by the sister chromatid exchange technique. These results are consistent with earlier negative reports in which the freezing and thawing of mammalian cells did not increase the frequency of micronuclei. No increases in the spontaneous mutation rates of several bacterial strains at different genetic loci were observed during the course of a number of years of storage at -196 degrees C. It is concluded that standard cryopreservation procedures are without genetic hazards. However, the well-documented effects of dimethyl sulfoxide on cell fusion and gene differentiation suggest caution in its use as a cryopreservative for animal and human embryos.