Aberrations In Chinese Hamster Ovary Cells Research Articles

Toxicity Studies of Lactobacillus plantarum PS128TM Isolated from Spontaneously Fermented Mustard Greens.

Probiotics are extensively available to consumers; however, the use of probiotics may not always be safe, and there are few reports on their side effects, including those of Lactobacillus. Lactobacillus plantarum strain PS128TM isolated from spontaneously fermented mustard greens in Taiwan was recently reported to exhibit probiotic properties. In this study, we aimed to assess the safety of strain PS128TM for use in humans via examining genotoxic and oral toxic effects using in vitro and in vivo testing. Five strains of Salmonella typhimurium were evaluated by the Ames test; no signs of increased reverse mutation were observed following exposure to PS128TM. Additional testing of Chinese hamster ovary (CHO) cells exposed to PS128TM revealed that the incidence of chromosomal aberrations in CHO cells had not increased. PS128TM treatment also did not affect the proportion of immature to total erythrocytes or the number of micronuclei in the immature erythrocytes of ICR mice. Moreover, following a 28 day study involving repeated oral dose toxicity tests (2400, 400, and 40 mg/kg body weight) utilizing an ICR mouse model, no observable adverse level (NOAEL) was found at any of the doses. PS128TM was sensitive to antibiotics; however, genes related to the production of biogenic amines were absent. While further research is required, these toxicological assessments suggest that PS128TM could be safe for human consumption.

Genotoxicity assessment of ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN)

Ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN) are relatively insensitive explosive compounds that are being explored as safe alternatives to other more sensitive compounds. When used in combination with other high explosives they are an improvement and may provide additional safety during storage and use. The genetic toxicity of these compounds was evaluated to predict the potential adverse human health effects from exposure by using a standard genetic toxicity test battery which included: a gene mutation test in bacteria (Ames), an in vitro Chinese Hamster Ovary (CHO) cell chromosome aberration test and an in vivo mouse micronucleus test. The results of the Ames test showed that EDDN increased the mean number of revertants per plate with strain TA100, without activation, at 5000μg/plate compared to the solvent control, which indicated a positive result. No positive results were observed with the other tester strains with or without activation in Salmonella typhimurium strains TA98, TA1535, TA1537, and Escherichia coli strain WP2 uvrA. DETN was negative for all Salmonella tester strains and E. coli up to 5000μg/plate both with and without metabolic activation. The CHO cell chromosome aberration assay was performed using EDDN and DETN at concentrations up to 5000μg/mL. The results indicate that these compounds did not induce structural chromosomal aberrations at all tested concentrations in CHO cells, with or without metabolic activation. EDDN and DETN, when tested in vivo in the CD-1 mouse at doses up to 2000mg/kg, did not induce any significant increase in the number of micronuclei in bone marrow erythrocytes. These studies demonstrate that EDDN is mutagenic in one strain of Salmonella (TA100) but was negative in other strains, for in vitro induction of chromosomal aberrations in CHO cells, and for micronuclei in the in vivo mouse micronucleus assay. DETN was not genotoxic in all in vitro and in vivo tests. These results show the in vitro and in vivo genotoxicity potential of these chemicals.

Genotoxicity assessment of an energetic propellant compound, 3-nitro-1,2,4-triazol-5-one (NTO)

3-Nitro-1,2,4-triazol-5-one (NTO) is an energetic explosive proposed for use in weapon systems, to reduce the sensitivity of warheads. In order to develop toxicity data for safety assessment, we investigated the genotoxicity of NTO, using a battery of genotoxicity tests, which included the Ames test, Chinese Hamster Ovary (CHO) cell chromosome aberration test, L5178Y TK+/− mouse lymphoma mutagenesis test and rat micronucleus test. NTO was not mutagenic in the Ames test or in Escherichia coli (WP2uvrA). NTO did not induce chromosomal aberrations in CHO cells, with or without metabolic activation. In the L5178Y TK+/− mouse lymphoma mutagenesis test, all of the NTO-treated cultures had mutant frequencies that were similar to the average frequencies of solvent control-treated cultures, indicating a negative result. Confirmatory tests for the three in vitro tests also produced negative results. The potential in vivo clastogenicity and aneugenicity of NTO was evaluated using the rat peripheral blood micronucleus test. NTO was administered by oral gavage to male and female Sprague–Dawley rats for 14 days at doses up to 2g/kg/day. Flow cytometric analysis of peripheral blood demonstrated no significant induction of micronucleated reticulocytes relative to the vehicle control (PEG-200). These studies reveal that NTO was not genotoxic in either in vitro or in vivo tests and suggest a low risk of genetic hazards associated with exposure.

Genotoxicity assessment of two hypergolic energetic propellant compounds

Recognition of the occupational hazards from exposure to the propellants hydrazine and monomethylhydrazine (MMH) has led to research into less toxic alternatives. Two hypergolic compounds, dimethylamino-2-ethylazide (DMAZ) and N,N,N′,N′-tetramethylethanediamine (TMEDA), have been identified as possible replacements for MMH. We have obtained genotoxicity data for these compounds from in vitro and in vivo studies. DMAZ did not produce any mutagenic effects at concentrations up to 5 mg/plate in the TA98 and TA1537 strains of Salmonella typhimurium and in an Escherichia coli (WP2 uvrA) strain, with or without metabolic activation, but did produce a positive response in the TA100 and TA1535 strains, both with and without metabolic activation. TMEDA was found not to be mutagenic in any of the bacterial strains tested ( Salmonella TA98, TA100, TA1535, TA1537 and E. coli, WP2 uvrA), with or without metabolic activation. DMAZ did not induce structural chromosomal aberrations at levels up to 5 mg/mL in Chinese hamster ovary (CHO) cells, with or without metabolic activation. TMEDA produced a positive response in this system, with or without metabolic activation, but only at the highest concentration, 5 mg/mL. However, according to the OECD guideline TG 473, the compound is considered to be negative in the CHO chromosomal aberration assay, since the compound was not clastogenic at 0.01 M (1.140 mg/mL). DMAZ and TMEDA, when tested in vivo in the CD-1 mouse at doses up to 500 and 250 mg/kg, respectively, did not induce micronuclei in bone marrow erythrocytes. These studies demonstrate that DMAZ is mutagenic in specific strains of Salmonella. However, both compounds were negative for induction of chromosomal aberrations in CHO cells in vitro and in the in vivo mouse micronucleus assay.

XRCC1 Protects against Particulate Chromate–Induced Chromosome Damage and Cytotoxicity in Chinese Hamster Ovary Cells

Water-insoluble hexavalent chromium compounds are well-established human lung carcinogens. Lead chromate, a model insoluble Cr(VI) compound, induces DNA damage, chromosome aberrations, and dose-dependent cell death in human and Chinese hamster ovary (CHO) cells. The relationship between lead chromate-induced DNA damage and chromosome aberrations is unknown. Our study focus was on examining the role of XRCC1 in lead chromate-induced cytotoxicity and structural chromosomal aberrations in CHO cells. Three different cell lines were used: AA8 (parental), EM9 (XRCC1 mutant), and H9T3 (EM9 complemented with human XRCC1 gene). Cytotoxicity was significantly higher in EM9 cells when compared to AA8 and H9T3 cells, indicating that XRCC1 is important for protecting cells from lead chromate particles-induced cell death. The frequency of damaged metaphase cells was not affected by XRCC1 deficiency. However, the total amount of Cr(VI)-induced chromosome damage was exacerbated by XRCC1 deficiency, and the spectrum of damage changed dramatically. Chromatid and isochromatid lesions were the most prominent aberrations induced in all cell lines. XRCC1 was essential to reduce the formation of chromatid lesions but not for isochromatid lesions. In addition, XRCC1 deficiency resulted in a dramatic increase in the number of chromatid exchanges, indicating that XRCC1 is involved in protection from lead chromate-induced chromosome instability.

XRCC1 Protects against Particulate Chromate–Induced Chromosome Damage and Cytotoxicity in Chinese Hamster Ovary Cells

Water-insoluble hexavalent chromium compounds are well-established human lung carcinogens. Lead chromate, a model insoluble Cr(VI) compound, induces DNA damage, chromosome aberrations, and dose-dependent cell death in human and Chinese hamster ovary (CHO) cells. The relationship between lead chromate-induced DNA damage and chromosome aberrations is unknown. Our study focus was on examining the role of XRCC1 in lead chromate-induced cytotoxicity and structural chromosomal aberrations in CHO cells. Three different cell lines were used: AA8 (parental), EM9 (XRCC1 mutant), and H9T3 (EM9 complemented with human XRCC1 gene). Cytotoxicity was significantly higher in EM9 cells when compared to AA8 and H9T3 cells, indicating that XRCC1 is important for protecting cells from lead chromate particles-induced cell death. The frequency of damaged metaphase cells was not affected by XRCC1 deficiency. However, the total amount of Cr(VI)-induced chromosome damage was exacerbated by XRCC1 deficiency, and the spectrum of damage changed dramatically. Chromatid and isochromatid lesions were the most prominent aberrations induced in all cell lines. XRCC1 was essential to reduce the formation of chromatid lesions, but not for isochromatid lesions. In addition, XRCC1 deficiency resulted in a dramatic increase in the number of chromatid exchanges, indicating that XRCC1 is involved in protection from lead chromate-induced chromosome instability.

Genotoxicity of Brosimum gaudichaudii measured by the Salmonella/microsome assay and chromosomal aberrations in CHO cells

The root bark of Brosimum gaudichaudii Trécul (Moraceae) is popularly used for treatment of vitiligo. In the present study the mutagenic activity of the aqueous and methanolic extract as well as of the n-butanolic fraction of this medicinal plant were evaluated using Salmonella typhimurium assays, TA100, TA98, TA102 and TA97a strains, while the clastogenic effect in Chinese hamster ovary (CHO) cells in the G1/S, S and G2/S phases of the cell cycle. The results showed mutagenic activity of the aqueous extract against TA102 in the presence of S9, and of methanolic extract, with and without metabolic activation. TA100 mutagenicity was only observed for the methanolic extract in the absence of S9. The n-butanolic fraction did not present mutagenic activity. In CHO cells only the methanolic extract induced a significant increase of chromosomal aberrations in the G1/S and S phases, whereas a decrease in the mitotic index was observed in the G1/S and G2/S phases. No clastogenicity was observed for the aqueous extract. The furocoumarins (psoralen and bergapten) presented in the extracts might contribute to the mutagenicity. The lower activity of the aqueous extract was probably due to the presence of smaller amount of furocoumarins compared to the methanolic extract.

Can a mixed damage interfere with DNA-protein cross-links repair?

Some photochemical and photobiological properties of 4,5',8-trimethylpsoralen (TMP) have been studied in comparison with 1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2 one (FQ) and 8-methoxypsoralen (8-MOP). TMP and FQ can photobind to mammalian cell DNA in vivo, by UVA irradiation, forming DNA-protein cross-links (DPC), but only TMP shows a strong capacity of inducing interstrand cross-links (ISC). The mechanism of DPC formation was studied using the double irradiation method in Chinese hamster ovary (CHO) cells, and DPC were detected by alkaline elution. Both TMP and FQ induce covalent diadducts linking together DNA and proteins. Studying the formation of double strand breaks (DSB) in CHO cells we observed that TMP induced a low amount of DSB, similar to 8-MOP. TMP and 8-MOP induced chromosomal aberrations in CHO cells to the same extent, while FQ appeared to be more active. Our data suggest that the ISC induced by TMP could trap enzymes involved in DPC repair.

The effect of 125I decay at different stages of S-phase on survival, expression of micronuclei and chromosome aberrations in CHO cells.

Chinese hamster ovary (CHO) cells were synchronized in M phase by mitotic selection, and then re-synchronized with aphidicolin at the G1/S phase border. The cells were labelled in early-S phase by 10 min exposure to 125I-iododeoxyuridine and then cultured (chased) in non-radioactive medium for 0.5, 3 or 5h, followed by harvesting and freezing to accumulate the desired number of 125I decays. Cell damage was assessed by evaluating colony formation, micronucleus formation and chromosome aberrations. These biological estimators of damage showed that the cytocidal effect of 125I decay increased with the duration of the post-labelling chase period: the highest level of damage was found in cells from the 5 h chase period and the lowest in the cells from the 0.5 h chase period. Survival curves for the three chase periods displayed low-dose hyper-radiosensitivity for 0 to 20 125I decays cell-1. The results indicate that the repair of DNA double-strand breaks (DSBs) may depend on the maturation stage of chromatin and an explanation of this finding is proposed which invokes the homologous recombination model for DSB repair.

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 B6C3F 1 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.

Induction of 6-thioguanine resistance, chromosome aberrations and SCE by dinitropyrenes in Chinese hamster ovary cells in vitro

The ability of Chinese hamster ovary (CHO) cells to convert dinitropyrenes (DNPs) to mutagenic species has been investigated by examining the effects of 1,6-DNP and 1,8-DNP on three distinct end-points in this cell line. At concentrations ranging from 0.05 to 5 μg/ml both analogues induced increases in the frequency of 6-thioguanine-resistant mutants in CHO cells when exposure was limited to 3 h. This treatment time was also sufficient to permit the induction of sister-chromatid exchange and chromosome aberrations in CHO cells. The results obtained suggest that CHO cells, unlike mouse lymphoma L5178Y cells, possess an endogenous metabolic activity which is capable of bringing about the conversion of DNPs to their mutagenic form without a requirement for prolonged exposure periods and as such may be a more suitable cell line for the study of this class of compounds.

Genetic effects of the flavonols quercetin, kaempferol, and galangin on Chinese hamster ovary cells in vitro

The genotoxicity of selected flavonols was evaluated by multiple endpoints in Chinese hamster ovary (CHO) cells. Chromosomal aberrations, sister-chromatid exchange (SCE), and forward mutation at 4 gene loci were measured in a single population of cells exposed to quercetin, kaempferol, or galangin for 15 h with and without metabolic activation. The incidence of chromosomal aberrations was significantly increased by quercetin in the absenve of activation and by kempferol and galangin with and without activation. Flavonol treatment affected SCE and mutation at the hgprt, aprt, orNa + /K + -ATPase loci only marginally, but significantly increased mutation frequencies at the tk locus. The response at the tk locus suggests that the CHO cells may behave similarly to L5178Y cells, in which the tk locus is thought to reflect chromosomal lesions in addition to point mutation. These results indicate that, at least under the conditions examined, flavonols induce chromosomal aberrations in CHO cells, but have little effect on point mutation or SCE.