Hamster Cell Hybrids Research Articles

Assessment of Genotoxic Effects by Constructing a 3D Cellular System with Highly Sensitive Mutagenic Human-Hamster Hybrid Cells.

Owing to complex microenvironmental conditions, it is challenging to reflect the actual biological responses of tissues or the body in a two-dimensional (2D) cellular system. In the present study, a low-attachment-cultivation technique was employed to establish a highly sensitive 3D human-hamster hybrid (AL) model to study the mutagenic effects of environmental pollutants. The results showed that the established 3D system has apparent organizational characteristics. The average diameter and average cell number of the 3D cells were approximately 240 μm and 1500, respectively. The expression of stemness and cell-junction genes (biomarkers for 3D cells) was higher than that in 2D cells. The present study analyzed the mutagenic effects of the environmental carcinogens arsenite and silver nanoparticles using the established 3D system to demonstrate its efficiency in mutagenic assessment. The results showed that the mutagenic effects of arsenite (10 μM) and silver nanoparticles (10 μg/mL) were 70 ± 3 and 99 ± 7 per 105 survivors, respectively. These values were much lower than those from 2D AL cells and comparable to those from the in vivo system. These results suggest that the developed 3D-cell-culture model based on the 2D AL cellular system more effectively reflects the actual gene-mutation frequency of mutagens in vivo.

Assessment of the cytotoxic and mutagenic potential of the Jialu River and adjacent groundwater using human-hamster hybrid cells

The Jialu River in China has been seriously polluted by the direct discharge of industrial and domestic wastewater. The predominant contaminants of the Jialu River and its adjacent groundwater were recently investigated. However, the potential genotoxic impact of polluted water on human health remains to be clarified. Here, we used human–hamster hybrid (AL) cells, which are sensitive for detecting environmental mutagens. We found that the cytotoxicity and mutagenicity of the groundwater in the Jialu River basin were influenced by the infiltration of the Jialu River. Hydrological periods significantly affected the cytotoxicity, but not the mutagenic potential, of surface and groundwater. Further, the mutagenic potential of groundwater samples located <1km from the Jialu River (SM-2 water samples) was detected earlier than that of groundwater samples located approximately 20km from the Jialu River (SN water samples). Because of high cytotoxicity, the mutagenic potential of water samples from the Jialu River (SM-1 water samples) was not significantly enhanced compared with that of untreated controls. To further assess the mutagenic dispersion potential, an artificial neural network model was adopted. The results showed that the highest mutagenic potential of groundwater was observed approximately 10km from the Jialu River. Although further investigation of mutagenic spatial dispersion is required, our data are significant for advancing our understanding of the origin, dispersion, and biological effects of water samples from polluted areas.

Mutagenicity of ZnO nanoparticles in mammalian cells: Role of physicochemical transformations under the aging process

Zinc oxide nanoparticles (ZnO NPs) potentially undergo physicochemical transformation in the environment, which may lead to unexpected environmental and health risks. The “aging” process is essential for better understanding the toxicity and fate of NPs in the environment. However, the mutagenic effects of aged ZnO NPs are still unexplored. The present study focused on investigating the physicochemical transformation during aging process and clarifying the mutagenicity of naturally aged ZnO NPs in human–hamster hybrid (AL) cells. It was found that ZnO NPs underwent sophisticated physicochemical transformations with aging regardless of original morphology or size, such as the microstructural changes, the formation of hydrozincite (Zn5(CO3)2(OH)6) and the release of free zinc ions. Interestingly, the aged ZnO NPs were investigated to be able to result in much lower cytotoxicity while relatively high degree mutation than fresh ZnO NPs. With characterization of the soluble and insoluble fractions of aged ZnO NPs suspension, together with the control measurements using metal chelator (TPEN) and endocytosis inhibitor (Nystatin), it was revealed that the release of zinc ions and nanoparticle uptake made significantly different contributions to the mutagenicity of fresh and aged ZnO NPs. This study clearly demonstrated that the physicochemical transformation of ZnO NPs with aging plays important and comprehensive roles in the ZnO NPs-induced mutagenicity in mammalian cells.

Role of nitric oxide in the genotoxic response to chronic microcystin-LR exposure in human–hamster hybrid cells

Microcystin-LR (MC-LR) is the most abundant and toxic microcystin congener and has been classified as a potential human carcinogen (Group 2B) by the International Agency for Research on Cancer. However, the mechanisms underlying the genotoxic effects of MC-LR during chronic exposure are still poorly understood. In the present study, human–hamster hybrid (AL) cells were exposed to MC-LR for varying lengths of time to investigate the role of nitrogen radicals in MC-LR-induced genotoxicity. The mutagenic potential at the CD59 locus was more than 2-fold higher (p<0.01) in AL cells exposed to a cytotoxic concentration (1μmol/L) of MC-LR for 30days than in untreated control cells, which was consistent with the formation of micronucleus. MC-LR caused a dose-dependent increase in nitric oxide (NO) production in treated cells. Moreover, this was blocked by concurrent treatment with the NO synthase inhibitor NG-methyl-l-arginine (l-NMMA), which suppressed MC-LR-induced mutations as well. The survival of mitochondrial DNA-depleted (ρ0) AL cells was markedly decreased by MC-LR treatment compared to that in AL cells, while the CD59 mutant fraction was unaltered. These results provided clear evidence that the genotoxicity associated with chronic MC-LR exposure in mammalian cells was mediated by NO and might be considered as a basis for the development of therapeutics that prevent carcinogenesis.

PFOS-induced apoptosis through mitochondrion-dependent pathway in human–hamster hybrid cells

Perfluorooctane sulfonate (PFOS) was listed as one of the persistent organic pollutants (POPs) in Stockholm Convention in 2009. Recent evidence showed that PFOS could induce apoptosis both in vivo and in vitro. However, the apoptotic mechanisms induced by PFOS as well as the possible relationship between apoptosis and other PFOS-induced endpoints, remain unclear. In the present study, normal human–hamster hybrid (AL) cells and mtDNA-depleted (ρ0 AL) cells were exposed to PFOS, and assayed for cytotoxicity, mutagenicity, and apoptosis (caspase-3/7, caspase-9 activities). Our results showed that PFOS decreased cell viability in a time- and concentration-dependent manner in AL cells, but not in ρ0 AL cells. However, long-term exposure to PFOS failed to induce the mutagenic effects at the CD59 locus in AL cells. Exposure to 200 μM PFOS significantly increased the activities of caspase-3/7 and caspase-9 in AL cells, but the activities of these caspases were not affected in ρ0 AL cells. In addition, PFOS increased the levels of reactive oxygen species (ROS), superoxide anion (O2−), as well as nitric oxide (NO), and decreased mitochondrial membrane potential (MMP) at the concentrations of 100 and 200μM in AL cells. On the other hand, exposure to PFOS had no effect on intracellular ROS, O2−, and NO production in ρ0 AL cells. Caspase-3/7 activity, which was increased by 200 μM PFOS, could be suppressed by ROS/O2− scavengers and nitric oxide synthases (NOSs) inhibitors in AL cells. These results implicate that PFOS-induced apoptosis and oxidative stress is mediated by a mitochondrion-dependent pathway and that the induction of apoptosis might be a protective function against mutagenesis in AL cells exposed to PFOS.

Cellular ATP content was decreased by a homogeneous 8.5 T static magnetic field exposure: Role of reactive oxygen species

The literature on the impact of strong static magnetic fields (SMF) on human health is vast and contradictory. The present study focused on the cellular effects of strong homogeneous SMF in human-hamster hybrid (A(L) ) cells, mitochondria-deficient (ρ(0) A(L) ) cells, and double-strand break (DSB) repair-deficient (XRS-5) cells. Adenosine triphosphate (ATP) content was significantly decreased in A(L) cells exposed to 8.5 Tesla (T) but not 1 or 4 T SMF for either 3 or 5 h. In addition, ATP content significantly decreased in the two deficient cell lines exposed to 8.5 T SMF for 3 h. With further incubation of 12 or 24 h without SMF exposure, ATP content could retrieve to the control level in the A(L) cells but not ρ(0) A(L) and XRS-5 cells. Under a fluorescence reader, the levels of reactive oxygen species (ROS) in the three cell lines were significantly increased by exposure to 8.5 T SMF for 3 h. Concurrent treatment with ROS inhibitor, DMSO, dramatically suppressed the ATP content in exposed A(L) cells. However, the CD59 mutation frequency and the cell cycle distribution were not significantly affected by exposure to 8.5 T SMF for 3 h. Our results indicated that the cellular ATP content was reduced by 8.5 T SMF for 3 h exposure, which was partially mediated by mitochondria and the DNA DSB repair process. Moreover, ROS were involved in the process of the cellular perturbations from the SMF.

Characterization and linkage mapping of ten sheep microsatellite markers derived from a sheep x hamster cell hybrid.

A cosmid library has been constructed from a sheep x hamster cell hybrid containing sheep chromosome t1, rob (6;24). Clones containing sheep DNA were identified by hybridizing to a total sheep genomic DNA probe. Small fragments (< 500 bp) containing (AC)n microsatellites were subcloned and sequenced. Ten microsatellite markers were characterized and six were mapped back to chromosomes 6 and 24. The remaining microsatellites mapped to chromosome 26, which was shown to be present in a small proportion of cells of the cell line.

Up-regulation of ROS by mitochondria-dependent bystander signaling contributes to genotoxicity of bystander effects

Genomic instability can be observed in bystander cells. However, the underlying mechanism(s) is still relatively unclear. In a previous study, we found that irradiated cells released mitochondria-dependent intracellular factor(s) which could lead to bystander γ-H2AX induction. In this paper, we used normal (ρ +) and mtDNA-depleted (ρ 0) human–hamster hybrid cells to investigate mitochondrial effects on the genotoxicity in bystander effect through medium transfer experiments. Through the detection of DNA double-strand breaks with γ-H2AX, we found that the fraction of γ-H2AX positive cells changed with time when irradiation conditioned cell medium (ICCM) were harvested. ICCM harvested from irradiated ρ + cells at 10 min post-irradiation (ρ + ICCM 10 min ) caused larger increases of bystander γ-H2AX induction comparing to ρ 0 ICCM 10 min , which only caused a slight increase of bystander γ-H2AX induction. The ρ + ICCM 10 min could also result in the up-regulation of ROS production (increased by 35% at 10 min), while there was no significant increase in cells treated with ρ 0 ICCM 10 min . We treated cells with dimethyl sulfoxide (DMSO), the scavenger of ROS, and quenched γ-H2AX induction by ρ + ICCM. Furthermore, after the medium had been transferred and the cells were continuously cultured for 7 days, we found significantly increased CD59 − gene loci mutation (increased by 45.9%) and delayed cell death in the progeny of ρ + ICCM-treated bystander cells. In conclusion, the work presented here suggested that up-regulation of the mitochondria-dependent ROS might be very important in mediating genotoxicity of bystander effects.

Response to comments on the paper “spindle disturbances in human hamster hybrid (AL) cells” by Mays Swicord

Response to comments on the paper “spindle disturbances in human hamster hybrid (A_L) cells” by Mays Swicord

Comment on “spindle disturbances in human hamster hybrid (AL) cells …” by T. Schrader et al.

BioelectromagneticsVolume 30, Issue 5 p. 422-422 Comment Comment on “spindle disturbances in human hamster hybrid (AL) cells …” by T. Schrader et al. Mays Swicord, Corresponding Author Mays Swicord mays.swicord@comcast.net Consultant, Ft Lauderdale, FloridaConsultant, Ft Lauderdale, Florida, 1033 NE 17th Way, Ft Lauderdale, FL 33304.Search for more papers by this author Mays Swicord, Corresponding Author Mays Swicord mays.swicord@comcast.net Consultant, Ft Lauderdale, FloridaConsultant, Ft Lauderdale, Florida, 1033 NE 17th Way, Ft Lauderdale, FL 33304.Search for more papers by this author First published: 24 March 2009 https://doi.org/10.1002/bem.20491AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume30, Issue5July 2009Pages 422-422 RelatedInformation

Activated toxicity of diesel particulate extract by ultraviolet a radiation in mammalian cells: role of singlet oxygen.

BackgroundDiesel exhaust [diesel exhaust particles (DEPs) and their extracts (DPE)] and ultraviolet A radiation (UVA) are two ubiquitous environmental factors that have been identified as essential risk factors for various benign or malignant human diseases, either alone or in combination with other agents.ObjectivesWe aimed to investigate the synergistic effects of DPE and UVA at low-dose exposures in human–hamster hybrid (AL) cells and their underlying mechanisms.MethodsWe exposed exponentially growing AL cells to DPE and/or UVA radiation with or without reactive oxygen species (ROS) quenchers and then assayed the cells for survival, mutation induction, apoptosis, and micronucleus generation. In addition, using a singlet oxygen (1O2) trapping probe, 2,2,6,6-tetramethyl-4-piperidone, coupled with electron paramagnetic resonance spectroscopy, we determined the production of 1O2.ResultsTreatment of AL cells with DPE + UVA induced significant cytotoxic and genotoxic damage. In contrast, we found no significant damage in cells treated with either UVA or DPE alone at the same doses. Mutation spectra of CD59− mutants showed that treatment with DPE + UVA easily induces multilocus deletions. Sodium azide significantly inhibited both cellular and DNA damage induced by DPE + UVA treatment, whereas other ROS inhibitors had little protecting effect. Furthermore, we found a significant increase of 1O2 in the cells that received DPE + UVA treatment.ConclusionThese findings suggest that UVA activated the genotoxicity and cytotoxicity of DPE in mammalian cells and that 1O2 played an important role in these processes.

Site-specific recombination in Schizosaccharomyces pombe and systematic assembly of a 400kb transgene array in mammalian cells using the integrase of Streptomyces phage ϕBT1

We have established the integrase of the Streptomyces phage ϕBT1 as a tool for eukaryotic genome manipulation. We show that the ϕBT1 integrase promotes efficient reciprocal and conservative site-specific recombination in vertebrate cells and in Schizosaccharomyces pombe, thus establishing the utility of this protein for genome manipulation in a wide range of eukaryotes. We show that the ϕBT1 integrase can be used in conjunction with Cre recombinase to promote the iterative integration of transgenic DNA. We describe five cycles of iterative integration of a candidate mouse centromeric sequence 80 kb in length into a human mini-chromosome within a human-Chinese hamster hybrid cell line. These results establish the generality of the iterative site-specific integration technique.

Mutagenicity of diesel exhaust particles mediated by cell–particle interaction in mammalian cells

Diesel exhaust particle (DEP) has been identified as a class 2A human carcinogen and closely related to the increased incidence of respiratory allergy, cardiopulmonary morbidity and mortality, and risk of lung cancer. However, the molecular mechanisms of DEP mutagenicity/carcinogenicity are still largely unknown. In the present study, we focused on the mutagenicity of DEPs in human–hamster hybrid (AL) cells and evaluated the role of cell–particle interaction in mediating mutagenic process. We found that DEPs formed micron-sized aggregates in the medium and located mainly in large cytoplasmic vacuoles of cells by 24h treatment. The cellular granularity was increased by DEP treatment in a dose-dependent manner. DEPs resulted in a dose-dependent increase of mutation yield at CD59 locus in AL cells, while inflicting minimal cytotoxicity. There was a more than two-fold increase of mutation yield at CD59 locus in AL cells exposed to DEPs at a dose of 50μg/ml. Such induction was significantly reduced by concurrent treatment with phagocytosis inhibitors, cytochalasin B and ammonium chloride (p<0.05). These results provided direct evidence that DEPs was mutagenic in mammalian cells and that cell–particle interaction played an essential role in the process.

Quantification of CD59− mutants in human–hamster hybrid (A L) cells by flow cytometry

Mutation assay is an important approach in evaluating the genotoxic risk of potentially harmful environmental chemicals. The human–hamster hybrid (A L) cell mutagenesis system, based on the complement/antibody-mediated cytotoxicity principle, has been used successfully to evaluate the mutagenic potential of a variety of environmental toxicants. The A L cells contain a standard set of CHO chromosomes and a single human chromosome 11, which expresses several cell surface proteins including CD59 encoded by the CD59 gene at 11p13.5. A modified mutation assay by flow cytometry was developed to determine the yield of CD59 − mutants after either radiation or chemical treatment. After incubation with phycoerythrin-conjugated mouse monoclonal anti- CD59 antibody, the CD59 − mutant yields were determined by quantifying the fluorescence of the cells using flow cytometry. This method is faster and eliminates the commonly encountered toxicity problems of the complements with the traditional complement/antibody assay. By comparing the mutant fractions of radiation or chemically treated A L cultures using the two methods, we show here that the flow cytometry assay is an excellent substitute in providing an efficient and highly sensitive method in mutant detection for the traditional complement/antibody assay.

Effects of irradiated medium with or without cells on bystander cell responses

Recent studies have indicated that extranuclear or extracellular targets are important in mediating the bystander genotoxic effects of α-particles. In the present study, human–hamster hybrid (A L) cells were plated on either one or both sides of double-mylar dishes 2–4 days before irradiation, depending on the density requirement of experiments. One side (with or without cells) was irradiated with α-particles (from 0.1 to 100 Gy) using the track segment mode of a 4 MeV Van de Graaff accelerator. After irradiation, cells were kept in the dishes for either 1 or 48 h. The non-irradiated cells were then collected and assayed for both survival and mutation. When one side with cells was irradiated by α-particles (1, 10 and 100 Gy), the surviving fraction among the non-irradiated cells was significantly lower than that of control after 48 h co-culture. However, such a change was not detected after 1 h co-culture or when medium alone was irradiated. Furthermore, co-cultivation with irradiated cells had no significant effect on the spontaneous mutagenic yield of non-irradiated cells collected from the other half of the double-mylar dishes. These results suggested that irradiated cells released certain cytotoxic factor(s) into the culture medium that killed the non-irradiated cells. However, such factor(s) had little effect on mutation induction. Our results suggest that different bystander end points may involve different mechanisms with different cell types.

Large-mutation spectra induced at hemizygous loci by low-LET radiation: evidence for intrachromosomal proximity effects.

A mathematical model is used to analyze mutant spectra for large mutations induced by low-LET radiation. The model equations are based mainly on two-break misrejoining that leads to deletions or translocations. It is assumed, as a working hypothesis, that the initial damage induced by low-LET radiation is located randomly in the genome. Specifically, we analyzed data for two hemizygous loci: CD59- mutants, mainly very large-scale deletions (>3 Mbp), in human-hamster hybrid cells, and data from the literature on those HPRT- mutants which involve at least deletion of the whole gene, and often of additional flanking markers (approximately 50-kbp to approximately 4.4-Mbp deletions). For five data sets, we estimated f, the probability that two given breaks on the same chromosome will misrejoin to make a deletion, as a function of the separation between the breaks. We found that f is larger for nearby breaks than for breaks that are more widely separated; i.e., there is a "proximity effect". For acute irradiation, the values of f determined from the data are consistent with the corresponding break misrejoining parameters found previously in quantitative modeling of chromosome aberrations. The value of f was somewhat smaller for protracted irradiation than for acute irradiation at a given total dose; i.e., the mutation data show a decrease that was smaller than expected for dose protraction by fractionation or low dose rate.

Induction of oxyradicals by arsenic: implication for mechanism of genotoxicity.

Although arsenic is a well-established human carcinogen, the mechanisms by which it induces cancer remain poorly understood. We previously showed arsenite to be a potent mutagen in human-hamster hybrid (A(L)) cells, and that it induces predominantly multilocus deletions. We show here by confocal scanning microscopy with the fluorescent probe 5',6'-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate that arsenite induces, within 5 min after treatment, a dose-dependent increase of up to 3-fold in intracellular oxyradical production. Concurrent treatment of cells with arsenite and the radical scavenger DMSO reduced the fluorescent intensity to control levels. ESR spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-1-hydroxypiperidine (TEMPOL-H) as a probe in conjunction with superoxide dismutase and catalase to quench superoxide anions and hydrogen peroxide, respectively, indicates that arsenite increases the levels of superoxide-driven hydroxyl radicals in these cells. Furthermore, reducing the intracellular levels of nonprotein sulfhydryls (mainly glutathione) in A(L) cells with buthionine S-R-sulfoximine increases the mutagenic potential of arsenite by more than 5-fold. The data are consistent with our previous results with the radical scavenger DMSO, which reduced the mutagenicity of arsenic in these cells, and provide convincing evidence that reactive oxygen species, particularly hydroxyl radicals, play an important causal role in the genotoxicity of arsenical compounds in mammalian cells.

A role for chromosomal instability in the development of and selection for radioresistant cell variants.

Chromosome instability is a common occurrence in tumour cells. We examined the hypothesis that the elevated rate of mutation formation in unstable cells can lead to the development of clones of cells that are resistant to the cancer therapy. To test this hypothesis, we compared chromosome instability to radiation sensitivity in 30 independently isolated clones of GM10115 human–hamster hybrid cells. There was a broader distribution of radiosensitivity and a higher mean SF 2 in chromosomally unstable clones. Cytogenetic and DNA double-strand break rejoining assays suggest that sensitivity was a function of DNA repair efficiency. In the unstable population, the more radioresistant clones also had significantly lower plating efficiencies. These observations suggest that chromosome instability in GM10115 cells can lead to the development of cell variants that are more resistant to radiation. In addition, these results suggest that the process of chromosome breakage and recombination that accompanies chromosome instability might provide some selective pressure for more radioresistant variants. © 2001 Cancer Research Campaign http://www.bjcancer.com

Fluorescence in situ hybridization of metaphase chromosomes in suspension

Purpose : To describe a new method for FISH analysis of metaphase chromosomes in suspension. Materials and methods : Metaphase chromosomes in suspension were isolated from a Chinese hamster human hybrid cell line, 314-2 (1) Y, and a human cell line, GM 130B. During suspension hybridization, specific chromosomes were labeled from these two cell lines using either biotin-labeled human genomic DNA, a directly labeled human pancentromere DNA probe or a chromosome #1 locus-specific probe. Results : The method allows, for the first time, recovery of large numbers of isolated individual hybridized chromosomes with good morphology for both human ×hamster hybrid and human cell lines. The results showed that 46-73% of the starting number of total chromosomes can be recovered after a FISH in suspension procedure. The well-preserved morphology of hybridized metaphase chromosomes allowed (1) rapid detection of individual human and hamster chromosome aberrations, (2) rapid counting of the painted human chromosomes and (3) fast, clear detection of chromosome region-specific probes. This method offers a new tool to assay chromosomes and DNA: it offers the possibility to develop new techniques for sorting chromosomes based on FISH signals, for early detection and screening of genetic diseases and for bulk measurement of both balanced or unbalanced chromosomal exchanges and rearrangements. Conclusion : The potential of the method described should facilitate fast, sensitive population monitoring, and increase sensitivity of the measurements in chromosome-based biodosimetry.

Using PCR Markers for Mapping Pig Chromosome 12

Using PCR analysis of pig–mink and pig–Chinese hamster hybrid cell lines and heterologous and homologous primers of various types, chromosomal and subchromosomal mapping of genesTOP2A, THRA, BRCA1, GAS, HLR1, MYL4, LIS1, MCP1, ENO3, CRYB1, P4HB, STAT5B, andH3F3Bto pig chromosome 12 was carried out. The efficiency of using different types of heterologous primers for pig chromosome mapping was compared.