Human Lymphoblastoid TK6 Cells Research Articles

Kinetics of γH2AX and phospho-histone H3 following pulse treatment of TK6 cells provides insights into clastogenic activity

The desire for in vitro genotoxicity assays to provide higher information content, especially regarding chemicals' predominant genotoxic mode of action, has led to the development of a novel multiplexed assay available under the trade name MultiFlow®. We report here on an experimental design variation that provides further insight into clastogens' genotoxic activity. First, the standard MultiFlow DNA Damage Assay-p53, γ H2AX, phospho-histone H3 was used with human TK6 lymphoblastoid cells that were exposed for 24 continuous hours to each of 50 reference clastogens. This initial analysis correctly identified 48/50 compounds as clastogenic. These 48 compounds were then evaluated using a short-term, 'pulse' treatment protocol whereby cells were exposed to test chemical for 4 h, a centrifugation/washout step was performed, and cells were allowed to recover for 20 h. MultiFlow analyses were accomplished at 4 and 24 h. The γ H2AX and phospho-histone H3 biomarkers were found to exhibit distinct differences in terms of their persistence across chemical classes. Unsupervised hierarchical clustering analysis identified three groups. Examination of the compounds within these groups showed one cluster primarily consisting of alkylators that directly target DNA. The other two groups were dominated by non-DNA alkylators and included anti-metabolites, oxidative stress inducers and chemicals that inhibit DNA-processing enzymes. These results are encouraging, as they suggest that a simple follow-up test for in vitro clastogens provides mechanistic insights into their genotoxic activity. This type of information will contribute to improve decision-making and help guide further testing.

Development and validation of the TGx-HDACi transcriptomic biomarker to detect histone deacetylase inhibitors in human TK6 cells.

Transcriptomic biomarkers can be used to inform molecular initiating and key events involved in a toxicant's mode of action. To address the limited approaches available for identifying epigenotoxicants, we developed and assessed a transcriptomic biomarker of histone deacetylase inhibition (HDACi). First, we assembled a set of ten prototypical HDACi and ten non-HDACi reference compounds. Concentration-response experiments were performed for each chemical to collect TK6 human lymphoblastoid cell samples after 4h of exposure and to assess cell viability following a 20-h recovery period in fresh media. One concentration was selected for each chemical for whole transcriptome profiling and transcriptomic signature derivation, based on cell viability at the 24-h time point and on maximal induction of HDACi-response genes (RGL1, NEU1, GPR183) or cellular stress-response genes (ATF3, CDKN1A, GADD45A) analyzed by TaqMan qPCR assays after 4h of exposure. Whole transcriptomes were profiled after 4h exposures by Templated Oligo-Sequencing (TempO-Seq). By applying the nearest shrunken centroid (NSC) method to the whole transcriptome profiles of the reference compounds, we derived an 81-gene toxicogenomic (TGx) signature, referred to as TGx-HDACi, that classified all 20 reference compounds correctly using NSC classification and the Running Fisher test. An additional 4 HDACi and 7 non-HDACi were profiled and analyzed using TGx-HDACi to further assess classification performance; the biomarker accurately classified all 11 compounds, including 3 non-HDACi epigenotoxicants, suggesting a promising specificity toward HDACi. The availability of TGx-HDACi increases the diversity of tools that can facilitate mode of action analysis of toxicants using gene expression profiling.

Differentiating between micronucleus dose-responses induced by whole cigarette smoke solutions with Benchmark Dose potency ranking

Dose-response modeling of in vitro micronucleus test (IVMNT) data was evaluated to determine if the approach has value in discriminating among different tobacco products. Micronucleus responses were generated in L5178Y/Tk+/− mouse lymphoma cells and TK6 human lymphoblastoid cells from a series of whole smoke solutions (WSSs) expected to have different levels of genotoxicity based on differences in their machine-generated smoke constituents. Eight WSSs were prepared by machine smoking different numbers (20 or 60) of two commercial cigarettes (Marlboro Silver or Red) under International Standardization Organization (ISO) or Health Canada Intense (HCI) smoking machine regimens and tested in the two cell lines with and without rat liver S9 activation. The S9-mediated IVMNT dose-response data from the WSSs were evaluated with PROAST software and Benchmark Doses (BMDs) and their upper and lower confidence intervals (CIs) were generated. IVMNT data differed based on the number and type of cigarettes smoked and smoking machine regimen. The IVMNT responses produced in mouse lymphoma cells generally were greater than in TK6 cells, but the ability of the two cell types to differentiate between WSSs was similar. The results indicate that BMD potency ranking was useful for differentiating between IVMNT responses.

Critical roles of Rad54 in tolerance to apigenin-induced Top1-mediated DNA damage.

Apigenin (APG), a flavone sub-class of flavonoids, possesses a diverse range of biological activities, including anti-cancer and anti-inflammatory effects. Previous studies identified the genotoxicity of APG in certain cancer cells, which may be associated with its anticancer effect. However, the DNA damage repair mechanism induced by APG has remained elusive. In order to clarify the molecular mechanisms, the present study determined the toxicity of APG to the wild-type (WT) DT40 chicken B-lymphocyte cell line, as well as to DT40 cells with deletions in various DNA repair genes, and their sensitivities were compared. It was demonstrated that cells deficient of Rad54, a critical homologous recombination gene, were particularly sensitive to APG. Cell-cycle analysis demonstrated that APG caused an increase in the G2/M-phase population of Rad54- / - cells that was greater than that in WT cells. Furthermore, it was demonstrated by immunofluorescence assay that Rad54- / - cells exhibited significantly increased numbers of γ-phosphorylated H2AX variant histone foci and chromosomal aberrations compared to the WT cells in response to APG. Of note, the in vitro complex of enzyme assay indicated that APG induced increased topoisomerase I (Top1) covalent protein DNA complex in Rad54- / - cells compared to WT cells. Finally, these results were verified using the TK6 human lymphoblastoid cell line and it was demonstrated that, as for DT40 cells, Rad54 deficiency sensitized TK6 cells to APG. The present study demonstrated that Rad54 was involved in the repair of APG-induced DNA damage, which was associated with Top1 inhibition.

Mutagenicity and genotoxicity assessments of some industrially processed meat products in Algeria

Processed meat products are presumptive sources of mutagens and genotoxins for consumers. Several epidemiological studies have reported that these products' high intakes have a positive link with cancer incidence. In Algeria, industrially processed meat products are widely consumed. However, there are no earlier studies involving their genotoxic activity. For this end, the current study aimed at evaluating the mutagenicity and the genotoxicity of some representative industrially processed meat products sold in popular supermarkets. All samples were extracted by established method, using both polar and non-polar solvents. The meat extracts mutagenicity was assessed by Ames test, using four strains of Salmonella typhimurium in the presence and absence of metabolic activation, and subsequently by treat and wash assay for extracts showing biologically significant results. The genotoxicity was determined in TK6 human lymphoblastoid cells using the in vitro micronucleus assay in micromethod. The results showed that all extracts analyzed induce no mutagenic activity. However, one of these extracts induced a biologically significant increase in the number of micronucleated cells. Our findings indicate the importance of the genetic damage detection for taking measures to suppress or reduce the exposure to harmful contaminants and encourage further research investigating genotoxic effects of industrially processed meat worldwide.

The genotoxicity potential of luteolin is enhanced by CYP1A1 and CYP1A2 in human lymphoblastoid TK6 cells

Luteolin (5,7,3′,4′-tetrahydroxyflavone) belongs to the flavone subclass of flavonoids. Luteolin and its glycosides are present in many botanical families, including edible plants, fruits, and vegetables. While the beneficial properties of luteolin have been widely studied, fewer studies have investigated its toxicity. In the present study, using human lymphoblastoid TK6 cells and our newly developed TK6-derived cell lines that each stably express a single human cytochrome P450 (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C18, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7), we systematically evaluated luteolin-induced cytotoxicity and genotoxicity, and the role of specific CYPs in the bioactivation of luteolin. Treatments with luteolin for 4−24 h induced cytotoxicity, apoptosis, DNA damage, and chromosome damage in a concentration-dependent manner. Subsequently, we observed that luteolin-induced cytotoxicity and genotoxicity, measured by the high-throughput micronucleus assay, were significantly increased in TK6 cells transduced with CYP1A1 and 1A2. In addition, key apoptosis and DNA damage biomarkers, including cleaved PARP-1, cleaved caspase-3, and phosphorylated histone 2AX (γH2A.X), were all significantly increased in the CYP1A1- and 1A2-expressing cells compared with the empty vector controls. Analysis by LC–MS/MS revealed that TK6 cells biotransformed the majority of luteolin into diosmetin, a less toxic O-methylated flavone, after 24 h; the presence of CYP1A1 and 1A2 partially reversed this process. Altogether, these results indicate that metabolism by CYP1A1 and 1A2 enhanced the toxicity of luteolin in vitro. Our results further support the utility of our TK6 cell system for identification of the specific CYPs responsible for chemical bioactivation and toxicity potential.

Weight of evidence approach using a TK gene mutation assay with human TK6 cells for follow-up of positive results in Ames tests: a collaborative study by MMS/JEMS

BackgroundConflicting results between bacterial mutagenicity tests (the Ames test) and mammalian carcinogenicity tests might be due to species differences in metabolism, genome structure, and DNA repair systems. Mutagenicity assays using human cells are thought to be an advantage as follow-up studies for positive results in Ames tests. In this collaborative study, a thymidine kinase gene mutation study (TK6 assay) using human lymphoblastoid TK6 cells, established in OECD TG490, was used to examine 10 chemicals that have conflicting results in mutagenicity studies (a positive Ames test and a negative result in rodent carcinogenicity studies).ResultsTwo of 10 test substances were negative in the overall judgment (20% effective as a follow-up test). Three of these eight positive substances were negative after the short-term treatment and positive after the 24 h treatment, despite identical treatment conditions without S9. A toxicoproteomic analysis of TK6 cells treated with 4-nitroanthranilic acid was thus used to aid the interpretation of the test results. This analysis using differentially expressed proteins after the 24 h treatment indicated that in vitro specific oxidative stress is involved in false positive response in the TK6 assay.ConclusionsThe usefulness of the TK6 assay, by current methods that have not been combined with new technologies such as proteomics, was found to be limited as a follow-up test, although it still may help to reduce some false positive results (20%) in Ames tests. Thus, the combination analysis with toxicoproteomics may be useful for interpreting false positive results raised by 24 h specific reactions in the assay, resulting in the more reduction (> 20%) of false positives in Ames test.

The use of benchmark dose uncertainty measurements for robust comparative potency analyses.

The Benchmark Dose (BMD) method is the favored approach for quantitative dose-response analysis where uncertainty measurements are delineated between the upper (BMDU) and lower (BMDL) confidence bounds, or confidence intervals (CIs). Little has been published on the accurate interpretation of uncertainty measurements for potency comparative analyses between different test conditions. We highlight this by revisiting a previously published comparative in vitro genotoxicity dataset for human lymphoblastoid TK6 cells that were exposed to each of 10 clastogens in the presence and absence (+/-) of low concentration (0.25%) S9, and scored for p53, γH2AX and Relative Nuclei Count (RNC) responses at two timepoints (Tian et al., 2020). The researchers utilized BMD point estimates in potency comparative analysis between S9 treatment conditions. Here we highlight a shortcoming that the use of BMD point estimates can mischaracterize potency differences between systems. We reanalyzed the dose responses by BMD modeling using PROAST v69.1. We used the resulting BMDL and BMDU metrics to calculate "S9 potency ratio confidence intervals" that compare the relative potency of compounds +/- S9 as more statistically robust metrics for comparative potency measurements compared to BMD point estimate ratios. We performed unsupervised hierarchical clustering that identified four S9-dependent groupings: high and low-level potentiation, no effect, and diminution. This work demonstrates the importance of using BMD uncertainty measurements in potency comparative analyses between test conditions. Irrespective of the source of the data, we propose a stepwise approach when performing BMD modeling in comparative potency analyses between test conditions.

Tyrosyl-DNA phosphodiesterases are involved in mutagenic events at a ribonucleotide embedded into DNA in human cells.

Ribonucleoside triphosphates are often incorporated into genomic DNA during DNA replication. The accumulation of unrepaired ribonucleotides is associated with genomic instability, which is mediated by DNA topoisomerase 1 (Top1) processing of embedded ribonucleotides. The cleavage initiated by Top1 at the site of a ribonucleotide leads to the formation of a Top1-DNA cleavage complex (Top1cc), occasionally resulting in a DNA double-strand break (DSB). In humans, tyrosyl-DNA phosphodiesterases (TDPs) are essential repair enzymes that resolve the trapped Top1cc followed by downstream repair factors. However, there is limited cellular evidence of the involvement of TDPs in the processing of incorporated ribonucleotides in mammals. We assessed the role of TDPs in mutagenesis induced by a single ribonucleotide embedded into DNA. A supF shuttle vector site-specifically containing a single riboguanosine (rG) was introduced into the human lymphoblastoid TK6 cell line and its TDP1-, TDP2-, and TDP1/TDP2-deficient derivatives. TDP1 and TDP2 insufficiency remarkably decreased the mutant frequency caused by an embedded rG. The ratio of large deletion mutations induced by rG was also substantially lower in TDP1/TDP2-deficient cells than wild-type cells. Furthermore, the disruption of TDPs reduced the length of rG-mediated large deletion mutations. The recovery ratio of the propagated plasmid was also increased in TDP1/TDP2-deficient cells after the transfection of the shuttle vector containing rG. The results suggest that TDPs-mediated ribonucleotide processing cascade leads to unfavorable consequences, whereas in the absence of these repair factors, a more error-free processing pathway might function to suppress the ribonucleotide-induced mutagenesis. Furthermore, base substitution mutations at sites outside the position of rG were detected in the supF gene via a TDPs-independent mechanism. Overall, we provide new insights into the mechanism of mutagenesis induced by an embedded ribonucleotide in mammalian cells, which may lead to the fatal phenotype in the ribonucleotide excision repair deficiency.

Expression of miR-34a is a sensitive biomarker for exposure to genotoxic agents in human lymphoblastoid TK6 cells

miR-34a has been identified as a tumor suppressor microRNA (miRNA) involved in the P53 network. Its expression levels correlate to carcinogenesis, which are generally lower in tumor tissue and higher in response to DNA damage. In this study, the response of miR-34a from exposure to genotoxic agents in human lymphoblastoid TK6 cells was evaluated to assess whether the expression of this miRNA could be used as an early indicator for genotoxic damage in mammalian cells. TK6 cells were treated with seven genotoxic agents with different mode-of-actions (cisplatin, N-ethyl-N-nitrosourea, etoposide, mitomycin C, methyl methanesulphonate, taxol, and X-ray radiation) and a non-genetic toxin (usnic acid) at different concentrations for four hours (except for X-rays) and the expression levels of miR-34a were measured 24 h after the beginning of the treatments. The expression levels of miR-34a were significantly increased by these genetic toxins in a dose-dependent manner, while no significant change in miRNA expression was found in the usnic acid-treated cells. These results suggest that miR-34a can respond to genotoxic insults sensitively; thus, miR-34a expression has the potential to be used to evaluate genotoxicity of agents.

Analysis of DNA Adducts and Mutagenic Potency and Specificity in Rats Exposed to Acrylonitrile.

Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in multiple organs/tissues of rats by unresolved mechanisms. For this report, evidence for ACN-induced direct/indirect DNA damage and mutagenesis was investigated by assessing the ability of ACN, or its reactive metabolite, 2-cyanoethylene oxide (CEO), to bind to DNA in vitro, to form select DNA adducts [N7-(2'-oxoethyl)guanine, N2,3-ethenoguanine, 1,N6-ethenodeoxyadenosine, and 3,N4-ethenodeoxycytidine] in vitro and/or in vivo, and to perturb the frequency and spectra of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene in rats exposed to ACN in drinking water. Adducts and frequencies and spectra of Hprt mutations were analyzed using published methods. Treatment of DNA from human TK6 lymphoblastoid cells with [2,3-14C]-CEO produced dose-dependent binding of 14C-CEO equivalents, and treatment of DNA from control rat brain/liver with CEO induced dose-related formation of N7-(2'-oxoethyl)guanine. No etheno-DNA adducts were detected in target tissues (brain and forestomach) or nontarget tissues (liver and spleen) in rats exposed to 0, 3, 10, 33, 100, or 300 ppm ACN for up to 105 days or to 0 or 500 ppm ACN for ∼15 months; whereas N7-(2'-oxoethyl)guanine was consistently measured at nonsignificant concentrations near the assay detection limit only in liver of animals exposed to 300 or 500 ppm ACN for ≥2 weeks. Significant dose-related increases in Hprt mutant frequencies occurred in T-lymphocytes from spleens of rats exposed to 33-500 ppm ACN for 4 weeks. Comparisons of "mutagenic potency estimates" for control rats versus rats exposed to 500 ppm ACN for 4 weeks to analogous data from rats/mice treated at a similar age with N-ethyl-N-nitrosourea or 1,3-butadiene suggest that ACN has relatively limited mutagenic effects in rats. Considerable overlap between the sites and types of mutations in ACN-exposed rats and butadiene-exposed rats/mice, but not controls, provides evidence that the carcinogenicity of these epoxide-forming chemicals involves corresponding mutagenic mechanisms.

Novel o-Toluidine Metabolite in Rat Urine Associated with Urinary Bladder Carcinogenesis.

o-Toluidine (o-Tol), a monocyclic aromatic amine, causes bladder cancer in humans and experimental animals and is therefore classified as a Group 1 carcinogen (IARC) in which the carcinogenicity of o-Tol is involved in metabolic activation, DNA damage, and DNA adduct formation. In the DNA adduct formation mechanism, o-Tol is metabolized by N-hydroxylation, N-acetoxylation, and then deacetoxylation to produce an electrophilic nitrenium ion, which is able to bind to a DNA base, such as dG-C8. Therefore, dG-C8-o-Tol is thought to be a plausible DNA adduct of o-Tol exposure. However, direct detection of dG-C8-o-Tol in biological samples has not been reported yet. Here, we show that a novel o-Tol metabolite, 2-methyl-N1-(2-methylphenyl)benzene-1,4-diamine (MMBD), a dimer by head-to-tail binding, was identified for the first time in o-Tol-exposed rat urine. MMBD was also detected in a reaction of o-Tol and S9 mix, indicating the formation was catalyzed by an enzymatic reaction. Moreover, MMBD showed a potent stronger mutagenicity in N-acetyltransferase overexpressed Salmonella typhimurium strains,and cytotoxicity in human bladder carcinoma T24 cells and human spleen lymphoblastoid TK6 cells compared with o-Tol. Furthermore, a DNA adduct (m/z 478.1) corresponding to dG-MMBD was detected in the reaction of calf thymus DNA with rat urine containing MMBD, and also in hepatic DNA of rats treated with o-Tol. These results therefore suggested that o-Tol-induced bladder carcinogenesis could be at least partly attributed to MMBD formation. The possible dimerization of monocyclic aromatic amines should be considered in the evaluation of the risk of bladder carcinogenesis after exposure.

Cytotoxic and genotoxic effects of e-liquids and their potential associations with nicotine, menthol and phthalate esters

In this study, we determined DNA damage and chromosome breakage (indicators of genotoxicity) and cell viability (an indicator of cytotoxicity) in human lymphoblastoid TK6 and Chinese hamster ovary (CHO) cells treated with 33 e-liquids using in vitro single cell gel (comet), micronucleus (MN), and trypan blue assays, respectively. We also measured the contents of nicotine, five phthalate esters, and DL-menthol in the e-liquids to examine their effects on DNA damage, chromosome breakage, and cell viability. Our chemical analyses showed that: (1) six e-liquids had nicotine ≥2-fold higher than the manufacture’s label claim (2–3.5 mg); (2) both dimethyl- and dibutyl-phthalate levels were >0.1 μg/g, i.e., their threshold limits as additives in cosmetics; and (3) the DL-menthol contents ranged from 0.0003 to 85757.2 μg/g, with those of two e-liquids being >1 mg/g, the threshold limit for trigging sensory irritation. Though all the e-liquids induced DNA damage in TK6 cells, 20 resulted in cell viabilities ≤75%, indicating cytotoxicity, yet the inverse relationship between cell viability and DNA damage (r = −0.628, p = 0.003) might reflect their role as pro-apoptotic and DNA damage inducers. Fifteen e-liquids induced MN% in TK6 cells ≥3-fold that of untreated cells. Some of the increase in %MN might be false due to high cytotoxicity, yet six brands showed acceptable cell viabilities (59–71%), indicating chromosome damage. DNA damage and %MN increased when the TK6 cells were exposed to metabolic activation. The CHO cells were less sensitive to the genotoxic effects of the e-liquids than the TK6 cells. DL-menthol was found to be associated with decreased cell viability and increased DNA damage, even at low levels. We cannot dismiss the presence of other ingredients in e-liquids with cytotoxic/genotoxic properties since out of the 63 different flavors, 47 induced DNA damage (≥3-folds), and 26 reduced cell viability (≤75%) in TK6 cells.

In vitro 50 Hz magnetic field long-term exposure: Cytogenetic tests on human lymphoblastoid TK6 cells and validation of the test environment

Potential health effects of extremely low-frequency (electro)magnetic fields (ELF-(E)MFs) have long been investigated, but the results are still inconclusive. With respect to genotoxicity, sound data related to the effects of long-term exposure to ELF-(E)MFs on the genetic material and the impact of long-term pre-exposure to ELF-(E)MFs on the sensitivity of cells to the damage induced by known mutagens are needed. In this manuscript, an optimized protocol for a combined in vitro comet/micronucleus study to investigate these effects in a human lymphoblastoid cell line (TK6) is provided including the description of a well-validated exposure system. Furthermore, the use of a shielding system to limit background ELF-MFs inside the incubator is described as well.•Optimized protocols for cytogenetic tests with ELF-MFs on TK6 cells ensure the reproducibility of test results.•Validation of exposure environment and exposure system are needed prior to performing tests with ELF-MFs.•A simple, but effective method to shield cells and reduce unintentional ELF-MF exposure consists of using the mu-metal cylinder. This is of particular interest when studying the effects of low exposure levels.

Effect of simulated microgravity and ionizing radiation on expression profiles of miRNA, lncRNA, and mRNA in human lymphoblastoid cells

In space, multiple unique environmental factors, particularly microgravity and space radiation, pose a constant threat to astronaut health. MicroRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are functional RNAs that play critical roles in regulating multiple cellular processes. To gain insight into the role of non-coding RNAs in response to radiation and microgravity, we analyzed RNA expression profiles in human lymphoblastoid TK6 cells incubated for 24 h under static or rotating conditions to stimulate microgravity in space, after 2-Gy γ-ray irradiation. The expression of 14 lncRNAs and 17 mRNAs (differentially-expressed genes, DEGs) was found to be significantly downregulated under simulated microgravity conditions. In contrast, irradiation upregulated 55 lncRNAs and 56 DEGs, whereas only one lncRNA, but no DEGs, was downregulated. Furthermore, two miRNAs, 70 lncRNAs, and 87 DEGs showed significantly altered expression in response to simulated microgravity after irradiation, and these changes were independently induced by irradiation and simulated microgravity. GO enrichment and KEGG pathway analyses indicated that the associated target genes showed similar patterns to the noncoding RNAs and were suggested to be involved in the immune/inflammatory response including LPS/TLR, TNF, and NF-κB signaling pathways. However, synergistic effects on RNA expression and cellular responses were also observed with a combination of simulated microgravity and irradiation based on microarray and RT-PCR analysis. Together, our results indicate that simulated microgravity and irradiation additively alter expression patterns but synergistically modulate the expression levels of RNAs and their target genes in human lymphoblastoid cells.

Carbon ion and proton beam irradiation of a normal human TK6 lymphoblastoid cell line within a magnetic field of 1.0 tesla.

BackgroundConsidering the increasing simultaneous application of magnetic resonance imaging (MRI) for more precise photon radiotherapy, it will be likely for particle radiotherapy to adopt MRI for future image guiding. It will then be imperative to evaluate the potential biological effects of a magnetic field (MF) on particle irradiation. This study explores such effects on the highly radiosensitive TK6 lymphoblastoid human cell line.MethodsThe following three parameters were measured after irradiation with either carbon ion or proton beams using spread out Bragg peaks and applying different doses within a perpendicular 1.0 T MF: (1) cell survival fraction (14 days postirradiation), (2) treatment-specific apoptosis, which was determined through the measurement of population in the sub-G1 phase, and (3) cell cycle progression by means of flow cytometry. These were compared to the same parameters measured without an MF.ResultsThe clonogenic assay in both treatment groups showed almost identical survival curves with overlapping error bars. The calculated α values with and without an MF were 2.18 (σ=0.245) and 2.17 (σ=0.234) for carbon ions and 1.08 (σ=0.138) and 1.13 (σ=0.0679) for protons, respectively. Similarly, the treatment-specific apoptosis and cell cycle progression showed almost identical curves with overlapping error bars. A two-sample, unpooled t-test analysis was implemented for comparison of all mean values and showed p-values >0.05.ConclusionNo statistically significant difference in biological response of the TK6 cells was observed when they were irradiated using spreadout Bragg peaks within a perpendicular 1.0 T MF as compared to those, which received the same dose without the MF. This should serve as another supporting piece of evidence toward the implementation of MRI in particle radiotherapy, though further research is necessary.

Comparative study of cytotoxic effects induced by environmental genotoxins using XPC- and CSB-deficient human lymphoblastoid TK6 cells

BackgroundThe human genome is constantly exposed to numerous environmental genotoxicants. To prevent the detrimental consequences induced by the expansion of damaged cells, cellular protective systems such as nucleotide excision repair (NER) exist and serve as a primary pathway for repairing the various helix-distorting DNA adducts induced by genotoxic agents. NER is further divided into two sub-pathways, namely, global genomic NER (GG-NER) and transcription-coupled NER (TC-NER). Both NER sub-pathways are reportedly involved in the damage response elicited by exposure to genotoxins. However, how disruption of these sub-pathways impacts the toxicity of different types of environmental mutagens in human cells is not well understood.ResultsTo evaluate the role of NER sub-pathways on the cytotoxic effects of mutagens, we disrupted XPC and CSB to selectively inactivate GG-NER and TC-NER, respectively, in human lymphoblastoid TK6 cells, a standard cell line used in genotoxicity studies. Using these cells, we then comparatively assessed their respective sensitivities to representative genotoxic agents, including ultraviolet C (UVC) light, benzo [a] pyrene (B(a)P), 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP), γ-ray, and 2-acetylaminofluorene (2-AAF). CSB−/− cells exhibited a hyper-sensitivity to UVC, B(a)P, and MeIQx. On the other hand, XPC−/− cells were highly sensitive to UVC, but not to B(a)P and MeIQx, compared with wild-type cells. In contrast with other genotoxins, the sensitivity of XPC−/− cells against PhIP was significantly higher than CSB−/− cells. The toxicity of γ-ray and 2-AAF was not enhanced by disruption of either XPC or CSB in the cells.ConclusionsBased on our findings, genetically modified TK6 cells appear to be a useful tool for elucidating the detailed roles of the various repair factors that exist to combat genotoxic agents, and should contribute to the improved risk assessment of environmental chemical contaminants.

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry.

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

Concentration-response studies of the chromosome-damaging effects of topoisomerase II inhibitors determined in vitro using human TK6 cells

Topoisomerase II (topo II) inhibitors are commonly used as chemotherapy to treat multiple types of cancer, though their use is also associated with the development of therapy related acute leukemias. While the chromosome-damaging effects of etoposide, a topo II poison, have been proposed to act through a threshold mechanism, little is known about the chromosome damaging effects and dose responses for the catalytic inhibitors of the enzyme. The current study was designed to further investigate the potencies and concentration-response relationships of several topoisomerase II inhibitors, including the topoisomerase II poison etoposide, as well as catalytic inhibitors aclarubicin, merbarone, ICRF-154 and ICRF-187 using both a traditional in vitro micronucleus assay as well as a flow-cytometry based version of the assay. Benchmark dose (BMD) analysis was used to identify models that best fit the data and estimate a BMD, in this case the concentration at which a one standard deviation increase above the control frequency would be expected. All of the agents tested were potent in inducing micronuclei in human lymphoblastoid TK6 cells, with significant increases seen at low micromolar, and in the cases of aclarubicin and etoposide, at low nanomolar concentrations. Use of the anti-kinetochore CREST antibody with the microscopy-based assay demonstrated that the vast majority of the micronuclei originated from chromosome breakage. In comparing the two versions of the micronucleus assay, significant increases in micronucleated cells were observed at similar or lower concentrations using the traditional microscopy-based assay. BMD modeling of the data exhibited several advantages and proved to be a valuable alternative for concentration-response analysis, producing points of departure comparable to those derived using traditional no-observed or lowest-observed genotoxic effect level (NOGEL or LOGEL) approaches.

The influence of a magnetic field on photon beam radiotherapy in a normal human TK6 lymphoblastoid cell line

BackgroundThe implementation of magnetic resonance imaging (MRI) guided radiotherapy (RT) continues to increase. Very limited in-vitro data on the interaction of ionizing radiation and magnetic fields (MF) have been published. In these experiments we focused on the radiation response in a MF of the TK6 human lymphoblastoid cells which are known to be highly radiosensitive due to efficient radiation-induced apoptosis.MethodsClonogenicity was determined 12–14 days after irradiation with 1–4 Gy 6 MV photons with or without a 1.0 Tesla MF. Furthermore, alterations in cell cycle distribution and rates of radiation induced apoptosis (FACS analysis of cells with sub-G1 DNA content) were analyzed.ResultsClonogenic survival showed an exponential dose-dependence, and the radiation sensitivity parameter (α = 1.57/Gy) was in accordance with earlier reports. Upon comparing the clonogenic survival between the two groups, identical results within error bars were obtained. The survival fractions at 2 Gy were 9% (without MF) and 8.5% (with MF), respectively.ConclusionA 1.0 Tesla MF does not affect the clonogenicity of TK6 cells irradiated with 1–4 Gy 6MV photons. This supports the use of MRI guided RT, however ongoing research on the interaction of MF and radiotherapy is warranted.