Human Lymphoblastoid TK6 Cells Research Articles

CDK4/6 inhibitor-induced bone marrow micronuclei might be caused by cell cycle arrest during erythropoiesis

BackgroundA micronucleus test is generally used to evaluate the genotoxic potential of chemicals. Exaggerated erythropoiesis, as occurs following bleeding, may induce an unexpected increase in micronucleus frequency. This false positive result would be typical in a genotoxicity study due to the enhanced progression of the cell cycle that restores decreased blood cells. The cyclin-dependent kinase (CDK) family is known to play an essential role in preventing genomic instability. Conversely, a selective CDK4/6 inhibitor PD0332991, clinically named Palbociclib, is reported to have genotoxic potential, shown by positive results in both in vitro and in vivo micronucleus studies. To clarify the mechanism by which cell cycle arrest induced by a CDK4/6 inhibitor increases micronucleus frequency, we investigated the positive results of the bone marrow micronucleus test conducted with PD0332991.ResultsRats treated with PD0332991 exhibited increased micronucleus frequency in an in vivo bone marrow micronucleus test whereas it was not increased by treatment in human lymphoblastoid TK6 cells. In addition, all other genotoxicity tests including the Ames test and the comet assay showed negative results with PD0332991. Interestingly, PD0332991 treatment led to an increase in erythrocyte size in rats and affected the size distribution of erythrocytes, including the micronucleus. The mean corpuscular volume of reticulocytes (MCVr) in the PD0332991 treatment group was significantly increased compared to that of the vehicle control (83.8 fL in the PD0332991, and 71.6 fL in the vehicle control.). Further, the average micronucleated erythrocytes (MNE) size of the PD0332991 group and vehicle control was 8.2 and 7.3 µm, respectively. In the histogram, the vehicle control showed a monomodal distribution with a peak near 7.3 µm. In contrast, the PD0332991 group showed a bimodal distribution with peaks around 7.5 and 8.5 µm. Micronucleated erythrocytes in the PD0332991 group were significantly larger than those in the vehicle control.These results suggest that the increase in micronucleus frequency induced by the CDK4/6 inhibitor is not due to genotoxicity, but is attributable to disturbance of the cell cycle, differentiation, and enucleation of erythroblasts.ConclusionsIt was suggested that the positive outcome of the in vivo bone marrow micronucleus test resulting from treatment with PD0332991 could not be attributed to its genotoxicity. Further studies to clarify the mechanism of action can contribute to the development of drug candidate compounds lacking intrinsic genotoxic effects.

MiR-7-5p targeted Rb regulating cell cycle is involved in hydroquinone-induced malignant progression in human lymphoblastoid TK6 cells

MiR-7-5p has been demonstrated to inhibit tumorigenesis by limiting tumor cell proliferation, migration and invasion. However, its role in countering hydroquinone (HQ)-induced malignant phenotype of TK6 cells has remained unclear. The present study aimed to investigate whether miR-7-5p overexpression could restrain the malignant phenotype in TK6 cells exposed to HQ. The results displayed that HQ suppressed the expression of miR-7-5p and promoted cell cycle progression. Further investigations confirmed that miR-7-5p could decelerate the cell cycle progression by targeting Rb after acute HQ exposure. Through the regulation of the Rb/E2F1 signaling pathway, the overexpression of miR-7-5p mitigated HQ-induced malignant phenotype in TK6 cells by impeding cell cycle progression. In conclusion, miR-7-5p overexpression appears to be involved in HQ-induced malignant transformation by suppressing Rb/E2F1 signaling pathway, resulting in a deceleration of the cell cycle progression.

Error-corrected duplex sequencing enables direct detection and quantification of mutations in human TK6 cells with strong inter-laboratory consistency.

Error-corrected duplex sequencing (DS) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DS to quantify induced mutation frequency (MF) and spectrum in human lymphoblastoid TK6 cells exposed to a prototypical DNA alkylating agent, N-ethyl-N-nitrosourea (ENU). Furthermore, we explored appropriate experimental parameters for this application, and assessed inter-laboratory reproducibility. In two independent experiments in two laboratories, TK6 cells were exposed to ENU (25-200µM) and DNA was sequenced 48, 72, and 96h post-exposure. A DS mutagenicity panel targeting twenty 2.4-kb regions distributed across the genome was used to sample diverse, genome-representative sequence contexts. A significant increase in MF that was unaffected by time was observed in both laboratories. Concentration-response in the MF from the two laboratories was strongly positively correlated (r=0.97). C:G>T:A, T:A>C:G, T:A>A:T, and T:A>G:C mutations increased in consistent, concentration-dependent manners in both laboratories, with high proportions of C:G>T:A at all time points. The consistent results across the three time points suggest that 48h may be sufficient for mutation analysis post-exposure. The target sites responded similarly between the two laboratories and revealed a higher average MF in intergenic regions. These results, demonstrating remarkable reproducibility across time and laboratory for both MF and spectrum, support the high value of DS for characterizing chemical mutagenicity in both research and regulatory evaluation.

Revisiting the mutagenicity and genotoxicity of N-nitroso propranolol in bacterial and human in vitro assays

Propranolol is a widely used β-blocker that can generate a nitrosated derivative, N-nitroso propranolol (NNP). NNP has been reported to be negative in the bacterial reverse mutation test (the Ames test) but genotoxic in other in vitro assays. In the current study, we systematically examined the in vitro mutagenicity and genotoxicity of NNP using several modifications of the Ames test known to affect the mutagenicity of nitrosamines, as well as a battery of genotoxicity tests using human cells. We found that NNP induced concentration-dependent mutations in the Ames test, both in two tester strains that detect base pair substitutions, TA1535 and TA100, as well as in the TA98 frameshift-detector strain. Although positive results were seen with rat liver S9, the hamster liver S9 fraction was more effective in bio-transforming NNP into a reactive mutagen. NNP also induced micronuclei and gene mutations in human lymphoblastoid TK6 cells in the presence of hamster liver S9. Using a panel of TK6 cell lines that each expresses a different human cytochrome P450 (CYP), CYP2C19 was identified as the most active enzyme in the bioactivation of NNP to a genotoxicant among those tested. NNP also induced concentration-dependent DNA strand breakage in metabolically competent 2-dimensional (2D) and 3D cultures of human HepaRG cells. This study indicates that NNP is genotoxic in a variety of bacterial and mammalian systems. Thus, NNP is a mutagenic and genotoxic nitrosamine and a potential human carcinogen.

An Evaluation of the Cytotoxic and Genotoxic Effects of the Marine Toxin C17-SAMT in Human TK6 and HepaRG Cell Lines.

This study investigates the genotoxicity and cytotoxicity of C17-sphinganine analog mycotoxin (C17-SAMT) using in vitro assays. C17-SAMT was previously identified as the cause of unusual toxicity in cultured mussels from the Bizerte Lagoon in northern Tunisia. While a previous in vivo genotoxicity study was inconclusive, in vitro results demonstrated that C17-SAMT induced an increase in micronucleus formation in human lymphoblastoid TK6 cells at concentrations of 0.87 µM and 1.74 µM. In addition, multiparametric cytotoxicity assays were performed in the human hepatoma HepaRG cell line, which showed that C17-SAMT induced mitochondrial dysfunction, decreased cellular ATP levels, and altered the expression of various proteins, including superoxide dismutase SOD2, heme oxygenase HO-1, and NF-κB. These results suggest that C17-SAMT is mutagenic in vitro and can induce mitochondrial dysfunction in HepaRG cells. However, the exact mode of action of this toxin requires further investigation. Overall, this study highlights the potential toxicity of C17-SAMT and the need for further research to better understand its effects.

Application of a new approach methodology (NAM)-based strategy for genotoxicity assessment of data-poor compounds.

The conventional battery for genotoxicity testing is not well suited to assessing the large number of chemicals needing evaluation. Traditional in vitro tests lack throughput, provide little mechanistic information, and have poor specificity in predicting in vivo genotoxicity. New Approach Methodologies (NAMs) aim to accelerate the pace of hazard assessment and reduce reliance on in vivo tests that are time-consuming and resource-intensive. As such, high-throughput transcriptomic and flow cytometry-based assays have been developed for modernized in vitro genotoxicity assessment. This includes: the TGx-DDI transcriptomic biomarker (i.e., 64-gene expression signature to identify DNA damage-inducing (DDI) substances), the MicroFlow® assay (i.e., a flow cytometry-based micronucleus (MN) test), and the MultiFlow® assay (i.e., a multiplexed flow cytometry-based reporter assay that yields mode of action (MoA) information). The objective of this study was to investigate the utility of the TGx-DDI transcriptomic biomarker, multiplexed with the MicroFlow® and MultiFlow® assays, as an integrated NAM-based testing strategy for screening data-poor compounds prioritized by Health Canada's New Substances Assessment and Control Bureau. Human lymphoblastoid TK6 cells were exposed to 3 control and 10 data-poor substances, using a 6-point concentration range. Gene expression profiling was conducted using the targeted TempO-Seq™ assay, and the TGx-DDI classifier was applied to the dataset. Classifications were compared with those based on the MicroFlow® and MultiFlow® assays. Benchmark Concentration (BMC) modeling was used for potency ranking. The results of the integrated hazard calls indicate that five of the data-poor compounds were genotoxic in vitro, causing DNA damage via a clastogenic MoA, and one via a pan-genotoxic MoA. Two compounds were likely irrelevant positives in the MN test; two are considered possibly genotoxic causing DNA damage via an ambiguous MoA. BMC modeling revealed nearly identical potency rankings for each assay. This ranking was maintained when all endpoint BMCs were converted into a single score using the Toxicological Prioritization (ToxPi) approach. Overall, this study contributes to the establishment of a modernized approach for effective genotoxicity assessment and chemical prioritization for further regulatory scrutiny. We conclude that the integration of TGx-DDI, MicroFlow®, and MultiFlow® endpoints is an effective NAM-based strategy for genotoxicity assessment of data-poor compounds.

Radiation-Induced Chromosome Instability in WTK1 and TK6 Human Lymphoblastoid Cells

The effects of ionizing radiation on single nucleotide polymorphism (SNP) copy number variations between TK6 and WTK1 cell lines are described herein. Specifically, the integrity of the chromosomes for two WIL2-derived lymphoblastoid cell lines (TK6 and WTK1) was analyzed in the presence and absence of ionizing radiation. WTK1 cells contain a p53 mutation, whereas the TK6 cell line has the native p53 tumor-suppressor gene. Each cell line was isolated post-irradiation for SNP analysis, which showed significant, genome-wide impacts on both cell lines; for the mutant WTK1 sample, there were a total of 48 gene deletions and no gene amplifications, whereas for the wild-type TK6 sample, there were 217 gene deletions and 9 gene amplifications. It appears that both cell lines are affected in the areas of cell-cycle control, but that other affected areas differ significantly between the two.

New Marine Diterpenoid from the Okinawan Soft Coral, Lobophytum sp.

A new lobane-type diterpenoid, loba-8,10,13(15)-triene-14,17,18-triol 17-acetate (1), and 5 known compounds, lobatriene (2), (17 R)-loba-8,10,13(15)-triene-17,18-diol (3), loba-8,10,13(15)-triene-14,17,18-triol 14,17-diacetate (4), loba-8,10,13(15)-triene-14,17,18-triol 14-acetate (5), and lobatrienetriol (6), were isolated from a population of Okinawan soft coral Lobophytum sp. The structures of these compounds were elucidated by spectroscopy, including nuclear magnetic resonance and high-resolution mass spectrometry. The cytotoxicity and genotoxicity of these compounds were tested using human lymphoblastoid TK6 cells.

The Genotoxicity of Acrylfentanyl, Ocfentanyl and Furanylfentanyl Raises the Concern of Long-Term Consequences.

Three fentanyl analogues Acrylfentanyl, Ocfentanyl and Furanylfentanyl are potent, rapid-acting synthetic analgesics that recently appeared on the illicit market of new psychoactive substances (NPS) under the class of new synthetic opioids (NSO). Pharmacotoxicological data on these three non-pharmaceutical fentanyl analogues are limited and studies on their genotoxicity are not yet available. Therefore, the aim of the present study was to investigate this property. The ability to induce structural and numerical chromosomal aberrations in human lymphoblastoid TK6 cells was evaluated by employing the flow cytometric protocol of the in vitro mammalian cell micronucleus test. Our study demonstrated the non-genotoxicity of Fentanyl, i.e., the pharmaceutical progenitor of the class, while its illicit non-pharmaceutical analogues were found to be genotoxic. In particular, Acrylfentanyl led to a statistically significant increase in the MNi frequency at the highest concentration tested (75 μM), while Ocfentanyl and Furanylfentnyl each did so at both concentrations tested (150, 200 μM and 25, 50 μM, respectively). The study ended by investigating reactive oxygen species (ROS) induction as a possible mechanism linked to the proved genotoxic effect. The results showed a non-statistically significant increase in ROS levels in the cultures treated with all molecules under study. Overall, the proved genotoxicity raises concern about the possibility of serious long-term consequences.

Evaluation of the mutagenic effects of Molnupiravir and N4-hydroxycytidine in bacterial and mammalian cells by HiFi sequencing.

Molnupiravir (MOV) is used to treat COVID-19. In cells, MOV is converted to the ribonucleoside analog N4-hydroxycytidine (NHC) and incorporated into the SARS-CoV-2 RNA genome during its replication, resulting in RNA mutations. The widespread accumulation of such mutations inhibits SARS-CoV-2 propagation. Although safety assessments by many regulatory agencies across the world have concluded that the genotoxic risks associated with the clinical use of MOV are low, concerns remain that it could induce DNA mutations in patients, particularly because numerous in vitro studies have shown that NHC is a DNA mutagen. In this study, we used HiFi sequencing, a technique that can detect ultralow-frequency substitution mutations in whole genomes, to evaluate the mutagenic effects of MOV in E.coli and of MOV and NHC in mouse lymphoma L5178Y cells and human lymphoblastoid TK6 cells. In all models, exposure to these compounds increased genome-wide mutation frequencies in a dose-dependent manner, and these increases were mainly composed of A:T → G:C transitions. The NHC exposure concentrations used for mammalian cells were comparable to those observed in the plasma of humans who received clinical doses of MOV.

Genotoxicity evaluation of nitrosamine impurities using human TK6 cells transduced with cytochrome P450s.

Many nitrosamines are recognized as mutagens and potent rodent carcinogens. Over the past few years, nitrosamine impurities have been detected in various drugs leading to drug recalls. Although nitrosamines are included in a 'cohort of concern' because of their potential human health risks, most of this concern is based on rodent cancer and bacterial mutagenicity data, and there are little data on their genotoxicity in human-based systems. In this study, we employed human lymphoblastoid TK6 cells transduced with human cytochrome P450 (CYP) 2A6 to evaluate the genotoxicity of six nitrosamines that have been identified as impurities in drug products: N-nitrosodiethylamine (NDEA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutanoic acid (NMBA), N-nitrosomethylphenylamine (NMPA), N-nitrosodiisopropylamine (NDIPA), and N-nitrosodibutylamine (NDBA). Using flow cytometry-based assays, we found that 24-h treatment with NDEA, NEIPA, NMBA, and NMPA caused concentration-dependent increases in the phosphorylation of histone H2A.X (γH2A.X) in CYP2A6-expressing TK6 cells. Metabolism of these four nitrosamines by CYP2A6 also caused significant increases in micronucleus frequency as well as G2/M phase cell-cycle arrest. In addition, nuclear P53 activation was found in CYP2A6-expressing TK6 cells exposed to NDEA, NEIPA, and NMPA. Overall, the genotoxic potency of the six nitrosamine impurities in our test system was NMPA > NDEA ≈ NEIPA > NMBA > NDBA ≈ NDIPA. This study provides new information on the genotoxic potential of nitrosamines in human cells, complementing test results generated from traditional assays and partially addressing the issue of the relevance of nitrosamine genotoxicity for humans. The metabolically competent human cell system reported here may be a useful model for risk assessment of nitrosamine impurities found in drugs.

SIRT1 regulated hexokinase-2 promoting glycolysis is involved in hydroquinone-enhanced malignant progression in human lymphoblastoid TK6 cells

Reprogramming of cellular metabolism is a vital event during tumorigenesis. The role of glycolysis in malignant progression promoted by hydroquinone (HQ), one of the metabolic products of benzene, remains to be understood. Recently, we reported the overexpression of sirtuin 1 (SIRT1) in HQ-enhanced malignant progression of TK6 cells and hypothesized that SIRT1 might contribute to glycolysis and favor tumorigenesis. Our data showed that acute exposure of TK6 cells to HQ for 48 h inhibited glycolysis, as indicated by reduction in glucose consumption, lactate production, hexokinase activity, and the expression of SIRT1 and glycolytic enzymes, including HIF-1α, hexokinase-2 (HK-2), ENO-1, glucose transporter 1 (Glut-1), and lactic dehydrogenase A (LDHA). Knockdown of SIRT1 or inhibition of glycolysis using the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) downregulated the levels of SIRT1 and glycolytic enzymes and significantly enhanced HQ-induced cell apoptosis, although knockdown of SIRT1 or 2-DG alone had little effect on apoptosis. Furthermore, immunofluorescence and Co-IP assays demonstrated that SIRT1 regulated the expression of HK-2, and HQ treatment caused a decrease in SIRT1 and HK-2 binding to mitochondria. Importantly, we found that glycolysis was promoted with increasing HQ treatment weeks. Long-term HQ exposure increased the expression of SIRT1 and several glycolytic enzymes and promoted malignant cell progression. Moreover, compared with the PBS group, glucose consumption and lactate production increased after 10 weeks of HQ exposure, and the protein levels of SIRT1 and HK-2 were increased after 15 weeks of HQ exposure, while those of Glut-1, ENO-1, and LDHA were elevated. In addition, SIRT1 knockdown HQ 19 cells exhibited decreased lactate production, glucose consumption, glycolytic enzymes expression, cell growth, and tumor formation in nude mice. Our findings identify the high expression of SIRT1 as a strong oncogenic driver that positively regulates HK-2 and promotes glycolysis in HQ-accelerated malignant progression of TK6 cells.

Thymidine Kinase+/- Mammalian Cell Mutagenicity Assays for Assessment of Nanomaterials.

The methods outlined here are part of a series of papers designed specifically for genotoxicity assessment of nanomaterials (NM). Common Considerations such as NM characterization, sample preparation and dose selection, relevant to all genotoxicity assays, are found in an accompanying paper. The present paper describes methods for evaluation of mutagenicity in the mammalian (mouse) thymidine kinase (Tk) gene occurring in L5178Y mouse lymphoma (ML) cells and in the designated TK gene in human lymphoblastoid TK6 cells. Mutations change the functional genotype from TK+/- to TK-/-, detectable as cells surviving on media selective for the lack of thymidine kinase (TK) function. Unlike cells with TK enzyme function, the TK-/- cells are unable to integrate the toxic selection agent, allowing these cells to survive as rare mutant colonies. The ML assay has been shown to detect a broad spectrum of genetic damage, including both small scale (point) mutations and chromosomal alterations. This assay is a widely used mammalian cell gene mutation assay for regulatory purposes and is included in the core battery of genotoxicity tests for regulatory decision-making. The TK6 assay is an assay using a human cell line derived similarly via mutagenic manipulations and optimal selection. Details are provided on the materials required, cell culture methods, selection of test chemical concentrations, cytotoxicity, treatment time, mutation expression, cloning, and data calculation and interpretation. The methods describe the microwell plate version of the assays without metabolic activation.

Actein contributes to black cohosh extract-induced genotoxicity in human TK6 cells.

Black cohosh extract (BCE) is one of the most popular botanical products for relieving menopausal symptoms. However, recent studies indicate that BCE is not only ineffective for menopausal therapy but also induces genotoxicity through an aneugenic mode of action (MoA). In this study, the cytotoxicity of five constituents of BCE was evaluated in human lymphoblastoid TK6 cells. Among the five constituents, actein (up to 50 μM) showed the highest cytotoxicity and was thus selected for further genotoxicity evaluations. Actein caused DNA damage proportionally to concentration as evidenced by the phosphorylation of the histone protein H2A.X (γH2A.X) and resulted in chromosomal damage as measured by the increased percentage of micronuclei (%MN) in cells. In addition, actein activated DNA damage response (DDR) pathway through induction of p-ATM, p-Chk1, and p-Chk2, which subsequently induced cell cycle changes and apoptosis. Moreover, both BCE and actein increased intracellular reactive oxygen species (ROS) production, decreased glutathione levels, and activated the mitogen-activated protein kinases (MAPK) signaling pathway. N-acetylcysteine, a ROS scavenger, attenuated BCE- and actein-induced ROS production, apoptosis, and DNA damage. These findings indicate that BCE- and actein-induced genotoxicity is mediated, at least partially, through oxidative stress. Taken together, our data show that actein is likely one of the major contributors to BCE-induced genotoxicity.

In vitro human cell-based aneugen molecular mechanism assay.

This laboratory previously described an in vitro human cell-based assay and data analysis scheme that discriminates common molecular targets responsible for chemical-induced in vitro aneugenicity: tubulin destabilization, tubulin stabilization, and inhibition of Aurora kinases (Bernacki et al., Toxicol. Sci. 170 [2019] 382-393). The current report describes updated procedures that simplify benchtop processing and data analysis methods. For these experiments, human lymphoblastoid TK6 cells were exposed to each of 25 aneugens over a range of concentrations in the presence of fluorescent paclitaxel (488 Taxol). After a 4h treatment period, cells were lysed and nuclei were stained with a nucleic acid dye and labeled with fluorescent antibodies against phospho-histone H3 (p-H3). Flow cytometric analyses revealed several unique signatures: tubulin stabilizers caused increased frequencies of p-H3-positive events with concentration-dependent increases in 488 Taxol-associated fluorescence; tubulin destabilizers caused increased frequencies of p-H3-positive events with concomitant decreases in 488 Taxol-associated fluorescence; and Aurora kinase B inhibitors caused reduced frequencies of p-H3-positive events and lower median fluorescent intensities of p-H3-positive events. These results demonstrate a simple rubric based on 488 Taxol- and p-H3-associated metrics can reliably discriminate between several commonly encountered aneugenic molecular mechanisms.

Replication-dependent cytotoxicity and Spartan-mediated repair of trapped PARP1-DNA complexes.

The antitumor activity of poly(ADP-ribose) polymerase inhibitors (PARPis) has been ascribed to PARP trapping, which consists in tight DNA-protein complexes. Here we demonstrate that the cytotoxicity of talazoparib and olaparib results from DNA replication. To elucidate the repair of PARP1-DNA complexes associated with replication in human TK6 and chicken DT40 lymphoblastoid cells, we explored the role of Spartan (SPRTN), a metalloprotease associated with DNA replication, which removes proteins forming DPCs. We find that SPRTN-deficient cells are hypersensitive to talazoparib and olaparib, but not to veliparib, a weak PARP trapper. SPRTN-deficient cells exhibit delayed clearance of trapped PARP1 and increased replication fork stalling upon talazoparib and olaparib treatment. We also show that SPRTN interacts with PARP1 and forms nuclear foci that colocalize with the replicative cell division cycle 45 protein (CDC45) in response to talazoparib. Additionally, SPRTN is deubiquitinated and epistatic with translesion synthesis (TLS) in response to talazoparib. Our results demonstrate that SPRTN is recruited to trapped PARP1 in S-phase to assist in the excision and replication bypass of PARP1-DNA complexes.

Follow-up genotoxicity assessment of Ames-positive/equivocal chemicals using the improved thymidine kinase gene mutation assay in DNA repair-deficient human TK6 cells.

Genotoxicity testing plays an important role in the safety assessment of pharmaceuticals, pesticides and chemical substances. Among the guidelines for various genotoxicity tests, the in vitro genotoxicity test battery comprises the bacterial Ames test and mammalian cell assays. Several chemicals exhibit conflicting results for the bacterial Ames test and mammalian cell genotoxicity studies, which may stem from the differences in DNA repair capacity or metabolism, between different cell types or species. For better understanding the mechanistic implications regarding conflict outcomes between different assay systems, it is necessary to develop in vitro genotoxicity testing approaches with higher specificity towards DNA-damaging reagents. We have recently established an improved thymidine kinase (TK) gene mutation assay (TK assay) i.e. deficient in DNA excision repair system using human lymphoblastoid TK6 cells lacking XRCC1 and XPA (XRCC1-/-/XPA-/-), the core factors of base excision repair (BER) and nucleotide excision repair (NER), respectively. This DNA repair-deficient TK6 cell line is expected to specifically evaluate the genotoxic potential of chemical substances based on the DNA damage. We focussed on four reagents, N-(1-naphthyl)ethylenediamine dihydrochloride (NEDA), p-phenylenediamine (PPD), auramine and malachite green (MG) as the Ames test-positive chemicals. In our assay, assessment using XRCC1-/-/XPA-/- cells revealed no statistically significant increase in the mutant frequencies after treatment with NEDA, PPD and MG, suggesting the chemicals to be non-genotoxic in humans. The observations were consistent with that of the follow-up in vivo studies. In contrast, the mutant frequency was markedly increased in XRCC1-/-/XPA-/- cells after treatment with auramine. The results suggest that auramine is the genotoxic reagent that preferentially induces DNA damages resolved by BER and/or NER in mammals. Taken together, BER/NER-deficient cell-based genotoxicity testing will contribute to elucidate the mechanism of genotoxicity and therefore play a pivotal role in the accurate safety assessment of chemical substances.

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.