An integrated in vitro carcinogenicity test that distinguishes between genotoxic carcinogens, non-genotoxic carcinogens, and non-carcinogens.

Chemical safety testing plays a crucial role in product and pharmacological development, as well as chemoprevention; however, in vitro genotoxicity safety tests do not always accurately predict the chemicals that will be in vivo carcinogens. If chemicals test positive in vitro for genotoxicity but negative in vivo, this can contribute to unnecessary testing in animals used to confirm erroneous in vitro positive results. Current in vitro tests typically evaluate only genotoxicity endpoints, which limits their potential to detect non-genotoxic carcinogens. The frequency of misleading in vitro positive results can be high, leading to a requirement for more informative in vitro tests. It is now recognized that multiple-endpoint genotoxicity testing may aid more accurate detection of carcinogens and non-carcinogens. The objective of this review was to evaluate the utility of our novel, multiple-endpoint in vitro test, which uses multiple cancer-relevant endpoints to predict carcinogenic potential. The tool assessed micronucleus frequency, p53 expression, p21 expression, mitochondrial respiration, cell cycle abnormalities and, uniquely, cell morphology changes in human lymphoblastoid cell lines, TK6 and MCL-5. The endpoints were used to observe cellular responses to 18 chemicals within the following categories: genotoxic carcinogens, non-genotoxic carcinogens, toxic non-carcinogens, and misleading in vitro positive and negative agents. The number of endpoints significantly altered for each chemical was considered, alongside the holistic Integrated Signature of Carcinogenicity score, derived from the sum of fold changes for all endpoints. Following the calculation of an overall score from these measures, carcinogens exhibited greater potency than non-carcinogens. Genotoxic carcinogens were generally more potent than non-genotoxic carcinogens. This novel approach therefore demonstrated potential for correctly predicting whether chemicals with unknown mechanism may be considered carcinogens. Overall, while further validation is recommended, the test demonstrates potential for the identification of carcinogenic compounds. Adoption of the approach could enable reduced animal use in carcinogenicity testing.

Understanding the impact of more realistic low-dose, prolonged engineered nanomaterial exposure on genotoxicity using 3D models of the human liver.

BACKGROUND: With the continued integration of engineered nanomaterials (ENMs) into everyday applications, it is important to understand their potential for inducing adverse human health effects. However, standard in vitro hazard characterisation approaches suffer limitations for evaluating ENM and so it is imperative to determine these potential hazards under more physiologically relevant and realistic exposure scenarios in target organ systems, to minimise the necessity for in vivo testing. The aim of this study was to determine if acute (24 h) and prolonged (120 h) exposures to five ENMs (TiO2, ZnO, Ag, BaSO4 and CeO2) would have a significantly different toxicological outcome (cytotoxicity, (pro-)inflammatory and genotoxic response) upon 3D human HepG2 liver spheroids. In addition, this study evaluated whether a more realistic, prolonged fractionated and repeated ENM dosing regime induces a significantly different toxicity outcome in liver spheroids as compared to a single, bolus prolonged exposure. RESULTS: Whilst it was found that the five ENMs did not impede liver functionality (e.g. albumin and urea production), induce cytotoxicity or an IL-8 (pro-)inflammatory response, all were found to cause significant genotoxicity following acute exposure. Most statistically significant genotoxic responses were not dose-dependent, with the exception of TiO2. Interestingly, the DNA damage effects observed following acute exposures, were not mirrored in the prolonged exposures, where only 0.2-5.0 µg/mL of ZnO ENMs were found to elicit significant (p ≤ 0.05) genotoxicity. When fractionated, repeated exposure regimes were performed with the test ENMs, no significant (p ≥ 0.05) difference was observed when compared to the single, bolus exposure regime. There was < 5.0% cytotoxicity observed across all exposures, and the mean difference in IL-8 cytokine release and genotoxicity between exposure regimes was 3.425 pg/mL and 0.181%, respectively. CONCLUSION: In conclusion, whilst there was no difference between a single, bolus or fractionated, repeated ENM prolonged exposure regimes upon the toxicological output of 3D HepG2 liver spheroids, there was a difference between acute and prolonged exposures. This study highlights the importance of evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

Multiple-endpoint in vitro carcinogenicity test in human cell line TK6 distinguishes carcinogens from non-carcinogens and highlights mechanisms of action.

Current in vitro genotoxicity tests can produce misleading positive results, indicating an inability to effectively predict a compound's subsequent carcinogenic potential in vivo. Such oversensitivity can incur unnecessary in vivo tests to further investigate positive in vitro results, supporting the need to improve in vitro tests to better inform risk assessment. It is increasingly acknowledged that more informative in vitro tests using multiple endpoints may support the correct identification of carcinogenic potential. The present study, therefore, employed a holistic, multiple-endpoint approach using low doses of selected carcinogens and non-carcinogens (0.001-770 µM) to assess whether these chemicals caused perturbations in molecular and cellular endpoints relating to the Hallmarks of Cancer. Endpoints included micronucleus induction, alterations in gene expression, cell cycle dynamics, cell morphology and bioenergetics in the human lymphoblastoid cell line TK6. Carcinogens ochratoxin A and oestradiol produced greater Integrated Signature of Carcinogenicity scores for the combined endpoints than the "misleading" in vitro positive compounds, quercetin, 2,4-dichlorophenol and quinacrine dihydrochloride and toxic non-carcinogens, caffeine, cycloheximide and phenformin HCl. This study provides compelling evidence that carcinogens can successfully be distinguished from non-carcinogens using a holistic in vitro test system. Avoidance of misleading in vitro outcomes could lead to the reduction and replacement of animals in carcinogenicity testing.

The effect of chronic dosing and p53 status on the genotoxicity of pro-oxidant chemicals in vitro.

In this study, we have studied the cytotoxicity and genotoxic potency of 3 pro-oxidants; H2O2, menadione and KBrO3 in different dosing scenarios, namely acute (1-day dosing) and chronic (5-days). For this purpose, relative population doubling (RPD%) and mononucleated micronucleus (MN) test were used. TK6 cells and NH32 were employed in in vitro experiments. In the study, the total acute dose was divided into 5 days for each prooxidant chemicals by dose fractionation (1/5th per day) method. Acute dosing was compared to chronic dosing. The oxidative stress caused by the exposure of cells with pro-oxidant chemicals to the cells was determined by an optimized 2',7'-dichlorofluorescein diacetate (DCFHDA) test method. The antioxidant levels of the cell lines were altered with buthionine sulfoxide (BSO) and N-acetyl cysteine (NAC), and the effect of antioxidant capacity on the MN formation in the cells was observed with this method. In the case of H2O2 and menadione, fractional dosing has been observed to result in lower toxicity and lower genotoxicity. But in the case of KBrO3, unlike the other 2 pro-oxidants, higher MN induction was observed with fractionated doses. DCFHDA test clearly demonstrated ROS induction with H2O2 and menadione but not with KBrO3. Unexpectedly, DCFHDA test demonstrated that KBrO3 did not cause an increase ROS levels in both acute and chronic dosing, suggesting an alternative ROS induction mechanism. It was also observed that, treatment with BSO and NAC, caused increasing and decreasing of MN fold change respectively, allowing further ROS specific mechanisms to be explored. Hence, dose fractionation expectedly caused less MN, cytotoxicity and ROS formation with H2O2 and menadione exposure, but not with KBrO3. This implies a unique mechanism of action for KBrO3 induced genotoxicity. Chronic dosing in vitro may be a valuable approach allowing better understanding of how chemicals damage DNA and pose human hazards.

Adaptation of the in vitro micronucleus assay for genotoxicity testing using 3D liver models supporting longer-term exposure durations.

Following advancements in the field of genotoxicology, it has become widely accepted that 3D models are not only more physiologically relevant but also have the capacity to elucidate more complex biological processes that standard 2D monocultures are unable to. Whilst 3D liver models have been developed to evaluate the short-term genotoxicity of chemicals, the aim of this study was to develop a 3D model that could be used with the regulatory accepted in vitro micronucleus (MN) following low-dose, longer-term (5 days) exposure to engineered nanomaterials (ENMs). A comparison study was carried out between advanced models generated from two commonly used liver cell lines, namely HepaRG and HepG2, in spheroid format. While both spheroid systems displayed good liver functionality and viability over 14 days, the HepaRG spheroids lacked the capacity to actively proliferate and, therefore, were considered unsuitable for use with the MN assay. This study further demonstrated the efficacy of the in vitro 3D HepG2 model to be used for short-term (24 h) exposures to genotoxic chemicals, aflatoxin B1 (AFB1) and methyl-methanesulfonate (MMS). The 3D HepG2 liver spheroids were shown to be more sensitive to DNA damage induced by AFB1 and MMS when compared to the HepG2 2D monoculture. This 3D model was further developed to allow for longer-term (5 day) ENM exposure. Four days after seeding, HepG2 spheroids were exposed to Zinc Oxide ENM (0-2 µg/ml) for 5 days and assessed using both the cytokinesis-block MN (CBMN) version of the MN assay and the mononuclear MN assay. Following a 5-day exposure, differences in MN frequency were observed between the CBMN and mononuclear MN assay, demonstrating that DNA damage induced within the first few cell cycles is distributed across the mononucleated cell population. Together, this study demonstrates the necessity to adapt the MN assay accordingly, to allow for the accurate assessment of genotoxicity following longer-term, low-dose ENM exposure.

Detection of urethane-induced genotoxicity in vitro using metabolically competent human 2D and 3D spheroid culture models.

In vitro genotoxicity studies are a quick and high throughput approach to assess the genotoxic potential of chemicals; however, the reliability of these tests and their relevance to in vivo effects depends on the choice of representative cell line and optimisation of assay conditions. For chemicals like urethane that require specific metabolic activation to cause genotoxicity, it is important that in vitro tests are conducted using cell lines exhibiting the activity and induction of CYP450 enzymes, including CYP2E1 enzyme that is important in the metabolism of urethane, at a concentration representing actual or perceived chemical exposure. We compared 2D MCL-5 cells and HepG2 cells with 3D HepG2 hanging drop spheroids to determine the genotoxicity of urethane using the micronucleus assay. Our 2D studies with MCL-5 did not show any statistically significant genotoxicity [99% relative population doubling (RPD)] compared to controls for concentrations and time point tested in vitro. HepG2 cells grown as 2D indicated that exposure to urethane of up to 30 mM for 23 h did not cause any genotoxic effect (102% RPD) but, at higher concentrations, genotoxicity was produced with only 89-85% RPD. Furthermore, an exposure of 20-50 mM for 23 h using 3D hanging drop spheroid assays revealed a higher MN frequency, thus exhibiting in vitro genotoxicity of urethane in metabolically active cell models. In comparison with previous studies, this study indicated that urethane genotoxicity is dose, sensitivity of cell model (2D vs. 3D) and exposure dependent.

Investigating FlowSight® imaging flow cytometry as a platform to assess chemically induced micronuclei using human lymphoblastoid cells in vitro.

Use of imaging flow cytometry to assess induced DNA damage via the cytokinesis block micronucleus (CBMN) assay has thus far been limited to radiation dosimetry in human lymphocytes using high end, 'ImageStream X' series imaging cytometers. Its potential to enumerate chemically induced DNA damage using in vitro cell lines remains unexplored. In the present manuscript, we investigate the more affordable FlowSight® imaging cytometry platform to assess in vitro micronucleus (MN) induction in the human lymphoblastoid TK6 and metabolically competent MCL-5 cells treated with Methyl Methane Sulfonate (MMS) (0-5 µg/ml), Carbendazim (0-1.6 µg/ml), and Benzo[a]Pyrene (B[a]P) (0-6.3 µg/ml) for a period of 1.5-2 cell-cycles. Cells were fixed, and nuclei and MN were stained using the fluorescent nuclear dye DRAQ5™. Image acquisition was carried out using a 20X objective on a FlowSight® imaging cytometer (Amnis, part of Merck Millipore) equipped with a 488 nm laser. Populations of ∼20000 brightfield cell images, alongside DRAQ5™ stained nuclei/MN were rapidly collected (≤10 min). Single, in-focus cells suitable for scoring were then isolated using the IDEAS® software. An overlay of the brightfield cell outlines and the DRAQ5 nuclear fluorescence was used to facilitate scoring of mono-, bi-, tri-, and tetra-nucleated cells with or without MN events and in context of the cytoplasmic boundary of the parent cell.To establish the potential of the FlowSight® platform, and to establish 'ground truth' cell classification for the supervised machine learning based scoring algorithm that represents the next stage of our project, the captured images were scored manually. Alongside, MN frequencies were also derived using the 'gold standard' light microscopy and manual scoring. A minimum of 3000 bi-nucleated cells were assessed using both approaches. Using the benchmark dose approach, the comparability of genotoxic potency estimations for the different compounds and cell lines was assessed across the two scoring platforms as highly similar. This study therefore provides essential proof-of-concept that FlowSight® imaging cytometry is capable of reproducing the results of 'gold standard' manual scoring by light microscopy. We conclude that, with the right automated scoring algorithm, imaging flow cytometry could revolutionise the reportability and scoring throughput of the CBMN assay.

A novel, integrated in vitro carcinogenicity test to identify genotoxic and non-genotoxic carcinogens using human lymphoblastoid cells.

Human exposure to carcinogens occurs via a plethora of environmental sources, with 70-90% of cancers caused by extrinsic factors. Aberrant phenotypes induced by such carcinogenic agents may provide universal biomarkers for cancer causation. Both current in vitro genotoxicity tests and the animal-testing paradigm in human cancer risk assessment fail to accurately represent and predict whether a chemical causes human carcinogenesis. The study aimed to establish whether the integrated analysis of multiple cellular endpoints related to the Hallmarks of Cancer could advance in vitro carcinogenicity assessment. Human lymphoblastoid cells (TK6, MCL-5) were treated for either 4 or 23 h with 8 known in vivo carcinogens, with doses up to 50% Relative Population Doubling (maximum 66.6 mM). The adverse effects of carcinogens on wide-ranging aspects of cellular health were quantified using several approaches; these included chromosome damage, cell signalling, cell morphology, cell-cycle dynamics and bioenergetic perturbations. Cell morphology and gene expression alterations proved particularly sensitive for environmental carcinogen identification. Composite scores for the carcinogens' adverse effects revealed that this approach could identify both DNA-reactive and non-DNA reactive carcinogens in vitro. The richer datasets generated proved that the holistic evaluation of integrated phenotypic alterations is valuable for effective in vitro risk assessment, while also supporting animal test replacement. Crucially, the study offers valuable insights into the mechanisms of human carcinogenesis resulting from exposure to chemicals that humans are likely to encounter in their environment. Such an understanding of cancer induction via environmental agents is essential for cancer prevention.

Recommendations, evaluation and validation of a semi-automated, fluorescent-based scoring protocol for micronucleus testing in human cells.

Micronucleus (MN) induction is an established cytogenetic end point for evaluating structural and numerical chromosomal alterations in genotoxicity testing. A semi-automated scoring protocol for the assessment of MN preparations from human cell lines and a 3D skin cell model has been developed and validated. Following exposure to a range of test agents, slides were stained with 4'-6-diamidino-2-phenylindole (DAPI) and scanned by use of the MicroNuc module of metafer 4, after the development of a modified classifier for selecting MN in binucleate cells. A common difficulty observed with automated systems is an artefactual output of high false positives, in the case of the metafer system this is mainly due to the loss of cytoplasmic boundaries during slide preparation. Slide quality is paramount to obtain accurate results. We show here that to avoid elevated artefactual-positive MN outputs, diffuse cell density and low-intensity nuclear staining are critical. Comparisons between visual (Giemsa stained) and automated (DAPI stained) MN frequencies and dose-response curves were highly correlated (R (2) = 0.70 for hydrogen peroxide, R (2) = 0.98 for menadione, R (2) = 0.99 for mitomycin C, R (2) = 0.89 for potassium bromate and R (2) = 0.68 for quantum dots), indicating the system is adequate to produce biologically relevant and reliable results. Metafer offers many advantages over conventional scoring including increased output and statistical power, and reduced scoring subjectivity, labour and costs. Further, the metafer system is easily adaptable for use with a range of different cells, both suspension and adherent human cell lines. Awareness of the points raised here reduces the automatic positive errors flagged and drastically reduces slide scoring time, making metafer an ideal candidate for genotoxic biomonitoring and population studies and regulatory genotoxic testing.

Assessing the transferability and reproducibility of 3D in vitro liver models from primary human multi-cellular microtissues to cell-line based HepG2 spheroids.

To reduce, replace, and refine in vivo testing, there is increasing emphasis on the development of more physiologically relevant in vitro test systems to improve the reliability of non-animal-based methods for hazard assessment. When developing new approach methodologies, it is important to standardize the protocols and demonstrate the methods can be reproduced by multiple laboratories. The aim of this study was to assess the transferability and reproducibility of two advanced in vitro liver models, the Primary Human multicellular microtissue liver model (PHH) and the 3D HepG2 Spheroid Model, for nanomaterial (NM) and chemical hazard assessment purposes. The PHH model inter-laboratory trial showed strong consistency across the testing sites. All laboratories evaluated cytokine release and cytotoxicity following exposure to titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles. No significant difference was observed in cytotoxicity or IL-8 release for the test materials. The data were reproducible with all three laboratories with control readouts within a similar range. The PHH model ZnO induced the greatest cytotoxicity response at 50.0 µg/mL and a dose-dependent increase in IL-8 release. For the 3D HepG2 spheroid model, all test sites were able to construct the model and demonstrated good concordance in IL-8 cytokine release and genotoxicity data. This trial demonstrates the successful transfer of new approach methodologies across multiple laboratories, with good reproducibility for several hazard endpoints.

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (≤14 days). These spheroid models (≥500 µm in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the 'gold standard' micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

A three-dimensional in vitro HepG2 cells liver spheroid model for genotoxicity studies.

The liver's role in metabolism of chemicals makes it an appropriate tissue for toxicity testing. Current testing protocols, such as animal testing and two-dimensional liver cell systems, offer limited resemblance to in vivo liver cell behaviour, in terms of gene expression profiles and metabolic competence; thus, they do not always accurately predict human toxicology. In vitro three-dimensional liver cell models offer an attractive alternative. This study reports on the development of a 3D liver model, using HepG2 cells, by a hanging-drop technique, with a focus on evaluating spheroid growth characteristics and suitability for genotoxicity testing. The cytokinesis-blocked micronucleus assay protocol was adapted to enable micronucleus (MN) detection in the 3D spheroid models. This involved evaluating the difference between hanging vs non-hanging drop positions for dosing of the test agents and comparison of automated Metafer scoring with manual scoring for MN detection in HepG2 spheroids. The initial seeding density, used for all experiments, was 5000 cells/20 µl drop hanging spheroids, harvested on day 4, with >75% cell viability. Albumin secretion (7.8 g/l) and both CYP1A1 and CYP1A2 gene expression were highest in the 3D environment at day 4. Exposure to metabolically activated genotoxicants for 24 h resulted in a 6-fold increase in CYP1A1 enzyme activity (3 µM B[a]P) and a 30-fold increase in CYP1A2 enzyme activity (5 µM PhIP) in 3D hanging spheroids. MN inductions in response to B[a]P or PhIP were 2-fold and 3-fold, respectively, and were greater in 3D hanging spheroids than in 2D format, showing that hanging spheroids are more sensitive to genotoxic agents. HepG2 hanging-drop spheroids are an exciting new alternative system for genotoxicity studies, due to their improved structural and physiological properties, relative to 2D cultures.

Reprint of: A three-dimensional in vitro HepG2 cells liver spheroid model for genotoxicity studies.

The liver's role in metabolism of chemicals makes it an appropriate tissue for toxicity testing. Current testing protocols, such as animal testing and two-dimensional liver cell systems, offer limited resemblance to in vivo liver cell behaviour, in terms of gene expression profiles and metabolic competence; thus, they do not always accurately predict human toxicology. In vitro three-dimensional liver cell models offer an attractive alternative. This study reports on the development of a 3D liver model, using HepG2 cells, by a hanging-drop technique, with a focus on evaluating spheroid growth characteristics and suitability for genotoxicity testing. The cytokinesis-blocked micronucleus assay protocol was adapted to enable micronucleus (MN) detection in the 3D spheroid models. This involved evaluating the difference between hanging vs non-hanging drop positions for dosing of the test agents and comparison of automated Metafer scoring with manual scoring for MN detection in HepG2 spheroids. The initial seeding density, used for all experiments, was 5000 cells/20 µl drop hanging spheroids, harvested on day 4, with >75% cell viability. Albumin secretion (7.8 g/l) and both CYP1A1 and CYP1A2 gene expression were highest in the 3D environment at day 4. Exposure to metabolically activated genotoxicants for 24 h resulted in a 6-fold increase in CYP1A1 enzyme activity (3 µM B[a]P) and a 30-fold increase in CYP1A2 enzyme activity (5 µM PhIP) in 3D hanging spheroids. MN inductions in response to B[a]P or PhIP were 2-fold and 3-fold, respectively, and were greater in 3D hanging spheroids than in 2D format, showing that hanging spheroids are more sensitive to genotoxic agents. HepG2 hanging-drop spheroids are an exciting new alternative system for genotoxicity studies, due to their improved structural and physiological properties, relative to 2D cultures.

A comparison of the genotoxicity of benzo[a]pyrene in four cell lines with differing metabolic capacity.

Benzo[a]pyrene (B[a]P) is a known genotoxin and carcinogen, yet its genotoxic response at low level exposure has not been determined. This study was conducted to examine the interplay of dose and metabolic capacity on genotoxicity of B[a]P. Investigating and better understanding the biological significance of low level chemical exposures will help improve human health risk assessments. The genotoxic and mutagenic effects of B[a]P were investigated using human cell lines (AHH-1, MCL-5, TK6 and HepG2) with differential expression of the CYP450 enzymes CYP1A1, 1B1 and1A2 involved in B[a]P metabolism. MCL-5 and HepG2 cells showed detectable basal expression and activity of CYP1A1, 1B1 and 1A2 than AHH-1 which only show CYP1A1 basal expression and activity. TK6 cells showed negligible expression levels of all three CYP450 enzymes. In vitro micronucleus and HPRT assays were conducted to determine the effect of B[a]P on chromosome damage and point mutation induction. After 24h exposure, linear increases in micronucleus (MN) frequency were observed in all cell lines except TK6. After 4h exposure, only the metabolically competent cell lines MCL-5 and HepG2 showed MN induction (with a threshold concentration at 25.5µM from MCL-5 cells) indicating the importance of exposure time for genotoxicity. The HPRT assay also displayed linear increases in mutant frequency in MCL-5 cells, after 4h and 24h treatments. Mutation spectra analysis of MCL-5 and AHH-1 HPRT mutants revealed frequent B[a]P induced G to T transversion mutations (72% and 44% of induced mutations in MCL-5 and AHH-1 respectively). This study therefore demonstrates a key link between metabolic capability, B[a]P exposure time and genotoxicity.

Pro-oxidant induced DNA damage in human lymphoblastoid cells: homeostatic mechanisms of genotoxic tolerance.

Oxidative stress contributes to many disease etiologies including ageing, neurodegeneration, and cancer, partly through DNA damage induction (genotoxicity). Understanding the i nteractions of free radicals with DNA is fundamental to discern mutation risks. In genetic toxicology, regulatory authorities consider that most genotoxins exhibit a linear relationship between dose and mutagenic response. Yet, homeostatic mechanisms, including DNA repair, that allow cells to tolerate low levels of genotoxic exposure exist. Acceptance of thresholds for genotoxicity has widespread consequences in terms of understanding cancer risk and regulating human exposure to chemicals/drugs. Three pro-oxidant chemicals, hydrogen peroxide (H(2)O(2)), potassium bromate (KBrO(3)), and menadione, were examined for low dose-response curves in human lymphoblastoid cells. DNA repair and antioxidant capacity were assessed as possible threshold mechanisms. H(2)O(2) and KBrO(3), but not menadione, exhibited thresholded responses, containing a range of nongenotoxic low doses. Levels of the DNA glycosylase 8-oxoguanine glycosylase were unchanged in response to pro- oxidant stress. DNA repair-focused gene expression arrays reported changes in ATM and BRCA1, involved in double-strand break repair, in response to low-dose pro-oxidant exposure; however, these alterations were not substantiated at the protein level. Determination of oxidatively induced DNA damage in H(2)O(2)-treated AHH-1 cells reported accumulation of thymine glycol above the genotoxic threshold. Further, the H(2)O(2) dose-response curve was shifted by modulating the antioxidant glutathione. Hence, observed pro- oxidant thresholds were due to protective capacities of base excision repair enzymes and antioxidants against DNA damage, highlighting the importance of homeostatic mechanisms in "genotoxic tolerance."