γH2AX and p53 responses in TK6 cells discriminate promutagens and nongenotoxicants in the presence of rat liver S9

Bernacki, Derek T.; Bryce, Steven M.; Bemis, Jeffrey C.; Kirkland, David; Dertinger, Stephen D.

doi:10.1002/em.22028

Cited by 28 publications

(20 citation statements)

References 45 publications

(34 reference statements)

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“…An in vitro multiplexed high-content screening assay (γH2AX and pH3) to distinguish genotoxicants from nongenotoxicants was used to study the genotoxicity potential of BEA and ENNB in vitro on HepaRG cells (Bernacki et al, 2016).…”

Section: Results Of the Multiparametric Genotoxicity Tests With Bea Amentioning

confidence: 99%

In vivo toxicity and genotoxicity of beauvericin and enniatins. Combined approach to study in vivo toxicity and genotoxicity of mycotoxins beauvericin (BEA) and enniatin B (ENNB)

Maranghi

Tassinari

Narciso

et al. 2018

EFS3

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Beauvericin (BEA) and Enniatins (ENN) are mycotoxins produced by Fusarium fungi detected in food and feed; there are insufficient data to establish their reference values. To evaluate BEA and ENN oral toxicity, an integrated approach was applied. Among ENN, Enniatin B (ENNB) was selected as test substance. The approach is composed by: i) in vitro and acute in vivo genotoxicity tests; ii) a repeated‐dose oral toxicity study focused on genotoxic, immune, endocrine, nervous endpoints and the reproductive/developmental toxicity screening. For BEA, all the genotoxicity endpoints yielded negative results excluding Comet assay in duodenum and kidney after repeated doses. BEA immunotoxicity was observed in female mice, concentrated in number and functional activity of effector T cells in the spleen. Based on the repeated‐dose BEA study, the No Observed Adverse Effect Level (NOAEL) for female mice is 1 mg/kg b.w. per day (increased thyroid pycnotic nuclei and endometrial hyperplasia). In males, the NOAEL is 0.1 mg/kg b.w. per day (reduced colloid and altered T4 serum levels). Maternal NOAEL is 0.1 mg/kg b.w. per day (increased thymus weight), developmental NOAEL is 10 mg/kg b.w. per day. For ENNB, the results support a genotoxic effect in bone marrow and liver cells after acute treatment, but not after repeated exposure. Immunotoxic ENNB effects were observed in both genders, suggestive of a suppressive/inhibiting activity particularly evident in males. Based on the repeated‐dose ENNB study, the NOAEL for females is 0.18 mg/kg b.w. per day (histomorphometrical effects on thymus, uterus and spleen). In male mice, the NOAEL is 1.8 mg/kg b.w. per day (enterocyte vacuolization in duodenum and increased Reactive Oxygen Species and reduced Glutathione brain levels). The maternal NOAEL is 1.8 mg/kg b.w. per day (decreased white pulp area and increased red/white pulp area ratio in spleen), developmental NOAEL is 18 mg/kg b.w. per day.

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Section: Results Of the Multiparametric Genotoxicity Tests With Bea Amentioning

confidence: 99%

In vivo toxicity and genotoxicity of beauvericin and enniatins. Combined approach to study in vivo toxicity and genotoxicity of mycotoxins beauvericin (BEA) and enniatin B (ENNB)

Maranghi

Tassinari

Narciso

et al. 2018

EFS3

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“…Representative bivariate graphs, gating logic, and position of regions were described in detail in earlier reports (Bernacki et al ; Bryce et al , ). Briefly, two biomarker measurements, γH2AX and p53, were based on the shift in median channel fluorescence intensity relative to same‐plate solvent controls.…”

Section: Methodsmentioning

confidence: 99%

“…Our laboratories have pursued the development and validation of a multiplexed flow cytometric assay that combines information from several biomarkers relevant to DNA damage response pathways and aneuploidy induction (Bryce et al , , , ; Bernacki et al ). This so‐called MultiFlow® DNA Damage Assay is formatted as an add‐and‐read test that efficiently prepares cells in microtiter plates for flow cytometric analysis.…”

Section: Introductionmentioning

confidence: 99%

Predictions of genotoxic potential, mode of action, molecular targets, and potency via a tiered multiflow® assay data analysis strategy

Dertinger

Kraynak

Wheeldon

et al. 2019

Environ and Mol Mutagen

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The in vitro MultiFlow® DNA Damage Assay multiplexes γH2AX, p53, phospho-histone H3, and polyploidization biomarkers into a single flow cytometric analysis. The current report describes a tiered sequential data analysis strategy based on data generated from exposure of human TK6 cells to a previously described 85 chemical training set and a new pharmaceutical-centric test set (n = 40). In each case, exposure was continuous over a range of closely spaced concentrations, and cell aliquots were removed for analysis following 4 and 24 hr of treatment. The first data analysis step focused on chemicals’ genotoxic potential, and for this purpose, we evaluated the performance of a machine learning (ML) ensemble, a rubric that considered fold increases in biomarkers against global evaluation factors (GEFs), and a hybrid strategy that considered ML and GEFs. This first tier further used ML output and/or GEFs to classify genotoxic activity as clastogenic and/or aneugenic. Test set results demonstrated the generalizability of the first tier, with particularly good performance from the ML ensemble: 35/40 (88%) concordance with a priori genotoxicity expectations and 21/24 (88%) agreement with expected mode of action (MoA). A second tier applied unsupervised hierarchical clustering to the biomarker response data, and these analyses were found to group certain chemicals, especially aneugens, according to their molecular targets. Finally, a third tier utilized benchmark dose analyses and MultiFlow biomarker responses to rank genotoxic potency. The relevance of these rankings is supported by the strong agreement found between benchmark dose values derived from MultiFlow biomarkers compared to those generated from parallel in vitro micronucleus analyses. Collectively, the results suggest that a tiered MultiFlow data analysis pipeline is capable of rapidly and effectively identifying genotoxic hazards while providing additional information that is useful for modern risk assessments—MoA, molecular targets, and potency.

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“…One line of investigation that has been pursued by our laboratories is the development of a multiplexed flow cytometric assay that combines several biomarkers relevant to DNA damage response pathways [Bernacki et al, ; Bryce et al, ]. This method involves a one‐step, add‐and‐read process that efficiently prepares samples in microtiter plate‐format for high throughput analysis via flow cytometry.…”

Section: Introductionmentioning

confidence: 99%

Interlaboratory evaluation of a multiplexed high information content in vitro genotoxicity assay

Bryce

Bernacki

Bemis

et al. 2017

Environ and Mol Mutagen

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We previously described a multiplexed in vitro genotoxicity assay based on flow cytometric analysis of detergent-liberated nuclei that are simultaneously stained with propidium iodide and labeled with fluorescent antibodies against p53, γH2AX, and phospho-histone H3. Inclusion of a known number of microspheres provides absolute nuclei counts. The work described herein was undertaken to evaluate the interlaboratory transferability of this assay, commercially known as MultiFlow™ DNA Damage Kit— p53, γH2AX, Phospho-histone H3. For these experiments seven laboratories studied reference chemicals from a group of 84 representing clastogens, aneugens, and non-genotoxicants. TK6 cells were exposed to chemicals in 96-well plates over a range of concentrations for 24 hrs. At 4 and 24 hrs cell aliquots were added to the MultiFlow reagent mix and following a brief incubation period flow cytometric analysis occurred, in most cases directly from a 96-well plate via a robotic walk-away data acquisition system. Multiplexed response data were evaluated using two analysis approaches, one based on global evaluation factors (i.e., cutoff values derived from all inter-laboratory data), and a second based on multinomial logistic regression that considers multiple biomarkers simultaneously. Both data analysis strategies were devised to categorize chemicals as predominately exhibiting a clastogenic, aneugenic, or non-genotoxic mode of action (MoA). Based on the aggregate 231 experiments that were performed, assay sensitivity, specificity, and concordance in relation to a priori MoA grouping were ≥ 92%. These results are encouraging as they suggest that two distinct data analysis strategies can rapidly and reliably predict new chemicals’ predominant genotoxic MoA based on data from an efficient and transferable multiplexed in vitro assay.

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γH2AX and p53 responses in TK6 cells discriminate promutagens and nongenotoxicants in the presence of rat liver S9

Cited by 28 publications

References 45 publications

In vivo toxicity and genotoxicity of beauvericin and enniatins. Combined approach to study in vivo toxicity and genotoxicity of mycotoxins beauvericin (BEA) and enniatin B (ENNB)

In vivo toxicity and genotoxicity of beauvericin and enniatins. Combined approach to study in vivo toxicity and genotoxicity of mycotoxins beauvericin (BEA) and enniatin B (ENNB)

Predictions of genotoxic potential, mode of action, molecular targets, and potency via a tiered multiflow® assay data analysis strategy

Interlaboratory evaluation of a multiplexed high information content in vitro genotoxicity assay

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