# Comparison of different techniques for γH2AX/pH3 biomarkers quantification for chemical genotoxicity assessment

Cynthia Recoules, Chloé Huertas, Julien Vignard, Marc Audebert

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Cynthia Recoules, Chloé Huertas, Julien Vignard, Marc Audebert. Comparison of different techniques for γH2AX/pH3 biomarkers quantification for chemical genotoxicity assessment. Mutation Research - Genetic Toxicology and Environmental Mutagenesis, 2025, 906, pp.503878. 10.1016/j.mrgentox.2025.503878. hal-05514863

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# Comparison of different techniques for γH2AX/pH3 biomarkers quantification for chemical genotoxicity assessment

Cynthia Recoules<fnref n="1" />, Chloé Huertas<fnref n="1" />, Julien Vignard*‡, Marc Audebert**‡

Toxalim, INRAE, INP-ENVT, INP-EI-Purpan, Université de Toulouse 3 Paul Sabatier, Toulouse, France

## ARTICLE INFO

### Keywords:

- Nuclear intensity
- Global intensity
- Foci
- Genotoxicity
- γH2AX
- PH3

## ABSTRACT

Chemical risk assessment relies on *in vitro* genotoxicity tests. Histone modifications (γH2AX and pH3) have emerged as valuable biomarkers for genotoxicity detection. In this study, we compared three parameters (global intensity, nuclear intensity, and foci number) for the γH2AX biomarker and two parameters (global intensity and % cell in mitosis) for the pH3 biomarker. These analyzes were performed in three cell lines: human osteosarcoma U2OS cells, human hepatocellular carcinoma HepG2 cells and rat intestinal epithelial IEC-6 cells. Cells were exposed for 24 h to four well-characterized hazardous substances: nocodazole (aneugen), etoposide (topoisomerase inhibitor), benzo[a]pyrene (DNA adducts inducer), and tunicamycin (apoptosis inducer). The Benchmark Concentration (BMC) approach indicated that the sensitivity of the technics varied depending on both the chemical compounds and the tested cell line. The γH2AX foci analysis provided the higher sensitivity for clastogenic compounds. For the aneugenic compound, the global intensity and the proportion of mitotic cells showed similar sensitivity. Following tunicamycin treatment, we only detected increase in γH2AX nuclear intensity in U2OS cell model, indicating that apoptosis does not interfere with γH2AX global intensity or foci number, thereby minimizing the risk of false positive results. Finally, we observed that compared to the other methods, global intensity permitted to monitor weaker fold inductions of the biomarkers. By comparing the different quantification methods of histone modifications used as genotoxicity biomarkers, this study highlights the most suitable parameters to be used.

## 1. Introduction

Humans are constantly exposed to hazardous environmental substances. In chemical risk assessment, genotoxicity testing is crucial as it represents a key characteristic of carcinogenic compounds [1]. Genotoxicity, defined as the ability of a substance to alter genetic integrity, is assessed through a battery of tests required from a regulatory standpoint [2]. According to OECD test guidelines, several cellular assays can be performed both *in vitro* and *in vivo*. Additionally, new approach methodologies (NAMs) for genotoxicity assessment have emerged [3].

Among OECD genotoxicity regulatory tests, the micronucleus assay enables the identification of clastogenic and aneugenic effects by detecting micronuclei, which result from lagging chromosome fragments or whole chromosomes, respectively [4]. Additionally, the chromosomal aberration assay identifies substances that induce structural chromosomal abnormalities in cultured mammalian cells [5]. Although

these techniques are standardized, they have some limitations, including challenges related to reproducibility and sensitivity. In the micronucleus assay, apoptosis may lead to false positive results [6,7]. The choice of cell lines also plays a crucial role in minimizing false positives results [8]. For the chromosomal aberration assay, responses can vary depending on the type of cells used (primary vs. non-primary cells), and reproducibility may be inconsistent, as it can fluctuate with concentration and experimental conditions [9].

In order to quantify DNA damage without a conventional approach, γH2AX signal can be evaluated by several approaches. H2AX histone is specifically phosphorylated at serine 139 following DNA damage, referred to as γH2AX, representing a well-established biomarker to identify clastogenic chemicals [10]. Several methods exist to detect and quantify γH2AX. Flow cytometry has been demonstrated to efficiently quantify γH2AX for genotoxicity assessment [11,12]. However, this technique is mostly dedicated to cells in suspension (e.g. TK6).

<footnote>* Correspondence to: INRAE-UMR1331, Toxalim, 180 chemin de Tournefeuille BP 93173, Toulouse Cedex 3 31027, France.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: julien.vignard@inrae.fr (J. Vignard), marc.audebert@inrae.fr (M. Audebert).</footnote>

<footnote n="1">¹ Equally contributing authors</footnote>
Additionally, liquid chromatography coupled with mass spectrometry provides absolute quantification of γH2AX [13]. Despite their precision, these two last methods have limitations, including high costs, low throughput due to a large number of cells needed, and the need for specialized expertise. Western blotting is another technique for measuring γH2AX levels, however it lacks the resolution to detect subtle differences in γH2AX induction [14]. Based on immunofluorescence technic, the γH2AX In-Cell Western (ICW) assay offers a highly sensitive and specific alternative for adherent cell models, allowing simultaneous evaluation of cytotoxicity and genotoxicity [15]. Performed in multi-well plates, ICW facilitates high-throughput genotoxicity screening across various compounds and cell lines [16]. Another approach is High-Content Screening (HCS), which employs automated high-content analysis (HCA) microscopy for rapid and precise data acquisition [17]. Similar to ICW, HCS allows for multi-compound screening in different cell lines within a single experiment. Using HCS, Garcia-Canton et al. (2013) demonstrated that the *in vitro* γH2AX assay is highly accurate, sensitive, and specific [18].

γH2AX quantification by its own is insufficient for a comprehensive genotoxicity assessment as it does not detect aneugenic events. Therefore, novel methodologies have been developed, notably the combined quantification of γH2AX with the phosphorylated histone H3 at serine 10 (pH3) biomarker [19]. This approach enables the detection of both clastogenic compounds (via γH2AX) and aneugenic compounds (via pH3). Histone H3 phosphorylation at serine 10 occurs during mitosis, making it a relevant marker for identifying aneugenic substances [20–22]. NAMs such as this method aim to modernize chemical toxicological testing by reducing reliance on animal models and overcoming limitations of conventional assays [23,24]. This study aims to compare different parameters used to measure γH2AX or pH3 biomarkers, either at the single-cell level by HCS or considering the entire cell population by ICW. For γH2AX biomarker, which is used to assess clastogenic compounds, three parameters were evaluated: global intensity by ICW, and nuclear intensity or the number of foci per cell by HCS. For pH3 biomarker, which is used to assess aneugenic compounds, two parameters were considered: global intensity by ICW and percentage of mitotic cells by HCS. The objective was to determine the most sensitive analysis method.

To achieve this goal, we used a small set of specific chemical: the clastogenic chemicals benzo[a]pyrene, which induces DNA adducts [25] and etoposide, a topoisomerase II inhibitor [26]. We tested nocodazole as model aneugenic chemical, which depolymerizes microtubules and induces mitotic arrest [27], and tunicamycin as pro-apoptotic compound inducing cytotoxicity but no genotoxicity [28–30].

Three well-characterized cellular models were selected, allowing to compare cancerous versus normal cells, as well as human versus rodent cell lines. HepG2 cells, derived from human hepatocellular carcinoma, are frequently used in hepatotoxicity studies due to their metabolic capacity [31,32]. U2OS cells, originating from human osteosarcoma, have been widely used in cancer research [33,34]. IEC-6 cells, derived from rat small intestinal epithelium, serve as a model for normal intestinal epithelial function and disease mechanisms [35–37]. The Benchmark Concentration (BMC) approach was used to compare the different parameters, employing mathematical modeling of concentration-response data as an alternative to the lowest observed genotoxic effect level [38–41].

# 2. Materials and methods

## 2.1. Chemicals and reagents

The chemicals benzo[a]pyrene (BaP; CAS 50–32–8), etoposide (CAS 33419–42–0), nocodazole (CAS 31430–18–9) and tunicamycin (CAS 11089–65–9) were purchased from Sigma-Aldrich (France) and dissolved in dimethyl sulfoxide (DMSO). Additional reagents, including penicillin-streptomycin, trypsin, PBS, RNase A and Triton X-100 were

obtained from Sigma-Aldrich. The blocking solution (Maxblock Blocking Medium) was purchased from Active Motif (Belgium).

## 2.2. Cell culture

In this study, we used three cell lines that were grown at 37°C in a humidified atmosphere containing 5 % CO2. HepG2 cells were cultured in MEM-alpha medium supplemented with 10 % fetal bovine serum (FBS, Gibco) and 1 % antibiotics (penicillin-streptomycin). IEC-6 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Life Technologies) supplemented with 10 % FBS, 1 % antibiotics and 0.1 U/mL insulin human solution (Sigma-Aldrich). U2OS cells were cultured in DMEM supplemented with 10 % FBS and 1 % antibiotics. Cells were seeded in Greiner 96-well transparent black-bottom plates compatible with fluorescence analysis, at a density of 32,000 cells per well for HepG2, 12,000 for IEC-6 and 10,000 for U2OS.

## 2.3. Cell treatments

Cells were treated with DMSO, ETO, BaP, nocodazole or tunicamycin at several concentrations separated by a one third dilution during 24 h. At least three independent experiments were performed.

## 2.4. Immunofluorescence

Cells were first fixed for 20 min in 4 % paraformaldehyde, then washed in PBS and permeabilized during 5 min in PST buffer (0.2 % Triton X-100 and 0.2 % FBS in PBS). Cells were then blocked for one hour at room temperature with MAXblock Blocking Medium supplemented with 0.1 % RNase and subsequently washed with PST. The primary antibody solution, containing antibodies directed against γH2AX (Merck/Millipore, 05–636–1) and pH3 (CellSignalling, D2C8), was incubated with cells for two hours. This was followed by three consecutive five-minute washes. Primary antibodies detection was performed for 1 h at room temperature using fluorescently labeled secondary antibodies: Alexa Fluor 647 Goat anti-mouse (Invitrogen, compatible with HCA and ICW systems), Alexa Fluor 546 anti-rabbit (Invitrogen, compatible with HCA system) and CF770 Goat anti-rabbit (Biotium, compatible with ICW system). This was followed by three consecutive five-minute incubations in washing buffer.

## 2.5. Sequential quantification of γH2AX/pH3 by HCA followed by ICW

After immunofluorescence, cells from the same 96-well plates were first analyzed by HCA and then by ICW, each method requiring a different DNA stain (Fig. 1A). Cells were first stained for 10 min with the DNA marker 4,6-diamino-2-phenyl indole (DAPI) at 100 nM, before to be imaged by HCA with the ImageXpress Micro Confocal High-Content Imaging System (MolecularDevices), with a 20x water immersion objective. DAPI-positive nuclei identification, γH2AX and pH3 signal analyzes were performed with the Metaxpress software. For γH2AX, the nuclear intensity and the number of nuclear foci were quantified. Background signal was subtracted using a no-primary-antibody control to ensure accurate measurement of γH2AX nuclear intensity. For pH3 biomarker, used to assess the proportion of cells in mitosis, a threshold value was determined manually. Cells displaying a pH3 signal above the threshold are considered in mitosis (% cell in mitosis). For each experiment, the level of each parameter (γH2AX nuclear intensity, γH2AX foci number and % cell in mitosis) was normalized to each control.

After HCA acquisition, cells were stained with SybrGold (Invitrogen) for 30 min to label DNA with a fluorophore compatible with the ICW system. DNA, γH2AX, and pH3 fluorescence signals were quantified simultaneously using the Sapphire Biomolecular Imager (Azure Biosystems, USA) and analyzed as previously reported [42]. Briefly, the background noise was deducted using the well without primary antibodies. Then, the measured signals in each condition were normalized to
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** γH2AX and pH3 immunostaining in response to clastogen or aneugen treatment. Panel A: Illustration of experimental approach with confocal microscope or Sapphire imager. Panel B: Representative images of γH2AX (green) and pH3 (red) immunostaining of cells exposed to DMSO vehicle, etoposide 1 µM or nocodazole 100 nM during 24 h vs control in U2OS, IEC-6 and HepG2 cells. Scale bar = 10 µm.</figcaption></figure>

the control.

Viability was assessed as previously reported [42] by comparing the relative DNA content of treated cells—correlated to cell count—to the DNA content of untreated control cells, expressed as % of relative cell count (% RCC).

## 2.6. Statistical analysis

The analyses from at least three independent experiments are presented as the mean ± standard error of the mean (SEM). Statistical significance of increase or decrease of γH2AX and pH3, compared to the DMSO vehicle or untreated controls, was determined using a two-sided Student's t-test (*P ≤ 0.05; **P ≤ 0.01). For γH2AX and pH3, a result was considered positive if the measured biomarker increased or decreased by more than 1.5-fold and cytotoxicity was less than 50 % relative to the control. If cytotoxicity exceeded 50 %, biomarker data were not displayed as in our previous studies [21,42,43].

## 2.7. BMC analysis

Positive results were analyzed using the Benchmark Concentration (BMC) covariate technique with the PROAST webtool (version 70.1-https://proastweb.rivm.nl/) following the instructions given by the European Food Safety Authority [44]. A Benchmark Response (BMR) value of 0.5 was used, representing a 50 % increase over the background signal, as suggested for in vitro genotoxicity data analysis [29,38,39].

# 3. Results

To evaluate genotoxicity, the γH2AX and pH3 biomarkers were quantified using two different methods (Fig. 1A). One approach involves acquiring and quantifying the immunofluorescence signal from the entire well (global intensity through the ICW technic), while with the HCA technic, we focused on measuring individual γH2AX nuclear intensity or foci number per nucleus, and determining the proportion of

pH3-positive mitotic cells. Well-characterized clastogenic or aneugenic compounds have been tested in three different cell lines: HepG2, U2OS and IEC-6 (Fig. 1B).

## 3.1. DNA damage after etoposide treatment

First, we tested etoposide, a genotoxic DNA topoisomerase II inhibitor, after 24 h of treatment on the three selected cell lines (Fig. 2 and Supplementary Figure 1). In HepG2 cells, cell viability, assessed by % relative cell count (% RCC) compared to control, decreased (52 %) at 10 µM but did not drop below the 50 % threshold (Fig. 2A). A statistically significant increase of γH2AX was observed from 33 nM using nuclear intensity and foci counting. Global intensity significantly increased from 100 nM. Fold induction reached 6.5 for global intensity, 7.7 for nuclear intensity, and 4.3 for γH2AX foci at the highest tested concentration (10 µM) (Fig. 2A). In U2OS cells, etoposide did not affect viability at the tested concentrations (Fig. 2B). A significant increase in γH2AX foci number was observed at 33 nM, whereas nuclear and global intensities significantly increased at 333 nM. Fold induction reached 7.3 for global intensity, 10.9 for nuclear intensity, and 33.3 for γH2AX foci at the highest tested concentration (10 µM) (Fig. 2B). In IEC-6 cells, cell viability decreased from 100 nM but remained above the 50 % threshold (Fig. 2C). A significant increase in γH2AX was observed at 100 nM for nuclear intensity and foci analysis, while global intensity only showed an increase from 333 nM. Fold induction reached 3.5 for global intensity, 6.9 for nuclear intensity, and 7.1 for γH2AX foci at the highest concentration.

To compare the three analysis methods used (γH2AX global intensity, nuclear intensity, and foci number) across cell lines, the BMC-covariate approach was applied (Fig. 2D). In IEC-6, the three analysis methods overlapped, indicating no significant differences. A slight difference was observed in HepG2 cells between global and nuclear intensity. Finally, in U2OS cells, γH2AX foci analysis resulted in a lower BMC compared to other applied methods.

The pH3 biomarker was also analyzed (Fig. 2E). In HepG2 cells, a
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** In vitro cytotoxicity and genotoxicity of etoposide tested in HepG2, U2OS and IEC-6 with γH2AX (global intensity, nuclear intensity and foci number) and pH3 (global intensity and % cell in mitosis). Panel A: γH2AX HepG2; Panel B: γH2AX U2OS. Panel C: γH2AX IEC-6. Panel D: γH2AX BMC analysis. Panel E: pH3 HepG2, U2OS, IEC-6. Viability is represented by the % RCC. Each value represents the mean ± SEM (n ≥ 3). Significant differences are noted (*p* < 0.05; **p* < 0.01; compared to control).</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** In vitro cytotoxicity and genotoxicity of benzo[a]pyrene tested in HepG2, U2OS and IEC-6 cells with γH2AX (global intensity, nuclear intensity and foci number) and pH3 (global intensity and % cell in mitosis). Panel A: γH2AX HepG2; Panel B: γH2AX U2OS. Panel C: γH2AX IEC-6. Panel D: γH2AX BMC analysis. Panel E: pH3 HepG2, U2OS, IEC-6. Viability is represented by the % RCC. Each value represents the mean ± SEM (n ≥ 3). Significant differences are noted (*p* < 0.05; **p* < 0.01; compared to control).</figcaption></figure>
significant decrease was observed at 100 nM for global intensity and 333 nM for % cell in mitosis. In U2OS cells, a significant decrease was observed at 333 nM for pH3 global intensity and % cell in mitosis. For IEC-6 cells, pH3 global intensity significantly decreased from 333 nM, while % cell in mitosis drops at 100 nM. These results indicate that whatever the cell line analyzed, the etoposide-induced increase of γH2AX is associated with a pH3 diminution.

## 3.2. DNA damage after BaP treatment

Next, cells have been exposed to BaP, a compound requiring bio-activation to be genotoxic (Figure 3 and Supplementary Figure 2). In HepG2 cells, viability was not affected except at the highest tested concentration (Fig. 3A). A significant increase of γH2AX foci number was detected from 100 nM, whereas nuclear increase intensity was detected from 333 nM and global intensity only from 1 µM. Fold induction reached 5.6 for global intensity, 5.4 for nuclear intensity, and 4.0 for γH2AX foci at the highest concentration tested (10 µM) (Fig. 3A). In U2OS cells, neither viability nor γH2AX biomarker showed significant changes (Fig. 3B). In IEC-6 cells, viability remained above 88 %. A significant increase in γH2AX was detected from 100 nM using γH2AX foci analysis, whereas global and nuclear intensities only increased from 333 nM (Fig. 3C). Fold induction reached 1.8 for global intensity, 2.0 for nuclear intensity, and 2.6 for foci number. Using the BMC-covariate approach for BaP treatment (Fig. 3D), the γH2AX foci analysis appears more sensitive in HepG2 cells, despite some overlap among methods. For IEC-6 cells, foci and nuclear intensity analyses resulted in a lower BMC compared to global intensity.

Concerning the pH3 biomarker, a significant decrease was observed in HepG2 at 3 µM for both parameters, whereas in IEC-6 cells a significant decrease was also observed at 3 µM but only for % cell in mitosis. For U2OS cells, no change was observed (Fig. 3E).

## 3.3. Proportion of mitotic cells after aneugen treatment: nocodazole

We next examined nocodazole, an aneogenic chemical that depolymerizes microtubules and induces cell cycle arrest in mitosis (Fig. 4). HepG2 cells viability was only weakly affected at the highest tested concentration (10 µM). From 33 nM of nocodazole, the pH3 signal detected by global intensity and the proportion of mitotic cells significantly increased (Fig. 4A). At the highest concentration, fold induction compared to control reached 4.0 for global intensity and 4.1 for % cell in mitosis. In U2OS cells, viability decreased from 33 nM but remained above the 50 % threshold (64 %). As for HepG2 cells, both analysis methods allowed detection of a significant increase from 33 nM of nocodazole (Fig. 4B). Fold induction compared to control reached 1.9 for global intensity, while mitotic cell proportion increased up to 13.8 at higher concentrations. In IEC-6 cells, viability dropped below the 50 % threshold from 333 nM. A significant decrease was observed at 33 nM for % cell in mitosis and at 100 nM for global intensity (Fig. 4C).

Using the BMC-covariate (Fig. 4D) approach for nocodazole treatment, global intensity and mitotic cell proportion overlapped for U2OS and HepG2 cells. Of note, a significant small increase of γH2AX nuclear intensity and foci number was detected at 10 µM only in U2OS cells (Fig. 4E).

## 3.4. Effect of a non-genotoxic treatment: tunicamycin

Finally, cells have been exposed to tunicamycin, a cytotoxic compound that induces apoptosis but no genotoxicity (Fig. 5 and Supplementary Figure 3). In HepG2 cells, neither viability nor γH2AX biomarkers showed significant changes, even at the highest tested concentration (10 µM) (Fig. 5A). In U2OS cells, viability decreased at high concentrations but remained above the 50 % threshold (69 %). No significant variations in γH2AX were observed using global intensity or foci analysis (Fig. 5B). However, nuclear intensity significantly increased at

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** In vitro cytotoxicity and genotoxicity of nocodazole tested in HepG2, U2OS and IEC-6 cells with pH3 (global intensity and % cell in mitosis) and γH2AX (global intensity, nuclear intensity and foci number). Panel A: pH3 HepG2; Panel B: pH3 U2OS. Panel C: pH3 IEC-6. Panel D: pH3 BMC analysis. Panel E: γH2AX HepG2, U2OS, IEC-6. Viability is represented by the % RCC. Each value represents the mean ± SEM (n ≥ 3). Significant differences are noted (*p* < 0.05; **p* < 0.01; compared to control).</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** In vitro cytotoxicity and genotoxicity of tunicamycin tested in HepG2, U2OS and IEC-6 cells with γH2AX (global intensity, nuclear intensity and foci number) and pH3 (global intensity and % cell in mitosis). Panel A: γH2AX HepG2; Panel B: γH2AX U2OS. Panel C: γH2AX IEC-6. Panel D: pH3 HepG2, U2OS, IEC-6. Viability is represented by the % RCC. Each value represents the mean ± SEM (n ≥ 3). Significant differences are noted (\*p < 0.05; \*\*p < 0.01; compared to control).</figcaption></figure>

10 µM (2.1-fold induction). In IEC-6 cells, viability decreased from 3 nM, falling below the threshold at 1 µM (Fig. 5C). Therefore, only the concentrations above this threshold (100 pM to 333 nM) were analyzed. γH2AX foci counting showed a significant decrease from 3 nM, whereas nuclear and global intensity analyses showed no significant changes.

When the pH3 biomarker was measured, a significant decrease of the global intensity was only observed at 100 nM in HepG2 cells (Fig. 5D). In U2OS cells, both % cell in mitosis and global intensity decreased from 333 nM. This tunicamycin-induced pH3 drop was more pronounced in IEC6 cells, beginning from 3 nM. Thus, in response to tunicamycin, loss of viability is associated with a decrease of mitotic cell population.

## 4. Discussion

The analysis of γH2AX and pH3 as genotoxicity biomarkers is attractive in chemical risk assessment, as it permits an accurate and reproductive detection of genetic damage resulting from exposure to toxic substances, and discriminates clastogen and aneugen compounds. In this regard, high-throughput assays offer a powerful alternative for rapid screening of numerous chemicals over a wide range of concentrations. The aim of this study was to compare different analyses methods, for γH2AX biomarker (global intensity, nuclear intensity and the number of γH2AX foci per cell), and for pH3 biomarker (global intensity and the proportion of mitotic cells).

In all cell lines, etoposide treatment induced the γH2AX biomarker. Regarding BMC analysis, γH2AX foci quantification was more sensitive in the U2OS cell line, whereas nuclear intensity measurement offered slightly higher sensitivity than global intensity in HepG2 cells. In contrast, in IEC-6 cells, all three analytical methods demonstrated comparable sensitivities. These differences could be explained by distinct baseline γH2AX levels between cell lines. Indeed, the mean baseline number of γH2AX foci per nucleus is lower in U2OS cells (2.6) compared to IEC-6 cells (6.7) and HepG2 cells (5.7). Moreover, the maximum number of detectable foci in U2OS cells is twice as high as in other cell lines (supplementary Figure 1), likely due to the larger size of the U2OS nucleus (Fig. 1). These two differences (lower mean baseline and maximum detectable number) may explain why the fold induction of γH2AX foci number was higher in U2OS cells, making γH2AX foci

quantification more effective in this cell line.

BaP must be metabolized to become genotoxic [45]. An increase in γH2AX levels was observed in HepG2 and IEC-6 cells, with foci quantification providing greater sensitivity than assessments based on nuclear and global intensity measurements. HepG2 cells are known to respond to BaP [46]. Polycyclic aromatic hydrocarbons, such as BaP, are biotransformed into genotoxic metabolites, notably through CYP1A1 and CYP1A2 [45,47]. This suggests that IEC-6 cells may have the ability to metabolize BaP through expression of these enzymes, similarly to other colon cell models [48,49]. In contrast, we found no significant γH2AX induction in U2OS cells. This suggests that U2OS cells may lack metabolic capacity.

In response to etoposide and BaP, a decrease in the pH3 biomarker was observed in all cell lines. Indeed, following DNA damage caused by clastogen compounds, cell cycle checkpoints are activated, resulting in cell cycle blockage before mitotic entry, which is reflected by a decrease in pH3 biomarker, as previously described [43,50].

For nocodazole treatment, both mitotic index and global intensity of pH3 signal yielded consistent BMC results in HepG2 and U2OS cell lines. However, the increase in fold induction was more pronounced when measured by mitotic cell percentage rather than global intensity, particularly in U2OS cells. This observation can be explained by baseline mitotic cell percentage. Indeed, U2OS cells exhibit a lower baseline mitotic index (2.1 %) compared to HepG2 (5.4 %) and IEC-6 cells (3.9 %), which may account for their stronger concentration-response induction. Aneugenic compounds arrest cells at mitosis. Once all cells are blocked in this phase, no further signal can be generated, resulting in a plateau in all quantification methods. In IEC-6 cells, a low decrease of mitosis cells was observed. This could be linked to cell death. Indeed, after an extended period of G2/M arrest, these cells may undergo cell death [51]. In light of this result, IEC-6 cells appear to have a faster cell cycle than HepG2 and U2OS cells and are highly sensitive to toxic chemicals [52], which may explain why at higher nocodazole concentrations, the proportion of viable cells drops dramatically. This cell line may therefore not be suitable for aneugenic chemical detection.

The pro-apoptotic compound tunicamycin induced, at the highest concentration in the U2OS cells, a significant increase in γH2AX nuclear intensity but not in γH2AX foci number or global intensity. This is
consistent with the fact that apoptotic processes can enhance γH2AX level [53], resulting in a specific pattern evolving from a ring- to a pan-staining of the nucleus, without foci formation [54]. This implies that to avoid any false positivity, γH2AX foci analysis should be performed rather than nuclear intensity in this cell line. Moreover, this increase was possibly related to cell line specific features, as previously described [29]. Additionally, a decrease in the number of mitotic cells (global intensity and mitotic cell percentage) in both IEC-6 and U2OS cell lines was observed. This is in agreement with the observation that tunicamycin induces ER stress, reducing the proportion of mitotic cells [30,55]. Thus, in response to tunicamycin, loss of viability is associated with a decrease of mitotic cell population, especially in the U2OS and IEC-6 cells.

Our findings indicate that sensitivity varies depending on both the analytical methodology employed and the cell line studied, as previously reported [21]. It is therefore essential to consider the limitations of each technique. In the global intensity approach, fluorescence was measured across the entire well, which contained at least 20,000 cells, depending on cell lines and treatment conditions. While this approach enables high-throughput screening and benefits from large sample size, it lacks resolution at the single-cell level. On the other hand, the nuclear intensity and the foci number approaches allowed for individual cell analysis, assessing approximately 1000 cells per experiment under our conditions. Despite considering fewer cells than global intensity, these methods exclude extranuclear background signals and provide greater analytical depth, allowing to measure additional parameters beyond intensity alone. However, it is important to note that nuclear intensity and foci number are time-consuming, both in terms of image acquisition and data processing compared to global intensity.

The γH2AX/pH3 method is faster than conventional assays such as micronucleus assay and chromosomal aberration. The quantification of γH2AX and pH3 biomarkers allows to detect clastogen and aneuge compounds in the same plate, resulting in significant time saving. Besides, in this study, γH2AX and pH3 signals were assessed through immunofluorescence. Of note, other non-fluorescent technics of detection have recently emerged for γH2AX, allowing to enhance sensitivity [56,57]. Finally, with the γH2AX foci analysis, no false positive chemical was detected, including the pro-apoptotic compound tunicamycin. In light of this, we have to note that micronucleus assay can misinterpret apoptotic bodies [6].

Through this study, we have demonstrated that all parameter provide comparable responses. Although HCS allows higher fold-change measurements, its sensitivity for detecting increases in the γH2AX biomarker can vary depending on the cell line and the selected parameter (nuclear intensity or γH2AX foci counting). The global intensity is well suited for large-scale screening, while other approaches provide more in-depth cellular analysis. These different parameters should therefore be considered in order to choose the most suitable approach when assessing γH2AX and pH3 biomarkers for genotoxicity screening.

### CRediT authorship contribution statement

**Julien Vignard:** Writing – review & editing, Project administration, Funding acquisition, Conceptualization. **Marc Audebert:** Writing – review & editing, Project administration, Funding acquisition, Conceptualization. **Cynthia Recoules:** Writing – review & editing, Writing – original draft, Formal analysis, Data curation, Conceptualization. **Huertas Chloe:** Writing – review & editing, Writing – original draft, Formal analysis, Data curation.

### Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

This project is funded by the European Partnership for the Assessment of Risks from Chemicals (PARC) under the Horizon Europe Research and Innovation Programme, Grant Agreement No.101057014, and from the French Agence Nationale de la Recherche (GenoMyc ANR-22-CE34).

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.mrgentox.2025.503878.

### Data availability

Data will be made available on request.

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