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.

Impact of indoor air pollution on DNA damage and chromosome stability: a systematic review.

Indoor air pollution is becoming a rising public health problem and is largely resulting from the burning of solid fuels and heating in households. Burning these fuels produces harmful compounds, such as particulate matter regarded as a major health risk, particularly affecting the onset and exacerbation of respiratory diseases. As exposure to polluted indoor air can cause DNA damage including DNA sd breaks as well as chromosomal damage, in this paper, we aim to provide an overview of the impact of indoor air pollution on DNA damage and genome stability by reviewing the scientific papers that have used the comet, micronucleus, and γ-H2AX assays. These methods are valuable tools in human biomonitoring and for studying the mechanisms of action of various pollutants, and are readily used for the assessment of primary DNA damage and genome instability induced by air pollutants by measuring different aspects of DNA and chromosomal damage. Based on our search, in selected studies (in vitro, animal models, and human biomonitoring), we found generally higher levels of DNA strand breaks and chromosomal damage due to indoor air pollutants compared to matched control or unexposed groups. In summary, our systematic review reveals the importance of the comet, micronucleus, and γ-H2AX assays as sensitive tools for the evaluation of DNA and genome damaging potential of different indoor air pollutants. Additionally, research in this particular direction is warranted since little is still known about the level of indoor air pollution in households or public buildings and its impact on genetic material. Future studies should focus on research investigating the possible impact of indoor air pollutants in complex mixtures on the genome and relate pollutants to possible health outcomes.

Is micronucleus assay a useful marker in gingiva, tongue, and palate for evaluating cytogenetic damage induced by chemical, physical, and biological agents in vivo? A systematic review with meta-analysis.

The present systematic review (SR) aims to evaluate manuscripts in order to help further elucidate the following question: is the micronucleus assay (MA) also a useful marker in gingiva, tongue, and palate for evaluating cytogenetic damage in vivo? A search was performed through the electronic databases PubMed/Medline, Scopus, and Web of Science, all studies published up to December 2023. The comparisons were defined as standardized mean difference (SMD), and 95% confidence intervals (CIs) were established. Full manuscripts from 34 studies were carefully selected and reviewed in this setting. Our results demonstrate that the MA may be a useful biomarker of gingival tissue damage in vivo, and this tissue could be a useful alternative to the buccal mucosa. The meta-analysis analyzing the different sites regardless of the deleterious factor studied, the buccal mucosa (SMD = 0.69, 95% CI, - 0.49 to 1.88, p = 0.25) and gingiva (SMD = 0.31, 95% CI, - 0.11 to 0.72, p = 0.15), showed similar results and different outcome for the tongue (SMD = 1.19, 95% CI, 0.47 to 1.91, p = 0.001). In summary, our conclusion suggests that the MA can be a useful marker for detecting DNA damage in gingiva in vivo and that this tissue could be effective site for smearing.

Assessment of imidacloprid induced genotoxicity in Pethia conchonius (Rosy barb), a common freshwater fish of India.

Imidacloprid is one of the highly efficient, globally used neonicotinoid groups of insecticides. The indiscriminate use of imidacloprid is contaminating large water bodies affecting not only the target organisms but also non-target organisms including fish. The present study aimed to assess the extent of nuclear DNA damage by imidacloprid in Pethia conchonius a freshwater fish in India using comet and micronucleus assays. The LC50 value of imidacloprid was estimated to be 227.33 mg L-1. Based on the LC50-96 h value, three sub-lethal concentrations of imidacloprid, SLC I -18.94 mg L-1, SLC II -28.41 mg L-1 and SLC III -56.83 mg L-1 were used to detect its genotoxic effect at DNA and cellular level. The imidacloprid exposed fishes exhibited higher DNA damage and nuclear abnormalities (p < 0.05) than the control. The %head DNA, %tail DNA, tail length and the frequency of micronuclei with other nuclear abnormalities like blebbed and notched nuclei were significantly higher than the control in a time and concentration-dependent manner. The DNA damage parameters such as %head DNA (29.107 ± 1.843), %tail DNA (70.893 ± 1.843), tail length (361.431 ± 8.455) micronucleus (1.300 ± 0.019), notched (0.844 ± 0.011) and blebbed (0.811 ± 0.011) nuclei were found to be highest for SLC III (56.83 mg L-1) at 96 h. The findings indicate that IMI is highly genotoxic in fish and other vertebrates leading to mutagenic/clastogenic effects. The study will be helpful in optimization of the imidacloprid use.

Enhanced antibacterial and genoprotective properties of nanoliposomal Satureja hortensis L. essential oil.

Nanoliposomes are drug delivery systems that improve bioavailability by encapsulating therapeutic agents. The main objective of this study was to investigate the effects of nanoliposomal (NL) formulation on enhancing the bioavailability of essential oil. The essential oil of Satureja hortensis (SHO) was encapsulated in nanoliposomes (SHNLs). Physicochemical characterizations of NL formulations (size, charge, polydispersity index [PDI]) were evaluated by dynamic light scattering technique. The nanoliposome encapsulation efficiency (EE) was calculated as 89.90%. The prepared bionanosystems demonstrated significant antibacterial activities against Escherichia coli ATCC 10536, Pseudomonas aeruginosa ATCC 15442, and Staphylococcus aureus ATCC as determined by the agar diffusion method and microdilution tests. Minimum inhibitory concentration (MIC) values for SHNLs were found to be 5.187 µg/µL for E. coli and 2.59 µg/µL for both P. aeruginosa and S. aureus. Importantly, despite the lower substance content, both SHNLs and SHO exhibited comparable antibacterial activity against all tested strains. Furthermore, in order to determine the toxicity profile and possible effects on DNA damage or repair both the genotoxic and antigenotoxic effects of SHNLs were assessed using the cytokinesis-blocked micronucleus (CBMN) method in human lymphocyte cultures. The experimental data collectively indicate that the NL formulation of the S. hortensis essential oil enhances antibacterial activities and provides genoprotective effects against DNA damage. This highlights the significance of liposomal formulations of antioxidants in augmenting their biological activity. The results indicate that SHNLs can be a safe antibacterial agent for the pharmaceutical industry.

In vitro evaluation of the selective cytotoxicity and genotoxicity of three synthetic ortho-nitrobenzyl derivatives in human cancer cell lines, with and without metabolic activation.

Although the presence of nitro groups in chemicals can be recognized as structural alerts for mutagenicity and carcinogenicity, nitroaromatic compounds have attracted considerable interest as a class of agents that can serve as source of potential new anticancer agents. In the present study, the in vitro cytotoxicity, genotoxicity, and mutagenicity of three synthetic ortho-nitrobenzyl derivatives (named ON-1, ON-2 and ON-3) were evaluated by employing human breast and ovarian cancer cell lines. A series of biological assays was carried out with and without metabolic activation. Complementarily, computational predictions of the pharmacokinetic properties and druglikeness of the compounds were performed in the Swiss ADME platform. The MTT assay showed that the compounds selectively affected selectively the cell viability of cancer cells in comparison with a nontumoral cell line. Additionally, the metabolic activation enhanced cytotoxicity, and the compounds affected cell survival, as demonstrated by the clonogenic assay. The comet assay, the cytokinesis-block micronucleus assay, and the immunofluorescence of the γ-H2AX foci formation assay have that the compounds caused chromosomal damage to the cancer cells, with and without metabolic activation. The results obtained in the present study showed that the compounds assessed were genotoxic and mutagenic, inducing double-strand breaks in the DNA structure. The high selectivity indices observed for the compounds ON-2 and ON-3, especially after metabolic activation with the S9 fraction, must be highlighted. These experimental biological results, as well as the theoretical properties predicted for the compounds have shown that they are promising anticancer candidates to be exploited in additional studies.

Is micronucleus assay in nasal mucosa cells an appropriate technique for detecting genotoxins by inhalation in humans? A systematic review.

This study aimed to evaluate the scientific literature on the micronucleus assay in nasal mucosa as an appropriate method for evaluating genotoxicity caused by chemical agents. According to the PRISMA guidelines, only in vivo human studies with micronucleus assays using nasal cells were considered. Reviews, case reports, editorials, letters to the editor, and articles not written in English were excluded. The following scientific databases/search engines were used: PubMed/MEDLINE, Scopus, and Web of Science. Results: This review included 13 studies. Four articles detected no statistical significance regarding the frequency of micronuclei while nine articles showed an increase in micronuclei in nasal cells. In the qualitative analysis, two articles were considered strong, eight were moderate and three were weak. The micronucleus assay using nasal mucosa cells is a sensitive and effective technique for assessing DNA damage and an appropriate method for monitoring humans continuously exposed to chemicals.

Application of the Cytokinesis-Block Micronucleus Assay for High-Dose Exposures Using Imaging Flow Cytometry.

The cytokinesis-block micronucleus assay is a well-established method to assess radiation-induced genetic damage in human cells. This assay has been adapted to imaging flow cytometry (IFC), allowing automated analysis of many cells, and eliminating the need to create microscope slides. Furthermore, to improve the efficiency of assay performance, a small-volume method previously developed was employed. Irradiated human blood samples were cultured, stained, and analyzed by IFC to produce images of the cells. Samples were run using both manual and 96-well plate automated acquisition. Multiple parameter-based image features were collected for each sample, and the results were compared to confirm that these acquisition methods are functionally identical. This paper details the multi-parametric analysis developed and the resulting calibration curves up to 10 Gy. The calibration curves were created using a quadratic random coefficient model with Poisson errors, as well as a logistic discriminant function. The curves were then validated with blinded, irradiated samples, using relative bias and relative mean square error. Overall, the accuracy of the dose estimates was adequate for triage dosimetry (within 1 Gy of the true dose) over 90% of the time for lower doses and about half the time for higher doses, with the lowest success rate between 5 and 6 Gy where the calibration curve reached its peak and there was the smallest change in MN/BNC with dose. This work describes the application of a novel multi-parametric analysis that fits the calibration curves and allows dose estimates up to 10 Gy, which were previously limited to 4 Gy. Furthermore, it demonstrates that the results from samples acquired manually and with the autosampler are functionally similar.

Genome instability in peripheral blood lymphocytes of patients with heart failure and reduced ejection fraction.

Heart failure (HF) is a complex clinical condition characterized by functional and structural defects of the myocardium, but genetic and environmental factors are considered to play an important role in the development of the disease. In the present study, we investigated the genome instability (DNA and chromosomal damage) in patients with heart failure with reduced ejection fraction (HFrEF) ≤40% and its association with risk factors. The studied population included 48 individuals, of which 29 HFrEF patients (mean age 57.41 ± 5.74 years) and 19 healthy controls (mean age 57.63 ± 6.09 years). The genetic damage index in peripheral blood lymphocytes was analyzed using the comet assay, while micronuclei frequency and nuclear division index were analyzed using the cytokinesis-block micronucleus assay. Our results showed that HFrEF patients had a significantly higher genetic damage index compared with the healthy controls (P < .001). Cytokinesis-block micronucleus assay showed that the average micronucleus frequency in peripheral blood lymphocytes of patients was significantly higher, while the nuclear division index values were significantly lower than in controls (P < .01). Using multiple linear regression analysis, pathological state, ejection fraction, creatinine, glucose, associated disease, residence, proBNP, troponin, urea, ACE-inhibitors, and length of the drug therapy were identified as predictors of DNA and/or chromosomal damage in HF patients. We can conclude that DNA and chromosomal damage was increased in patients with HF, which may be a consequence of disease and/or drug therapy.

High-throughput micronucleus assay using three-dimensional HepaRG spheroids for in vitro genotoxicity testing.

The in vitro micronucleus (MN) assay is a component of most test batteries used in assessing potential genotoxicity. Our previous study adapted metabolically competent HepaRG cells to the high-throughput (HT) flow-cytometry-based MN assay for genotoxicity assessment (Guo et al. in J Toxicol Environ Health A 83:702-717, 2020b, https://doi.org/10.1080/15287394.2020.1822972 ). We also demonstrated that, compared to HepaRG cells grown as two-dimensional (2D) cultures, 3D HepaRG spheroids have increased metabolic capacity and improved sensitivity in detecting DNA damage induced by genotoxicants using the comet assay (Seo et al. in ALTEX 39:583-604, 2022, https://doi.org/10.14573/altex.22011212022 ). In the present study, we have compared the performance of the HT flow-cytometry-based MN assay in HepaRG spheroids and 2D HepaRG cells by testing 34 compounds, including 19 genotoxicants or carcinogens and 15 compounds that show different genotoxic responses in vitro and in vivo. 2D HepaRG cells and spheroids were exposed to the test compounds for 24 h, followed by an additional 3- or 6-day incubation with human epidermal growth factor to stimulate cell division. The results demonstrated that HepaRG spheroids showed generally higher sensitivity in detecting several indirect-acting genotoxicants (require metabolic activation) compared to 2D cultures, with 7,12-dimethylbenzanthracene and N-nitrosodimethylamine inducing higher % MN formation along with having significantly lower benchmark dose values for MN induction in 3D spheroids. These data suggest that 3D HepaRG spheroids can be adapted to the HT flow-cytometry-based MN assay for genotoxicity testing. Our findings also indicate that integration of the MN and comet assays improved the sensitivity for detecting genotoxicants that require metabolic activation. These results suggest that HepaRG spheroids may contribute to New Approach Methodologies for genotoxicity assessment.

Evaluation of in vitro cytotoxic and genotoxic effects of the 3-(3,4-dihydroxyphenyl)-8-hydroxycoumarin.

A wide variety of natural and synthetic coumarins present therapeutic potential. Therefore, the assessment of their safety for humans is essential. 3-(3,4-Dihydroxyphenyl)-8-hydroxycoumarin is a coumarin derivative with antioxidant properties, among other biological activities. The aim of this study is to evaluate the cytotoxic and genotoxic potential of this molecule on peripheral blood mononuclear cells (PBMC) and human hepatocellular carcinoma cells (HepG2/C3A). The results obtained for the cytotoxicity assays, evaluated by the trypan blue staining assay, using concentrations between 0.1 and 20 µg/mL, showed that there is no decrease in cell viability for both cell lines. The MTT assay showed a significant decrease in the viability of HepG2/C3A cells at the highest concentrations tested, after 48 h, for all the tested concentrations, after 72 h of exposure. Regarding the genotoxic assays, the data obtained by the comet assay and the micronucleus test, up to the tested concentration of 10 µg/mL, do not show significant DNA damage and/or chromosomal mutations, for both cell lines. However, at the highest tested concentration of 20 µg/mL, a small but significant genotoxic effect was observed in PBMC. In view of the observed results, it can be concluded that the 3-(3,4-dihydroxyphenyl)-8-hydroxycoumarin, up to a concentration of 10 µg/mL, does not present genotoxic effects in human cells with and without liver enzymes metabolism. Additional studies with higher concentrations of this molecule need to be performed to address its complete biosafety.

Genotoxic effects of particulate matter on larvae of a common and widespread butterfly along an urbanization gradient.

Biodiversity is currently declining worldwide. Several threats have been identified such as habitat loss and climate change. It is unknown if and how air pollution can work in addition or in synergy to these threats, contributing to the decline of current species and/or local extinction. Few studies have investigated the effects of particulate matter (PM), the main component of air pollution, on insects, and no studies have investigated its genotoxic effects through Micronucleus assay. Butterflies play an important role in the environment, as herbivores during larval stages, and as pollinators as adults. The aim of this study was to evaluate the genotoxic effects of PM10 from different sites along a gradient of population urbanization, on a common cabbage butterfly species (Pieris brassicae). PM10 was collected from April to September in an urban (Turin, Italy), a suburban (Druento, Italy) and a mountain site (Ceresole Reale, Italy) with different urbanization levels. P. brassicae larvae (n = 218) were reared in the laboratory under controlled conditions (26 °C, L:D 15:9) on cabbage plants (average 9.2 days), and they were exposed to PM10 organic extracts (20 and 40 m3/mL) or dimethyl sulfoxide (controls) through vaporization. After exposure, larvae were dissected and cells were used for the Micronucleus (MN) assay. Results showed that all PM extracts induced significant DNA damage in exposed larvae compared to controls, and that increasing the PM dose (from 20 to 40 m3/mL) increased genotoxic effects. However, we did not detect any significant differences between sites with different urbanization levels. In conclusion, PM at different concentrations induced genotoxic effects on larvae of a common butterfly species. More alarmingly, PM could work in addition to and/or in synergy with other compounds (e.g. pesticides) and, especially on species already threatened by other factors (e.g. fragmentation), thus affecting the vitality of populations, leading to local extinctions.

Modulatory effect of myricitrin against chromosome instability and cytostasis induced by bleomycin and oxaliplatin in CHO-K1 cells.

Myricitrin (MYR), a flavonol consumed in the leaves and fruits of plants of the Myrtaceae family, presents anti-proliferative, anti-inflammatory, anti-diabetic, and antioxidant properties in humans. However, there are few studies regarding the cyto-genotoxicity and the chemopreventive potential of MYR. Using the in vitro Micronucleus test, the cytostasis, mutagenicity, and modulatory effect of MYR in CHO-K1 cells were assessed. The concentrations of 39 and 78 µg/mL (p < 0.001.) of MYR decrease the cytokinesis-block proliferation index (CBPI) in the short exposure treatment (4 h), while in the extended treatment (24 h), concentrations of 4.8, 9.7, 19.5, 39 and 78 µg/mL (p < 0.001.) decreased the CBPI. MYR associated with oxaliplatin decreased CBPI at all tested concentrations in the pre-(p < 0.001) and post-treatments (p < 0.001), but there was no decrease when associated with bleomycin. As for chromosome instability, MYR did not increase the frequency of micronuclei (MNi), nucleoplasmic bridges (NPBs), or nuclear buds (NBUDs) in the 4 h exposure time, however, in the 24 h treatment, MYR increased the frequency of MNi and NPBs at concentration 19.5 µg/mL (p < 0.001). As for the modulatory effect, MYR associated with bleomycin decreased the frequency of MNi, NPBs, and NBUDs at all concentrations in the pretreatment (MNi and NPBs p < 0.001, NBUDs p < 0.05) and simultaneously (MNi, NPBs and NBUDs p < 0.001). When associated with oxaliplatin, the simultaneous treatment decreased the frequency of MNi (p < 0.001) and NBUDs (p < 0.01) at all concentrations, however, in the post-treatment, MYR increased MNi (p < 0.001) and NPBs p < 0.05) in CHO-K1 cells, when compared to oxaliplatin alone. The results demonstrated that MYR could modulate the mutagenic and cytostatic actions of bleomycin and oxaliplatin, demonstrating distinct behaviors, depending on the mechanism of action of the chemotherapeutic agent.

Biocompatibility of bulk-fill resins in vitro.

OBJECTIVES: This study aims to evaluate the cytotoxicity and genotoxicity of three different extracts obtained from Filtek™ One Bulk Fill, Tetric Evoceram® Bulk Fill and Coltene Fill-Up! resins. MATERIALS AND METHODS: The cytotoxicity was determined on 3T3 fibroblast cells using the MTT and crystal violet assays. The genotoxicity was determined using a cytokinesis-block micronucleus assay. RESULTS: The cytotoxicity of the resin extracts on 3T3 mouse fibroblasts was found to be dose-dependent with both the MTT and crystal violet assays. Extracts concentrated above 1% were cytotoxic according to the MTT assay. The Filtek™ One Bulk Fill, Tetric Evoceram® Bulk Fill, and Coltene Fill-Up! resins reached the LD50 at concentrations of 60%, 50%, and 20%, respectively, and showed genotoxicity rates that were 2-5 times, 3-8 times, and 4-15 times higher than the negative control, respectively. CONCLUSIONS: Coltene Fill-Up! resin extracts were the most cytotoxic and genotoxic, followed by Tetric Evoceram® Bulk Fill and Filtek™ One Bulk Fill. CLINICAL RELEVANCE: The analyzed bulk-fill resins showed differences in in vitro biocompatibility, and the Filtek™ One Bulk Fill was found to be the safest for clinical use.

Atorvastatin Can Modulate DNA Damage Repair in Endothelial Cells Exposed to Mitomycin C.

HMG-CoA reductase inhibitors (statins) are widely used in the therapy of atherosclerosis and have a number of pleiotropic effects, including DNA repair regulation. We studied the cytogenetic damage and the expression of DNA repair genes (DDB1, ERCC4, and ERCC5) in human coronary artery (HCAEC) and internal thoracic artery endothelial cells (HITAEC) in vitro exposed to mitomycin C (MMC) (positive control), MMC and atorvastatin (MMC+Atv), MMC followed by atorvastatin treatment (MMC/Atv) and 0.9% NaCl (negative control). MMC/Atv treated HCAEC were characterized by significantly decreased micronuclei (MN) frequency compared to the MMC+Atv group and increased nucleoplasmic bridges (NPBs) frequency compared to both MMC+Atv treated cells and positive control; DDB1, ERCC4, and ERCC5 genes were upregulated in MMC+Atv and MMC/Atv treated HCAEC in comparison with the positive control. MMC+Atv treated HITAEC were characterized by reduced MN frequency compared to positive control and decreased NPBs frequency in comparison with both the positive control and MMC/Atv group. Nuclear buds (NBUDs) frequency was significantly lower in MMC/Atv treated cells than in the positive control. The DDB1 gene was downregulated in the MMC+Atv group compared to the positive control, and the ERCC5 gene was upregulated in MMC/Atv group compared to both the positive control and MMC+Atv group. We propose that atorvastatin can modulate the DNA damage repair response in primary human endothelial cells exposed to MMC in a cell line- and incubation scheme-dependent manner that can be extremely important for understanding the fundamental aspects of pleoitropic action of atorvastatin and can also be used to correct the therapy of patients with atherosclerosis characterized by a high genotoxic load.

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.

Transferability and reproducibility of the EpiSkin™ Micronucleus Assay.

A novel in vitro 3D micronucleus assay was developed in China using the EpiSkin™ 3D human skin model. This EpiSkin™ Micronucleus Assay showed good predictivity and reproducibility during internal validation and is expected to contribute to in vitro genotoxicity testing as a follow-up for positive results from 2D micronucleus assay. Having developed the assay in one laboratory, further work focused on the transferability and inter-laboratory reproducibility in two additional Chinese authority laboratories (Guangdong Provincial Center for Disease Control and Prevention and Zhejiang Institute for Food and Drug Control). Formal training was provided for both laboratories, which resulted in good transferability based on the results of two positive compounds, such as mitomycin C and vinblastine. Independent experiments were then performed, and inter-laboratory reproducibility was checked using 2-acetylaminofluorene, 5-fluorouracil, 2,4-dichlorophenol, and d-limonene. The dose-responses of the positive control chemical, mitomycin C, were similar to those of the developing laboratory, and all test chemicals were correctly classified by all laboratories. Overall, there was a good transferability as well as intra- and inter-laboratory reproducibility of the EpiSkin™ Micronucleus Assay. This study further confirmed the assay's robustness and provided confidence to enter following validation stages for scientific acceptance.

A novel in vitro 3D model of the human bone marrow to bridge the gap between in vitro and in vivo genotoxicity testing.

The regulatory 2D in vitro micronucleus (MN) assay is part of a battery of tests, used to test for genotoxicity of new and existing compounds before they are assessed in vivo (ICH S2). The 2D MN assay consists of a monolayer of cells, whereas the in vivo bone marrow (BM) setting comprises a multicellular environment within a three-dimensional extracellular matrix. Although the in vitro MN assay follows a robust protocol set out by the Organisation for Economic Co-operation and Development (OECD) to comply with regulatory bodies, some compounds have been identified as negative genotoxicants within the in vitro MN assay but marginally positive when assessed in vivo. The glucocorticoids, which are weakly positive in vivo, have generally been suggested to pose no long-term carcinogenic risk; however, for novel compounds of unknown activity, improved prediction of genotoxicity is imperative. To help address this observation, we describe a novel 3D in vitro assay which aims to replicate the results seen within the in vivo BM microenvironment. AlgiMatrix scaffolds were optimized for seeding with HS-5 human BM stromal cells as a BM microenvironment, to which the human lymphoblast cell line TK6 was added. An MN assay was performed aligning with the 2D regulatory assay protocol. Utilizing this novel 3D in vitro model of the BM, known genotoxicants (mitomycin C, etoposide, and paclitaxel), a negative control (caffeine), and in vivo positive glucocorticoids (dexamethasone and prednisolone) were investigated for the induction of MN. It was found, in agreement with historical in vivo data, that the model could accurately predict the in vivo outcome of the glucocorticoids, unlike the regulatory 2D in vitro MN assay. These preliminary results suggest our 3D MN assay may better predict the outcome of in vivo MN tests, compared with the standard 2D assay.

Impact of experimental design factors on the potency of genotoxicants in in vitro tests.

Previous studies have shown that differences in experimental design factors may alter the potency of genotoxic compounds in in vitro genotoxicity tests. Most of these studies used traditional statistical methods based on the lowest observed genotoxic effect levels, whereas more appropriate methods, such as the benchmark dose (BMD) approach, are now available to compare genotoxic potencies under different test conditions. We therefore investigated the influence of two parameters, i.e. cell type and exposure duration, on the potencies of two known genotoxicants [aflatoxin B1 and ethyl methanesulfonate (EMS)] in the in vitro micronucleus (MN) assay and comet assay (CA). Both compounds were tested in the two assays using two cell types (i.e. CHO-K1 and TK6 cells). To evaluate the effect of exposure duration, the genotoxicity of EMS was assessed after 3 and 24 h of exposure. Results were analyzed using the BMD covariate approach, also referred to as BMD potency ranking, and the outcome was compared with that of more traditional statistical methods based on lowest observed genotoxic effect levels. When comparing the in vitro MN results obtained in both cell lines with the BMD covariate approach, a difference in potency was detected only when EMS exposures were conducted for 24 h, with TK6 cells being more sensitive. No difference was observed in the potency of both EMS and aflatoxin B1 in the in vitro CA using both cell lines. In contrast, EMS was more potent after 24 h exposure compared with a 3 h exposure under all tested conditions, i.e. in the in vitro MN assay and CA in both cell lines. Importantly, for several of the investigated factors, the BMD covariate method could not be used to confirm the differences in potencies detected with the traditional statistical methods, thus highlighting the need to evaluate the impact of experimental design factors with adequate approaches.

Characterization of benzo[a]pyrene and colchicine based on an in vivo repeat-dosing multi-endpoint genotoxicity quantitative assessment platform.

Two prototypical genotoxicants, benzo[a]pyrene (B[a]P) and colchicine (COL), were selected as model compounds to deduce their quantitative genotoxic dose-response relationship at low doses in a multi-endpoint genotoxicity assessment platform. Male Sprague-Dawley rats were treated with B[a]P (2.5-80 mg/kg bw/day) and COL (0.125-2 mg/kg bw/day) daily for 28 days. The parameters included were as follows: comet assay in the peripheral blood and liver, Pig-a gene mutation assay in the peripheral blood, and micronucleus test in the peripheral blood and bone marrow. A significant increase was observed in Pig-a mutant frequency in peripheral blood for B[a]P (started at 40 mg/kg bw/day on Day 14, started at 20 mg/kg bw/day on Day 28), whereas no statistical difference for COL was observed. Micronucleus frequency in reticulocytes of the peripheral blood and bone marrow increased significantly for B[a]P (80 mg/kg bw/day on Day 4, started at 20 mg/kg bw/day on Days 14 and 28 in the blood; started at 20 mg/kg bw/day on Day 28 in the bone marrow) and COL (started at 2 mg/kg bw/day on Day 14, 1 mg/kg bw/day on Day 28 in the blood; started at 1 mg/kg bw/day on Day 28 in the bone marrow). No statistical variation was found in indexes of comet assay at all time points for B[a]P and COL in the peripheral blood and liver. The dose-response relationships of Pig-a and micronucleus test data were analyzed for possible point of departures using three quantitative approaches, i.e., the benchmark dose, breakpoint dose, and no observed genotoxic effect level. The practical thresholds of the genotoxicity of B[a]P and COL estimated in this study were 0.122 and 0.0431 mg/kg bw/day, respectively, and our results also provided distinct genotoxic mode of action of the two chemicals.