Exploring cytokinesis block micronucleus assay in Croatia: A journey through the past, present, and future in biomonitoring of the general population.

In this study, we used the cytokinesis-block micronucleus (CBMN) assay to evaluate the background frequency of cytogenetic damage in peripheral blood lymphocytes of the general population concerning different anthropometric data and lifestyle factors. The background frequency of CBMN assay parameters was analysed in 850 healthy, occupationally non-exposed male and female subjects (average age, 38±11 years) gathered from the general Croatian population from 2000 to 2023. The mean background values for micronuclei (MNi) in the whole population were 5.3±4.3 per 1000 binucleated cells, while the mean frequency of nucleoplasmic bridges (NPBs) was 0.7±1.3 and of nuclear buds (NBUDs) 3.1±3.2. The cut-off value, which corresponds to the 95th percentile of the distribution of 850 individual values, was 14 MNi, 3 NPBs, and 9 NBUDs. Results from our database also showed an association of the tested genomic instability parameters with age and sex but also with other lifestyle factors. These findings underscore the importance of considering several anthropometric and lifestyle factors when conducting biomonitoring studies. Overall, the normal and cut-off values attained here present normal values for the general population that can later serve as baseline values for further human biomonitoring studies either in Croatia or worldwide.

Adapting the in vitro micronucleus assay (OECD Test Guideline No. 487) for testing of manufactured nanomaterials: recommendations for best practices.

The current Organisation for Economic Co-Operation and Development test guideline number 487 (OECD TG No. 487) provides instruction on how to conduct the in vitro micronucleus assay. This assay is one of the gold standard approaches for measuring the mutagenicity of test items; however, it is directed at testing low molecular weight molecules and may not be appropriate for particulate materials (e.g. engineered nanoparticles [ENPs]). This study aimed to adapt the in vitro micronucleus assay for ENP testing and underpins the development of an OECD guidance document. A harmonized, nano-specific protocol was generated and evaluated by two independent laboratories. Cell lines utilized were human lymphoblastoid (TK6) cells, human liver hepatocytes (HepG2) cells, Chinese hamster lung fibroblast (V79) cells, whole blood, and buffy coat cells from healthy human volunteers. These cells were exposed to reference ENPs from the Joint Research Council (JRC): SiO2 (RLS-0102), Au5nm and Au30nm (RLS-03, RLS-010), CeO2 (NM212), and BaSO4 (NM220). Tungsten carbide-cobalt (WC/Co) was used as a trial particulate positive control. The chemical controls were positive in all cell cultures, but WC/Co was only positive in TK6 and buffy coat cells. In TK6 cells, mutagenicity was observed for SiO2- and both Au types. In HepG2 cells, Au5nm and SiO2 showed sub-two-fold increases in micronuclei. In V79 cells, whole blood, and buffy coat cells, no genotoxicity was detected with the test materials. The data confirmed that ENPs could be tested with the harmonized protocol, additionally, concordant data were observed across the two laboratories with V79 cells. WC/Co may be a suitable particulate positive control in the in vitro micronucleus assay when using TK6 and buffy coat cells. Detailed recommendations are therefore provided to adapt OECD TG No. 487 for testing ENP.

The Micronucleus Assay on Cryopreserved Whole Blood.

The in vitro cytokinesis-block micronucleus (CBMN) assay is a widely used technique in radiobiology research, biological dosimetry, genotoxicity studies, and in vitro radiosensitivity testing. This cytogenetic method is based on the detection of micronuclei in binucleated cells resulting from chromosomal fragments lagging during cell division. Fresh whole blood samples are the most preferred sample type for the CBMN assay. However, the disadvantages of working with fresh blood samples include immediate processing after blood collection and the limited number of repeated analyses that can be performed without extra blood sampling. As the need for fresh blood samples can be logistically challenging, CBMN assay on cryopreserved whole blood samples would be of great advantage, especially in large-scale patient studies. This paper describes a protocol to freeze whole blood samples and to perform the CBMN assay on these frozen blood samples. Blood samples from healthy volunteers have been frozen and thawed at different time points and then, subjected to a modified micronucleus assay protocol. The results demonstrate that this optimized procedure allows the performance of the CBMN assay on frozen blood samples. The described cryopreservation protocol may also be very useful for other cytogenetic assays and a variety of functional assays requiring proliferating lymphocytes.

Investigations on the genotoxic potential of styrene in Fischer 344 rats using multiple endpoints.

Genotoxicity of styrene monomer was evaluated in male Fischer 344 rats using the alkaline comet assay for DNA damage, micronucleus assay for cytogenetic damage and the Pig-a assay for gene mutations. In a dose range finding (DRF) study, styrene was administered by oral gavage in corn oil for 28 consecutive days at 0, 100, 500, and 1000 mg/kg/day. The bioavailability of styrene was confirmed in the DRF by measuring its plasma levels at approximately 7- or 15-min following dosing. The 1000 mg/kg/day group exceeded the maximum tolerated dose based on body weight and organ weight changes and signs of central nervous system depression. Based on these findings, doses of 0, 100, 250, and 500 mg/kg/day (for 28 or 29 days) were selected for the genotoxicity assays. Animals were sacrificed 3-4 h after treatment on Day 28 or 29 for assessing various genotoxicity endpoints. Pig-a mutant frequencies and micronucleus frequencies were determined in peripheral blood erythrocytes. The comet assay was conducted in the glandular stomach, duodenum, liver, lung, and kidney. These studies were conducted in accordance with the relevant OECD test guidelines. Oral administration of styrene did not lead to genotoxicity in any of the investigated endpoints. The adequacy of the experimental conditions was assured by including animals treated by oral gavage with the positive control chemicals ethyl nitrosourea and ethyl methane sulfonate. Results from these studies supplement to the growing body of evidence suggesting the lack of in vivo genotoxic potential for styrene.

Comparative analysis of micronucleus induction and DNA damage biomarkers in TK6 and A375 cells using flow cytometry.

Previously, we introduced an alternative adherent A375 cell line for clastogenicity and aneugenicity testing using a high content imaging platform. To further characterize the performance of A375 cells, we investigated the sensitivity and specificity of A375 and TK6 cells by directly comparing micronucleus (MN) induction, cytotoxicity (relative cell counts, viability, and apoptosis), clastogenicity (γH2AX), and aneuploidy markers (pH 3, MPM-2, and polyploidy) using flow cytometric methods. We evaluated 14 compounds across different mechanisms (non-genotoxic apoptosis inducers, clastogens, and aneugens with either tubulin binding or aurora kinase inhibiting phenotypes) at 4-h and 24-h post treatment. Both aneugens and clastogens tested positive for micronucleus induction in both cell lines. Apoptosis continued to be a confounding factor for flow cytometry-based micronuclei assessment in TK6 cells as evidenced by positive responses by the three cytotoxicants. Conversely, A375 cells were not affected by apoptosis-related false positive signals and did not produce a positive response in the in vitro micronucleus assay. Benchmark dose response (BMD) analysis showed that the induction of micronuclei and biomarkers occurred at similar concentrations in both cell lines for clastogens and aneugens. By showing that A375 cells have similar sensitivity to TK6 cells but a greater specificity, these results provide additional support for A375 cells to be used as an alternative adherent cell line for in vitro genetic toxicology assessment.

Veterinarians exposed to inhaled anesthetic present chromosome damage, apoptosis and cell cycle changes.

This cross-sectional study evaluated, for the first time, DNA damage, viability, and cell death of lymphocytes and cell cycle phases of mononuclear and polymorphonuclear cells in veterinarians exposed to the volatile anesthetic isoflurane. Veterinarians who were occupationally exposed to isoflurane (exposed group; n = 20) and matched-unexposed individuals (volunteers without occupational exposure; n = 20) were enrolled in the study. DNA damage was assessed in lymphocytes by micronucleus (MN) and phosphorylated histone gamma-H2AX (γ-H2AX). Cell viability, cytotoxicity, and the cell cycle were evaluated by flow cytometry. Isoflurane was detected in urine samples by headspace gas chromatography-mass spectrometry. Compared with unexposed subjects, veterinarians occupationally exposed to isoflurane (25.7 ± 23.7 µg/L urine) presented statistically higher MN frequencies, lymphocytic apoptosis rates, and numbers of polymorphonuclear cells in the G0/G1 stage. Additionally, the exposed group presented statistically lower proportions of viable lymphocytes and G2/M polymorphonuclear cells. Our findings indicate that veterinarians who are frequently exposed to inhaled anesthetic exhibit chromosomal and cell damage in addition to changes in peripheral blood cell proliferation.

Cytogenotoxicity effects in addicts with multidrug consumption.

Drug abuse is considered a global health problem with serious social impact. In recent decades, changes in drug consumption patterns have shown a clear rising trend in the use of multiple drugs. Although the buccal micronucleus cytome (BMCyt) assay has evaluated cytotoxicity in drug abuse, there has not been an approach that takes into account this pattern of multiple drug use. Therefore, in this study, we evaluate for the first time the cytogenotoxic effects in multidrug users, and its correlation with the amount consumed and years of abuse. This study was conducted on 166 individuals by the BMCyt assay. A total of 83 individuals with a history of multiple licit (alcohol and tobacco) and at least one illicit drug abuse (marijuana, methamphetamines, cocaine, and/or inhalants), and 83 healthy individuals, non-drug abusers were analyzed. The results showed that drug abusers had higher frequencies of nuclear abnormalities nuclear buds, binucleated cells, pyknotic nuclei (PNs), karyorrhexis (KX), and abnormally condensed chromatin when compared with healthy controls. Moreover, results suggests that the use of licit and illicit drugs is related to cytogenotoxic damage, as was shown by an upward trend in the frequency of nuclear abnormalities identified in groups 1 (alcohol + tobacco + at least one illicit drug) and 2 (tobacco + at least one illicit drug). Furthermore, a positive correlation was found in the different groups, between the years and the amount of consumption of some drugs (alcohol, methamphetamine, and tobacco) with cytotoxicity markers such as KL, KX, and PNs.

Assessment of buccal mucosa genotoxicity in insecticide-exposed urban fumigators in Cali, Colombia.

OBJECTIVES: This study aimed to evaluate cytogenetic damage in the buccal mucosa of non-exposed subjects (N = 33) and insecticide-exposed fumigators (N = 31) in the urban area of Cali, Colombia. MATERIAL AND METHODS: Through a questionnaire sociodemographic data, anthropometric measurements, state of health, and lifestyle were collected. Buccal micronucleus cytome (BMCyt) assay was using for evaluate cytogenetic damage. RESULTS: The study showed that all fumigators used adequate personal protective equipment (PPE) and had low alcohol consumption. The authors did not find significant differences in BMCyt biomarkers between the groups (p > 0.05). Multivariate analysis showed a 13% increase in micronucleus (MN) frequency for every year of increasing age (OR = 1.13, p = 0.029), and higher MN with the decrease in daily fruit consumption (OR = 4.71, p = 0.084), without statistical significance. CONCLUSIONS: The results between groups could be related to healthy habits and PPE use among the subjects. Int J Occup Med Environ Health. 2024;37(1):128-37.

Evidence on the genotoxic and ecotoxic effects of PFOA, PFOS and their mixture on human lymphocytes and bacteria.

Considering that the PFOA and PFOS are widely spread chemicals with harmful effects in human and environmental health as well as the increasing interest of the scientific community in the implications that might present especially when they co-exist, this study aims to assess their harmful impacts, both individually and as a mixture on human lymphocytes and aquatic microorganisms. The cytokinesis-block micronucleus (CBMN) assay was used to examine their potential for cytotoxicity and genotoxicity towards human cells, and Microtox assay using Aliivibrio fischeri assay was used to estimate the environmental risk. Regarding the human lymphocytes, the tested concentrations ranged between 250 and 1000 µg L-1, for all cases. PFOA increased slightly the frequency of micronuclei (MN) but without statistical significance. In the case of PFOS, our results showed a dose-dependent increase in the frequency of micronuclei which showed a statistically significant difference (p < 0.001) at 1000 µg L-1, which is the highest studied concentration. Regarding the CBPI index, statistically significant (p < 0.05, p < 0.01, and p < 0.001 respectively) differences were observed at all studied concentrations of PFOS, compared to the control. The mixture was found to be more cytotoxic and genotoxic than the individual tested compounds, causing a higher decrease at the CBPI index even in lower concentrations and increase at the MN frequencies. Aliivibrio fischeri was exposed to various concentrations in the range of 0.5 µg L-1- 20 mg L-1, for 5 and 15 min and significant increase in the inhibition percentage at the highest tested concentration of their mixture after 15 min was observed.

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.

Study of the DNA damage and cell death in human peripheral blood mononuclear and HepG2/C3A cells exposed to the synthetic 3-(3-hydroxyphenyl)-7-hydroxycoumarin.

Hydroxycoumarins are an important source of biologically active compounds. Previous studies have shown that the number and position of the hydroxyl substituents in the scaffold play an important role for the observed biological activity. In the present study, 3-(3-hydroxyphenyl)-7-hydroxycoumarin was synthesized, and potential cytogenotoxic effects determined in human HepG2/C3A cells displaying phase 1 and phase 2 enzymes (metabolizing cell ability) and compared to human peripheral blood mononuclear cells (PBMC) without xenobiotics metabolizing capacity. Cell viability was determined with concentrations between 0.01 and 10 µg/ml of 3-(3-hydroxyphenyl)-7-hydroxycoumarin using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) and trypan blue tests. Genotoxicity was determined utilizing the comet assay, and the clastogenic/aneugenic potential employing the micronucleus (MN) test. The results of the in vitro cytotoxicity assays showed a significant decrease in cell viability of PBMC following exposure to 10 µg/ml concentration of the studied compound after 48 and 72 hr. Comet assay observations noted significant DNA damage in PBMC after 4 hr treatment. No marked cytogenotoxic effects were found in HepG2/C3A cells. No chromosomal mutations were observed in both cell lines. It is important to note that 3-(3-hydroxyphenyl)-7-hydroxycoumarin may exert beneficial pharmacological actions at the low micromolar range and with half-life less than 24 hr. Therefore, the results obtained encourage the continuation of studies on this new molecule for medicinal purposes, but its potential toxicity at higher concentrations and longer exposure times needs to be investigated in further studies.

Genotoxicity assessment of 1,4-anhydro-4-seleno-D-talitol (SeTal) in human liver HepG2 and HepaRG cells.

1,4-Anhydro-4-seleno-D-talitol (SeTal) is a highly water-soluble selenosugar with interesting antioxidant and skin-tissue-repair properties; it is highly stable in simulated gastric and gastrointestinal fluids and is a potential pharmaceutical ingredient that may be administered orally. Hepatic toxicity is often a major problem with novel drugs and can result in drug withdrawal from the market. Predicting hepatotoxicity is therefore essential to minimize late failure in the drug-discovery process. Herein, we report in vitro studies to evaluate the cytotoxic and genotoxic potential of SeTal in HepG2 and hepatocyte-like differentiated HepaRG cells. Except for extremely high concentrations (10 mM, 68 h-treatment in HepG2), SeTal did not affect the viability of each cell type. While the highest examined concentrations (0.75 and 1 mM in HepG2; 1 mM in HepaRG) were observed to induce primary DNA damage, SeTal did not exhibit clastogenic or aneugenic activity toward either HepG2 or HepaRG cells. Moreover, no significant cytostasis variations were observed in any experiment. The clearly negative results observed in the CBMN test suggest that SeTal might be used as a potential active pharmaceutical ingredient. The present study will be useful for the selection of non-toxic concentrations of SeTal in future investigations.

Effect of iron and calcium on radiation sensitivity in prostate cancer patients relative to controls.

High intake of red meat and/or dairy products may increase the concentration of iron and calcium in plasma-a risk factor for prostate cancer (PC). Despite our understandings of nutrients and their effects on the genome, studies on the effects of iron and calcium on radiation sensitivity of PC patients are lacking. Therefore, we tested the hypothesis that high plasma levels of iron and calcium could increase baseline or radiation-induced DNA damage in PC patients relative to healthy controls. The present study was performed on 106 PC patients and 132 age-matched healthy individuals. CBMN assay was performed to measure mi-cronuclei (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBuds) in lymphocytes. Plasma concentrations of iron and calcium were measured using inductively coupled plasma atomic emission spectroscopy. MN, NPBs, and NBuds induced by radiation ex vivo were significantly higher in PC patients with high plasma iron (P = .004, P = .047, and P = .0003, respectively) compared to healthy controls. Radiation-induced MN and NBuds frequency were also significantly higher in PC patients (P = .001 and P = .0001, respectively) with high plasma calcium levels relative to controls. Furthermore, radiation-induced frequency of NBuds was significantly higher in PC patients (P < .0001) with high plasma levels of both iron and calcium relative to controls. Our results support the hypothesis that high iron and calcium levels in plasma increases the sensitivity to radiation-induced DNA damage and point to the need of developing nutrition-based strategies to minimize DNA damage in normal tissue of PC patients undergoing radiotherapy.

Assessment of Micronuclei Frequency in the Peripheral Blood of Adult and Pediatric Patients Receiving Fractionated Total Body Irradiation.

The cytokinesis-block micronucleus (CBMN) assay is an established method for assessing chromosome damage in human peripheral blood lymphocytes resulting from exposure to genotoxic agents such as ionizing radiation. The objective of this study was to measure cytogenetic DNA damage and hematology parameters in vivo based on MN frequency in peripheral blood lymphocytes (PBLs) from adult and pediatric leukemia patients undergoing hematopoietic stem cell transplantation preceded by total body irradiation (TBI) as part of the conditioning regimen. CBMN assay cultures were prepared from fresh blood samples collected before and at 4 and 24 h after the start of TBI, corresponding to doses of 1.25 Gy and 3.75 Gy, respectively. For both age groups, there was a significant increase in MN yields with increasing dose (p < 0.05) and dose-dependent decrease in the nuclear division index (NDI; p < 0.0001). In the pre-radiotherapy samples, there was a significantly higher NDI measured in the pediatric cohort compared to the adult due to an increase in the percentage of tri- and quadri-nucleated cells scored. Complete blood counts with differential recorded before and after TBI at the 24-h time point showed a rapid increase in neutrophil (p = 0.0001) and decrease in lymphocyte (p = 0.0006) counts, resulting in a highly elevated neutrophil-to-lymphocyte ratio (NLR) of 14.45 ± 1.85 after 3.75 Gy TBI (pre-exposure = 4.62 ± 0.49), indicating a strong systemic inflammatory response. Correlation of the hematological cell subset counts with cytogenetic damage, indicated that only the lymphocyte subset survival fraction (after TBI compared with before TBI) showed a negative correlation with increasing MN frequency from 0 to 1.25 Gy (r = -0.931; p = 0.007). Further, the data presented here indicate that the combination of CBMN assay endpoints (MN frequency and NDI values) and hematology parameters could be used to assess cytogenetic damage and early hematopoietic injury in the peripheral blood of leukemia patients, 24 h after TBI exposure.

Blood molecular profile to predict genotoxicity from exposure to antineoplastic drugs.

Genotoxicity is an important information that should be included in human biomonitoring programmes. However, the usually applied cytogenetic assays are laborious and time-consuming, reason why it is critical to develop rapid and economic new methods. The aim of this study was to evaluate if the molecular profile of frozen whole blood, acquired by Fourier Transform Infrared (FTIR) spectroscopy, allows to assess genotoxicity in occupational exposure to antineoplastic drugs, as obtained by the cytokinesis-block micronucleus assay. For that purpose, 92 samples of peripheral blood were studied: 46 samples from hospital professionals occupationally exposed to antineoplastic drugs and 46 samples from workers in academia without exposure (controls). It was first evaluated the metabolome from frozen whole blood by methanol precipitation of macromolecules as haemoglobin, followed by centrifugation. The metabolome molecular profile resulted in 3 ratios of spectral bands, significantly different between the exposed and non-exposed group (p < 0.01) and a spectral principal component-linear discriminant analysis (PCA-LDA) model enabling to predict genotoxicity from exposure with 73 % accuracy. After optimization of the dilution degree and solution used, it was possible to obtain a higher number of significant ratios of spectral bands, i.e., 10 ratios significantly different (p < 0.001), highlighting the high sensitivity and specificity of the method. Indeed, the PCA-LDA model, based on the molecular profile of whole blood, enabled to predict genotoxicity from the exposure with an accuracy, sensitivity, and specificity of 92 %, 93 % and 91 %, respectively. All these parameters were achieved based on 1 µL of frozen whole blood, in a high-throughput mode, i.e., based on the simultaneous analysis of 92 samples, in a simple and economic mode. In summary, it can be conclude that this method presents a very promising potential for high-dimension screening of exposure to genotoxic substances.

DNA damage in foundry workers using non-invasive micronucleus cytome assay.

Workers in the foundry industry are exposed to hazardous chemical agents such as metal fumes, gases, vapor of molten metal, and respirable dust and hazardous physical agents such as heat, noise, and electromagnetic fields. Co-exposures to hazardous physical and chemical agents in foundry workplaces may cause DNA damage in workers. This study aimed to evaluate DNA damage in foundry workers. Thirty-three exposed foundry workers as a exposure groups and 33 non-exposed individuals as a control groups participated in this study. Buccal micronucleus cytome (BMCyt assay) assay was used to assess DNA damage. Results showed that foundry workers were under exposure to hazardous chemical and physical agents such as metal fumes and noise. The percentage of micronucleus (MN) cells in exposure group (0.59 ± 0.93 %) were statistically higher than control group (0.23 ± 0.23 %) (P < 0.05) %). Also, the percentage of nuclear bud cells and binucleated cells in exposure group were statistically higher than control group (P < 0.05). The percentage of differentiated normal cells were significantly higher in the control group compared to the exposed group (P < 0.05). Foundry workers are at risk of DNA damage; therefore, prevention measures need to be implemented to reduce exposure to air pollutants in foundry workplaces.

Micronuclei in human peripheral blood and bone marrow as genotoxicity markers: A systematic review and meta-analysis.

Can human peripheral blood cells be used as a surrogate for bone marrow cells, in evaluating the genotoxic effects of stressors? We searched the Pubmed/Medline and PubChem databases to identify publications relevant to this question. Micronucleus formation was the genotoxicity endpoint. Three publications comparing exposed vs. non-exposed individuals are included in this analysis; the exposures were to ethylene oxide or ionising radiation (atomic bomb, thorotrast, or radioiodine therapy). Information was extracted on the types of exposure, the numbers of participants, and the micronucleus frequencies. Relative differences (odds ratios) and absolute differences (risk differences) in the numbers of micronuclei between exposed and non-exposed persons were calculated separately for individual cell types (peripheral blood and bone marrow). Random effects meta-analyses for the relative differences in cell abnormalities were performed. The results showed very small differences in the frequencies of micronuclei between exposed and non-exposed individuals, as measured in either peripheral blood or bone marrow cell populations, on both absolute and relative scales. No definite conclusion concerning the relative sensitivities of bone marrow and peripheral blood cells can be made, based on these publications.

Genotoxicity assessment in HepaRG™ cells as a new approach methodology follow up to a positive response in the human TK6 cell micronucleus assay: Naphthalene case study.

We are evaluating the use of metabolically competent HepaRG™ cells combined with CometChip® for DNA damage and the micronucleus (MN) assay as a New Approach Methodology (NAM) alternative to animals for follow up genotoxicity assessment to in vitro positive genotoxic response. Naphthalene is genotoxic in human TK6 cells inducing a nonlinear dose-response for the induction of micronuclei in the presence of rat liver S9. of naphthalene. In HepaRG™ cells, naphthalene genotoxicity was assessed using either 6 (CometChip™) or 12 concentrations of naphthalene (MN assay) with the top dose used for assessment of genotoxicity for the Comet and MN assay was 1.25 and 1.74 mM respectively, corresponding to approximately 45% cell survival. In contrast to human TK6 cell with S9, naphthalene was not genotoxic in either the HepaRG™ MN assay or the Comet assay using CometChip®. The lack of genotoxicity in both the MN and comet assays in HepaRG™ cells is likely due to Phase II enzymes removing phenols preventing further bioactivation to quinones and efficient detoxication of naphthalene quinones or epoxides by glutathione conjugation. In contrast to CYP450 mediated metabolism, these Phase II enzymes are inactive in rat liver S9 due to lack of appropriate cofactors causing a positive genotoxic response. Rat liver S9-derived BMD10 over-predicts naphthalene genotoxicity when compared to the negative genotoxic response observed in HepaRG™ cells. Metabolically competent hepatocyte models like HepaRG™ cells should be considered as human-relevant NAMs for use genotoxicity assessments to reduce reliance on rodents.

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.

Hepatocyte proliferation activity in untreated rats, measured by immunohistochemical detection of Ki-67: The effect of age on the repeated-dose liver micronucleus assay.

The repeated-dose liver micronucleus (RDLMN) assay is a widely accepted method for detecting genotoxic substances. We investigated the effect of animal age on this assay. Proliferation activity in the liver tissue of untreated rats at age = 3.5, 6, 8, 10, or 12 weeks was measured via immunohistochemical expression of Ki-67 protein. The percentage of Ki-67-positive hepatocytes decreased markedly with age, reaching very low levels after 10 weeks, indicating decline with age of proliferative capacities in the liver. We calculated the area under the curve (AUC) of the approximate curve generated from the percentage of Ki-67-positive cells, to estimate the hepatocyte proliferation activity over the dosing period in the two regimens of the 4-week RDLMN assay: dosing initiated at age = 6 or 8 weeks. Hepatocyte proliferation activity of the former regimen was approximately double that of the latter. We also calculated the AUC for the juvenile-rat method, in which rats are treated for two days at age = 3.5 weeks. The AUC calculated for that method was approximately half of that for the 4-week repeated-dosing regimen initiated at 6 weeks of age. These findings suggest that the 4-week RDLMN assay with dosing initiated at age = 6 weeks could be approximately twice as sensitive as the other two methods.