Micronuclei Count Research Articles

Comparative analysis of chlorhexidine and listerine mouthwashes on buccal cell health: A clinical and cytological investigation

Mouthwashes are used routinely as adjunct to scaling and root planing. Chlorhexidine mouthwash is known as gold standard while Listerine is a widely used essential oil mouthwash. This study was done to evaluate and compare the genotoxic effect of Listerine with Chlorhexidine mouthwash on buccal cells.It was a parallel design study with 20 participants in each group (Group 1: Chlorhexidine mouthwash and Group 2: Listerine mouthwash). Gingival index was recorded and buccal cells scraping were taken at baseline and 3 weeks after use of respective mouthwashes. The buccal cells were assessed for cytotoxicity using micronuclei test (under microscope at 100 x magnification) using Papanicolaou Stain.There was a statistically significant reduction seen in gingival index from baseline to 3 weeks in both the groups (p< 0.001). There was a statistically significant increase in micronuclei count and number of Micronucleated cells in both the groups from baseline to 3 weeks (p< 0.001). The intergroup analysis showed highly significant increase in micronuclei and Micronucleated cells numbers in Chlorhexidine mouthwash group as compared to Listerine group (p< 0.001).After a 3 weeks exposure, Listerine mouthwash showed less cytotoxicity on buccal cells as compared to Chlorhexidine mouthwash.

Exploratory Study on Micronuclei and Metanuclear Abnormalities in Exfoliated Buccal Cells of COVID-19 Suspected Patients.

SARS-CoV-2 virus causes COVID-19 by infecting nasal and oral cavities primarily by attaching its spike proteins to ACE 2 receptors expressed in epithelial cells. This study was done to evaluate the micronucleated cell count, metanuclear abnormalities, and genotoxic factor in exfoliated buccal mucosal cell among the COVID-19 suspected patients. This cross-sectional study was conducted at AIIMS, Mangalagiri, between August and October 2022. One hundred COVID-19 suspected patients were recruited for this study after obtaining informed and written consent; buccal smear was obtained and stained for papanicolaou test (PAP). The PAP-stained slides were analyzed for micronuclei (MN), pyknotic, karyolytic, and karyorrhexic cell count, respectively. Based on their reverse transcription-polymerase chain reaction (RT-PCR) report, the patients were grouped into COVID-19 positive and negative groups. The genotoxicity factor was calculated using the micronucleated cell count from both the groups using mean and standard deviation. The MN, micronucleated cell, pyknotic, karyolitic, and karyorrhexic cell count in COVID-19 positive patients were 24.12, 15.24, 3.08, 2.88 and 4.40, respectively, than COVID-19 negative patients 5.69, 8.17, 1.08, 1.00 and 2.43, respectively. The genotoxicity factor for SARS-CoV-2 was 2.68 which is a positive genotoxic effect on buccal mucosal cells. SARS-CoV-2 increases the expression of micronucleated cells, pyknotic cells, karyolytic cells, and karyorhexic cells and concludes SARS-CoV-2 is having cytogenotoxic effect on the buccal mucosal cells. This can be used as a reliable marker in identifying the early carcinogenic effects of virus causing COVID-19.

Morphological markers of chromosomal instability as predictors of malignancy in pleural effusion.

Malignancy in pleural effusion is an indication of poor prognosis. The distinction between malignant cells and reactive mesothelial cells in effusion cytology is sometimes difficult and requires ancillary techniques. Evaluation of morphological indicators of chromosomal instability (CI) like micronuclei (MN), chromatin bridging (CB), nuclear budding (NB), and multipolar mitosis (MM) on routine cytology smears is a promising tool to distinguish malignant from benign ascitic fluids. However, it has been scarcely evaluated in pleural effusions. The present study was conducted to evaluate the diagnostic value of these markers in differentiating between malignant and benign pleural fluids. It is a cross sectional study in which a total of 72 pleural fluid samples over a period of 2 years received in the cytology department of the hospital were evaluated. The cytological analysis was done by two independent cytopathologists and interpreted as either malignant or benign. Four morphological markers of CI were counted in the May-Grünwald Giemsa (MGG) stained smears of all the cases and the score was compared with the conventional cyto-morphological diagnosis. Out of 72 cases, there were 42 malignant and 30 benign effusions on cytological examination. The mean score of micronuclei count, nuclear budding, chromatin bridging and multipolar mitosis in malignant effusions were 7.26 ± 2.74, 9.55 ± 5.53, 1.83 ± 1.17, and 2.21 ± 1.62 respectively that was significantly higher than the benign effusions (1 ± 0.71, 1.1 ± 0.86, 0.38 ± 0.50, and 0.15 ± 0.37 respectively) (p < .05). On Receiver operating characteristic (ROC) curve analysis, a cut-off of 5 for the MN count had a sensitivity of 88% and specificity of 100% in detecting malignant pleural effusion [Area under curve (AUC) 95.8%, p < .001]. Evaluation of morphological indicators of CI on routine MGG stained smears is a simple and cost-effective method to differentiate between benign and malignant pleural fluids.

DNA Damage Estimation after Chronic and Combined Exposure to Endocrine Disruptors: An In Vivo Real-Life Risk Simulation Approach.

Exposure to chemical substances has always been a matter of concern for the scientific community. During the last few years, researchers have been focusing on studying the effects resulting from combined exposure to different substances. In this study, we aimed to determine the DNA damage caused after chronic and combined exposure to substances characterized as endocrine disruptors using comet and micronuclei assays, specifically glyphosate (pure and commercial form), bisphenol A, parabens (methyl-, propyl- and butylparaben), triclosan and bis (2-ethylhexyl) phthalate. The highest mean tail intensity was observed in the group exposed to a high-dose (10 × ADI) mixture of substances (Group 3), with a mean value of 11.97 (11.26-13.90), while statistically significant differences were noticed between the groups exposed to low-dose (1 × ADI) (Group 2) and high-dose (10 × ADI) (Group 3) mixtures of substances (p = 0.003), and between Group 3 and both groups exposed to high doses (10 × ADI) of the pure and commercial forms of glyphosate (Groups 4 (p = 0.014) and 5 (p = 0.007)). The micronuclei assay results were moderately correlated with the exposure period. Group 5 was the most impacted exposure group at all sampling times, with mean MN counts ranging between 28.75 ± 1.71 and 60.75 ± 1.71, followed by Group 3 (18.25 ± 1.50-45.75 ± 1.71), showing that commercial forms of glyphosate additives as well as mixtures of endocrine disruptors can enhance MN formation. All exposure groups showed statistically significant differences in micronuclei counts with an increasing time trend.

CONNOTATION OF MICRONUCLEUS AS A BIOMARKER IN CERVICAL INTRAEPITHELIAL LESIONS AND CARCINOMA

Introduction: The Pap smears study is the most rampant and cost-effective method for cervical cancer screening. The role of micronuclei in exfoliated cells of pap smears is the greater beneficial tool used in the screening of cervical cancers. They are intracytoplasmic inclusions made up of chromatin fragments that suggest instability in the chromosomes and they are found in great numbers in carcinogen-exposed tissues long before any clinical signs occur. Aims: To evaluate the predominance of Micronuclei as a biomarker in various premalignant and malignant lesions of cervixand to explore the association between various grades of cervical lesions and micronuclei frequency. Materials &amp;Methods:A prospective study was done in the cytopathology section on 250 cervical pap smears collected in the Pathology laboratoryRajarajeswari medical college, Bangalore. The study period was from January 2021 to June 2021. Slides containing the cell material were stained with Papanicolaou stain, The results were analyzed as per the Micronuclei Scoring done per 1000 exfoliated squamous cells under light microscopy Results: In this study of 250 cervical pap smear screening, there was a stepwise gradual increase in micronuclei count(MN score)from Inflammatory lesions to carcinoma cervix. Inflammatory 2.04 ± 0.43, Atypicalsquamous cells of undetermined significance 3.9 ± 1.73, Atypical squamous cells - High grade5.64 ±1.83,Low grade squamous intraepithelial lesion 7.68 ±1.48, High grade squamous intraepithelial lesion08 12.32 ± 2.68and Intraepithelial Carcinoma 18.60 ± 2.83. Conclusion: MN scoring on cervix squamous epithelial cells is a simple and reliable objective test that may be performed on routine Pap-stained smears and can be used as adjunctive to the screening of cervical cancers and provide a potential pathway for disease monitoring in the future.

Evaluation of Micronuclei in Buccal Smears of Mobile Phone Users: A Comparative Study

Background: The mobile uses have been increased drastically, creating increased health concern about radiation effects emitted from mobile phone. Hence, this study aimed to evaluate the micronuclei (MN) frequency in exfoliated oral mucosal cells in high and low mobile users. Materials and Methods: The total of 50 subjects was divided into two major groups: low mobile phone users and high mobile phone users. Further, subjects who use mobile phone for more than 10 h a week were considered high talk time users and &lt;4–5 h a week was low talk time users. The buccal mucosa cells are extracted by scraping the oral cavity with a wooden spatula. MN was evaluated in 1000 cells per individual at the microscope. Results: There was a significantly increase in MN count in high mobile users compared to low mobile users. There was a highly significant difference in the mean MN count of participants using code division multiple access than global system for mobiles mobile phones. The MN mean count was found to be significantly increased in nonheadphone users in comparison to headphone users. Furthermore, the MN count on the frequently used side of mobile phone was found to be statistically significantly elevated compared to the opposite side. Conclusions: Mobile phone radiation, even in the permissible range when used for a longer duration, can cause significant genotoxicity. The genotoxicity is noted when the mobile phone is used more frequently on the same side, which may be due to more amount of radiation and increase in the temperature. Headphone usage reduces the genotoxicity of mobile phone radiation to some extent.

Detection of Mitogenic and Genotoxic Effects of the Turkey Tail Medicinal Mushroom (Trametes versicolor, Agaricomycetes) Extracts from Mexico on Human Lymphocyte Cultures

Diseases caused by a compromised immune system, characterized by decreased production and diversification of T lymphocytes, such as immunodeficiencies or chronic infections, are becoming increasingly prominent. These diseases lead to increased vulnerability to infections caused by parasites, viruses, bacteria, fungi, and other microorganisms. According to various articles, &lt;i&gt;Trametes versicolor&lt;/i&gt; has been used as immunotherapy and cancer treatment due to its polysaccharides, which have shown their value in traditional medicine. However, most of the studies have been done with Asian samples. For this reason, the aim of this study was to evaluate the effect of samples of Mexican &lt;i&gt;T. versicolor&lt;/i&gt; on human lymphoid cells. Of various extracts, the one with the best T cell proliferative response was the extract produced by maceration in water at room temperature, but all treatments in aqueous and ethanolic extracts increased the lymphocyte count, showing that extracts of Mexican &lt;i&gt;T. versicolor&lt;/i&gt; also have compounds that stimulate T cells. Unfortunately, genetic damage expressed as an increment in micronuclei count was identified, so using these fungus extracts in traditional medicine would require careful control of recommended doses.

Cytotoxic and genotoxic effects of cigarette and waterpipe tobacco smoking on buccal mucosa: A systematic review and meta-analysis.

Waterpipe tobacco smoking (WTS) is an issue all over the world, although it is particularly prevalent in the Middle East, and Southeast Asia. The genotoxic effects of smoking were reported to be associated with nucleus abnormalities such as micronuclei (MN), karyorrhexis (KR), karyolysis, pyknosis, binucleates, broken eggs, condensed chromatin in exfoliated buccal mucosal cells, and was believed to be associated with apoptosis of cells and was not correlated to the exposure time. The aim of this study was to evaluate and compare the cytotoxic and genotoxic effects of cigarette and WTS on buccal mucosa. The pertinent search was done through the computerized literature on MEDLINE, EMBASE, and PUBMED databases, which included case-control, clinical and observational studies regarding the mutagenic effects of cigarettes and WTS in oral tissues. The retraction of data in this study was undertaken from May 2010 to May 2022. A total of 60 articles from the search data were retrieved. This investigation was registered with the research center of Riyadh Elm University for institution review board approval (IRB) and obtained the IRB number "FRP/2021/448/733/707 and the systematic review registration number with respect to PROSPERO is 345417. After the removal of duplicates, 32 were evaluated for the inclusion and exclusion criteria. Out of 32 articles, twenty studies were evaluated for cytogenetic abnormalities in buccal mucosal cells of waterpipe tobacco smokers (WTS) and cigarette smokers, and 12 were excluded. The mean MN levels in the oral tissues of WTS were more (1.94 ± 0.39) than in non-smokers (1.68 ± 0.35). Therefore, we conclude that the MN count can be employed as a biomarker and preliminary signal for the identification of changes in oral mucosa among smokers, which develop towards cancer formation.

Correlation of the activities of antioxidant enzymes superoxide dismutase and glutathione reductase with micronuclei counts among areca nut chewers of Manipuri population using exfoliative cytology: A preliminary study.

Areca nut (AN) is a potent cytotoxic and genotoxic agent. Oxidative stress-induced by chewing of AN can cause DNA damage leading to nuclear anomalies such as micronuclei (MN) and also alters antioxidant defense mechanisms, leading to genomic instabilities and the development of oral cancer. The aim of this study is to study the correlation between the levels of glutathione reductase (GR) and superoxide dismutase (SOD) in exfoliated buccal mucosal cells and the genotoxicity levels (MN count) in chronic AN chewers. The present study was conducted with the approval of the Research Ethics Board in 60 individuals; 40 cases (Group I-20 raw AN chewers, Group II-20 dried areca with tobacco chewers), and 20 controls as Group III in the age group of 18-68 years who attended the outpatient department of our college. Estimation of SOD and GR and MN assessment was done using buccal exfoliated cells. Statistical analysis was performed using the Student's t-test and one-way ANOVA. Antioxidant levels were found to be significantly reduced in both Group I and Group II in comparison to the control group. Group II showed significantly reduced level of GR in comparison to Group I. The MN count was significantly increased in Group II in comparison to Group I. The MN counts showed an inverse correlation to the activities of the antioxidant enzymes. Greater activities of antioxidant enzymes correlated with decreased MN counts. Detection of MN in AN chewers with or without tobacco can be a useful biomarker for clinical screening procedures that may be used as a risk marker for oral cancer. It is important to increase the awareness programs to educate the public about the deleterious effects of AN chewing, emphasize on early intervention of AN chewing habit and thus prevent the development of oral cancer.

Reply to Takeshita, W.M.; Ribeiro, D.A. Comment on "Pop et al. Early Diagnosis of Oral Mucosal Alterations in Smokers and E-Cigarette Users Based on Micronuclei Count: A Cross-Sectional Study among Dental Students. Int. J. Environ. Res. Public Health 2021, 18, 13246".

Thank you for your interest [...].

Comment on Pop et al. Early Diagnosis of Oral Mucosal Alterations in Smokers and E-Cigarette Users Based on Micronuclei Count: A Cross-Sectional Study among Dental Students. Int. J. Environ. Res. Public Health 2021, 18, 13246.

We read the manuscript recently published in International Journal of Environmental Research and Public Health titled "Early diagnosis of oral mucosal alterations in smokers and e-cigarette users based on micronuclei count: a cross-sectional study among dental students" by Pop et al. [...].

Comparison of Repair Index in Cigarette and Waterpipe Smokers: A Bio-Monitoring Assessment Using Human Exfoliated Buccal Mucosa Cells.

Background:Repair index (RI) using DNA changes reveals the activity of carcinogenesis. Cigarette and waterpipe smoking are important contributors to oral cavity malignancy. The RI in cigarette and waterpipe smokers has not been compared so far. The aim of this study was to compare the RI in cigarette and waterpipe smokers using the human exfoliated buccal mucosa cells.Methods:The exfoliated buccal mucosa cells of 60 cigarette and waterpipe smokers and 20 nonsmokers were evaluated in a case-control study. The number of micronuclei (MN), broken egg (BE), karyorrhexis (KR), and karyolysis (KL) were counted in 1000 cells from randomly selected fields. The RI = (KL + KR)/(MN + BE) was calculated and compared between subjects and controls. Data were analyzed by one-way analysis of variance (ANOVA), Tukey's Honest Significant Difference (HSD), and Spearman's correlation coefficient test at P < 0.05 probability level.Results:The difference of MN (P < 0.0001), BE (P < 0.0001), KR (P < 0.0001), and KL (P < 0.0001) count was significant between cigarette smokers, waterpipe smokers, and nonsmokers. The RI was significantly different between groups (P = 0.007). The RI was significantly higher in cigarette smokers compared to waterpipe smokers (P = 0.04) and nonsmokers (P = 0.009).Conclusions:The RI was significantly higher in cigarette smokers compared to waterpipe smokers. The finding suggests that due to higher interrupted cellular hemostasis, the risk of carcinoma in waterpipe smokers can be greater than that in cigarette smokers.

Early Diagnosis of Oral Mucosal Alterations in Smokers and E-Cigarette Users Based on Micronuclei Count: A Cross-Sectional Study among Dental Students.

The presence of micronuclei in oral epithelial cells is considered a marker of genotoxicity, which can be identified using exfoliative cytology. The aim of this study was to investigate cytotoxic damage through the evaluation of micronuclei in the oral mucosa of smokers and e-cigarette users compared to nonsmokers. We obtained smears from the buccal mucosa of 68 participants divided in 3 groups (smokers, e-cigarette users and nonsmokers), which were further processed with Papanicolaou stain. The frequencies of micronuclei and micronucleated cells were recorded and statistically analyzed at a level of significance of p < 0.05. The mean micronuclei values per 1000 cells were 3.6 ± 1.08 for smokers, 3.21 ± 1.12 for e-cigarette users and 1.95 ± 1.05 for nonsmokers. The mean values of micronucleated cells per 1000 cells were 2.48 ± 0.91 for smokers, 2.39 ± 1.07 for e-cigarette users and 1.4 ± 0.68 for nonsmokers. Smokers and e-cigarette users had significantly higher values of micronuclei and micronucleated cells compared to nonsmokers, but there were no significant differences between smokers and e-cigarette users. We concluded that the micronuclei count can be used as an early indicator for alterations of oral mucosa and exfoliative cytology represents an accessible tool which could be applied for mass screening.

Stress response markers in the blood of São Tomé green sea turtles (Chelonia mydas) and their relation with accumulated metal levels

Metals are persistent worldwide being harmful for diverse organisms and having complex and combined effects with other contaminants in the environment. Sea turtles accumulate these contaminants being considered good bioindicator species for marine pollution. However, very little is known on how this is affecting these charismatic animals. São Tomé and Príncipe archipelago harbours important green sea turtle (Chelonia mydas) nesting and feeding grounds. The main goal of this study was to determine metal and metalloid accumulation in the blood of females C. mydas nesting in São Tomé Island, and evaluate the possible impacts of this contamination by addressing molecular stress responses. Gene expression analysis was performed in blood targeting genes involved in detoxification/sequestration and metal transport (mt, mtf and fer), and in antioxidant and oxidative stress responses (cat, sod, gr, tdx, txrd, selp and gclc). Micronuclei analysis in blood was also addressed as a biomarker of genotoxicity. Present results showed significant correlations between different gene expressions with the metals evaluated. The best GLM models and significant relationships were found for mt expression, for which 78% of the variability was attributed to metal levels (Al, Cu, Fe, Hg, Pb and Zn), followed by micronuclei count (65% - Cr, Cu, Fe, Hg, Mn and Zn), tdx expression (52% - Cd, Fe, Mn, Pb and Se), and cat expression (52% - As, Fe, Se and Cd x Hg). Overall, this study demonstrates that these green sea turtles are trying to adapt to the oxidative stress and damage produced by metals through the increased expression of antioxidants and other protectors, which raises concerns about the impacts on these endangered organisms' fitness. Furthermore, promising biomarker candidates associated to metal stress were identified in this species that may be used in future biomonitoring studies using C. mydas’ blood, allowing for a temporal follow-up of the organisms.

Characteristic differences in radiation-induced DNA damage response in human papillomavirus-negative and human papillomavirus-positive head and neck cancers with accumulation of fractional radiation dose.

Superior treatment responses by patients with human papillomavirus (HPV) positive head and neck squamous cell carcinoma (HNSCC), compared to patients with HNSCC from other causes, drive biomarker research to optimize treatment. Most HNSCC patients receive radiation therapy delivered as a fractionated course. Changing HPV status in HNSCC from a positive prognostic marker to a predictive one requires biomarkers that capture cellular radiation response to cumulative dose. Nuclear enlargement, γH2AX expression and micronuclei count, were studied in six HNSCC cell lines after 4 Gy fractionated X-irradiation. All HNSCC cell lines displayed altered cellular responses, indicating increasing inability to repair radiation damage with subsequent radiation fractions. Increases in nuclear area were significantly greater among HPV positive cell lines (207% and 67% for the HPV positive and HPV negative groups, respectively). A different character of DNA repair dysfunction in the HPV positive group suggests greater chromosomal translocation with accumulated radiation dose.

Abstract 2042: A cytoskeletal function for PBRM1: reading methylated microtubules to maintain genomic stability

Abstract The chromatin modifier SETD2 often mutated in clear cell renal cell carcinoma (ccRCC), was recently shown to be a dual-function methyltransferase that “writes” methyl marks on both chromatin and microtubules, revealing α-tubulin methylation as a new posttranslational modification of the mitotic spindle. Here, we report that the polybromo protein PBRM1, the 2nd most mutated gene in ccRCC, is a “reader” for this SETD2-dependent methyl mark on α-tubulin. PBRM1 is a component of the PBAF (Polybromo BRG1 associated factor) chromatin remodeler complex. Our western and immunocytochemistry data in multiple kidney-derived cell lines, including HEK293T, HKC and 786-O, revealed that PBRM1 binds to methylated α-tubulin and localizes to the mitotic spindle and spindle pole during cell division. PBRM1 has six bromo domains, two bromo-associated homology (BAH) domains and one HMG domain. While PBRM1 is known to bind acetylated histones via its bromo domains, our GST pull down assays showed that PBRM1 binds methylated α-tubulin via its two BAH domains. Additional western and immunocytochemical experiments following knockout or re-expression of PBRM1 revealed that PBRM1 recruits other PBAF components to the mitotic spindle to maintain genomic stability. Two clinically established ccRCC mutations (P1048R and C1233W) in PBRM1 BAH domains result in loss of microtubule binding, mislocalization of PBAF, and the inability of PBRM1 to maintain genomic stability, as assessed by increased lagging chromosomes, chromosome bridges, multipolar spindles and micronuclei count. A third pathogenic ccRCC mutation (T1202K) in the PBRM1 BAH domain did not affect microtubule binding and consequently was not associated with mitotic spindle defects or genomic instability. Mass spectrometry and RNASeq confirmed BAH domain mutant PBRM1 still assembled a transcriptionally competent PBAF complex, clearly distinguishing the cytoskeletal from the chromatin impact of these mutations. These data reveal a previously unknown function of PBRM1 beyond reading acetylated histones, and expand the repertoire of chromatin remodelers acting on the cytoskeleton to maintain genomic stability. Citation Format: Menuka Karki, Rahul Jangid, Ramakrishnan Anish, Riyad N. Seervai, Jean-Philippe Bertocchio, Takashi Hotta, Pavlos Msaouel, Sung Y. Jung, Sandra L. Grimm, Cristian Coarfa, Bernard E. Weissman, Ryoma Ohi, Kristen J. Verhey, Courtney H. Hodges, Ruhee Dere, In Young Park, B. V. Venkataram Prasad, W. Kimryn Rathmell, Cheryl L. Walker, Durga N. Tripathi. A cytoskeletal function for PBRM1: reading methylated microtubules to maintain genomic stability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2042.

Validation of a biomarker tool capable of measuring the absorbed dose soon after exposure to ionizing radiation

A radiological or nuclear attack could involve such a large number of subjects as to overwhelm the emergency facilities in charge. Resources should therefore be focused on those subjects needing immediate medical attention and care. In such a scenario, for the triage management by first responders, it is necessary to count on efficient biological dosimetry tools capable of early detection of the absorbed dose. At present the validated assays for measuring the absorbed dose are dicentric chromosomes and micronuclei counts, which require more than 2–3 days to obtain results. To overcome this limitation the NATO SPS Programme funded an Italian–Egyptian collaborative project aimed at validating a fast, accurate and feasible tool for assessing the absorbed dose early after radiation exposure. Biomarkers as complete blood cell counts, DNA breaks and radio-inducible proteins were investigated on blood samples collected before and 3 h after the first fraction of radiotherapy in patients treated in specific target areas with doses/fraction of about: 2, 3.5 or > 5 Gy and compared with the reference micronuclei count. Based on univariate and multivariate multiple linear regression correlation, our results identify five early biomarkers potentially useful for detecting the extent of the absorbed dose 3 h after the exposure.

The 3D reconstructed skin micronucleus assay using imaging flow cytometry and deep learning: A proof-of-principle investigation

The reconstructed skin micronucleus (RSMN) assay was developed in 2006, as an in vitro alternative for genotoxicity evaluation of dermally applied chemicals or products. In the years since, significant progress has been made in the optimization of the assay, including publication of a standard protocol and extensive validation. However, the diverse morphology of skin cells makes cell preparation and scoring of micronuclei (MN) tedious and subjective, thus requiring a high level of technical expertise for evaluation. This ultimately has a negative impact on throughput and the assay would benefit by the development of an automated method which could reduce scoring subjectivity while also improving the robustness of the assay by increasing the number of cells that can be scored. Imaging flow cytometry (IFC) with the ImageStream®X Mk II can capture high-resolution transmission and fluorescent imagery of cells in suspension. This proof-of-principle study describes protocol modifications that enable such automated measurement in 3D skin cells following exposure to mitomycin C and colchicine. IFC was then used for automated image capture and the Amnis® Artificial Intelligence (AAI) software permitted identification of binucleated (BN) cells with 91% precision. On average, three times as many BN cells from control samples were evaluated using IFC compared to the standard manual analysis. When IFC MNBN cells were visually scored from within the BN cell images, their frequency compared well with manual slide scoring, showing that IFC technology can be applied to the RSMN assay. This method enables faster time to result than microscope-based scoring and the initial studies presented here demonstrate its capability for the detection of statistically significant increases in MNBN frequencies. This work therefore demonstrates the feasibility of combining IFC and AAI to automate scoring for the RSMN assay and to improve its throughput and statistical robustness.

Structure-dependent genotoxic potencies of selected pyrrolizidine alkaloids in metabolically competent HepG2 cells

1,2-unsaturated pyrrolizidine alkaloids (PAs) are natural plant constituents comprising more than 600 different structures. A major source of human exposure is thought to be cross-contamination of food, feed and phytomedicines with PA plants. In humans, laboratory and farm animals, certain PAs exert pronounced liver toxicity and can induce malignant liver tumors in rodents. Here, we investigated the cytotoxicity and genotoxicity of eleven PAs belonging to different structural classes. Although all PAs were negative in the fluctuation Ames test in Salmonella, they were cytotoxic and induced micronuclei in human HepG2 hepatoblastoma cells over-expressing human cytochrome P450 3A4. Lasiocarpine and cyclic diesters except monocrotaline were the most potent congeners both in cytotoxicity and micronucleus assays with concentrations below 3 μM inducing a doubling in micronuclei counts. Other open di-esters and all monoesters exhibited weaker or much weaker geno- and cytotoxicity. The findings were in agreement with recently suggested interim Relative Potency (iREP) factors with the exceptions of europine and monocrotaline. A more detailed micronuclei analysis at low concentrations of lasiocarpine, retrorsine or senecionine indicated that pronounced hypolinearity of the concentration–response curves was evident for retrorsine and senecionine but not for lasiocarpine. Our findings show that the genotoxic and cytotoxic potencies of PAs in a human hepatic cell line vary in a structure-dependent manner. Both the low potency of monoesters and the shape of prototype concentration–response relationships warrant a substance- and structure-specific approach in the risk assessment of PAs.

Estragole: DNA adduct formation in primary rat hepatocytes and genotoxic potential in HepG2-CYP1A2 cells

Estragole is a natural constituent in herbs and spices and in products thereof such as essential oils or herbal teas. After cytochrome P450-catalyzed hydroxylation and subsequent sulfation, estragole acts as a genotoxic hepatocarcinogen forming DNA adducts in rodent liver. Because of the genotoxic mode of action and the widespread occurrence in food and phytomedicines a refined risk assessment for estragole is needed. We analyzed the time- and concentration-dependent levels of the DNA adducts N2-(isoestragole-3‘-yl)-2‘-desoxyguanosine (E3′N2dG) and N6-(isoestragole-3‘-yl)-desoxyadenosine (E3′N6dA), reported to be the major adducts formed in rat liver, in rat hepatocytes (pRH) in primary culture after incubation with estragole. DNA adduct levels were measured via UHPLC-ESI-MS/MS using stable isotope dilution analysis. Both adducts were formed in pRH and could already be quantified after an incubation time of 1 h (E3′N6dA at 10 μM, E3′N2dG at 1μM estragole). E3′N2dG, the main adduct at all incubation times and concentrations, could be detected at estragole concentrations < 0.1 μM after 24 h and < 0.5 μM after 48 h. Adduct levels were highest after 6 h and showed a downward trend at later time-points, possibly due to DNA repair and/or apoptosis. While the concentration-response characteristics of adduct formation were apparently linear over the whole concentration range, strong indication for marked hypo-linearity was obtained when the modeling was based on concentrations < 1 μM only. In the micronucleus assay no mutagenic potential of estragole was found in HepG2 cells whereas in HepG2-CYP1A2 cells 1 μM estragole led to a 3.2 fold and 300 μM to a 7.1 fold increase in micronuclei counts. Our findings suggest the existence of a ‘practical threshold’ dose for DNA adduct formation as an initiating key event of the carcinogenicity of estragole indicating that the default assumption of concentration-response-linearity is questionable, at least for the two major adducts studied here.