Современные подходы к выявлению и изучению эпигенетически активных ксенобиотиков

Objective. Review of the modern methodological approaches for testing and studying the epigenetic activity of xenobiotics.Materials and methods. In preparing the review, we used information databases of biomedical literature SciVerse Scopus (538), PubMed (746), Web of Science (625), RSCI (45). To obtain full-text documents, electronic resources of PubMed Central (PMC), Research Gate, RSCI, CyberLeninka were used. In the text of the review, 87 modern publications (2010–2019) were cited, as well as 17 earlier articles published by the founders of the methods, which are used today.Results. In the review, current data on epigenetic regulation for gene expression at the level of DNA methylation and histone modification are discussed, in vitro model systems and model organisms are described, and modern methods for screening of epigenetically active xenobiotics are presented.Conclusion. Modern data concerning the mechanisms of epigenetic regulation of gene expression, the usage of existing model systems and model organisms, as well as the application of various methodological approaches and techniques, allow extensive screening of xenobiotics (including drugs and compounds synthesized for national economic tasks) for epigenetic activity. The identification of epigenetically active compounds is important in terms of improving the prevention and treatment of a number of diseases and, in particular, malignant neoplasms.Цель исследования – анализ существующих методических подходов к тестированию и изучению эпигенетической активности ксенобиотиков.Материалы и методы. При подготовке обзора были использованы информационные базы биомедицинской литературы SciVerse Scopus (538), PubMed (746), Web of Science (625), РИНЦ (45). Для получения полнотекстовых документов использованы электронные ресурсы PubMed Central (PMC), Research Gate, РИНЦ, КиберЛенинка. В тексте обзора процитированы 87 современных публикаций (2010–2019 гг.), а также 17 статей основоположников анализируемых в обзоре методик, которые используются и в наши дни.Результаты. В обзоре рассмотрены современные данные по основным закономерностям функционирования системы регуляции экспрессии генов на уровне метилирования ДНК и модификации гистонов, описаны модельные организмы, используемые как при тестировании ксенобиотиков на эпигенетическую активность, так и для детального изучения их конкретных эффектов на отдельные механизмы эпигенетической регуляции транскрипции. Поскольку в настоящее время не разработаны тест-системы, позволяющие интегрально оценить эффекты ксенобиотиков на эпигенетическую систему регуляции транскрипции, в обзоре представлены методы оценки выявления влияний различных факторов окружающей среды, в том числе различных ксенобиотиков, на метилирование ДНК и модификацию гистонов.Заключение. Современные представления о механизмах эпигенетической регуляции экспрессии генов, использование существующих модельных систем и модельных организмов, а также применение различных методологических и методических подходов позволяют проводить широкое тестирование ксенобиотиков, в том числе медицинских препаратов и соединений, синтезированных для выполнения народнохозяйственных задач, на эпигенетическую активность. Выявление эпигенетически активных соединений важно в плане совершенствования профилактики и лечения ряда заболеваний и, прежде всего, злокачественных новообразований

Нарушение метилирования ДНК при злокачественных новообразованиях

DNA methylation is a chromatin modification that plays an important role in the epigenetic regulation of gene expression. Changes in DNA methylation patterns are characteristic of many malignant neoplasms. DNA methylation is occurred by DNA methyltransferases (DNMTs), while demethylation is mediated by TET family proteins. Mutations and changes in the expression profile of these enzymes lead to DNA hypo- and hypermethylation and have a strong impact on carcinogenesis. In this review, we considered the key aspects of the mechanisms of regulation of DNA methylation and demethylation, and also analyzed the role of DNA methyltransferases and TET family proteins in the pathogenesis of various malignant neoplasms.During the preparation of the review, we used the following biomedical literature information bases: Scopus (504), PubMed (553), Web of Science (1568), eLibrary (190). To obtain full-text documents, the electronic resources of PubMed Central (PMC), Science Direct, Research Gate, CyberLeninka were used. To analyze the mutational profile of epigenetic regulatory enzymes, we used the cBioportal portal (https://www.cbioportal.org / ), data from The AACR Project GENIE Consortium (https://www.mycancergenome.org / ), COSMIC, Clinvar, and The Cancer Genome Atlas (TCGA).Метилирование ДНК представляет собой модификацию хроматина, которая играет важную роль в эпигенетической регуляции экспрессии генов. Изменение паттернов метилирования ДНК характерно для многих злокачественных новообразований. Метилирование ДНК осуществляется ДНК-метилтрансферазами (DNMTs), в то время как деметилирование происходит под действием метилцитозиновых диоксигеназ, или белков семейства TET. Мутации и изменение профиля экспрессии данных ферментов, приводящие к гипо- и гиперметилированию ДНК, могут оказывать сильное влияние на канцерогенез.В обзоре рассмотрены ключевые аспекты механизмов регуляции метилирования и деметилирования ДНК, а также проведен анализ роли ДНК-метилтрансфераз и белков семейства ТЕТ в патогенезе различных злокачественных новообразований.При подготовке обзора были использованы информационные базы биомедицинской литературы Scopus (504), PubMed (553), Web of Science (1568), eLibrary (190), для получения полнотекстовых документов – электронные ресурсы PubMed Central (PMC), Science Direct, Research Gate, КиберЛенинка, для анализа мутационного профиля эпигенетических регуляторных ферментов – портал cBioportal (https://www.cbioportal.org / ), данные проекта The AACR Project GENIE Consortium (https://www.mycancergenome.org / ), базы данных COSMIC, Clinvar и Атласа генома рака (The Cancer Genome Atlas, TCGA)

Modern approaches for the screening of epigenetically active xenobiotics

Objective. Review of the modern methodological approaches for testing and studying the epigenetic activity of xenobiotics.Materials and methods. In preparing the review, we used information databases of biomedical literature SciVerse Scopus (538), PubMed (746), Web of Science (625), RSCI (45). To obtain full-text documents, electronic resources of PubMed Central (PMC), Research Gate, RSCI, CyberLeninka were used. In the text of the review, 87 modern publications (2010–2019) were cited, as well as 17 earlier articles published by the founders of the methods, which are used today.Results. In the review, current data on epigenetic regulation for gene expression at the level of DNA methylation and histone modification are discussed, in vitro model systems and model organisms are described, and modern methods for screening of epigenetically active xenobiotics are presented.Conclusion. Modern data concerning the mechanisms of epigenetic regulation of gene expression, the usage of existing model systems and model organisms, as well as the application of various methodological approaches and techniques, allow extensive screening of xenobiotics (including drugs and compounds synthesized for national economic tasks) for epigenetic activity. The identification of epigenetically active compounds is important in terms of improving the prevention and treatment of a number of diseases and, in particular, malignant neoplasms

HeLa TI cell-based assay as a new approach to screen for chemicals able to reactivate the expression of epigenetically silenced genes

Chemicals reactivating epigenetically silenced genes target diverse classes of enzymes, including DNMTs, HDACs, HMTs and BET protein family members. They can strongly influence the expression of genes and endogenous retroviral elements with concomitant dsRNA synthesis and massive transcription of LTRs. Chemicals reactivating gene expression may cause both beneficial effects in cancer cells and may be hazardous by promoting carcinogenesis. Among chemicals used in medicine and commerce, only a small fraction has been studied with respect to their influence on epigenetic silencing. Screening of chemicals reactivating silent genes requires adequate systems mimicking whole-genome processes. We used a HeLa TSA-inducible cell population (HeLa TI cells) obtained by retroviral infection of a GFP-containing vector followed by several rounds of cell sorting for screening purposes. Previously, the details of GFP epigenetic silencing in HeLa TI cells were thoroughly described. Herein, we show that the epigenetically repressed gene GFP is reactivated by 15 agents, including HDAC inhibitors–vorinostat, sodium butyrate, valproic acid, depsipeptide, pomiferin, and entinostat; DNMT inhibitors–decitabine, 5-azacytidine, RG108; HMT inhibitors–UNC0638, BIX01294, DZNep; a chromatin remodeler–curaxin CBL0137; and BET inhibitors–JQ-1 and JQ-35. We demonstrate that combinations of epigenetic modulators caused a significant increase in cell number with reactivated GFP compared to the individual effects of each agent. HeLa TI cells are competent to metabolize xenobiotics and possess constitutively expressed and inducible cytochrome P450 mono-oxygenases involved in xenobiotic biotransformation. Thus, HeLa TI cells may be used as an adequate test system for the extensive screening of chemicals, including those that must be metabolically activated. Studying the additional metabolic activation of xenobiotics, we surprisingly found that the rat liver S9 fraction, which has been widely used for xenobiotic activation in genotoxicity tests, reactivated epigenetically silenced genes. Applying the HeLa TI system, we show that N-nitrosodiphenylamine and N-nitrosodimethylamine reactivate epigenetically silenced genes, probably by affecting DNA methylation. © 2021 Maksimova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

EPIGENETIC EFFECTS OF ENZASTAURIN – A NEW ASPECT IN THE MECHANISM OF ACTION OF AN ANTICANCER DRUG FROM PROTEIN KINASE INHIBITORS

The purpose of the study was to analyze the ability of five antitumor drugs from the pharmaceutical group of protein kinase inhibitors (gefitinib, imatinib, pazopanib, ponatinib and enzastaurin) to reactivate the expression of the epigenetically silenced GFP in HeLa TI cells, and to estimate the effect of epigenetically active drugs on: 1) acetylation and methylation of histones H3 and H4; 2) integral DNA methylation; 3) activity of HAT and HDAC1 enzymes; 4) expression levels of the genes encoding epigenetic regulation enzymes (DNMT1, DNMT3A, DNMT3B; SIRT1, HDAC1; SETD1A, SETD1B, SUV420H1, SUV420H2, SUV39H1, SUV39H2). Material and Methods. The epigenetic activity of antitumor drugs was determined using the HeLa TI test system, a population of HeLa cells with the retroviral vector containing the epigenetically silenced GFP. The level of integral DNA methylation was analyzed using MspI/HpaII methyl-sensitive restriction analysis. Histone modifications were analyzed by Western blotting with antibodies to acetylated and methylated histones H3 and H4. The total activity of HAT enzymes was analyzed using Histone Acetyltransferase Activity Assay Kit. Expression of the epigenetic enzyme genes was analyzed using real-time quantitative RT-PCR. Results. It was shown that only the enzyme inhibitor Cβ protein kinase enzastaurin had the ability to reactivate the expression of epigenetically silenced GFP in the HeLa TI cells. We showed that under the action of enzastaurin, the level of integral DNA methylation and expression of DNMT3A and DNMT3B DNA methyltransferase genes decreased. It was also found that enzastaurin reduced the expression levels of histone deacetylases HDAC1 and SIRT1, but did not affect the activity and expression levels of histone acetylases, the level of histone methylation (H3K4me3, H3K9me3, H3K27me3, H4K20me3), and the level of expression of the histone methyltransferases (SUV39H1, SUV39H2, SUV420H1, SUV420H2, SETD1A и SETD1B). Conclusion. The data obtained are important for clarifying the mechanisms of action of 5 protein kinase inhibitors, in particular with respect to enzastaurin, the protein kinase Cβ inhibitor, for which the ability to reactivate epigenetically silent genes due to the effect on DNA methylation and histone acetylation was demonstrated

Stop COVID Cohort: An Observational Study of 3480 Patients Admitted to the Sechenov University Hospital Network in Moscow City for Suspected Coronavirus Disease 2019 (COVID-19) Infection

Background: The epidemiology, clinical course, and outcomes of patients with coronavirus disease 2019 (COVID-19) in the Russian population are unknown. Information on the differences between laboratory-confirmed and clinically diagnosed COVID-19 in real-life settings is lacking. Methods: We extracted data from the medical records of adult patients who were consecutively admitted for suspected COVID-19 infection in Moscow between 8 April and 28 May 2020. Results: Of the 4261 patients hospitalized for suspected COVID-19, outcomes were available for 3480 patients (median age, 56 years; interquartile range, 45-66). The most common comorbidities were hypertension, obesity, chronic cardiovascular disease, and diabetes. Half of the patients (n=1728) had a positive reverse transcriptase-polymerase chain reaction (RT-PCR), while 1748 had a negative RT-PCR but had clinical symptoms and characteristic computed tomography signs suggestive of COVID-19. No significant differences in frequency of symptoms, laboratory test results, and risk factors for in-hospital mortality were found between those exclusively clinically diagnosed or with positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RT-PCR. In a multivariable logistic regression model the following were associated with in-hospital mortality: older age (per 1-year increase; odds ratio, 1.05; 95% confidence interval, 1.03-1.06), male sex (1.71; 1.24-2.37), chronic kidney disease (2.99; 1.89-4.64), diabetes (2.1; 1.46-2.99), chronic cardiovascular disease (1.78; 1.24-2.57), and dementia (2.73; 1.34-5.47). Conclusions: Age, male sex, and chronic comorbidities were risk factors for in-hospital mortality. The combination of clinical features was sufficient to diagnose COVID-19 infection, indicating that laboratory testing is not critical in real-life clinical practice