Genomic imprinting and environmental disease susceptibility

Genomic imprinting is one of the most intriguing subtleties of modern genetics. The term "imprinting" refers to parent-of-origin-dependent gene expression. The presence of imprinted genes can cause cells with a full parental complement of functional autosomal genes to specifically express one allele but not the other, resulting in monoallelic expression of the imprinted loci. Genomic imprinting plays a critical role in fetal growth and behavioral development, and it is regulated by DNA methylation and chromatin structure. This paper summarizes the Genomic Imprinting and Environmental Disease Susceptibility Conference held 8-10 October 1998 at Duke University, Durham, North Carolina. The conference focused on the importance of genomic imprinting in determining susceptibility to environmentally induced diseases. Conference topics included rationales for imprinting: parental antagonism and speciation; methods for imprinted gene identification: allelic message display and monochromosomal mouse/human hybrids; properties of the imprinted gene cluster human 11p15.5 and mouse distal 7; the epigenetics of X-chromosome inactivation; variability in imprinting: imprint erasure, non-Mendelian inheritance ratios, and polymorphic imprinting; imprinting and behavior: genetics of bipolar disorder, imprinting in Turner syndrome, and imprinting in brain development and social behavior; and aberrant methylation: methylation and chromatin structure, methylation and estrogen exposure, methylation of tumor-suppressor genes, and cancer susceptibility. Environmental factors are capable of causing epigenetic changes in DNA that can potentially alter imprint gene expression and that can result in genetic diseases including cancer and behavioral disorders. Understanding the contribution of imprinting to the regulation of gene expression will be an important step in evaluating environmental influences on human health and disease.     

Influence of epigenetic changes during oocyte growth on nuclear reprogramming after nuclear transfer

Genomic imprinting is the epigenetic mechanism that distinguishes whether the loci that are inherited from the maternal or paternal genome lead to parent-specific gene expression. The mechanism also regulates development in mammalian embryos. Genomic imprinting is established after implantation according to the specific markers that are imposed on the genome during gametogenesis; the allele-specific gene expression is then maintained throughout embryogenesis. The genomic imprinting markers are erased and renewed on an own-sex basis only in cells that differentiate into germline cells. This report shows that the epigenetic modifications that occur during oogenesis perform the crucial function of establishing the allele-specific expression of imprinted genes, and also suggests that the epigenetic DNA modification is related to the reprogramming and aberrant development seen in manipulated embryos.     

人卵巢组织冷冻对印迹基因IGF-2/H19甲基化程度及表达的影响

目的：探讨程序化冷冻和固相表面玻璃化冷冻对人卵巢组织中印迹基因胰岛素样生长因子2（IGF-2）/H１９（一种非可读框架RNＡ）差异性甲基化区（DMR）CpG岛甲基化程度及两基因表达的影响。方法：15例因其他疾病而手术的患者,将病理检查剩余的卵巢组织切割成皮质小条后随机分配到新鲜组（F组）、程序化冷冻组 （SF组）和固相表面玻璃化组（SSV组）。通过甲基化聚合酶链反应（MS-PCR）检测各组卵巢组织中IGF-2/H19 CpG岛甲基化程度,并分别采用免疫组化法和原位杂交方法检测各组卵泡中IGF-2蛋白和H19 RNA的表达情况。结果：F组、SF组和SSV组卵巢组织甲基化程度分别为26.46%,41.02%和33.09%,虽然SF组和SSV组的甲基化程度略高于F组,但差异无统计学意义。在新鲜和冻融后的卵巢组织中,各级别卵泡的卵母细胞和颗粒细胞胞质中均有IGF-2蛋白阳性表达,3组之间原始和初级卵泡IGF-2的表达差异无统计学意义（P＞0.05）。原始卵泡的颗粒细胞和卵母细胞均可见到H19 RNA阳性表达,3组之间原始卵泡H19 RNA的表达差异无统计学意义（P＞0.05）。结论：未检测到慢速程序化冷冻和快速固相表面玻璃化冷冻对印迹基因IGF-2/H19 CpG岛甲基化程度的影响,亦未发现2种冷冻方法对原始和初级卵泡中IGF-2蛋白和H19 RNA表达的影响。     

男性焦虑和压力对精子印记基因甲基化的影响

目的 近年来,心理健康问题发病率不断增高,有研究报道,母亲妊娠期间因承受压力通过改变胎儿发育的编程而影响下一代的发育,而父源性的负性心理健康因素对后代的影响则研究甚少。本课题通过对男性负性心理健康因素与精子质量以及印记基因甲基化改变的相关性研究,以探讨心理因素对精子质量和印记基因甲基化水平的影响,为促进生育健康和预防不良出生结局提供依据。方法 采用横断面方法调查山西省太原市2所医院620名男性门诊就诊者。包括相关问卷调查,焦虑自评量表和知觉压力量表,收集精液,并进行精子质量分析和印记基因H19、PEG3和MEG3的甲基化水平分析。结果 被研究人群有压力的占27.4%,焦虑的占7.08%。焦虑组精液量低于非焦虑组(P<0.05)。尽管压力组和非压力组在精子质量各项指标方面未发现差异,但压力组精子印记基因PEG3甲基化区的7个CpG位点的平均甲基化水平低于非压力组(P<0.05)。焦虑和压力量表得分与PEG3甲基化区的7个CpG位点的平均甲基化水平之间存在负相关关系,而H19和MEG3与焦虑和压力量表得分均未发现相关。结论 心理健康影响男性的精子质量,并与精子印记基因甲基化水平的改变相关。     

印迹基因H19在早孕绒毛组织中印迹状态的研究

目的检测早孕绒毛组织中印迹基因H19印迹的状态,探讨其印迹状态与滋养细胞浸润能力之间的联系。方法PCR-RFLP方法检测93例正常早孕绒毛组织中H19的印迹状态。结果在93例正常早孕绒毛组织中,有42例为杂合子,杂合子基因型中有11例为双等位基因的表达,其中所有双等位基因表达均在孕5～9周,而孕10～12周未发现有双等位基因的表达。结论在早孕期间,H19基因在绒毛组织中存在双等位基因的表达,主要集中在孕10周前,提示H19基因在滋养细胞中可能存在有动态变化,并与滋养细胞的浸润能力的变化有一定的联系。     

A Lack of Contact of Sperm with Accessory Sex Gland Secretions Deregulates DNA Methylation and Imprinted Gene Expression in Rodent Embryos

Increased oxidative DNA damage is observed in sperm devoid of contact with accessory sex gland (ASG) secretion. After fertilization, these sperm may produce abnormal embryos. In this study, we investigated the possibility that the pattern of DNA methylation and imprinted gene expression in these embryos may be perturbed. Epididymal sperm, uterine sperm, and embryonic day 13 (E13) embryos were collected from hamster and mouse. The extent of global DNA methylation was determined with an antibody against methylcytosine using an embryo DNA dot. The sperm and embryo Gtl2 promoter and H19 differential methylated region (DMR) were subject to bisulfite sequencing. Expression of their reciprocally activated genes Dlk1 and Igf2 was quantified by real-time PCR. Genome-wide DNA hypo-methylation in both hamster and mouse embryos sired by males without ASG was observed. The imprinting pattern of fetal mouse Gtl2 promoter and fetal hamster H19 DMR were also disrupted while the expression of Dlk1 and Igf2 was dysregulated in the hamster embryo. This study suggests that a lack of contact of sperm with the ASG secretion disrupts genome-wide DNA methylation and also affects the DNA methylation pattern of imprinted genes in embryos.     

Imprinted genes as potential genetic and epigenetic toxicologic targets

Genomic imprinting is an epigenetic phenomenon in eutherian mammals that results in the differential expression of the paternally and maternally inherited alleles of a gene. Imprinted genes are necessary for normal mammalian development. This requirement has been proposed to have evolved because of an interparental genetic battle for the utilization of maternal resources during gestation and postnatally. The nonrandom requisite for monoallelic expression of a subset of genes has also resulted in the formation of susceptibility loci for neurobehavioral disorders, developmental disorders, and cancer. Since imprinting involves both cytosine methylation within CpG islands and changes in chromatin structure, imprinted genes are potential targets for dysregulation by epigenetic toxicants that modify DNA methylation and histone acetylation.     

叶酸缺乏对胚胎停育患者绒毛印记基因甲基化的影响

目的 探讨叶酸缺乏对胚胎停育患者绒毛印记基因甲基化的影响.方法 选取2015年4月至2017年4月在陆军总医院263临床部妇科门诊224例胚胎停育患者(研究组)以及同期同比例自愿来陆军总医院263临床部人工流产孕妇(对照组),按照是否叶酸缺乏分为叶酸正常亚组和叶酸缺乏亚组,分析不同组别胰岛素生长因子2、父系表达基因3以及生长因子受体结合蛋白10甲基化状态.结果 对照组叶酸正常亚组IGF2、GRB10的甲基化指数均低于研究组叶酸缺乏亚组、叶酸正常亚组(t值分别为8.21、7.54、6.12、5.29,均P<0.05).对照组与研究组IGF2、GRB10异常甲基化率比较,差异有统计学意义(χ2值分别为6.11、6.34,均P<0.05).研究组组内叶酸缺乏亚组IGF2、GRB10异常甲基化率显著高于叶酸正常亚组(χ2值分别为5.02、5.44,均P<0.05);对照组叶酸正常亚组IGF2、GRB10异常甲基化率均显著低于研究组叶酸缺乏亚组(χ2值分别为6.17、6.43,均P<0.05).结论 叶酸缺乏可能导致IGF2、PEG3基因甲基化程度以及异常甲基化率增高,从而引起胚胎停育.     

Treating donor cells with 2-PCPA corrects aberrant histone H3K4 dimethylation and improves cloned goat embryo development

Epigenetic modifications extensively occur in mammalian embryonic development and cell differentiation process. They play an essential role in the reprogramming of nuclei during somatic cell nuclear transfer (SCNT) and subsequent in vitro embryonic development. Recently, SCNT embryos have been verified to contain a subnormal level of histone H3K4 dimethylation (H3K4me2) in contrast to in vitro fertilized embryos. This finding suggested that increasing H3K4me2 levels may ameliorate the aberrant development of cloned embryos. In this study, we investigated the influence of treating donor cells with trans-2-Phenylcyclopropylamine (2-PCPA), a specific inhibitor of lysine-specific demethylase 1 (LSD1), on embryogenesis, H3K4me2 level, and gene expression in cloned goat embryos. Treated goat fetal fibroblast cells (GFFs) with 2-PCPA served as donor cells for subsequent SCNT. Results showed that H3K4me2 levels in treated GFFs increased gradually with the increasing 2-PCPA concentration (p < 0.05) and had no obvious influence in cell viability. The 2-PCPA-induced up-regulation of H3K4me2 levels led to G0/G1 cell cycle arrest and the difference was significant at 2μM compared with the control group (p < 0.05). Interestingly, the development rate of goat SCNT embryos in vitro was significantly improved and aberrant H3K4me2 levels were effectively corrected in 2-PCPA-treated SCNT embryos in contrast to that in SCNT control embryos. Moreover, 2-PCPA treatment promoted the mRNA expression of key developmental genes Oct4 and Sox2 (p < 0.05) without affecting the expression levels of imprinted genes IGF2R and H19 in goat SCNT embryos. These results indicated that abnormal H3K4me2 status can be corrected and SCNT embryo development can be promoted through treatment of donor cells with 2-PCPA.

Abbreviations: SCNT: somatic cell nuclear transfer; H3K4me2: H3K4 dimethylation; 2-PCPA: trans-2-Phenylcyclopropylamine; LSD1: lysine-specific demethylase 1; GFFs: goat fetal fibroblast cells; IVF: in vitro fertilization; iPS: induced pluripotent stem; PBS: phosphate-buffered saline; IVM: in vitro maturation; RNAPII: RNA polymerase II; HMTs: histone methyltransferase     

Putative imprinted gene expression in uniparental bovine embryo models

Altered patterns of gene expression and the imprinted status of genes have a profound effect on cell physiology and can markedly alter embryonic and fetal development. Failure to maintain correct imprinting patterns can lead to abnormal growth and behavioural problems, or to early pregnancy loss. Recently, it has been reported that the Igf2R and Grb10 genes are biallelically expressed in sheep blastocysts, but monoallelically expressed at Day 21 of development. The present study investigated the imprinting status of 17 genes in in vivo, parthenogenetic and androgenetic bovine blastocysts in order to determine the prevalence of this unique phenomenon. Specifically, the putatively imprinted genes Ata3, Impact, L3Mbtl, Magel2, Mkrn3, Peg3, Snrpn, Ube3a and Zac1 were investigated for the first time in bovine in vitro fertilised embryos. Ata3 was the only gene not detected. The results of the present study revealed that all genes, except Xist, failed to display monoallelic expression patterns in bovine embryos and support recent results reported for ovine embryos. Collectively, the data suggest that monoallelic expression may not be required for most imprinted genes during preimplantation development, especially in ruminants. The research also suggests that monoallelic expression of genes may develop in a gene- and time-dependent manner.     

硅转化BALB/c-3T3细胞基因组DNA异常甲基化的研究

目的 对硅转化细胞基因组DNA异常甲基化进行研究，探讨硅的表遗传致癌机制。方法 从结晶型硅(Si)转化BALB/c-3T3细胞中提取基因组DNA，经Msel(甲基化非敏感性酶)单独消化或Msel和BstU1(甲基化敏感性酶)联合消化，消化产物用甲基化敏感性内切酶指纹法(MSRF)进行分析，差异显示出异常甲基化基因片段，进一步将异常甲基化DNA片段亚克隆和序列测定，再与基因文库中的基因进行类比分析。结果发现硅转化细胞存在6条异常甲基化DNA(其中1条为高甲基化，5条有低甲基化现象)，序列测定显示这些异常甲基化基因片段似乎来源于一些RNA转录和蛋白质翻译等基因家族。结论DNA异常甲基化会导致基因表达激活或抑制，因此硅转化细胞基因组某些功能基因DNA异常甲基化导致的异常表达，可能间接是硅诱导细胞转化及其致癌作用的一种表遗传机制。     

5-氮-2'脱氧胞苷逆转人卵巢癌细胞系OVCAR3和Anglne中RIZ1基因的表达

目的:观察甲基转移酶抑制剂5-氮-2'脱氧胞苷(5-Aza-CdR)对人卵巢癌细胞系OVCAR3和Anglne RIZ1基因启动子区甲基化状态及表达的影响,探讨人卵巢癌细胞系OVCAR3和AnglneRIZ1基因失活的机制及5-Aza-CdR对RIZ1基因表达的调控。方法:用5-Aza-CdR(浓度为5μmol/L)处理体外培养的人卵巢癌细胞系OVCAR3和Anglne后,采用MSP法检测用药前后细胞中RIZl基因的甲基化状态,RT-PCR及Western-blot法检测用药前后细胞中RIZ1基因mRNA及蛋白表达的变化。结果:在人卵巢癌细胞系OVCAR3和Anglne中,RIZ1基因启动子区呈异常甲基化状态,RIZ1基因mRNA及蛋白表达缺失,经过5-Aza-CdR处理后,RIZ1基因启动子区呈非甲基化状态,其mRNA及蛋白重表达。结论:5-Aza-CdR可改变人卵巢癌细胞系OVCAR3和Anglne中RIZ1基因甲基化状态,从而调控癌细胞中RIZ1基因的表达。     

Ex vivo early embryo development and effects on gene expression and imprinting

The environment to which the mammalian embryo is exposed during the preimplantation period of development has a profound effect on the physiology and viability of the conceptus. It has been demonstrated that conditions that alter gene expression, and in some instances the imprinting status of specific genes, have all previously been shown to adversely affect cell physiology. Thus, questions are raised regarding the aetiology of abnormal gene expression and altered imprinting patterns, and whether problems can be averted by using more physiological culture conditions. It is also of note that the sensitivity of the embryo to its surroundings decreases as development proceeds. Post compaction, environmental conditions have a lesser effect on gene function. This, therefore, has implications regarding the conditions used for IVF and the culture of the cleavage stage embryo. The developmental competence of the oocyte also impacts gene expression in the embryo, and therefore superovulation has been implicated in abnormal methylation and imprinting in the resultant embryo. Furthermore, the genetics and dietary status of the mother have a profound impact on embryo development and gene expression. The significance of specific animal models for human assisted reproductive technologies (ART) is questioned, given that most cattle data have been obtained from in vitro-matured oocytes and that genes imprinted in domestic and laboratory animals are not necessarily imprinted in the human. Patients treated with ART have fertility problems, which in turn may predispose their gametes or embryos to greater sensitivities to the process of ART. Whether this is from the drugs involved in the ovulation induction or from the IVF, intracytoplasmic sperm injection or culture procedures themselves remains to be determined. Alternatively, it may be that epigenetic alterations are associated with infertility and symptoms are subsequently revealed through ART. Whatever the aetiology, continued long-term monitoring of the children conceived through ART is warranted.     

辅助生殖技术中基因印迹分析

人类辅助生殖临床数据已经显示,辅助生殖技术（ART）与自发流产、早产和围生期死亡、低体质量儿以及一些印迹疾病有关。在配子及胚胎早期发育过程中,基因印迹需经历印迹擦除、重建和维持过程,其中任何一个环节出错都可能导致胚胎发育缺陷,甚至死亡。ART恰施于这一表观遗传重编程的关键时期。因此,这些异常结局可能与ART导致的印迹基因的异常表达有关。而ART中主要的治疗手段有促排卵、体外受精、胞浆内单精子注射（ICSI）和体外培养。这些操作通过干扰基因印迹的重建和维持,影响基因表达和表型,进而影响配子和早期胚胎的发育,从而影响子代的生长发育潜能。     

镉转化细胞DNA异常甲基化对肿瘤相关基因表达的影响

目的 对镉转化细胞DNA异常甲基化及其对肿瘤相关基因表达的影响进行研究，探讨镉的外遗传致癌机制。方法 从CdCl2转化BAIB／c—3T3细胞中提取基因组DNA，经甲基化非敏感性酶(Mse1)单独消化或Mse1和甲基化敏感性酶(BstUl)联合消化，消化产物用甲基化敏感性内切酶指纹法(MSRF)进行分析，差异显示出异常甲基化基因片段，进一步以异常甲基化DNA为探针进行Southern分子杂交加以证实，并进行DNA序列测定，与基因文库中的基因进行类比分析。结果 发现镉转化细胞存在异常甲基化DNA，其中一个甲基化DNA片段为p16抑癌基因。结论 DNA高甲基化会导致基因表达抑制，因此，p16基因高甲基化会导致其抑癌功能减弱或丧失，这可能是镉诱导细胞转化及其致癌作用的一种外遗传机制。     

DNA methylation: a marker for carcinogen exposure and cancer risk

Cancers arise as a consequence of multiple genetic and epigenetic alterations. Many genes aberrantly methylated in cancers have been identified in recent years, and their use in cancer diagnosis and therapy is currently under investigation. During our genome-wide screening for a novel tumor-suppressor gene in gastric cancers, we found that only a small amount of aberrant methylation was present, even in non-cancerous gastric mucosae. A subsequent large-scale analysis of the gastric mucosae of healthy individuals and gastric cancer patients using quantitative methylation-specific PCR (qMSP) revealed that Helicobacter pylori infection potently induced aberrant DNA methylation in non-cancerous gastric mucosae and that these high methylation levels can decrease following cessation of the H. pylori infection. Helicobacter pylori infection induced the methylation of specific genes among 48 genes that can be methylated in gastric cancer cell lines. Most importantly, the methylation levels in the gastric mucosae of individuals without H. pylori infection correlated with their risk of gastric cancer. These findings show that a field for cancerization is formed by H. pylori infection and that this field can be measured using DNA methylation as a marker. The concept of an “epigenetic field for cancerization” has been also demonstrated for colon and breast cancers, and it is possibly present for other cancers and other diseases. Applied knowledge of epigenetic changes in human diseases has now started to make an impact on the prevention, diagnostics, and therapeutics of these diseases.     

Parent-of-origin, imprinting, mitochondrial, and X-linked effects in traits related to alcohol dependence: presentation Group 18 of Genetic Analysis Workshop 14

The participants of Presentation Group 18 of Genetic Analysis Workshop 14 analyzed the Collaborative Study on the Genetics of Alcoholism data set to investigate sex-specific effects for phenotypes related to alcohol dependence. In particular, the participants looked at imprinting (which is also known as parent-of-origin effect), differences between recombination fractions for the two sexes, and mitochondrial and X-chromosomal effects. Five of the seven groups employed newly developed or existing methods that take imprinting into account when testing for linkage, or test for imprinting itself. Single-marker and multipoint analyses were performed for microsatellite as well as single-nucleotide polymorphism markers, and several groups used a sex-specific genetic map in addition to a sex-averaged map. Evidence for paternal imprinting (i.e., maternal expression) was consistently obtained by at least two groups at genetic regions on chromosomes 10, 12, and 21 that possibly harbor genes responsible for alcoholism. Evidence for maternal imprinting (which is equivalent to paternal expression) was consistently found at a locus on chromosome 11. Two groups applied extensions of variance components analysis that model a mitochondrial or X-chromosomal effect to latent class variables and electrophysiological traits employed in the diagnosis of alcoholism. The analysis, without using genetic markers, revealed mitochondrial or X-chromosomal effects for several of these traits. Accounting for sex-specific environmental variances appeared to be crucial for the identification of an X-chromosomal factor. In linkage analysis using marker data, modeling a mitochondrial variance component increased the linkage signals obtained for autosomal loci.     

抑癌基因RIZ1启动子区甲基化与卵巢癌的关系

目的:探讨抑癌基因RIZ1启动子区甲基化与卵巢上皮性癌发生的关系。方法:以卵巢癌组织及卵巢癌细胞系为研究对象。MSP检测RIZ1基因甲基化状况;RT-PCR、蛋白印迹分别检测RIZ1mRNA及蛋白表达;细胞增殖实验检测5-杂氮-2'-脱氧胞苷处理卵巢癌细胞前后细胞的增殖状况。结果:卵巢癌组织、卵巢癌细胞系、良性卵巢肿瘤组织及正常卵巢组织中的甲基化率分别为25.8%、40%、0%和0%。5-杂氮-2'-脱氧胞苷处理RIZ1基因发生甲基化的卵巢癌细胞系后,RIZ1基因重新获得表达;且去甲基化处理后,卵巢癌细胞生长均减慢。结论:抑癌基因RIZ1启动子区甲基化与上皮性卵巢癌的发生有关,DNA甲基化是其表达缺陷的原因之一。     

Embryonale Stammzellen der Maus Eigenschaften, Potenzial und Verwendung

During the last few years research on embryonic stem cells has received much public attention due to the fact that these cells are able to differentiate in vitro into many specialized cells and thus may serve as a source for a variety of tissues. The following article focuses on mouse embryonic stem cells (murine ES cells), because research on these cells has given insight into the potential of embryonic stem cells. Murine ES cells are permanent cell lines established from the inner cell mass (ICM) of early embryos (blastocysts). ES cells are undifferentiated pluripotent cells that are able to undergo an unlimited number of cell divisions without loosing the undifferentiated phenotype. The same is true for mouse primordial germ cell lines (murine EG cell lines), that where established from the fetal progenitor cells of primordial germ cells. Mouse embryonic stem cells are used for different purposes. In basic research they are used to study the consequences of mutations within genes that control embryonic development and/or the development of diseases. Because of their ability to differentiate into a variety of specialized cell types, murine ES cells also serve as model systems to establish specific differentiation protocols. In the last few years protocols were established for the in vitro development of undifferentiated embryonic stem cells into differentiated cardiac, skeletal muscle, neural, adipogenic, haematopoietic, endothelial, chondrogenic or vascular smooth muscle cells. Last but not least, studies on mouse ES cells have demonstrated that embryonic cells and their differentiated derivatives can be used to analyse the effects of toxic substances or of pharmaceutical drugs.     

Epigenetic modulation of BRCA1 and BRCA2 gene expression by equol in breast cancer cell lines

S-Equol is a metabolite resulting from the conversion of daidzein, a soya phyto-oestrogen, by the gut microflora. The potential protective effects of equol in breast cancer are still under debate. Consequently, we investigated the effects of equol on DNA methylation of breast cancer susceptibility genes (BRCA1 and BRCA2) and oncosuppressors in breast cancer cell lines (MDA-MB-231 and MCF-7) and in a dystrophic breast cell line (MCF-10a) following exposure to S-equol (2?μm) for 3 weeks. We demonstrated by quantitative analysis of methylated alleles a significant decrease in the methylation of the cytosine phosphate guanine (CpG) islands in the promoters of BRCA1 and BRCA2 after the S-equol treatment in MCF-7 and MDA-MB-231 cells and a trend in MCF-10a cells. We also showed that S-equol increases BRCA1 and BRCA2 protein expression in the nuclei and the cytoplasm in MCF-7, MDA-MB-231 and MCF-10a cell lines by immunohistochemistry. The increase in BRCA1 and BRCA2 proteins was also found after Western blotting in the studied cell lines. In summary, we demonstrated the demethylating effect of S-equol on the CpG islands inside the promoters of BRCA1 and BRCA2 genes, resulting in an increase in the level of expressed oncosuppressors in breast cancer cell lines.