Subclone Eradication Analysis Identifies Targets for Enhanced Cancer Therapy and Reveals L1 Retrotransposition as a Dynamic Source of Cancer Heterogeneity.

Treatment-eradicated cancer subclones have been reported in leukemia and have recently been detected in solid tumors. Here we introduce Differential Subclone Eradication and Resistance (DSER) analysis, a method developed to identify molecular targets for improved therapy by direct comparison of genomic features of eradicated and resistant subclones in pre- and posttreatment samples from a patient with BRCA2-deficient metastatic prostate cancer. FANCI and EYA4 were identified as candidate DNA repair-related targets for converting subclones from resistant to eradicable, and RNAi-mediated depletion of FANCI confirmed it as a potential target. The EYA4 alteration was associated with adjacent L1 transposon insertion during cancer evolution upon treatment, raising questions surrounding the role of therapy in L1 activation. Both carboplatin and enzalutamide turned on L1 transposon machinery in LNCaP and VCaP but not in PC3 and 22Rv1 prostate cancer cell lines. L1 activation in LNCaP and VCaP was inhibited by the antiretroviral drug azidothymidine. L1 activation was also detected postcastration in LuCaP 77 and LuCaP 105 xenograft models and postchemotherapy in previously published time-series transcriptomic data from SCC25 head and neck cancer cells. In conclusion, DSER provides an informative intermediate step toward effective precision cancer medicine and should be tested in future studies, especially those including dramatic but temporary metastatic tumor regression. L1 transposon activation may be a modifiable source of cancer genomic heterogeneity, suggesting the potential of leveraging newly discovered triggers and blockers of L1 activity to overcome therapy resistance. SIGNIFICANCE: Differential analysis of eradicated and resistant subclones following cancer treatment identifies that L1 activity associated with resistance is induced by current therapies and blocked by the antiretroviral drug azidothymidine.

In utero exposure to chlordecone affects histone modifications and activates LINE-1 in cord blood.

Environmental factors can induce detrimental consequences into adulthood life. In this study, we examined the epigenetic effects induced by in utero chlordecone (CD) exposure on human male cord blood as well as in blood-derived Ke-37 cell line. Genome-wide analysis of histone H3K4me3 distribution revealed that genes related to chromosome segregation, chromatin organization, and cell cycle have altered occupancy in their promoters. The affected regions were enriched in ESR1, SP family, and IKZF1 binding motifs. We also observed a global reduction in H3K9me3, markedly in repeated sequences of the genome. Decrease in H3K9me3 after CD exposure correlates with decreased methylation in LINE-1 promoters and telomere length extension. These observations on human cord blood were assessed in the Ke-37 human cell line. H3K4me3 and the expression of genes related to immune response, DNA repair, and chromatin organization, which were affected in human cord blood were also altered in CD-exposed Ke-37 cells. Our data suggest that developmental exposure to CD leads to profound changes in histone modification patterns and affects the processes controlled by them in human cord blood.

The effects of flavonoids, green tea polyphenols and coffee on DMBA induced LINE-1 DNA hypomethylation.

The intake of carcinogenic and chemopreventive compounds are important nutritional factors related to the development of malignant tumorous diseases. Repetitive long interspersed element-1 (LINE-1) DNA methylation pattern plays a key role in both carcinogenesis and chemoprevention. In our present in vivo animal model, we examined LINE-1 DNA methylation pattern as potential biomarker in the liver, spleen and kidney of mice consuming green tea (Camellia sinensis) extract (catechins 80%), a chinese bayberry (Morella rubra) extract (myricetin 80%), a flavonoid extract (with added resveratrol) and coffee (Coffee arabica) extract. In the organs examined, carcinogen 7,12-dimethylbenz(a)anthracene (DMBA)-induced hypomethylation was prevented by all test materials except chinese bayberry extract in the kidneys. Moreover, the flavonoid extract caused significant hypermethylation in the liver compared to untreated controls and to other test materials. The tested chemopreventive substances have antioxidant, anti-inflammatory properties and regulate molecular biological signaling pathways. They increase glutathione levels, induce antioxidant enzymes, which decrease free radical damage caused by DMBA, and ultimately, they are able to increase the activity of DNA methyltransferase enzymes. Furthermore, flavonoids in the liver may inhibit the procarcinogen to carcinogen activation of DMBA through the inhibition of CYP1A1 enzyme. At the same time, paradoxically, myricetin can act as a prooxidant as a result of free radical damage, which can explain that it did not prevent hypomethylation in the kidneys. Our results demonstrated that LINE-1 DNA methylation pattern is a useful potential biomarker for detecting and monitoring carcinogenic and chemopreventive effects of dietary compounds.

The Retrotransposition of L1 is Involved in the Reconsolidation of Contextual Fear Memory in Mice.

BACKGROUND: The long interspersed element-1 (L1) participates in memory formation, and DNA methylation patterns of L1 may suggest resilience or vulnerability factors for Post-Traumatic Stress Disorder (PTSD), of which the principal manifestation is a pathological exacerbation of fear memory. However, the unique roles of L1 in the reconsolidation of fear memory remain poorly understood. OBJECTIVE: The study aimed to investigate the role of L1 in the reconsolidation of context-dependent fear memory. METHODS: Mice underwent fear conditioning and fear recall in the observation chambers. Fear memory was assessed by calculating the percentage of time spent freezing in 5 min. The medial prefrontal cortex (mPFC) and hippocampus were removed for further analysis. Open Reading Frame 1 (ORF1) mRNA and ORF2 mRNA of L1 were analyzed by real-time quantitative polymerase chain reaction. After reactivation of fear memory, lamivudine was administered and its effects on fear memory reconsolidation were observed. RESULTS: ORF1 and ORF2 mRNA expressions in the mPFC and hippocampus after recent (24 h) and remote (14 days) fear memory recall exhibited augmentation via different temporal and spatial patterns. Reconsolidation of fear memory was markedly inhibited in mice treated with lamivudine, which could block L1. DNA methyltransferase mRNA expression declined following lamivudine treatment in remote fear memory recall. CONCLUSION: The retrotransposition of L1 participated in the reconsolidation of fear memory after reactivation of fear memory, and with lamivudine treatment, spontaneous recovery decreased with time after recent and remote fear memory recall, providing clues for understanding the roles of L1 in fear memory.

Adipose-Derived Mesenchymal Stem Cells do not Affect the Invasion and Migration Potential of Oral Squamous Carcinoma Cells.

Mesenchymal stem cells (MSCs) are commonly isolated from bone marrow and adipose tissue. Depending on the tissue of origin, MSCs have different characteristics and physiological effects. In various cancer studies, MSCs have been found to have either tumor-promoting or tumor-inhibiting action. This study investigated the effect of adipose tissue-MSCs (AT-MSCs) and bone marrow-MSCs (BM-MSCs) on global long interspersed nuclear element-1 (LINE-1) methylation, the expression level of microenvironment remodeling genes and cell proliferation, migration and invasion of oral tongue squamous cell carcinoma (OTSCC). Additionally, we studied the effect of human tongue squamous carcinoma (HSC-3)-conditioned media on LINE-1 methylation and the expression of microenvironment remodeling genes in AT-MSCs and BM-MSCs. Conditioned media from HSC-3 or MSCs did not affect LINE-1 methylation level in either cancer cells or MSCs, respectively. In HSC-3 cells, no effect of MSCs-conditioned media was detected on the expression of ICAM1, ITGA3 or MMP1. On the other hand, HSC-3-conditioned media upregulated ICAM1 and MMP1 expression in both types of MSCs. Co-cultures of AT-MSCs with HSC-3 did not induce proliferation, migration or invasion of the cancer cells. In conclusion, AT-MSCs, unlike BM-MSCs, seem not to participate in oral cancer progression.

Oral cedazuridine/decitabine for MDS and CMML: a phase 2 pharmacokinetic/pharmacodynamic randomized crossover study.

This phase 2 study was designed to compare systemic decitabine exposure, demethylation activity, and safety in the first 2 cycles with cedazuridine 100 mg/decitabine 35 mg vs standard decitabine 20 mg/m2 IV. Adults with International Prognostic Scoring System intermediate-1/2- or high-risk myelodysplastic syndromes (MDS) or chronic myelomonocytic leukemia (CMML) were randomized 1:1 to receive oral cedazuridine/decitabine or IV decitabine in cycle 1, followed by crossover to the other treatment in cycle 2. All patients received oral cedazuridine/decitabine in subsequent cycles. Cedazuridine and decitabine were given initially as separate capsules in a dose-confirmation stage and then as a single fixed-dose combination (FDC) tablet. Primary end points: mean decitabine systemic exposure (geometric least-squares mean [LSM]) of oral/IV 5-day area under curve from time 0 to last measurable concentration (AUClast), percentage long interspersed nuclear element 1 (LINE-1) DNA demethylation for oral cedazuridine/decitabine vs IV decitabine, and clinical response. Eighty patients were randomized and treated. Oral/IV ratios of geometric LSM 5-day AUClast (80% confidence interval) were 93.5% (82.1-106.5) and 97.6% (80.5-118.3) for the dose-confirmation and FDC stages, respectively. Differences in mean %LINE-1 demethylation between oral and IV were ≤1%. Clinical responses were observed in 48 patients (60%), including 17 (21%) with complete response. The most common grade ≥3 adverse events regardless of causality were neutropenia (46%), thrombocytopenia (38%), and febrile neutropenia (29%). Oral cedazuridine/decitabine (100/35 mg) produced similar systemic decitabine exposure, DNA demethylation, and safety vs decitabine 20 mg/m2 IV in the first 2 cycles, with similar efficacy. This study is registered at www.clinicaltrials.gov as #NCT02103478.

Long INterspersed element-1 mobility as a sensor of environmental stresses.

Long INterspersed element (LINE-1, L1) retrotransposons are the most abundant transposable elements in the human genome, constituting approximately 17%. They move by a "copy-paste" mechanism, involving reverse transcription of an RNA intermediate and insertion of its cDNA copy at a new site in the genome. L1 retrotransposition (L1-RTP) can cause insertional mutations, alter gene expression, transduce exons, and induce epigenetic dysregulation. L1-RTP is generally repressed; however, a number of observations collected over about 15 years revealed that it can occur in response to environmental stresses. Moreover, emerging evidence indicates that L1-RTP can play a role in the onset of several neurological and oncological diseases in humans. In recent years, great attention has been paid to the exposome paradigm, which proposes that health effects of an environmental factor should be evaluated considering both cumulative environmental exposures and the endogenous processes resulting from the biological response. L1-RTP could be an endogenous process considered for this application. Here, we summarize the current understanding of environmental factors that can affect the retrotransposition of human L1 elements. Evidence indicates that L1-RTP alteration is triggered by numerous and various environmental stressors, such as chemical agents (heavy metals, carcinogens, oxidants, and drugs), physical agents (ionizing and non-ionizing radiations), and experiential factors (voluntary exercise, social isolation, maternal care, and environmental light/dark cycles). These data come from in vitro studies on cell lines and in vivo studies on transgenic animals: future investigations should be focused on physiologically relevant models to gain a better understanding of this topic.

AhR ligands reactivate LINE-1 retrotransposon in triple-negative breast cancer cells MDA-MB-231 and non-tumorigenic mammary epithelial cells NMuMG.

Breast cancer is the most common cancer type in females worldwide. Environmental exposure to pesticides affecting hormonal homeostasis does not necessarily induce DNA mutations but may influence gene expression by disturbances in epigenetic regulation. Expression of long interspersed nuclear element-1 (LINE-1) has been associated with tumorigenesis in several cancers. In nearly all somatic cells, LINE-1 is silenced by DNA methylation in the 5́'UTR and reactivated during disease initiation and/or progression. Strong ligands of aryl hydrocarbon receptor (AhR) activate LINE-1 through the transforming growth factor-ß1 (TGF-ß1)/Smad pathway. Hexachlorobenzene (HCB) and chlorpyrifos (CPF), both weak AhR ligands, promote cell proliferation and migration in breast cancer cells, as well as tumor growth in rat models. In this context, our aim was to examine the effect of these pesticides on LINE-1 expression and ORF1p localization in the triple-negative breast cancer cell line MDA-MB-231 and the non-tumorigenic epithelial breast cell line NMuMG, and to evaluate the role of TGF-ß1 and AhR pathways. Results show that 0.5 µM CPF and 0.005 µM HCB increased LINE-1 mRNA expression through Smad and AhR signaling in MDA-MB-231. In addition, the methylation of the first sites in 5́'UTR of LINE-1 was reduced by pesticide exposure, although the farther sites remained unaffected. Pesticides modulated ORF1p localization in MDA-MB-231: 0.005 µM HCB and 50 µM CPF increased nuclear translocation, while both induced cytoplasmic retention at 0.5 and 5 µM. Moreover, both stimulated double-strand breaks, enhancing H2AX phosphorylation, coincidentally with ORF1p nuclear localization. In NMuMG similar results were observed, since they heighten LINE-1 mRNA levels. CPF effect was through AhR and TGF-ß1 signaling, whereas HCB action depends only of AhR. In addition, both pesticides increase ORF1p expression and nuclear localization. Our results provide experimental evidence that HCB and CPF exposure modify LINE-1 methylation levels and induce LINE-1 reactivation, suggesting that epigenetic mechanisms could contribute to pesticide-induced breast cancer progression.

Effects of prenatal exposure to air particulate matter on the risk of preterm birth and roles of maternal and cord blood LINE-1 methylation: A birth cohort study in Guangzhou, China.

BACKGROUND: Epidemiological studies have found that increased risk of preterm birth (PTB) is associated with higher prenatal exposure to PM10 and PM2.5, but few studies have been conducted to assess the impacts of extremely fine particulate matter (PM1) which may have more toxic effects than other types of ambient particulate air pollution (PM). Several studies have separately investigated the associations between DNA methylation and PTB risk and PM. Maternal LINE-1 methylation level negatively correlated with prenatal exposure to PM and risk of PTB. A comprehensive picture is lacking regarding the associations between prenatal exposure to PM, LINE-1 methylation, and risk of PTB. OBJECTIVES: This study aimed to estimate the effects of exposure to ambient PM (PM10, PM2.5, and PM1) of different sizes during pregnancy on risk of PTB, identify susceptible exposure windows, and illustrate the roles of LINE-1 methylation in the associations between PM and PTB risk. METHODS: The Birth Cohort Study on Prenatal Environments and Offspring Health (PEOH) has been ongoing since 2016 in Guangzhou, China. A total of 4928 pregnant women were recruited during early pregnancy, and 4278 (86.8%) were successfully followed-up. Each individual weekly exposure to PM10 and PM2.5 from 3 months before pregnancy to childbirth was assessed using a spatiotemporal land use regression model, and the weekly PM1 exposure was estimated by employing a generalized additive model. Maternal and cord blood LINE-1 methylation levels (%5mC) were tested using bisulfite-PCR pyrosequencing. A distributed lag nonlinear model incorporated with a Cox proportional hazard model was applied to assess the effect of weekly-specific maternal PM exposure on PTB risk, and a multiple-linear regression model was employed to investigate the associations between PM exposure and LINE-1 methylation levels of maternal and cord bloods. We also assessed the associations between LINE-1 methylation levels and PTB risk by using a logistic regression model. RESULTS: The risk of PTB was positively associated with PM2.5 and PM1 concentrations during the 12th to 20th gestational weeks, and the strongest association was in the fourth quartile (Q4) versus the first quartile (Q1) and observed during the 16th gestational week (PM2.5: harzard ratio [HR] = 1.18, 95%CI: 1.04-1.35, IQR = 11.94 µg/m3. PM1: HR = 1.20, 95%CI: 1.03-1.39, IQR = 11.36 µg/m3). We observed significantly negative associations of PM10(ß = -0.51%5mC per 10 µg/m3, P = 0.014), PM2.5 (ß = -0.66%5mC per 10 µg/m3, P = 0.032) and PM1 (ß = -0.67%5mC per 10 µg/m3, P = 0.032) concentrations with cord blood LINE-1 methylation levels, and a negative association between PM1 concentration and maternal LINE-1 methylation level (ß = -0.86%5mC per 10 µg/m3, P = 0.034). CONCLUSION: Higher prenatal exposure to PM1 and PM2.5 during the 12th to 20th gestational weeks was associated with increased risk of PTB. Maternal and fetal LINE-1 methylation alternation might be an underlying mechanism of PM that increasing the risk of PTB.

DNA methylome-wide alterations associated with estrogen receptor-dependent effects of bisphenols in breast cancer.

BACKGROUND: Bisphenol A (BPA), an estrogen-like endocrine disruptor used in plastics, has been associated with development and promotion of breast cancer, so plastic manufacturers shifted towards less-studied analogs, BPF and BPS. Studying the associated DNA methylome-wide mechanisms of these derivatives is timely, particularly in comparison with BPA. METHODS: We assessed proliferation, cell cycle, and migration of breast cancer cells (estrogen receptor (ER)-positive: MCF-7 and ER-negative: MDA-MB-231) treated with BPF and BPS ± estrogen receptor inhibitor (ERI) in comparison to BPA ± ERI. RNA expression and activity of DNA (de)methylation enzymes and LINE-1 methylation were quantified. DNA methylome-wide analysis was evaluated in bisphenol-exposed cells and compared to clinical breast cancer data. RESULTS: The three bisphenols caused ER-dependent increased proliferation and migration of MCF-7 but not MDA-MB-231 cells, with BPS being 10 times less potent than BPA and BPF. Although they have similar chemical structures, the three bisphenols induced differential DNA methylation alterations at several genomic clusters of or single CpG sites, with the majority of these being ER-dependent. At equipotent doses, BPA had the strongest effect on the methylome, followed by BPS then BPF. No pathways were enriched for BPF while BPA- and BPS-induced methylome alterations were enriched in focal adhesion, cGMP-PKG, and cancer pathways, which were also dysregulated in methylome-wide alterations comparing ER-positive breast cancer samples to adjacent normal tissues. CONCLUSIONS: The three bisphenols have important epigenetic effects in breast cell lines, with those of BPA and BPS overlapping with cancer-related pathways in clinical breast cancer models. Hence, further investigation of their safety is warranted.

The prevalence and persistence of aberrant promoter DNA methylation in benzene-exposed Chinese workers.

Aberrant DNA methylation patterns are common in cancers and environmental pollutant exposed subjects. Up to date, few studies have examined the aberrant DNA methylation patterns in benzene exposed workers. We recruited 141 benzene-exposed workers, including 83 benzene-exposed workers from a shoe factory in Wenzhou and 58 workers from a painting workshop in Wuhu, 35 workers in Wuhu were followed from 2009 to 2013, and 48 indoor workers as controls from Wenzhou. We used high-resolution melting (HRM) to quantitate human samples of DNA methylation in long interspersed nuclear element-1 (LINE-1), (6)-methylguanine-DNA methyltransferase (MGMT), and DNA mismatch repair gene human mutator L homologue 1 (hMLH1). AML-5 cells were treated with benzoquinone (BQ) and hydroquinone (HQ), and the promoter methylation of MGMT and hMLH1 was detected using the bisulfite sequencing PCR method. The degree of LINE-1 methylation in benzene-exposed workers was significantly lower than that of the controls (p<0.001), and the degree of MGMT (p<0.001) and hMLH1 (p = 0.01) methylation was significantly higher than that of the controls. The in vitro study validated the aberrant hypermethylation of hMLH1 after treatment with BQ. Among the cohort workers who were followed from 2009 to 2013, the LINE1 methylation elevated in 2013 than 2009 (p = 0.004), and premotor methylation in hMLH1 reduced in 2013 than 2009 (p = 0.045) with the reduction of the benzene exposure. This study provides evidence that benzene exposure can induce LINE-1 hypomethylation and DNA repair gene hypermethylation.

N-acetylcysteine reverses the decrease of DNA methylation status caused by engineered gold, silicon, and chitosan nanoparticles.

Introduction: Engineered nanoparticles (ENPs) are one of the most widely used types of nanomaterials. Recently, ENPs have been shown to cause cellular damage by inducing ROS (reactive oxygen species) both directly and indirectly, leading to the changes in DNA methylation levels, which is an important epigenetic mechanism. In this study, we investigated the effect of ENP-induced ROS on DNA methylation. Materials and methods: Human embryonic kidney and human keratinocyte (HaCaT) cells were exposed to three different types of ENPs: gold nanoparticles, silicon nanoparticles (SiNPs), and chitosan nanoparticles (CSNPs). We then evaluated the cytotoxicity of the ENPs by measuring cell viability, morphology, cell apoptosis, cell proliferation, cell cycle distribution and ROS levels. Global DNA methylation levels was measured using 5-methylcytosine immunocytochemical staining and HPLC analysis. DNA methylation levels of the transposable elements, long interspersed element-1 (LINE-1) and Alu, were also measured using combined bisulfite restriction analysis technique. DNA methylation levels of the TEs LINE-1 and Alu were also measured using combined bisulfite restriction analysis technique. Results: We found that HaCaT cells that were exposed to SiNPs exhibited increased ROS levels, whereas HaCaT cells that were exposed to SiNPs and CSNPs experienced global and Alu hypomethylation, with no change in LINE-1 being observed in either cell line. The demethylation of Alu in HaCaT cells following exposure to SiNPs and CSNPs was prevented when the cells were pretreated with an antioxidant. Conclusion: The global DNA methylation that is observed in cells exposed to ENPs is associated with methylation of the Alu elements. However, the change in DNA methylation levels following ENP exposure is specific to particular ENP and cell types and independent of ROS, being induced indirectly through disruption of the oxidative defense process.

Synthesis and Characterization of Specific Reverse Transcriptase Inhibitors for Mammalian LINE-1 Retrotransposons.

Retrotransposons are a type of transposable element (TE) that have amplified to astonishing numbers in mammalian genomes, comprising more than a third of the human and mouse genomes. Long interspersed element class 1 (LINE-1 or L1) retrotransposons are abundant and currently active retroelements in the human and mouse genomes. Similarly, long terminal repeat (LTR)-containing retrotransposons are abundant in both genomes, although only active in mice. LTR- and LINE-1-retroelements use different mechanisms for retrotransposition, although both involve the reverse transcription of an intermediate retroelement-derived RNA. Retrotransposon activity continues to effect the germline and somatic genomes, generating interindividual variability over evolution and potentially influencing cancer and brain physiology, respectively. However, relatively little is known about the functional consequences of retrotransposition. In this study, we have synthesized and characterized reverse transcriptase inhibitors specific for mammalian LINE-1 retrotransposons, which might help deciphering the functional impact of retrotransposition in vivo.

Soy Isoflavone Supplementation Increases Long Interspersed Nucleotide Element-1 (LINE-1) Methylation in Head and Neck Squamous Cell Carcinoma.

AIM: Soy isoflavones have been suggested as epigenetic modulating agents with effects that could be important in carcinogenesis. Hypomethylation of LINE-1 has been associated with head and neck squamous cell carcinoma (HNSCC) development from oral premalignant lesions and with poor prognosis. To determine if neoadjuvant soy isoflavone supplementation could modulate LINE-1 methylation in HNSCC, we undertook a clinical trial. METHODS: Thirty-nine patients received 2-3 weeks of soy isoflavone supplements (300 mg/day) orally prior to surgery. Methylation of LINE-1, and 6 other genes was measured by pyrosequencing in biopsy, resection, and whole blood (WB) specimens. Changes in methylation were tested using paired t tests and ANOVA. Median follow up was 45 months. RESULTS: LINE-1 methylation increased significantly after soy isoflavone (P < 0.005). Amount of change correlated positively with days of isoflavone taken (P = 0.04). Similar changes were not seen in corresponding WB samples. No significant changes in tumor or blood methylation levels were seen in the other candidate genes. CONCLUSION: This is the first demonstration of in vivo increases in tissue-specific global methylation associated with soy isoflavone intake in patients with HNSCC. Prior associations of LINE-1 hypomethylation with genetic instability, carcinogenesis, and prognosis suggest that soy isoflavones maybe potential chemopreventive agents in HNSCC.

Relationship between LINE-1 methylation pattern and pesticide exposure in urban sprayers.

Recently a relationship has been reported between pesticide exposure and changes in global DNA methylation patterns. Urban sprayers are a particularly vulnerable population because of the high risk of pesticide exposure that their work implies. Therefore, the aim of this study was to estimate the changes in the Long Interspersed Nucleotide Element (LINE-1) in urban sprayers and its relationship with pesticide exposure. The study population consisted of 190 individuals stratified into three study groups: no occupational pesticide exposure; moderate exposure, and high exposure. Pesticide exposure and other external factors such as diet, lifestyle, and others were evaluated through a validated questionnaire, and the butyrylcholinesterase enzyme activity was evaluated spectrophotometrically and used as exposure biomarker. DNA methylation was evaluated by pyrosequencing on bisulfite-treated DNA. The results showed a significant decrease of %5mC in both the moderate- and high-exposure groups with respect to the non-exposed group (p < 0.05). In addition, alcohol intake was associated with a higher percentage of LINE- 1 methylation. In conclusion, our results suggest that occupational pesticide exposure and external factors appears to modify the DNA methylation pattern measured through LINE-1.

Gender-specific association of exposure to non-dioxin-like polychlorinated biphenyls during pregnancy with methylation levels of H19 and long interspersed nuclear element-1 in cord blood in the Hokkaido study.

BACKGROUND: Associations between prenatal exposure to polychlorinated biphenyls (PCBs) and reduced birth-size, and between DNA methylation of insulin-like growth factor-2 (IGF-2), H19 locus, and long interspersed nuclear element-1 (LINE-1) and reduced birth-size are well established. To date, however, studies on the associations between prenatal exposure to PCBs and alterations in methylation of IGF-2, H19, and LINE-1 are lacking. Thus, in this study, we examined these associations with infant-gender stratification. METHODS: We performed a prospective birth cohort study using the Sapporo cohort from the previously described Hokkaido Birth Cohort Study on Environment and Children's Health conducted between 2002 and 2005 in Japan. In the final 169 study participants included in this study, we measured the concentrations of various non-dioxin-like PCBs in maternal blood during pregnancy using high-resolution gas chromatography/high-resolution mass spectrometry. IGF-2, H19 and LINE-1 methylation levels in cord blood were measured using the bisulfite pyrosequencing methods Finally, we assessed the associations between prenatal exposure to various PCBs and the gene methylation levels using multiple regression models stratified by infant gender. RESULTS: We observed a 0.017 (95% confidence interval [CI]: 0.003-0.031) increase in the log10-transformed H19 methylation levels (%) in cord blood for each ten-fold increase in the levels of decachlorinated biphenyls (decaCBs) in maternal blood among all infants. Similarly, a 0.005 (95% CI: 0.000-0.010) increase in the log10-transformed LINE-1 methylation levels (%) in cord blood was associated with each ten-fold increase in heptachlorinated biphenyls (heptaCBs) in maternal blood among all infants. In particular, we observed a dose-dependent association of the decaCB levels in maternal blood with the H19 methylation levels among female infants (P value for trend=0.040); likewise a dose-dependent association of heptaCB levels was observed with LINE-1 methylation levels among female infants (P value for trend=0.015). Moreover, these associations were only observed among infants of primiparous women. CONCLUSION: Our results suggest that the dose-dependent association between prenatal exposure to specific non-dioxin-like PCBs and increases in the H19 and LINE-1 methylation levels in cord blood might be more predominant in females than in males.

Simultaneous Modeling of Biomarker and Toxicity Response Predicted Optimal Regimen of Guadecitabine (SGI-110) in Myeloid Malignancies.

Guadecitabine (SGI-110) is a novel next-generation hypomethylating agent (HMA) administered as s.c. injection with extended decitabine exposure. Dose/exposure-response analyses of longitudinal measures of long interspersed nucleotide element-1 (LINE-1) methylation and absolute neutrophil counts (ANC) pooled from 79 and 369 patients in 2 phase I/II trials, respectively, were performed to assist, through modeling and simulation, the selection of dosing regimens for phase III. Simulation of ANC predicted a decrease after a 5-day regimen of 60 mg/m2 with partial recovery before the next cycle, whereas the nadir of 90 mg/m2 on the same schedule was below 100/µl. ANC following a 60 mg/m2 10-day regimen was predicted to be suppressed below 100/µl as long as treatment continued without recovery. The developed models provided useful tools to assist simultaneous evaluation of the relative dynamics of the two effects (DNA demethylation and the effect on ANC).

Analysis of global DNA methylation changes in primary human fibroblasts in the early phase following X-ray irradiation.

Epigenetic alterations may contribute to the generation of cancer cells in a multi-step process of tumorigenesis following irradiation of normal body cells. Primary human fibroblasts with intact cell cycle checkpoints were used as a model to test whether X-ray irradiation with 2 and 4 Gray induces direct epigenetic effects (within the first cell cycle) in the exposed cells. ELISA-based fluorometric assays were consistent with slightly reduced global DNA methylation and hydroxymethylation, however the observed between-group differences were usually not significant. Similarly, bisulfite pyrosequencing of interspersed LINE-1 repeats and centromeric α-satellite DNA did not detect significant methylation differences between irradiated and non-irradiated cultures. Methylation of interspersed ALU repeats appeared to be slightly increased (one percentage point; p = 0.01) at 6 h after irradiation with 4 Gy. Single-cell analysis showed comparable variations in repeat methylation among individual cells in both irradiated and control cultures. Radiation-induced changes in global repeat methylation, if any, were much smaller than methylation variation between different fibroblast strains. Interestingly, α-satellite DNA methylation positively correlated with gestational age. Finally, 450K methylation arrays mainly targeting genes and CpG islands were used for global DNA methylation analysis. There were no detectable methylation differences in genic (promoter, 5' UTR, first exon, gene body, 3' UTR) and intergenic regions between irradiated and control fibroblast cultures. Although we cannot exclude minor effects, i.e. on individual CpG sites, collectively our data suggest that global DNA methylation remains rather stable in irradiated normal body cells in the early phase of DNA damage response.

Multiple endpoints to evaluate pristine and remediated titanium dioxide nanoparticles genotoxicity in lung epithelial A549 cells.

Titanium dioxide nanoparticles (TiO2 NP) are broadly used in a wide range of applications. Several studies have reported that TiO2 NP possess cytotoxic and genotoxic properties that could induce adverse health effects in humans. The FP7 Sanowork project was aimed to minimize occupational hazard and exposure to engineered nanomaterials (ENM), including TiO2 NP, through the surface modification in order to avoid possible adverse toxic effects for humans. In this study we investigated cytotoxicity, genotoxicity and epigenetic properties of TiO2 NP uncoated and coated with silica or citrate, as well as of the benchmark material P25. We used a panel of in vitro assays in the human lung epithelial cell line A549, in order to better understand if the remediation strategy adopted was able to counteract possible toxic effects of uncoated TiO2 NP. Our results showed that the uncoated TiO2 NP were both cytotoxic and genotoxic, and the remediation strategy adopted did not reduce the adverse effects of uncoated TiO2 NP. In particular, the presence of citrate was able to increase their cytotoxicity and genotoxicity, exerting also epigenotoxic effects, as evaluated by the marked reduction of LINE-1 methylation levels.

Sodium bisulfite pyrosequencing revealed that developmental exposure to environmental contaminant mixtures does not affect DNA methylation of DNA repeats in Sprague-Dawley rats.

Hypomethylation of DNA repeats has been linked to diseases and cancer predisposition. Human studies suggest that higher blood concentrations of environmental contaminants (EC) correlate with levels of hypomethylation of DNA repeats in blood. The objective of this study was to examine the effect of in utero and/or lactational exposure to EC on the methylation of DNA repeats (LINE-1 and identifier element) in Sprague-Dawley rat pups at birth, at postnatal day (PND) 21, and in adulthood (PND78-86). From gestation day 0 to PND20, dams were exposed to a mixture "M" of polychlorinated biphenyls (PCB), pesticides, and methylmercury (MeHg), at 0.5 or 1 mg/kg/d (0.5M and M). At birth, some control (C) and M litters were cross-fostered to create the following in utero/postnatal exposure groups: C/C, M/C, C/M, M/M. Additional dams received 1.8 ng/kg/d of a mixture of aryl-hydrocarbon receptor (AhR) agonists (non-ortho-PCB, PC-dibenzodioxins, and PC-dibenzofurans) without or with 0.5M (0.5MAhR). Measurements of EC residue levels confirmed differences in their accumulation across treatments, age, and tissues. Although induction of hepatic detoxification enzyme activities (cytochrome P-450) demonstrated biological effects of treatments, the assessment of methylation in DNA repeats by sodium bisulfite pyrosequencing of liver, spleen, and thymus samples revealed no marked treatment-related effects but significant tissue- and age-related methylation differences. Further studies are required to determine whether absence of significant observable treatment effects on methylation of DNA repeats in the rat relate to tissue, strain, or species differences.