scholarly journals Measurement of oxidized and methylated DNA bases by HPLC with electrochemical detection

1996 ◽  
Vol 318 (1) ◽  
pp. 21-23 ◽  
Author(s):  
Harparkash KAUR ◽  
Barry HALLIWELL
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Damage ◽  
Electrochemical Detection ◽  
Oxidative Dna Damage ◽  
The Other ◽  
Dna Bases ◽  
Cancer Development ◽  
Methylated Dna ◽  
Important Contributor

Oxidative DNA damage is thought to be an important contributor to cancer development and to be affected by dietary constituents, so its accurate measurement is important. DNA methylation is recognized as an important mechanism affecting gene expression. In the present paper we describe an HPLC-with-electrochemical-detection procedure that allows rapid and sensitive measurement of four oxidized (2,6-diamino-4-hydroxy-5-formamidopyrimidine, 5-hydroxyuracil, 8-hydroxyguanine, 8-hydroxyadenine) and three methylated (7-methylguanine, 1-methylguanine, O6-methylguanine) bases in acid hydrolysates of DNA. Guanine was also detected, but was clearly separated from the other bases.

scholarly journals Changes of DNA methylation and hydroxymethylation in plant protoplast cultures.

2013 ◽  
Vol 60 (1) ◽  
Author(s):  
Pavla Moricová ◽  
Vladan Ondřej ◽  
Božena Navrátilová ◽  
Lenka Luhová
Keyword(s):  
Dna Methylation ◽  
Dna Damage ◽  
Gene Regulation ◽  
Oxidative Dna Damage ◽  
Cytosine Methylation ◽  
Protoplast Culture ◽  
The Other ◽  
Plant Protoplast ◽  
Higher Eukaryotes ◽  
Methylation Patterns

Cytosine methylation patterns in higher eukaryotes are important in gene regulation. Along with 5-methylcytosine (5-mC), a newly discovered constituent of mammalian DNA, 5-hydroxymethylcytosine (5-hmC), is the other modified base in higher organisms. In this study we detected 5-hmC in plant protoplast DNA and demonstrated its increasing content during the first 72 hrs. of protoplast cultivation. In contrast to 5-hmC, the amount of 5-mC decreased during protoplast cultivation. It was also found that 5-hmC did not primarily arise as a product of oxidative DNA damage following protoplast culture.

scholarly journals Genotoxicity and Gene Expression in the Rat Lung Tissue following Instillation and Inhalation of Different Variants of Amorphous Silica Nanomaterials (aSiO2 NM)

Nanomaterials ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1502
Author(s):  
Fátima Brandão ◽  
Carla Costa ◽  
Maria João Bessa ◽  
Elise Dumortier ◽  
Florence Debacq-Chainiaux ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Amorphous Silica ◽  
Oxidative Dna Damage ◽  
Intratracheal Instillation ◽  
Dna Lesions ◽  
Lung Cells ◽  
Rat Lung ◽  
Inhalation Study

Several reports on amorphous silica nanomaterial (aSiO2 NM) toxicity have been questioning their safety. Herein, we investigated the in vivo pulmonary toxicity of four variants of aSiO2 NM: SiO2_15_Unmod, SiO2_15_Amino, SiO2_7 and SiO2_40. We focused on alterations in lung DNA and protein integrity, and gene expression following single intratracheal instillation in rats. Additionally, a short-term inhalation study (STIS) was carried out for SiO2_7, using TiO2_NM105 as a benchmark NM. In the instillation study, a significant but slight increase in oxidative DNA damage in rats exposed to the highest instilled dose (0.36 mg/rat) of SiO2_15_Amino was observed in the recovery (R) group. Exposure to SiO2_7 or SiO2_40 markedly increased oxidative DNA lesions in rat lung cells of the exposure (E) group at every tested dose. This damage seems to be repaired, since no changes compared to controls were observed in the R groups. In STIS, a significant increase in DNA strand breaks of the lung cells exposed to 0.5 mg/m3 of SiO2_7 or 50 mg/m3 of TiO2_NM105 was observed in both groups. The detected gene expression changes suggest that oxidative stress and/or inflammation pathways are likely implicated in the induction of (oxidative) DNA damage. Overall, all tested aSiO2 NM were not associated with marked in vivo toxicity following instillation or STIS. The genotoxicity findings for SiO2_7 from instillation and STIS are concordant; however, changes in STIS animals were more permanent/difficult to revert.

scholarly journals Oxidative DNA Damage Modulates DNA Methylation Pattern in Human Breast Cancer 1 (BRCA1) Gene via the Crosstalk between DNA Polymerase β and a de novo DNA Methyltransferase

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 225 ◽  
Author(s):  
Zhongliang Jiang ◽  
Yanhao Lai ◽  
Jill M. Beaver ◽  
Pawlos S. Tsegay ◽  
Ming-Lang Zhao ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Dna Methylation ◽  
Dna Damage ◽  
Dna Polymerase ◽  
Dna Methyltransferase ◽  
Oxidative Dna Damage ◽  
De Novo ◽  
Brca1 Gene ◽  
Methylation Pattern ◽  
Dna Polymerase Β

DNA damage and base excision repair (BER) are actively involved in the modulation of DNA methylation and demethylation. However, the underlying molecular mechanisms remain unclear. In this study, we seek to understand the mechanisms by exploring the effects of oxidative DNA damage on the DNA methylation pattern of the tumor suppressor breast cancer 1 (BRCA1) gene in the human embryonic kidney (HEK) HEK293H cells. We found that oxidative DNA damage simultaneously induced DNA demethylation and generation of new methylation sites at the CpGs located at the promoter and transcribed regions of the gene ranging from −189 to +27 in human cells. We demonstrated that DNA damage-induced demethylation was mediated by nucleotide misincorporation by DNA polymerase β (pol β). Surprisingly, we found that the generation of new DNA methylation sites was mediated by coordination between pol β and the de novo DNA methyltransferase, DNA methyltransferase 3b (DNMT3b), through the interaction between the two enzymes in the promoter and encoding regions of the BRCA1 gene. Our study provides the first evidence that oxidative DNA damage can cause dynamic changes in DNA methylation in the BRCA1 gene through the crosstalk between BER and de novo DNA methylation.

DNA Methylation and Gene Expression Analysis in a Phase II Randomized Study of Decitabine Vs. Decitabine Plus Valproic Acid in MDS and AML.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3808-3808
Author(s):  
Ryan J Castoro ◽  
Noel J Raynal ◽  
Xuelin Huang ◽  
Carlos E. Bueso-Ramos ◽  
Guillermo Garcia-Manero ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Valproic Acid ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Response Rate ◽  
Randomized Study ◽  
Fold Increase ◽  
The Other ◽  
Dna Methylation Inhibitors

Abstract Abstract 3808 Poster Board III-744 DNA methylation is a common epigenetic mechanism of gene silencing in patients with the Myelodysplastic Syndrome (MDS) and Acute Myelogenous Leukemia (AML). Epigenetic therapy with drugs which inhibit DNA methylation such as 5-azacytidine and 5-aza-2'-deoxycytidine (decitabine) have proven to be clinically potent in MDS and AML. In addition to DNA methylation inhibitors, histone deacetylase inhibitors (HDACi) have activity in leukemias, and at low doses show epigenetic synergy with DNA methylation inhibitors. To test this synergy in the clinic, we designed a phase II randomized study comparing decitabine alone (20 mg/m2 IV daily x 5 every 4 weeks) to decitabine (same dose) plus valproic acid (50 mg/kg PO daily for 7 days started at the same time as decitabine). We have previously reported interim results from this study, showing an overall response rate of 64% in MDS/CMML (CR in 39%) and 46% in AML (CR in 25%) with no significant differences in response or survival between the two arms. We now report on molecular analyses in this trial. We have studied DNA methylation of ALOX12, LINE1, MapK15, miR124a-1 and 3 and P15 using bisulfite-pyrosequencing, and expression of ATM, mi124a, p15 and p21 by qPCR at baseline and at days 5, 12 and 30 after initiation of therapy in 60 (32 for expression) patients treated on the study (33 received decitabine, 27 received decitabine + valproic acid, overall there were 31CRs or HI's and 28 NRs, 1 patient was inevaluable for response). Global methylation (measured by LINE1) decreased at day 5 by an average of 6.8 ±1.8% in the DAC arm and 3.5 ± 1.2% in the DAC/VPA arm (p=0.20). At day 12, the decrease (from baseline) was by 10.2 ± 2.2% in the DAC arm and 7.0 ± 1.5% in the DAC/VPA arm (p=0.32). At day 30 we observed a decrease of 6.4 ± 1.4% in the DAC arm and 4.8 ±2.2% in the DAC/VPA arm. We found no statistical differences between the two arms in any of the other genes studied for hypomethylation. By qPCR, expression of p15 at day 5 increased by 1.2±0.6 fold in the DAC arm and by 2.5±0.7 fold in the DAC/VPA arm (p=0.01). We found no differences in the other 3 genes studied between the two arms. We next asked about correlations between epigenetic modulation and response. There was no association between LINE1 methylation change at days 5, 12 or 30 and response. By contrast, sustained hypomethylation of miR124a1 correlated with response; at day 5, miR124a methylation had changed by -17.9 ± 3.7% in responders vs. -15.2 ± 5.8% in non-responders, while at day 30, methylation decreased further to -24±6.5%% in responders, but had already partially recovered to -5.3 ±5.8% of baseline in non-responders (p=0.029 for a comparison between responders and non-responders). Additionally we found that responders hypomethylated miR124a-3 faster by a change in methylation of -34.1 ± 6.3% at day 5 compared to non-responders who had -14.8 ± 6.3% at day 5 (p= 0.039). However there was no difference at day 30. By qPCR we studied the same genes as previously listed. We found that responders had a larger induction of p15 gene expression at day 5, 2.3 ± 0.75 fold compared to non-responders who had a 0.91 ± 0.66 fold increase (p=0.018). We also found a similar pattern in expression induction in the ATM gene, where responders at day 5 had a 1.92 ± 0.51 fold increase as compared to non-responders who had a 0.3 ± 0.64 fold increase (p=0.034). Similarly, for the mature miR124a locus, responders had a 2.91 ± 0.88 fold increase in expression at day 30 compared to non-responders who had 1.1 ± 0.24 fold change in gene expression (p=0.03). In conclusion, we found that adding Valproic acid to decitabine enhances activation of P15, but also shows trends for reducing hypomethylation induction, which is consistent with in-vitro studies. These opposing trends may explain why the response rate is not dramatically different in the two arms. We also found that sustained gene specific hypomethylation correlates with response, as does induction of expression of P15, miR124 and ATM, which confirms and extends our prior studies. Thus, modulation of DNA methylation and gene expression appears to be associated with response to decitabine, and testing whether histone deacetylase inhibitors enhance this response will require non-overlapping dosing regimens and, likely, more potent HDAC inhibitors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals EGCG Prevents High Fat Diet-Induced Changes in Gut Microbiota, Decreases of DNA Strand Breaks, and Changes in Expression and DNA Methylation ofDnmt1andMLH1in C57BL/6J Male Mice

2017 ◽  
Vol 2017 ◽  
pp. 1-17 ◽  
Author(s):  
Marlene Remely ◽  
Franziska Ferk ◽  
Sonja Sterneder ◽  
Tahereh Setayesh ◽  
Sylvia Roth ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Damage ◽  
Gut Microbiota ◽  
High Fat Diet ◽  
Dna Methyltransferase ◽  
Dna Methyltransferase 1 ◽  
Male Mice ◽  
High Fat ◽  
Methyltransferase 1

Obesity as a multifactorial disorder involves low-grade inflammation, increased reactive oxygen species incidence, gut microbiota aberrations, and epigenetic consequences. Thus, prevention and therapies with epigenetic active antioxidants, (-)-Epigallocatechin-3-gallate (EGCG), are of increasing interest. DNA damage, DNA methylation and gene expression ofDNA methyltransferase 1,interleukin 6, andMutL homologue 1were analyzed in C57BL/6J male mice fed a high-fat diet (HFD) or a control diet (CD) with and without EGCG supplementation. Gut microbiota was analyzed with quantitative real-time polymerase chain reaction. An induction of DNA damage was observed, as a consequence of HFD-feeding, whereas EGCG supplementation decreased DNA damage. HFD-feeding induced a higher inflammatory status. Supplementation reversed these effects, resulting in tissue specific gene expression and methylation patterns ofDNA methyltransferase 1andMutL homologue 1. HFD feeding caused a significant lower bacterial abundance. TheFirmicutes/Bacteroidetesratio is significantly lower in HFD + EGCG but higher in CD + EGCG compared to control groups. The results demonstrate the impact of EGCG on the one hand on gut microbiota which together with dietary components affects host health. On the other hand effects may derive from antioxidative activities as well as epigenetic modifications observed on CpG methylation but also likely to include other epigenetic elements.

Different Mechanisms Between Copper and Iron in Catecholamines-Mediated Oxidative DNA Damage and Disruption of Gene Expression In Vitro

2010 ◽  
Vol 20 (1) ◽  
pp. 84-92 ◽  
Author(s):  
Yoshihiko Nishino ◽  
Motozumi Ando ◽  
Rena Makino ◽  
Koji Ueda ◽  
Yoshinori Okamoto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Oxidative Dna Damage

scholarly journals Molecular Alterations in Chronic Myelomonocytic Leukemia Monocytes: Transcriptional and Methylation Profiling

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3889-3889
Author(s):  
Anca Franzini ◽  
Jamshid S Khorashad ◽  
Hein Than ◽  
Anthony D. Pomicter ◽  
Dongqing Yan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Damage ◽  
Transcription Factors ◽  
Chronic Myelomonocytic Leukemia ◽  
Healthy Controls ◽  
Age Related ◽  
Dna Methylation Profiling ◽  
Myelomonocytic Leukemia ◽  
Methylation Profiling

Abstract Chronic myelomonocytic leukemia (CMML) is a genetically heterogeneous hematopoietic stem cell disorder that combines features of a myelodysplastic syndrome and a myeloproliferative neoplasm and exhibits a strong bias towards older age. The prognosis of CMML is poor, with overall survival of less than 3 years in most studies, however recurrent somatic mutations explain only 15-24% of the clinical heterogeneity of CMML (Elena C. et al. Blood 128:1408-17, 2016). The extreme skewing of the CMML age distribution suggests that CMML reflects the malignant conversion of the myelomonocytic-biased differentiation characteristic of an aged hematopoietic system. We hypothesized that separating the contribution of the normal aging process from bona fide CMML-specific alterations will improve the molecular characterization and biological understanding of CMML. We decided to focus on monocytes as the phenotypic minimal common denominator of genetically heterogeneous diseases. CD14+ monocytes were sorted from the blood of untreated CMML patients (N=12, median age 77 years, range 61-90), age-matched healthy controls (old controls: N=12, median age 68 years, range 62-74) and young healthy controls (young controls: N=16, median age 29 years, range 24-44) and subjected to RNA sequencing and DNA methylation profiling. Differentially expressed genes in CMML monocytes compared to healthy controls were identified with DESeq2 using a 1% false discovery rate (FDR) and a fold-change cutoff set at >│2│ (Figure 1A). We identified the 2480 CMML-specific genes by subtracting all genes with significant differences in the young controls vs. old controls comparison from the CMML vs. old controls comparison. The top-25 most significantly upregulated genes (Figure 1B) included transcription factors, TNFα signaling genes, genes that regulate genomic stability, and genes involved in apoptosis. The most significantly downregulated transcripts were genes involved in response to DNA damage, RNA binding, monocyte differentiation and mediators of inflammatory process. To link these observations to function, we imputed the 2480 CMML-specific differentially expressed genes into the ingenuity pathway analysis (IPA) application. This analysis uncovered significant enrichment of pathways involved in: mitotic roles of Polo-like kinase, G2/M DNA damage checkpoint regulation, lymphotoxin β receptor signaling, IL-6 signaling and ATM signaling (Figure 1C). DNA methylation profiling revealed 909 differentially methylated regions (DMRs) between CMML and age-matched controls, with most regions being hypermethylated in CMML monocytes. Of these, 37% of the DMRs were intronic, 22% were exonic, 14 % were in the promoter region (Figure 1D), 10% were downstream, 10% were upstream, the remainder were 3' and 5'-overlaps. We also performed integrated analysis using the promoter DMRs and the gene expression profile to identify CMML-associated genes that are likely to be regulated by specific changes in methylation. We observed concomitant changes in CMML-specific mRNA transcripts and DNA methylation promoter regions in the CMML vs. old controls contrast for 10 genes (Figure 1E). AOAH, SERINC5, TAF3 and AHCYL1 were downregulated and hypermethylated; MS4A3, TNF, VCAM1, and IFT80, were upregulated and hypermethylated; TUBA1B was upregulated and hypomethylated and PITPNA was downregulated and hypomethylated. Our study is the first to combine transcriptional and methylation profiling for molecular characterization of CMML monocytes. Conclusions: (i) age-related gene expression changes contribute significantly to the CMML transcriptome; (ii) the CMML-specific transcriptome is characterized by differential regulation of transcription factors, inflammatory response genes and anti-apoptotic pathway genes; (iii) differences in promoter methylation represent only a small proportion of overall differences in methylation, suggesting that intragenic or intronic methylation is a major contributor to the leukemic phenotype; (iv) age-related changes may be necessary, but are not sufficient to realize the CMML phenotype. Figure 1. Figure 1. Disclosures Deininger: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.

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