Chemical tolerance related to the ABC transporter gene and DNA methylation in cladocera (Daphnia magna).

We performed multigenerational tests to clarify the chemical tolerance mechanisms of a nontarget aquatic organism, Daphnia magna. We continuously exposed D. magna to a carbamate insecticide (pirimicarb) at lethal or sublethal concentrations (0, 3.8, 7.5, and 15 µg/L) for 15 generations (F0-F14). We then determined the 48 h-EC50 values and mRNA expression levels of acetylcholinesterase, glutathione S-transferase, and ATP (Adenosine triphosphate)-binding cassette transporter (ABCt) in neonates (<24 h old) from F0, F4, F9, and F14. To ascertain the effects of DNA methylation on pirimicarb sensitivity, we measured 5-methylcytosine levels (DNA methylation levels) in neonates of parents in the last generation (F14). In addition, we cultured groups exposed to 0 and 7.5 µg/L (the latter of which acquired chemical tolerance to pirimicarb) with or without 5-azacytidine (de-methylating agent) and determined methylation levels and 48 h-EC50 values in neonates (<24 h old) from the treated parents. The EC50 values (30.3-31.6 µg/L) in F14 of the 7.5 and 15 µg/L groups were approximately two times higher than that in the control (16.0 µg/L). A linear mixed model analysis showed that EC50 and ABCt mRNA levels were significantly increased with generational alterations; further analysis showed that the ABCt mRNA level was positively related to the EC50 . Therefore, ABCt may be associated with altered pirimicarb sensitivity. In addition, the EC50 value and DNA methylation levels in pirimicarb-tolerant clones decreased after exposure to 5-azacytidine, suggesting that DNA methylation contributes to chemical tolerance. These findings improved our knowledge regarding the acquisition of chemical tolerance in aquatic organisms.

Extended Prophylactic Effect of N-tert-Butyl-α-phenylnitron against Oxidative/Nitrosative Damage Caused by the DNA-Hypomethylating Drug 5-Azacytidine in the Rat Placenta.

Antioxidant N-tert-Butyl-α-phenylnitron (PBN) partly protected embryos from the negative effects of a DNA demethylating drug 5-azacytidine during pregnancy. Our aim was to investigate PBN's impact on the placenta. Fischer rat dams were treated on gestation days (GD) 12 and 13 by PBN (40 mg/kg), followed by 5azaC (5 mg/kg) after one hour. Global methylation was assessed by pyrosequencing. Numerical density was calculated from immunohistochemical expression in single cells for proliferating (PCNA), oxidative (oxoguanosine) and nitrosative (nitrotyrosine) activity. Results were compared with the PBN-treated and control rats. PBN-pretreatment significantly increased placental weight at GD15 and GD20, diminished by 5azaC, and diminished apoptosis in GD 20 placentas caused by 5azaC. Oxoguanosine expression in placentas of 5azaC-treated dams was especially high in the placental labyrinth on GD 15, while PBN-pretreatment lowered its expression on GD 15 and GD 20 in both the labyrinth and basal layer. 5azaC enhanced nitrotyrosine level in the labyrinth of both gestational stages, while PBN-pretreatment lowered it. We conclude that PBN exerted its prophylactic activity against DNA hypomethylating agent 5azaC in the placenta through free radical scavenging, especially in the labyrinthine part of the placenta until the last day of pregnancy.

The DNMT1 inhibitor GSK-3484862 mediates global demethylation in murine embryonic stem cells.

BACKGROUND: DNA methylation plays an important role in regulating gene expression in mammals. The covalent DNMT1 inhibitors 5-azacytidine and decitabine are widely used in research to reduce DNA methylation levels, but they impart severe cytotoxicity which limits their demethylation capability and confounds interpretation of experiments. Recently, a non-covalent inhibitor of DNMT1 called GSK-3484862 was developed by GlaxoSmithKline. We sought to determine whether GSK-3484862 can induce demethylation more effectively than 5-azanucleosides. Murine embryonic stem cells (mESCs) are an ideal cell type in which to conduct such experiments, as they have a high degree of DNA methylation but tolerate dramatic methylation loss. RESULTS: We determined the cytotoxicity and optimal concentration of GSK-3484862 by treating wild-type (WT) or Dnmt1/3a/3b triple knockout (TKO) mESC with different concentrations of the compound, which was obtained from two commercial sources. Concentrations of 10 µM or below were readily tolerated for 14 days of culture. Known DNA methylation targets such as germline genes and GLN-family transposons were upregulated within 2 days of the start of GSK-3484862 treatment. By contrast, 5-azacytidine and decitabine induced weaker upregulation of methylated genes and extensive cell death. Whole-genome bisulfite sequencing showed that treatment with GSK-3484862 induced dramatic DNA methylation loss, with global CpG methylation levels falling from near 70% in WT mESC to less than 18% after 6 days of treatment with GSK-3484862. The treated cells showed a methylation level and pattern similar to that observed in Dnmt1-deficient mESCs. CONCLUSIONS: GSK-3484862 mediates striking demethylation in mESCs with minimal non-specific toxicity.

Development of a new in vitro assay system for evaluating the effects of chemicals on DNA methylation.

Epigenetic toxicity, a phenomenon in which chemicals exert epigenetic effects and produce toxicity, has been attracting attention in recent years due to advances in toxicology accompanying the development of life sciences. However, it has been difficult to identify epigenetic toxicants due to the lack of a simple experimental system to evaluate epigenetic toxicity. In this study, we developed a prototype of an in vitro reporter assay system for assessing the effects of chemicals on DNA methylation using two promoters showing different degrees of DNA methylation, Agouti IAP and Daz1 promoters, and a luciferase reporter. The system successfully detected DNA demethylating activity using 5-azacytidine, a chemical having DNA demethylation activity, as a positive control chemical, and demethylation of cytosine of CpG in the promoter was confirmed by pyrosequencing analysis. Next, in order to improve the detection sensitivity of the DNA demethylating activity of this system, we tried to increase the basal level of methylation of the Daz1 promoter by pre-methylase treatment of the reporter vectors. As a result, the detection sensitivity of the system was successfully improved in cells where the basal level of methylation was indeed increased by methylase treatment. Thus, the developed assay system here is effective for the simple evaluation of chemicals that affect DNA methylation.

Treatment of human cells with 5-aza-dC induces formation of PARP1-DNA covalent adducts at genomic regions targeted by DNMT1.

The nucleoside analog 5-aza-2'-deoxycytidine (5-aza-dC) is used to treat some hematopoietic malignancies. The mechanism of cell killing depends upon DNMT1, but is otherwise not clearly defined. Here we show that PARP1 forms covalent DNA adducts in human lymphoblast or fibroblasts treated with 5-aza-dC. Some adducts recovered from 5-aza-dC-treated cells have undergone cleavage by apoptotic caspases 3/7. Mapping of PARP1-DNA adducts, by a new method, "Adduct-Seq", demonstrates adduct enrichment at CpG-dense genomic locations that are targets of maintenance methylation by DNMT1. Covalent protein-DNA adducts can arrest replication and induce apoptosis, and these results raise the possibility that induction of PARP1-DNA adducts may contribute to cell killing in response to treatment with 5-aza-dC.

Protective effect of bleomycin on 5-azacitidine induced cytotoxicity and apoptosis in mice hematopoietic stem cells via Bcl-2/Bax and HMGB1 signaling pathway.

Antineoplastic drugs cause severe cytotoxicity for normal cells, especially hematopoietic stem cells (HSCs). However, bleomycin (BLM) is glycopeptide antibiotic that is effective on various cancers and has either low or no myelosuppression effects. The aim of the present study was to investigate the effect of BLM on 5-Azacitidine (5-AZA) induced cytotoxicity in bone marrow HSCs. 5-AZA reduced HSC cell viability in a time and dose-dependent manner with an IC50 value of 16 µM. However, pretreatment of the cells with BLM for 4 h induced an antagonistic cytotoxicity with an increased IC50 of 64 µM. 5-AZA decreased the colony formation ability of HSC cells in semi-solid agar culture and this effect was attenuated by BLM. 5-AZA significantly downregulated high mobility group Box1 (HMGB1) and Bcl-2 gene expression but upregulated Bax gene expression, while BLM impeded the action of 5-AZA. Pretreatment with BLM remarkably decreased HMGB1 release into culture media that was induced by 5-AZA. The cells were distribution at the sub/G1 phase. Annexin/PI staining of the cells, poly (ADP-ribose) polymerase (PARP) cleavage, and anion superoxide production indicated that BLM limited 5-AZA induced apoptotic cell death. In conclusion, BLM in combination with 5-AZA effectively reduces the adverse cytotoxic effects of 5-AZA on bone marrow hematopoietic stem cells, providing a new chemotherapeutic strategy.

Gas Chromatography-Mass Spectrometry Profiling of Volatile Compounds Reveals Metabolic Changes in a Non-Aflatoxigenic Aspergillus flavus Induced by 5-Azacytidine.

Aspergillus flavus is one of the most opportunistic pathogens invading many important oilseed crops and foodstuffs with such toxic secondary metabolites as aflatoxin (AF) and Cyclopiazonic acid. We previously used the DNA methylation inhibitor 5-azacytidine to treat with an AF-producing A. flavus A133 strain, and isolated a mutant (NT) of A. flavus, which displayed impaired abilities of AF biosynthesis and fungal development. In this study, gas chromatography-mass spectrometry (GC-MS) analysis was used to reveal the metabolic changes between these two strains. A total of 1181 volatiles were identified in these two strains, among which 490 volatiles were found in these two strains in vitro and 332 volatiles were found in vivo. The NT mutant was found to produce decreasing volatile compounds, among which most of the fatty acid-derived volatiles were significantly downregulated in the NT mutant compared to the A133 strain, which are important precursors for AF biosynthesis. Two antioxidants and most of the amino acids derived volatiles were found significantly upregulated in the NT mutant. Overall, our results reveal the difference of metabolic profiles in two different A. flavus isolates, which may provide valuable information for controlling infections of this fungal pathogen.

Lobular neutrophilic panniculitis associated with DNA methyltransferase inhibitors in the treatment of myeloid disease.

Cutaneous toxicities to DNA methyltransferase inhibitors are variable and include localized injection site reactions, ecchymoses, maculopapular eruptions, and neutrophilic dermatoses including pyoderma gangrenosum, Sweet syndrome, and neutrophilic eccrine hidradenitis. This series describes two patients diagnosed with lobular neutrophilic panniculitis arising during treatment of acute myelogenous leukemia with "hypomethylating drugs," including the first report of its occurrence with a next-generation agent. Differential diagnoses, histopathologic characteristics, treatment considerations, and proposed pathogenesis will be discussed.

A Free Radical Scavenger Ameliorates Teratogenic Activity of a DNA Hypomethylating Hematological Therapeutic.

The spin-trap free radical scavenger N-tert-butyl-α-phenylnitron (PBN) ameliorated effects of several teratogens involving reactive oxygen species (ROS). We investigated for the first time whether PBN could ameliorate teratogenesis induced by a DNA hypomethylating hematological therapeutic 5-azacytidine (5azaC). At days 12 and 13 of gestation, Fisher rat dams were pretreated by an i.v. injection of PBN (40 mg/kg) and 1 h later by an i.p. injection of 5azaC (5mg/kg). Development was analyzed at gestation day 15 in embryos and day 20 in fetuses. PBN alone did not significantly affect development. PBN pretreatment restored survival of 5azaC-treated dams' embryos to the control level, restored weight of embryos and partially of fetuses, and partially restored crown-rump lengths. PBN pretreatment converted limb adactyly to less severe oligodactyly. PBN pretreatment restored global DNA methylation level in the limb buds to the control level. Cell proliferation in limb buds of all 5azaC-treated dams remained significantly lower than in controls. In the embryonic liver, PBN pretreatment normalized proliferation diminished significantly by 5azaC; whereas in embryonic vertebral cartilage, proliferation of all 5azaC-treated dams was significantly higher than in PBN-treated dams or controls. Apoptotic indices significantly enhanced by 5azaC in liver and cartilage were not influenced by PBN pretreatment. However, PBN significantly diminished ROS or reactive nitrogen species markers nitrotyrosine and 8-hydroxy-2'deoxyguanosine elevated by 5azaC in embryonic tissues, and, therefore, activity of this DNA hypomethylating agent was associated to the activation of free radicals. That pretreatment with PBN enhanced proliferation in the liver and not in immature tissue is interesting for the treatment of 5azaC-induced hepatotoxicity and liver regeneration.

5-Azacitidine Induces Cell Death in a Tissue Culture of Brachypodium distachyon.

Morphological and histological observations revealed that, at a concentration of 50 µM, 5-azacitidine (5-azaC) totally inhibited the induction of embryogenic masses (EM), while the cultivation of explants (zygotic embryos; ZEs) in the presence of 5 µM of 5-azaC led to the formation of a callus with EM in 10% of the cases. Transmission electron microscopy (TEM) analyzes revealed the presence of the morphological and ultrastructural features that are typical for the vacuolar type of cell death in the callus cells that were treated. A TUNEL assay confirmed the presence of DNA double-strand breaks for the callus cells that had been treated with both 5 and 50 µM 5-azaC concentrations. Analysis of the gene expression of selected cell death markers demonstrated a reduced expression of metacaspase, protein executer 1 (EX1), and thioredoxin (TRX) in the callus cells that had been treated compared to the control culture. The strongest increase in the gene activity was characteristic for glutathione S-transferase (GST). Our studies also included an analysis of the distribution of some arabinogalactan proteins (AGPs) and extensin epitopes, which can be used as markers of cells that are undergoing death in a Brachypodium distachyon tissue culture.