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The Plant Journal : For Cell and... Sep 2023DNA glycosylases remove mispaired or modified bases from DNA initiating the base excision repair (BER) pathway. The DNA glycosylase MBD4 (methyl-CpG-binding domain...
DNA glycosylases remove mispaired or modified bases from DNA initiating the base excision repair (BER) pathway. The DNA glycosylase MBD4 (methyl-CpG-binding domain protein 4) has been functionally characterized in mammals, but not yet in plants, where it is called MBD4-like (MBD4L). Mammalian MBD4 and Arabidopsis recombinant MBD4L excise U and T mispaired with G, as well as 5-fluorouracil (5-FU) and 5-bromouracil (5-BrU) in vitro. Here, we investigate the ability of Arabidopsis MBD4L to remove some of these substrates from the nuclear genome in vivo in coordination with uracil DNA glycosylase (AtUNG). We found that mbd4l mutants are hypersensitive to 5-FU and 5-BrU, as they displayed smaller size, less root growth, and higher cell death than control plants in both media. Using comet assays, we determined BER-associated DNA fragmentation in isolated nuclei and observed reduced DNA breaks in mbd4l plants under both conditions, but particularly with 5-BrU. The use of ung and ung x mbd4l mutants in these assays indicated that both MBD4L and AtUNG trigger nuclear DNA fragmentation in response to 5-FU. Consistently, we here report the nuclear localization of AtUNG based on the expression of AtUNG-GFP/RFP constructs in transgenic plants. Interestingly, MBD4L and AtUNG are transcriptionally coordinated but display not completely overlapping functions. MBD4L-deficient plants showed reduced expression of BER genes and enhanced expression of DNA damage response (DDR) gene markers. Overall, our findings indicate that Arabidopsis MBD4L is critical for maintaining nuclear genome integrity and preventing cell death under genotoxic stress conditions.
Topics: Animals; Arabidopsis; DNA; DNA Damage; DNA Repair; Fluorouracil; Mammals; Uracil-DNA Glycosidase
PubMed: 37278489
DOI: 10.1111/tpj.16344 -
Environmental Science & Technology Jun 2023Constant efforts have been devoted to exploring new disinfection byproducts in drinking water causally related to adverse health outcomes. In this study, five...
Constant efforts have been devoted to exploring new disinfection byproducts in drinking water causally related to adverse health outcomes. In this study, five halogenated nucleobases were identified as emerging disinfection byproducts in drinking water, including 5-chlorouracil, 6-chlorouracil, 2-chloroadenine, 6-chloroguanine, and 5-bromouracil. We developed a solid phase extraction-ultraperformance liquid chromatography-tandem mass spectrometry method with the limits of detection (LOD) and recoveries ranging between 0.04-0.86 ng/L and 54-93%, respectively. The detection frequency of the five halogenated nucleobases ranged from 73 to 100% with a maximum concentration of up to 65.3 ng/L in the representative drinking water samples. The cytotoxicity of the five identified halogenated nucleobases in Chinese hamster ovary (CHO-K1) cells varied with great disparity, in which the cytotoxicity of 2-chloroadenine (IC = 9.4 μM) is appropriately three times higher than emerging DBP 2,6-dichloro-1,4-benzoquinone (IC = 42.4 μM), indicating the significant toxicological risk of halogenated nucleobase-DBPs. To the best of our knowledge, this study reports the analytical method, occurrence, and toxicity of halogenated nucleobase-DBPs for the first time. These findings will provide a theoretical basis for further research on probing the relationship between its mutagenicity and human health risk.
Topics: Cricetinae; Animals; Humans; Disinfection; Drinking Water; CHO Cells; Halogenation; Cricetulus; Water Pollutants, Chemical; Water Purification; Disinfectants
PubMed: 37232460
DOI: 10.1021/acs.est.3c01484 -
Physical Chemistry Chemical Physics :... Jan 2024Core (C, N, and O 1s regions) and valence band electronic structures of bulk uracil and 5-fluoro-, -chloro-, and -iodouracils were investigated using X-ray photoemission...
Core (C, N, and O 1s regions) and valence band electronic structures of bulk uracil and 5-fluoro-, -chloro-, and -iodouracils were investigated using X-ray photoemission spectroscopy and comprehensively compared with those of 5-bromouracil measured under the same experimental conditions before. The halogenation of uracil shifted the core peaks of the 5-position carbons toward the higher binding energy side and reduced the ionization potentials depending on the type of halogen. Theoretical calculations supported these results. The alterations of electronic properties induced by the halogenation would result in the characteristic properties of 5-halouracils.
PubMed: 38240048
DOI: 10.1039/d3cp05932d -
Scientific Reports Aug 20236-Thioguanine is an immunosuppressive drug, an analogue of guanine, applied to treat acute leukemia and inflammatory bowel disease. Excessive use of 6-thioguanine during...
6-Thioguanine is an immunosuppressive drug, an analogue of guanine, applied to treat acute leukemia and inflammatory bowel disease. Excessive use of 6-thioguanine during clinical treatment may cause side effects. Moreover, providing a dose too low will be ineffective. Therefore, there is a critical need for a rapid, selective and routine approach to quantifying 6-thioguanine in body fluids to support a clinical application. A fully validated HPLC method has been developed to determine 6-thioguanine in whole blood samples using 5-bromouracil as an internal standard. 6-Thioguanine nucleotides were released from erythrocytes by perchloric acid, and then hydrolysed at 100 °C to the parent thiopurine, 6-thioguanine. The following validation parameters of the method were determined: specificity/selectivity, linearity range (479-17,118 ng/mL, R > 0.992), limits of detection (150 ng/mL) and quantification (479 ng/mL), accuracy (- 5.6 < Bias < 14.7), repeatability (CV 1.30-3.24%), intermediate precision (CV 4.19-5.78%), extraction recovery (79.1-103.6%) and carryover. Furthermore, the stability of the drug in whole blood samples under various storage conditions was investigated. The suggested method is suitable for determining 6-thioguanine in whole blood erythrocyte samples for drug level monitoring, thus correct dosing.
Topics: Thioguanine; Chromatography, High Pressure Liquid; Erythrocytes; Body Fluids; Bromouracil
PubMed: 37644112
DOI: 10.1038/s41598-023-41426-5