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Chemical Research in Toxicology Feb 2022Apurinic/apyrimidinic (AP) sites appear in DNA spontaneously and as intermediates of base excision DNA repair. AP sites are noninstructive lesions: they strongly block...
Apurinic/apyrimidinic (AP) sites appear in DNA spontaneously and as intermediates of base excision DNA repair. AP sites are noninstructive lesions: they strongly block DNA polymerases, and if bypassed, the nature of the incorporated dNMP is mostly guided by the interactions within the polymerase-DNA active site. Many DNA polymerases follow the "A-rule", preferentially incorporating dAMP opposite to natural AP sites. Methoxyamine (MX), a small molecule, efficiently reacts with the aldehyde moiety of natural AP sites, thereby preventing their cleavage by APEX1, the major human AP endonuclease. MX is currently regarded as a possible sensitizer of cancer cells toward DNA-damaging drugs. To evaluate the mutagenic potential of MX, we have studied the utilization of various dNTPs by five DNA polymerases of different families encountering MX-AP adducts in the template in comparison with the natural aldehydic AP site. The Klenow fragment of DNA polymerase I strictly followed the A-rule with both natural AP and MX-adducted AP sites. Phage RB69 DNA polymerase, a close relative of human DNA polymerases δ and ε, efficiently incorporated both dAMP and dGMP. DNA polymerase β mostly incorporated dAMP and dCMP, preferring dCMP opposite to the natural AP site and dAMP opposite to the MX-AP site, while DNA polymerase λ was selective for dGMP, apparently via the primer misalignment mechanism. Finally, translesion DNA polymerase κ also followed the A-rule for MX-AP and additionally incorporated dCMP opposite to a natural AP site. Overall, the MX-AP site, despite structural differences, was similar to the natural AP site in terms of the dNMP misincorporation preference but was bypassed less efficiently by all polymerases except for Pol κ.
Topics: DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA-Directed DNA Polymerase; Humans; Hydroxylamines
PubMed: 35089032
DOI: 10.1021/acs.chemrestox.1c00359 -
PloS One 2018To investigate the photochemical degradation of trypan blue (TB) and to identify decomposition products.
PURPOSE
To investigate the photochemical degradation of trypan blue (TB) and to identify decomposition products.
METHODS
Defined solution samples of TB and a mixture with lutein/zeaxanthin were exposed to blue light. Thermal degradation processes were ruled out using controls not subjected to irradiation. All samples were analyzed using optical microscopy, UV/Vis spectroscopy, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and nuclear magnetic resonance (NMR) spectrometry. Degradation kinetics were determined based on changes in absorbance; intermediates were identified by analyzing mass differences of characteristic fragment ion peaks within the fragmentation patterns, and assignments were verified by NMR.
RESULTS
TB demonstrated a photochemical degradation, which can be triggered by lutein/zeaxanthin. Intermediates vary depending on the presence of lutein/zeaxanthin. The self-sensitized photodegradation of TB occurs under generation of dimethyl sulfate and presumed formation of phenol. In contrast, within the presence of lutein/zeaxanthin the decomposition of TB indicates the formation of methoxyamine and sulfonyl arin. Thermal degradation processes were not observed.
CONCLUSIONS
TB demonstrated a photodegradation that may be triggered by lutein/zeaxanthin and results in the formation of cytotoxic decomposition products. Our findings contribute to understand degradation mechanisms of TB and may elucidate previous clinical and experimental observations of cellular toxicity after TB application.
Topics: Kinetics; Light; Lutein; Photochemistry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypan Blue; Zeaxanthins
PubMed: 29634764
DOI: 10.1371/journal.pone.0195849 -
Experimental Biology and Medicine... Jun 2016The purpose of this study was to evaluate the combination effect of resveratrol and methoxyamine on radiosensitivity of iododeoxyuridine in spheroid culture of U87MG...
The purpose of this study was to evaluate the combination effect of resveratrol and methoxyamine on radiosensitivity of iododeoxyuridine in spheroid culture of U87MG glioblastoma cell line using colony formation and alkaline comet assays. Spheroids on day-20 with 350 µm diameters were treated with 20 µM resveratrol and/or 6 mM methoxyamine and/or 1 µM iododeoxyuridine for one volume doubling time (67 h), and then irradiated with 2 Gy gamma-radiation ((60)Co) in different groups. After treatment, viability of the cells, colony forming ability and DNA damages were obtained by blue dye exclusion, colony formation and alkaline comet assay, respectively. Our results showed that methoxyamine and resveratrol could significantly reduce colony number and induce the DNA damages of glioblastoma spheroid cells treated with iododeoxyuridine in combination with gamma-rays. Therefore, methoxyamine as base excision repair inhibitor and resveratrol as hypoxia inducible factor 1-alpha inhibitor in combination with iododeoxyuridine as radiosensitizer enhanced the radiosensitization of glioblastoma spheroid cells.
Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Colony-Forming Units Assay; Comet Assay; Gamma Rays; Humans; Hydroxylamines; Idoxuridine; Neuroglia; Radiation Tolerance; Radiation-Sensitizing Agents; Resveratrol; Stilbenes
PubMed: 26748400
DOI: 10.1177/1535370215622583 -
Radiotherapy and Oncology : Journal of... Nov 2016The anti-folate pemetrexed is a radiosensitizer. In pre-clinical models, pemetrexed is more effective along with the base-excision-repair inhibitor methoxyamine. We...
BACKGROUND AND PURPOSE
The anti-folate pemetrexed is a radiosensitizer. In pre-clinical models, pemetrexed is more effective along with the base-excision-repair inhibitor methoxyamine. We tested whether methoxyamine enhances pemetrexed-mediated radiosensitization of lung adenocarcinoma cells and xenografts.
MATERIALS AND METHODS
A549 and H1299 cells were evaluated for cell cycle distribution by flow cytometry, radiosensitization by clonogenic assay, and DNA repair by neutral comet assay and repair protein activation. H460 cells were included in some studies. Xenografts in nude mice received drug(s) and/or radiation, and tumor growth was monitored by caliper and in vivo toxicity by animal weight.
RESULTS
Exposure to pemetrexed/methoxyamine for 24 (H1299, H460) or 48 (A549)hours before irradiation resulted in accumulation of cells near the radiosensitive G1/S border; dose-enhancement factors of 1.62±0.19, 1.97±0.25, and 1.67±0.30, respectively; reduction of mean inactivation dose by 32%, 30%, and 46%, respectively; and significant reductions of SF2 and SF4 (p<0.05). Radiosensitization was associated with rapid DNA double-strand-break rejoining and increased levels of DNA-PKcs. Both tumor-growth rate and tumor-growth delay were significantly improved by adding methoxyamine to pemetrexed pre-irradiation (p<0.0001); no mice lost weight during treatment.
CONCLUSIONS
Addition of methoxyamine to pemetrexed and fractionated radiotherapy may improve outcome for patients with locally advanced non-squamous non-small-cell lung cancer.
Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; DNA Breaks, Double-Stranded; DNA Repair; DNA-Activated Protein Kinase; Dose Fractionation, Radiation; Drug Synergism; Female; Humans; Hydroxylamines; Lung Neoplasms; Mice, Nude; Nuclear Proteins; Pemetrexed; Radiation Tolerance; Radiation-Sensitizing Agents; Xenograft Model Antitumor Assays
PubMed: 27838149
DOI: 10.1016/j.radonc.2016.10.007 -
Investigational New Drugs Feb 2021Temozolomide (TMZ) generates DNA adducts that are repaired by direct DNA and base excision repair mechanisms. Methoxyamine (MX, TRC-102) potentiates TMZ activity by...
Temozolomide (TMZ) generates DNA adducts that are repaired by direct DNA and base excision repair mechanisms. Methoxyamine (MX, TRC-102) potentiates TMZ activity by binding to apurinic and apyrimidinic (AP) sites after removal of N-methyladenine and N-methylguanine, inhibiting site recognition of AP endonuclease. We conducted a phase I trial to determine the maximum tolerated dose and dose-limiting toxicities (DLTs) of intravenous MX when given with oral TMZ. Patients with advanced solid tumors and progression on standard treatment were enrolled to a standard 3 + 3 dose escalation trial assessing escalating doses of TMZ and MX. Tumor response was assessed per RECIST and adverse events (AEs) by CTCAEv3. Pharmacokinetics (PK) of MX and COMET assays on peripheral blood mononuclear cells were performed. 38 patients were enrolled-median age 59.5 years (38-76), mean number of cycles 2.9 [1-13]. No DLTs were observed. Cycle 1 grade 3 AEs included fatigue, lymphopenia, anemia, INR, leukopenia, neutropenia, allergic reaction, constipation, psychosis and paranoia. Cycle 2-13 grade 4 AEs included thrombocytopenia and confusion. A partial response was seen in 1 patient with a pancreatic neuroendocrine tumor (PNET) and six additional patients, each with different tumor types, demonstrated prolonged stable disease. MX PK was linear with dose and was not affected by concomitant TMZ. TMZ 200 mg/m daily × 5 may be safely administered with MX 150 mg/m intravenously once on day 1 with minimal toxicity. Further studies assessing this drug combination in select tumor types where temozolomide has activity may be warranted.
Topics: Adult; Aged; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; DNA Repair; Dose-Response Relationship, Drug; Drug Synergism; Female; Half-Life; Humans; Hydroxylamines; Male; Maximum Tolerated Dose; Metabolic Clearance Rate; Middle Aged; Neoplasms; Temozolomide
PubMed: 32556884
DOI: 10.1007/s10637-020-00962-x -
Antibiotics (Basel, Switzerland) Mar 2019Fluorescent probes are widely used for imaging and measuring dynamic processes in living cells. Fluorescent antibiotics are valuable tools for examining...
Fluorescent probes are widely used for imaging and measuring dynamic processes in living cells. Fluorescent antibiotics are valuable tools for examining antibiotic⁻bacterial interactions, antimicrobial resistance and elucidating antibiotic modes of action. Profluorescent nitroxides are 'switch on' fluorescent probes used to visualize and monitor intracellular free radical and redox processes in biological systems. Here, we have combined the inherent fluorescent and antimicrobial properties of the fluoroquinolone core structure with the fluorescence suppression capabilities of a nitroxide to produce the first example of a profluorescent fluoroquinolone-nitroxide probe. Fluoroquinolone-nitroxide (FN) exhibited significant suppression of fluorescence (>36-fold), which could be restored via radical trapping (fluoroquinolone-methoxyamine ) or reduction to the corresponding hydroxylamine . Importantly, FN was able to enter both Gram-positive and Gram-negative bacterial cells, emitted a measurable fluorescence signal upon cell entry (switch on), and retained antibacterial activity. In conclusion, profluorescent nitroxide antibiotics offer a new powerful tool for visualizing antibiotic⁻bacterial interactions and researching intracellular chemical processes.
PubMed: 30836686
DOI: 10.3390/antibiotics8010019 -
Bioorganic & Medicinal Chemistry May 2017Human apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1) is a multifunctional protein which is essential in the base excision repair (BER) pathway... (Review)
Review
Human apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1) is a multifunctional protein which is essential in the base excision repair (BER) pathway of DNA lesions caused by oxidation and alkylation. This protein hydrolyzes DNA adjacent to the 5'-end of an apurinic/apyrimidinic (AP) site to produce a nick with a 3'-hydroxyl group and a 5'-deoxyribose phosphate moiety or activates the DNA-binding activity of certain transcription factors through its redox function. Studies have indicated a role for APE1/Ref-1 in the pathogenesis of cancer and in resistance to DNA-interactive drugs. Thus, this protein has potential as a target in cancer treatment. As a result, major efforts have been directed to identify small molecule inhibitors against APE1/Ref-1 activities. These agents have the potential to become anticancer drugs. The aim of this review is to present recent progress in studies of all published small molecule APE1/Ref-1 inhibitors. The structures and activities of APE1/Ref-1 inhibitors, that target both DNA repair and redox activities, are presented and discussed. To date, there is an urgent need for further development of the design and synthesis of APE1/Ref-1 inhibitors due to high importance of this protein target.
Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Enzyme Inhibitors; Humans; Neoplasms; Oxidation-Reduction
PubMed: 28161249
DOI: 10.1016/j.bmc.2017.01.028 -
Journal of Bioscience and Bioengineering Dec 2017A novel derivatization method for gas chromatography/mass spectrometry (GC/MS)-based metabolomics was developed, based on solid-phase analytical derivatization (SPAD)...
A novel derivatization method for gas chromatography/mass spectrometry (GC/MS)-based metabolomics was developed, based on solid-phase analytical derivatization (SPAD) with methoximation followed by trimethylsilylation. This SPAD method realized derivatization on solid phases combining strong anion exchange with strong cation exchange. To omit a sample condensation process, GC/MS injection was performed using a large-volume injection mode. This mode uses a stomach-shaped insert, and enables a large quantity of sample to be vaporized and introduced into the GC/MS system. In the present study, several parameters were investigated for each SPAD step. The optimal derivatization conditions were determined to be 3-min-methoximation with 5 μL of >5% methoxyamine solution, and 10-min-trimethylsilylation with 25 μL of N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA). Derivatized analytes were effectively eluted with 25 μL of n-hexane. The influences of coexisting substances were also investigated. Coexisting saccharides did not significantly affect the derivatization of analytes. Moreover, saccharides were efficiently washed out using 80% (v/v) acetonitrile in water. The influences of coexisting sodium chloride were negated by dilution of the sample solution with water. The developed method enables the derivatization of both anionic and cationic metabolites, and high-throughput sample preparation. The coverage of detectable metabolites for the developed method was similar to that of the conventional method. This is the first report of a SPAD-based human plasma metabolome analysis protocol.
Topics: Acetamides; Fluoroacetates; Gas Chromatography-Mass Spectrometry; Hexanes; Humans; Metabolome; Metabolomics; Trimethylsilyl Compounds
PubMed: 28800906
DOI: 10.1016/j.jbiosc.2017.07.006 -
European Journal of Pharmaceutical... Oct 2020Atomoxetine (ATX), a selective and potent inhibitor of the presynaptic norepinephrine transporter, is used mainly to treat attention-deficit hyperactivity disorder....
Atomoxetine (ATX), a selective and potent inhibitor of the presynaptic norepinephrine transporter, is used mainly to treat attention-deficit hyperactivity disorder. Although multiple adverse effects associated with ATX have been reported including severe liver injuries, the mechanisms of ATX-related toxicity remain largely unknown. Metabolism frequently contributes to adverse effects of a drug through reactive metabolites, and the bioactivation status of ATX is still not investigated yet. Here, we systematically investigated ATX metabolism, bioactivation, species difference in human, mouse, and rat liver microsomes (HLM, MLM, and RLM) and in mice using metabolomic approaches as mice and rats are commonly used animal models for the studies of drug toxicity. We identified thirty one ATX metabolites and adducts in LMs and mice, 16 of which are novel. In LMs, we uncovered two methoxyamine-trapped aldehydes, two cyclization metabolites, detoluene-ATX, and ATX-N-hydroxylation for the first time. Detoluene-ATX and one cyclization metabolite were also observed in mice. Using chemical inhibitors and recombinant CYP enzymes, we demonstrated that CYP2C8 and CYP2B6 mainly contribute to the formation of aldehyde; CYP2D6 is the dominant enzyme for the formation of ATX cyclization and detoluene-ATX; CYP3A4 is major enzyme responsible for the hydroxylamine formation. The findings concerning aldehydes should be very useful to further elucidate the mechanistic aspects of adverse effects associated with ATX from metabolic angles. Additionally, the species differences for each metabolite should be helpful to investigate the contribution of specific metabolites to ATX toxicity and possible drug-drug interactions in suitable models.
Topics: Animals; Atomoxetine Hydrochloride; Attention Deficit Disorder with Hyperactivity; Metabolomics; Mice; Microsomes, Liver; Norepinephrine; Rats
PubMed: 32712217
DOI: 10.1016/j.ejps.2020.105488 -
PloS One 2015Indoleamine 2,3-dioxygenase-1 (IDO) is an immune regulatory enzyme expressed by most human tumors. IDO levels in tumor cells correlate with increased metastasis and poor...
Indoleamine 2,3-dioxygenase-1 (IDO) is an immune regulatory enzyme expressed by most human tumors. IDO levels in tumor cells correlate with increased metastasis and poor patient outcome and IDO is linked to tumor cell resistance to immunotherapy, radiation therapy, and chemotherapy. Knowledge of tumor cell-autonomous effects of IDO, independent of its well-known role in regulating and suppressing anti-tumor immune responses, is limited. Clonal populations of A549 human lung adenocarcinoma cells stably transfected with anti-IDO shRNA or scrambled control shRNA were used to study IDO effects on drug sensitivity and resistance. IFNγ was used to induce IDO in those cells. We show, for the first time, that IDO mediates human tumor cell resistance to the candidate anticancer drugs FK866 (an NAD+ inhibitor), methoxyamine (MX, a base excision repair [BER] inhibitor) and approved anticancer drugs pemetrexed (a folate anti-metabolite) and gemcitabine (a nucleoside analogue), and combined treatment with pemetrexed and MX, in the absence of immune cells. Concurrent knockdown of IDO and thymidylate synthase (TS, a key rate-limiting enzyme in DNA synthesis and repair) sensitizes human lung cancer cells to pemetrexed and 5FUdR to a greater degree than knockdown of either target alone. We conclude that BER in IDO-expressing A549 cells plays a major role in mediating resistance to a range of approved and candidate anticancer drugs. IDO inhibitors are undergoing clinical trials primarily to improve antitumor immune responses. We show that targeting IDO alone or in combination with TS is a potentially valuable therapeutic strategy for cancer treatment, independent of immune activity and in combination with conventional chemotherapy.
Topics: Acrylamides; Animals; BRCA2 Protein; Cell Line, Tumor; Clone Cells; DNA Repair; Deoxycytidine; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Induction; Floxuridine; Gene Knockdown Techniques; Humans; Hydroxylamines; Indoleamine-Pyrrole 2,3,-Dioxygenase; Mice, SCID; NAD; Pemetrexed; Piperidines; RNA, Small Interfering; Thymidylate Synthase; Xenograft Model Antitumor Assays; Gemcitabine
PubMed: 26579709
DOI: 10.1371/journal.pone.0143435