The human MutT homolog 1 (hMTH1, human NUDT1) hydrolyzes oxidatively damaged nucleoside triphosphates and is the main enzyme responsible for nucleotide sanitization. hMTH1 recently has received attention as an anticancer target because hMTH1 blockade leads to accumulation of oxidized nucleotides in the cell, resulting in mutations and death of cancer cells. Unlike Escherichia coli MutT, which shows high substrate specificity for 8-oxoguanine nucleotides, hMTH1 has broad substrate specificity for oxidized nucleotides, including 8-oxo-dGTP and 2-oxo-dATP. However, the reason for this broad substrate specificity remains unclear. Here, we determined crystal structures of hMTH1 in complex with 8-oxo-dGTP or 2-oxo-dATP at neutral pH. These structures based on high quality data showed that the base moieties of two substrates are located on the similar but not the same position in the substrate binding pocket and adopt a different hydrogen-bonding pattern, and both triphosphate moieties bind to the hMTH1 Nudix motif (i.e. the hydrolase motif) similarly and align for the hydrolysis reaction. We also performed kinetic assays on the substrate-binding Asp-120 mutants (D120N and D120A), and determined their crystal structures in complex with the substrates. Analyses of bond lengths with high-resolution X-ray data and the relationship between the structure and enzymatic activity revealed that hMTH1 recognizes the different oxidized nucleotides via an exchange of the protonation state at two neighboring aspartate residues (Asp-119 and Asp-120) in its substrate binding pocket. To our knowledge, this mechanism of broad substrate recognition by enzymes has not been reported previously and may have relevance for anticancer drug development strategies targeting hMTH1.

In vivo electron spin resonance (ESR) spectroscopy is a noninvasive technique that measures the oxidative stress in living experimental animals. The rate of decay of the ESR signal right after an injection of nitroxyl radical has been measured to evaluate the oxidative stress in animals, although the probe's disposition could also affect this rate. Because the amount of probes forming the redox pair of hydroxyl amine and its corresponding nitroxyl radical was shown to be nearly constant in most organs or tissues 10min after the injection of 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) in mice, we evaluated the oxidative stress in sepsis model mice induced by lipopolysaccharide (LPS) by intravenously injecting ACP as a precursor of redox probes. The in vivo ESR signal increased up to 7-8min after the ACP injection and then decreased. Decay of the in vivo signal in LPS-treated mice was significantly slower than that in healthy mice, whereas no significant difference was observed in the rate of change in the total amount of redox probes in the blood and liver between these groups. ESR imaging showed that the in vivo signals observed at the chest and upper abdomen decayed slowly in LPS-treated mice. Suppression of the decay in LPS-treated mice was canceled by the administration of a combination of pegylated superoxide dismutase and catalase, or an inhibitor of nitric oxide synthase, or gadolinium chloride. These results indicate that the LPS-treated mouse is under oxidative stress and that reactive oxygen species, such as superoxide and peroxynitrite, related to macrophages are mainly involved in the oxidative stress.

Background: In order to investigate the combination effect of anticancer drugs and X-ray irradiation on neurotoxic side-effects (neurotoxicity), a method that provides homogeneously X-ray-irradiated cells was newly established. Materials and Methods: PC12 cell suspension was irradiated by X-ray (0.5 Gy) in serum-supplemented medium, immediately inoculated into 96-microwell plates and incubated overnight. The medium was replaced with fresh serum-depleted medium containing 50 ng/ml nerve growth factor to induce differentiation toward nerve-like cells with characteristic neurites according to the overlay method without changing the medium. The differentiated cells were treated by anticancer drugs as well as antioxidants, oxaliplatin or bortezomib, and the viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide method. Results: Antioxidants and anticancer drugs were cytotoxic to differentiating PC12 cells. Combination of anticancer drugs and X-ray irradiation slightly reduced cell viability. Conclusion: The present 'population irradiation method' may be useful for the investigation of the combination effect of X-ray irradiation and any pharmaceutical drug. X-Rays are used for many purposes in various fields. This includes the visualization of the distribution and therapeutic effects of drugs by X-ray computed tomography (1); the assessment of chemical element changes by energy dispersive X-ray spectrometry (2); the analysis of crystal structure by Xray diffraction (3); the analysis of oral mucosal distribution of trace metal elements by X-ray fluorescence with synchrotron radiation and particle-induced X-ray emission, and the estimation of chemical states of elements by X-ray absorption fine-structure analysis (4); the visual inspection of passenger baggage in airports by X-ray images (5); and therapeutic applications in medicine (6). Radiation, such as X-rays, and drugs, are reported to exert dose-dependent biphasic effects (7). X-Ray irradiation has been reported to stimulate the production of reactive oxygen-species such as superoxide and nitrite (8, 9), the secretion of Fas ligand (10), the accumulation of cells in the G 2 +M phase in the cell cycle (11), and the shortening of telomeres (12). On the other hand, at lower doses of X-irradiation, beneficial effects (known as hormesis) can be induced. For example, Xirradiation at 10 mGy (but not 50 or 100 mGy) reduced the frequency of micronucleated cells in human lymphocytes to below the spontaneous level (13). Recently, we discovered that among anticancer drugs, platinum preparations (cisplatin, carboplatin, oxaliplatin) and proteasome inhibitor bortezomib showed potent cytotoxicity not only against normal oral keratinocytes (14) but also against PC12 nerve-like cells (Iijima et al., unpublished data). The neurotoxicity of anticancer agents and X-ray irradiation is a recent research topic in today's aging society. In the present study, we investigated whether cytotoxic doses of X-ray irradiation further augment neurotoxicity...

Background/Aim: Chitosan-coated iron oxide nanoparticles (Chi-NP) have gained attention because of their biocompatibility, biodegradability, low toxicity and targetability under magnetic field. In this study, we investigated various biological properties of Chi-NP. Materials and Methods: Chi-NP was prepared by mixing magnetic NP with chitosan FL-80. Particle size was determined by scanning and transmission electron microscopes, cell viability by MTT assay, cell cycle distribution by cell sorter, synergism with anticancer drugs by combination index, PGE 2 production in human gingival fibroblast was assayed by ELISA. Results: The synthetic process of Chi-NP from FL-80 and magnetic NP increased the affinity to cells, up to the level attained by nanofibers. Upon contact with the culture medium, Chi-NP instantly formed aggregates and interfered with intracellular uptake. Aggregated Chi-NP did not show cytotoxicity, synergism with anticancer drugs, induce apoptosis (accumulation of subG1 cell population), protect the cells from X-ray-induced damage, nor affected both basal and IL-1β-induced PGE 2 production. Conclusion: Chi-NP is biologically inert and shows high affinity to cells, further confirming its superiority as a scaffold for drug delivery.

Human MutT homolog 1 (MTH1), also known as Nudix‐type motif 1 (NUDT1), hydrolyzes 8‐oxo‐dGTP and 2‐oxo‐dATP with broad substrate recognition and has attracted attention in anticancer therapeutics. Previous studies on MTH1 have proposed that the exchange of the protonation state between Asp119 and Asp120 is essential for the broad substrate recognition of MTH1. To understand the relationship between protonation states and substrate binding, we determined the crystal structures of MTH1 at pH 7.7–9.7. With increasing pH, MTH1 gradually loses its substrate‐binding ability, indicating that Asp119 is deprotonated at pH 8.0–9.1 in 8‐oxo‐dGTP recognition and Asp120 is deprotonated at pH 8.6–9.7 in 2‐oxo‐dATP recognition. These results confirm that MTH1 recognizes 8‐oxo‐dGTP and 2‐oxo‐dATP by exchanging the protonation state between Asp119 and Asp120 with higher pKa.