Irradiation with high linear energy transfer α-emitters, like the clinically used Ra-223 dichloride, severely damages cells and induces complex DNA damage including closely spaced double-strand breaks (DSBs). As the hematopoietic system is an organ-at-risk for the treatment, knowledge about Ra-223-induced DNA damage in blood leukocytes is highly desirable. Therefore, 36 blood samples from six healthy volunteers were exposed ex-vivo (in solution) to different concentrations of Ra-223. Absorbed doses to the blood were calculated assuming local energy deposition of all α- and β-particles of the decay, ranging from 0 to 142 mGy. γ-H2AX + 53BP1 co-staining and analysis was performed in leukocytes isolated from the irradiated blood samples. For DNA damage quantification, leukocyte samples were screened for occurrence of α-induced DNA damage tracks and small γ-H2AX + 53BP1 DSB foci. This revealed a linear relationship between the frequency of α-induced γ-H2AX damage tracks and the absorbed dose to the blood, while the frequency of small γ-H2AX + 53BP1 DSB foci indicative of β-irradiation was similar to baseline values, being in agreement with a negligible β-contribution (3.7%) to the total absorbed dose to the blood. Our calibration curve will contribute to the biodosimetry of Ra-223-treated patients and early after incorporation of α-emitters.

Mouse aldehyde oxidase (mAOX1) forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Eschericia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site.

Background: Cancer patients are increasingly treated with alpha-particle-emitting radiopharmaceuticals. At the subcellular level, alpha particles induce densely spaced ionizations and molecular damage. Induction of DNA lesions, especially clustered DNA double-strand breaks (DSBs), threatens a cell’s survival. Currently, it is under debate to what extent the spatial topology of the damaged chromatin regions and the repair protein arrangements are contributing. Methods: Super-resolution light microscopy (SMLM) in combination with cluster analysis of single molecule signal-point density regions of DSB repair markers was applied to investigate the nano-structure of DNA damage foci tracks of Ra-223 in-solution irradiated leukocytes. Results: Alpha-damaged chromatin tracks were efficiently outlined by γ-H2AX that formed large (super) foci composed of numerous 60–80 nm-sized nano-foci. Alpha damage tracks contained 60–70% of all γ-H2AX point signals in a nucleus, while less than 30% of 53BP1, MRE11 or p-ATM signals were located inside γ-H2AX damage tracks. MRE11 and p-ATM protein fluorescent tags formed focal nano-clusters of about 20 nm peak size. There were, on average, 12 (±9) MRE11 nanoclusters in a typical γ-H2AX-marked alpha track, suggesting a minimal number of MRE11-processed DSBs per track. Our SMLM data suggest regularly arranged nano-structures during DNA repair in the damaged chromatin domain.

Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a molybdo-flavoprotein that is highly homologous to the homodimeric mammalian xanthine oxidoreductase. However, the bacterial enzyme has an (␣␤) 2 heterotetrameric structure, and the cofactors were identified to be located on two different polypeptides. We have analyzed the mechanism of cofactor insertion and subunit assembly of R. capsulatus XDH, using engineered subunits with appropriate substitutions in the interfaces. In an (␣␤) heterodimeric XDH containing the XdhA and XdhB subunits, the molybdenum cofactor (Moco) was shown to be absent, indicating that dimerization of the (␣␤) subunits has to precede Moco insertion. In an (␣␤) 2 XDH heterotetramer variant, including only one active Moco-center, the active (␣␤) site of the chimeric enzyme was shown to be fully active, revealing that the two subunits act independent without cooperativity. Amino acid substitutions at two cysteine residues coordinating FeSI of the two [2Fe-2S] clusters of the enzyme demonstrate that an incomplete assembly of FeSI impairs the formation of the XDH (␣␤) 2 heterotetramer and, thus, insertion of Moco into the enzyme. The results reveal that the insertion of the different redox centers into R. capsulatus XDH takes place sequentially. Dimerization of two (␣␤) dimers is necessary for insertion of sulfurated Moco into apo-XDH, the last step of XDH maturation. Xanthine oxidoreductases (XORs)2 are the best studied enzymes of the small but important class of molybdenum-containing iron-sulfur flavoproteins, containing two non-identical [2Fe2S] clusters, FAD, and the sulfurated form of the molybdenum cofactor (Moco) as catalytically acting units (1-3).Mammalian XORs catalyze the hydroxylation of hypoxanthine and xanthine, the last two steps in the formation of urate, and exist originally as the dehydrogenase form (XDH, EC 1.17.1.4) but can be converted to the oxidase form (XO, EC 1.1.3.22) either reversibly by oxidation of sulfhydryl residues of the protein molecule or irreversibly by proteolysis (4). XDH shows a preference for NAD ϩ reduction at the FAD reaction site, whereas XO exclusively uses dioxygen as a terminal electron acceptor, leading to the formation of superoxide and hydrogen peroxide (5). The enzyme has been implicated in diseases characterized by oxygen radical-induced tissue damage, such as postischemic reperfusion injury (6). The oxidation of xanthine takes place at the molybdenum center, and the electrons thus introduced are rapidly distributed to the other centers according to their relative redox potentials (1). The re-oxidation of the reduced enzyme by the oxidant substrate, either NAD ϩ or molecular oxygen, occurs through FAD (7). The two [2Fe2S] clusters (FeSI and FeSII) are indistinguishable in terms of their absorption spectra, but the midpoint redox potential of FeSII is generally more positive than that of the FeSI center (8, 9). The FeS centers from enzymes of the XO family have been characterized earlier by EPR (10 -12). The FeSI center of eukaryotic XOR exh...

Purpose The aim of this study was to investigate the time- and dose-dependency of DNA double-strand break (DSB) induction and repair in peripheral blood leucocytes of prostate cancer patients during therapy with 177 Lu-PSMA. Methods Blood samples from 16 prostate cancer patients receiving their first 177 Lu-PSMA therapy were taken before and at seven time-points (between 1 h and 96 h) after radionuclide administration. Absorbed doses to the blood were calculated using integrated time–activity curves of the blood and the whole-body. For DSB quantification, leucocytes were isolated, fixed in ethanol and immunostained with γ-H2AX and 53BP1 antibodies. Colocalizing foci of both DSB markers were manually counted in a fluorescence microscope. Results The average number of radiation-induced foci (RIF) per cell increased within the first 4 h after administration, followed by a decrease indicating DNA repair. The number of RIF during the first 2.6 h correlated linearly with the absorbed dose to the blood ( R 2 = 0.58), in good agreement with previously published in-vitro data. At late time-points (48 h and 96 h after administration), the number of RIF correlated linearly with the absorbed dose rate ( R 2 = 0.56). In most patients, DNA DSBs were repaired effectively. However, in some patients RIF did not disappear completely even 96 h after administration. Conclusion The general pattern of the time- and dose-dependent induction and disappearance of RIF during 177 Lu-PSMA therapy is similar to that of other radionuclide therapies.