4D Super-Resolution Microscopy with Conventional Fluorophores and Single Wavelength Excitation in Optically Thick Cells and Tissues

Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample

DNA repair synthesis induced by <em>N</em>-hydroxyurea, acetohydroxamic acid, and <em>N</em>-hydroxyurethane in primary rat hepatocyte cultures: Comparative evaluation using the autoradiographic and the bromodeoxyuridine density-shift method.

N-Hydroxyurea and two structurally related compounds, acetohydroxamic acid and N-hydroxyurethane, were investigated for their potential to induce DNA repair synthesis in primary rat hepatocyte cultures. Repair was determined as repair replication by means of the bromodeoxyuridine density-shift method and, in the same cell preparations, as unscheduled DNA synthesis (UDS) by autoradiography. For all 3 compounds, a clear concentration-dependent induction of DNA repair replication could be demonstrated. Interpretation of the UDS data, however, depended on the mode whereby the results were evaluated. Expression of the results as net grains per nucleus after subtraction of cytoplasmic from nuclear grain counts yielded statistically significant increases over the control values for all compounds. In contrast, no significant changes of the nuclear labeling were obtained when nuclear and cytoplasmic grain counts were plotted separately. These findings demonstrate that the two modes to present UDS data may lead to different conclusions, a consequence of the uncertainty regarding the origin and importance of the cytoplasmic background. The observation that both hydroxyurea and the structurally related compounds acetohydroxamic acid and N-hydroxyurethane induce DNA repair in primary hepatocyte cultures suggests that metabolism-dependent genotoxicity may be a common property of aliphatic hydroxamic acids

Background DNA repair synthesis in rat hepatocyte cultures used for genotoxicity testing.

DNA repair synthesis in primary rat hepatocyte cultures (HPC) was investigated using the bromodeoxyuridine (BrdUrd) density-shift method and autoradiography. Analysis by density-gradient centrifugation of DNA labelled with BrdUrd and [3H]deoxycytidine ([63H]dCyd) for 18-20 hr showed that considerable DNA repair replication occurs in HPC even in the absence of an added genotoxic agent. Repair was demonstrated to be most probably a consequence of DNA damage caused by the collagenase perfusion of the liver during hepatocyte isolation, and by &beta;-decay of the 3H-labelled DNA precursor. Autoradiographic analysis of the distribution of silver grains over nuclei in intact non-S-phase cells, and over isolated nuclei from HPC exposed to [3H]dCyd for 18-20 hr, showed that the vast majority of the radioactivity was incorporated into the nuclei themselves, and not into overlying cytoplasm or mitochondria. In addition, direct localization of mitochondria were actually located over the nucleus. The results suggest that cytosolic labelling of control HPC in autoradiographs is mainly caused by mitochondrial DNA synthesis whereas nuclear labelling essentially reflects repair synthesis. They call into question the commonly applied practice of evaluating unscheduled DNA synthesis (UDS) in HPC by subtracting the number of cytosolic silver grains from nuclear grains and expressing repair synthesis as net grains per nucleus

Comparison of the continuous rat hepatoma cell line 2sFou with primary rat hepatoyctes cultures for the induction of DNA repair synthesis by nitrosamines, benzo[<em>a</em>]pyrene and hydroxyurea.

We have examined the suitability of the continuous rat hepatoma cell line 2sFou for testing the genotoxicity of chemicals in comparison with that of primary rat hepatocyte cultures (HPC). The capacity of the cells for metabolic activation was assessed by measuring induction of DNA-repair synthesis and inhibition of replicative DNA synthesis by the test compounds dimethylnitrosamine (DMN), diethylnitrosamine (DEN), hydroxyurea (HU) and benzo[a]pyrene (BaP), which are substrates for major hepatic and extrahepatic forms of cytochrome P-450 dependent monooxygenases. The cellular capacity for DNA-repair synthesis was assessed using UV-light as a DNA-damaging agent. Repair-specific incorporation of [3H]deoxycytidine (3H-dCyd) caused by UV-light was higher in 2sFou cells than in HPC. In contrast, background repair incorporation of 3H-dCyd in 2sFou cells was only 1/3 that found in HPC. All the test agents induced DNA repair and inhibited DNA synthesis in both 2sFou cells and HPC. The two nitrosamines were more effective in HPC than in 2sFou cells. HU and BaP affected DNA repair and DNA synthesis in the two cell systems at similar range of concentrations. In general, DNA repair in the 2sFou cells increased near linearly with the concentrations of the test compounds. The data indicate that 2sFou cells are capable of activating hepatotropic pro-mutagens/carcinogens such as dialkylnitrosamines, and are sensitive indicators of DNA damage. In contrast, BaP, a non-hepatotoxic compound, caused only little DNA repair in these cells. Thus, continuously growing cells, such as 2sFou, show a qualitatively similar response to genotoxic chemicals as HPC and offer a potential alternative to HPC for genotoxicity testing

Thiourea induces DNA repair synthesis in primary rat hepatocyte cultures and gene mutations in V79 Chinese hamster cells.

Thiourea was investigated for its capacity to cause DNA alterations in cultured mammalian cells. The induction of DNA repair in primary rat hepatocyte cultures and of gene mutations in V79 Chinese hamster cells were used as biological endpoints. In hepatocytes, thiourea elicited a linear increase in DNA repair replication in the concentration range tested (5-25 mM). In V79 cells, thiourea (10-40 mM) significantly increased the frequency of 8-azaguanine-resistant mutants. The present results show that thiourea is weakly, but definitely, genotoxic and mutagenic in cultured mammalian cells

In Vitro-Testsysteme zur Identifizierung gentoxischer Chemikalien in der Umwelt des Menschen: Der Nachweis der DNA-Reparatursynthese in Leberzellen

Copy held by FIZ Karlsruhe; available from UB/TIB Hannover / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman