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 ([³H]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 β-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 in hepatocytes using the mitochondrion-specific dye rhodamine 123 showed that very few 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.     

Optical single-channel resolution imaging of the ryanodine receptor distribution in rat cardiac myocytes

We have applied an optical super-resolution technique based on single-molecule localization to examine the peripheral distribution of a cardiac signaling protein, the ryanodine receptor (RyR), in rat ventricular myocytes. RyRs form clusters with a mean size of approximately 14 RyRs per cluster, which is almost an order of magnitude smaller than previously estimated. Clusters were typically not circular (as previously assumed) but elongated with an average aspect ratio of 1.9. Edge-to-edge distances between adjacent RyR clusters were often <50 nm, suggesting that peripheral RyR clusters may exhibit strong intercluster signaling. The wide variation of cluster size, which follows a near-exponential distribution, is compatible with a stochastic cluster assembly process. We suggest that calcium sparks may be the result of the concerted activation of several RyR clusters forming a functional “supercluster” whose gating is controlled by both cytosolic and sarcoplasmic reticulum luminal calcium levels.     

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.