The genetic consequences of nucleotide precursor pool imbalance in mammalian cells

The existing DNA damage detection technology cannot meet the current detection requirements. It is critical to build new methods and discover novel biomarkers. In this study, alkaline comet and 8-OHDG ELISA assays were used to identify DNA damage in HT-1080 cells exposed to K₂Cr₂O₇, and electrochemical behaviors of HT-1080 cells with DNA damage was studied. With an increase in K₂Cr₂O₇ exposure time, two electrochemical signals from HT-1080 cells at 0.69 and 1.01 V steadily grew before decreasing after reaching their highest values. The electrochemical signal's initial response time and peak time decreased as the concentration of K₂Cr₂O₇ increased. The duration of the high dose group was 0.5 and 1 h, while the low dose group was 1.5 and 6 h. Western blotting analysis revealed that DNA damage increased the expression of proteins involved in catabolism and de novo purine synthesis, particularly de novo purine synthesis. Expressions of PRPP amidotransferase, IMPDH, and ADA were all higher than those of ADSS, XOD, and GDA, which resulted in larger concentrations of hypoxanthine, guanine, and xanthine, and in turn improved electrochemical signaling. These findings suggest that intracellular purine identified by linear scan voltammetry is predicted to evolve as a marker of early DNA damage.

CTP synthase (CTPS) has been demonstrated to form evolutionarily-conserved filamentous structures termed cytoophidia whose exact cellular functions remain unclear, but they may play a role in intracellular compartmentalization. We have previously shown that the mammalian target of rapamycin complex 1 (mTORC1)–S6K1 pathway mediates cytoophidium assembly in mammalian cells. Here, using the fission yeast Schizosaccharomyces pombe as a model of a unicellular eukaryote, we demonstrate that the target of rapamycin (TOR)-signaling pathway regulates cytoophidium formation (from the S. pombe CTPS ortholog Cts1) also in S. pombe. Conducting a systematic analysis of all viable single TOR subunit–knockout mutants and of several major downstream effector proteins, we found that Cts1 cytoophidia are significantly shortened and often dissociate when TOR is defective. We also found that the activities of the downstream effector kinases of the TORC1 pathway, Sck1, Sck2, and Psk1 S6, as well as of the S6K/AGC kinase Gad8, the major downstream effector kinase of the TORC2 pathway, are necessary for proper cytoophidium filament formation. Interestingly, we observed that the Crf1 transcriptional corepressor for ribosomal genes is a strong effector of Cts1 filamentation. Our findings connect TOR signaling, a major pathway required for cell growth, with the compartmentalization of the essential nucleotide synthesis enzyme CTPS, and we uncover differences in the regulation of its filamentation among higher multicellular and unicellular eukaryotic systems.

Pendant longtemps, il a été admis qu’il n’existait pas de seuil en mutagenèse : un produit était ou non mutagène, ce qui signifie que, quelle que soit la dose, il existait toujours un risque puisque, au moins théoriquement, une molécule pouvait réagir avec une base et provoquer ainsi une mutation. Cependant depuis ces dernières années, des travaux ont montré que pour certains mécanismes d’action mutagène, il était possible de démontrer un seuil essentiellement dans le cas de produits ne réagissant pas directement avec l’ADN. Il existe différents types de seuils et le simple seuil statistique apparaît comme étant insuffisant en terme d’évaluation du risque, il faut que l’on puisse établir un seuil biologique reposant sur des mécanismes cohérents avec un seuil. Parmi ces mécanismes, on peut retenir comme étant parfaitement démontré à seuil : les effets des aneugènes, les effets des inhibiteurs des topoisomérases, les inhibiteurs d’ADN polymérases, les produits provoquant une inhibition de la fidélité de la réplication de l’ADN ou certains produits dont le métabolisme est différent à faible et forte dose. En revanche, pour certains mécanismes, la démonstration d’un seuil est moins bien établie, c’est le cas des produits induisant des déséquilibres des pools de nucléotides ou les produits provoquant une inhibition de la réparation de l’ADN. Dans certains cas, des mécanismes impliquant des systèmes existant de façon redondante dans la cellule, tels que la réparation de l’ADN des lésions oxydatives, laissent penser que des seuils pourraient exister. Certains auteurs tentent même de démontrer qu’il existerait un seuil pour les agents alkylants. Si la plupart des seuils ont été démontrés in vitro, il a pu être démontré également in vivo que certains mécanismes tels que les perturbations physiologiques comme l’hypo ou l’hyperthermie induisant des micronoyaux dans la moelle de rongeurs présentaient un seuil.

L’utilisation d’une dose seuil pour l’évaluation du risque implique de retenir le marqueur le plus sensible, par exemple la non-disjonction dans le cas des aneugènes, afin d’éliminer les facteurs confondants comme l’apoptose et de prendre en compte les différences de sensibilité entre espèce, mais aussi de démontrer que, si ce mécanisme à seuil existe, il est le seul à l’origine des effets mutagènes observés.

La démonstration de tels seuils présente un intérêt particulier pour l’évaluation du risque pour l’homme des produits mutagènes et des cancérogènes génotoxiques.

During years, it has been widely admitted in the scientific community that there was no threshold in mutagenesis : a compound was or not a mutagen. The meaning of such a proposition was that a risk existed at all exposure level, because, at least theorically, one molecule is sufficient to cause the formation of a DNA adduct which is able to induce a mutation. However, works carried out in the last few years have shown that in the case of some specific mechanisms of mutagenesis, a threshold could be demonstrated essentially in the case of compounds that do not react directly with DNA. Several types of thresholds exist, and the simple statistical threshold is not sufficient in terms of risk assessment. A biological threshold that is consistent with a mechanism of action of the mutagen should be established. Amongst these mechanisms, we can mention some mechanism with a demonstrated threshold: effects of aneugens, effects of topoisomerases inhibitors, effects of DNA polymerases inhibitors, effects of compounds with a different metabolism at high doses compared to low doses. On the contrary, for some mechanisms, the demonstration of the mechanism is suspected, but not totally demonstrated. It is the case of compounds which induce nucleotides pool imbalance or compounds which are DNA repair inhibitors. In some cases, when a redundancy exists in the repair of damages, like oxydative DNA damage, a threshold is suspected. Some authors even recently proposed the possibility of a threshold in the case of alkylating agents. The majority of mutagenic thresholds were demonstrated in vitro, however some mechanisms were demonstrated in vivo, for example in the case of micronucleus induction by hypo or hyperthermia in rodents bone marrow.

The use of threshold in risk assessment requires the use of the most sensitive endpoint for example, non disjunction in the case of aneugens, confusing factors like apoptosis should be eliminated and species sensitivities should be taken into account. A very important point to consider is to demonstrate that the mechanism with threshold was really thee only one involved in the mutagenic effect.

The demonstration of such thresholds is of particular interest for human risk assessment in the case of mutagens and of genotoxic carcinogens.

Terminal deoxynucleotidyl transferase (TdT) catalyzes the condensation of deoxyribonucleotides on 3′-hydroxyl ends of DNA strands in a template-independent manner and adds N-regions to gene segment junctions during V(D)J recombination. Although TdT is able to incorporate a few ribonucleotides in vitro, TdT discrimination between ribo- and deoxyribonucleotides has never been studied. We found that TdT shows only a minor preference for incorporation of deoxyribonucleotides over ribonucleotides on DNA strands. However, incorporation of ribonucleotides alone or in the presence of deoxyribonucleotides generally leads to premature chain termination, reflecting an impeded accommodation of ribo- or mixed ribo/deoxyribonucleic acid substrates by TdT. An essential catalytic aspartate in TdT was identified, which is a first step toward understanding the apparent lack of sugar discrimination by TdT.

The influence of commercial inosine triphosphate (ITP) on the chromosome aberration rate, the mitotic rate, sister-chromatid exchange (SCE) frequency, and the proportion of first (X1), second (X2) and third (X3) division metaphases was investigated in 72 h cultures of human peripheral lymphocytes. The blood donors had mild inactive arthrosis and a normal health check-up. All cultures of each volunteer were set-up simultaneously.

In contrast to a previous report [Arch. Biochem. Biophys. 278 (1990) 238–244], it was demonstrated in two preliminary studies (number of subjects, n=5 each) that ITP at a final concentration of 100 μM does not induce chromosomal aberrations and, furthermore, that not ITP concentrations higher than 100 μM but ITP doses higher than 3.8 mM prohibit culture growth.

Based on these results, cultures with a final ITP concentration of 3.6 mM (max.) and 1.8 mM (max./2) were compared with control cultures (number of subjects n=10; three males and seven females, mean age x=57.6 years). Whereas no increase in the chromosomal breakage rate was observed in cultures with an ITP concentration of 1.8 mM and only a marginally significant one (P=0.048) for 3.6 mM ITP cultures, a highly significant induction of SCEs, not only at an ITP concentration of 3.6 mM (P<0.0001) but also at 1.8 mM (P<0.0001) was seen. The increase in the SCE frequency was not linear, but steeper from 0 to 1.8 mM than from 1.8 to 3.6 mM. Nevertheless, the difference between 1.8 and 3.6 mM cultures was significant (P=0.027). The distribution of the number of SCEs per metaphase as well as the distribution of SCEs per chromosome correspond to the expected Poisson values. The investigation of the cytotoxic effect of the studied ITP concentrations revealed a highly significant reduction of the mitotic rate from 0 to 1.8 mM as well as from 1.8 to 3.6 mM in the aberration studies (all P values are equal to smallest possible one for a sample size of 10, namely, 0.002), and in the SCE studies there is a significant decrease in the X3 frequency when ITP is increased (0–1.8 mM: P=0.0061 and 1.8–3.6 mM: P<0.0001). The proportion of X1 within all X1 and X2 metaphases changes significantly only at the second dose step (0–1.8 mM ITP: P=0.22 and 1.8–3.6 mM ITP: P<0.0001). The results are discussed.

Ribonucleotide reductases (RNRs) catalyze all new production in nature of deoxyribonucleotides for DNA synthesis by reducing the corresponding ribonucleotides. The reaction involves the action of a radical that is produced differently for different classes of the enzyme. Class I enzymes, which are present in eukaryotes and microorganisms, use an iron center to produce a stable tyrosyl radical that is stored in one of the subunits of the enzyme. The other classes are only present in microorganisms. Class II enzymes use cobalamin for radical generation and class III enzymes, which are found only in anaerobic organisms, use a glycyl radical. The reductase activity is in all three classes contained in enzyme subunits that have similar structures containing active site cysteines. The initiation of the reaction by removal of the 3′-hydrogen of the ribose by a transient cysteinyl radical is a common feature of the different classes of RNR. This cysteine is in all RNRs located on the tip of a finger loop inserted into the center of a special barrel structure. A wealth of structural and functional information on the class I and class III enzymes can now give detailed views on how these enzymes perform their task. The class I enzymes demonstrate a sophisticated pattern as to how the free radical is used in the reaction, in that it is only delivered to the active site at exactly the right moment. RNRs are also allosterically regulated, for which the structural molecular background is now starting to be revealed.