Kinetics of the in vivo genotoxic and radioprotective effects of methyl gallate and epigallocatechin gallate.

The aim of this study was to compare the kinetics of the in vivo action of equimolar doses of methyl gallate (MG) and epigallocatechin gallate (EGCG) on their capacity to induce DNA damage and to protect against DNA damage induced by 60 Co gamma rays. DNA-damaged cells were determined by single-cell gel electrophoresis (comets) in murine peripheral blood leukocytes. The maximum radioprotective effects of MG and EGCG (approximately 70%) occurred at 15 min after administration when their effect was determined 2 min following irradiation. MG and EGCG have similar radioprotective indexes, which due to their fast response indicate that they are involved in free radical scavenging. Due to the similar radioprotective activities of MG and EGCG, the in vivo radioprotective effects of these agents do not seem to be dependent on the number of hydroxyl groups present in their structures but instead on the presence of the galloyl radical. EGCG induces an early, significant, and persistent increase in the number of DNA-damaged cells and a later and more important increase in the number of damaged cells, suggesting that it has two mechanisms by which it can induce DNA damage. MG at the same molar dose as EGCG caused a significant and persistent increase in DNA damaged cells but to a much lesser extent to that induce by EGCG, suggesting that the galloyl radical is not involved in the mechanism of DNA breaks induction.

Induction and assessment of persistent radioresistance in murine leukocytes in vivo.

The aim of the present study was to investigate whether weekly exposure to gamma rays causes a persistent increase in the number of radioresistant leukocytes in mice in vivo. Using the comet assay, 1 Gy radiation exposure decreased the percentage of leukocytes with less than 5% DNA in the tail (<5% DNAT), and we propose that radioresistance induction might increase the number of cells with <5% DNAT after radiation exposure. We exposed mice to 1 Gy gamma rays weekly for four weeks or 2 Gy per week for nine weeks. We observed a significant increase in cells with <5% DNAT after the third week and up to nine weeks of exposure. We exposed animals to gradually increasing radiation doses and finally challenged the lymphocytes with 1 Gy radiation both in vivo and in vitro. We observed increased radioresistance in vitro, providing evidence that a cellular process is involved. However, more radioresistance was observed in vivo than in vitro, suggesting a physiological effect. Cells challenged in vitro were maintained on ice during and after exposure, which likely caused a reduction in DNA repair. Radioresistance induction likely arose from mutation selection in stem cells because leukocytes are unable to proliferate in peripheral blood.

In vivo Characterization of the Radiosensitizing Effect of a Very Low Dose of BrdU in Murine Cells Exposed to Low-Dose Radiation.

The aim of the present study was to characterize the in vivo radiosensitizing effect of a very low dose of bromodeoxyuridine (BrdU) in mice exposed to low-dose radiation by establishing the following: (1) the radiosensitizing effect during DNA synthesis using single-cell gel electrophoresis (SCGE) in murine bone marrow cells, and (2) the number and timing of the mechanisms of genotoxicity and cytotoxicity, as well as the correlation of both end points, using flow cytometry analysis of the kinetics of micronucleus induction in reticulocytes. Groups of mice received intraperitoneal injections of 0.125 mg/g of BrdU 24 h prior to irradiation with 0.5 Gy of 60 Co gamma rays. DNA breaks measured using SCGE were determined at 30 min after exposure to radiation. The kinetics of micronucleated reticulocyte (MN-RET) induction was determined every 8 h after irradiation up to 72 h. The results from both experimental models indicated that low-level BrdU incorporation into DNA increased the sensitivity to 0.5 Gy of radiation, particularly in the S phase. The formation of micronuclei by gamma rays was produced at three different times using two main mechanisms. In the BrdU-substituted cells, the second mechanism was associated with a high cytotoxic effect that was absent in the irradiated BrdU-unsubstituted cells. The third mechanism, in which micronucleus formation was increased in irradiated substituted cells compared with the irradiated nonsubstituted control cells, was also related to an increase in cytotoxicity. Environ. Mol. Mutagen. 60:534-545, 2019. © 2019 Wiley Periodicals, Inc.

Genotoxicity kinetics in murine normoblasts as an approach for the in vivo action of difluorodeoxycytidine.

PURPOSE: This study analyzed the kinetics of in vivo micronucleus induction in normoblasts by determining the kinetics of difluorodeoxycytidine (dFdC)-induced micronucleated polychromatic erythrocytes (MN-PCEs) in the peripheral blood of mice. The kinetic indexes of MN-PCE induction of dFdC were correlated with the previously reported mechanisms DNA damage induction by this compound. In general, this study aimed to establish an in vivo approach for discerning the processes underlying micronucleus induction by antineoplastic agents or mutagens in general. METHODS: The frequencies of PCEs and MN-PCEs in the peripheral blood of mice were determined prior to treatment and after treatment using dFdC at doses of 95, 190, or 380 µmol/kg at 8 h intervals throughout a 72 h post-treatment. RESULTS: The area beneath the curve (ABC) for MN-PCE induction as a function of time, which is an index of the total effect, indicated that the dose response was directly proportional and that the effect of dFdC on micronucleus induction was reduced compared with that of aneuploidogens and monofunctional and bifunctional alkylating agents but increased compared with that of promutagens, which is consistent with our previous results. The ABC showed a single peak with a small broadness index, which indicates that dFdC has a single mechanism or concomitant mechanisms for inducing DNA breaks. The time of the relative maximal induction (T rmi) indicated that dFdC requires more time to achieve MN-PCE induction compared with aneugens and monofunctional and bifunctional alkylating agents, although it requires a similar time to achieve MN-PCE induction as azacytidine, which is consistent with evidence showing that both agents must be incorporated into DNA for their action to be realized. The timing of maximal cytotoxicity observed with the lowest dFdC dose was correlated with the timing of the main genotoxic effect. However, early and late cytotoxic effects were detected, and these effects were independent of the genotoxic response. CONCLUSIONS: A correlation analysis indicated that dFdC appears to induce MN-PCEs through only one mechanism or mechanisms that occur concomitantly, which could be explained by the previously reported concurrent inhibitory effects of dFdC on DNA polymerase alpha, polymerase epsilon, and/or topoisomerase. The timing of maximal cytotoxicity was correlated with the timing of maximal genotoxicity; however, an early cytotoxic effect that appeared to occur prior to the incorporation of dFdC into DNA was likely related to a previously reported inhibitory effect of dFdC on thymidylate synthase and/or ribonucleotide reductase.

Kinetic of genotoxic expression in the pharmacodynamics of busulfan.

BACKGROUND: Busulfan (BUS) is a highly toxic antineoplastic bifunctional-alkylating agent and has a narrow therapeutic window. Our previous study revealed a narrow dose-range of BUS, which causes a sudden dose-dependent transition from early- to late-expressing micronucleus induction and from a non-cytotoxic to a cytotoxic effect. In the present study, the kinetics of DNA-damaged cell induction by BUS and its dose-effect relationship were established. METHODS: This was achieved by using the kinetics of DNA-damaged cell induction, determined by the comet assay in murine peripheral blood leukocytes of mice, after the intraperitoneal exposure to 16, 30, 45, 60 or 80 micromol/kg of BUS. RESULTS: Doses of 15 or 30 micromol/kg of BUS were able to increase DNA-damaged cell frequency, but doses of 45 micromol/kg body weight or higher caused a sudden drop in this frequency. CONCLUSIONS: This suggests that higher doses cause lesions that inhibit the expression of damage as comets, i.e., DNA-protein or interstrand crosslinks. The latter could be explained by sudden monoadduct-to-crosslink transformation due to a DNA conformational change induced by monoadduct accumulation that facilitates crosslink formation. This narrow dose-dependent transition could contribute to the narrow therapeutic window of BUS.

Kinetics of micronucleated polychromatic erythrocyte (MN-PCE) induction in vivo by aneuploidogens.

The aim of the present study was to make inferences about the cytotoxic and genotoxic action of the antineoplastic aneuploidogens, vinblastine and vincristine, by analyzing the kinetics of MN-PCE induction in mice in vivo. The kinetics of MN-PCE induction was assessed by taking blood samples from the tail, before the single i.p. injection of different doses of vinblastine or vincristine and every 8h after that. The analysis was done in groups consisting of three or four animals. The results indicate that both agents have similar kinetics of MN-PCE induction which differs from the kinetics previously obtained for colchicine in the following aspects: (i) vinblastine and vincristine cause a longer delay after exposure, (ii) they produce a higher maximal velocity of induction, and (iii) higher doses give rise to more than one peak in the curve of MN-PCE frequency versus time. The results of the present study indicate that the different mechanisms of action of vinca alkaloids and colchicine are reflected in their kinetics of MN-PCE induction, and that such mechanisms could also explain the differences in their efficiency. Vinca alkaloids seem to block the cell division immediately, but the cell appears to be capable of reverting the blockage during the period of time corresponding to the first division. Moreover, evidence was obtained indicating that high doses could induce a long lasting aneuploidogen effect, probably related to the accumulation of vinca alkaloids that are either free or associated to tubulin.