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.

Population-Level Effectiveness of an Inactivated Whole-Virion COVID-19 Vaccine: A Test Negative Case-Control Study in the Dominican Republic.

Background: A continuing nationwide vaccination campaign began in the Dominican Republic on February 16, 2021 to prevent severe consequences of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Estimates of vaccine effectiveness under real-world conditions are needed to support policy decision making and inform further vaccine selection. Methods: We conducted a test-negative case-control study to assess the real-world effectiveness of nationwide coronavirus disease 2019 (COVID-19) vaccination program using an inactivated vaccine (CoronaVac) on preventing symptomatic SARS-CoV-2 infections and hospitalizations from August to November 2021 in the Dominican Republic. Participants were recruited from 10 hospitals in 5 provinces to estimate the effectiveness of full immunization (≥14 days after receipt of the second dose) and partial immunization (otherwise with at least 1 dose ≥14 days after receipt of the first dose). Results: Of 1078 adult participants seeking medical care for COVID-19-related symptoms, 395 (36.6%) had positive polymerase chain reaction (PCR) tests for SARS-CoV-2; 142 (13.2%) were hospitalized during 15 days of follow up, including 91 (23%) among 395 PCR-positive and 51 (7.5%) among 683 PCR-negative participants. Full vaccination was associated with 31% lower odds of symptomatic infection (odds ratio [OR], 0.69; 95% confidence interval [CI], 0.52-0.93) and partial vaccination was associated with 49% lower odds (OR, 0.51; CI, 0.30-0.86). Among 395 PCR-positive participants, full vaccination reduced the odds of COVID-19-related hospitalization by 85% (OR, 0.15; 95% CI, 0.08-0.25) and partial vaccination reduced it by 75% (OR, 0.25; 95% CI, 0.08-0.80); full vaccination was associated with reduced use of assisted ventilation by 73% (OR, 0.27; 95% CI, 0.15-0.49). Conclusions: Given the ancestral and delta viral variants circulating during this study period, our results suggest that the inactivated COVID-19 vaccine offered moderate protection against symptomatic SARS-CoV-2 infections and high protection against COVID-19-related hospitalizations and assisted ventilation. This is reassuring given that, as of August 2022, an estimated 2.6 billion inactivated CoronaVac vaccine doses had been administered worldwide. This vaccine will become a basis for developing multivalent vaccine against the currently circulating omicron variant.

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.

La región sudeste del Gran Buenos Aires frente a la pandemia Evaluación de la ampliación del sistema de salud en el marco de la COVID 19 / The Southeastern region of the Buenos Aires Province metropolitan area in the face of the pandemic: evaluation of the expansion of the health system in the framework of COVID-19

INTRODUCCIÓN: La región sudeste del Gran Buenos Aires (GBA) reformuló el sistema público de salud por la pandemia de COVID19. Entre las medidas que se tomaron, está la ampliación del número de camas mediante la construcción y puesta en marcha de tres hospitales. OBJETIVO: Evaluar el impacto de la ampliación del número de camas en los resultados de internación de los pacientes asistidos por los efectores públicos de salud durante el período de estudio (8 de abril de 2020 al 11 de septiembre de 2020). MÉTODOS: Estudio descriptivo a partir de información registrada en el Tablero COVID-19, software de gestión desarrollado por el equipo del Instituto del Cálculo de la Universidad de Buenos Aires, en el que se obtienen datos de cada paciente internado en la red de efectores de salud; se evalúan los resultados del efecto del aumento de la capacidad instalada. RESULTADOS: Se registraron 2 306 pacientes internados, de los cuales 266 (11,54%) requirieron internación en unidad de cuidados intensivos (UCO), 1 786 (77,4%) en cuidados intermedios y 254 (11%) pacientes en sala general. La media de edad fue de 50,63 y los pacientes de sexo masculino representaron el 55,5% del total. Se produjeron 253 muertes (10,97%), de las cuales el 64% fueron hombres. El 58,3% del total tenían enfermedades preexistentes, estos tienen un riesgo 90% más alto que quienes no las tenían. El promedio total de ocupación de camas en UCI fue del 40,7%, mientras que el de ocupación en cuidados intermedios fue de 61,5%. Sin los hospitales nuevos, 169 pacientes (9,46%) no hubieran tenido camas en cuidados intermedios y 31 pacientes (11,6%) no hubieran tenido cama en la UCI. DISCUSIÓN: El sistema de salud de la región sudeste del GBA se preparó de manera adecuada gracias a la ampliación del número de camas de internación.

In vivo kinetics of the genotoxic and cytotoxic activities of cladribine and clofarabine.

The aim of the present in vivo study was to determine the kinetics of the genotoxic and cytotoxic activities of cladribine and clofarabine in mouse normoblasts using flow cytometry. Mice in groups of five were treated with cladribine or clofarabine. Blood samples were obtained from the mouse tails before treatment and every 8 hr posttreatment for 72 hr. These samples were cytometrically scored for micronucleated reticulocytes (RETs), and the percentage of RETs was determined. The results showed that clofarabine and cladribine have early cytotoxic effects that are related to the genotoxic effects reported in previous studies; the drugs have both complex long-lasting genotoxic and cytotoxic kinetic activity, with similar profiles that suggest a relationship between the genotoxic and cytotoxic parameters. The initial genotoxkinetics timing of clofarabine is equivalent to those of difluorodeoxycytidine, likely because both agents inhibit DNA polymerase. Clofarabine shows a higher genotoxic and cytotoxic efficiency than cladribine, in agreement with previous results.

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.

The OECD's micronucleus test guideline for single exposure to an agent and the genotox-kinetic alternative.

The 'Organization for Economic Co-operation and Development (OECD) guidelines for the Testing of Chemicals' aims to identify whether a chemical is a genotoxic hazard, and these guidelines 'are periodically reviewed in the light of scientific progress, changing regulatory needs and animal welfare considerations'. OECD published a mammalian erythrocyte micronucleus test guideline for testing chemicals (1) that proposes: 'Animals are treated with the test chemical once…Samples of peripheral blood are taken at least twice (from the same group of animals), starting not earlier than 36 h after treatment, with appropriate intervals following the first sample, but not extending beyond 72 h'. This guidelines are base on the report by the Collaborative Study Group for the Micronucleus Test (CSGMT), which was based on the sampling of mice peripheral blood every 24 h We investigated the kinetics of micronucleus induction by taking samples prior to administration and every 8 or 10 h after single treatment. The comparisons suggest that 24-h sampling could cause not only an underestimation of the responses to various agents but also a misestimation of the time of maximal induction. We proposed that samples of peripheral blood must be collected at two different times during an optimal 25-h sampling range (from 25 to 50 h). Besides, we hypothesize that the time of maximal EPC-MN induction depends on the time required for the mechanisms involved in micronucleus production; and we suggest that a kinetic analysis of MN-PCE induction by several agents with well-known mechanisms of micronuclei induction would allow derivation of a specific relationship between the kinetics of MN-PCE induction and some process of DNA breaks and/or micronuclei induction.

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.

Kinetics of micronucleus induction and cytotoxicity caused by distinct antineoplastics and alkylating agents in vivo.

This mini-review aims to compare the differences in the kinetics of the induction of micronucleated polychromatic erythrocytes (MN-PCE) and cytotoxicity by distinct antineoplastic and genotoxic agents in murine peripheral blood in vivo and to correlate these kinetics with the underlying processes. Comparisons were carried out using our previously obtained data with nominal doses causing similar levels of cytotoxicity, as measured in terms reduction of PCE. The aneuploidogens caused the most rapid induction of MN-PCEs and had the highest rates of cytotoxicity and genotoxicity. The promutagens cyclophosphamide and dimethylnitrosamine showed the most delayed responses and had the lowest genotoxic and cytotoxic efficiencies. DNA crosslinking agents had a similar delay of 4-5 h, greater than those of aneuploidogens, but differed in their cytotoxic and genotoxic efficiencies. Methylnitrosourea and 5-aza-cytidine caused greater delays than crosslinking agents. These delays can be due to the methylnitrosourea-mediated induction of formation of mono alkyl adducts which are interpreted as mismatches during DNA duplication, whereas 5-aza-cytidine requires incorporation into the DNA to induce breakage. This review allows us to conclude that the requirement for metabolic activation and the mechanisms of DNA breakage and of micronucleus induction are the main factors that affect the time of maximal MN-PCE induction.

Detection of disseminated tumor cells in locally advanced breast cancer patients before primary systemic therapy.

AIM: To assess the prevalence and prognostic power of disseminated tumor cells (DTC) in patients with locally advanced breast cancer (LABC) before primary systemic therapy (PST). MATERIALS AND METHODS: LABC patients attending our Breast Unit were studied between 2002 and 2012, all of them being considered for PST. To determine the presence of DTC, posterior iliac crest aspirates were obtained and marrow samples were processed by gradient separation with Ficoll (Lymphoprep(®)) and immunohistochemical staining using the antiCK A45-B/B3 (EPIMET) antibody. Clinicopathologic variables were recorded before and after PST to assess response. Disease-free survival (DFS) and overall survival (OS) were determined after follow-up. The presence of DTC as a predictor of response to PST and as a prognostic tool for OS and DSF was evaluated. RESULTS: DTC were observed in 26% of 47 patients included in the study. PST consisted of chemotherapy in 94% and hormone therapy in 6%. Breast-conserving therapy was attained in 33%. Mean follow-up was 68 months. Complete clinical response (CR) after PST was seen in 26%, disease recurrence in 38%, and cancer-related death in 8%; tumor size and negative estrogen receptors were significant predictors of CR and mastectomy was associated with DFS. Persistent axillary disease after PST and previous recurrence were predictive of OS. DTC were detected more often in patients who did not achieve CR and those who presented recurrence. DTC detection was a significant prognostic factor for a worse OS (OR = 7.62; CI95%: 1.46-39.61; p = 0.009) and a decreased survival time (62 versus 82 months, p = 0.004). CONCLUSION: Presence of DTC before PST was found in a significant number of patients with LABC. DTC were found to be a significant prognostic factor for cancer-related death. DTC could be a surrogate predictor of response to PST and also of disease recurrence in LABC patients.

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.

In vivo kinetics of micronuclei induction by bifunctional alkylating antineoplastics.

The aim of the present study was to determine in vivo the kinetics of micronucleated polychromatic erythrocyte (MN-PCE) induction in mice, as an approach for studying the mechanism of micronuclei induction by mitomycin C, cis-diamine dichloroplatinum, busulfan and bis-chloroethylnitrosourea, bifuctional alkylating antineoplastic agents having different patterns of crosslink induction. The kinetics of MN-PCE induction was established by scoring the frequency of MN-PCE in 2000 PCE in peripheral blood, for periods of 8 or 10 h after acute treatment and up to 80 h, with different doses of the agent. The kinetics of MN-PCE induction and particularly the times of maximal induction by different bifunctional alkylating agents were compared with the kinetics previously obtained for ethylnitrosourea, methylnitrosourea and 6-mercaptopurine, agents that cause MN-PCE mainly in the first, second and third divisions after exposure, respectively. The results obtained in the present study allow us to conclude that: (i) bifunctional alkylating agents have very different efficiencies of genotoxic and cytotoxic action; (ii) all assayed bifunctional alkylating agents induced micronuclei during the first cell division, owing to the mistaken repair of primary lesions, e.g. excision; (iii) busulfan and bis-chloroethylnitrosourea showed an additional late mechanism of micronuclei induction, which is expressed at the third division and seems to be related to the mismatch repair process.