In Vivo Cytotoxic, Genotoxic and Radiosensitizing Effects of Clofarabine.

BACKGROUND/AIM: ClFdA is a second-generation antineoplastic agent that has demonstrated significant anticancer activity, particularly against acute lymphoblastic leukemia and has been shown to have radiosensitizing activity. The aim of the study was to explore the genotoxic, cytotoxic and radiosensitizing effects of clofarabine (ClFdA) on bone marrow cells (BMCs), normoblasts and leukocytes of mice in vivo. MATERIALS AND METHODS: Cytotoxicity was determined by the reduction in reticulocytes (RET), and genotoxicity was determined by the induction of micronucleated reticulocytes (MN-RET) in the peripheral blood and by DNA break induction in leukocytes determined by single-cell gel electrophoresis (SCGE). The radiosensitizing capacity of ClFdA was determined in leukocytes and BMCs by SCGE. RESULTS: Two mechanisms of MN-RET induction were identified according to the antecedents, that could be due to inhibition of DNA synthesis and demethylation of G-C regions, and subsequent chromosome fragility. ClFdA cytotoxicity causes two contiguous peaks, an early peak that seems to inhibit MN-RET induction and a second peak that seems to be caused by ribonucleotide reductase (RR) and/or DNA synthesis inhibitions. ClFdA induced early DNA damage in noncycling leukocytes, and also radiosensitizes leukocytes immediately after treatment. ClFdA-ionizing radiation (IR) causes two time-dependent episodes of DNA damage, the latest after 80 min triggers a major breakage of DNA. In terms of the number of damaged cells, leukocytes and BMCs are similarly sensitive to ionizing radiation; BMCs are slightly more sensitive than leukocytes to ClFdA, but BMCs are doubly sensitive to combined treatment. CONCLUSION: ClFdA causes early DNA damage and radiosensitivity in non-proliferating leukocytes, which rules out the most favored hypotheses of the participation of RR and DNA polymerase inhibition.

In Vivo Genotoxicity and Cytotoxicity Kinetics of Trimethoprim Sulfamethoxazole in Well-nourished and Undernourished Young Rats.

BACKGROUND/AIM: Undernutrition is a serious health problem prevalent in poor countries, affecting millions of people worldwide, especially young children, pregnant women, and sick elderly individuals. This condition increases vulnerability to infections, leading to widespread use of antibiotic treatments in undernourished populations. The objective of the present study was to determine the in vivo genotoxic and cytotoxic effects of trimethoprim-sulfamethoxazole (TMP-SMX) treatment according to nutritional conditions. MATERIALS AND METHODS: The effects of TMP-SMX treatment were measured by analyzing the kinetics of micronucleated reticulocytes (MN-RET) induced in the peripheral blood of young, well-nourished (WN) and undernourished (UN) rats. RESULTS: In the WN group, two distinct peaks of MN-RET were observed, while the UN group had a significantly higher basal frequency of MN-RET compared to the WN group and only a later peak. Reticulocyte (RET) frequency slightly decreased in WN, indicating a poor cytotoxic effect. In contrast, in the UN, the treatment caused a significant increase in RET frequency. The results indicate that SMX's aromaticity index decreases when formed with TMP, suggesting potentially fewer toxic effects. CONCLUSION: In vivo TMP-SMX produces two MN-RET induction peaks in WN animals, indicating two DNA damage induction mechanisms and consequent micronucleus production. The UN rats did not display the two peaks, indicating that the first MN induction mechanism did not occur in UN, possibly due to pharmacokinetic effects, decreased metabolism or effects on cell proliferation. TMP-SMX has a slight cytotoxic effect on WN. In contrast, in the UN, the antibiotic treatment seems to favor early erythropoiesis.

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.

Assessment of micronucleus and oxidative stress in peripheral blood from malnourished children.

INTRODUCTION: malnutrition is one of the most common health problems among children in underdeveloped countries, including Mexico. Previous studies have indicated increased genetic damage in malnourished humans and animal models, but the essential mechanisms remain unclear. In the present study, we assessed the effects of malnutrition on the frequency of micronucleus (MN) in reticulocytes (RET) from the peripheral blood of well-nourished uninfected (WN), well-nourished infected (WNI), moderately malnourished infected (UNM) and severely malnourished infected (UNS) children. Moreover, lipid peroxidation and the antioxidant status were evaluated to investigate the role of oxidative processes in malnutrition-associated genotoxicity. METHODS: the antioxidant status of the study population was determined by measuring superoxide dismutase (SOD) in the red blood cells and glutathione peroxidase (GPX) in whole blood. RESULTS: the UNS and UNM groups have increased percentages of MN-RET compared to the WNI group. Moreover, the data showed a significant increase in lipid peroxidation and a decrease in erythrocyte SOD activity and GPX activity in the malnourished group compared to the well-nourished infected children. CONCLUSION: the data suggest that the antioxidant system was impaired in the cells of malnourished children and that oxidative stress causes a significant increase in DNA damage, as evaluated by the MN-RET frequency.

Assessment of micronucleus and oxidative stress in peripheral blood from malnourished children / Evaluación de micronúcleos y estrés oxidante en sangre periférica de niños desnutridos

Introduction: malnutrition is one of the most common health problems among children in underdeveloped countries, including Mexico. Previous studies have indicated increased genetic damage in malnourished humans and animal models, but the essential mechanisms remain unclear. In the present study, we assessed the effects of malnutrition on the frequency of micronucleus (MN) in reticulocytes (RET) from the peripheral blood of well-nourished uninfected (WN), well-nourished infected (WNI), moderately malnourished infected (UNM) and severely malnourished infected (UNS) children. Moreover, lipid peroxidation and the antioxidant status were evaluated to investigate the role of oxidative processes in malnutrition-associated genotoxicity. Methods: the antioxidant status of the study population was determined by measuring superoxide dismutase (SOD) in the red blood cells and glutathione peroxidase (GPX) in whole blood. Results: the UNS and UNM groups have increased percentages of MN-RET compared to the WNI group. Moreover, the data showed a significant increase in lipid peroxidation and a decrease in erythrocyte SOD activity and GPX activity in the malnourished group compared to the well-nourished infected children. Conclusion: the data suggest that the antioxidant system was impaired in the cells of malnourished children and that oxidative stress causes a significant increase in DNA damage, as evaluated by the MN-RET frequency

Introducción: la desnutrición es uno de los principales problemas de salud entre los niños de los países en desarrollo, incluido México. Estudios previos han mostrado que existe un incremento en el daño genético en humanos y modelos animales desnutridos, pero los mecanismos por los que se desencadena aún son poco claros. En el presente estudio, evaluamos los efectos de la desnutrición en la frecuencia de micronúcleos (MN) en reticulocitos (RET) de sangre periférica de niños bien nutridos no infectados (WN), bien nutridos infectados (WNI), desnutridos moderados infectados (UNM) y con desnutrición severa e infecciones (UNS). Asimismo, se evaluaron la lipoperoxidación y la capacidad antioxidante para investigar el papel del proceso oxidativo en la genotoxicidad asociada a la desnutrición. Métodos: la capacidad antioxidante de la población de estudio fue determinada midiendo la superóxido-dismutasa (SOD) en los eritrocitos y glutatión peroxidasa (GPX) en sangre completa. Resultados: los niños UNS y UNM tienen alto porcentaje de MN-RET comparados con el grupo WNI. Además, los datos mostraron un incremento significativo en la lipoperoxidación y disminución en la actividad de SOD y GPX en el grupo de niños desnutridos comparados con el grupo de niños bien nutridos infectados. Conclusión: los datos sugieren que el sistema antioxidante está deteriorado en las células de los niños desnutridos y que el estrés oxidante causa un incremento significativo en el daño al ADN, el cual se refleja en el incremento en la frecuencia de RET-MN

Trimethoprim-sulfamethoxazole treatment increases the Pig-a mutant frequency in peripheral blood from severely malnourished rats.

Severe malnutrition is a complex condition that increases susceptibility to infections. Thus, drugs are extensively used in malnutrition cases. In the present study, we assessed the mutagenic effects of combined trimethoprim and sulfamethoxazole (TMP-SMX) treatment in undernourished (UN) and well-nourished (WN) rats. Six-week-old UN and WN Han-Wistar rats were treated with TMP-SMX at a daily dose of 10 mg/kg/d TMP and 50 mg/kg/d SMX for 5 or 10 days. Blood was collected from the tail vein one day before (day -1) and 15, 30, and 45 days after TMP-SMX administration. The Pig-a mutant frequencies (MFs) in peripheral blood reticulocytes (RETs) and erythrocytes (RBCs) were measured through flow cytometry. Severe malnutrition increased the basal MFs in RETs (RET CD59-) and RBC (RBCs CD59-). These findings support the hypothesis that severe malnutrition is mutagenic even in the absence of exposure to an exogenous mutagen. UN and WN rats treated for 5 or 10 consecutive days with TMP-SMX had significantly increased and sustained Pig-a mutant frequencies, demonstrating the mutagenic effects of this drug.

Moderate malnutrition in rats induces somatic gene mutations.

The relationship between malnutrition and genetic damage has been widely studied in human and animal models, leading to the observation that interactions between genotoxic exposure and micronutrient status appear to affect genomic stability. A new assay has been developed that uses the phosphatidylinositol glycan class A gene (Pig-a) as a reporter for measuring in vivo gene mutation. The Pig-a assay can be employed to evaluate mutant frequencies (MFs) in peripheral blood reticulocytes (RETs) and erythrocytes (RBCs) using flow cytometry. In the present study, we assessed the effects of malnutrition on mutagenic susceptibility by exposing undernourished (UN) and well-nourished (WN) rats to N-ethyl-N-nitrosourea (ENU) and measuring Pig-a MFs. Two week-old UN and WN male Han-Wistar rats were treated daily with 0, 20, or 40mg/kg ENU for 3 consecutive days. Blood was collected from the tail vein one day before ENU treatment (Day-1) and after ENU administration on Days 7, 14, 21, 28, 35, 42, 49, 56 and 63. Pig-a MFs were measured in RETs and RBCs as the RET(CD59-) and RBC(CD59-) frequencies. In the vehicle control groups, the frequencies of mutant RETs and RBCs were significantly higher in UN rats compared with WN rats at all sampling times. The ENU treatments increased RET and RBC MFs starting at Day 7. Although ENU-induced Pig-a MFs were consistently lower in UN rats than in WN rats, these differences were not significant. To understand these responses, further studies should use other mutagens and nucleated surrogate cells and examine the types of mutations induced in UN and WN rats.

Malnutrition and infection influence the peripheral blood reticulocyte micronuclei frequency in children.

Malnutrition is a serious public health problem that affects approximately one third of all children. Developing countries have the highest incidence of malnourished children, and approximately 60% of deaths that occur in children under five are directly related to malnutrition and associated diseases. The relationship between malnutrition and genetic damage has been widely studied in humans and animal models. The micronucleus (MN) assay is useful in detecting chromosome damage induced by several factors. The aim of this study was to evaluate the effects of infection and malnutrition on the frequency of MN in erythrocytes from the peripheral blood of well-nourished, uninfected (WN) and well-nourished, infected (WNI) children, and moderately malnourished (UNM) and severely malnourished (UNS) children, both with infection, using a flow cytometric analysis technique. The percentage of reticulocytes (RETs) was significantly higher (1.5-fold) in WNI children than well-nourished controls. In addition, the UNS group had a 2.2-fold increase in the percentage of RETs compared to the WNI group. The frequency of micronucleated reticulocytes (MN-RETs) was 2.5 times greater, in WNI group compared to the WN group. These frequencies were significantly higher (1.7- and 2.1-fold) in UNM and UNS, respectively, compared to the WNI group. The results suggest that infection and malnutrition induce DNA damage in children.