RESUMO
Nigella sativa (N. sativa) is a medicinal plant used for its therapeutic pharmacological effects such as anti-inflammatory, antioxidant, anticancer, antidiabetic, and immunomodulation. This study explored the anti-cytotoxic and anti-genotoxic effect of N. sativa through a micronucleus test (MNT) of BALB/c mice peripheral blood. Using 6-to-8-week-old healthy male BALB/c mice, four groups were formed: (1) Control (sterile water), single-dose 2 mg/kg/intraperitoneal (i.p); (2) N. sativa oil, 500 mg/kg/24 h/7 days/i.p; (3) Cisplatin (CP), single-dose 2 mg/kg/subcutaneous (s.c); (4) N. sativa + CP with their respective dosage. When evaluating polychromatic erythrocytes (PCE), a biomarker of cytotoxicity, the group treated with N. sativa + CP experienced an increase in the frequency of PCE, which demonstrated the recovery of bone marrow and modulation of cell proliferation. The analysis of micronucleated polychromatic erythrocytes (MNPCE), an acute genotoxicity biomarker, showed similar frequency of MNPCE within the groups except in CP, but, in the N. sativa + CP group, the frequency of MNPCE decreased and then regulated. Finally, the frequency of micronucleated erythrocytes (MNE), a biomarker of genotoxicity, the supplementation of N. sativa oil did not induce genotoxic damage in this model. Thus, we conclude that N. sativa has both cytoprotective, genoprotective effects and modulates cell proliferation in BALB/c mice.
Assuntos
Cisplatino/toxicidade , Citoproteção/efeitos dos fármacos , Eritroblastos/efeitos dos fármacos , Testes para Micronúcleos/métodos , Nigella sativa/química , Óleos de Plantas/farmacologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cisplatino/administração & dosagem , Masculino , Camundongos Endogâmicos BALB C , Óleos de Plantas/administração & dosagem , Óleos de Plantas/isolamento & purificaçãoRESUMO
Reversing the developmental switch from fetal hemoglobin (HbF, α2γ2) to adult hemoglobin (HbA, α2ß2) is an important therapeutic approach in sickle cell disease (SCD) and ß-thalassemia. In healthy individuals, SCD patients, and patients treated with pharmacologic HbF inducers, HbF is present only in a subset of red blood cells known as F cells. Despite more than 50 years of observations, the cause for this heterocellular HbF expression pattern, even among genetically identical cells, remains unknown. Adult F cells might represent a reversion of a given cell to a fetal-like epigenetic and transcriptional state. Alternatively, isolated transcriptional or posttranscriptional events at the γ-globin genes might underlie heterocellularity. Here, we set out to understand the heterogeneity of HbF activation by developing techniques to purify and profile differentiation stage-matched late erythroblast F cells and non-F cells (A cells) from the human HUDEP2 erythroid cell line and primary human erythroid cultures. Transcriptional and proteomic profiling of these cells demonstrated very few differences between F and A cells at the RNA level either under baseline conditions or after treatment with HbF inducers hydroxyurea or pomalidomide. Surprisingly, we did not find differences in expression of any known HbF regulators, including BCL11A or LRF, that would account for HbF activation. Our analysis shows that F erythroblasts are not significantly different from non-HbF-expressing cells and that the primary differences likely occur at the transcriptional level at the ß-globin locus.
Assuntos
Eritroblastos/metabolismo , Hemoglobina Fetal/biossíntese , Hemoglobina A/metabolismo , Adulto , Anemia Falciforme/sangue , Anemia Falciforme/genética , Linhagem Celular , Separação Celular/métodos , Células Cultivadas , Eritroblastos/classificação , Eritroblastos/efeitos dos fármacos , Células Eritroides/classificação , Células Eritroides/metabolismo , Hemoglobina Fetal/genética , Perfilação da Expressão Gênica , Hemoglobina A/genética , Humanos , Hidroxiureia/farmacologia , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Talidomida/análogos & derivados , Talidomida/farmacologiaRESUMO
Some of the most polluting activities occur in bovine skin processing. Tannery generates effluents containing high concentrations of heavy metals and organic compounds. The phases composing the leather production process generate a large volume of tannery effluents that are often discarded in aquatic environments without any previous treatment. However, the effect these xenobiotics have on adult representatives belonging to the class Amphibia remains unknown. Thus, the aim of the present study is to assess the geno- and cytotoxic effects of tannery effluent on adult male bullfrogs (Lithobates castesbeianus) exposed to it. Accordingly, the animals were divided into the following groups: negative control (tannery effluent-free water), positive control (cyclophosphamide), and effluent (water added with 5% tannery effluent). The animals were euthanized for blood collection, and erythrocyte analyses were conducted after 35 and 90 days of exposure. The micronuclei (MN) frequency and the frequency of other nuclear abnormalities in each of the animals in the experimental groups were assessed in 2000 erythrocytes. According to the present results, the exposure to tannery effluents increased MN frequency as well as other nuclear abnormalities (i.e., lobed nuclei, binucleated cell, kidney-shaped nuclei, notched nuclei, and apoptotic cell) in the erythrocytes of animals in the effluent group and in the positive control group after 35 and 90 exposure days. Thus, the current study corroborated the hypothesis that the tannery effluent has aneugenic and clastogenic potential in adult male bullfrogs (L. castesbeianus). The present study is the first to report such effect.
Assuntos
Eritrócitos/efeitos dos fármacos , Metais Pesados/toxicidade , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Mutagênicos/toxicidade , Curtume , Poluentes Químicos da Água/toxicidade , Animais , Dano ao DNA , Relação Dose-Resposta a Droga , Eritroblastos/química , Eritroblastos/efeitos dos fármacos , Eritroblastos/patologia , Eritrócitos/química , Eritrócitos/patologia , Eritrócitos Anormais/química , Eritrócitos Anormais/efeitos dos fármacos , Eritrócitos Anormais/patologia , Resíduos Industriais/análise , Masculino , Metais Pesados/análise , Testes para Micronúcleos , Estrutura Molecular , Mutagênicos/análise , Rana catesbeiana , Fatores de Tempo , Poluentes Químicos da Água/análiseRESUMO
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.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/toxicidade , Desoxicitidina/análogos & derivados , Eritroblastos/efeitos dos fármacos , Mutagênicos/toxicidade , Animais , Azacitidina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Desoxicitidina/farmacologia , Desoxicitidina/toxicidade , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Cinética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes para Micronúcleos , GencitabinaRESUMO
BACKGROUND: During pregnancy, glucocorticoids are frequently used to accelerate fetal lung maturation in preterm delivery. However, prenatal administration of glucocorticoids has been shown to affect organs such as fetal liver, an important hematopoietic organ during fetal development. OBJECTIVE: The aim of this study was to document the qualitative and quantitative changes in erythroid and megakaryocytic cell populations found in fetal livers as well as the hematology profile in neonates after maternal glucocorticoid treatment in rats. METHODS: Pregnant female Wistar rats were treated with dexamethasone 21-phosphate from days 13 to 16 of gestation. On the 17th day of pregnancy, the fetuses were collected and their livers processed for light and transmission electron microscopy. Glycol methacrylate-embedded sections were stained with PAS to determine the erythroblast and megakaryocytic cell frequencies. Fetal liver pieces embedded in Spurr resin were analyzed by transmission electron microscopy for morphologic changes. A standard hematology profile was evaluated in neonatal rats. RESULTS: In the fetuses from treated dams, the total cell number of erythroid cells in livers was significantly reduced compared to control fetuses (P < .001), but erythroblasts did not present ultrastructural abnormalities. The degree of maturation in the megakaryocyte series tended to be increased. In neonates, there were elevated numbers of nucleated RBCs (P = .002), along with a higher HCT and HGB (P = .02). In addition, the platelet concentration was also significantly increased (P < .007). CONCLUSION: These results suggest that maternal dexamethasone treatment has quantitative effects on erythroid and megakaryocytic cells in fetal liver and the neonatal hematology profile in rats.
Assuntos
Dexametasona/análogos & derivados , Desenvolvimento Fetal/efeitos dos fármacos , Glucocorticoides/efeitos adversos , Fígado/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Plaquetas/efeitos dos fármacos , Dexametasona/efeitos adversos , Dexametasona/uso terapêutico , Eritroblastos/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Feminino , Glucocorticoides/uso terapêutico , Testes Hematológicos/veterinária , Troca Materno-Fetal , Megacariócitos/efeitos dos fármacos , Microscopia Eletrônica de Transmissão/veterinária , Gravidez , Ratos , Ratos WistarRESUMO
Methods for visualizing DNA damage at the microscopic level are based on treatment of cell nuclei with saline or alkaline solutions. These procedures for achieving chromatin dispersion produce halos that surround the nuclear remnants. We improved the fast halo assay for visualizing DNA breakage in cultured cells to create a simplified method for detection and quantitative evaluation of DNA breakage. Nucleated erythrocytes from chicken blood were selected as a model test system to analyze the production of nuclear halos after treatment with X-rays or H(2)O(2). After staining with ethidium bromide or Wright's methylene blue-eosin solution, nuclear halos were easily observed by fluorescence or bright-field microscopy, respectively, which permits rapid visualization of DNA breakage in damaged cells. By using image processing and analysis with the public domain ImageJ software, X-ray dose and H(2)O(2) concentration could be correlated well with the size of nuclear halos and the halo:nucleus ratio. Our results indicate that this simplified nuclear halo assay can be used as a rapid, reliable and inexpensive procedure to detect and quantify DNA breakage induced by ionizing radiation and chemical agents. A mechanistic model to explain the differences between the formation of saline or alkaline halos also is suggested.
Assuntos
Cromatina/efeitos dos fármacos , Cromatina/efeitos da radiação , Quebras de DNA , Dano ao DNA , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/efeitos da radiação , Galinhas , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/efeitos da radiação , Eritroblastos/efeitos dos fármacos , Eritroblastos/efeitos da radiação , Peróxido de Hidrogênio/toxicidade , Técnicas In Vitro , Microscopia de Fluorescência , Modelos Biológicos , Coloração e RotulagemRESUMO
This study is a follow-up of previous research in which we described the frequency of spontaneous micronucleated erythrocytes (MNE) in the Goodeid Xenotocoa melanosoma collected from Lake La Alberca, located in the state of Michoacan, Mexico. In the present work, we measured micronuclei (MN) and nuclear abnormalities (NA) in erythrocytes of peripheral blood. Bioassays taken at 24 or 96 hours in either the cyclophosfamide (CP) or colchicine (COL) showed a significant increase in MN and BC (P values ranging from 0.0499 to 0.0036) compared with information from wild organisms collected over 3 years. Concentrationdependent and time-dependent responses support the proposal of using endemic Xenotoca melanosoma as a bioindicator of genotoxicity and cytotoxicity with a high transcendence for the health of the entire ecosystem and evaluation of the Lerma-Chapala watershed.
Assuntos
Aneugênicos/toxicidade , Colchicina/toxicidade , Ciclofosfamida/toxicidade , Ciprinodontiformes , Eritroblastos/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Mutagênicos/toxicidade , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/patologia , Relação Dose-Resposta a Droga , Monitoramento Ambiental , Eritroblastos/fisiologia , Feminino , Água Doce , Masculino , México , Testes para MicronúcleosRESUMO
Acute toxicity, genotoxicity, and cytotoxicity of the pirimicarb-containing commercial-formulation carbamate insecticide Aficida(R) (50% pirimicarb) were evaluated on Rhinella arenarum (Anura, Bufonidae) tadpoles exposed under laboratory conditions. Lethal and sublethal effects were employed as bioassays for acute toxicity, whereas micronuclei (MNi) induction and alterations in the ratio erythrocytes:erythroblasts were employed as end-points for genotoxicity and cytotoxicity, respectively. Cr(VI) (23 mg L(-1)) and cyclophosphamide (40 mg L(-1)) were employed as positive controls for toxicity and geno-cytotoxicity assays, respectively. In Gosner stage 25 (STD25), the results revealed mean values of 402.0 and 223.6 mg Aficida L(-1) for LC-50(24)(h) and LC-50(96)(h), respectively. When STD37-39 tadpoles were exposed, the LC-50(24)(h) and LC-50(96)(h) reached values of 239.4 and 181.7 mg Aficida L(-1), respectively. Sublethal effects revealed a mean EC-50(96)(h) of 133.85 and 104.2mg Aficida in those STD25 and STD37-39 treated tadpoles, respectively. The results demonstrated that in 48-h-exposed tadpoles, a MNi increase was found only in those 80.0 mg L(-1) Aficida-treated individuals. When tadpoles were exposed to Aficida for 96h, only the 160 mg L(-1)-treated individuals showed a significant increase in MNi frequency. Concentrations ranging from 80.0 to 250.0mg Aficida L(-1) resulted in cellular cytotoxicity, revealed by a decreased proportion of circulating erythrocytes and an enhancement of erythroblasts. Accordingly, this species could provide a suitable and useful experimental model for biomonitoring aquatic ecosystems.
Assuntos
Bufo arenarum/crescimento & desenvolvimento , Carbamatos/toxicidade , Inseticidas/toxicidade , Pirimidinas/toxicidade , Animais , Ciclofosfamida/toxicidade , Citotoxinas/toxicidade , Eritroblastos/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Larva/efeitos dos fármacos , Dose Letal Mediana , Testes de Mutagenicidade , Testes de Toxicidade AgudaRESUMO
In this study, we describe the presence of P2 receptor subtypes and Ca2+ signaling in erythroblasts. ATP and ADP produced a biphasic increase of intracellular Ca2+ concentration ([Ca2+]i), with an initial transient phase followed by a sustained phase. Reverse transcription polymerase chain reaction (RT-PCR) showed the expression of P2Y1, P2Y2 and P2Y12. The selective P2Y1 receptor antagonist 2'-deoxy-N6-methyl-adenosine-3',5'-diphosphate (MRS2179) and the G(i) protein inhibitor pertussis toxin blocked Ca2+ increase. The initial transient [Ca2+]i increase phase was sensitive to the 1,4,5-inositol trisphosphate (IP3) receptor blocker 2-aminoethoxy-diphenylborate (2-APB), while the sustained phase was sensitive to the protein kinase C (PKC) inhibitor 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF109203X) and calcium calmodulin kinase II (CaMKII) inhibitor 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62). In addition, the PKC activator phorbol-12,13-dibutyrate (PDBu) produced increase of [Ca2+]i. Flow cytometry analysis showed the expression of Ca2+-dependent PKC alpha, betaI, gamma and phospho-CaMKII. These results suggest that the activation of the P2Y1 receptor triggers two different [Ca2+]i increase pathways, one IP3-dependent and the other kinase-dependent.
Assuntos
Sinalização do Cálcio , Eritroblastos/metabolismo , Receptores Purinérgicos P2/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Compostos de Boro/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Relação Dose-Resposta a Droga , Eritroblastos/efeitos dos fármacos , Feminino , Indóis/farmacologia , Receptores de Inositol 1,4,5-Trifosfato , Maleimidas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y1RESUMO
In previous studies, we inferred some pharmacokinetic and pharmacodynamic parameters of alkylating agents and antimetabolites by comparing their kinetics of micronucleated polychromatic erythrocyte (MN-PCE) induction with the one obtained after the exposure to gamma rays in peripheral blood of mice, assuming that radiation acts immediately because it does not require absorption and distribution in the organism. According to our earlier studies, the kinetics of MN-PCE induction depends mainly on the following: (i) the cytotoxic effects that in turn could affect the duration of cell division; (ii) the pharmacokinetics including the metabolic activation requirement; and (iii) the mechanism of MN induction. The aim of the present study was to analyze the kinetics of MN-PCE induction by an aneuploidogen that induces micronuclei by acting on the achromatic spindle. The kinetics of MN-PCE induction by colchicine, as well as the reduction in the PCE frequency over time was determined in peripheral blood of mice treated with different doses of the aneuploidogen. The genotoxic effect, established as the area beneath the curve (ABC) of MN-PCE versus time-response, indicates an almost directly proportional relationship with respect to dose. Similarly, the relationship between dose and cytotoxic effect determined as the ABC of PCE versus time was inversely proportional, suggesting a relationship between both endpoints and doses administered. However, the number of cells affected by these two phenomena indicates that cytotoxicity is not necessarily caused, or at least not only by genotoxicity. The analysis of the kinetics of MN-PCE induction after the treatment with non-cytotoxic dose of colchicine, indicates that the MN-PCE appear in the blood stream at almost the same time, as occurs after the exposure to gamma rays; in spite of the differences in the cell cycle stage in which they can cause micronucleus (MN). Perhaps the fact that cells are not synchronized does not permit one to observe some difference in the time they appear in the blood. These results suggest that colchicine acts rapidly after exposure. The elimination half-life of colchicine is 17h, suggesting that colchicne is disposable for long time. With high doses of colchicine the pharmacokinetic parameters increases substantially. These data imply that low doses of colchicine are slightly cytotoxic, and that under this circumstances colchicines arrives rapidly to hemopoyetic tissues and acts for several hours.