RESUMEN
Alzheimer's disease (AD) is a chronic neurodegenerative disease leading to progressive dementia in elderly people. The disease is characterized, among others, by formation of amyloid-ß (Aß) polypeptide plaques in the brain. Although etiology of the disease is not fully understood, recent research suggest that nanomaterials may affect AD development. Here, we described the consequences of exposure of mouse BV-2 microglia to silver nanoparticles (AgNPs, 50 µg/mL), cerium oxide nanoparticles (CeO2NPs, 100 µg/mL), and cadmium telluride quantum dots (CdTeQDs, 3 or 10 µg/mL) in the context of its ability to clear Aß plaques. The brain microglial cells play an important role in removing Aß plaques from the brain. Cell viability and cycle progression were assessed by trypan blue test and propidium iodide binding, respectively. The uptake of Aß and NPs was measured by flow cytometry. Secretion of proinflammatory cytokines was measured with the use of cytometric bead array. Aß (0.1 µM) did not affect viability, whereas NPs decreased microglia growth by arresting the cells in G1 phase (CdTeQDs) or in S phase (AgNPs and CeO2NPs) of cell cycle. The uptake of Aß was significantly reduced in the presence of AgNPs and CeO2NPs. In addition, the least toxic CeO2NPs induced the release of proinflammatory cytokine, tumor necrosis factor α. In summary, each of the NPs tested affected either the microglia phagocytic activity (AgNPs and CeO2NPs) and/or its viability (AgNPs and CdTeQDs) that may favor the occurrence of AD and accelerate its development.
Asunto(s)
Péptidos beta-Amiloides/efectos de los fármacos , Péptidos beta-Amiloides/metabolismo , Cerio/toxicidad , Nanopartículas del Metal/toxicidad , Microglía/efectos de los fármacos , Puntos Cuánticos/toxicidad , Plata/toxicidad , Anciano , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Animales , Femenino , Humanos , Masculino , Ratones , Modelos AnimalesRESUMEN
Compounds with the structural motif of 1,4-dihydropyridine display a broad spectrum of biological activities, often defined as bioprotective. Among them are L-type calcium channel blockers, however, also derivatives which do not block calcium channels exert various effects at the cellular and organismal levels. We examined the effect of sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate (denoted here as DHP and previously also as AV-153) on X-ray-induced DNA damage and mutation frequency at the HGPRT (hypoxanthine-guanine phosphoribosyl transferase) locus in Chinese hamster ovary CHO-K1 cells. Using formamido-pyrimidine glycosylase (FPG) comet assay, we found that 1-h DHP (10nM) treatment before X-irradiation considerably reduced the initial level of FPG-recognized DNA base damage, which was consistent with decreased 8-oxo-7,8-dihydro-2'-deoxyguanosine content and mutation frequency lowered by about 40%. No effect on single strand break rejoining or on cell survival was observed. Similar base damage-protective effect was observed for two calcium channel blockers: nifedipine (structurally similar to DHP) or verapamil (structurally unrelated). So far, the specificity of the DHP-caused reduction in DNA damage - practically limited to base damage - has no satisfactory explanation.
Asunto(s)
Antimutagênicos/farmacología , Daño del ADN/efectos de los fármacos , Dihidropiridinas/farmacología , Rayos X/efectos adversos , Animales , Células CHO/efectos de la radiación , Bloqueadores de los Canales de Calcio/farmacología , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , MutaciónRESUMEN
The genotoxic activity of microcystin-LR (MC-LR) is a matter of debate. MC-LR is known to be a phosphatase inhibitor and it may be expected that it is involved in the regulation of the activity of DNA-dependent protein kinase (DNA-PK), the key enzyme involved in the repair of radiation-induced DNA damage. We studied the effect of MC-LR on the repair capacity of radiation-induced DNA damage in human lymphocytes and human glioblastoma cell lines MO59J and MO59K. A dose of 0.5 microg/ml of MC-LR was chosen because it induced very little early apoptosis which gives no false positive results in the comet assay. Human lymphocytes in G0-phase of the cell cycle were pre-treated with MC-LR for 3 h and irradiated with 2 Gy of gamma radiation. The kinetics of DNA repair was assessed by the comet assay. In addition the frequencies of chromosomal aberrations were analysed. The pre-treatment with MC-LR inhibited the repair of radiation-induced damage and lead to enhanced frequencies of chromosomal aberrations including dicentric chromosomes. The results of a split-dose experiment, where cells were exposed to two 1.5 Gy doses of radiation separated by 3 h with or without MC-LR, confirmed that the toxin increased the frequency of dicentric chromosomes. We also determined the effect of MC-LR and ionizing radiation on the frequency of gamma-H2AX foci. The pre-treatment with MC-LR resulted in reduced numbers of gamma-H2AX foci in irradiated cells. In order to elucidate the impact of MC-LR on DNA-PK we examined the kinetics of DNA repair in human glioblastoma MO59J and MO59K cells. Both cell lines were exposed to 10 Gy of X-rays and DNA repair was analysed by the comet assay. A strong inhibitory effect was observed in the MO59K but not in the MO59J cells. These results indicate that DNA-PK might be involved in DNA repair inhibition by MC-LR.
Asunto(s)
Daño del ADN , Reparación del ADN , Inhibidores Enzimáticos/efectos adversos , Rayos gamma/efectos adversos , Péptidos Cíclicos/efectos adversos , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Aberraciones Cromosómicas , Ensayo Cometa , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Proteína Quinasa Activada por ADN/metabolismo , Citometría de Flujo , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Glioblastoma/radioterapia , Histonas/metabolismo , Humanos , Cinética , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/efectos de la radiación , Toxinas Marinas/efectos adversos , Microcistinas , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Receptores de Neuropéptido Y/antagonistas & inhibidores , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Fase de Descanso del Ciclo Celular/efectos de la radiaciónRESUMEN
Curcumin, a yellow pigment from Curcuma longa, exhibits anti-inflammatory, antitumor, and antioxidative properties. Although its precise mode of action has not been elucidated so far, numerous studies have shown that curcumin may induce apoptosis in normal and cancer cells. Previously, we showed that in Jurkat cells curcumin induced nontypical apoptosis-like pathway, which was independent of mitochondria and caspase-3. Now we show that the inhibition of caspase-3 by curcumin, which is accompanied by attenuation of internucleosomal DNA fragmentation, may be due to elevation of glutathione, which increased in curcumin-treated cells to 130% of control. We have demonstrated that glutathione depletion does not itself induce apoptosis in Jurkat cells; though, it can release cytochrome c from mitochondria and caspase-3 from inhibition by curcumin, as shown by Western blot. The level of Bcl-2 protein was not affected by glutathione depletion even upon curcumin treatment. Altogether, our results show that in Jurkat cells curcumin prevents glutathione decrease, thus protecting cells against caspase-3 activation and oligonucleosomal DNA fragmentation. On the other hand, it induces nonclassical apoptosis via a still-unrecognized mechanism, which leads to chromatin degradation and high-molecular-weight DNA fragmentation.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Inhibidores de Caspasas , Curcumina/farmacología , Glutatión/metabolismo , Bisbenzimidazol , Caspasa 3 , Caspasas/metabolismo , Grupo Citocromo c/metabolismo , Citometría de Flujo , Radicales Libres/metabolismo , Humanos , Células Jurkat/efectos de los fármacos , Células Jurkat/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Azul de TripanoRESUMEN
The two L5178Y (LY) sublines bear a heterozygous Tp53 mutation that affects its transactivation function. LY-S (radiation-sensitive) cells are deficient in double strand break (DSB) repair by non-homologous end-joining (NHEJ) and do not express p21WAF1 (Cdkna1) either constitutively or after x-irradiation, in contrast to their radiation-resistant counterpart LY-R cells, which express p21WAF1 constitutively. Radiation-induced G2 arrest in LY-S cells is very long (11 h/Gy) but 2 mM caffeine treatment shortens it, decreases the fraction of G2 cells and increases the fraction of apoptotic cells. The treatment also increases the DNA damage that is estimated with the comet assay 18 h after irradiation with 5 Gy (ca. 23% of the initial value for x-rays and ca. 47% for x-rays plus caffeine). This indicates that either the repair has not been completed or the apoptotic DNA fragmentation has been initiated (or both). The same treatment applied to x-irradiated (5 Gy) LY-R cells (G2 arrest, 4 h/Gy) has no radiosensitising effect, induces no apoptosis and does not alter the amount of DNA damage left unrepaired (ca. 28%). The results are compatible with the assumption that inhibition of the Atm-dependent homologous recombination repair by caffeine, brings differential effects in LY sublines because of the defect of the alternative DNA repair system (NHEJ) in LY-S cells.
Asunto(s)
Cafeína/farmacología , Estimulantes del Sistema Nervioso Central/farmacología , Reparación del ADN , Fase G2/efectos de la radiación , Animales , Apoptosis , Western Blotting , Ensayo Cometa , Ciclina B/metabolismo , Ciclina B1 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Fragmentación del ADN , Electroforesis en Gel de Poliacrilamida , Rayos gamma , Ratones , Modelos Biológicos , Proteína de Retinoblastoma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Células Tumorales Cultivadas , Rayos XRESUMEN
We examined apoptosis and expression of p53, E2F-1, bax, bclx(L) and bc12 proteins in two L5178Y (LY) murine lymphoma sublines, LY-R and LY-S, which differ in radiosensitivity and double-strand break (DSB) repair. Both sublines are heterozygous for a p53 mutation in codon 170 that precludes the transactivation function. Accordingly, there is no G1/S arrest after irradiation. We found that there is no change in expression of E2F-1, bax, bclx(L) or bc12 proteins in both LY sublines after x-irradiation. LY-R cells do not constitutively express bc12, whereas both sublines show high bax content. Radiation induces delayed apoptosis to a greater extent in LY-S than in LY-R cells. The apoptosis can be seen 24 h after irradiation (2 Gy) of LY-S cells, with a maximum at 48 h. LY-R cells need 5 Gy and 72 h post-irradiation incubation to show marked apoptosis (identified by the TUNEL method). The reported observations support the assumption that differential radiosensitivity of LY sublines is associated with the induction of apoptosis that is not related to transactivation by p53 and is primarily related to differential DNA repair ability.
Asunto(s)
Apoptosis/efectos de la radiación , Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Genes p53 , Mutación , Animales , Ciclo Celular/efectos de la radiación , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Genes p53/genética , Leucemia L5178 , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Tolerancia a Radiación , Proteína 1 de Unión a Retinoblastoma , Factores de Transcripción/genética , Células Tumorales Cultivadas , Rayos X , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
We examined the response to hydrogen peroxide of two L5178Y (LY) sublines which are inversely cross-sensitive to hydrogen peroxide and X-rays: LY-R cells are radio-resistant and hydrogen peroxide-sensitive, whereas LY-S cells are radiosensitive and hydrogen peroxide-resistant. Higher initial DNA breaks and higher iron content (potentially active in the Fenton reaction) were found in the hydrogen peroxide sensitive LY-R cells than in the hydrogen peroxide resistant LY-S cells, whereas the antioxidant defence of LY-R cells was weaker. In particular, catalase activity is twofold higher in LY-S than in LY-R cells. The content of monobromobimane-reactive thiols is 54% higher in LY-S than in LY-R cells. In contrast, the activity of glutathione peroxidase (GPx) is about two times higher in LY-R than in LY-S cells; however, upon induction with selenium the activity increases 15.6-fold in LY-R cells and 50.3-fold in LY-S cells. Altogether, the sensitivity difference is related to the iron content, the amount of the initial DNA damage, as well as to the efficiency of the antioxidant defence system. Differential nuclear translocation of p65-NF-kappaB in LY sublines is due to the more efficient antioxidant defence in LY-S than in LY-R cells.
Asunto(s)
Transporte Activo de Núcleo Celular , Antioxidantes/metabolismo , Núcleo Celular/metabolismo , Hierro/metabolismo , Linfoma/metabolismo , FN-kappa B/metabolismo , Estrés Oxidativo , Animales , Daño del ADN , Relación Dosis-Respuesta a Droga , Humanos , Peróxido de Hidrógeno/farmacología , Modelos Biológicos , Temperatura , Factores de Tiempo , Factor de Transcripción ReIA , Células Tumorales Cultivadas , Rayos XRESUMEN
We examined the induction of nuclear translocation of the p65 subunit of NF-kappaB in L5178Y (LY) cells. We used two LY sublines which are inversely cross-sensitive to hydrogen peroxide and x-rays: LY-R cells are radioresistant and oxidant-sensitive, whereas LY-S cells are radiosensitive and oxidant-resistant. Hydrogen peroxide, phorbol ester and x-rays caused a marked translocation of p65-NF-kappaB in LY-R cells and a weak translocation in LY-S cells. By manipulating the antioxidant defense status, we obtained an alteration in the p65-NF-kappaB translocation induction in LY-R cells. A similar effect was achieved with lovastatin pretreatment (25 microM, 24 h, 37 degrees C). The response of LY-S cells under all these conditions was considerably weaker. We conclude that differential nuclear translocation of p65-NF-kappaB in LY sublines is not related to the lethal effect of the activating, damaging agent; rather it is due to the more efficient antioxidant defense in LY-S than in LY-R cells.
Asunto(s)
Antioxidantes/farmacología , Leucemia L5178/genética , FN-kappa B/genética , Translocación Genética/efectos de los fármacos , Translocación Genética/efectos de la radiación , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Western Blotting , Muerte Celular/efectos de los fármacos , ADN/efectos de los fármacos , ADN/efectos de la radiación , Peróxido de Hidrógeno/farmacología , Leucemia L5178/tratamiento farmacológico , Leucemia L5178/radioterapia , Lovastatina/farmacología , Ratones , FN-kappa B/efectos de los fármacos , FN-kappa B/efectos de la radiación , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/efectos de la radiaciónRESUMEN
The processes involved in cell response to camptothecin (CPT) were investigated in two sublines of L5178Y (LY) murine lymphoma; LY-R, resistant and LY-S, sensitive to X-irradiation, which are inversely cross-sensitive to the drug. The cells were pulse-treated with 2 microM CPT for 1 h; this resulted in equal numbers of replication-related DNA double-strand breaks (DSBs) in both sublines. After drug removal, at different time points up to 24 h, the levels of DSBs were measured by using field inversion gel electrophoresis (FIGE) and comet assay at neutral pH. Both methods revealed faster DSBs repair in LY-S than in LY-R cells, in contrast with X-ray-induced DSBs. This however, was followed by the appearance of secondary breaks in the former subline. The cell cycle arrest was at S/G2 phase and comprised equal numbers of cells in LY-S and LY-R populations. In both sublines formation of giant cells took place, as well as delayed apoptosis starting about 20 h post-CPT incubation and proceeding with similar intensity. At the same time, the total number of necrotic cells appearing during post-exposure incubation in the LY-R subline exceeded that in the LY-S subline. We suggest that, beside previously documented higher susceptibility of topoisomerase I (Topo I) from LY-R cells to CPT, a higher initial rate of replication-related DSBs repair, but not lower propensity to apoptosis, may contribute to the relative CPT resistance of LY-S versus LY-R cells.
Asunto(s)
Apoptosis/fisiología , Camptotecina/farmacología , Ciclo Celular/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Leucemia L5178/patología , Animales , Apoptosis/efectos de los fármacos , Replicación del ADN , Electroforesis en Gel de Agar , Citometría de Flujo , Concentración de Iones de Hidrógeno , Células Tumorales CultivadasRESUMEN
Two L5178Y (LY) murine lymphoma cell sublines, LY-R, resistant, and LY-S, sensitive, to X-irradiation display inverse cross-sensitivity to camptothecin (CPT): LY-R cells were more susceptible to this specific topoisomerase I inhibitor than LY-S cells. After 1 h incubation with CPT, the doses that inhibited growth by 50 per cent (ID50) after 48 h of incubation were 0.54 microM for LY-R cells and 1.25 microM for LY-S cells. Initial numbers of DNA-protein crosslinks (DPCs) measured at this level of growth inhibition were two-fold higher in LY-R (5.6 Gray-equivalents) than in LY-S cells (3.1 Gray-equivalents), which corresponds well with the greater in vitro sensitivity of Topo I from LY-R cells to CPT. Conversely, the initial levels of single-strand DNA breaks (SSBs) and double-strand DNA breaks (DSBs) were lower in LY-R cells (4.2 Gray-equivalent SSBs and 5.8 Gray equivalent DSBs) than in LY-S cells (8.0 Gray-equivalent SSBs and 12.0 Gray-equivalent DSBs). Dissimilarity in the replication-dependent DNA damage observed after 1 h of treatment with CPT was not due to a difference in the rate of DNA synthesis between the two cell lines, but may have arisen from a substantially slower repair of DNA breaks in LY-S cells. Release from G2 block by caffeine co-treatment significantly increased cell killing in the LY-S subline, and only slightly inhibited growth of LY-R cells. These results show that after CPT treatment cells arrest in G2, allowing them time to repair the long-lived DSBs. As LY-S cells are slower in repairing the DSBs, they were more susceptible to CPT in the presence of caffeine.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Camptotecina/uso terapéutico , Daño del ADN , Inhibidores Enzimáticos/uso terapéutico , Leucemia L5178/tratamiento farmacológico , Inhibidores de Topoisomerasa I , Animales , Supervivencia Celular/genética , Resistencia a Antineoplásicos , Leucemia L5178/genética , Leucemia L5178/patología , Ratones , Células Tumorales CultivadasRESUMEN
The L5178Y (LY) murine lymphoma subline, LY-R, is more radioresistant and more sensitive to camptothecin (CPT, inhibitor of topisomerase I) than the second subline used in our investigation, LY-S. Post-irradiation treatment with 3 microM CPT enhanced the radiosensitivity of LY-S cells (D0 decrease from 0.52 to 0.34 Gy), but did not change it in LY-R cells. Treatment with 2 mM benzamide [BZ, inhibitor of poly (ADP-ribosylation)] before x-rays and CPT increased the radiosensitivity of LY-R cells (D0 decrease from 1.15 to 0.52) without further modification of radiosensitivity of LY-S cells. Activity of topoisomerase I was diminished 10 min after x-irradiation (5 Gy) in LY-S, but not in LY-R cells. The data on DNA damage (fluorescent halo or comet assays) showed that the ultimate fate of the cells did not depend on the DNA damage pattern estimated immediately after treatment (e.g. the damage was greater in x-rays plus CPT than in BZ plus x-rays plus CPT treated LY-R cells, although the radiosensitivity was less). Aphidicolin (inhibitor of DNA polymerases alpha and delta) applied concomitantly with CPT in cells not pretreated with BZ prevented the increase in DNA damage in LY-R cells, but was without effect in LY-S cells. Taking into account the differential inhibition by x-rays of DNA synthesis in LY sublines and its reversion by BZ in LY-S but not in LY-R cells, we conclude that the pattern of DNA damage observed by the methods applied depended on the status of DNA replication.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Benzamidas/farmacología , Camptotecina/farmacología , Daño del ADN , Tolerancia a Radiación , Inhibidores de Topoisomerasa I , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Replicación del ADN , Leucemia L5178/patología , Leucemia L5178/terapia , Ratones , Poli Adenosina Difosfato Ribosa/metabolismo , Células Tumorales Cultivadas , Rayos XRESUMEN
The murine L5178Y (LY) lymphoma sublines, LY-R (radiation resistant) and LY-S (radiation sensitive) display a difference in susceptibility to camptothecin (CPT): LY-S cells are less sensitive to killing by this inhibitor of topoisomerase I than LY-R cells. Post-treatment (CPT present until 3 h after irradiation) sensitizes only LY-S cells. In agreement with this, only in LY-S cells is the relative number of DNA-protein cross-links formed after treatment with CPT + X higher than expected for additivity of X-ray and CPT-induced damage. The pattern of changes in the labelling indices and cell cycle distribution in cells that underwent combined treatment is essentially like that seen for single-agent treatment: for LY-S cells like that for radiation, for LY-R cells like that for CPT. We found no direct relation between the patterns of cell cycle distributions and the enhancement of the lethal effect of X-irradiation by CPT post-treatment. The sublines are not markedly differentially sensitive to beta-lapachone, which modifies topoisomerase I activity, and not sensitized to X-rays by post-irradiation treatment with the drug.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Camptotecina/farmacología , Leucemia L5178/tratamiento farmacológico , Leucemia L5178/radioterapia , Naftoquinonas/farmacología , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Inhibidores de Topoisomerasa I , Animales , Terapia Combinada , Ensayos de Selección de Medicamentos Antitumorales , Leucemia L5178/enzimología , Ratones , Células Tumorales CultivadasRESUMEN
After feeding rats a vitamin B-6-deficient diet, we observed a decrease in pyridoxal 5'-phosphate concentrations in intestinal mucosa cells to 32 and 48% of control in cytoplasm and cell nuclei, respectively. Correlation analysis suggested that there were two pyridoxal 5'-phosphate pools in the nuclei: a "mobile" pool (equivalent to about 5% the concentration of the cytoplasmic pyridoxal 5'-phosphate), and a "stable" pool, which was independent of cytoplasmic fluctuations of pyridoxal 5'-phosphate (about 9 pmol pyridoxal 5'-phosphate/mg DNA). Reduction in pyridoxal 5'-phosphate content in the cells of vitamin B-6-deficient animals was accompanied by a substantial increase in 1,25-dihydroxyvitamin D-receptor ligand concentration in the cell nuclei (76.6 +/- 19.7 vs 762 +/- 291 fmol/mg DNA, mean +/- SEM). The degree of 1,25-dihydroxyvitamin D accumulation in the nuclei appeared to be an exponential function of the "mobile" nuclear pyridoxal 5'-phosphate concentration. Semilogarithmic transformation of the data yielded a straight line, representing an inverse correlation between the cytoplasm-related nuclear pool of pyridoxal 5'-phosphate and the logarithm of the 1,25-dihydroxyvitamin D concentration in the nuclei (r = -0.95). These data suggest that pyridoxal 5'-phosphate may be related to 1,25-dihydroxyvitamin D retention in the nuclei, possibly through interaction of the pyridoxal 5'-phosphate with the vitamin D receptor protein in the nuclei.
Asunto(s)
Calcitriol/metabolismo , Mucosa Intestinal/metabolismo , Fosfato de Piridoxal/metabolismo , Receptores de Calcitriol/metabolismo , Fosfatasa Alcalina/sangre , Animales , Peso Corporal/efectos de los fármacos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Ligandos , Masculino , Ratas , Ratas Wistar , Receptores de Calcitriol/agonistas , Deficiencia de Vitamina B 6/metabolismoRESUMEN
Nitracrine (Ledakrin, 1-nitro-9-(3,3-N,N-dimethylaminopropylamino)-acridine) is of interest as a DNA intercalator and alkylator with very high cytotoxic potency, especially against hypoxic cells. DNA-DNA crosslinks [Konopa et al., Chem Biol Interact 43: 175-197, 1983; Pawlak et al., Cancer Res 44: 4289-4296, 1984] or DNA-protein crosslinks (DPCs) [Woynarowski et al., Biochem Pharmacol 38: 4095-4101, 1989; Szmigiero and Studzian, Biochim Biophys Acta 1008: 339-345, 1989] are related to the toxicity of the drug. The cytotoxic effect of and DNA damage induced by nitracrine were measured in two sublines of mouse lymphoma L5178Y, LY-R (resistant to ionizing radiation) and LY-S (sensitive to ionizing radiation). LY-R cells were more sensitive to nitracrine (D10 = 0.11 microM) than LY-S (D10 = 0.35 microM) when treated for 1 hr at 37 degrees. To a DNA-DNA crosslinking agent, mitomycin C, the comparative sensitivity was opposite. LY-R cells were more resistant to this drug than LY-S cells (D10 = 7.1 vs 2.3 microM). DNA damage induced by nitracrine was measured by the alkaline elution method and by nitrocellulose filter binding assay. Nitracrine treatment with biologically relevant concentrations (0.1-3.0 microM, 1 hr, 37 degrees) induced only DPCs. Interstrand crosslinks and DNA breaks were not detected. Nitracrine produced about two times more DPCs in LY-R cells than in LY-S cells. Both sublines removed 50% of initial lesions during 2 hr post-treatment incubation. The greater sensitivity of LY-R cells to nitracrine is thus not related to the efficiency of DNA repair, but may be a consequence of enhanced initial damage in the form of DPCs. This finding is consistent with the latter lesion being responsible for the cytotoxicity of nitracrine.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN/efectos de los fármacos , ADN/efectos de los fármacos , Nitracrina/toxicidad , Animales , Supervivencia Celular/efectos de los fármacos , Reparación del ADN , Ratones , Células Tumorales CultivadasRESUMEN
Growth rate of five children with vitamin D-dependent rickets was analyzed during the long-term treatment with an active analog of vitamin D3. Considerable increase in growth rate together with the improvement of biochemical values and radiological pattern took place during the initial phase of administration of 1-hydroxyvitamin D3. During the maintenance treatment of long duration with 1-hydroxyvitamin D3 both the acceleration of growth and catch-up growth persisted. However, in 4 among 5 children studied an inhibition of growth was observed during different periods of time. Only in one boy was this connected with the conclusion of the process of physiological growth. In three remaining children a slow-down in growth rate appeared during the pre-pubertal period or was the effect of lowering the dose of 1-hydroxyvitamin D3 as an countermeasure to hypercalciuria. In such cases inhibition of growth was caused by the administration of too small a dose of 1-hydroxyvitamin D3 in relation to the requirement. In all cases the appearance of biochemical features of rickets aggravation, such as low blood serum phosphate concentration and elevated alkaline phosphatase activity, preceded the observable inhibition of growth. The results obtained allow us to conclude that the inhibition of growth observed during the long-term treatment of rickets with 1-hydroxyvitamin D3 may be regarded as the first signal of inadequate dosage of 1-hydroxy vitamin D3.
Asunto(s)
Crecimiento/efectos de los fármacos , Hidroxicolecalciferoles/efectos adversos , Raquitismo/tratamiento farmacológico , Adolescente , Fosfatasa Alcalina/metabolismo , Calcio/sangre , Calcio/orina , Niño , Preescolar , Esquema de Medicación , Femenino , Humanos , Hidroxicolecalciferoles/administración & dosificación , Masculino , Raquitismo/fisiopatologíaRESUMEN
A simple method has been employed to prepare crude nuclear extract from rat thymus, using hypertonic buffer after previous treatment with hypotonic buffer. The preparation is free from serum vitamin D-binding protein and contains a 3.7 S receptor molecule, which specifically binds 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3). The receptor is of high affinity (KD = 0.85 X 10(-11) M at O degrees C) and low capacity (260-460 fmol/g tissue). The Scatchard analysis of ligand binding results in a concave downward curve. The Hill analysis of the same data gives good linear fitting (r = +0.971) with the Hill coefficient nH = 1.63. These facts indicate positive cooperativity between two ligand binding sites of the rat thymus 1,25-(OH)2D3 receptor. The preparation was used in a competitive protein binding assay of 1,25-(OH)2D in serum extracts, purified on Sep-Pak C18 followed by silica Sep-Pak cartridges. The method was sensitive to 0.5 pg/tube (2.0 ng/l) when 1 ml of serum was extracted. Intra- and interassay coefficients of variation were 9% and 14%, respectively. The serum 1,25-(OH)2D concentration estimated in 33 children (mean age 6.5 +/- 3 years) was 46.6 +/- 18.4 ng/l (mean +/- S.D.).