The secretome of non-tumorigenic mammary cells MCF-10A elicits DNA damage in MCF-7 and MDA-MB-231 breast cancer cells.

Radiotherapy is used in breast cancer to destroy tumor cells lingering after surgery. It is accepted that lethal effects of ionizing radiation occur as a result of damage to DNA in irradiated (IR) cells. However, response mechanisms may promote cell survival with efficient DNA repair or genomic alterations. Chromosomal aberrations are frequent in surviving cells and may enhance chromosomal instability (CIN) which is associated with increased risk of recurrence and metastasis. Intercellular communication can affect the response in IR cells and cause damage in non-irradiated (N-IR) cells. We evaluated the effect of the secretome of non-tumorigenic mammary cells (MCF-10A) on proliferation and DNA damage in breast cancer cells (MCF-7 and MDA-MB-231). Results showed that conditioned media from IR and N-IR MCF-10A cells produced cycles of DNA double-strand breaks in N-IR and IR tumor cells leaving them with residual damage. CIN markers (micronuclei, nucleoplasmic bridges, nuclear buds) were also increased in IR and N-IR tumor cells, being the effect of conditioned media from IR MCF-10A greater in many cases. The inhibition of phosphorylation/activation of Src kinase in cancer cells hindered CIN markers' increment. Besides, clonogenic survival of tumor cells was differentially modulated by conditioned media from MCF-10A: decreased in MCF-7 and enhanced in MDA-MB-231 cells. These results signal the relevance of tumor-host interaction in tumor behavior and the response to radiotherapy.

Modulation of hepatic ABC transporters by Eruca vesicaria intake: Potential diet-drug interactions.

The aim of this work was to evaluate whether oral administration of Eruca vesicaria, a species of rocket cultivated in Argentina, could modify cyclophosphamide (CP)-induced genotoxicity through modulation of hepatic ABC transporters. Daily oral administration of E. vesicaria fresh leaves juice (1.0, 1.4 and 2.0  g/kg) for 14 days did not alter genotoxicity biomarkers -alkaline comet assay and micronucleus test -in neither male nor female mice. Instead, repeated intake of this cruciferous decreased CP-induced DNA damage dose-dependently and it caused hepatic overexpression of P-glycoprotein (P-gp; 1.4 and 2.0  g/kg) and multidrug resistance protein 2 (MRP2; 2.0  g/kg), but not breast cancer resistance protein (Bcrp). The antigenotoxic effect of E. vesicaria was prevented by 50 mg/kg verapamil (P-gp inhibitor) or 10 mg/kg indomethacin (MRP2 inhibitor). In turn, CP-induced cytotoxicity (10 mM, 24 h) on human hepatoma cells (HepG2/C3A) was significantly reduced by preincubation with E. vesicaria (1.4 mg/ml; 48 h); this effect was absent when CP was coincubated with 35 µM verapamil, 80 µM indomethacin or 10 µM KO-143 (BCRP inhibitor). Altogether, these results allow us to demonstrate that repeated intake of E. vesicaria exhibited antigenotoxicity, at least in part, by induction of hepatic ABC transporters in vivo in mice as well as in vitro in human liver cells. This could account for other diet-drug interactions.

In vivo antigenotoxic activity of Diplotaxis tenuifolia against cyclophosphamide-induced DNA damage: Relevance of modulation of hepatic ABC efflux transporters.

Several epidemiological studies have demonstrated that a diet with high contents of cruciferous vegetables (which belong to the Brassicaceae family) may reduce the incidence of cancer and neurodegenerative diseases. However, some authors have postulated that they might bring about toxic effects. Therefore, the aim of this study was to assess the effects of chronic administration of Diplotaxis tenuifolia (wild rocket), a species found in Argentina, concerning its putative genotoxicity or antigenotoxicity against the DNA damage inducer cyclophosphamide, and its ability to modulate the hepatic expression of ABC efflux transporters on mice. The alkaline comet assay and the micronucleus test were used as genotoxicity biomarkers, and the ABC transporter expression was analyzed by Western-blotting. D. tenuifolia juice exhibited no genotoxicity in any of the three tested doses (p > 0.05), showing instead a protective effect against genotoxic damage induced by cyclophosphamide (p < 0.001) in a dose-dependent behavior. Furthermore, hepatic expression of ABCB1 remained unchanged in both sexes at every dose, whereas ABCG2 expression increased in females (p < 0.05) and males (p < 0.01) at the highest dose. Regarding ABCC2, sex-related differences were observed (p < 0.05), its expression decreasing in females (p < 0.05) and increasing in males (p < 0.05). The modulation of these transporters may contribute to the antigenotoxic effects of D. tenuifolia since they act as universal detoxifiers, excreting xenobiotics to the cellular exterior. Phytochemicals present in the juice such as glucosinolates, quercetin and kaempherol may be responsible for these beneficial effects.

Assessment of the health status and risk of genotoxic and cytotoxic damage in Argentinian adolescents living near horticultural crops.

In some rural areas in Argentina, adolescents may be considered as a group indirectly exposed to agrochemicals because their parents plant small crops near their homes. This could become a health risk to children and adolescents who may be more sensitive to exposure to chemicals than adults. The objective of this study was to evaluate the health status of two different groups of Argentinian adolescents using biochemical parameters, dietary information, and cytogenetic biomarkers of genotoxicity and cytotoxicity. The study groups included 32 adolescents from Montecarlo, who were indirectly exposed to agrochemicals, and 30 unexposed adolescents from Exaltación de la Cruz. The values of total cholesterol, LDL cholesterol, triglycerides, gamma glutamyltransferase, and butyrylcholinesterase (BuChE) were higher (p < 0.05) in males from Exaltación de la Cruz compared with those from Montecarlo. The BuChE activity was also higher (p < 0.05) in females from this region. Furthermore, the consumption of citrus, vegetable-like fruits, tubers, and red meat was more frequent (p < 0.05) in Montecarlo. On the other hand, differences in frequency of biomarkers of genetic damage in lymphocytes were not found (p > 0.05). However, the cytome assay in buccal cells showed that karyorrhectic and pyknotic cells were more frequent (p < 0.05) in the Montecarlo group; whereas, the frequencies of cells with nuclear buds, condensed chromatin and karyolysis were higher (p < 0.05) in the Exaltación de la Cruz group. Despite the differences between the parameters and biomarkers evaluated, the adolescents of Montecarlo did not present health impairment probably due to the type and level of exposure to agrochemicals.

Modulator effect of watercress against cyclophosphamide-induced oxidative stress in mice.

Watercress (Nasturtium officinale, Cruciferae; W. Aiton) is a vegetable widely consumed in our country, with nutritional and potentially chemopreventive properties. Previous reports from our laboratory demonstrated the protective effect of watercress juice against DNA damage induced by cyclophosphamide in vivo. In this study, we evaluated the in vivo effect of cress plant on the oxidative stress in mice. Animals were treated by gavage with different doses of watercress juice (0.5 and 1g/kg body weight) for 15 consecutive days before intraperitoneal injection of cyclophosphamide (100 mg/kg body weight). After 24 h, mice were killed by cervical dislocation. The effect of watercress was investigated by assessing the following oxidative stress biomarkers: catalase activity, superoxide dismutase activity, lipid peroxidation, and glutathione balance. Intake of watercress prior to cyclophosphamide administration enhanced superoxide dismutase activity in erythrocytes with no effect on catalase activity. In bone marrow and liver tissues, watercress juice counteracted the effect of cyclophosphamide. Glutathione balance rose by watercress supplementation and lipid oxidation diminished in all matrixes when compared to the respective control groups. Our results support the role of watercress as a diet component with promising properties to be used as health promoter or protective agent against oxidative damage.

Modulator effect of watercress against cyclophosphamide-induced oxidative stress in mice

Watercress (Nasturtium officinale, Cruciferae; W. Aiton) is a vegetable widely consumed in our country, with nutritional and potentially chemopreventive properties. Previous reports from our laboratory demonstrated the protective effect of watercress juice against DNA damage induced by cyclophosphamide in vivo. In this study, we evaluated the in vivo effect of cress plant on the oxidative stress in mice. Animals were treated by gavage with different doses of watercress juice (0.5 and 1g/kg body weight) for 15 consecutive days before intraperitoneal injection of cyclophosphamide (100 mg/kg body weight). After 24 h, mice were killed by cervical dislocation. The effect of watercress was investigated by assessing the following oxidative stress biomarkers: catalase activity, superoxide dismutase activity, lipid peroxidation, and glutathione balance. Intake of watercress prior to cyclophosphamide administration enhanced superoxide dismutase activity in erythrocytes with no effect on catalase activity. In bone marrow and liver tissues, watercress juice counteracted the effect of cyclophosphamide. Glutathione balance rose by watercress supplementation and lipid oxidation diminished in all matrixes when compared to the respective control groups. Our results support the role of watercress as a diet component with promising properties to be used as health promoter or protective agent against oxidative damage.

El berro (Nasturtium officinale, crucíferas; W. Aiton) es una hortaliza ampliamente consumida en nuestro país, con valor nutricional y propiedades potencialmente quimiopreventivas. En trabajos previos demostramos que el jugo de berro tiene efecto protector in vivo contra el daño del ADN inducido por ciclofosfamida en tejidos del ratón. En el presente trabajo evaluamos, también in vivo, los efectos del jugo sobre el estrés oxidativo en diferentes tejidos del ratón. Los siguientes biomarcadores fueron investigados: actividad de superóxido dismutasa, actividad de catalasa, peroxidación lipídica y balance de glutatión. Los animales fueron tratados con diferentes dosis de jugo (0.5 y 1 g/kg de peso corporal) por alimentación forzada durante 15 días consecutivos antes de la inyección intraperitoneal con ciclofosfamida (100 mg/kg). La ingesta de berro antes de la administración de ciclofosfamida mejoró la actividad de superóxido dismutasa en los eritrocitos sin efecto sobre la actividad de la catalasa. En médula ósea e hígado, el jugo de berro contrarrestó el efecto deletéreo de la ciclofosfamida. En todas las matrices, el balance de glutatión fue mayor y la oxidación de lípidos menor que los valores encontrados en los grupos control. Nuestros resultados demuestran que el berro es un componente de la dieta con propiedades prometedoras como promotor de la salud o como agente protector contra el daño oxidativo.

In vivo antigenotoxic activity of watercress juice (Nasturtium officinale) against induced DNA damage.

The present study was carried out to investigate the genotoxicity as well as possible protective activity against damage induced by cyclophosphamide (CP) of the aqueous juice of watercress (Nasturtium officinale, W.T. Aiton) in vivo. Male and female Swiss mice 7-8 weeks old (N = 48) were treated by gavage with 1 g kg(-1) body weight and 0.5 g kg(-1) body weight of watercress juice during 15 consecutive days. Genotoxicity and its possible protective effect were tested by the comet assay in peripheral blood cells and the micronucleus test in bone marrow. In addition, biopsies of the bladder, epididymis and testicles of mice were performed to extend the experimental design. Watercress juice per se did not induce genetic damage according to the comet assay and micronucleus study, exhibiting a protective activity against CP (P < 0.05 and P < 0.001, respectively). The comparative analysis of bladder histological changes obtained in the watercress plus CP group against those treated with CP alone suggests a probable protective effect. Further studies are needed in order to establish the protective role of watercress juice against DNA damage.

DNA single strand breaks in peripheral blood lymphocytes induced by three nitroimidazole derivatives.

Tinidazole (TNZ), ornidazole (ONZ) and metronidazole (MTZ) are antiparasitic drugs (nitroimidazole derivatives) that have proven to be effective against Trichomonas vaginalis, Entoamoeba histolytica, Giardia lamblia and Helicobacter pylori. The reduction of the nitro group and the generation of short-lived reactive intermediates are the basis of its parasiticidal activity. This reduction is associated with its mutagenic activity in bacteria, although in mammalian cells DNA damage seems to be related to the production of reactive oxygen species (ROS). Using alkaline single cell electrophoresis, a significant increase in single strand breaks and alkali labile sites in human peripheral blood lymphocytes (PBL) exposed to MTZ, ONZ and TNZ at 10, 100 and 500 microg/ml is observed. MTZ causes less damage, especially at higher concentrations, when compared with TNZ, the most harmful of the drugs tested. These findings suggest that primary damage is induced under aerobic conditions and confirms that these nitroimidazoles are DNA damaging agents.

Mutagenicidad quínica y evaluación de daño potencial mediante ensayos de corto plazo (ECT) / Chemical mutagenesis and evaluatial of potential damage by short term test (STT)

Los estudios del daño al DNA por agentes químicos se realizan en distintos niveles según el sistema experimental que se utilice. Es así que los ensayos propuestos para el monitoreo biológico se presentan en cuatro niveles diferentes: a) primer nivel: ensayos moleculares o en bacterias; b) segundo nivel: pruebas in vitro en células de cultivo; c) tercer nivel: análisis in vivo ; d) estudios en poblaciones expuestas. Se pueden evaluar tanto agentes químicos o físicos, como muestras de suelo, agua o aire (mezclas complejas). Se trabajó con ensayos de corto plazo (ECPs) para evaluar el daño genético potencial de diferentes agente químocos, productos naturales o contaminantes ambientales. En la actualidad, el interés particular consiste en caracterizar de modo exhaustivo diferentes derivados nitroimidazólicos como mebendazol (MBZ), tiabendazol (TBZ), metronidazol (MTZ) y ornidazol (ONZ), por su uso muy difundido como drogas antiparasitarias. El trabajo fue desarrollado con un amplio espectro de biomarcadores: aberraciones cromosónicas, cinética de proliferación celular, intercambio de cromátidas hermanas, retraso en anafase, micronúcleos, mutaciones de punto, recombinación somática, etcétera. Los estudios se han realizado en niveles crecientes de complejidad: nivel II (estudios in vitro en cultivos de célñulas de roedores y primates), nivel III (estudios in vivo en células de discos imaginales de insectos, células de médula ósea de roedores y linfocitos de primates) y nivel IV (estudios epidemiológicos en poblaciones humanas expuestas). Estos estudios que conllevan un enfoque multidisciplinario sobre un problema mediante el uso de distintos modelos animales son herramientas muy valiosas y pueden aplicarse al estudio de distintos contaminantes ambientales o a mezclas complejas.

Mutagenicidad quínica y evaluación de daño potencial mediante ensayos de corto plazo (ECT) / Chemical mutagenesis and evaluatial of potential damage by short term test (STT)

Los estudios del daño al DNA por agentes químicos se realizan en distintos niveles según el sistema experimental que se utilice. Es así que los ensayos propuestos para el monitoreo biológico se presentan en cuatro niveles diferentes: a) primer nivel: ensayos moleculares o en bacterias; b) segundo nivel: pruebas in vitro en células de cultivo; c) tercer nivel: análisis in vivo ; d) estudios en poblaciones expuestas. Se pueden evaluar tanto agentes químicos o físicos, como muestras de suelo, agua o aire (mezclas complejas). Se trabajó con ensayos de corto plazo (ECPs) para evaluar el daño genético potencial de diferentes agente químocos, productos naturales o contaminantes ambientales. En la actualidad, el interés particular consiste en caracterizar de modo exhaustivo diferentes derivados nitroimidazólicos como mebendazol (MBZ), tiabendazol (TBZ), metronidazol (MTZ) y ornidazol (ONZ), por su uso muy difundido como drogas antiparasitarias. El trabajo fue desarrollado con un amplio espectro de biomarcadores: aberraciones cromosónicas, cinética de proliferación celular, intercambio de cromátidas hermanas, retraso en anafase, micronúcleos, mutaciones de punto, recombinación somática, etcétera. Los estudios se han realizado en niveles crecientes de complejidad: nivel II (estudios in vitro en cultivos de célñulas de roedores y primates), nivel III (estudios in vivo en células de discos imaginales de insectos, células de médula ósea de roedores y linfocitos de primates) y nivel IV (estudios epidemiológicos en poblaciones humanas expuestas). Estos estudios que conllevan un enfoque multidisciplinario sobre un problema mediante el uso de distintos modelos animales son herramientas muy valiosas y pueden aplicarse al estudio de distintos contaminantes ambientales o a mezclas complejas. (AU)

Aplicaciones de la citogenética en bioquímica clínica: marcadores biológicos tempranos en la exposición laboral inadecuada a óxido de etileno / Laboral ethylene oxide exposure: early biochemical and cytogenetic biomarkers applications

El óxido de etileno (OE), compuesto de amplio uso en la esterilización de materiales médico-quirúrgicos, es un agente alquilante con actividad mutagénica en distintos sistemas experimentales y probable carcinógeno en humanos. El objetivo de este trabajo es evaluar la sensibilidad de parámetros bioquímicos y citogenéticos en la determinación de daño inducido por OE en 12 técnicos de una central de esterilización con sus correspondientes controles. Los marcadores biológicos incluyeron: hemograma; electroforesis de hemoglobina; separación electroforética de proteínas séricas; determinación de la concentración sérica de bilirrubina: total y directa; determinación de la actividad de aminotransferasas: ALAT y ASAT, lactato deshidrogenasa y fosfatasa alcalina; fraccionamiento electroforético de fosfatasa alcalina. Al mismo tiempo en cultivo de linfocitos de sangre periférica se determinó la frecuencia de aberraciones cromosómicas/célula (AC), la variación del índice mitótico (IM) y la cinética de proliferación celular expresado como índice de replicación (IR). El análisis del fraccionamiento de la fosfatasa alcalina, reveló la presencia de fosfatasa alcalina placentaria en seis de los individuos expuestos. Asimismo, en los estudios de genotoxicidad, se observó un incremento estadísticamente significativo de la frecuencia de AC (p < 0,01) y una disminución tanto del IM (p < 0,0001) como del IR (0,001). Estos datos corroboran los efectos tóxicos del OE y demuestran la importancia de implementar una batería de marcadores biológicos en los estudios de monitoreo humano

Aplicaciones de la citogenética en bioquímica clínica: marcadores biológicos tempranos en la exposición laboral inadecuada a óxido de etileno / Laboral ethylene oxide exposure: early biochemical and cytogenetic biomarkers applications

El óxido de etileno (OE), compuesto de amplio uso en la esterilización de materiales médico-quirúrgicos, es un agente alquilante con actividad mutagénica en distintos sistemas experimentales y probable carcinógeno en humanos. El objetivo de este trabajo es evaluar la sensibilidad de parámetros bioquímicos y citogenéticos en la determinación de daño inducido por OE en 12 técnicos de una central de esterilización con sus correspondientes controles. Los marcadores biológicos incluyeron: hemograma; electroforesis de hemoglobina; separación electroforética de proteínas séricas; determinación de la concentración sérica de bilirrubina: total y directa; determinación de la actividad de aminotransferasas: ALAT y ASAT, lactato deshidrogenasa y fosfatasa alcalina; fraccionamiento electroforético de fosfatasa alcalina. Al mismo tiempo en cultivo de linfocitos de sangre periférica se determinó la frecuencia de aberraciones cromosómicas/célula (AC), la variación del índice mitótico (IM) y la cinética de proliferación celular expresado como índice de replicación (IR). El análisis del fraccionamiento de la fosfatasa alcalina, reveló la presencia de fosfatasa alcalina placentaria en seis de los individuos expuestos. Asimismo, en los estudios de genotoxicidad, se observó un incremento estadísticamente significativo de la frecuencia de AC (p < 0,01) y una disminución tanto del IM (p < 0,0001) como del IR (0,001). Estos datos corroboran los efectos tóxicos del OE y demuestran la importancia de implementar una batería de marcadores biológicos en los estudios de monitoreo humano (AU)