María del Carmen García-Rodríguez

Antigenotoxic Effects of (–)-Epigallocatechin-3-Gallate (EGCG), Quercetin, and Rutin on Chromium Trioxide-Induced Micronuclei in the Polychromatic Erythrocytes of Mouse Peripheral Blood

This study was conducted to investigate the modulating effects of (–)-epigallocatechin-3-gallate (EGCG), quercetin, and rutin on the genotoxic damage induced by Cr(VI) in polychromatic erythrocytes of CD-1 mice. The animals were divided into the following groups: (i) vehicle only; (ii) flavonoids (10 mg/kg EGCG, 100 mg/kg quercetin, 625 mg/kg rutin, or 100–625 mg/kg quercetin–rutin); (iii) Cr(VI) (20 mg/kg of CrO3); and (iv) flavonoids concomitantly with Cr(VI). All of the treatments were administered intraperitoneally (ip). The genotoxic damage was evaluated based on the number of micronucleated polychromatic erythrocytes (MN-PCE) obtained from the caudal vein 0, 24, 48, and 72 h after treatment. Groups treated with EGCG and quercetin exhibited no significant statistical changes in induction of MN-PCE. However, CrO3 treatment significantly increased MN-PCE induction 24 and 48 h after injection. Treatment with flavonoids prior to CrO3 exposure decreased MN-PCE induction compared with CrO3 only. The magnitudes of the potency of flavonoids were in the following order: rutin (82%) > quercetin (64%) > quercetin–rutin (59%) and EGCG (44%). The group treated with rutin significantly reduced genotoxic damage in mice treated with Cr(VI) (antioxidant effect). However rutin exerted a marginal genotoxic effect when administered alone (pro-oxidant effect). Our findings suggest protective effects of EGCG, quercetin, and rutin against genotoxic damage induced by Cr(VI).

Effect of chlorophyllin on chromium trioxide-induced micronuclei in polychromatic erythrocytes in mouse peripheral blood.

The effect of chlorophyllin on micronucleated polychromatic erythrocytes (MN-PCE) induction by chromium trioxide (CrO(3)) exposure in peripheral blood of mice was studied. Animals were treated with a single intraperitoneal dose of chlorophyllin (CHL) (20mg/kg), CrO(3) (20mg/kg), and CHL (20mg/kg) 4h before (CHL-CrO(3)) or 4h before and 20h after chromium treatments (20mg/kg; CHL-CrO(3)-CHL). Peripheral blood samples were drawn from the caudal vein at 0, 12 and 48h, and analyzed by the acridine orange (AO) technique. The results obtained in present study shown that CHL injection did not modify the number of MN-PCE. CrO(3) treatment resulted in a significantly increases 12 and 48h after the injection, reaching a four-fold increase 48h after CrO(3) administration. Whereas treatment with 20mg/kg of CHL prior to chromium, decreased the MN frequency induced by chromium in the 12h samples. When the samples were analyzed 48h after CrO(3) injection, no significant differences between CHL-CrO(3) and CHL-CrO(3)-CHL in comparison with CrO(3) treatment, were observed. These results indicate that increase of MN-PCE by CrO(3) is CHL-blocked in both protocols used (CHL-CrO(3) and CHL-CrO(3)-CHL) at 12h after treatment, but it was unable to modify the frequency of MN-PCE measured 48h after CrO(3) injection. The absence of a protective effect by CHL in the MN-PCE induction by CrO(3) at 48h, show that CHL has action only on one of the times of MN induction and suggests the possible action of CrO(3) by two different mechanisms, and not by CHL time-limited in vivo.

Antigenotoxic and Apoptotic Activity of Green Tea Polyphenol Extracts on Hexavalent Chromium-Induced DNA Damage in Peripheral Blood of CD-1 Mice: Analysis with Differential Acridine Orange/Ethidium Bromide Staining

This study was conducted to investigate the modulating effects of green tea polyphenols on genotoxic damage and apoptotic activity induced by hexavalent chromium [Cr (VI)] in CD-1 mice. Animals were divided into the following groups: (i) injected with vehicle; (ii) treated with green tea polyphenols (30 mg/kg) via gavage; (iii) injected with CrO3 (20 mg/kg) intraperitoneally; (iv) treated with green tea polyphenols in addition to CrO3. Genotoxic damage was evaluated by examining micronucleated polychromatic erythrocytes (MN-PCEs) obtained from peripheral blood at 0, 24, 48, and 72 h after treatment. Induction of apoptosis and cell viability were assessed by differential acridine orange/ethidium bromide (AO/EB) staining. Treatment of green tea polyphenols led to no significant changes in the MN-PCEs. However, CrO3 treatment significantly increased MN-PCEs at 24 and 48 h after injection. Green tea polyphenols treatment prior to CrO3 injection led to a decrease in MN-PCEs compared to the group treated with CrO3 only. The average of apoptotic cells was increased at 48 h after treatment compared to control mice, suggesting that apoptosis could contribute to eliminate the DNA damaged cells induced by Cr (VI). Our findings support the proposed protective effects of green tea polyphenols against the genotoxic damage induced by Cr (VI).

In Vivo Effects of Vanadium Pentoxide and Antioxidants (Ascorbic Acid and Alpha-Tocopherol) on Apoptotic, Cytotoxic, and Genotoxic Damage in Peripheral Blood of Mice

This study was conducted to investigate the effects of vanadium pentoxide (V2O5), ascorbic acid (AA), and alpha-tocopherol (α-TOH) on apoptotic, cytotoxic, and genotoxic activity. Groups of five Hsd:ICR mice were treated with the following: (a) vehicle, distilled water; (b) vehicle, corn oil; (c) AA, 100 mg/kg intraperitoneally (ip); (d) α-TOH, 20 mg/kg by gavage; (e) V2O5, 40 mg/kg by ip injection; (f) AA + V2O5; and (g) α-TOH + V2O5. Genotoxic damage was evaluated by examining micronucleated polychromatic erythrocytes (MN-PCE) obtained from the caudal vein at 0, 24, 48, and 72 h after treatments. Induction of apoptosis and cell viability were assessed at 48 h after treatment in nucleated cells of peripheral blood. Treatment with AA alone reduced basal MN-PCE, while V2O5 treatment marginally increased MN-PCE at all times after injection. Antioxidants treatments prior to V2O5 administration decreased MN-PCE compared to the V2O5 group, with the most significant effect in the AA + V2O5 group. The apoptotic cells increased with all treatments, suggesting that this process may contribute to the elimination of the cells with V2O5-induced DNA damage (MN-PCE). The necrotic cells only increased in the V2O5 group. Therefore, antioxidants such as AA and α-TOH can be used effectively to protect or reduce the genotoxic effects induced by vanadium compounds like V2O5.

Modulation of hexavalent chromium-induced genotoxic damage in peripheral blood of mice by epigallocatechin-3-gallate (EGCG) and its relationship to the apoptotic activity

ABSTRACT This study was conducted to investigate the relationship between modulation of genotoxic damage and apoptotic activity in Hsd:ICR male mice treated with (–)-epigallocatechin-3-gallate (EGCG) and hexavalent chromium [Cr(VI)]. Four groups of 5 mice each were treated with (i) control vehicle only, (ii) EGCG (10 mg/kg) by gavage, (iii) Cr(VI) (20 mg/kg of CrO3) intraperitoneally (ip), and (iv) EGCG in addition to CrO3 (EGCG-CrO3). Genotoxic damage was evaluated by examining presence of micronucleated polychromatic erythrocytes (MN-PCE) obtained from peripheral blood of the caudal vein at 0, 24, 48, and 72 h after treatment. Induction of apoptosis and cell viability were assessed by differential acridine orange/ethidium bromide (AO/EB) staining. EGCG treatment produced no significant changes in frequency of MN-PCE. However, CrO3 treatment significantly increased number of MN-PCE at 24 and 48 h post injection. Treatment with EGCG prior to CrO3 injection decreased number of MN-PCE compared to CrO3 alone. The MN-PCE reduction was greater than when EGCG was administered ip. The frequency of early apoptotic cells was elevated at 48 h following EGCG, CrO3, or EGCG-CrO3 exposure, with highest levels observed in the combined treatment group, while the frequencies of late apoptotic cells and necrotic cells were increased only in EGCG-CrO3 exposure. Our findings support the view that EGCG is protective against genotoxic damage induced by Cr(VI) and that apoptosis may contribute to elimination of DNA-damaged cells (MN-PCE) when EGCG was administered prior to CrO3. Further, it was found that the route of administration of EGCG plays an important role in protection against CrO3-induced genotoxic damage.

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.

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.

The effect of intragastric and ad libitum administration of red wines (Cabernet Sauvignon, Merlot, Carménère and Cabernet Sauvignon/Nebbiolo) and alcohol in the cyto/genotoxicity evaluated in the peripheral blood of CD-1 mice

Se evaluo la cito/genotoxicidad en sangre periferica de ratones CD-1 expuestos a diferentes variedades de vino tinto ( Cabernet Sauvignon , Merlot , Carmenere y Cabernet Sauvignon/Nebbiolo ). Grupos de cinco organismos fueron tratados: a) con el vehiculo, b) vino tinto o etanol (diluidos al 75 y 10%, respectivamente) ad libitum , y c) vino tinto sin diluir o etanol diluido al 13.5% (dos dosis diarias-0.25 ml, via i.g.). La genotoxicidad se evaluo con los micronucleos (MN) y la citotoxicidad con la relacion entre eritrocitos policromaticos/normocromaticos (EPC/ENC) a las 0, 24, 48 y 72 h. Solo se observaron incrementos significativos en los MN con los tratamientos i.g de Cabernet Sauvignon , Merlot y etanol, siendo mayor para el etanol. La administracion ad libitum de los vinos tintos redujo los MN. Nuestros hallazgos sugieren que los efectos genotoxicos del vino tinto podrian estar relacionados con la biodisponibilidad, farmacodinamica y farmacocinetica del etanol. No se observaron efectos citotoxicos.espanolSe evaluo la cito/genotoxicidad en sangre periferica de ratones CD-1 expuestos a las variedades de vino tinto Cabernet Sauvignon, Merlot, Carmenere y Cabernet Sauvignon/ Nebbiolo. Grupos de cinco organismos fueron tratados: a) con el vehiculo, b) vino tinto o etanol (diluidos al 75% y 10%, respectivamente) ad libitum, y c) vino tinto sin diluir o etanol diluido al 13.5% (dos dosis diarias-0.25 ml, intragastricamente [i.g.]). La cito/ genotoxicidad se evaluo mediante el ensayo de micronucleos (MN) y la relacion entre eritrocitos policromaticos/normocromaticos (EPC/ENC) a las 0 h, 24 h, 48 h y 72 h. La administracion i.g. de Cabernet Sauvignon, Merlot y etanol mostraron ligeros incrementos en los MN que resultaron significativos; sin embargo, la administracion ad libitum de los vinos mostro una tendencia a reducirlos. Nuestros hallazgos sugieren que los MN podrian estar relacionados con la biodisponibilidad, farmacodinamica y farmacocinetica del etanol y de los antioxidantes del vino tinto. No se observaron efectos citotoxicos. EnglishWe evaluated the cyto/genotoxicity in peripheral blood of CD-1 mice exposed to different varieties of red wine Cabernet Sauvignon, Merlot, Carmenere and Cabernet Sauvignon/Nebbiolo. Groups of five CD-1 mice were treated with: a) vehicle only; b) red wine or ethanol ad libitum (diluted 75% and 10%, respectively); c) undiluted red wine or ethanol diluted to 13.5% (two daily doses-0.25 ml via intragastric [i.g.]). The cyto/genotoxicity was evaluated by micronucleus (MN) assay, and by the relationship between polychromatic/monochromatic erythrocytes at 0 h, 24 h, 48 h and 72 h. Significant but slight increases of MN were observed only in the treatments i.g. of Cabernet Sauvignon, Merlot, and ethanol. However, the administration ad libitum of red wines tended to decrease the MN. Our findings suggest that the MN could be related with the bioavailability, pharmacodynamics, and pharmacokinetics of ethanol and the antioxidants of red wine. No cytotoxic effects were observed.