Micronuclei in embryos of eight seabird species in northwestern Mexico: A biomarker of exposure to coastal pollution?

The micronucleus (MN) test may be used to evaluate genome instability in birds and the potential of different species to function as biomarkers of genotoxicity. However, little is known regarding genome instability in seabird embryos or the instability present among embryonic development stages. Therefore, the present study aimed to describe the frequencies of micronucleated erythrocytes (MNE) and micronucleated polychromatic erythrocytes (MNPCE) in blood samples collected from the embryos of eight seabird species nesting on the coast of Sinaloa, Mexico. An additional description of blood cell maturation along with embryo development during incubation was conducted based on the proportion of polychromatic erythrocytes (PCE), and the potential relationships between metals (Hg and Cd concentrations in egg content) and the MN frequencies in embryo blood were evaluated. The PCE proportion appears to decline as incubation advances (initial stage > intermediate stage > advanced stage), and the values varied between species (Suliformes/Pelecaniformes < Charadriiformes: Laridae), which may be related to differences among incubation periods and reproductive strategies. Interspecific variation in the MNPCE frequency was found in embryos showing advanced development, which could be related to both variations in life-history traits and ecological factors and not Hg or Cd exposure. The genomic instability values in this study are the first to be reported for embryos of seabird species nesting in a subtropical coastal region.

[Radioprotective effects of gallic acid on bone marrow cells in mice].

OBJECTIVE: To investigate the radioprotective effect of gallic acid(GA) on mouse bone marrow cells. METHODS: Healthy male ICR mice were randomly divided into saline control group, GA control group, X-ray irradiation group and GA protection group, with 10 mice in each group. X-ray irradiation group and normal saline control group were given 0.01 mL/g normal saline gavage, GA control group and GA protection group were given 200 mg/kg GA(20 mg/mL) gavage once a day for 14 consecutive days. On the 15 th day, 4 X-ray irradiation groups and 4 GA protection groups were given one-time X-ray irradiation to the whole body of the mice, and the absorbed doses were 1.0, 2.0, 3.0 and 4.0 Gy, respectively. The saline control group and the GA control group were not irradiated. After irradiation, detected the whole blood catalase(CAT), superoxide dismutase(SOD), malondialdehyde(MDA) and micronucleus frequency of polychromatic erythrocyte in bone marrow(MN-PCE), and use flow cytometry to detect bone marrow cell cycle, early apoptosis rate and late apoptosis rate. RESULTS: The CAT activities in the serum of mice in the 1.0, 2.0, 3.0 and 4.0 Gy GA protection groups were 2.13, 1.74, 1.49 and 1.15 U/mL, respectively, which were significantly increased compared with the corresponding X-ray irradiation group(P<0.01). SOD activities were 184.69, 156.92, 139.17 and 107.15 U/mL, which were significantly increased compared with the corresponding X-ray irradiation group(P<0.01). The contents of MDA were 3.92, 4.20, 6.32 and 9.31 nmol/mL, which were significantly lower than those of the corresponding X-ray irradiation group(P<0.05, P<0.01). The bone marrow MN-PCE rate of the mice in the 1.0, 2.0, 3.0 and 4.0 Gy GA protection groups were 4.35‰, 8.00‰, 12.90‰ and 3.80‰, respectively, which were significantly lower than those in the corresponding X-ray irradiation group(P<0.01). The proportions of G_0/G_1 phase cells of bone marrow cells in the 1.0, 2.0, 3.0 and 4.0 Gy GA protection group were 81.00%, 86.28%, 92.04% and 93.15%, respectively, which were significantly reduced compared with the corresponding X-ray irradiation group(P<0.01). The proportions of G_2/M phase cells were 4.51%, 3.05%, 2.35% and 1.81%, which were significantly increased compared with the corresponding X-ray irradiation group(P<0.05, P<0.01). The proportions of S phase cells were 15.32%, 11.36%, 5.96% and 4.92%, which were significantly increased compared with the corresponding X-ray irradiation group(P<0.01). The early apoptosis rate of bone marrow cells of mice in the 1.0, 2.0, 3.0 and 4.0 Gy GA protection groups were 3.32%, 8.96%, 12.11% and 2.26%, respectively, which were significantly reduced compared with the corresponding X-ray irradiation group(P<0.01). The late apoptosis rates of bone marrow cells were 7.21%, 11.73%, 17.11% and 19.36%, which were significantly reduced compared with the corresponding X-ray irradiation group(P<0.05, P<0.01). CONCLUSION: GA can reduce the oxidative damage, DNA damage and bone marrow cell cycle arrest of the bone marrow cells of radiation-damaged mice, inhibit the apoptosis of bone marrow cells, and have radioprotective effects on the bone marrow cells of mice.

Safety assessment of a novel thermostable phytase.

A novel 6-phytase (Phytase TSP, trade name OptiPhos® PLUS) with improved thermostability has been developed for use in animal feed. The safety of the new phytase was evaluated by testing for genotoxicity and subchronic toxicity. In in vitro and in vivo genotoxicity assays Phytase TSP concentrate was not mutagenic and did not induce biologically or statistically significant increases in the frequency of micronucleated polychromatic erythrocytes. In a subchronic toxicity study, male and female rats administered 100, 500 or 1000 mg/kg body weight/day of Phytase TSP concentrate via oral gavage for 90 days had no mortalities, and no treatment-related effects on body weight, food consumption, clinical observations or ophthalmology. Furthermore, there were no changes in haematology, clinical chemistry, urinalysis, gross pathology, organ weights or histopathology that could be attributed to the test article. Several endpoints exhibited statistically significant effects, but none was dose-related or considered to be of toxicological relevance. Based on these results, Phytase TSP concentrate (OptiPhos® PLUS) was not genotoxic and the No Observed Adverse Effect Level (NOAEL) for male and female rats was 1000 mg/kg body weight/day.

Genotoxicity of mixture of imidacloprid, imazalil and tebuconazole.

Genotoxicity of the mixture of generic pesticides imidacloprid + imazalil + tebuconazole in a ratio of 14.0/1.7/1.0 by weight was assessed using Ames test (Salmonella typhimurium) and micronucleus test in vivo on mammalian bone marrow erythrocytes (CD-1 mice) supporting the data creation for the Real Life Risk Simulation (RLRS) approach. This pesticides' combination is used in the commercial formulation for seed treatment in advance of or immediately before sowing. Tested pesticides' technical grade active ingredients (TGAIs) showed no evidence of genotoxicity upon separate treatments. In combination, the three pesticides demonstrated negative results in the Ames test but induced a statistically significant, dose-depended increase in MN-PCEs in mice bone marrow at doses lower than those used separately. The observed effect may be mediated by the synergistic action of the tested TGAIs, their metabolites or impurities.

Evaluación genotóxica de las moléculas con efectos tripanocida y leishmanicida cantin-6-ona y 5-metoxicantin-6-ona en células de médula ósea de ratones / Genotoxic evaluation of the molecules with trypanocidal and leishmanicidal effects, canthin-6-one and 5-methoxycanthin-6-one in mouse bone marrow cells

La tripanosomiasis americana y la leishmaniasis son problemas de salud pública relevantes en Iberoamérica. Las drogas utilizadas actualmente para el tratamiento de estas enfermedades poseen efectos colaterales tóxicos severos. Varios grupos de investigación están abocados a la búsqueda de productos naturales y sintéticos para encontrar nuevos agentes terapéuticos efectivos que no presenten reacciones colaterales adversas. En la evaluación de compuestos de la especie vegetal Zanthoxylum chiloperone (Rutaceae), se demostró que compuestos aislados del extracto presentaban actividad leishmanicida, tripanocida y antifúngica in vivo. Teniendo como antecedentes estos resultados, en el presente estudio se evaluaron los efectos genotóxico y citotóxico del cantín-6-ona y del 5-metoxicantin-6-ona, moléculas aisladas de la planta, en células de médula ósea de animales tratados. El estudio de los efectos genotóxicos se hizo a través del ensayo de modificaciones en la frecuencia de micronúcleos y el efecto citotóxico por modificaciones en la relación entre eritrocitos policromáticos y eritrocitos normocromáticos. Se realizaron 2 ensayos independientes y en cada ensayo los animales fueron divididos en tres grupos de tratamiento: GRUPO I: control negativo que recibió 200 uL de agua y 2.1% de DMSO, vía oral, GRUPO II: compuesto a ser evaluado (canthin-6-ona o 5-methoxicantin-6-ona) con 2.1% de DMSO, y GRUPO III: control positivo que recibió ciclofosfamida 50mg/kg/peso del animal, vía intraperitoneal. El análisis estadístico mostró que ambos compuestos no presentaron efectos genotóxicos ni citotóxicos. Estos resultados permiten proponer a estas moléculas como candidatas a ser sometidas a estudios más detallados como potenciales fármacos contra estas dos enfermedades

American trypanosomiasis and leishmaniasis are relevant public health problems in Latin America. The drugs currently used to treat these diseases have severe toxic side effects. Several research groups are dedicated to the search of natural and synthetic products to find new effective therapeutic agents that do not present adverse collateral reactions. In the evaluation of compounds of the plant species Zanthoxylum chiloperone (Rutaceae), it was shown that isolated compounds of the extract had leishmanicidal, trypanocidal and antifungal in vivo activities. Based on these results, the genotoxic and cytotoxic effects of canthin-6-one and 5-methoxycanthin-6-one, molecules isolated from the plant, on bone marrow cells of treated mice were evaluated in the present study. The study of genotoxic effects was made through the test of modifications in the frequency of micronuclei and the cytotoxic effects by modifications in the relationship between polychromatic erythrocytes and normochromic erythrocytes. Two independent assays were performed and in each assay the animals were divided into three treatment groups: GROUP I: negative control that received 200 µL of water and 2.1% of DMSO, orally, GROUP II: compound to be evaluated (canthin-6 -one or 5-methoxycanthin-6-one) with 2.1% DMSO, and GROUP III: positive control that received cyclophosphamide 50mg /kg animal weight, intraperitoneal. Statistical analysis showed that both compounds had neither genotoxic nor cytotoxic effects. These results allow these molecules to be proposed as candidates to be subjected to more detailed studies as potential drugs against these two diseases

In Vivo Micronucleus Assay in Mouse Bone Marrow.

The presence of genotoxic agents in the environment may cause chromosomal mutations through different mechanisms, which are associated with serious health effects. Genotoxicity is commonly evaluated for the chemical safety assessment, in which the in vivo micronucleus test is paid more attention in the field of genotoxicity as compared to other toxicological endpoints. This assay is an in vivo cytogenetic test which uses erythrocytes in the bone marrow of rodents to detect chemical damage to the chromosomes or mitotic apparatus of mammalian cells. At the time of erythroblast development into a polychromatic erythrocyte (PCEs) in bone marrow, the main nucleus is extruded, so any micronucleus (MN) that has been formed may remain behind in the otherwise anucleated cytoplasm. The damage in the chromosome appears as a small additional nucleus and is readily identifiable by light microscope. An increase in the frequency of micronucleated polychromatic erythrocytes (MN PCEs) in treated animals is an indication of genotoxicity.

Maximum tolerated doses and erythropoiesis effects in the mouse bone marrow by 79 pesticides' technical materials assessed with the micronucleus assay.

Effects of technical materials of pesticide active ingredients, belonging to various chemical classes, on erythropoiesis in mouse bone marrow were studied as part of the research on the pesticide mutagenic activity in micronucleus test. The purpose of the present study was to estimate the toxic action of the test substances on the target organ and the validity of the results of the micronucleus assay under conditions of erythropoiesis suppression. It was demonstrated that intragastrically administrated triazole pesticides reached bone marrow (target organ where micronucleus induction was assessed) and exerted an inhibitory effect on erythropoiesis. The effects of triazole pesticides were enhanced in the following order: difenoconazole ≤ tebuconazole < cyproconazole < flutriafol. Furthermore, an association between structural features of molecules and specific target organ activity of the test pesticides was observed. Based on the data on the general toxicity and the results of the evaluation of the effects on erythropoiesis, the maximum tolerated doses (MTDs) of 79 different technical materials of pesticides for CD-1 mice were determined.

Magnesium stearate, a widely-used food additive, exhibits a lack of in vitro and in vivo genotoxic potential.

Magnesium stearate is widely used in the production of dietary supplement and pharmaceutical tablets, capsules and powders as well as many food products, including a variety of confectionery, spices and baking ingredients. Although considered to have a safe toxicity profile, there is no available information regarding its potential to induce genetic toxicity. To aid safety assessment efforts, magnesium sulfate was evaluated in a battery of tests including a bacterial reverse mutation assay, an in vitro chromosome aberration assay, and an in vivo erythrocyte micronucleus assay. Magnesium stearate did not produce a positive response in any of the five bacterial strains tested, in the absence or presence of metabolic activation. Similarly, exposure to magnesium stearate did not lead to chromosomal aberrations in CHL/IU Chinese hamster lung fibroblasts, with or without metabolic activation, or induce micronuclei in the bone marrow of male CD-1 mice. These studies have been used by the Japanese government and the Joint FAO/WHO Expert Committee on Food Additives in their respective safety assessments of magnesium stearate. These data indicate a lack of genotoxic risk posed by magnesium stearate consumed at current estimated dietary exposures. However, health effects of cumulative exposure to magnesium via multiple sources present in food additives may be of concern and warrant further evaluation.

Genotoxicity evaluation of alpha-linolenic acid-diacylglycerol oil.

The alpha-linolenic acid (ALA)-diacylglycerol (DAG) oil is an edible oil enriched with DAG (>80%) and ALA (>50%). Although DAG oil, which mainly consists of oleic and linoleic acids has no genotoxic concerns, the fatty acid composition could affect the chemical property of DAG. Therefore, the purpose of this study was to evaluate the genotoxicity of ALA-DAG oil using standard genotoxicity tests in accordance with the OECD guidelines. ALA-DAG oil showed negative results in the bacterial reverse mutation test (Ames test) and in vitro micronucleus test in cultured Chinese hamster lung cells with and without metabolic activation, and in the in vivo bone marrow micronucleus test in mice. Our results did not show any genotoxicity, suggesting that the fatty acid composition had no deleterious effects. We conclude that ALA-DAG oil had no genotoxicity concerns under the testing conditions.

Clastogenic effects of Trypanosoma brucei brucei and Trypanosoma evansi mixed infection in bone marrow of Wistar rats.

The clastogenic effect of mixed infection of Trypanosoma evansi and Trypanosoma brucei brucei in the bone marrow (BM) cells of Wistar albino rats was investigated. Clastogenic effects were observed in the BM cells using the micronucleus assay. The findings indicate that T. evansi, T. b. brucei and mixed infection with both parasites induced the formation of micronucleated polychromatic erythrocyte (MN-PCEs) in the BM cells significantly (P < 0.05) by 60, 63 and 81 micronuclei/1000 PCE respectively. Mixed infection induced formation of MN-PCEs increase by about 1.33 fold when compared with single infections of T. b. brucei and T. evansi. These data give a preliminary evidence of possible genotoxic effects in trypanosomiasis.

Avaliação da genotoxicidade da Ilex paraguariensis (erva mate) pelo teste do micronúcleo / Evaluation of the genotoxicity of Ilex paraguariensis (yerba mate) by micronucleus test

A Ilex paraguariensis é espécie nativa da América do Sul. O consumo de erva mate tem sido associado ao aumento nas taxas de câncer oral, de orofaringe, esôfago e laringe. O objetivo deste estudo foi investigar o potencial genotóxico da exposição a dose única de Ilex paraguariensis através do teste do micronúcleo. Para este estudo, foram utilizados 32 ratos Wistar albinos machos e adultos, divididos em 4 grupos: A - Composto por 8 ratos que receberam infusão de chá preparado na concentração de 5% de erva mate (concentração usualmente encontrada no chá de consumo humano); B - Composto por 8 ratos que receberam chá preparado por imersão em água fria na concentração de 5% de erva mate; C - Composto por 8 ratos, os quais receberam ciclofosfamida em dose única subcutânea (50mg/kg) no primeiro dia do experimento (grupo controle positivo); D - Composto por 8 ratos, os quais receberam somente água (grupo controle negativo). Todos os animais receberam ração ad libitum. Os animais dos grupos A, B e D foram submetidos à eutanásia 48 horas após o início do experimento e os do grupo C, 24 horas após. Foi coletado material da medula óssea de cada rato após a eutanásia para realização do teste do micronúcleo em eritrócito policromático, para avaliação do grau de genotoxicidade. A mediana de micronúcleos para o grupo A (chá mate preparado com infusão) foi de 0,00, do grupo B (chá mate em imersão em água fria) foi de 0,00, do grupo C (ciclofosfamida - controle positivo) foi de 9,00, e no grupo D (controle negativo) foi de 0,00. Não se observou genotoxicidade da Ilex paraguariensis, em ambas as formas de preparo do chá, através do teste de micronúcleo, ao nível de significância de 5%.

The Ilex paraguariensis is a native species of South America. Yerba mate consumption has been associated with increased rates of oral, oropharynx, esophagus and larynx cancer. The aim of this study was to investigate the genotoxic potential of the exposure to a single dose of Ilex paraguariensis by the micronucleus test. For this study, 32 male, adult Wistar rats were used, divided into 4 groups: A - Consisting of 8 rats, which received an infusion of tea prepared at a concentration of 5% of mate (concentration usually found in human consumption of tea); B - Consisting of 8 rats, which received tea prepared immersed in cold water at a concentration of 5% of mate; C - Consisting of 8 rats, which received a single subcutaneous dose of cyclophosphamide (50mg/kg) on the first day of the experiment (positive control group); D - Consisting of 8 rats, which received only water (negative control group). All animals received food ad libitum. The animals in groups A, B and D were sacrificed 48 hours after the beginning of the experiment and group C, 24 hours after. Material from the bone marrow of each rat was collected after euthanasia to perform the micronucleus test in polychromatic erythrocyte to assess the degree of genotoxicity. The median of micronuclei for group A (mate tea prepared with infusion) was 0.00, for group B (mate tea immersed in cold water) was 0.00, for group C (cyclophosphamide - positive control) was 9.00 and for group D (negative control) was 0.00. No genotoxicity of Ilex paraguariensis was observed in both tea preparation methods by the micronucleus test at a significance level of 5%.

Correlation analysis on MN-RET from peripheral blood and MN-PCE from bone marrow in mice following exposure to irradiation / 中华放射医学与防护杂志

objective To study the changes of reticulocyte micronueleus(MN-RET)from peripherai blood and polychromatic erythrocyte mieronucleUS(MN-PCE)from bone marrow in mice following exposure to X-rays in order to provide an experimental basis for exploring possible hish-throughput radiation biodosimeter.Methods Male ICR mice were whole-body irradiated with 0,0.5,1,2,4 and 5 Gy at a dose rate of 0.488 Gy/min.MN-RET from peripheral blood wag scored with FCM and MN-PCE from bone marrow was scored with manual microscopy at 24,48 and 72 h post-irradiation.Results Both MN-RET and MN-PCE rates increaged with doses in the range of 0-5 Gy at 24,48 and 72 h after WBI.The dose-response relationship can be fit with linear equations(t=10.26-25.77,P<0.05).The correlation coeffcients between MN-RET from peripheral blood and MN-PCE from bone mallow were highly significant(r=0.986-0.996,P<0.05).Conclusions In view of its simplicity,accuracy and high throughput capacity,FCM scoring of peripheral blood MN-RET may be a candidate for radiation biodosimetry,More work should be carried out on human specimens to investigate this possibility.