Cytokinesis-block Micronucleus Test Research Articles

In vitro Radio Protective Effects of Metformin on Human Lymphocytes Irradiated with Gamma Rays

Radio protective effects of metformin and its ability to alter the spontaneous and induced genotoxic and cytotoxic levels effects on human peripheral blood lymphocytes were investigated in this study. Metformin, a hypoglycemic oral drug, is a biguanide derived from Galega officinalis that is widely utilized in controlling type 2 diabetes mellitus. Whole blood samples from 10 healthy donors (5 males and 5 females) were exposed to two doses of gamma-rays (1 and 2 Gray). Lymphocytes in cultures were treated with metformin (10 and 50µM) before gamma-irradiation. Cytokinesis-block micronucleus test was used to evaluate the protective effects of metformin on radiation induced genomic damage, cytostasis and cytotoxicity, via scoring micronuclei and nucleoplasmic bridges in once divided binucleated cells as well as counting nuclear division index. The results of the current study revealed that the increase in micronuclei and nucleoplasmic bridges rate is associated with the decrease of nuclear division index in human lymphocytes exposed to gamma radiation in a dose dependent manner. Metformin effectively decreased the rate of spontaneous micronuclei and nucleoplasmic bridges paralleled with the control as well as increased nuclear division index. Moreover, treatment of whole blood samples with metformin (10 and 50 µM), 2 h preceding to irradiation, remarkably reduced rate of micronuclei and nucleoplasmic bridges accompanied with an increase in nuclear division index rate. The results introduced metformin to be an effective radio protector against DNA damage induced by gamma radiation in human lymphocytes and that it can be used to develop radio protective materials for protection cells of cancer patients from the genomic damage prompted via radiotherapy.

Biological response to the continuous occupational exposure to antineoplastic drugs and radionuclides

Purpose Antineoplastic drugs and radioiodine are recognized occupational risk factors affecting the genetic material of exposed persons. To assess cytogenetic damage and evaluate the presence of chromosomal instability during occupational exposure, a biomonitoring study was performed using a chromosomal aberration assay and a cytokinesis-block micronucleus (CBMN) test. Materials and Methods Blood samples from 314 healthy donors divided into 3 groups (control, exposed to antineoplastic drugs and exposed to radioiodine) were collected and cytogenetically analyzed. Results There was an increase in almost all analyzed parameters registered in the exposed persons. Chromatid breaks were higher in the subjects exposed to antineoplastic drugs, while dicentrics and premature centromere division (PCD) parameters were higher in nuclear medicine workers. The total number of micronuclei was higher in both groups of the exposed. The correlation analysis indicated the association of dicentrics, acentrics, chromosome and chromatid break with PCDs in both groups of the exposed, and micronuclei and nucleoplasmic bridges with PCDs in the subjects exposed to radioiodine. The discriminant analysis marked off PCD1-5 as the best predictor of exposure. Age, sex, sampling season and duration of exposure significantly influenced the analyzed parameters, while smoking habits did not show any influence. Conclusion Based on the observed results, premature centromere division can be considered a valuable parameter of genotoxic risk for individuals occupationally exposed to low doses of ionizing radiation.

PIG-A gene mutation as a mutagenicity biomarker among coke oven workers.

PIG-A gene mutations can be detected in humans, and PIG-A assays can potentially predict the risk of exposure to carcinogens. However, extensive, population-based studies to validate this are lacking. We studied a cohort of occupational coke oven workers with chronic high exposure to carcinogenic polycyclic aromatic hydrocarbons, which are well-studied genotoxins classified by the IARC as carcinogenic to humans. Peripheral blood erythrocytes of workers were assessed for gene mutations using a PIG-A assay, and chromosome damage using the cytokinesis-block micronucleus test with lymphocytes. Two sample populations from a non-industrialized city and new employees in industrial plants were selected as controls. We observed a significantly elevated PIG-A mutation frequency (MF) and increased frequencies of micronuclei (MN) and nuclear buds (NBUDs) in coke oven workers, compared with levels in the control groups. We found that the coke oven workers with different lengths of service had a relatively high mutation frequency. Overall, the study findings showed that occupational exposure of coke oven workers increases the genetic damage and the PIG-A MF could be a potential biomarker for risk assessment of carcinogen exposure.

Re-evaluation of CBMN test reference values of persons continuously occupationally exposed to low doses of ionizing radiation in Serbia.

When established, cytokinesis-block micronucleus (CBMN) test reference values should be periodically evaluated according to the recommendations of reference documents. The biodosimetry cytogenetic laboratory of the Serbian Institute of Occupational Health established the CBMN test reference range for people occupationally exposed to ionizing radiation in 2016. Since then, new occupationally exposed persons have been subjected to micronucleus testing, resulting in the need for re-evaluation of existing CBMN test values. The examined population comprised 608 occupationally exposed subjects - 201 from the previous laboratory database and 407 newly examined. Comparison of groups based on gender, age and cigarette consumption did not show significant differences, although certain CBMN values differed significantly between the old and new groups. Duration of occupational exposure, gender, age and smoking habit influenced micronuclei frequency in all three analyzed groups, while no relation was found between type of work and micronucleus test parameters. Since the mean values of all tested parameters in the new group of examinees are within previously established reference ranges, existing values can be used in further research.

Gallic acid a flavonoid isolated from Euphorbia hirta antagonizes gamma radiation induced radiotoxicity in lymphocytes in vitro.

The current study was executed to isolate and evaluate gallic acid from Euphorbia hirta for in vitro radioprotective potentials against gamma irradiation caused radiotoxicity in human lymphocytes. The defatted E.hirta plant material was treated to methanol extraction using the soxhlet device. Bioflavonoids were isolated from the E.hirta methanol extract using column chromatography. In human cells exhibited to gamma radiation, separated flavonoid gallic acid was examined for in vitro radioprotective potentials using the micronucleus test, DNA fragmentation assay, superoxide free radical scavenging method, and apoptic assay. The frequency of micronuclei was considerably declined when cells were preprocessed with gallic acid (25g/mL) before being exhibited to 2Gy gamma radiation, as determined by the cytokinesis blocked micronucleus test. Similarly, pre-gamma radiation treatment of human cells with gallic acid led in markedly less DNA injury, as assessed by comet metrics like olive tail moment and percent tail DNA. Gallic acid (25g/mL) given to lymphocytes prior to gamma irradiation considerably decreased the percentage of apoptotic bodies. Gallic acid also considerably lowered the reactive oxygen species concentrations elicited by gamma radiation. Our findings showed that gallic acid protects lymphocytes isolated from human blood from gamma radiation-induced DNA destruction and anti-apoptotic activity, which could be because of inhibition of free radicals formed by gamma radiation as well as the decline of gamma radiation-induced oxidative stress.

Assessment of Protective Effects of Carvacrol on Haloperidol-Induced Oxidative Stress and Genotoxicity in Human Peripheral Blood Lymphocytes.

Haloperidol is a first-generation antipsychotic drug that has several indications in a wide range of mental conditions. The extensive prescription of haloperidol is correlated with some less-known adverse effects such as genotoxicity. Carvacrol is a monoterpenoid mainly found in oregano and thyme. It has the potential to scavenge free radicals in addition to increasing antioxidant defense enzyme activities and glutathione levels. In this study, we attempted to explore the possible potential of haloperidol in inducing genotoxicity in human peripheral lymphocytes as well as the protective role of carvacrol against this effect. The lymphocytes were divided into separate groups as follows: control group (cosolvent and NS); carvacrol group (5 μM); haloperidol group (25, 50, and 100 ng/ml); haloperidol (25, 50, and 100 ng/ml) + carvacrol (5 μM); positive control (0.8 μg/ml Cisplatin). After 24 hours of treatment, we conducted a cytokinesis-Block micronucleus test and an alkaline comet assay in order to determine genetic damage. Additionally, we measured glutathione and MDA levels as the biomarkers associated with oxidative stress. Significant increases in the levels of genotoxicity biomarkers (micronucleus frequency, DNA percentage in tail and tail moment) were observed in haloperidol-treated cells. The result of our oxidative stress tests also demonstrated that haloperidol had the potential to induce oxidative stress via reducing the levels of glutathione and increasing lipid peroxidation. Treatment with carvacrol significantly decreased the genotoxic events. It can be presumed that the induction of oxidative stress by haloperidol is the critical event associated with haloperidol-mediated genotoxicity. Therefore, using carvacrol as a natural antioxidant protected human lymphocytes against haloperidol genetic damage.

Genotoxic potential of different nano-silver halides in cultured human lymphocyte cells

Most antibacterial applications in nanotechnology are carried out using silver nanoparticles (AgNPs). However, there is a dearth of information on the biological effects of AgNPs on human blood cells. In this study, the cytotoxic and genotoxic potentials of ionic silver (Ag+), AgNP, silver bromide (AgBr), silver chloride (AgCl), and silver iodide (AgI) were evaluated through chromosome aberration (CA) test and cytokinesis-blocked micronucleus (CBMN) test in human cultured lymphocytes in vitro. Furthermore, the potential damages that can cause to DNA were evaluated through alkaline single cell gel electrophoresis (Comet) assay on isolated lymphocytes. The results showed that AgNPs exerted cytotoxic effects by reducing the cytokinesis-block proliferation index and mitotic index at 24 and 48 h. AgNPs also increased micronucleus (MN) formation at both exposure times in the cultured cells. Meanwhile, AgCl had no genotoxic effects on the human lymphocyte cultured cells but had a cytotoxic effect at high doses. AgNP, Ag+, AgBr, and AgI caused substantial DNA damage by forming DNA strand breaks. They may also have clastogenic, genotoxic and cytotoxic effects on human lymphocyte cells. Based on the foregoing findings, silver nanomaterials may have genotoxic and cytotoxic potentials on human peripheral lymphocytes in vitro.

Irradiation Consequences from Chromosome Damages to Triggering of Diverse Immunological Responses

Present work was designed to investigate DNA damages post irradiation via cytokinesis-block micronucleus (CBMN) test and its corresponding immunological response. Determination of interferon- α, β for innate, interferon-γ for acquired response, TNF- α and immunoglobulin concentration IgG & IgM. Six human blood samples were divided into 4 groups (control & 3irradiated) which exposed to doses (0.5 - 2 and 4 Gy). Triplet blood samples (control and irradiated groups) were cultured for 72 hours after 1 hour of irradiation. γ - Irradiation induced significant increase of IFN-α (innate immunology hallmark) in all experimental doses (0.5 -2.0 and 4.0 Gy). IFN-β also recorded significant increase with control at dose 4.0 Gy. The results showed significant increase in IFN-γ representing acquired immune response at 2.0 and 4.0 Gy. These results confirmed by exhibits increase in the level of IgG and IgM production. TNF-α late immune response started to give significant increase at 2.0 and 4.0 Gy. TNF-α and IFN-β recorded significant difference when compared with control at 1.0 and 4Gy exposure also 4Gy group recorded significant increase compared with 1.0 Gy exposure. INF-α recorded significant increase at all doses when compared with control and each other. IFN-γ recorded significant increase in 1.0 and 4.0 Gy when compared with control with no significant difference between them. We conclude that immune system can sense when cells damaged. Mni which come from a variety of sources such as irradiation can lead to an immune response similar to that observed during viral infection.

PIG-A gene mutation as a genotoxicity biomarker in polycyclic aromatic hydrocarbon-exposed barbecue workers

BackgroundThe PIG-A gene mutation assay is a valuable tool for measuring in vivo gene mutations in blood cells. The human PIG-A assay, used as a potential genotoxicity biomarker, is minimally invasive, sensitive, and cost-efficient; however, the relationship between carcinogen exposure and PIG-A mutations is not well understood.MethodsWe investigated the genotoxic effect of red blood cells using PIG-A assay and lymphocyte cytokinesis-block micronucleus test in barbecue restaurant workers (N = 70) exposed to polycyclic aromatic hydrocarbons (PAHs) and self-identified healthy control subjects (N = 56). Urinary PAH metabolites were measured to evaluate internal exposure levels.ResultsMultivariate Poisson regression showed that the PAH-exposed workers exhibited significantly higher PIG-A mutant frequency (MF) (8.04 ± 6.81 × 10− 6) than did the controls (5.56 ± 5.26 × 10− 6) (RR = 0.707, 95% CI: 0.615–0.812, P < 0.001). These results indicate that PAH exposure is a risk factor for elevated PIG-A MF. The frequencies of micronuclei (MN) and nuclear buds (NBUD) in the PAH-exposed workers (MN: 3.06 ± 2.07 ‰, NBUD: 1.38 ± 1.02 ‰) were also significantly higher than in the controls (MN: 1.46 ± 0.64 ‰, P < 0.001; NBUD: 0.70 ± 0.60 ‰, P < 0.001). Additionally, PIG-A MFs showed better associations with several urinary hydroxylated PAH metabolites (P2-OH-Flu = 0.032, r2-OH-Flu = 0. 268; P2-OH-Phe = 0.022, r2-OH-Phe = 0.286; P3-OH-Phe = 0.0312, r3-OH-Phe = 0.270; P4-OH-Phe = 0.018, r4-OH-Phe = 0.296), while the increase in MN, NPB, and NBUD frequencies was not associated with any OH-PAH metabolites; and high-PAH-exposed workers showed the highest PIG-A MFs. Furthermore, there was a significant association between PIG-A MF and PAH exposure levels (Chi-square test for trend, P = 0.006).ConclusionsOur results indicate that an increase in PIG-A MF in barbecue workers could reflect the response to PAH exposure, providing evidence of its potential as a genotoxicity biomarker in human risk assessment.

In Vitro Assessment of the Genotoxic Potential of Pristine Graphene Platelets.

(1) Background: Graphene is a two-dimensional atomic structure with a wide range of uses, including for biomedical applications. However, knowledge of its hazards is still limited. This work brings new cytotoxic, cytostatic, genotoxic and immunotoxic data concerning the in vitro exposure of human cell line to two types of graphene platelets (GP). It also contributes to the formation of general conclusions about the health risks of GP exposure. (2) Methods: In vitro exposure of a THP-1 cell line to three concentrations of two GP over 40 h. The cytotoxic potential was assessed by the measurement of LDH and glutathione (ROS) and by a trypan blue exclusion assay (TBEA); the cytostatic and genotoxic potential were assessed by the cytokinesis-block micronucleus (CBMN) test; and the immunotoxic potential was assessed by the measurement of IL-6, IL-10 and TNF-α. (3) Results: We found a significant dose-dependent increase in DNA damage (CBMN). The lowest observed genotoxic effect levels (LOGEL) were 5 µg/mL (GP1) and 30 µg/mL (GP2). We found no significant leaking of LDH from cells, increase in dead cells (TBEA), induction of ROS, increased levels of cytostasis, or changes in IL-6, IL-10 and TNF-α levels. (4) Conclusions: The genotoxicity increased during the short-term in vitro exposure of THP-1 to two GP. No increase in cytotoxicity, immunotoxicity, or cytostasis was observed.

In vitro evaluation of chromosomal damage and DNA strand breaks after treatment with the poppy seed alkaloid thebaine

Thebaine is an alkaloid and can be found in poppy seeds in relatively high concentrations. Acute toxicity of thebaine is fairly high, but not much is known about chronic toxicity. To investigate the genotoxicity of thebaine, cytokinesis-block micronucleus test and comet assay were conducted in TK6 cells. In addition, effects of putative thebaine metabolites were analysed using metabolically active HepG2 cells and TK6 cells with S9 mix. FDA test and trypan blue test were used together with the frequency of mitotic and apoptotic cells to assess potential cytotoxicity of thebaine treatment. Micronucleus induction was observed after high doses (150 and 500 μM) of thebaine without metabolic activation in the presence of slight to moderate cytotoxicity. No effects were observed in the comet assay or after metabolic activation up to the highest dose of 500 μM. A potential protective effect on micronucleus induction after thebaine treatment was investigated via co-treatment with MMC and BaP in TK6 cells. Only after co-treatment with MMC, a reduction of micronucleus frequency was found. Overall, this study shows a potential of thebaine to induce genotoxic effects at high concentrations. The observation of cytotoxicity at these concentrations supports the hypothesis that genotoxicity may be caused by cytotoxic effects. Further studies will need to elucidate whether these effects are directly genotoxic or indeed result from cytotoxicity.

Suitability of the In Vitro Cytokinesis-Block Micronucleus Test for Genotoxicity Assessment of TiO2 Nanoparticles on SH-SY5Y Cells.

Standard toxicity tests might not be fully adequate for evaluating nanomaterials since their unique features are also responsible for unexpected interactions. The in vitro cytokinesis-block micronucleus (CBMN) test is recommended for genotoxicity testing, but cytochalasin-B (Cyt-B) may interfere with nanoparticles (NP), leading to inaccurate results. Our objective was to determine whether Cyt-B could interfere with MN induction by TiO2 NP in human SH-SY5Y cells, as assessed by CBMN test. Cells were treated for 6 or 24 h, according to three treatment options: co-treatment with Cyt-B, post-treatment, and delayed co-treatment. Influence of Cyt-B on TiO2 NP cellular uptake and MN induction as evaluated by flow cytometry (FCMN) were also assessed. TiO2 NP were significantly internalized by cells, both in the absence and presence of Cyt-B, indicating that this chemical does not interfere with NP uptake. Dose-dependent increases in MN rates were observed in CBMN test after co-treatment. However, FCMN assay only showed a positive response when Cyt-B was added simultaneously with TiO2 NP, suggesting that Cyt-B might alter CBMN assay results. No differences were observed in the comparisons between the treatment options assessed, suggesting they are not adequate alternatives to avoid Cyt-B interference in the specific conditions tested.

Antiproliferative and Genotoxic Action of an Underexploited Organoteluran Derivative on Sarcoma 180 Cells.

The search for novel metallic chemical compounds with toxicogenic effects has been of great importance for more efficient cancer treatment. The study evaluated the cytotoxic, genotoxic and mutagenic activity of organoteluran RF07 in the S-180 cell line. The bioassays used were cell viability with 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test, evaluation of apoptosis and necrosis using fluorescence and flow cytometry, cytokinesisblock micronucleus test and comet assay. The compound was tested at 1; 2.5 and 5μM. The results showed the cytotoxicity of RF07 at concentrations of 2.5, 5, 10 and 20μM when compared to the negative control. For genotoxicity tests, RF07 showed effects in all concentrations assessed by increased index and frequencies of damage and mutagenic alterations. The compound was also cytotoxic due to the significant decrease in the nuclear division index, with significant values of apoptosis and necrosis. The results of fluorescence and flow cytometry showed apoptosis as the main type of cell death caused by RF07 at 5μM, which is thought to avoid an aggressive immune response of the organism. In addition to cytotoxic and genotoxic effects, RF07 creates good perspectives for future antitumor formulations.

Attenuation of oxidative stress and chromosomal aberrations in cultured macrophages and pulmonary cells following self-sustained high temperature synthesis of asbestos

Inhalation of asbestos fibres can cause lung and pleural diseases in humans and constitutes a severe public health threat worldwide. The aim of the present study was to assess the biological effects induced in both pulmonary cells (A549) and monocyte/macrophage (RAW 264.7) cell lines by combustion slags obtained from asbestos through a self-sustained high-temperature synthesis (SHS) reaction. The SHS reaction involves rapid thermal treatment and displays great ability to neutralise asbestos. Cytotoxicity, redox status imbalance, lipid peroxide production, DNA strand breaks (comet assay) and chromosomal aberrations (cytokinesis block micronucleus test) were evaluated in cells exposed either to untreated asbestos fibres or to grinded SHS-generated slags of different granulometry, tested in cultured cells at varying doses and for varying exposure times. Our results show that asbestos fibres cause redox status imbalance, especially in monocyte/macrophage cell lines. Moreover, they promote lipid peroxidation and trigger genomic alterations. When the cells were exposed to slag powders, which are the products of SHS asbestos treatment, generation of lipid peroxides and induction of DNA strand breaks still persisted, due to the high content in iron and other metals detected in these samples. However, there was an attenuation of redox status imbalance and an absence of chromosomal aberrations, which probably reflects the loss of the asbestos fibrous structure following SHS reaction, as demonstrated by electron microscopy analyses. In conclusions, SHS-treated asbestos wastes can potentially have deleterious health effects due to the oxidative stress induced by inhaled powders but they loose the asbestos ability to induce chromosomal alterations.

Micronucleus assay for predicting side effects of radiotherapy for cervical cancer

ABSTRACT Radiotherapy (RT) is an important treatment for cervical cancer. The quality of life of patients undergoing RT may be compromised during and following treatment by nausea, diarrhea, vomiting, burns, erythema and fistula. Cytokinesis-block micronucleus (CBMN) assays may be useful for predicting adverse effects of RT for cancer. The CBMN test is easy to perform and is reproducible for screening subjects exposed to ionizing radiation. We investigated the use of the frequency of micronuclei (MN) from peripheral blood samples, irradiated in vitro, as a possible biomarker to predict the side effects of RT in patients with cervical cancer. We used 10 patients with cervical cancer receiving RT and chemotherapy. We found a strong relation between the frequency of MN and the appearance of acute side effects of RT for cervical cancer. We suggest that the methodology presented here may be useful for predicting side effects of RT for patients affected by cervical cancer and who have undergone chemotherapy.

The potential application of human PIG-A assay on azathioprine-treated inflammatory bowel disease patients.

The rodent Pig-a assay has been used extensively as a potential regulatory assay for evaluating the in vivo mutagenicity of test substances. Although the assay can be conducted in different mammalian species, there have been only a few reports describing its use in humans, and rarely in genotoxicant-exposed human populations. In this study, PIG-A mutation frequencies (MFs) were evaluated in 36 azathioprine (AZA; human carcinogen)-treated inflammatory bowel disease (IBD) patients and 36 healthy volunteers. IBD patients exhibited a slight but statistically higher MF (6.10 ± 4.44 × 10-6 ) than healthy volunteers (4.97 ± 2.74 × 10-6 ) (P = 0.0489). The estimated relative risk for the exposed patients was 1.22 which indicated that AZA is a risk factor for inducing PIG-A mutation. However, the PIG-A MF showed no associations with AZA treatment duration or total AZA exposure. In addition, we performed the cytokinesis-block micronucleus test on the same samples. The frequencies of micronuclei (MN) and nuclear buds (NBUD) in IBD patients (MN: 4.70 ± 2.86‰; NBUD: 1.89 ± 0.95‰) were significantly higher than in healthy volunteers (MN: 1.47 ± 0.77‰, P < 0.001; NBUD: 0.90 ± 0.58‰, P = 0.004). MN frequency also had significant correlations with AZA treatment duration (P = 0.011) and total AZA exposure (P = 0.018). Our findings indicate that AZA-treated IBD patients have only a marginally significant increase in PIG-A MF; in contrast, a much stronger AZA-associated increase in genotoxicity was detected with the lymphocyte MN assay. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

In Vitro Genotoxicity of Polystyrene Nanoparticles on the Human Fibroblast Hs27 Cell Line.

Several studies have provided information on environmental nanoplastic particles/debris, but the in vitro cyto-genotoxicity is still insufficiently characterized. The aim of this study is to analyze the effects of polystyrene nanoparticles (PNPs) in the Hs27 cell line. The viability of Hs27 cells was determined following exposure at different time windows and PNP concentrations. The genotoxic effects of the PNPs were evaluated by the cytokinesis-block micronucleus (CBMN) assay after exposure to PNPs. We performed ROS analysis on HS27 cells to detect reactive oxygen species at different times and treatments in the presence of PNPs alone and PNPs added to the Crocus sativus L. extract. The different parameters of the CBMN test showed DNA damage, resulting in the increased formation of micronuclei and nuclear buds. We noted a greater increase in ROS production in the short treatment times, in contrast, PNPs added to Crocus sativus extract showed the ability to reduce ROS production. Finally, the SEM-EDX analysis showed a three-dimensional structure of the PNPs with an elemental composition given by C and O. This work defines PNP toxicity resulting in DNA damage and underlines the emerging problem of polystyrene nanoparticles, which extends transversely from the environment to humans; further studies are needed to clarify the internalization process.

Bacterial cellulose membrane functionalized with hydroxiapatite and anti-bone morphogenetic protein 2: A promising material for bone regeneration.

Bone tissue engineering seeks to adequately restore functions related to physical and biological properties, aiming at a repair process similar to natural bone. The use of compatible biopolymers, such as bacterial cellulose (BC), as well as having interesting mechanical characteristics, presents a slow in vivo degradation rate, and the ability to be chemically modified. To promote better bioactivity towards BC, we synthesized an innovative BC membrane associated to hydroxyapatite (HA) and anti-bone morphogenetic protein antibody (anti-BMP-2) (BC-HA-anti-BMP-2). We present the physical-chemical, biological and toxicological characterization of BC-HA-anti-BMP-2. Presence of BC and HA components in the membranes was confirmed by SEM-EDS and FTIR assays. No toxic potential was found in MC3T3-E1 cells by cytotoxicity assays (XTT Assay and Clonogenic Survival), genotoxicity (Comet Assay) and mutagenicity (Cytokinesis-blocked micronucleus Test). The in vitro release kinetics of anti-BMP-2 antibodies detected gradually reducing antibody levels, reducing approximately 70% in 7 days and 90% in 14 days. BC-HA-anti-BMP-2 increased SPP1, BGLAP, VEGF, ALPL, RUNX2 and TNFRSF11B expression, genes involved in bone repair and also increased mineralization nodules and phosphatase alcalin (ALP) activity levels. In conclusion, we developed BC-HA-anti-BMP-2 as an innovative and promising biomaterial with interesting physical-chemical and biological properties which may be a good alternative to treatment with commercial BMP-2 protein.

The size-dependent genotoxic potentials of titanium dioxide nanoparticles to endothelial cells.

Despite intensive research activities, there are still many major knowledge gaps over the potential adverse effects of titanium dioxide nanoparticles (TiO2 -NPs), one of the most widely produced and used nanoparticles, on human cardiovascular health and the underlying mechanisms. In the present study, alkaline comet assay and cytokinesis-block micronucleus test were employed to determine the genotoxic potentials of four sizes (100, 50, 30, and 10 nm) of anatase TiO2 -NPs to human umbilical vein endothelial cells (HUVECs) in culture. Also, the intracellular redox statuses were explored through the measurement of the levels of reactive oxygen species (ROS) and reduced glutathione (GSH) with kits, respectively. Meanwhile, the protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) were also detected by western blot. The results showed that at the exposed levels (1, 5, and 25 μg/mL), all the four sizes of TiO2 -NPs could elicit an increase of both DNA damage and MN frequency in HUVECs in culture, with a positive dose-dependent and negative size-dependent effect relationship (T100 < T50 < T30 < T10). Also, increased levels of intracellular ROS, but decreased levels of GSH, were found in all the TiO2 -NP-treated groups. Intriguingly, a very similar manner of dose-dependent and size-dependent effect relationship was observed between the ROS test and both comet assay and MN test, but contrary to that of GSH assay. Correspondingly, the levels of Nrf2 protein were also elevated in the TiO2 -NP-exposed HUVECs, with an inversely size-dependent effect relationship. These findings indicated that induction of oxidative stress and subsequent genotoxicity might be an important biological mechanism by which TiO2 -NP exposure would cause detrimental effects to human cardiovascular health.

Craterellus cornucopioides Edible Mushroom as Source of Biologically Active Compounds

Here we determined the phenolic compounds and antioxidant, antimicrobial, genotoxic, and anticancer potential of edible mushrooms Craterellus cornucopioides. The phytochemical analysis was evaluated using high-performance liquid chromatography. Antioxidant activity was evaluated by free radical scavenging, superoxide anion scavenging, and reducing power. Craterellus cornucopioides extract had potent antioxidant activity. Further, the antimicrobial potential was determined by a microdilution method, where minimum inhibitory concentration values ranged from 0.1 to 10 mg/mL. Genotoxic potential was determined by cytokinesis block micronucleus test. The separate treatment did not show genotoxic effect, whereas the combined treatment with mitomycin C significantly reduced the micronuclei frequency in a dose-dependent manner. The highest concentration significantly reduced nuclear division index in comparison to untreated human peripheral blood lymphocytes, whereas in combined treatment, the extract did not significantly affect this parameter. Finally, the cytotoxic activity was tested using microculture tetrazolium test where measured IC50 values ranged from 65.5 to 131.7 μg/mL.