Cytokinesis-block Micronucleus Assay Research Articles

Calcium-dependent cyto- and genotoxicity of nickel metal and nickel oxide nanoparticles in human lung cells

BackgroundGenotoxicity is an important toxicological endpoint due to the link to diseases such as cancer. Therefore, an increased understanding regarding genotoxicity and underlying mechanisms is needed for assessing the risk with exposure to nanoparticles (NPs). The aim of this study was to perform an in-depth investigation regarding the genotoxicity of well-characterized Ni and NiO NPs in human bronchial epithelial BEAS-2B cells and to discern possible mechanisms. Comparisons were made with NiCl2 in order to elucidate effects of ionic Ni.MethodsBEAS-2B cells were exposed to Ni and NiO NPs, as well as NiCl2, and uptake and cellular dose were investigated by transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS). The NPs were characterized in terms of surface composition (X-ray photoelectron spectroscopy), agglomeration (photon cross correlation spectroscopy) and nickel release in cell medium (ICP-MS). Cell death (necrosis/apoptosis) was investigated by Annexin V-FITC/PI staining and genotoxicity by cytokinesis-block micronucleus (cytome) assay (OECD 487), chromosomal aberration (OECD 473) and comet assay. The involvement of intracellular reactive oxygen species (ROS) and calcium was explored using the fluorescent probes, DCFH-DA and Fluo-4.ResultsNPs were efficiently taken up by the BEAS-2B cells. In contrast, no or minor uptake was observed for ionic Ni from NiCl2. Despite differences in uptake, all exposures (NiO, Ni NPs and NiCl2) caused chromosomal damage. Furthermore, NiO NPs were most potent in causing DNA strand breaks and generating intracellular ROS. An increase in intracellular calcium was observed and modulation of intracellular calcium by using inhibitors and chelators clearly prevented the chromosomal damage. Chelation of iron also protected against induced damage, particularly for NiO and NiCl2.ConclusionsThis study has revealed chromosomal damage by Ni and NiO NPs as well as Ni ionic species and provides novel evidence for a calcium-dependent mechanism of cyto- and genotoxicity.

Diosgenin induces genotoxic and mutagenic effects on HepG2 cells

Yam roots and other plants from Dioscorea genus have cultural, nutritional and economic importance to tropical and subtropical regions and have a great amount of diosgenin in its composition. In the present study the cytotoxic, genotoxic and mutagenic potential of diosgenin on HepG2 cells was investigated. Cytotoxicity was assessed using MTT and clonogenic assay. Genotoxic and mutagenic effects were performed using single cell gel electrophoresis and cytokinesis-block micronucleus assay, respectively. A reduction on cell viability was observed due to diosgenin treatment at concentrations higher than 30 μM. A genotoxic effect was shown by comet assay and CBMN. Besides, an increase in micronucleus frequency along with a significant cytostatic effect were observed. Diosgenin elicited DNA damage on HepG2 cells which could not be efficiently repaired contributing to the mutagenic effect observed. Those results suggest that diosgenin deleterious effect could take place through genetic instability, fact that affects the normal cell cycle, leading to cell's death.

Combined effects of co-exposure to formaldehyde and acrolein mixtures on cytotoxicity and genotoxicity in vitro.

FA (formaldehyde) and ACR (acrolein) are common pollutants in environment, which often occur together in air. So, adverse health effects may not only result from their individual toxicity but also from the combined toxicity. While often studied alone, combination effects of these pollutants are inconclusive. Here, we examined the combined cytotoxicity and genotoxicity of FA and ACR on A549 cells based on CCK-8 assay, comet assay, and cytokinesis-block micronuclei assay. FA and ACR mixtures showed significant cytotoxicity and genotoxicity even at NOECs (no observed effect concentrations). Moreover, FA and ACR administrated jointly at doses from NOECs to sub-cytotoxic concentrations demonstrated significant interactions in cytotoxicity, DNA strand breaks, and chromosome damage. Co-exposure to FA and ACR significantly showed a lower responses on DNA strand break and chromosome damage than those expected for additivity, while an opposite result was obtained on cytotoxicity. Taken together, these results indicated that there was significant interactions on cytotoxicity and genotoxicity for FA and ACR, and prolonged exposure to mixtures of FA and ACR below sub-cytotoxic concentrations can produce a serious threat in human's health.

Shelterin differentially respond to oxidative stress induced by TiO2-NPs and regulate telomere length in human hepatocytes and hepatocarcinoma cells in vitro

Titanium dioxide nanoparticles (TiO2-NPs) have raised serious attention for their widely use and potential adverse effects on human mainly due to producing ROS. However, the influence of TiO2-NPs on telomere maintaining has not been studied clearly. Shelterin plays core roles in telomere length (TL) regulation. Abnormal TL are associated with chromosome instability (CIN) and high risk of diseases. This study investigated whether TiO2-NPs affect TL to induce CIN through ROS generation and the possible mechanisms. Human hepatocyte L-02 and hepatocarcinoma cells QGY were exposed to TiO2-NPs (0, 40, 80 μg/mL) for 72 h. The intracellular hydrogen dioxide (H2O2) concentration were measured. The TL, Nrf-2, and three core shelterin components (TRF1, TRF2, and POT1) transcription level were determined by quantitative real-time PCR. CIN was measured by cytokinesis-block micronucleus assay. TiO2-NPs exposure increased intracellular H2O2 in both L-02 and QGY cells, and induced Nrf-2, TRF1, TRF2, POT1 downregulated transcription compared with control (P < 0.001) in L-02 but all upregulated (P < 0.05) in QGY. Significant TL shortening (P < 0.001) and CIN increase (P < 0.01) in L-02 cells were observed but not in QGY cells. The differentially responses of the tested components of shelterin and Nrf-2 to oxidative stress induced by TiO2-NPs led to the weakened telomere protection in normal cells and effective telomere maintenance in cancer cells, respectively. The upregulation of Nrf-2 and shelterin could protect TL and chromosome stability against TiO2-NPs exposure.

In vitro testing of estragole in HepG2 cells: Cytokinesis-block micronucleus assay and cell-cycle analysis

The alkenylbenzene estragole (systematic name, 1-allyl-4-methoxybenzene) is a natural component of essential oils from various spices and herbs, including fennel, and it is used as a food and beverage flavouring agent. Estragole has been reported to be hepatocarcinogenic at high doses in rodents. However, in a previous in vitro study, we found that estragole did not exhibit cytotoxic effects after 4 hours of exposure, nor did it induce DNA damage or apoptosis in human HepG2 hepatoblastoma cells. As fennel tea is widely used for symptomatic treatment of spasmodic gastrointestinal conditions in infants, we aimed at further assessing its safety in a different experimental setting. We thus searched for possible cytogenetic effects and interference with cell-cycle progression in the same human hepatoblastoma cell line. Estragole did not show any clastogenic/aneugenic activities in the cytokinesis-block micronucleus assay, and no effects on cell-cycle checkpoints were observed.

The cytokinesis-blocked micronucleus assay as a novel biomarker for selection of lung cancer screening participants.

Despite the promising results of the National Lung Screening Trial in reducing lung cancer mortality among high risk smokers, several challenges remain to be addressed. These include the high false positive rates and the large number of smokers screened in order to prevent one lung cancer death. In addition, host genetic susceptibility has not been integrated into selection of who should be screened. These challenges highlight the need to develop robust ways to identify susceptible smokers for appropriate screening. We used the cytokinesis block micronucleus (CBMN) assay to assess smoking induced genetic instability among NLST participants. Blood cultures were prepared at time of entry into the screening study and DNA damage was recorded as the frequency of binucleated nucleoplasmic bridges and micronuclei. Low dose CT (LDCT) and chest X-ray (CXR) image findings were available upon unblinding of the NLST study and imaging data were merged with blood marker data for statistical analysis. A total of 641 participants were included in this study. The frequency of the CBMN endpoints at time of entry into the study was significantly higher among study participants who had a positive finding during the 3-year screening or reported lung cancer at the end of the follow-up period as compared to participants who were negative. Growth curve models were used to compare trajectories of change in CBMN endpoints between entry into the study and end-of-screening period. A statistically significant increase was predicted for CBMN endpoints among the study participants who were positive versus those who remained negative at the end-of-screening period (P<0.001). Genetic instability biomarkers have the potential of facilitating the identification of genetically susceptible high-risk smokers who would benefit from targeted lung screening programs.

DNA damage and repair in individuals with ataxia-telangiectasia and their parents

Ataxi A-T elangiectasia (AT) is a multisystem, complex and rare disease inherited in an autosomal recessive manner. Homozygous individuals have a variety of pathological manifestations, however, heterozygotes only present a higher risk of developing cancer. We evaluated the background levels of DNA damage (basal damage) and cell response to bleomycin or ionizing radiation using Comet assay and the cytokinesis-block micronucleus (CBMN) test in individuals with AT, their parents and controls. To evaluate DNA repair, the challenge experiment with ionizing radiation was performed using Comet assay, and different recovery times were evaluated. Results showed that basal MN frequencies differ between patients, parents and controls. Meanwhile, using the Comet assay, the results from the basal analysis do not differ between the groups, but monitoring the kinetics of DNA repair, we verified that the group of patients showed a delay in repair, compared to controls. Another finding was the nuclear bud (NBUD) frequency: spontaneous and induced cell cultures (with bleomycin and radiation) showed clear differences between patients, parents and controls. The CBMN assay and repair measurement with the Comet assay can help in the diagnosis of AT patients and ATM gene carriers, as complementary methods. The use of genomic instability evaluation techniques for the identification of the heterozygotes in families, where at least one member is affected, may be of great clinical importance.

Construction of dose response curves up to 6 Gy for Micronucleus and Dicentric Chromosome Aberration Assay with 6 MV X-ray Beam

In-vitro dose response curves were built with 6 MV X-ray for a dose range of 0.1–6 Gy, at a dose rate of 300 cGy/min, using Linear Accelerator (LINAC, SIMENS PRIMUS) for Cytokinesis-Block Micronucleus (CBMN) and Dicentric Chromosome Aberration assay (DCA). The dose response curves constructed up to 6 Gy and also up to 4 Gy using the manual method were compared to those curves obtained with the software program such as CABAS (V.2) and Dose Estimate software (V.5.2). The alpha and beta coefficients for MN and CA obtained for the curves constructed up to 6 Gy showed variation compared to those obtained in dose response curve constructed up to 4 Gy. However, the doses estimated based on these coefficients did not show much variation. The results are discussed in this paper.

Hypermethylation of CpG islands is associated with increasing chromosomal damage in chinese lead-exposed workers.

Lead is a widely existing environmental pollutant with potential carcinogenicity. To investigate the association of blood lead level (B-Pb) with potential chromosomal damage and cancer, we analyzed micronucleus (MN) frequency of peripheral blood lymphocytes (PBLs) and the methylation status of six human tumor suppressor genes (TSGs) post lead exposure. In the study, 147 lead-exposed workers were divided into two groups according to their B-Pb P50 value, with other 50 lead-unexposed workers as a control group. The cytokinesis-blocked micronucleus (CBMN) assay was performed to detect chromosomal damage of PBLs of both lead-exposed and -unexposed workers. The methylation-specific polymerase chain reaction (MSP-PCR) was further used to examine the methylation status of six TSGs (GSTP1, hMLH1, MGMT, p14, p15, and p16). Results showed that MN frequencies of high B-Pb workers 8.1 ± 3.1‰ and low B-Pb workers 5.7 ± 2.3‰ were significantly higher than that of control group 2.8 ± 1.9‰ (P < 0.01), while the MN frequency of high B-Pb workers was also higher than that of the low B-Pb workers (P < 0.01). The MN frequency in PBLs of lead-exposed group with the methylated TSGs was significantly higher than that in PBLs with the unmethylated TSGs (P < 0.05). Notably, the CpG island methylator phenotype (CIMP) correlated with chromosome damage (P < 0.05). Additionally, workers with high B-Pb had higher chromosome damage than those with low B-Pb (P < 0.05). Taken altogether, the results suggest that lead-exposed workers with CIMP positive and high B-Pb have a higher risk of being vulnerable to tumorigenesis. Environ. Mol. Mutagen. 59:549-556, 2018. © 2018 Wiley Periodicals, Inc.

Validity of the Lymphocyte Cytokinesis-Block Micronucleus Assay (L-CBMN) as biomarker for human exposure to chemicals with different modes of action: A synthesis of systematic reviews

Recently fourteen systematic reviews applying the same selection and evaluation criteria analyzed the induction of micronuclei in lymphocytes as biomarker for DNA damage induced by human exposure to a given chemical or chemical mixture. The results obtained in the individual reviews were summarized to evaluate the validity of the Cytokinesis-Block-Micronucleus assay in lymphocytes (L-CBMN) and propose recommendations for its use in occupational and environmental exposure studies. All systematic reviews found consistent increases of MN frequencies in exposed subjects versus controls in all genotoxic compounds or group of chemicals investigated, in the following decreasing order: As/Cr/Ni, vinyl chloride, formaldehyde, Hg/Pb/Cd, “miscellaneous”, pesticides, cytostatics/antineoplastics, anaesthetic gasses, dust/asbestos/other fibers, polycyclic aromatic hydrocarbons, ethylene oxide, butadiene, styrene and petroleum/derivatives. Two reviews compared the results with the recommended exposure limits. For styrene, MN was found not to be induced under the recommended threshold limit. For vinyl chloride the safe exposure limit based on the L-CBMN data is lower than the current one. The L-CBMN thus appears to be a valid biomarker to assess DNA damage in populations exposed to genotoxic chemicals. Many shortcomings have been reported in assessment of confounding factors, such as lifestyle patterns, in particular diet and the major one the exposure assessment. All these factors together with methodological variables may contribute to the large variability in MN frequencies, also in controls. Information on frequency and origin of MN in more than one tissue (e.g. lymphocytes and buccal cells) in parallel, may provide better understanding of the mechanisms involved. Use of automated MN scoring systems to increase numbers of cells scored and facilitate screening more individuals would increase data reliability and provide information on the link between mutagenicity and carcinogenicity, if the studies are done prospectively. Efforts should be made to unravel the genotoxic effects induced when chronic and/or mixed exposures are involved.

The potential for complete automated scoring of the cytokinesis block micronucleus cytome assay using imaging flow cytometry

The lymphocyte Cytokinesis-Block Micronucleus (CBMN) assay was originally developed for the measurement of micronuclei (MN) exclusively in binucleated (BN) cells, which represent the population of cells that can express MN because they completed nuclear division. Recently the assay has evolved into a comprehensive cytome method to include biomarkers that measure chromosomal instability and cytotoxicity by quantification of nuclear buds (NBUDs), nucleoplasmic bridges (NPBs) and apoptotic/necrotic cells. Furthermore, enumeration of mono- and polynucleated cells allows for computation of the nuclear division index (NDI) to assess mitotic activity. Typically performed by manual microscopy, the CBMN cytome assay is laborious and subject to scorer bias and fatigue, leading to inter- and intra-scorer variability. Automated microscopy and conventional flow cytometry methods have been developed to automate scoring of the traditional and cytome versions of the assay. However, these methods have several limitations including the requirement to create high-quality microscope slides, lack of staining consistency and sub-optimal nuclear/cytoplasmic visualization. In the case of flow cytometry, stripping of the cytoplasmic membrane makes it impossible to measure MN in BN cells, calculate the NDI or to quantify apoptotic or necrotic cells. Moreover, the absence of cellular visualization using conventional flow cytometry, makes it impossible to quantify NBUDs and NPBs. In this review, we propose that imaging flow cytometry (IFC), which combines high resolution microscopy with flow cytometry, may overcome these limitations. We demonstrate that by using IFC, images from cells in suspension can be captured, removing the need for microscope slides and allowing visualization of intact cytoplasmic membranes and DNA content. Thus, mono-, bi- and polynucleated cells with and without MN can be rapidly and automatically identified and quantified. Finally, we present high-resolution cell images containing NBUDs and NPBs, illustrating that IFC possesses the potential for completely automated scoring of all components of the CBMN cytome assay.

Zoledronic acid induces micronuclei formation, mitochondrial-mediated apoptosis and cytostasis in kidney cells

AimsZoledronic acid (ZA), a FDA approved drug has used widely in the treatment of bone metastasis complications, has been linked to renal toxicity with unclear mechanism. The present study is aimed at investigating the genotoxic and cytotoxic effects of ZA in renal epithelial cells. Main methodsThe genotoxic effect of ZA in Vero and MDCK cells determined by cytokinesis block micronucleus (CBMN) assay. The cytotoxic effect assessed by analysing cell cycle profile, cell death and mitochondrial membrane potential by flow cytometry using propidium iodide, AnnexinV-FITC/PI and JC1 dye staining, respectively, BAX and Bcl-2 expression by Western blotting and caspase activity by spectrofluorimetry. Key findingsThe cytotoxic effect of ZA based on MTT assay revealed variable sensitivities of Vero and MDCK cells, with IC50 values of 7.41 and 109.58 μM, respectively. The CBMN assay has shown prominent dose-dependent (IC10-50) induction of micronuclei formation in both cells, indicating ZA's clastogenic and aneugenic potential. Further, the ZA treatment led the cells to apoptosis, evident from dose-dependent increase in the percentage of cells in subG1 phase and display of membranous phosphatidylserine translocation. Studies also confirmed apoptosis through mitochondria, evident from the prominent increase in BAX/Bcl-2 ratio, mitochondrial membrane depolarization and caspase-3/7 activity. In addition, ZA reduces cytokinetic activity of renal cells, evident from dose-wise lowered replicative indices. SignificanceThe study depict ZA's potential genotoxic effect along with cytotoxic effect in renal epithelial cells, could be key factors for the development of renal complications associated with it, which prompts renal safety measures in lieu with ZA usage.

Polyphenolic contents of Teucrium polium L. and Teucrium scordium L. associated with their protective effects against MMC-induced chromosomal damage in cultured human peripheral blood lymphocytes.

Teucrium species have been used in traditional medicine for treatment of different diseases. The aim of this study was to investigate polyphenolic contents by high-performance liquid chromatography (HPLC), and the genotoxic effect of methanolic extracts of Teucrium polium and Teucrium scordium using the cytokinesis-block micronucleus (CBMN) assay on human peripheral blood lymphocytes (PBLs) from healthy donors. The HPLC analysis showed that extracts consist of phenolic acid (gallic, vanillic, caefic, chlorogenic, p-coumaric, sinapic) and flavonoids (catechin, rutin, myricetin, luteolin, quercetin and apigenin). Cultures were treated with extracts of both plants separately and in combinations with mitomycin C (MMC). In separate treatments, both herbal extracts significantly induced micronucleus (MN) frequency only at the highest concentrations. All concentrations of T. scordium , except the lowest, and all concentrations of T. polium extracts in combined treatment with MMC significantly reduced the frequency of MN. The extract of T. polium did not significantly aefct the nuclear division index (NDI), whereas T. scordium in higher concentrations, separately and in combined treatment with MMC, significantly decreased the NDI value. Our results suggest that both herbal extracts in combination with MMC have antimutagenic (T. polium) and proapoptotic effects (T. scordium), which indicates their protective effects in PBLs.

Assessment of genotoxic activity of oleoresins and leaves extracts of six Copaifera species for prediction of potential human risks

Ethnopharmacological relevanceCopaifera species are used in folk medicine for a wide variety of pharmacological properties. This paper reports the cytotoxic and genotoxic analyses of oleoresins and leaves extracts of Copaifera species: C. duckei, C. multijuga, C. paupera, C. pubiflora, C. reticulata and C. trapezifolia. Materials and methodsIn vitro assays were performed using Chinese hamster lung fibroblasts (V79 cells). The clonogenic efficiency and cytokinesis-block micronucleus assays were employed for the cytotoxicity and genotoxicity assessment, respectively. The mouse bone marrow micronucleus test was used for in vivo studies. ResultsThe cytotoxicity results using the clonogenic efficiency assay showed IC50 values ranging from 9.8 to 99.2 µg/mL for oleoresins and 66.4–721.5 for leaves extracts. However, no cytotoxic effect was observed in the in vivo studies. Additionally, the treatments with oleoresins and leaves extracts did not significantly increase the frequency of micronuclei in both in vitro and in vivo mammalian cells. The UPLC-MS/MS and CG/MS analyses of Copaifera oleoresins allowed the identification of 10 acid diterpenes and 11 major volatile sesquiterpenes. Leaves are rich in phenolic compounds including two flavonoid heterosides and 16 galloylquinic acid derivatives. ConclusionsThe oleoresins and leaves extracts of studied Copaifera species were not cytotoxic in vivo, as well as not genotoxic in both in vitro and vivo assays, under the experimental conditions used. Therefore, the obtained results should be sufficient to demonstrate the absence of significant genotoxic risk of these Copaifera products for human use in the evaluated concentrations range.

Differences in the Effects of EGCG on Chromosomal Stability and Cell Growth between Normal and Colon Cancer Cells.

The tea catechin epigallocatechin-3-gallate (EGCG) proved to be the most potent physiologically active tea compound in vitro. It possesses antioxidant as well as pro-oxidant properties. EGCG has the effect of inducing apoptosis of tumor cells and inhibiting cell proliferation. Whether this effect is associated with the antioxidant or pro-oxidative effects of EGCG affecting the genome stability of normal and cancer cells has not been confirmed. Here, we selected Human normal colon epithelial cells NCM460 and colon adenocarcinoma cells COLO205 to investigate the effects of EGCG (0–40 μg/mL) on the genome stability and cell growth status. Chromosomal instability (CIN), nuclear division index (NDI), and apoptosis was measured by cytokinesis-block micronucleus assay (CBMN), and the expression of core genes in mismatch repair (hMLMLH1 and hMSH2) was examined by RT-qPCR. We found that EGCG significantly reduced CIN and apoptosis rate of NCM460 at all concentrations (5–40 μg/mL) and treatment time, EGCG at 5 μg/mL promoted cell division; EGCG could significantly induce chromosome instability in COLO205 cells and trigger apoptosis and inhibition of cell division. These results suggest that EGCG exhibits different genetic and cytological effects in normal and colon cancer cells.

Genotoxicity and osteogenic potential of sulfated polysaccharides from Caulerpa prolifera seaweed

Marine algae are sources of novel bioactive molecules and present a great potential for biotechnological and biomedical applications. Although green algae are the least studied type of seaweed, several of their biological activities have already been described. Here, we investigated the osteogenic potential of Sulfated Polysaccharide (SP)-enriched samples extracted from the green seaweed Caulerpa prolifera on human mesenchymal stem cells isolated from Wharton jelly (hMSC-WJ). In addition, the potential genotoxicity of these SPs was determined by cytokinesis-block micronucleus (CBMN) assay. SP-enriched samples did not show significant cytotoxicity towards hMSCs-WJ at a concentration of up to 10μg/mL, and after 72h of exposure. SP enrichment also significantly increased alkaline phosphatase (ALP) activity, promoting calcium accumulation in the extracellular matrix. Among the SP-enriched samples, the CP0.5 subfraction (at 5μg/mL) presented the most promising results. In this sample, ALP activity was increased approximately by 60%, and calcium accumulation was approximately 6-fold above the negative control, indicating high osteogenic potential. This subfraction also proved to be non-genotoxic, according to the CBMN assay, as it did not induce micronuclei. The results of this study highlight, for the first time, the potential of these SPs for the development of new therapies for bone regeneration.

Successful proof of concept of a micronucleus genotoxicity assay on reconstructed epidermis exhibiting intrinsic metabolic activity

We investigated the commercially available Episkin LM™ reconstructed epidermis test system as a potential 3D model for human genotoxicity assessment by cytokinesis-block micronucleus assay to mitigate limitations of the currently accepted micronucleus test.We established appropriate culture conditions for cytokinesis-block micronucleus assay in maximizing the frequency of binucleated cells by choice of culture medium and calibration of the system exposure to the cytokinesis inhibitor Cytochalasin B, without affecting the basal frequency of micronuclei in the model. We confirmed that the application of the classic solvents had no significant effect on this basal level of micronuclei.We determined the performance of cytokinesis-block micronucleus assay in Episkin LM™ reconstructed epidermis to predict in vivo genotoxins by testing the genotoxicity potential of 17 well known in vivo genotoxic, progenotoxic and non-genotoxic reference chemicals over a 48 h and 72 h exposure period.We found that cytokinesis-block micronucleus assays in Episkin™ reconstructed epidermis following the 48 h-topical regimen had a specificity of 60–75% and a sensitivity of 83–85%, resulting in an overall accuracy of 76–82% for genotoxicity assessment in tissues depending on the assessment of the reference chemicals with equivocal genotoxic profiles in the literature.The positive micronucleus test results obtained without addition of any exogenous metabolic activation system confirmed the ability of Episkin LM™ reconstructed epidermis to intrinsically bioactivate progenotoxic chemicals. The evidence showed that the 72-h exposure protocol significantly improved the detection of progenotoxins.Taken together, our data demonstrated that the Episkin LM™ reconstructed epidermis system is a relevant in vitro tool in the study of genetic toxicology.

The genotoxic and antigenotoxic potential of the methanolic root extract of Glycyrrhiza glabra L. on human peripheral blood lymphocytes

Glycyrrhiza glabra L. (licorice) is one of the most important medicinal plants, which is widely used throughout the world both in traditional and contemporary medical industries. This study was undertaken to investigate the potential genotoxic activity of G. glabra methanolic root extract, and its possible antigenotoxic properties against mitomycin C (MMC)-induced DNA damage in in vitro chromosome aberrations (CAs) and cytokinesis-block micronucleus (CBMN) assays in human peripheral blood lymphocytes (PBLs). Lymphocytes were treated with 25, 50, and 100 µg/ml G. glabra methanolic root extract alone as well as in combination with MMC (0.1 µg/ml) for 24 and 48 h treatment periods. It was found that there were no statistically significant differences between the negative control and the groups treated with all concentrations of G. glabra root extract of alone (p > 0.05), demonstrating the absence of genotoxic effects at both 24 and 48 h treatment periods. Besides, the co-treatment of G. glabra methanolic root extract and MMC significantly decreased the percentage of structural CAs and MN formation when compared with the culture treated with MMC alone (p < 0.001). In addition, the negative interaction factor (IF) values obtained for all combinations represent an antagonistic effect of G. glabra versus MMC. We can state that this extract acts as an antagonist and markedly decreased MMC-induced cytogenotoxicity. In conclusion, the present results demonstrate that in the tested experimental conditions, G. glabra methanolic root extract is not genotoxic in cultured human PBLs and has also antigenotoxic activity against MMC, which is widely used in chemotherapy against cancer.

Correlation between Serum Amyloid A-Low Density Lipoprotein and Genotoxicity in Smokers.

To investigate the relation between serum amyloid A-low density lipoprotein (SAA-LDL) and genotoxicity in smokers. An experimental study. Army Medical College, Rawalpindi and National Institute of Health (NIH), Islamabad, from June 2014 to February 2015. Seventy healthy Sprague Dawley rats were purchased from NIH and exposed to cigarette smoke in smoke chamber for three months. Blood samples were drawn from each rat at the end of the study period. SAA-LDL was determined by enzyme-linked immunosorbent assay (ELISA). Genotoxicity was assessed by cytokinesis block micronucleus (CBMN) assay. Pearson correlation was used to find correlation between SAA-LDL and genotoxicity. Strong positive correlation was found between SAA-LDL and micronuclei frequency in smoke-exposed rats (r=0.799, N=70, p <0.01). Statistically significant strong positive correlation between SAA-LDL and genotoxicity in smoke-exposed rats shows that changes in one is associated with changes in other and vice versa.

Whole thorax irradiation of non-human primates induces persistent nuclear damage and gene expression changes in peripheral blood cells.

We investigated the cytogenetic and gene expression responses of peripheral blood cells of non-human primates (NHP, Macaca mulatta) that were whole-thorax irradiated with a single dose of 10 Gy. In this model, partial irradiation of NHPs in the thoracic region (Whole Thorax Lung Irradiation, WTLI) allows the study of late radiation-induced lung injury, while avoiding acute radiation syndromes related to hematopoietic and gastrointestinal injury. A transient drop in circulating lymphocytes and platelets was seen by 9 days, followed by elevations in respiratory rate, circulating neutrophils, lymphocytes, and monocytes at 60–100 days, corresponding to computed tomography (CT) and histologic evidence of pneumonitis, and elective euthanasia of four animals. To evaluate long-term DNA damage in NHP peripheral blood lymphocytes after 10 Gy WTLI, we used the cytokinesis-block micronucleus (CBMN) assay to measure chromosomal aberrations as post-mitotic micronuclei in blood samples collected up to 8 months after irradiation. Regression analysis showed significant induction of micronuclei in NHP blood cells that persisted with a gradual decline over the 8-month study period, suggesting long-term DNA damage in blood lymphocytes after WTLI.We also report transcriptomic changes in blood up to 30 days after WTLI. We isolated total RNA from peripheral blood at 3 days before and then at 2, 5 and 30 days after irradiation. We identified 1187 transcripts that were significantly changed across the 30-day time course. From changes in gene expression, we identified biological processes related to immune responses, which persisted across the 30-day study. Response to oxygen-containing compounds and bacteria were implicated by gene-expression changes at the earliest day 2 and latest, day 30 time-points. Gene expression changes suggest a persistent altered state of the immune system, specifically response to infection, for at least a month after WTLI.