Aberrant Metaphases Research Articles

PIWIL1 is recruited to centrosomes during mitosis in colorectal cancer cells and is linked to cell cycle progression

PIWI proteins, traditionally associated with germline development, have recently gained attention for their expression in various cancers, including colorectal cancer. However, the molecular mechanisms underlying their reactivation and impact on cancer initiation and progression remain elusive. Here, we found that PIWIL1 is expressed at relatively high levels in CRC-derived samples and cell lines, where it undergoes a dynamic relocalization to the centrosome during mitosis. Knockdown of PIWIL1 induces G2/M arrest associated with disruption of the mitotic spindle and aberrant metaphase events, highlighting its role in cell cycle progression. We also found that the expression of PIWIL1 is lost during the differentiation of Caco-2 cells into enterocytes and that PIWIL1 is expressed in cells at the base of the intestinal crypts in normal human colon tissue, where intestinal stem cells are known to reside. Thus, it is possible that the presence of PIWIL1 in cancer cells reflects a physiological role of this protein in stem cell maintenance, which would argue in favor of the proposed stem cell origin of CRC. Supporting this view, dedifferentiation of human fibroblasts into induced pluripotent stem cells (iPSCs) involves the reactivation of PIWIL2 expression, another member of the PIWI protein family. Overall, our findings suggest a role of PIWIL1 in mediating cell cycle dynamics, both in colorectal cancer cells and possibly also in intestinal stem cells. In a broader aspect, we provide evidence supporting an involvement of PIWI proteins in somatic stem cell maintenance, thus expanding the known non-gonadal functions of this protein family.

Abstract A017: Comprehensive evaluation of the therapeutic effects of whole-body hyperthermia for pancreatic ductal adenocarcinoma (PDAC) and the potential synergy with standard-of-care chemotherapeutics

Abstract Whole-body hyperthermia (WBHT) is a promising therapeutic approach that leverages elevated temperatures to enhance cancer treatment efficacy. By inducing controlled hyperthermia in the range of 39-42.5°C, cellular processes are disrupted and cancer cells can be sensitized to standard-of-care chemotherapies. However, the precise cellular and molecular mechanisms by which WBHT exerts its effects are not fully understood. Elucidating these mechanisms is particularly crucial for optimizing treatment protocols and improving outcomes in aggressive malignancies such as pancreatic ductal adenocarcinoma (PDAC). This study explores the impact of WBHT at 41.5°C on the cell cycle and associated molecular pathways in PDAC cells, providing insights into its therapeutic value and potential for combination regimens. PDAC cell lines, BxPC-3 and AsPC-1, were subjected to WBHT at 41.5°C for 1-6 hours and analyzed immediately, 24h and 48h post-WBHT Cell proliferation was assessed using the 5-ethynyl-2'-deoxyuridine (EdU) base-incorporation assay and proliferating cell nuclear antigen (PCNA) staining for immunofluorescence detection. Morphological changes post-WBHT were monitored using confocal fluorescence microscopy. Total RNA was extracted and sequenced using the Illumina platform. An in-house pipeline was used for QC, alignment and differential expression analysis. Enrichment analysis was performed to identify affected biological processes and pathways. In vivo experiments employed the chorioallantois membrane (CAM) assay per protocol published in STAR protocols. WBHT at 41.5°C significantly disrupted cell cycle progression in PDAC cell lines. The EdU incorporation assay revealed a dose-dependent decrease in DNA synthesis during and following WBHT, indicating cell cycle arrest in the S-phase. Decreases in Ki67, cyclin B1 and PCNA intensities further confirmed this reduction in cell proliferation. Moreover, cells showed notable signs of cellular and mitotic stress, such as the formation of multi-nucleated giant cells, aberrant metaphase morphology and micronuclei. Bulk RNA-seq confirmed alterations in gene expression profiles relating to the cell cycle immediately after HT exposure. Genes associated with the E2F targets and G2/M checkpoint pathways were negatively enriched, whereas genes involved in the Hallmark apoptosis pathway were positively enriched. Preliminary data from in ovo CAM experiments showed a significant inhibitory effect of WBHT monotherapy on growth of BxPC-3 tumors. Our data show that WBHT severely disrupts the cell cycle at the S- and M-phases, via multiple pathways involved in cell proliferation and apoptosis. These cancer hallmarks are targeted by conventional chemotherapeutics, paving the way for combination therapy as one-two punch cancer treatments. The synergy between WBHT with cisplatin and oxaliplatin, but also with gemcitabine and 5-fluorouracil (5-FU) could be optimized based on the improved understanding of the WBHT effects over time, in terms of WBHT duration, sequence of WBHT and chemo and the exact treatment schedule. Citation Format: Robin Colenbier, Tine Logghe, Gaëlle Boulet, Johannes Bogers, Jean-Pierre Timmermans. Comprehensive evaluation of the therapeutic effects of whole-body hyperthermia for pancreatic ductal adenocarcinoma (PDAC) and the potential synergy with standard-of-care chemotherapeutics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A017.

Enhancement of the Clastogenic Effects of Topotecan In Vivo by Tyrosyl-DNA Phosphodiesterase 1 Inhibitors.

Topotecan administered intraperitoneally at single doses of 0.25, 0.5, and 1 mg/kg induced chromosomal aberrations in bone marrow cells of F1(CBA×C57BL/6) hybrid mice in a dose-dependent manner. A tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitor, an usnic acid derivative OL9-116 was inactive in a dose range of 20-240 mg/kg, but enhanced the cytogenetic effect of topotecan (0.25 mg/kg) at a dose of 40 mg/kg (per os). The TDP1 inhibitor, a coumarin derivative TX-2552 (at doses of 20, 40, 80, and 160 mg/kg per os), increased the level of aberrant metaphases induced by topotecan (0.25 mg/kg) by 2.1-2.6 times, but was inactive at a dose of 10 mg/kg. The results indicate that TDP1 inhibitors enhance the clastogenic activity of topotecan in mouse bone marrow cells in vivo and are characterized by different dose profiles of the co-mutagenic effects.

Prevention of the Genotoxic Effects of Doxorubicin with Anthocyanin-Containing Complex from Sorbus aucuparia L. Fruit.

We studied the effect of an anthocyanin-containing complex from the fruits of S. aucuparia L. on doxorubicin-induced genotoxicity in bone marrow cells of C57BL/6 mice. The complex reduced the genotoxic effect doxorubicin in metaphase plates of bone marrow cells in 24, 48 h, and 10 days after the administration of the cytostatic. The mean number of single fragments and the fraction of cells with gaps and aberrant metaphases also decreased.

L-Ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations

The objective of the present study was to with all details explain of the efficiency of L-ascorbic acid (L-AsA) also known as vitamin C on cytotoxicity and genotoxicity induced by salt stress in the barley apical meristems. As a result of the statistical analysis salt stress caused a significant (P ≤ 0.05) decrease in mitotic index of barley seeds depending on concentration increase, while the frequency of chromosomal aberration (CA) increased. In addition, it was determined that mitotic index value was decreased by 46% with 1 μM L-AsA supplementation as compared to control and chromosomal abnormalities were increased by 8.96% as well as. However, in the case of simultaneously application of 1 μM L-AsA and different salt concentrations, the high salt concentrations exhibited an excellent success according to low salt concentrations in alleviating the mitodepressive effect of salt stress. Moreover, the frequency of chromosomal aberrations in the root meristem cells of those seeds with 1 μM L-AsA supplementation germinated at different salt concentrations was substantially reduced compared to own control group (alone 1 μM L-AsA pretreatment). The 1 μM L-AsA pretreatment at the highest salt concentration (at 0.40 M) was showed an excellent success by reducing the frequency of the chromosomal aberrations by approximately 90 %. Different salt concentrations and/or 1 μM L-AsA supplementation caused micronuclei and granulation as well as various chromosomal aberrations in prophase, metaphase, anaphase and telophase.

Chronic dust bronchitis: composition of the sputum bacterial microbiome and its association with chromosome damage in blood lymphocytes

BACKGROUND: Recent studies show that the bacterial microbiome of the respiratory tract can influence the development of a number of diseases of the human respiratory system. Changes in the composition of the microbiome in patients are associated with dysbiosis, and in addition, many bacteria have a genotoxic potential and can directly or indirectly damage the genome in the cells of the host organism.
 AIM: The aim of the study was to analyze the composition of the sputum microbiome and its relationship with chromosome damage in the blood leukocytes of patients with chronic dust bronchitis (CDB).
 MATERIALS AND METHODS: The taxonomic composition of the sputum microbiome of 22 patients with CKD and 22 sputum donors from the control group was studied using next-generation sequencing (NGS) technology of 16S rRNA of bacterial genes. At the same time, the basic frequencies of chromosomal aberrations and micronuclei were determined in blood leukocytes.
 RESULTS: The sputum microbiome of chronic dust bronchitis patients had a significant reduction in alpha and beta diversity parameters compared to healthy study participants. In addition, an increase in the relative abundance of the genus Streptococcus (29.97 3.03 vs. 18.78 2.47; p = 0.003) was found in the sputum of CP patients compared with the control. Thus, the results of metagenome sequencing indicate a common dysbiotic process with a predominance of one dominant genus of bacteria in this pulmonary pathology. The results of cytogenetic analysis of blood leukocytes showed a significant increase in the proportion of aberrant metaphases in CKD patients compared with healthy donors (3.41% vs. 1.84%; p 0.01) and the absence of significant differences in frequency leukocytes with micronuclei between the compared groups (1.28% vs. 1.11%). Correlation analysis revealed the presence of significant direct relationships between the frequency of aberrant metaphases and the percentage of representatives of the genera Bacteroides in the sputum of patients with chronic dust bronchitis (r = 0.471; p = 0.031); Lachnoanaerobaculum (r = 0.446; p = 0.043) and Alloprevotella (r = 0.444; p = 0.044). Further studies should be devoted to the search for possible mechanisms of influence of these bacteria on clastogenic effects in the cells of the host organism.

Хромосомна нестабільність чистопородних і кросбредних корів молочного напряму продуктивності

The article presents the results of research on the karyotype characteristics variability in purebred and crossbred dairy cows. The material for the research was peripheral blood samples of purebred firstborn cows of the Ukrainian red-spotted dairy and Ukrainian black-spotted dairy breeds, as well as crossbred cows obtained from crossing Ukrainian red-spotted with Montbeliard bulls (DG “Nyva” SE of the Institute of Animal Breeding and Genetics named after M. V. Zubets NAAS. Preparation of cytogenetic preparations, analysis of morphology, identification and classification of chromosome aberrations were carried out according to generally accepted methods. We performed cell analysis with the Axiostar plus microscope (Carl Zeiss, Germany) under immersion magnification of 1000 times and took microphotographs. In all studies, the frequency of aberrant metaphases and the spectrum of chromosomal aberrations were determined as parameters of chromosomal instability. The following signs were taken into account: the frequency of aneuploid and polyploid cells, cells with premature separation of the centromeric regions of chromosomes (CRC), cells with structural aberrations of chromosomes (breaks, fragments and associations of non-homologous chromosomes). As a result of the analysis of karyotypes of firstborn cows of purebred and crossbred origin, it was established that the proportion of diploid cells in the norm is on average 85%. The remaining almost 15% are somatic cells with numerical and structural abnormalities. Crossbred cows have significantly higher frequencies (P<0.001) of aneuploid (one and a half times), polyploid (by 27%), structural aberrations of chromosomes (by 20%) than in purebred cows of the Ukrainian red-spotted and black-spotted dairy breeds. 15–20% more cells containing chromosomes with breaks and fragments were also found in crossbred firstborns. The results of the cytogenetic study indicate greater chromosomal instability in crossbred cows compared to purebred cows. One of the reasons for this phenomenon may be the influence of breeding methods, in particular crossbreeding.

Evidence for Recombinant GRP78, CALR, PDIA3 and GPI as Mediators of Genetic Instability in Human CD34+ Cells.

Simple SummaryFactors secreted from irradiated mesenchymal stromal cells (MSC) may exert genotoxic effects in human CD34+ cells, thereby potentially contributing to the pathogenesis of hematologic disorders such as leukemias. In a proteomics approach, we recently identified four key proteins in the secretome of X-ray-irradiated MSC, among them the three chaperones GRP78, CALR, and PDIA3, and one glycolytic enzyme GPI. Here, we demonstrate that recombinant GRP78, CALR, PDIA3 and GPI induce significant levels of genetic instability in human CD34+ cells. Our data suggest that GRP78, CALR, PDIA3 and GPI released from irradiated MSC act as mediators of genetic instability in human CD34+ cells with potential implications for radiation-induced hematologic disorders.Soluble factors released from irradiated human mesenchymal stromal cells (MSC) may induce genetic instability in human CD34+ cells, potentially mediating hematologic disorders. Recently, we identified four key proteins in the secretome of X-ray-irradiated MSC, among them three endoplasmic reticulum proteins, the 78 kDa glucose-related protein (GRP78), calreticulin (CALR), and protein disulfide-isomerase A3 (PDIA3), as well as the glycolytic enzyme glucose-6-phosphate isomerase (GPI). Here, we demonstrate that exposition of CD34+ cells to recombinant GRP78, CALR, PDIA3 and GPI induces substantial genetic instability. Increased numbers of γH2AX foci (p < 0.0001), centrosome anomalies (p = 0.1000) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells upon incubation with these factors. Specifically, γH2AX foci were found to be induced 4–5-fold in response to any individual of the four factors, and centrosome anomalies by 3–4 fold compared to control medium, which contained none of the recombinant proteins. Aberrant metaphases, not seen in the context of control medium, were detected to a similar extent than centrosome anomalies across the four factors. Notably, the strongest effects were observed when all four factors were collectively provided. In summary, our data suggest that specific components of the secretome from irradiated MSC act as mediators of genetic instability in CD34+ cells, thereby possibly contributing to the pathogenesis of radiation-induced hematologic disorders beyond direct radiation-evoked DNA strand breaks.

Genotoxic Bystander Signals from Irradiated Human Mesenchymal Stromal Cells Mainly Localize in the 10-100 kDa Fraction of Conditioned Medium.

Genotoxic bystander signals released from irradiated human mesenchymal stromal cells (MSC) may induce radiation-induced bystander effects (RIBEs) in human hematopoietic stem and progenitor cells (HSPC), potentially causing leukemic transformation. Although the source of bystander signals is evident, the identification and characterization of these signals is challenging. Here, RIBEs were analyzed in human CD34+ cells cultured in distinct molecular size fractions of medium, conditioned by 2 Gy irradiated human MSC. Specifically, γH2AX foci (as a marker of DNA double-strand breaks) and chromosomal instability were evaluated in CD34+ cells grown in approximate (I) < 10 kDa, (II) 10–100 kDa and (III) > 100 kDa fractions of MSC conditioned medium and un-/fractionated control medium, respectively. Hitherto, significantly increased numbers of γH2AX foci (p = 0.0286) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells grown in the (II) 10–100 kDa fraction (0.67 ± 0.10 γH2AX foci per CD34+ cell ∨ 3.8 ± 0.3 aberrant metaphases per CD34+ cell sample; mean ± SEM) when compared to (I) < 10 kDa (0.19 ± 0.01 ∨ 0.3 ± 0.2) or (III) > 100 kDa fractions (0.23 ± 0.04 ∨ 0.4 ± 0.4) or un-/fractionated control medium (0.12 ± 0.01 ∨ 0.1 ± 0.1). Furthermore, RIBEs disappeared after heat inactivation of medium at 75 °C. Taken together, our data suggest that RIBEs are mainly mediated by the heat-sensitive (II) 10–100 kDa fraction of MSC conditioned medium. We postulate proteins as RIBE mediators and in-depth proteome analyses to identify key bystander signals, which define targets for the development of next-generation anti-leukemic drugs.

Biomarkers of geno- and cytotoxicity in the native broad-snouted caiman (Caiman latirostris): Chromosomal aberrations and mitotic index

We evaluated the sensitivity of the chromosomal aberration (CA) and mitotic index (MI) assays on peripheral blood lymphocytes (PBLs) of Caiman latirostris, following ex vivo exposure to the alkylating agent, MMS. Two concentrations of MMS were tested in cultured peripheral blood. Relative to controls, MMS exposure reduced the number of metaphases observed, but both the numbers of cells with MN and the percentages of aberrant metaphases increased. The types of CA identified were chromosome and chromatid breaks, chromosomal rearrangements, monosomies, and nullisomies, with significantly higher values in the MMS-exposed groups. The incorporation of the MI and CA tests in C. latirostris can provide information on damage caused by xenobiotic exposures.

The frequency of chromosomal aberrations in individuals chronically in contact with excess dosages of fluorides

BACKGROUND: Chronic excess human exposure to fluorides is an underestimated problem. There is information that fluorides are capable of exerting a genotoxic effect in vitro and in vivo. The data obtained on human populations are limited and contradictory. More research is needed.&#x0D; MATERIALS AND METHODS: The material for the study was peripheral venous blood taken from residents of the city of Novokuznetsk. The material was selected in four groups: workers of the Novokuznetsk Aluminum Plant (NkAZ) with fluorosis, NkAZ workers without pathology, residents of the area adjacent to NkAZ, residents of the city living away from the industrial zone. The frequency of chromosomal abnormalities in blood leukocytes was estimated using the method of accounting for metaphase chromosomal aberrations, as well as the concentration of fluoride ion in blood plasma by direct potentiometry.&#x0D; RESULTS: A significant increase in the frequency of aberrant metaphases in blood leukocytes was observed among workers at an aluminum smelter compared to control, workers with fluorosis compared to workers without pathology, and residents of the area adjacent to the plant compared to residents of the city living far from the industrial zone. A correlation of the frequency of chromosomal aberrations with the concentration of fluoride ion in blood plasma was noted.&#x0D; CONCLUSION: The results obtained confirm the ability of the industrial factor of the aluminum smelter to exert a genotoxic load on the human population. The presence of an association of fluoride factor with chromosomal abnormalities in blood leukocytes is likely.

Chromosomal aberrations in coal mine workers with lung diseases

Introduction. It is known that despite the achievements of modern science and technology and the introduction of modern safe technologies in coal mining enterprises, working in coal mines remains one of the most dangerous for health. It is established that one of the most important and signifi cant causes of occupational diseases in miners is respiratory pathology. Miners working in underground conditions are exposed to a long-term set of harmful physical and chemical factors that contribute not only to increasing the risk of various chronic occupational diseases, but also to genotoxic risk. Accounting for chromosomal aberrations is one of the most highly sensitive methods of biological indication, which allows us to assess the General patterns and specifi cs of the impact of genotoxic exposure. The aim of the study is to conduct a chromosomal analysis of somatic cells of workers of Kuzbass coal mines who are suffering from occupational pulmonary pathologies. Materials and methods. To assess the genotoxic impact of the production environment of coal mines, a cytogenetic analysis of chromosomal aberrations in the blood lymphocytes of 169 miners with lung diseases was performed. Of these, 93 people were diagnosed with “chronic dust bronchitis”, and 76 people — “anthracosilicosis”. For comparison, we used data from cytogenetic analysis of 376 conditionally healthy miners, without signs of lung diseases. As a control, 250 male individuals who did not work in industrial enterprises were studied. The material for the study was whole peripheral blood. Blood cells were cultured using a standard semi-micrometer method. The proportion of aberrant metaphases was determined by calculating the frequency of metaphases with chromosome aberrations (as a percentage of the studied number of cells). Results. It was found that the frequency of chromosome aberrations in the group of miners with lung diseases (5.04±2.40%) and healthy miners (4.12±2.32%) was signifi cantly higher than in the group of individuals who did not work in production (1.78±1.26%, p &lt;0.00001). There was a signifi cant increase in aberrant cells, single fragments, and chromatid-type aberrations in miners with pulmonary pathologies compared to healthy miners. Conclusions. The results of the study indicate a high genotoxic hazard of working conditions in coal mines. Chronic lung diseases in miners caused by prolonged contact with production factors are accompanied by a statistically signifi cant increase in chromosomal instability in peripheral blood lymphocytes. The highest values of the frequency of chromosomal abnormalities were recorded in miners performing basic production operations. Data were obtained on the modifying eff ect of work experience on the frequency of certain types of chromosomal aberrations in miners. It is shown that smoking miners with more than 20 years of experience registered an increase in the level of chromosomal aberrations .

In vivo assessment of genotoxic potential of brown shammah (smokeless tobacco) in bone marrow cells of mice

This study was aimed to assess the genotoxicity of brown shammah (BS), a local form of smokeless tobacco, popular in Middle East countries including Yemen, Saudi Arabia and Sudan. The genotoxicity was explored using in vivo chromosomal aberration (CA), micronucleus (MN) and sperm abnormality (SA) assays. In addition, oxidative stress was also determined using various hepatic markers. Swiss albino mice were selected for the study, which were divided in to 5 groups of six animals each. They include, negative control (NC, received only vehicle) as well as positive control group (PC, received vehicle for 2 weeks followed by administration of cyclophosphamide, CP). Depending upon their dose, three BS treated animal groups were BS-100, 300 and 900 mg/kg. Doses of BS were obtained by suspending BS in 0.5% CMC (carboxy methyl cellulose) and orally administered once a day for 2 weeks. Significant augmentation of the average percentage of aberrant metaphase (AM), CA per cells and suppressed mitotic activity was observed on post administration of BS. In addition, BS increased the occurrence of MNPCEs (micronucleated polychromatic erythrocytes) formation, induced cytotoxicity and increased percentage of abnormal sperms as compared to NC. Moreover, BS also induced oxidative stress as the activities of hepatic superoxide dismutase (SOD) and glutathione (GSH) were reduced and malondialdehyde (MDA) content were increased by BS. Cyclophosphamide was utilized as clastogen, showed anticipated positive results and confirmed the sensitivity of test system. Therefore, it may be deduced from the study that the BS possesses genotoxic effects on mice bone marrow and germ cells in vivo.

Mitotic motor CENP-E cooperates with PRC1 in temporal control of central spindle assembly.

Error-free cell division depends on the accurate assembly of the spindle midzone from dynamic spindle microtubules to ensure chromatid segregation during metaphase–anaphase transition. However, the mechanism underlying the key transition from the mitotic spindle to central spindle before anaphase onset remains elusive. Given the prevalence of chromosome instability phenotype in gastric tumorigenesis, we developed a strategy to model context-dependent cell division using a combination of light sheet microscope and 3D gastric organoids. Light sheet microscopic image analyses of 3D organoids showed that CENP-E inhibited cells undergoing aberrant metaphase–anaphase transition and exhibiting chromosome segregation errors during mitosis. High-resolution real-time imaging analyses of 2D cell culture revealed that CENP-E inhibited cells undergoing central spindle splitting and chromosome instability phenotype. Using biotinylated syntelin as an affinity matrix, we found that CENP-E forms a complex with PRC1 in mitotic cells. Chemical inhibition of CENP-E in metaphase by syntelin prevented accurate central spindle assembly by perturbing temporal assembly of PRC1 to the midzone. Thus, CENP-E-mediated PRC1 assembly to the central spindle constitutes a temporal switch to organize dynamic kinetochore microtubules into stable midzone arrays. These findings reveal a previously uncharacterized role of CENP-E in temporal control of central spindle assembly. Since CENP-E is absent from yeast, we reasoned that metazoans evolved an elaborate central spindle organization machinery to ensure accurate sister chromatid segregation during anaphase and cytokinesis.

Genotoxicity of yellow shammah (smokeless tobacco) in murine bone marrow cells in vivo

Shammah is a traditional form of smokeless tobacco (ST) that is manufactured and used locally by people of Middle East with highest usage in Saudi Arabia, Yemen and Sudan. In Saudi Arabia, shammah comes in three variants: white, brown and yellow. In the present study, we investigated the genotoxicity of yellow shammah (YS) on bone marrow (BM) cells in vivo using mice. Bone marrow (BM) chromosomal aberration (CA) and micronucleus (MN) assay were performed and hepatic markers of oxidative stress were determined. Swiss albino mice were divided into five groups (n = 6) including negative control (NC) and positive control (PC) groups. The three treated groups included YS-100, 200 and 300 mg/kg, doses freshly prepared in 0.5% carboxymethyl cellulose (CMC) and administered orally once a day for 2 weeks. PC animals were administered cyclophosphamide (CP) at a dose of 40 mg/kg body weight, 24 h before termination. Two weeks continuous treatment of YS induced a dose dependent and significant increase in aberrant metaphases (AM), CA per cell and depression in mitotic activity. In micronucleus assay, YS treatment increased the percentage of micronucleated polychromatic erythrocytes (MNPCE) frequency and showed statistically significant reduction in polychromatic erythrocyte/normochromatic erythrocyte ratio at all doses, as compared to NC. YS also markedly inhibited the activities of superoxide dismutase, reduced glutathione and increased malondialdehyde content. CP was used as clastogen (positive control) and yielded the expected positive results. Therefore, it may be concluded that YS has genotoxic and cytotoxic effects for BM cells of mice in vivo.

Safety assessment of Gossence™ (galactooligosaccharides): Genotoxicity and general toxicity studies in Sprague Dawley rats

Galactooligosaccharides (GOS) used as prebiotics are one of the major constituents of the infant milk formulas. GOS (Gossence™) is produced by a patented process of biotransformation of lactose; hence toxicology studies were carried out to assess its safety. The objective of the present study was to evaluate the general and genetic toxicity of Gossence™. In 14-day and subchronic (90-day) oral toxicity studies in Sprague Dawley rats, daily administration of GOS at dose levels of 1000, 2000, or 5000 mg/kg (equivalent to 1347, 2694, and 6735 mg/kg/day of Gossence™, respectively) did not cause any mortality, or clinical signs, and changes in body weights, feed consumption, hematology, clinical chemistry, and urinalysis. In 90-day study, no changes in ophthalmological and neurological findings were observed. Significant increases in the cecum weights (with and/or without content) at dose levels of ≥2000 mg/kg were observed in both 14-day and 90-day studies. Based on the results of 90-day study, the no-observed-adverse-effect-level for GOS is 5000 mg/kg/day which is equivalent to 6735 mg Gossence™/kg/day. In the bacterial reverse mutation test, there was no significant increase in the mean numbers of revertants at the tested concentrations. Gossence™ was not mutagenic up to 5000 µg/plate. In chromosomal aberration test, there was no statistically significant increase in the number of percent aberrant metaphase for the Gossence™. Gossence™ is non-clastogenic (negative) in the in vitro chromosomal aberration test using human peripheral blood lymphocyte during short and prolonged treatment.

Poor prognosis of chromosome 7 clonal aberrations in Philadelphia-negative metaphases and relevance of potential underlying myelodysplastic features in chronic myeloid leukemia.

Clonal chromosome abnormalities in Philadelphia-negative cells could concern chronic myeloid leukemia patients treated by tyrosine kinase inhibitors. The European LeukemiaNet distinguishes -7/del(7q) abnormalities as a “warning”. However, the impact of clonal chromosome abnormalities, and specifically those of -7/del(7q), in Philadelphia-negative cells on clinical outcomes is unclear and based on case-reports showing morphological dysplasia and increased risk of acute myeloid leukemia, suggesting the coexistence of chronic myeloid leukemia and high-risk myelodysplastic syndrome. The aim of this study was to determine whether the impact of -7/del(7q) clonal chromosome abnormalities in Philadelphia-negative cells on the clinical outcome is different from that of other types of abnormalities, and we argue for an underlying associated high-risk myelodysplastic syndrome. Among 102 chronic myeloid leukemia patients with clonal chromosome abnormalities in Philadelphia-negative cells with more than a median of 6 years of follow up, patients with -7/del(7q) more frequently had signs of dysplasia, a lower cumulative incidence of deep molecular response and often needed further treatment lines, with the consequent impact on event-free and progression-free survival. Morphological features of dysplasia are associated with myelodysplastic syndrome/acute myeloid leukemia mutations and compromise the optimal response to tyrosine kinase inhibitors, irrespectively of the type of clonal chromosome abnormalities in Philadelphia-negative cells. However, mutation patterns determined by next-generation sequencing could not clearly explain the underlying high-risk disease. We hereby confirm the pejorative prognostic value of -7/del(7q) clonal chromosome abnormalities in Philadelphia-negative cells and suggest that myelodysplastic features constitute a warning signal that response to tyrosine kinase inhibitors may be less than optimal.

Comparative studies on the effect of environmental pollution on secondary metabolite contents and genotoxicity of two plants in Asir area, Saudi Arabia

Purpose: To investigate the phytochemical contents and genotoxic effects of Ficus carica and Schinus molle grown in healthy and polluted environments in Asir area, Saudi Arabia. Methods: Extracts of the aerial parts of the plants were screened for phytochemical constituents. Genotoxicity tests were carried out in mice using Comet assay, micronucleus test and chromosomal analysis. Results: Extracts of the two plants grown in polluted soil showed elevations in phytochemical and heavy metal contents, when compared with extracts from non-polluted sites. In genotoxicity tests, F. carica and S. molle extracts produced significant increases in the number of micro-nucleated cells in mice, compared to control. Cytotoxicity tests showed that extracts from plants grown in non-polluted environments did not decrease polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) ratio in bone marrow cells, relative to control. Mice in the vehicle control group showed some aberrant metaphases and a few aberrations per hundred metaphases. Conclusion: Pollution has significant effects on phenolic compounds, total flavonoids, and genotoxic potential of the two plants. Keywords: Ficus carica , Schinus molle , Pollution, Genotoxicity, Total phenolic compounds, Total flavonoids

Evaluation of chromosomal aberrations induced by 188Re-dendrimer nanosystem on B16f1 melanoma cells

Purpose: To study the rhenium-188 labeling of polyamidoamine (PAMAM) generation 4 (G4) dendrimer and its evaluation on biodistribution and chromosomal aberrations in melanoma cells induced by ionizing radiation as potential treatment agent.Materials and methods: Dendrimers were first conjugated with Suc-HYNIC (succinimidyl 6-hydrazinopyridine-3-carboxylic acid hydrochloride). Dendrimer-HYNIC was then incubated with 188ReO4−. Biodistribution was performed administrating 188Re-dendrimer to normal (NM) or melanoma-bearing mice (MBM). Chromosome aberration test was conducted in order to measure treatment capacity of 188Re-dendrimer in melanoma cells.Results: Radiolabeling yield of dendrimer was approx. 70%. Biodistribution studies in NM showed blood clearance with hepatic and renal depuration. MBM showed a similar pattern of biodistribution with tumor uptake of 6% of injected dose. Aberrant metaphases quantified in control cells were 7%, increasing to 29.5% in cells treated with 15μCi (0.555 MBq) of 188Re-dendrimer for 24 h.Conclusions: 188Re-dendrimer can produce double-stranded breaks in DNA induced by ionizing radiation in melanoma cells in vitro.

Ketu mutant mice uncover an essential meiotic function for the ancient RNA helicase YTHDC2

Mechanisms regulating mammalian meiotic progression are poorly understood. Here we identify mouse YTHDC2 as a critical component. A screen yielded a sterile mutant, 'ketu', caused by a Ythdc2 missense mutation. Mutant germ cells enter meiosis but proceed prematurely to aberrant metaphase and apoptosis, and display defects in transitioning from spermatogonial to meiotic gene expression programs. ketu phenocopies mutants lacking MEIOC, a YTHDC2 partner. Consistent with roles in post-transcriptional regulation, YTHDC2 is cytoplasmic, has 3'→5' RNA helicase activity in vitro, and has similarity within its YTH domain to an N6-methyladenosine recognition pocket. Orthologs are present throughout metazoans, but are diverged in nematodes and, more dramatically, Drosophilidae, where Bgcn is descended from a Ythdc2 gene duplication. We also uncover similarity between MEIOC and Bam, a Bgcn partner unique to schizophoran flies. We propose that regulation of gene expression by YTHDC2-MEIOC is an evolutionarily ancient strategy for controlling the germline transition into meiosis.