Polyploid superficial cells that maintain the urothelial barrier are produced via incomplete cytokinesis and endoreplication

Summary: The urothelium is an epithelia barrier lined by a luminal layer of binucleated, octoploid, superficial cells. Superficial cells are critical for production and transport of uroplakins, a family of proteins that assemble into a waterproof crystalline plaque that helps protect against infection and toxic substances. Adult urothelium is nearly quiescent, but rapidly regenerates in response to injury. Yet the mechanism by which binucleated, polyploid, superficial cells are produced remains unclear. Here, we show that superficial cells are likely to be derived from a population of binucleated intermediate cells, which are produced from mononucleated intermediate cells via incomplete cytokinesis. We show that binucleated intermediate and superficial cells increase DNA content via endoreplication, passing through S phase without entering mitosis. The urothelium can be permanently damaged by repetitive or chronic injury or disease. Identification of the mechanism by which superficial cells are produced may be important for developing strategies for urothelial repair. : Binucleated superficial cells are critical for urothelial barrier function. Wang et al. show that they derive from binucleated intermediate cells that form via incomplete cytokinesis. Both superficial and intermediate cells increase ploidy via endoreplication, a feature likely to be important for repair and response to environmental changes. Keywords: endoreplication, urothelium, polyploidy, epithelial barrier, regeneratio

Low doses of ochratoxin A induce micronucleus formation and delay DNA repair in human lymphocytes

The contamination of food commodities by fungal toxins has attracted great interest because many of these mycotoxins are responsible for different diseases, including cancer and other chronic illnesses. Ochratoxin A (OTA) is a mycotoxin naturally present in food, and long-term exposure to food contaminated with low levels of OTA has been associated with renal cancer. In the present study, the cytotoxicity, cytostaticity, and genotoxicity of OTA (0.075-15 μM) in human lymphocytes were evaluated. A comet assay, a modified comet assay (DNA repair assay), which uses N-hydroxyurea (NHU) to detect nonrepaired lesions produced by OTA, and a cytokinesis-blocked micronucleus assay were used. Treatments with OTA were not cytotoxic, but OTA caused a cytostatic effect in human lymphocytes at a concentration of 15 μM. OTA (0.075-5 μM) produced a slight increase in the percentage of DNA in the comets and a delay in the DNA repair capacity of the lymphocytes. Micronucleus (MN) induction was observed at OTA concentrations of 1.5 and 5 μM. Our results indicate that OTA induces DNA stable damage at low doses that are neither cytotoxic nor cytostatic, and OTA delays the DNA repair kinetics. These findings indicate that OTA affects two pivotal events in the carcinogenesis pathway.The authors are grateful to the Spanish (Project AGL2011-24862) and Catalonian (XaRTA-Reference Network on Food Technology) Governments for their financial support. C.A. González-Arias thanks the Secretaria de Universitats i Recerca del Departament de Economia i Coneixement of the Generalitat de Catalunya for the pre-doctoral grant. The authors also thank Q.F.B. Guillermina Vázquez Estrada, Francisco Alberto Verdín Betancourt, and Carlos Alberto Martínez Delgado for their technical assistance

Increased chromosomal radiosensitivity in asymptomatic carriers of a heterozygous BRCA1 mutation

Background: Breast cancer risk increases drastically in individuals carrying a germline BRCA1 mutation. The exposure to ionizing radiation for diagnostic or therapeutic purposes of BRCA1 mutation carriers is counterintuitive, since BRCA1 is active in the DNA damage response pathway. The aim of this study was to investigate whether healthy BRCA1 mutations carriers demonstrate an increased radiosensitivity compared with healthy individuals. Methods: We defined a novel radiosensitivity indicator (RIND) based on two endpoints measured by the G2 micronucleus assay, reflecting defects in DNA repair and G2 arrest capacity after exposure to doses of 2 or 4 Gy. We investigated if a correlation between the RIND score and nonsense-mediated decay (NMD) could be established. Results: We found significantly increased radiosensitivity in the cohort of healthy BRCA1 mutation carriers compared with healthy controls. In addition, our analysis showed a significantly different distribution over the RIND scores (p = 0.034, Fisher’s exact test) for healthy BRCA1 mutation carriers compared with non-carriers: 72 % of mutation carriers showed a radiosensitive phenotype (RIND score 1–4), whereas 72 % of the healthy volunteers showed no radiosensitivity (RIND score 0). Furthermore, 28 % of BRCA1 mutation carriers had a RIND score of 3 or 4 (not observed in control subjects). The radiosensitive phenotype was similar for relatives within several families, but not for unrelated individuals carrying the same mutation. The median RIND score was higher in patients with a mutation leading to a premature termination codon (PTC) located in the central part of the gene than in patients with a germline mutation in the 5′ end of the gene. Conclusions: We show that BRCA1 mutations are associated with a radiosensitive phenotype related to a compromised DNA repair and G2 arrest capacity after exposure to either 2 or 4 Gy. Our study confirms that haploinsufficiency is the mechanism involved in radiosensitivity in patients with a PTC allele, but it suggests that further research is needed to evaluate alternative mechanisms for mutations not subjected to NMD

HIPK2 and extrachromosomal histone H2B are separately recruited by Aurora-B for cytokinesis

Cytokinesis, the final phase of cell division, is necessary to form two distinct daughter cells with correct distribution of genomic and cytoplasmic materials. Its failure provokes genetically unstable states, such as tetraploidization and polyploidization, which can contribute to tumorigenesis. Aurora-B kinase controls multiple cytokinetic events, from chromosome condensation to abscission when the midbody is severed. We have previously shown that HIPK2, a kinase involved in DNA damage response and development, localizes at the midbody and contributes to abscission by phosphorylating extrachromosomal histone H2B at Ser14. Of relevance, HIPK2-defective cells do not phosphorylate H2B and do not successfully complete cytokinesis leading to accumulation of binucleated cells, chromosomal instability, and increased tumorigenicity. However, how HIPK2 and H2B are recruited to the midbody during cytokinesis is still unknown. Here, we show that regardless of their direct (H2B) and indirect (HIPK2) binding of chromosomal DNA, both H2B and HIPK2 localize at the midbody independently of nucleic acids. Instead, by using mitotic kinase-specific inhibitors in a spatio-temporal regulated manner, we found that Aurora-B kinase activity is required to recruit both HIPK2 and H2B to the midbody. Molecular characterization showed that Aurora-B directly binds and phosphorylates H2B at Ser32 while indirectly recruits HIPK2 through the central spindle components MgcRacGAP and PRC1. Thus, among different cytokinetic functions, Aurora-B separately recruits HIPK2 and H2B to the midbody and these activities contribute to faithful cytokinesis

In Vitro Mutagenic and Genotoxic Assessment of a Mixture of the Cyanotoxins Microcystin-LR and Cylindrospermopsin

The co-occurrence of various cyanobacterial toxins can potentially induce toxic effects different than those observed for single cyanotoxins, as interaction phenomena cannot be discarded. Moreover, mixtures are a more probable exposure scenario. However, toxicological information on the topic is still scarce. Taking into account the important role of mutagenicity and genotoxicity in the risk evaluation framework, the objective of this study was to assess the mutagenic and genotoxic potential of mixtures of two of the most relevant cyanotoxins, Microcystin-LR (MC-LR) and Cylindrospermopsin (CYN), using the battery of in vitro tests recommended by the European Food Safety Authority (EFSA) for food contaminants. Mixtures of 1:10 CYN/MC-LR (CYN concentration in the range 0.04-2.5 µg/mL) were used to perform the bacterial reverse-mutation assay (Ames test) in Salmonella typhimurium, the mammalian cell micronucleus (MN) test and the mouse lymphoma thymidine-kinase assay (MLA) on L5178YTk± cells, while Caco-2 cells were used for the standard and enzyme-modified comet assays. The exposure periods ranged between 4 and 72 h depending on the assay. The genotoxicity of the mixture was observed only in the MN test with S9 metabolic fraction, similar to the results previously reported for CYN individually. These results indicate that cyanobacterial mixtures require a specific (geno)toxicity evaluation as their effects cannot be extrapolated from those of the individual cyanotoxins.España Ministerio de Economía y Competitividad AGL2015-64558-

Chromosomal integrity after UV irradiation requires FANCD2-mediated repair of double strand breaks

Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress.Fil: Federico, Maria Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Vallerga, María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Radl, Daniela Betiana. Autoridad Regulatoria Nuclear; ArgentinaFil: Paviolo, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Bocco, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Di Giorgio, Marina. Autoridad Regulatoria Nuclear; ArgentinaFil: Soria, Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Gottifredi, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Effect of vitamin C and quercetin treatment on the liver histopathologic profile in congenital lead exposed male rat pups

Introduction: Lead is one of the most important environmental pollutants due to its vast use in various industries. Lead accumulation in different organs, especially the brain, liver and kidneys can cause serious health problems. Lead exposure is more dangerous during fetal period and childhood. Materials and Methods: Timed pregnant female rats divided into 6 groups. Group 1served as control group and received tap water, group 2 received 500 mg/liter lead acetate in the drinking water from 5th day of gestation up to 25th day post-partum, group 3 received the same dose of lead acetate along with daily IP injection of 40mg/kg quercetin, Group 4 received the same dose of lead acetate along with 2g/liter vitamin C, groups 5 and 6 received vitamin C and quercetin respectively like groups 2 and 3 but without lead acetate. On the 25th day postpartum, 6 male pups in each group were deeply anesthetized by chloroform; livers were removed and processed for Hematoxyline- Eosin staining. The microscopic slides were photographed and liver tissue morphological characteristics were evaluated. Results: Lead exposure caused extensive histopathologic changes in liver tissue including hepatocyte degradation, cell nucleus bifurcation and inflammation around hepatic veins. Quercetin and vitamin C treatment could prevent these pathologic changes to a considerable extent. Conclusion: Vitamin C in drinking water and quercetin via IP injection could protect the liver tissue against lead hepatotoxic effects. © 2015, Iranian Society of Physiology and Pharmacology. All rights reserved

Direct MN Test on Peripheral Blood to Detect Chromosomal Breakage: Application in Smokers

The purpose was to assess chromosomal damage in blood mononuclear cells of smokers. Smoker’s peripheral blood samples were screened for micronuclei. Samples from smokers who had an illness were excluded. From each sample, 500 swelled mononuclear leucocytes were screened using a light microscope, with 400x magnification. Frequency distribution of subjects having 0, 1, 2, 3, 4, and 5 micronuclei (MN) according to age and condition were tabulated. From the 102 samples, 5 were excluded, and only 97 were analyzed. There was an increase in MN count in 12.8%, 12.9%, 33.3%, and 25% of normal smokers living in unpolluted area, hypertensive smokers living in unpolluted area, normal smokers living in polluted area, and hypertensive smokers living in polluted area, respectively. Therefore, there was a tendency of increasing MN count in smokers in the productive age group, hypertensive people, and people living in polluted area.&nbsp

The micronucleus assay as a biological dosimeter of in vivo ionising radiation exposure

Biological dosimetry, based on the analysis of micronuclei (MN) in the cytokinesis-block micronucleus (CBMN) assay can be used as an alternative method for scoring dicentric chromosomes in the field of radiation protection. Biological dosimetry or Biodosimetry, is mainly performed, in addition to physical dosimetry, with the aim of individual dose assessment. Many studies have shown that the number of radiation-induced MN is strongly correlated with dose and quality of radiation. The CBMN assay has become, in the last years, a thoroughly validated and standardised technique to evaluate in vivo radiation exposure of occupational, medical and accidentally exposed individuals. Compared to the gold standard, the dicentric assay, the CBMN assay has the important advantage of allowing economical, easy and quick analysis. The main disadvantage of the CBMN assay is related to the variable micronucleus ( MN) background frequency, by which only in vivo exposures in excess of 0.2-0.3 Gy X-rays can be detected. In the last years, several improvements have been achieved, with the ultimate goals (i) of further increasing the sensitivity of the CBMN assay for low-dose detection by combining the assay with a fluorescence in situ hybridisation centromere staining technique, (ii) of increasing the specificity of the test for radiation by scoring nucleoplasmic bridges in binucleated cells and (iii) of making the assay optimally suitable for rapid automated analysis of a large number of samples, viz. in case of a large-scale radiation accident. The development of a combined automated MN-centromere scoring procedure remains a challenge for the future, as it will allow systematic biomonitoring of radiation workers exposed to low-dose radiation