Genotoxic Exposure Research Articles

Vitamin D insufficient levels during pregnancy and micronuclei frequency in peripheral blood T lymphocytes mothers and newborns (Rhea cohort, Crete)

Vitamin D deficiency is common among pregnant women and may be associated with several adverse health outcomes including cancer. Micronuclei frequency is a biomarker of early genetic effects and has been used to examine the association between genotoxic exposures and cancer. We examined maternal vitamin D levels during pregnancy in associations with micronuclei frequency in maternal blood and in cord blood. 173 mothers and 171 newborns born between 2007 and 2008 in Heraklion (Crete, Greece) were included in the study. Between 14th and 18th weeks of gestation we collected information on maternal diet using food frequency questionnaires (FFQs). We measured maternal serum concentrations of 25-hydroxyvitamin D [25(OH)D] between the first and second trimester of pregnancy. We estimated dietary vitamin D intake using information from FFQ. After delivery we collected cord blood and maternal peripheral blood. We used the cytokinesis-block micronucleus (CBMN) assay to assess the frequencies of micronucleated cells in binucleated T lymphocytes (MNBN). Maternal insufficient serum levels of 25(OH)D (<50nmol/L) during pregnancy were associated with increased MNBN frequency in cord blood [IRR=1.32 (95%CI: 1.00, 1.72)]. This increase was higher for newborns with birth weight above the third quartile [≥3.500kg; IRR=2.21 (1.26, 3.89)]. Similarly, low levels of dietary vitamin D were associated with increased MNBN frequency in cord blood [middle tertile IRR=1.08 (0.78, 1.47), lower tertile IRR=1.51 (1.06, 2.14)]. Insufficient levels of vitamin D were not associated with MNBN in mothers. Our results suggest that vitamin D deficiency during pregnancy increases genotoxic risks in newborns. The prevalence of vitamin D deficiency globally is high and it is important to further investigate whether vitamin D supplementation or similar interventions during pregnancy could prevent DNA damage at early stages of life.

Frequency of micronuclei and other biomarkers of DNA damage in populations exposed to dusts, asbestos and other fibers. A systematic review.

Airborne particles are small, solid particles projected into the air either by natural forces, or by mechanical or man-made processes, and include fibers and dusts. Their toxicity is usually subsequent to inhalation and can lead to pulmonary dysfunctions and diseases, including cancer. Cytochalasin B blocked micronucleus assay in lymphocytes (L-CBMN) has been shown as a sensitive and reliable technique in assessing genotoxic exposure, An extensive search of the PubMed and Web of Science databases allowed retrieval of 18 articles on occupational or environmental exposure evaluating L-CBMN in subjects exposed to fibers or dusts (asbestos, silica, rockwool, beryllium, tobacco, and wood). For each study, mean L-CBMN levels were compared in exposed subjects vs. unexposed controls providing a point estimate, the Mean Ratio (MR). The high heterogeneity among retrieved studies and their relatively limited number did not allow a quantitative meta-analysis. However, the inter-quartile range of all MRs fell within the interval between 1.25 and 2.23, supporting the hypothesis that exposure to airborne particles increases DNA damage, although mechanisms of genotoxicity should be further investigated. A borderline significant correlation was found with SCE, but not with chromosome aberrations or comet assay. Future research should focus on exposure assessment, in order to perform proper dose-response studies and disentangle the effect of different compounds in mixed exposures. To fully exploit the cytome assay, L-CBMN frequency should be integrated with other endpoints, such as nucleoplasmic bridges and nuclear buds. The use of alternative tissues, such as nasal and buccal mucosa, and the implementation of other cytogenetic assay, may help to understand the effects of this exposure.

Sister chromatid exchange assay as a predictor of tumor chemoresponse

Sister Chromatid Exchanges (SCEs) are known to enhance as a consequence of exposure to various mutagenic agents and appear to indicate DNA damaging effects and/or subsequent repair by homologous recombination (HR). DNA damage plays an interesting role in the majority of mechanisms underlying the effects of antitumor drugs, since the genetic activity of the plethora of these agents is due to their ability to damage the DNA. The DNA-effects of antitumor agents towards normal cells (genotoxicity) are great drawbacks of antitumor therapy and are connected to important adverse health effects in cancer patients undergoing chemotherapy. On the other hand, failure of chemotherapy in many cases is due to the DNA repair ability which cancer, like normal cells, also possess. As both DNA repair and genotoxic exposure are expected to vary among patients, correlating SCEs frequencies with only individual repair capacity may be feasible to predict. Cancer risk has not been observed to be associated with high SCEs levels. Since the administration of effective antitumor drugs with limited adverse effects is of great importance in the success of anticancer therapy, a lot of interest has been directed toward the development of methods and approaches that would enable the correct selection of appropriate drugs prior to the initiation of therapy on an individual basis. To this effect, more than 30 years ago, an investigation of the ability of the in vitro and the in vivo SCEs-assay to predict the in vitro and in vivo sensitivity of tumor cells to newly synthesized drugs or to those already in use began. In this short review a critical appraisal of the SCEs-assay as an important biomarker used for predicting cancer chemo-response as well as a summary of the key findings from several studies published within the last 20 years in this field is performed.

Moderate malnutrition in rats induces somatic gene mutations

The relationship between malnutrition and genetic damage has been widely studied in human and animal models, leading to the observation that interactions between genotoxic exposure and micronutrient status appear to affect genomic stability. A new assay has been developed that uses the phosphatidylinositol glycan class A gene (Pig-a) as a reporter for measuring in vivo gene mutation. The Pig-a assay can be employed to evaluate mutant frequencies (MFs) in peripheral blood reticulocytes (RETs) and erythrocytes (RBCs) using flow cytometry. In the present study, we assessed the effects of malnutrition on mutagenic susceptibility by exposing undernourished (UN) and well-nourished (WN) rats to N-ethyl-N-nitrosourea (ENU) and measuring Pig-a MFs. Two week-old UN and WN male Han-Wistar rats were treated daily with 0, 20, or 40mg/kg ENU for 3 consecutive days. Blood was collected from the tail vein one day before ENU treatment (Day-1) and after ENU administration on Days 7, 14, 21, 28, 35, 42, 49, 56 and 63. Pig-a MFs were measured in RETs and RBCs as the RETCD59− and RBCCD59− frequencies. In the vehicle control groups, the frequencies of mutant RETs and RBCs were significantly higher in UN rats compared with WN rats at all sampling times. The ENU treatments increased RET and RBC MFs starting at Day 7. Although ENU-induced Pig-a MFs were consistently lower in UN rats than in WN rats, these differences were not significant. To understand these responses, further studies should use other mutagens and nucleated surrogate cells and examine the types of mutations induced in UN and WN rats.

The γH2AX DNA damage assay from a drop of blood

DNA double-strand breaks (DSB) and blocked replication forks activate the DNA damage response (DDR), a signaling pathway marked by phosphorylation of histone 2AX (H2AX). The phosphorylated form, γH2AX, accumulates at the site of damage and can be detected as foci by immunocytochemistry. Therefore, γH2AX is a sensitive and robust biomarker of DNA damage, notably DSB. Cells from peripheral blood are often used for studies on genotoxic exposure of humans. They are limited, however, by the amount of blood required and the costly blood purification method. Here, we present a method that enables the detection of DNA damage by the analysis of γH2AX foci in a drop of blood. The blood drop method (BDM) is simple, fast, inexpensive and allows large series of blood sampling and storage over time. It can be combined with genotoxic treatment of cells in the collected blood sample for experimental purposes on DNA damage induction and repair. The BDM is suitable for rapid and large-scale screenings of genetic damage in human and animal populations.

Micronucleus as a Non-invasive Biomarker – A Review

Oral Cancer has a remarkably high incidence worldwide and a significant decrease in its mortality and morbidity rates has been established if it is diagnosed in early stages. There has been always a strong need to develop new, objective, non-invasive methods for its early detection. Micronucleus has come up in the recent past as non-invasive biomarker for diagnosis of not only malignant and pre-malignant lesions but also many other significant diseases. Micronucleus (“MN) is defined as microscopically visible, round or oval cytoplasmic chromatin mass next to the nucleus. Micronuclei Review Article Dev et al.; JAMPS, 6(4): 1-9, 2016; Article no.JAMPS.24136 2 (“MNi”) originate from aberrant mitoses and consist of eccentric chromosomes, chromatid fragments or whole chromosomes that have failed to be incorporated into the daughter nuclei during mitosis. The MN assay has been widely accepted as an in vitro genotoxicity test and a biomarker assay for genotoxic exposure and effect in humans. An attempt has been made to review the related studies, utilizing micronucleus assay of buccal cells as a novel marker of genotoxicity in head and neck region.

Micronucleated Erythrocytes in Peripheral Blood from Neonate Rats Exposed by Breastfeeding to Cyclophosphamide, Colchicine, or Cytosine-Arabinoside.

Genotoxic exposure to chemical substances is common, and nursing mothers could transmit harmful substances or their metabolites to their offspring through breast milk. We explored the possibility of determining genotoxic effects in the erythrocytes of breastfeeding rat pups whose mothers received a genotoxic compound while nursing. Ten groups of female rats and five pups per dam were studied. The control group received sterile water, and the experimental groups received one of three different doses of cyclophosphamide, colchicine, or cytosine-arabinoside. Blood smears were prepared from samples taken from each dam and pup every 24 h for six days. There were increased numbers of micronucleated erythrocytes (MNEs) and micronucleated polychromatic erythrocytes (MNPCEs) in the samples from pups in the experimental groups (P < 0.02) and increased MNPCE frequencies in the samples from the dams (P < 0.05). These results demonstrate the vertical transmission of the genotoxic effect of the compounds tested. In conclusion, assessing MNEs in breastfeeding neonate rats to assess DNA damage may be a useful approach for identifying genotoxic compounds and/or cytotoxic effects. This strategy could help in screening for therapeutic approaches that are genotoxic during the lactation stage and these assessments might also be helpful for developing preventive strategies to counteract harmful effects.

Replication stress in MLL-rearrangements.

Hematopoietic stem cells (HSC) are the only cells capable of self-renewal throughout the individual's lifetime and generate the whole spectrum of blood cells. Therefore genome aberrations in HSC can result in hematopoiesis failure or leukemic transformation. Chromosomal translocations, inversions, amplifications and complex rearrangements at the 11q human genomic locus encoding mixed lineage leukemia gene (MLL) are the hallmark of several blood malignancies including infant, therapy-induced, donor - and de novo leukemias. The vast majority of these 11q aberrations fall within a 7.3kb MLL breakpoint cluster region (MLLbcr) with a particular hotspot at the intron11-exon12 boundary [1]. Intriguingly, a large variety of genotoxic, cytotoxic and biological stimuli were connected with MLLbcr breakage pointing to the existence of several DNA cleavage and repair mechanisms acting at this locus [1, 2]. From the broad spectrum of stimuli triggering cleavage in concert with diverse mutagenic outcomes at the locus it is tempting to seek for a common molecular process engaged. Based on our and others’ experimental evidences, we postulate that replication stress in HSC can be responsible for MLL rearrangements (Figure ​(Figure1).1). Thus, our data revealed MLLbcr breakage upon mere replication blockage via DNA polymerase inhibition or upon exposure to the nucleoside analog 5-fluorouracil [2]. Induction of HSC's specific replication stress can be linked to many agents and conditions implicated in MLL leukemias. Normally, quiescence of HSC with only rare replication cycles accompanied by low metabolic activity and ROS levels contributes to minimize the mutational load under homeostatic conditions [3, 4]. In contrast, forcing HSC into excessive cycling by chronic stimulation with physiological triggers mimicking inflammation, bleeding or cytopenia provokes a robust DDR that drives both HSC death and mutagenesis of the survivors. Thus, Walter et al. [4] detected DDR markers associated with replication fork stalling and collapse such as DNA breaks and nuclear γ H2AX, 53BP1 and FANCD2 foci upon enforced HSC exit from quiescence. Transplantation induces rapid cycling of normally dormant HSC that can be exacerbated by donor immunosuppression, damaged microenvironment and altered cytokine profile. Signs of endogenous DNA damage upon serial transplantation of HSC are well documented in both humans and mice with evidence for altered DNA replication dynamics, chromosome gaps and breaks indicative of replication stress [3, 5]. We suggest that exhaustion or failure of replication stress-associated high fidelity repair pathways under transplantation challenge can be implicated in donor cell-derived acute leukemia with MLL translocations in patients who received HSC transplant [6]. Given the fact that replication stress in HSC is associated with aging [3] one can hypothesize that MLL rearrangements, particularly amplifications often associated with complex rearrangements [7], observed in de novo AML in the elderly are the consequence of replication stress associated DNA repair failures. Figure 1 MLL rearrangements can result from failure to correctly resolve replication stress in HSC Induction of replication stress in HSC can also be linked to agents and conditions associated with common solid cancer therapies. Indeed, topoisomerase II inhibitors and cytostatics with a different mode-of-action such as alkylating agents or 5-fluorouracil [1, 8] but all implicated in the etiology of therapy-induced leukemia or MLL rearrangements can recruit dormant HSC into the replication cycle as a result of chemotherapy-associated cytopenia. Moreover, fetal HSC which are the infant leukemia cell-of-origin are highly cycling populations and thus collision of replication forks with lesions can cause overwhelming replicative stress. Altogether, replication stress may represent the integrating signal in HSC following genotoxic exposure of the fetus and of patients undergoing radio- or chemotherapy, in HSC undergoing excessive self-renewal in the fetus or upon transplantation as well as in HSC from aged individuals suffering from the exhaustion of replication factors [3]. The extraordinary susceptibility of the MLLbcr to replication stress-induced breakage may stem from its secondary structure resulting in the collision of transcription and replication machineries recruiting nucleases such as Endonuclease G in decondensed chromatin [1](Figure ​](Figure11). To summarize, replication stress response plays a key role in regulating HSC function. We anticipate that deeper understanding of associated molecular mechanisms responsible for MLLbcr cleavage and subsequent repair in HSC can hold the key for future chemoprevention and anti-aging modalities.

DNA Double Strand Break Response and Limited Repair Capacity in Mouse Elongated Spermatids.

Spermatids are extremely sensitive to genotoxic exposures since during spermiogenesis only error-prone non homologous end joining (NHEJ) repair pathways are available. Hence, genomic damage may accumulate in sperm and be transmitted to the zygote. Indirect, delayed DNA fragmentation and lesions associated with apoptotic-like processes have been observed during spermatid elongation, 27 days after irradiation. The proliferating spermatogonia and early meiotic prophase cells have been suggested to retain a memory of a radiation insult leading later to this delayed fragmentation. Here, we used meiotic spread preparations to localize phosphorylate histone H2 variant (γ-H2AX) foci marking DNA double strand breaks (DSBs) in elongated spermatids. This technique enabled us to determine the background level of DSB foci in elongated spermatids of RAD54/RAD54B double knockout (dko) mice, severe combined immunodeficiency SCID mice, and poly adenosine diphosphate (ADP)-ribose polymerase 1 (PARP1) inhibitor (DPQ)-treated mice to compare them with the appropriate wild type controls. The repair kinetics data and the protein expression patterns observed indicate that the conventional NHEJ repair pathway is not available for elongated spermatids to repair the programmed and the IR-induced DSBs, reflecting the limited repair capacity of these cells. However, although elongated spermatids express the proteins of the alternative NHEJ, PARP1-inhibition had no effect on the repair kinetics after IR, suggesting that DNA damage may be passed onto sperm. Finally, our genetic mutant analysis suggests that an incomplete or defective meiotic recombinational repair of Spo11-induced DSBs may lead to a carry-over of the DSB damage or induce a delayed nuclear fragmentation during the sensitive programmed chromatin remodeling occurring in elongated spermatids.

Elevated APEX1 Disrupts G2/M Checkpoint, Contributing to Evolution and Survival of Myeloma Cells

Cell cycle checkpoints provide the cell with time to repair chromosomal DNA damage before its replication (G1) and also prior to its segregation (G2), thus ensuring integrity, maintenance and protection of genome. Although proper functioning of both checkpoints is essential, G2/M has a special significance as a potentially lethal double-strand break in DNA escape repair and persist from G2 into mitosis, it may recombine in G1 to produce gene rearrangements. Moreover, G2 is the phase where homologous recombination (HR) can utilize a sister chromatid as a template to provide error-free repair. There is ample evidence that supports the role of defective G2/M checkpoint and dysregulated HR in genomic rearrangements and evolution in cancer. Previously, we have shown that elevated APEX1 contributes to dysregulated HR and genome stability in multiple myeloma (MM), and its upregulation leads to genomic instability and tumorigenesis in animal model. To further understand the role of APEX1 in myeloma, we investigated the impact of elevated APEX1 on cell cycle checkpoint/s and in the cellular response to genotoxic exposure. Our investigation using antibody array and subsequent confirmation with immunoprecipitation experiments demonstrated that APEX1 interacts with cyclin B and PLK in myeloma cells. A key step in progression from G2 to mitosis is the activation of cyclin B-CDK1 complex, which subsequently activates PLK to ensure G2/M progression. Based on observed interaction of APEX1 with cyclin B/PLK, we hypothesized that elevated APEX1 disrupts G2 checkpoint by mediating progression into mitosis. To test this, we inhibited APEX1 in myeloma cells by a small molecule as well as by shRNA targeting this gene, and investigated the impact on cell cycle checkpoints using a unique phospho-antibody array which allows investigation of 238 relevant proteins and their phosphorylation status. APEX1 inhibition by small molecule led to downregulation (> 2-fold) of many proteins/phosphorylations involved in the activation of cyclin B-CDK1 complex and other mediators of G2/M progression (including CDC25A, CDC25A, CDK1, ABL1), and upregulation of proteins/phosphorylations involved in G2/M arrest, including CDK1-phospho-Tyr15, 14-3-3 zeta-phospho-Ser58, p53-phospho-Ser15, and MYT and WEE which are involved in negative regulation of cyclin B-CDK1 complex. To further investigate the role of APEX1 in G2/M progression, myeloma cell lines (ARP, RPMI 8226, MM1S, LR5, H929) were treated with APEX1 inhibitor and subjected to cell cycle analysis using flow cytometery. Compared to control cells, all five APEX1-inhibitor treated cell lines showed a strong G2/M block, ranging from 5- to 10-fold increase in the fraction of cells in G2 phase in a dose dependent manner. The G2/M cell cycle arrest of APEX1-treated myeloma cells was further supported by reduced cell viability of treated myeloma cells (RPMI, H929, MM1S, ARP and U266); IC50 of inhibitor in myeloma cell lines ranged from 1.2 to 4 µM. Co-treatment with APEX1 inhbitor also sensitized myeloma cells to Melphalan. Consistent with these data, shRNA-mediated knockdown (KD) of APEX1 in RPMI cells was associated with 4-fold increase in the fraction of cells in G2, relative to control cells. APEX1-KD was also associated with reduction in cell viability (by 40%) and sensitization to melphalan. Our results therefore suggest that elevated APEX1 disrupts G2 checkpoint and sets a stage for genomic rearrangements by allowing persistance of DNA damage from G2 into mitosis. Dysfunctional G2 checkpoint, combined with APEX1-mediated dysregulation of HR, could be attributed to APEX1 associated genomic instability and oncogenic transformation. Therefore, inhibitors of APEX1, alone or in combination with other agents, have potential to make myeloma cells static. DisclosuresNo relevant conflicts of interest to declare.

Human Genotoxic Study Carried Out Two Years after Oil Exposure during the Clean-up Activities Using Two Different Biomarkers

Micronuclei, comet and chromosome alterations assays are the most widely used biomarkers for determining the genotoxic damage in a population exposed to genotoxic chemicals. While chromosome alterations are an excellent biomarker to detect short- and long-term genotoxic effects, the comet assay only measures early biological effects, and furthermore it is unknown whether nuclear abnormalies, such as those measured in the micronucleus test, remain detectable long-term after an acute exposure. In our previous study, an increase in structural chromosome alterations in fishermen involved in the clean-up of the Prestige oil spill, two years after acute exposure, was detected. The aim of this study is to investigate whether, in lymphocytes from peripheral blood, the nuclear abnormalies (micronucleus, nucleoplasmic bridges and nuclear buds) have a similar sensitivity to the chromosome damage analysis for genotoxic detection two years after oil exposure in the same non-smoker individuals and in the same peripheral blood extraction. No significant differences in nuclear abnormalies frequencies between exposed and non-exposed individuals were found (p &gt; 0.05). However, chromosome damage, in the same individuals, was higher in exposed vs. non-exposed individuals, especially for chromosome lesions (p &lt; 0.05). These findings, despite the small sample size, suggest that nuclear abnormalities are probably less-successful biomarkers than are chromosome alterations to evaluate genotoxic effects two or more years after an exposure to oil. Due to the great advantage of micronucleus automatic determination, which allows for a rapid study of hundreds of individuals exposed to genotoxic chemical exposure, further studies are needed to confirm whether this assay is or is not useful in long-term genotoxic studies after the toxic agent is no longer present.

Uso de marcadores genotoxicológicos para la evaluación de agricultores expuestos a plaguicidas organofosforados

Introducción: Los plaguicidas organofosforado son compuestos utilizados para el control de plagas en actividades agrícolas; genera exposición ocupacional asociada a posibles intoxicaciones agudas y crónicas que se manifiestan por alteraciones a nivel bioquímico, molecular y genético, los cuales pueden ser evaluados mediante el uso de marcadores genotoxicológicos. Objetivo: Determinar el uso de marcadores genotoxicológicos para la evaluación de agricultores expuestos a plaguicidas organofosforados. Diseño: Observacional, analítico, transversal con grupo control. Lugar: Empresa de agroexportación de espárragos en el Distrito de Virú, La Libertad. Intervenciones: Se determinó la actividad enzimática de la colinesterasa sérica (BChE) y eritrocitaria (AChE), y se hizo ensayos de genotoxicidad (aberraciones cromosómicas, ensayo cometa y micronúcleos) en 59 trabajadores expuestos a plaguicidas (grupo expuesto) y 50 personas sin exposición a plaguicidas (grupo control). Principales medidas de resultados: Actividad enzimática de la colinesterasa sérica (BChE) y eritrocitaria (AChE), y ensayos de genotoxicidad (aberraciones cromosómicas, ensayo cometa y micronúcleos). Resultados: Las edades promedio del grupo expuesto y control fueron de 39,6±10,8 y 34,0±11,5 años, respectivamente. En referencia a la actividad BChE, se encontró diferencia significativa (p&amp;lt;0,001) entre el grupo expuesto (4 733.0±1 350.1 U/L) y control (7 075.0±1 674.0 U/L). Caso contrario se presentó en la actividad AChE entre el grupo expuesto (4 867,0±632,2 U/L) y control (5 051,0±505,5 U/L). Además, se evidenció la inhibición de ambas enzimas en 9 trabajadores expuestos por debajo del valor de referencia. No se evidenció diferencias significativas en los marcadores genotoxicológicos, a excepción del recuento de micronúcleos, puentes nucleoplásmicos, gemaciones, binucleaciones celulares y cariorexis de células de epitelio bucal; y en la evaluación del daño al ADN mediante el ensayo cometa (p&amp;lt;0,05) se obtuvo promedios de intensidad de cola de 8,28±1,85 (Rango: 6,06-15,75) y 7,30±1,25 (Rango: 4,04-9,41) para el grupo expuesto y control, respectivamente. Conclusiones: El uso de marcadores genotoxicológicos aporta información relevante como herramienta que permite predecir el riesgo asociado a cáncer, considerando que el evento inicial es el daño al ácido desoxiribonucléico (ADN) en cualquier etapa del ciclo celular; además, existió asociación significativa entre el efecto genotóxico y la exposición a plaguicidas organofosforados evidenciada por la inhibición de la BChE.

Genotoxic damage and occupational exposure to formaldehyde in anatomic pathology laboratory workers

Genotoxic damage and occupational exposure to formaldehyde in anatomic pathology laboratory workers

Anti-genotoxic ability of α-tocopherol and Anthocyanin to counteract fish DNA damage induced by musk xylene.

Many compounds released into the environment are able to interact with genetic material. The main purpose of genetic toxicology is to investigate the adverse effects of genotoxic molecules such as reduced fitness, changes in gene frequencies and their impact on genetic diversity in populations following genotoxic exposure. However, the ecological effects of many genotoxic compounds remain poorly understood. The aim of this research was to evaluate the genotoxic activity of an artificial musk (musk xylene, MX) and the potential anti-genotoxicity against this chemical compound of two antioxidant substances (α-tocopherol and an anthocyanins enriched extract). The studies were performed both in vivo and in vitro, using the teleost Danio rerio and the DLEC (Dicentrarchus labrax embryonic cells) cell line. We carried out the exposure to these substances at different times. DNA and cell damage and their possible repair were detected by various experimental approaches: DNA strand breaks (Comet Assay), degree of apoptosis (Diffusion Assay) and molecular alterations at the genomic level (RAPD-PCR technique). Data were collected and analyzed for statistical significance using the Student's t test. The results of this study showed that MX exhibited a genotoxic activity even after short exposure times. The anti-genotoxicity experiments evidenced that both α-tocopherol and Anthocyanin were able to contrast the genotoxic effects induced by MX, both in vivo and in vitro.

Sensitivity to chromosomal breakage as risk factor in young adults with oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) may develop in young adults. In contrast to older patients, the well-known etiological factors, exposure to tobacco and alcohol, play a minor role in the carcinogenesis in this patient group. It has been suggested that an intrinsic susceptibility to environmental genotoxic exposures plays a role in the development of OSCC in these patients. The hypothesis was tested whether young OSCC patients have an increased sensitivity to induced chromosomal damage. Fourteen OSCC patients with an average age of 32 years (range 20-42) were selected. Peripheral blood lymphocytes and skin fibroblasts of patients and 14 healthy controls were subjected to the chromosome breakage test with Mitomycin C. This test is routinely used to identify Fanconi anemia patients, who are well-known for their inherited high sensitivity to this type of DNA damage, but also for the high risk to develop OSCC. Human papilloma virus status of the carcinomas was also determined. None of the 14 young patients with OSCC had an increased response in the MMC-chromosomal breakage test. All tumors tested negative for human papilloma virus. No evidence was obtained for the existence of a constitutional hypersensitivity to DNA chromosomal damage as a potential risk factor for OSCC in young adults.

Abstract 3024: Lack of CK1δ increases DNA damage and genomic instability due to defects in DNA repair and mitotic checkpoints

Abstract Casein kinase 1 delta (CK1δ) is a conserved serine/threonine protein kinase that regulates diverse cellular processes including vesicle trafficking and circadian rhythm. We previously reported that CK1δ is a mediator of Wnt-3a-dependent neurite outgrowth (Greer and Rubin, JCB, 2011) and ciliogenesis (Greer et al. MBoC, 2014). Mice that lack Csnk1d exhibit a perinatal lethal phenotype and typically weigh 30-50% less than their wild type littermates, however, the exact causes of death and small size are unknown. We hypothesized that the absence of CK1δ contributes to cellular stresses that adversely affect cell survival. Mouse embryonic fibroblasts (MEFs) were collected from mice homozygous for a Csnk1d floxed allele, and MEFCsnk1d null cells were generated by infection with adenovirus (Ad) expressing Cre. As control, MEFCtl. cells were produced by infection with Ad-GFP. In addition, MEFCtl cells were treated with CK1δ siRNA reagents to transiently suppress endogenous expression. Cells were analyzed by confocal microscopy, western blotting and flow cytometry. At early passage (P2), many MEFCsnk1d null cells had detached from the culture dish, raising the possibility of cell death. Subsequent MTS assay confirmed a significantly lower cell viability of MEFCsnk1d null cells compared to MEFCtl cells. MEFCsnk1d null cells (P3) exhibited a larger sub-Go fraction (64%) than MEFCtl cells (34%) in flow cytometric analysis. γ-H2AX staining and comet assay confirmed significant DNA damage in MEFCsnk1d null cells. MEFCsnk1d null cells exhibited micronuclei, a characteristic of genomic instability. Similarly, abrogation of CK1δ expression in MEFCtl cells induced micronuclei formation in response to DNA damage triggered by doxorubicin, suggesting that the absence of CK1δ increases susceptibility to genotoxic exposure. Chk1, a kinase with a major role in DNA damage response, and its downstream substrate, Cdc2/CDK1 kinase, were inhibited in MEFCsnk1d null cells as well as in MEFCtl cells transfected with CK1δ siRNA, suggesting that CK1δ is required for the G2/M mitotic checkpoint. Hydroxyurea-induced Chk1 activation was also impaired in cells lacking CK1δ, and re-expression of CK1δ rescued the phenotype. Together, the data indicate that CK1δ is required for efficient repair of DNA damage and the proper functioning of mitotic checkpoints. The mechanism(s) responsible for these activities are currently under investigation. Citation Format: Yoshimi E. Greer, Bo Gao, Yingzi Yang, Stanley Lipkowitz, Jeff S. Rubin. Lack of CK1δ increases DNA damage and genomic instability due to defects in DNA repair and mitotic checkpoints. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3024. doi:10.1158/1538-7445.AM2015-3024

Chemically induced DNA damage and sperm and oocyte repair machinery: the story gets more interesting.

Assisted reproductive technologies have allowed us to bypass elusive questions of how spermatogenesis truly works so that end results, namely conception, can be achieved quickly and efficiently. Yet, reproductive biology, andrology, and molecular mutagenesis have not completely abandoned unanswered questions about how spermatogenesis unfolds, where things can go wrong in the process, and what are the impacts on the conceptus when sperm are somehow defective. Additional questions stem from the recognition that model systems are only approximations of how human reproduction actually happens in a world replete with extraneous exposures, from micronutrients to everyday chemicals. New evidence is adding to our understanding of how exceptionally stage-dependent spermatogenesis is, and how genotoxic exposures at exact stages differentially affect sperm cells. Recently reported new mouse model evidence demonstrates that postfertilization, oocytes misrepair sperm DNA defects that allow chromosomal structural aberrations to persist in the conceptus. These findings give new perspectives on both intrinsic actions of DNA repair checkpoints and on critical windows of sperm vulnerability to mutagenicity and toxicity.

Evaluating microRNA profiles reveals discriminative responses following genotoxic or non-genotoxic carcinogen exposure in primary mouse hepatocytes.

Chemical carcinogenesis can be induced by genotoxic (GTX) or non-genotoxic (NGTX) carcinogens. GTX carcinogens have a well-described mode of action. However, the complex mechanisms by which NGTX carcinogens act are less clear and may result in conflicting results between species [e.g. Wy-14,643 (Wy)]. We hypothesise that common microRNA response pathways exist for each class of carcinogenic agents. Therefore, this study compares and integrates mRNA and microRNA expression profiles following short term acute exposure (24 and 48h) to three GTX [aflatoxin B1 (AFB1), benzo[a]pyrene (BaP) and cisplatin (CisPl)] or three NGTX (2,3,7,8-tetrachloordibenzodioxine (TCDD), cyclosporine A (CsA) and Wy) carcinogens in primary mouse hepatocytes. Discriminative gene sets, microRNAs (not for 24h) and processes were identified following 24 and 48h of exposure. From the three discriminative microRNAs found following 48h of exposure, mmu-miR-503-5p revealed to have an interaction with mRNA target gene cyclin D2 (Ccnd2 - 12444) which was involved in the discriminative process of p53 signalling and metabolism. Following exposure to NGTX carcinogens Mmu-miR-503-5p may have an oncogenic function by stimulating Ccnd2 possibly leading to a tumourigenic cell cycle progression. By contrast, after GTX carcinogen exposure it may have a tumour-suppressive function (repressing Ccnd2) leading to cell cycle arrest and to increased DNA repair activities. In addition, compound-specific microRNA-mRNA interactions [mmu-miR-301b-3p-Papss2 (for AFB1), as well as mmu-miR-29b-3p-Col4a2 and mmu-miR-24-3p-Flna (for BaP)] were found to contribute to a better understanding of microRNAs in cell cycle arrest and the impairment of the DNA damage repair, an important hallmark of GTX-induced carcinogenesis. Overall, our results indicate that microRNAs represent yet another relevant intracellular regulatory level in chemical carcinogenesis.

Radiation preconditioning in vivo increases the resistance of mouse retina to genotoxic exposure and stimulates its regeneration

Exposure to a low level of stress, so-called preconditioning, has been shown to have a protective effect on the retina. Our previous studies demonstrated that there are nonlinear relationships between the doses of ionizing radiation and N-methyl-N-nitrosourea (MNU) and their cytotoxic effects on mouse retina; moreover, treatment with subcytotoxic doses of MNU increased the tolerance of the retina to the challenge-doses of the agent (Tronov et al., 2012). These results indicate the existence of a regeneration mechanism in the retina. In this work, we used an in vivo mouse model system for MNU-induced retinal degeneration to investigate the adaptive response of the retina to proton irradiation and a role of Muller glial cells in this process. We found that retina recovery following exposure to genotoxic stress that induced its degeneration was associated with an increased efficiency of DNA repair and a decrease in retinal photoreceptor death.

Spatial positioning of all 24 chromosomes in the lymphocytes of six subjects: evidence of reproducible positioning and spatial repositioning following DNA damage with hydrogen peroxide and ultraviolet B.

The higher-order organization of chromatin is well-established, with chromosomes occupying distinct positions within the interphase nucleus. Chromatin is susceptible to, and constantly assaulted by both endogenous and exogenous threats. However, the effects of DNA damage on the spatial topology of chromosomes are hitherto, poorly understood. This study investigates the organization of all 24 human chromosomes in lymphocytes from six individuals prior to- and following in-vitro exposure to genotoxic agents: hydrogen peroxide and ultraviolet B. This study is the first to report reproducible distinct hierarchical radial organization of chromosomes with little inter-individual differences between subjects. Perturbed nuclear organization was observed following genotoxic exposure for both agents; however a greater effect was observed for hydrogen peroxide including: 1) More peripheral radial organization; 2) Alterations in the global distribution of chromosomes; and 3) More events of chromosome repositioning (18 events involving 10 chromosomes vs. 11 events involving 9 chromosomes for hydrogen peroxide and ultraviolet B respectively). Evidence is provided of chromosome repositioning and altered nuclear organization following in-vitro exposure to genotoxic agents, with notable differences observed between the two investigated agents. Repositioning of chromosomes following genotoxicity involved recurrent chromosomes and is most likely part of the genomes inherent response to DNA damage. The variances in nuclear organization observed between the two agents likely reflects differences in mobility and/or decondensation of chromatin as a result of differences in the type of DNA damage induced, chromatin regions targeted, and DNA repair mechanisms.