Cytogenetic and oxidative effects of three lichen extracts on human peripheral lymphocytes.

In the present study, we investigated cytogenetic and oxidative [total antioxidant capacity (TAC), total oxidant status (TOS)] effects of methanol and water extracts of Cladonia chlorophaea (Flörke ex Sommerf.) Sprengel, Dermatocarpon miniatum (L.) W.Mann and Parmelia saxatilis (L.) Ach. on cultured human lymphocytes. In addition, different phenolic compounds in the extracts were quantified by high performance liquid chromatography (HPLC) analysis. As a result of HPLC analysis, methanol extracts of all lichen species tested had higher phenolic compounds. Likewise, methanol extracts of each lichen increased TAC levels in lymphocytes more than water extracts. The TOS levels of the cells treated with different concentrations (1-100 mg/L) of the extracts decreased due to the increasing concentration of the extracts. Genotoxicity experiments revealed that the tested lichen extracts did not significantly increase (p > 0.05) the level of genotoxicity on human peripheral lymphocyte culture compared to the negative control group. The results showed that C. chlorophaea, D. miniatum and P. saxatilis lichens, which were found to be a rich source of phenolic compounds, might be of interest in the pharmaceutical and food industries.

In vivo bioavailability and in vitro toxicological evaluation of the new butyric acid releaser N-(1-carbamoyl-2-phenyl-ethyl) butyramide.

A large body of evidence suggests that supplementation of butyric acid exerts beneficial intestinal and extra-intestinal effects. Unfortunately, unpleasant sensorial properties and unfavourable physico-chemical properties strongly limit its use in food supplements and foods for medicinal purposes. N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA) is a new butyric acid releaser in solid form with neutral sensorial properties. The aim of this investigation is to provide preliminary information on its pharmacokinetic and toxicological properties through the study of a) in vivo bioavailability of FBA administered by oral gavage to male and female Swiss CD1 mice in comparison with sodium butyrate, b) the influence of digestion on FBA stability through an in vitro simulated oro-gastro-duodenal digestion process, and c) in vitro toxicological profile by means of the Ames Test and Micronucleus Test. The results reveal that FBA is a good butyric acid releaser, being able to increase butyrate serum concentration in a dose and time dependent manner in both male and female mice with a pharmacokinetic profile similar to that obtained from sodium butyrate as such. These data are confirmed by investigating the influence of digestion on FBA, which undergoes extensive hydrolysis following oro-gastro-duodenal digestion, especially in duodenal conditions, with a residual concentration of less than 10% of the initial FBA concentration. Finally, in the Ames and Micronucleus Tests, FBA does not show any in vitro genotoxicity as it is non mutagenic in the Ames Test and results to be unable to induce chromosome breaks in the Micronucleus Test. In conclusion, FBA is a new butyric acid releaser that can overcome the disadvantages of butyric acid while maintaining the same pharmacokinetic properties and safety profile, as shown by the results of the preliminary in vitro toxicological studies performed in this investigation.

(E)4-[4-N,N-dimethylaminophenyl]but-3-en-2-one mitigates radiation-induced chromosome damage in BALB/c mouse bone marrow.

We examined the effects of administration of (E) 4-[4-N,N-dimethylaminophenyl]but-3-en-2-one (DMAP) on radiation-induced chromosome damage in mice. Mice were whole-body exposed to γ-rays, 0-4 Gy, and then immediately administered DMAP, 20 mg/kg. After 24 h, mice were sacrificed, femora were removed, marrow was extracted, and chromosome aberrations were scored in the bone marrow cells. With vehicle-only (saline or oil) treatment, radiation dose-dependent damage was seen in aberrant cells, chromosome breaks, chromatid breaks, centric rings, di-, tri-, and tetracentrics, acentric fragments, total aberrations, polyploidy, and pulverization. Post-administration of DMAP was protective as it reduced chromosome damage. DMAP treatment may be a useful protective agent following radiation accidents or radiotherapy.

Replication Stress Induces Global Chromosome Breakage in the Fragile X Genome.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene and deficiency of a functional FMRP protein. FMRP is known as a translation repressor whose nuclear function is not understood. We investigated the global impact on genome stability due to FMRP loss. Using Break-seq, we map spontaneous and replication stress-induced DNA double-strand breaks (DSBs) in an FXS patient-derived cell line. We report that the genomes of FXS cells are inherently unstable and accumulate twice as many DSBs as those from an unaffected control. We demonstrate that replication stress-induced DSBs in FXS cells colocalize with R-loop forming sequences. Exogenously expressed FMRP in FXS fibroblasts ameliorates DSB formation. FMRP, not the I304N mutant, abates R-loop-induced DSBs during programmed replication-transcription conflict. These results suggest that FMRP is a genome maintenance protein that prevents R-loop accumulation. Our study provides insights into the etiological basis for FXS.

Participation of TDP1 in the repair of formaldehyde-induced DNA-protein cross-links in chicken DT40 cells.

Proteins are covalently trapped on DNA to form DNA-protein cross-links (DPCs) when cells are exposed to DNA-damaging agents. Aldehyde compounds produce common types of DPCs that contain proteins in an undisrupted DNA strand. Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs topoisomerase 1 (TOPO1) that is trapped at the 3'-end of DNA. In the present study, we examined the contribution of TDP1 to the repair of formaldehyde-induced DPCs using a reverse genetic strategy with chicken DT40 cells. The results obtained showed that cells deficient in TDP1 were sensitive to formaldehyde. The removal of formaldehyde-induced DPCs was slower in tdp1-deficient cells than in wild type cells. We also found that formaldehyde did not produce trapped TOPO1, indicating that trapped TOPO1 was not a primary cytotoxic DNA lesion that was generated by formaldehyde and repaired by TDP1. The formaldehyde treatment resulted in the accumulation of chromosomal breakages that were more prominent in tdp1-deficient cells than in wild type cells. Therefore, TDP1 plays a critical role in the repair of formaldehyde-induced DPCs that are distinct from trapped TOPO1.

Application of the adverse outcome pathway framework to genotoxic modes of action.

In May 2017, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee hosted a workshop to discuss whether mode of action (MOA) investigation is enhanced through the application of the adverse outcome pathway (AOP) framework. As AOPs are a relatively new approach in genetic toxicology, this report describes how AOPs could be harnessed to advance MOA analysis of genotoxicity pathways using five example case studies. Each of these genetic toxicology AOPs proposed for further development includes the relevant molecular initiating events, key events, and adverse outcomes (AOs), identification and/or further development of the appropriate assays to link an agent to these events, and discussion regarding the biological plausibility of the proposed AOP. A key difference between these proposed genetic toxicology AOPs versus traditional AOPs is that the AO is a genetic toxicology endpoint of potential significance in risk characterization, in contrast to an adverse state of an organism or a population. The first two detailed case studies describe provisional AOPs for aurora kinase inhibition and tubulin binding, leading to the common AO of aneuploidy. The remaining three case studies highlight provisional AOPs that lead to chromosome breakage or mutation via indirect DNA interaction (inhibition of topoisomerase II, production of cellular reactive oxygen species, and inhibition of DNA synthesis). These case studies serve as starting points for genotoxicity AOPs that could ultimately be published and utilized by the broader toxicology community and illustrate the practical considerations and evidence required to formalize such AOPs so that they may be applied to genetic toxicity evaluation schemes. Environ. Mol. Mutagen. 61:114-134, 2020. © 2019 Wiley Periodicals, Inc.

A method for the cell-cycle-specific analysis of radiation-induced chromosome aberrations and breaks.

The classical G2-assay is widely used to assess cell-radiosensitivity and cancer phenotype: Cells are exposed to low doses of ionizing-radiation (IR) and collected for cytogenetic- analysis ˜1.5 h later. In this way, chromosome-damage is measured in cells irradiated in G2-phase, without retrieving information regarding kinetics of chromosome-break-repair. Modification of the assay to include analysis at multiple time-points after IR, has enabled kinetic-analysis of chromatid-break-repair and assessment of damage in a larger proportion of G2-phase cells. This modification, however, increases the probability that at later time points not only cells irradiated in G2-phase, but also cells irradiated in S-phase will reach metaphase. However, the response of cells irradiated in G2-phase can be mechanistically different from that of cells irradiated in S-phase. Therefore, indiscriminate analysis may confound the interpretation of experiments designed to elucidate mechanisms of chromosome-break-repair and the contributions of the different DSB-repair-pathways in this response. Here we report an EdU based modification of the assay that enables S- and G2-phase specific analysis of chromatid break repair. Our results show that the majority of metaphases captured during the first 2 h after IR originate from cells irradiated in G2-phase (EdU- metaphases) in both rodent and human cells. Metaphases originating from cells irradiated in S-phase (EdU+ metaphases) start appearing at 2 h and 4 h after IR in rodent and human cells, respectively. The kinetics of chromatid-break-repair are similar in cells irradiated in G2- and S-phase of the cell-cycle, both in rodent and human cells. The protocol is applicable to classical-cytogenetic experiments and allows the cell-cycle specific analysis of chromosomal-aberrations. Finally, the protocol can be applied to the kinetic analysis of chromosome-breaks in prematurely-condensed-chromosomes of G2-phase cells. In summary, the developed protocol provides means to enhance the analysis of IR-induced-cytogenetic-damage by providing information on the cell-cycle phase where DNA damage is inflicted.

In vivo genotoxicity of 1,4-dioxane evaluated by liver and bone marrow micronucleus tests and Pig-a assay in rats.

1,4-Dioxane, used widely as a solvent in the manufacture of chemicals and as a laboratory reagent, induced liver adenomas and carcinomas in mice and rats, and nasal tumors in rats in several long-term studies. 1,4-Dioxane has been reported to be non-genotoxic in vitro, and there is no clear conclusion concerning its in vivo genotoxicity in rodents. In the present study, we investigated the ability of 1,4-dioxane to induce micronuclei in the liver and bone marrow of rats. For the liver micronucleus test, we performed the juvenile animal method and two methods using partial hepatectomy (PH), dosing before PH or dosing after PH. We also evaluated the in vivo mutagenicity of 1,4-dioxane by Pig-a gene mutation assay using rat peripheral blood. As a result, all methods of liver micronucleus test showed an increase in the frequency of micronucleated hepatocytes by 1,4-dioxane. The dosing before PH, a suitable method for detecting structural chromosome aberration inducers, showed the clearest response for micronucleated hepatocytes induction among the three methods. This finding is consistent with a previous report that 1,4-dioxane induces mainly chromosome breakage in the liver. Negative results were obtained in the bone marrow micronucleus test and Pig-a gene mutation assay in our study. These results suggested that 1,4-dioxane is clastogenic in the liver but not genotoxic in the bone marrow of rats.

Matrix association region/scaffold attachment region: the crucial player in defining the positions of chromosome breaks mediated by bile acid-induced apoptosis in nasopharyngeal epithelial cells.

BACKGROUND: It has been found that chronic rhinosinusitis (CRS) increases the risk of developing nasopharyngeal carcinoma (NPC). CRS can be caused by gastro-oesophageal reflux (GOR) that may reach nasopharynx. The major component of refluxate, bile acid (BA) has been found to be carcinogenic and genotoxic. BA-induced apoptosis has been associated with various cancers. We have previously demonstrated that BA induced apoptosis and gene cleavages in nasopharyngeal epithelial cells. Chromosomal cleavage occurs at the early stage of both apoptosis and chromosome rearrangement. It was suggested that chromosome breaks tend to cluster in the region containing matrix association region/scaffold attachment region (MAR/SAR). This study hypothesised that BA may cause chromosome breaks at MAR/SAR leading to chromosome aberrations in NPC. This study targeted the AF9 gene located at 9p22 because 9p22 is a deletion hotspot in NPC. METHODS: Potential MAR/SAR sites were predicted in the AF9 gene by using MAR/SAR prediction tools. Normal nasopharyngeal epithelial cells (NP69) and NPC cells (TWO4) were treated with BA at neutral and acidic pH. Inverse-PCR (IPCR) was used to identify chromosome breaks in SAR region (contains MAR/SAR) and non-SAR region (does not contain MAR/SAR). To map the chromosomal breakpoints within the AF9 SAR and non-SAR regions, DNA sequencing was performed. RESULTS: In the AF9 SAR region, the gene cleavage frequencies of BA-treated NP69 and TWO4 cells were significantly higher than those of untreated control. As for the AF9 non-SAR region, no significant difference in cleavage frequency was detected between untreated and BA-treated cells. A few breakpoints detected in the SAR region were mapped within the AF9 region that was previously reported to translocate with the mixed lineage leukaemia (MLL) gene in an acute lymphoblastic leukaemia (ALL) patient. CONCLUSIONS: Our findings suggest that MAR/SAR may be involved in defining the positions of chromosomal breakages induced by BA. Our report here, for the first time, unravelled the relation of these BA-induced chromosomal breakages to the AF9 chromatin structure.

Bioflavonoids promote stable translocations between MLL-AF9 breakpoint cluster regions independent of normal chromosomal context: Model system to screen environmental risks.

Infant acute leukemias are aggressive and characterized by rapid onset after birth. The majority harbor translocations involving the MLL gene with AF9 as one of its most common fusion partners. MLL and AF9 loci contain breakpoint cluster regions (bcrs) with sequences hypothesized to be targets of topoisomerase II inhibitors that promote translocation formation. Overlap of MLL bcr sequences associated with both infant acute leukemia and therapy-related leukemia following exposure to the topoisomerase II inhibitor etoposide led to the hypothesis that exposure during pregnancy to biochemically similar compounds may promote infant acute leukemia. We established a reporter system to systematically quantitate and stratify the potential for such compounds to promote chromosomal translocations between the MLL and AF9 bcrs analogous to those in infant leukemia. We show bioflavonoids genistein and quercetin most biochemically similar to etoposide have a strong association with MLL-AF9 bcr translocations, while kaempferol, fisetin, flavone, and myricetin have a weak but consistent association, and other compounds have a minimal association in both embryonic stem (ES) and hematopoietic stem cell (HSC) populations. The frequency of translocations induced by bioflavonoids at later stages of myelopoiesis is significantly reduced by more than one log. The MLL and AF9 bcrs are sensitive to these agents and recombinogenic independent of their native context suggesting bcr sequences themselves are drivers of illegitimate DNA repair reactions and translocations, not generation of functional oncogenic fusions. This system provides for rapid systematic screening of relative risk, dose dependence, and combinatorial impact of multitudes of dietary and environmental exposures on MLL-AF9 translocations. Environ. Mol. Mutagen. 60: 154-167, 2019. © 2018 Wiley Periodicals, Inc.

Essential Role of BRCA2 in Ovarian Development and Function.

The causes of ovarian dysgenesis remain incompletely understood. Two sisters with XX ovarian dysgenesis carried compound heterozygous truncating mutations in the BRCA2 gene that led to reduced BRCA2 protein levels and an impaired response to DNA damage, which resulted in chromosomal breakage and the failure of RAD51 to be recruited to double-stranded DNA breaks. The sisters also had microcephaly, and one sister was in long-term remission from leukemia, which had been diagnosed when she was 5 years old. Drosophila mutants that were null for an orthologue of BRCA2 were sterile, and gonadal dysgenesis was present in both sexes. These results revealed a new role for BRCA2 and highlight the importance to ovarian development of genes that are critical for recombination during meiosis. (Funded by the Israel Science Foundation and others.).

Generation of Advanced Glycation End-Products (AGEs) by glycoxidation mediated by copper and ROS in a human serum albumin (HSA) model peptide: reaction mechanism and damage in motor neuron cells.

Glucose, in the presence of reactive oxygen species (ROS), acts as an as an oxidative agent and drives deleterious processes in Diabetes Mellitus. We have studied the mechanism and the toxicological effects of glucose-dependent glycoxidation reactions driven by copper and ROS, using a model peptide based on the exposed sequence of Human Serum Albumin (HSA) and containing a lysine residue susceptible to copper complexation. The main products of these reactions are Advanced Glycation End-products (AGEs). Carboxymethyl lysine and pyrraline condensed on the model peptide, generating a Modified Peptide (MP). These products were isolated, purified, and tested on cultured motor neuron cells. We observed DNA damage, enhancement of membrane roughness, and formation of domes. We evaluated nuclear abnormalities by the cytokinesis-blocked micronucleus assay and we measured cytostatic and cytotoxic effects, chromosomal breakage, nuclear abnormalities, and cell death. AGEs formed by glycoxidation caused large micronucleus aberrations, apoptosis, and large-scale nuclear abnormalities, even at low concentrations.

Alternative DNA structure formation in the mutagenic human c-MYC promoter.

Mutation 'hotspot' regions in the genome are susceptible to genetic instability, implicating them in diseases. These hotspots are not random and often co-localize with DNA sequences potentially capable of adopting alternative DNA structures (non-B DNA, e.g. H-DNA and G4-DNA), which have been identified as endogenous sources of genomic instability. There are regions that contain overlapping sequences that may form more than one non-B DNA structure. The extent to which one structure impacts the formation/stability of another, within the sequence, is not fully understood. To address this issue, we investigated the folding preferences of oligonucleotides from a chromosomal breakpoint hotspot in the human c-MYC oncogene containing both potential G4-forming and H-DNA-forming elements. We characterized the structures formed in the presence of G4-DNA-stabilizing K+ ions or H-DNA-stabilizing Mg2+ ions using multiple techniques. We found that under conditions favorable for H-DNA formation, a stable intramolecular triplex DNA structure predominated; whereas, under K+-rich, G4-DNA-forming conditions, a plurality of unfolded and folded species were present. Thus, within a limited region containing sequences with the potential to adopt multiple structures, only one structure predominates under a given condition. The predominance of H-DNA implicates this structure in the instability associated with the human c-MYC oncogene.

Persistence of Breakage in Specific Chromosome Bands 6 Years after Acute Exposure to Oil.

BACKGROUND: The identification of breakpoints involved in chromosomal damage could help to detect genes involved in genetic disorders, most notably cancer. Until now, only one published study, carried out by our group, has identified chromosome bands affected by exposure to oil from an oil spill. In that study, which was performed two years after the initial oil exposure in individuals who had participated in clean-up tasks following the wreck of the Prestige, three chromosomal bands (2q21, 3q27, 5q31) were found to be especially prone to breakage. A recent follow-up study, performed on the same individuals, revealed that the genotoxic damage had persisted six years after oil exposure. OBJECTIVES: To determine whether there exist chromosome bands which are especially prone to breakages and to know if there is some correlation with those detected in the previous study. In addition, to investigate if the DNA repair problems detected previously persist in the present study. DESIGN: Follow-up study performed six years after the Prestige oil spill. SETTING: Fishermen cooperatives in coastal villages. PARTICIPANTS: Fishermen highly exposed to oil spill who participated in previous genotoxic study six years after the oil. MEASUREMENTS: Chromosome damage in peripheral lymphocytes. For accurate identification of the breakpoints involved in chromosome damage of circulating lymphocytes, a sequential stain/G-banding technique was employed. To determine the most break-prone chromosome bands, two statistical methods, the Fragile Site Multinomial and the chi-square tests (where the bands were corrected by their length) were used. To compare the chromosome lesions, structural chromosome alterations and gaps/breaks between two groups of individuals we used the GEE test which takes into account a possible within-individual correlation. Dysfunctions in DNA repair mechanisms, expressed as chromosome damage, were assessed in cultures with aphidicolin by the GEE test. RESULTS: Cytogenetic analyses were performed in 47 exposed individuals. A total of 251 breakpoints in exposed individuals) were identified, showing a non-uniform distribution in the human ideogram. Ten chromosome bands were found to be especially prone to breakage through both statistical methods. By comparing these bands with those observed in certain exposed individuals who had already participated the previous study, it was found in both studies that four bands (2q21, 3q27, 5q31 and 17p11.2) are particularly sensitive to breakage. Additionally, the dysfunction in DNA repair mechanisms was not significantly higher in oil-exposed individuals than in non-exposed individuals. LIMITATIONS: The sample size and the possibility of some kind of selection bias should be considered. Genotoxic results cannot be extrapolated to the high number of individuals who participated occasionally in clean-up tasks. CONCLUSION: Our findings show the existence of at least four target bands (2q21, 3q27, 5q31 and 17p11.2) with a greater propensity to break over time after an acute exposure to oil. The breaks in these bands, which are commonly involved in hematological cancer, may explain the increase of cancer risk reported in chronically benzene-exposed individuals. In addition, a more efficiency of the DNA repair mechanisms has been detected six years after in fishermen who were highly exposed to the oil spill. To date, only this study, performed by our group on the previous and present genotoxic effects, has analyzed the chromosomal regions affected by breakage after an acute oil exposure.

Tetratricopeptide repeat factor XAB2 mediates the end resection step of homologous recombination.

We examined the influence of the tetratricopeptide repeat factor XAB2 on chromosomal break repair, and found that XAB2 promotes end resection that generates the 3' ssDNA intermediate for homologous recombination (HR). Namely, XAB2 is important for chromosomal double-strand break (DSB) repair via two pathways of HR that require end resection as an intermediate step, end resection of camptothecin (Cpt)-induced DNA damage, and RAD51 recruitment to ionizing radiation induced foci (IRIF), which requires end resection. Furthermore, XAB2 mediates specific aspects of the DNA damage response associated with end resection proficiency: CtIP hyperphosphorylation induced by Cpt and BRCA1 IRIF. XAB2 also promotes histone acetylation events linked to HR proficiency. From truncation mutation analysis, the capacity for XAB2 to promote HR correlates with its ability to form a complex with ISY1 and PRP19, which show a similar influence as XAB2 on HR. This XAB2 complex localizes to punctate structures consistent with interchromatin granules that show a striking adjacent-localization to the DSB marker γH2AX. In summary, we suggest that the XAB2 complex mediates DNA damage response events important for the end resection step of HR, and speculate that its adjacent-localization relative to DSBs marked by γH2AX is important for this function.

Dysregulation of the DNA Damage Response and KMT2A Rearrangement in Fetal Liver Hematopoietic Cells.

Etoposide, a topoisomerase 2 (TOP2) inhibitor, is associated with the development of KMT2A (MLL)-rearranged infant leukemia. An epidemiological study suggested that in utero exposure to TOP2 inhibitors may be involved in generation of KMT2A (MLL) rearrangement. The present study examined the mechanism underlying the development of KMT2A (MLL)-rearranged infant leukemia in response to in utero exposure to etoposide in a mouse model. Fetal liver hematopoietic stem cells were more susceptible to etoposide than maternal bone marrow mononuclear cells. Etoposide-induced Kmt2a breakage was detected in fetal liver hematopoietic stem cells using a newly developed chromatin immunoprecipitation (ChIP) assay. Assessment of etoposide-induced chromosomal translocation by next-generation RNA sequencing (RNA-seq) identified several chimeric fusion messenger RNAs that were generated by etoposide treatment. However, Kmt2a (Mll)-rearranged fusion mRNA was detected in Atm-knockout mice, which are defective in the DNA damage response, but not in wild-type mice. The present findings suggest that in utero exposure to TOP2 inhibitors induces Kmt2a rearrangement when the DNA damage response is defective.

Curcumin-Mediated Reversal of p15 Gene Promoter Methylation: Implication in Anti-Neoplastic Action against Acute Lymphoid Leukaemia Cell Line.

Curcumin has been documented to exert anticancer effects by interacting with altered proliferative and apoptotic pathways in cancer models. In this study, we evaluated the potential of curcumin to reverse promoter methylation of the p15 gene in Raji cells and its ability to induce apoptosis and genomic instability. Anti-neoplastic action of curcumin showed an augmentation in reactive oxygen species (ROS) and cell cycle arrest in G1 phase. Subsequently, curcumin- exposed Raji cells showed structural abnormalities in chromosomes. These observations suggest that curcumin also causes ROS-mediated apoptosis and genomic instability. The treatment of Raji cell line with 10 µM curcumin caused hypomethylation of the p15 promoter after six days. Hypomethylation of p15 was further found to be favoured by downregulation of DNA methyltransferase 1 after 10 µM curcumin treatment for six days. Methylation-specific PCR suggested demethylation of the p15 promoter. Demethylation was further validated by DNA sequencing. Reverse-transcription PCR demonstrated that treatment with curcumin (10 µM) for six days led to the up-regulation of p15 and down-regulation of DNA methyltransferase 1. Furthermore, curcumin- mediated reversal of p15 promoter methylation might be potentiated by down-regulation of DNA methyltransferase 1 expression, which was supported by cell cycle analysis. Furthermore, curcumin acts as a double-pronged agent, as it caused apoptosis and promoter hypomethylation in Raji cells.

Environmental and chemotherapeutic agents induce breakage at genes involved in leukemia-causing gene rearrangements in human hematopoietic stem/progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) give rise to all of the cells that make up the hematopoietic system in the human body, making their stability and resilience especially important. Damage to these cells can severely impact cell development and has the potential to cause diseases, such as leukemia. Leukemia-causing chromosomal rearrangements have largely been studied in the context of radiation exposure and are formed by a multi-step process, including an initial DNA breakage and fusion of the free DNA ends. However, the mechanism for DNA breakage in patients without previous radiation exposure is unclear. Here, we investigate the role of non-cytotoxic levels of environmental factors, benzene, and diethylnitrosamine (DEN), and chemotherapeutic agents, etoposide, and doxorubicin, in generating DNA breakage at the patient breakpoint hotspots of the MLL and CBFB genes in human HSPCs. These conditions represent exposure to chemicals encountered daily or residual doses from chemotherapeutic drugs. Exposure of HSPCs to non-cytotoxic levels of environmental chemicals or chemotherapeutic agents causes DNA breakage at preferential sites in the human genome, including the leukemia-related genes MLL and CBFB. Though benzene, etoposide, and doxorubicin have previously been linked to leukemia formation, this is the first study to demonstrate a role for DEN in the generation of DNA breakage at leukemia-specific sites. These chemical-induced DNA breakpoints coincide with sites of predicted topoisomerase II cleavage. The distribution of breakpoints by exposure to non-cytotoxic levels of chemicals showed a similar pattern to fusion breakpoints in leukemia patients. Our findings demonstrate that HSPCs exposed to non-cytotoxic levels of environmental chemicals and chemotherapeutic agents are prone to topoisomerase II-mediated DNA damage at the leukemia-associated genes MLL and CBFB. These data suggest a role for long-term environmental chemical or residual chemotherapeutic drug exposure in generation of DNA breakage at sites with a propensity to form leukemia-causing gene rearrangements.

Effect of sodium benzoate preservative on micronucleus induction, chromosome break, and Ala40Thr superoxide dismutase gene mutation in lymphocytes.

Sodium benzoate is food preservative that inhibits microbial growth. The effects of sodium benzoate preservative on micronucleus induction, chromosome break, and Ala40Thr superoxide dismutase gene mutation in lymphocytes were studied. Sodium benzoate concentrations of 0.5, 1.0, 1.5, and 2.0 mg/mL were treated in lymphocyte cell line for 24 and 48 hrs, respectively. Micronucleus test, standard chromosome culture technique, PCR, and automated sequencing technique were done to detect micronucleus, chromosome break, and gene mutation. The results showed that, at 24- and 48-hour. incubation time, sodium benzoate concentrations of 1.0, 1.5, and 2.0 mg/mL increased micronucleus formation when comparing with the control group (P < 0.05). At 24- and 48-hour. incubation time, sodium benzoate concentrations of 2.0 mg/mL increased chromosome break when comparing with the control group (P < 0.05). Sodium benzoate did not cause Ala40Thr (GCG→ACG) in superoxide dismutase gene. Sodium benzoate had the mutagenic and cytotoxic toxicity in lymphocytes caused by micronucleus formation and chromosome break.

Acute toxic and genotoxic activities of widely used cytostatic drugs in higher plants: Possible impact on the environment.

Cytostatic drugs are highly toxic pharmaceuticals and it was repeatedly postulated that they may cause adverse effects in ecosystems. The acute toxic and genotoxic properties of these drugs have not been adequately investigated in higher plants so far; therefore, we studied the most widely used drugs (5-flurouracil, 5FU; etoposide, Et; cisplatin, CisPt; carboplatin, CaPt; vincristine sulfate, VinS and cyclophosphamide monohydrate, CP) in micronucleus (MN) assays with meiotic pollen tetrad cells of Tradescantia and with root cells from Allium cepa. MNi are formed as a consequence of chromosome breaks and aneuploidy. We monitored also the acute toxic properties of the drugs, i.e. inhibition of cell division (mitotic indices and retardation of root growth) in the latter species. All compounds caused in both indicator plants genotoxic effects. The order of genotoxic potencies expressed as NOELs in µM was CisPt (0.1)≥ Et (0.5)>CP (1.0)>CaPt (10)>5FU (30)>VinS (100) in Tradescantia. A similar order was seen in Allium MN but Et was less active (5.0µM). Four compounds caused alterations of the mitotic indices under the present conditions namely CisPt (0.5), Et (10.0), 5FU (10.0) and VinS (100). Inhibition of root growth decreased in the order CisPt (0.5)>Et (1.0)≥VinS (1.0)>5FU (5.0)>CaPt (33.0)>CP (>1000). Comparisons of the NOELs with the predicted environmental concentrations (PEC) show that the latter values are at least 5 orders of magnitude lower and indicate that it is unlikely that their release in the environment may cause adverse effects in higher plants. However, it is notable that the levels of both platinum compounds and of 5FU in hospital effluents may reach levels which may induce damage of the genetic material.