The Role of Telomerase in Radiation-Induced Genomic Instability.

Findings from previous studies have suggested that the telomerase system is involved in radiation-induced genomic instability. In this study, we investigated the involvement of telomerase in the development and processing of chromosomal damage at different cell cycle stages after irradiation of human fibroblasts. Several response criteria were investigated, including cell survival, chromosomal damage (using the micronucleus assay), G2-induced chromatid aberrations (using the conventional G2 assay as well as a chemically-induced premature chromosome condensation assay) and DNA double-strand breaks (DSBs; using γ-H2AX, 53BP1 and Rad51) in an isogenic pair of cell lines: BJ human foreskin fibroblasts and BJ1-hTERT, a telomerase-immortalized BJ cell line. To distinguish among G1, S and G2 phase, cells were co-immunostained for CENP-F and cyclin A, which are tightly regulated proteins in the cell cycle. After X-ray irradiation at doses in the range of 0.1-6 Gy, the results showed that for cell survival and micronuclei induction, where the overall effect is dominated by the cells in G1 and S phase, no difference was observed between the two cell types; in contrast, when radiation sensitivity at the G2 stage of the cell cycle was analyzed, a significantly higher number of chromatid-type aberrations (breaks and exchanges), and higher levels of γ-H2AX and of Rad51 foci were observed for the BJ cells compared to the BJ1-hTERT cells. Therefore, it can be concluded that telomerase appears to be involved in DNA DSB repair processes, mainly in the G2 phase. These data, taken overall, reinforce the notion that hTERT or other elements of the telomere/telomerase system may defend chromosome integrity in human fibroblasts by promoting repair in G2 phase of the cell cycle.

Health effects of desalinated water: Role of electrolyte disturbance in cancer development.

This review contends that "healthy" water in terms of electrolyte balance is as important as "pure" water in promoting public health. It considers the growing use of desalination (demineralization) technologies in drinking water treatment which often results in tap water with very low concentrations of sodium, potassium, magnesium and calcium. Ingestion of such water can lead to electrolyte abnormalities marked by hyponatremia, hypokalemia, hypomagnesemia and hypocalcemia which are among the most common and recognizable features in cancer patients. The causal relationships between exposure to demineralized water and malignancies are poorly understood. This review highlights some of the epidemiological and in vivo evidence that link dysregulated electrolyte metabolism with carcinogenesis and the development of cancer hallmarks. It discusses how ingestion of demineralized water can have a procarcinogenic effect through mediating some of the critical pathways and processes in the cancer microenvironment such as angiogenesis, genomic instability, resistance to programmed cell death, sustained proliferative signaling, cell immortalization and tumorigenic inflammation. Evidence that hypoosmotic stress-response processes can upregulate a number of potential oncogenes is well supported by a number studies. In view of the rising production and consumption of demineralized water in most parts of the world, there is a strong need for further research on the biological importance and protean roles of electrolyte abnormalities in promoting, antagonizing or otherwise enabling the development of cancer. The countries of the Gulf Cooperative Council (GCC) where most people consume desalinated water would be a logical place to start this research.

Verification by the FISH translocation assay of historic doses to Mayak workers from external gamma radiation.

The aim of this study was to apply the fluorescence in situ hybridization (FISH) translocation assay in combination with chromosome painting of peripheral blood lymphocytes for retrospective biological dosimetry of Mayak nuclear power plant workers exposed chronically to external gamma radiation. These data were compared with physical dose estimates based on monitoring with badge dosimeters throughout each person's working life. Chromosome translocation yields for 94 workers of the Mayak production association were measured in three laboratories: Southern Urals Biophysics Institute, Leiden University Medical Center and the former Health Protection Agency of the UK (hereinafter Public Health England). The results of the study demonstrated that the FISH-based translocation assay in workers with prolonged (chronic) occupational gamma-ray exposure was a reliable biological dosimeter even many years after radiation exposure. Cytogenetic estimates of red bone marrow doses from external gamma rays were reasonably consistent with dose measurements based on film badge readings successfully validated in dosimetry system "Doses-2005" by FISH, within the bounds of the associated uncertainties.

Causes of genome instability: the effect of low dose chemical exposures in modern society.

Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genome's integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus, genome instability can be defined as an enhanced tendency for the genome to acquire mutations; ranging from changes to the nucleotide sequence to chromosomal gain, rearrangements or loss. This review raises the hypothesis that in addition to known human carcinogens, exposure to low dose of other chemicals present in our modern society could contribute to carcinogenesis by indirectly affecting genome stability. The selected chemicals with their mechanisms of action proposed to indirectly contribute to genome instability are: heavy metals (DNA repair, epigenetic modification, DNA damage signaling, telomere length), acrylamide (DNA repair, chromosome segregation), bisphenol A (epigenetic modification, DNA damage signaling, mitochondrial function, chromosome segregation), benomyl (chromosome segregation), quinones (epigenetic modification) and nano-sized particles (epigenetic pathways, mitochondrial function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make scientists aware of the increasing need to unravel the underlying mechanisms via which chemicals at low doses can induce genome instability and thus promote carcinogenesis.

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.

FISH analysis of translocations induced by chronic exposure to Sr radioisotopes: second set of analysis of the Techa River Cohort.

Fluorescent in situ hybridisation analysis of stable translocations was performed for 26 residents living along the Techa River (Russia), who were predominantly (95%) exposed to ingested strontium radioisotopes ((89)Sr and (90)Sr) resulting in exposure of their red bone marrow (RBM). Analysis was conducted at the Urals Research Center for Radiation Medicine, Public Health England and Leiden University Medical Center. Each laboratory scored 1000 cells per donor, which resulted in ∼1000 genome equivalents (GE) per donor. The age-dependent spontaneous level of translocations for each donor was evaluated on the basis of data published by Sigurdson et al. (International study of factors affecting human chromosome. Mutat. Res. 2008;652: :112-121). Reconstruction of doses was performed with the 'Techa River Dosimetry System' developed in 2009. In the studied donors, the range of individual cumulated RBM dose was from 0.3 to 3.7 Gy. Analysis of the yield of stable translocations dependent on the individual RBM dose from (89,90)Sr showed a linear dose-response relationship of 0.007 ± 0.002 translocation/GE cell/Gy (R = 0.61, p = 0.001). This set of results was in a good agreement with the previous data reported for 18 donors by Vozilova et al. (Preliminary FISH-based assessment of external dose for residents exposed on the Techa River.

What radiation dose does the FISH translocation assay measure in cases of incorporated radionuclides for the Southern Urals populations?

The fluorescence in situ hybridisation (FISH) technique is now well established for retrospective dosimetry in cases of external radiation exposure that occurred many years ago. However, the question remains as to whether FISH provides valid estimates of cumulative red bone marrow radiation doses in cases of incorporation of radionuclides or combined external and internal exposures. This question has arisen in connection with the interpretation of results of dose assessments for epidemiological studies of plutonium workers at the Russian Mayak plant and of members of the public exposed to strontium radioisotopes and external radiation as a result of discharges from Mayak to the Techa River. Exposures to penetrating external radiation result in fairly uniform irradiation of body tissues, and hence similar doses to all tissues, for which FISH dosimetry can provide a reliable measure of this whole body dose. However, intakes of radionuclides into the body by inhalation or ingestion may result in retention in specific organs and tissues, so that the distribution of dose is highly heterogeneous. For radionuclides emitting short-range radiations (e.g. alpha particles), this heterogeneity can apply to dose delivery within tissues and between cells within tissues. In this paper, an attempt is made to address the question of what FISH measures in such circumstances by considering evidence regarding the origin and lifetime dynamics of lymphocyte subsets in the human body in relation to the localised delivery of dose from the internal emitters (90)Sr and (239)Pu, which are of particular interest for the Southern Urals Mayak and Techa River populations, and for which most evidence is available in these populations. It is concluded that the FISH translocation assay can be usefully applied for detecting internal and combined external gamma and internal doses from internally deposited (90)Sr, albeit with fairly large uncertainties. The same may be true of (239)Pu, as well as other radionuclides, although much work remains to be done to establish dose-response relationships.

Metakaryotic stem cell nuclei use pangenomic dsRNA/DNA intermediates in genome replication and segregation.

Bell shaped nuclei of metakaryotic cells double their DNA content during and after symmetric and asymmetric amitotic fissions rather than in the separate, pre-mitotic S-phase of eukaryotic cells. A parsimonious hypothesis was tested that the two anti-parallel strands of each chromatid DNA helix were first segregated as ssDNA-containing complexes into sister nuclei then copied to recreate a dsDNA genome. Metakaryotic nuclei that were treated during amitosis with RNase A and stained with acridine orange or fluorescent antibody to ssDNA revealed large amounts of ssDNA. Without RNase treatment metakaryotic nuclei in amitosis stained strongly with an antibody complex specific to dsRNA/DNA. Images of amitotic figures co-stained with dsRNA/DNA antibody and DAPI indicated that the entire interphase dsDNA genome (B-form helices) was transformed into two dsRNA/DNA genomes (A-form helices) that were segregated in the daughter cell nuclei then retransformed into dsDNA. As this process segregates DNA strands of opposite polarity in sister cells it hypothetically offers a sequential switching mechanism within the diverging stem cell lineages of development.

Detection and quantification of a radiation-associated mitochondrial DNA deletion by a nested real-time PCR in human peripheral lymphocytes.

In this study we implemented a new assay using a nested real-time polymerase chain reaction (PCR) to detect radiation-induced common deletion (CD) in mitochondrial DNA (mtDNA) of human peripheral lymphocytes. A standard curve for real-time PCR was established by applying a plasmid DNA containing human normal mtDNA or mutated mtDNA. Human peripheral lymphocyte DNA was amplified and quantified by real-time PCR using primer sets for total damaged or mutated mtDNA, plus probes labeled with the fluorescent dyes. The first-round PCR generated multiple products were used as the template for a second-round PCR. We herein describe a nested real-time PCR assay capable of quantifying mtDNA bearing the CD in human peripheral lymphocytes following exposure (in vitro) to (137)Cs γ-rays in a dose range of 0.5 up to 5Gy. The reproducibility of this assay was evident for both unirradiated and irradiated samples by examining human blood lymphocytes from 14 donors. This technique was sensitive enough to detect deletions in mtDNA at low dose levels, as low as 0.5Gy, and higher levels of CD mtDNA were evident at higher doses (≥1Gy), however, there was no consistent dose-response relationship.

Potent cytotoxic effects of Calomeria amaranthoides on ovarian cancers.

BACKGROUND: Ovarian cancer remains the leading cause of death from gynaecological malignancy. More than 60% of the patients are presenting the disease in stage III or IV. In spite of combination of chemotherapy and surgery the prognosis stays poor for therapy regimen. METHODS: The leaves of a plant endemic to Australia, Calomeria amaranthoides, were extracted and then fractionated by column chromatography. In vitro cytotoxicity tests were performed with fractions of the plant extract and later with an isolated compound on ovarian cancer cell lines, as well as normal fibroblasts at concentrations of 1-100 µg/mL (crude extract) and 1-10 µg/mL (compound). Cytotoxicity was measured after 24, 48 and 72 hours by using a non-fluorescent substrate, Alamar blue.In vivo cytotoxicity was tested on ascites, developed in the abdomen of nude mice after inoculation with human OVCAR3 cells intraperitoneally. The rate of change in abdomen size for the mice was determined by linear regression and statistically evaluated for significance by the unpaired t test. RESULTS: Two compounds were isolated by chromatographic fractionation and identified by 1H-NMR, 13C-NMR and mass spectrometry analyses, EPD, an α-methylene sesquiterpene lactone of the eremophilanolide subtype, and EPA, an α-methylene carboxylic acid.Cytotoxicity of EPD for normal fibroblasts at all time points IC50 was greater than 10 µg/mL, whereas, for OVCAR3 cells at 48 hours IC50 was 5.3 µg/mL (95% confidence interval 4.3 to 6.5 µg/mL).Both, the crude plant extract as well as EPD killed the cancer cells at a final concentration of 10 µg/mL and 5 µg/mL respectively, while in normal cells only 20% cell killing effect was observed. EPA had no cytotoxic effects.Changes in abdomen size for control versus Cisplatin treated mice were significantly different, P = 0.023, as were control versus EPD treated mice, P = 0.025, whereas, EPD versus Cisplatin treated mice were not significantly different, P = 0.13. CONCLUSIONS: For the first time both crude plant extract from Calomeria amaranthoides and EPD have been shown to have potent anti-cancer effects against ovarian cancer.

Human fetal/tumor metakaryotic stem cells: pangenomic homologous pairing and telomeric end-joining of chromatids.

Metakaryotic cells and syncytia with large, hollow, bell-shaped nuclei demonstrate symmetrical and asymmetrical amitotic nuclear fissions in microanatomical positions and numbers expected of stem cell lineages in tissues of all three primordial germ layers and their derived tumors. Using fluorescence in situ hybridization, mononuclear metakaryotic interphase cells have been found with only 23 centromeric and 23 telomeric staining regions. Syncytial bell-shaped nuclei found approximately during weeks 5-12 of human gestation display 23 centromeric and either 23 or 46 telomeric staining regions. These images suggest that (1) homologous chromatids pair at centromeres and telomeres, (2) all paired telomeres join end-to-end with other paired telomeres in all mononuclear and some syncytial metakaryotic cells, and (3) telomere junctions may open and close during the syncytial phase of development. Twenty-three telomeric joining figures could be accounted by 23 rings of one chromatid pair each, a single pangenomic ring of 23 joined chromatid pairs, or any of many possible sets of oligo-chromatid pair rings. As telomeric end-joining may affect peri-telomeric gene expression, a programmed sequence of telomeric end-joining associations in metakaryotic stem cells could guide developmental arboration and errors in, or interruptions of, this program could contribute to carcinogenesis.

PCC and COBRA-FISH a new tool to characterize primary cervical carcinomas: to assess hall-marks and stage specificity.

A newly developed assay based on chemically induced premature chromosome condensation (PCC) and multi-color combined binary ratio labeling (COBRA) fluorescence in situ hybridization (FISH) techniques have been implemented in order to investigate for the first time for recurrent cytogenetic aberrations in primary cervical carcinoma (derived directly from biopsies) at different stages of progression. The cytogenetic profiles of 17 biopsies derived from 14 and 3 cervical cancer patients with squamous-cell carcinomas (Sq) and with adenocarcinomas (Ad), respectively, were assessed. Frequencies of both structural as well as numerical aberrations were found to be higher in Sq than in Ad. The analysis revealed that even in early tumors stages (IB1) have a higher frequency of chromosome-losses and -gains as well as chromosomal alterations as compared to normal cells. A positive trend was found between stage advancement of cervical tumors and the frequency of numerical and structural aberrations. No specific and common chromosomal abnormality (e.g. distinct clones of translocation) was found among cervical carcinoma at the different stages (IB1, IIA and IIB). However, a distinct difference was found between stage IIIB and lower tumor stages, as all analyzed IIIB samples revealed a near tetraploid karyotype. Furthermore, all studied metaphases were aberrant and had a high frequency of translocations. PCC-COBRA-FISH characterization of a common type of an established culture from cervical carcinoma CSCC-1 revealed a triploidy/tetraploidy karyotype with several structural aberrations. In general, no similarity was found between this model and early stages of primary tumors. The newly established assay has a novel potential and can reveal the original status of primary tumors at different stages.

International study of factors affecting human chromosome translocations.

Chromosome translocations in peripheral blood lymphocytes of normal, healthy humans increase with age, but the effects of gender, race, and cigarette smoking on background translocation yields have not been examined systematically. Further, the shape of the relationship between age and translocation frequency (TF) has not been definitively determined. We collected existing data from 16 laboratories in North America, Europe, and Asia on TFs measured in peripheral blood lymphocytes by fluorescence in situ hybridization whole chromosome painting among 1933 individuals. In Poisson regression models, age, ranging from newborns (cord blood) to 85 years, was strongly associated with TF and this relationship showed significant upward curvature at older ages versus a linear relationship (p<0.001). Ever smokers had significantly higher TFs than non-smokers (rate ratio (RR)=1.19, 95% confidence interval (CI), 1.09-1.30) and smoking modified the effect of age on TFs with a steeper age-related increase among ever smokers compared to non-smokers (p<0.001). TFs did not differ by gender. Interpreting an independent effect of race was difficult owing to laboratory variation. Our study is three times larger than any pooled effort to date, confirming a suspected curvilinear relationship of TF with age. The significant effect of cigarette smoking has not been observed with previous pooled studies of TF in humans. Our data provide stable estimates of background TF by age, gender, race, and smoking status and suggest an acceleration of chromosome damage above age 60 and among those with a history of smoking cigarettes.

How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: Report of an ECVAM Workshop.

Workshop participants agreed that genotoxicity tests in mammalian cells in vitro produce a remarkably high and unacceptable occurrence of irrelevant positive results (e.g. when compared with rodent carcinogenicity). As reported in several recent reviews, the rate of irrelevant positives (i.e. low specificity) for some studies using in vitro methods (when compared to this "gold standard") means that an increased number of test articles are subjected to additional in vivo genotoxicity testing, in many cases before, e.g. the efficacy (in the case of pharmaceuticals) of the compound has been evaluated. If in vitro tests were more predictive for in vivo genotoxicity and carcinogenicity (i.e. fewer false positives) then there would be a significant reduction in the number of animals used. Beyond animal (or human) carcinogenicity as the "gold standard", it is acknowledged that genotoxicity tests provide much information about cellular behaviour, cell division processes and cellular fate to a (geno)toxic insult. Since the disease impact of these effects is seldom known, and a verification of relevant toxicity is normally also the subject of (sub)chronic animal studies, the prediction of in vivo relevant results from in vitro genotoxicity tests is also important for aspects that may not have a direct impact on carcinogenesis as the ultimate endpoint of concern. In order to address the high rate of in vitro false positive results, a 2-day workshop was held at the European Centre for the Validation of Alternative Methods (ECVAM), Ispra, Italy in April 2006. More than 20 genotoxicity experts from academia, government and industry were invited to review data from the currently available cell systems, to discuss whether there exist cells and test systems that have a reduced tendency to false positive results, to review potential modifications to existing protocols and cell systems that might result in improved specificity, and to review the performance of some new test systems that show promise of improved specificity without sacrificing sensitivity. It was concluded that better guidance on the likely mechanisms resulting in positive results that are not biologically relevant for human health, and how to obtain evidence for those mechanisms, is needed both for practitioners and regulatory reviewers. Participants discussed the fact that cell lines commonly used for genotoxicity testing have a number of deficiencies that may contribute to the high false positive rate. These include, amongst others, lack of normal metabolism leading to reliance on exogenous metabolic activation systems (e.g. Aroclor-induced rat S9), impaired p53 function and altered DNA repair capability. The high concentrations of test chemicals (i.e. 10 mM or 5000 microg/ml, unless precluded by solubility or excessive toxicity) and the high levels of cytotoxicity currently required in mammalian cell genotoxicity tests were discussed as further potential sources of false positive results. Even if the goal is to detect carcinogens with short in vitro tests under more or less acute conditions, it does not seem logical to exceed the capabilities of cellular metabolic turnover, activation and defence processes. The concept of "promiscuous activation" was discussed. For numerous mutagens, the decisive in vivo enzymes are missing in vitro. However, if the substrate concentration is increased sufficiently, some other enzymes (that are unimportant in vivo) may take over the activation-leading to the same or a different active metabolite. Since we often do not use the right enzyme systems for positive controls in vitro, we have to rely on their promiscuous activation, i.e. to use excessive concentrations to get an empirical correlation between genotoxicity and carcinogenicity. A thorough review of published and industry data is urgently needed to determine whether the currently required limit concentration of 10mM or 5000 microg/ml, and high levels of cytotoxicity, are necessary for the detection of in vivo genotoxins and DNA-reactive, mutagenic carcinogens. In addition, various measures of cytotoxicity are currently allowable under OECD test guidelines, but there are few comparative data on whether different measures would result in different maximum concentrations for testing. A detailed comparison of cytotoxicity assessment strategies is needed. An assessment of whether test endpoints can be selected that are not intrinsically associated with cytotoxicity, and therefore are less susceptible to artefacts produced by cytotoxicity, should also be undertaken. There was agreement amongst the workshop participants that cell systems which are p53 and DNA-repair proficient, and have defined Phase 1 and Phase 2 metabolism, covering a broad set of enzyme forms, and used within the context of appropriately set limits of concentration and cytotoxicity, offer the best hope for reduced false positives. Whilst there is some evidence that human lymphocytes are less susceptible to false positives than the current rodent cell lines, other cell systems based on HepG2, TK6 and MCL-5 cells, as well as 3D skin models based on primary human keratinocytes also show some promise. Other human cell lines such as HepaRG, and human stem cells (the target for carcinogenicity) have not been used for genotoxicity investigations and should be considered for evaluation. Genetic engineering is also a valuable tool to incorporate missing enzyme systems into target cells. A collaborative research programme is needed to identify, further develop and evaluate new cell systems with appropriate sensitivity but improved specificity. In order to review current data for selection of appropriate top concentrations, measures and levels of cytotoxicity, metabolism, and to be able to improve existing or validate new assay systems, the participants called for the establishment of an expert group to identify the in vivo genotoxins and DNA-reactive, mutagenic carcinogens that we expect our in vitro genotoxicity assays to detect as well as the non-genotoxins and non-carcinogens we expect them not to detect.

Role of Artemis in DSB repair and guarding chromosomal stability following exposure to ionizing radiation at different stages of cell cycle.

We analyzed the phenotype of cells derived from SCID patients with different mutations in the Artemis gene. Using clonogenic survival assay an increased sensitivity was found to X-rays (2-3-fold) and bleomycin (2-fold), as well as to etoposide, camptothecin and methylmethane sulphonate (up to 1.5-fold). In contrast, we did not find increased sensitivity to cross-linking agents mitomycin C and cis-platinum. The kinetics of DSB repair assessed by pulsed-field gel electrophoresis and gammaH2AX foci formation after ionizing irradiation, indicate that 15-20% of DSB are not repaired in Artemis-deficient cells. In order to get a better understanding of the repair defect in Artemis-deficient cells, we studied chromosomal damage at different stages of the cell cycle. In contrast to AT cells, Artemis-deficient cells appear to have a normal G(1)/S-block that resulted in a similar frequency of dicentrics and translocations, however, frequency of acentrics fragments was found to be 2-4-fold higher compared to normal fibroblasts. Irradiation in G(2) resulted in a higher frequency of chromatid-type aberrations (1.5-3-fold) than in normal cells, indicating that a fraction of DSB requires Artemis for proper repair. Our data are consistent with a function of Artemis protein in processing of a subset of complex DSB, without G(1) cell cycle checkpoint defects. This type of DSB can be induced in high proportion and persist through S-phase and in part might be responsible for the formation of chromatid-type exchanges in G(1)-irradiated Artemis-deficient cells. Among different human radiosensitive fibroblasts studied for endogenous (in untreated samples) as well as X-ray-induced DNA damage, the ranking order on the basis of higher incidence of spontaneously occurring chromosomal alterations and induced ones was: ligase 4> or =AT>Artemis. This observation implicates that in human fibroblasts following exposure to ionizing radiation a lower risk might be created when cells are devoid of endogenous damage.

Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), two naturally occurring benzoxazinones contained in sprouts of Gramineae are potent aneugens in human-derived liver cells (HepG2).

Benzoxazinoids (BAs) are toxic constituents of sprouts of Gramineae such as wheat, maize and rye and are part of the plant defence system against pests. In the last years, sprouts have been increasingly consumed as health foods and are also used for the production of dietary supplements. In the present study we investigated the mutagenic activities of the two most abundant BAs, namely 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) in the Salmonella/microsome assay and additionally, in micronucleus (MN) assay and single cell gel electrophoresis (SCGE) assay in a human-derived liver cell line (HepG2). DIBOA caused significant induction of his(+) revertants in all three strains in the range between 0.02 and 0.50 mg/plate; the highest activity was observed in TA100 (fivefold increase over the background at the highest dose level). The effect in YG1024 (a derivative of TA98 with increased acetyltransferase activity) was only slightly higher than the effect in the parental strain indicating that acetylation plays no crucial role in the activation of this BA. DIMBOA was in general less active and a positive result was only seen in the base substitution strain (TA100). Addition of rat liver homogenate (S9-mix) led to a significant (ca. twofold) increase of the mutagenic activities of both BAs. In SCGE assays with HepG2 cells consistently negative results were obtained with both compounds whereas in MN assays significant dose dependent effects were observed under similar experimental conditions. DIMBOA caused significant effects already at concentrations > or =1 microM; at the highest dose (20 microM) the MN frequency was sevenfold higher than the background level. DIBOA caused weaker effects and was positive at doses > or =2.5 microM, the maximal induction (twofold over background) was observed with 20 microM. Overall, DIMBOA was ca. 30-fold more active as DIBOA. Subsequent experiments with pancentromeric probes showed that >80% of the MN induced at the highest doses gave a centromere positive signal indicating that both BAs are aneugenic. This is an interesting observation as it is assumed that aneuploidy is a key event in cancer induction and at present no other aneugenic plant-derived substances of dietary relevance are known.

Interphase chromosome positioning affects the spectrum of radiation-induced chromosomal aberrations.

In interphase, chromosomes occupy defined nuclear volumes known as chromosome territories. To probe the biological consequences of the described nonrandom spatial positioning of chromosome territories in human lymphocytes, we performed an extensive FISH-based analysis of ionizing radiation-induced interchanges involving chromosomes 1, 4, 18 and 19. Since the probability of exchange formation depends strongly on the spatial distance between the damage sites in the genome, a preferential formation of exchanges between proximally positioned chromosomes is expected. Here we show that the spectrum of interchanges deviates significantly from one expected based on random chromosome positioning. Moreover, the observed exchange interactions between specific chromosome pairs as well as the interactions between homologous chromosomes are consistent with the proposed gene density-related radial distribution of chromosome territories. The differences between expected and observed exchange frequencies are more pronounced after exposure to densely ionizing neutrons than after exposure to sparsely ionizing X rays. These experiments demonstrate that the spatial positioning of interphase chromosomes affects the spectrum of chromosome rearrangements.

Mismatch repair protein Msh2 contributes to UVB-induced cell cycle arrest in epidermal and cultured mouse keratinocytes.

Nucleotide excision repair (NER), cell cycle regulation and apoptosis are major defence mechanisms against the carcinogenic effects of UVB radiation. NER eliminates UVB-induced DNA photolesions via two subpathways: global genome repair (GGR) and transcription-coupled repair (TCR). In a previous study, we found UVB-induced accumulation of tetraploid (4N) keratinocytes in the epidermis of Xpc(-/-) mice (no GGR), but not in Xpa(-/-) (no TCR and no GGR) or in wild-type (WT) mice. We inferred that this arrest in Xpc(-/-) mice is caused by erroneous replication past photolesions, leading to 'compound lesions' known to be recognised by mismatch repair (MMR). MMR-induced futile cycles of breakage and resynthesis at sites of compound lesions may then sustain a cell cycle arrest. The present experiments with Xpc(-/-)Msh2(-/-) mice and derived keratinocytes show that the MMR protein Msh2 indeed plays a role in the generation of the UVB-induced arrested cells: a Msh2-deficiency lowered significantly the percentage of arrested cells in vivo (40-50%) and in vitro (30-40%). Analysis of calyculin A (CA)-induced premature chromosome condensation (PCC) of cultured Xpc(-/-) keratinocytes showed that the delayed arrest occurred in late S phase rather than in G(2)-phase. Taken together, the results indicate that in mouse epidermis and cultured keratinocytes, the MMR protein Msh2 plays a role in the UVB-induced S-phase arrest. This indicates that MMR plays a role in the UVB-induced S-phase arrest. Alternatively, Msh2 may have a more direct signalling function.

Genotoxic effects of dietary and lifestyle related carcinogens in human derived hepatoma (HepG2, Hep3B) cells.

Aim of the study was to investigate the usefulness of two human derived hepatoma cell lines (HepG2 and Hep3B) for the detection of dietary and lifestyle related DNA-reactive carcinogens. Comparisons of the sensitivity of HepG2 cells of different origin towards benzo[a]pyrene (B(a)P) showed that strong differences exist in the induction of micronuclei (MN). The most sensitive was used for all further experiments, in which we investigated the effects of aflatoxin B(1) (AFB(1)), B(a)P, As(2)O(3), CdCl(2), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), ethanol, acetaldehyde and caffeic acid in micronucleus (MN) tests. Dose dependent effects were detected in HepG2 with AFB(1) (0.2microM), CdCl(2) (2.2microM), As(2)O(3) (8.1microM), B(a)P (22.7microM), PhIP (35.7microM), NDMA (22.7mM), acetaldehyde (11.2mM) and ethanol (442.2mM). Numbers in parentheses indicate the C(D) values (concentration that induced a two-fold increase over the background). NNK and caffeic acid gave negative results under all conditions. In Hep3B cells, the effects were generally weaker. With PhIP, As(2)O(3) and NDMA negative results were obtained; with caffeic acid and NPYR marginal but significant induction of MN was observed. Enzyme measurements showed that both cell lines possess CYP1A1, glutathione-S-transferase (three-fold higher in HepG2) as well as N-acetyltransferase (NAT) 1 and sulfotransferases (SULT1A1 and SULT1A3; two- and seven-fold higher in HepG2); other cytochrome P450 enzymes (CYP1A2, 2B1, 2E1) and NAT2 were not detectable. The differences in the activities of the various enzymes may explain the contrasting results obtained in the MN experiments. Overall, our results indicate that the HepG2 line is more sensitive towards dietary genotoxins than Hep3B, and support the assumption that the HepG2/MN assay enables the detection of genotoxic carcinogens which give negative results in other currently used in vitro assays.

Premature chromosome condensation revisited: a novel chemical approach permits efficient cytogenetic analysis of cancers.

Chemical induction of premature chromosome condensation (PCC) was investigated and optimized to be able to analyze the chromosomal constitution of cancer cells independent of mitosis and with minimal culture artifacts. A potent protein phosphatase inhibitor, calyculin A, was used to induce PCC in normal diploid cells, in several established human tumor cell lines, and in cells isolated from freshly dissected adenomatous polyps of a patient with hereditary colorectal cancer. In parallel, mitotic arrest was pursued by use of Colcemid. In cell lines, a difference of up to 10-fold was found between frequency of cells with PCC induced by calyculin A (PCC index) and the mitotic index after treatment with Colcemid. In the fresh tumor specimens, Colcemid failed to result in metaphase formation, whereas a regimen of 80 nM calyculin A for 75 min, after only 2 days of culturing, resulted in a PCC index of 2-5%. pq-COBRA-FISH (COmbined Binary RAtio labeling-fluorescence in situ hybridization) was used for a detailed analysis of four cell lines treated with calyculin A, which proved that PCC spreads are amenable to molecular karyotyping, and a comparison between PCC spreads and metaphases from mitotic arrest revealed no discrepancies in karyotypes. pq-COBRA-FISH on PCC spreads from fresh colon tumor samples revealed only numerical and no structural abnormalities. Calyculin A-induced PCC combined with multicolor FISH gives a new opportunity for analysis of the chromosomal constitution of G(1) and G(2) cancer cells and may find application in the study of the role of chromosome instability in cancer development.