Fluorescence in-situ hybridisation on biopsies from clam ileocystoplasties and on a clam cancer.

The incidence of carcinoma following an enterocystoplasty increases with time and is a major concern after such procedures. The aim of this study was to investigate genetic instability (in the form of numerical chromosomal aberrations) at the enterovesical anastomosis in patients who had undergone a clam ileocystoplasty using fluorescent in-situ hybridisation (FISH). Fluorescent in-situ hybridisation was performed on touch preparation samples prepared from fresh endoscopic biopsies obtained from the enterovesical anastomosis and native bladder remnant (control specimens) of 15 patients who had undergone a clam ileocystoplasty. Fluorescent in-situ hybridisation was also performed on one squamous cell cancer specimen. Significant aneusomic changes were found at the enterovesical anastomosis in all 15 patients. Alterations in chromosome 18 copy number were the most frequent abnormal finding (trisomy 18, n=8; monosomy 18, n=7). Nine patients were monosomic for chromosome 9. Isolated monosomy 8 and trisomy 8 were each found in one patient. The control specimens were all normal. An unusually high incidence of polysomic cells was found in the clam tumour specimen, reflecting the aggressive nature of this cancer. Chromosomal numerical abnormalities occur at the enterovesical anastomosis following a clam ileocystoplasty and chromosome 18 appears to be a particularly good marker of genetic instability. The results of this study indicate that morphologically normal tissue obtained from the enterovesical anastomosis displays evidence of chromosomal instability that may predispose to tumour formation. However, further prospective, blinded, longitudinal studies are required to establish whether predetermined FISH signal patterns in enterocystoplasty cells in urine or obtained by biopsy predict the presence or absence of tumour.

Molecular cytogenetic insights into the ageing syndrome Hutchinson-Gilford Progeria (HGPS).

Hutchinson-Gilford Progeria Syndrome (HGPS) is an extremely rare genetic disorder characterized by premature ageing in childhood and serves as a valuable model for the human ageing process in general. Most recently, point mutations in the lamin A (LMNA) gene on chromosome 1q have been associated with the disease, however how these mutations relate to the complex phenotype of HGPS remains to be established. It has been shown that fibroblasts from HGPS patients are frequently resistant to immortalization with telomerase (hTERT), consistent with the idea that the loss of a dominant acting HGPS gene is a pre-requisite for immortalization. In this study we report the first detailed cytogenetic analysis of hTERT-immortalised HGPS cell lines from three patients and one corresponding primary fibroblast culture. Our results provide evidence for a cytogenetic mosaicism in HGPS with a distinctive pattern of chromosome aberrations in all the HGP clones. Chromosome 11 alterations were observed at a high frequency in each immortalised HGPS cell line but were also present at a lower frequency in the corresponding primary cells. Moreover, we were able to identify the 11q13-->q23 region as a potential site of breakage. Our results are therefore consistent with a role of chromosome 11 alterations in the escape from senescence observed in HGPS cells. In addition to this defined rearrangement, we consistently observed complex chromosomal rearrangements, suggesting that HGPS displays features of chromosomal instability.

Fluorescence in situ hybridisation analysis of chromosomal aberrations in gastric tissue: the potential involvement of Helicobacter pylori.

In this series of experiments, a novel protocol was developed whereby gastric cells were collected using endoscopic cytology brush techniques, and prepared, such that interphase fluorescence in situ hybridization (FISH) could be performed. In total, 80 distinct histological samples from 37 patients were studied using four chromosome probes (over 32,000 cells analysed). Studies have previously identified abnormalities of these four chromosomes in upper GI tumours. Using premalignant tissues, we aimed to determine how early in Correa's pathway to gastric cancer these chromosome abnormalities occurred. Aneuploidy of chromosomes 4, 8, 20 and 17(p53) was detected in histologically normal gastric mucosa, as well as in gastritis, intestinal metaplasia, dysplasia and cancer samples. The levels of aneuploidy increased as disease severity increased. Amplification of chromosome 4 and chromosome 20, and deletion of chromosome 17(p53) were the more common findings. Hence, a role for these abnormalities may exist in the initiation of, and the progression to, gastric cancer. Helicobacter pylori infection was determined in premalignant tissue using histological analysis and PCR technology. Detection rates were comparable. PCR was used to subtype H. pylori for CagA status. The amplification of chromosome 4 in gastric tissue was significantly more prevalent in H. pylori-positive patients (n=7) compared to H. pylori-negative patients (n=11), possibly reflecting a role for chromosome 4 amplification in H. pylori-induced gastric cancer. The more virulent CagA strain of H. pylori was associated with increased disease pathology and chromosomal abnormalities, although numbers were small (CagA+ n=3, CagA- n=4). Finally, in vitro work demonstrated that the aneuploidy induced in a human cell line after exposure to the reactive oxygen species (ROS) hydrogen peroxide was similar to that already shown in the gastric cancer pathway, and may further strengthen the hypothesis that H. pylori causes gastric cancer progression via an ROS-mediated mechanism.

Generation of locus-specific probes for interphase fluorescence in situ hybridisation--application in Barrett's esophagus.

Despite the wide range of probes commercially available for interphase fluorescence in situ hybridisation (FISH), the supply of locus-specific probes is limited to genes or chromosomal regions commonly altered in genetic diseases or during carcinogenesis. Generation of these probes is therefore desirable to accommodate individual research requirements. Hence, we detail the methodology required to design and produce custom locus-specific interphase FISH probes for any human genomic region of interest and their application was illustrated in cytogenetic investigations of Barrett's tumourigenesis. Previously utilising FISH, we observed that Barrett's tissues demonstrated chromosome 4 hyperploidy [Gut 52 (2003) 623], but as centromeric probes were used in this analysis, it was not known if the whole chromosome was amplified. We consequently generated single-copy sequence probes for the 4p16.3 and 4q35.1 subtelomeric loci. Multicolour FISH was subsequently performed on interphase preparations originating from patients with Barrett's esophagus at varying histological grades, thus demonstrating the whole region of chromosome 4 was amplified within the tissues. Additionally, probes for the DNA methyltransferase genes were produced to determine if gene dosage alterations were responsible for increasing methylation activity during Barrett's neoplastic progression. No significant alterations at the DNMT1 and DNMT3a loci were detected. An increased copy number of these genes is therefore not the basis for the hypermethylation commonly observed in this premalignant lesion.

Investigations into the biological relevance of in vitro clastogenic and aneugenic activity.

In the current study we present a view of events leading to chemically induced DNA damage in vitro from both a cytogenetic and molecular aspect, focusing on threshold mediated responses and the biological relevance of DNA damaging events that occur at low and high cellular toxicity levels. Current regulatory mechanisms do not take into account chemicals that cause significant DNA damage only at high toxicity. Our results demonstrate a defined threshold for micronucleus induction after insult with the alkylating agent MMS. Other results define a significant change in gene expression following treatment with chemicals that give rise to structural DNA damage only at high toxicity. Pairs of chemicals with a similar mode of action but differing toxicity levels were chosen, the chemicals that demonstrated structural DNA damage only at high levels of toxicity showed an increase in heat shock protein gene expression whereas the chemicals causing DNA damage events at all levels of toxicity did not induce changes in heat shock gene expression at identical toxicity levels. The data presented indicates that there are a number of situations where the linear dose response model is not appropriate for risk estimation. However, deviation from linear risk models should be dependent upon the availability of appropriate experimental data such as that shown here.

Characterisation of p53 status at the gene, chromosomal and protein levels in oesophageal adenocarcinoma.

p53 mutations and loss of heterozygosity have been commonly associated with oesophageal adenocarcinoma. In this investigation, the p53 status of a Welsh population of Barrett's-associated oesophageal adenocarcinomas were fully characterised at the gene sequence, chromosomal, mRNA and protein levels. In total, 31 tumours were examined for p53 gene sequence mutations using RFLP with sequencing, allelic loss of the gene was characterised by FISH, mRNA expression by p53 pathway signalling arrays and protein levels by p53 immunohistochemistry. In all, 9.6% of adenocarcinomas harboured p53 mutations, 24% displayed p53 allelic loss and 83% exhibited p53 protein accumulation. Point mutations and deletions of the gene did not coexist within the same samples. All samples containing p53 mutations also displayed positive immunostaining; however; in the majority of cases, p53 protein accumulation developed in the absence of mutations. The gene expression analysis demonstrated no differences in p53 and mdm-2 transcription levels between the p53 immunonegative and immunopositive samples, indicating other mechanisms underlie the proteins' overexpression. In conclusion, p53 mutations and deletions do not appear to be frequent events in oesophageal adenocarcinomas; however, abnormal accumulation of the protein is present in a vast majority of cases. P53 gene mutations are not the primary cause of protein overexpression--an alternative mechanism is responsible for the positive p53 immunohistochemistry detected.

Detection of p53 mutations in precancerous gastric tissue.

Intestinal-type gastric cancer is preceded by gastritis and intestinal metaplasia. There is uncertainty regarding the stage at which genetic alterations in the p53 gene occur. Reactive oxygen species (ROS) may participate in the production of mutations and the inactivation of p53 is due to infection by the bacterium Helicobacter pylori. We have investigated whether alterations of the p53 gene can be detected in gastritis and intestinal metaplasia using the restriction site mutation assay. We also assessed the potential contribution of ROS to p53 inactivation using electron spin resonance spectroscopy (ESR) and correlated with the presence of H. pylori. In all, 35% of the gastritis samples and 45% of the intestinal metaplasia samples were found to contain mutations in exons 5-8 of the p53 gene. Electron spin resonance spectroscopy analysis showed a significant increase in free radical levels in gastritis samples compared with normal, intestinal metaplasia and cancer samples, suggesting that free radicals present in gastritis may contribute to p53 mutations. There was no significant difference in free radical levels between the H. pylori-positive and -negative groups. However, a small subpopulation of the H. pylori-negative patients had much higher levels of free radicals. This suggests a more prominent role for other factors in ROS production.

Chromosome 4 hyperploidy represents an early genetic aberration in premalignant Barrett's oesophagus.

BACKGROUND AND AIMS: Characterisation of the underlying molecular mechanisms that promote Barrett's progression may ultimately lead to identification of potential predictive genetic markers that classify patients' malignant risk. In an attempt to understand these causative pathways, fluorescence in situ hybridisation (FISH) was used in this study to determine when specific genetic alterations arise during Barrett's associated neoplastic progression. METHODS: Endoscopic cytology brushings were obtained from 28 patients with Barrett's metaplasia, 28 with dysplasia (20 low grade dysplasia (LGD) and eight with high grade dysplasia (HGD)), and seven with adenocarcinoma, together with paired control brushings from regions of normal proximal squamous cell epithelium. The exfoliated epithelial cells were washed and deposited onto slides. Probes specific for the centromeres of chromosomes 4, 8, 20, and Y, and locus specific probes for the tumour suppressor genes p16, p53, and Rb were subsequently hybridised. RESULTS: Aneuploidy was found early in progression, with metaplastic tissues displaying increased copy numbers of chromosomes 4 and 8. Chromosome 4 hyperploidy was found in 89%, 90%, 88%, and 100% of metaplasias, LGD, HGD, adenocarcinomas, respectively, while chromosome 8 hyperploidy occurred in 71%, 75%, 100%, and 100% of patients with the respective staging. Loss of the p16 tumour suppressor gene also presented in metaplastic epithelium (7%) but most other genetic aberrations were only seen in HGD. CONCLUSIONS: Genetic instability arises well before dysplasia in Barrett's oesophagus, with chromosome 4 and 8 hyperploidy representing the earliest and most common alterations identified. As these aberrations are widespread at all the premalignant stages, there may be genes on chromosomes 4 and 8 that are involved in both the initiation and progression of Barrett's oesophagus.

Detection and characterization of mechanisms of action of aneugenic chemicals.

A comprehensive evaluation of the genotoxic potential of chemicals requires the assessment of the ability to induce gene mutations and structural chromosome (clastogenic activity) and numerical chromosome (aneugenic activity) aberrations. Aneuploidy is a major cause of human reproductive failure and an important contributor to cancer and it is therefore important that any increase in its frequency due to chemical exposures should be recognized and controlled. The in vitro binucleate cell micronucleus assay provides a powerful tool to determine the ability of a chemical to induce chromosome damage. The application of an anti-kinetochore antibody to micronuclei allows their classification into kinetochore-positive and kinetochore-negative, indicating their origin by aneugenic or clastogenic mechanisms, respectively. The availability of chromosome-specific centromere probes allows the analysis of the segregation of chromosomes into the daughter nuclei of binucleate cells to evaluate chromosome non-disjunction. Quantitative relationships between the two major causes of aneuploidy, chromosome loss and non-disjunction, can be determined. The mechanisms leading to chromosome loss and non-disjunction can be investigated by the analysis of morphological and structural changes in the cell division apparatus by the application of specific stains and antibodies for various cell division components. We illustrate such analyses by the demonstration of the interaction of the monomer bisphenol-A with the centrosome of the mitotic spindle and the folic acid antagonist pyrimethamine with the centromeres of chromosomes. Both types of modifications lead to the induction of aneuploidy in exposed cells. Our studies also implicate the products of the p53 and XPD genes in the regulation of the fidelity of chromosome segregation at mitosis.

Spontaneous and induced aneuploidy, considerations which may influence chromosome malsegregation.

Aneuploidy plays a major role in the production of human birth defects and is becoming increasingly recognised as a critical event in the etiology of a wide range of human cancers. Thus, the detection of aneuploidy and the characterisation of the mechanisms which lead to chromosome malsegregation is an important area of genotoxicological research. As an aid to aneuploidy research, methods have been developed to analyse the mechanisms of chromosome malsegregation and to investigate the role of aneuploidy in tumour progression. The presence of aneuploid cells is a common characteristic of many of tumour cell types as illustrated by the wide range of chromosome number changes detected in post-menopausal breast tumours. To investigate the time of occurrence of aneuploidy during tumour progression, we have studied the chromosome number status of Syrian hamster dermal (SHD) cells cultures progressing to morphological transformation. The production of both polyploid and aneuploid cells is a common feature of progressing cells in this model. The elevation of both progression to morphological transformation and aneuploid frequencies can be produced by exposure to a diverse range of carcinogens and tumour promoters. Analysis of the genotoxic activity of the hormone 17-beta oestradiol demonstrated its ability to induce both chromosome loss and non-disjunction in human lymphoblastoid cells implicating aneugenic activity in hormone related cancers. Mutations in the p53 tumour suppressor gene introduced into human fibroblasts produced modifications in chromosome separation at mitosis which may lead to the production of both aneuploidy and polyploid cells. Our studies indicate that the production of aneuploid cells can be influenced by both endogenous and exogenous factors and occur throughout the progression of normal cells to a malignant phenotype.

The detection of mutations induced in vitro in the human p53 gene by hydrogen peroxide with the restriction site mutation (RSM) assay.

We have analysed five mutation hotspots within the p53 gene (codons 175, 213, 248, 249, and 282) for mutations induced by hydrogen peroxide (H(2)O(2)), employing the restriction site mutation (RSM) assay. In addition, four other restriction sites covering non-hotspot codons of exons 5-9 of the p53 gene (codons 126, 153/54, 189 and the 3' splice site of exon 9) were analysed by the RSM assay for H(2)O(2)-induced mutations. Two cell types were concurrently analysed in this study, i.e. primary fibroblast cells and a gastric cancer cell line. Using the RSM assay, H(2)O(2)-induced mutations were only detected in exon 7 of the p53 gene. This was true for both cell types. These mutations were mainly induced in the Msp I restriction site (codon 247/248) and were predominantly GC to AT transitions (71%). Hence these GC to AT mutations were presumably due to H(2)O(2) exposure, possibly implicating the 5OHdC adduct, which is known to induce C to T mutations upon misreplication. Importantly, this study demonstrates that the RSM methodology is capable of detecting rare oxidative mutations within the hotspot codons of the p53 tumour suppressor gene. Hence, this methodology may allow the detection of early p53 mutations in pre-malignant tissues.

Multicolour FISH detection of radioactive iodine-induced 17cen-p53 chromosomal breakage in buccal cells from therapeutically exposed patients.

Simultaneous labelling of 17cen and the p53 locus by multicolour FISH was used to monitor radioactive iodine-induced structural and numerical chromosome abnormalities in buccal cells from 29 hyperthyroidism and thyroid cancer patients sampled before and after therapeutic treatment. This novel methodology allowed the efficient detection of 17p deletions leading to p53 allelic deletions, 17p gains and whole chromosome 17 numerical abnormalities in epithelial cells. Highly significant increases in the frequency of cells with (i) 17p abnormalities (1.8-fold; P < 0.001), including p53 monoallelic deletions (2.1-fold; P < 0.001) and 17p gains (3.5-fold; P < 0.001); (ii) chromosome 17 numerical abnormalities (2-fold; P < 0.001); and (iii) simultaneous 17p breakage and chromosome 17 numerical abnormalities (2.3-fold; P < 0.001), were observed after radioactive iodine treatment. As expected, the major contribution to these increases was detected in hyperthyroidism patients compared with thyroid cancer patients who suffered thyroidectomy before radioactive iodine exposure and, therefore, experienced a rapid elimination of the radioisotope. Considering that both the genetic endpoints and the target tissue are extremely relevant in carcinogenesis, it is suggested that the observed genetic damage could contribute to the reported increase in cancer risk of people therapeutically or accidentally exposed to radioactive iodine.

In vitro and in vivo extrapolations of genotoxin exposures: consideration of factors which influence dose-response thresholds.

The concept of a threshold of activity of a genotoxic agent is primarily based upon considerations of protective mechanisms and multiple cellular targets, which require inactivation before a toxic response is produced. In this paper, we have considered and evaluated the influences of compound metabolism, DNA lesion formation, mutation induction and sequence content, aneuploidy induction and the influence of repair enzymes upon genetic endpoints produced by both DNA reactive chemicals and by those chemicals which modify non-DNA cellular targets. Thresholds of activity have been evaluated by critical analysis of the published literature and original data analysing both the role of sequence context upon point mutation induction and DNA repair mechanisms upon the sensitivity of cultured cells to the induction of aneuploidy. In the case of DNA reactive chemicals, the presence of a threshold of chemical activity will be dependent upon cellular activities such as those of the Phase II enzymes reducing the activity of chemicals before lesion formation takes place and/or those of the DNA repair enzymes which reduce the proportion of DNA lesions which are processed into DNA sequence changes. Under such conditions, a given exposure of a DNA reactive chemical does not produce a linear or semi-linear increase in DNA lesions or in mutation frequency. However, even when these protective mechanisms are overwhelmed by the high exposures of genotoxic chemicals the biological effects of a genotoxin may be influenced by the sequence context of the gene under consideration. Here, we demonstrate that point mutations are detected at relatively higher frequencies in the non-coding introns compared with the coding exons. Many of the base changes detected in the exons do not produce amino acid changes in the proteins coded for by the genes being monitored for mutation induction. Both sequence context and the types of base changes induced may provide a "buffering" effect reducing the biological consequences of mutation induction. Spindle damaging chemicals, such as colcemid and vinblastine, induce aneuploidy by modifying the numbers of spindle fibres which regulate the segregation of chromosomes during mitosis and meiosis. The redundancy of spindle fibres in the dividing mammalian cell leads to the prediction that only chemical exposures which damage most, if not all, of the fibres will lead to the induction of polyploidy and/or aneuploidy. Such predicted thresholds of chemical activity can be observed when both chromosome loss and non-disjunction are measured in wild type cultures. However, we observed a substantial increase in sensitivity to aneugenic chemicals when measurements were made in primary cell cultures derived from xerodoma pigmentosum and trichothiodystrophy patients. Further studies are necessary to evaluate the consequences of the genetic background of tester strains upon the nature of the dose-response curve of aneugenic chemicals.

The application of comparative genomic hybridization and fluorescence in situ hybridization to the characterization of genotoxicity screening tester strains AHH-1 and MCL-5.

AHH-1 TK+/- is a human B cell-derived lymphoblastoid cell line that constitutively expresses a high level of the cytochrome CYP1A1. The MCL-5 cell line was developed by transfection of AHH-1 with cDNAs encoding the human cytochrome P450s, CYP1A2, CYP2A6, CYP2E1, CYP3A4 and microsomal epoxide hydrolase carried in plasmids. The metabolic components of these cell lines make them a useful screening tool for use in mutagenicity studies. Although AHH-1 and MCL-5 are closely related, the two cell lines show differences which cannot be attributed to transfection. In the present study both cell lines were investigated for chromosome stability by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) using whole chromosome probes and telomeric probes. Amplification in chromosomes 4q, 3q and 9p was observed in both cell lines. To compare the cell lines directly, AHH-1 and MCL-5 DNAs were co-hybridized on the same metaphases using a modified CGH technique. The only difference observed between AHH-1 and MCL-5 was the degree of amplification involving the subtelomeric region of chromosome 4; the additional telomeric region (4q) was translocated onto chromosome 11 and/or chromosome X. FISH was use to show the presence of isochromosomes 3q and 9p in both cell lines with a chromosome number of 48 or higher. These data demonstrate that CGH and FISH with chromosome-specific probes are able to resolve complex karyotypes and to highlight subchromosomal regions involved in rearrangements and potential chromosome fragile sites. Analyses such as those described here may be of considerable value in the determination of the stability of a variety of the cell lines used in the mutagenicity testing of chemicals.

Chemically induced aneuploidy: investigations into chromosome specific effects in mitosis.

Genotoxicity studies of aneuploidy may potentially produce different results depending upon the chromosome selected for analysis if chromosome-specific sensitivities to chemical exposure exist. Any chromosome specificity characteristics that predispose to aneuploidy might interact with environmental exposures in additional different ways related to the mechanism of aneuploidy induction. Thus, we have undertaken an investigation of chromosome-specific effects using morphologically distinct chromosomes in a hybrid cell line. We were able to identify eight different chromosomes simultaneously by dual colour FISH analysis in controls and in cells exposed to a range of griseofulvin concentrations. Certain chromosomes were more frequently involved in aneuploidy, but no simple relationship between chromosome organisation and sensitivity emerged apart from the over-representation of the alien human chromosome. Aneuploidy was detected at higher frequencies in interphase cells compared with metaphase cells. Overall the data indicate that chemically induced aneuploidy may be detected for a variety of chromosomes and cell types using both interphase and metaphase protocols. However, the data obtained should be used with care in the hazard evaluation of chemical aneugens.

Segregational fidelity of chromosomes in human thyroid tumour cells.

Using fluorescence in situ hybridisation (FISH) we have analysed the segregational fidelity of all the human chromosomes during mitotic cell division. The losses and gains of chromosomes were analysed in human polyploid cell lines derived from a well-differentiated papillary thyroid cancer. These thyroid cells can be cultured for more than 300 population doublings. For the purpose of our study the polyploid nature of the cells may act as a protective buffer against the cell-lethal effects of the loss of individual chromosomes. To evaluate the role of the p53 gene product in maintaining the fidelity of chromosome segregation we compared the frequencies of chromosome loss and gain in cultures with wild-type p53 activity (K1E7neo3) and cultures transfected with plasmids expressing a mutant p53 product (K1E7scx6). Cultures were analysed for the presence of both structurally normal and rearranged chromosomes at both early and late passages. Cell cultures with defective p53 activity showed progressive chromosome loss from a median chromosome number of 87-97 to 75-86. Cell growth in cultures with wild-type p53 activity showed the loss of chromosomes 6, 7, and 8 and the gain of 17 and 20. Cultures expressing mutant p53 activity showed the loss of chromosomes 2, 5, 14 and 17 and the gain of 4 and 22. The combination of defective p53 and growth resulted in further destabilisation with the additional losses of chromosomes 3, 11, 15, 16 and 21. Chromosomes 1, 9, 10, 12, 13, 18, 19, X and Y segregated stably under all the culture conditions as did the structurally rearranged marker chromosomes. The study has demonstrated variation in the fidelity of mitotic chromosome segregation and the influence of p53 gene activity upon the segregation of individual human chromosomes.

Characterization of highly radiosensitive cell lines from a human ovarian small-cell cancer.

Cells were obtained at paracentesis from a patient with a rapidly growing ovarian tumor. A monolayer cell line (V7S), a xenograft tumor line (V7), and subsequently a xenograft-derived monolayer cell line (V7M) were established. Histological and immunohistochemical studies of the original tumor, xenograft, and cell lines provided a diagnosis of small-cell carcinoma of the ovary-which is consistent with the clinical course of the patient. V7S and V7M had a predominantly hypodiploid karyotype with a small tetraploid population. The V7M, which has been in long-term culture, also showed a nonrandom translocation involving chromosomes 1 and 14 and monosomy of X. Radiobiologically, V7S, V7, and V7M showed marked radiosensitivity with surviving fractions at 2 Gy, measured by clonogenic assay, of between 0.022 and 0.147. Split-dose experiments provided evidence that this radiosensitivity was not due to an inability in cellular repair. In vivo data from the xenograft (V7) revealed a highly radiosensitive tumor, corroborating the in vitro studies.

Biomonitoring study of a group of workers potentially exposed to traffic fumes.

Risk assessment of environmental pollutants is concerned with the identification of compounds in the environment that might be hazardous to human health: measuring exposure levels, measuring cellular damage and then estimating the probability of harm occurring. The feasibility of such a comprehensive approach has been explored in this study of two groups of workers, one of which may be occupationally exposed to exhaust fumes. No statistically significant difference in cellular damage, as measured by the lymphocyte micronucleus assay, was found between these two groups of workers, although clear differences in exposure levels to volatile organic compounds were detected. A number of other factors identified in the study did show clear effects on micronucleus frequency.

A study of the aneugenic activity of trichlorfon detected by centromere-specific probes in human lymphoblastoid cell lines.

The potential of the pesticide trichlorfon to induce mitotic aneuploidy has been investigated in genetically engineered human lymphoblastoid cell lines. Trichlorfon failed to induce micronuclei in the AHH-1 and MCL-5 cell lines when treated in media at normal cell culture pH (pH 7.3). Under a treatment pH of 5.5, trichlorfon exposures resulted in the induction of both chromosome loss and chromosome non-disjunction as measured by fluorescence in situ hybridisation (FISH) using a pan-centromeric probe for all human centromeres and centromere probes specific for chromosomes 2, 7 and 18. At treatment concentrations greater than 20 micrograms/ml trichlorfon also induced structural chromosome damage resulting in the production of centromere negative micronuclei.

A comparison of conventional metaphase analysis of Giemsa-stained chromosomes with multi-colour fluorescence in situ hybridization analysis to detect chromosome aberrations induced by daunomycin.

Chromosome aberrations induced by daunomycin, a widely used positive control compound for in vitro cytogenetics assays, were identified by multi-colour fluorescence in situ hybridization with probes for chromosomes 1, 2 and 3. The frequency and distribution of aberration types were compared to conventional metaphase analysis of Giemsastained chromosomes from parallel human lymphocyte cultures. Multi-colour chromosome painting was a more sensitive method for detecting daunomycin-induced chromosome aberrations compared with conventional metaphase analysis because: (i) a higher level of statistical significance was achieved at low doses; and (ii) the increases in aberration frequencies compared with controls were greater. The majority of exchanges identified by Giemsastaining were unstable and were likely to lead to cell death. In contrast, those detected by FISH were mostly stable exchanges which may be transmitted to cell progeny. Multicolour FISH using whole chromosome probes may provide an elegant solution to the problem of identifying non-lethal, heritable exchange events. The benefit of this technique is the quantification of a cytogenetic endpoint directly associated with carcinogenesis.