James M. Parry

Radiation-induced transgenerational alterations in genome stability and DNA damage

Mutation induction in directly exposed cells is currently regarded as the main component of the genetic risk of ionizing radiation for humans. However, recent data on the transgenerational increases in mutation rates in the offspring of irradiated parents indicate that the genetic risk could be greater than predicted previously. Here, we have analysed transgenerational changes in mutation rates and DNA damage in the germline and somatic tissues of non-exposed first-generation offspring of irradiated inbred male CBA/Ca and BALB/c mice. Mutation rates at an expanded simple tandem repeat DNA locus and a protein-coding gene (hprt) were significantly elevated in both the germline (sperm) and somatic tissues of all the offspring of irradiated males. The transgenerational changes in mutation rates were attributed to the presence of a persistent subset of endogenous DNA lesions (double- and single-strand breaks), measured by the phosphorylated form of histone H2AX (γ-H2AX) and alkaline Comet assays. Such remarkable transgenerational destabilization of the F1 genome may have important implications for cancer aetiology and genetic risk estimates. Our data also provide important clues on the still unknown mechanisms of radiation-induced genomic instability.

A comparison of two in vitro mammalian cell cytogenetic assays for the detection of mitotic aneuploidy using 10 known or suspected aneugens

Two in vitro cytogenetic assays were evaluated for their ability to detect aneugenic and polyploidy-inducing agents using a battery of 10 known or suspected aneugens supplied as part of the EEC 4th Environmental Research and Development Programme. The compounds tested were colchicine, vinblastine, chloral hydrate, thiabendazole, hydroquinone, thimerosal, cadmium chloride, econazole nitrate, pyrimethamine and diazepam. The cell division aberration assay employed a differential chromosome/spindle staining procedure to detect perturbations of the mitotic division apparatus. This assay was carried out in two pulmonary-derived Chinese hamster cell lines; the immortal DON:Wg3h culture and a low passage LUC2 culture. The second assay involved quantification of metaphase chromosomes, for which only the LUC2 cell line was used, due to the stability of its diploid karyotype. All the chemicals induced spindle disturbances in the immortal line. In addition, all the compounds except cadmium chloride yielded positive results in the LUC2 culture, although many were not as potent. In the low passage line, 8 of the compounds (colchicine, vinblastine, chloral hydrate, thiabendazole, thimerosal, econazole nitrate, pyrimethamine and diazepam) induced aneuploidy and/or tetraploidy. Cadmium chloride was negative in the chromosome enumeration assay and hydroquinone yielded inconclusive results. The study of cell division aberrations was much less time-consuming and technically complex than the counting of metaphase chromosomes. In addition, it provided a degree of mechanistic understanding of the mode of action of some aneugenic and polyploidy-producing agents. However, the enumeration of chromosomes provides a more definitive data set for the evaluation of a chemicals aneugenic potential.

The effects of benzodiazepines upon the fidelity of mitotic cell division in cultured Chinese hamster cells.

4 benzodiazepine sedatives, namely diazepam, medazepam, midazolam and bromazepam were investigated for their effects upon the fidelity of cell division in both low passage number and immortalised Chinese hamster cell lines. The study revealed substantial differences in the effect of these structurally related drugs upon mitosis, which may reflect different mechanisms of action of the drugs in cultured cells. Diazepam and medazepam exposure of immortal and low passage number cells resulted in the formation of monopolar mitotic spindles and subsequent metaphase arrest. The production of these spindles may be explained by the inhibition or centriole separation . In contrast, midazolam and bromazepam failed to produce observable changes in spindle structure. All 4 benzodiazepines produced significant toxicity in low passage number cells whereas, immortalised cells were more resistant to their toxic effects. They all induced metaphase chromosome dislocations in immortalised cells, whereas only diazepam and medazepam produced such effects in the low passage number cell line. In general, immortal cells appeared to be less sensitive to the toxic effects of benzodiazepines than the low passage number cells.

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 32u2009000 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 Correas 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. Helicobactor 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.

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.

The detection, definition and regulation of aneugenic chemicals

The stability of the genome (i.e. of chromosome number and structure) is dependant upon the coordinated functioning of the mitotic and meiotic cell division cycles. Events of significance in stability include both chromosome replication and segregation. These processes involve the functioning of a series of inter-related cell organelles and coordinated activities such as the synthesis and functioning of the proteins of the cell division spindle and the attachment and movement of the chromosomes on the spindle apparatus. Modifications of the functioning of the cell division apparatus may lead to aberrations of chromosome segregation and the production of cells with abnormal chromosome numbers, whether a multiple of the complete karyotype (polyploidy) or deviations from the normal number of individual chromosomes (aneuploidy). When aberrant segregation is symmetrical and produces progeny cells in which one cell gains a chromosome (eg. 2n+1 or n+1) and the other looses a chromosome (eg. 2n-1 or n-1) the event is called non-disjunction. However, non-disjunction is only one of a number of events which result in numerical change and in aneuploidy screening systems reciprocal products are only rarely detected.