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J M Parry

Publications and source records attributed to J M Parry.

At least 55 records · Page 3Linked to original sources
PubMed2000

The mammalian gene mutation database.

The Mammalian Gene Mutation Database (MGMD) is a comprehensive collection of published mutation data from the open literature on mammalian cell-based gene model mutation detection systems. The database currently contains approximately 30000 comprehensively described mutant spectra records and it is maintained and up- dated on a daily basis. The major objectives of the MGMD were (i) to provide an Internet-accessible database (http://lisntweb.swan.ac. uk/cmgt/index.htm) for chemically induced and spontaneous mutation types and spectra in selected genes; (ii) to standardize the reporting of mutations within different genes where ambiguity exists in the literature; and (iii) to provide interactive and user-friendly access to the information. A multi-option search facility has been included that allows the user to search the database for parameters such as mutagen, gene or cell type of interest. The structure of the database permits easy retrieval of specific mutation data for further analysis. Thus, the MGMD should become a useful and necessary reference source and provides an analysis tool for genetic toxicologists.

Animals↗
PubMed2000

PCR-based methodology for the determination of the photoprotection afforded by sunscreen application.

We have applied a PCR-based methodology to study the DNA damage induced by UV-A, UV-B and sunlight itself. Our results, employing a cell-free system, indicate that UV-B (310 nm) is approximately 30-fold more potent at inhibiting DNA synthesis than UV-A (365 nm). We were also able to show that 20 min of sunlight exposure on a summer day induced DNA damage capable of inhibiting DNA synthesis. Hence, this methodology has a sensitivity suitable to detect biologically relevant doses of UV light. In addition, we propose that this technique may be suitable to assess the relative photoprotection of commercially available sunscreens. We present here preliminary data on the photoprotection afforded by the topical application of sunscreen. This photoprotection was measured by a reduction in the subsequent UV-B- and UV-A-induced DNA damage when sunscreen was applied. Our results demonstrate that the particular sunscreen tested was effective against both UV-B and UV-A. However, the estimated photoprotective factor of the sunscreen (against both UV-A and UV-B) was approximately tenfold less than the stated Sun Protection Factor (SPF) of 25. This methodology may also be useful in identifying new photoprotective agents by assessing their relative value as UV-B and UV-A absorbing agents.

Animals↗
PubMed2000

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.

Adult↗
PubMed1999

An assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in selected invertebrate and vertebrate species.

The grounding of the Sea Empress oil tanker resulted in the release of 72,000 tonnes of crude oil into Milford Haven, Wales, UK. Our initial studies indicated that this contamination resulted in elevated levels of DNA adducts in one of the area's native marine species Lipophrys pholis [B.P. Lyons, J.S. Harvey, J.M. Parry, An initial assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in the intertidal teleost Lipophrys pholis, Mutat. Res. 390 (1997) 263-268]. These original studies were extended and the genotoxic impact of the oil contamination was investigated in the invertebrates Halichondria panicea and Mytilus edulis, along with the vertebrate fish species L. pholis, Pleuronectes platessa and Limanda limanda. DNA adduct levels were assessed in these species over a period of 2-17 months after the incident. The studies indicate differences in the impact of acute oil contamination upon vertebrate and invertebrate species. The oil contamination did not induce any detectable elevations in adduct levels in the invertebrate species H. panicea and M. edulis. In contrast, the oil contamination did appear to induce adducts in the vertebrate teleost species L. pholis, P. platessa and Lim. limanda. Despite some difficulties in sampling, the data obtained 12-17 months after the spill suggested that the affected species recovered from the oil contamination. While the studies indicate that the genetic impact of the oil contamination was less severe than might have been expected, it remains possible that the DNA adducts detected in the teleosts could lead to genetic changes in these species in the future.

Animals↗
PubMed1999

P53 integrity in the genetically engineered mammalian cell lines AHH-1 and MCL-5.

Recently, a C to T transition mutation in exon 8 of the p53 gene has been identified in a subculture of the genetically engineered human lymphoblastoid cell line AHH-1 and this mutation was proposed to cause a loss of function of the p53 suppressor protein and may limit the use of this cell culture in genotoxicology test assays. This led us to investigate early passage cultures of AHH-1 and its derivative MCL-5 to determine the distribution of the mutation. In order to characterise the presence of mutations at the p53 locus, exon 8 was analysed using restriction enzyme analysis and automated sequencing to locate possible changes of sequence. Mutations were identified at codon 282, and treatment with the Msp1 restriction enzyme led to incomplete digestion suggesting the presence of heterozygosity at the site which was confirmed by sequencing. Our results indicate that the p53 gene is heterozygous at the interface between the codons 281 and 282 in both AHH-1 and MCL-5. An Annexin V labeling study was carried out and both AHH-1 and MCL-5 cell lines were shown to undergo DNA damage induced cell death after a 1-h exposure to MNNG.

Animals↗
PubMed1999

Influence of volume of work on the outcome of treatment for patients with colorectal cancer.

BACKGROUND: Recent recommendations for the reorganization of cancer services emphasize the importance of a 'minimal acceptable volume of work'. The influence of both hospital and surgical workload has been examined using a population-based series of patients with colorectal cancer. METHODS: Nine hundred and twenty-seven patients with primary colorectal cancer diagnosed during the period 1 January to 30 June 1993 were identified from the North Western Regional Cancer Registry. Case notes were reviewed for information on patient age and sex, histological diagnosis, disease stage, degree of tumour differentiation, mode of admission, identity of operating surgeon, timing of operative procedure, and use of radiotherapy and/or chemotherapy. A multivariate Cox proportional hazards model was then constructed to examine, simultaneously, the effects of patient-, disease- and health service-related variables on survival. RESULTS: Age, tumour stage and differentiation, and mode of admission were revealed as significant independent prognostic variables. After adjusting for these variables, neither operator grade (consultant versus junior), consultant workload nor hospital throughput were identified as independently influencing patient survival. CONCLUSION: The results of this study do not support an association between volume of work and patient outcome.

Adult↗
PubMed1999

Inverse restriction site mutation (iRSM) analysis. Mutation detection involving the formation of restriction enzyme sites in target genes.

This paper describes a rapid screening procedure for the detection of DNA sequence changes resulting in the creation of new restriction enzyme sites. The basic methodology involves the identification of the conversion of one restriction site into another by mutagenesis. The selective removal of the wild-type sequences by digestion with a restriction enzyme acting on the wild-type sequence increases the sensitivity beyond that of PCR-RFLP analysis (10(-4)-10(-5) detectable here). In this paper we describe the rapid detection of induced in vivo mutations transforming the ApaI restriction site present in intron 6 of the mouse p53 gene to a unique AvaII site. The potential application of this method in other genes and organisms as a rapid screen for induced mutations is discussed.

Animals↗
PubMed1999

Anaphase aberrations in the embryos of the marine tubeworm Pomatoceros lamarckii (Polychaeta: Serpulidae): a new in vivo test assay for detecting aneugens and clastogens in the marine environment.

The marine environment receives a wide variety of chemical inputs, many of which have the potential to damage DNA or interfere with the process of cell division. Here we describe a new assay based on the early embryo and larval stages of a planktonic spawning, tube dwelling marine worm, Pomatoceros lamarckii, which for experimental purposes has the advantage of producing large numbers of ripe gametes throughout the majority of the year. One of the most promising end-points is the use of dividing cells to detect anaphase aberrations such as lagging chromosomes, tripolar anaphases, acentric fragments and chromosome bridges. Apart from the reference mutagens mitomycin C and cyclophosphamide and the well-documented spindle poison colchicine, we tested the fungicide carbendazim, a primary metabolite of the fungicide benomyl, and thiabendazole, a pesticide and antihelminthic drug; both of which are known to act as aneugens in other test systems. In addition we tested sodium hypochlorite, a widely used oxidizing agent and disinfectant, di-butylphthalate, a commercial plasticizer and suspected aneugen, and sodium chloride, a recognized non-genotoxin. Significant increases in the frequency of anaphase abnormalities occurred with most test compounds at relatively low concentrations, confirming the sensitivity of the new assay. Sodium chloride yielded a negative response except at the highest non-relevant concentrations, where some chromatid stickiness was observed. In addition, the developmental consequences of exposure to these compounds were assessed in 4-8 cell embryos and at 48 h once the embryos had metamorphosed into free swimming larvae. Mitotic inhibition and anaphase aberrations were found to be a more sensitive indicator of genotoxic exposure than larval development, although there was a suggestion of a possible mechanistic link between aneugenicity/clastogenicity and larval fitness. The new test assay provides a rapid and inexpensive method for screening chemicals and effluents destined for release into the marine environment for potential gamete effects.

Animals↗
PubMed1999

The restriction site mutation assay: a review of the methodology development and the current status of the technique.

The restriction site mutation (RSM) assay has been employed in our laboratory, as a mutation detection system, since its first description in 1990. In principle the technique is capable of detecting mutations in ubiquitous restriction enzyme sites and is, therefore, readily applicable to any sequenced gene and/or organism. The RSM assay has been applied in our laboratory in various species, detecting rare mutations induced in mouse, rat, Xenopus, flatfish and human cells and tissues. This paper reviews the data accumulated by the RSM methodology in our hands and charts the developmental processes which have steadily improved the technique such that it is now applicable as a sensitive genotypic mutation detection system. This paper also includes PCR primer sequences and restriction enzymes employed in mutational analyses performed in the various species studied. We detail a variety of problems associated with the assay and the steps taken to solve them. The specific hurdles which have been overcome include the lack of quantitative data, the question of the contribution of DNA adducts to the induced mutation profile and the presence of false positives. Finally, the methods which have been developed to increase the sensitivity of the assay are also detailed. This paper describes our recommended RSM methodology, as it is routinely employed in our laboratory, which enables the analyses of mutations induced by chemical exposures and spontaneous endogenous processes. Our aim in presenting the developmental data on the RSM assay is to provide other researchers with sufficient information about the RSM methodology to facilitate its application in mutation analysis in other genes and organisms.

Animals↗
PubMed1999

An investigation of male-mediated F1 effects in mice treated acutely and sub-chronically with urethane.

In order to investigate the alleged potential of paternally administered urethane to cause foetal abnormalities and heritable tumours, male CD-1 mice were treated with urethane, either acutely by intraperitoneal injection at doses of 1.25 and 1.75 g/kg bodyweight (bwt) or sub-chronically in the drinking water at 1.25 for 10 weeks, and 3.75 mg/ml for 9 weeks or vehicle for the control groups. They were mated to untreated females 1 week later. Uterine contents of half the pregnant females were examined just before full term, while the remaining females were allowed to deliver their litters. The resulting F1 offspring were observed for approximately 18 months and 12 months for acute and sub-chronic exposures respectively and subjected to necropsy examination. Some of the mice treated acutely with 1.75 g/kg bwt exhibited partial infertility but none of those treated with 1.25 g/kg bwt had an adverse effect on their reproductive ability. There was no genetic effect of acute urethane treatment on male germ cells as indicated by dominant lethality. After birth, there was an increase (P < 0.05) in post-implantation deaths possibly due to perinatal mortality. There was an increased incidence and earlier onset of liver tumours induced in F1 male offspring from F0 males treated with 1.75 g/kg bwt, (20.7% vs. 10.1%, P = 0.026) but not in the female offspring. F1 males from both treatment groups had mean bodyweights significantly higher than controls (P < 0.01). Some males from each dose group of the acute study were examined using the restriction site mutation assay involving analysis of exon sequences. No mutations were identified in testes, liver or spleen of DNA isolated from the urethane-treated animals. No reproductive or genetic effects were seen with sub-chronic treatment at either 1.25 or 3.75 mg/ml urethane nor was there any predisposition of F1 animals to tumours although observation times were shorter.

Abnormalities, Drug-Induced↗
PubMed1999

A study of ENU-induced mutagenesis in the mouse using the restriction site mutation (RSM) assay.

We report here the application of the restriction site mutation (RSM) assay to study the induction of mutations by the alkylating agent ENU. Specifically, mutations were sought in the spleen and bone marrow of mice 3, 10, and 100 days after being treated with ENU; this was compared to data previously published from our laboratory on ENU-induced testes mutations. It was found that the ENU-induced mutations were all at GC bases implicating the O(6)-ethylguanosine adduct. The mutations detected reached a peak at day 10 in the spleen and were detectable to a lesser extent at 100 days, which is similar to the testes data. In the bone marrow, the mutation level rose until day 100, although the level remained below that of the spleen and testes. However, by studying the mutations detected in control animals, it was found that spontaneous mutational events were detectable at the day 100 time point in all three tissues. Hence the spleen, testes, and bone marrow mutations at day 100 in the ENU-treated samples were probably spontaneous mutational events with very few genuine ENU-induced mutations remaining in any of these tissues after 100 days. This paper also demonstrates the applicability of the inverse RSM methodology in the detection of ENU-induced mutations, whereby mutations can be detected by the conversion of one restriction site to another. The iRSM assay appears to be particularly suitable to studying alkylating agents due to their known sequence specific mutation induction. We also show a comparison of the bone marrow micronucleus data with the RSM assay and show that both assays are capable of detecting the genotoxicity of ENU to the mouse bone marrow in vivo.

Animals↗
PubMed1998

Mutation analysis using the restriction site mutation (RSM) assay.

The restriction site mutation (RSM) assay (see Steingrimsdottir et al. [H. Steingrimsdottir, D. Beare, J. Cole, J.F.M. Leal, T. Kostic, J. Lopez-Barea, G. Dorado, A.R. Lehmann, Development of new molecular procedures for the detection of genetic alteration in man, Mutat. Res. 353 (1996) pp. 109-121] for a review) has been developed as a genotypic mutation detection system capable of identifying mutations occurring in restriction enzyme sites of genomic DNA. Here we will report the steps taken to overcome some of the initial problems of the assay, namely the lack of quantitative data and limited sensitivity, the aim being to achieve a methodology suitable for the study of low dose chemical exposures. Quantitative data was achieved in the RSM assay by the inclusion of an internal standard molecule in the PCR amplification stage, thus allowing the calculation of both spontaneous and induced mutation frequencies. The sensitivity of the assay was increased through the discovery that intron sequences of genomic DNA accumulated more mutations in vivo compared to the exons, presumably due to differential selective pressure within genes [G.J.S. Jenkins, I.deG. Mitchell, J.M. Parry, Enhanced restriction site mutation (RSM) analysis of 1, 2-dimethylhydrazine-induced mutations, using endogenous p53 intron sequences, Mutagenesis 12 (1997) pp. 117-123]. This increased sensitivity was examined by applying the RSM assay to analyse the persistence of N-ethyl-N-nitrosourea (ENU)-induced mutations in mice testes. Germ line mutations were sought in testes DNA 3, 10 and 100 days after ENU treatment. Mutations were detected in exons and especially intron regions, the intron mutations were more persistent, still being detected 100 days post-chemical treatment. Assignment of these mutations as ENU induced was complicated in some cases where the spontaneous mutation level was high. This theme of mutation persistence was further investigated by studying the presence of 4-nitroquinoline-1-oxide (4-NQO)-induced DNA mutations in vitro. This study also analysed the relationship between DNA adduct formation and DNA mutation induction by the concurrent RSM analysis and 32P post-labelling analysis of 4-NQO treated human fibroblasts. The results demonstrated that early DNA mutations detected 4 days post-treatment by the RSM assay were probably ex vivo mutations induced by Taq polymerase misincorporation of 4-NQO adducted DNA, due to the maximum levels of 4-NQO adducts being present at this time point. A later mutational peak, after the adduct level had declined, was assumed to be due to DNA sequence changes produced in the fibroblasts by the in vivo processing of DNA adducts.

4-Nitroquinoline-1-oxide↗
PubMed1998

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.

Aneuploidy↗
PubMed1998

The analysis of DNA adduct formation, removal and persistence in the common mussel Mytilus edulis exposed to 4-nitroquinoline 1-oxide.

32P-postlabelling was used for the detailed analysis of 4-nitroquinoline 1-oxide (4-NQO) induced DNA adduct formation, removal and persistence in the marine shellfish Mytilus spp. The results had a number of important implications concerning the use of such DNA adducts as dosimeters of environmental genotoxin exposures. Our studies indicated that the maintenance of the Mytilus specimens under controlled laboratory conditions can result in the induction of 'stress-related adducts' seemingly related to the nature of the experimental set-up. The studies also indicated that the absorption and activation of genotoxins in this species appear to affect the rate of adduct formation, and that the maximum levels of adducts may not necessarily be induced immediately after the cessation of a genotoxin exposure. In addition, Mytilus specimens were shown to possess a significant capacity to remove these genotoxin-induced DNA adducts. The removal of these adducts appeared to be biphasic in nature, with the rapid removal of a large proportion of adducts occurring within 48 h of the cessation of the exposure, followed by a slow rate of adduct removal over the remaining period of the studies. Despite the relatively efficient removal of the majority of these genotoxin-induced DNA adducts, a proportion remained up to 56 days after the initial exposure. The persistence of these genotoxin-DNA adducts, combined with the information on the rates of adduct removal, indicated that under well-defined conditions, such adducts could serve as suitable biomarkers of environmental contamination.

4-Nitroquinoline-1-oxide↗
PubMed1998

Evaluation of the comet assay as a method for the detection of DNA damage in the cells of a marine invertebrate, Mytilus edulis L. (Mollusca: Pelecypoda).

The potential application of the comet assay for monitoring the effect of DNA damaging agents on the marine mussel, Mytilus edulis (an important pollution indicator organism), was explored. A detailed investigation of the baseline levels of single-strand breaks in isolated gill cells, and how they were affected by age/size of animal, time since collection, feeding regime, in vivo vs. in vitro exposure conditions, and by antioxidant supplementation was undertaken. The level of cometing in untreated controls was found to be highly variable over time (fluctuations between low and very high DNA damage occurred over just 14 days post collection). No difference was observed between age/size and feeding regime of the animals. On exposure to 0, 100, 500 and 1000 microM H2O2, it was observed that the in vitro exposure produced a markedly more homogeneous dose response compared to the in vivo studies (where gill cells were exposed as a tissue). An important finding of our research was the effect of prior supplementation of the animals' diet with 1 mg/ml alpha-tocopherol acetate (vitamin E compound), which resulted in a marked reduction in the levels of DNA damage expressed by the negative controls, without influencing the actual response to H2O2 (0, 5, 25, and 100 microM) and N-nitrosodimethylamine, NDMA (0, 5, 25, and 100 mM). The effect of vitamin E supplementation was to increase the sensitivity of the comet assay at the lower end of the dose range. This study demonstrated the potential application of the comet assay to the gill cells of the mussel, M. edulis. Although preliminary findings suggest that antioxidant supplementation can improve the sensitivity of the assay by lowering the baseline damage in untreated animals, our conclusion is that the assay has more potential for use in an in vitro context for the screening of agents destined for release or disposal into the marine environment.

Animals↗
PubMed1998

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.

Chromosome Segregation↗