Mutagenesis, Vol. 14, No. 6, 639-656,
November 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Meeting Report |
Abstracts of the United Kingdom Environmental Mutagen Society 23rd Annual General Meeting, June 23–25, University of Ulster, Coleraine, Northern Ireland, UK
1. Novel role for DNA repair in the genotoxic effects of haloalkanesNieves Abril and Geoff Margison
CRC Section of Genome Damage and Repair, Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Manchester M20 4BX, UK
Halogenated aliphatic hydrocarbons such as 1,2-dibromoethane (DBE) and dibromomethane (DBM) have been widely used as pesticides, solvents and intermediates in agricultural and industrial chemistry. They are mutagens and are carcinogenic, teratogenic and embryotoxic in laboratory animals. The two principal routes of metabolic transformation of DBA in mammalian cells are GST-mediated conjugation with GSH and cyP450-mediated oxidation.
The effect of expression of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, on the genotoxic effects of DBM and DBE was determined in Chinese hamster lung fibroblasts transfected with and expressing high levels of Escherichia coli alkyltransferase (ATase) genes. These included the ogt gene and complete or truncated versions of the E.coli ada gene encoding either O6-alkylguanine (O6-alkG) or alkylphosphotriester (alkPT) ATase activities. As previously observed, the O6-alkG ATase in these cells protect against the growth inhibitory effects of the methylating and chloroethylating agents. However, cells expressing the full length or the O6-alkG ATase region, but not the alkPT ATase region, of Ada were found to be more sensitive to the growth inhibitory effects of the DBA; ogt expression sensitized cells to DBM but not significantly to DBE. Addition of DBA to cell extracts depleted O6-alkG ATase activity on the methylated DNA substrate, but was without effect on alkPT ATase activity. This suggests that ATase-mediated sensitization of the intact cells may be related to the inactivation of the ATase protein. Addition to the cell culture medium of GSH or buthionine sulphoximine to respectively augment or deplete cellular GSH had no marked effect on the ATase-mediated sensitisation to DBA. The effect may therefore be mediated by a DNA adduct caused by the oxidative metabolic pathway but the mechanism has yet to be defined. We conclude that ATase may have an unexpected detrimental effect on mammalian cell sensitivity to environmentally relevant alkylating agents.
2. DNA repair in plants studied by the comet assay
K.J.Angelis
Institute of Experimental Botany ASCR, Na Karlovce 1, 160 00 Praha 6, Czech Republic
By using isolated nuclei instead of whole cells we have adapted a comet assay for virtually any plant. In this study we compared Vicia faba, a `standard' plant for cytogenetic studies, and Arabidopsis thaliana, which because of its small genome size, genetic characterisation, possibility of genetic manipulation and modest requirement for propagation is at present the most widely used model plant. Moreover, availability of well characterized mutants and of assays for mutagenesis makes this plant particularly suitable for the study of DNA damage and repair.
We used N'-methyl-N'-nitrosourea to show rapid repair of DNA damage detected under neutral conditions and of AP-sites, contrary to persistence of DNA damage for more than 24 h under alkaline assay conditions.
We also show that the phenomenon of clastogenic adaptation described in V.faba can be also detected by the comet assay. Surprisingly `clastogenic' adaptation followed by the comet assay can be induced in Arabidopsis.
3. DNA damage and apoptosis
Colin Arlett
MRC Cell Mutation Unit, University of Sussex, Brighton BN1 9RR, UK
Apoptosis is frequently invoked as a cancer avoidance mechanism whereby cells carrying potentially carcinogenic mutations are voided from the system. We questioned whether the induction of apoptosis is a universal response of human cells to two proven mutagens: ultra-violet 280–315 nm (UVB) and ionising (
) radiation using two assay systems: in situ end-binding and time lapse videomicroscopy (VDM). Two categories of cells were examined, fibroblasts or keratinocytes derived from normal or radiation hypersensitive DNA repair donors. We were able to utilise SV40-T immortalised fibroblasts or transformed keratinocytes as surrogate cancer cells.
UVB induces apoptosis in transformed but not primary normal and xeroderma pigmentosum (XP) fibroblasts. The XP fibroblasts are not more sensitive than normal cells. Both primary and transformed keratinocytes are susceptible to UVB-induced apoptosis. KB cells are significantly more sensitive than untransformed keratinocytes and the response of the p53–/–HaCaT cell line is very similar to untransformed cells confirming the existence of a p53-independent pathway of UVB-induced apoptosis. Keratinocytes examined in ex vivo experiments on skin from XP patients from complementation groups A, C and D are significantly more sensitive to the induction of apoptosis than cells from normal donors. These observations show that XP patients do not owe their cancer proneness to a lack of apoptosis.
For -irradiation, no apoptosis is seen in either untransformed normal or ataxia-telangiectasia (A-T) fibroblasts, there is a weak positive response in SV40-T transformed fibroblasts from A-T but not normal donors. Transformed but not primary keratinocytes are susceptible to -irradiation-induced apoptosis. This was confirmed by their response to the topoisomerase II inhibitor mitoxantrone which, like -irradiation, generates DNA double strand breaks. Both -irradiation and mitoxantrone-induced apoptosis becomes apparent 40 h later than with UVB suggesting different and possibly cell-cycle-related processes dependent upon the nature of DNA damage.
These results reveal the heterogeneity of the apopogenic response showing differences between cell types, transformation status and classes of DNA damage.
4. Two forms of coal tar genotoxicity and mutagenicity evaluation and analysis of their phototoxicity and photogenotoxicity
C.Baudouin, C.Vaissière, M.Charvéron and Y.Gall
Institut de Recherche Pierre Fabre, Laboratoire de Biologie Cellulaire Cutanée, Faculté de Médecine Rangueil, 133 Route de Narbonne, 31062 Toulouse, France
Coal tars are used in dermatology in the treatment of skin disorders like eczema and psoriasis. In addition, distilled or refined tars are included in the composition of dermocosmetic products. Coal tars are composed of numerous particularly toxic polyaromatic molecules like benzo(a)pyrene [B(a)P] which has been widely reported to be genotoxic and carcinogenic.
In order to evaluate the carcinogenic power of two forms of coal tar (topical, CTT, and distillate, CTD), we have determined their genotoxicity and mutagenicity in comparison with B(a)P. The genotoxicity and mutagenesis analyses have been investigated on Chinese hamster ovary cell line (treated by a metabolic activator) using the comet assay and the mutagenesis test based on selective cloning on the hypoxanthine phosphoribosyl transferase (HPRT) locus, respectively. Comparing tail moment, we have shown that B(a)P was powerfully genotoxic (from 1 µg/ml), CTT was genotoxic (from 1 µg/ml) and CTD was not genotoxic. Analysing the number of HPRT–mutants/106 viable cells, we have clearly demonstrated that B(a)P was very mutagenic (from 0.01 µg/ml), CTT was mutagenic (from 0.3 µg/ml) and CTD was not mutagenic.
The chemical structure of the polyaromatic compounds contained in coal tars can be modified, in particular during ultraviolet irradiation. Once photo-activated, these molecules acquire new physical–chemical properties and become very often phototoxic, photogenotoxic and can induce increased photomutagenesis responsible for carcinogenesis promotion. Using a well-known photosensitiser (chlorpromazine) as positive control, we have evaluated the phototoxicity and photogenotoxicity of the two forms of coal tars (CTT and CTD). To assess their phototoxicity, we have used a protocol validated by the European Community and based on a comparison between the cytotoxicity (revealed by neural red) of the irradiated coal tar with that of the non-irradiated molecule. The ratio of IC50 (–UVA)/IC50 (+UVA) has demonstrated that the CTT and CTD were both phototoxic. The photogenotoxicity was evaluated using the comet assay and an in vitro technique indirectly detecting DNA damage on plasmid DNA. But using the two genotoxicity tests, only CTT was photogenotoxic. Indeed, photoactivation by UVA and UVB of CTD did not induce DNA damage.
In conclusion, there are notable differences in the toxic effect induced by the two forms of coal tar: all products are toxic and phototoxic but only CTT is genotoxic, photogenotoxic and mutagenic. CDD is devoid of genotoxicity, photogenotoxicity and mutagenicity.
5. Lack of genotoxic activity of trichloroethylene and its metabolite S-(1,2-dichlorovinyl)-L-cysteine in rat renal proximal tubular cells in vivo using the comet assay
Philip Clay, Barry Elliott, Eryl Jones, Trevor Green and Rob Lee
Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK
Low incidences of renal tumours have been reported in several lifetime studies in which rats were exposed to high doses of trichloroethylene (TCE) either by gavage or by inhalation. A minor metabolic pathway of TCE (<0.05% of the dose) has been reported which leads to the formation of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a nephrotoxin in the rat and a bacterial mutagen in Salmonella typhimurium. DCVC is activated by the renal enzyme ß-lyase which is localised in the pars recta of the proximal tubule. The in vivo comet assay is designed to detect DNA breaks following treatment of animals with a test substance causing DNA damage. In this study, the genotoxicity of both TCE and DCVC has been evaluated in renal proximal tubular cells following in vivo exposure of rats. The formation of comets was assessed in cells isolated from rats immediately following five, daily, 6 h inhalation exposures to a range of concentrations of TCE using whole body inhalation chambers. Similar assessments were made 2 and 16 h after a single oral dose of DCVC. The highest exposure concentration used for TCE was four-fold higher than that known to cause kidney tumours in lifetime bioassays, and the highest dose of DCVC was approximately three orders of magnitude higher than the amounts of DCVC formed following exposure of rats to TCE. No substantial increases in tail length, % tail DNA or tail moment were seen for any of the groups treated with TCE. Isolated, small increases were seen in some animals treated with DCVC but these were insufficient to indicate a positive response in this assay. These data lend support to the hypothesis that TCE-related tumours in the renal proximal tubules of the rat are produced by a non-genotoxic mechanism
6. Evaluation of the direct count scoring method for the autoradiographic unscheduled DNA synthesis assay
Philip Clay, Mandy Lane, Eryl Jones and Barry Elliott
Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK
The endpoint of the UDS assay involves assessing the number of silver grains present in the emulsion overlying the cells under investigation. Previously, most users of the assay have estimated the number of grains by an area count method of image analysis as recommended by Kennelly et al. (1993). This method has been preferred historically due to the low resolution of the video image and the technology did not allow efficient separation of touching objects. Improvements in image analysis now allow accurate scoring of slides by the direct count method. To take advantage of the latest technology, we have developed a UDS scoring and data capture system in collaboration with Perceptive Instruments Ltd which has been designed to comply with current GLPs and the FDA ruling on electronic signatures (FDA 21 CFR 11). We have scored a range of slides by both methods and compared results with the definitive eye counts. The differences observed between the mean net nuclear grain counts achieved by area count and direct count methods were small and would not affect the qualitative result. Where differences did occur (generally with the higher counts seen on positive slides), the direct counts more accurately reflected the definitive eye counts than did the area counts. Using the direct count method offers significant savings in time by eliminating the need to calibrate each slide and has been shown, with this system, to be at least as accurate as the area count method. It is therefore considered that using available technology, the scoring of autoradiographic UDS slides is best achieved by the direct count method.
Reference
Kennelly et al. (1993) In vivo rat liver UDS assay. In Supplementary Mutagenicity Tests: UKEMS recommended procedures, Cambridge University Press, Cambridge, UK.
7. PRINS localisation of the telomeric sequence (TTAGGG)n in the rat karyotype indicates the presence of telomeric-like sites at the centromeres of some chromosomes
Chiara Corso and James M.Parry
Centre for Molecular Genetics and Toxicology, University of Wales Swansea, Swansea SA2 8PP, UK
The rat is an important model organism in biomedical research because it provides a well-characterized system for the broad spectrum of pathological traits, including cancer and inherited disease (Greenhouse et al., 1990). However, the limited knowledge of the rat genome and the lack of availability of rat probes restricts the analysis of the genetic aspects of the research. Telomeres of vertebrate organisms end in (TTAGGG)n repeat sequences of variable length. The presence of telomeric-like sequences has been documented at paracentromeric and intrachromosomal regions (interstitial sites, IS) in many vertebrates (Mayne et al., 1990). Meyne and co-workers, using FISH (fluorescence in situ hybridisation) with a synthetic probe for the telomeric regions, found a telomere-only pattern in Rattus norvegicus. However, in Rattus rattus an additional signal in the long arm of chromosome 2 was detected by the same group. In this study we used the PRINS (primed in situ synthesis) technique with (TTAGGG) primers in order to localise the presence of telomeric-like sequences in the rat genome. This approach permitted us to visualize, in the R.rattus karyotype, some paracentromeric regions not previously detected. Experimental evidence indicates that interstitial telomeric repeat sites are hot spots for chromosomal aberrations (Hastie and Allshire, 1989) suggesting a potential role in directing non-random patterns of genetic rearrangements. Moreover, our findings suggest the possibility of the isolation of these sequences and their eventual cloning as chromosome-specific probes. The analysis of telomeric-like sequences in the R.norvegicus genome is in process.
References
Greenhouse et al. (1990) In Hedrich,H. and Adams,M. (eds) Genetic Monitoring of Inbred Strains of Rats. Manual on Colony Management, Basic Monitoring Technique, and Genetic Variants of the Laboratory Rat 411-480, Gustav Fisher Verlag.
Meyne et al. (1990) Chromosoma, 99, 3–10.
Hastie and Allshire (1989) Trends Genet., 10, 326–331.
8. Alkali sensitive DNA strand breaks and evidence of repair in primary cells and tissue comparable cell lines after UV and clastogen exposure
S.Devery, W.Qin and P.T.Tomkins1
Toxicology Unit, Athlone Institute of Technology, Athlone and 1Bioserv Ltd, AIT Campus, Athlone, Ireland
As part of a larger programme to develop rapid organotypic, mechanistic screening assays for biomaterials toxicity we are evaluating the ability of DNA strand breakage and mutation detection assays to report damage in primary liver and skin cells and their cell line counterparts following exposure to established genotoxicants. The single cell gel electrophoresis assay (SCGE) was optimised for primary mouse hepatocytes and keratinocytes, the human hepatoma HepG2 cell line and the mouse keratinising teratoma XB-2 cell line using hydrogen peroxide and UV irradiation as positive control agents. Resultant DNA comets were stained with ethidium bromide and the tail moment resolved by image analysis (Perceptive Instruments). Both primary and immortalised cells exhibited similar basal control levels of strand breakage, but primary mouse hepatocytes were 1.5x less sensitive to H2O2 than HepG2 cells (EC50 402 and 300 µM, respectively, P < 0.01). This differential was even more pronounced in the keratinising epithelial cells where XB-2 cells were >6x more sensitive to H2O2 clastogenesis (EC50 287 and 47 µM, respectively, P < 0.001). These differences may reflect differences in compound permeability or metabolism or DNA repair.
Parallel studies of BrdUrd uptake in S-phase-inhibited cells did not suggest significant differences in repair capability between primary and immortalised cells. A similar pattern was seen when cells were exposed to 320 µW/cm2 for 0–20 min although prolonged exposure abolished both responses due to cytotoxicity. PCR based mutation screening of a 1100 bp region on human chromosome 17q is reporting similar dose-dependent trends which are proportional to the obviously restricted genome target size.
9. DNA damage, toxic vents and environmental contamination
D.R.Dixon, J.T.Wilson, V.Bombail and L.R.J.Dixon1
Southampton Oceanography Centre, Empress Dock, Southampton S014 3ZH and 1Marine Biological Association, Citadel Hill, Plymouth PL1 2PB, UK
Deep-sea hydrothermal vents are arguably one of the most toxic environments on the face of the planet. Apart from the high temperature and pressure, vent organisms are exposed to elevated levels of toxic heavy metals and radionuclides (arsenic, cadmium, mercury and radon), substances which have well-documented mutagenic and carcinogenic properties. Recent work carried out as part of the NERC-BRIDGE Special Topic and EU-funded VENTOX project has revealed surprisingly high levels of DNA damage in the cells of vent mussels (Bathymodiolus), which correlated with emission toxicity at specific vent fields on the mid-Atlantic Ridge (LUCKY STRIKE and RAINBOW). It would appear that, despite their long evolutionary association with environmental contamination (fossil evidence can be traced back to the Mesozoic), vent organisms are not fully resistant to the damaging effects of their toxic environment. Older (i.e. larger) mussels exhibited higher levels of DNA damage in their cell nuclei compared with younger (smaller) animals, indicating that the latter may be more efficient at repairing DNA lesions. Paradoxically, the deep-sea hydrothermal environment is characterised by extremely high growth rates fuelled mainly by bacterial chemosynthesis linked with the vent chemistry (hydrogen sulphide and methane). Rapid growth and early reproduction, previously thought of as characteristics which enable vent species to cope with the disjunct and temporally unstable nature of venting along the ridge axis, can equally well be seen as adaptations which allow them to survive in this highly toxic environment, e.g. through an increase in DNA repair activity linked with certain enzymes (telomerases) which are normally only active during embryogenesis. Investigations of the cellular and molecular biology adaptations of vent organisms hold great potential for biotechnological discoveries, particularly in the areas of DNA repair and bioremediation. Given their long geological history, deep-sea vents provide a unique evolutionary insight into the ways that marine organisms are able to adapt to extremely long-term contaminant exposure.
10. Uracil misincorporation in human DNA detected using a novel variation of the comet assay
Susan J.Duthie, Sabrina Narayanan and Gillian M.Brand
Rowett Research Institute, Greenburn Road, Aberdeen AB21 9SB, UK
Poor folate status has been implicated in the development of cancer. Folate deficiency may alter DNA stability by increasing uracil misincorporation, DNA double strand breakage, chromosome instability and ultimately cancer (Blount et al., 1997). We have modified the comet assay expressly to detect uracil misincorporation in isolated cells (Duthie and McMillan, 1997).
Human lymphocytes and human colonocytes (HCEC) carry uracil in their DNA. Folate deficiency in vitro induces uracil misincorporation in these cells (Table I
). Moreover, folate deficiency in vivo significantly increases the level of uracil in lymphocytes isolated from rats fed a folate-free diet for 8 weeks (Table I).
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This assay will prove valuable in determining the role of folic acid in the area of nutrient/gene interactions and DNA stability.
References
Blount et al. (1997) Proc. Natl Acad. Sci. USA, 94, 3290.
Duthie and McMillan (1997) Carcinogenesis, 18, 1709.
11. The polymerase arrest assay
P.M.Evans, L.Penrose, G.Jenkins and B.Burlinson
Glaxo Wellcome Research and Development, Ware, Herts, UK
The polymerase arrest assay (PAA) is currently being developed to detect DNA adducts using murine p53 gene sequences. This technique relies upon the behaviour of DNA polymerase enzymes when encountering DNA adducted bases. In general, polymerases either misinsert bases opposite sites of adduction, creating mutations, or the polymerase pauses at such sites, unable to insert a base. The latter behaviour is exploited by the polymerase arrest assay, which relies upon the detected reduction in PCR amplification efficiency associated with adducted DNA. The aim of the project is to develop a high throughput-screening assay for genotoxic chemical agents.
Several crucial factors in polymerase arrest became apparent when conducting optimisation experiments. These were: the choice of polymerase, the DNA concentration during treatment, the Mg/dNTP concentration during the PCR and the thermal profile of the PCR.
The assay has been optimised in order to improve sensitivity and has been shown to be able to detect UV adducts induced by doses of <100 J/M2.
In order to detect chemical agents, an immobilised polymerase arrest assay has been developed, facilitating the necessary washing steps to remove the chemical without losing the adducted DNA or affecting its concentration. Optimisation of this immobilised method has allowed the detection of ENU, MNU, EMS and MMS induced adducts.
12. The mouse lymphoma assay (MLA) using the microtitre methodology: historical data and cell cleansing
M.Fellows, J.Clements, J.Wilkinson and D.Kirkland
Covance Laboratories Limited, Harrogate, UK
Our laboratory has many years experience performing the MLA using the microtitre methodology. In light of the recent ICH recommendation indicating a preference for this protocol, we considered it would be of interest to present a review of our extensive historical data for negative and positive controls. The historical data presented show that a wide range of spontaneous mutant frequencies have been observed in our laboratory at different times (ranging from as low as ~60 to as high as ~300 mutants per 106 viable cells). Furthermore, on a day-to-day basis, high spontaneous mutant frequencies often tend to correlate with high positive control responses. Confidence limits for control responses have been calculated. The results show the importance of using historical data as a basis for assay acceptance criteria in relation to the positive response. We have also investigated how both the spontaneous and small colony mutant frequencies alter with time in culture. Following continuous culturing of cells without re-cleansing to remove TK–/– mutants, the spontaneous mutant frequency increases but the proportion of small colonies decreases. The effect of cleansing is to reverse this trend.
13. Mutagenicity of 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) in the germ cells of male lacZ transgenic mice (MutaTMMouse)
S.Fernando, A.Lynch1, A.Boobis and D.Davies
Department of Clinical Pharmacology, Imperial College, Ducane Road, London W12 ONN and 1SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, Herts AL6 9AR, UK
The genotoxic carcinogen PhIP, has been investigated for its mutagenicity in germ cells at the lacZ locus of male transgenic mice (MutaTMMouse). In an initial study male mice were administered 20 mg/kg p.o for 4 days and sacrificed 7 days later. High molecular weight testicular DNA was extracted, and the lambda vectors containing the lacZ reporter gene were recovered by in vitro packaging. Bacteriophage plaques were analysed after infecting Escherichia coli C (lacZ–galE–) under a positive selection system in the presence of phenyl-ß-galactoside. Suspected mutants were confirmed by re-plating with E.coli C (lacZ–) and X-gal. The mutation frequency (MF) was calculated for the PhIP-treated, and vehicle control groups. Initial analysis of the data revealed that PhIP caused a 2.5-fold increase in MF compared to the vehicle control, which is marginally positive under these experimental conditions. We investigated further these initial findings in a second study, undertaken with a similar treatment regime, but extended to included a longer manifestation period of 25 days (c.f. 7 days) to allow for germ cell maturation. The results obtained from this second study showed that PhIP caused only a 1.3-fold increase in MF above background. Our experiments suggest that on sub-acute exposure PhIP is equivocal as a germ cell mutagen.
14. Inducible global genomic repair but constitutive transcription-coupled DNA repair in human cells
Philip C.Hanawalt
Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA
There are two branches of excision repair, global genomic repair (GGR) that deals with lesions throughout the genome and transcription-coupled repair (TCR) that specifically targets lesions in the transcribed strand of expressed genes (Hanawalt, 1994). In mammalian cells only those genes transcribed by RNA polymerase II are subject to TCR and the mechanism is thought to involve the initial arrest of the translocating polymerase at the lesion site. The efficiency of TCR does not vary over the cell cycle for a gene that is continually expressed (Lommel et al., 1995). Efficient GGR of UV-induced cyclobutane pyrimidine dimers (CPDs) has been shown to require activation of the p53 tumor suppressor, although the efficient repair of the more structure-distorting 6-4 photoproduct is much less dependent upon p53 induction (Ford et al., 1995Ford et al., 1997). There is a remarkable parallelism between the regulation of GGR by the inducible SOS response in bacteria and the p53-dependent pathway on human cells (Crowley and Hanawalt, 1998). Recent studies have shown that, like p53-defective cells, xeroderma pigmentosum group E cells are deficient in GGR of CPDs, but proficient in TCR. The expression of the p48 xeroderma pigmentosum gene is dependent upon p53 and appears to be the link between p53 and GGR (Hwang et al., 1999). The implications of p53-dependent GGR and constitutive TCR will be discussed in terms of implications for cell survival, mutagenesis and oncogenic transformation. These findings may be relevant to the use of rodent models in carcinogenicity testing since the rodent cells are deficient in the inducible GGR of CPDs and perhaps some other types of DNA lesions.
References
Hanawalt (1994) Science, 266, 1957–1958.
Lommel et al. (1995) Mutat. Res., 336, 181–192.
Ford and Hanawalt (1995) Proc. Natl Acad. Sci. USA, 92, 8876–8880.
Ford and Hanawalt (1997) J. Biol. Chem., 272, 28073–28080.
Crowley and Hanawalt (1998) J. Bacteriol., 180, 3345–3352.
Hwang et al. (1999) Proc. Natl Acad. Sci. USA, 96, 424–428.
15. The Mammalian Gene Mutation Database
J.S.Harvey, E.Waters and J.M.Parry
Centre for Molecular Genetics and Toxicology, School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, USA
The Mammalian Gene Mutation Database (MGMD) is being constructed to provide a comprehensive collection of data on somatic mutations found in mammalian cells that are associated with chemical and physical mutagen exposure. The database is comprised of published single base pairs substitutions, deletions, duplications, insertions and more complex rearrangements in mammalian nuclear genes. The database will be available online and will be accessible via the Internet where the user will be able to search for specific information relevant to their interests. The MGMD mutation entries will include a unique identifier, a standard unambiguous mutation description, amino acid changes (if applicable), mutation frequency descriptions, the reference abstract source and access to the gene's cDNA sequence. The mutation spectra information in the database will be used to study causal relationships that exist between the mutations induced in specific genes by environmental mutagens and the induction of human genetic disease. These analyses will attempt to answer questions such as (i) what parameters influence the induction of a gene's mutational spectra, (ii) whether laboratory induced mutation profiles compare with those found in tumours, or (iii) why specific mutation hot spots exist in target genes. While the database is being developed for research purposes it is hoped that when complete it will acquire a broader utility among researchers in the field of genetic toxicology.
16. Preliminary investigation into blood biomarkers of human exposures to N-nitroso compounds
A.L.Hewitt, S.J.Gant, A.J.Turnbull1, S.Kelly1, D.J.Alexander1, J.G.Hoggett and R.C.Garner
Department of Biology, University of York, York YO1 5YW and 1York District Hospital, York Y03 7HE, UK
N-nitroso compounds (NOC) have been implicated as causative agents of gastric cancer, although there is insufficient evidence to support their role in human gastric carcinogenesis. One such NOC is N-methyl-N-nitrosourea (MNU), which is produced in the stomach by the nitrosation of methylurea. MNU breaks down to form reactive alkylating agents which attack DNA to yield methyl adducts. In order to investigate the role of MNU in human carcinogenicity a clinical study was undertaken using accelerator mass spectrometry to measure DNA adduct formation. [14C]methylurea (370 kBq, 0.185 µmol) was administered to patients undergoing gastric surgery and DNA adducts were detected in the gastric tissue of all patients involved. Levels of adducts ranged from 36 to 426 adducts/1012 nucleotides and 709 to 9337 adducts/1012 nucleotides in gastrectomy and ERCP patients, respectively. This wide variation in adduct levels reflects the inter-individual diversity of patients and differences in sampling techniques. In order to identify possible biomarkers of this tissue DNA damage, DNA was extracted from whole blood. Preliminary data suggests a relationship between the number of adducts in whole blood DNA and tissue DNA. Further work will consider the potential of albumin and haemoglobin as biomarkers of exposure to NOC.
17. Toxicological evaluation of a herbal product
I.Horan and P.T.Tomkins1
Toxicology Unit, Athlone Institute of Technology, Athlone and 1Bioserv Ltd, AIT Campus, Athlone, Ireland
A herbal medicine is defined as any drug containing active substances exclusively of plant origin. The complex multicomponent nature of herbal medicines with their potential for constituent interactions and the reduced precision inherent in their composition means that evaluation of their toxicology and target affinity cannot simply be considered another aspect of natural product chemistry. The herbal medicines business is estimated to be worth more than US$6 billion per annum in Europe and the lack of harmonisation between member states in assessing toxicology and efficacy of herbal products is an issue of growing concern (Benzi, 1998). In evaluating a herbal product intended for topical application, we have developed a hierarchical strategy for the analytical and toxicological investigation for such complex mixtures. Interpretation of toxicological data in the absence of some understanding of the composition of the material is difficult (Shelby et al., 1997). Protein at a concentration of 25.17 ± 1.3 mg/ml in the extract and 0.044 ± 0.01 mg/ml for the finished product was determined and in all subsequent experiments the concentration of test mixture was expressed relative to the protein content. The protein content was further characterised by exclusion chromatography, ultrafiltration and SDS–PAGE. The mixture was further fractionated and analysed by FTIR, UV/VIS, GC-MS and polarimetry. Composition analysis predicted low genotoxicity but could give no immediate indication of interactive effects. Cytotoxicity was evaluated by combined neutral red, SRB and MTT in a keratinising cell line, XB-2 and NRK cells in the presence and absence of S9. The cytotoxicity data was used to select doses for assessment in a six strain Ames and TK assays. Dermal toxicity and inflammatory mediator release was evaluated in a 3D skin equivalent model. Free radical studies have permitted further characterisation of its genotoxic potential.
References
Benzi (1998) Regl. Rev., 1, 8–16.
Shelby et al. (1997) Mutat. Res., 390, R5.
18. The development of the polymerase inhibition assay for the sensitive detection of DNA damage induced by test chemical agents
G.J.S.Jenkins, P.Evans1, B.Burlinson1 and J.M.Parry
School of Biological Sciences, University of Wales Swansea, Swansea SA2 8PP and 1Genetic Toxicology, GlaxoWellcome Pharmaceuticals, Ware, Hertfordshire SG12 0DP, UK
The polymerase inhibition assay exploits the fact that DNA lesions impair the efficient replication of DNA. Hence damaged DNA is a poor target for PCR, the presence of DNA damage and hence the identification of DNA damaging agents can be inferred from a subsequent reduction in PCR efficiency. Here we demonstrate the suitability of the polymerase inhibition assay for detecting DNA damaging agents. A PCR target of 1.8 kb of the mouse p53 gene was chosen. The assay conditions were optimised through the detection of UV-C induced DNA damage. This identified several key parameters in the successful detection of DNA damage, the choice of polymerase employed being the most important. After optimisation, the assay was successfully employed to detect DNA damage induced by MNU, ENU, B[a]P, 2-AAF and AFB1. The assay has also been adapted to a microtitre format, allowing high throughput analysis of test compounds.
19. The effect of acetonitrile on the incidence of micronucleated polychromatic erythrocytes in mouse bone marrow and peripheral blood
Eryl Jones, Ginny Fox, Barry Elliott, Elaine Ogden and Nigel Moore1
Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ and 1BP Chemicals, Research and Engineering Centre, Chertsey Road, Sunbury on Thames, Middlesex TW16 7LN, UK
Acetonitrile was tested for its ability to induce clastogenic and aneugenic effects in a mouse bone marrow and peripheral blood micronucleus test. Groups of five male and five female NMRI mice were administered a single intraperitoneal dose of acetonitrile, corresponding to the maximum tolerated dose of 100 or 125 mg/kg body wt for males and females, respectively. Bone marrow was sampled at 18, 24 or 36 h after treatment, while peripheral blood was sampled before treatment, and 24, 48, 72 and 96 h after treatment. Positive controls were administered cyclophosphamide (65 mg/kg i.p.). Acetonitrile did not increase the incidence of micronucleated polychromatic erythrocytes in either bone marrow or peripheral blood in male mice, or in peripheral blood in females. A small increase in micronucleated polychromatic erythrocytes was observed in female bone marrow at the 36 h time point, but it was within the range of control data. Therefore, although this increase was statistically significant (P < 0.05), it is not considered to be of biological significance. Cyclophosphamide increased the incidence of micronucleated polychromatic erythrocytes in bone marrow and peripheral blood of both sexes. Acetonitrile increased the incidence of polychromatic erythrocytes in male and female bone marrow and peripheral blood, confirming that the bone marrow had been stressed by treatment. It is concluded that acetonitrile is neither clastogenic nor aneugenic in the bone marrow of the mouse at the maximum tolerated dose.
20. Interaction between DNA mismatch repair and damaged DNA in human cells
Peter Karran
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts EN6 3LD, UK
Mismatch repair (MMR) is a DNA replication editing function which protects the cell against spontaneous mutations. Defective MMR is associated with a mutator phenotype and cancer. In the hereditary non-polyposis colorectal cancer (HNPCC) syndrome, individuals with germline mutations in MMR genes are predisposed to colorectal and other malignancies. Tumors which arise in HNPCC individuals, and a significant fraction of apparently sporadic tumors, are defective in MMR.
In addition to preventing mutation, MMR contributes to the cytotoxicity of methylating agents which are used as antitumor drugs. Cultured cells which are selected for resistance to methylating agents frequently have defective MMR. The mechanism of toxicity of methylating agents is not fully elucidated but it is known to require processing of the DNA lesion O6-methylguanine by MMR. DNA O6-methylguanine forms anomalous base pairs which interact with mismatch recognition factors.
MMR defects are also associated with resistance to cisplatin and doxorubicin, chemotherapeutic drugs which are structurally unrelated to each other and to the methylating agents. It has been proposed that MMR acts as a general sensor of persistent DNA damage and, perhaps by interacting with cell cycle checkpoint functions, modulates the cell's sensitivity to diverse types of DNA damage. The properties of xeroderma pigmentosum cells with defects in both nucleotide excision repair and MMR indicate that MMR is unlikely to be a significant general sensor of persistent DNA damage. Evidence will be presented that, unlike methylating agents, much of the drug resistance of MMR defective cells may not be a direct consequence of their MMR defects.
21. The single-laser flow cytometric micronucleus test: a timecourse study using colchicine and urethane in rat and mouse peripheral blood
Steve Kenny, Geoffrey Hynes and David Gatehouse
Genetic Toxicology Department, Glaxo Wellcome Research and Development, Park Road, Ware, Herts, UK
The micronucleus test in rodent bone marrow or peripheral blood is an internationally recognised valid system for the detection of compounds that are potentially clastogenic/aneugenic. However, conventional manual analysis currently employed within most pharmaceutical industries is both time consuming and labour intensive.
In a paper published by Dertinger et al. (1996), a methodology was described that could resolve both micronucleated reticulocytes (RET) and normochromatic erythrocytes (NCE) within mouse peripheral blood using a single-laser flow cytometer. With this procedure, fluorescein-conjugated antibodies were used to bind to the CD71-defined antigen (the transferrin receptor) or RET, thus rendering them identifiable by flow cytometric analysis. By degrading the RNA content of RET and simultaneously labelling them with the antibody, propidium iodide could be used to label the DNA containing cells, i.e. micronucleated cells, thus clearly resolving the different cell types. Flow cytometry provides obvious advantages to manual analysis, i.e. high speed, larger sample sizes, and hence the potential to improve sensitivity and consistency. Furthermore, the use of this approach with rat peripheral blood may allow integration of these analyses into routine toxicology assays thus maximising animal usage, as has recently been suggested by Wakata et al. (1998).
Accurate and reliable flow cytometer set-up was achieved by using malarial-infected erythrocytes (Tometsko et al., 1993). These mimic micronucleated erythrocytes and are prevalent at 10–20% in the cells of interest (compared with 0.2% in micronucleated erythrocyte control blood). This enabled comparison of data obtained on different days or from different experiments. Preliminary experiments using cyclophosphamide in the mouse have shown high data correlation in collaborative work with Litron Laboratories in New York.
Colchicine and urethane were tested in a timecourse study, tracking the incidence of micronucleated RET in the peripheral blood pool of rats and mice over 24, 48 and 72 h. Approximately 20 000 RET were analysed using flow cytometry.
Earlier studies on colchicine using conventional microscopic analysis yielded inconclusive results in rat peripheral blood, although clearly positive effects were obtained in the mouse. Splenic removal within the rat of reticulocytes containing large micronuclei derived from aneugenic events has been suggested as a possible reason for this weak/negative result (Wakata et al., 1998). Flow cytometry allows the analysis of a larger RET sample size. Furthermore, focusing on the more immature RET population may enable micronuclei arising from aneugenic events to be clearly detected before removal by the rat spleen. This study will provide a more conclusive result between rat and mouse peripheral blood using colchicine.
References
Dertinger et al. (1996) Mutat. Res., 371, 283–292.
Tometsko et al. (1993) Mutat. Res., 292, 129–135.
Wakata et al. (1998) Environ. Mol. Mutagen., 32, 84–100.
22. The interpretation of the biological relevance of genotoxicity test results: the importance of thresholds
David Kirkland
Covance Laboratories, Otley Road, Harrogate HG3 1PY, UK
Refinement of genotoxicity protocols has led to an increase in the occurrence of positive results, particularly in vitro, yet very few of these are accompanied by positive responses in vivo. Although these positive results may not be biologically relevant for humans, they can cause delays in drug development both within the company and in response to questions posed by the regulatory authorities. How then should we determine `biological relevance'? Chemicals that produce thresholded dose–responses may well not pose a genotoxic risk at low (relevant to human) exposures, but it is not enough just to `see' apparent thresholds in graphs of results; there must be an explanation and understanding of the underlying mechanism. Extremes of pH, ionic strength and osmolality have already been identified as being indirect causes of genotoxicity in vitro. In addition, indirect genotoxicity may result from interaction of chemicals (or their metabolites) with non-DNA targets, from chemicals/metabolites which are inherently genotoxic but which, at therapeutic concentrations, are effectively conjugated and unable to form adducts with DNA, and from production of specific metabolites under in vitro conditions that are not formed in rodents or humans in vivo. We have developed a three-point approach (see Kirkland and Müller, Interpretation of the Biological Relevance of Genotoxicity Test Results: The Importance of Thresholds, Mutat. Res., in press; Müller and Kasper, Human Biological Relevance and the Use of threshold Arguments in Regulatory Genotoxicity Assessment: Experience with Pharmaceuticals, Mutat. Res., in press) for evaluating these mechanistic data, together with negative in vivo results and supporting animal and human exposure data, to make risk assessments. This approach has speeded up the development of many products (particularly drugs) by enabling in-house decisions to be made earlier, and by avoiding regulatory questions at the time of clinical trial or marketing applications.
23. Mutations in the XPD DNA repair/transcription gene: site of mutation determines cellular defect and clinical outcome
A.R.Lehmann, B.C.Broughton, E.M.Taylor, M.Berneburg, E.Botta1 and M.Stefanini1
MRC Cell Mutation Unit, Sussex University, Falmer, Brighton BN1 9RR, UK and 1Istituto di Genetica, Biochimica ed Evolutionistica CNR, Pavia, Italy
Defects in the XPD gene are associated with three clinical phenotypes, (i) xeroderma pigmentosum (XP), a highly cancer-prone disorder, (ii) trichothiodystrophy (TTD), which is cancer-free, or (iii) the combined features of XP and Cockayne Syndrome (CS). The XPD protein is a subunit of transcription factor TFIIH, which has two roles, in basal transcription and in nucleotide excision repair (NER). We have shown that the site of each mutation in XPD in affected individuals is associated with a specific phenotype. Thus, 80% of XP patients have one or both alleles mutated at arg683, whereas the majority of TTD patients have mutations at arg112, arg658, arg722 or arg730 (Taylor et al., 1997). Based on our findings, our colleagues in Rotterdam have generated a mouse containing a mutation at arg722, and these mice showed features very similar to those of TTD patients (De Boer et al., 1998). This proves that the site of the mutation determines the phenotype, and is consistent with a model in which mutations causing deficient DNA repair result in XP, but if the mutation also causes subtle defects in transcription, TTD ensues. Within the sub-group of TTD patients with the arg112his mutation, individuals who are homozygous for this mutation have a mild phenotype, whereas those who are compound heterozygotes expressing only one allele mutated at this site have much more severe features. This implicates gene dosage as a further factor determining the clinical outcome (Botta et al., 1998).
The two known XP-D patients with the combined features of XP and CS are mutated at gly602 and gly675, respectively. No XP or TTD patients have mutations at these sites. Cells from both these patients are extremely sensitive to cell killing by UV, and they appear to be totally defective in their ability to repair UV-induced photoproducts in cellular DNA. Nevertheless, unlike other XP-Ds, the XP/CS cells show near normal incision following UV-irradiation, but this appears to be uncoupled from the rest of the NER process and may even occur at sites distant from the DNA damage.
References
Botta et al. (1998) Am. J. Hum. Genet., 63, 1036–1048.
De Boer et al. (1998) Mol. Cell, 1, 981–990.
Taylor et al. (1997) Proc. Natl Acad. Sci. USA, 94, 8658–8663.
24. The comet assay and male infertility
Sheena E.M.Lewis
Department of Obstetrics and Gynaecology, The Queen's University of Belfast, Northern Ireland, UK
Sperm DNA integrity is crucial for accurate transfer of information to the next generation. Surprisingly, little is known about it; of how resistant it is to damage or how it can be protected during preparation for assisted conception techniques. Our group has modified the single cell gel electrophoresis assay for use with human sperm and has found the assay to be highly reproducible (coefficients of variation of 4–9%). We have studied ejaculated and, more recently, testicular and epididymal sperm from fertile and infertile men using a variety of conditions. Although there is little difference in baseline DNA, fertile sperm appear to be more resistant to induced damage than infertile samples. A major source of damage to DNA is reactive oxygen species. Sperm are uniquely susceptible to oxidative damage because of their high content of polyunsaturated fatty acids and lack of repair mechanisms. They are initially protected from oxidant attack by seminal plasma but during preparation for assisted conception this is removed, making them vulnerable. By supplementing sperm preparation media with individual antioxidants such as ascorbate and 
-tocopherol, sperm DNA was protected from subsequent damage whereas additions in combination induced further damage. With the recent advent of new assisted conception techniques, previously untreatable men can be helped using non-ejaculated, immature sperm. We have compared testicular and epididymal sperm DNA integrities to those of ejaculated sperm from fertile and infertile men and found that epididymal sperm DNA has significantly more damage. Since these procedures are invasive, cryopreservation of surplus sperm is routinely performed. The comet assay has again be useful in determining DNA status in sperm before and after freeze-thawing.
25. The effect of chlorophyllin on the formation of PhIP-DNA adducts in humans
T.J.Lightfoot, J.M.Coxhead, Y.Tanaka1, D.Russell, B.C.Cupid, S.H.Leveson2, D.A.Alexander2 and R.C.Garner
JBUEC, Department of Biology, University of York, Heslington, York YO1 5YW, UK, 1Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan and 2York District Hospital, Wigginton Road, York YO3 7HE, UK
PhIP (2-amino-1-methyl-6-phenylimadazo[4,5-b]pyridine) is a putative carcinogenic heterocyclic amine (HA) formed during the cooking of red meat. It is believed that HAs are involved in human colon cancer and are known to be carcinogenic in rodents. We have previously shown the formation of PhIP-DNA and albumin adducts in humans. However, it is possible that other dietary factors may influence the metabolism of HAs and thus affect DNA adduct formation. Chlorophyllin (CHL) has been shown to reduce the formation of PhIP-induced aberrant crypts and DNA adducts in F344 rats and mammary tumours in female F344 rats. Preliminary data following the co-administration of PhIP and CHL to Sprague–Dawley rats showed a lower level of DNA and albumin adducts in rats treated with both compounds. No significant effect was seen on urinary or faecal excretion of PhIP in the presence of CHL. Subsequently, patients undergoing colo-rectal surgery were orally co-administered 20 µg of [14C]PhIP (4.91 µCi: specific activity 50 mCi/mmol) with a tablet containing 250 mg CHL or placebo. DNA and albumin were extracted from colon tissue and blood, respectively, and analysed by AMS. Urine was collected for 24 h post dose and analysed by HPLC for PhIP and PhIP metabolites. No difference in the % urinary recovery of PhIP was observed in the patients given CHL or placebo in combination with PhIP (52.5 ± 8.6 and 51.2 ± 7.3%, respectively).
26. The polymerase stop assay for the determination of DNA adduct frequency on the human p53 gene exposed to increasing doses of UVB and UVC radiation
Margaret Linley, Gareth Jenkins and James M.Parry
Centre for Molecular Genetics and Toxicology, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK
The polymerase chain reaction (PCR) is an important exponential amplification technique which results in a sufficiently elevated concentration of target region DNA for the assessment of DNA damage. Generally, the reaction is manipulated to allow preferential amplification of rare mutated target sequences. In contrast, the polymerase stop assay relies on a loss of PCR product to give evidence of adduct formation. The polymerase stop assay exploits the inability of Taq (Thermus aquaticus) DNA polymerase to circumvent large bulky adducts, the subsequent aborted polymerisation interrupts the full potential of the PCR reaction by creating shorter length extension products which cannot act as substrates for the PCR and result in a loss of PCR product. A quantitative approach was achieved by the stringent assessment of initial genomic DNA concentration by the Hoechst 33258 dye binding assay (Labarca and Paigen, 1980). Graphic representation of DNA product during cycle optimisation ensured that the final PCR product was generated during the exponential phase of the reaction and a range of DNA concentrations were shown to be linear with PCR product (McCarthy et al., 1996). The combined polymerase stop assay/quantitative PCR was applied to various regions within the human p53 tumour suppressor gene exposed to increasing doses of UVB and UVC radiation.
References
Labarca and Paigen (1980) Anal. Biochem., 2, 344–352.
McCarthy et al. (1996) Mutat. Res., 3, 57–66.
27. Exfoliated breast milk cells in the comet assay: a novel and non-invasive means of assessing genotoxicity in mammary epithelial cells
Francis L.Martin, J.Andrew Williams, Barbara C.Millar, Kathleen J.Cole, Philip L.Grover and David H.Phillips
Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey SM2 5NG, UK
Epidemiological studies suggest that environmental/dietary factors play an important role in the aetiology of breast cancer. The use of breast milk cells in the single-cell gel (comet) assay, and the ability of such cells to activate extracts of breast milk to DNA-damaging species were investigated. UK-resident women were recruited as donors (n = 16) and extracts, prepared by a solid-phase procedure, and cells were obtained from freshly-expressed milk. Exfoliated breast milk cells were prepared by centrifuging milk diluted 50:50 with RPMI 1640 medium and washing resultant cell pellets twice with RPMI medium. The proportion of epithelial cells was measured by flow cytometry using a monoclonal anti-human epithelial cell antibody. Freshly isolated cell preparations were found to consist of 17 ± 9% epithelial cells and were 81–94% viable as measured by trypan blue exclusion. The cells were either examined immediately for pre-existing DNA damage or were cultured for 1 week in complete RPMI medium, which was found to enrich the epithelial cell proportion to 47%. DNA single strand breaks were detected using the comet assay with alkaline electrophoresis (pH 12.3) and quantified by measuring comet tail length (CTL) (µm). Cell preparations (n = 16) examined immediately in the comet assay exhibited a large interindividual variation in median CTL (range 2.5–37.5 µm) in the presence of the DNA repair inhibitors, hydroxyurea and cytosine arabinoside. In nine cases, the median CTL was greater than the arbitrary figure of 15.0 µm (a figure not exceeded in controls): the values being 16.0, 18.0, 22.5, 23.5, 28.5, 32.0, 37.0 and 37.5 µm. This suggests an in vivo exposure to genotoxic agents in some individuals, as seen in mammary epithelial cells obtained following surgery (Martin et al., 1997). The level of pre-existing DNA damage decreased in all individual cell preparations cultured, suggesting either DNA repair or death of DNA-damaged cells. Genotoxins detected in human mammary lipid (Martin et al., 1996) may be present in breast milk. Cultured cell preparations were susceptible to breast milk extracts previously found to be genotoxic and statistically significant increases in median CTLs were observed. Genotoxicity occurred in the absence of appreciable cytotoxicity with 87–95% viability after treatment with breast milk extracts, including the donors' own milk extracts. This study suggests that exfoliated breast milk cells provide a novel and non-invasive in vitro system for determining in vivo genotoxic exposures. These observations could have important implications for studies on the aetiology and epidemiology of sporadic breast cancer.
References
Martin et al. (1996) Cancer Res., 56, 5342–5346.
Martin et al. (1997) Carcinogenesis, 18, 2299–2305.
28. The comet assay as a pre-screen for potential L5178Y positives
J.Mattison, L.Penrose and B.Burlinson
Genetic Toxicology, Glaxo Wellcome, Park Road, Ware, Hertfordshire, UK
Combinatorial chemistry and high throughput screening (HTS) for pharmacological activity will dramatically increase the amount of potential drug candidates requiring testing for genotoxicity. In order to meet this demand various HTSs have been developed. In general however, these are bacterial assays and therefore may miss clastogens or other classes of genotoxin which are active only in eukaryotic systems.
The aim of this series of experiments was to investigate the use of the comet assay as a predictor of potential mouse lymphoma assay positives. Data from a series of compounds will be given which show a 100% concordance between L51Y and comet assay results with both direct and indirect acting materials. If, as expected, the comet assay in L5178Y cells fulfills its promise as a good predictor of activity in the mouse lymphoma assay then a major reduction in time and resources will be gained. For example, a current pre-screen using the standard mouse lymphoma assay requires 3 weeks and ~300 mg of material, the comet assay, described here, will use around 75–100 mg of material and take 2 days.
29. High voltage electric field induced DNA damage, nuclear lamin disassembly and cell cycle dependent apoptosis
Rosalind A.Meldrum, Elizabeth Deacon, Timothy Cross and Janet M.Lord
School of Biochemistry, University of Birmingham and Birmingham University Medical School, Edgbaston, Birmingham B15 2TT, UK
We previously established that a high voltage electric pulse could induce DNA damage in mammalian cells. The number of DNA breaks induced was proportional to the field strength and the length of time of the electric pulse. A simple method has now been devised to assess permeabilisation and cell viability in electroporated cells so that the procedure may be applied to introduce biochemical intermediates into live cells and maintain viability.
Further observations revealed that a high voltage electric pulse can cause disassembly of nuclear lamins which form part of the nuclear matrix. Gross disruption of the nuclear lamina takes place at high field strengths which also induce DNA damage. At moderate field strengths, which do not induce DNA damage, an electric pulse can induce cells in mitosis to enter apoptosis. At higher field strengths, an electric pulse induces apoptosis in cells in G1 and necrosis in cells in all other phases of the cell cycle. Molecular evidence for apoptosis in electroporated cells was demonstrated by the cleavage of the lamin proteins from the native 67 kDa protein to a characteristic 46 kDa fragment. Apoptosis consists of a commitment phase which can be variable in time and an execution phase which commonly occurs in ~1 h. During this time effector caspases, including caspase 3, are activated. Caspase 3 in turn cleaves and activates several protein kinases, including protein kinase C-
. These studies showed that an inhibitor of PKC prevented the electroporation-induced apoptosis, confirming that the pro-apoptotic kinase PKC- is activated during electroporation-induced apoptosis. Nuclear translocation of PKC- and its association with lamin proteins has been detected during apoptotic induction by other agents. Cells in mitosis were most sensitive to the electric charge, and moderate levels of charge at which DNA damage had not been detected induced apoptosis in mitotic cells. In mitosis, the lamina proteins disassemble and locate in the cytoplasm and are held there by hyperphosphorylation by PKCßII. The electric charge may modify the lamins and interfere with the normal reassembly process, leading to apoptosis.
30. The immunological consequences of UVB-induced DNA damage
Mary Norval
Department of Medical Microbiology, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, UK
Exposure to UVB has genotoxic effects but also results in suppression of some cell-mediated immune responses to antigens encountered shortly after the irradiation. The latter change has critical implications for the control of skin tumours and various infectious diseases. It is likely that a cutaneous chromophore is involved as the initiating step in the complex series of changes resulting in immunomodulation. Three approaches have provided evidence that DNA can act as one important initiator of this process. Firstly mouse models have been developed in which suppression of contact hypersensitivity (CH) occurs when the sensitiser is applied to the skin after the UV exposure, followed by challenge several days later. If the cutaneous thymine dimers, induced by UV, are repaired with T4 endonuclease V or photolyase plus light, then the suppression in CH does not occur. The effect is not confined to the skin as some dendritic cells which accumulate in lymph nodes as a result of UV exposure display DNA damage: antigen presentation to T lymphocytes or the local cytokine milieu may be changed as a result. Secondly various strains of nucleotide repair deficient mice (XPA, XPC, TTD, CSB) have been generated recently. Here the minimal dose of UVB necessary to produce erythema (MED), the minimal dose necessary to suppress CH (MID) and the tendency to develop skin cancers have been compared. Perhaps surprisingly, these three parameters do not correlate; for example, the XPA knockout mice have a low MED and and a low MID in comparison with the parent strain, while the CSB knockout mice have a low MED but a high MID. Thirdly UV-induced DNA damage has been shown to lead directly to changes in the production of various immunological mediators by keratinocytes, such as the induction of interleukin-10, the suppression of interferon- stimulated ICAM-1 expression, and the up-regulation of interleukin-6.
31. Are aneugens mutagenic in the mouse lymphoma TK assay?
M.R.O'Donovan, M.G.Clare, J.Clements1, M.Fellows1, J.Oliver2, D.Ong2 and D.Gatehouse2
AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leics LE11 5RH, 1Covance Laboratories Ltd, Otley Road, Harrogate, Yorks HG3 1PY and 2Pre-Clinical Safety Sciences, Glaxo-Wellcome R&D, Park Road, Ware, Herts SG12 0DP, UK
Aneugens including colchicine and vinblastine sulphate are reported to be mutagenic at the tk locus in mouse lymphoma L5178Y cells using 24 h, but not 3–4 h, periods of exposure (Honma et al., 1999). This ability to detect aneugens is one of the reasons the recent ICH guideline recommends that tests without metabolic activation should be conducted using exposure periods of both 3–4 and 24 h duration (ICH, 1997). However, in these laboratories we consistently see no evidence of mutagenicity with colchicine, even using concentrations resulting in <5% survival. We are able to detect some agents known to be aneugenic in other systems, including carbendazim and griseofulvin, but a significant proportion of the induced mutants are large colonies, rather than small colonies as might be expected. These data indicate that the mouse lymphoma assay, even using 24 h exposure, cannot detect all aneugens.
References
Honma et al. (1999) Mutagenesis, 14, 23–30.
ICH Topic S2B (1997) Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. Step 4, Consensus Guideline, July 1997.
32. The use of flow cytometry as a predictor of cytotoxicity
J.Oliver, F.Rossin, A.Goodchild, R.Fagg and D.Gatehouse
Glaxo Wellcome R&D, Genetic Toxicology Department, Park Road, Ware, Herts, UK
Flow cytometric measurements for cytotoxicity have been assessed in an attempt to increase the efficiency of compound testing in the mouse lymphoma L5178Y assay (MLA), by streamlining concentration selection and thus reducing compound requirement.
The ability of the FACS to accurately count cells was seen as a potential time-saving method. Exponentially growing cells were counted using a coulter counter. A range of cell densities was provided for cell counting on the FACS.
Samples from nine tests following a 3 h treatment period conducted in the MLA where a range of cytotoxicity profiles was produced, have been analysed using FACS. Samples were assessed at three time points; immediately post-treatment, 24 and 48 h after treatment. Samples (500 µl) were removed from cultures being assessed in the MLA. The samples had been aspirated and so were single cell suspensions. Coulter counts and cloning efficiency assessments were performed as part of the standard MLA protocol. All of these measurements for cytotoxicity were compared to the viability counts obtained from the flow cytometer.
Where coulter counts performed immediately post-treatment reduced in a dose-dependent manner, reflecting the amount of cell lysis that occurred during treatment, the viability counts produced by the flow cytometer immediately post-treatment remained around 90% and above. This finding is very similar to that found when trypan blue has been assessed as a potential cytotoxicity measure.
However, viability counts performed on the FACS 24 h after treatment showed a reduction in a dose-dependent manner. When compared with the relative survival data obtained during the course of the MLA, the data correlated very well.
33. Detection of oxidative DNA damage with the comet assay: utilisation as a biomarker for potential cancer prevention by nutrients
B.L.Pool-Zobel
Department of Nutritional Toxicology, Institute for Nutrition, Friedrich-Schiller University of Jena, Dornburger Strasse 25, 07743 Jena, Germany
One recent important achievement of biomedical research has been the recognition that high vegetable and fruit consumption is significantly associated with a reduced tumour risk in multiple tissues. Phytoprotectants are implicated as contributing to risk reduction by different mechanisms, but it has not been possible to actually pinpoint individual compounds as the responsible factors. In order to enhance our knowledge on anticancer mechanisms of nutritional factors, we are determining several types of protective effects of isolated compounds and of food products which contain these phytoprotectants. For this we are using various in vitro methods with human cells and dietary human intervention studies. Since antioxidative effects are expected to be a protective mechanism for many plant ingredients, the detection of oxidative DNA damage with the comet assay is a valuable technique in the strategy. A reduction of oxidative damage should indicate a reduced exposure to risk factors which lead to the generation of reactive oxygen species. In this context, we have shown that the consumption of tomato juice and, even more so, carrot juice reduce DNA damage in peripheral lymphocytes. Subsequent in vitro studies in human lymphocytes with major carotinoid-ingredients of the juices (lycopene, ß-carotene) revealed that all-trans-ß-carotene reduced DNA breaks and oxidised DNA bases in lymphocytes from eight subjects, whereas lycopene was less effective, thus reflecting the activities of the whole foods. An alternative approach is being used to investigate phytoprotectants (e.g. polyphenolics) in cells of tumour target tissues, such as the colon. For this, the modulation of H2O2-induced and steady state levels of oxidative DNA damage by individual polyphenolics are being measured in a human colon tumour cell line in vitro. Results are compared with similar activities of faecal water from humans who consumed foods rich with the respective polyphenolics. Alternatively, oxidative DNA damage may also be detected in colon cells isolated from biopsies. Altogether, the comet assay is a useful technique enabling us to efficiently monitor oxidative DNA damage and the modulation thereof in human cells in vitro and in vivo. The reduction of oxidative stress is a reflection of a reduced exposure load to reactive oxygen species and is meaningful for cancer prevention strategies.
34. Comparison of acute and sub-acute dosing of 2-acetylaminofluorene (AAF) in the rat bone marrow and peripheral blood micronucleus test
Raymond Proudlock, Claire Bradley, Kevin Adams and Wayne Howard
Huntingdon Life Sciences, PO Box 2, Huntingdon, Cambridgeshire PE18 6ES, UK
The relative sensitivity of bone marrow and blood to micronucleus induction, following acute and sub-acute oral exposure to AAF was assessed as part of the inter-laboratory trial co-ordinated by the Japanese Environmental Mutagen Society. If the sub-acute dosing regime is found to be sufficiently sensitive, the eventual aim is to incorporate analysis of either bone marrow (at termination) or blood into on-going standard toxicology studies. This would result in a considerable reduction in the number of animals used in research and would allow consideration of toxicokinetic data, which are usually only available for the rat.
Dose levels used were based on the published results of acute rat micronucleus tests on AAF. Blood was sampled at various times during the course of the 28 day study to establish the kinetics of any response, bone marrow was sampled at termination. Bone marrow samples were also collected from satellite group animals 24 and 48 h after a single administration of AAF to allow a direct comparison with the conventional assay. Acridine orange staining was used throughout.
Only marginal increases in the incidence of micronucleated immature erythrocytes were obtained for the blood on days 2, 3, 4 and 7, but much more marked (up to 9-fold) dose-related increases were seen on days 14, 21 and 28.
AAF causes liver damage in the rat, and analysis of blood in this study was complicated by the presence of pale-staining bodies (probably anaemia-related) in all peripheral blood erythrocytes in higher level groups. In addition, high level group animals also showed a few erythrocytes with inclusions, which stained similarly to micronuclei but were generally larger and less regular in shape
Spleens of AAF-treated animals were enlarged, and it is not clear whether the increase in micronuclei in the peripheral blood at day 14 onwards was, in part, due to increased cell turn-over as a result of anaemia and a decreased ability of the spleen to filter micronucleated cells.
35. Preferential rejoining of -radiation induced DNA strand breaks in the p53 domain of J82 bladder carcinoma cells
N.F.Rajab and V.J.McKelvey-Martin
Cancer and Ageing Research Group, University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK
Combination of the single cell gel electrophoresis (comet) assay to separate break-free and broken DNA domains and fluorescent in situ hybridisation (FISH) localisation of specific sequences in the head or tail of the comet provides the opportunity to determine whether the domains containing sequences of interest are or are not broken. In this study we have used the previously reported p53 FISH/alkaline comet assay (McKelvey-Martin et al., 1998) to follow DNA damage and repair in the mutant p53 domain of invasive J82 bladder carcinoma cells following -irradiation. Cells embedded in low melting point agarose on frosted microscope slides were treated with 0, 2, 5 and 10 Gy -irradiation at 2 cGy/s. Cells were then lysed and their DNA allowed to unwind, electrophoresed and neutralised, as previously described, before being subjected to FISH. J82 cells irradiated with 5 Gy were allowed to repair at 37°C in RPM1 medium on the slides and were then harvested at 10, 30 and 60 min following irradiation. FISH, using the p53 (LS1) Spectrum Orange DNA probe (which comprised randomly sheared 50–200 bp lengths of DNA covering ~200 kb around and including the p53 locus) (Vysis), was performed on stabilised comet gel preparations by heat denaturation (74 ± 1°C) followed by overnight hybridisation. After further processing the cells were stained with 4'6-diamidino-2-phenylindole (DAPI) (Vysis) and examined using a fluorescence microscope equipped with a double interface filter set, tuned for the simultaneous detection of DAPI and Spectrum Orange label. The % tail DNA was quantitated by image analysis using the Fenestra Comet v 3.1 Software (Kinetic Imaging Ltd) and the numbers of head and tail spots per cell were counted manually. Six hundred cells were analysed for each dose/time point studied. A clear dose response was observed as measured by mean % tail DNA and by the mean number of spots per cell. Rejoining of strand breaks was evidenced over the observed repair period following 5 Gy irradiation by decreasing mean % tail DNA and by a decrease in the mean number of spots per cell. Interestingly, it was also observed that at all the dose and time points the number of cells showing DNA damage (% tail DNA >10%) notably exceeded the number of cells displaying p53 spots in the comet tail. This was particularly marked in the cell populations having undergone 10, 30 and 60 min repair. Following 5 Gy irradiation, 50% of cells showed comet tails but only 19.7% cells showed p53 tail spots. At 10 min repair 38% of cells had comet tails but only 3.33% of cells showed tail spots. After 30 min repair 25% cells had comet tails whilst 1.72% cells had tail spots and at 60 min repair 16% cells had comet tails with only 0.91% cells having tail spots. These results, although preliminary, indicate clearly that the p53 domain in J82 cells has preferential rejoining of -irradiation-induced DNA strand breaks. Experiments are currently underway to further elucidate these findings.
Reference
McKelvey-Martin et al. (1998) Mutagenesis, 13, 1–8.
36. The endogenous hormone 17ß-estradiol at low concentrations induces DNA damage in MCF-7 breast cancer cells
Nissanka Rajapakse and Andreas Kortenkamp
Centre for Toxicology, School of Pharmacy, University of London, London, UK
We have utilized the single cell gel electrophoresis (comet) assay to investigate the potential of estrogens to induce DNA damage in MCF-7 breast cancer cells. It has been hypothesized that in the majority of cases the multi-step process of breast carcinogenesis results from exposure to endogenous or exogenous estrogens that alter normal estrogen metabolism. Estrogens appear to work bifunctionally, via hormonal and genetic pathways. Binding of an estrogen to the estrogen receptor or to a downstream component of the proliferative pathway may result in aberrant cell division. This mitogenic effect has been demonstrated for 17ß-estradiol and other environmental estrogens such as op'-DDT in many laboratories. The genetic pathways, however, have not been investigated thoroughly. Metabolic activation of 17ß-estradiol to catechol estrogens which may then undergo redox cycling to produce reactive oxygen species could potentially damage DNA. The ability of catechol estrogens or estradiol semiquinones to induce DNA damage in breast cell lines has been reported. However, the dosage levels of these compounds was extremely high. The results presented here demonstrate clearly that exposure of MCF-7 cells to the endogenous parent hormone 17ß-estradiol at concentrations as low as 10 nM causes DNA fragmentation. To date this is the lowest concentration shown to induce DNA damage. Our observation that DMSO suppressed the ability of 17ß-estradiol to form comets indicates that metabolic activation of the compound resulting in the formation in DNA-damaging reactive oxygen species may be involved.
37. Mutagenesis and repair of 8-oxoguanine in human cells
Alain Sarasin, Florence Le Page, Priscilla Cooper1 and Alain Gentil
Laboratory of Molecular Genetics, UPR 42, CNRS, Villejuif, France and 1Life Science Division, Department of Radiation Biology and DNA repair, Berkeley, CA, USA
The aerobic metabolism is accompanied by the formation of reactive free radicals of oxygen that can damage cellular components and DNA. The ease with which the 8-oxoguanine is formed by numerous oxidizing agents suggests that this lesion could make a significant contribution to mutagenesis. In both bacteria and mammalian cells, repair enzymes have been discovered which possess activities towards 8-oxoguanine (Fpg, OGG1). The possible mispairing between 8-oxoguanine and adenine allows us to think that this lesion can give rise to G
T transversion in the absence of efficient repair. Therefore, we investigate the mutation frequency and types of mutations induced by a unique 8-oxoguanine residue in different human cell lines.
Our results show that replication of the modified base in a single-stranded or a double-stranded plasmid in repair-proficient human cells and repair-deficient cells from classical xeroderma pigmentosum patients induces a similar mutation frequency of ~1–3%.
On the other hand, the base mispair GO:C as well as the base mispair GO:A have also been shown to be intermediates of replication of the altered base. Studies concerning the biological consequences of these base pairs show an efficient repair of the GO:C leading to a low mutation frequency while the absence of efficient repair of GO:A in human cells, under our experimental conditions, explains the high mutational potency of 8-oxoguanine and also the observed mutation spectrum (GT transversions in 95% of mutants).
Cells isolated from Cockayne's syndrome patients or xeroderma pigmentosum patients also exhibiting Cockayne symptoms are unable to repair the 8-oxoguanine leading, therefore, to at least 10 times more GT transversions than in normal cells. The low repair level is associated with mutations on the XPB, XPD, XPG or CSB gene. Full complementation with the expression of the wild-type gene indicates that this high mutation frequency is due to the genetic defects of the repair gene. This high mutagenic potency of 8-oxoguanine is only observed when it is located on the transcribed strand while normal repair is observed when the lesion is located on the non-transcribed strand. This absence of 8-oxoguanine repair may explain the severe neurological deteriorations observed in these patients, due to the accumulation of lesions on the transcribed strand of brain cells.
38. PhIP mutagenicity in the kidney of male MutaTMMouse mice
C.C.Smith, G.E.Archer, D.S.Davies1, A.R.Boobis1, R.W.Rees and A.M.Lynch
SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, Herts AL6 9AR and 1Department of Clinical Pharmacology, ICSM, Hammersmith Hospital, DuCane Road, London W12 OTE, UK
The mutagenicity of the most common heterocyclic amine found in cooked meat, 2-amino-1-methyl-6-phenylimidazo[4,5,b]pyridine (PhIP), a multi-organ rodent carcinogen, was investigated using transgenic MutaTMMouse mice. Male mice (23–30 g, n = 5 per group) were dosed with 20 mg/kg PhIP p.o. in DMSO and water (1:1) daily for 4 days. Animals dosed with PhIP or vehicle control were killed 25 days after final administration. In parallel, a positive control group (n = 3) was treated with a single administration of ethylnitrosourea (ENU, 100 mg/kg, i.p.) on day 4 only. Genomic DNA was extracted from the kidney and mutation frequencies (MF) were determined at the transgenic lacZ locus using a positive selection method (Dean et al., 1994). The control MF (mean: 48.3x10–6) was similar to that seen in the literature. Statistical analysis of the mutation data using a generalised linear mixed effects model (Fung, 1998) showed that there was no extra binomial variation in the data at the animal level, and only limited variation at the packaging level (variance 0.07). Compared with vehicle control, there was a 2-fold increase in the mean MF following treatment with PhIP (t statistic –3.98, P < 0.001) and a 6-fold increase in the mean MF following treatment with ENU (t statistic –9.44, P < 0.001). This positive finding is in contrast with previous studies (Lynch et al., 1996), which reported PhIP as non-mutagenic in the kidney of animals receiving identical treatment and sampled only 7 days after the final dose. The differences in the outcome of these studies, therefore, show the importance of sampling time on organ-specific mutation in transgenic assays.
References
Dean and Myhr (1994) Mutagenesis, 9, 183–185.
Fung (1998) Environ. Mol. Mutagen., 31, 48–54.
Lynch et al. (1996) Mutagenesis, 11, 505–509.
39. Comparative study of the p53 gene and LacZ gene mutations in MutaTMMouse testes treated with ENU
Honglin Song, Gareth J.S.Jenkins and James M.Parry
School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK
Transgenic mouse modelling has proved to be a powerful approach to explore the various steps involved in spontaneous and induced carcinogenesis. It has provided unprecedented access to rodent somatic and germline tissues for the study of gene mutation in vivo (Douglas et al., 1997). Here, restriction site mutation (RSM) assay was employed to study the mutation induced in endogenous p53 gene and LacZ gene in MutaTMMouse testes treated with ENU. The aim of this experiment was to compare the mutation susceptibility between endogenous p53 gene and integrated LacZ gene in transgenic mouse.
The RSM assay is a genotypic mutation detection method (Jenkins et al., 1998). It can identify mutations occurring in restriction enzyme cutting sites of genomic DNA. MutaTMMouse were given a single dose i.p. injection of ENU (150 mg/kg) and control animals were given i.p. injection of saline (10 ml/kg). Testes were removed 102 days post exposure. RSM analysis was performed on the LacZ gene (four restriction sites) and five p53 gene regions, namely exons 4, 5 and 7 and introns 6 and 8. One GCAT mutation was detected in the LacZ gene on treated samples. In the p53 gene, two restriction sites with a total of five mutations were detected in exon 5 in treated samples. A total of five mutations were revealed in intron 8, among them, two mutations from control samples and three from treated samples. No mutations were detected in exons 4 or 7 or intron 6.
The results confirmed that ENU was a potent chemical mutagen in spermatogonial stem cells (Favor et al., 1990). The predominant mutation detected was GCAT transition which result from mispairing of O6-alkylguanine, followed by ATGC (mispairing of O4-alkylthymidine) transitions and GCTA, ATTA transversions. This was compatible with the results of others (van Zeeland et al., 1990). The results implied that the p53 gene was perhaps more susceptible to mutagen ENU than the LacZ gene in MutaTMMouse testes. This means that the integrated LacZ gene might not be necessarily be representative for the endogenous mutation targets of environmental carcinogens.
References
Favor et al. (1990) Mutat. Res., 229, 105–114.
Douglas et al. (1997) Mutat. Res., 388, 197–212.
Jenkins et al. (1998) Mutat. Res., 405, 209–220.
Van Zeeland et al. (1990) Mutat. Res., 231, 55–62.
Vijg et al. (1998) Mutat. Res., 400, 337–354.
40. Genetic effects of oxidative stress
Günter Speit, Claudia Dennog and Andreas Rothfuß
Abteilung Medizinische Genetik, Universitätsklinikum Ulm, Ulm, Germany
In order to study the genetic effects of oxidative stress in humans, we exposed volunteers to hyperbaric oxygen (HBO) treatment (i.e., exposure to 100% oxygen at a pressure of 2.5 ATA for a total of 3x20 min periods). HBO leads to increased levels of reactive oxygen species (ROS) and caused clear and reproducible DNA effects in the comet assay with leukocytes. Using FPG protein we detected significant oxidative DNA base damage. HBO-induced DNA damage did not lead to chromosome aberrations (micronucleus test) and gene mutations (HPRT T-cell cloning) possibly due to effective repair of primary DNA damage.
To further evaluate the mutagenic potential of HBO, we established an in vitro HBO model with human blood and V79 cells. Unexpectedly, the exposure conditions for the induction of DNA damage in blood cells in vitro had to be higher than those used in vivo. V79 cells showed a higher sensitivity towards HBO-induced DNA damage and increased HBO exposure caused a dose-related increase in the frequency of micronuclei but did not lead to clear induction of HPRT mutations. These findings support the view that pre-mutagenic oxidative base damage is efficiently repaired in mammalian cells.
As DNA damage was induced in vivo only after the first HBO treatment and not after further treatments under the same conditions, we supposed an increase in antioxidant defences. However, determination of the `antioxidant status' did not reveal any difference before and after HBO. But we can show that blood taken 24 h after HBO is well protected against the induction of DNA damage by hydrogen peroxide (H2O2). This protective effect lasted for several days. Experiments with isolated lymphocytes gave similar results, indicating that the adaptive response is an intracellular effect. The cells were not comparably protected against the genotoxic effects of -irradiation, suggesting increased scavenging or reduced production of ROS distant from nuclear DNA. Recent results indicate overexpression of heme oxygenase-1 after HBO, suggesting increased sequestration of iron by ferritin as a probable cause for the adaptive antioxidant defence.
41. N-acetyltransferases (NAT) and sulphotransferases activate heterocyclic amines in the human breast, but NAT mRNA levels are not influenced by NAT genotype
E.M.Stone, J.A.Williams and D.H.Phillips
Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton, Surrey SM2 5NG, UK
Humans are exposed to a wide variety of potential mammary carcinogens in the environment including the heterocyclic amines (HAs), such as PhIP and IQ, which are procarcinogens formed when protein-rich food is cooked at high temperatures. HAs are also present in cigarette smoke and are rodent mammary carcinogens at high doses. In the human mammary gland these agents are thought to be metabolically activated via CYP1A1- and CYP1B1-catalysed hydroxylation, followed by esterification catalysed by phase II enzymes including N-acetyltransferases (NAT) and sulphotransferases. We have shown using 32P-postlabelling that DNA adducts are formed in primary cultures of human mammary epithelial cells (HMECs) by 500 µM PhIP (mean 1.0/108 nucleotides, n = 26) and 500 µM IQ (mean 7.4/108 nucleotides, n = 36) and their N-hydroxy intermediates, 20 µM N-OH-PhIP (mean 155.8/108 nucleotides, n = 18) and 20 µM N-OH-IQ (mean 6.6/108 nucleotides, n = 18). The NAT1 and NAT2 loci are both polymorphic. We determined the NAT1 and NAT2 genotypes using PCR–RFLP methods and have shown using semi-quantitative RT–PCR, relative to ß-actin mRNA levels expressed as standard units (SU), that both NAT1 and NAT2 mRNA are expressed in both human mammary tissues (HMT, mean 1.30 and 0.43 SU, respectively, n = 20) and in HMECs (mean 1.90 and 0.89 SU, respectively, n = 10). In both HMT and HMECs the levels of NAT1 mRNA expression were found to be 2–4-fold higher than levels of NAT2 mRNA expression and these differences were statistically significant (P = 0.0001 for HMT, P = 0.0078 for HMECs). The relationship between NAT genotype and expression was compared but no significant association was detected. Preliminary studies were performed using specific enzyme inhibitors to assess the relative contributions of sulphotransferases and N-acetyltransferases to the metabolic activation of heterocyclic amines in human mammary cytosols. These have shown that N-OH-PhIP-DNA adduct formation is inhibited to a greater extent than N-OH-IQ-DNA adduct formation by the estrogen sulphotransferase inhibitor 17ß-estradiol (100 µM) and that the NAT/sulphotransferase inhibitor pentachlorophenol (PCP, 100 µM) appears to inhibit both N-OH-IQ and N-OH-PhIP adduct formation. Our results indicate that NAT mRNA expression levels may not be linked to genotype and that the metabolic activation of PhIP and IQ may proceed via slightly different mechanisms.
42. An investigation into the effects of colchicine on chromosome loss and non-disjunction in cell lines with varying p53 status
J.C.Strefford, E.M.Parry and J.M.Parry
Centre for Molecular Genetics and Toxicology, School of Biological Sciences, University of Wales, Swansea, UK
This study demonstrates that p53 status affects the frequencies of chromosome loss (CL) and non-disjunction (ND) observed in diploid human fibroblasts after treatment with colchicine. Three cell lines (Neo-control, Scx-mutant p53 codon 143 & E6-HPV-16 viral oncogene fragments) were treated with a dose range of colchicine and the levels of CL and ND were measured using the in vitro micronucleus assay in conjunction with centromeric FISH probes. Relative to the control cell line, elevated frequencies of CL were observed at all doses in the Scx cell line and at higher doses (4–10 µg/ml) in the E6 cell line, suggesting a different underlying molecular mechanism for Scx and E6. At lower doses (2–4 µg/ml) elevated levels of ND were observed in both abnormal cell lines. Specific CL and ND were measured for chromosomes 1, 7 and 17 using three-colour centromeric FISH probes. Chromosome 1 was shown to be the most stable, showing no increase in CL and ND in the Scx and E6 cell lines, while chromosome 17 demonstrated dramatic increases in CL and ND in both abnormal cell lines. Chromosome 7 demonstrated elevated levels of ND in the two p53 deficient cell lines but no increase in CL. These FISH data demonstrate that p53 has little or no effect on the stability of chromosome 1, while it does affect the stability of the other two chromosomes. This work illustrates a function of wild-type p53 at mitosis as well as preferential effects on chromosome-specific stability.
43. Antioxidant/pro-oxidant effects of dietary components: in vitro assessment using the comet assay
Y.T.Szeto and I.F.F.Benzie
Department of Nursing and Health Sciences, Hong Kong Polytechnic University, Hong Kong SAR, China
The aim of this study was to assess in vitro DNA protective or damaging effects of various dietary antioxidants, including purified tea and wine polyphenols, `whole' green and black tea, and the antioxidant vitamins C (ascorbate) and E (-tocopherol).
Working solutions of testing agents were prepared as needed in PBS (0.01 mol/l, pH 7.4) from freshly made aqueous or ethanolic stock solutions. Human lymphocytes were harvested from heparinised venous blood, washed in PBS and then incubated in PBS, with or without test agent at known concentration, at 37°C for 30 min. Cells were washed in PBS and oxidative stress was induced by exposure of cells to H2O2 (5 min on ice at final concentrations of 0, 30, 45 and 60 µmol/l H2O2 in PBS). Cells were washed and the comet assay was performed on the treated, untreated, stressed and unstressed cells in parallel. Fifty cells were scored per treatment in each of three independent experiments. Analysis of comets was performed by measuring the tail moment using a computerised image analysis system (Komet 3.0, Kinetic Imaging, Liverpool, UK).
Less DNA damage was seen in H2O2-stressed cells which had been pre-incubated in quercetin (P < 0.001) or caffeic acid (P < 0.05) at up to 100 µmol/l, and these agents did not themselves cause DNA damage. Protection by 50–200 µmol/l -tocopherol was seen (P < 0.001) at the highest H2O2 stress only. Ascorbate showed no significant protective effect over this range. No DNA damage was caused by -tocopherol or by ascorbate at 
200 µmol/l, however, cells incubated with higher concentrations of ascorbate showed a dose–response increase in DNA damage. This was significant (P < 0.001) at >1000 µmol/l. Catechin, catechin gallate and epicatechin at 100 µmol/l did not damage DNA, but no DNA protection was seen in the pre-treated cells after H2O2 challenge. Epigallocatechin, epigallocatechin gallate and infusions of `whole' green and black tea at all concentrations tested caused increased DNA damage in treated, unstressed cells (P < 0.001), and these agents did not protect against H2O2-induced oxidant stress. A clear dose–response damaging effect was seen for teas and for resveratrol.
Results indicate that the protective effect of antioxidant-rich diets may be mediated by a small number of antioxidants only. Furthermore, some antioxidants may have undesirable DNA damaging effects. The damaging effect of ascorbate at high concentration is particularly interesting, as intracellular ascorbate can reach millimolar levels in vivo. The effect of dietary antioxidants, alone and in combination with other antioxidants, deserves further study.
44. An investigation of the effects of MDR-1 expression in the haemopoietic compartment on etoposide-induced genotoxicity
S.D.Turner1, L.J.Fairbairn1, J.A.Rafferty1, H.Tinwell3, J.Ashby3, W.Ostertag4, C.Baum4 and L.S.Lashford1,2
1Section of Haemopoietic Cell and Gene Therapeutics and 2Paterson Institute for Cancer Research, Academic Department of Paediatric Oncology, Christie Hospital, Manchester M20 4BX, 3Zeneca Central Toxicology Laboratory, Alderley Park, Cheshire, UK and 4Heinrich-Pette Institute, Hamburg, Germany
Etoposide is commonly used to treat a variety of cancers and exposure is often associated with severe myelotoxicity due to the interaction of etoposide with topoisomerase II, resulting in DNA strand breaks. Etoposide exposure in some cases also results in the development of secondary leukaemia that is both cumulative dose- and schedule-dependent. Transduction of the human MDR-1 gene into mouse bone marrow results in protection of the haemopoietic compartment against myelotoxicity. However, the effects of such an approach on the chronic toxicity have not been previously investigated. The BMMN assay has been employed to determine the genotoxicity associated with etoposide exposure in the absence of an assay that is able to directly measure chromosomal mutations in vivo and is able to detect clastogenic activity 24 h after exposure with a significant increase between the doses of 0.1–1.0 mg/kg i.p.; at higher doses there is a depression in the frequency of micronuclei that correlates with a perturbation of erythropoiesis. An increase in the frequency of micronucleated polychromatic erythrocytes was seen when etoposide was administered in various serial schedules. On this basis we conclude that the BMMN assay is a robust test by which to monitor the frequency of clastogenic events that occur following etoposide exposure. This assay was therefore employed to determine if the frequency of clastogenic events could be reduced in mice reconstituted with MDR-1-transduced bone marrow compared with mice reconstituted with mock-transduced bone marrow. Mice in which 15% of marrow colony forming cells were positive for exogenously expressed (human) MDR1 were significantly protected against MN induction. It was possible to select for the transduced cells such that they represented 35% and 64% of the marrow following a single or double dose of 50 mg/kg of etoposide respectively. However, there was no increase in protection from further MN-inducing doses of etoposide in these mice, indicating that there is an upper limit to the protection possible using the BMMN assay. Further work is underway investigating the relationship between etoposide-induced micronuclei and mutations using an APRT knockout mouse model.
45. The BrdU–comet assay: a novel assay for the detection of replication in individual cells
G.R.Wasson, A.P.McGlynn, V.J.McKelvey-Martin, G.McKerr, F.J.Mullan1, J.M.Scott2, D.G.Weir2 and C.S.Downes
Cancer and Ageing Research Group, School of Biomedical Science, University of Ulster, Coleraine, Northern Ireland, 1Coleraine Hospital, 28A MountSandel Road, Coleraine, Northern Ireland, UK and 2Biochemistry Department, Trinity College, Dublin, Ireland
The BrdU–comet assay is a novel technique developed to combine the standard alkaline comet assay with bromodeoxyuridine (BrdU) labeling of recently replicated DNA followed by immunolocalisation of the BrdU incorporated using a fluorescein tagged antibody. This assay allows the detection of single strand breaks in the newly replicated DNA of individual cells. The validity of this assay was demonstrated using SVM muntjac fibroblasts which are rendered replication incompetent following UV irradiation. Data obtained using the BrdU–comet assay was found to be consistent with data described earlier using a range of biochemical techniques to detect and quantify DNA damage-induced gaps in recently replicated DNA.
The BrdU–comet assay was used to calibrate the replicative integrity of DNA in four colon carcinoma cell lines with or without known mutational defects in post replication repair. Pulse–chase experiments were carried out using a short pulse of BrdU which was then chased for various time periods by growing the cells in medium supplemented with normal nucleotides. All of the cell lines demonstrated a gradual reduction of tail moment over an 80 min chase period, indicative of DNA maturation and repair, with the repair-defective cell lines showing a delayed and more incomplete reduction in tail moment.
The BrdU–comet assay is also suitable for use in the assessment of replicative integrity in single cells derived from human small endoscopic biopsies. In addition, entrapment of the biopsy cells in agarose microbeads has proved a successful method for transportation and further manipulation of the cells, for example, for enzyme digestion.
Other potential applications of the BrdU–comet assay include the examination of the effects of folate deficiency on DNA replication in colon cells. Folate deficiency has previously been implicated in colon carcinogenesis and our results indicate that altered DNA replicative integrity is found in folate-deprived cells in culture.
46. Benign prostatic tissues express CYP1 and NAT genes, and activate potential prostate carcinogens
J.A.Williams, F.L.Martin, G.H.Muir1, B.Blomeke2, P.L.Grover and D.H.Phillips
Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey SM2 5NG, 1Department of Surgery, King's College Hospital, London SE5 9RS, UK and 2Department of Dermatology, University of Aachen, Germany
The prevalence of, and mortality from, prostate cancer in the Western world is increasing. It arises most frequently in elderly men, and is most common in North America and in Scandinavia, but is relatively rare in Asia. The incidence of prostate cancer is highest in those countries with a high risk of developing benign prostatic hyperplasia (BPH). In this pilot study, BPH tissues obtained by transurethral or radical retropubic prostatectomy from otherwise healthy individuals (n = 18) were analysed for their ability to metabolically activate the heterocyclic amines (HAs) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, 500 µM, n = 9), N-hydroxy PhIP (20 µM, n = 8), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ, 500 µM, n = 5) or the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (B[a]P, 50 µM, n = 5). After incubation (22 h, 37°C) DNA was isolated and HA– and PAH–DNA adducts were quantified by 32P-postlabelling analysis. DNA adduct formation was measurable in all tissue samples incubated with PhIP (mean, 3 adducts/108 nucleotides), N-hydroxy-PhIP (2736/108) or B[a]P (1/108). IQ–DNA adducts were measurable in four out of five tissues (mean, 1/108). Addition of the CYP1 inhibitor -naphthoflavone (10 µM) reduced B[a]P–DNA adduct formation in tissues from two individuals by 96% and 64%, respectively. Semi-quantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis of CYP1 expression in BPH tissues (in four of four individuals) revealed expression of CYP1A2 mRNA transcripts (which are usually confined to the liver), as well as mRNA transcripts from the following genes: CYP1A1, CYP1B1 and the DNA excision repair gene XPD. Quantitative real-time RT–PCR analysis of N-acetyltransferase (NAT) gene expression in tissues from three individuals showed mean levels of NAT1 mRNA were 10-fold higher than NAT2. This pilot study shows that human prostate tissue can metabolically activate typical `cooked meat' carcinogens which may contribute to the overall risk of prostate cancer development. Further preliminary experiments show that primary cultures of BPH tissue-derived cells (48 h post-prostatectomy) retain the ability to activate IQ. Primary cell cultures may therefore offer a useful system for investigating mechanisms of prostate carcinogenesis involving DNA modification and repair.
47. The use of primary human cell types for aneugen screening in the in vitro cytokinesis blocked micronucleus assay
Justine Williamson, Elizabeth M.Parry and James M.Parry
Centre for Molecular Genetics and Toxicology, University of Wales Swansea, Swansea SA2 8PP, UK
The in vitro cytokinesis blocked micronucleus assay (Fenech and Morley, 1985) is currently being considered for inclusion into the genetic toxicology test battery as both a quicker and simpler alternative/supplement to the chromosome aberration assay and as a test for the detection of aneugenic compounds (aneuploidy being a genotoxic end-point currently not covered by regulatory guidelines). However, before this can be incorporated, the test's full validation is required, and several international collaborative studies are in progress to this end.
The work discussed assesses the suitability of using different primary human cell types (fibroblasts and lymphocytes) in the in vitro micronucleus assay and in particular the ability of the assay to detect aneugenic compounds. Several known aneugens have been tested in the primary cell types for both micronucleus induction and for their ability to induce micronuclei containing whole chromosomes. Acridine orange staining and antikinetochore antibody labelling being the respective methods employed.
Reference
Fenech and Morley (1985) Mutat. Res., 147, 29–36.
48. Fluorescence in situ hybridisation applied to the embryos of the marine polychaete: Pomatoceros lamarckii
James T.Wilson and David R.Dixon
Southampton Oceanography Centre, Empress Dock, Southampton SO14 3ZH, UK
The marine environment receives a wide variety of anthropogenic and natural inputs, many of which have the potential to damage DNA or interfere with the process of cell division. At present there is a shortage of relevant screening methods for determining (i) the presence of low levels of genotoxins in the marine environment and (ii) the possible consequences for exposed individuals and populations. Here we describe an application of the fluorescence in situ hybridisation (FISH) method to the early embryonic/larval stages of a common marine worm Pomatoceros lamarckii. This method, which involves the use of biotin-labelled rDNA probes, opens up the possibility for the identification of specific chromosomes or arm regions, previously not possible using conventional karyotyping techniques. An additional advantage of this molecular method is that it allows visualisation of clastogenic and aneuploidic aberrations in interphase nuclei, therefore reducing the possibility of artefacts often associated with in vivo metaphase preparations. By utilising the developing egg cell as an integrator of mutagen exposure during gametogenesis (3–6 months), this FISH method provides a sensitive indicator of chronic mutagen exposure and offers the potential for following induced chromosomal aberrations into the next generation. The advent of molecular techniques and their application to aquatic subjects offers the potential for an array of novel test protocols applicable to compound screening and environmental monitoring.
49. The heterocyclic amine PhIP causes S phase cell cycle delay, apoptosis and hprt gene mutations in human lymphoblastoid TK6 cells
H.Zhu, A.R.Boobis and N.J.Gooderham
Clinical Pharmacology and Molecular Toxicology, Imperial College School of Medicine, London, UK
The heterocyclic amine 2-amino-1-methyl-6-phenylimidazol[4,5-b]pyridine (PhIP) is a rodent procarcinogen, being formed in cooked meat at parts per billion levels. It is efficiently activated to its N-hydroxy genotoxic derivative by human cytochrome P4501 (CYP1) family enzymes. In order to better understand the genetic toxicity of PhIP, we have used human lymphoblastoid cells (TK6) as target to examine cellular events which may be important in PhIP-induced DNA damage, including cell cycle arrest, apoptotic cell death and induced mutation. Chinese hamster cell line XEMh1A2-MZ cells, which have been genetically engineered to express human CYP1A2, were cocultured with TK6 cells in the presence of PhIP (0–10 µg/ml). TK6 cells were harvested at various times, fixed and stained with propidium iodide for the examination of cell cycle and apoptosis by fluorescence activated flow cytometry. After 20 h of treatment, a slight S-phase delay was observed at all concentrations of PhIP, although the cell growth cycle recovered after PhIP withdrawal. When PhIP treatment was extended to 40 h, S-phase arrest was more striking and cell cycling failed to recover after PhIP withdrawal. This pronounced S-phase arrest was followed by the appearance of a subG1 population (indicative of apoptotic cell death) which ranged from 12 to 54%, compared with 9% in the vehicle-treated controls. A concomitant increase in mutation frequency at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus, assessed by colony formation assay, was detected after 40 h, but not 20 h, PhIP treatment, range 31–50x10–6 compared with 10x10–6 hprt mutants in vehicle control. These studies with human cells show that, upon activation, PhIP induces early cellular events which are frequently associated with DNA damage. However some cells escaped the cell cycle checkpoint and survived, but with an increased frequency of gene mutation.
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