Mutagenesis

This Article Extract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Google Scholar Search for Related Content Social Bookmarking          What's this?

Mutagenesis, Vol. 16, No. 6, 557-570, November 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press

Meeting report

Abstracts of the United Kingdom Environmental Mutagen Society 25^th Annual Meeting, July 2–4, University of Nottingham, Nottingham, UK

1. Transformation in hTERT transfected human lines

A.C. Riches¹, C. Peddie¹, P. Bryant¹, G. Bakirtzis¹,

H. Zitzelsberger², L. Heiber²

¹School of Biology, University of St. Andrews, St. Andrews KY16 9TS, UK and

²Institute of Radiobiology, GSF, Neuherberg, Germany

Rodent cell lines, such as the C3H10T1/2 cell line, proved useful models to investigate transformation but have a number of disadvantages. Attempts to utilise human primary cultures were limited by cell senescence. Human cell lines were then immortalised using a variety of viral constructs and enabled studies on radiation and chemical carcinogenesis to be undertaken. Cell lines immortalised in this way are often genetically unstable, with variable chromosome numbers. Human cell lines have now been immortalised using vectors expressing hTERT (the catalytic subunit of telomerase). A human retinal pigment epithelial cell line (340RPE-T53 hTERT) has been utilised for studies on radiation-induced carcinogenesis. Following exposure to fractionated doses of gamma irradiation a marked increase in anchorage independent growth was observed. Cloned lines derived from these colonies exhibited serum independent growth, increased anchorage independent growth and were tumourigenic in athymic nude mice. The parent cell line did not produce tumours in immunosuppressed mice and retained a diploid karyotype whereas structural aberrations of different complexity were observed in the irradiated clones. The primary RPE cell line without the hTERT insert did not exhibit anchorage independent growth following fractionated radiation exposure. Exposure to fractionated doses of UVA or UVB induced an increase in anchorage independent growth but cell lines derived from these colonies did not form tumours in athymic nude mice. A variety of human cell lines including fibroblasts, keratinocytes, mammary epithelial cells and endothelial cells have been immortalised using hTERT. These essentially provide a source of human cells immortalised by a physiological mechanism utilised by stem cell populations to maintain their self renewal capacity. This promises to be a useful addition to the models available for investigating mechanisms of carcinogenesis.

2. The ILSI Consortium on Application of Genomics to Risk Assessment

J.D. Tugwood

Molecular Toxicology Department, Safety Assessment UK, AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK

The ILSI Health and Environmental Sciences Institute Collaborative Programme `Application of Genomics to Mechanism Based Risk Assessment' has been operating since September 1999, and has two principal aims: (i) to establish the usefulness of genomics technology to toxicology and safety assessment, and (ii) to construct a gene expression database with the intellectual and practical input of the Programme participants, which will be publicly available. A large number of industrial laboratories are participating (from pharmaceutical, agrochemical, chemical, and consumer product companies), together with government representatives and academic advisors.

Three experimental `working groups' were established, namely Genotoxicity, Hepatotoxicity, and Nephrotoxicity. Participating laboratories undertook to perform experiments to generate material for gene expression studies which were then distributed to other labs. These labs and others also used one or more `transcript profiling' technologies to obtain large scale gene expression information. The results were then shared with the wider group for analysis and discussion.

The Genotoxicity Working Group elected to study fourteen direct-acting mutagens and clastogens, with several in vitro systems. Common compounds, experimental protocols and genotoxicity end-points were agreed amongst labs in a `pair-wise' fashion, i.e. pairs of labs elected to perform experiments with the same cells, compounds and doses, so that comparable data sets from two different sites could be generated. Five different technical platforms for gene profiling are involved.

At the Programme's plenary meeting in Washington 22^nd –23^rd May 2001, the individual working groups presented an initial analysis of the data sets from their respective areas. This presentation will give an overview of progress to date, and present some of the early messages coming out of this large, multinational collaborative exercise.

3. Telomere dynamics, immortalisation, and cancer progression in mouse and man

Jerry Shay

University of Texas Southwestern Medical Center at Dallas, Department of Cell Biology, 5323 Harry Hines Boulevard, Dallas, Texas, USA 75390-9039

There is substantial evidence in human cells that replicative senescence (Mortality Stage 1 or M1) is induced as a G1, p53-dependent cell cycle checkpoint growth arrest when one or a few short telomeres are recognized as damaged DNA. Senescence can be overcome by viral oncogenes (such as SV40 large T-antigen) that can inactivate both p53 and p16^Ink4a/pRB checkpoint pathways. Such cells are not immortal but continue to divide and lose telomeric repeats. Terminal telomere shortening eventually causes a second growth arrest stage termed crisis or Mortality Stage 2 (M2). In contrast to mouse cells, escape from M2/crisis is a very rare event in human cells and is almost always accompanied by activation of telomerase, a cellular reverse transcriptase complex which synthesizes and extends telomeric repeats.

The expression of the catalytic subunit of human telomerase (hTERT), circumvents the induction of both senescence and crisis. hTERT immortalises a variety of normal human cell types. Such `telomerized' cells have normal cell cycle controls, functional p53, p21^Cip1, and p16^Ink4a/pRB checkpoints, are contact inhibited, anchorage-dependent, require growth factors for proliferation, and possess a normal karyotype. In addition, hTERT does not affect the ability of cells to differentiate in a variety of systems (vascular endothelium, muscle satellite cells, adrenal cortical cells, corneal epithelial cells, and skin keratinocytes). Breast epithelial (p53+/–) cells derived from patients with Li-Fraumeni syndrome, and fibroblasts derived from patients with familial adenomatous polyposis (APC +/–), basal cell nevus syndrome, ataxia telangiectasia, xeroderma pigmentosum, progeria, and Robert's, Werner's and Bloom's syndrome can also be immortalised by hTERT. In these instances the characteristic phenotypic properties of these cells are not altered by hTERT-induced immortalisation. The development of these cell reagents represents a substantial improvement over the biological uncertainties associated with the use of viral oncogenes for the establishment of human cell lines.

In cells with long telomeres and functional cell cycle checkpoints, UV-B and gamma irradiation, oxidative damage, overexpression of H-ras V12 and Raf-1 leads to an irreversible growth arrest state (often called premature-senescence). In these instances, the growth arrest is not caused by short telomeres. Many stimuli may cause 'stress' or DNA damage to cells and produce phenotypes that overlap with telomere-based replicative senescence. In addition, a variety of reports now claim that inactivation of the p16^INK4a/pRB pathway is required in addition to telomere maintenance for the immortalisation of cells such as skin keratinocytes and breast epithelial cells. The premature growth arrest of breast and skin epithelial cells grown on plastic substrates may be due to an inadequate culture environment. Cultivating telomerase-expressing breast epithelial cells or keratinocytes on feeder layers avoids the premature growth arrest associated with increased p16^INK4a. In summary, published results do not support a telomere-independent mechanism of replicative aging.

Telomere length homeostasis can be maintained by telomerase or by a recombination based alternative lengthening of telomeres (ALT) mechanism. Since a subset of telomeres in ALT cells become extremely short, ALT cells may be faced with a continuous state of crisis. Expression of telomerase in a cell line that uses the ALT pathway, VA13, results in telomerase maintaining the length of the shortest telomeres and in some cases the long heterogeneous telomeres are completely lost and the cells contain short homogeneous telomeres. In addition, there are reductions in the fraction of cells containing ALT-associated PML bodies and extrachromosomal telomere repeats. However, no alterations in the rate of sister chromatid exchange in ALT cells expressing telomerase was observed. This demonstrates that ALT cells retain factors required for telomerase to maintain telomeres and once the telomerase-based pathway of telomere length regulation is engaged, recombination-based elongation is inhibited.

References

Bodnar, A.G., Ouellete, M., Frolkis, M., Holt, S.E., Chiu, C-P., Morin, G.B., Harley, C.B., Shay, J.W., Lichtsteiner, S., and Wright, W.E. (1998) Extension of lifespan by introduction of telomerase in normal human cells. Science, 279, 349–352.[Abstract/Full Text]

Ford, L.P., Zou, Y., Pongracz, K, Gryaznov, S.M., Shay, J.W. and Wright W.E. (in press, 2001).Telomere maintenance by telomerase can inhibit the ALT pathway in human cells J. Biol. Chem.

Ouellette, M.M., McDaniel, L., Wright, W.E., Shay, J.W., and Shultz, R. (2000) The establishment of telomerase-immortalized cell lines representing human chromosome instability syndromes. Human Mol. Genetics, 9, 403–411.[Abstract/Full Text]

Ramirez, R.D., Passons, C., Rohde, J., Morales, C.P., Herbert, B-S., Shay, J.W. and Wright, W.E. (2001) Putative telomere-independent mechanisms of replicative aging reflect inadequate growth conditions. Genes and Dev., 15, 398–403.[Abstract/Full Text]

Shay, J.W. and Wright, W.E. (2001) When do telomere matter? Science, 291, 839–840.[Full Text]

Wright, W.E. and Shay, J.W. (2000) Telomere dynamics in cancer progression and prevention: fundamental differences in human and mouse telomere biology, Nature Medicine, 6, 849–851.[Medline]

Wright, W.E. and Shay, J.W. (2001) Cellular senescence as a tumor-protection mechanism: the essential role of counting. Cur. Opin. Gen. Dev., 11,98–103.

4. The role of telomerase in the malignant transformation of human cells

William C. Hahn

Department of Adult Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115 USA

The cancer cell arises from normal precursors through the accumulation of genetic mutations that permit uncontrolled proliferation. Several lines of evidence now implicate telomeres as a major molecular mechanism that regulates replicative lifespan. We and others have shown that ectopic expression of the telomerase catalytic subunit, hTERT, extends the lifespan of both pre-senescent and post-senescent, pre-crisis cells. These observations establish that repression of telomerase plays an important role in regulating both senescence and crisis. Corroborating these studies, disruption of telomere maintenance by expression of a dominantly-acting, catalytically inert mutant of hTERT in human cancer cell lines led to inhibition of telomerase activity, telomeric shortening, and widespread apoptosis. These observations support the notion that the up-regulation of hTERT observed in human tumors directly allows cancer cells to divide beyond the replicative capacity of normal cells. To investigate the role of hTERT in the transformation of human cells, we co-expressed combinations of hTERT, the simian virus 40 early region (ER) and an oncogenic allele of H-ras in normal human embryonic kidney epithelial, mammary epithelial, small airway epithelial cells and human foreskin fibroblasts. Only cells co-expressing all three genetic elements were immortal, maintained stable telomere length, exhibited anchorage-independent growth, and formed tumors in animal hosts. These results demonstrate that telomerase cooperates in the direct tumorigenic conversion of human cells. Expression of hTERT in human cells that utilize the telomerase-independent alternative (ALT) mechanism to maintain telomere length enhanced tumorigenic growth. Using SV40 large T antigen mutants, we have begun to dissect the intracellular pathways that cooperate with telomerase and oncogenic ras to convert normal cells to tumorigenicity. The SV40 ER contributes to tumorigenic transformation by perturbing three intracellular pathways through the actions of the SV40 large T antigen (LT) and the SV40 small t antigen (ST). LT simultaneously disables the retinoblastoma (pRB) and p53 tumor suppressor pathways; however, complete transformation requires the additional perturbation of protein phosphatase 2A by ST. Taken together, these studies identify a limited set of genetic alterations that cooperate to program the conversion of normal human cells to the tumorigenic state.

5. Regulation and Higher Order Structure of Human Telomerase

Joachim Lingner

Swiss Institute for Experimental Cancer Research (ISREC), CH-1066 Epalinges, Switzerland

Telomerase is required for the complete replication of chromosomal ends. In tumours, the telomerase reverse transcriptase subunit (hTERT) is up-regulated thereby removing a critical barrier for unlimited cell proliferation. Microcell transfer of a normal copy of chromosome-3 into several human cancer cell lines abolishes telomerase activity and induces senescence. We measured hTERT RNA levels by quantitative reverse transcriptase (RT)-PCR (A.L. Ducrest, M. Amacker, Y. Matthieu and M. Nabholz, in collaboration with A.P. Cuthbert, D.A. Trott and R.F. Newbold (Brunel University)). Telomerase-positive cell lines were found to contain between 0.2 and 4 molecules of hTERT RNA per cell, whereas no hTERT RNA was detectable in telomerasenegative monochromosome-3:21NT hybrids. Because direct upregulation of hTERT through c-Myc overexpression had previously been reported, we investigated whether molecules of the c-Myc family could be involved in the down-regulation of hTERT by chromosome 3. A > 60 fold reduction of hTERT RNA was observed following the introduction of normal chromosome 3, but expression levels of c-Myc and c-Myc target genes were unchanged. Our results suggest that telomerase is regulated primarily at the level of hTERT transcription and that the putative hTERT repressor on chromosome 3 is unlikely to regulate the expression of hTERT through c-Myc or one of its co-regulators.

Telomerase uses a short stretch of its intrinsic RNA molecule as template for telomere repeat synthesis. Reverse transcription of the RNA template is catalyzed by the TERT protein subunit. We have determined that human telomerase reconstituted from recombinant TERT and telomerase RNA runs as a dimer on a gel filtration column and that it contains two telomerase RNA molecules (C. Wenz, M. Amacker and C. Kelleher in collaboration with B. Enenkel and K. Damm (Boehringer-Ingelheim)). Significantly, a telomerase heterodimer reconstituted from wild type and mutant telomerase RNA is barely active when compared to the wild type homodimer. We conclude that the telomerase RNA templates in the active enzyme are interdependent and functionally cooperate with each other.

6. p53: at the cross-roads of senescence and cancer

D. Wynford-Thomas, D. Kipling

Cancer Research Campaign Laboratories, Department of Pathology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN

In addition to regulation by extracellular factors, most normal mammalian cells possess intrinsic controls capable of limiting the number of times the cell can divide. Such `proliferative lifespan barriers' (PLB) are likely to have evolved as an innate anti-cancer mechanism but are also a potential source of organ failure with increasing age.

The classic PLB, as seen in normal human fibroblasts (NHF) and several other cell types, appears to be mediated by telomere erosion acting as a biological clock triggering G1 cell cycle arrest once a critical threshold has been reached. We and others have shown that a key signalling intermediate in this pathway is the tumour suppressor protein p53. Thus, gene transfer and micro-injection studies have established that p53 function is essential for induction of senescence in NHF, and surprisingly also for its maintenance (the latter pointing to a potential therapeutic route to reversal of age-related tissue dysfunction). Consistent with a direct signalling role, p53 transactivation activity (though interestingly not protein level) correlates well with growth arrest at senescence and is accompanied by a spectrum of post-translational changes consistent with activation. Some of the observed changes in phosphorylated residues (notably at S15 and T18), though not others (for example at S20 and S392), are also seen in response to ionising radiation, suggesting that telomere erosion generates a signal similar to that of strand-break DNA damage. Consistent with this, telomere stabilisation by forced activation of telomerase in NHF leads to permanent escape from senescence and prevention of all senescence-associated changes in p53. In contrast, escape from senescence by abrogation of p53 function alone is only temporary, cells subsequently entering a second, senescence-like arrest after a further 15-25 population doublings. This state, which we have termed `M^int' (to distinguish it from M1 senescence and M2 crisis), is associated with further elevation of the CDK inhibitor p16^INK4a, which is probably necessary (though we believe not sufficient) to account for growth arrest. The nature of this second PLB is currently of major interest given its potential as a tumour suppressor mechanism.

In many other human cell types, the rate-limiting controls on proliferative lifespan appear to differ (at least in tissue culture). Thus in astrocytes, abrogation of p53 function leads to temporary escape from M1 senescence ending in M^int state similar to NHF, but forced telomerase activation is without effect, indicating that p53 is being activated by a telomere-independent signal. In thyroid epithelial cells, neither loss of p53 nor activation of telomerase is sufficient to escape growth arrest, which in this case appears to require, in addition, targetting of the Rb pathway.

Such variations in the nature and timing of PLBs between different cell types may be an important explanation for the differences in `selection' of tumour-suppressor gene loss observed in the corresponding human cancers.

7. Mechanisms of human keratinocyte senescence and immortalisation

K. Parkinson

CRC Beatson Laboratories, Garscube Estate, Switchback Road, Glasgow G61 1BD

In contrast to normal human keratinocytes many neoplastic squamous lesions are immortal. Immortality is not especially associated with advanced tumours, since around 40% of premalignant lesions are immortal but is tightly associated with the dysfunction of p16INK4A and p53 and the presence of high levels of telomerase. The dividing layers of stratified epithelia have been reported to express the telomerase component genes, hTERT and hTR, and to possess telomerase activity. It is therefore unclear whether the telomerase positive tumours result from the selection of pre-existing telomerase positive cells (selection), or arise following genetic/epigenetic alteration, following a period clonal proliferation as a telomerase-negative population (re-activation). One prediction of the re-activation hypothesis is that human tumour cells should exist, which although they have bypassed senescence, still have insufficient telomerase to maintain telomere function and should eventually enter crisis. We present evidence that at least some squamous neoplasms display crisis properties (normal BrdU incorporation, short telomeres, low telomerase, frequent dicentric chromosomes, low cloning efficiency, high TUNEL staining) and that the defects can be corrected by the ectopic expression of hTERT. Control immortal lines were unaffected by the same manipulation. However, only 2 out of 39 cultured biopsies displayed features of crisis, leading us to question why crisis is so rare. It is possible that the telomerase re-activation pathway is very rare and most squamous neoplasms arise from pre-existing telomerase positive cells. However, it is also possible that the combination of critically short telomeres and a dysfunctional p53 gene creates such great genetic instability that telomerase is soon re-activated in epithelial populations in crisis. Three lines of evidence support the latter possibility. Firstly, the number of chromosomal gains and losses in immortal cancer cells greatly exceeds that of mortal cancer cells, and the chromosomes of immortal squamous lines show evidence of telomeric associations. Secondly, immortal squamous cell lines show gains of 5p15 and 3q26-q27, which carry the genes hTERT and hTR respectively, and loss of 3p, which carries a suppressor of telomerase activity. Thirdly, the transfer of chromosome 3 into immortal squamous lines inhibits telomerase activity and elicits telomeric attrition. Overall, our results support the hypothesis that normal keratinocytes have not enough telomerase to maintain telomere function and that most immortal neoplastic squamous cells re-activate telomerase following clonal expansion as a telomerase-negative population of cells.

8. Mouse models to study the role of telomeres and telomerase in tumour suppression and carcinogenesis

E. Gónzalez-Súarez¹, F.A. Goytisolo¹, E. Samper¹, A. Ramírez², J. Martín-Caballero¹, J.M. Flores³, P. Finnon⁴, S.D. Bouffler⁴, J.L. Jorcano², M.A. Blasco¹

¹ Department of Immunology and Oncology, Centro Nacional de Biotecnología-CSIC, Madrid

²Project on Cell and Molecular Biology and Gene Therapy, CIEMAT, Madrid

³Department of Animal Pathology II, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid

⁴Radiation Effects Department, National Radiological Protection Board, Oxfordshire

Continued cell division in the absence of telomerase is a potent inducer of chromosomal instability, which is thought to be a key event in cellular transformation. Hence, mice genetically deficient for telomerase activity provide a unique opportunity to understand the role of telomere maintenance in oncogenic transformation in vivo. Mice that lack the mouse telomerase RNA (mTR) are viable for only four to six generations depending on the genetic background. We have used late generation mTR–/– mice for chemical carcinogenic studies on the skin; these mice have a normal phenotype even though their telomeres are very short. The study shows that late generation mTR–/– mice have a highly significant low appearance of skin papilomas when compared to the wild type mice. Conversely, over-expression of TERT in mouse keratinocytes causes an increase in skin tumors upon induction by chemical carcinogens. We will also present evidence that mice deficient for Ku86–/– are also resistant to chemically induced skin carcinogenesis; Ku86–/– is a protein involved in non-homologous end joining and which plays a very important role in stabilising the telomere. We have also shown that when late generation mTR–/– mice are treated with DNA damaging agents that normally induce carcinogenesis, the result is death of the organism. The results suggest that telomere shortening may in fact protect the organism from tumor progression. Also critically short telomeres enhance the sensitivity of the organism towards DNA damaging agents. These results may have important implications for radiotherapy as tumors treated with telomerase inhibitors could lead to a telomere loss, which is likely to increase the sensitivity of the tumors to radiotherapy.

9. Public Attitudes to Genetic Technologies

Martin Richards

Director, Centre for Family Research, Faculty of Social and Political Sciences, University of Cambridge, Free School Lane, Cambridge CB2 3RF

The paper will discuss the extent to which genetic information may be distinctive in comparison with other kinds of biological information and some of the particular ethical issues its collection, storage and use may create.

Drawing on a number of sources of data, public attitudes towards the use of genetic technologies will be discussed. While there is strong public support for the use of DNA technologies for the diagnosis and treatment of disease, about a third of the population agrees with the statement that `research on human genetics is tampering with nature and is therefore unethical'. It is argued that at least, in part, this concern relates to the wide use by biologists of misleading metaphors for the genome and its function. Surveys indicate that, in general, the public greatly over-estimates the extent to which genetic technologies may already play a significant role in health care. This might suggest there is a danger of unreal expectations of the `genetic revolution', at least for the medium term.

In other areas attitudes may be more influenced by ends rather than means. So, for example, the strong public support for forensic DNA databases may reflect concerns about serious crime and the need to apprehend those responsible, rather than anything related to DNA technology. Similarly, the majority, who do not think that insurance companies should have access to genetic test information, may simply be expressing general attitudes to the insurance industry. Though again in the contexts such as these, the use of metaphors such as `the essence of humanity', `the secret of life' or `the book of life' may serve to increase anxieties about the use of genetic information by third parties.

10. From hazard identification to risk assessment, the evolution of the science of genetic toxicology

James M Parry

Centre for Molecular Genetics and Toxicology, University of Wales Swansea, Swansea SA2 8PP UK

The standard in vitro mutagenicity test battery recommended by the UK Advisory Committee on Mutagenicity is made up of a package of methods capable of identifying the potential mutagenic hazard of chemicals (COM 2000). However, risk assessment of mutagenic activity requires more comprehensive mechanistic information which can be used to assess the significance and thus the relevance of mutagenic hazards detected in vitro to intact animals including humans. Considerable efforts have been made to develop methods which aid in risk assessment.

The in vitro micronucleus assay provides an example of a method with both the capacity to identify modifications of chromosome integrity and the ability to distinguish between clastogenic and aneugenic activity. When aneugenic activity is detected the analysis of chromosome segregation patterns in binucleate cells provides the ability to quantify both chromosome loss and non-disjunction. Model point mutation systems such as the analysis of the SupF containing plasmids and the restriction site mutation assay provide methods to identify the specific type of mutations induced by a chemical and the position in the DNA sequence of mutagen interaction. Similarly, molecular cytogenetics now provides a range of methods which can be used to compare the profiles of chromosome changes observed in both spontaneous and chemically induced tumours. Combinations of sequence and chromosome analysis provide powerful tools for identifying unique compound related patterns of genetic change. Metabolically active cell lines also provide tools for the evaluation of the role of variations in biotransformations in the assessment of genotoxic risk.

Thus, molecular methods provide information which allow comparisons between the qualitative and quantitative effects of mutagens. Of particular importance, when molecular information is available on the specificity of induction of point and chromosomal mutations, is the relationship between spontaneous and test chemical profiles of activity and dose response relationships. Thus the emphasis of our science changes to identifying and quantifying real risk rather than potential but unquantified hazard.

The past 10 years has also seen the development and application of highly sensitive methods for detecting and quantifying the presence of DNA adducts which act as the basis for the initiation of mutagenic changes. However, such methods are currently hazard based and provide measures of exposure rather than quantitative indicators of genetic change and care should be used in their application in genotoxic risk assessment.

Reference

COM (2000). Guidance on a strategy for testing of chemicals for mutagenicity. UK Department of Health.Advisory Committee on the Mutagenicity of Chemicals.

11. Distinguishing between chalk and cheese: verification of the validity of cell line designation by DNA profiling

C.F. Arlett

Department of Biological Sciences, Brunel University, Uxbridge, Middlesex, UP8 3PH

A survey of two issues of Cancer Research revealed that the majority of papers reported on studies with cells of human origin. Very few gave adequate information on the provenance of their working material and virtually none provided evidence of testing for mycoplasma infections. Instances of mycoplasma infection are well known and since these organisms are known to perturb many cellular functions it is regrettable that the relatively simple tests for their presence do not appear to be in wide spread use. Mislabelling and contamination between cell lines is a long-standing and frequent cause of scientific confusion and misrepresentation. Indeed, estimates from national testing services indicate that up to 36% of cell lines may be of a different origin or species to that claimed. Well known examples of contamination concern HeLa a keratin-forming human tumour cell line of cervical epithelial origin. Despite its apparent basis in HeLa contamination a cell line KB is still commonly presented as a separate entity, with over 380 papers referring to KB cells listed in Medline since the link to HeLa was discovered. In order to test a standard method of cell line authentication, 253 coded human cell lines from cell banks and research institutes worldwide were analyzed by short tandem repeat (STR) profiling. The STR profile is a simple numerical code that is reproducible between laboratories, inexpensive and can provide an international reference standard for every cell line. Its use is not restricted to cells of human origin. If DNA profiling of cell lines is accepted and demanded internationally, scientific mis-representation due to cross-contamination can be largely eliminated. I recommend that its use should be mandatory in GLP.

12. Short-term exposure to carbendazim induces micronuclei in cultured human peripheral blood lymphocytes

Riaz Mahmood^{1, 2} and James M. Parry¹

¹Centre for Molecular Genetics and Toxicology, School of Biological Sciences, University of Wales, Singleton Park, Swansea SA2 8PP UK

²Department of Biotechnology, Kuvempu University, Gnanasahyadri, Shankarghatta-577 451. Shimoga Dist. Karnataka, India

Among all the chemical pollutants synthetic pesticides pose a major threat to the living systems. Apart from producing general health hazards they may also induce aneuploidy and chromosomal aberrations. Aneuploidy is considered as being the basis of a number of genetic diseases. It is therefore, important to detect the aneugenic effects of widely used pesticides. Carbendazim (methyl benzamidazol-2-ylcarbamate), a highly active metabolite of benomyl and a known fungicide has been used in the present experiments. Although, its aneugenic effects causing micronuclei and non-disjunction have been studied by Bentley et al., (2000) in cultured human lymphocytes, their studies were performed using long durations of chemical exposure (48h). To model conditions closer to potential human exposure, we have made an attempt to detect its ability to induce micronuclei using very short exposures of 1 and 2 hours after 24 h of the initiation of cultures. The cytokinesis blocked in vitro micronucleus assay was performed using 72 h human blood lymphocyte cultures. Five different doses of carbendazim ranging from 0.5 µg/ml – 8.0 µg/ml were employed. After the chemical treatment, cultures were washed and fresh medium was added. Micronuclei were scored from both mono- and binucleated cells. The data suggest that at almost all concentrations, carbendazim produced significant number of micronuclei when compared to the control cultures for both the treatment schedules of 1h and 2h. The present study clearly indicates that the fungicide carbendazim is a very active compound and has the ability to induce aneuploidy even at the shortest exposure time of 1h. These results indicate that there is a need to analyse the genotoxic and aneugenic effects of commonly used chemicals at shorter exposure times, since the duration of cellular exposure may vary from chemical to chemical. Furthermore, the understanding of the relationships between dose and exposure time could be more useful in the safe handling of the hazardous chemicals in day-to-day life.

13. Genotoxic agents in human milk

L.M.C. Wheat, F.L. Martin, D.H. Phillips

Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton, Surrey, SM2 5NG, UK

Genotoxic agents of dietary and/or environmental origin may play a role in the aetiology of breast cancer. Breast milk is known to contain components capable of causing DNA damage in in vitro assays and is potentially a means of investigating the exposure of breast epithelial cells to environmental agents. In the present study, extracts of human milk (8–48 g-equiv.) were examined for their ability to cause DNA single-strand breaks in MCL-5 cells (`Comet' assay) in the presence of DNA repair inhibitors hydroxyurea and cytosine arabinoside, and to cause mutations in Salmonella typhimurium strain YG1019 (Ames assay) in the presence of Aroclor 1254-induced rat liver S9. Cows' milk was a negative control. Extracts of milk were prepared by a solid-phase tandem extraction (64 g-equiv.) and fractionated (8 fractions, 10 mls) by reverse-phase HPLC. Fraction 3 was significantly positive for comet formation (16 g-equiv., median comet tail length = 121.5 µm), compared with control (15.0 µm), (P<0.0001, Mann-Whitney test) and also significantly mutagenic in YG1019, (880 ± 47 revertants/plate, 16g-equiv., compared with 21 ± 4 for controls). In all experiments, genotoxicity was observed in the absence of cytotoxicity. Furthermore, fractionated blue rayon extracts from pooled milk samples gave rise to additional positive fractions (3, 4 and 6) over a dose range of 2.5 – 25 g-equiv. milk/plate in the Ames assay. In further studies, partitioning of activity between lipid and skimmed milk was examined. Milk phases, separated by centrifugation, were extracted. After HPLC, approximately two-thirds of the total Ames activity (fraction 3) partitioned in the aqueous and one-third in the lipid phase when compared with whole milk, suggesting that the agent/s responsible for the observed genotoxicity are moderately polar. This type of approach may provide new clues as to the role of environmental agents in the initiation of breast cancer.

14. Ageing and genetic alteration in the male germ-line

M.H. Brinkworth, T.E. Schmid

Department of Biomedical Sciences, University of Bradford, Bradford BD7 1DP, UK

The greatest mutational risk to the human population is the ageing male (Crow, 1997), partly because the spontaneous mutation rate in the male germ-line increases with age. The reason for this is not know but is likely to involve an age-related degeneration in the efficacy of cellular processes. One such process that might be responsible is apoptosis and mutation rates are higher in spermatogenic-cell types that rarely undergo apoptosis than in those where it is common (Walter et al., 1998). Suppression of spermatogenic cell apoptosis can lead to the fixation of mutations in the germ-line (Brinkworth, 2000). In order to investigate this, 2-month and 12-month old male MF-1 mice were examined using FISH to assess sperm aneuploidy and a modified TUNEL technique for levels of apoptosis among testicular cells. Sperm were labelled with probes to chromosomes 8, X and Y and 20,000 sperm scored from 5 animals each per group. A significant increase of gonosomal disomy was found in the aged mice (0.053% vs. 0.034% in the control group, p=0.041). Aneuploid sperm with X-X-8 were significantly higher (2-fold) in the old mice (0.028% vs. 0.013% in the controls, p=0.018). The data suggest that ageing affects segregation during the second meiotic division. No higher diploidy or autosomal disomy rate was observed in the older group. Apoptosis (apoptotic cells per seminiferous tubule cross-section per animal per group) was higher in the old mice (1.44 n = 4) than controls (0.92 n = 5) but not significantly. The rise in levels of sperm aneuploidy is novel for 1-year old mice and confirms the genotoxic effect of age. Since apoptosis usually eliminates unrepaired damage the apoptotic response in older mice may be compromised. This is unproven but the data show no dramatic increase in apoptosis with the significantly elevated rate of genetic damage.

References

Brinkworth, M.H. (2000) Int J Androl. 23, 123–35[Medline]

Crow, J. F. (1997) Proc Natl Acad Sci U S A, 94, 8380–6[Abstract/Full Text]

Walter, C.A., Intano, G.W., McCarrey, J.R., McMahan, C.A., Walter, R.B. (1998) Proc Natl Acad Sci USA, 95, 10015–9[Abstract/Full Text]

15. Chinese herbs nephropathy and urothelial carcinoma in the UK

V.M. Arlt^1,2, H.H. Schmeiser², J.L. Nortier³, J.-L. Vanherweghem³, T. Cook⁴, G. Williams⁴, C.D. Pusey⁴ and G.M. Lord⁴

¹Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey SM2 5NG, UK

²Division of Molecular Toxicology, German Cancer Research Center, Heidelberg, Germany

³Department of Nephrology, Hôpital Erasme, Brussels, Belgium

⁴Division of Renal Medicine and Transplantation, Hammersmith Hospital NHS Trust, London W12 0NN, UK

Chinese herbs nephropathy (CHN), a unique type of nephropathy, associated with the prolonged intake of Chinese herbs during a slimming regimen, was observed for the first time in Belgium in 1991. The toxic effects have been traced to Aristolochia fangchi containing nephrotoxic aristolochic acid (AA) inadvertently included in the weight-reducing pills. In a large cohort of 39 Belgian CHN patients with end-stage renal disease we recently found 18 cases with urothelial carcinoma (UC) (prevalence 46%) and 19 cases showed mild to moderate dysplasia (Nortier et al., 2000). Logistic regression analysis predicted that the cumulative dose of Aristolochia fangchi was associated with a significantly higher probability of developing UC (P=0.045). Using the ³²P-postlabelling method we found specific AA-DNA adducts, described biomarkers of AA exposure and associated with AAs carcinogenic and mutagenic activity (Arlt et al., 2000, 2001), in all urothelial tissues analyzed. The major adenosine adduct of aristolochic acid I (dA-AAI), the major component of the plant extract AA, was detectable in renal (RAL from 0.12 to 16.5 per 10⁸ nucleotides, n=61) and ureteral (RAL from 0.25 to 3.0 per 10⁸ nucleotides, n=17) tissues even 7 years after the patients stopped taking the herbal drugs. Since the onset of Belgian cases similar clinical presentations have been reported in other European and in Asian countries. Previously, we reported two CHN patients in the UK who had taken herbal preparations containing AA prescribed for eczema (Lord et al., 1999). We now report the development of invasive urothelial carcinoma in one of these two English CHN patients associated with the presence of AA-DNA adducts (RAL: 0.38 and 4.0 dA-AAI-adducts per 10⁸ nucleotides in renal and ureteral tissue, respectively). This is the first direct demonstration of the human carcinogenicity of AA outside of the Belgian cohort. In conclusion our results highlight the role of AA in the causation of this nephropathy. Moreover, our data clearly demonstrate that exposure to AA and in particular the formation of AA-DNA adducts is involved in the development of urothelial cancer in CHN patients.

References

Arlt et al (2000)Carcinogenesis, 21, 235–242.[Abstract/Full Text]

Arlt et al (2001) Carcinogenesis, 22, 133–140.[Abstract/Full Text]

Lord et al (1999) Lancet, 354, 481–482.[Medline]

Nortier et al (2000) New Engl. J. Med., 342, 1686–1692.[Abstract/Full Text]

16. Association of polymorphisms in the glutathione S-transferases with risk of non-melanoma skin cancer in renal transplant recipients

T.J. Lovatt¹, H.M. Ramsay², A.A. Fryer¹, R.C. Strange¹, C.M. Hawley⁴, D. Nicol⁴, P.N. Harden³

Departments of Cell and Molecular Medicine¹, Dermatology ²and Nephrology³ North Staffordshire Hospital, Stoke on Trent, United Kingdom; and Renal Transplant Unit⁴, Princess Alexandra Hospital, Brisbane, Queensland, Australia

Non-melanoma skin cancer (NMSC) represents a significant cause of morbidity and mortality among renal transplant recipients, with tumors behaving more aggressively than those in non-transplant patients. However, not all immunosuppressed patients develop NMSC and in those that do, the rate of accrual of lesions varies considerably. Though UV is critical, it is unlikely that this alone explains the observed phenotypic diversity, suggesting the possible involvement of genetic factors. We have previously shown that polymorphism in members of the glutathione S-transferase (GST) supergene family is associated with altered NMSC risk in non-transplant patients. Accordingly, we examined allelism in GSTM1, GSTP1, GSTM3 and GSTT1 in 398 renal transplant recipients recruited from Queensland (mean age at transplantation; 41.0±14.2 years, mean follow up; 8.6±7.4). Structured interview and full skin examination was performed; 198 developed 4080 histologically-proven NMSC. 1834 invasive squamous cell carcinoma (SCC) arose in 139 patients, 1029 in situ SCC in 123, 976 basal cell carcinoma (BCC) in 151, 234 keratoacanthoma in 65, and 1 sweat gland tumour. NMSC was associated with fair skin, red/blonde hair, hazel/blue eyes, duration of immunosuppression, male gender, and outdoor occupation. Negative binomial regression analysis showed that, compared with GSTM1 AB, GSTM1 null was associated with increased numbers of both SCC (p=0.008, rate ratio(RR)=7.14, 95% CI=1.67-33.33) and BCC (p=0.004, RR=7.69, 95% CI=1.89-33.33). GSTT1 null was associated with fewer SCC (p=0.023, RR=0.48, 95% CI=0.26-0.90) while GSTM3 AA was associated with reduced numbers of SCC (p=0.010, RR=0.47, 95% CI=0.27-0.83). These data, together with our previous findings in UK transplant and non-transplant patients, suggest that genetic factors mediating protection against oxidative stress are important in NMSC development in immunosuppressed patients.

17. Susceptibility to basal cell carcinoma: Comparison of characteristics in cases with and without squamous cell cancers

Tracy Lovatt¹, Andrew Smith², John Lear², Bill Bowers³, Peter W Jones⁴, Anthony A Fryer¹, Richard C Strange¹, Sudarshan Ramachandran⁵

¹Clinical Biochemistry Research Group, School of Postgraduate Medicine, North Staffordshire Hospital, Staffordshire, ²Department of Dermatology, North Staffordshire Hospital, Stoke-on-Trent, Staffordshire, ³Department of Dermatology, Royal Cornwall Hospitals, Truro, Cornwall, England, ⁴Department of Mathematics, Keele University, Staffordshire, ⁵Department of Biochemistry, Good Hope Hospital, Sutton Coldfield, West Midlands, B75 7RR, England

Cutaneous basal cell carcinoma (BCC) is the most common cancer in Caucasians. Patients with this cancer demonstrate considerable variation in phenotype. For example, tumor accrual differs markedly between patients. Thus, while most patients present with relatively few lesions, a minority develop numerous primary tumors. We found that all patients with more than six primary BCC demonstrated one or both of two phenotypes. The first phenotype, termed multiple presentation phenotype (MPP) is characterised by the development of clusters of BCC. The second phenotype, the development of tumors on the trunk, was also associated with more BCC than other patients. Available evidence indicates that these phenotypes are associated with distinct protective and risk genetic factors. A further aspect of phenotypic diversity in BCC patients is a predisposition to develop other cutaneous malignancies. The factors that determine why most patients develop only BCC while a minority suffer a BCC and, an SCC are not known though we speculate that these patients comprise a high-risk group with a strong genetic predisposition. Thus, a group comprising patients with a BCC and SCC might include a high proportion of patients with risk phenotypes such as male gender, the MPP or presentation with truncal tumors. They might also be expected to demonstrate high rates of BCC accrual. We describe studies in 140 patients with at least one BCC and one SCC to firstly, compare the clinical characteristics of these patients with those of 1041 cases who developed BCC only. We found that BCC accrual was similar in the two groups and there was no relationship between BCC and SCC numbers in individual patients. Similarly, we found that the influence of genetic polymorphism on BCC phenotype was similar in the groups. The data suggest that development of both SCC and BCC in individual patients is not defined by high-risk characteristics but rather the two tumours develop by random events.

18. The strong relationship between

Helicobacter pylori caga and vaca genotypes and disease outcome in a Saudi Arabian population

H. Ismail¹, N. Balaratnam¹, G. Jenkins¹, N. Ismail^3, B.M. Al Sallami⁴, J.G. Williams² and J.M. Parry¹

Schools of Biological Sciences¹ & Postgraduate Studies², University of Wales Swansea, Swansea and Department of Microbiology³, King Abdul Aziz University Hospital & Department of Gastroenterology, King Fahad General Hospital⁴, Jeddah, Saudi Arabia

Helicobacter pylori (HP), a spiral shaped gastric organism, is the commonest human chronic bacterial infection. It is associated with chronic atrophic gastritis, peptic ulcer disease and gastric carcinoma. IARC has classified HP as a class 1 carcinogen. The presence of the cagA gene and different vacA subtypes (virulence genes) of HP are associated with disease outcome. This relationship and the predominant HP strain in circulation varies in different geographical regions. Here, we describe for the first time the relationship of the cagA and vacA virulence genes to clinical outcome in a Saudi Arabian population. HP isolates were obtained from 92 infected patients undergoing endoscopy at the King Abdul Aziz University Hospital & King Fahad General Hospital (Jeddah, Saudi Arabia). DNA extracted was analysed by the polymerase chain reaction with primers for the flagellin, vacA and cagA genes. Histology was reported by a single pathologist. The results were coded. The mean age of patients was 41 years. The cagA prevalence was 50%. Histology revealed chronic atrophic gastritis (45 cases), intestinal metaplasia [IM] (20), peptic ulcer disease [PUD] (16), gastric adenocarcinoma [GA] (10) and normal (1). The cagA gene was present in 20%, 65%, 94%, 80% and 0% respectively. 94% of patients with IM, PUD and GA possessed the S1 subtype of the vacA gene, and of those 80% exhibited the cagA gene. 4% were mixed S1 & S2 HP infections. However, only 58% of cases with chronic atrophic gastritis had the S1 subtype (20% with the cagA gene). 59% of the S1 subtype in total possessed the cagA gene compared to 20% with the S2 subtype. A strong relationship exists between the presence of the cagA gene and vacA S1 subtype, and a more serious clinical outcome in this population.

19. Diversity of the Helicobacter pyloricaga gene in a Welsh population

N. Balaratnam¹, H. Ismail¹, G. Jenkins¹, P. Herron¹, J.G. Williams², and J.M. Parry¹

Schools of Biological Sciences¹ and Postgraduate Studies², University of Wales Swansea, Swansea SA2 8PP

Helicobacter pylori (HP) is a clinically important bacterium, because it is associated in the pathogenesis of peptic ulcer disease and gastric carcinoma. The cytotoxic associated gene (cagA) is a marker for a genomic pathogenicity island. The presence of this island is associated with a more serious clinical outcome in most populations. Despite overall conservation of most genes, HP is a highly diverse bacterial species. Their population structure indicates that they are freely recombining. The nucleotide sequence of the cagA gene contains internal duplication of a 102bp fragment. Differences (possibly by a mechanism to generate antigenic diversity) have been suggested to generate proteins with different sizes and immunogenicities. Here, we evaluate whether there is a biological significance to variability in the 5' region of the cagA gene in a Welsh population; a population with a moderately high prevalence (77%) of isolates containing the cagA gene. Nineteen HP cagA positive strains isolated from dyspeptic patients were studied for diversity in the cagA gene. The nucleotide and amino acid sequence homology between all samples were 94.8% and 92.3% respectively. Sequence changes of the transition type were on average six-fold more frequent than transversion changes. Non-synonymous substitutions were more common than synonymous substitutions. Conservative amino acid changes were 1.5 times as common as non-conservative changes. AT to GC and/or CG to TA changes were the most frequently encountered. The average GC content of the samples was 38%. Four sequences had an insertion leading to a frameshift. The sequence of the HP cagA gene in this population appears to be reasonably well conserved.

20. Potential genotoxic risk for humans by the environmental contaminant 3-nitrobenzanthrone

V.M. Arlt^1,2, C.A. Bieler¹, M. Wiessler¹, D.H. Phillips² and H.H. Schmeiser¹

¹Division of Molecular Toxicology, German Cancer Research Center, Heidelberg, Germany, ²Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, UK

Diesel exhaust is known to induce tumours in animals and is suspected of being carcinogenic in humans. Of the compounds found in diesel exhaust and in airborne particulate matter 3-nitrobenzanthrone (3-NBA) is a particularly powerful mutagen and was shown to be genotoxic in vitro (Bieler et al., 1999) and in vivo in rats (Arlt et al., 2001) by forming DNA adducts. In this study a panel of V79 Chinese hamster fibroblast cell lines, expressing various human cytochrome (CYP) P450 enzymes (CYPh1A1, CYPh1A2, CYPh3A4) and/or human NADPH:CYP P450 oxidoreductase (CYPhOR) was used to identify enzymes involved in the metabolic activation of 3-NBA in humans. We analysed the formation of specific 3-NBA-adducts by ³²P-postlabelling after exposing cells to 1 µM 3-NBA. In all cell lines tested, an identical pattern with a total of four distinct 3-NBA-adducts was found similar to those found in vitro using xanthine oxidase or rat liver S9 as the activating system and in rats in vivo. On TLC plates all adducts migrated primarily along a diagonal zone, typical for DNA adducts derived from extracts of airborne particulate matter. Total adduct levels ranged from 75 to 220 adducts per 10⁸ nucleotides using either the nuclease P1 or butanol enrichment, respectively. Comparison of activation of the parental cell line V79MZ with activation in cells expressing CYPhOR alone or expressing both CYPhOR and CYPh3A4 demonstrated that both enzymes were involved in the metabolic activation of 3-NBA. Furthermore, in V79NH cells expressing high activities of nitroreductase and N,O-acetyltransferase (NAT2), high adduct levels of up to 1 adduct per 10⁴ nucleotides were detected. When patterns produced in V79MZ cells were compared to those in V79NH cells no additional adducts were obtained. Our results demonstrate that 3-NBA binds covalently to DNA after metabolic activation, forming multiple DNA adducts that are all products derived from reductive metabolites. These results further suggest that nitroreduction is the major pathway in the bioactivation of 3-NBA. Moreover, acetylation of the initially formed N-hydroxy arylamine intermediates may contribute to the high genotoxic potential of 3-NBA.

References

Arlt et al (2001) Int. J. Cancer, in press.

Bieler et al (1999) Mutat. Res., 439, 307–311.[Medline]

21. The versatility of the comet assay; new applications to studying DNA repair in human populations and individual genes

Andrew R. Collins, ¹Eva Horvathova and ²Maria Dusinska

Rowett Research Institute, Aberdeen AB21 9SB, Scotland, UK

¹Cancer Research Institute, 833 91 Bratislava, Slovakia

²Institute of Preventive and Clinical Medicine, 833 01 Bratislava, Slovakia

Measurement of individual DNA repair capacity requires a rapid, simple, sensitive and reliable assay suitable for use in population studies. We have modified the comet assay to measure repair activity in extracts of human lymphocytes. Extract is incubated with substrate in the form of agarose-embedded nucleoids from cells pre-treated with specific DNA-damaging agent (for instance, reactive oxygen generated by photosensitiser plus light); during incubation, the damage is incised and DNA breaks accumulate. The rate of incision on DNA carrying oxidative base damage varies significantly between individuals, while samples from the same individual taken four months apart show similar activity. The comet assay has also been adapted to examine repair of particular regions of DNA, by combining it with fluorescent in situ hybridisation (FISH). Comets are prepared from cells treated with hydrogen peroxide, which causes DNA breaks, or with photosensitiser plus light, to introduce 8-oxoguanine. Formamidopyrimidine DNA glycosylase is used to convert 8-oxoguanine to breaks. After electrophoresis as usual, DNA loops containing strand breaks appear in the comet tail. The comet DNA is hybridised with oligonucleotide probes complementary to terminal exons of specific genes, tagged with either biotin or FITC. Following antibody amplification, culminating in Texas red or FITC fluorescent labels, the two ends of the gene are visualised by fluorescence microscopy. Occurrence of red or yellow/green spots in the tail indicates that a break is present in the DNA loop containing the gene. If cells are incubated after damage, DNA breaks (or oxidised bases) are repaired and comet tails become less pronounced. The rate of `retreat' of the gene-specific signals from the tail, relative to the rate of reduction of total tail DNA, indicates the gene-specific repair rate. Repair of DHFR, p53 and the alkyl transferase gene has been followed in CHO cells/human lymphocytes.

22. Lysis of whole blood in vitro causes DNA strand breaks in human lymphocytes

S. Narayanan¹, M.R. O'Donovan² and S.J. Duthie¹

¹Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB

²Safety Assessment, AstraZeneca R&D, Bakewell Road, Loughborough, Leics. LE11 5RH

Human peripheral blood lymphocytes (PBL) have been used to monitor environmentally induced genetic damage by a variety of methods involving their culture either as separated mononuclear cell fractions or in whole blood. Despite extensive use of lymphocytes in these systems, little work has addressed how time in culture or the system employed (whole blood or separated lymphocytes) influences DNA damage. In this study, we analysed DNA damage in lymphocytes over time in various culture systems using the single cell gel electrophoresis or `Comet' assay. DNA damage in lymphocytes was greatly increased by the concurrent lysis of whole blood both in freshly isolated samples and in PHA-stimulated cultures over a period of seven days. Further, there was a marked progressive increase in DNA damage with time in PHA-stimulated lymphocytes cultured in whole blood even when the lymphocytes were isolated before analysis. No such increase was seen in lymphocytes cultured as separated mononuclear cell fractions suggesting that there are components in whole blood that cause the DNA damage. Granulocytes and lysis of red blood cells are likely candidates, and DNA damage was significantly reduced in granulocyte-depleted whole blood cultures. However, even in these, significant damage was observed at later sampling times. It was concluded that careful sample preparation is of paramount importance if the comet assay is to be successfully used to assess DNA damage in human PBL. Moreover, the progressive increase in DNA damage in whole-blood cultures may influence other methods using lymphocytes for population biomonitoring, and may be significant for in vitro genotoxicity testing.

23. The Single-Laser Flow Cytometric Cytotoxicity Assay

Geoffrey Hynes, Brian Burlinson, David Gatehouse

Genetic Toxicology Dept., GlaxoSmithKline, Park Rd, Ware, Herts., UK

The Comet Assay provides a rapid, visual and quantitative method for measuring DNA strand breaks and alkali-labile sites. The comet assay is a particularly valuable technique in that it allows the detection of DNA damage and repair in virtually any eukaryotic cell population that can be obtained as a single cell suspension (Singh et al, 1988; Ostling et al, 1984; Tice et al, 2000). Within GlaxoSmithKline, the Comet assay has been further developed to meet the need to test larger numbers of compounds per week than the existing genotoxicity tests and has replaced the L5178Y Mouse Lymphoma candidate selection assay. The current Comet Assay guideline requires a means of measuring cytotoxicity in the Comet Assay. To this end we have chosen to investigate the use of flow cytometry using a BD FACSCalibur flow cytometer, and compared cytotoxicity and tail moment in the Comet Assay with relative survival in the L5178Y Mouse Lymphoma Assay.

24. Further Development of the In Vitro Micronucleus Test

Charlotte Fifield, Jo Oliver

Genetic Toxicology Dept., GlaxoSmithKline, Park Road, Ware, Herts., UK

The in vitro micronucleus test (ivMNT) is capable of identifying both clastogens and aneugens. The ivMNT has been recognised by ICH (1997) as `a promising test for the screening of aneugens'. Therefore, the need to further develop the existing protocol into a robust high throughput, micro scale screen has been identified. Using Nesslany and Marzin's paper (Nesslany and Marzin, 1999) on the 96-well method as a guide, a 24-well protocol is being developed using L5178Y mouse lymphoma cells. Flow cytometry has been used to assess cytotoxicity both post treatment and post harvest, with Acridine Orange being used to stain the cells for scoring. Known clastogens have been used to validate the system; in the absence of S9-mix (3 and 24 hours), and in the presence of S9-mix (3 hours). The results generated so far correlate well with existing data using the current in house 'macro' method.

References

ICH (1997) Genotoxicity: A Standard Battery for Genotoxicity testing of Pharmaceuticals. Department of Health and Human Service, Food and Drug Administration.

Nesslany, F. and Marzin, D. (1999) Mutagenesis, 14, 403–410.[Abstract/Full Text]

25. A Flow Cytometric Method for the Analysis of Micronuclei in vitro using L5178Y cells

E.N. Nicholas¹, T.R. Lambert¹, A.M. Lynch¹, I. Marshall², S. Newman², A. Siddiqui¹, M.D. Smith¹, S.D. Thomas¹, S.A. Walker¹, M. Wilkinson² and R.W. Rees¹

¹Genetic Toxicology, GlaxoSmithKline, The Frythe, Welwyn, Hertfordshire, AL6 9AR

²Analytical Sciences, New Frontiers Science Park, GlaxoSmithKline, Harlow, Essex

Due to the implementation of technologies such as combinatorial chemistry and genomic sciences there is increased demand for lead optimisation of candidate drugs entering development. Current in vitro and in vivo cytogenetic assays used for regulatory assessment of development compounds are labour intensive and inefficient for lead optimisation. The aim of this study was to develop a medium throughput flow cytometric screen, based on the analysis of micronuclei (MN) in L5178Y cells, to use for early development selection. Ethidium bromide was used to stain DNA in cell lysates and the dimensions of the ethidium bromide fluorescence signal combined with light scattering properties were used to distinguish MN from cell nuclei and debris. A series of clastogenic or aneugenic compounds were selected based on their ability to induce micronuclei and several non-mutagenic compounds were used as controls. Manual scoring of the samples was used to confirm the trend for MN induction observed with flow cytometry (FC). The results of this validation study suggest that the use of FC is suitable for the rapid identification and measurement of MN in cultured cells.

26. Genetic Toxicology of Gene Therapy: Current issues

C.C. Smith¹, A.M. Lynch², N.J. Gooderham¹

¹Molecular Toxicology, Imperial College School of Medicine, London

²Genetic Toxicology, Glaxo SmithKline, The Frythe, Welwyn, Herts.

Approximately 300 clinical trials of gene therapy have been approved Worldwide (Mountain, 2000), however, unlike drug approval, no conventional pre-clinical toxicological studies currently exist for DNA-based products. Existing methods do not address the potential hazards of gene therapy, and are likely to be open to question. Therefore the FDA have proposed in-house studies to examine the long-term safety of DNA-based products, particularly insertional mutagenesis. To address this issue, we have examined a model non-viral gene therapy approach in a V79 Chinese hamster cell line with hprt as a surrogate target gene. Using a plasmid based vector (pL3312BSKS) incorporating the reporter gene green fluorescent protein, and the non-liposomal lipid effectene as vector delivery facilitator, we have obtained transient transfection efficiencies ranging from 7–94%. At these transfection efficiencies, cytotoxicity was low and subsequent stable transfection (gene therapy) was achieved, but at very low level (<1%). As expected, the effect of the treatment on the mutation frequency of the hprt gene was minimal. In these experiments, the delivered gene was not targeted for site directed integration, but was dependent upon random insertion, and the absolute mutation frequency was therefore described by the level of stable transfection. Whether the same is true of other non-viral vector systems is currently under investigation, and targeted gene delivery is likely to introduce further variables into the system. However, given the limited level of sophistication of the current approach our preliminary experiments suggest that such safety assessment procedures for these novel medicinal therapies are inadequate and a new generation of reporting systems are required.

Reference

Mountain, A. (2000) TIBTECH, 18, 119–128

27. Development of a novel site-selective mutagenesis assay using LC-MS

K.I.E. McLuckie¹, D.J.L. Jones¹, J.K. Sandhu¹, G.C.K. Roberts², P.B. Farmer¹, E.A. Martin^1,3

¹MRC Toxicology Unit, University of Leicester, Lancaster Road, Leicester, LE1 9HN, UK

²NMR Center, University of Leicester, Leicester, LE1 9HN, UK

³Current address: Genetic Toxicology Department, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire, LE11 5RH, UK

We have developed a system for investigating the site-selective nature of mutagen reaction with DNA using the pSP189 plasmid shuttle vector (Parris & Seidman, 1992) in human adenovirus transformed kidney cells (Ad293). Single-stranded deoxyoligonucleotides containing a single deoxyguanosine are reacted with a mutagenic compound which forms DNA adducts with deoxyguanosine. These are then annealed with their complementary deoxyoligonucleotide strands to form a dsDNA insert. This insert is ligated into the supF gene of the pSP189 shuttle vector and transfected into Ad293 cells to allow replication of the plasmid, where either repair or mutagenesis will occur. Correctly ligated plasmid and insert will result in a mutant white colony when screened in indicator E.coli, whereas plasmids not containing the insert will be blue. White colonies are picked and PCR is used to amplify the deoxyoligonucleotide inserts. The PCR products are visualized using gel electrophoresis and then digested using Bpm1 restriction enzyme to yield small (16bp) deoxyoligonucleotides. LC-MS is used to determine the masses of these deoxyoligonucleotides, and consequently their sequence can be deduced (Laken et al., 1998). Due to the mass difference of all four bases it is possible to determine whether the deoxyguanosine-DNA adducts have been repaired or mutated in the cell system. Initially we have been using this assay to investigate the mutagenic potential of -acetoxytamoxifen, a model of the reactive intermediate of the breast cancer drug tamoxifen that causes liver cancer in rats and endometrial tumours in women. To date deoxyoligonucleotides have been treated with -acetoxytamoxifen, and ligated plasmids have been recovered. Single chromatographic peaks have been observed with corresponding masses for deoxyoligonucleotide standards. The limit of detection of the deoxyoligonucleotides by the system is now being investigated.

References

Laken et al. (1998) Nature Biotechnology, 16, 1352–1356.[Medline]

Parris & Seidman, (1992) Gene, 117, 1–5.[Medline]

28. Perspectives in research on metal toxicity and carcinogenicity by In vitro advanced methods (cell cultures)

E. Sabbioni, S. Fortaner, F. Mazzotti and M. Balls

European Commission, JRC-Ispra, IHCP, ECVAM Unit, 21020 Ispra (Varese), Italy

Specific metal compounds, particularly those of arsenic, beryllium, cadmium, chromium and nickel are occupational carcinogens. They could also pose some risk as environmental carcinogens as industrial activities redistribute them into the environment. Much of the information we have now on the carcinogenic effects of metals have been obtained by bioassays using animal models. However, interpreting animal data in experimental metal carcinogenesis with respect to the human situation is very difficult, both with regard the extrapolation from one species to another and extrapolation for low doses. The development of in vitro morphological transformation of mammalian cells in culture has greatly facilitated the study of metal carcinogenesis providing some crucial evidence specific to the carcinogenic potential of a metal which cannot be supplied by genotoxic testing, and data on the molecular mechanisms involved in carcinogenesis. The objectives of this work are: a) to review the scientific database and the state of development of cell transformation assays and their potential role in the study of metal carcinogenesis (Syrian hamster embryo (SHE) primary cell culture model, BALB/3T3 mouse fibroblast and C3H/10 T1/2 mouse embryo fibroblast), b) to discuss some research priorities that include: the organisation of a focused interlaboratory study involving one or more rodent cell-based transformation assays (prevalidation study); the need of mechanistic studies of metal carcinogenesis; the development of a reliable human cell transformation assay system; and the implementation of an in vitro testing strategy for the identification of metal-induced genotoxic and non-genotoxic events leading to malignancy, c) to present experimental results concerning the current use of the BALB/3T3 transformation assay in the context of the IMETOX project (In Vitro MEtal TOXicology) at ECVAM, in order to study the quantitative response of concurrent induction of cytotoxicity and neoplastic transformation by metal compounds of environmental, occupational and biomedical interest. Metals considered will include As, Cr, Pt, V.

29. Influence of metal speciation on cytotoxicity and carcinogenic potential induced by Pt-compounds in BALB/3T3 cell line

F. Mazzotti, J. Ponti, M. Ghiani, S. Fortaner and E. Sabbioni

European Commission, JRC-Ispra, Institute for Health & Consumer Protection (IHCP), ECVAM Unit, 21020 Ispra (Varese), Italy

The steadily increase of human exposure to metal compounds represents an utmost concern with respect to environmental, occupational and consumer safety. In this context, the assessment of the risk on human health involves also the evaluation of the potential carcinogenic effects induced by metal exposure, a problem that is complicated by the ability of many metals to exist in various chemical forms. This paper reports the results on cytotoxicity and carcinogenic potential of selected metal compounds obtained by using an in vitro rodent model, the BALB/3T3 cell transformation assay. A first screening of 53 metals of environmental, occupational and biomedical interest, shows different dose-response curves concerning six metal species (Cr (III) and (VI), Metallic- and organic-Hg, Ir (III) and (IV), Te (IV) and (VI), Pd (II) and (IV), Pt(II) and (IV)) when tested in different oxidation states. The influence of metal speciation on the biological response was particularly evident for Pt-compounds, as showed by the different degree of cytotoxicity and carcinogenic potential of their anionic, cationic and organometallic forms. The decreasing order of potency was: PtCl₂ > cisPt >> carbo-Pt > PtCl₄ > (NH₄)₂PtCl₆ >> (NH₄)₂PtCl₄ for the cytotoxicity, whereas the carcinogenic potential was ranked as: cisPt >> carbo-Pt > PtCl₄ > PtCl₂ > (NH₄)₂PtCl₆ >> (NH₄)₂PtCl₄. The determination of Pt uptake by ICP-MS (Inductively Coupled Plasma – Mass Spectrometry) analysis in the BALB/3T3 cells showed for each compound a linear increase of Pt content as a function of exposure concentration. These results suggest that Pt-compounds are able to enter the cells in a similar way. Cultured subclones originated by Pt-transformed BALB/3T3 cells have been obtained and are under characterisation. They will give new insights for investigations of specific mechanisms related to the action of different metal species.

Present address: STEM CELL Research Institute DIBIT Parco Scientifico San Raffaele, 20132 (Milano), Italy.

30. Induction of aneuploidy by xenobiotic diacylglycerols in cultured mammalian cell

Mahmood A. Kayani and James M. Parry.

Centre for Molecular Genetics and Toxicology, School of Biological Sciences, University of Wales, Swansea. SA2 8PP

The occurrence of most xenobiotic lipids has been reported as an interesting minor metabolite detected in the course of mandatory metabolism and elimination studies: few investigators have proceeded to investigate possible toxic consequences of their conjugates. Nevertheless, a number of isolated instances of experimental toxicity has been reported. At this stage there appears to be sufficient evidence to warrant serious investigations of protein kinase C stimulation by xenobiotic diacylglycerols, of the precipitation of xenobiotic cholesterol esters in tissues and of potential membrane disruption by amphipasthic xenobiotic lipids (Dodds, 1995). The present study was carried out in order to investigate the genetic consequences of exposure of mammalian cells to seven xenobiotic diacylglycerols which were synthesized in vitro in Wye College, London. The major aim was to evaluate directly the potential of these xenobiotic compounds to induce numerical chromosomal changes. Studies were undertaken to assess the production of both chromosome loss and non-disjunction in genetically engineered cell line MCL-5 which expresses selected human xenobiotic metabolizing enzymes. Xenobiotic diacylglycerols used in this study were MCPP-DAG, MCPB-DAG, 2,4 DAB-DAG, Indomethacin-DAG, Saliscylic acid-DAG, Ibuprofen-DAG and Fenbufen-DAG. These compounds were initially screened for micronucleus formation using the Cytokinesis blocked micronucleus assay (CBMN), the compounds found positive for micronucleus induction were then evaluated for the presence of whole chromosome or chromosomal fragments by analysis with antikinetochore antibodies. Non-disjunction was then determined for Chromosomes 10, 17 and 18 using specific centromeric probes. In our experiments Ibuprofen-DAG and Fenbufen-DAG produced significantly higher (P<0.001) micronuclei which were found to be due to both aneugenic and clastogenic mechanisms, respectively, using antikinetochore antibodies. MCPP-DAG and 2,4 DAB-DAG were found to be weakly positive for micronucleus formation, but the difference was statistically non-significant. Using Chromosome specific centromeric probes we found that Ibuprofen-DAG produces significantly higher (P<0.001) non-disjunction for all three chromosomes. Apoptosis and Necrosis was also measured using the criteria described by Fenech et al (1999). MCPP-DAG produced statistically significant (P<0.001) apoptosis, whereas Fenbufen-DAG produced a significantly higher apoptosis at lower doses which decreased with the increase of dose.

References

Dodds, P. F. (1995) Prog. Lipid Res., 34(3), 219–247.

Fenech, M., Crott, J., Turner, J. and S. Brown. (1999) Mutagenesis, 14(6), 605–612.

31. Comparison of the SOS/umu Assay and the SOS-Lux test

Leigh Penrose, Brian Burlinson, David Gatehouse

Genetic Toxicology Dept., GlaxoSmithKline, Park Rd, Ware, Herts., UK

The SOS/umu assay was developed to meet the need to test larger numbers of compounds than the existing microbial genotoxicity tests. This test is based on the ability of DNA damaging agents to induce the expression of the umu operon. The tester strain Salmonella typhimurium TA1535/pSK1002 (carrying a fused umuC'-`lacZ) enables us to monitor the levels of umu operon expression by measuring the intracellular ß-galactosidase activity, by a colourmetric method (Miller et al., 1972). Recently, a new test to detect genotoxicity, the SOS-Lux test, has been developed. The tester strain Salmonella typhimurium TA1535 carries a plasmid containing the entire information for the synthesis of bacterial luciferase and its substrate. This assay is based on the receptor reporter principle with the SOS system as a reporter sensitive to DNA damage and the bioluminescence system giving an optical signal, which is measured on an appropriate detector. Bioluminescence as a response to the presence of DNA damaging agents is brought about by the induction of the promotorless luxCDABFE genes of photobacterium leiognathi as a reporter component under the control of a strong SOS-promoter. As a consequence of exposure to genotoxic agents the intensity of the emitted light is proportional to the concentration of the compound tested. 15 compounds with varying structures and modes of action were tested in both the SOS/umu and SOS-Lux tests. The purpose of the study was to compare the sensitivity and reproducibility of the two methods. In the majority of cases the SOS-Lux test was found to be more sensitive, both in terms of minimum concentration required to produce a doubling response and magnitude of response obtained.

32. A High-Throughput Genotoxicity and Cytotoxicity Screen using Eukaryotic Cells

N. Billinton, P. Cahill, A.W. Knight, and R.M. Walmsley

Gentronix Ltd, Fairbairn Building, Sackville Street, Manchester, M60 1QD

Current microbial genotoxicity assays monitor either mutagenicity or induction of DNA repair in prokaryotes including Salmonella, Escherichia and Vibrio. These bacterial cells are not particularly robust, and they differ from (eukaryotic) mammalian cells in such factors as uptake, metabolism, chromosome structure and DNA repair processes. Such tests can be a misleading source of information on the mutagenic and carcinogenic potency of a substance in mammals. We are developing a genotoxicity and cytotoxicity test using yeast cells, which are both robust and eukaryotic. Repair activity is linked to synthesis of the Green Fluorescent Protein (GFP), which is stable and can be estimated non-invasively. A preliminary validation study of 80 compounds has revealed a strong correlation between positive results and mammalian carcinogenicity. A number of compounds known to require exogenous metabolic activation in other tests correctly gave positive results in our test, which relies on endogenous activities. We have demonstrated that strains expressing human cytochrome P450 genes are able to metabolise known pro-mutagens to DNA-damaging species. The test can be automated for use in medium- and high-throughput screening using standard laboratory robotics.

33. Analysis of mutations at the cII locus in transgenic Big Blue Mouse embryonic fibroblasts

K.L. Brockhurst, A.M. Lynch, M. Burman and R.W. Rees

Genetic Toxicology, GlaxoSmithKline, The Frythe, Welwyn, Hertfordshire, AL6 9AR

The transgenic Select-cII^TM gene mutation assay may be used with Muta^TMMouse and Big Blue® rodent tissues and/or the Big Blue® cell lines, and therefore has potential for comparative studies between all three systems. However, it has been suggested that -based transgenic systems are insensitive to clastogens (Suzuki et al, 1993; Gahlmann, 1993) because large deletions in DNA inhibit successful phage packaging which is essential to these assays (Gossen et al, 1995). To investigate these potential limitations we treated Big Blue® Mouse embryonic fibroblasts with two known clastogenic genotoxins, bleomycin and camptothecin, for 4 hours. Positive controls were ethylnitrosourea (ENU) and methylnitrosourea (MNU), treated for 30 mins. Cells were harvested 48 hours after treatment and clastogenicity was determined by assessing the induction of micronuclei (MN). Gene mutation frequencies at the cII locus were determined following extraction of genomic DNA. Initial results show all compounds induced significant increases in %MN compared with negative control cultures.

References

Gahlmann R. (1993) Journal of Experimental Animal Science, 35, 232–243[Medline]

Gossen et al, (1995) Mutation Research, 331, 89–97[Medline]

Suzuki et al, (1993) Mutation Research, 285, 219–224[Medline]

34. Comparative Genomic Hybridization (CGH) discloses recurrent gains of chromosome X in Gastro-Intestinal MNNG-induced tumours in rat

Chiara Corso and James M. Parry

Centre for Molecular Genetics and Toxicology, University of Wales Swansea, SA2 8PP

The genetic characterization of experimental tumours is essential in order to evaluate their relevance as appropriate animal models for human neoplasms. N-Methyl-N'-Nitrosoguanidine (MNNG) is known to induce cancers in different animal models and one of its preferential targets is the Gastro-Intestinal tract. Despite these observations, the genomic alterations that contribute to the pathogenesis of MNNG induced stomach cancer of the rat have remained largely undefined. CGH provides a powerful tool to screen entire genomes for subchromosomal copy number changes and have significantly contributed to the analysis of many human and experimental solid tumours. Here we report an investigation of 8 MNNG induced gastric tumours of the rat by Comparative Genomic Hybridization (CGH). Forestomach tumours were induced by a single dose of MNNG whereas pyloric tumours were induced by chronic exposure. The CGH technique disclosed copy number deviation in each tumour. Frequent gains occurred in specific regions of chromosome 2, 9, 10, 12, 15, 16, 19, 20, X and Y. Chromosome 10 in particular is involved in copy number deviation in seven out of eight cases. Homology studies between rat and human genome demonstrate that chromosome 10 contains the Mrp (Multidrug resistance-associate protein) gene at the 10q11-q12 band that was found amplified in two cases of gastric tumour in this investigation. The Gh1 (Growth Hormone 1) and the TP53 genes are also located in the same chromosome. The bands 9q11-q12 and Xq11-q12 have been found involved in gains in the majority of the cases. These results suggest the involvement of the ETS oncogene and probably of the HRAS-2 oncogene, whose human homologue resides in the X chromosome. These findings could provide some insights on the presence of putative gene playing a key role in the MNNG-induced gastric cancer initiation and or progression in rat.

35. A critique of the ILSI study on alternatives to the carcinogenicity bioassay from a scientific and animal welfare perspective

R.D. Combes

FRAME , Nottingham NG1 4EE, UK

The interim results of the ILSI collaborative study on alternatives to the carcinogenicity bioassay were discussed at a workshop in Leesberg, VA., USA (November, 2000). This study was initiated in 1996 to explore the possibility of replacing the lifetime mouse bioassay with a shorter, more relevant test(s). Data obtained for 20 different test chemicals in 7 assay models were compared. Five of the models were transgenic mouse strains (Tg-rasH2; Tg,AC; p53+/–; XPA–/–; XPA–/– p53+/–), sensitive to different types of carcinogenic mechanism. One model was a neonatal mouse model, and the remaining model was the SHE cell transformation assay. Test substances covered 5 different classes of chemicals: genotoxic human carcinogens (3); one immunosuppressant human carcinogen; hormones (2); rodent carcinogens/putative human non-carcinogens (5); rodent carcinogens/putative human non-carcinogens (6) and 3 non-carcinogens. The transgenic mouse models used exhibit enhanced sensitivity compared with wild-type strains to carcinogens, with a short latency time for tumour development. Ideally, these transgenic strains should express the relevant transgene widely to enable the detection of tissue-specific carcinogens. However, before exposure to a carcinogen, the strains should be genetically stable, phenotypically normal, and exhibit low levels of spontaneous tumorigenesis. During the ILSI study, several problems with the experimental design and the overall objectives of the work emerged. Some models exhibited instability and insensitivity to various chemicals, as a consequence of which protocols were lengthened to include full histopathological analyses and animal numbers increased. Thus, one of the main original objectives of the work, to develop short-term animal tests with high sensitivities, has been compromised. The workshop concluded: (a) there is an overlap in the activities of genotoxic and non-genotoxic chemicals in several assays, implying a lack of specificity; (b) the basis for enhanced tumorigenesis in some models cannot be explained; (c) more work is needed to optimise and standardise protocols; (d) regulatory agencies differ in their attitudes toward using these models, and (e) no single assay, or group of assays, emerged as suitable for regulatory carcinogen detection. This unfortunate outcome is due in part to the fact protocols were altered and they varied between some laboratories, and insufficient non-carcinogens were used. The scientific justification for continuing to develop rodent transgenic strains to detect carcinogens of human relevance is questionable, especially when their performance is being measured against rodent data. Already, >$30m and some 26,000 animals have been used to validate systems that might provide an irrelevant result for human hazard more quickly than obtained via the conventional approach. The only in vitro system (the SHE cell transformation assay) detects non-carcinogens, and performed well. However, the assay was given insufficient attention at the workshop due it being a non-animal method. It might, perhaps, therefore be better to focus more on developing human cell-based transformation systems, and use the resulting data in conjunction with other chronic and sub-chronic toxicity to identify carcinogens of human importance. This combined approach might be preferable to relying on rodent-only systems for detecting carcinogens, to reduce the need for species-extrapolation.

36. Collaborating to achieve reduction – the work of the FRAME Reduction Committee

S. Vaughan¹, M. Festing² and R. Combes¹

¹FRAME , Nottingham NG1 4EE, UK

²MRC Toxicology Unit, Leicester, UK

The Three Rs concept of Russell & Burch (replacement, reduction and refinement of laboratory animal experimentation) has been widely accepted as a means of improving scientific research and animal welfare¹. Reduction can be defined as the use of fewer animals in each experiment without compromising scientific output and quality, and without compromising animal welfare. In 1998, following an ECVAM workshop on Reduction², FRAME (Fund for the Replacement of Animals in Medical Experiments) formed a Reduction Committee comprising nine experts from industry, academia, regulatory bodies, animal welfare and Government in statistics, experimental design, animal welfare and alternatives. The four principal ways for achieving reduction are: (a) implementing better research strategies; (b) having better control of experimental variation; (c) using better and more appropriate statistics, and (d) harmonising international testing guidelines. Progress in each of these areas is varied, and there is evidence that the number of laboratory animals used in experiments can be reduced further, while still providing statistically valid data. The committee is undertaking a diverse range of research projects, including the creation and distribution of journal editor guidelines, an analysis of the Home Office statistical returns for animal procedures, an examination of laboratory animal group sizes according to species being used, and the use of inbred versus outbred strains of animals. Also, a comprehensive directory of training material on experimental design and the statistical analysis of laboratory animal experiments has been compiled, and ongoing reviews of computer statistical packages are being undertaken. It would seem that many who use animals require more training in experimental design and statistics. Accordingly, the Reduction Committee has also recently organised a training workshop on Reduction, in conjunction with the Laboratory Animal Science Association (LASA). Other similar activities are planned for the future. Some of this work and information is directly relevant to those using in vivo assays in genotoxicity testing, and further details are available on the FRAME website (www.frame.org.uk).

References

¹ Balls, M. et al. (1995) Alternatives to Laboratory Animals, 23, 838–866.[Medline]

² Festing, M. et al. (1998) Alternatives to Laboratory Animals, 26, 283–301.

CiteULike    Connotea    Del.icio.us    What's this?

This Article Extract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Google Scholar Search for Related Content Social Bookmarking          What's this?

Online ISSN 1464-3804 - Print ISSN 0267-8357

Copyright © 2009 United Kingdom Environmental Mutagen Society

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics