Use of a modified hemi-body irradiation technique for metastatic carcinoma of the prostate: report of a 10-year experience.

AIMS: To determine whether patients receiving hemi-body irradiation required further treatment to painful bone sites out with the radiation field (skull or lower leg), whether patients required further treatment to areas within the treated radiation field for pain or new skeletal events, and whether the treatment outcome was successful in terms of pain control. Toxicities, the need for transfusions and survival were also analysed. MATERIALS AND METHODS: In our retrospective review, 103 men aged 50-87 years, with skeletal metastases from prostate cancer, received modified hemi-body irradiation (HBI) during a consecutive 10-year period, using the same radiotherapy technique and dose. The upper HBI field excluded the region above the ramus of the mandible and the lower HBI field excluded the lower limb below the knee. A successful outcome was determined by assessing the pain response in combination with a change in analgesic intake. RESULTS: Twenty patients received upper HBI; 17/20 (85%) had a successful outcome at the 6-week review, sustained in 94.1% at the final follow-up with no need for radiotherapy to the skull. Thirty-eight patients received lower HBI; 26/38 (68.4%) had a successful outcome at the 6-week review, sustained in 80.8% at the final follow-up with no need for radiotherapy to the lower leg. Forty-five patients received sequential HBI; 33/45 (73.3%) had a successful outcome at the 6-week review, sustained in 87.9% at the final follow-up, with three patients requiring further radiotherapy to the skull (2/45) or lower leg (1/45). Only 5/103 patients (4.8%) developed new skeletal events in the treated area. Toxicity and transfusion requirements were minimal. CONCLUSIONS: Modifying the field size for single-fraction HBI does not have a significant effect on the final outcome of treatment, namely pain control and a need for additional radiotherapy. In our experience, modified HBI should be considered in patients with multiple bone pain sites, especially if they will probably require several visits for localised radiotherapy to single painful bone sites within a short period of time.

Radiation-induced transgenerational alterations in genome stability and DNA damage.

Mutation induction in directly exposed cells is currently regarded as the main component of the genetic risk of ionizing radiation for humans. However, recent data on the transgenerational increases in mutation rates in the offspring of irradiated parents indicate that the genetic risk could be greater than predicted previously. Here, we have analysed transgenerational changes in mutation rates and DNA damage in the germline and somatic tissues of non-exposed first-generation offspring of irradiated inbred male CBA/Ca and BALB/c mice. Mutation rates at an expanded simple tandem repeat DNA locus and a protein-coding gene (hprt) were significantly elevated in both the germline (sperm) and somatic tissues of all the offspring of irradiated males. The transgenerational changes in mutation rates were attributed to the presence of a persistent subset of endogenous DNA lesions (double- and single-strand breaks), measured by the phosphorylated form of histone H2AX (gamma-H2AX) and alkaline Comet assays. Such remarkable transgenerational destabilization of the F(1) genome may have important implications for cancer aetiology and genetic risk estimates. Our data also provide important clues on the still unknown mechanisms of radiation-induced genomic instability.

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

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

Testing strategies in mutagenicity and genetic toxicology: an appraisal of the guidelines of the European Scientific Committee for Cosmetics and Non-Food Products for the evaluation of hair dyes.

The European Scientific Committee on Cosmetics and Non-Food Products (SCCNFP) guideline for testing of hair dyes for genotoxic/mutagenic/carcinogenic potential has been reviewed. The battery of six in vitro tests recommended therein differs substantially from the batteries of two or three in vitro tests recommended in other guidelines. Our evaluation of the chemical types used in hair dyes and comparison with other guidelines for testing a wide range of chemical substances, lead to the conclusion that potential genotoxic activity may effectively be determined by the application of a limited number of well-validated test systems that are capable of detecting induced gene mutations and structural and numerical chromosomal changes. We conclude that highly effective screening for genotoxicity of hair dyes can be achieved by the use of three assays, namely the bacterial gene mutation assay, the mammalian cell gene mutation assay (mouse lymphoma tk assay preferred) and the in vitro micronucleus assay. These need to be combined with metabolic activation systems optimised for the individual chemical types. Recent published evidence [D. Kirkland, M. Aardema, L. Henderson, L. Müller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] suggests that our recommended three tests will detect all known genotoxic carcinogens, and that increasing the number of in vitro assays further would merely reduce specificity (increase false positives). Of course there may be occasions when standard tests need to be modified to take account of special situations such as a specific pathway of biotransformation, but this should be considered as part of routine testing. It is clear that individual dyes and any other novel ingredients should be tested in this three-test battery. However, new products are formed on the scalp by reaction between the chemicals present in hair-dye formulations. Ideally, these should also be tested for genotoxicity, but at present such experiences are very limited. There is also the possibility that one component could mask the genotoxicity of another (e.g. by being more toxic), and so it is not practical at this time to recommend routine testing of complete hair-dye formulations as well. The most sensible approach would be to establish whether any reaction products within the hair-dye formulation penetrate the skin under normal conditions of use and test only those that penetrate at toxicologically relevant levels in the three-test in vitro battery. Recently published data [D. Kirkland, M. Aardema, L. Henderson, L. Müller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] suggest the three-test battery will produce a significant number of false as well as real positives. Whilst we are aware of the desire to reduce animal experiments, determining the relevance of positive results in any of the three recommended in vitro assays will most likely have to be determined by use of in vivo assays. The bone marrow micronucleus test using routes of administration such as oral or intraperitoneal may be used where the objective is extended hazard identification. If negative results are obtained in this test, then a second in vivo test should be conducted. This could be an in vivo UDS in rat liver or a Comet assay in a relevant tissue. However, for hazard characterisation, tests using topical application with measurement of genotoxicity in the skin would be more appropriate. Such specific site-of-contact in vivo tests would minimise animal toxicity burden and invasiveness, and, especially for hair dyes, be more relevant to human routes of exposure, but there are not sufficient scientific data available to allow recommendations to be made. The generation of such data is encouraged.

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

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

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

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

Development of screening tests for aneuploidy induction by environmental pollutants.

When legally required mutagenicity testing of chemicals is undertaken, the important genetic end point of aneuploidy is not included because validated test methods are lacking. Therefore, the Commission of the European Communities (CEC) has funded a research program to develop and validate tests for aneuploidy induction. Ten chemicals, selected on the basis of their ability to interact with cell organelles relevant for aneuploidy induction, were tested in 11 laboratories. The assays ranged from in vitro tubulin assembly studies to in vivo germ-cell tests. The results allow several conclusions: a) Fungal aneuploidy tests are not capable of detecting inhibitors of mammalian tubulin polymerization such as colchicine and vinblastine. Therefore, they will not play a role in screening for aneuploidy but are of value for studying the relationship between induced aneuploidy and recombination. b) Chemicals that induce aneuploidy in mammalian germ cells are readily detected in the in vitro mammalian cell systems. Some chemicals such as thiabendazole and thimerosal induce aneuploidy in vitro but do not appear to be very effective in vivo. c) Cell division aberrations induced in mammalian cells in vitro seem to be predictive for aneuploidy induction in the same cell type. Likewise, c-mitotic effects and cell cycle delay in vivo in mitotic and meiotic cells correlate with aneuploidy induction in the respective tissue. A second CEC Aneuploidy Program has started recently to refine the most promising test protocols, to provide understanding of variety of mechanisms by which chemicals induce aneuploidy, and to establish a data base for aneugens among environmental pollutants.

Detection of mitotic and meiotic aneuploidy in the yeast Saccharomyces cerevisiae.

A number of genetic systems are described which involve the use of the yeast Saccharomyces cerevisiae. The systems may be used to detect the production of aneuploid cells produced during both mitotic and meiotic cell division in the presence of genetically active chemicals. During mitotic cell division, monosomic colonies (2n - 1) may be detected by plating upon selective medium. Increases in such monosomic colonies are produced by exposure of cells to a number of chemical mutagens such as ethyl methane-sulfonate and mitomycin C. More importantly, monosomic colonies are also induced by nonmutagens such as sulfacetamide and saccharin, which suggests that such chemicals are capable of inducing aneuploidy (aneugenic) in the absence of mutagenic activity. Genetic analysis of aneuploid colonies produced on nonselective medium indicate that at least a proportion of the monosomic colonies were the result of mitotic nondisjunction. During meiotic cell division, disomic cells (n + 1) produced by chromosome nondisjunction may be detected by plating on selective media. The frequency of disomic cells has been shown to increase after exposure to p-fluorophenylalanine.

Severe clinical conditions associated with Bacillus cereus and the apparent involvement of exotoxins.

Twenty-one cases of infection with Bacillus cereus are summarised. The histories supplied showed that at least 15 of these were associated with severe or potentially severe symptoms including two deaths. Analysis of the production of exotoxins, including haemolysin and phospholipase, by these strains is given, and the relevance of these metabolites to the severity of the condition is discussed. Three incidents of bovine mastitis resulting from B. cereus and involving three deaths are also included. The observations presented here together with those of previous reports which are reviewed indicate that B. cereus may be of clinical importance, not just an opportunist but also as an agent of potentially severe infections in its own right.

Site distribution of oesophagogastric cancer.

AIMS: It has been suggested that adenocarcinomas of the lower oesophagus and gastric cardia should be reclassified as oesophagogastric junction (OGJ) cancers. This study aimed to define the frequency of OGJ cancers in a geographically defined population of 4.3 million people. METHODS: All cases of oesophageal and gastric cancer occurring in 1993 were identified by the North Western Regional Cancer Registry. A total of 1192 hospital case notes were reviewed and a study group of 1067 patients was defined. Tumour involvement was documented at individual subsites in the oesophagus and stomach, allowing for tumour presence in more than one oesophageal/gastric subsite. RESULTS: There were 627 tumours in men and 440 in women. The tumour was confined to the oesophagus in 281 (26.3%) cases and to the stomach in 454 (42.6%) cases. The tumour encroached upon or crossed the OGJ in 332 (31.1%) cases. Overall, tumours involved the cardia, OGJ, or lower oesophagus in 633 (59.3%) cases; in 179 (18.5%) cases the tumour involved the lower oesophagus but not the OGJ, and in another 122 (11.4%) cases the cardia was involved but not the OGJ. CONCLUSIONS: Oesophagogastric cancers in this population predominantly involve the OGJ, lower oesophagus, and/or cardia.

Research on the mechanisms of action of aneugenic chemicals and regulatory approaches for their control in the European Communities.

The European Communities have developed a wide range of regulatory instruments for the control of chemical products sold and used within its geographical area. An important part of the testing requirements for most chemicals within the European Communities is the preparation of an information package on the potential mutogen properties of each chemical. Currently, no test requirements specify a unique test for aneugenic activity, although current methods such as in vitro cytogenetic and bone marrow micronucleus assays provide some useful indirect information on aneugenic activity. During the past 15 years the European Communities supported a series of collaborative research projects that have investigated the mechanisms by which chemicals induce aneuploidy and developmental studies of test methods for the detection of aneugenic chemicals. These projects led to the development of in vitro methods for the detection and quantification of induced nondisjunction and chromosome loss and the measurement of aneuploidy in rodent bone marrow. The European Communities projects have demonstrated the aneugenic potential of a diverse range of chemicals and their potential role in inherited disease and tumour induction. However, regulatory guidelines have yet to be modified to take advantage of the methods developed for the detection and evaluation of aneugenic chemicals.

Use of multicolour chromosome painting to identify chromosomal rearrangements in human lymphocytes exposed to bleomycin: a comparison with conventional cytogenetic analysis of Giemsa-stained chromosomes.

Exchange aberrations induced by bleomycin were identified by multicolour fluorescence in situ hybridisation (FISH) with probes for chromosomes 1, 2, and 3. The frequency and distribution of aberration types were compared to conventional metaphase analysis of Giemsa-stained chromosomes from the same human lymphocyte cultures. The total percentage of exchanges detectable by painting three pairs of chromosomes with separate colours was calculated as 40%. Giemsa staining revealed predominantly asymmetric chromosome exchanges, which are expected to comprise 50% of the total induced exchanges. Genomic exchange frequencies were, therefore, determined by multiplying the observed frequencies from FISH analysis by 2.5 and the number of asymmetric exchanges identified in Giemsa-stained slides by 2.0. By these calculations, the genomic exchange frequency calculated from chromosome painting exceeded that estimated by Giemsa-staining. This difference was due to the identification by chromosome painting of a unique class of cells in which chromosomes had undergone complex exchanges (nonreciprocal exchanges involving multiple mutual sites). The percentage of cells exhibiting exchanges was similar for both methods.

Testing for Helicobacter pylori in primary care: trouble in store?

STUDY OBJECTIVE: To assess the role of testing for Helicobacter pylori in the management of dyspeptic patients in primary care. DESIGN: Selective review of literature frequently quoted to support use of H pylori testing. MAIN RESULTS: Testing for H pylori and referral of only positive cases for endoscopy aims to reduce the number of "unnecessary" endoscopies. Patients with negative results may receive short-term reassurance and subsequently place fewer demands on health services. However, studies to date have only assessed this practice in secondary care settings. Given the relatively high prevalence of both dyspepsia and H pylori infection, the transfer of this practice to primary care may lead to a paradoxical increase in endoscopy referrals. Identification of H pylori and prescribing of eradication treatment also aims to reduce endoscopy referrals. No primary care trials have yet assessed this approach. Given that fewer than one in four of dyspeptic patients have peptic ulceration, a high proportion may fail to respond to eradication treatment and subsequently require referral for endoscopy. The longer term clinical and psychosocial sequelae of treating or labelling patients with an infection associated with gastric cancer remain unknown. CONCLUSIONS: Given uncertainty concerning the possible adverse effects of H pylori testing in primary care, we suggest a moratorium on its use in this setting until results from relevant clinical trials become available.

A comparative study of the use of primary Chinese hamster liver cultures and genetically engineered immortal V79 Chinese hamster cell lines expressing rat liver CYP1A1, 1A2 and 2B1 cDNAs in micronucleus assays.

Liver microsome preparations (S9 mix) have been extensively used for in vitro genotoxicity studies to provide the capacity for the activation of indirect genotoxins. However, the use of S9 preparations with mammalian cell cultures has raised considerable toxicity problems which limit their use to exposure times which are only a small fraction of the cell cycle. In addition, false negative results may be obtained if reactive metabolites are unable to penetrate the cell membrane or have short half-lives. The generation and detection of a promutagen within a single cell would therefore be advantageous. To this end, we have studied the bioactivation of a panel of promutagens (benzo[a]pyrene, cyclophosphamide, 2-aminoanthracene and sterigmatocystin) in low passage Chinese hamster fibroblasts of hepatic origin (LiC2 cells) and in a series of V79 Chinese hamster cell lines genetically engineered to express rat liver cytochrome P450 cDNAs. These include strains XEM2 (expresses CYP1A1), SD1 (CYP2B1) and strains XEMd-MZ and XEMd-NH which express CYP1A2. The end point selected for study was the induction of micronuclei. The protocol incorporated a cytochalasin B-induced cytokinesis block and the enumeration of micronuclei in the resulting binucleate cells which have undergone one nuclear division following the induction of chromosome damage. Micronuclei containing whole chromosomes and chromosome fragments were distinguished by the use of CREST antibody specific for kinetochore protein as a measure for the presence of centromeres. Micronuclei were induced by the test agents in low passage liver fibroblasts and in immortal V79 cultures only in the presence of Aroclor-induced S9 preparations. The data obtained from micronucleus assays of the genetically engineered V79 cell lines demonstrated the utility of each strain for the optimal detection and quantification of the activity of the individual test compounds. Kinetochore antibody demonstrated differences in the kinetics of induction of micronuclei containing chromosome fragments and whole chromosomes with chemicals such as benzo[a]pyrene. As part of this cytogenetic study, we also conducted karyotypic analyses and spindle fidelity assays of the V79 cell lines to investigate the presence of chromosomal instabilities which may arise as a consequence of the genetic engineering procedure. Such studies represent an important quality control step in the validation of the suitability of each cell line prior to their use in genotoxicity studies.

Molecular analysis of the chemoprotective effects of topical sunscreen and vitamin C in preventing UV-induced and reactive oxygen species-induced DNA damage, respectively, using the PCR inhibition methodology.

BACKGROUND: We have developed a PCR-based technique which has previously been shown to be capable of detecting DNA damage (Adducts, strand breaks) induced by carcinogens. We wanted to explore the possibility of detecting anti-carcinogens with this methodology through a corresponding reduction in DNA damage induction. MATERIALS AND METHODS: We have used a PCR-based system which relies on the fact that Taq polymerase cannot amplify damaged DNA efficiently. We immobilised DNA in microtitre plates and exposed them to UV-B light and hydrogen peroxide. In addition, we tested the capacity of sunscreens and ascorbate (vitamin C) to ameliorate the DNA damage caused by UV-B and hydrogen peroxide, respectively. RESULTS: We have shown that in the case of UV-B, sunscreens can effectively reduce the amount of DNA damage induced. This appears to be dependent upon the Sun Protection Factor of the sunscreen, with SPF 35 sunscreens protecting DNA particularly well. We found that both hydrogen peroxide and ascorbate were capable, at high doses, of inducing DNA damage. When mixed, ascorbate effectively increased the DNA damaging effect of hydrogen peroxide. CONCLUSION: We have shown that the PCR inhibition method may be suitable for untangling the complex carcinogenic/anticarcinogenic aspects of factors in the diet and environment. However, the DNA damaging effect of ascorbate must be viewed in the context of the high concentrations used here.

The equivalence of assays within individual guidelines for the testing of the potential mutagenicity of chemicals: problems associated with battery selection.

Many individual Mutagenicity Guidelines contain suggested test systems with choices of such parameters as strains, cell types and even endpoint assayed. Comparisons have been made of data obtained from variants of yeast assays for the induction of mitotic recombination, in vitro assays for the induction of chromosome aberrations and assays for the induction of cell transformation. Individual test variants included in guidelines of the EEC and OECD show considerable qualitative and quantitative variability of response to potential mutagens and carcinogens. Such variability between assays within the same guideline raises considerable problems in the selection of test batteries chosen from published Mutagenicity Guidelines. Improved battery selection is dependent upon the reduction of choice within guidelines to those assays which produce consistent and reproducible results.

An evaluation of the use of in vitro tubulin polymerisation, fungal and wheat assays to detect the activity of potential chemical aneugens.

The test chemicals included in the EC Aneuploidy Project were evaluated for their ability to induce aneuploidy or aneuploidy related endpoints in assays using in vitro tubulin polymerisation, fungi and wheat. The results obtained demonstrated considerable qualitative and quantitative differences between the responses of the assays to the 10 test chemicals. Fungal assays failed to respond to the potent mammalian spindle poisons colchicine and vinblastine and only three chemicals were positive in all three fungal test systems i.e. chloral hydrate, thimerosol and thiabendazole. The in vitro tubulin polymerisation assays produced unambiguous positive results with three chemicals i.e. colchicine, thimerosol and vinblastine sulphate. The hexaploid wheat assay produced a positive response with 8 of the test chemicals i.e. colchicine, econazole, thimerosol, pyrimethamine, thiabendazole, cadmium chloride, vinblastine and diazepam. However, the wheat assay was relatively insensitive to the potent spindle poison colchicine.

Reflections on the implications of thresholds of mutagenic activity for the labelling of chemicals by the European Union.

During the course of the safety evaluation and regulatory control of chemicals it is important to distinguish between "potential hazard" and "actual risk" of exposure to toxins. In the case of DNA reactive chemicals, it has been prudent to assume that hazard is expressed as risk at low exposure concentrations. However, analysis of the dose-response relationships of both DNA reactive and non-DNA reactive genotoxins (e.g., aneugens) indicate that there are exposure concentrations below which protective mechanisms such as DNA repair activity and the presence of multiple targets may lead to the prediction of no risk until threshold concentrations are achieved. Current European Union management procedures for mutagenic chemicals are based predominantly upon hazard assessment rather than assessment of actual risk under likely exposure scenarios. As our knowledge of protective mechanisms increases, the time is now appropriate to undertake a re-evaluation of European Union criteria and to base the clarification mutagenic chemical more firmly upon the basis of actual risks to the human population and to the environment.

The effects of BC, 4CMB and 4HMB upon the induction of mitotic gene conversion in yeast.

Both BC and 4CMB but not 4HMB were shown to be capable of inducing mitotic gene conversion in exponential phase cultures of the JDI strain of the yeast Saccharomyces cerevisiae. The results obtained indicate that in terms of the relative frequency of genetic events per lethal event 4CMB was more active than BC in this test system.

The use of yeast cultures for the detection of environmental mutagens using a fluctuation test.

A microbial fluctuation test, modified for the detection of environmental mutagens has been evaluated using a number of strains of the yeast Saccharomyces cerevisiae. Auxotrophic diploid cultures of yeast which produce prototrophic colonies by both mitotic gene conversion and mutation have been extensively utilized for the detection and evaluation of chemicals showing genetic activity. A number of the yeast strains utilized were shown to be suitable for use in the fluctuation test although the time scales of the experiments were considerably extended (up to 16 days) compared to those involving bacteria. The yeast strains respond to doses of mutagens at least a 100-fold lower than that required in a conventional short exposure treat and plate experiment. In experiments involving the induction of mitotic gene conversion at the tryptophan-5 and histidine-4 loci in the fluctuation test significant increases in prototrophic cells were produced in the presence of the insecticide Lindex (0.05 microng/ml), the preservative Thiomersal (0.0001 microng/ml), a mahogany hair dye (0.01 microng/ml), the herbicide Paraquat (0.02 microng/ml) and the alkylating agent ethyl methane sulphonate (0.1 microng/ml). The results demonstrate that the fluctuation test provides an extremely sensitive assay for the detection of chemicals which show genetic activity in yeast at non-toxic concentrations.