Flow cytometric enumeration of micronucleated reticulocytes: high transferability among 14 laboratories.

This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.

Comparison of the sensitivities of Salmonella typhimurium strains TA102 and TA2638A to 16 mutagens.

The qualitative and quantitative sensitivity of the genetically related, histidine-auxtrophic Salmonella typhimurium strains TA102 and TA2638a to 16 compounds was examined. The compounds were mainly cross-linking and oxidising mutagens, the effects of which were known to be detected by strain TA102 preferentially or by a combination of Escherichia coli WP2 (pkM101) and uvrA/pkM101. The morphology and number of spontaneous revertants was also compared. Fourteen of the 16 compounds caused reversion in both strains. Bleomycin and streptomycin induced reversion in strain TA102 but not TA2638a. The greater sensitivity of TA102 to these compounds may be associated with the extrachromosomal location of the target genes. The overall quantitative sensitivity of the two strains was similar for the other compounds. The number of compounds that caused reversions at lower doses or produced greater proportional increases were the same in TA102 as in TA2638a. The spontaneous number of revertants, without and with metabolic activation, respectively, was 98 and 130 for TA2638a and 322 and 465 for TA102. Strain TA2638a formed larger, more uniform colonies than TA102. The present results together with those of previous studies indicate a high degree of concordance between the sensitivity of strains TA102 and TA2638 for the detection of mutagens. The uniform colony size and lower spontaneous reversion frequency seen with strain TA2638a compared with TA102 would make it more reliable and convenient for routine testing. It is concluded that strain TA2638a should be considered as an alternative to TA102 and included, as well as the two E.coli strains, in the set of bacterial strains used in the standard test battery for mutagenicity testing.

Mouse lymphoma thymidine kinase locus gene mutation assay: International Workshop on Genotoxicity Test Procedures Workgroup Report.

The Mouse Lymphoma Assay (MLA) Workgroup addressed and reached consensus on a number of issues. Discussion focused on five areas: (1) acceptable assay versions; (2) cytotoxicity measure; (3) 24-hr treatment; (4) microwell colony counting and sizing; and (5) data acceptability/statistical analysis. Although the International Conference on Harmonisation (ICH) indicated a preference for the microwell over the soft agar method, all of the workgroup members agreed that both versions of the MLA are equally acceptable. The workgroup agreed that it is desirable for both assay versions to use the same measure of cytotoxicity to define the acceptable and required concentration range. Currently, laboratories using the microwell version use the relative survival (RS) determined by cloning immediately after the treatment. Laboratories using the soft agar method do not obtain an RS but use the relative total growth (RTG), a combination of the relative suspension growth (RSG) during the expression period and the relative cloning efficiency determined at the time of mutant selection. The workgroup agreed to investigate the RSG, the RS, and the RTG and to develop further guidance. In the interim, the workgroup reached consensus that the RTG be used as the standard measure of cytotoxicity. The ICH recommended a 24-hr treatment in the absence of S9 when negative results are obtained with short (3-4 hr) treatments. The workgroup agreed to retain this requirement but acknowledged that more data are needed prior to making final recommendations concerning the need for and the specific protocol for the 24-hr treatment. Environ. Mol. Mutagen. 35:185-190, 2000 Published 2000 Wiley-Liss, Inc.

Mutational specificity of aflatoxin B1. Comparison of in vivo host-mediated assay with in vitro S9 metabolic activation.

An intrasanguineous host-mediated assay was used to determine the pattern of mutagenesis induced by the carcinogen aflatoxin B1 in the lacI gene of Escherichia coli recovered from rat liver. To investigate the influence of different types of metabolic activation, the mutation spectrum induced by AFB1 activated in vitro by a commercially prepared S9 microsomal fraction from Aroclor 1254-treated rats was also obtained. A total of 281 forward mutations affecting the N-terminal region of the lacI gene were characterized by DNA sequencing analysis. AFB1 induced similar type of mutations with similar site specificity when activated by the standard S9 fraction or by employing a rat host-mediated assay. These results indicate the ability of the in vitro S9 fraction to mimic the in vivo metabolism, suggesting that the same active metabolite, presumably AFB1 8,9-epoxide, is responsible for generating a similar pattern of DNA damage, as reflected in the similarity of mutational spectra. For both activation systems, most mutations (>90%) were base substitutions that occurred primarily at G:C pairs. Somewhat over one-half of G:C targeted substitutions were GC>TA transversions, other mutations being evenly divided between G:C>AT transitions and GC>CG transversions. The mutational specificity exhibited by activated AFB1 can be explained by incorporation of different bases opposite a single type of non-instructive lesion during error-prone DNA synthesis. To what extent the mutations are due to the main adduct (AFB1-N7-Gua), its imidazole-ring-opened derivative or an apurinic site remains unknown.

Mammalian cell gene mutation assays working group report.

As part of the International Workshop on Standardization of Genotoxicity Test Procedures, in Melbourne, 27-28 February 1993, various international guidelines were examined with respect to protocol issues in the area of mammalian cell gene mutation assays. The working group on mammalian cell gene mutation assays discussed a wide range of protocol issues related to study design; in most cases the recommendations are reasonably consistent with existing guidelines. Agreement was reached on several issues as follows. The upper limit of concentration for testing non-toxic substances should be 10 mM or 5 mg/ml, whichever is lower. For testing toxic substances the criteria of an acceptable upper limit of concentration should yield 10-20% survival. Any of several established mammalian cell mutation assays (L5178Y TK+/-, CHO/HPRT, AS52/XPRT, V79/HPRT) can be used to evaluate mutagenesis in mammalian cells; the ouabain (Na/K-ATPase) system is not an acceptable mutation assay for routine evaluation of mutagenesis in mammalian cells. Ability to recover small colonies must be convincingly demonstrated when using the L5178Y TK+/- mouse lymphoma assay. In the mouse lymphoma assay (L5178Y TK+/-), colonies in positive controls and at least two (if available) representative positive doses of the test compound should be sized if a positive response is seen; in the event of a negative response due to the test compound, colony sizing of the positive control is necessary to validate the conduct of the assay. Testing both in the presence and absence of S9 metabolic activation is necessary. It was not possible to come to a firm conclusion about the length of treatment. There was a general agreement that extended treatment times (> 2 cell cycles) often bear more disadvantages than advantages and should only be used with adequate justification. It is not necessary to repeat clear positive or clear negative tests when the assay has been adequately performed; this recommendation differs significantly from the UK guidelines. If treatment groups are not replicated, the numbers of doses tested should be increased; this recommendation differs significantly from the UK guidelines. Each laboratory should establish a historical database for the performance of a given assay in that laboratory.

The ECITTS integrated toxicity testing scheme: The application of in vitro test systems to the hazard assessment of chemicals.

A multicentre project has been designed, with the purpose of developing the concept of integrated in vitro testing. The aim is to use non-animal methods to assess the toxicological properties of chemicals, and to improve this assessment through the use of knowledge of mechanisms of toxic action. A number of tests or test batteries were selected within eight areas: basal cytotoxicity, irritancy, developmental toxicology, hepatotoxicity, nephrotoxicity, immunotoxicity, neurotoxicity and biokinetics. For each area, a number of calibration chemicals were selected. In an initial pilot study, the neurotoxic properties of five chemicals will be studied in combination with a biokinetic analysis, in which blood and brain concentrations will be predicted from biokinetic modelling.

An evaluation of the E. coli K-12 uvrB/recA DNA repair host-mediated assay. II. In vivo results for 36 compounds tested in the mouse.

The aim of this study was to further evaluate the E. coli K-12 DNA repair host-mediated assay, as a short-term in vivo genotoxicity test, to be used as a complement to the micronucleus test in the routine testing of chemicals and drugs. The assay involves the administration of the test substance to mice by the route of choice, followed by the intravenous administration of a mixture of DNA repair deficient and proficient derivatives of E. coli K-12. After an incubation period the relative survival of the two strains was determined in blood, liver, lungs, kidneys and testes of the host. A significant preferential reduction of the DNA repair deficient strain in any organ indicates that the test substance possesses genotoxic properties. A total of 36 substances, 26 carcinogens, 4 weak or non-carcinogens and 6 unclassified substances, were tested in this assay. Positive results were obtained for 23 compounds. Of the carcinogens 18 were positive and of the non-carcinogens 3 were negative. The overall concordance between the assay and carcinogenicity was 72%. In general, alkylating agents and direct-acting nitroso compounds showed genotoxic activity in all organs tested, while the other substances were positive in a limited number of organs. With oral administration, which was the most commonly used administration route in the study, the organ showing a positive response most often was the blood. The results from the present study were compared with results from the micronucleus test, which were available for 26 of the substances. Results were in agreement for 15 of the substances, while 8 substances were positive in the present assay and negative in the micronucleus test: 4-aminobiphenyl, 2-anisidine, epichlorohydrin, formaldehyde, 1- and 2-naphthylamine, 2-nitrophenylenediamine and 4-nitroquinoline-N-oxide. The substances negative in the E. coli DNA repair host-mediated assay, but positive in the micronucleus test were: benzene, catechol and cyclophosphamide. It is concluded from this evaluation that the E. coli K-12 DNA repair host-mediated assay detects a number of carcinogens that are negative in the micronucleus test, while detecting most of the compounds that are positive in the latter. The advantages of this test are that differential DNA repair measures a broad spectrum of genetic damage, an in vitro/in vivo comparison is possible with the same test organisms, results can be obtained from various organs and the test is rapid.(ABSTRACT TRUNCATED AT 400 WORDS)

An evaluation of the E. coli K-12 uvrB/recA DNA repair host-mediated assay. I. In vitro sensitivity of the bacteria to 61 compounds.

A differential DNA repair test was evaluated in vitro, using derivatives of E. coli K-12 343/113 with the genotype uvrB-/recA- and uvrB+/recA+. The aim of this study was to characterize the sensitivity of the assay to different compounds in vitro and thereby provide information on the usefulness of this end-point as an indicator of genotoxicity in a host-mediated assay. Sixty-one compounds from diverse chemical groups were tested and of these 32 gave a positive result. The results obtained were compared with results from the Ames test and were in agreement for 49 out of the 61 compounds tested. Chemicals that were detected in this test but negative in the Ames test were 4-aminophenol, catechol, diethylstilbestrol, thioacetamide and thiourea. Seven of the compounds tested gave a negative result in E. coli but were positive in Salmonella. These were 4-aminobiphenyl, benzo[a]pyrene, cyclophosphamide, 1-naphthylamine, N-nitrosobutylpropylamine, quinoline and 2-toluidine. The performance of the in vitro test and reasons for the discrepant results with the Ames test are discussed. The overall concordance between the two tests was about 80%. On the basis of these results we consider these bacterial strains, and differential DNA repair as an end-point, to be sufficiently accurate as an indicator of genotoxicity in vitro and thereby also in vivo.

Evaluation of a genotoxicity test measuring DNA-strand breaks in mouse lymphoma cells by alkaline unwinding and hydroxyapatite elution.

A rapid genotoxicity test, based on the measurement of the proportion of single- to double-stranded DNA by alkaline unwinding and hydroxyapatite elution in mouse lymphoma cells treated in vitro with various chemicals, was evaluated. Seventy-eight compounds from diverse chemical groups, including commonly tested mutagens, toxic compounds not usually tested for genotoxicity and non-toxic compounds not thought to be genotoxic were tested. The results obtained were compared with those from the mouse lymphoma TK locus forward-mutation assay, providing a basis for assessing the relative sensitivity of the 2 assays using the same cells exposed to chemicals under similar conditions. Clear evidence of DNA-damaging activity was obtained with 43 of the compounds, while 4 gave equivocal results. Of the remaining 31 compounds, 14 were toxic without inducing DNA damage while the rest were non-toxic and did not induce any DNA damage. Results were available from both the alkaline unwinding assay and the mouse lymphoma assay for 61 compounds; they showed a concordance between the 2 assays of 77%. Of the 47 compounds that were positive or equivocal in the alkaline unwinding assay, only carbon tetrachloride and prednisolone were negative in the mouse lymphoma assay, while 12 of the 19 compounds that were negative in the alkaline unwinding assay were positive in the mouse lymphoma assay. These included 3 compounds that interfere with nucleic acid metabolism, and 3 crosslinking agents, which would be expected to produce mutations to a greater extent than strand breaks. The other 6 compounds were anthranilic acid, benzoquinone, p-chloroaniline, diethylmaleate, glucose and procarbazine HCl. Of these only the last is a known carcinogen. It is concluded from the present study that there was good overall agreement between the results of the DNA alkaline unwinding and mouse lymphoma TK locus assays, but that the sensitivity of the alkaline unwinding assay is lower for some classes of compounds. Bearing this in mind, the alkaline unwinding assay is considered suitable as a rapid screen for genotoxic activity in eukaryotic cells.

Mouse lymphoma L5178Y thymidine kinase locus assay of 50 compounds.

Mutagenicity results are presented for 50 compounds tested in the mouse lymphoma TK+/(-)----TK-/- forward mutation assay. Test compounds were mostly from chemical classes not previously tested, to provide new information on the sensitivity of the assay; chemicals of low toxicity or thought to be non-carcinogenic and metabolic inhibitors, to indicate whether and under what conditions the assay can generate so-called false positive results. Twelve compounds that have been tested previously were included in this study to provide an indication of the reproducibility of the assay. Concordant results were obtained for nine of these, while disagreeing, positive results were seen with aniline, fluorene and pyrene. The following compounds belonging to the noncarcinogen category were positive at concentrations in the range 0.02-1 mol/l: dimethyl sulphoxide, EDTA, glucose, polyethyleneglycol, sodium chloride, sodium nitrite and urea. Measurements of the osmotic pressure indicated a lack of a simple relationship to mutagenic effects for these compounds. While the potent mutagenic/carcinogenic compounds tested gave greater than 4-fold increases in the mutation frequency, weak carcinogens or compounds not known to be carcinogenic that were positive in the assay gave increases of between 2- and 4-fold. Exceptions were aldehyde derivatives and chemicals that can lead to oxidative stress, which were detected with exaggerated sensitivity by the assay. Among the metabolic inhibitors tested, positive results were obtained with actinomycin D, cycloheximide, diethyl maleate, hydroxyurea and ouabain. Negative results were found with antimycin A. On the basis of the present results and previously published data it is concluded that a maximum limit for the test compound concentration can be set at 20 mmol/l and that testing to 20% total growth is adequate, with certain stipulations, to detect the mutagenic activity of test compounds. A similar analysis of the available test data shows that less than 4-fold increases in the mutation frequency have a lower predictivity for carcinogenicity.

Effect of pH shifts on the mutant frequency at the thymidine kinase locus in mouse lymphoma L5178Y TK+/- cells.

Evidence has been accumulating that conditions of nonphysiological pH may affect the results of in vitro genetic tests by mechanisms unrelated to the chemical being tested. Medium was pH-adjusted with HCl, NaOH or with organic buffers (Good's zwitterions). In the absence of S9 mix, no changes in mutant frequency were observed over a pH range of 6.4-9.2; a small, 1.9-fold increase was observed for a moderately toxic treatment (24% relative growth) at pH 6.3. However, in the presence of S9 mix, the mutant frequency increased sharply for pH values below 6.8. At pH 6.4, a 4-fold increase was induced, and pH 6.0 resulted in a 10-fold increase in mutant frequency. Basic pH shifts in the presence of S9 mix caused no changes in mutant frequency up to pH 8.0; treatment with pH 8.8 was highly toxic (5.3% relative growth) and caused a 3-fold increase in mutant frequency. Thirteen mutant clones induced at pH 6.0 with S9 mix were challenged with trifluorothymidine after their expansion in nonselective medium and all retained their resistance; another 14 clones were tested for thymidine utilization and all incorporated only 0.1-5.5% of the 14C-labeled thymidine used by the parental line. The induced mutants were primarily of the small-colony phenotype, which indicated clastogenic activity. This was confirmed with chromosome studies which showed a large increase in cells with aberrations consisting of chromatid breaks and complex rearrangements. The results show that the combination of weak acidity (pH 6-6.8) and S9 mix is mutagenic and clastogenic to L5178Y TK+/- cells.

Genetic toxicity of dopamine.

The genetic toxicity of dopamine was studied in a battery of test systems including DNA single-strand break analysis in cultured human skin fibroblasts, the Salmonella/mammalian-microsome mutagenicity test, sister-chromatid exchange analysis in human lymphocytes, the mouse-lymphoma forward mutation assay, the sex-linked recessive lethal test in Drosophila melanogaster and the micronucleus test in mouse and rat. Dopamine at concentrations of 50-300 micrograms/ml induced DNA strand breaks in human fibroblasts. It also gave a positive response in the mouse-lymphoma forward mutation assay, where a dose-dependent increase in the frequency of mutant cells was observed in the presence of dopamine, 94-750 micrograms/ml. All other tests showed no response to dopamine. The dopamine-induced DNA strand breaks in human fibroblasts were inhibited by superoxide dismutase or dithiothreitol. Furthermore, dopamine caused nicking of circular Col El DNA and bound to calf thymus DNA in vitro. It is suggested that this genetic activity of dopamine in vitro relates to oxidation of dopamine and the generation of reactive oxygen radicals, semiquinones and quinones. It is unlikely that similar reactions would occur and cause genotoxic activity of dopamine in vivo.

Control of transfer RNA synthesis in the presence of inhibitors of protein synthesis.

The rate of synthesis of transfer RNA in suspension cultures of Chang's liver cells, has been examined in the presence of various inhibitors of protein synthesis with different modes of action. Inhibitors of polypeptide chain elongation such as cycloheximide and emetine stimulated the rate of synthesis of transfer RNA at concentrations that inhibited protein synthesis by 60-90%. Trichodermin, an inhibitor of the elongation and termination steps in protein synthesis, had as effect similar to that of cycloheximide and emetine. On the other hand verrucarin, an inhibitor of initiation, and puromycin, an analogue of the aminoacyl terminus of tRNA, had little effect of the synthesis of transfer RNA at low concentrations. At high concentrations these compounds inhibited transfer RNA synthesis. Inhibitors of protein synthesis can be divided in two groups based on their effect on the polysom pattern. The first group increased the number of large polysomes, while verrucarin and puromycin lead to a break down of large polysomes and to an accumulation of monosomes and small polysomes. Thus, there appears to be a correlation between the effect of these inhibitors of protein synthesis on the aggregational state of the polysomes and their effect on the rate of transfer RNA synthesis.