International Workshop on Standardisation of Genotoxicity Test Procedures. Summary of major conclusions.
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Animals↗
Biomedical subjects
S M Galloway
Publications and source records attributed to S M Galloway.
Report from working group on in vitro tests for chromosomal aberrations.
The following summary represents a consensus of the working group except where noted. The items discussed are listed in the order in which they appear in the OECD guideline (473) for easy reference. Metabolic activation. S9 from animals induced either with Aroclor 1254 or with the combination of phenobarbital with beta-naphthoflavone is acceptable, and other systems could be used with suitable justification. Exposure concentrations. The upper limit of testing should be 10 mM (or 5 mg/ml where molecular weight is not known or mixtures are being tested), whichever is lower. Where this limit is inappropriate the investigator should give detailed justification of the choice of top concentration. Cytotoxicity should be measured not only in range-finding tests but also concurrently with the assay for chromosomal aberrations. Cytotoxicity should be assessed by measurements of cell growth such as cell counts or confluence estimation. Mitotic index data alone are not a sufficient measure of cytotoxicity, except in the case of blood cultures for which other methods are impractical. Cytotoxicity at the top dose should be greater than 50% of concurrent negative/solvent controls, if this can be achieved without exceeding a concentration limit of 10 mM or 5 mg/ml. There should be at least three concentrations scored for aberrations (each with and without S9), covering a toxicity range down to a concentration giving little or no cytotoxicity. This will usually mean that the concentrations scored will be quite closely spaced. It was not possible to reach a consensus on the issue of solubility limits. The group did not agree on whether (a) solubility rather than cytotoxicity should be the limiting factor, such that only one top dose with evident precipitate should be scored even if toxicity is not observed, or (b) several concentrations with evident precipitate should be scored for aberrations if this were necessary to obtain cytotoxicity. It was agreed that evidence of precipitation should be determined in the final culture medium. Controls. Concurrent positive controls are required but the working group thought it inappropriate to specify the control chemicals or the degree of response that should be obtained, leaving it up to the test laboratory to demonstrate that the system was working adequately based on historical data within the laboratory. It is not necessary to include both negative and solvent controls concurrently with the aberration test; solvent controls alone are acceptable provided that the laboratory has data to demonstrate that there is no effect of the solvent on baseline values. Preparation of cultures.(ABSTRACT TRUNCATED AT 400 WORDS)
Aneuploidy↗
Replicate flasks are not necessary for in vitro chromosome-aberration assays in CHO cells.
Some recommended protocols for in vitro chromosome-aberration assays call for two flasks per dose group. Use of replicate flasks allows for possible variation in percent aberrant cells (ABR) between flasks. We studied the magnitude of variation between replicate flasks of Chinese hamster ovary (CHO) cells using data from 211 assays from three laboratories, in order to assess the effect on assay sensitivity. Based on all 403 pairs of replicate "control" flasks, there was almost no excess variability between flasks. The standard deviation (SD) was only 4% larger than the value expected purely from sampling cells (P > 0.05). Data from all 366 pairs of replicate "treated" flasks showed that between-flask variation increased with the average percent aberrant cells (P < 0.001). The SD for 60 pairs of flasks with 3.0-7.5% ABR cells was 32% larger than the expected value. However, computer simulations based on these data showed use of replicate flasks has little effect on assay false-positive or true-positive rates. All assays with replicate treated flasks and at least three dose groups including control were re-analyzed as "single-flask" experiments. A "single-flask" experiment was defined by taking both control flasks but only one treated flask per dose. For each assay, all possible single-flask experiments were re-analyzed and the percent with positive results recorded. For most assays, conclusions were the same regardless of which treated flasks were selected, in spite of the fact that these single-flask experiments had only half as many cells scored per active dose group. For a very few assays with marginal results, the conclusion could change depending on which set of flasks was chosen, but these were such borderline results that a repeat assay was required in any case. Repeating the assay is a better way to resolve marginal results than examining replicate flasks. From our re-examination of the experimental data and from the computer simulation, we conclude that, while flask-to-flask variability exists, it has no practical effect on the test outcome, so that use of replicate flasks is not necessary for this assay.
Analysis of Variance↗
A panel of RFLP markers from sheep genomic DNA.
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Animals↗
Evaluation of the genetic toxicity of the peroxisome proliferator and carcinogen methyl clofenapate, including assays using Muta Mouse and Big Blue transgenic mice.
The rodent liver carcinogen and hepatic peroxisome proliferator methylclofenapate (MCP) has been evaluated for genetic toxicity in a range of in vitro and rodent genotoxicity assays. It gave a negative response in each of the following assays: mutagenicity to S. typhimurium and E. coli (+/- S9 mix, plate and pre-incubation assays), clastogenicity to cultured human lymphocytes and CHO cells (+/- S9 mix), a mouse bone marrow micronucleus assay (24h and 48h sampling), a rat liver assay for UDS in vivo (12h sampling), assays for lac I (Big Blue) and lac Z (Muta Mouse) mutations in the liver of transgenic mice, and an assay of the ability of MCP to modify the mutagenicity to the liver of dimethylnitrosamine in both transgenic mutation assays. The micronucleus and UDS assays were conducted using a single administration of MCP at its maximum tolerated dose, while the transgenic assays were conducted using nine daily administrations of MCP at its cancer bioassay dose level. These nine daily administrations were shown to double the weight of the liver of non-transgenic, Big Blue and Muta Mice, as well as leading to a dramatic proliferation of peroxisomes (electron microscopy) in the livers of each strain. These changed parameters had returned to control levels when the mutation analyses were conducted (10 days after the final dose of MCP). Despite the liver enlargement observed following MCP administration, no evidence of mitotic activity was observed in treated livers, although an increased number of cells were undergoing replicative DNA synthesis during the final 3 days of the 9 days of administration (BUdR assessment of S-phase). Liver biochemistry parameters (ALT, AST, AP, CK, GGT and albumin) were unaffected by the chronic (9 day) administration of MCP indicating an absence of hepatic toxicity. These combined observations favour a non-genotoxic mechanism of action for the hepatic carcinogenicity of MCP. The clastogenicity in vitro of the perixisome proliferator Wyeth 14,643 has been confirmed in CHO cells, but it is noted that this chemical is more soluble than is MCP. In particular, at the highest dose level at which MCP could be tested, Wy 14,643 was also non-clastogenic.
Animals↗
Ovine microsatellites at the OarFCB5, OarFCB19, OarFCB20, OarFCB48, OarFCB129 and OarFCB226 loci.
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Animals↗
UDP-N-acetylglucosamine acyltransferase of Escherichia coli. The first step of endotoxin biosynthesis is thermodynamically unfavorable.
UDP-N-acetylglucosamine acyltransferase of Escherichia coli catalyzes the reaction, UDP-GlcNAc + R-3-hydroxymyristoyl-ACP--> UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc + ACP. Using Matrex Gel Green A and heparin-agarose, we have purified the enzyme to near homogeneity from a strain that overproduces it 474-fold. The subunit molecular mass determined by SDS-gel electrophoresis is approximately 30 kDa, consistent with results of previous radiolabeling experiments in mini-cells. The amino-terminal sequence (Met-Ile-Asp-Lys-Ser-Ala-Phe-Val-His-Pro) and the amino acid composition of the purified protein are consistent with DNA sequencing (Coleman, J., and Raetz, C. R. H. (1988) J. Bacteriol. 170, 1268-1274). At saturating concentrations of the second substrate, the apparent Km values for UDP-GlcNAc and R-3-hydroxymyristoyl-ACP are 99 and 1.6 microM, respectively. There is an absolute requirement for the R-3-hydroxy moiety of the fatty acyl-ACP substrate; myristoyl-ACP binds effectively (IC50 = 2 microM) but is inactive (< 0.01%) as an alternate substrate. The most remarkable feature of the reaction is its unfavorable equilibrium constant, Keq approximately equal to 0.01, which is not predicted by model S-->O acyl transfer reactions. Thus, although UDP-GlcNAc acyltransferase catalyzes the first unique step of lipid A biosynthesis, it is the second enzyme (the deacetylase) that commits the substrates to this pathway. The specific activity of the deacetylase is elevated approximately 5-fold when lipid A synthesis is inhibited.
Acyltransferases↗
Activation of immediate-early gene expression by peroxisome proliferators in vitro.
The hepatocarcinogenicity of peroxisome proliferators (PPs) in rodents has been attributed both to oxidative DNA damage resulting from excessive leakage of peroxisomal H2O2 and to increased hepatocellular replication that may be independent of peroxisome proliferation. Because of the growing association between tumor promotion and alterations in growth-regulatory signal transduction pathways, we investigated whether PPs can modulate these pathways in a mouse liver epithelial cell line, BNL-CL.2. We tested two PPs that differ markedly in rodent tumorigenicity for their ability to activate immediate-early proto-oncogene expression. 4-Chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (Wy-14643), a highly tumorigenic PP, was an exceptionally strong inducer of c-fos expression. Wy-14643 was also stronger than DEHP in stimulating c-jun expression, whereas both PPs were fairly strong inducers of jun-B and jun-D. The induction of fos and jun expression by Wy-14643 was specifically inhibited by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperizine dihydrochloride (H-7). DEHP-induced gene expression was strongly inhibited by H-7, but was also partially inhibited by an inhibitor of protein kinase A. The activation of fos and jun gene expression by PPs was independent of peroxisome proliferation since it was an immediately-early response not requiring protein synthesis and since the cell lines used in this study do not undergo peroxisome proliferation. Our r results raise the possibility that the carcinogenicity of PPs may be due, in part, to epigenetic modulation of growth-regulatory signal transduction pathways.
3T3 Cells↗
Measurement of plasma angiotensin II: purification by cation-exchange chromatography.
Measurement of plasma angiotensin II (AII) by radioimmunoassay (RIA) usually requires prior purification of the plasma to remove substances that cross-react in the RIA, most notably angiotensin III (AIII). Purification of AII is generally accomplished by solid-phase extraction (SPE) followed by reverse-phase HPLC, with tedious evaporation and resuspension steps in between, and requires collection of many HPLC fractions per sample for RIA. In this report, we describe a rapid two-step SPE procedure for the purification of plasma AII, including an improved protease inhibitor cocktail for preventing the formation or degradation of AII in vitro. Plasma is first extracted on an S-Sepharose cation-exchange column, in which AII is separated from AIII by virtue of their difference in net charge, and then extracted on a C8 SPE column, without need for intermediate sample handling. The two-step SPE method is fast, results in only a single fraction for RIA per sample, and yields consistently high recoveries (77-86%) of AII, reducing the volume of plasma needed from 2 to 0.5 ml. Rat plasma was used in the present study, but the complete conservation of angiotensin peptide sequences (except angiotensinogen) in mammals suggests that this method will be applicable for other species including humans. In summary, the two-step SPE method offers the speed and simplicity of solid phase extraction while achieving a purity in AII (i.e., free of AIII) previously only obtained by laborious procedures involving HPLC.
Amino Acid Sequence↗
Microsatellites and associated repetitive elements in the sheep genome.
To determine the frequency and clustering of a variety of simple di- and trinucleotide repeats, an Artiodactyl short interspersed element (SINE), an ovine satellite repeat, and a human Alu 1 repeat were used to screen a random selection of cosmids containing inserts of ovine genomic DNA. In total, 197 individual cosmids were digested with EcoRI and the fragments separated on 0.7% agarose gels. Southern blots of these gels were then sequentially probed with (AC)7, (CT)9, and (CAC)6 oligonucleotides, and the repeats described above. The frequency at which (AC)n, (CT)n, and (CAC)n repeats were found in the cosmids indicated that they occurred at average intervals of 65 kb, 367 kb, and 213 kb respectively within the ovine genome. The Artiodactyl SINE was the most common, occurring at an average interval of 20 kb. No human Alu 1 sequences were detected. There was a significant positive association between the (AC)n and the Artiodactyl SINE. This association is quite strong as there was significant clustering of the two repeats both within cosmids and also within the EcoRI fragments of the digested genomic fragments. With the exception of the sheep satellite sequence, which occurs in tandem arrays, none of the other repeats showed significant clustering within the 41-kb (average size) cosmid inserts. The first 25 ovine microsatellites we characterized had an average polymorphic information content (PIC) of 0.65. The different microsatellite types, containing either perfect, imperfect, or compound repeats, had similar average PICs of 0.64, 0.65, and 0.66 respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Evaluation of the need for a late harvest time in the assay for chromosome aberrations in Chinese hamster ovary cells.
Harvest time is one of the most important variables in the assessment of whether a compound is clastogenic and in establishing a dose relation. In CHO cells we have found that for a variety of chemicals one harvest time near 20 h is optimal following a 3-h treatment (Bean et al., 1992). However, some guidelines for testing for regulatory purposes recommend an additional late harvest time 24 h after the first. We tested 10 diverse chemicals in CHO-WBL cells harvested 20-21 h and 42-44 h from the beginning of a 3-hr treatment. We added BrdUrd after treatment and recorded the total% of aberrant cells, and the proportions of aberrations (abs) in first (M1), second (M2) or later metaphases. The chemicals fell into 3 categories: ab yield greatly decreased at 44 h: benzo[a]pyrene, cadmium sulfate, chlorambucil, 2,6-diaminotoluene, 4-nitroquinoline N-oxide and mitomycin C (e.g., 37.0% cells with abs at 20 h and 1.0% at 44 h); ab yields similar at 20 and 44 h: 2-aminobiphenyl, eugenol and 8-hydroxyquinoline (e.g., 8.5% at 20 h and 7.0% at 44 h); and one, dimethylnitrosamine (DMN), which was detected at both times but gave a stronger response at 44 h than at 20 h (e.g., at 10 mM: 6.2% at 20 h and 25.0% at 44 h). This DMN effect was not seen in normal diploid human cells. For DMN the higher ab levels at 44 h than at 20 h were contributed by abs in M3 cells. Thus, while for some chemicals ab yields decrease with successive division, further increases can be seen in CHO in later metaphases, notably for DMN. Overall, however, after a 3-h pulse treatment of CHO cells a positive ab result could be obtained at the early harvest time (20 h) for all 10 chemicals.
Animals↗
2,4,6-Trichlorophenol (TCP) induces chromosome breakage and aneuploidy in vitro.
2,4,6-Trichlorophenol (2,4,6-TCP), a non-mutagen to Salmonella, was reportedly negative in tests for chromosome breakage in vitro, but did produce numerical chromosome changes and micronuclei in V79 cells (Jansson and Jansson, 1992). This apparent specific ability to induce aneuploidy is of interest since aneuploidy testing is not part of routine genotoxicity test procedures. Here we show 2,4,6-TCP clearly induces structural chromosome aberrations in CHO cells and in V79 cells using a 3-h treatment and 20-h sampling time (17-h recovery). The isomers 2,4,5- and 2,3,6-TCP were also clastogenic in this protocol. There was no increase in aberrations when we used the protocol of Jansson and Jansson (1992), i.e., a 24-h treatment with sampling either immediately, or with a 24-h recovery period. However, positive results were obtained when a recovery time of 4-12 h was allowed after the 24-h treatment with 2,4,6-TCP. Previous negative aberration tests of 2,4,6-TCP (Galloway et al., 1987; Ishidate, 1988) are also likely due to inappropriate protocols. All these results were obtained without S9 metabolic activation. We also found positive results in CHO cells when 2,4,6-TCP was tested with S9. The present study demonstrates that 2,4,6-TCP induces both structural and numerical aberrations, and underscores the importance of protocol design, in particular the appropriate recovery time after treatment, for detecting clastogenic activity in vitro.
Aneuploidy↗
Micronuclei induced in peripheral blood of E mu-PIM-1 transgenic mice by chronic oral treatment with 2-acetylaminofluorene or benzene but not with diethyl-nitrosamine or 1,2-dichloroethane.
Micronucleus induction in peripheral blood was examined during carcinogenicity assays of the genotoxic carcinogens 2-acetylaminofluorene (2-AAF), benzene, diethylnitrosamine (DEN) and 1,2-dichloroethane (1,2-DCE) in lymphoma prone E mu-PIM-1 transgenic mice. In both sexes, micronuclei were increased in polychromatic (PCE) and normochromatic (NCE) erythrocytes after 14 weeks of oral treatment with 75 mg/kg 2-AAF or 50 and 100 mg/kg benzene. The micronucleus frequencies induced by benzene were higher in males than in females. There was no apparent treatment related suppression of erythropoiesis by 2-AAF or by benzene. Blood micronucleus frequencies induced by benzene were similar in transgenic mice and their non-transgenic litter mates. There was no micronucleus induction or PCE suppression detected in the blood of either sex after treatment with 1 and 3 mg/kg DEN or 100 to 300 mg/kg 1,2-DCE. At 40 weeks bone marrow was sampled from mice given 100 mg/kg benzene, and it was confirmed that micronucleated PCE frequencies in blood were an accurate reflection of those induced in bone marrow. However, the spontaneous and induced frequencies of micronucleated cells in blood were slightly higher in PCE than in NCE suggesting that a small degree of selective removal of micronucleated cells occurs in this mouse strain. Control micronucleus frequencies in E mu-PIM-1 mice appeared comparable to those in other, non-transgenic mouse strains. Thus micronuclei are readily detectable in blood during chronic exposure to the bone-marrow clastogens 2-AAF and benzene, but not to DEN and 1,2-DCE, probably because active species do not reach the bone marrow in sufficient concentrations to induce increases in micronuclei.
2-Acetylaminofluorene↗
The ovine Booroola fecundity gene (FecB) is linked to markers from a region of human chromosome 4q.
The autosomal Booroola fecundity gene (FecB) mutation in sheep increases ovulation rate and litter size, with associated effects on ovarian physiology and hormone profiles. Analysis of segregation in twelve families (379 female progeny) identified linkage between the mutation, two microsatellite markers (OarAE101 and OarHH55, Zmax > 9.0) and epidermal growth factor (EGF) from human chromosome 4q25 (Zmax > 3.0). The marker OarAE101 was linked to secreted phosphoprotein 1 (SPP1, which maps to chromosome 4q21-23 in man) in the test pedigrees and independent families (Zmax > 9.7). The identification of linkage between the FecB mutation and markers from human chromosome 4q is an important step towards further understanding the control of ovulation rates in mammals.
Animals↗
A BglII RFLP at the ovine locus for insulin-like growth factor 1 (IGF1).
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Animals↗
Effect of sampling time on chromosome aberration yield for 7 chemicals in Chinese hamster ovary cells.
Choice of harvest time is one of the most important variables in the assessment of whether a compound is clastogenic and in establishing a dose relation. We examined the effects of sampling time on aberration yield for 7 diverse chemicals in CHO-WBL cells by harvesting at intervals from 9 to 30 h after treatment for 3 h with or without S9 metabolic activation. We observed both the percentage of aberrant cells and the total number of aberrations. Our data suggest that for most compounds a single harvest time approximately 17-21 h after the beginning of a 3-h treatment is optimal for aberration detection in CHO cells. Maximal aberration yields were observed for 2,4-diaminotoluene, 2,6-diaminotoluene and cytosine beta-D-arabinofuranoside from 17 to 21 h, eugenol from 15 to 21 h, cadmium sulfate from 15 to 24 h and 2-aminobiphenyl, from 17 to 24 h. For adriamycin at 1 microM, the % aberrant cells remained elevated throughout the period from 9 to 29 h, while small increases at 0.1 microM ADR were found only at 13 and at 25 h. For most chemicals the maximal aberration yield occurred at a different time for each concentration tested. However, the use of 3 or more closely spaced concentrations, carefully selected to yield up to 50% toxicity, allowed detection of a positive response at a single harvest time for all 7 chemicals.
Aminobiphenyl Compounds↗
A quantitative assessment of the cytotoxicity associated with chromosomal aberration detection in Chinese hamster ovary cells.
Regulatory guidelines suggest testing chemicals up to cytotoxic doses in chromosomal-aberration assays. To investigate the utility and limitations of various cytotoxicity indicators we used Chinese hamster ovary (CHO) cells to test 8 chemicals with differing ratios of cytotoxicity to clastogenicity. We measured immediate or delayed cell killing and growth inhibition (ATP levels, cell counts, colony-forming efficiency, CFE) and cell-cycle perturbations (mitotic index, MI; average generation time, AGT). Aberrations (abs) were scored 10 and 24 h from the beginning of the 3-h treatment. All 8 compounds induced abs at concentrations that reduced cell growth at 24 h by 50% or less. Concentrations of each chemical which induced at least 15% cells with abs, gave little loss of CFE (0-20%) for mitomycin C, adriamycin, cadmium sulfate and 2,6-diaminotoluene in contrast to the marked loss of CFE (70-80%) for eugenol (EUG), 2-aminobiphenyl and 8-hydroxyquinoline (8-HQ). 2,4-Diaminotoluene (2,4-DAT) was intermediate. Higher aberration yields were found at 24 h than at 10 h, even when minimal cell-cycle delay was detected by AGT estimates from BrdUrd-labeled cells. Cells with multiple abs were seen at 24 but not at 10 h, and often confirmed clastogenicity when there was only a weak increase in the percentage of cells with aberrations. Total ATP per culture did not always correlate with cell number, especially at later times after treatment. This is likely due to metabolic perturbations or altered cell biomass that are known to affect cell ATP content. MI suppression often did not correlate with AGT, e.g., only small increases in AGT were seen for 8-HQ, 2,4-DAT and EUG despite severe mitotic suppression at 10 h. By 24 h the MI for all chemicals had recovered, sometimes exceeding control levels. Marked mitotic accumulation was seen at 10 h for 2,4-DAT, indicating cell synchrony. Thus, the MI has limited value for dose selection. In conclusion, even weakly active chemicals were detected at a single time without exceeding a 50% growth reduction at 24 h.
Aminobiphenyl Compounds↗
Genotoxicity hazard assessment of Caramel Colours III and IV.
Results from a battery of short-term tests in vitro and in vivo used to assess the genotoxicity of caramel colours are presented and discussed in relation to reports from the literature. No evidence of genotoxicity was found in the Salmonella plate incorporation test using five standard strains or in the Saccharomyces cerevisiae gene conversion assay using strain D4, either with or without S-9 for activation. A weak clastogenic effect for a sample of Caramel Colour III in CHO cells was abolished in the presence of S-9. Two samples of Caramel Colour IV were not clastogenic in CHO cells. Salmonella pre-incubation tests without S-9 also failed to reveal any mutagenic activity for any of the caramel colours tested. The Caramel Colour III sample that showed clastogenic activity in CHO cells in vitro did not induce micronuclei when evaluated in a mouse bone marrow assay. These results are in general agreement with reports in the literature regarding the genotoxicity of caramel colours, and support the conclusion that caramel colours do not pose a genotoxic hazard to humans.
Animals↗