Dominant lethal assay of some hair-dye components in random-bred male rats.

Male rats were exposed to maximally tolerated doses of 5 hair-dye components in a dominant lethal test. Each component was tested at 3 dosage levels with 15 random-bred male rats per level. The highest dose, selected on the basis of subacute toxicity testing, generally reduced weight gains without being lethal. Freshly prepared solutions were injected i.p. at 1 ml/kg 3 times a week for 10 weeks. Rats injected with dimethylsulfoxide and triethylenemelamine served as solvent and positive controls, respectively. A majority of rats survived the treatment at the levels tested and were mated to two virgin females each per week for 2 weeks. The females were sacrificed at midterm of pregnancy and examined for live and dead implants. Dominant lethality was evaluated on the basis of 4 criteria: dead implants per pregnant female, dead implants per total implants, proportion of females with one or more dead implants, and proportion of females with two or more dead implants. 2-Nitro-p-phenylenediamine, 2,4-diaminoanisole sulfate and 2,5-diaminoanisole sulfate produced negative responses, whereas m-phenylenediamine and 4-nitro-o-phenylenediamine induced weak dominant lethality in the first trial. On retesting these weakly positive components, both m-phenylenediamine and 4-nitro-o-phenylenediamine produced negative responses.     

A simple trypsin resistance assay for muscle and other cell fusion

Muscle cells fusing in vitro have long provided biologists with a tool to study development and gene expression. However, many such studies used morphological assays of cell fusion. We present here a method for assaying fusion at a specific, operationally defined step. Muscle cells grown in monolayer are exposed to trypsin-EDTA solution at 37 degrees C; the trypsin is inactivated, the cells fixed in Lugol's iodine, and 200 to 300 nuclei are counted as being single or multiple. The presence of EDTA is important under standard conditions for muscle culture; however, little difference is seen in divalent cation-depleted cultures. Therefore, for consistency EDTA can be included in all assays. Samples are stable for over 24 hr, with no cell loss from trypsinization or fixation. This assay exploits a specific stage of muscle fusion, trypsin-resistant contact, to provide a rapid, simple, and observer-independent assay for an early state of muscle fusion. The assay can be used to measure fusion between any nucleated cells.     

Mutagenicity monitoring in humans by autoradiographic assay for mutant T lymphocytes

Somatic cell mutation which occurs in vivo in humans can be determined by measurement of the frequency of the 6-thioguanine-resistant (TGr) T lymphocytes in samples of peripheral blood. This frequency can be determined by either a short-term autoradiographic methodology or a longer cell-cloning methodology. The advantage of the former is the relative simplicity of the assay, while the latter allows recovery of mutant clones for further characterization. This report presents results of a longitudinal study of cancer chemotherapy nurses and other health care personnel by use of the autoradiography assay. The use of this assay in human mutagenicity monitoring and the analysis of the TGr cell frequencies are discussed in terms of age effects and validation of 'elevated' frequencies by use of the clonal assay. This report then presents evidence that both assays yield similar TGr cell frequencies in two groups of 'normal adults'. The mean variant frequency (+/- S.D.) for 82 autoradiographic assays was 8.7 (+/- 6.1) X 10(-6), while the mean mutant frequency (+/- S.D.) for 115 clonal assays was 6.5 (+/- 4.8) X 10(-6). In addition, concurrent autoradiographic and clonal assays on 33 individuals yielded mean values (+/- S.D.) of 8.4 (+/- 8.5) X 10(-6) and 10.5 (+/- 6.3) X 10(-6), respectively.     

Dominant lethal test in the mouse for mutagenic effects of saccharine

Summary Twenty male NMRI mice received 5 g saccharine per kilogram body weight by the oral route daily for 5 successive days. After the last dose each male was mated with 3 untreated females. For fractionated examination with regard to successive germ cell stages, each week 3 other untreated females were placed with each male for mating. The whole mating period was 8 weeks. The uteri of the females were inspected on the 14th day of gestation and pre-implantative and post-implantative loss determined from the numbers of corpora lutea, implantations, live and dead implants.The treatment did not damage the males and did not impair their mating capacity or their fertility.Post-implantative loss remained unaffected by the saccharine treatment compared with parallel controls.Pre-implantative loss persisted in the saccharine-treated group throughout the 8 weeks in the normal range of the strain. A statistically significant difference between saccharine group and control group in the 3rd week of mating after treatment was without biological relevance.Our investigations revealed no indication of a mutagenic action of saccharine in terms of an induction of dominant lethal mutations.This is in keeping with cytogenetic in vitro findings of other authors on human leukocytes.

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Zusammenfassung 20 männliche NMRI-Mäuse erhielten täglich je 5 g Saccharin per os pro Kilogramm Körpergewicht an 5 aufeinanderfolgenden Tagen. Nach der letzten Applikation wurde jedes Männchen mit 3 unbehandelten Weibchen gepaart. Zur fraktionierten Untersuchung der aufeinanderfolgenden Keimzellstadien der Männchen wurden jede Woche 3 neue, unbehandelte Weibchen zu jedem Bock gesetzt und besamen lassen, insgesamt über 8 Wochen.Die Uteri der Weibchen wurden am 14. Tag der Trächtigkeit untersucht, und der präimplantative under postimplantative Verlust wurden an Hand der Corpora lutea, der Implantationen und der lebenden und toten Keimlinge ermittelt.Die Behandlung schädigte die Männchen nicht und beeinträchtigte nicht ihre Deckfreudigkeit und Fertilität.Der postimplantative Verlust blieb im Vergleich zu der parallel durchgeführten Kontrolle unbeeinflußt durch die Behandlung mit Saccharin.Der präimplantative Verlust der mit Saccharin behandelten Gruppe lag während aller 8 Versuchswochen im Bereich der Norm des Stammes. Eine in der 3. Paarungswoche nach den Applikationen aufgetretene statistische Signifikanz zwischen den Verlusten der Saccharin-Gruppe und der Kontroll-Gruppe war ohne biologische Relevanz.Unsere Untersuchungen erbrachten keinen Hinweis für eine mutagene Wirkung von Saccharin im Sinne der Induktion dominanter Letalmutationen.Dies steht im Einklang mit cytogenetischen in vitro-Befunden anderer Autoren an menschlichen Leukocyten.

     

Assessment of reference values for DNA damage detected by the comet assay in human blood cell DNA

Genotoxicity measured by the comet assay is expressed by different researchers using parameters that are not easy to conceptualize, except for percent tail DNA (%T) or visual score (arbitrary units). A total of 125 publications have reported genotoxicity as DNA damage (representing strand breaks, alkaline labile sites, and transient repair sites), endonuclease III (ENDOIII), or formamidopyrimidine DNA glycosylase (FPG) sensitive sites. I have recalculated the visual score so that it is expressed in the range of 0-100, similar to that of %T. Similar values were obtained for DNA damage and ENDOIII sites, regardless of whether of the data were reported as %T or visual score. Thus, these endpoints can be used interchangeably, assuming that the visual score is expressed in the 0-100 range. Pooled analysis of %T and visual score data showed that the median (25-75%) values of DNA damage, ENDOIII, and FPG sites were 8.6 (4.4-14.5), 11.0 (4.2-19.5), 7.6 (3.2-14.2), respectively. The duration of alkaline treatment and electrophoresis had no significant effect on the level of DNA damage. There was a positive correlation between age and the level of DNA damage. A sub-analysis of DNA damage obtained from European countries showed a negative correlation with latitude. In conclusion, reference values for DNA lesions measured by the comet assay are around 7-11 %T or arbitrary units.     

Dominant lethal mutations induced by MMS and mitomycin C in the fish Oryzias latipes

Males of the fish Oryzias latipes were treated with various chemicals and then mated with normal females. The fertility and hatchability of the eggs laid by the parents were examined, and the dominant lethal effects were estimated. Mitomycin C induced dominant lethals in the fish spermatids and spermatocytes after the males had been treated with concentrations of 2.5 and 25 micrograms/ml. Methyl methanesulfonate (MMS) induced dominant lethals in spermatozoa and spermatozoa and spermatids after the injection of 200 and 400 mg/kg. These results are in good agreement with the results obtained with mice. However, the effects of ethyl methanesulfonate (EMS) were not clear on spermatogenic cells at any stage. We could not recognize any significant induction of dominant lethals by urethanes, bleomycin, caffeine, and two kinds of food-color additives, at least under the present experimental conditions.     

Conditional lethal mutants of Chinese hamster cells: mutants requiring exogenous carbondioxide for growth

A series of Chinese hamster cell lines were tested and found to be able to proliferate in the absence of added bicarbonate and carbondioxide if hypoxanthine and uridine were present in the medium. Conversely, cells incapable of salvaging one of these precursors, such as hypoxanthine-guanine phosphoribosyltransferase (HGPRT^?) deficient cells did not multiply under these conditions. We describe another variant capable of utilizing hypoxanthine and uridine which has an absolute requirement for exogenous CO₂/NaHCO₃ for growth. These cells appear to be defective in the complete oxidation of pyruvate to carbondioxide, and indications are that the entry of pyruvate into the Krebs cycle is affected.     

An enzymatic electrochemiluminescence assay for the lethal factor of anthrax

The lethal factor (LF) of anthrax toxin is the toxic component of the exotoxin (lethal toxin) secreted by toxic strains of Bacillus anthracis. The lethal factor is a zinc-dependent metalloprotease that specifically cleaves the mitogen-activated protein kinase kinase (MAPKK) family of enzymes. We took advantage of this substrate specificity to develop an electrochemiluminescence (ECL) peptide cleavage assay. The ECL assay uses the stable ruthenium (Ru) metal chelate that, in the presence of tripropylamine, generates a light reaction triggered by the application of an electric potential. The Ru label is specifically incorporated into the C-terminal CYS residue of a synthetic peptide (23mer) containing the MAPKK2 cleavage sequence of LF. Streptavidin-coated paramagnetic beads were the solid phase and facilitated separation and characterization of the enzymatic reaction products based upon N-terminal biotinylation of the peptide substrate. Intact peptide bound via the biotin moiety generated high signal due to the Ru label, whereas binding of the cleaved peptide fragment devoid of Ru label reduced the ECL signal. The proposed assay provides a novel opportunity for the screening of potential therapeutics against anthrax.     

cdc9 ligase-defective mutants of Saccharomyces cerevisiae exhibit lowered resistance to lethal effects of bleomycin

Conditional ligase-deficient mutants of Saccharomyces cerevisiae were more sensitive than their parental (CDC9) strain to dose-dependent killing by bleomycin, even when mutant cells were pregrown and exposed to the antibiotic at permissive temperatures. Pretreatment incubation at the restrictive temperature (37 degrees C) under growing or nongrowing conditions enhanced bleomycin killing of both cdc9-1 and cdc9-9 mutants. This sensitization could be relieved by incubation at the permissive temperature before treatment.