Mutagenicity of methylisocyanate and its reaction products to cultured mammalian cells

Methylisocyanate (MIC) induced mutagenic responses in the absence of exogenous activation in the mouse lymphoma cell forward mutation assay at concentrations as low as 8-24 microM. MIC produced predominantly small mutant colonies, suggesting the possibility of clastogenic activity. The intermediate hydrolysis product, methylamine, was also mutagenic without exogenous activation but required several hundred-fold higher concentrations (ca. 3 mM). N,N'-Dimethylurea, the final product in the reaction of methylisocyanate and water, was totally refractory in either the presence or absence of S9 for concentrations up to 57 mM (5 mg/ml). The ethyl ester of N-methylcarbamic acid was also tested since it was the only available analogue to the highly reactive N-methylcarbamic acid intermediate. This compound was mutagenic only in the presence of S9 at doses exceeding 5-40 microM, which suggested the possibility that the free acid, produced by enzymatic hydrolysis, is also mutagenic. The mutagenic activity of the ester resulted solely in the production of small mutant colonies.     

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 toxicology evaluation of commercial beers. II. Mutagenic activity of commercial beer products in Salmonella typhimurium strains TA98, TA100 and TA102

5 concentrated extracts of commercial beers were prepared using XAD-2 resin. The residues were subjected to evaluation for mutagenic activity in Salmonella typhimurium strains TA98, TA100 and TA102. The tests were conducted using preincubation protocols including provisions for S9 metabolic activation. Although the extracts did produce moderate toxicity to the Salmonella organisms used in the assays, none of the residues were found to induce mutation up to their maximum testable concentrations.     

Genotoxicity of theobromine in a series of short-term assays

Theobromine (3,7-dimethylxanthine) was evaluated for genotoxic activity in a series of in vitro assays. Theobromine was not mutagenic in the Ames assay up to a maximum concentration of 5000 micrograms/plate either with or without S9 activation. The compound also failed to induce significant levels of chromosome aberrations in CHO cells (with and without S9 activation) or transformation in Balb/c-3T3 cells. At the maximum tolerated concentration theobromine increased the frequency of TK-/- mutants in mouse lymphoma L5178Y cells. Increased frequencies were observed both with and without S9 activation and they were reproducible in 2 independent experiments. Statistically significant increases in SCEs were obtained in human lymphocytes and in CHO cells under nonactivation test conditions. The spectrum of results in this battery of tests indicate that theobromine treatment results in the expression of genotoxic potential in some assays and the observed activity appears qualitatively and quantitatively similar to that of caffeine, a closely related methylxanthine.     

Ultraviolet mutagenesis of normal and xeroderma pigmentosum variant human fibroblasts

The mutabilities of normal and xeroderma pigmentosum variant (XP4BE) human fibroblasts by ultraviolet light (UV) were compared under conditions of maximum expression of the 6-thioguanine resistance (TGr) phenotype. Selection was with 20 micrograms TG/ml on populations reseeded at various times after irradiation. Approx. 6--12 days (4--8 population doublings), depending on the UV dose, were necessary for complete expression. The induced mutation frequencies were linear functions of the UV dose but the slope of the line for normal cells extrapolated to zero induced mutants at 3 J/m2. The postreplication repair-defective XP4BE cells showed a higher frequency of TGr colonies than normal fibroblasts when compared at equal UV doses or at equitoxic treatments. The induced frequency of TGr colonies was not a linear function of the logarithm of survival for either cell type. Instead, the initial slope decreased to a constant slope for survivals less than about 50%. The UV doses and induced mutation frequencies corresponding to 37% survival of cloning abilities were 6.7 J/m2 and 6.2 X 10(-5), respectively, for normal cells and 3.75 J/m2 and 17.3 X 10(-5) for the XP4BE cells. The lack of an observable increase in the mutant frequency for normal fibroblasts exposed to slightly lethal UV doses suggests that normal postreplication repair of UV-induced lesions is error-free (or nearly so) until a threshold dose is exceeded.     

Requirement for Cell Dispersion Prior to Selection of Induced Azaguanine-Resistant Colonies of Chinese Hamster Cells

With V79 Chinese hamster cell cultures treated with a mutagen, the maximum frequency of colonies resistant to 8-azaguanine (AZG) was attained when the cells were dispersed after a suitable expression time before adding the selection medium. V79–4 cells were exposed to 500 µM MMS, 7 µM AFAA, or 10 µM MNNG and allowed to multiply before being reseeded at 4 x 10⁴ cells/60 mm dish and selected with 10 µg/ml AZG. Maximum frequencies of 4 x 10^-5, 4 x 10^-4, and 2.4 x 10^-3 were obtained about 100, 130, and 200 hrs after exposure to MMS, AFAA, and MNNG, respectively. The maximum frequencies following MMS or MNNG treatments were about 10-fold greater than those obtained when induction and selection of AZG-resistant colonies were performed in the same culture dish. The reseeding of treated cells eliminated the possibility of metabolic cooperation within mosaic colonies of wild-type and mutant cells and achieved expression of the induced changes before intercolony crossfeeding reduced the frequency of resistant colonies.—AZG-resistant colonies were selected in medium containing dialyzed fetal bovine serum, and the selection medium was replaced at least twice. Both serum dialysis and selection medium replacement were necessary for consistent achievement of background frequencies of resistant colonies near 10^-6. Reconstruction experiments with AZG-resistant V79 lines showed that the efficiency of recovery of resistant cells in the selection medium was constant over a range of 0–20 colonies observed/dish. A mixed population of V79 and AZG-resistant cells was also correctly analyzed by the procedure used in mutagenesis studies.     

Label‐free analysis of human cerebrospinal fluid addressing various normalization strategies and revealing protein groups affected by multiple sclerosis

The aims of the study were to: (i) identify differentially regulated proteins in cerebrospinal fluid (CSF) between multiple sclerosis (MS) patients and non‐MS controls; (ii) examine the effect of matching the CSF samples on either total protein amount or volume, and compare four protein normalization strategies for CSF protein quantification. CSF from MS patients (n = 37) and controls (n = 64), consisting of other noninflammatory neurological diseases (n = 50) and non neurological spinal anesthetic subjects (n = 14), were analyzed using label‐free proteomics, quantifying almost 800 proteins. In total, 122 proteins were significantly regulated (p < 0.05), where 77 proteins had p‐value <0.01 or AUC value >0.75. Hierarchical clustering indicated that there were two main groups of MS patients, those with increased levels of inflammatory response proteins and decreased levels of proteins involved in neuronal tissue development (n = 30), and those with normal protein levels for both of these protein groups (n = 7). The main subgroup of controls clustering with the MS patients showing increased inflammation and decreased neuronal tissue development were patients suffering from chronic fatigue. Our data indicate that the preferable way to quantify proteins in CSF is to first match the samples on total protein amount and then normalize the data based on the median intensities, preferably from the CNS‐enriched proteins.     

Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.     

中国的炭疽杆菌DNA分型及其地理分布

炭疽广泛分布于中国各地,特别是西部地区,并经常造成人畜疾病,在一项合作研究中,用多位点VNTR分析（MLVA）对从1952-1998年自中国主要地理流行区域分离的病人,病畜和土壤等来源的炭疽杆菌进行了基因分型,MLVA分析结果揭示了21种新的基因型,其等位基因组合在以前世界范围分离物的研究中未曾发现,此外,分离物的分群显示,A3b组是地理上最广泛分布的基因组,说明该组可能是中国的“地方流行株”。而来自古丝绸之路重要贸易中心新疆的大量分离株其基因型特别分散。