Utilization of rat lymphocytes for the in vitro chromosomal aberration assay

In vitro cytogenetic assays are widely conducted to assess the mutagenic potential of chemicals. Chinese hamster ovary (CHO) cells or human lymphocytes are often used for these assays; however, these cell types have certain limitations. In order to evaluate an alternate cell system, cultured rat lymphocytes were treated for 4 h at 48 h of incubation with a variety of direct- and indirect-acting clastogens in the presence or absence of an exogenous mammalian activation system. Cytogenetic effects of in vitro physiological alterations such as medium hypertonicity or pH changes were also evaluated. The background aberration rate of rat lymphocytes is approximately 2%, and they respond positively to both direct- and indirect-acting clastogens. In contrast to CHO cells, however, neither the hyperosmolality nor pH changes in the treatment media have significant effect on background aberrations. Unlike samples of human blood, rat blood can be collected under well-controlled environmental conditions. Because of the easy access to rat blood samples, the simplicity of culture, the reproducible nature of its in vitro growth, the positive response to known clastogens and negative response to media pH changes or hyperosmolalities, the rat lymphocyte in vitro chromosomal assay presented is an optimal system to assess the mutagenic potential of chemicals.     

Evaluation of a poliovirus-binding inhibition assay as an alternative to the virus neutralization test.

An enzyme-linked immunosorbent assay (ELISA)-based poliovirus-binding inhibition (PoBI) test to detect and quantify antibodies to polioviruses was optimized and evaluated for use in population studies as an alternative to the virus neutralization test (NT) in tissue culture. The sensitivities of the inhibition ELISA compared with the NT in an inactivated poliovirus vaccine (IPV)-vaccinated population were 98.6, 97.4, and 92.1% for serotypes 1, 2, and 3, respectively. The specificities of the PoBI test, as determined with sera from nonvaccinated persons, were also high for all three serotypes (99.0, 95.8, and 100%, respectively). Antibodies to other enteroviruses did not cross-react in the serotype 1 and 3 PoBI, and only levels of cross-reactivity were found for serotype 2. We found high correlations between the PoBI and NT titers for serotypes 1 and 2 in IPV-vaccinated blood donors (0.97 and 0.95), in oral poliovirus vaccine (OPV)-vaccinated blood donors (0.91 and 0.95), and in naturally immune persons (0.90 and 0.87). The correlation coefficient for serotype 3, however, was significantly lower in OPV-vaccinated blood donors (0.73) and in naturally immune persons (0.76) than in IPV-vaccinated persons (0.94; P < 0.01). These results indicate that the antibody response to serotype 3 poliovirus in IPV recipients is different from that in OPV recipients and naturally infected persons. We conclude that the PoBI test is a suitable alternative to the NT for estimating the seroprevalence of neutralizing antibodies to poliovirus, especially in large-scale population studies.     

Procarbazine is a potent mutagen at the heterozygous thymidine kinase (tk+/-) locus of mouse lymphoma assay

Procarbazine (Natulan®) is a potent inducer of gene mutationsat the heterozygous tk^+/– locus in L5178Y mouse lymphomacells in the presence of Aroclor-induced rat liver s9 metabolicactivation ({small tilde} 10^–3 mutant frequency at 10µg/ml) while exerting a far weaker effect in the absenceof s9. This mutagenicity is fairly robust with respect to thequantitative composition of the s9 mix and to variations inmouse lymphoma assay protocols (soft agar cloning versus ‘microwell’assays). The high proportion of small colony tk^–/–mutants induced by procarbazine together with the far weakermuta-genic response at the hemizygous hgprt locus in these samecells is interpreted in terms of a chromosomal or mufti-genemutational mechanism. Although procarbazine is clastogenic invivo, it does not appear to be so under standard protocols usingcultured human lymphocytes (±s9). It is not yet clearwhy this should be so, especially in light of its apparent clasto-genicityin mouse lymphoma cells.     

An in vitro haemolysis test as an alternative to the draize test for ocular irritation

A haemolysis test has been evaluated as an alternative to the Draize eye test (Draize et al. 1944) for ocular irritancy. Haemolysis was seen with corrosive/ severe and severe/moderate eye irritants but was negligible with non-irritant substances.Although not quite as sensitive as dog blood to irritants, human blood gave comparable results and was also more easily available in quantity. As with other in vitro tests, some false-positive results were given by the haemolysis test and also a few false-negative results. However, in all cases these were explicable given the physical and chemical properties of the materials. pH of test solutions did not seem to be a complicating factor.There was good correlation between the results of this test and published in vivo data. Although it may not yet entirely replace the in vivo model, the haemolysis test is a valuable preliminary stage in a tiered approach to eye irritancy screening.     

Evaluation of the thymidine kinase (tk) locus as an insertion site in the highly attenuated vaccinia MVA strain

Summary The highly attenuated modified vaccinia Ankara (MVA) strain is a potential live vaccine vector. Insertional inactivation of the tk-gene resulted in viruses difficult to purify. Co-integration of a functional fowlpox virus tk-gene allowed easy generation of recombinants, indicating that the genetically stable tk-gene region is a suitable insertion site, if tk-gene activity is substituted.     

Evaluation of di-(2-ethylhexyl)phthalate and its major metabolites in the ames test and L5178Y mouse lymphoma mutagenicity assay

Di-(2-ethylhexyl)phthalate (DEHP) and its two major metabolites, mono-(2-ethylhexyl)phthalate (MEHP) and 2-ethylhexanol (EH), were tested for genetic activity in both the Salmonella/mammalian microsome mutagenicity (Ames) assay and the L5178Y TK^+/— mouse lymphoma cell mutagenicity assay. All chemicals were tested in both the presence and absence of Aroclor-induced liver microsomes prepared from male Sprague-Dawley rats. Dose levels for both assays were selected from preliminary toxicity studies for each chemical. Neither DEHP, MEHP, nor EH exhibited any significant mutagenic activity in strains TA-98, TA-100, TA-1535, TA-1537, and TA-1538 in the Ames test or when tested in the L5178Y TK^+/ — mouse lymphoma cell mutagenicity assay.     

An alternative linear trend analysis for assessing the results of the mouse lymphoma assay

As recommended by the mouse lymphoma assay (MLA) Workgroup of the International Workshop on Genotoxicity Testing (Aberdeen, 2003), a trend test is critical if an induced mutant frequency (MF) of at least 126 × 10(-6) (global evaluation factor, GEF) is achieved at one or more test concentrations. Only those responses that both achieve the GEF and a significant trend are biologically relevant. While no specific trend test was recommended by the Workshop, a trend test was recommended by the UK Environmental Mutagen Society (1989). The test uses MF (untransformed) averaged over replicate cultures following a consistency test (against a historical heterogeneity factor) in a weighted linear regression with chi-square (χ(2)) test for slope and returns significant results in virtually all cases that are positive for the GEF, including those with no apparent dose-response. We have explored an alternative method where the natural logarithm of MF and its variance are estimated for each replicate culture separately and used in a weighted ordinary linear regression with t-test for slope. Using test cases positive for the GEF, the P-value from this model is shown to be sensitive to changes in the number of replicates, the shape and magnitude of mutant induction, in contrast to the χ(2) model. Cases with no apparent dose-response and thereby questionable biological significance are tested negative by our method but positive by the χ(2) model. Our method is thus straight-forward and provides a meaningful complement to the GEF in assessing the biological significance of the MLA results.     

The spectral karyotype of L5178Y TK+/− mouse lymphoma cells clone 3.7.2C and factors affecting mutant frequency at the thymidine kinase (tk) locus in the microtitre mouse lymphoma assay

There has been much discussion on acceptable spontaneous mutant frequencies in the mouse lymphoma assay (MLA). This culminated in the International Workshop on Genotoxicity Testing (IWGT) recommended control limits for the microtitre version of 50–170 mutants/10⁶ viable cells, which has now been included in the draft Organization for Economic Co‐Operation and Development guideline for assays investigating mammalian cell gene mutation at the tk locus. Some of the factors affecting mutant frequency have been investigated. It was shown that when culturing methotrexate cleansed TK^+/? cells, a spontaneous mutant frequency of ～100 mutants/10⁶ viable cells was achieved after only 26 doublings. However, after further culturing for ～6 months the spontaneous mutant frequency only gradually increased. Culturing for this time did not affect the karyotype of the cell in so much as the modal chromosome number remained stable. The spontaneous mutant frequency could effectively be manipulated by cleansing with various concentrations of methotrexate. The necessity for using appropriately heat‐inactivated horse serum was confirmed. Finally, following treatment with 4‐nitroquinoline‐N‐oxide, cells did not preferentially survive when plated at high cell densities (1.6 cells plus 2,000 feeder cells/well) versus cells at low density (1.6 cells/well). It was considered that these findings confirm that the dynamics of spontaneous mutant formation in the MLA are complex. However, the karyotype of L5178Y cells is remarkably stable and assuming investigators are using cells with appropriate provenance and good culturing technique, it is clear that the IWGT recommendations are achievable. Environ. Mol. Mutagen. 55:35–42, 2014. © 2013 Wiley Periodicals, Inc.     

Fragile site at Xq21 in Chinese hamster and its implications for the in vitro chromosomal aberration test

Normal Chinese hamster primary fibroblasts were exposed to methotrexate,an antifolate agent known to induce fragile sites. In a totalof 156 banded metaphases prepared from methotrexate-treatedcells, 93 breaks and gaps were observed and mapped to the individualhamster chromosomes and their bands. About two-thirds (69%)of all breaks and gaps mapped to the band Xq21, which is significantlymore than expected. Some other bands exhibited fragility butthe effect was not significantly. In 6% of cells, homozygousfragility of Xq21 was observed, suggesting that this is a commonfragile site. It has been reported previously that various chemicalsinduce preferential localized fragility of Xq in Chinese hamsterovary (CHO) and V79 cell lines. Our results suggest that preferentialfragility of Xq in the presence of chemical compounds representsexamples of fragile-site induction. A tendency of the in vitrochromosomal aberration test to yield positive results in theabsence of positive data in other test systems, such as theAmes test and the mouse bone marrow micro-nuceleus test mightbe due to fragile-site expression induced by chemical compoundsunder study. ¹To whom correspondence should be addressed     

Responses of the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay: III. 72 coded chemicals

Seventy-two chemicals were tested for their mutagenic potential in the L5178Y tk+/- mouse lymphoma cell forward mutation assay, using procedures based upon those described by Clive and Spector (Mutat Res 44:269-278, 1975) and Clive et al. (Mutat Res 59:61-108, 1979). Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 micrograms/ml. The chemicals were tested at least twice. Significant responses were obtained with allyl isothiocyanate, p-benzoquinone dioxime, benzyl acetate, 2-biphenylamine HCl, bis(2-chloro-1-methylethyl)ether, cadmium chloride, chlordane, chlorobenzene, chlorobenzilate, 2-chloroethanol, chlorothalonil, cytarabine.HCl, p,p'-DDE, diazinon, 2,6-dichloro-p-phenylenediamine, N,N-diethylthiourea, diglycidylresorcinol ether, 2,4-dimethoxy aniline.HCl, disperse yellow 3, endosulfan, 1,2-epoxyhexadecane, ethyl acrylate, ethyl benzene, ethylene thiourea, F D and C yellow Number 6, furan, heptachlor, isophorone, mercuric chloride, 4,4'-methylenedianiline.2 HCl, methyl viologen, nickel sulfate.6H2O, 4,4'-oxydianiline, pentachloroethane, piperonyl butoxide, propyl gallate, quinoline, rotenone, 2,4,5,6-tetrachloro-4-nitro-anisole, 1,1,1,2-tetrachloroethane, trichlorfon, 2,4,6-trichlorophenol, 2,4,5-trimethoxybenzaldehyde, 1,1,3-trimethyl-2-thiourea, 1-vinyl-3-cyclopetene dioxide, vinyl toluene, and ziram. Apart from 2-biphenylamine.HCl, 2-chloroethanol, disperse yellow 3, ethylene thiourea, FD and C yellow number 6, phenol, and 1,1,2-tetrachloroethane, rat liver S9 mix was not a requirement for these compounds. Chemicals not identified as mutagens were acid red, 11-aminoudecanoic acid, boric acid, 5-chloro-o-toluidine, coumaphos, cyclohexanone, decabromodiphenyl oxide, di(2-ethylhexyl)adipate, ferric chloride, fluometuron, melamine, monuron, phenesterin, phthalimide, reserpine, sodium dodecyl sulfate, 4,4-sulfonyldianiline, tetrachloroethylene, and zearalenone. The assay was incapable of providing a clear indication of whether some chemicals were mutagens; these were benzyl alcohol, 1,4-dichlorobenzene, phenol, succinic acid-2,2-dimethyl hydrazide, and toluene.     

Responses of the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay. II: 18 coded chemicals

Eighteen chemicals were tested for their mutagenic potential in the L5178Y tk+/- mouse lymphoma cell forward mutation assay by the use of procedures based upon those described by Clive and Spector [Mutat Res 44:269-278, 1975] and Clive et al [Mutat Res 59:61-108, 1979]. Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 micrograms/ml. The chemicals were tested at least twice. Significant responses were obtained with benzofuran, benzyl chloride, bromodichloromethane, butylated hydroxytoluene, chlorendic acid, o-chlorobenzalmalonitrile, 1,2,3,4-diepoxybutane, dimethyl formamide, dimethyl hydrogen phosphite, furfural, glutaraldehyde, hydroquinone, 8-hydroxyquinoline, and resorcinol. Apart from bromodichloromethane, butylated hydroxytoluene and dimethyl hydrogen phosphite, rat liver S9 mix was not a requirement for the activity of any of these compounds. Chemicals not identified as mutagens were water, tert-butyl alcohol, pyridine, and witch hazel.     

An ELISA-based method for measurement of food-specific IgE antibody in mouse serum: an alternative to the passive cutaneous anaphylaxis assay

Passive cutaneous anaphylaxis (PCA) assay has been a gold standard method to measure allergen-specific IgE antibody (ASIgE Ab) levels in allergy mouse models. Many factors including stringent guidelines for laboratory animal use make PCA a difficult choice. Therefore, alternative methods are needed that can be readily applied for measurement of specific IgE antibody levels in mouse serum. Herein we describe a novel ELISA-based method that is more sensitive in comparison to PCA, IgE isotype-specific (because it has little cross-reactivity with IgG1 or IgG2a isotype) and highly reproducible (<10% inter- or intra-assay variation). Furthermore, we demonstrate the utility of this assay to measure specific IgE Ab against a variety of food extracts including chicken egg, peanut, almond, filbert/hazelnut and sweet potato. These findings are of particular interest to those who are seeking (i) to measure food-extract-specific IgE antibody in animal models and (ii) an alternative to the animal-based PCA method to measure mouse IgE antibodies.     

Evaluation of alternating consecutive maximum contractions as an alternative test of neuromuscular function

The standard strength test (SST) has been based only on sustained maximum forces, as well as on relatively large number trials needed to record the maximum forces (F) and their rate of development (RFD). The aim of this study was to extend our recent research on alternating consecutive maximum contractions (ACMC) performed by antagonist muscles. Instead of varying the frequency, we explored the properties of ACMC performed at the self-selected frequency and compared it with SST. Knee extensors and flexors were tested in 64 participants. Within-session reliability of F and RFD of the two muscles evaluated through a single ACMC trial proved to be high (ICC ≥ 0.8), as well as their concurrent validity regarding the SST (r ≥ 0.7). Mainly strong relationships (r > 0.50) with the maximum performance tests also suggested moderate-to-high external validity of ACMC variables. Finally, the same variables were also able to distinguish among the participants of different training and physical activity history (p < 0.05). Taken together, these data suggest that ACMC could have the properties of reliability, external validity, and sensitivity similar to SST. However, since ACMC still retains some important advantages over SST (e.g., being based on a brief and fatigue free procedure for testing two antagonistic muscles, and exposing the muscle and joint tissues to relatively low and transient forces), one could conclude that ACMC performed at the self-selected frequency could be developed into a test of muscle function that could be either alternative or complementary to the SST.     

Responses of the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay to coded chemicals. I: Results for nine compounds

Nine substances were tested for their mutagenic potential in the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay, by means of procedures based upon those described by Clive and Spector (Mutat Res 44:269-278, 1975) and Clive et al (Mutat Res 59:61-108, 1979). Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 micrograms/ml. The coded chemicals were tested at least twice. Significant responses were obtained with calcium chromate, 3-(chloromethyl)pyridine, 1,2-epoxybutane, monochloroacetic acid, dicyclohexylthiourea, 2,4-diaminophenol hydrochloride, and pentachloroanisole. Apart from pentachloroanisole, rat liver S9 mix was not a requirement for the clearly mutagenic activity of any of these compounds. Compounds not identified as mutagens were 3-amino-1,2,4-triazole and sucrose.