Four widely used in vitro assays for genetic toxicity were evaluated for their ability to predict the carcinogenicity of selected chemicals in rodents. These assays were mutagenesis in Salmonella and mouse lymphoma cells and chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells. Seventy-three chemicals recently tested in 2-year carcinogenicity studies conducted by the National Cancer Institute and the National Toxicology Program were used in this evaluation. Test results from the four in vitro assays did not show significant differences in individual concordance with the rodent carcinogenicity results; the concordance of each assay was approximately 60 percent. Within the limits of this study there was no evidence of complementarity among the four assays, and no battery of tests constructed from these assays improved substantially on the overall performance of the Salmonella assay. The in vitro assays which represented a range of three cell types and four end points did show substantial agreement among themselves, indicating that chemicals positive in one in vitro assay tended to be positive in the other in vitro assays.
The development of automated flow cytometric (FCM) methods for evaluating micronucleus (MN) frequencies in erythrocytes has great potential for improving the sensitivity, reproducibility, and throughput of the traditional in vivo rodent MN assay that uses microscopy-based methods for data collection. Although some validation studies of the FCM evaluation methods have been performed, a comprehensive comparison of these two data collection methods under routine testing conditions with a variety of compounds in multiple species has not been conducted. Therefore, to determine if FCM evaluation of MN frequencies in rodents was an acceptable alternative to traditional manual scoring methods in our laboratory, we conducted a comparative evaluation of MN-reticulocyte (MN-RET) frequencies determined by FCM- and microscopy-based scoring of peripheral blood and bone marrow samples from B6C3F1 mice and Fisher 344 rats. Four known inducers of MN (cyclophosphamide, ethyl methanesulfonate, vincristine sulfate, acrylamide) were assayed in bone marrow and peripheral blood of both mice and rats. In addition, MN-RET frequencies were measured in bone marrow (microscopy) and peripheral blood (FCM) of mice treated with five nongenotoxic chemicals (S-adenosylmethionine chloride, cefuroxime, diphenolic acid, 3-amino-6-methylphenol, pentabromodiphenyl oxide). No significant differences were observed between results obtained by the two methods in either species. These results support the use of FCM for determining MN-RET frequency in rodents after chemical exposure.
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 μg/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‐epoxyhexa‐decane, 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‐trimetho‐xybenzaldehyde, 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‐aminoundecanoic 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.
The in vivo micronucleus (MN) assay has proven to be an effective measure of genotoxicity potential. However, sampling a single tissue (bone marrow) for a single indicator of genetic damage using the MN assay provides a limited genotoxicity profile. The in vivo alkaline (pH>13) Comet assay, which detects a broad spectrum of DNA damage, can be applied to a variety of rodent tissues following administration of test agents. To determine if the Comet assay is a useful supplement to the in vivo MN assay, a combined test protocol (MN/Comet assay) was conducted in male B6C3F1 mice and F344/N rats using four model genotoxicants: ethyl methanesulfonate (EMS), acrylamide (ACM), cyclophosphamide (CP), and vincristine sulfate (VS). Test compounds were administered on 4 consecutive days at 24-hour intervals (VS was administered to rats for 3 days); animals were euthanized 4 hours after the last administration. All compounds induced significant increases in micronucleated reticulocytes (MN-RET) in the peripheral blood of mice, and all but ACM induced MN-RET in rats. EMS and ACM induced significant increases in DNA damage, measured by the Comet assay, in multiple tissues of mice and rats. CP-induced DNA damage was detected in leukocytes and duodenum cells. VS, a spindle fiber disrupting agent, was negative in the Comet assay. Based on these results, the MN/Comet assay holds promise for providing more comprehensive assessments of potential genotoxicants, and the National Toxicology Program is presently using this combined protocol in its overall evaluation of the genotoxicity of substances of public health concern.
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.
Forty-one chemicals were tested for their abilities to induce trifluorothymidine resistance in L5178Y mouse lymphoma (MOLY) cells. These chemicals were included in the National Toxicology Program's evaluation of four in vitro short-term toxicity assays for predicting carcinogenicity in the rodent bioassay. Of the 41 chemicals examined for this report, 8 were equivocal in the rodent bioassay, and 7 were questionable in- the MOLY assay. If these chemicals are eliminated from an analysis of concordance, the remaining 26 chemicals lead to a concordance of 69% with a sensitivity of 71%. The specificity could not be determined because only two non-carcinogens were detected.
Lipid-soluble spin labels were incorporated into human lymphocytes and mouse L-cells and the resulting electron spin resonance spectra were compared with spectra obtained from similarly labeled human erythrocytes. Spin labels were found in all subcellular fractions of the nucleated cells that contained membranes. Spinlabeled cells remained viable and capable of replicating in vitro. Electron spin resonance signals from spin-labeled nucleated cells underwent a time-and temperaturedependent decay that was reversed by bathing the cells in K3Fe(CN)6. The demonstration of a relative cell impermeability to ferricyanide, as measured by both colorimetric and radioisotopic label methods, indicated that only spin-labeled molecules in the surface membrane were reactivated when ferricyanide was added to spin-labeled cells after the electron spin resonance signal had decayed.Electron spin resonance (ESR) spectroscopy and spin-label techniques have been used recently to probe the structure of artificial membranes (1, 2) and membranes of erythrocytes (3), neurospora (4), viruses (5), lobster walking nerves (6), sarcoplasmic reticulum (7), and mycoplasma (8). However, information is not available regarding application of spinlabel techniques to studies with intact nucleated mammalian cells. A major problem in the application of ESR techniques to studies of surface membrane phenomena in intact cells is differentiating between labels attached to cell surface and those bound to internal membrane structures.This report describes a technique for differential detection of surface-membrane signals from spin-labeled, intact nucleated mammalian cells. The cellular distribution and spectral characteristics of various lipid-soluble spin labels incorporated in membranes of mouse L-cells and human lymphocytes are described. ESR spectra of these spin-labeled nucleated cells are compared with spectra of spin-labeled human erythrocytes. In addition, factors affecting signal stability and the effect of label incorporation on cell viability and replication are reported. MATERIALS AND METHODSSpin Labels. All spin labels were purchased from Syva Assoc., Palo Alto, Calif. The spin labels used were ( Fig. 1 in ME medium to 50% of the original volume of homogenate, the nuclear pellet was given three additional strokes in the Duall homogenizer and again centrifuged. Supernates were combined and centrifuged at 25,000 X g for 10 min to obtain a mitochondrial fraction (M), and at 48,000 X g for 60 min to obtain a microsomal fraction (P). The final supernate was the soluble fraction (S). N, M, and P fractions were concentrated by suspending them to 50% or 25% of the homogenate's original volume. Relative concentrations of label in each of the subcellular fractions were determined. 66
Experimental data from the testing of 31 chemicals for mutagenicity at the TK locus in L5178Y mouse lymphoma cells are presented and evaluated. If mutagenic activity was not obtained for the chemical added to suspension cultures for 4 hr, then the testing was repeated in the presence of hepatic S9 mix prepared from Aroclor 1254-induced male Fischer 344 rats. Multiple trials were performed for each chemical, and mutagenic treatments were analyzed for the induction of small and large mutant colony populations. Twelve chemicals were not detected as mutagenic, one (ascorbic acid) was questionable, and 18 were evaluated as mutagenic. These results were used in the evaluations presented by Tennant et al. [Science 236:933-941, 1987] in a critical comparison of four in vitro genotoxicity assays with rodent carcinogenicity results. The mouse lymphoma assay results were in general agreement with the carcinogenicity studies. Discordant evaluations with respect to carcinogenicity (four false negatives and six false positives) were discussed from the standpoint of how the predictive performance of the in vitro mutation assay might be improved.
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