ABSTRACT
Almost all new drugs must be tested for carcinogenicity at some point during their development, and ultimately, a lifetime in vivo assay, usually in rodents, must be performed. Many in vitro assays of carcinogenicity have been developed for use before short- or long-term in vivo testing in order to remove from the development stream those drugs that are likely to produce tumors in vivo. This review discusses in vitro assays that are required by the International Conference on Harmonization, followed by a discussion of in vitro carcinogenicity assays, which are currently in use, but are not specifically required. The concluding section is devoted to a discussion of high-throughput compatible carcinogenicity screens and potential human cell-based high-throughput compatible screens with reference to future methods in silico.
Subject(s)
Carcinogenicity Tests/methods , Carcinogenicity Tests/trends , Drug Screening Assays, Antitumor/methods , Drug Screening Assays, Antitumor/trends , Mutagenicity Tests/methods , Mutagenicity Tests/trends , Animals , Chemistry, Pharmaceutical , HumansABSTRACT
A new in vitro test for identifying carcinogens is evaluated against a testing database of 100 chemicals including the following groups: steroids, antineoplastics, PCBs, dioxins, alkyl halides, aromatic amines, nitrogen heterocycles, polyaromatic hydrocarbons, mustards, and benzodioxoles. The assay uses focus formation in a stable, BPV-1-DNA-carrying C3H/10T 1/2 mouse embryo fibroblast cell line (T1), which does not require transfection, infection with virus, or isolation of primary cells from animals. For this group of chemicals, the T1 assay correctly predicted the rodent carcinogenicity or noncarcinogenicity of 77% of the chemicals for which carcinogenicity is reported. Based on published data the bacterial mutagenicity assay would have correctly predicted carcinogenicity or noncarcinogenicity of 53% of the chemicals. The Syrian hamster embryo test would have correctly predicted carcinogenicity or noncarcinogenicity of 61% of the chemicals. We also demonstrate dose--response relationships for two of the chemicals. We report the responses of T1 cells to the group of chemicals used in the International Life Sciences Institute's program for screening of alternative methods of predicting carcinogenicity.
Subject(s)
Bovine papillomavirus 1/genetics , Carcinogenicity Tests/methods , Cell Line/drug effects , Animals , Cell Line/virology , Dose-Response Relationship, Drug , Mice , Predictive Value of TestsABSTRACT
A new in vitro test for predicting rodent carcinogenicity is evaluated against a testing database of 64 chemicals including both genotoxic and nongenotoxic carcinogens and carcinogens that normally require addition of an S-9 microsomal fraction for detection in the bacterial mutagenicity assay. The assay uses focus formation in a stable, bovine papillomavirus type 1 (BPV-1) DNA carrying C3H/10T(1/2) mouse embryo fibroblast cell line (T1) that does not require transfection, infection with virus, isolation of primary cells from animals, or addition of a microsomal fraction. Of a total database of 64 compounds, 92% of the carcinogens, promoters, or noncarcinogens were correctly predicted. Based on previously reported results, the test of bacterial mutagenicity would have correctly predicted 58% of carcinogens, promoters or noncarcinogens and the Syrian hamster embryo test would have correctly predicted 87% of carcinogens, promoters, or noncarcinogens of this database. Of carcinogens that normally require addition of an S-9 fraction, T1 cells correctly predicted rodent carcinogenicity of polyaromatic hydrocarbons, aflatoxins, azo-compounds, nitrosamines, and hydrazine without the addition of an S-9 fraction. Of nongenotoxic carcinogens, T1 cells correctly predicted diethylstilbestroel, diethylhexylphthalate, acetamides, alkyl halides, ethyl carbamate, and phorbol ester tumour promoters.
Subject(s)
Bovine papillomavirus 1/genetics , Carcinogens/toxicity , DNA, Viral/drug effects , Animals , Carcinogenicity Tests , Cattle , Cell Line , Coculture Techniques , Cricetinae , Mice , Mice, Inbred C3H , Reproducibility of ResultsABSTRACT
Tumor promoters such as phorbol esters, teleocidin and okadaic acid increase the numbers of multilayered, transformed foci produced by BPV DNA-transfected C3H/10T1/2 cells. We questioned whether arsenic and chromium, which are known human carcinogens also enhance transformation of BPV DNA-transfected C3H/10T1/2 cells. Cr(III) potassium sulfate at 100 microM enhanced transformation by 1.4-fold, but Cr(VI) as potassium chromate did not enhance transformation, although toxicity of potassium chromate may have prevented enhancement of transformation. Sodium arsenite (As(III) at 5 microM and sodium arsenate (As(V)) at 25 microM both enhanced neoplastic transformation by 6-fold. By comparison, in previous studies, sodium orthovanadate (V(IV)) or vanadyl sulfate (V(IV)) at 4 microM enhanced numbers of transformed foci by 25-50-fold. The comparatively strong enhancement of transformation by vanadium and phorbol esters suggests that neoplastic transformation may occur by mechanisms that are common to these compounds including alteration of tyrosine phosphorylation.
Subject(s)
Arsenates/toxicity , Arsenites/toxicity , Bovine papillomavirus 1/genetics , Carcinogens/toxicity , Cell Transformation, Neoplastic/drug effects , Chromium Compounds/toxicity , Potassium Compounds/toxicity , Sodium Compounds/toxicity , Sulfates/toxicity , Animals , Cattle , Cell Line , Chromates/toxicity , DNA, Viral , Drug Synergism , Humans , Mice , Mice, Inbred C3H , Transfection , Vanadates/pharmacology , Vanadium Compounds/pharmacologyABSTRACT
Bovine papillomavirus (BPV) DNA-transfected C3H/10T1/2 cells respond to tumor promoters by enhanced production of transformed foci. Vanadate, a suspected carcinogen, is a mitogen, generates active oxygen species and alters phosphorylation of proteins. We investigated whether vanadate would enhance transformation of BPV DNA-transfected C3H/10T1/2 cells. Transformed foci in BPV DNA transfected C3H/10T1/2 cells exposed continuously to vanadate for 21 days increased in a dose-dependent manner to 50-fold at 4 microM vanadate. This increase was not due to enhanced uptake of BPV DNA post transfection. Neither catalase nor superoxide dismutase inhibited the vanadate-mediated increase in transformed foci but this does not necessarily rule out the involvement of intracellular active oxygen species. At vanadate concentrations greater than 6 microM, cells lost adherence to the Petri plates. We conclude that vanadate is capable of enhancing BPV DNA-mediated cell transformation. Possible mechanisms may involve active oxygen species or altered patterns of protein phosphorylation.
Subject(s)
Cell Transformation, Neoplastic , Cell Transformation, Viral/drug effects , DNA, Viral , Papillomaviridae/genetics , Tumor Virus Infections , Vanadates/pharmacology , Animals , Catalase/pharmacology , Cell Line , Mice , Mice, Inbred C3H , Superoxide Dismutase/pharmacology , TransfectionABSTRACT
Rabbit antisera to newt (T. cristatus) globin were produced by repeated injections of globin and antiglobin antibodies purified by chromatography on globin-Sepharose 4B. Ouchterlony and SDS PAGE analysis indicated that the material eluted from the affinity column was rabbit IgG. The antiglobin antibodies tested by immunodiffusion and ELISA cross-reacted with native hemoglobin and globin from T. cristatus and to varying extents with globins of N. viridescens, R. pipiens and X. laevis, but not with human globin. The degree of cross-reactivity appeared to parallel the evolutionary relatedness of these species, suggesting common antigenic determinants among globins of various vertebrate species.
