Overexpression of human H-ras transgene is responsible for tumors induced by chemical carcinogens in mice

Level of human prototype H-ras transgene expression in tumors induced by chemical carcinogens (N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea) was analyzed in human H-ras transgenic mice (CB6F1-TgrasH2 Jic mice). All forestomach tumors examined revealed about 2-fold overexpression of the human H-ras transgene with or without point mutation at codon 12 or codon 61. However, endogenous mouse H- and K-ras genes exhibited neither point mutation nor overexpression. These results suggested that increased levels of ras gene products in the cell played an important role in facilitating chemical carcinogenesis in transgenic mice.     

An evaluation of 6 short-term tests for detecting organic chemical carcinogens.

A number of tests have been described which are thought to be capable of identifying carcinogens without using the actual induction of cancer as an endpoint. This study compared the performance of 6 such tests on a selection of 120 organic chemicals. The tests studies were: (1) mutation of Salmonella typhimurium; (2) cell transformation; (3) degranulation of endoplasmic reticulum; (4) sebaceous gland suppression; (5) tetrazolium reduction and (6) subcutaneous implant. A further 4 tests were examined briefly, but were not included in the complete evaluation. The chemicals were classified into carcinogens (58) and non-carcinogens (62) on the basis of published experimental data, and into 1 of 4 broad chemical classes. There was considerable variation between tests in their ability to predict carcinogenicity, with the cell-transformation test and the bacterial-mutation test being the most accurate (94% and 93% accurate respectively). These 2 tests were considered to be of general use in screening, since they were clearly more accurate than the others. Statistical consideration of various combinations of these tests showed that the use of cell transformation and bacterial mutation together, provide an advantage over the use of either test alone. The inclusion of the other 4 tests in a screening battery predictably resulted in a great increase in overall inaccuracy and loss of discrimination, even though the detection of carcinogens is improved. All the tests were shown to generate both false positive and false negative results, a situation which may be controlled by the use, where possible, of appropriate chemical-class controls, to identify the test which is optimal for the class of chemical under test. Structural analogy may have a part to play in the rapid detection of environmental carcinogens, and some general guidelines for its use are given.     

Protein adducts in the molecular dosimetry of chemical carcinogens

Genotoxic carcinogens form covalent bonds with proteins as well as with DNA. The adducts which result are useful for assessing exposure to the carcinogen, determining inter-individual differences in metabolism and other carcinogen processing, and perhaps in risk assessment. This commentary reviews the development of molecular dosimetry based on protein adducts and describes some of the principles involved. Also described are studies of the binding of bulky lipophilic carcinogens to proteins, which clearly indicate that a high degree of specificity is characteristic of many carcinogen-protein interactions. Studies which have been conducted with human populations are summarized and some proposals for future studies are made.     

Invasive tumors derived from xenotransplanted, immortalized human cells after in vivo exposure to chemical carcinogens

Several chemicals that are found in cigarette smoke or dieseloil engine exhausts, such as benzo[a]pyrene (B[a]P) and 1, 6-dinitropyrene(DNP) are carcinogenic in experimental animal models. In thepresent study, we have exposed in vivo the xenotransplantedimmortalized human bronchial epithelial cell line BEAS-2B tothe ultimate carcinogen of B[a]P, benzo[a]pyrene diolepoxide(BPDE), to DNP or to the benzo[e]pyrene, a less active compoundthat has tumor-promoting abilities in mouse skin carcinogenesisbioassays. All three compounds were administered using slow-releasebeeswax pellets. After a 6 month exposure, BPDE produced twotumors in seven transplants, four tumors were seen in 10 transplantstreated with DNP and one tumor was observed in five trachealgrafts exposed to B[a]P. All the neoplasms were well-differentiatedinvasive adenocarcinomas. Tracheal transplants exposed to beeswaxwithout carcinogen did not show any evidence of neoplastic growth,and their luminal surfaces were lined by a single or doublelayer of cuboidal cells. All lines derived from the adenocarcinomasshowed increased in vitro resistance to serum-induced terminaldifferentiation, gelatinolytic activity, s.c. tumorigenicityand invasive growth in an in vivo assay. When these cell lineswere compared with previously described tumor cell lines derivedfrom xenotransplants exposed to cigarette smoke condensate,it became clear that the latter exhibited a more aggressiveinvasive behavior. Nevertheless treatment with the three chemicalsgave rise to tumor cell lines that exhibited a similar invasivebehavior in vivo, and were able to penetrate early into thewall of the tracheal transplants in which they were seeded.These data indicate that this system based on xenotransplantedbronchial epithelial cells is a very relevant model to identifyhuman carcinogens and to study mechanisms of bronchogenic cancerpathogenesis.     

Role of depurination in mutagenesis by chemical carcinogens

The effect of modifying phi chi 174 viral DNA by the chemical carcinogens beta-propiolactone, N-acetoxyacetylaminofluorene and anti-benzo[a]pyrene diol-epoxide was investigated by transfecting the modified DNA into Escherichia coli spheroplasts. Modification of the DNA in vitro by each of these agents was mutagenic for the phi chi 174 amber mutants am3 and am86. Mutagenicity depended on the induction of the "SOS" response in the host spheroplasts. Heating beta-propiolactone-treated DNA at neutral pH caused strong inactivation such that the number of lethal hits was increased 4-fold. Sucrose gradient analysis showed the induction of alkali-labile sites in the heated DNA. The "nicked circle assay" with double-stranded phi chi 174 DNA showed greater than 70% of these sites to be apurinic sites. Concomitantly with the production of these new sites, a strong increase in the mutation frequency was observed. This mutagenesis also depended upon the induction of the error-prone SOS response in the spheroplasts, as was previously shown to be the case for mutagenesis at putative apurinic sites induced directly by acid-heat treatment. These results suggest that depurination may be of importance to the mechanism of mutagenesis by beta-propiolactone and other carcinogens.     

Induction of different genetic changes by different classes of chemical carcinogens during progression of mouse skin tumors

By analysis of skin tumors from F₁ hybrid mice we demonstrated that the genetic events that occur during tumor progression depend on the type of chemical carcinogenesis protocol used to induce tumor growth. More than 95% of tumors induced by initiation with 7, 12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) exhibited mutations in Ha-ras and trisomy of chromosome 7. Carcinomas induced with multiple DMBA treatments had a lower frequency of alterations on chromosome 7 (50%), but only in tumors with Ha-ras mutations, and had a much wider spectrum of alterations, including trisomy, mitotic recombination, deletion, and gene duplication. Carcinomas induced with multiple N-methyl-N-nitro-N-nitrosoguanidine treatments only rarely exhibited alterations on chromosome 7 (8%), even if they contained mutant Ha-ras. More frequent numerical alterations of chromosome 11 were also seen in TPA-promoted tumors (23%) than in tumors induced by multiple carcinogen treatments (8%). These results show that postinitiation events are nonrandom and fit a model in which promoting agents induce numerical chromosomal alterations but in which mutagens cause more directed mutational events. ©1994 Wiley-Liss, Inc.     

Characterization of a quantitative assay for the in vitro transformation of normal human diploid fibroblasts to anchorage independence by chemical carcinogens

We have characterized an assay for the quantitative measurement of the frequency of conversion to anchorage-independent growth of N-acetoxy-2-acetylaminofluorene-treated normal human diploid fibroblasts. We investigated the effects of the following parameters on the absolute number and on the frequency of anchorage-independent colonies scored: (a) the number of cells seeded per dish; (b) the type of posttreatment medium; (c) the number of population doublings allowed posttreatment prior to seeding in suspension; and (d) the carcinogen dose. The assay was linear over the range of 1.9 X 10(3) to 3.8 X 10(4) cells seeded per 6-mm dish for both total colonies scored and the induced frequency of anchorage-independent growth. The medium used posttreatment affected both the frequency and the kinetics of appearance of the anchorage-independent phenotype. The number of population doublings and the number of days allowed posttreatment prior to assaying for anchorage-independent growth potential also influenced the frequency of recovery of this phenotype. Under standardized conditions, the assay yielded a dose-response relationship for transformation to anchorage independence over the concentration range of 0 to 10 microM N-acetoxy-2-acetylaminofluorene.     

Interaction of bulky chemical carcinogens with DNA in chromatin

DNA damage is an important effect of treatment of cells andorganisms with chemical carcinogens. Although much has beenlearned from in vitro studies of the reaction of carcinogenswith purified DNA, in vivo the DNA is associated with a varietyof histone and non-histone proteins in a complex and dynamicstructure known as chromatin. The covalent interactions of bulkychemical carcinogens with chromatin are reviewed. Differencesfrom bulk genomic DNA in adduct density are found in replicating,transcribing and nuclear matrix-bound DNA regions, and betweenDNA in nucleosome cores and linker DNA regions. These differencesrange from 2- to 3-fold for linker versus core, to