The use of short-term tests and limited bioassays in carcinogenicity testing.

A brief discussion is given of the use of short-term in vivo and in vitro tests in carcinogenicity testing. Data are presented on the performance of nine such tests and five limited bioassays, as measured in terms of their sensitivity, specificity, accuracy, and predictive value. It is concluded that tests are available which, when used in combinations such as have been proposed by various authors, are capable of correctly identifying carcinogens and noncarcinogens with high confidence. A discussion of the statistics of batteries of tests is given. This is followed by a semiquantitative graphical representation and a discussion of how, on proceeding through a testing scheme such as that proposed by J. H. Weisburger and G. M. Williams (1981, Science 214, 401-407), the cumulative cost and the probability of correctly identifying a carcinogen or a noncarcinogen change as results become available from each of the stages of such a testing scheme.

Fecal steroid 21-dehydroxylase, a potential marker for colorectal cancer.

Eubacterium lentum and phenotypically similar organisms synthesize a steroid 21-dehydroxylase which converts biliary tetrahydrodeoxycorticosterone to pregnanolone. Tetrahydrodeoxycorticosterone, in contrast to pregnanolone, is carcinogenic for hamster embryonic cells (HECT test). In patients with recently diagnosed, untreated sigmoidal or rectal cancer the fecal concentration of 21-dehydroxylating organisms is reduced by more than 99% as compared with age-matched controls. The lack of fecal 21-dehydroxylating organisms, therefore, is a potential marker for the disorder. The role of steroid 21-dehydroxylase in the pathogenesis of colorectal cancer is unknown.

Cell transformation by chemical agents--a review and analysis of the literature. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

The literature on cell transformation by chemical carcinogens has been critically reviewed. This subject is highly relevant to carcinogenesis in vivo, because the phenotypic changes that are collectively referred to as cell transformation usually involve the acquisition of tumorigenicity on inoculation into suitable rodent hosts. The systems chosen for review fall into 3 categories: cell strains (cells with a limited lifespan); cell lines (cells with an unlimited lifespan); and oncogenic viral-chemical interactions involving cells (Fischer rat embryo cells expressing an endogenous retrovirus, mouse embryo cells expressing the AKR leukemia virus, chemical enhancement of a simian adenovirus, SA7 transformation of Syrian hamster or rat embryo cells). Of the entire literature reviewed, 117 papers have been accepted for data abstraction by pre-defined criteria; these include 41 references to cell strains, 40 in cell lines, and 38 in viral-chemical interactions including cells. Because different systems have been reviewed, it would be meaningless to group all the compounds. The overall summary of the systems is as follows (many compounds have been tested in more than one system and, hence, are duplicated in these totals). (Chart: see text) In general, there is a reasonably good correlation between the results of the cell transformation systems and in vivo carcinogenesis. However, the many deficiencies of the EPA Merged Carcinogen List preclude definitive comparisons. Moreover, a number of 'false negatives' were obtained in systems that did not employ external metabolic activation. Further validation of all systems is required, but it seems very probable that several cell transformation systems will become valuable in assaying (with reasonable time and cost) the carcinogenic potential of environmental chemicals.

Comparative neoplastic transformation responses of Balb/3T3 cells, Syrian hamster embryo cells, and Rauscher murine leukemia virus-infected Fischer 344 rat embryo cells to chemical compounds.

This study provides a preliminary comparative evaluation of the responses to a series of 49 chemicals, in in vitro transformation assays, of Balb/3T3 cells, Syrian hamster embryo cells, and Fischer 344 rat embryo cells infected with Rauscher murine leukemia virus. The chemicals assayed included aromatic amines; polycyclic aromatic hydrocarbons; alkylating agents; nitrosamines, hydrazines, and related compounds; heterocyclic compounds; amides, ureas, and acylating agents; inorganic compounds; and hormones. In all three assays 37 of the chemicals were tested. The most uniform test responses were obtained with the polycyclic aromatic hydrocarbons and inorganic compounds With the other groups of chemicals, more variation in response was observed. This study expands the base of information on the potential of these in vitro transformation systems, and the lack of responses with some of the chemicals underscores the need for incorporation of exogenous metabolic activating systems into these assay systems.

Transformation of hamster embryo cells by aromatic amines.

a mammalian cell transformation system that required cryopreserved primary cultures of Syrian golden hamster embryo cells was used in the evaluation of the carcinogenic potential of 46 structurally related aromatic amines. The results generally correlated with those obtained with animal bioassay systems. However, several carcinogenic compounds required the addition of an exogenous metabolic activation system provided by hamster liver S9 homogenate enzymes or cultured hepatocytes to transform the hamster embryo cells.

Greater effectiveness of hepatocyte and liver S9 preparations from hamsters than rat preparations in activating N-nitroso compounds to metabolites mutagenic to Salmonella.

A comparison was made of the ability of liver S9 and hepatocyte preparations from noninbred Syrian golden hamsters and noninbred Sprague-Dawley rats to metabolically activate a number of nitroso compounds in the Salmonella mutagenesis assay. The liver S9 and hepatocyte preparations from hamsters were consistently more effective than were preparations from rats in metabolizing nitrosodimethylamine (NDM), nitrosodiethylamine, nitrosodiallylamine, nitrosopyrrolidine (NP), nitrosomorpholine (NM), nitrosodiethylmethylurea (NDEMU), and nitrosodimethyl-ethylurea (NDMEU) to mutagenic forms. The use of hamster S9 preparations with NP and NM resulted in up to 14 times the number of revertant colonies obtained with rat preparations; in the presence of hamster hepatocytes, up to 32 times the number of revertants were obtained. The S9 preparations from male hamsters not treated with the enzyme inducers phenobarbital and Aroclor 1254 were more effective than were those from female hamsters for activating NP, NM, and NDM, NDEMU and NDMEU, which have been reported to be carcinogens but not mutagens, were mutagenic in the presence of induced liver S9 or hepatocyte preparations from hamsters but not from rats. When tested with any of the S9 or hepatocyte preparations, nitrosodiphenylamine and nitrosomethylaniline, also reported to be carcinogens but not mutagens, gave no mutagenic responses. Nitrosodioctyl-amine, which has been reported to be noncarcinogenic, was also not mutagenic.

Transformation of hamster embryo cells by neutral sterols and bile acids.

Toxicity and cell transformation of Syrian hamster embryo cells in culture by certain neutral sterols and bile acids show interesting trends related to their structures: cholesterol-alpha-epoxide and cholestan-3 beta, 5 alpha, 6 beta-triol were more toxic and induced transformation to these cells, whereas their metabolic precursor, cholesterol, was inactive. The secondary bile acids, lithocholic and deoxycholic acids, were more toxic than their primary bile acid precursors, cholic and chenodeoxycholic acids and transformed the cells. These data suggest that mammalian cell transformation is a useful short-term assay to measure the potential toxicity and carcinogenicity of steroid derivatives.

Metabolic activation by hamster and rat hepatocytes in the Salmonella mutagenicity assay.

Intact and homogenized hepatocytes from untreated or Aroclor 1254-treated male and female noninbred Sprague-Dawley rats and noninbred Syrian golden hamsters were compared for their ability to metabolize chemicals in the Salmonella-mammalian microsome mutagenesis assay. The following chemicals were used: two aromatic amines, 2-amino-anthracene and N-2-fluorenylacetamide; two polycyclic aromatic hydrocarbons, 3-methylcholanthrene and benzo[a]pyrene (BP); and one nitrosamine, diethylnitrosamine (DENA). With one exception, hepatocytes from hamsters were more active than were hepatocytes from rats in the activation of these mutagens. The homogenized preparations from Aroclor 1254-treated rats were slightly more active with BP than was the equivalent hamster preparation. Intact hepatocytes from Aroclor 1254-treated hamsters were more efficient at metabolizing the aromatic amines and DENA, whereas homogenates were more effective with the hydrocarbons. Results were similar with the rat preparations, except that only large quantities of Aroclor 1254-treated intact male rat hepatocytes appeared to activate DENA. These results suggest that, in the choice of an activation system, the kind of chemical being evaluated should be considered.

Correlation between transformation potential and inducible enzyme levels of hamster embryo cells.

When benzo(a)pyrene was used to evaluate the transformability of 129 hamster embryo cell preparations from pooled or individual embryos, approximately 50% of the cultures were transformable. A transformable and a non-transformable cell culture were further tested with other carcinogens (3-methylcholanthrene [MCA], benzyl chloride, ethyl-p-toluenesulfonate, 2-naphthylamine, and aflatoxin B1). The transformable culture responded to all of the carcinogens while the non-transformable culture always gave negative results. Aryl hydrocarbon hydroxylase (AHH) and epoxide hydrase (EH) levels were compared in the two cell cultures using beta-naphthoflavone (BNF), benz(a)anthracene (BA), sodium phenobarbital (PB) or MCA as microsomal enzyme-inducing agents. It was found that AHH levels and the degree of induction following treatment of the cells with BNF or BA were consistently higher in the transformable than in the non-transformable cells following treatment with either BNF, BA, PB or MCA. Inducible AHH and EH levels might, therefore, be useful as predictors of the transformation potential of hamster embryo cell cultures.

Detection of urinary metabolites of 2-acetylaminofluorene by cultured hamster embryo cells and high performance liquid chromatography.

Urine was collected from Sprague-Dawley rats injected intraperitoneally with a suspension of 2-acetylaminofluorene (AAF) or with an inert suspension vehicle. Morphological transformation was observed in clonal cultures of secondary passage hamster embryo cells (HEC) after the addition of AAF-treated rat urine or concentrated urine extracts prepared by incubating the urine with beta-glucuronidase. No morphological alteration was observed in cultures treated with urine (or urine extracts) from rats injected with the suspension vehicle alone. The presence of N-hydroxy- and ring-hydroxylated metabolites of AAF in the AAF-treated concentrated urine extract was confirmed by high-performance liquid chromatography (HPLC).

Effect of fetal bovine serum on 3-methylcholanthrene-induced transformation of hamster cells in vitro.

Eighteen lots of fetal bovine serum were tested for their ability to support clonal growth and 3-methylcholanthrene-induced morphological transformation of hamster embryo cells in vitro. Most of them supported cloning efficiencies of over 11%. However, cloning efficiency alone was an inadequate criterion for selecting serum for transformation studies, since no transformation was observed with some lots, even though their cloning efficiencies were over 16%. This shows the importance of pretesting serum for its ability to support morphological transformation before it is used in mammalian cell carcinogenesis tests.

Use of hamster hepatocytes to metabolize carcinogens in an in vitro bioassay.

Adult Syrian golden hamster hepatocytes were shown to have a high capacity to metabolize carcinogens, such as diethylnitrosamine, 2-nitrofluorene, and 4-aminoazobenzene, to a form that transforms mammalian cells. The addition of hepatocytes to the hamster embryo cell transformation system extends its range of usefulness for detecting carcinogens that require metabolic activation.

Two-stage malignant transformation in hamster embryo cells.

Transformation of primary hamster embryo cells was investigated using 3-methylcholanthrene (MCA), a combination of MCA and 12-O-tetradecanoylphorbol-13-acetate (TPA), and initiation with MCA or dibenz(a,h)anthracene (DBA) followed by promotion with TPA. Evidence for transformation was (a) abnormal cellular morphology, (b) increased lifespan, (c) growth in soft agar, and (d) tumour induction by s.c. inoculation into suckling hamsters.Cells treated with either MCA or MCA+TPA showed the same latent period to morphological transformation, although their tumorigenic potential varied. Cells did not form tumours when TPA was administered 7 days after treatment with either MCA or DBA. However, when administration of TPA was delayed to 27 days after treatment with a transforming dose of MCA or a subthreshold dose of DBA, the cells transformed and produced tumours in hamsters.Our results show that TPA may act as an inhibitor or promoter, depending on the length of time between treatment of the hamster embryo cells with the carcinogen and administration of the TPA. It appears that treatment of cells with TPA before the initiating event is complete inhibits or delays the development of their ability to induce tumours in animals or grow in soft agar. However, with a sufficient interval between the application of the initiating carcinogen and the promoter, transformation occurs, and the ability of cells treated with subthreshold doses of DBA to form tumours is enhanced.

Malignant transformation of cryopreserved early passage syrian golden hamster cells by 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide.

2-(2-Furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2) induced the malignant transformation of secondary cultures of Syrian golden hamster embryo cells prepared from cryopreserved primary cells. Transformed cells grew in semi-solid agar medium and formed sarcomas when inoculated subcutaneously into non-immunosuppressed suckling hamsters.