Cell Transformation Assay Research Articles

Assessment of chemical carcinogen-induced transforming activity using BALB/c-3T3 cells

The BALB/c-3T3 cell transformation assay evaluates the morphologic transforming potential of test chemicals using cultured mammalian cells as targets. The assay is semiquantitative and employs a contact-inhibited clone of cells derived from the original BALB/c-3T3 mouse cell line. Transforming activity, or a focus, is recognized as a dense layer of morphologically altered cells superimposed on a monolayer of the normal cell phenotype. The induction of transforming activity in a standard assay has been reported to correlate well with the carcinogenicity of many test chemicals in rodent bioassay; however, the standard assay does not detect carcinogens of all chemical classes. This report describes an operational protocol for the standard BALB/c-3T3 cell transformation assay. In addition, it provides assay acceptance criteria and a discussion of a method to evaluate transforming activity data.

Mammalian cell transformation assays: What role can they play in the identification of carcinogens?

Mammalian cell transformation assays: What role can they play in the identification of carcinogens?

Initiation of in vitro cell transformation by formaldehyde and acetaldehyde as measured by attachment-independent survival of cells in aggregates.

The ability of formaldehyde and acetaldehyde to initiate transformation of a rat kidney cell line has been studied using a newly developed two-stage in vitro cell transformation assay. The assay is based on measurements of attachment-independent survival of cells in aggregates. Short treatment with non-cytotoxic doses of formaldehyde and acetaldehyde did not affect survival of the cells in the aggregate assay system. However, when the aldehyde treatment was followed by exposure of the cells to the tumor promoters TPA and PDD, a considerable increase in the number of viable cells was observed. On a molar basis, formaldehyde was about 100 times more potent than acetaldehyde in initiation of cell transformation. The data showed that cells derived from aggregates of cultures treated with formaldehyde or acetaldehyde followed by exposure to TPA possessed a considerably higher ability to form colonies in soft agar than untreated control cells.

Recognising cardiac arrest and providing basic life support

The wild-type E6 and E7 genes of human papillomavirus type 16 (HPV16) can cooperate to immortalize normal human keratinocytes in culture. The E6 open reading frame of HPV16 and other HPV types highly associated with cervical cancer has the potential of encoding both full-length E6 and two truncated E6* proteins, the latter being generated via splicing within the E6 open reading frame portion of the E6-E7 polycistronic transcript. Those types, such as HPV6, that are infrequently associated with cervical carcinoma lack the splice site and encode only a full-length E6. We have now found that, in addition to cooperating with E7 to immortalize keratinocytes, HPV16 E6 can induce anchorage-independent growth in NIH 3T3 cells and trans-activate the adenovirus E2 promoter. HPV6 E6 was also able to trans-activate the adenovirus E2 promoter, although it was inactive in both cell transformation assays. An HPV16 splice site mutant which expressed only the full-length HPV16 E6 was active in all three assays, indicating that the E6* proteins are not required for these activities. The plasmid which encodes the E6* proteins was inactive and did not potentiate the activity of the HPV16 splice site mutant. The mutation that prevented splicing in E6-E7 mRNA severely reduced the level of E7 protein and increased E6 protein. Taken together, the results suggest that the primary function of the splice within E6 is to facilitate the translation of E7 and reduce translation of full-length E6, rather than to generate biologically active E6* proteins. Images

Genotoxicity of cocoa in a series of short-term assays

Cocoa powder was evaluated for genotoxic activity and found to be inactive in the Ames assay, the mouse lymphoma assay, cytogenetic assays measuring chromosome breakage and SCE, and a cell transformation assay using Balb/c-3T3 cells. Although pure theobromine has been shown to be active in some of these test procedures, the levels of this methylxanthine present in cocoa powder were insufficient to elicit responses in this battery of tests.

Effect of mouse skin tumor promoters upon [3H]uridine exchange and focus formation in cultures of C3H/10T1/2 mouse fibroblasts.

The abilities of 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (4-O-methyl-TPA) and mezerein to promote the process of transformation were evaluated in cultures of C3H/10T1/2 mouse embryo fibroblasts treated with N-methyl-N'-nitro-N-nitrosoguanidine. Mezerein was found to be as potent as the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) for the promotion of focus formation, eliciting a promotion response at concentrations that ranged from 100 to 2500 ng/ml. 4-O-Methyl-TPA (25-2500 ng/ml) did not promote focus formation, but was mitogenic for confluent cultures. The effects of promoting and non-promoting compounds upon intercellular communication were then evaluated to determine if a rapid assay for the inhibition of communication might serve as a surrogate for the relatively long term, labor-intensive cell transformation assay. Inhibited intercellular communication, as measured by inhibition of [3H]uridine exchange between cells, appeared to correlate with the ability of phorbol related compounds to promote transformation. However, the potent promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin did not inhibit [3H]uridine transfer. Inhibition of intercellular communication may thus be diagnostic of the promoting potential of phorbol-related compounds in C3H/10T1/2 cultures, but may not be an appropriate endpoint for the study of carcinogenic dioxins.

Welding fumes and chromium compounds in cell transformation assays

Fumes generated from mild steel and stainless steel welding were collected on paper filters and tested in the BHK and SHE cell transformation assays. Fumes from the manual metal arc welding of stainless steel (MMA/SS) had a toxic and transforming effect attributable to their Cr(VI) content. The fumes from metal inert gas stainless steel (MIG/SS) welding also had a toxic effect but this was 2-3 times greater than that expected from their soluble Cr(VI) content based on the activity of soluble Cr(VI) from pure chromium compounds. When collected in an impinger, the fumes from MIG/SS were found to contain approximately 10 times the soluble Cr(VI) content of samples collected on filters. This additional Cr(VI), when collected in a water impinger, also exhibited a greater toxicity compared with that found for the additional Cr(VI) collected in an impinger filled with growth medium. This comparison implies the presence of a short-lived biologically active Cr(VI) species usually lost in conventional sampling techniques. It also implies that there is a detoxification step associated with the formation of Cr(VI) organic complexes. Relatively insoluble Cr(VI) compounds showed a higher toxic and transforming effect in the BHK assay than could be ascribed to the soluble Cr(VI) content of the medium, indicating the importance of phagocytosis as a pathway for the uptake of Cr(VI) and other toxic substances from particulates.

Balb c/3T3 cell transformation response to extracts of organic air samples as seen by their survival in aggregate form

Extracts of organic matter from samples of airborne particulate matter have been shown to possess components capable of transforming mammalian cells. This study was done to determine if Balb c/3T3 cells exposed to extracts of air samples could, unlike their normal counterparts, in the absence of a surface for attachment, divide on agar to form aggregates, and if these cells would demonstrate a dose-response phenomenon. Untreated and solvent treated control cells failed to form large aggregates and showed a decline in viable cell number over a 6-day period. Cells treated with either cyclohexane or acetone extracts of airborne particulate matter showed a dose-response increase in cell number along with the formation of progressively larger aggregates, findings similar to those seen with the positive, benzo[ a]pyrene (BaP), control. Furthermore, these findings are in direct agreement with those in the simultaneously performed standard cell transformation assay which required 21 days to perform. Results show that survival in aggregate form is a rapid in vitro test system capable of detecting potentially carcinogenic activity in complex environmental mixtures.

In vitro transformation of [formula omitted] cells by chlorinated ethanes and ethylenes

Eight chlorinated ethanes and 3 chlorinated ethylenes were tested in the BALB c -3T3 cell transformation assay. Under the conditions of the assay, vinyl chloride and 1,1,1-trichloroethane induced a clear positive transformation response while 1,1,2-trichloroethane and trichloroethylene were weakly positive. Chloroethane, 1,1- and 1,2-dichloroethane, 1,1,1,2- and 1,1,2,2-tetrachloroethane, hexachloroethane and tetrachloroethylene were all negative in the assay conducted in the absence of an exogenous metabolic activation system. These results suggest that the BALB c -3T3 cells possess capability to activate some, but not all, of the chlorinated hydrocarbons which exhibit species specificity in producing carcinogenicity in mice but not in rats.

Effect of process distillation on mutagenicity and cell-transformation activity of solvent-refined, coal-derived liquids

Blended SRC-II process streams, representing a full boiling range distillate material, were fractionally distilled into non-overlapping 50 °F cuts with boiling points between 300 and 850 °F. Another set of 18 distillate cuts were obtained with boiling points ranging between 138 and 1055 °F. Distillate cuts were assayed for mutagenic activity using the histidine reversion assay with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, as well as for mammalian-cell transformation activity in the Syrian hamster embryo test, and DNA damage in the prophage induction assay. Samples were also separated into chemical class fractions by alumina column chromatography and analysed by high resolution gas chromatography so that the chemical composition of the cuts could be related to their relative activity in the different assays. In the mammalian cell transformation and microbial mutagenicity assays, significant activity was found almost exclusively in distillate cuts with components boiling > 700 °F, with the highest activity in the transformation assay observed for cuts > 800 °F. All of the distillate cuts showed increased levels of DNA damage as expressed by lambda prophage induction in Escherichia coli 8177. However, the greatest activity was associated with distillate cuts with boiling points in the 800 °F + range. Chemical analysis of the 50 °F distillate cuts showed a variety of polycyclic aromatic hydrocarbons (PAH) and amino-PAH compounds to be present in the distillate cuts boiling > 700 °F and essentially absent from cuts boiling < 700 °F. The sample set of non-overlapping (50 °F) cuts were reblended according to the proportions of each cut found in the original blend material. These reblended composites were then assayed to compare their activity with that predicted from the activities of the component distillate cuts. The reblending experiments indicated the microbial mutagenicity response was essentially additive. Mammalian cell transformation activities were non-additive, indicating a compositional effect on the expression of transforming agents in the complex mixture.

Cytotoxicity and negligible genotoxicity of borax and borax ores to cultured mammalian cells.

The cytotoxicity and genotoxicity of refined borax and borax ores were studied in cultured mammalian cells. In V79 Chinese hamster cells, C3H/10T1/2 mouse embryo fibroblasts, and diploid human foreskin fibroblasts, crude borax ore, kernite ore, and refined borax were all cytotoxic. The lowest concentrations at which cytotoxicity was observed were 0.02 mg/ml and 0.1 mg/ml for borax ore in C3H/10T1/2 and human fibroblasts, respectively, 0.2 mg/ml for kernite ore in both cell types, and 0.1 mg/ml for refined borax in both C3H/10T1/2 and human fibroblasts. The cytotoxicity was dose dependent above these concentrations. The concentrations of borax ore, kernite ore, and refined borax that reduced the relative plating efficiency to 50% were approximately 3.2, 1.6, and 0.8 mg/ml, respectively, in human fibroblasts and were 0.8 mg/ml for all three substances in C3H/10T1/2 cells. All three borax samples were not significantly mutagenic in assays for mutation to ouabain resistance in human fibroblasts an C3H/10T1/2 cells and were at most only weakly mutagenic in an assay for mutation to 8-azaguanine resistance in V79 Chinese hamster cells. Refined borax did not induce neoplastic transformation in C3H/10T1/2 cells. Crude borax ore and kernite ore induced weak transformation that was not dose-dependent and was not reproducible in another experiment. Therefore, borax and its ores are cytotoxic to mammalian cells at high (mg/ml) concentrations and are at most weakly mutagenic but not significantly oncogenic as measured in a cell transformation assay.

S-9 Metabolic activation enhances aflatoxin-mediated transformation of C3H/10T 1/2 cells

The use of a rat liver S-9 metabolic activation system to enhance aflatoxin B 1-mediated transformation of C3H/10T 1/2 cells was studied. Under conditions of metabolic activation, the cytotoxicity of Aflatoxin B 1 (AFB 1) was increased approximately 10-fold over that observed in the absence of activation. Similarly, activation increased the transformation of these cells treated with 1 μg/ml AFB 1 greater than 10-fold when cells were treated in the absence of activation with 1 μg/ml AFB 1 for 3 hr, and four-to-fivefold over the transformation observed when cells were treated for 48 hr with 1 μg/ml AFB 1. This same activation procedure also induced the transformation of these cells treated with 10 and 20 μg/ml cyclophosphamide, whereas no transformed foci were induced by these concentrations of cyclophosphamide in the absence of activation. The use of S-9 metabolic activation greatly increased the sensitivity of AFB 1-mediated transformation of C3H/10T 1/2 cells, and should expand the range of chemical carcinogens, requiring metabolic activation, in particular mycotoxins related to AFB 1, that can be effectively detected and studied in the C3H/10T 1/2 cell transformation assay.

In vitro cell transformation by air pollutants

The present study demonstrates that extracts of air pollution and emission from wood combustion contain relatively high concentrations of substances able to induce morphological transformation of Syrian hamster embryo cells. Samples corresponding to 1–2 m 3 ambient air from the center of Oslo and combustion from 1.5 mg burned wood per mL of incubation medium were shown to give response in the present transformation assay. The highest transforming activity following fractionation of emissions from wood burning was obtained in a fraction containing polar derivatives of polyaromatic hydrocarbons including aza-arenes and possibly aromatic amines and highly oxygenated compounds. The Syrian hamster embryo cell transformation assay has been found to respond to substaances with low or no genotoxic activity such as metal salts and tumor promoters, and may thus be a promising supplement to tests measuring genotoxic activity in studies of air pollutants.

Characterization of an unscheduled DNA synthesis assay with Syrian hamster embryo cells

The Syrian hamster embryo cell transformation assay is widely used for studies of carcinogenesis. The characterization of an unscheduled DNA synthesis (UDS) assay for these cells in reported, Benzol[α]pyrene, aflatoxin B 1 and UV light induced UDS in the cells in a dose-dependent manner without exogenous metabolic activity. Nitrosopiperidine induced UDS as well as gene mutations and cell transformations only in the presence of an exogeneous metabolic activation system. The utility of this UDS assay with these cells is discussed.

A technically simple "non-lethal" vital staining procedure for viral plaque and cell transformation assays. Brief report.

The widely used tetrazolium dye, MTT, has several advantages as a vital stain in the identification of viral plaques. First, since the yellow colored dye MTT stains live cells dark blue, viral plaques can be counted without removal of the phenol red containing agar overlay. Secondly, the high contrast between live and dead cells afforded by MTT permits one to readily detect small plaques at an early time after infection. Thirdly, since cells intensely stained with MTT remain viable, MTT vital staining can be used to isolate cells which are either transformed or resistant to viruses. Thus, MTT vital staining should be useful in several types of viral plaque assays.

Bacterial Mutagenesis and Cell Transformation Assays of Pyrvinium Pamoate (Povan), An Antiparasitic Agent

Mutagenicity of pyrvinium pamoate formulations was determined in various Salmonella typhimurium strains in a well test and plate incorporation assays in the presence and absence of liver homogenate (S9) from Aroclor-1254-induced rats or from normal human liver. Strains TA98 and TA100 were reverted to histidine independence by bulk pyrvinium pamoate over a concentration range of 500 to 2500 μg/plate, particularly in the presence of rat S9. Purified pyrvinium pamoate was mutagenic over a concentration range of 5.0 to 25 μg/plate in the presence of both rat and human liver S9. Urine concentrates from human subjects given 10 mg/kg and mice given drug up to 100 mg/kg were not significantly mutagenic even after S9 activation and/or β-glucuronidase-sulfatase treatment. These results confirm earlier observations of mutagenic activity in Salmonella, but, unlike several antiparasitic agents, mutagenic metabolites do not appear in urine. In vitro mouse cell (C3H10T1/2) transformation assays of pyrvinium pamoate were negative. The effects of pyrvinium pamoate in Salmonella are not paralleled by effects in mammalian cell systems. In the absence of quantitative human population data for risk assessments, the evidence of carcinogenic potential of Povan is not defined.

Effects of diazepam in mutation test systems in vitro and in the BHK 21 cell transformation assay

Compounds of various chemical classes were comparatively assayed in the Ames reversion test with his− S. typhimurium strains TA1535, TA1537, TA1538, TA98, TA100 and, in part, TA97, and in a DNA-repair test with trp- E. coli strains WP2 (repair-proficient), WP67 (uvrA- polA-) and CM871 (uvrA- recA- lexA-). A liquid micromethod procedure for the assessment of the minimal inhibitory concentration (MIC) of test compounds, using the same reagents as the Ames test, was set up and calibrated in its technical details. Other techniques (spot test and treat-and-plate method) were applied to a number of compounds in order to obtain more complete information on their DNA-damaging activity in E. coli.From a qualitative standpoint, the results obtained in the reversion test and in the DNA-repair test (liquid micromethod) were overlapping for 96 (59 positive and 37 negative) out of 135 compounds (71.1%). There was disagreement for 39 compounds (28.9%), 9 of which were positive only in the reversion test (8 requiring metabolic activation and 5 genotoxic in the treat-and-plate method). 30 compounds were positive only in the lethality test, showing a direct DNA-damaging activity, which in half of the cases was completely eliminated by S9 mix. Although the experimental protocol intentionally included several compounds already reported as nonmutagenic carcinogens or as noncarcinogenic mutagens, the overall accuracy was 64.5% for the reversion test and 72.4% for the DNA-repair test, as evaluated for 75 compounds classified according to their carcinogenic activity.Quantitation of results was obtained in the Ames test by relating the net number of revertants to nmoles of compound and in the DNA-repair test by means of a formula relating the difference and ratio of MICs in repair-proficient and -deficient bacteria to nmoles of compound. Following these criteria, the genotoxic potency varied over a 4.5 × 107-fold range among compounds positive in the reversion test and over a 6 × 109-fold range among compounds damaging E. coli DNA. The genotoxic potencies in the two bacterial systems were correlated within the majority of the chemical classes under scrutiny.

A comparison of the predictive values of the Salmonella/microsome mutation and BHK21 cell transformation assays in relation to dyestuffs and similar materials

A group of 22 dye-related compounds were selected for testing in two short-term predictive tests for carcinogenicity. The group of compounds was made up of nine established animal carcinogens and 13 chemicals for which there was substantial evidence of non-carcinogenicity. The materials were coded and used to assess the predictive value of the Salmonella/microsome reverse mutation assay and the BHK21 cell transformation test. The overall predictive value with these compounds obtained for the Salmonella microsome reverse mutation assay was 86% and it is concluded that because of the good predictive value and the relative ease of experimental procedure, the Salmonella mutation assay is a useful first step in any proposed series of toxicological bioassays for the identification of genotoxic agents in the dyestuffs industry. The cell transformation test on the other hand was difficult to conduct and interpret. The interpretation of the coded data as judged by the IRI scientists was that of the 22 'unknown' compounds, eight results were judged to be correct, six were wrong and seven were doubtful. However, when the same data were re-evaluated uncoded by ICI staff, 15 results were judged to be correct and six were wrong. These results serve to exemplify the difficulties encountered with cell transformation assays and it is concluded that the system should not be used as a routine test for dyestuffs and related compounds.

Interaction of acute transforming retroviruses with murine hematopoietic cells.

There is emerging evidence that several retroviral onc gene products possess functional, structural, and amino acid sequence homology. Rous sarcoma virus (RSV), Abelson-murine leukemia virus (Abelson-MuLV), and Snyder-Thei1en feline sarcoma virus (ST-FeSV) code for related phosphoproteins that have associated tyrosine-specific protein kinase activity (Barbacid et al., 1 980; Witte et al., 1980; Reddy et al., 1983). BALB-, Harvey-, and Kirsten-murine sarcoma viruses (MSVs) are members of another family whose 21,000 dalton transforming proteins possess GDP binding and autophosphorylation activity (Shih et al., 1979; Andersen et al., 1981). Investigation of the diversity of hematopoietic target cells susceptible to neoplastic transformation by specific retroviruses might be expected to provide insights into the relationship of the differentiated state of a cell and its susceptibility to the action of a family of onc genes. As an approach to this question, we took advantage of We availability of hematopoietic cell transformation assay systems to analyze the array of cellular targets whose growth and differentiation could be altered by different retroviral onc genes.KeywordsTerminal Deoxynucleotidyl TransferaseRous Sarcoma VirusCompact ColoniLymphoid Progenitor CellMurine Hematopoietic CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Induction of Neoplastic Transformation and DNA Single-Strand Breaks in C3H/10T1/2 Clone 8 Cells by Polycyclic Hydrocarbons and Alkylating Agents&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

The standard C3H/10T1/2 clone 8 (C3H/10T1/2 CL8) cell transformation assay was tested for its ability to identify a variety of polycyclic hydrocarbons and alkylating agents. Dose-dependent morphologic transformation occurred with benzo[a]pyrene (BaP), 3-methylcholanthrene (MCA), 7,12-dimethylbenz[a]anthracene, BaP-7,8-dihydroxy-7,8-dihydrodiol (BaP-7,8-diol), as well as with the relatively weak in vivo carcinogen benzo[e]pyrene. Dibenz[a,h]anthracene yielded a relatively weak response, whereas anthracene and phenanthrene were negative. In contrast, treatment of C3H/10T1/2 CL8 cells with two directly acting alkylating agents, N-nitroso-N-methylnitroguanidine (MNNG) and styrene oxide, gave no transformation, whereas a third alkylating agent, ethyl methanesulfonate (EMS), gave a weak response. Treatment with MCA (2.5 micrograms/ml) yielded a reproducible positive response and, therefore, served as a positive control for routine use of the C3H/10T1/2 CL8 assay. When cells treated with the hydrocarbons BaP, BaP-7,8-diol, or MCA were analyzed for nonspecific DNA damage (single-strand breaks or alkaline-labile sites) by alkaline elution techniques, little if any DNA damage was observed. In contrast, the alkylating agents MNNG, styrene oxide, and EMS yielded substantial numbers of single-strand breaks.