Application of the Improved BALB/c 3T3 Cell Transformation Assay to the Examination of the Initiating and Promoting Activities of Chemicals: The Second Inter-laboratory Collaborative Study by the Non-genotoxic Carcinogen Study Group of Japan

Abstract

The Non-genotoxic Carcinogen Study Group in the Environmental Mutagen Society of Japan organised the second step of the inter-laboratory collaborative study on one-stage and two-stage cell transformation assays employing BALB/c 3T3 cells, with the objective of confirming whether the respective laboratories could independently produce results relevant to initiation or promotion. The method was modified to use a medium consisting of DMEM/F12 supplemented with 2% fetal bovine serum and a mixture of insulin, transferrin, ethanolamine and sodium selenite, at the stationary phase of cell growth. Seventeen laboratories collaborated in this study, and each chemical was tested by three to five laboratories. Comparison between the one-stage and two-stage assays revealed that the latter method would be beneficial in the screening of chemicals. In the test for initiating activity with the two-stage assay (post-treated with 0.1μg/ml 12-O-tetradecanoylphorbol-13-acetate), the relevant test laboratories all obtained positive results for benzo[a]pyrene and methylmethane sulphonate, and negative results for phenanthrene. Of those laboratories assigned phenacetin for the initiation phase, two returned positive results and two returned negative results, where the latter laboratories tested up to one dose lower than the maximum dose used by the former laboratories. In the exploration of promoting activity with the two-stage assay (pretreated with 0.2μg/ml 3-methylcholanthrene), the relevant test laboratories obtained positive results for mezerein, sodium orthovanadate and TGF-β1, and negative results for anthralin, phenacetin and phorbol. Two results returned for phorbol 12,13-didecanoate were positive, but one result was negative — again, the maximum dose to achieve the latter result was lower than that which produced the former results. These results suggest that this modified assay method is relevant, reproducible and transferable, provided that dosing issues, such as the determination of the maximum dose, are adequately considered. The application of this two-stage assay for screening the initiating and promoting potential of chemicals is recommended for consideration by other research groups and regulatory authorities.

Mechanisms of Chemical Carcinogenesis in the Kidneys

“…These projects include the EU funded ACuteTox [89,90], Predictomics [91] and carcinoGENOMICS [92], programs, which were implemented to assess the use of cell culture based organ-specific toxicity screening platforms, thereby reducing the animal burden involved in that aspect of REACH implementation. Alternative methods assessing the physiological impact of carcinogen exposure are currently being investigated in tandem [93,94], such as deciliation following carcinogen exposure [92], or colony formation using the cell transformation assay (CTA) [95,96]. The scale of these efforts highlights the gravity of the threat posed to the kidney by carcinogenic substances, and the seriousness with which these threats should be taken.…”

Detection of Cell Carcinogenic Transformation by a Quadruplex DNA Binding Fluorescent Probe

“…The formation of foci is an important hallmark of carcinogenesis [41] , [42] . Thus, the model showing distinct frequency of foci formation was established [28] , [31] , [32] . Foci appeared in the groups that were treated with high MCA doses and long culture periods, and the foci increased not only in size but also in numbers ( Figure 3a ).…”

“…Cells were maintained in culture for 2–5 weeks, with medium change twice a week [29] , [31] . The scoring of foci was performed according to the established guidelines [28] , [31] , [32] . Only foci classified as type III which featured characteristics such as basophilic, dense multilayered, cells randomly orientated at the focus edge, invasion into the monolayer, and spindle-shaped, were scored as positive and counted.…”

The Use ofIn VitroSystems to Assess Cancer Mechanisms and the Carcinogenic Potential of Chemicals

“…Based on the performances of the three cell transformation assays evaluated in the OECD report, the reliability of these systems was considered satisfactory for the screening of chemicals with unknown carcinogenicity (40). A modified protocol for the detection of the initiating and promoting activities of chemicals by using the 3T3 cell transformation assay, has recently been described (43). The results of the interlaboratory study were promising, and this two-stage model could potentially be used for chemical screening in the future.…”