Transformation of BALB/c-3T3 Cells: IV. Rank-Ordered Potency of 24 Chemical Responses Detected in a Sensitive New Assay Procedure

Abstract

This report introduces an improved method of detecting chemical-induced morphological transformation of A-31-1-13 BALB/c-3T3 cells. The new procedure uses an increased target cell population to assess chemical-induced damage by increasing the initial seeding density and by delaying the initiation time of chemical treatment. Furthermore, a newly developed co-culture clonal survival assay was used to select chemical doses for the transformation assay. This assay measured the relative cloning efficiency (RCE) of chemical treatments in high-density cell cultures. In addition, transformation assay sensitivity was enhanced through the use of improved methods to solubilize many chemicals. From a group of 24 chemicals tested in at least two trials, clear evidence of chemical-induced transformation was detected for 12 chemicals (aphidicolin, barium chloride-2H2O, 5-bromo-2'-deoxyuridine, C.I. direct blue 15, trans-cinnamaldehyde, cytosine arabinoside, diphenylnitrosamine, manganese sulfate-H2O, 2-mercaptobenzimidazole, mezerein, riddelliine, and 2,6-xylidine); 2 chemicals had equivocal activity [C.I. direct blue 218 and mono(2-ethylhexyl)phthalate], 9 chemicals were inactive [carisoprodol, chloramphenicol sodium succinate, 4-chloro-2-nitroaniline, C.I. acid red 114, isobutyraldehyde, mono(2-ethylhexyl)adipate, sodium fluoride, and 12-O-tetradecanoylphorbol-13-acetate), and 1 chemical had an indeterminate response (2,6-dinitrotoluene). All positive responses were detected in the absence of an exogenous activation system and exhibited significant activity at two or more consecutive doses. This report also presents a mathematical method that uses t-statistics for rank-ordering the potency of chemical-induced transformation responses. This model detects sensitivity differences in experiments used to evaluate chemical-induced transformation. Furthermore, it provides a method to estimate a chemical's transformation response in terms of the historical behavior of the assay, as well as its future activity. The most active of the 24 chemicals was mezerein, and the least active chemical was diphenylnitrosamine.

Transformation of BALB/c-3T3 Cells: II. Investigation of Experimental Parameters That Influence Detection of Benzo[a]pyrene-Induced Transformation

“…Briefly, each transformation assay contained three components: a standard clonal survival assay (13,15), a co-culture clonal survival assay (13,17), and a transformation assay (13,15). In each experiment, BaP-induced transformation was detected in the positive control, which consisted of 20 vessels seeded with 3.2 x 104 cells/vessel.…”

“…BaP-induced transformation of BALB/c-3T3 cells was evaluated in a standard transformation assay protocol that has been reported in detail (13) and is summarized in these investigations in part IV (15). Briefly, each transformation assay contained three components: a standard clonal survival assay (13,15), a co-culture clonal survival assay (13,17), and a transformation assay (13,15).…”

“…BaP treatments were terminated by removal of the treatment medium, washing the culture vessels twice with Hank's balanced salt solution (HBSS; Quality Biologicals, Gaithersburg, MD), and fed with culture medium. After a total culture period of 8 days, the vessels were washed, fixed in methanol, stained with Giemsa, and colonies of cells were hand tabulated according to the procedure described in part IV of these investigations (15).…”

“…In each experiment, BaP-induced transformation was detected in the positive control, which consisted of 20 vessels seeded with 3.2 x 104 cells/vessel. BaP doses were applied to cell cultures for 48 hr, day 2-4, using standard procedures (13,15).…”

Two new approaches to improve the analysis of BALB/c 3T3 cell transformation assay data

“…Some authors analyse the number of foci per dish, e.g. by means of linear models and analysis of variance methods (often combined with pairwise t-tests) after data transformation [3,4], by means of the non-parametric Mann-Whitney U-test [5] or by a parametric approach using a possibly modified Poisson distribution [6]. Others calculate the proportions of dishes showing foci and apply a test for proportions, e.g.…”