Transformation of BALB/c-3T3 cells: III. Development of a co-culture clonal survival assay for quantification of chemical cytotoxicity in high-density cell cultures.

Abstract

A co-culture clonal survival assay was developed to measure the cytotoxicity of test chemical treatments to BALB/c-3T3 cells because the standard clonal survival assay using 200 wild type (WT) cells frequently overestimates chemical cytotoxicity when compared with identical treatment doses in high-density cultures. The assay used co-cultures of 3.2 x 10(4) WT cells, the same seeding density used in the transformation assay, and 200 ouabain resistant (OUAr) cells. After a 48-hr test chemical treatment, co-cultured cells were fed with culture medium containing 4 mM ouabain. The test chemical was cytotoxic to an equal percentage of WT and OUAr cells. Ouabain treatments killed the remaining WT cells. Thus, OUAr cells surviving the test chemical treatment measured the relative cloning efficiency (RCE) of all treated cells in the high-density cell co-culture. The co-culture assay succeeded because metabolic cooperation at the OUAr locus was not detected in BALB/c-3T3 cells. Five chemicals induced comparable cytotoxic responses in both assays, including actinomycin D, 5-bromo-2'-deoxyuridine, N'-methyl-N-nitro-N'-nitrosoguanidine, dimethyl sulfoxide and sodium chloride. In contrast, chemical cytotoxic responses detected in the standard and co-culture assays differed by > or = 10-fold for 11-aminoundecanoic acid, benzo[a]pyrene, cytosine arabinoside, and 3-methyl-cholanthrene and differed by > 2-fold for 2-acetylaminofluorene and dimethylnitrosamine. Detection of 11-aminoundecanoic acid-induced transformation was shown to be dependent on selecting treatment doses from the co-culture assay data. Thus, this method permits more accurate assessment of both chemical-induced cytotoxicity and transformation.

Transformation of BALB/c-3T3 cells: IV. Rank-ordered potency of 24 chemical responses detected in a sensitive new assay procedure.

“…This investigation reports the results of using a new sensitive method to detect cytotoxic (17) and transformation responses (10,11) of 24 test chemicals using a clone A31-1-13 of BALB/-3T3 cells (13)(14)(15) (Tables 2 and 3). …”

“…The materials and methods used to culture the cells have been previously reported in detail (15) and are summarized in part I of these investigations (17).…”

“…The procedure used for the co-culture clonal survival assay has been previously reported in detail (11,13) and is summarized in part III of this series (17).…”

“…The transformation assay culture vessels were seeded with 3 to assess the reproducibility of dose-related increases of BaP-induced cytotoxic and transforming activities (17). A total of three to six test chemicals were included in each transformation experiment, and each chemical was tested at four treatment doses in two or more independent trials.…”

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.…”