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

Matthews, Edwin J.

doi:10.2307/3431403

Cited by 2 publications

(7 citation statements)

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“…The new method has enhanced sensitivity for detecting chemical-induced transformation compared to published procedures (2,4,5,27-29), and the enhanced sensitivity of the transformation assay procedure is due to three procedural changes from published procedures. First, the published method of detecting chemical-induced cytotoxic effects on cells in culture uses a standard clonal survival assay (1-3) employing 200 WT cells, and this method inaccurately measures the cytotoxic responses of chemicals in highdensity cell cultures (17). For most chemicals, the clonal survival assay using 200 WT cells overestimates the chem-ical's cytotoxic activity when it is applied to transformation assays (17), as well as assays for detecting cell to cell communication activities in cultured mammalian cells (30).…”

Section: Discussionmentioning

confidence: 99%

“…First, the published method of detecting chemical-induced cytotoxic effects on cells in culture uses a standard clonal survival assay (1-3) employing 200 WT cells, and this method inaccurately measures the cytotoxic responses of chemicals in highdensity cell cultures (17). For most chemicals, the clonal survival assay using 200 WT cells overestimates the chem-ical's cytotoxic activity when it is applied to transformation assays (17), as well as assays for detecting cell to cell communication activities in cultured mammalian cells (30). This investigation used a co-culture clonal survival assay that quantitatively and accurately measured the RCE of chemical-treated cels at the same cell densities used to measure the induction of the transformed cell phenotype (14).…”

Section: Discussionmentioning

confidence: 99%

“…The investigations in this report used the 1-13 clone ofA31 BALB/c-3T3 cells (13,14). 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).…”

Section: Cell Culturementioning

confidence: 99%

“…The co-culture clonal survival assay was used to select chemical treatment doses for transformation assays, assess the reproducibility of chemical-induced cytotoxic responses, and verify that the test chemical and positive control treatment doses were cytotoxic in the transformation assay. 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).…”

Section: Co-culture Clonal Survival Assaymentioning

confidence: 99%

“…The positive control and test chemical treatment sets had 20 60-mm culture dishes (18 25-cm2 culture flasks), and the negative control (NC-1) had 40-80 60-mm culture dishes (36-72 culture flasks). The positive control for each assay was benzo[a]pyrene (BaP; Sigma, St. Louis, MO), and it was tested at doses of 0.200 and 0.0633 [Lg/mL 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.…”

Section: Transformation Assaymentioning

confidence: 99%

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

Matthews¹,

Spalding²,

Tennant³

1993

Environmental Health Perspectives

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%