Chemical enhancement of survival in aggregation of retrovirus‐infected rat cells: An interlaboratory comparison

Abstract

This study provides a comparative evaluation among three independent laboratories of the responses to 16 chemicals in the retrovirus (Rauscher leukemia) infected Fischer rat embryo (RIFRE) cell-survival-in-aggregation assay. When suspended in liquid media above an agar base, control cells showed a rapid decline in cell survival, whereas cells that had previously been treated with chemical carcinogen survive in suspension longer than control cells. The endpoint, survival in aggregation, is measured by counting viable cells dissociated from aggregates in suspension for 4 days. By modifying previously reported procedures, we have improved the system so that a clear differential (positive or negative) response is achieved by cells treated with either a known carcinogen or known noncarcinogen. Using procedures designed to minimize assay variability, replicate assays were performed and the data analyzed for inter- and intralaboratory concordance. The RIFRE cell-survival-in-aggregation assay demonstrated a high degree of interlaboratory reproducibility in assessing the overall positive or negative responses of known carcinogens and noncarcinogens, and good qualitative reproducibility in assessing compounds tested under code. The assay could discriminate between known carcinogens and noncarcinogens. All chemicals were tested without the addition of a metabolic activation system. Cells exhibiting carcinogen-induced enhancement of survival in aggregation, when plated back onto a solid substrate and carried in culture, subsequently expressed transformation-associated changes in their cellular morphology, growth in semisolid media, and tumorigenicity in nude mice. These results indicate that retrovirus-infected Fischer rat embryo cells detect a carcinogen-mediated early event that progresses to neoplastic phenotypes. Survival in aggregation appears to require the presence of the exogenous retrovirus, since uninfected cells did not show a differential survival response when carcinogen-treated, noncarcinogen-treated, or control cells were compared. This system provides a reproducible method of detecting carcinogenic chemicals based on their ability to induce enhanced survival in aggregation of treated cells.

The use of survival in aggregation to detect potential carcinogens with retrovirus-infected fischer rat embryo cells

“…This end point has been shown to be relevant for determining the carcinogenic potential of a variety of agents (6)(7)(8)10). Moreover, the induction of survival in cellular aggregates can be correlated with the expression of progression to neoplastic phenotypes in the same cells {2,6,7,10).…”

“…The assay system has demonstrated a high degree of reproducibility in assessing the responses of known carcinogens and noncarcinogens and good qualitative reproducibility in assessing coded compounds (7,8,10). This bioassay has the ability to discriminate between structurally related chemicals, without the addition of exogenous activation (2,10). However, the lack or variability or both of responses with some chemicals may indicate the need for incorporating a metabolic activating system.…”

“…The aggregation assay uses this event of enhanced cell survival to indicate the carcinogenic potential of a given agent. The assay system has demonstrated a high degree of reproducibility in assessing the responses of known carcinogens and noncarcinogens and good qualitative reproducibility in assessing coded compounds (7,8,10). This bioassay has the ability to discriminate between structurally related chemicals, without the addition of exogenous activation (2,10).…”

Potential carcinogenicity of Aza‐aromatic hydrocarbons: Azabenz(A)anthracenes

“…Several studies indicate that chemical carcinogens induce increased aggregation and anchorage-indepen-dent survival of certain cells in vitro (Traul et al, 1979;Suk et al, 1983, 198%;Poiley et al, 1984;Eker and Sanner, 1983;Rhim et al, 1981;Steuer et al, 1977;Putnam et al, 1977). The enhancement of anchorage-independent survival (survival in aggregation) has been proposed as an in vitro assay system for the identification of potential chemical carcinogens (Suk et al, 19856;Shin et al, 1975). The 2FR4 cell system has been shown to be responsive to a wide range of chemical carcinogens, including DMBA, 4NQ0, benzo(a)pyrene, 3methylcholanthrene, 2-acetylaminofluorene, NiS04, urethane, and diethylnitrosamine (Traul et al, 1979;Suk, 1985;Suk et al, 19856).…”

“…The enhancement of anchorage-independent survival (survival in aggregation) has been proposed as an in vitro assay system for the identification of potential chemical carcinogens (Suk et al, 19856;Shin et al, 1975). The 2FR4 cell system has been shown to be responsive to a wide range of chemical carcinogens, including DMBA, 4NQ0, benzo(a)pyrene, 3methylcholanthrene, 2-acetylaminofluorene, NiS04, urethane, and diethylnitrosamine (Traul et al, 1979;Suk, 1985;Suk et al, 19856). The 2FR4 system does not require the addition of a metabolic activation system.…”