C. Shopsis

Rapid cytotoxicity testing using a semi-automated protein determination on cultured cells

A rapid cytotoxicity screening procedure that measures the inhibition of protein synthesis in cultured cells is described. Cells are cultured in 96 microwell plates, test agents are added to the cultures, and the protein content of the cultures is then determined in the microwells using a modification of the Coomassie blue dye complex method. Absorbances in the wells are determined by scanning the plate in a microplate reader. The method is sensitive, rapid, reproducible and inexpensive, enabling the large-scale screening of potential toxicants. Results of the application of the procedure to the measurement of toxicity of 6 surfactants and 6 divalent metal ions are presented.

In vitro cytotoxicity testing of aquatic pollutants (cadmium, copper, zinc, nickel) using established fish cell lines

The cytotoxicity of cadmium toward cultured bluegill fry (BF-2) cells was determined using several assay endpoints. The concentrations of cadmium causing a 50% decrease in colony formation, cell replication, uptake of neutral red, population growth (as determined by protein analysis), and uptake of [3H]uridine and 50% detachment of cells (as determined by protein analysis) were 0.03, 0.04, 0.08, 0.09, 0.12, and 0.21 mM cadmium, respectively. The neutral red assay was used to compare the relative sensitivities of bluegill BF-2 cells and RTG-2 cells, derived from the rainbow trout, toward four metals. The concentrations of cadmium, zinc, copper, and nickel causing a 50% reduction in the uptake of neutral red were 0.08, 0.19, 0.55, and 2.0 mM, respectively, with the BF-2 cells and 0.18, 0.64, 1.45, and greater than 10.0 mM, respectively, with the RTG-2 cells. The RTG-2 cells were less sensitive to the metals, in particular to nickel. The less stringent temperature requirements for growth, their greater sensitivity to pollutants, and their markedly shorter doubling time in vitro make the BF-2 cells the preferable cell line for ecotoxicity screening of aquatic pollutants.

Toxicity monitored with a correlated set of cell-culture assays

A set of assays for toxicity has been developed in which cell cultures serve as an alternative to toxicity testing in vivo. One test is the assessment of the highest concentration of toxicant which produces minimal morphological alterations in cell cultures, followed by the determination of the amount of neutral red dye uptake by the cells. A second test is based on 50% inhibition of uptake of [3H]uridine after incubation of the cultures with the toxicant. There is good agreement between these assays in the rank correlation of a broad spectrum of compounds tested, as well as with the data from Draize rabbit eye irritancy tests in vivo.

A semi-automated protein assay for cell cultures

A semi-automated modification of the protein determination procedure of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951, J. Biol. Chem. 193, 265-275) is described. The assay is well suited to the analysis of the protein of adherent cultured cells. The procedure is carried out in 96-well microtest plates on protein solutions of 50 microliter or less, and can detect less than 0.5 micrograms of protein (equivalent to about 10(3) cultured cells). Optical densities are read and printed by an automatic microplate reader capable of processing 96 samples in less than 2 min.

A battery of potential alternatives to the Draize test: Uridine uptake inhibition, morphological cytotoxicity, macrophage chemotaxis and exfoliative cytology

Four assays that may serve as components of a battery of alternatives to the conventional Draize test were described. The exfoliative cytology assay is a refinement of the Draize test that may provide a more sensitive and more objective end-point. The macrophage migration assay addresses the inflammatory aspects of the physiological response to irritation. The uridine uptake inhibition assay uses a quantitative, reversible end-point to detect the short-term action of agents on cell membranes and cell phosphorylative potential. Finally, the cytological assay serves as a rapid, easily performed general indicator of cytotoxic action. The two latter assays have been demonstrated to correlate very well with Draize test results and with each other for a wide range of test agents.

Inhibition of uridine uptake in cultured cells: a rapid sublethal cytotoxicity test

A cytotoxicity assay is described that utilizes measurement of inhibition of the rate of uptake of [3H]uridine by cultured cells as a sensitive endpoint. BALB/c 3T3 cells are exposed to test agents for 4 h, and their capacity to take up uridine (largely into trichloroacetic acid soluble pools) over a subsequent 15-min period is then determined. The concentration of toxicant required to inhibit uptake by 50% is used to rank toxicity. The relative toxicity rankings of the test compounds obtained with this sublethal endpoint correlate well with the rankings of their ocular irritancies.

Cadmium-nickel toxicity interactions towards a bacterium, filamentous fungi, and a cultured mammalian cell line

The response of the biota to exposure to individual metals may differ from its response to multiple metals, as mixtures of metals may interact antagonistically or synergistically in their resultant toxicity. The present study evaluated the effects of a combination of Cd and Ni on the freshwater bacterium, Aeromonas hydrophila, the terrestrial fungi, Trichodema viride and Aspergillus niger, and the mammalian cell line, BALB/c mouse 3T3 fibroblasts. This particular spectrum of target cells was selected because studies in the literature show a wide variety of possible interactions between Cd and Ni in their combined toxicities towards bacteria cyanobacteria, slime molds, isolated rat hepatocytes, and rats.

DEVELOPING ALTERNATIVE ASSAY SYSTEMS FOR TOXICITY TESTING

Our present understanding of biomedicine and toxicology is strongly based on knowledge obtained through experimentation using living animals. The safety and potency of established and new biological, chemical, and pharmaceutical products are also routinely ascertained using in vivo toxicology tests. Despite these accomplishments, the combination of heightened public concern about the possible adverse effects of consumer products and the desire to improve humane use of laboratory animals has created demands for new toxicological The development of new techniques that would better detect the potential toxicity of substances which are made available to the public, is thus desirable. Procedures that reduce the use of animals would be welcomed by the scientific community and the general public. Any new toxicology assay must produce results that lead to an equivalent conclusion with the same or greater degree of confidence as the assay being replaced. Acceptable safety evaluation and consumer protection mandate the continued use of the best available technology. The focus of much of what has come to be called “alternatives research” in our and other laboratories is to determine if suitable cell culture technology can be better utilized to develop new acceptable in vitro toxicologic assays. Many in vitro assay techniques have been developed to replace in vivo assays for specific and well defined chemicals. Over the past two decades, in vitro immune assays have virtually replaced in vivo assays for specific hormone and vitamin quantitation. Cell cultures have been used instead of animal inoculation for isolation, growth, and identification of certain microbes while histocompatability antigens originally found through tissue transplantation can now be detected in assays using mixed lymphocyte cultures. Microbiologic and cell culture mutagenicity assays are being utilized in some laboratories to replace animal inoculation in the evaluation of potential carcinogens. The essential requirements for the development of in vitro assay systems include the understanding of critical biochemical aspects of the biologic response observed in the whole animal and technical advances that enable the routine performance of the appropriate experimental procedure. The development of human pregnancy testing provides an illuminating example of this process. Prior to 1929 satisfactory diagnostic tests for early human pregnancy were unavailable. In that year Friedman showed that an undefined hormonal component of the urine of pregnant women would induce the formation of a corpus Iuteum in the ovary of