E. Borenfreund

Toxicity determined in vitro by morphological alterations and neutral red absorption.

A method is described which combines the use of a visual morphological cytotoxicity assay with a quantitative neutral red (NR) spectrophotometric test, for the assessment of the effect of toxic agents on 3T3 cells in culture. These sensitive and reproducible assays lend themselves to a screening procedure of potential toxicants which can help reduce the use of animals for toxicity testing.

A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90)

A sensitive quantitative procedure for assaying viable cells in monolayer cultures is described. The two-component test involves (a) microscopic screening for morphological alterations after an experimental protocol for cytotoxicity studies and (b) quantitation of surviving cells by incubation with the supravital dye neutral red, followed by colorimetric analysis of the dye extracted from the lysosomes of the viable cells. Both assays, conveniently carried out within the same culture, have been standardized for use with 96-well microtiter plates and automatic reading with a Dynatech spectrophotometric microplate reader. The test can be adapted for use with conventional spectrophotometers.

Comparisons of two in vitro cytotoxicity assays—The neutral red (NR) and tetrazolium MTT tests

The neutral red (NR) and tetrazolium MTT in vitro cytotoxicity assays were compared for 28 test agents of widely varying potency using the BALB/c mouse 3T3 fibroblast cell line as the bioindicator. For any given cell density in the microtitre plate well, the optical density absorbance with the NR assay was about twice that obtained with the MTT assay. However, there was good agreement (r = 0.939) between the ranking of the test agents on the basis of midpoint cytotoxicity values (NR(50) and MTT(50)), although the assays were based on different physiological endpoints. Nevertheless, the two assays were found to differ in sensitivity for a few test agents, such as chloroquine sulphate and several antineoplastic drugs. Both assays were capable of demonstrating a reduction in the cytotoxicity of methotrexate by leucovorin.

Cytotoxicity and genotoxicity assays with cultured fish cells: A review

Cultured fish cells can be used in a variety of cytotoxicity and genotoxicity assays for the preliminary testing of environmental chemical hazards that may be hazardous to the aquatic biota. Such assays can also be used to evaluate synergistic and antagonistic interactions between combinations of test agents and to establish structure-activity relationships for series of related chemicals. A range of fish cell lines are available for use in such assays and a variety of endpoints may be used. To detect toxicants that require bioactivation the chosen cell line must have significant P-450 activity, or a metabolizing component must be incorporated into the assay. Fish cells in culture respond to the same chemical mutagens and clastogens that are genotoxic to mammalian cells in culture. However, since fish cells in culture are eurythermic, they represent a unique system for studying temperature as a parameter in mediating the genotoxicity and the cytotoxicity of a test agent.

Rapid chemosensitivity assay with human normal and tumor cells in vitro

SummaryNeutral red assay, as an index of cytotoxicity, has been applied to predictive screening of chemotherapeutic agents. Human hepatoma and melanoma tumor cells and normal melanocytes, keratinocytes and fibroblasts were incubated for 2, 24, and 48 h with graded concentrations of cis-platinum (0.1 to 80 μM), doxorubicin (0.01 to 100 μM), and 5-fluorouracil (1 to 1000 μM). Cells were most sensitive after 48 h. Tumor cells, based on 50% toxicity values, were 2–4 times more sensitive than the normal cells, except for cis-platinum, where only melanoma cells, as compared to normal melanocytes, showed a marked difference in cytotoxic response. Methotrexate (1 to 10 μM) toxicity could be reversed in the presence of 100 μM of leucovorin. This sensitive, rapid, and economical assay is suitable for preclinical screening and drug development.

In vitro cytotoxicity assays. Potential alternatives to the Draize ocular allergy test.

A short-term cytotoxicity assay carried out in multiwell test plates and a supplementary colony forming assay are both useful for screening and range finding of toxic concentrations of test agents. The highest tolerated dose (HTD), a concentration at which only minimal morphological changes were observed, was designated as endpoint in the assay. Epithelial rabbit cornea cells, murine fibroblasts, Chinese hamster lung cells, human hepatoma cells and mouse macrophage cultures were used as targets. Several of the alcohols tested at HTD in the colony forming assay were found to inhibit colony formation. An ID50 of colony formation was used as a quantitative corroborating test. The ranking of 34 toxicants was found to be virtually the same with all cell types examined. This easily reproducible, rapid in vitro test is cost-effective and can be used for preliminary large scale screening of potential toxicants.

In vitro cytotoxicity of metals to bluegill (BF-2) cells

BF-2 cells, an established fibroblastic cell line derived from the caudal fin of bluegill sunfish (Lepomis macrochirus), were exposed to 18 metal salts. Cytotoxieity was assayed by the neutral red (NR) technique. Based on the concentration of metal that reduced the uptake of neutral red by 50% (NR50), the rank order of Cytotoxieity for the cationic metals was silver > mercury > cadmium > zinc > copper > cobalt > nickel > lead > tin > manganese > chromium (trivalent), and for the anionic metal complexes it was arsenite > dichromate > chromate > arsenate > selenite > permanganate > selenate. There was a strong correlation (r = 0.909) between the NR50 ranking for the divalent metal cations and their chemical softness parameters (σp). A good correlation (r=0.833) was also found between thein vitro NR50 Cytotoxieity values for the cationic metals and thein vivo water-borne LC50 values for bluegill. There was no correlation, however, between NR50 and LC50 data for the anionic metal complexes, primarily due to the unusual tolerance, as reported in the literature, of fish for hexavalent chromium salts.

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.

Acute cytotoxicities of polynuclear aromatic hydrocarbons determined in vitro with the human liver tumor cell line, HepG2.

The neutral red in vitro cytotoxicity assay was adapted for use with the human hepatocellular tumor cell line HepG2 to detect the cytotoxic potencies of polynuclear aromatic hydrocarbons (PAHs). Using benzo[a]pyrene (B[a]P) as the representative PAH, it was determined that a 3-day exposure was the most suitable for detecting cytotoxic potency and that preexposure to S μg/ ml Arochlor enhanced the sensitivity of the HepG2 cells to the toxicant. Such enhanced sensitivity probably reflected increased metabolic conversion of the B[a]P to active metabolites after culturing the cells in the presence of Arochlor. This was shown by a 3-fold increase in the activity of 7-ethoxycoumarin deethylase, an indicator of mixed-function oxygenase activity. Furthermore, a reduction in sensitivity to B[a]P occurred when the cells were cultured in the presence of α-napthoflavone, an inhibitor of aryl hydrocarbon hydroxylase activity. When Arochlor-induced cells were transferred to medium lacking Arochlor, the level of 7-ethoxycoumarin deethylase quickly declined to basal levels. Arochlor-induced cells were also able to detect the cytotoxic potencies of benzo[k]fluoranthene, benzo[b]-fluoranthene, chrysene, benzo[a]anthracene pyrene, phenanthrene, and fluoranthene, whereas fluorene, anthracene, acenaphthene, and acenaphthylene were not cytotoxic.