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Protocol no. 64
THE NEUTRAL RED CYTOTOXICITY ASSAY

The cytotoxic effect of chemicals upon mammalian cells, such as BALB/c 3T3 and HepG2, in culture is measured by highest tolerated dose (HTD), cell viability (Neutral Red) and total cell protein (coomassie blue).

CONTACT

Dr Ellen Borenfreund
Laboratory Animal Research Center
The Rockefeller University
1230 York Avenue New York, NY 10021-6399 United States
Tel: 212-327-8475 Fax: 212-327-7974
Dr Harvey Babich
Department of Biological Sciences Stern College
Yeshiva University
245 Lexington Avenue New York, NY 10016-4699 United States
Tel: 212-340-7739 Fax: 212-340-7788

RATIONALE

The neutral red (NR) cytotoxicity assay procedure is a cell survival/viability chemosensitivity assay, based on the ability of viable cells to incorporate and bind neutral red, a supravital dye. NR is a weak cationic dye that readily penetrates cell membranes by non-ionic diffusion, accumulating intracellularly in lysosomes, where it binds with anionic sites in the lysosomal matrix. Alterations of the cell surface or the sensitive lysosomal membrane lead to lysosomal fragility and other changes that gradually become irreversible. Such changes brought about by the action of xenobiotics result in a decreased uptake and binding of NR. It is thus possible to distinguish between viable, damaged, or dead cells, which is the basis of this assay.

BASIC PROCEDURE

BALB/c 3T3 or HepG2 cells are maintained in culture and exposed to test compounds over a broad range of concentrations. The cultures are visually examined after 24 hours and the highest tolerated dose (HTD) estimated which is then used to establish a narrower range of concentrations for the Neutral Red assay and/or the total cell protein assay. The nature of these assays are such that both may be used on the same cultures provided that the Neutral Red uptake (an indication of the number of viable cells) determination is performed first. Range Finder - HTD Before performing the NR assay, a preliminary, range-finding analysis of the potential toxicity of the test agent is conducted. This preliminary highest tolerated dose (HTD) determination is defined as the highest concentration of toxicant that is tolerated by the cells, at which only minimal morphological alterations are evident. An HTD value has been shown to be equivalent to an NR90 value, i.e. the concentration of toxicant that causes a 10% reduction in the uptake of NR when compared to the controls. The unknown chemical is tested over a wide range of concentrations over 24 hours. The plate is then visually examined under an inverted microscope and each well is scored for cytotoxic effect. The HTD is determined by establishing which concentration causes only minimal morphological changes compared to the control. Scoring each well provides an insight into the slope of the resulting dose response curve. Neutral Red Assay An accurate number of cells are seeded into individual wells of a 96-well microtitre tissue culture plate to achieve 60-70% confluence at the time of the addition of the test agents. The medium is then replaced with various concentrations of test agent, 4-8 wells per concentration. For direct-acting toxicants, the plate is incubated for 1 day, but for test agents which require metabolic activation the incubation may need to be extended to 3-6 days to enable metabolic conversion of the agent to occur. Alternatively, hepatic S9 microsomal fraction may be added directly to the target cells in the presence of test chemical and incubated over 24 hours. The medium is then replaced with NR-containing medium, and after incubation for an additional 3 hours to allow for uptake of the dye, the cells are rapidly washed with a solution of 0.5% formaldehyde:1% calcium chloride. Damaged or dead cells lose their ability to retain NR, which is then removed during this wash/fixation procedure. The dye is then extracted from the intact, viable cells with a solution of 1% acetic acid:50% ethanol. The plate is left to stand at room temperature for 10-15 minutes, then agitated on a microplate shaker for 30 minutes. The absorbance of solubilised dye is then determined using a spectrophotometer equipped with a 540nm filter. Quantitation of the extracted NR by spectrophotometry has been correlated with cell numbers, both by direct counting with a haemocytometer and by protein determination of cell populations. Total Cell Protein Measurement After the absorbance of the NR has been established the cells are washed first with PBS then distilled water. NaOH is added to each well to lyse and solubilise the cells. The plates are incubated for 3 hours or overnight before 200ml of coomassie blue dye is added to each well. The plate is incubated for 30 minutes at room temperature and then agitated on a plate shaker. The absorbance is read at 630nm, reference filter 410nm. Data are presented either as a percentage of the control or read from a standard curve of BSA. The versatility of this system enables the HTD, the NR assay and the protein determination to be performed on the same cells.

CRITICAL ASSESSMENT

Studies with the NR assay have been extensive, using a variety of bio-indicators, including mammalian cells, derived both from laboratory animals and from human beings, as well as fish cells. The spectrum of chemicals tested includes inorganic metals, organometals, surfactants, cancer chemotherapeutics, and other pharmaceutical agents, food additives, preservatives and anti-bacterial agents, pesticides, phthalates, toluenes, benzenes, anilines, phenolics, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), complex mixtures (e.g. shampoos), and a variety of miscellaneous chemical test agents. Cell culture procedure The maintenance and culture of mammalian cell lines such as BALB/c 3T3 and HepG2 cells is a relatively simple and inexpensive technique. The application of such cultures to determine cytotoxicity enables the rapid, highly reproducible, testing of many chemicals on a routine basis. a) Use of BALB/c 3T3 cells for direct-acting cytotoxicants Initially, this assay was developed as an in vitro alternative to the Draize rabbit eye irritancy test. The cytotoxic effects of a series of surfactants were examined with several mammalian cell types, including freshly isolated rabbit corneal epithelial cells and various cell lines (i.e. BALB/c mouse 3T3 fibroblasts; Chinese hamster V79 lung fibroblasts; mouse RAW 264.7 macrophages; human hepatoma HepG2 cells), based on HTD analyses. The primary task was to determine whether low passage rabbit corneal cells were an essential requirement for an in vitro cytotoxicity assay designed to determine the cell corrosion aspect of the Draize rabbit eye test, or whether readily available established cell lines could be used instead. The study showed that although the low passage corneal cells were more sensitive than were the immortalized cell lines, the overall rankings of the test agents were equivalent with each cell type. There was, therefore, no apparent need for the repeated isolation and propagation of fresh rabbit corneal cells for use in the NR cytotoxicity assay. Because of their stability, uniformity of cell type, and ready availability (e.g. American Type Culture Collection, Rockville, MD, USA), the BALB/c mouse 3T3 cell line was selected as the representative cell type for further use in the NR assay. However, in later studies, cells derived from human tissues have been used (keratinocytes, fibroblasts, melanocytes, and melanoma cells) including three-dimensional substrates containing several cell layers. Subsequent studies with the NR assay confirmed that, although different cell types and cell lines exhibit differential sensitivities to test agents, the overall potency ranking of the chemicals was approximately equivalent. It would therefore appear that for the assessment of direct-acting cytotoxicants the overall ranking of the test agents is independent of the particular cell type of cell line used as the target in the NR assay. b) Use of HepG2 cells for indirect-acting cytotoxicants It is critical when performing a cytotoxicity assay to have an understanding of the metabolic capacity of the cell line that is intended to be the target cell. Cells which lack significant xenobiotic metabolising capacity will underestimate the cytotoxicity of some test agents which act indirectly by causing toxicity through their metabolites. The human hepatoma cell line, HepG2, has been well characterised and has been shown to produce most of the plasma proteins and biosynthetic capabilities to that of normal human hepatocytes. The most important factor for their use in in vitro cytotoxicity assays is that they retain some of the drug metabolising capability of normal hepatocytes. This can be further enhanced by culturing the cells in the presence of inducers, such as benzo[a]anthracene (B[a]A), 3-methylcholanthrene (3-MC), and aroclor. However, the incubation period of the assay has to be extended to compensate for the low metabolic activity of these cells which perhaps could be viewed as a disadvantage but the possibility of using a cell line of human origin may provide a system which is more relevant for assessment of risk to human health. c) Use of S9 mix for indirect-acting cytotoxicants An alternative approach to the use of a cell line with inherent metabolic activity is to include a hepatic S9 microsomal fraction from Aroclor-induced rats or hamsters along with the test agent. This approach shortens the exposure period required to 24 hours and, furthermore, makes the choice of target cell of secondary importance. Neutral Red Assay Lysosomal integrity, with the concomitant binding of the neutral red dye, is a highly sensitive indicator of cell viability. The assay quantitates cell viability and can be used to measure cell replication, cytostatic effects, or cell death depending on the seeding density. The test is very sensitive, specific, and readily quantifiable. The protein assay, in contrast, measures total proteinaceous material, which could be due to retention of dead cells in the wells or to cellular debris or proteinaceous exudates. Many laboratories have been unable to record a complete cytotoxicity curve with this assay, since some proteinaceous material (although no viable cells) was always present. The assay can be quite readily modified to test highly volatile compounds by sealing the 96-well plate with pressure sensitive film (Flacon 3073, Becton Dickinson). Coloured compounds do not pose a particular problem with this assay because the medium with the test agent(s) is removed and the cells are washed prior to the addition of NR. It is important to emphasise that the use of the NR assay is not limited to the cell lines used as examples in this protocol. The assay has been used as an endpoint measurement in other human cell lines, including melanoma cells, neuroblastoma cells, melanocytes, and fibroblasts, and low passage cells, such as normal keratinocytes and endothelial cells, with other rodent cells lines, such as mouse neuroblastoma cells, and with fish cell lines, including fibroblast, epithelioid, and hepatoma cell types by numerous laboratories. Applications of the NR assay The NR assay has a number of applications in acute toxicity:- 1) the ranking of chemicals according to their potencies and the correlation of in vitro ranking with in vivo ranking; 2) the evaluation and selective sensitivities of normal and malignant cells to cancer chemotherapeutics; 3) the formulation of structure-activity relationships (SARs) for series of related chemicals; 4) the elucidation of antagonistic/synergistic interactions between combinations of test agents; 5) the study of metabolism-mediated cytotoxicity, both for detoxification and toxification reactions; 6) the determination of toxicity-temperature interactions, both for direct-acting and indirect-acting cytotoxicants; 7) the study of the potential phototoxic potency of various forms of radiation, alone or in combination with chemical test agents. This assay provides information on the general, overall, toxic potential of a chemical but cannot give an insight into the specific physiological or secretory system of a particular organ. Comparison with other tests The NR assay has been compared to the MTT cytotoxicity assay, which is based on the reduction of a tetrazolium dye to a formazan product by mitochondrial enzymes. Although both assays yielded comparable ranking of cytotoxicity data, the optical density absorbance with the NR assay was about twice that obtained with the MTT assay, and thus required fewer cells for analysis. Chemicals which have a direct effect on lysosomes (e.g. chloroquine sulphate) or interfere with mitochondrial enzymes (e.g. ascorbic acid) may exhibit greater toxicity in the NR and MTT assays, respectively.

TEST STATUS

The NR assay is undergoing validation as an in vitro alternative to the Draize test in a number of internationally validation programmes such as those organised by the Commission of the European Communities (CEC); the Cosmetics, Toiletries and Fragrance Association (CTFA), and Soaps and Detergent Association (SDA) of the United States. In addition, the NR assay is an accepted in vitro cytotoxicity test that is in use in many research and industrial laboratories.
OTHER ORGANISATIONS USING THE TEST
A large number of laboratories in the United States, Europe and Asia are currently using the NR assay with numerous publications in peer-reviewed journals.

CHEMICALS TESTED

Inorganic metals
Organometals
Surfactants
Cancer therapeutics
Pharmaceutical agents
Food additives
Preservatives and antibacterial agents
Pesticides
Phthalates
Toluenes
Benzenes
Anilines
Phenolics
Polycyclic aromatic hydrocarbons
Polychlorinated biphenyls (PCBs)
Complex mixtures (e.g. shampoos)

REFERENCES

  1. Babich, H. & Borenfreund, E. (1987) Structure-activity relationship (SAR) models established in vitro with the neutral red cytotoxicity assay. Toxicology In Vitro, 1, 3-9.
  2. Babich, H. & Borenfreund, E. (1990) Applications of the neutral red cytotoxicity assay to in vitro toxicology (Review). Alternatives to Laboratory Animals, 18, 129-144.
  3. Babich, H. & Borenfreund, E. (1991) Cytotoxicity of T-2 toxin and its metabolites determined with the neutral red cell viability assay. Applied Environmental Microbiology, 57, 2101-2103.
  4. Babich, H. & Borenfreund, E. (1992) Neutral Red assay for toxicology in vitro. In: In Vitro Methods of Toxicology, (editor R.R. Watson), pp 237-251, Chapter 17. CRC Press, Boca Raton, Ann Arbor, London, Tokyo. Babich, H. & Borenfreund E. (1992) Cytotoxic and morphologic effects of penylpropanolamine, caffeine, nicotine, and some of their metabolites studied in vitro. Toxicology In Vitro (in press).
  5. Babich, H., Martin-Alguacil, N. & Borenfreund, E. (1987/1988) Mediating role of metabolic activation in in vitro cytotoxicity assays. Molecular Toxicology, 1, 363-372. Babich, H., Martin-Alguacil, N., Raul, C., Rosenberg, D.W. & Borenfreund, E. (1991) Response of human cell cultures to cytotoxicants requiring metabolic activation. In: Alternatives Methods in Toxicology, volume 8 (editor, A.M. Goldberg), pp 263-276.
  6. Mary Ann Liebert, Inc., Publ., New York. Babich, H., Puerner, J.A. & Borenfreund, E. (1986) In vitro cytotoxicity of metals to bluegill (BF-2) cells. Archives of Environmental Contamination and Toxicology, 15, 31-37.
  7. Babich, H., Rosenberg, D.W. & Borenfreund, E. (1991) In vitro cytotoxicity studies with the fish hepatoma cell line, PLHC-1 (Poeciliopsis lucida). Ecotoxicology and Environmental Safety, 21, 327-336.
  8. Babich, H., Sardana, M.K. & Borenfreund, E. (1988) Acute cytotoxicities of polynuclear aromatic hydrocarbons determined in vitro with the human liver tumour line, HepG2. Cell Biology and Toxicology, 4, 295-309.
  9. Borenfreund, E. & Babich, H. (1987) In vitro cytotoxicity of heavy metals, acrylamide, and organotin salts to neural cells and fibroblasts. Cell Biology and Toxicology, 3, 63-73.
  10. Borenfreund, E., Martin-Alguacil, N. & Babich, H. (1988) Comparisons of two in vitro cytotoxicity assays: the neutral red (NR) and tetrazolium MTT tests. Toxicology In Vitro, 2, 1-6.
  11. Borenfreund, E. & Puerner, J.A. (1984) A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). Journal of Tissue Culture Methods, 9, 7-9.
  12. Borenfreund, E. & Puerner, J.A. (1985) Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicology Letters, 24, 119-124.
  13. Borenfreund, E. & Puerner, J.A. (1986) Cytotoxicity of metals, metal-metal and metal-chelator combinations assay in vitro. Toxicology, 39, 121-134.
  14. Borenfreund, E. & Puerner, J.A. (1987) Short-term quantitative in vitro cytotoxicity assay involving an S9 activating system. Cancer Letters, 34, 243-248.
  15. Borenfreund, E. & Shopsis, C. (1985) Toxicity monitored with a correlated set of cell-culture assays. Xenobiotica, 15, 705-711.
  16. Shopsis, C. & Eng, B. (1985) Rapid cytotoxicity testing using a semi-automated protein determination on cultured cells. Toxicology Letters, 26, 1-8.

IP-64 © December 1992