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

Toxic. in Vitro Vol. 2, No. 1, pp. 1-6, 1988 Printed in Great Britain. All rights reserved

088%2333/88 $3.00+ 0.00 Copyright © 1988 PergamonJournals Ltd

COMPARISONS OF TWO I N VITRO CYTOTOXICITY ASSAYS--THE N E U T R A L RED (NR) A N D TETRAZOLIUM MTT TESTS E. BORENFREUND, H. BABICH and N. MARTIN-ALGUACIL Laboratory Animal Research Center, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA (Received 29 June 1987; revisions received 21 August 1987)

Abstract--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 (NRs0 and MTTs0), 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.

INTRODUCTION

Considerable interest has focused recently on the development of short-term in vitro cytotoxicity assays with cultured cells for the evaluation of the acute toxicities of chemical agents. Such assays would not only curtail the use of animals for LDso and similar tests, but would serve as an economic approach for the rapid screening of xenobiotics. Furthermore, such assays would be applicable to pilot studies in drug development, to measure the effectiveness of novel drugs and to assess the chemosensitivities of different cell types to drugs. This paper compares two recently developed in vitro cytotoxicity tests---the neutral red (NR) and the tetrazolium MTT assays. Both assays use colorimetric measurements for the quantitation of viable cells after their incubation with toxicants. The N R assay is based on the uptake of neutral red, a supravital dye, and its accumulation in the lysosomes of viable uninjured cells (Borenfreund & Puerner, 1984 & 1985). The N R assay has been used to measure the relative cytotoxicities of a spectrum of agents, including surfactants (Borenfreund & Puerner, 1985), pharmaceuticals (Borenfreund & Puerner, 1987; Hockley & Baxter, 1986; Riddeli et al. 1986; Stark et al. 1986), shampoos (Stark et al. 1985), preservatives and antibacterial agents (Shopsis & Borenfreund, 1985), industrial chemicals (Babich & Borenfreund, 1987), inorganic metals (Borenfreund & Puerner, 1986), organotins and other neurotoxins (Borenfreund & Babich, 1987), and aquatic pollutants (Babich & Borenfreund, 1987a; Babich et al.

DMEM = Dulbecco's modified Eagles medium; DMSO = dimethylsulphoxide; EDTA = ethylenediaminetetraacetic acid; NR = neutral red; PBS = phosphate-buffered saline.

Abbreviations:

T.I.V 2/I--A

1986). The N R assay has been modified to include, if needed, a protein determination on the cells after their incubation with toxicants and extraction of the dye (Babich & Borenfreund, 1987; Borenfreund & Puerner, 1987; Riddell et al. 1986). More recently a hepatic S-9 microsomal fraction has been included to detect those agents that require biotransformation in order to exert cytotoxic effects (Babich & Borenfreund, 1987c; Borenfreund & Puerner, 1987). Although fibroblasts have been used most extensively for the assay, target cultures have included liver cells, keratinocytes, neural cells, corneal epithelium and tumour cells. The MTT assay is based on the reduction of the soluble yellow MTT tetrazolium salt to a blue insoluble MTT formazan product by mitochondrial succinic dehydrogenase (Mosmann, 1983). Since its development, this assay has been modified by various investigators (Carmichael et al. 1987a; Denizot & Lang, 1986; Maehara et al. 1987) and has been used primarily with tumour cells and, to a lesser extent, with fibroblast cell lines, to evaluate the cytotoxicities of chemotherapeutic agents (Carmichael et al. 1987a; Cole, 1986). The MTT assay has also been adapted for detecting lymphotoxins (Green et al. 1984) and for measuring cell activation (Gerlier & Thomasett, 1986) and radiation effects (Carmichael et aL 1987b). In the study presented herein, the protocol for the MTT assay was adapted from Mosmann (1983) and Denizot & Lang (1986). M A T E R I A L S A N D METHODS

Chemicals. Most of the test agents and antibiotics and the dyes were obtained from Sigma Chemical Co. (St Louis, MO) and were stored as stock solutions at 4°C at 100 times their highest test concentrations; fresh solutions were, in general, prepared at l-2-wk

E. BORENFREUNDet al.

intervals. The pesticides were obtained from the US Environmental Protection Agency Repository for Toxic and Hazardous Materials and the triorganotins were obtained from the National Bureau of Standards through the courtesy of Dr Frederick Brinckman. Care was taken that final concentrations of the solvent--usually methanol or, less frequently, dimethylsulphoxide (DMSO)---were kept below the cytotoxic effect levels. Fungizone was from E. R. Squibb & Sons (Princeton, N J). Cell line. BALB/c 3T3 fibroblasts (American Type Culture Collection) were grown in a humidified 5.5% CO2 atmosphere at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum, 100U penicillin G/ml 100#g streptomycin/ml and 1.25 #g Fungizone/ml. The cells were dissociated in a solution containing 0.5 g trypsin and 0.2 g ethylenediaminetetraacetic acid (EDTA) in 100 ml phosphate-buffered saline (PBS). Experimental design. Individual wells of a 96-well tissue-culture microtitre plate were inoculated with 0.2 ml medium containing 9 x 103 cells. Plates were prepared in parallel for the N R assay and the MTT assay. The plates were incubated for 24 hr to achieve approximately 60-70% confluency in each well. Thereafter, the medium was removed and the cells were re-fed with unmodified medium (control) or with medium modified with various concentrations of test agents. Identical dilutions of the test agents were prepared for each plate. After incubation for another 24 hr, the medium was removed and the plates were prepared for the N R and MTT assays. N R assay. To each well was added 0.2 ml medium containing 50/~g NR/ml. [The NR-containing medium had been pre-incubated overnight at 37°C and was centrifuged prior to use to remove fine precipitates of dye crystals.] The plate was returned to the incubator for another 3 hr to allow for the uptake of the vital dye into the lysosomes of viable uninjured cells. Thereafter the medium was removed, the cells were washed rapidly with a mixture of 1% formaldehyde-l% CaCI 2 and then 0.2ml of a solution of 1% acetic acid-50% ethanol was added to each well to extract the dye. After an additional l0 min at room temperature and a brief agitation on a microtitre-plate shaker, the plate was transferred to a microplate reader equipped with a 540 nm filter to measure absorbance of the extracted dye. This absorbance has shown a linear relationship with the number of surviving cells (Borenfreund & Puerner, 1984 & 1985). M T T a s s a y . MTT tetrazolium salt was prepared as a stock solution of 5 mg/ml in PBS, stored in the dark at 4°C and filter-sterilized; before use it was diluted l:10 into D M E M containing 1% foetal calf serum but no phenol red. MTT-containing medium was added to each well in a volume of 0.1 ml and the plates were covered with foil and incubated at 37°C for an additional 3 hr. Thereafter, the supernates were removed by inverting the plates and 0.1 ml of a solution of l N-HCl-isopropanol (1:24, v/v) was added to extract and solubilize the formazan. After standing for 10 min at room temperature and a brief agitation on a microtitre plate shaker, the plates were transferred to a microplate reader and the optical density of each well was measured using a 550 nm

test wavelength and a 700 nm reference wavelength (Denizot & Lang, 1986; Mosmann, 1983). Statistics. All experiments were performed at least four times, using four or eight wells for each concentration of test agent. Data for the dose-response cytotoxicity curves were presented as the arithmetic mean + SEM. Linear regression analysis was used to compute the concentration of toxicant needed to reduce absorbance of the NR or MTT by 10% (NRg0 and MTTg0 values, respectively) and by 50% (NRso and MTTs0, respectively). RESULTS For the BALB/c mouse 3T3 fibroblasts seeded at a density of 9 x 103 cells per well, 2 days' growth in control medium yielded optical density measurements between 0.9 and 1.0 in the NR assay and between 0.4 and 0.5 in the MTT assay. Low sensitivity in the MTT assay has been noted by other investigators (Carmichael et al. 1987a; Denizot & Lang, 1986). The cytotoxicity data in both assays were standardized by expressing absorbance data in the presence of toxicants as a percentage of that in control medium. Using the triorganotins as the representative toxicants, Figs 1 & 2 illustrate typical dose-response cytotoxicity curves obtained with both assays. For the six triorganotins tested, cytotoxicity was detected with lower concentrations in the N R than in the MTT assay. However, the ranking of the triorganotins in order of their potency was similar in both assays: triphenyltin hydroxide > tripropyltin chloride > tributyltin chloride > tricyclohexyltin bromide > triethyltin bromide > trimethyltin hydroxide. In lieu of constructing dose-response cytotoxicity curves for each test agent, the data were reduced to values indicative of initial toxicity (NRg0 and MTTg0 values, the concentration of toxicant that caused a 10% decrease in absorbance) and of midpoint toxicity (NRs0 and MTTs0 values). Such cytotoxicity data for the organotins and for three chlorinated pesticides are presented in Table 1. On the basis of midpoint cytotoxicity values, the sequence of potency for the chlorinated pesticides was chlordane> heptachlor ~ aldrin, with no marked differences in sensitivity between the NR and MTT assays.

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Fig. 1. Cytotoxicities of the triorganotins trimethyltin hydroxide (11), triethyltin bromide (A), tricyclohexyltin bromide (O) and triphenyltin hydroxide (@), in the neutral red ( ) and tetrazolium ( - - - - ) assays. Data are means + SEM (within the dimensions of the symbols).

Neutral red and MT'I" assays: cytotoxicity studies

these agents in the N R and M'IT assays, there was, for the most part, good agreement between the two assays. Thus, in both assays, oligomycin, antimycin and p-chloromercuribenzoic acid were highly toxic (NRso and MTTso < 100/~M), propanolol, chloroquine and phenylbutazone were moderately toxic (NRs0 and MTTs0~> 100#M< 1000#M), sodium azide, 2,4-dinitrophenol and L-ascorbic acid were slightly toxic (NRs0 and MTTs0 > 1000 #M < 2000 tiM or only just outside that range) and phenobarbital and caffeine were nontoxic (NRs0 and MTTs0 > 2500 #ra). Some of the test agents studied (oligomycin, antimycin, sodium azide and 2,4-dinitrophenol) affect oxidative phosphorylation and were selected to determine whether the mitochondrial-based MTT assay would be more sensitive than the NR assay to such mitochondrial poisons. However, as noted in Table 2, both assays exhibited similar sensitivities to these toxicants. Conversely, chloroquine, an anti-inflammatory cationic amphiphilic drug, was selected as it is known to cause

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Fig. 2. Cytotoxicities of the triorganotins tributyltin chloride ( O ) and tripropyltin chloride (O), in the neutral red ( ) and tetrazolium MTT ( - - - - ) assays. Data are means + SEM (within the dimensions of the symbols).

Table 2 lists cytotoxicity data for 11 test agents with widely differing potencies. Although some differences were noted in the cytotoxicity rankings of

Table 1. Comparative cytotoxicity of triorganotin compounds and pesticides, as determined by the neutral red (NR) and tetrazolium MTT assay NR assay Test agent* TriphenylTripropylTricyclohexylTributylTriethylTrimethylChlordane Heptachlor Aldrin

MTT assay

NR~t

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24 30 33

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47 59 55

*Both the organotins and the pesticides are listed according to their NRso values. tMean concentrations (~M) required to reduce absorbance by 10% (NRso and MTTgo) and by 50% (NRso and MTTs0). Standard deviations were < + 10%. Table 2. Comparative cytotoxicity of two groups of compounds, including drugs for cancer chemotherapy, as determined by the neutral red (NR) and tetrazolium MTT assay NR assay Test agent* Oligomycin p-Chloromercuribenzoic acid Antimycin Chloroquine sulphate Propanolol HCI Phenylbutazone L-Ascorbic acid 2,4-Dinitrophenol Sodium azide Caffeine Phenobarbital Actinomycin D Methotrexate Cytosine arabinoside Adriamycin Mitomycin C 5-Fluorouracil Azacytidine Leucovorin

NP~t

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MTT assay MTTgot

MT%ot

Test compounds---group 3 0.001 13 20 42 0.004 75 59 98 46 174 195 519 256 840 109 1358 106 1538 550 3000 1077 6459

0.001 15 0.004 57 42 145 256 109 85 500 646

13 36 66 359 212 422 1419 1630 1500 3500 4306

Cancer chemotherapeutic agents 0.002 0.2 0.06 0.9 0.8 8.0 0.2 8.0 2.0 14.0 0.6 15.0 4.0 102.0 1750.0 > 10,000.0

0.002 0.02 2.0 0.06 0.3 0.8 2.0 1600.0

0,2 44,0 12.0 1.0 9.0 27.0 62.0 > 10,000.0

*In each of the two groups, compounds are listed according to their NRs0 values. tMean concentrations (#M) required to reduce absorbance by 10% (NRg0 and MTT~) and by 50% (NRs0 and MTTs0). Standard deviations were < + 10%.

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Fig. 3. Chemicals---(a) chloroquine, (b) methotrexate and (c) cytosine arabinoside--showing cytotoxicity at lower concentrations in the neutral red ( ) than in the tetrazolium MTT (. . . . . ) assay. Data are means + SEM.

labilization of lysosomes (Hruban, 1984). As expected, chloroquine was more cytotoxic in the N R than in the MTT assay, with NRs0 and MTT~0 values of 98 and 359/~M, respectively (Table 2, Fig. 3). For phenobarbital, the MTT assay was the more sensitive (Table 2; Fig. 4). A group of drugs of particular interest for in vitro screening are those used in cancer chemotherapy (Table 2). Within this particular group of test agents there were distinctions in cytotoxicities as noted with the two assays. Although actinomycin D and cytosine arabinoside exhibited approximately equivalent potencies in both assays, methotrexate (Fig. 3) and 5-fluorouracil were cytotoxic at lower drug concentrations in the NR assay, and adriamycin (Fig. 4) and azacytidine were more readily cytotoxic in the MTT assay. In contrast to these antineoplastic drugs, leucovorin, a reduced hydrofolate used as a 'rescue' drug in cancer chemotherapy (Bertino, 1977), was not toxic in either assay (Table 2). For the 28 chemicals evaluated in this study, there was good correlation between the potency rankings (NRs0 v. MTTs0 values, in #M) in the two assays. The correlation coefficient for such ranking according to midpoint potency in both assays was 0.939. Another application of in vitro cytotoxicity assays is to discern synergistic or antagonistic interactions between chemicals applied in combination (Borenfreund & Puerner, 1986). We compared the combined toxicities of two agents, leucovorin and methotrexate. As shown in Fig. 5, 0.5 and 1.0 #M methotrexate were

cytotoxic, but the toxicity was reduced in combination with 50/~M leucovorin. This interaction between leucovorin and methotrexate, which has been demonstrated in vivo (Bertino, 1977), was observed with both the N R and MTT assays. DISCUSSION

When cells are seeded at a density so as to be subconfluent (c. 60-75% confluency with the 3T3 cells) at the time of inoculation of the test agents, the assays are suitable for measuring both the cytocidal and the cytostatic (growth inhibitory) effects of the agents. Furthermore, exposure and observation times can be varied to fit the experimental design by adjusting the initial number of cells seeded. For example, with both the N R assay (Babich & Borenfreund, 1987d) and the MTT assay (Carmichael et al. 1987b), exposure times could be extended to 7 days when the initial seeding was at a low density and the cells had a moderate replication time. For any cell density per well, the optical density measurements for the NR assay were, as noted earlier, about twice those for the MTT assay. Doubling the initial inoculation of cells into the wells of 96-well plates in order to achieve higher absorbance values with MTT assay was not feasible, since such an inoculum would have exceeded confluence at the time of the addition of test agents. The lower sensitivity of the MTT assay may be due to the poor solubilization of the formazan product (Dcnizot &

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Fig. 4. Chemicals--(a) phenobarbital, (b) adriamycin and (c) mitomycin showing cytotoxicity at lower concentrations in the tetrazolium MTT ( - - - ) than in the neutral red ( ) assay. Data are means + SEM.

Neutral red and MTr assays: cytotoxicity studies

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Fig. 5. Reduction in the cytotoxicity of methotrexate by leucovorin, as detected with (a) the neutral

red assay and (b) the tetrazolium MTT assay. Data are means + SEM for 50 #~l leucovorin ([:3), 0.5 or 1.0 #M methotrexate (Mx) as indicated (Fq) and the corresponding concentrations of methotrexate and leucovorin (11). Lang, 1986) as well as to the fact that the amount of iological endpoint, the assays yield comparable data. N R dye taken up by lysosomes was larger than the Conversely, the differential sensitivities between the amount of tetrazolium salt reduced by mitochondria. N R and MTT assays for specific test agents, such as Apparently, the MTT assay is still in the devel- chloroquine sulphate and some of the antineoplastic opmental stages, with various modifications being drugs, might prompt the inclusion of both assays in recommended for formazan solubilization. F o r ex- a battery of test methods. ample, suggested solvents include HCl-isopropanol (Mosmann, 1983), ethanol (Denizot & Lang, 1986), REFERENCES HCl-sodium dodecyl sulphate (Tada et al. 1986), trichloroacetic acid-acetone (Maehara et al. 1987), Babich H. & Borenfreund E. (1987a). In vitro cytotoxicity of organic pollutants to bluegill sunfish (BF-2) cells. DMSO and mineral oil (Carmichael et al. 1987a). Envir. Res. 42, 229-237. Retention of formazan within the cells can lead at times to an inability to measure cell survival under Babich H. & Borenfreund E, (1987b). Structure-activity relationship (SAR) models established in vitro with the highly toxic conditions. Since the MTT assay does neutral red cytotoxicity assay. Toxic. in Vitro 1, 3-9. not include a fixation step, cells tend to detach from Babich H. & Borenfreund E. (1987c). Polycyclic aromatic the surface of the culture plate during the formazanhydrocarbon in vitro cytotoxicity to bluegill BF-2 cells: solubilization procedure. mediation by S-9 microsomal fraction and temperature. For analysis of the cytotoxicity data it was apparToxicology Left. 36, 107-116. ently necessary to determine the concentrations of Babich H. & Borenfreund E. (1987d). In vitro cytotoxicity of polychlorinated biphenyls (PCBs) and toluenes to test agents causing initial cytotoxicity (NR90 and cultured bluegill sunfish BF-2 cells. In Aquatic Toxicology MTTg0 values) and those causing midpoint cytoand Hazard Assessment. American Society for Testing and toxicity (NR50 and MTTs0 values). Such comparative Materials. Philadelphia, PA. In press. data were not only indicative of the kinetics of the Babich H., Puerner J. A. & Borenfreund E. 0986). In vitro cytotoxicity curves but may also serve to distinguish cytotoxicity of metals to bluegill (BF-2) cells. Archs envir. between subtle responses of lysosomes (NR assay) Contain. Toxicol. 15, 31-37. and mitochondria (MTT assay) to low levels of toxic Bertino J. R. (1977). "Rescue" techniques in cancer chemotherapy: use of leucovorin and other rescue agents after agents. For example, although the midpoint cytomethotrexate treatment. Semin. Oncol. 4, 203-216. toxicity value for heptachlor was similar in both assays (59 #M), initial toxicity was detected at a lower Borenfreund E. & Babich H. 0987). In vitro cytotoxicity of heavy metals, acrylamide, and organotin salts to neural concentration in the MTT assay (MTTgo, 20 #M) than cells and fibroblasts. Cell Biol. ToxicoL 3, 63-73. in the N R assay (NRgo, 30 pM). Conversely, whereas Borenfreund E. & Puerner J. A. 0984). A simple quanmethotrexate showed approximately similar initial titative procedure using monolayer culture for cytocytotoxicities in the two assays (0.06 and 0.02 #~l for toxicity assays (HTD/NR-90). J. Tiss. Cult. Meth. 9, 7-9. the N R ~ and MTTg0, respectively), the midpoint Borenfreund E. & Puerner J. A. 0985). Toxicity determined cytotoxicity values differed greatly (NRs0, 0.9#M; in vitro by morphological alterations and neutral red absorption. Toxicology Lett. 24, 119-124. MTTs0, 44.0 #M). Such differential responses both in initial and midpoint cytotoxicity values demonstrate Borenfreund E. & Puerner J. A. (1986). Cytotoxicity of metals, metal-metal, and metal-chelator combinations the uniqueness of the assays. assayed in vitro. Toxicology 39, 121-134. The studies reported here demonstrate that both' tile N R and MTT in vitro cytotoxicity assays can be Borenfreund E. & Puerner J. A. 0987). Short-term quantitative in vitro cytotoxicity assays involving an S-9 activaapplied both to screening the acute toxicities of ting system. Cancer Lett. 34, 243-248. chemical test agents and to identifying interactions Carmichael J., DeGraff W. G., Gazdar A. F., Minna J. D. when combinations of agents are used. The good & Mitchell J. B. (1987a). Evaluation of a tetrazoliumagreement (r = 0.939) between the ranking of the based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47, 936-942. chemicals according to their NR50 and MTTs0 values suggests that although each test has a different phys- Carmichael J., DeGraff W. G., Gazdar A. F., Minna J. D.

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dehydrogenase inhibition test for predicting cell viability. Eur. J. Cancer olin. Oncol. 23, 273-276. Mosmann T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. lmmun. Meth. 65, 55-63. Riddell R. J., Clothier R. H. & Balls M. (1986). An evaluation of three in vitro cytotoxicity assays. Fd Chem. Toxic. 24, 469-471. Shopsis C. & Borenfreund E. (1985). In vitro cytotoxicity assays: potential alternatives to the Draize test. In Evaluation des Effects Cosmetiques Methodes D'Aujourd'hui et de Demain. XVemes J. Int. de Dermacosmetologic de Lyon,

pp. 170-195. Stark D. M., Borenfreund E., Walberg J. & Shopsis C. (1985). Comparison of several alternative assays for measuring potential toxicants. In Alternative Methods in Toxicology. Vol. 3, Edited by A. M. Goldberg, pp. 373-390. Mary Ann Liebert, New York. Stark D. M., Shopsis C., Borenfreund E. & Babich H. (1986). Progress and problems in evaluating and validating alternative assays in toxicology. Fd Chem. Toxic. 24, 449-455. Tada H., Shiho O., Kuroshima K.-I., Koyama M. & Tsukamota K. (1986). An improved colorimetric assay for interleukin 2. J. Immun. Meth. 93, 157-165.