APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1991, p. 2101-2103
0099-2240/91/072101-03$02.00/0
Vol. 57, No. 7
Copyright © 1991, American Society for Microbiology
Cytotoxicity of T-2 Toxin and Its Metabolites Determined with the Neutral Red Cell Viability Assay H.
BABICH' 2* AND E.
BORENFREUND'
Laboratory Animal Research Center, The Rockefeller University, 1230 York Avenue, New York, New York 10021,1* and Department of Biological Sciences, Stern College, Yeshiva University, New York, New York 100162 Received 1 February 1991/Accepted 15 April 1991
The neutral red (NR) cell viability assay was used with various cell types of human origin to quantitate the potency of T-2 mycotoxin and its metabolites. The human melanoma SK-Mel/27 cell line was the most sensitive, with a midpoint cytotoxicity value of 2.8 ng of T-2 per ml. With the human hepatoma cell line, HepG2, the sequence of potency for a series of mycotoxins was T-2 > HT-2 > T-2 triol > T-2 tetraol.
Whole-animal bioassays, such as the rabbit skin irritancy test (9) and the rodent 50% lethal dose determination (28), have been used for the detection and quantitation of mycotoxins. However, the current concern to reduce the use of animals in acute-toxicity screenings, coupled with the required use of large numbers of animals and the lack of accurate quantitation in such bioassays, has prompted the development of alternative bioassays (29). In one such alternative approach, cytotoxicity assays involving mammalian cells in culture have been developed for the toxicological evaluation of mycotoxins. Some of these cytotoxicity assays, however, are tedious and time-consuming. For example, Oldham et al. (18) used a cell survival assay, which required the counting of viable cells with a hemacytometer, and a cloning assay, which required 10 to 14 days of incubation, to assess the relative cytotoxicities of T-2 mycotoxin and its metabolite, T-2 tetraol. Other cell culture techniques involve the quantitation of the toxicities of mycotoxins by measuring protein (24, 25) and DNA (20, 23)
larly to sites of the lysosomal matrix (16). Xenobiotics that injure the plasma or lysosomal membrane decrease the uptake and subsequent retention of the dye. Dead or damaged cells cannot retain the dye after the washing and fixation procedures. After NR has been extracted from the lysosomes, it is quantitated spectrophotometrically and the amount is compared with the amount of dye extracted from control cell cultures. Quantitation of the extracted dye has been shown to be linear with cell numbers, both by direct cell counts and by protein determination of cell populations (6, 7). The NR assay has been found to be more sensitive than the MTT assay (5, 11). The focus of this report is on the mycotoxin T-2 and some of its metabolites, HT-2, T-2 triol, and T-2 tetraol (19, 27). Previous studies with the NR assay have shown its sensitivity in distinguishing parent compounds from their intermediate metabolites (3). T-2 is one of the more toxic of the 12,13-epoxytrichothecene mycotoxins produced by Fusarium species. Such mycotoxins have been the agents of illness and death in animals and humans ingesting moldy agricultural products. T-2 mycotoxin is a potent inhibitor of protein and scheduled DNA synthesis (18, 26) and has genotoxic properties, including unscheduled DNA repair synthesis (18) and chromosomal aberrations (14). All cell types used in this study are of human origin. A foreskin fibroblast cell line, designated HFF, and the hepatoma cell line HepG2 were grown in Dulbecco minimal essential medium supplemented with 2% fetal bovine serum and 10% Serum Plus (Hazelton Research Products, Lenexa, Kans.). The human melanoma cell line SK-Mel/27, the nonmalignant keratinocyte cell line HPK-1A, and normal kidney primary cell cultures, designated NK cells, were grown in Dulbecco minimal essential medium supplemented with 10% fetal bovine serum. The renal carcinoma cell line CAKI was grown in minimal essential medium supplemented with 10% fetal bovine serum, and normal umbilical vein endothelial primary cell cultures, designated HEC cells, were grown in medium 199 supplemented with 20% fetal bovine serum in plates treated with 50 ,ug of fibronectin per ml. All of the above-noted media were supplemented with 100 U of penicillin G per ml, 100 ,ug of streptomycin per ml, and 2.5 ,ug of amphotericin B (Fungizone) per ml. All cultures were passaged by dissociation in a solution of 0.05% trypsin-0.02% EDTA. Secondary cultures of normal breast keratinocytes, designated NHEK cells, were obtained from Clonetics Corp., San Diego, Calif., as was the serumfree, biochemically defined keratinocyte growth medium
syntheses by radiolabeling procedures. Such techniques, however, in addition to requiring expensive laboratory equipment and radioisotopes, generate radioactive refuse. A more practical approach to the use of mammalian cell culture cytotoxicity assays for quantitating the potencies of mycotoxins has been the application of the MTT colorimetric cell viability assay. In this assay, cells seeded into a 96-well microtiter plate are exposed to various concentrations of the test agent, with cell survival based on the ability of mitochondrial enzymes in viable cells to chemically reduce a yellow tetrazolium salt (MTT) to a purple formazan dye. Color formation is read on a microplate spectrophotometer (enzyme-linked immunosorbent assay [ELISA] reader) (12, 13, 21, 22). The MTT assay, initially developed by Mosmann (17), has undergone many modifications (8, 10) because of the less than optimal sensitivity and the difficulty in solubilizing the final formazan product. We now report on the use of a different colorimetric cell viability assay, the neutral red (NR) assay, to quantitate the potency of mycotoxins by using immortalized human cell lines and lowpassage human cell cultures as the bioindicators. The NR assay is based on the incorporation of the supravital dye, NR, into the lysosomes of viable cells after their incubation with toxic chemicals. This weakly cationic dye penetrates cell membranes by nonionic diffusion and binds intracellu* Corresponding author. 2101
2102
APPL. ENVIRON.
NOTES
TABLE 2. Comparison of mycotoxin toxicities in vitro and in vivo
TABLE 1. Comparative toxicities of T-2 toxin to human cell types Cell line
SK-Mel/27 NHEK HepG2 CAKI HPK-1A HFF HEC NK
Description
Melanoma cell line Second-passage keratinocyte Hepatoma cell line Renal carcinoma cell line Keratinocyte cell line Fibroblast cell line Second-passage endothelial cell Second-passage kidney cell
NR50 (ng/ml)a
2.8 5.6 9.8 35.4 35.7 42.3 44.8 72.1
MICROBIOL.
0.53 0.66 1.10 2.10 2.82 6.62 3.08 5.71
a NR50, midpoint cytotoxicity value after a 48-h exposure to T-2 mycotoxin. Results are the mean and standard deviation of several determinations.
(KGM). The NHEK cells were dissociated with 0.025% trypsin-0.01% EDTA for dispersal. All cell types were maintained at 37°C in a humidified incubator under 5.5% CO2.
Individual wells of a 96-well tissue culture microtiter plate inoculated with 0.2 ml of the appropriate media containing cells (usually 104 cells) to achieve a 65 to 75% confluence after attachment and addition of the test agents.
were
This allows for the target cells to complete an additional replication cycle; as such, the NR assay measures both cytotoxic and proliferative effects. After 1 to 2 days of incubation, the media were removed and replaced with unamended (control) medium or with medium amended with varied concentrations of the mycotoxin to be tested. After 2 days of exposure to the mycotoxin, the media were removed and replaced with media containing 40 ,ug of NR per ml. The NR-containing media had been preincubated overnight at 37°C and centrifuged (1,500 x g for 10 min) prior to use to remove fine precipitates of dye crystals. The assay plate was then returned to the incubator for another 3 h to allow for uptake of the supravital dye into the lysosomes of viable cells. Thereafter, the media were removed and the cells were rapidly washed with 0.5% formaldehyde-1% CaCl2 followed by 0.2 ml of a solution of 1% acetic acid-50% ethanol to extract the dye from the cells. After 10 min at room temperature and a brief but rapid agitation on a microtiter plate shaker, the plates were transferred to a microplate spectrophotometer equipped with a 540-nm filter to measure the absorbance of the extracted dye (6, 7). The toxicities of the various mycotoxins were compared with those of control cultures by computing, by linear regression analysis, the concentration of test agent needed to reduce the absorbance of the dye by 50% (i.e., the midpoint cytotoxicity [NR50] value). All experiments were performed at least four times, with six to eight wells for each concentration of test agent. The mycotoxins (all from Sigma) were solubilized in ethanol; the concentration of ethanol in the test media was always below 1% to avoid cytotoxicity of the solvent. The responses of the various human cell types to T-2 mycotoxin are presented in Table 1. The melanoma SKMel/27 cell line was the most sensitive, with an NR50 of 2.8 ng of T-2 mycotoxin per ml, and the secondary-passage kidney NK cells were the least sensitive, with an NR50 of 72.1 ng of T-2 mycotoxin per ml. This range of cytotoxicity
data for T-2, obtained with the NR assay, is within the range determined for other human cell lines with other cytotoxicity assays. Oldham et al. (18), using cell survival as the cytotoxicity end point with primary cultures of human foreskin fibroblasts, noted a midpoint cytotoxicity value of 70 ng of
afterL:
In vitro toxicity (ng/ml)
Mycotoxin
NR50
(HepG2)a T-2 HT-2 T-2 triol T-2 tetraol
9.8 15.8 398.4 445.3
+ 1.10
± 1.44 ± 27.54 ± 43.62
PSI50
(VERO)b 6.7 27.0 428.0
1,747.0
s.c.
i.p.
inoculation
inoculation
3.3 6.7 75.3 15.7
9.1 10.2
11.0
a Human hepatoma cell line; neutral red assay. b African green monkey cell line; protein synthesis inhibition (PSI) assay
(25).
c In vivo data from Thompson and Wannemacher (25). LD50, dose. s.c., subcutaneous; i.p., intraperitoneal.
50% lethal
T-2 per ml. Thompson and Wannemacher (24), using a protein synthesis inhibition assay, observed a midpoint cytotoxicity value of 17.3 ng of T-2 per ml for the human cervical carcinoma HeLa cell line and 12.5 ng of T-2 per ml for the human A-549 lung carcinoma cell line. Holt et al. (13), using the mitochondrially based MTT assay, derived a midpoint cytotoxicity value of 10.5 ng of T-2 per ml for the HeLa cell line. The mechanisms responsible for the differential sensitivities of the human cell types to T-2 mycotoxin are unknown. T-2 mycotoxin has both cytotoxic and proliferative effects (15). The greater susceptibility of the SK-Mel/27 melanoma cells to T-2 mycotoxin was not due to their proliferative capacity, as under the conditions of the assay, the cells were replicating only about every 30 h. T-2 toxin can be metabolized to a number of different intermediates by cells cultured in vitro (27). As noted by Holt et al. (13), who also observed distinctions in the sensitivity of human and other mammalian cell lines to T-2, the differences in sensitivity may partially reflect the differential abilities of the cells to act on and alter the toxin. The human hepatoma cell line HepG2 was used to differentiate among the cytotoxicities of T-2 mycotoxin and its metabolites, HT-2, T-2 triol, and T-2 tetraol. HepG2 was selected because it maintains elevated indigenous levels of enzymatic xenobiotic-metabolizing capacity (4). T-2 was more cytotoxic to the HepG2 cells than were the intermediate metabolites, as has been noted for other in vitro cytotoxicity assays involving human, monkey, and rodent cell lines (1, 18, 20, 25). On the basis of NR50, the sequence of cytotoxicity to HepG2 was T-2 > HT-2 > T-2 triol > T-2 tetraol (Table 2). This differential sequence of cytotoxicity was similar to that noted by Thompson and Wannemacher (25) when using a protein inhibition assay with the green monkey VERO cell line as the bioindicator. The T-2 parent compound therefore appears to be more toxic than its metabolites. It must be noted that the sequence of cytotoxicity determined in vitro, either with the NR assay or with the protein inhibition assay, did not completely parallel the in vivo sequence of toxicity. Thus, Thompson and Wannemacher (25) noted that after subcutaneous inoculation, the sequence of potency, based on mouse 50% lethal dose determinations, was T-2 > HT-2 > T-2 tetraol > T-2 triol. The mode of inoculation, however, greatly influenced the degree of acute toxicity (Table 2). The NR assay has been applied to evaluating and ranking the cytotoxicity of a spectrum of chemicals, including inorganic metals, organometals, and various industrial, pharma-
VOL. 57, 1991
ceutical, and environmental organics (see reference 2 for a review). This report described the first application of the NR assay to the assessment of natural toxins. The advantages of the NR assay, which include its simplicity, speed, reproducibility of data, low cost, and sensitivity to test agents, make it a useful tool for detecting and quantitating the potencies of chemical test agents, including natural toxins. Great appreciation is expressed to M. Eisinger, Memorial SloanKettering Cancer Center, New York, N.Y., for the HFF and SK-Mel/27 cells; to B. B. Knowles, Wistar Institute, Philadelphia, Pa., for the HepG2 cells; to J. S. Rhim, National Cancer Institute, Bethesda, Md., for the HPK-1A cells; to N. H. Bander, Memorial Sloan-Kettering Cancer Center, for the NK cells; to L. Pfeffer, The Rockefeller University, for the CAKI cells; to W. A. Muller, The Rockefeller University, for the HEC cells; and to Clonetics Corp., San Diego, Calif., for the NHEK cells and KGM medium. This research was supported, in part, by Hoffmann-LaRoche.
REFERENCES 1. Abbas, H. K., W. T. Shier, and C. J. Mirocha. 1984. Sensitivity of cultured human and mouse fibroblasts to trichothecenes. J. Assoc. Off. Anal. Chem. 67:607-610. 2. Babich, H., and E. Borenfreund. 1990. Applications of the neutral red cytotoxicity assay to in vitro toxicology. ATLA 18:129-144. 3. Babich, H., N. Martin-Alguacil, and E. Borenfreund. 1987/1988. Mediating role of metabolic activation in in vitro cytotoxicity assays. Mol. Toxicol. 1:363-372. 4. Babich, H., M. K. Sardana, and E. Borenfreund. 1988. Acute cytotoxicities of polynuclear aromatic hydrocarbons determined in vitro with the human liver tumor cell line, HepG2. Cell Biol. Toxicol. 4:295-309. 5. Borenfreund, E., H. Babich, and N. Martin-Alguacil. 1988. Comparisons of two in vitro cytotoxicity assays-the neutral red (NR) and tetrazolium MTT tests. Toxicol. In Vitro 2:1-6. 6. Borenfreund, E., and J. A. Puerner. 1984. A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR90). J. Tissue Cult. Methods 9:7-9. 7. Borenfreund, E., and J. A. Puerner. 1985. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24:119-124. 8. Carmichael, J., W. G. DeGraff, A. F. Gazdar, J. D. Minna, and J. B. Mitchell. 1987. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47:936-942. 9. Chung, C. W., M. W. Trucksess, A. L. Giles, and L. Friedman. 1974. Rabbit skin test for estimation of T-2 toxin and other skin-irritating toxins in contaminated corn. J. Assoc. Off. Anal.
Chem. 57:1121-1127. 10. Denziot, F., and R. Lang. 1986. Rapid colorimetric assay for cell growth and survival. J. Immunol. Methods 89:271-277. 11. Hoh, A., K. Maier, and R. M. Dreher. 1987-1988. Multilayered keratinocyte culture used for in vitro toxicology. Mol. Toxicol. 1:537-546. 12. Holt, P. S., S. Buckley, and J. R. DeLoach. 1987. Detection of the lethal effects of T-2 mycotoxin on cells using a rapid colorimetric viability assay. Toxicol. Lett. 39:301-312.
NOTES
2103
13. Holt, P. S., S. Buckley, J. 0. Norman, and J. R. DeLoach. 1988. Cytotoxic effect of T-2 mycotoxin on cells in culture as determined by a rapid colorimetric bioassay. Toxicon 26:549-558. 14. Hsia, C.-C., Y. Gao, J.-L. Wu, and B.-L. Tzian. 1986. Induction of chromosome aberrations by Fusarium T-2 toxin in cultured human peripheral blood lymphocytes and Chinese hamster fibroblasts. J. Cell. Physiol. 4(Suppl.):65-72. 15. Hsia, C.-C., B.-L. Tzian, and C. C. Harris. 1983. Proliferative and cytotoxic effects of Fusarium T2 toxin on cultured human fetal esophagus. Carcinogenesis 4:1101-1107. 16. Lulimann-Rauch, R. 1979. Drug-induced lysosomal storage disorders, p. 49-129. In J. T. Dingle, P. J. Jacques, and I. H. Shaw (ed.), Lysosomes in applied biology and therapeutics. NorthHolland Publishing Co., Amsterdam. 17. Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55-63. 18. Oldham, J. W., L. E. Allred, G. E. Milo, 0. Kindig, and C. C. Capen. 1980. The toxicological evaluation of the mycotoxins T-2 and T-2 tetraol using normal human fibroblasts in vitro. Toxicol. Appl. Pharmacol. 80:377-385. 19. Pace, J. G., M. R. Watts, E. P. Burrows, R. E. Dinterman, C. Matson, E. C. Hauer, and R. W. Wannemacher. 1985. Fate and distribution of 3H-labeled T-2 mycotoxin in guinea pigs. Toxicol. Appl. Pharmacol. 80:377-385. 20. Porcher, J.-M., C. LaFarge-Frayssinet, C. Frayssinet, A. Nurie, D. Melcion, and D. Richard-Molard. 1987. Determination of cytotoxic trichothecenes in corn by cell culture toxicity assay. J. Assoc. Off. Anal. Chem. 70:844-849. 21. Reubel, G. H., M. Gareis, and W. M. Amselgruber. 1987.
Cytotoxicity evaluation of mycotoxins by an MTT-bioassay. Mycotoxin Res. 3:85-96. 22. Reubel, G. H., M. Gareis, and W. M. Amselgruber. 1989. Effects of the Fusarium mycotoxins, zearalenone and deoxynivalenol, on the mitochondrial methylthiazotetrazolium-cleavage activity of monolayer cells. Toxicol. In Vitro 3:311-316. 23. Robbana-Barnat, S., C. LaFarge-Frayssinet, and C. Frayssinet. 1989. Use of cell cultures for predicting the biological effects of mycotoxins. Cell Biol. Toxicol. 5:217-226. 24. Thompson, W. L., and R. W. Wannemacher. 1984. Detection and quantitation of T-2 mycotoxin with a simplified protein synthesis inhibition assay. Appl. Environ. Microbiol. 48:11761180. 25. Thompson, W. L., and R. W. Wannemacher. 1986. Structurefunction relationships of 12,13-epoxytrichothecene mycotoxins in cell culture: comparison to whole animal lethality. Toxicon 24:985-994. 26. Thompson, W. L., and R. W. Wannemacher. 1990. In vivo effects of T-2 mycotoxin in synthesis of proteins and DNA in rat tissues. Toxicol. Appl. Pharmacol. 105:483-491. 27. Trusal, L. R. 1986. Metabolism of T-2 mycotoxin by cultured cells. Toxicon 24:597-603. 28. Ueno, Y., Y. Ishikawa, M. Nakajima, K. Sakai, K. Ishii, H. Tsunoda, M. Saito, M. Enomoto, K. Ohtsubo, and M. Umeda. 1971. Toxicological approaches to the metabolites of fusaria. I. Screening of toxic strains. Jpn. J. Exp. Med. 41:257-272. 29. Watson, D. H., and D. G. Lindsay. 1982. A critical review of biological methods for the detection of fungal toxins in foods and foodstuffs. J. Sci. Food Agric. 33:59-67.
0099-2240/91/072101-03$02.00/0
Vol. 57, No. 7
Copyright © 1991, American Society for Microbiology
Cytotoxicity of T-2 Toxin and Its Metabolites Determined with the Neutral Red Cell Viability Assay H.
BABICH' 2* AND E.
BORENFREUND'
Laboratory Animal Research Center, The Rockefeller University, 1230 York Avenue, New York, New York 10021,1* and Department of Biological Sciences, Stern College, Yeshiva University, New York, New York 100162 Received 1 February 1991/Accepted 15 April 1991
The neutral red (NR) cell viability assay was used with various cell types of human origin to quantitate the potency of T-2 mycotoxin and its metabolites. The human melanoma SK-Mel/27 cell line was the most sensitive, with a midpoint cytotoxicity value of 2.8 ng of T-2 per ml. With the human hepatoma cell line, HepG2, the sequence of potency for a series of mycotoxins was T-2 > HT-2 > T-2 triol > T-2 tetraol.
Whole-animal bioassays, such as the rabbit skin irritancy test (9) and the rodent 50% lethal dose determination (28), have been used for the detection and quantitation of mycotoxins. However, the current concern to reduce the use of animals in acute-toxicity screenings, coupled with the required use of large numbers of animals and the lack of accurate quantitation in such bioassays, has prompted the development of alternative bioassays (29). In one such alternative approach, cytotoxicity assays involving mammalian cells in culture have been developed for the toxicological evaluation of mycotoxins. Some of these cytotoxicity assays, however, are tedious and time-consuming. For example, Oldham et al. (18) used a cell survival assay, which required the counting of viable cells with a hemacytometer, and a cloning assay, which required 10 to 14 days of incubation, to assess the relative cytotoxicities of T-2 mycotoxin and its metabolite, T-2 tetraol. Other cell culture techniques involve the quantitation of the toxicities of mycotoxins by measuring protein (24, 25) and DNA (20, 23)
larly to sites of the lysosomal matrix (16). Xenobiotics that injure the plasma or lysosomal membrane decrease the uptake and subsequent retention of the dye. Dead or damaged cells cannot retain the dye after the washing and fixation procedures. After NR has been extracted from the lysosomes, it is quantitated spectrophotometrically and the amount is compared with the amount of dye extracted from control cell cultures. Quantitation of the extracted dye has been shown to be linear with cell numbers, both by direct cell counts and by protein determination of cell populations (6, 7). The NR assay has been found to be more sensitive than the MTT assay (5, 11). The focus of this report is on the mycotoxin T-2 and some of its metabolites, HT-2, T-2 triol, and T-2 tetraol (19, 27). Previous studies with the NR assay have shown its sensitivity in distinguishing parent compounds from their intermediate metabolites (3). T-2 is one of the more toxic of the 12,13-epoxytrichothecene mycotoxins produced by Fusarium species. Such mycotoxins have been the agents of illness and death in animals and humans ingesting moldy agricultural products. T-2 mycotoxin is a potent inhibitor of protein and scheduled DNA synthesis (18, 26) and has genotoxic properties, including unscheduled DNA repair synthesis (18) and chromosomal aberrations (14). All cell types used in this study are of human origin. A foreskin fibroblast cell line, designated HFF, and the hepatoma cell line HepG2 were grown in Dulbecco minimal essential medium supplemented with 2% fetal bovine serum and 10% Serum Plus (Hazelton Research Products, Lenexa, Kans.). The human melanoma cell line SK-Mel/27, the nonmalignant keratinocyte cell line HPK-1A, and normal kidney primary cell cultures, designated NK cells, were grown in Dulbecco minimal essential medium supplemented with 10% fetal bovine serum. The renal carcinoma cell line CAKI was grown in minimal essential medium supplemented with 10% fetal bovine serum, and normal umbilical vein endothelial primary cell cultures, designated HEC cells, were grown in medium 199 supplemented with 20% fetal bovine serum in plates treated with 50 ,ug of fibronectin per ml. All of the above-noted media were supplemented with 100 U of penicillin G per ml, 100 ,ug of streptomycin per ml, and 2.5 ,ug of amphotericin B (Fungizone) per ml. All cultures were passaged by dissociation in a solution of 0.05% trypsin-0.02% EDTA. Secondary cultures of normal breast keratinocytes, designated NHEK cells, were obtained from Clonetics Corp., San Diego, Calif., as was the serumfree, biochemically defined keratinocyte growth medium
syntheses by radiolabeling procedures. Such techniques, however, in addition to requiring expensive laboratory equipment and radioisotopes, generate radioactive refuse. A more practical approach to the use of mammalian cell culture cytotoxicity assays for quantitating the potencies of mycotoxins has been the application of the MTT colorimetric cell viability assay. In this assay, cells seeded into a 96-well microtiter plate are exposed to various concentrations of the test agent, with cell survival based on the ability of mitochondrial enzymes in viable cells to chemically reduce a yellow tetrazolium salt (MTT) to a purple formazan dye. Color formation is read on a microplate spectrophotometer (enzyme-linked immunosorbent assay [ELISA] reader) (12, 13, 21, 22). The MTT assay, initially developed by Mosmann (17), has undergone many modifications (8, 10) because of the less than optimal sensitivity and the difficulty in solubilizing the final formazan product. We now report on the use of a different colorimetric cell viability assay, the neutral red (NR) assay, to quantitate the potency of mycotoxins by using immortalized human cell lines and lowpassage human cell cultures as the bioindicators. The NR assay is based on the incorporation of the supravital dye, NR, into the lysosomes of viable cells after their incubation with toxic chemicals. This weakly cationic dye penetrates cell membranes by nonionic diffusion and binds intracellu* Corresponding author. 2101
2102
APPL. ENVIRON.
NOTES
TABLE 2. Comparison of mycotoxin toxicities in vitro and in vivo
TABLE 1. Comparative toxicities of T-2 toxin to human cell types Cell line
SK-Mel/27 NHEK HepG2 CAKI HPK-1A HFF HEC NK
Description
Melanoma cell line Second-passage keratinocyte Hepatoma cell line Renal carcinoma cell line Keratinocyte cell line Fibroblast cell line Second-passage endothelial cell Second-passage kidney cell
NR50 (ng/ml)a
2.8 5.6 9.8 35.4 35.7 42.3 44.8 72.1
MICROBIOL.
0.53 0.66 1.10 2.10 2.82 6.62 3.08 5.71
a NR50, midpoint cytotoxicity value after a 48-h exposure to T-2 mycotoxin. Results are the mean and standard deviation of several determinations.
(KGM). The NHEK cells were dissociated with 0.025% trypsin-0.01% EDTA for dispersal. All cell types were maintained at 37°C in a humidified incubator under 5.5% CO2.
Individual wells of a 96-well tissue culture microtiter plate inoculated with 0.2 ml of the appropriate media containing cells (usually 104 cells) to achieve a 65 to 75% confluence after attachment and addition of the test agents.
were
This allows for the target cells to complete an additional replication cycle; as such, the NR assay measures both cytotoxic and proliferative effects. After 1 to 2 days of incubation, the media were removed and replaced with unamended (control) medium or with medium amended with varied concentrations of the mycotoxin to be tested. After 2 days of exposure to the mycotoxin, the media were removed and replaced with media containing 40 ,ug of NR per ml. The NR-containing media had been preincubated overnight at 37°C and centrifuged (1,500 x g for 10 min) prior to use to remove fine precipitates of dye crystals. The assay plate was then returned to the incubator for another 3 h to allow for uptake of the supravital dye into the lysosomes of viable cells. Thereafter, the media were removed and the cells were rapidly washed with 0.5% formaldehyde-1% CaCl2 followed by 0.2 ml of a solution of 1% acetic acid-50% ethanol to extract the dye from the cells. After 10 min at room temperature and a brief but rapid agitation on a microtiter plate shaker, the plates were transferred to a microplate spectrophotometer equipped with a 540-nm filter to measure the absorbance of the extracted dye (6, 7). The toxicities of the various mycotoxins were compared with those of control cultures by computing, by linear regression analysis, the concentration of test agent needed to reduce the absorbance of the dye by 50% (i.e., the midpoint cytotoxicity [NR50] value). All experiments were performed at least four times, with six to eight wells for each concentration of test agent. The mycotoxins (all from Sigma) were solubilized in ethanol; the concentration of ethanol in the test media was always below 1% to avoid cytotoxicity of the solvent. The responses of the various human cell types to T-2 mycotoxin are presented in Table 1. The melanoma SKMel/27 cell line was the most sensitive, with an NR50 of 2.8 ng of T-2 mycotoxin per ml, and the secondary-passage kidney NK cells were the least sensitive, with an NR50 of 72.1 ng of T-2 mycotoxin per ml. This range of cytotoxicity
data for T-2, obtained with the NR assay, is within the range determined for other human cell lines with other cytotoxicity assays. Oldham et al. (18), using cell survival as the cytotoxicity end point with primary cultures of human foreskin fibroblasts, noted a midpoint cytotoxicity value of 70 ng of
afterL:
In vitro toxicity (ng/ml)
Mycotoxin
NR50
(HepG2)a T-2 HT-2 T-2 triol T-2 tetraol
9.8 15.8 398.4 445.3
+ 1.10
± 1.44 ± 27.54 ± 43.62
PSI50
(VERO)b 6.7 27.0 428.0
1,747.0
s.c.
i.p.
inoculation
inoculation
3.3 6.7 75.3 15.7
9.1 10.2
11.0
a Human hepatoma cell line; neutral red assay. b African green monkey cell line; protein synthesis inhibition (PSI) assay
(25).
c In vivo data from Thompson and Wannemacher (25). LD50, dose. s.c., subcutaneous; i.p., intraperitoneal.
50% lethal
T-2 per ml. Thompson and Wannemacher (24), using a protein synthesis inhibition assay, observed a midpoint cytotoxicity value of 17.3 ng of T-2 per ml for the human cervical carcinoma HeLa cell line and 12.5 ng of T-2 per ml for the human A-549 lung carcinoma cell line. Holt et al. (13), using the mitochondrially based MTT assay, derived a midpoint cytotoxicity value of 10.5 ng of T-2 per ml for the HeLa cell line. The mechanisms responsible for the differential sensitivities of the human cell types to T-2 mycotoxin are unknown. T-2 mycotoxin has both cytotoxic and proliferative effects (15). The greater susceptibility of the SK-Mel/27 melanoma cells to T-2 mycotoxin was not due to their proliferative capacity, as under the conditions of the assay, the cells were replicating only about every 30 h. T-2 toxin can be metabolized to a number of different intermediates by cells cultured in vitro (27). As noted by Holt et al. (13), who also observed distinctions in the sensitivity of human and other mammalian cell lines to T-2, the differences in sensitivity may partially reflect the differential abilities of the cells to act on and alter the toxin. The human hepatoma cell line HepG2 was used to differentiate among the cytotoxicities of T-2 mycotoxin and its metabolites, HT-2, T-2 triol, and T-2 tetraol. HepG2 was selected because it maintains elevated indigenous levels of enzymatic xenobiotic-metabolizing capacity (4). T-2 was more cytotoxic to the HepG2 cells than were the intermediate metabolites, as has been noted for other in vitro cytotoxicity assays involving human, monkey, and rodent cell lines (1, 18, 20, 25). On the basis of NR50, the sequence of cytotoxicity to HepG2 was T-2 > HT-2 > T-2 triol > T-2 tetraol (Table 2). This differential sequence of cytotoxicity was similar to that noted by Thompson and Wannemacher (25) when using a protein inhibition assay with the green monkey VERO cell line as the bioindicator. The T-2 parent compound therefore appears to be more toxic than its metabolites. It must be noted that the sequence of cytotoxicity determined in vitro, either with the NR assay or with the protein inhibition assay, did not completely parallel the in vivo sequence of toxicity. Thus, Thompson and Wannemacher (25) noted that after subcutaneous inoculation, the sequence of potency, based on mouse 50% lethal dose determinations, was T-2 > HT-2 > T-2 tetraol > T-2 triol. The mode of inoculation, however, greatly influenced the degree of acute toxicity (Table 2). The NR assay has been applied to evaluating and ranking the cytotoxicity of a spectrum of chemicals, including inorganic metals, organometals, and various industrial, pharma-
VOL. 57, 1991
ceutical, and environmental organics (see reference 2 for a review). This report described the first application of the NR assay to the assessment of natural toxins. The advantages of the NR assay, which include its simplicity, speed, reproducibility of data, low cost, and sensitivity to test agents, make it a useful tool for detecting and quantitating the potencies of chemical test agents, including natural toxins. Great appreciation is expressed to M. Eisinger, Memorial SloanKettering Cancer Center, New York, N.Y., for the HFF and SK-Mel/27 cells; to B. B. Knowles, Wistar Institute, Philadelphia, Pa., for the HepG2 cells; to J. S. Rhim, National Cancer Institute, Bethesda, Md., for the HPK-1A cells; to N. H. Bander, Memorial Sloan-Kettering Cancer Center, for the NK cells; to L. Pfeffer, The Rockefeller University, for the CAKI cells; to W. A. Muller, The Rockefeller University, for the HEC cells; and to Clonetics Corp., San Diego, Calif., for the NHEK cells and KGM medium. This research was supported, in part, by Hoffmann-LaRoche.
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