- Email: [email protected]
Evidence for delayed cytotoxicity effects following exposure of rat hepatoma-derived Fa32 cells: implications for predicting human acute toxicity
Toxicology in Vitro 17 (2003) 797–801 www.elsevier.com/locate/toxinvit
Evidence for delayed cytotoxicity effects following exposure of rat hepatoma-derived Fa32 cells: implications for predicting human acute toxicity Paul J. Dierickx* Instituut voor Volksgezondheid, Afdeling Toxikologie, Laboratorium Biochemische Toxikologie, Wytsmanstraat 14, B-1050 Brussels, Belgium Accepted 30 June 2003
Abstract The delayed cytotoxicity of the Multicentre Evaluation of In vitro Cytotoxicity (MEIC) reference chemicals was investigated in rat hepatoma-derived Fa32 cells. The cells were treated for 24 h with the test chemicals, and were than further cultured for 5 days in normal culture medium. The cytotoxicity was measured by the neutral red uptake inhibition, and the results were quantified by determining the NI50del. This is the concentration of test compound required to decrease the neutral red uptake with 50% compared with control cells. The results were compared with the acute NI50, the corresponding value measured immediately after 24 h treatment of the cells. On a total of 44 chemicals, nine showed delayed cytotoxicity (NI50del lower than or equal to NI50), 11 a probably delayed, and 24 no delayed cytotoxicity (NI50del more than 1.5NI50). When the NI50del was compared with human toxicity, a correlation coefficient r2=0.761 was obtained. For the same series of 44 chemicals this correlation was clearly higher than that for human hepatoma-derived Hep G2 cells (r2=0.695). The Hep G2 assay was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. Consequently, the delayed cytotoxicity assay on cultured Fa32 cells has the best prediction value so far obtained for the human toxicity. # 2003 Elsevier Ltd. All rights reserved. Keywords: Fa32 cells; Delayed cytotoxicity; Neutral red uptake; MEIC; Correlation human toxicity
1. Introduction The Multicentre Evaluation of In vitro Cytotoxicity (MEIC) programme was initiated by the Scandinavian Society of Cell Toxicology in order to evaluate the relevance of in vitro toxicity assays for acute human toxicity (Bondesson et al., 1989). The final results have now been published in extenso (Clemedson et al., 1996a,b, 1998; Ekwall et al., 1998a,b, 2000) and are summarized in two other papers (Clemedson and Ekwall, 1999; Ekwall, 1999). One of the problems in foregoing in vitro/in vivo comparisons was that quantitative, mutually comparable acute human toxicity data were not available. In vitro toxicity data were, therefore, compared with aniAbbreviations: MEIC, Multicentre Evaluation of In vitro Cytotoxicity; PBS, phosphate buffered saline. * Tel.: +32-2-642-5107; fax: +32-2-642-5224. E-mail address: [email protected] (P.J. Dierickx). 0887-2333/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0887-2333(03)00124-3
mal toxicity data. However, the use of cytotoxicity assays for the prediction of the toxicity is in the first place orientated towards toxicity in humans. An important accomplishment of the MEIC study was that acute toxicity data for humans were established (Ekwall et al., 1998b). All available data from handbooks, poison centres, etc. were gathered and processed as described (Ekwall et al., 1998b). The peak concentration of the approximate LC50 curve, expressed as the logM, appeared to be the best measure for human acute toxicity. Another important conclusion from the MEIC study is that in vitro systems predict the acute human toxicity better than rat or mice models (Ekwall et al., 1998a). We participated in the MEIC programme with a 24 h test on human hepatoma-derived Hep G2 cells, measuring the total protein content. This assay was shown to be the best acute in vitro test for the prediction of the human toxicity (Fig. 9 in Ekwall et al., 2000). Later, we investigated the cytotoxicity of the MEIC reference
798
P.J. Dierickx / Toxicology in Vitro 17 (2003) 797–801
chemicals in rat hepatoma-derived Fa32 cells in six different assays (Dierickx, 2000), in the presence of vitamin E (Dierickx et al., 2001) and by the neutral red uptake inhibition in adhered and adhering Fa32 cells (Dierickx and Scheers, 2002). These studies provided interesting information about the toxic mechanism of several chemicals, but the correlation with human toxicity was almost always identical to that in Hep G2 cells (Ekwall, 1999), except for the neutral red uptake after 24 h treatment of fixed cells, which showed a slightly higher correlation (Dierickx and Scheers, 2002). In order to improve the correlation between the in vitro results and logM, we hypothesized that a longer assay schedule could increase the correlation. Indeed, besides the above mentioned 24 h test on Hep G2 cells, we participated in the MEIC programme with a longterm (6 weeks) cytotoxicity test on human embryonic lung cells (Dierickx and Ekwall, 1992, 1993). Surprisingly, this chronic cytotoxicity test gave the best correlation with human acute toxicity of all assays submitted (Fig. 9 in Ekwall et al., 2000), but it was withdrawn from the list of candidates for a battery because it was not an acute, and thus more laborious assay (Ekwall et al., 2000). In this study, the Fa32 cells were treated for 24 h with the MEIC chemicals, and than further cultured in normal culture medium for 5 days. The cytotoxicity measured after this total period of 6 days is called the delayed cytotoxicity. We compared it with the acute toxicity and as a parameter for the prediction of human toxixity.
2. Materials and methods The MEIC reference chemicals were of the same origin as described (Clemedson et al., 1996a, 1998). Established Fa32 cells, derived from a rat hepatoma, were provided by Professor Szpirer (Universite´ Libre de Bruxelles, Brussels, Belgium). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% foetal calf serum, 100 units/ml penicillin, and 100 mg/ml streptomycin (complete medium). The cells were cultured at 37 C in a humidified 5% CO2 atmosphere. They were grown in 50 cm2 Petri dishes (Nunc, Roskilde, Denmark) and were harvested by trypsinisation. The cytotoxicity was measured using the slightly modified (Brandao et al., 1992) neutral red uptake inhibition assay (Borenfreund and Shopsis, 1985). Briefly, 60 000 cells were seeded in each of the 64 wells of a microtitre plate (Nunc). After 24 h, the growing cellsfixed on the bottom, were treated with different concentrations of the freshly prepared test compounds in complete medium (eight wells/concentration) for 24 h. For the chemicals solubilized in 2% ethanol the control
wells contained the same amount of ethanol, which had no influence on the growth of the cells in comparison with cells cultured in normal complete medium. After 24 h the cells were washed with 200 ml phosphate buffered saline (PBS)/well and than received 200 ml complete medium for 5 further days. The relative toxicity of the test compounds was established by the determination of the concentration of test compound required to induce a 50% inhibition in neutral red uptake as compared with control cells. The 50% inhibitory concentrations were extrapolated from dose–response effect curves by linear regression analysis. In order to characterize quantitatively the two different assays, the following symbols are used: NI50: the concentration (in mM) of test compound required to reduce the neutral red uptake with 50% as compared with the control cells, when the cells were treated for 24 h (Dierickx and Scheers, 2002). NI50del: the concentration (in mM) of test compound required to reduce the neutral red uptake with 50% as compared with the control cells, when the cells were first treated for 24 h with the test compounds and than further cultivated in normal complete medium for 5 days. The reproducibility of the assays was examined on several occasions. Because of the small standard deviations most experiments were performed only twice. The results of the cytotoxicity experiments are expressed in millimolar concentrations. These values were transformed to logM values for correlation calculations with human acute toxicity (Ekwall et al., 1998b) by linear regression analysis.
3. Results Some chemicals were not tested, mainly because of solubility limitations. Nevertheless, the chemical numbering for the complete series of the MEIC reference chemicals is followed in this paper in order to facilitate comparisons with other publications. The results of the complete series of tested chemicals are summarized in Table 1, comparing the delayed with the acute cytotoxicity. The effects of the different treatment schedules on the neutral red uptake inhibition are visualized in Fig. 1. For malathion, potassium cyanide, arsenic trioxide, mercuric chloride and chloroquine (chemicals 15, 19, 26, 28, 41) a lower NI50del was observed than the NI50. This means that these chemicals are more toxic when further cultivated for 5 days in normal culture medium after the normal 24 h treatment, and thus that these chemicals have a delayed cytotoxic effect. The same holds true for carbon chloride, orphenadrine, amphetamine and potassium chloride (chemicals
799
P.J. Dierickx / Toxicology in Vitro 17 (2003) 797–801 Table 1 The cytotoxicity of the MEIC reference chemicals in Fa32 cells, measured by the neutral red uptake inhibition MEIC compound number
Compound
N150 (24 h)a (nM)
N150dele (nM)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Paracetamol Acetylsalicyclic acid Ferrous sulfate 7H2Ob Diazepam Amitriptyline HCl Digoxin Ethylene glycol Methanol Ethanol Isopropanol 1,1,1-Trichloroethanec Phenol Sodium chloride Sodium fluoride Malathionc 2,4-Dichlorophenoxyacetic acid Xylene 1-Nicotine Potassium cyanide Lithium sulfate H2O Theophylline d-Propoxyphene. HCl dl-Propranolol. HCl Phenobarbital (sodium salt) Paraquat Arsenic trioxide Cupric sulfate. 5H2O Mercuric chloride Thioridazine. HCl Thallium(I)sulfate Warfarin (sodium salt) Lindane Chloroformc Carbon tetrachloridec Isoniazid Dichloromethanec Barium nitrate Hexachlorophenec Pentachlorophenol Verapamil. HClb Chloroquine (diphosphate salt) Orphenadrine. HCl Quinidine sulfate. 2H2O Phenytoin (sodium salt) Chloramphenicol Sodium oxalateb dl-Amphetamine sulfate Caffeine Atropine sulfate Potassium chloride
21 11 7.5 —d 0.046 —d 1604 1948 671 316 6.0 3.75 84 2.4 0.34 4.5 —d 2.9 9.7 57 6.5 0.29 0.064 9.6 4.1 0.107 0.91 0.047 0.014 0.66 1.8 —d 18 7.8 93 105 —d 0.019 0.108 0.162 0.029 0.258 0.158 —d 4.3 16 1.66 6.1 0.78 102
432 27 (24–30) 9.1 (8.7–9.5) —d 0.101 (0.094–0.108) —d 2082 (1955–2209) 2559 (2320–2798) 873 (831–915) 53314 36 (32–40) 8.9 (7.9–9.9) 178 (163–193) 4.4 (4.0–4.8) 0.23 (0.19–0.27) 7.6 (6.9–8.3) —d 4.90.3 2.6 (2.2–3.0) 1134 33.3 (29.8–36.8) 0.39 (0.37–0.41) 0.102 (0.097–0.107) 26 (22–30) 5.9 (5.2–6.6) 0.068 (0.064–0.072) 2.53 (2.9–2.67) 0.037 (0.032–0.042) 0.017 (0.014–0.020) 2.0 (1.7–2.3) 8.45 (8.04–8.86) —d 24 (22–26) 7.7 (7.1–8.3) 125 (117–133) 232 (214–250) —d 0.164– (0.147–0.181) 0.573 (0.531–0.615) 0.238 (0.215–0.261) 0.022 (0.019–0.025) 0.2660.011 0.237 (0.221–0.253) —d 7.4 (6.8–8.0) 24.7 (23.3–26.1) 1.71 (1.41–2.01) 25.7 (23.1–28.3) 1.06 (0.98–1.14) 1055
a b c d e
Results taken from Dierickx and Scheers (2002). Tested as a suspension. Solubilized in 2% ethanol. Not tested because of solubility limitations. Standard deviations are given for assays performed in triplicate, lowest and highest values are given for assays performed in duplicate.
800
P.J. Dierickx / Toxicology in Vitro 17 (2003) 797–801
Fig. 1. The delayed cytotoxicity of the MEIC reference chemicals in Fa32 cells as compared with the acute cytotoxicity.
34, 42, 47, 50) for which the NI50del was almost equal to the NI50. The NI50del of 16 chemicals was 2–8.6 higher than the NI50 (chemicals 1, 2, 5, 11, 12, 13, 20, 21, 24, 27, 30, 31, 36, 38, 39, 48), suggesting that these chemicals did not have a delayed cytotoxic effect under these experimental conditions. The NI50del of the remainder 19 chemicals was 1–2 higher than the NI50.
4. Discussion From the protocol it is evident that chemicals without delayed cytotoxic effect have higher NI50del than NI50 values. It is not evident, however, how much the NI50del value must increase as compared with the NI50 to judge if a chemical has delayed cytotoxicity or not. For 11 of the above mentioned series of 19 chemicals with a 1–2 higher NI50del, the latter is increased only with 20–50%: ferrous sulfate, ethylene glycol, methanol, ethanol, d-propoxyphene, paraquat, thioridazine, chloroform, isoniazid, verapamil and quinidine sulfate (chemicals 3, 7, 8, 9, 22, 25, 29, 33, 35, 40, 43). Therefore, these chemicals probably also have a delayed cytotoxic effect. The other eight chemicals of this series of 19 chemicals can be considered as showing an impaired recovery, while the 16 chemicals with a NI50del 2–8.6 higher than the NI50 were fully recovering from the 24 h toxic insult after five additional days in normal complete culture medium. Altogether, 20 of the 44 investigated MEIC chemicals show delayed cytotoxicity, at least when a NI50del lower than 1.5 the 24 h acute NI50 can be considered as the limit value. We hypothesized that delayed cytotoxicity could have a better predictive value for human toxicity than acute
Fig. 2. Correlation between the delayed cytotoxicty of the MEIC reference chemicals in Fa32 cells and human toxicity.
cytotoxicity. A good correlation was observed between acute and delayed cytotoxicity as measured by the neutral red uptake in Fa32 cells (r2=0.955). As already mentioned, our previous 24 h total protein content assay in Hep G2 cells (Clemedson et al., 1996a, 1998) was the best acute in vitro test for the prediction of human toxicity within the MEIC study (Ekwall, 1999). When comparing the logNI50 in Fa32 cells with the human toxicity, expressed as the peak concentration of the approximate LC50 curve (Ekwall et al., 1998a), a correlation coefficient r2=0.722 (n=44) was previously obtained for exactly the same series of chemicals as investigated in this study (Dierickx and Scheers, 2002). This value was slightly higher than the corresponding
P.J. Dierickx / Toxicology in Vitro 17 (2003) 797–801
one for Hep G2 cells (r2=0.695), indicating that rat hepatoma-derived Fa32 cells can be used as well as human hepatoma-derived Hep G2 cells for the prediction of human toxicity. The correlation between the NI50del and the peak logM was better than any other obtained before: r2=0.761 (n=44, Fig. 2). This means that the delayed cytotoxicity in Fa32 cells has the best prediction value so far obtained for the human toxicity. The very high prediction value of the delayed cytotoxicity assay described in this study is very encouraging for further research. The correlation can probably further increase by introducing other parameters, such as bioavailability, which plays an important role on the cytotoxicity of MEIC and other chemicals (Gu¨lden et al., 2002; Seibert et al., 2002).
Acknowledgements I thank Roel Anthonissen for his excellent technical assistance.
References Brandao, J.C., Bohets, H.H.I., Van de Vyver, I.E., Dierickx, P.J., 1992. Correlation between the in vitro cytotoxicity to cultured fathead minnow fish cells and fish lethality data for 50 chemicals. Chemosphere 25, 553–562. Bondesson, I., Ekwall, B., Hellberg, S., Romert, L., Stenberg, K., Walum, E., 1989. MEIC- A new international multicenter project to evaluate the relevance to human toxicity of in vitro cytotoxicity tests. Cell Biology and Toxicology 5, 331–347. Borenfreund, E., Shopsis, C., 1985. Toxicity monitored with a correlated set of cell-culture assays. Xenobiotica 15, 705–711. Clemedson, C., McFarlane-Abdulla, E., Andersson, M., Barile, F.A., Calleja, M.C., Chesne´, C., Clothier, R., Cottin, M., Curren, R., Daniel-Szolgay, E., Dierickx, P., Ferro, M., Fiskesjo¨, G., GarzaOcanas, L., Gomez-Lechon, M.J., Gu¨lden, M., Isomaa, B., Janus, J., Judge, P., Kahru, A., Kemp, R.B., Kerszman, G., Kristen, U., Kunimoto, M., Ka¨renlampi, S., Lavrijsen, K., Lewan, H., Ohno, T., Persoone, G., Roguet, R., Romert, L., Sawyer, T.W., Seibert, H., Shrivastava, R., Stammati, A., Tanaka, N., Torres-Alanis, O., Voss, J.-U., Wakuri, S., Walum, E., Wang, X., Zucco, F., Ekwall, B., 1996a. MEIC evaluation of acute systemic toxicity. Part I. Methodology of 68 in vitro toxicity assays used to test the first 30 reference chemicals. ATLA 24, 251–272. Clemedson, C., McFarlane-Abdulla, E., Andersson, M., Barile, F.A., Calleja, M.C., Chesne´, C., Clothier, R., Cottin, M., Curren, R., Dierickx, P., Ferro, M., Fiskesjo¨, G., Garza-Ocanas, L., GomezLechon, M.J., Gu¨lden, M., Isomaa, B., Janus, J., Judge, P., Kahru, A., Kemp, R.B., Kerszman, G., Kristen, U., Kunimoto, M., Ka¨renlampi, S., Lavrijsen, K., Lewan, H., Lilius, H., Malmsten, A., Ohno, T., Persoone, G., Pettersson, R.G., Roguet, R., Romert, L., Sandberg, M., Sawyer, T.W., Seibert, H., Shrivastava, R., Sjo¨stro¨m, M., Stammati, A., Tanaka, N., Torres-Alanis, O., Voss, J.-U.,
801
Wakuri, S., Walum, E., Wang, X., Zucco, F., Ekwall, B., 1996b. MEIC evaluation of acute systemic toxicity. Part II. In vitro results from 68 toxicity assays used to test the first 30 reference chemicals and a comparative cytotoxicity analysis. ATLA 24, 273–311. Clemedson, C., Andersson, M., Aoki, Y., Barile, F.A., Bassi, A.M., Calleja, M.C., Castano, A., Clothier, R., Dierickx, P., Ekwall, B., Ferro, M., Fiskesjo¨, G., Garza-Ocanas, L., Gomez-Lechon, M.J., Gu¨lden, M., Hall, T., Imai, K., Isomaa, B., Kahru, A., Kerszman, G., Kjellstrand, P., Kristen, U., Kunimoto, M., Ka¨renlampi, S., Lewan, H., Lilius, H., Loukianov, A., Monaco, F., Ohno, T., Persoone, G., Romert, L., Sawyer, T.W., Segner, H., Seibert, H., Shrivastava, R., Sjo¨stro¨m, M., Stammati, A., Tanaka, N., Thuvander, A., Torres-Alanis, O., Valentino, M., Wakuri, S., Walum, E., Wang, X., Wieslander, A., Zucco, F., Ekwall, B., 1998. MEIC evaluation of acute systemic toxicity. Part IV. In vitro results from 67 toxicity assays used to test reference chemicals 31–50 and a comparative cytotoxicity analysis. ATLA 26, 131–183. Clemedson, C., Ekwall, B., 1999. Overview of the final MEIC results: I. The in vitro–in vivo evaluation. Toxicology In Vitro 13, 657–663. Dierickx, P.J., 2000. The cytotoxicity of the MEIC reference chemicals in rat hepatoma-derived Fa32 cells. Toxicology 150, 159–169. Dierickx, P.J., Ekwall, B., 1992. Long-term cytotoxicity testing of the first twenty MEIC chemicals by the determination of the protein content in human embryonic lung cells. ATLA 20, 285–289. Dierickx, P.J., Ekwall, B., 1993. Long-term cytotoxicity testing of the MEIC chemicals 21–50 by the determination of the protein content in human embryonic lung cells. AATEX 2, 33–36. Dierickx, P.J., Scheers, E.M., 2002. Neutral red uptake inhibition in adhered and adhering rat hepatoma-derived Fa32 cells to predict human toxicity. Journal of Applied Toxicology 22, 61–65. Dierickx, P.J., Smit, C., Scheers, E.M., 2001. Cytotoxicity of the MEIC reference chemicals in antioxidant-enriched rat hepatomaderived Fa32 cells. ATLA 29, 217–223. Ekwall, B., 1999. Overview of the final MEIC results: II. The in vitro– in vivo evaluation, including the selection of a practical battery of cell tests for prediction of acute lethal blood concentrations in humans. Toxicology In Vitro 13, 665–673. Ekwall, B., Barile, F.A., Castano, A., Clemedson, C., Clothier, R., Dierickx, P., Ekwall, B., Ferro, M., Fiskesjo¨, G., Garza-Ocanas, L., Gomez-Lechon, M.J., Gu¨lden, M., Hall, T., Isomaa, B., Kahru, A., Kerszman, G., Kristen, U., Kunimoto, M., Ka¨renlampi, S., Lewan, H., Loukianov, A., Ohno, T., Persoone, G., Romert, L., Sawyer, T.W., Shrivastava, R., Segner, H., Stammati, A., Tanaka, N., Valentino, M., Walum, E., Zucco, F., 1998a. MEIC evaluation of acute systemic toxicity. Part VI. The prediction of human toxicity by rodent LD50 values and results from 61 in vitro methods. ATLA 26, 617–658. Ekwall, B., Clemedson, C., Crafoord, B., Ekwall, B., Hallander, S., Walum, E., Bondesson, I., 1998b. MEIC evaluation of acute systemic toxicity. Part V. Rodent and human toxicity data for the 50 reference chemicals. ATLA 26, 571–616. Ekwall, B., Ekwall, B., Sjo¨stro¨m, M., 2000. MEIC evaluation of acute systemic toxicity. Part VIII. Multivariate partial least squares evaluation, including the selection of a battery of cell lines with a good prediction of human acute lethal peak blood concentrations for 50 chemicals. ATLA 28, 201–234. Gu¨lden, M., Mo¨rchel, S., Tahan, S., Seibert, H., 2002. Impact of protein binding on the availability and cytotoxic potency of organochlorine pesticides and chlorophenols in vitro. Toxicology 177, 201–213. Seibert, H., Mo¨rchel, S., Gu¨lden, M., 2002. Factors influencing nominal effective concentrations of chemical compounds in vitro: medium protein concentration. Toxicology In Vitro 16, 289–297.
Evidence for delayed cytotoxicity effects following exposure of rat hepatoma-derived Fa32 cells: implications for predicting human acute toxicity Paul J. Dierickx* Instituut voor Volksgezondheid, Afdeling Toxikologie, Laboratorium Biochemische Toxikologie, Wytsmanstraat 14, B-1050 Brussels, Belgium Accepted 30 June 2003
Abstract The delayed cytotoxicity of the Multicentre Evaluation of In vitro Cytotoxicity (MEIC) reference chemicals was investigated in rat hepatoma-derived Fa32 cells. The cells were treated for 24 h with the test chemicals, and were than further cultured for 5 days in normal culture medium. The cytotoxicity was measured by the neutral red uptake inhibition, and the results were quantified by determining the NI50del. This is the concentration of test compound required to decrease the neutral red uptake with 50% compared with control cells. The results were compared with the acute NI50, the corresponding value measured immediately after 24 h treatment of the cells. On a total of 44 chemicals, nine showed delayed cytotoxicity (NI50del lower than or equal to NI50), 11 a probably delayed, and 24 no delayed cytotoxicity (NI50del more than 1.5NI50). When the NI50del was compared with human toxicity, a correlation coefficient r2=0.761 was obtained. For the same series of 44 chemicals this correlation was clearly higher than that for human hepatoma-derived Hep G2 cells (r2=0.695). The Hep G2 assay was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. Consequently, the delayed cytotoxicity assay on cultured Fa32 cells has the best prediction value so far obtained for the human toxicity. # 2003 Elsevier Ltd. All rights reserved. Keywords: Fa32 cells; Delayed cytotoxicity; Neutral red uptake; MEIC; Correlation human toxicity
1. Introduction The Multicentre Evaluation of In vitro Cytotoxicity (MEIC) programme was initiated by the Scandinavian Society of Cell Toxicology in order to evaluate the relevance of in vitro toxicity assays for acute human toxicity (Bondesson et al., 1989). The final results have now been published in extenso (Clemedson et al., 1996a,b, 1998; Ekwall et al., 1998a,b, 2000) and are summarized in two other papers (Clemedson and Ekwall, 1999; Ekwall, 1999). One of the problems in foregoing in vitro/in vivo comparisons was that quantitative, mutually comparable acute human toxicity data were not available. In vitro toxicity data were, therefore, compared with aniAbbreviations: MEIC, Multicentre Evaluation of In vitro Cytotoxicity; PBS, phosphate buffered saline. * Tel.: +32-2-642-5107; fax: +32-2-642-5224. E-mail address: [email protected] (P.J. Dierickx). 0887-2333/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0887-2333(03)00124-3
mal toxicity data. However, the use of cytotoxicity assays for the prediction of the toxicity is in the first place orientated towards toxicity in humans. An important accomplishment of the MEIC study was that acute toxicity data for humans were established (Ekwall et al., 1998b). All available data from handbooks, poison centres, etc. were gathered and processed as described (Ekwall et al., 1998b). The peak concentration of the approximate LC50 curve, expressed as the logM, appeared to be the best measure for human acute toxicity. Another important conclusion from the MEIC study is that in vitro systems predict the acute human toxicity better than rat or mice models (Ekwall et al., 1998a). We participated in the MEIC programme with a 24 h test on human hepatoma-derived Hep G2 cells, measuring the total protein content. This assay was shown to be the best acute in vitro test for the prediction of the human toxicity (Fig. 9 in Ekwall et al., 2000). Later, we investigated the cytotoxicity of the MEIC reference
798
P.J. Dierickx / Toxicology in Vitro 17 (2003) 797–801
chemicals in rat hepatoma-derived Fa32 cells in six different assays (Dierickx, 2000), in the presence of vitamin E (Dierickx et al., 2001) and by the neutral red uptake inhibition in adhered and adhering Fa32 cells (Dierickx and Scheers, 2002). These studies provided interesting information about the toxic mechanism of several chemicals, but the correlation with human toxicity was almost always identical to that in Hep G2 cells (Ekwall, 1999), except for the neutral red uptake after 24 h treatment of fixed cells, which showed a slightly higher correlation (Dierickx and Scheers, 2002). In order to improve the correlation between the in vitro results and logM, we hypothesized that a longer assay schedule could increase the correlation. Indeed, besides the above mentioned 24 h test on Hep G2 cells, we participated in the MEIC programme with a longterm (6 weeks) cytotoxicity test on human embryonic lung cells (Dierickx and Ekwall, 1992, 1993). Surprisingly, this chronic cytotoxicity test gave the best correlation with human acute toxicity of all assays submitted (Fig. 9 in Ekwall et al., 2000), but it was withdrawn from the list of candidates for a battery because it was not an acute, and thus more laborious assay (Ekwall et al., 2000). In this study, the Fa32 cells were treated for 24 h with the MEIC chemicals, and than further cultured in normal culture medium for 5 days. The cytotoxicity measured after this total period of 6 days is called the delayed cytotoxicity. We compared it with the acute toxicity and as a parameter for the prediction of human toxixity.
2. Materials and methods The MEIC reference chemicals were of the same origin as described (Clemedson et al., 1996a, 1998). Established Fa32 cells, derived from a rat hepatoma, were provided by Professor Szpirer (Universite´ Libre de Bruxelles, Brussels, Belgium). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% foetal calf serum, 100 units/ml penicillin, and 100 mg/ml streptomycin (complete medium). The cells were cultured at 37 C in a humidified 5% CO2 atmosphere. They were grown in 50 cm2 Petri dishes (Nunc, Roskilde, Denmark) and were harvested by trypsinisation. The cytotoxicity was measured using the slightly modified (Brandao et al., 1992) neutral red uptake inhibition assay (Borenfreund and Shopsis, 1985). Briefly, 60 000 cells were seeded in each of the 64 wells of a microtitre plate (Nunc). After 24 h, the growing cellsfixed on the bottom, were treated with different concentrations of the freshly prepared test compounds in complete medium (eight wells/concentration) for 24 h. For the chemicals solubilized in 2% ethanol the control
wells contained the same amount of ethanol, which had no influence on the growth of the cells in comparison with cells cultured in normal complete medium. After 24 h the cells were washed with 200 ml phosphate buffered saline (PBS)/well and than received 200 ml complete medium for 5 further days. The relative toxicity of the test compounds was established by the determination of the concentration of test compound required to induce a 50% inhibition in neutral red uptake as compared with control cells. The 50% inhibitory concentrations were extrapolated from dose–response effect curves by linear regression analysis. In order to characterize quantitatively the two different assays, the following symbols are used: NI50: the concentration (in mM) of test compound required to reduce the neutral red uptake with 50% as compared with the control cells, when the cells were treated for 24 h (Dierickx and Scheers, 2002). NI50del: the concentration (in mM) of test compound required to reduce the neutral red uptake with 50% as compared with the control cells, when the cells were first treated for 24 h with the test compounds and than further cultivated in normal complete medium for 5 days. The reproducibility of the assays was examined on several occasions. Because of the small standard deviations most experiments were performed only twice. The results of the cytotoxicity experiments are expressed in millimolar concentrations. These values were transformed to logM values for correlation calculations with human acute toxicity (Ekwall et al., 1998b) by linear regression analysis.
3. Results Some chemicals were not tested, mainly because of solubility limitations. Nevertheless, the chemical numbering for the complete series of the MEIC reference chemicals is followed in this paper in order to facilitate comparisons with other publications. The results of the complete series of tested chemicals are summarized in Table 1, comparing the delayed with the acute cytotoxicity. The effects of the different treatment schedules on the neutral red uptake inhibition are visualized in Fig. 1. For malathion, potassium cyanide, arsenic trioxide, mercuric chloride and chloroquine (chemicals 15, 19, 26, 28, 41) a lower NI50del was observed than the NI50. This means that these chemicals are more toxic when further cultivated for 5 days in normal culture medium after the normal 24 h treatment, and thus that these chemicals have a delayed cytotoxic effect. The same holds true for carbon chloride, orphenadrine, amphetamine and potassium chloride (chemicals
799
P.J. Dierickx / Toxicology in Vitro 17 (2003) 797–801 Table 1 The cytotoxicity of the MEIC reference chemicals in Fa32 cells, measured by the neutral red uptake inhibition MEIC compound number
Compound
N150 (24 h)a (nM)
N150dele (nM)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Paracetamol Acetylsalicyclic acid Ferrous sulfate 7H2Ob Diazepam Amitriptyline HCl Digoxin Ethylene glycol Methanol Ethanol Isopropanol 1,1,1-Trichloroethanec Phenol Sodium chloride Sodium fluoride Malathionc 2,4-Dichlorophenoxyacetic acid Xylene 1-Nicotine Potassium cyanide Lithium sulfate H2O Theophylline d-Propoxyphene. HCl dl-Propranolol. HCl Phenobarbital (sodium salt) Paraquat Arsenic trioxide Cupric sulfate. 5H2O Mercuric chloride Thioridazine. HCl Thallium(I)sulfate Warfarin (sodium salt) Lindane Chloroformc Carbon tetrachloridec Isoniazid Dichloromethanec Barium nitrate Hexachlorophenec Pentachlorophenol Verapamil. HClb Chloroquine (diphosphate salt) Orphenadrine. HCl Quinidine sulfate. 2H2O Phenytoin (sodium salt) Chloramphenicol Sodium oxalateb dl-Amphetamine sulfate Caffeine Atropine sulfate Potassium chloride
21 11 7.5 —d 0.046 —d 1604 1948 671 316 6.0 3.75 84 2.4 0.34 4.5 —d 2.9 9.7 57 6.5 0.29 0.064 9.6 4.1 0.107 0.91 0.047 0.014 0.66 1.8 —d 18 7.8 93 105 —d 0.019 0.108 0.162 0.029 0.258 0.158 —d 4.3 16 1.66 6.1 0.78 102
432 27 (24–30) 9.1 (8.7–9.5) —d 0.101 (0.094–0.108) —d 2082 (1955–2209) 2559 (2320–2798) 873 (831–915) 53314 36 (32–40) 8.9 (7.9–9.9) 178 (163–193) 4.4 (4.0–4.8) 0.23 (0.19–0.27) 7.6 (6.9–8.3) —d 4.90.3 2.6 (2.2–3.0) 1134 33.3 (29.8–36.8) 0.39 (0.37–0.41) 0.102 (0.097–0.107) 26 (22–30) 5.9 (5.2–6.6) 0.068 (0.064–0.072) 2.53 (2.9–2.67) 0.037 (0.032–0.042) 0.017 (0.014–0.020) 2.0 (1.7–2.3) 8.45 (8.04–8.86) —d 24 (22–26) 7.7 (7.1–8.3) 125 (117–133) 232 (214–250) —d 0.164– (0.147–0.181) 0.573 (0.531–0.615) 0.238 (0.215–0.261) 0.022 (0.019–0.025) 0.2660.011 0.237 (0.221–0.253) —d 7.4 (6.8–8.0) 24.7 (23.3–26.1) 1.71 (1.41–2.01) 25.7 (23.1–28.3) 1.06 (0.98–1.14) 1055
a b c d e
Results taken from Dierickx and Scheers (2002). Tested as a suspension. Solubilized in 2% ethanol. Not tested because of solubility limitations. Standard deviations are given for assays performed in triplicate, lowest and highest values are given for assays performed in duplicate.
800
P.J. Dierickx / Toxicology in Vitro 17 (2003) 797–801
Fig. 1. The delayed cytotoxicity of the MEIC reference chemicals in Fa32 cells as compared with the acute cytotoxicity.
34, 42, 47, 50) for which the NI50del was almost equal to the NI50. The NI50del of 16 chemicals was 2–8.6 higher than the NI50 (chemicals 1, 2, 5, 11, 12, 13, 20, 21, 24, 27, 30, 31, 36, 38, 39, 48), suggesting that these chemicals did not have a delayed cytotoxic effect under these experimental conditions. The NI50del of the remainder 19 chemicals was 1–2 higher than the NI50.
4. Discussion From the protocol it is evident that chemicals without delayed cytotoxic effect have higher NI50del than NI50 values. It is not evident, however, how much the NI50del value must increase as compared with the NI50 to judge if a chemical has delayed cytotoxicity or not. For 11 of the above mentioned series of 19 chemicals with a 1–2 higher NI50del, the latter is increased only with 20–50%: ferrous sulfate, ethylene glycol, methanol, ethanol, d-propoxyphene, paraquat, thioridazine, chloroform, isoniazid, verapamil and quinidine sulfate (chemicals 3, 7, 8, 9, 22, 25, 29, 33, 35, 40, 43). Therefore, these chemicals probably also have a delayed cytotoxic effect. The other eight chemicals of this series of 19 chemicals can be considered as showing an impaired recovery, while the 16 chemicals with a NI50del 2–8.6 higher than the NI50 were fully recovering from the 24 h toxic insult after five additional days in normal complete culture medium. Altogether, 20 of the 44 investigated MEIC chemicals show delayed cytotoxicity, at least when a NI50del lower than 1.5 the 24 h acute NI50 can be considered as the limit value. We hypothesized that delayed cytotoxicity could have a better predictive value for human toxicity than acute
Fig. 2. Correlation between the delayed cytotoxicty of the MEIC reference chemicals in Fa32 cells and human toxicity.
cytotoxicity. A good correlation was observed between acute and delayed cytotoxicity as measured by the neutral red uptake in Fa32 cells (r2=0.955). As already mentioned, our previous 24 h total protein content assay in Hep G2 cells (Clemedson et al., 1996a, 1998) was the best acute in vitro test for the prediction of human toxicity within the MEIC study (Ekwall, 1999). When comparing the logNI50 in Fa32 cells with the human toxicity, expressed as the peak concentration of the approximate LC50 curve (Ekwall et al., 1998a), a correlation coefficient r2=0.722 (n=44) was previously obtained for exactly the same series of chemicals as investigated in this study (Dierickx and Scheers, 2002). This value was slightly higher than the corresponding
P.J. Dierickx / Toxicology in Vitro 17 (2003) 797–801
one for Hep G2 cells (r2=0.695), indicating that rat hepatoma-derived Fa32 cells can be used as well as human hepatoma-derived Hep G2 cells for the prediction of human toxicity. The correlation between the NI50del and the peak logM was better than any other obtained before: r2=0.761 (n=44, Fig. 2). This means that the delayed cytotoxicity in Fa32 cells has the best prediction value so far obtained for the human toxicity. The very high prediction value of the delayed cytotoxicity assay described in this study is very encouraging for further research. The correlation can probably further increase by introducing other parameters, such as bioavailability, which plays an important role on the cytotoxicity of MEIC and other chemicals (Gu¨lden et al., 2002; Seibert et al., 2002).
Acknowledgements I thank Roel Anthonissen for his excellent technical assistance.
References Brandao, J.C., Bohets, H.H.I., Van de Vyver, I.E., Dierickx, P.J., 1992. Correlation between the in vitro cytotoxicity to cultured fathead minnow fish cells and fish lethality data for 50 chemicals. Chemosphere 25, 553–562. Bondesson, I., Ekwall, B., Hellberg, S., Romert, L., Stenberg, K., Walum, E., 1989. MEIC- A new international multicenter project to evaluate the relevance to human toxicity of in vitro cytotoxicity tests. Cell Biology and Toxicology 5, 331–347. Borenfreund, E., Shopsis, C., 1985. Toxicity monitored with a correlated set of cell-culture assays. Xenobiotica 15, 705–711. Clemedson, C., McFarlane-Abdulla, E., Andersson, M., Barile, F.A., Calleja, M.C., Chesne´, C., Clothier, R., Cottin, M., Curren, R., Daniel-Szolgay, E., Dierickx, P., Ferro, M., Fiskesjo¨, G., GarzaOcanas, L., Gomez-Lechon, M.J., Gu¨lden, M., Isomaa, B., Janus, J., Judge, P., Kahru, A., Kemp, R.B., Kerszman, G., Kristen, U., Kunimoto, M., Ka¨renlampi, S., Lavrijsen, K., Lewan, H., Ohno, T., Persoone, G., Roguet, R., Romert, L., Sawyer, T.W., Seibert, H., Shrivastava, R., Stammati, A., Tanaka, N., Torres-Alanis, O., Voss, J.-U., Wakuri, S., Walum, E., Wang, X., Zucco, F., Ekwall, B., 1996a. MEIC evaluation of acute systemic toxicity. Part I. Methodology of 68 in vitro toxicity assays used to test the first 30 reference chemicals. ATLA 24, 251–272. Clemedson, C., McFarlane-Abdulla, E., Andersson, M., Barile, F.A., Calleja, M.C., Chesne´, C., Clothier, R., Cottin, M., Curren, R., Dierickx, P., Ferro, M., Fiskesjo¨, G., Garza-Ocanas, L., GomezLechon, M.J., Gu¨lden, M., Isomaa, B., Janus, J., Judge, P., Kahru, A., Kemp, R.B., Kerszman, G., Kristen, U., Kunimoto, M., Ka¨renlampi, S., Lavrijsen, K., Lewan, H., Lilius, H., Malmsten, A., Ohno, T., Persoone, G., Pettersson, R.G., Roguet, R., Romert, L., Sandberg, M., Sawyer, T.W., Seibert, H., Shrivastava, R., Sjo¨stro¨m, M., Stammati, A., Tanaka, N., Torres-Alanis, O., Voss, J.-U.,
801
Wakuri, S., Walum, E., Wang, X., Zucco, F., Ekwall, B., 1996b. MEIC evaluation of acute systemic toxicity. Part II. In vitro results from 68 toxicity assays used to test the first 30 reference chemicals and a comparative cytotoxicity analysis. ATLA 24, 273–311. Clemedson, C., Andersson, M., Aoki, Y., Barile, F.A., Bassi, A.M., Calleja, M.C., Castano, A., Clothier, R., Dierickx, P., Ekwall, B., Ferro, M., Fiskesjo¨, G., Garza-Ocanas, L., Gomez-Lechon, M.J., Gu¨lden, M., Hall, T., Imai, K., Isomaa, B., Kahru, A., Kerszman, G., Kjellstrand, P., Kristen, U., Kunimoto, M., Ka¨renlampi, S., Lewan, H., Lilius, H., Loukianov, A., Monaco, F., Ohno, T., Persoone, G., Romert, L., Sawyer, T.W., Segner, H., Seibert, H., Shrivastava, R., Sjo¨stro¨m, M., Stammati, A., Tanaka, N., Thuvander, A., Torres-Alanis, O., Valentino, M., Wakuri, S., Walum, E., Wang, X., Wieslander, A., Zucco, F., Ekwall, B., 1998. MEIC evaluation of acute systemic toxicity. Part IV. In vitro results from 67 toxicity assays used to test reference chemicals 31–50 and a comparative cytotoxicity analysis. ATLA 26, 131–183. Clemedson, C., Ekwall, B., 1999. Overview of the final MEIC results: I. The in vitro–in vivo evaluation. Toxicology In Vitro 13, 657–663. Dierickx, P.J., 2000. The cytotoxicity of the MEIC reference chemicals in rat hepatoma-derived Fa32 cells. Toxicology 150, 159–169. Dierickx, P.J., Ekwall, B., 1992. Long-term cytotoxicity testing of the first twenty MEIC chemicals by the determination of the protein content in human embryonic lung cells. ATLA 20, 285–289. Dierickx, P.J., Ekwall, B., 1993. Long-term cytotoxicity testing of the MEIC chemicals 21–50 by the determination of the protein content in human embryonic lung cells. AATEX 2, 33–36. Dierickx, P.J., Scheers, E.M., 2002. Neutral red uptake inhibition in adhered and adhering rat hepatoma-derived Fa32 cells to predict human toxicity. Journal of Applied Toxicology 22, 61–65. Dierickx, P.J., Smit, C., Scheers, E.M., 2001. Cytotoxicity of the MEIC reference chemicals in antioxidant-enriched rat hepatomaderived Fa32 cells. ATLA 29, 217–223. Ekwall, B., 1999. Overview of the final MEIC results: II. The in vitro– in vivo evaluation, including the selection of a practical battery of cell tests for prediction of acute lethal blood concentrations in humans. Toxicology In Vitro 13, 665–673. Ekwall, B., Barile, F.A., Castano, A., Clemedson, C., Clothier, R., Dierickx, P., Ekwall, B., Ferro, M., Fiskesjo¨, G., Garza-Ocanas, L., Gomez-Lechon, M.J., Gu¨lden, M., Hall, T., Isomaa, B., Kahru, A., Kerszman, G., Kristen, U., Kunimoto, M., Ka¨renlampi, S., Lewan, H., Loukianov, A., Ohno, T., Persoone, G., Romert, L., Sawyer, T.W., Shrivastava, R., Segner, H., Stammati, A., Tanaka, N., Valentino, M., Walum, E., Zucco, F., 1998a. MEIC evaluation of acute systemic toxicity. Part VI. The prediction of human toxicity by rodent LD50 values and results from 61 in vitro methods. ATLA 26, 617–658. Ekwall, B., Clemedson, C., Crafoord, B., Ekwall, B., Hallander, S., Walum, E., Bondesson, I., 1998b. MEIC evaluation of acute systemic toxicity. Part V. Rodent and human toxicity data for the 50 reference chemicals. ATLA 26, 571–616. Ekwall, B., Ekwall, B., Sjo¨stro¨m, M., 2000. MEIC evaluation of acute systemic toxicity. Part VIII. Multivariate partial least squares evaluation, including the selection of a battery of cell lines with a good prediction of human acute lethal peak blood concentrations for 50 chemicals. ATLA 28, 201–234. Gu¨lden, M., Mo¨rchel, S., Tahan, S., Seibert, H., 2002. Impact of protein binding on the availability and cytotoxic potency of organochlorine pesticides and chlorophenols in vitro. Toxicology 177, 201–213. Seibert, H., Mo¨rchel, S., Gu¨lden, M., 2002. Factors influencing nominal effective concentrations of chemical compounds in vitro: medium protein concentration. Toxicology In Vitro 16, 289–297.