Combinatorial chemistry has increased the number of compounds available for efficacy and safety assessment by several orders of magnitude and has made high throughput assays essential. To test whether higher throughput toxicity assays could be of utility in screening compounds in early development, a selected set of combinatorial chemistry compounds was screened for induction of 70-Kd heat shock protein (HSP70) and 45-Kd growth arrest and DNA damage protein (GADD45) mRNA levels as well as cytotoxicity, in HepG2 cells, using a 96-well microtiter plate format. Both assays, the branched DNA (Quantigene) assay for mRNA levels and MTT for cytotoxicity, were robust enough to be incorporated into a screening format using a single replicate and a single concentration of compound. Significantly, a structure/toxicity correlation was established with this set of compounds with cytotoxicity and gene induction patterns linked to compound structure. Therefore, this type of early screening may be useful in identifying toxic substituents, enabling the design of libraries with less potential for toxicity. While structure/toxicity correlations were observed, no relationship was observed between GADD45 gene induction and mutagenesis as measured by the Ames bacterial reverse mutation assay.
References
1.
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Meth.65:55-60.
2.
Wang, H.Z., Chang, C.H., Lin, C.P., Tsai, M.C. (1996). Using MTT viability assay to test the cytotoxicity of antibiotics and steroid to cultured porcine corneal endothelial cells. J. Ocul. Pharmacol. Ther.12:35-43.
3.
Sudo, K., Konno, K., Yokota, T., Shigeta, S. (1995). A screening system for antiviral compounds against herpes simplex virus type 1 using the MTT method with L929 cells. Tohoku J. Exp. Med.176:163-171.
4.
Pelham, H.R.B. (1990). Functions of the hsp70 protein family: An overview. In Stress Proteins in Biology and Medicine. R.I. Morimoto, A. Tissieres, and C. Georgopoulos (eds.), pp. 287-299. Cold Spring Harbor Press, Cold Spring Harbor, NY.
5.
Rassow, J., Voos, W., Pfanner, N. (1995). Partner proteins determine multiple functions of Hsp70. Trends Cell Biol.5:207-209.
6.
Ito, H., Okamoto, K., Kato, K. (1998). Enhancement of expression of stress proteins by agents that lower the levels of glutathione in cells. Biochem. Biophys. Acta1397:223-230.
7.
Liu, H.Lightfoot, R., Stevens, J.L. (1996). Activation of heat shock factor by alkylating agents is triggered by glutathione depletion and oxidation of protein thiols. J. Biol. Chem.271:4805-4812.
8.
Walsh, A.C., Michaud, S.G., Malossi, J.A., Lawrence, D.A. (1995). Glutathione depletion in human T lymphocytes: Analysis of activation-associated gene expression and the stress response. Toxicol. Appl. Pharmacol.133:249-261.
9.
Smith, M.L., Chen, I.-T., Zhan, Q., Bae, I., Chen, C.-Y., Gilmer, T.M., Kastan, M.B., O'Connor, P.M., Fornace, A.J., Jr. (1994). Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science266:1376-1380.
10.
Smith, M.L., Kontny, H.U., Zhan, Q., Sreenath, A., O'Connor, P.M., Fornace, A.J., Jr. (1996). Antisense GADD45 expression results in decreased DNA repair and sensitizes cells to u.v.-irradiation or cisplatin. Oncogene13:2255-2263.
11.
Sanchez, Y., Elledge, S.J. (1995). Stopped for repairs. Bioessays17:545-548.
Wilber J.C. (1997). Branched DNA for quantification of viral load. Immunol. Invest.26:9-13.
14.
Todd, M.D., Ludtke, D.N., Oshidari, F., Johnson, D., Wolfgang G.H.I.The utility of branched DNA (bDNA) assays in high throughput toxicity screening. In Approaches to High Throughput Screening in Toxicity Testing. Atterwill, Goldfarb, Purcell (eds.), Taylor & Francis Ltd., London (in press).
15.
Ames, B.N., McCann, J., Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Res.31:347-364.
16.
Sugimura, T., Sato, S., Nagao, M., Yahagi, T., Matsushima, T., Seino, Y., Takeuehi, M., Kawachi, T. (1976). Overlapping of carcinogens and mutagens. In Fundamentals of Cancer Prevention. P.E. Magee, et al (eds.), pp. 191-215. Tokyo/University Park Press, Baltimore.
17.
Maron, D.M., Ames, B.N. (1983). Revised methods for the Salmonella mutagenicity test. Mutation Res.113:173-179.
18.
Snee, R.D., Irr, J.D. (1981). Design of a statistical method for the analysis of mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase locus of cultured Chinese hamster ovary cells. Mutation Res.85:77-93.
19.
Clive, D. (1985). Mutagenicity in drug development: interpretation and significance of test results. Regul. Toxicol. Pharmacol.5:79-100.
