FUNDAMENTAL ANDAPPLIEDTOXICOLOGY 9,541-549 (1987) Evaluation of the Interaction of Three Genotoxic Agents in Eliciting Sister- Chromatid Exchanges Using Response Surface Methodology RICHARDP.SOLANA,*VERNONM.CHINCHILLI,*'$JOHN D.WILSON,P WALTER H. CARTER, JR.,* ANDRICHARD A. CARCHMAN*@ *Department of Pharmacology and Toxicology, TDepartment of Radiology, Box 40, MCVStation, and SDepartment of Biostatistics, Box 32, MCV Station, Medical College of Virginia, Richmond, Virginia 23298 Evaluation of the Interaction of Three Genotoxic Agents in Eliciting Sister-Chromatid Ex- changes Using Response Surface Methodology. SOLANA, R. P., CHINCHILLI, V. M., WILSON, J. D., CARTER, W. H., JR.. AND CARCHMAN. R. A. (1987). Fundam. Appl. Toxicol. 9, 541- 549. Chinese hamster cells (V79) were treated with ethylnitrosourea (ENU), bischloroethylni- trosourea (BCNU), and cis-diamminedichloroplatinum(II) (DDP) alone and in combination. Sister chromatid exchanges (SCEs) were quantitated as measures of genotoxicity of the three agents. The combination experiment employed a factorial design in which cells were treated, in various concentration combinations, with all agents simultaneously. Response surface method- ology using a polynomial model in treatment variables to approximate the mean the distribution of SCE events was employed for analysis of the interactions of the three genotoxic agents. Due to unequal variances of the number of SCEs in the various treatment groups, a weighted least- squares analysis was used to estimate the parameters of the dose-response relationship. The single-agent results suggest that the DDP concentration-response curve has a much steeper slope. than the ENU and BCNU curves, and is concave downward as compared to the relatively linear concentration-response curves of ENU and BCNU. The combination results suggest that ENU and DDP are involved in a negative interaction. The BCNU/DDP interaction, the ENU/BCNU interaction, and the three-factor interaction are not statistically significant. The analysis ofthese data demonstrates the usefulness of a statistical procedure for evaluating the biological effects resulting from exposure to multiple cytotoxic agents. The methodology can be used with many other types of endpoints and is not limited by the number of treatment agents. o 1987 society of Toxicology. The evaluation of effects elicited by agents in combination is, at present, difficult. Very few methods are currently available to analyze the interacting effects of multiple variables. Isobolograms can be readily utilized with two-agent combinations, yet lack a statisti- cally valid interpretation and may not be ap- plicable to combinations of agents with dis- similar dose-response curves (Loewe, 1953). Analysis of variance (ANOVA) is a statistical technique capable of detecting a statistically significant interaction between multiple agents in combination, yet it cannot describe the interaction. Response surface methodol- ’ To whom all correspondence should be addressed. ogy (RSM), due to its regression component, has descriptive and predictive capabilities and permits the evaluation of combinations of agents with dissimilar dose-response curves. RSM is not limited by the number of agents it can evaluate. Response surface methods are just begin- ning to be utilized in the biological sciences, having been used for decades in other fields. RSM permits the estimation of the combina- tion of all variables being addressed which optimizes the effect being measured. This op- timization feature has been utilized by the chemical industry and in the agricultural sci- ences (Giovanni, 1983; Schutz, 1983). In ag- riculture, RSM has been used to determine the amount of each of the multiple nutrients 541 0272X)590/87 $3.00 Copyright 0 1987 by the Society of Toxicology. All rights of reproduction in any form reserved.