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Experimental Reactivity Parameters for Toxicity Modeling: Application to the Acute Aquatic Toxicity of SN2 Electrophiles to Tetrahymena pyriformis David W. Roberts,† T. Wayne Schultz,‡ Erika M. Wolf,‡ and Aynur O. Aptula*,§ School of Pharmacy and Chemistry, LiVerpool John Moores UniVersity, Byrom Street, LiVerpool L3 3AF, England, Department of ComparatiVe Medicine, College of Veterinary Medicine, The UniVersity of Tennessee, 2407 RiVer DriVe, KnoxVille, Tennessee 37996-4543, and SEAC, UnileVer Colworth, Sharnbrook, Bedford, MK44 1LQ, England ReceiVed October 2, 2009
A diverse set of 60 haloaliphatic compounds were evaluated for reactivity with cysteine thiol groups in the previously described RC50 assay using glutathione (GSH) as a model nucleophile. Reactivity was quantified by the RC50 value, the concentration of test compound that produced 50% reaction of the GSH thiol groups in 120 min. Under standard conditions, RC50 values are mathematically proportional to reciprocal rate constants. Quantitative structure-activity relationship (QSAR) analysis correlating acute aquatic toxicity (IGC50) to Tetrahymena pyriformis with RC50 values was carried out. It was found that subdivision of the compounds into subdomains according to their reaction mechanism characteristics enabled toxicity-reactivity relationships to be identified. The largest subdomain consisting of 22 compounds in which a primary halogen is R to a carbonyl or other electronegative unsaturated group and which can be confidently assigned as SN2 electrophiles fits the equation pIGC50 (mM) ) 0.94 ((0.07) pRC50 (mM) + 1.34 ((0.07), n ) 22, r2 ) 0.889, r2(adj) ) 0.884, s ) 0.27, and F ) 161. Compounds in which the halogen is not R to an unsaturated group are not reactive in the GSH assay and do not exhibit reactive toxicity to T. pyriformis. Compounds tested in which the halogen is R to an unsaturated nonelectronegative group were found to be less toxic in the assay than predicted by the above QSAR equation. Within a subdomain of 21 compounds having a halogen R to an electronegative unsaturated group that, in the absence of experimental evidence, could not be confidently assigned as SN2 electrophiles, 2-bromoalkanoates of general structure R1CHBrCO2R2, 2-bromopropionamide, and 2-haloalkanoic acids of general formula R1CHXCO2H (nine compounds in total) are all well-predicted by the above equation. Of the other 12 compounds of this subdomain, eight are substantially less toxic than predicted by the above equation and are considered to react differently, whereas the R-halonitriles (four compounds) are more toxic than predicted and fit a correlation of their own: pIGC50 ) 1.01 ((0.05) pRC50 + 2.04 ((0.05), n ) 4, r2 ) 0.995, r2(adj) ) 0.992, s ) 0.08, and F ) 381, with a similar slope but larger intercept. An explanation in terms of their physical chemistry and possible involvement of released cyanide ion is suggested. Introduction It is well-known that acute aquatic toxicity is a multipathway phenomenon where the biological mode of toxic action is downstream of the initial chemico-biological interaction (1). Chemistry and physics alone determine the molecular mechanism of action. While general narcosis is a mode of action with a single mechanism, electrophilic toxicity is a phenomenon with a multiplicity of molecular mechanisms (2). However, in each case, the electrophilic interaction results in covalent modification of those nucleophiles in biological systems, whose modification initiates the cascade of events along the pathway leading to the adverse outcome. The ability to group electrophilic chemicals into mechanisticbased structural domains for protein binding (3) has the potential to lead to filling of data gaps by read-across or quantitative * To whom correspondence should be addressed. Tel: +44/1234-264823. Fax: +44/1234-264722. E-mail:
[email protected]. † Liverpool John Moores University. ‡ The University of Tennessee. § Unilever Colworth.
mechanistic model predictions for several regulatory end points (4-7) without the need for additional in vivo testing. The simplest structure-based classifications (e.g., aldehydes, phenols, and organic halides) do not generally map onto the mechanistic classification. However, expanded structural alerts in the form of conjugated chemical functionality (e.g., R,β-unsaturated esters) have been shown to be useful in identifying electrophilic toxicity (8). The objective of structure-toxicity analyses is to predict toxic activity from information on molecular structure. This information can be gained from physicochemical properties (e.g., octanol-water partition coefficient, log P) or quantum chemical calculations (e.g., energy of the lowest unoccupied molecular orbital, ELUMO). Although they can be very useful, such parameters are not always adequate to model the toxic end point and, in such cases, may need to be supplemented by experimental chemistry data, such as measurement of in chemico reactivity parameters (9) used in this work. In chemico reactivity testing is not novel, and several protocols are available that quantify either depletion of a reactant or formation of a product or adduct (9). Methods include (1)
10.1021/tx9003648 2010 American Chemical Society Published on Web 11/23/2009
Experimental ReactiVity Parameters for Toxicity Modeling
full kinetic assays, which take concentration measurements at several time intervals often with several initial concentrations of electrophile; (2) assays determining the concentration of electrophile giving 50% reaction of model nucleophile after a fixed time, which uses several initial (stoichiometric excess) concentrations of electrophile and a standard concentration of nucleophile; or (3) assays determining the extent of reaction after a fixed time with one initial concentration of electrophile (5). While full kinetic assays provide the highest quality data, their time and resource needs are traditionally higher than either of the other two methods, although a recently reported high throughput kinetic profiling approach combining time-response and dose-response data generation (10) may change this scenario. In the context of toxicity prediction, experimental measurements of the chemical reactivity of electrophiles with model nucleophiles such as cysteine have earlier focused on characterizing the Michael acceptor mechanistic domain (6, 8, 11). Among the other biologically relevant chemical mechanisms that can result in the covalent modification of proteins at thiol groups are nucleophilic substitution reactions, particularly the SN2 mechanism (12-14). Many, but not all, haloaliphatic compounds can form covalent adducts at the thiol of cysteine with the concomitant loss of the halogen (15). While different nucleophiles and different protocols can result in different absolute reactivities toward a given electrophile, relative reactivity between different electrophiles is typically similar over a range of nucleophiles, especially ones with similar softness or hardness (2, 5). The relative reactivity obtained from different protocols is typically well-correlated as long as comparisons are made within the same chemical mechanism (10). Thus, structure-reactivity relationships once determined from experimental data should be maintained regardless of the protocol by which they are obtained, always provided of course that the same type of reaction is being measured. This study was designed to build on previous work on R-halosubstituted carbonyl-containing compounds (15). The purpose of it was to quantify chemical reactivity between selected haloaliphatic compounds and glutathione (GSH), in particular chemicals able to react by an SN2 mechanism and to assess the applicability of the so-obtained reactivity indices to modeling a toxic end point, chosen for the purposes of this study as acute aquatic toxicity to Tetrahymena pyriformis.
Materials and Methods Test Chemicals. In this work, we studied a range of 60 chemicals all containing an aliphatic halogen that, in principle, could react by SN2 displacement of the halide anion. The selection of the haloaliphatic compounds was also based on commercial availability and the intention to cover this structural domain as widely as possible. All test substances were purchased from commercial sources (SigmaAldrich.com or Alfa.com) in the highest purity available (95% minimum) and were not further purified prior to testing. GSH reactivity data (RC50, the concentration giving 50% reaction in a fixed time of 2 h) for 22 compounds reported in the literature (15, 16) were confirmed. An additional 38 chemicals were selected and tested in the GSH reactivity assay. The goal was to better define and more uniformly cover the structural domain of halogenated aliphatic chemicals which are reactive with thiol groups. Measurement of Reactivity. To measure reactivity with the thiol group of GSH, a simple and rapid (120 min duration) spectrophotometric-based assay was used as outlined by Schultz et al. (16). The free SH group was quantified by its reaction with DTNB, measuring the absorption of the product at 412 nm. Briefly, 1.375 mM GSH was freshly prepared by dissolving 0.042 g of reduced
Chem. Res. Toxicol., Vol. 23, No. 1, 2010 229 GSH into 100 mL of phosphate buffer at pH 7.4. Stock solutions of test compound were prepared by dissolving them in DMSO; subsequently, phosphate buffer was added. The proper aliquots of GSH solution, test compound stock solution, and buffer were added to bring the final concentration of thiol to 0.1375 mM, in a manner so the concentration of DMSO in the final solution was always
