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Chem. Res. Toxicol. 2007, 20, 558-562
Abiotic Sulfhydryl Reactivity: A Predictor of Aquatic Toxicity for Carbonyl-Containing r,β-Unsaturated Compounds Jason W. Yarbrough and T. Wayne Schultz* Department of ComparatiVe Medicine, College of Veterinary Medicine, The UniVersity of Tennessee, 2407 RiVer DriVe, KnoxVille, Tennessee 37996-4543 ReceiVed December 1, 2006
A diverse series of aliphatic R,β-unsaturated esters, ketones, and aldehydes were evaluated for reactivity with the model nucleophile sulfhydryl group in the form of the cysteine residue of the tripeptide glutathione; the reactive end point (RC50) was then related to aquatic toxicity (IGC50) assessed in the Tetrahymena pyriformis population growth impairment assay. The substructure specific to all tested reactive substances, an olefin conjugated to a carbonyl group, is inherently electrophilic and conveys the potential to act by way of Michael-type nucleophilic addition. All such unsaturated compounds are inherently acutely toxic. However, their toxicity is difficult to model with conventional descriptors since toxicity is independent of both hydrophobicity and molecular orbital electrophilicity but dependent on the specific molecular structure. While methacrylates typically did not attain an RC50 value at saturation, a linear relationship [log (IGC50-1) ) 0.936[log (RC50-1)] + 0.508, where n ) 41, r2 ) 0.846, q2 ) 0.832, s ) 0.35, F ) 214, and Pr > F ) 0.0001] was observed between aquatic toxicity and reactivity for the other carbonylcontaining R,β-unsaturated chemicals. Introduction Recent efforts by our group to use regression analysis with traditional physicochemical and quantum chemical descriptors to model the toxic potency of carbonyl-containing R,β-unsaturated compounds, including aldehydes, esters, and ketones, to the ciliate Tetrahymena pyriformis have met with mixed results (1). Although methacrylates and other esters, such as the crotonates and tiglates, exhibit toxicity that is hydrophobicitydependent and similar to that predicted by baseline narcosis, some R,β-unsaturated ketones, acrylates, and ethynylenecontaining esters exhibit toxicity in excess of baseline predictions, but independent of hydrophobicity and one another. Moreover, efforts to develop a single model encompassing all carbonyl-containing R,β-unsaturated compounds were unsuccessful. If a CdC bonded vinyl or vinylene group or CtC bonded acetylenic or ethynylene group is conjugated to a carbonyl group, the result is a polarized R,β-unsaturated compound. These particular molecular substructures have the capacity to act via Michael-type nucleophilic addition (2-4). Michael-type addition is thought to be a two-step process that provides a means of forming covalent adducts at a soft electrophilic center without the presence of a leaving group in the molecule. Specifically, the molecular mechanism is deemed to be nucleophilic addition via an SH addition to the outer or β-C-atom of the CdC or CtC moiety. Toxicity of such substances (1, 5) typically is enhanced over baseline and is thought to be related to reactivity (6-8). Comparison of toxicity (9) and reactivity (8) among carbonylcontaining R,β-unsaturated compounds with and without methyl substitution on one of the C-atoms in a vinyl or vinylene group revealed that reduced reactivity and reduced toxicity is consistently observed upon methyl substitution on either of the vinyl * Corresponding author: e-mail
[email protected]; phone (865) 974-5826; fax (865) 974-5640.
C-atoms, with substitution on the β-C-atom exhibiting the greater inhibition. These results along with the QSAR1 studies (1, 3) suggest methacrylates and other carbonyl-containing R,βunsaturated compounds with methyl substitution on one of the C-atoms in the olefin group do not act (or at most are very slow reacting) as Michael-type acceptors. Specifically, the alkyl group substituted on the β-carbon atom in the CdC changes the electron density and thus opposes the withdrawing effect of the carbonyl group. The net result is that the kinetics of electro(nucleo)philic reactions is so slow (8) that the primary mechanism of toxic action is reversible narcosis. Papirmeister et al. (10) reviewed the hypothesis for the toxicity of Michael-type electrophiles. Briefly, toxicity is considered to be initiated by the alkylation of GSH and its subsequent depletion, making other thiol groups, especially ones in Ca2+ translocases, susceptible to attack. The consequence is a disruption in Ca2+ homeostasis, disruption of the cytoskeleton, and loss of plasma membrane integrity. GSH is a tripeptide in the form of L-γ-glutamyl-L-cysteinylglycine. It is the cysteinyl moiety that provides the critical SH moiety, which is the key functional element facilitating transport and elimination of many reactive chemicals via conjugation (11). Spontaneous conjugation of selected soft electrophiles with the thiol in GSH protects the thiol groups of enzymes and structural proteins. Since this GSH reactivity is responsible for the detoxification, the relationships between molecular structure and thiol reactivity seem to be pivotal to explaining structure-toxicity relationships for carbonyl-containing R,β-unsaturated chemicals (8). The purpose of this study was to quantify the chemical reactivity of selected R,β-unsaturated aliphatic esters, ketones, and aldehydes toward the SH group of the model nucleophile 1 Abbreviations: RC , 50% reactive concentration; IGC , 50% inhibi50 50 tory growth concentration; SH, sulfhydryl group; GSH, glutathione (reduced form); QSAR, quantitative structure-activity relationship; DTNB, 5,5′dithiobis(2-nitrobenzoic acid); DMSO, dimethyl sulfoxide; SAS, statistical analysis system; r2, coefficient of determination; s, square root of the mean square of error; F, Fisher statistic; NRAS, not reactive at saturation.
10.1021/tx600344a CCC: $37.00 © 2007 American Chemical Society Published on Web 02/24/2007
QSAR for Polarized R,β-Unsaturated Chemicals
Chem. Res. Toxicol., Vol. 20, No. 3, 2007 559 Table 1. Repeatability of Sulfhydryl Reactivity with Acrolein and Methyl Methacrylate replicate
Figure 1. Common formula of the compounds used in this study. These include R,β-unsaturated aldehydes (R1 ) H and R2 ) CnH(2n+1)), R,βunsaturated ketones (R1 ) R2 ) CnH(2n+1)), and R,β-unsaturated esters (R1 ) OCnH(2n+1), R2 ) CnH(2n+1), R3 ) H or CH3).
GSH and compare that reactivity with previously reported aquatic toxicity data (1). The specific aims were to (1) determine the reactive potency of a series of carbonyl-containing R,βunsaturated chemicals to GSH and (2) examine the relationship between reactivity and aquatic toxicity. We hypothesized that reactivity would be quantitatively related to toxicity for chemicals acting by Michael addition.
Materials and Methods Chemicals. The 44 tested carbonyl-containing R,β-unsaturated chemicals 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. The test chemicals included acrolein, 25 esters, 11 ketones, and 7 aldehydes. The esters included members from a variety of subclasses, including 10 acrylates and 5 methacrylates, as well as 5 containing a vinylene group and 5 containing an ethynylene group. The common formula of these compounds is shown in Figure 1. Caution: These chemicals are hazardous and should be handled carefully. As reported (1), seVeral of these chemicals haVe significant acute toxicity. In addition, many of them are potential mutagens (12) and skin sensitizers (13). Sulfhydryl Reactivity Data. Reactivity data for 17 compounds were secured from Schultz et al. (8). The spectrophotometric-based, concentration-response assay for reactivity with the SH group of glutathione as outlined by Schultz et al. (8) was used to test the remaining compounds. By use of absorbance at 412 nm, free thiol groups were quantified by their reaction with 5,5′-dithiobis(2nitrobenzoic acid). Briefly, 1.375 mM of reduced glutathione was freshly prepared. Toxicant stock solutions were prepared by dissolving them in dimethyl sulfoxide (DMSO); subsequently, a phosphate buffer was added to the toxicant/DMSO solution. The proper aliquots of glutathione solution, toxicant 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 >20% and typically >5%. Following 120 min, the reactivity was determined by calculation of the RC50 value [the concentration (millimolar) resulting in 50% binding of available sulfhydryl groups] (8). To get a sense of the repeatability of the sulfhydryl assay, fastreacting acrolein and slow-reacting methyl methacrylate were assessed multiple times by several different operators. Toxicity Test Data. Population growth impairment data for Tetrahymena pyriformis for 30 compounds were taken from Schultz and Yarbrough (9). For the remaining chemicals, toxicity assessments were conducted by use of the protocol outlined by Schultz (14). Briefly, this is a static 40-h assay, which uses population density measured spectrophotometrically at 540 nm as the end point in a concentration-response scenario. The toxicity was determined by calculation of the IGC50 value [the concentration (millimolar) resulting in 50% inhibition of population growth compared to controls] (14). Data Analyses. The effect levels were determined from nominal chemical concentrations. The RC50 and IGC50 values were determined by Probit Analysis by use of SAS software (15). In both cases, chemical concentration was the dependent variable, and absorbance normalized to control was the independent variable. The
1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7
RC50 value 0.094 0.081 0.085 0.086 0.092 0.074 0.084 0.085 0.088 0.068 0.10 75 77 73 66 63 61 75
date Acrolein 02-10-05 02-18-05 02-18-05 02-19-05 03-03-05 03-06-05 03-14-05 03-21-05 12-21-06 01-03-07 01-05-07
Methyl Methacrylate 08-24-05 08-24-05 12-17-06 12-18-06 12-18-06 12-22-06 01-02-07
lot
operator
1 1 1 1 1 1 1 1 2 2 2
A A B B A A A A C D D
1 1 2 2 2 2 2
A A D C D A C
reported RC50 values were the means of at least two replicate tests reported to two significant digits. QSARs were developed by use of least-squares regression (regression procedure of MINITAB release 13.1) with log (IGC50-1) as the dependent variable and log (RC50-1) as the independent variable. The resulting model was measured for fit by its r2 value. The uncertainty in the model was noted as the s-value, while the predictivity of the model was noted as the q2 value by the leaveone-out method. The F-values and probability greater than F also were noted. Outliers had residual values outside the 95% confidence interval of the model.
Results In Table 1 are reported the RC50 values for independent replicates of the GSH assay for acrolein and methyl methacylate. The mean and standard deviation of the 11 RC50 values for acrolein is 0.085 ( 0.009, while the mean and standard deviation of the 7 RC50 values for methyl methacrylate is 70 ( 6.5. The assay is repeatable. In Table 2 the chemical group, name, CAS number, reactivity, and toxicity are reported for each compound assessed. Within each class (i.e., esters, ketones, and aldehydes) and subclass, small homologous series were tested. Except for methyl methacrylate, the RC50 values for simple methacrylates were outside the aquatic solubility range. However, these three methacrylates were the only compounds where an RC50 value was not attained. The plot in Figure 2 shows the relationship between reactivity and toxicity for these carbonyl-containing R,β-unsaturated chemicals. The regression analysis between SH reactivity [log (RC50-1)] and aquatic toxicity [log (IGC50-1)] yields the relationship
log (IGC50-1) ) [0.936 (( 0.055)]log (RC50-1) + [0.508 (( 0.064)] (1) where n ) 41, r2 ) 0.846, q2 ) 0.832, s ) 0.35, F ) 214, and Pr > F ) 0.0001. For eq 1, three compounds were identified as statistical outliers. Methyl trans-2-octenonate was observed to be more toxic than predicted, while tert-butyl acrylate and 3-penten-2-one were observed to be less toxic than predicted. Removal of these outliers and subsequent reanalysis (data not shown) improved the fit but did not change the basic nature of the equation.
560 Chem. Res. Toxicol., Vol. 20, No. 3, 2007
Yarbrough and Schultz
Table 2. Thiol Reactivity and Toxicity of Selected r,β Unsaturated Esters, Ketones, and Aldehydes group
CAS number
RC50 (mM)
IGC50 (mM)
acrolein
107-02-8 0
0.086
0.013
methyl acrylate ethyl acrylate vinyl acrylate n-propyl acrylate allyl acrylate propargyl acrylate n-butyl acrylate tert-butyl acrylate n-pentyl acrylate isopentyl acrylate
Acrylate Esters 96-33-3 140-88-5 2177-18-6 925-60-0 999-55-3 10477-47-1 141-32-2 1663-39-4 2998-23-4 4245-35-6
0.55 0.52a 0.11a 0.80 3.5 0.21 0.80 1.5 0.81 0.93
0.28 0.31b 0.038 0.30b 0.21b 0.088b 0.30b 4.4b 0.29b 0.39b
methyl methacrylate ethyl methacrylate vinyl methacrylate n-propyl methacrylate isopropyl methacrylate
Methacrylate Esters 80-62-6 97-63-2 4245-37-8 2210-28-8 4655-34-9
76a NRASc 4.5 NRAS NRAS
Vinylene-Containing Esters methyl crotonate 623-43-8 25a ethyl crotonate 623-70-1 23a vinyl crotonate 14861-06-4 4.3a methyl tiglate 6622-76-0 6.1 methyl trans-2-octenoate 7367-81-9 5.8 methyl propiolate ethyl propiolate methyl tetrolate ethyl tetrolate methyl 2-octynoate 1-penten-3-one 1-hexen-3-one 1-octen-3-one
22 8.6b 4.2b 4.5b 7.6b 8.3b 5.8b 1.4b 6.1b 0.17b
Ethynylene-Containing Esters 922-67-8 0.095 623-47-2 0.091a 23326-27-4 1.27 4341-76-8 1.31 111-12-6 0.26
0.017 0.020b 0.40 0.31b 0.091b
Ketones: 1-Alken-3-ones 1629-58-9 0.051a 1629-60-3 0.052a 4312-99-6 0.051
0.030b 0.022 0.012b
Ketones: 3-Alken-2-ones et al. 3-buten-2-one 78-94-4 0.090 3-penten-2-one 625-33-2 0.11 4-hexan-3-one 2497-21-4 0.34a 3-octen-2-one 1669-44-9 0.46 4-methyl-3-penten-2-one 141-79-7 28a 3-methyl-3-penten-2-one 565-62-8 10a
0.031b 0.29b 0.12 0.18b 4.4b 2.2b
Ketones: 3-Alkyn-2-ones 1423-60-5 0.057 1679-36-3 0.12
0.011b 0.048
Aldehydes: 2-Alkenals 123-73-9 0.22 1576-87-0 0.78a 6728-26-3 0.76a 623-36-9 21a 5362-56-1 1.1a 142-83-6 1.52 2548-87-0 0.28
0.088 0.22b 0.17 5.8 0.15 0.18 0.063b
3-butyn-2-one 3-hexyn-2-one 2-butenal 2-pentenal 2-hexenal 2-methyl-2-pentenal 4-methyl-2-pentenal 2,4-hexadienal 2-octenal a
Reference 8. b Reference 9. c NRAS, not reactive at saturation.
Discussion Modeling approaches to toxicity-based relationships include the mechanism of action approach (16, 17). Historically, problems associated with a priori assignment of mechanism of action have discouraged the use of this approach (18). The aquatic toxicity of nonreactive compounds is largely independent of specific molecular substructure (19). The dramatic difference in potency when the toxicity values of ethyl acrylate and methyl methacylate are compared (1, 9) suggests that in the case of carbonyl-containing R,β-unsaturated compounds, a minor alteration in molecular structure, which is independent of either hydrophobicity or electrophilicity, can result in a significant alteration in toxicity.
Figure 2. Plot of toxicity [log (IGC50-1)] versus thiol reactivity [log (RC50-1)] for selected R,β-unsaturated compounds.
This alteration in toxic potency is thought to be due to molecular reactivity. Reactivity, as it relates to toxicity, is the irreversible reaction of a toxicant (or a metabolite) with a biological macromolecule (20). That reaction leads to a series of biochemical and physiological processes, ultimately resulting in a measured toxic end point. Regardless of the adverse effect, the molecular initiating event is binding to a specific molecular site. It is the nature of the toxicant that determined the selectivity of the molecular site of action (20). The best explanation for this selectivity is achieved by classifying electrophiles and nucleophiles according to their chemical “hardness” and “softness” (21), a classification that forms a continuum and is a function of the polarizability of the electro(nucleo)philic center. Soft nucleophiles such as SH groups are easily oxidized, readily polarizable, and have low electronegativity. The goal of any chemical classification scheme is to assemble toxicants into appropriate groups for classification, labeling, and risk assessment. It would be advantageous to be able to do this a priori, especially on the basis of two-dimensional structure and/or functional groups (22). The net result for some aquatic toxicity categories (e.g., nonpolar narcosis) is accurate, transparent, and well-understood models; whereas in other cases such models are less accurate general response surfaces or are sharply limited to a simple chemical class (1, 23). This lack of accuracy is in part because little effort has been put forth to evaluate chemical reactivity as a means of aiding in the development of a classification scheme for grouping chemicals based on mechanism of action, especially for electrophiles. However, recent efforts are changing this perception (20, 24). Irreversible toxic reactions result in chemical alterations in biological systems. Such reactions include a number of competing processes and different chemical reactive mechanisms. Many of these alterations involve alkylation or arylation to protein and peptide-related cellular nucleophiles, in particular thiol moieties (10). Such chemical reactions include conjugated addition or Michael-type addition, a toxic mechanism associated with the presence of a polarized R,β-unsaturated toxicophore like that associated with most of the esters, ketones, and aldehydes examined in this study (4). The olefin in the R,β-unsaturated compounds examined here can be observed in one of three possible configurations: (1) branched or alkyl-substituted, such as with the methacrylates; (2) unbranched and in the terminal position, such as with acrylates; and (3) unbranched and in an internal position, such as with crotonates. As demonstrated in this investigation, the specific structural arrangement affects SH reactivity and thus
QSAR for Polarized R,β-Unsaturated Chemicals
also affects toxic potency as well as the most likely molecular mechanism of action. Simple CdC bonds are electron-rich and thus not subject to nucleophilic attack; however, if an electron-withdrawing carbonyl group is conjugated to a CdC bond, then the electron density of the double bond is lower, giving the compound a greater likelihood of undergoing nucleophilic attack (4). Therefore, nucleophilic addition to R,β-unsaturated compounds should be examined in terms of the entire conjugated system. The propensity for carbonyl-containing R,β-unsaturated chemicals to undergo nucleophilic addition is aided by the electronwithdrawing ability of the carbonyl group. This withdrawing ability leads to the nucleophilic addition occurring at the β-Catom of the CdC or CtC moiety. Regardless of the location of the CdC or CtC moiety (i.e., terminal or interior), the preferred biological nucleophile for such a Michael addition is the thiol group (2, 4). CtC-containing substances are typically more reactive and more toxic than CdC-containing ones, and compounds with a terminally situated CtC or CdC group are more reactive and toxic than ones with the CtC or CdC moiety imbedded within the molecule. All are significantly reactive with thiol and are proposed to exhibit toxicity by way of the Michael-addition mechanism. All the polarized R,β-unsaturates with methyl substitution on one of the C-atoms in the olefinic groups (i.e., branched compounds) are less reactive. Moreover, greater reduction in reactivity, especially for the lower molecular weight compounds, is concomitant with methyl substitution on the R-C-atom. The alkyl group substituted on the R-C-atom in the olefinic moiety releases electron density and thus counters the withdrawing effect of the carbonyl group. The net result is a much slower rate of addition of the SH nucleophile. Therefore, the mechanisms of toxic action are mixed but are dominated by narcosis. This supposition is supported by the facts that the RC50 values for the methacrylates are typically extrapolated beyond aquatic solubility, the toxicity of the methacrylates is directly related to hydrophobicity, and the observed toxicity is not significantly different than predicted by baseline models (1). Equation 1 shows a slope of 1, which indicates a simple and direct relationship between toxicity and abiotic SH reactivity. For the direct-acting electrophiles examined in this investigation, biouptake and metabolism are not important to determining acute toxicity. Rather, the molecular initiating event, the covalent alteration of cellular proteins via the molecular mechanism of Michael addition, is the sole determining factor in toxicity. The intercept of nearly 0.5 is less significant and probably represents the specifics of the two protocols, in particular, the ratio of available SH-based nucleophiles to toxicants. In the case of the toxicity test, these nucleophiles include ones found in the cells as well as those available in the complex medium. Population growth kinetics experiments (25) show that Tetrahymena exposed to classic Michael-type accepting electrophiles exhibit a concentration- and time-dependent partial mortality of the inoculum followed by normal population growth rates for the surviving ciliates. These results are consistent with the idea that Michael-type acceptors as electrophiles induce survival effects. In conclusion, a series of aliphatic, polarized R,β-unsaturated esters, ketones, and aldehydes were evaluated. In chemico reactivity was used to segregate chemicals into those acting as Michael-type acceptors and those acting by nonreactive mechanisms. By use of the T. pyriformis population growth impairment assay, toxic potency for all tested chemicals was deter-
Chem. Res. Toxicol., Vol. 20, No. 3, 2007 561
mined. Reactivity and toxic potency were found to be dependent on the specific molecular structure. Between homologues, the CtC-substituted derivatives are more reactive and more toxic than the CdC-substituted ones. Between homologues, derivatives with a terminally located olefin are more reactive and more toxic than ones with an internally located olefin. Methyl substitution on an olefinic C-atom, particularly on the R-C-atom, reduces both reactivity and toxicity, especially for low molecular weight compounds. Compounds with a highly branched alkyl group substituted next to the ester linkage were less reactive and less toxic than unbranched derivatives. A strong linear relationship was demonstrated between reactivity and toxicity for carbonyl-containing R,β-unsaturated compounds. Finally, these results suggest that in chemico evaluation for reactivity has the potential to be a rapid and inexpensive means of classifying chemicals, especially for selected soft electrophilic mechanisms of action. Acknowledgment. This work was supported in part by the U.S. Army Medical Research and Material Command under Contract W81XWH-050C-0017. The views, opinions and/or findings contained in this report are those of the authors and should not be construed as an official Department of the Army position, policy or decision unless otherwise so designated by other documentation.
References (1) Schultz, T. W., Netzeva, T. I., Roberts, D. W., and Cronin, M. T. D. (2005) Structure-toxicity relationships for carbonyl-containing R,βunsaturated aliphatic chemicals evaluated with Tetrahymena pyriformis. Chem. Res. Toxicol. 17, 330-341. (2) Friedman, M., Cavins, J. F., and Wall, J. S. (1965) Relative nucleophilic reactivities of amino groups and mercaptide ions in addition reactions with R,β-unsaturated compounds. J. Am. Chem. Soc. 87, 3672-3682. (3) Freidig, A. P., Verhaar, H. J. M., and Hermens, J. L. M. (1999) Quantitative structure property relationships for the chemical reactivity of acrylates and methacrylates. EnViron. Toxicol. Chem. 18, 11331139. (4) Aptula, A. O., Patlewicz, G. Y., and Roberts, D. W. (2005) Skin sensitization: Reaction mechanistic applicability domains for structureactivity relationships. Chem. Res. Toxicol. 18, 1420-1426. (5) Greim, H., Ahlers, J., Bias, R., Broecker, B., Hollander, H., Gelbke, H. P., Jacobi, S., Klimisch, H. J., Mangelsdorf, I., Mayr, W., Schon, N., Stropp, G., Stahnecker, P., Vogel, R., Weber, C., ZieglerSkylakakis, K., and Bayer, E. (1995) Assessment of structurally related chemicals: Toxicity and ecotoxicity of acrylic acid and acrylic acid alkyl esters (acrylates), methacrylic acid and methacrylic acid alkyl esters (methacrylates). Chemosphere 31, 2637-2659. (6) Osman, R., Namboodiri, K., Weistein, H., and Rabinowitz, J. (1988) Reactivities of acrylic and methacrylic acids in a nucleophilic addition model of their biological activity. J. Am. Chem. Soc. 110, 1701-1707. (7) McCarthy, T. J., Hayes, E. P., Schwartz, C. S., and Witz, G. (1994) The reactivity of selected acrylate esters toward glutathione and deoxyribonucleosides in vitro: Structure-activity relationships. Fundam. Appl. Toxicol. 22, 543-548. (8) Schultz, T. W., Yarbrough, J. W., and Johnson, E. L. (2005) Structureactivity relationships for glutathione reactivity of carbonyl-containing compounds. SAR QSAR EnViron. Res. 16, 313-322. (9) Schultz, T. W., and Yarbrough, J. W. (2004) Trends in structuretoxicity for carbonyl-containing R,β-unsaturated compounds. SAR QSAR EnViron. Res. 15, 139-146. (10) Papirmeister, B., Feister, A. F., Robinson, S. I., and Ford, R. D. Medical Defense Against Mustard Gas: Toxic Mechanisms and Pharmacological Implications. CRC Press, Boca Raton, FL, 1991. (11) Reed, D. J. (1990) Glutathione: Toxicological Implications. Annu. ReV. Pharmacol. Toxicol. 30, 603-631. (12) Benigni, R., Passerini, L., and Rodomonte, A. (2003) Structureactivity relationships for the mutagenicity and carcinogenicity of simple and alpha-beta unsaturated aldehydes. EnViron. Mol. Mutagen. 42, 136-143. (13) Gerberick, G. F., Ryan, C. A., Kern, P. S., Schlatter, H., Dearman, R. J., Kimber, I., Patlewicz, G. Y., and Basketter, D. A. (2005) Compilation of historical local lymph node data for evaluation of skin sensitization alternative methods. Dermatitis 16, 157-202.
562 Chem. Res. Toxicol., Vol. 20, No. 3, 2007 (14) Schultz, T. W. (1997) TETRATOX: Tetrahymena pyriformis population growth impairment endpoint-A surrogate for fish lethality. Toxicol. Methods 7, 289-309. (15) SAS Institute Inc. (1989) SAS/STAT User’s Guide, v. 6, 4th ed., Vol. 2, p 846, SAS, Cary, NC. (16) Cronin, M. T. D. (2003) Quantitative structure-activity relationships for acute aquatic toxicity: the role of mechanism of toxic action in successful modeling. In QuantitatiVe Structure-ActiVity Relationship (QSAR) Models of Mutagens and Carcinogens (Benigni, R., Ed.) pp 235-258, CRC Press, Boca Raton, FL. (17) Bradbury, S. P., Russom, C. L., Ankley, G. T., Schultz, T. W., and Walker, J. D. (2003) Overview of data and conceptual approaches for derivation of QSARs for ecotoxicological effects of organic chemicals. EnViron. Toxicol. Chem. 22, 1789-1798. (18) Bradbury, S. P. (1994) Predicting modes of toxic action from chemical structure: An overview. SAR QSAR EnViron. Res. 2, 89-104. (19) Escher, B. I., and Hermens, J. L. M. (2002) Modes of action in ecotoxicology: Their role in body burden, species sensitivity, QSARs, and mixture effects. EnViron. Sci. Technol. 36, 4201-4217. (20) Schultz, T. W., Carlson. R. E., Cronin, M. T. D., Hermens, J. L. M., Johnson, R., O’Brien, P. J., Roberts, D. W., Siraki, A., Wallace, K. D., and Veith, G. D. (2006) A conceptual framework for predicting
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toxicity of reactive chemicals: Models for soft electrophilicity. SAR QSAR EnViron. Res. 17, 413-428. Parson, R. G., and Songstad, J. (1967) Application of the principle of hard and soft acids and bases to organic chemistry. J. Am. Chem. Soc. 89, 1827-1836. Schultz, T. W., and Cronin, M. T. D. (2003) Essential and desirable characteristics of ecotoxicity quantitative structure-activity relationships. EnViron. Toxicol. Chem. 22, 599-607. Schultz, T. W., Cronin, M. T. D., Netzeva, T. I., and Aptula, A. O. (2002) Structure-toxicity relationships for aliphatic chemicals evaluated with Tetrahymena pyriformis. Chem. Res. Toxicol. 15, 1602-1609. Aptula, A. O., and Roberts, D. W. (2006) Mechanistic applicability domains for nonanimal-based prediction of toxicological end points: General principles and application to reactive toxicity. Chem. Res. Toxicol. 19, 1097-1105. Bearden, A. P., Sinks, G. D., and Schultz, T. W. (1999) Population growth kinetics of Tetrahymena pyriformis exposed to selected electrophiles. In EnVironmental Toxicology and Risk Assessment, Vol. 8 (Henshel, D., Black, M. C., and Harrass, M. C., Eds.) ASTM STP 1364, pp 319-328, American Society for Testing and Materials, West Conshohocken, PA.
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