Food Safety Assessment - ACS Publications - American Chemical

Chapter 18 Predicting Quantitative

Chemical

M u t a g e n i c i t y by

Using

Structure—Activity Relationships

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 12, 2016 | http://pubs.acs.org Publication Date: February 14, 1992 | doi: 10.1021/bk-1992-0484.ch018

Alan J. Shusterman Department of Chemistry, Reed College, Portland, OR 97202-8199

Molecular orbital calculations have been performed for several classes of chemicals that are mutagenic in the Ames test in Salmonella typhimurium. The results of these calculations, along with the hydrophobicity of the mutagens, allows construction of quantitative structure-activity relationships (QSAR) for mutagenicity and prediction of mutagenicity over a very wide range of structure and activity. The QSARs also provide insight into the mechanisms responsible for the metabolic activation of these mutagens. QSARs for the action of nitro-polycyclic aromatic hydrocarbons, phenyland heteroaromatic triazenes, and aminoimidazoles such as IQ and MeIQ will be presented.

The public has become increasingly aware of the chemical basis, both real and perceived, of many diseases. Consequently, the public has also begun to demand information regarding the toxicity of chemicals found in the public domain. The desire to avoid unnecessary chemical contamination of basic substances such as food, water, and air is especially pronounced. Scientists are in a poor position, however, to provide toxicity information because of the huge number of untested chemicals, both synthetic and naturally occurring, the lack of unambiguous biological tests for establishing toxicity, and the expense of performing these tests. Mathematical models that can predict several types of toxicity on the basis of chemical structure without the need for biological testing are, therefore, highly desirable. Quantitative structure-activity relationships (QS AR) for biological systems have had a long and successful history as tools for the study of biochemical reaction mechanisms, and for the rational design of therapeutic drugs. QS ARs have also been used to study several types of toxicity, and this work has been the subject of a recent review (7). This paper describes recent work devoted to the development of QSARs for chemical mutagenicity in Ames bacteria, S. typhimurium (2-4). Most of this work has been carried out in collaboration with Dr. Corwin Hansch and his associates at Pomona College. 0097-6156/92/0484-0181S06.00/0 © 1992 American Chemical Society In Food Safety Assessment; Finley, John W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

FOOD SAFETY ASSESSMENT

182


QSAR Methodology Before one can construct a quantitative structure-activity relationship for a set of chemicals the following must exist: a quantitative test for the biological activity of interest, a quantitative description of chemical structure or some structure-dependent property, and a mathematical formalism for relating structure and activity. While each of these areas continues to be the subject of vigorous research, the approach taken here is a traditional one. Activity is defined using the experimental dose-response curves observed for different chemicals acting on various strains of S. typhimurium as measured by the Ames test (5). The test has the advantage of being readily quantifiable, and reasonably reproducible from laboratory to laboratory. On the other hand, the results of the Ames test should not be construed as representing carcinogenicity, since the formation of cancer involves a significantly more complicated chain of events. The mutagen families described here all require metabolic activation of some sort. It is also anticipated that certain types of metabolic transformations may render the molecule inactive. Therefore, a reasonable set of structure descriptors would be those properties most closely related to chemical reactivity, particularly the processes involved in (de)activation. Such descriptors include the electronic and steric/shape properties of the molecule. Another key factor, often overlooked by chemists, is the relative hydrophobicity of the molecule. The hydrophobic properties of a mutagen affect its penetration of biological membranes, and its binding to metabolic enzymes. Following accepted practice, log Ρ has been employed as a measure of relative hydrophobicity where Ρ is the mutagen's octanol-water partition coefficient. Finally, multiple linear regression equations are used to correlate activity with the relevant structural properties. As described below, great care must be taken to guarantee that each term occurring in the regression equation is justified both statistically and chemically. There is a great potential for confounding variables, which accidentally parallel the behavior of more meaningful structural parameters (at least for the limited set of compounds under consideration), to lead to QS ARs that either lack generality or whose apparent chemical interpretation is unrealistic (see below). Quantum Chemical Calculations Hammett substituent constants, σ have traditionally been used to describe the elec tronic properties of different chemical structures. This approach has restricted the range of chemical structures that can be studied to those that can be described by the interchange of different substituent groups on a common skeleton. This limitation on structure also affects one's ability to manipulate other properties such as hydropho bicity, and limits the range of biological activity that can be studied. We have attempted to overcome these problems by undertaking the use of quantum chemical calculations, such as the semi-empirical M N D O and A M I methods, to describe electronic properties related to chemical reactivity. χ

6

7

In Food Safety Assessment; Finley, John W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

18. SHUSTERMAN

Predicting Chemical Mutagenicity

183


We have made use of several quantum chemical indices including molecular orbital energies (q), electron densities associated with particular atoms (qj), and calculated reaction enthalpies (AEj), as measures of a given mutagen's electronic properties. As shown below, it is quite common for several parameters to be collinear, and QS ARs can be derived for a given family of mutagens using a variety of electronic descriptors. However, given this high degree of collinearity, it is essential that mechanistic conclusions not be based solely on the presence of a given electronic descriptor in the QS AR. Triazenes Venger et al., initially reported a QS A R (Equation 1 ) for 17 aryltriazenes, 1, acting on S. typhimurium strain TA92 (2c). The triazenes do not exhibit any activity except in the presence of the S9 fraction obtained from rat liver microsomes. Thus, it was concluded that cytochrome P-450 activation of the triazenes was essential.

1 Mutagenic activity was defined as log 1/C, where C is the molar concentration of triazene causing 30 revenants above background/10 TA92 bacteria. A low mutation rate was chosen so that the cytotoxicity of the mutagen would not interfere with the test results. The statistical parameters associated with this equation are n, the number of data points; r, the correlation coefficient; and s, the standard deviation. Figures in parenthesis are for construction of the 95% confidence intervals. 8

log 1/C = 1.04 (±0.17) log Ρ - 1.63 (±0.35) σ+ + 3.06 η = 17, r = 0.974, s = 0.315

(D

Several points concerning this equation are noteworthy, the first being that the quality of the correlation is unusually high. This can be attributed, in part, to the fact that all of the data were collected in a single laboratory where the testing of each mutagen could be carried out in a reproducible fashion. Also interesting are the appearance of both hydrophobic and electronic terms in the equation, and the coefficients associated with each. Triazene mutagenic activity is increased by attaching substituents to the benzene ring that either render the triazene more hydrophobic (larger log P), or more electron-rich (smaller σ+). Cytochrome P-450 is known to activate more hydrophobic substrates preferentially, and so the coefficient with log Ρ may reflect this selectivity, or it may reflect the ease with which more hydrophobic substances can penetrate the bacterium. Likewise, electron donor substituents are expected to facilitate oxidation of the triazene by P-450 (2a).


184



We have followed up on this study by preparing several more triazenes in which Ζ is a heterocyclic ring (2a). Since σ+ constants are not available for most of these rings, we have also performed M N D O calculations on the parent triazenes in order to describe their electronic properties. One triazene (Z = 1,2,4-triazole) is inactive, while another triazene (Z = 2-dibenzofuran) is more active than any of the aryltriazenes originally studied by Venger et al. Two QS ARs, Equations 2 and 3, were found that correlated the behavior of the 17 aryltriazenes along with 4 of the heterocyclic triazenes. log 1/C = 0.95 (± 0.25) log Ρ + 2.22 (± 0.88) η = 21, r = 0.919, s = 0.631

EHOMO

+ 22.69

(2)

log 1/C = 0.97 (± 0.24) log Ρ - 7.76 (± 2.73) q Mo + 5.96 η = 21, r = 0.931, s = 0.585

(3)

HO

is the energy of the highest occupied molecular orbital (HOMO) in eV. q oMo is the electron density on the alkyl Ν in the HOMO. The relationship between hydrophobicity and activity defined by these equations is essentially identical to that discovered earlier by Venger et al. At the same time, the electronic terms in these equations can be interpreted in a fashion consistent with that given for Equation 1. As the H O M O rises in energy, i.e., £HOMO becomes less negative, the molecule becomes easier to oxidize and activity increases. Equation 3, on the other hand, would appear to indicate that higher activity is associated with lower electron density on N . A closer inspection of the H O M O , however, shows that this orbital is largely concentrated on Z, i.e., the aromatic or heterocyclic ring, and not on the triazene. Electron-donor groups, while making the triazene easier to oxidize, also concentrate more of the electron density of the H O M O on Z, hence the paradoxical correlation between activity and qHOMO- It is important to remember that the electron density on an atom in a particular orbital is not necessarily indicative of the chemical reactivity of that site in the molecule. Equations 2 and 3 predict the 1,2,4-triazole to be 10 -10 -fold less active than the 2-dibenzofuran in accord with the observed inactivity of the triazole. The activity of one heterocyclic triazene, Ζ=2-thiazole, is greatly underpredicted by Equations 2 and 3. In this case, a σ* value for the thiazole ring is available, and Equation 1 also underpredicts the activity of this triazene. Since the M N D O and Hammett parameters give a consistent prediction for this triazene it is reasonable to believe that an additional, unknown mutation mechanism is acting in this case. EHOMO

H

6

7

Nitro-Polycyclic Aromatic Hydrocarbons Nitroarenes are mutagenic in both S. typhimurium strain TA98 and T A 100, and do not require the presence of S9 microsomes. QS ARs describing the mutagenic activity of these two systems are given in Equations 4 (3a) and 5 (3b).


18. SHUSTERMAN

185

Predicting Chemical Mutagenicity p

log TA98 = 0.65 (±0.16) log Ρ - 2.90 (±0.59) log (β10 8 + 1) (4) -1.38 (±0.25) e + 1.88 (±0.29) I - 2.89 (±0.81) I - 4.15 (±0.58) η = 188, r = 0.900, s = 0.886, log P = 4.93, log β = -5.48 1ο

LUMO

L

a

0

log TA100 = 1.36 (±0.20) log Ρ -1.98 (±0.39) e - 7.01 (±1.2) η = 47, r = 0.911, s = 0.737

(5)


LUMO

TA98 and TA100, the mutagenic activity of the nitroarene in each strain of the Ames bacteria, are defined as revertants/nmol mutagen. e is the energy of the lowest unoccupied molecular orbital in eV ( A M 1 for TA98, M N D O for T A 100). The negative coefficient with £ o in each equation indicates that the nitroarene becomes more active as this empty orbital falls in energy, i.e., the compound becomes a better electron acceptor. This relationship is consistent with the generally accepted activation mechanism for nitroarenes which postulates initial reduction of the nitro group to give a hydroxylamine intermediate, which ultimately reacts with D N A . L U M 0

L U M

The TA98 QS A R shows a two-term dependence of activity on log P. These two terms describe a bilinear function; activity rises as 0.65 log Ρ for log Ρ < 4.93 and then falls as -2.25 log Ρ for log Ρ > 4.93. The simple linear relationship seen for TA100 and log Ρ may simply be due to the lack of more hydrophobic compounds in the smaller T A 100 data set. The appearance of a bilinear activity-hydrophobicity relationship indicates that there is an optimal log Ρ for mutagenic compounds (log P ). More hydrophilic compounds are less likely to penetrate the bacterial membrane, while more hydrophobic compounds are likely to become sequestered in lipid phases before they can react with D N A . G

Equation 4 also contains two indicator variables. These variables are set equal to either 0 or 1 depending on the absence or presence of a particular structural feature. I = 1 signifies the presence of 3 or more aromatic rings in the nitroarene. Compounds containing 3 or more rings are 76 times more active than 1 or 2 ring compounds other factors being equal. This jump in activity may be due to an increased ability for larger compounds to intercalate into bacterial D N A . I = 1 indicates the arene belongs to the acenthrylene family. The negative coefficient with I shows that these compounds are all much less active than would be predicted on the basis of their hydrophobic and electronic properties alone. L

a

a

Equations 4 and 5 span a tremendous range in structural types and in mutagenic activity (ca. 10 revertants/nmol). Thus, these equations are powerful predictors of nitroarene behavior. Inspection of the TA98 data set shows that there is also a significant variation in each of the structural properties; variation in e o spans a 2.84 eV range accounting for an 8000-fold range in activity, while log Ρ varies from -0.02 to 7.84 accounting for 1700-fold (P < P ) and 3.5 χ lO^fold (P > P ) ranges in activity. 8

LUM

0

0


186



IQ-type Mutagens The IQ-type mutagens, i.e., 2-amino derivatives of imidazo[4,5-f]quinoline, 2, are especially potent mutagens which occur as pyrolysates in various types of cooked food (4,8-12). The compounds all require the presence of the S9 microsomal fraction, and lose most of their activity when either the 2-amino or 3-methyl group is missing, indicating that the key mutagenic processes involve this part of the skeleton. Unfortunately, the compounds isolated from food make a poor data set from the standpoint of QS A R development since their mutagenic activities are all nearly the same, and their hydrophobic and electronic properties span a relatively narrow range (-8.60 < ε Μο ^ -8.22 eV, 1.01 < log Ρ < 2.62). Expansion of the data set is essential in order to sort out the structural factors responsible forrelative mutagenicity. Debnath et al. have synthesized 5- (Y = H) and 6-substituted (X = H) derivatives of 1-methyl2-aminobenzimidazole, 3 (4). These compounds, like the IQ derivatives, require S9 microsome activation, and are inactive when either the 2-amino or 1-methyl group is absent. It is reasonable, therefore, to assume that these compounds act by the same mechanism as the IQ-type mutagens. ΗΟ

CH 2

CH

3

3

3

In order to facilitate the study of this enlarged data set we have excluded several compounds whose mutation rates in TA98 are known. Compounds with 1 or 2 methyl substituents on the exocyclic Ν were excluded, since it was assumed that cytochrome P-450-catalyzed N-demethylation would occur, and the observed mutation rates would not be representative of the parent compound. Similarly, two imidazonaphthalene derivatives were found to be much less active than predicted and were excluded. A QSAR describing the remaining compounds in our data set is given in Equation 6. log TA98 = 1.31 (±0.74) log Ρ - 0.30 (±0.09) Δ Ε η = 22, r = 0.906, s = 1.011

Ν Η

+ + 64.92

(6)

In this equation, log TA98 is the number of revertants/nmol mutagen, and Δ Ε + is the reaction enthalpy (in kcal/mol) for conversion of a hydroxylamine intermediate into a putative nitrenium ion, Equation 7 (geometries and energies calculated using AMI). Ν Η

Imid-NHOH -> Imid-NH+ + O H -


(7)


18. SHUSTERMAN


187

A positive relationship between hydrophobicity and activity is observed, in accord with the need for P-450 activation and membrane penetration. Activity also increases with increasing ease of nitrenium ion formation (smaller ΔΕΝΗ+). Several points concerning this QSAR are noteworthy. First, all of the terms in Equation 6 are significant at die 99% confidence level according to the t-test. On the other hand, the confidence interval for the log Ρ coefficient is rather large. Indeed, removal of one compound from the data set, 2-amino-5-hydroxy-lmethylbenzimidazole, slightly improves the quality of the correlation (r = 0.914, s = 0.853), but at the expense of the log Ρ term, which is only significant at the 87% confidence level. If log Ρ is removed from the equation altogether, then r = 0.901 for this smaller data set. Since this one compound is significantly more hydrophilic than any of the others in the data set not much trust can be placed in the log Ρ term until its role is validated by the testing of more compounds. Second, since nitrenium ion formation is believed to be essential before reaction can occur with D N A , it is very interesting to find a correlation between activity and ΔΕΝΗ+· On the other hand, it is important to keep in mind the possibility of collinear variables. In this case, the calculated ionization potential of the parent amine is strongly correlated with ΔΕΝΗ+ (r = 0.887). Thus, the dominant electronic factor may be ease of amine oxidation to generate a hydroxylamine, conversion of the hydroxylamine to a nitrenium ion, or even some still undetected process. Nevertheless, we believe that Equation 6 is a good starting point for designing and testing more compounds belonging to this critical family of mutagens. Inspection of the molecular and electronic structure of the nitrenium ions related to 2 and 3 is also revealing. The A M I geometry for the imidazole and adjacent benzene ring of the nitrenium ion clearly shows a pattern of alternating single and double bonds, similar to the imine resonance structure, 5. Inspection of bond orders shows that pi bond localization is predicted to occur for the nitrenium ion.

Ctv CH

3

4 minor

major

While it has been generally assumed that such a nitrenium ion would react with D N A exclusively at the exocyclic N , it would appear that other sites for nucleophilic attack are also available. For example, the nitrenium ion might transfer a methyl group to the D N A (Figure 1). This might serve to explain the need for a methyl group on the imidazole ring. Another intriguing possibility is nucleophilic attack on the benzene ring at C-6. If the nucleophile is water, rearrangement would yield a much less active phenol




5 nitrenium ion

\

CH

3

Figure 2. Mechanism of phenol formation from a nitrenium ion.


18. SHUSTERMAN


189

(Figure 2). Some support for a deactivation reaction occurring at C-6 can be found by comparing the mutagenic activities of the 5-CN and 6-CN derivatives of 3. The 6-CN derivative is 41 times more mutagenic, possibly due to a steric effect; the substituent blocks attack of water on the benzeneringof the nitrenium ion, preventing phenol formation and deactivation, and enhancing mutagenicity. Metabolic conver sion of food mutagens to analogous phenols appears to occur in vivo as well.


Conclusion The Ames test provides useful quantitative data which can be correlated using traditional QS AR techniques. The resultant QS AR equations can be used to correlate, and potentially, to predict mutagenic activity over a very wide activity range, and can accommodate a broad range of chemical structures. Consideration of relative hydrophobicity, as reflected in log P, is often essential in order to correctly account for the influence of structure on activity. Variations in chemical structure that can not be treated using Hammett substituent constants appear to be well treated using a variety of parameters derived from semi-empirical quantum chemical calculations. However, care must be exerted in the interpretation of the resultant QS ARs due to the great potential for collinear variables. Acknowledgment This research was supported by a grant for fundamental studies in toxicologyfromthe R. J. Reynolds Company. The support and advice of Corwin Hansen is gratefully acknowledged. The assistance of Asim Debnath in communicating unpublished results concerning the mutagenic activities and log Ρ values of substituted 1-methyl2-aminobenzimidazoles is gratefully acknowledged. Literature Cited 1. 2.

3.

4. 5.

Hansch,C.; Kim, D.; Leo, A.J.; Novellino, E.; Silipo, C .; Vittoria, A. CRC Crit. Rev. Toxicol.l989, 19, 185-226. (a) Shusterman, A.J.; Debnath, A.K.; Hansch, C.; Horn, G.W.; Fronczek, F.R.; Greene, A.C.; Watkins, S.F. Mol. Pharm. 1989, 36, 939-944. (b) Shusterman, A.J.; Johnson, A.S.; Hansch, C. Int. J. Quant. Chem. 1989, 36, 19-33. (c) Venger, B.H.; Hansch,C.;Hatheway, G.J.; Amrein, Y.U. J. Med. Chem. 1979, 22, 473-476. (a) Debnath, A.K.; Compadre, R.L.L.; Debnath, G.; Shusterman, A.J.; Hansch, C. J. Med. Chem. 1990, in press, (b) Compadre, R.L.L.; Debnath, A.K.; Shusterman, A.J.; Hansch, C. Environ. Mol. Mutagen. 1990, 15, 4455. (c) Compadre, R.L.L.; A. J. Shusterman, C. Hansch, Int. J. Quant. Chem. 1988, 34, 91-101. Debnath, A.K.; Shusterman, A.J.; Raine, G.P.; Hansch, C. unpublished results. Maron, D.; Ames, B.N. Mutat. Res. 1983, 113, 173-215.


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6. 7.

Dewar, M.J.S.; Thiel, W. J. Am. Chem. Soc. 1977, 99, 4899-4907. Dewar, M.J.S.; Zoebisch, E.G.; Healy, E.F.; Stewart, J.J.P. J. Am. Chem. Soc. 1985, 107, 3902-3909. 8. Kaiser, G.; Harnasch, D.; King, M.T.; Wild, D. Chem.-Biol. Interact. 1986, 57, 97-106. 9. Sugimura, T. Science 1986, 233, 312-318. 10. Jagerstad, M.; Grivas, S. Mutat. Res. 1985, 144, 131-136. 11. Nagao, M.; Wakabayashi, K.; Kasai, H.; Nishimura, S.; Sugimura, T. Carcinogenesis 1981, 2, 1147-1149. 12. Alexander, J.; Wallin, H.; Holme, J.A.; Brunborg, G.; Soderlund, E.J.; Becher, G.; Mikalsen, Α.; Hongslo, J.K. Mutation in the Environment, Part E; Wiley-Liss: New York, 1990, p. 159. RECEIVED November 18, 1991


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