Chem. Res. Toxicol. 1994, 7, 73-76
73
Structure-Genotoxicity Relationships of Allylbenzenes and Propenylbenzenes: A Quantum Chemical Study Ruey-Shiuan Tsai,? Pierre-Alain Carrupt,t Bernard Testa,*pt and John Caldwell* Institut de Chimie Thkrapeutique, Ecole de Pharmacie, Uniuersitk de Lausanne, BEP, CH-1015 Lausanne, Switzerland, and Department of Pharmacology and Toxicology, St. Mary's Hospital Medical School, Norfolk Place, London W2 IPG, U.K. Received August 26, 1993" Quantum mechanical calculations a t the semiempirical level (AM1 method) were conducted for estragole (l), methyleugenol (2), safrole (3), a-asarone (41, 8-asarone (5), elemicin (6), allylbenzene (7),eugenol (€9,trans-anethole (9), isosafrole (lo),and myristicin (ll),and the results compared with the known genotoxicity of 1-6 and the absence of genotoxicity of 7-1 1 (unscheduled DNA synthesis assay). The various compounds showed no significant differences in the relative stability of the radical species formed as intermediates in C-sp3 hydroxylation ( P H ~ ( ~ ~ d and i ~ d in ) ) the corresponding enthalpy of activation (AH*). In contrast, the carbonium ions of the genotoxic congeners 1-6 were shown to be comparatively more stable than those of the inactive compounds 7-1 1,with the exception of eugenol (8). The inactivity of this compound could be due to a very rapid stabilization of the carbonium ion by deprotonation to form a quinone methide, as suggested by quantum chemical calculations. The relative stability of the carbonium ion thus appears to be one of the key factors in the genotoxicity of allylbenzenes and propenylbenzenes.
Introduction Allylbenzenesand propenylbenzenes form an important group of flavor constituents found in a variety of essential oils. It has been shown that estragole (11,methyleugenol (2), safrole (3), a-asarone (a), and 8-asarone (5) are hepatocarcinogenic in the pre-weanling mouse model (13) and are genotoxic in the unscheduled DNA synthesis (UDS) assay using freshly isolated rat hepatocytes in primary culture (4-6), while allylbenzene (71, eugenol (8), trans-anethole (9),isosafrole (lo),and myristicin (11)show little or no genotoxicity or carcinogenicity (Figure 1).In contrast, elemicin (6) has no detectable hepatocarcinogenic activity in the pre-weanling mouse model (I), but is genotoxic in the UDS assay (6). Metabolic oxidation of the allyl or propenyl side chains of these compounds may proceed via epoxidation of the or via hydroxylation of the C-sp3carbon double bond (7,8) a to t h e double bond (8-10) (benzylic or terminal hydroxylation, respectively). The primary mechanism of allylbenzenes genotoxicity is believed to involve benzylic hydroxylation followed by 0-sulfation of the alcoholic metabolite and heterolytic cleavage of the 0-sulfate moiety to form an electrophilic carbonium ion reactive toward a variety of nucleophilic sites in DNA (Figure 2) (11,12). Similarly, the w-hydroxylation and subsequent sulfation of propenylbenzenes could also lead to formation of carbonium ions (Figure 2), thus offering a mechanistic model for the genotoxic effects of some of these compounds. Structure-genotoxicity relationships among the compounds in Figure 1 are not immediately obvious. Allylbenzene congeners with 4-methoxy, 3,4-dimethoxy, 3,4methylenedioxy, or 3,4,54rimethoxy substituents on the
* Correspondence should be addressed to this author; FAX:
4121/
692-2880.
Universite de Lausanne. St. Mary's Hospital Medical School. a Abstract published in Aduance ACS Abstracts, December 15,1993. 7
0893-228x/94/2707-0073$04.50/0
phenyl ring show a higher genotoxicity than the unsubstituted allylbenzene,the phenolic eugenol,and myristicin. In contrast, the propenylbenzenes, except for cy- and P-asarone, are relatively non-genotoxic. However, no mechanistic hypothesis exists that would explain these differences in genotoxicity. The goal of this study is to search for a mechanistic model that would make sense of the biological data. The side-chain hydroxylation of allylbenzenes and propenylbenzenes is catalyzed by cytochrome P450, which acts here by an oxygen-rebound mechanism (C-H homolytic cleavage followed by radical recombination) (13). Since the rate-limiting step in this reaction is the C-H homolytic cleavage, we first examined the influence of aromatic and side-chain substituents on the C-H bond dissociation energy, using quantum chemical calculations. The relative stability of the carbonium ions resulting from C-0 heterolytic cleavage was also examined and found to offer a fair discrimination between the active and inactive compounds.
Methods All calculations were performed with the semiempirical AM1 method (14) in the MOPAC 5.0 software (15) runningon aSilicon Graphics Personal Iris 4D/25workstation or a Sun Sparc 2 workstation. All geometry optimizations were performed with the PRECISE keyword allowing similar resultson both machines. The reaction profile representing the possible metabolic activation of allylbenzenes and propenylbenzenes can be simplified as shown in Figure 3. Since there exists a linear relationship between the enthalpy of activation (AH')and the relative stability of the radical (AI&) in hydrogen abstraction reactions, particularly in the case of congeneric reactants, we assessed the C-H homolytic cleavage first by MR. In addition, the bond dissociation energies were also evaluated following the approach of Korzekwa et al. (16),whoused thep-nitrosophenoxyl radical to model P450-mediated hydrogen abstraction and calculated the corresponding enthalpies of activation according to eq 1: 0 1994 American Chemical Society
74 Chem. Res. Toxicol., Vol. 7,No. 1, 1994
Tsai et al.
I
0’
‘0
1 Estragole
‘0
2 Methyleugenol
7 Allylbenzene
3 Safrole
8 Eugenol
‘ 0
4 a-Asarone
9 trens-Anethole
‘ 0
5 B-Asarone
10 bosafrole
6 Elemicin
11 Myristicin
Figure 1. Chemical structure of the allylbenzenes and propenylbenzenes investigated here.
I
C-H homolytic cleavage
C-H homolytic cleavage
[ - - R a y
Benzylic hydroxylation ’
Sulfation
1
I
w Hydroxylation
I
TruJtbDltpte
i
j Sulfation
i
OH
OSOj
Figure 3. Simplified reaction profile of allylbenzenes and propenylbenzenes and corresponding thermochemical parameters. is the enthalpy of activation of the C-H homolytic cleavage, A H R ( ~ ~is & the~relative ) stability of the radical species, and A H ~ ( ~ b o , , is i ~the ) relative stability of the carbonium ion.
I Elecuophilic atrack on DNA
Figure 2. Postulated mechanisms of metabolic activation of allylbenzenesand propenylbenzenesleadingto their genotoxicity. Dotted arrows indicate hypothetical routes. 0.22AHR’ + 2.38IP + 2.60
n = 19
(1)
r2 = 0.94
where n is the number of compounds examined, and r2 is the squared correlation coefficient. This equation combines the stability of the radical relative to that of the p-nitrosophenoxyl radical (AHR’)as well as ita ionization potential (IP)to assess the enthalpic change in C-H homolytic cleavage. The heats of formation of the p-nitrosophenoxyl radical and p-nitrosophenol were 19.04 and -14.65 kcal/mol, respectively (16).
As for the carbonium ions, their relative stabilities A H R ( ~ ~ ~ ) were calculated as the heat of formation of the carbonium ion minus that of the alcoholicmetabolite. A more rigorous approach would have been to consider C-0 heterolytic cleavage of the 0-sulfate ester. However, the sulfate group creates difficulties with the semiempirical calculations and would only affect the A H R ( values ~ ~ ~by ~a constant ~ ) increment.
Results and Discussion T h e calculated heat of formation (A&) for the substrates, t h e radicals, t h e alcohols, and t h e carbonium ions a n d the ionization potential of the radicals are listed in Table 1. Some of t h e calculated thermochemical parameters together with t h e toxicity d a t a (4-6) are shown in Table 2. Globally, t h e average AHp, value of radicals for t h e genotoxic compounds 1-6 (14.8 f 2.9 kcal/mol) is
Structure-Genotoxicity Relationships of Alkenylbenzenes
Chem. Res. Toxicol., Vol. 7,No. 1, 1994 76
Table 1. Calculated Heats of Formation for Allylbenzenes Interestingly, the genotoxic compounds 1-6 have AHk and Propenylbenzenes and Their Radicals, Alcoholic (carbonium)values below 231.0 kcal/mol, while for the inactive Metabolites, and Carbonium Ions* compounds 7 and 9-1 1,the corresponding values are above substrate Ahh,hb)* “m M f ( h h d d “hiu Ipfm, e kcal/mol (Table 2). The only exception is eugenol 231.0 -4.7 8.4 -45.6 179.6 8.3 1,estragole (81, whose case will be discussed later. In fact, the average -38.9 -26.4 -79.9 145.6 8.3 2,methyleugenol M R ( c a r b o n i u ) Value Of the genOtOXiC compounds is 227.7 3,safrole -25.6 -13.1 -66.2 164.7 8.5 f 2.2 kcal/mol, while for the inactive compounds (excluding -63.3 -124.4 103.4 8.1 4,a-asarone -80.5 eugenol) it is 233.9 f 3.2 kcal/mol. This difference, while -59.3 -122.9 106.6 8.1 5,B-asarone -28.8 111.4 8.4 -61.4 -116.1 6,elemicin -75.2 not considerable, is nevertheless genuine and is seen as 33.2 46.8 -7.6 225.1 8.5 7,allylbenzene suggesting that the stability of the carbonium ions may -36.3 -89.7 137.9 8.4 -48.6 8,eugenol be one of the determinants for the genotoxicity of allyl179.6 8.3 8.4 -53.1 9,trans-anethole -9.3 10,isosafrole -30.1 -13.1 -73.9 164.7 8.6 and propenylbenzenes. Thus, the non-genotoxicity of -48.1 -100.7 130.9 8.6 -60.3 11, myristicin some allyl- and propenylbenzenes could be due to a greater difficulty of forming its carbonium species rather than to Heats of formation were calculated by the semiempirical AM1 method and are in kcal/mol. b Heat of formation of the substrate. a particular inertness of hydroxylation. As far as strucHeat of formation of the radical. Heat of formation of the alcoholic ture-reactivity relationships are concerned, a p-methoxy, metabolite. e Heat of formation of the carbonium ion. f Ionization o-methoxy, or p-hydroxy substituent on the phenyl ring potential of the radical. Table 2. Calculated Thermochemical Parameters for Allylbenzenes and Propenylbenzenes and Their Radicals and Carbonium Ions*
1, estragole 2,methyleugenol 3,safrole 4,a-sarone 5,j3-asarone 6,elemicin 7,allylbenzene 8,eugenol 9,trans-anethole 10,isosafrole 11, myristicine
2.68i 0.93 2.16 i 0.47f 2.01 f 0.448 3.75 i 0.7% 5.86 i 0.788 2.57 i 1.6W -1 (inactive) -1 (inactive) -1 (inactive) -1 (inactive) -1 (inactive)
13.0 12.5 12.5 17.2 19.5 13.8 13.6 12.3 17.7 17.1 12.2
17.7 17.7 18.2 18.3 18.8 18.2 18.5 17.8 18.8 19.3 18.4
225.2 225.5 230.9 227.8 229.5 227.5 232.7 227.6 232.7 238.6 231.6
Thermochemical parameters are in kcal/mol. Induction of unscheduled DNA synthesis (UDS) measured by the ratio of the incorporated [aHIthymidine into the hepatocyte nuclear DNA in the presence and absence of the compound (4-6). Relative stability of the radical, m f ( r a d i 4 ) - A&(8ubtrat,+ Calculated energies of activation based on eq 1. e Relative stability of the carbonium ion, AHf(carbonium) - mf(+&ol). f Observed maximal UDS response at 1 mM. At concentrations higher than 1 mM, no UDS response was observed, and the compound was highly cytotoxic (4-6). e Observed maximal UDS response at 0.5 mM. At concentrations higher than 0.5 mM, no UDS response was observed, and the compound was highly cytotoxic (4-6).
identical to that of the inactive compounds 7-11 (14.6 f 2.6 kcal/mol). Similarly, the average calculated enthalpies of activation AHs (radical) cannot distinguish between genotoxic (mean value = 18.2 i 0.4 kcal/mol) and inactive compounds (mean value = 18.6 f 0.6 kcal/mol). It should be noted that the calculated enthalpies of activation for the benzylic hydroxylation of allylbenzenes or w-oxidation of propenylbenzenes appear to be low compared to other substrates of cytochrome P450 (16),implying that the allyland propenylbenzenes are very good substrates of the enzyme as far as electronic factors are concerned. This is confirmed by the fact that the alcohols are indeed urinary metabolites of these substrates (17-19). It should be noted that the identical geometry and heat of formation (AHf)of the radical species of estragole (1) and trans-anethole (91, and of safrole (3) and isosafrole (10) (Table 11, result from the delocalization of the unpaired electron in this type of radicals (Figure 2). Although some propenyl metabolites were detected when allylbenzene, safrole, or eugenol was given to rats ( I 7,20, 21 1, double bond migration was either nonexistent or negligible in the metabolites of propenylbenzenes (18-19, 22).
with electron-releasingmesomeric effects tends to stabilize the formation of the carbonium ion, as seen with the lower M R ( c a r b o n i u ) values of 1-3,6,8, and 11 as compared to 7, or those of 4 and 5 as compared to 9. However, bridging of the methoxy substituents in 3 and 11 in comparison with 2 and 6, respectively, results in a decrease of carbonium stability. The fact that the carbonium species of allylbenzenes and propenylbenzenescan readily interconvert as indicated by the gas-phase calculations, i.e., 1 and 9 as well as 3 and 10 having identical geometry and energy (Table 11,suggests that the formation of DNA adducts with the carbonium ions is a concerted rather than two-step reaction. In addition, the non-genotoxicity of the 3‘-hydroxy metabolites of 9 and 10 could also be due to their fast subsequent biooxidation, resulting in the formation of cinnamic acids and benzoic acids (18, 22-23). The case of eugenol (8) warrants special consideration since this compound stands out as a remarkable exception. One possible explanation could be that eugenol is the only compound in the series that can be detoxified by glucuronidation or sulfation of a phenolic group (24). However, another mechanism also exists to account for its lack of genotoxicity, namely, the formation of a quinone methide species either from its radical by a second hydrogen abstraction or from the carbonium ion by deprotonation (Figure 4). The AHfvalue of the quinone methide species was calculated as -7.2 kcal/mol, indicating that its formation could either be competitive to that of the carbonium ion or could allow instantaneous detoxication of the latter by deprotonation (Figure 4). Eugenol is the only compound in the series having a p-hydroxy group and thus being able to form a quinone methide. In addition, indirect experimental proof exists for the formation of the quinone methide as an intermediate in the metabolism of eugenol, since the oxidation of this compound with rat liver or rat lung microsomes yielded glutathione conjugates at both the benzylic and w-carbon atoms (25). The computational results reported here suggest that the relative energy of the carbonium ion is a major factor accounting for the genotoxicity, or lack thereof, of allylbenzenes and propenylbenzenes. Equally, the calculations are consistent with the carbonium ion being the actual genotoxic species. Indeed, the genotoxicity of both allylbenzenes (1-3 and 6) and propenylbenzenes (4 and 5) can be satisfactorily explained on this basis, while a relatively stable quinone methide either prevents forma-
76 Chem. Res. Toxicol.,Vol. 7,No.1, 1994 OH
(*)
w
H’
1
j‘-c +29.1 kcaVmol
+174.2 kcUmol
t OH
-145.1 kcaVmol
f
+\ Figure 4. Postulated reaction pathways of eugenol (8) leading to the formation of a quinone methide species. (A) Formation from the radical by a secondhydrogen abstraction. (B)Formation from the carbonium ion by deprotonation. The numbers are differences in enthalpy.
tion of the carbonium ion of eugenol or allows its instantaneous deactivation by deprotonation. Nevertheless, because other routes of biotransformation obviously influence the carcinogenicity of this class of compounds, the results reported here can only be interpreted qualitatively and not quantitatively. Thus, quantum chemical calculations, while not offering compelling evidence, nevertheless allow valuable insights into molecular mechanisms of genotoxication of some allylbenzenes and propenylbenzenes. Acknowledgment. B.T. and P.A.C. are indebted to the Swiss National Science Foundation for support.
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