Benzo[a]pyrene and Benz[c]phenanthrene: The ... - ACS Publications

Chem. Res. Toxicol. 2002, 15, 1069-1079

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Benzo[a]pyrene and Benz[c]phenanthrene: The Effect of Structure on the Binding of Water Molecules to the Diol Epoxides Katrina W. Brown,† Stephen B. Little,‡ and James R. Rabinowitz*,‡ United States Environmental Protection Agency, NHEERL/ECD, Research Triangle Park, North Carolina 27711, and Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7270 Received February 6, 2002

The interactions with water of the diol epoxides (DEs) of both a planar and a nonplanar PAH have been examined using molecular dynamics. To determine probable water locations around the DE for later use in the study of DE protonation, molecular dynamics simulations using the OPLS force field were carried out on diol epoxides surrounded by a 22 Å box of explicit water molecules. Results for 30 ps simulations indicate that 10-60% of the time, depending strongly on the conformation and type of the DE, there is a water molecule forming a hydrogen bond with the epoxide oxygen. The patterns seen in the frequency at which a DE binds a water molecule reflect patterns seen in the relationship between the type of PAH DE and amount of DNA adduct formation. Examination of the orientations and arrangements of the water and DEs during the simulations showed that the bound waters existed in several preferred configurations which are also dependent upon the PAH DE geometry.

Introduction 1

Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous organic chemicals to which the human population and the environment are chronically exposed. There are many of these types of compounds, and they cause a number of different biological effects. Some PAHs are powerful mutagens and animal carcinogens, while other molecules in this class show no similar activity after considerable testing. Metabolic activation of PAHs is a prerequisite for carcinogenesis, and in a number of examples a bay region diol epoxide metabolite (DE) has been demonstrated to be the ultimate carcinogen (1-3). Benzo[a]pyrene (BaP) is an extensively studied PAH in which the bay region DE has two diastereomeric forms, one in which the distal hydroxyl group and the epoxide oxygen are anti and one in which they are syn. For each of these two conformations, there are two low-energy possibilities for the orientations of the hydroxyl groups relative to one another: one arrangement in which the hydroxyl groups are nearly in the plane of the remainder of the molecule (quasi-diequatorial) and one in which they are nearly perpendicular to the plane (quasi-diaxial), giving rise to four possible BaP DE conformations (see Figure 1). Experimental evidence shows that the syn and anti conformations are quasi-diaxial and quasi-diequatorial, respectively (4), corresponding to the two conformations calculated to be of the lowest energy (5). Benzo[c]phenanthrene (BcPh), unlike BaP, is a nonplanar PAH due to crowding in the fjord region, and this †

University of North Carolina at Chapel Hill. United States Environmental Protection Agency, NHEERL/ECD. Abbreviations: BaP, benzo[a]pyrene; BcPh, benzo[c]phenanthrene; DE, diol epoxide; PAH, polycyclic aromatic hydrocarbon; ae, anti-quasidiequatorial; iae, in-anti-quasi-diequatorial; ise, in-syn-quasi-diequatorial; isx, in-syn-quasi-diaxial; oae, out-anti-quasi-diequatorial; sx, syn-quasi-diaxial. ‡

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nonplanarity gives rise to another structural relationship that is relevant when considering the possible conformations of its bay region DE (Figure 2) (6). The epoxide oxygen can be on the same side of the saturated ring as the distal ring of the remaining conjugated system (in) or on the opposite side (out) so that for each BaP conformation, BcPh has two possible related structures. Experimental evidence indicates that in BcPh both the syn and anti structures take the quasi-diequatorial form (7), and theoretical calculations have shown the in-synquasi-diequatorial and the in-anti-quasi-diequatorial structures are the lowest energy syn and anti structures (6). Once the PAH DE is formed by metabolic processes, there are several pathways through which it can potentially advance, possibly resulting in a tetrol or a DNA adduct. Tetrols can arise both before the PAH DE has interacted with DNA and after the formation of a noncovalent PAH DE-DNA complex (8). They can be produced after the PAH DE hydrolyzes, either through a spontaneous reaction or, in the proton-rich environment of the polyanionic DNA polymer, through an acidcatalyzed reaction (9, 10). The hydrolysis of the epoxide ring can occur through the attack of a water molecule on the epoxide ring or through a DNA-mediated, acidcatalyzed reaction in which initial attack by a proton on the epoxide oxygen opens the epoxide ring, forming a carbocation that can then react with water to form a tetrol. Alternatively, the PAH DE may react with DNA to form an adduct. This can occur after the hydrolysis of the PAH DE if a nucleophilic site on DNA is more readily available to the carbocation than a water molecule. Adducts may also be formed by direct interaction between the PAH DE and nucleophilic sites in DNA. DNA adducts often arise from a reaction of the PAH DE with the exocyclic amine groups of adenine and

10.1021/tx020009+ CCC: $22.00 © 2002 American Chemical Society Published on Web 06/29/2002

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Figure 1. 7,8-Dihydroxy-9,10-epoxide benzo[a]pyrene (BaPDE). The distal hydroxyl group can be syn or anti to the epoxide group, and the two hydroxyl groups can be either axial or equatorial to the saturated ring, yielding four possible low-energy isomers.

Figure 2. 3,4-Dihydroxy-1,2-epoxide benzo[c]phenanthrene (BcPhDE). In BcPhDE, as in BaPDE, the distal hydroxyl group can be syn or anti to the epoxide group, and the two hydroxyl groups can be either axial or equatorial to the saturated ring. Figure 2b and Figure 2c show an example of the additional conformational possibilities introduced due to the nonplanarity of the BcPhDE where, as shown, the distal ring of an anti-quasidiequatorial DE can be either ‘in’ or ‘out’ with respect to the epoxide oxygen.

guanine. The location and structure of the adducts are, however, dependent not only on the type of PAH but also

on the isomeric and enantiomeric form of the DE. Experimentally, it is seen that the percentage of the PAH DEs that bind to the DNA is larger if the DE is nonplanar or has four rings (11). Additionally, it has also been observed that nonplanar PAH DEs such as BcPh form more adducts with adenine (11-13) whereas adducts of planar PAHs form more adducts with guanine. Although it is obvious that the ratio of adduct formation to tetrol formation is dependent upon the structure of the PAH DE, the factors which determine this dependence are not at all well understood. We are currently using computational methods to investigate the underlying forces that determine the relative capacity of a PAH DE to bind to DNA rather than to form a tetrol. Fundamental to this work is determining the ease with which the epoxide ring is opened by neutral and charged species, such as water and H3O+, and how the energetics of this ring opening are dependent upon the PAH DE structure. Before we can explore this question, we must consider how and where water molecules approach the diol epoxides. In the present study, we discuss molecular dynamics simulations we have conducted to determine orientations of water molecules around BaP and BcPh DEs. Molecular dynamics simulations were performed to determine the frequency of water molecules bound to the epoxide oxygens and the probable positions of the water molecules around four different diol epoxides of BcPh and two of BaP. The in-syn-quasi-diequatorial (BcPhDEise), in-syn-quasi-diaxial (BcPhDEisx), in-anti-quasi-diequatorial (BcPhDEiae), and out-anti-quasi-diequatorial conformations (BcPhDEoae) of BcPh as well as the antiquasi-diequatorial (BaPDEae) and syn-quasi-diaxial (BaPDEsx) conformations of BaP were examined. The two selected BaPDEs were chosen because they are the

Water Binding to Diol Epoxides

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Table 1. Close Approaches of Water Molecules to Diol Epoxidesa PAH DE

no. of incidences of r(Oep-Ow) < 3 Åb

no. of responsible O atomsc

no. of incidences of r(Oep-Hw) < 2 Åd

no. of responsible H atomsc

no. of unique configurationse

BaPDEae BaPDEsx BcPhDEise BcPhDEisx BcPhDEoae BcPhDEiae

29621 15394 24265 30332 19202 32128

8 7 8 8 9 4

15082 3270 7551 4551 5966 18536

8 9 11 10 14 7

3 3 4 3 4 3

a Table 1 shows the number of occasions during the 30 402 time steps examined (step size of 0.5 fs and time step of 1 fs) in which water oxygen atoms and hydrogen atoms are close to the epoxide oxygen atom. b The number of occasions there was a vicinal water molecule (occurrences when water molecules had an oxygen atom, but not necessarily a hydrogen atom, close to the epoxide oxygen). In some of the individual snapshots, there were several water molecules with oxygen atoms within the 3 Å cutoff of the epoxide oxygen, hence the number of occurrences where r(Oep-Ow) < 3 Å can be larger than the number of time steps examined. c The number of distinct closeapproaching water oxygen and hydrogen atoms. d The number of occurrences where a hydrogen atom on the vicinal water was also close to the epoxide oxygen atom. e The number of unique configurations seen in the two simulations that appear in more than 1% of the snapshots.

lowest energy structures for the anti and syn conformations and are the forms observed experimentally. These structures along with the selected BcPhDE conformers will allow us to make comparisons between a planar and a nonplanar DE as well as between a four-ring and a fivering DE. Additionally, the selected BcPhDEs will enable us to examine effects of the syn and anti structures, as well as the quasi-diequatorial and quasi-diaxial structures, on the surrounding water molecules.

Methods Version 5.11 of HyperChem was used for all simulations (14). A 22 Å box was initially constructed around the DE, containing approximately 350 water molecules. A geometry optimization was performed using the OPLS (15) force field in which the water was free to move but the DE had a fixed position and geometry. This constraint of the DE was necessary because energy minimizations of the DEs using the OPLS force field yield a different energy ordering of the conformers from that found in the quantum mechanical studies (5, 6), the latter of which agreed with existing experimental information. (The other force fields examined, MM+ and AMBER, were also unable to give the correct energy ordering of the different DEs.) The geometries for the DEs were therefore taken from previous quantum mechanical calculations (5, 6), as were the potential derived charges (6). TIP3P was used as the model for the water molecules (16). The optimized water-DE system was used as the starting point for the dynamics simulations which once again used the OPLS force field and were conducted at 320 K. There was a heat-up time of 0.2 ps, and then each simulation was run for 30 ps with a 0.0005 ps step size. Data, including the system temperature and energy as well as the three-dimensional positions and orientations of each molecule, were collected every 0.001 ps. This yielded 30 200 time steps of the simulation where each time step includes all the pertinent information about the system such that if the time steps are replayed in order, they reproduce the simulation. The potential energy was monitored throughout the simulation, and if it did not reach a steady state after the first 15 ps, the process was repeated. As in the optimization, the geometry of the DE was held fixed during the dynamics simulation, thus keeping the correct conformation of the DE while still allowing for electrostatic interactions between the DE and water molecules. The distances between the epoxide oxygen and the oxygen atoms in each of the water molecules were measured in every time step. The water molecules whose oxygen atom came within 3 Å of the epoxide oxygen during the last 15.2 ps of the simulation were thus determined. These close approaching water molecules were labeled so that they could be distinguished in all time steps. The distances between the hydrogen atoms of a labeled water molecule and the epoxide oxygen were also measured for each time step. Time steps from the last 15.2 ps

of the simulation in which a water hydrogen to epoxide oxygen distance was less than 2 Å (those within a good hydrogen bonding distance) were then reexamined, and three-dimensional geometries and orientations of the bound water and the DE were extracted. We refer to this arrangement of the two molecules as the configuration of the bound water and the DE. In addition to the hydrogen-bound water, any water molecules, the oxygen of which was consistently closer than 3 Å to the epoxide oxygen, were also retained in the extracted configuration. This entire process was repeated twice for each DE examined.

Results The occurrences of water oxygen and hydrogen atoms close to the epoxide oxygen in the simulations are tabulated in Table 1 for all of the diol epoxides examined. The second column in the table shows the combined number (from both simulations for each DE) of occurrences in which a water oxygen atom (Ow) was within 3 Å of the epoxide oxygen atom (Oep). In some of the individual time steps, there were several water molecules with oxygen atoms within the 3 Å cutoff of the epoxide oxygen; hence, the number of occurrences where r(Oe-Ow) < 3 Å can be larger than the number of time steps examined. However, for all of the DEs, there were only 2-5 different water molecules whose oxygen atom was ever within 3 Å of the epoxide oxygen during the course of the 15.2 ps simulation. Due to the distinguishability of the water molecules, the snapshots could be reexamined in sequence while monitoring the distance between the epoxide oxygen and each of the hydrogens on the vicinal water molecules. The number of occurrences when a water molecule’s hydrogen atom came within 2 Å of the epoxide oxygen was found and is given in column 4 of Table 1. This gives an indication that the water molecule was close enough, and oriented such that it could form a hydrogen bond with the epoxide oxygen. Comparisons of the figures in column 2 with those in column 4 give an idea of how often the nearby water molecules bind, or do not bind, with the epoxide oxygen. For example, in the case of BaPDEsx, only 21% of the time steps that had an oxygen atom within 3 Å also had a hydrogen atom within 2 Å, indicating that the majority of the time a water molecule was actually close enough to bind to the epoxide group, its hydrogen atoms were not oriented such that it could. Even a smaller percentage, 15%, of the close approaching water molecules actually formed bonds in the BcPhDEisx simulations whereas in the BcPhDEiae and BaPDEae simulations when a water was close enough to bind to

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the epoxide oxygen, it did indeed form a hydrogen bond over half of the time. As discussed in the previous section, the system at each time step could be examined to determine the configuration, or three-dimensional orientation, of a bound water molecule with respect to the DE. The spatial configurations of bound water molecules extracted from each time step in which both distance criteria were met, namely, r(Oep-Ow) < 3 Å and r(Oep-Hw) < 2 Å, were compared, and commonly occurring spatial configurations were then determined. A representative time step where the water and DE were in a specific, commonly occurring, orientation/conformation was chosen to illustrate the geometry of the molecular system (see Figure 3). It should be noted that several of the common configurations extracted from the first simulation for a given DE were the same as, or very similar to, configurations extracted from the second simulation for the same DE. Consequently, the configurations extracted from the two individual simulations were compared, and if the RMS deviation of the water oxygen and bound hydrogen positions was less than 0.8 Å, then those configurations were considered equivalent. The last column of Table 1 gives the total number of unique water bound configurations found in the two simulations for each DE having accounted for the redundant cases. The percentages of time for which each of the configurations existed during the simulations are shown in Table 2 as well as the total percentage of time that a DE had a bound water. The representative snapshots showing the orientations of the water molecules around the DEs for the two most commonly occurring configurations for each DE, and their frequencies of occurrence, are shown in Figure 3. As stated above, for each simulation, an extracted configuration represents a specific orientation of the water oxygen and the epoxide-bound hydrogen. Whereas the positions of the water molecule would fluctuate slightly from time step to time step, the snapshots in Figure 3 show atom positions found in a typical time step and are representative of the general configuration. In Tables 2 and 1, only those configurations that occurred in more than 1% of the snapshots are included, yet this cutoff excludes only two configurations at most for a given simulation. It was occasionally seen that two water molecules, represented by two different extracted configurations, would both be within binding distance of the epoxide oxygen in the same snapshot. However, this situation never occurred for a majority of time that either configuration existed, so the overlap of the configurations was ignored. Table 3 shows, for each of the DEs examined, the total number of individual water molecules from both of the simulations that have contributed to the occurrence of an extracted configuration, and it can be seen that at most there were only three pertinent water molecules. In other words, up to three water molecules independently adopted the same orientation around a DE when bound to the epoxide oxygen. However, for a given DE, there were also only a total of four to seven molecules that were responsible for all of the common configurations extracted for that DE. This means that there were no more than seven water molecules that individually bound to the DE during the simulations. Although water molecules from the two separate DEwater simulations would often adopt the same orientation

Brown et al.

(cases in Table 3 where the number of water molecules contributing to the configuration is greater than or equal to 2), there was only one case, that of configuration oaeA, where more than one water molecule from a single simulation adopted the same orientation around the epoxide. There was also only one occurrence, during the first BcPhDEisx simulation, in which one water molecule was responsible for more than one extracted configuration (isxA and isxC). In this case, the position of the water molecule fluctuated such that the two hydrogens were each oriented toward the epoxide oxygen but in distinctive ways and at different times during the simulation. It was frequently found that the position of a water molecule fluctuated such that during the course of the simulation both hydrogens had at some point acquired the same orientation near the epoxide. In these cases, the extracted configuration is then representative of the orientation of one water oxygen and either of its hydrogen atoms, when they are within a good hydrogen bonding distance. For these instances, the number of contributing water molecules listed in Table 3 will be less than the number of contributing hydrogen atoms. Comparing column 3 of Table 1 (the number of water oxygens that were close to the epoxide) to the rows in Table 3 showing the number of water molecules represented by the extracted configurations, it can be seen that not all of the water oxygens that were within 3 Å of the epoxide oxygen are represented in the extracted configurations. This is due to the fact that in many cases a water molecule that was close to the epoxide oxygen [where r(Oep-Ow) < 3 Å] never had a hydrogen that was within 2 Å of the epoxide oxygen. Some of these water molecules were bound to the hydroxyl oxygens, and if this placed the water oxygen close to the epoxide oxygen for the majority of the time during which a configuration existed, then the hydroxyl-bound water is included in the extracted configuration, as seen in Figure 3. Figure 3 shows the water orientations in the two most common configurations found in the simulations for each of the diol epoxides. To give an idea of the spatial orientation of the water molecules, Table 4 details the position a bound water molecule adopts around the epoxide oxygen for each of the configurations. Its orientation is described relative to the epoxide oxygen in three linearly independent directions: (1) either on the side of the epoxide oxygen that is closer to or on the side that is further from the conjugated rings; (2) either on the side of the epoxide oxygen that is closer to or on the side that is further from the plane of the saturated ring; and (3) either ‘below’ the epoxide oxygen (between the epoxide oxygen and the saturated ring) or ‘above’ the epoxide oxygen (the epoxide oxygen is between the saturated ring and the water oxygen) as shown in Figure 4. Some general trends can be seen in the positions of the water molecules such as the propensity of the water to be above the epoxide oxygen. Figure 3a shows the most common orientation of the water molecule found in the BaPDEae-water simulations, aeA. In this case, there was a second water molecule that was bound to the neighboring hydroxyl group, regularly close to the epoxide oxygen, and this water molecule has therefore been included in the extracted configuration. The water molecule bound to the epoxide is not over the saturated ring and is slightly toward the conjugated ring side of the epoxide oxygen. This orientation of the water molecule places it less than



Figure 3. Representative snapshots of the two most frequently occurring water orientations for each of the diol epoxides studied. The percentages are the percent of time, during the 30.4 ps examined, that the configuration existed. The figures are all viewed from above, as defined in Figure 4, where the epoxide oxygen is out of the page toward the viewer. The angle given in each figure is defined in Figure 3m. It is measured as the angle between the midpoint of the two carbons of the epoxide group (×) and the oxygen atom of the water when the epoxide oxygen is the origin. These values give an idication of how high (out of the page) the water oxygen is from the epoxide carbons as compared to the epoxide oxygen atom. If a water oxygen atom were the same distance above the epoxide carbons as the epoxide oxygen, the angle would be 90°, and if a water oxygen were directly above the epoxide oxygen, the angle would be 180°. It should be recalled that all of the water hydrogen atoms involved in a bond with the epoxide oxygen are less than 2 Å from the epoxide oxygen.

3 Å from H10 (see Figure 1) such that electrostatic interactions between the water oxygen and DE hydrogen could help stabilize the configuration. Figure 3b is a

representative snapshot of the second most common configuration, the aeC configuration, which is found far less frequently in the simulations. In this configuration,

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Chem. Res. Toxicol., Vol. 15, No. 8, 2002 Table 2. Times of Existence for Configurationsa

configuration BaPDE aeA BaPDE aeB BaPDE aeC BaPDE sxA BaPDE sxB BaPDE sxC

% of time in existence 41 2 6

% of bound waters that adopt configurationb 84 4 12 49c

4.5 5 1

43 48 9 10.5c

BcPhDE iseA BcPhDE iseB BcPhDE iseC BcPhDE iseD

2.5 3 15.5 2.5

10 13 66 11 23.5c

BcPhDE isxA BcPhDE isxB BcPhDE isxC

8.5 3 3.5

57 20 23 15c

BcPhDE oaeA BcPhDE oaeB BcPhDE oaeC BcPhDE oaeD

7 3.5 2 6.5

37 18 11 34 19c

BcPhDE iaeA BcPhDE iaeB BcPhDE iaeC

7.5 30 23.5

12 49 39 61c

a Table 2 shows the percentage of time in the last 15.2 ps of both of the molecular dynamics simulations for each diol epoxide that the representative configuration existed. b Percentage indicates how frequently the bound water molecules were oriented as in the extracted configurations. c Total percent of time with a bound water.

the bound water still favors the conjugated ring side of the epoxide group but is over the C10-C10a bond of the saturated ring. The aeB configuration (not shown) was not as common as these but was interesting because in addition to being involved in a hydrogen bond with the epoxide oxygen, the water molecule is also in a hydrogen bond with a hydroxyl group of the DE where the water’s oxygen atom is bound to the hydroxyl’s hydrogen atom. In the BaPDEsx-water simulations, there was a bound water molecule only 10.5% of the time, less than for any other DE. The BaPDEsx molecule was also the only DE examined for which none of the extracted configurations had a water molecule on or even close to the side of the epoxide oxygen that is nearer the saturated ring. The second most frequently seen configuration which accounts for 43% of all of the bound waters is the BaPDEsxA configuration, a representative snapshot of which is shown in Figure 3d. In this arrangement, a second water molecule was commonly found bound to the distal hydroxyl group but oriented such that it was frequently close to the epoxide oxygen, and it is therefore included in the extracted configuration. Figure 3c shows the extracted configuration sxB which occurs slightly more often than the sxA configuration. The sxB configuration is the only one in all of the extracted configurations, regardless of the DE examined, in which the bound water is seen to go beneath the epoxide oxygen or, in other words, the water molecule can be found between the epoxide oxygen and the plane of the saturated ring. This places the water molecule in such a position that the water oxygen is within 3 Å of both the H9 and H10 atoms on the DE, resulting in possible electrostatic interactions that may stabilize the configuration.

Brown et al.

In the BcPhDEise simulations, the iseC configuration, shown in Figure 3e, appeared far more frequently than the others did, accounting for two-thirds of the bound water molecules. In this arrangement, the bound water is over the C1-C1a bond of the saturated ring, and due to the twist of the molecule, this puts the hydrogen across the fjord region, H12, within 3 Å of the water oxygen, possibly accounting for the frequency of this water orientation. The second most common configuration, iseB, is shown in Figure 3f, and in this orientation, the water is almost directly over the epoxide oxygen. For the BcPhDEisx simulations, the most common configuration was isxA, shown in Figure 3g, which accounts for 57% of the bound water molecules. In this configuration, the water molecule is essentially in the plane of the epoxide ring, above the oxygen and C10. A representative snapshot of the second most common configuration, isxC, is shown in Figure 3h. In both of these configurations, the orientation of the water is such that the water oxygen is within 3 Å of the H12 atom across the fjord region on the DE, an interaction that could possibly help stabilize the configurations. This potential interaction arises from the nonplanarity of the BcPhDE which causes the H12 atom to be on the same side of the saturated ring as the epoxide oxygen, bringing it closer to any water molecules that are over the epoxide oxygen. The two most common configurations from the waterBcPhDEoae simulations, which occur for approximately equal amounts of time and account for a total of 71% of the waters bound to the epoxide oxygen, are shown in Figure 3i,j. While the water molecule in configuration oaeA is not over the saturated ring, the water molecule in configuration oaeD (Figure 3j) is essentially centered over the saturated ring such that the oxygen atom of the water is about 2.6 Å from the distal hydrogen (H4) on the saturated ring of the DE. Overall, about half of the bound water molecules are on the side of the epoxide oxygen nearer the saturated ring, and about a third are on the side of the epoxide oxygen that is nearer to the conjugated ring system. In the BcPhDEiae-water simulations, there were fewer distinct water molecules that came close to the epoxide oxygen than there were for any other DE simulations (see Table 1). These four water molecules did, however, bind more often since there was a bound water 61% of the time in the in-anti-quasi-diequatorial simulations, a greater frequency than in any of the other simulations. There were two orientations of the water around the epoxide oxygen that were quite common: iaeB and iaeC shown in Figure 3k,l, respectively. None of the BcPhDEiae-bound waters, in any of the extracted configurations, favored the conjugated ring side of the epoxide group. However, half of the bound waters were found on the side of the epoxide oxygen that is nearer to the saturated ring; in fact, in the iaeB configuration, the water molecule is nearly centered over the saturated ring whereas in the iaeC configuration it is not over the saturated ring at all. As previously stated, we have found from the simulations that water molecules orient themselves around the epoxide groups on the PAH DEs in usually only four or fewer distinctly different ways. There is also occasionally a water molecule that is close to the epoxide group but bound to a hydroxyl group instead. Comparisons can be made between the ways the waters tend to orient around



Table 3. Number of Hydrogen Atoms and Water Molecules Represented by the Extracted Configurationsa BaPDEae

BaPDEsx

BcPhDEise

BcPhDEisx

BcPhDEoae

BcPhDEiae

aeA aeB aeC sxA sxB sxC iseA iseB iseC iseD isxA isxB isxC oaeA oaeB oaeC oaeD iaeA iaeB iaeC no. of H contributing to configuration no. of waters contributing to configuration total no. of waters represented by all configurations

4

1

2

4

2

1

3

1

2

1

4

3

1

5

2

2

2

2

2

2

2

1

1

2

2

1

2

1

1

1

2

2

1

3

2

1

1

2

1

1

4

5

5

4

7

4

a Table 3 gives the total number of hydrogen atoms and water molecules from the two simulations for each DE that are represented by the extracted configuration. Note that the number of water molecules represented refers only to the water molecules bound to the epoxide oxygen and not those bound to the diol oxygens.

Table 4. Positions of the Water Molecules around the Diol Epoxidea

DE-water configuration

on side of epoxide oxygen nearer conjugated ring system

above epoxide oxygen

on side of epoxide oxygen nearer saturated ring

BaPDEae A BaPDEae B BaPDEae C BaPDEsx A BaPDEsx B BaPDEsx C BcPhDEise A BcPhDEise B BcPhDEise C BcPhDEise D BcPhDEisx A BcPhDEisx B BcPhDEisx C BcPhDEoae A BcPhDEoae B BcPhDEoae C BcPhDEoae D BcPhDEiae A BcPhDEiae B BcPhDEiae C

yes no yes ∼ ∼ no no ∼ yes no yes no yes ∼ yes yes ∼ no ∼ ∼

yes yes yes yes ∼ yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes

no yes yes no no no no yes yes yes ∼ no no no yes no yes no yes no

a The positions of the water molecules around the diol epoxide for each of the common orientations around the diol epoxides. The location of the water molecule is described relative to the epoxide oxygen (see Figure 4 also). A ∼ indicates that the water molecule is centered about the epoxide oxygen in the direction examined.

the different diol epoxides to elucidate the effects of the DE structure on its interactions with water molecules. BcPhDEiae vs oae. By comparing the water simulations of the iae and oae BcPhDE conformations, we can examine the influences of the in and out structural forms on the water orientations around the BcPhDEs. Similarities are seen between the orientations the water molecules acquire around the BcPhDE in-anti-quasi-diequatorial and out-anti-quasi-diequatorial conformations. The two most frequently seen configurations of the water molecules around the in-anti-quasi-diequatorial conformation are the same as the two most frequently seen configurations of the out-anti-quasi-diequatorial conformation: the oaeA configuration is the same as the iaeC configuration, and the oaeD configuration is the same as the iaeB configuration. Despite the similarities between the water molecule orientations around the two different conformations, there is an obvious difference in the frequency at which the water molecules are close to the DE (Table 2). Even though there was a greater number of individual water molecules that came close to the epoxide group in the out-anti-quasi-diequatorial simulations, they did so less frequently than in the in-anti-quasi-diequatorial simulations. In the BcPhDEiae simulations, there was a water

Figure 4. The figure shows an idealized schematic of BaPDE as viewed from ‘above’ such that the dark circle between C9 and C10, the epoxide oxygen, is out of the page toward the viewer. The three Cartesian dimensions are defined as follows relative to the epoxide oxygen atom as the origin: (1) the shaded and nonshaded areas where the shaded area is on the side of the epoxide group away from the saturated ring and the nonshaded area is on the side of the epoxide group nearer the saturated ring; (2) the horizontal and vertical lines where the horizontal lines cover the area that is on the side of the epoxide group nearer the conjugated rings and the vertical lines cover the area that is on the side of the epoxide group away from the conjugated rings; and (3) the third dimension above and below the epoxide oxygen where this figure is viewed from above.

molecule vicinal to the epoxide oxygen 3 times more often than in the BcPhDEoae water simulations. In further contrast, only 31% of those water molecules close enough to potentially bind, did bind in the BcPhDEoae simulations versus 60% in the BcPhDEiae simulations. It appears that the out conformation does not alter the way in which the water molecules bind to the epoxide group, merely how frequently they do so. BcPhDEise vs isx. To examine the possible changes in water molecule orientations due to the equatorial and axial structural forms in the BcPhDEs, we can compare the results of the BcPhDEise water simulations to those of the BcPhDEisx water simulations. Looking at the frequency of vicinal water molecules (Table 1), it can be seen that the in-syn-quasi-diaxial conformation has a nearby water molecule 25% more often than does the insyn-quasi-diequatorial conformation. However, more of the nearby waters bind to the in-syn-quasi-diequatorial DE than to the in-syn-quasi-diaxial DE as can be seen in column 4 of Table 1. As stated earlier, in three of the four configurations extracted from the BcPhDEise simulations, which account for 90% of all of the bound waters from those simulations, the water is over the saturated ring. Of these bound waters, only those in configuration iseC, the most common configuration, appear toward the conjugated ring side of the DE, or equivalently 66% of the bound waters are on the conjugated ring side of the epoxide oxygen and close to the H12 of the DE.

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In the BcPhDEisx water simulations, on the other hand, the bound water molecule is never fully or consistently over the saturated ring. Both of the two most common BcPhDEisx water configurations, however, have the bound water on the conjugated ring side of the epoxide oxygen, or, equivalently, 80% of the bound waters are toward the conjugated ring side of the epoxide oxygen. So, whereas in the in-syn-quasi-diequatorial conformations it is very probable to find a bound water over the saturated ring and slightly less likely to find it toward the conjugated ring system of the DE, in the in-syn-quasidiaxial conformation, it is very probable to find the bound water molecule toward the conjugated ring system but very seldom over the saturated ring. BcPhDEise vs iae. The results of the BcPhDEiae water simulations can be compared to those of the BcPhDEise water simulations to determine the possible effects of the anti and syn structural forms on the orientations of the surrounding water molecules. This is an additionally interesting comparison to make since these are the lowest energy anti and syn conformations of the BcPhDE, respectively. In the in-anti-quasi-diequatorial DE simulations, there were half as many distinct water molecules that came close to the epoxide oxygen as in the in-syn-quasidiequatorial simulations. However, there were one-third more time steps in which the oxygen atoms of these closeapproaching water molecules were less than 3 Å from the epoxide oxygen in the in-anti-quasi-diequatorial water simulations than in the in-syn-quasi-diequatorial simulations. Additionally, when the water molecules did come close to the DE, a larger fraction of them were aligned well enough to form a bond in the in-anti-quasidiequatorial simulations than in the in-syn-quasi-diequatorial simulations. Overall, there was a bound water molecule 61% of the time in the in-anti-quasi-diequatorial simulations compared to only 23.5% of the time in the in-syn-quasi-diequatorial simulations. In addition to making comparisons in the frequencies of bound waters, we can also examine the orientations of the bound water molecules. The epoxide-bound water molecule is over the saturated ring about 90% of the time it is bound during the BcPhDEise water simulations. However, in the BcPhDEiae water simulations, the bound water is over the saturated ring in only one of the three configurations, iaeB, which is the most commonly occurring configuration accounting for half of the bound water molecules. Thus, we can remark that, although a water is bound to the syn DE less frequently than the anti DE, when it is bound to the syn DE the water is only likely to appear over the saturated ring. In contrast, when a water is bound to the anti DE, it is just as likely to be over the saturated ring as it is not. It can also be seen from the comparisons of the simulations that the water molecules bound to the BcPhDEiae conformer essentially show no preference for being toward the conjugated ring side of the molecule or away from it. From the BcPhDEise simulations, we see, however, that the water molecule favors the conjugated ring side of the in-syn-quasi-diequatorial diol epoxide in the configuration which is by far the most common, iseC. We can therefore state that although it is probable that the bound water molecule will be toward the conjugated ring side of the epoxide group when the BcPhDE has the in-syn-quasi-diequatorial structure, it will favor neither

Brown et al.

side when bound to a DE with the in-anti-quasi-diequatorial structure. BaPDEsx vs ae. In BaPDE simulations, the antiquasi-diequatorial conformation has a water molecule bound to the epoxide oxygen 49% of the time, far more regularly than the syn-quasi-diaxial DE which has a water molecule bound for only 10.5% of the time. This disparity is also seen in the number of time steps with vicinal, but not necessarily bound, water molecules as there were twice as many occurrences of close-approaching water molecules in the BaPDEae simulations. Of the three configurations extracted from the BaPDEae water simulation, the two most common ones, accounting for 96% of the bound water molecules, show the bound water to favor the side of the epoxide group nearer the conjugated ring system. Only in the most common configuration, aeA, which accounts for 83% of the bound waters, was the water also on the side of the epoxide group away from the saturated ring. Comparatively, in the BaPDEsx water simulations, the bound water was never seen over the saturated ring, and it never favored the side of the epoxide group nearer the conjugated ring system. So a water molecule bound to the epoxide oxygen of either the anti-quasi-diequatorial or the syn-quasi-diaxial BaPDE conformer will not be found on the side of the epoxide group nearer the saturated ring, but in the syn-quasi-diaxial case, it will also not generally be found on the side of the epoxide ring closer to the conjugated ring system whereas in the antiquasi-diequatorial DE water system this latter statement is not the case. BaPDE vs BcPhDE. The water molecule orientations around the BcPhDE in-anti-quasi-diequatorial and insyn-quasi-diequatorial conformers, the lowest energy anti and syn BcPhDEs, respectively, can be compared to the water orientations around the BaPDE anti-quasi-diequatorial and syn-quasi-diaxial conformers, the lowest energy anti and syn BaPDEs, respectively, to examine the differences in the water behavior due to the different PAH DEs. For both PAHDEs, the simulations with the low-energy anti conformers resulted in a higher occurrence of vicinal water molecules than the simulations with the low-energy syn conformers. This trend paralleled the results seen in the frequency of bound water molecules where the BcPhDEiae conformer had a bound water molecule more than twice as often as the BcPhDEise conformer and the BaPDEae conformer had a bound water molecule almost five times more often than the BaPDEsx conformer. In fact, the largest percentages of vicinal waters that resulted in bonds with the epoxide oxygen for any of the PAHDEs examined were for the BcPhDEiae (58%) and BaPDEae (51%) conformers. The in-anti-quasi-diequatorial conformer, in fact, had more occurrences of both vicinal and bound water molecules than any other DE examined (see columns 2 and 4 of Table 1). For both low-energy anti conformations of the PAH DEs, the bound water molecules were seen over the saturated ring, albeit more frequently in the BcPhDEiae conformation than in the BaPDEae conformation. In the in-anti-quasi-diequatorial BcPhDE simulations, the bound water molecules did not favor being on the side of the epoxide oxygen nearer the conjugated ring system. Conversely, in the BaPDEae simulation, the bound water molecules were almost always on the side of the epoxide group toward the conjugated ring system. In fact, the


most frequent configuration found from the BaPDEae water simulation had a bound water molecule that was close to the H10 atom of the diol epoxide, placing the water molecule lower in relation to the epoxide oxygen and closer to the carbon atoms of the epoxide group than was seen in any of the extracted BcPhDEiae configurations. In the BaPDEsx conformer, the bound water never appears over the saturated ring whereas in the BcPhDEise water simulations the water molecule is almost always over the saturated ring. In the most common configuration extracted from the BcPhDEise water simulations, the bound water molecules were on the side of the epoxide group nearer the conjugated ring system of the DE. However, none of the configurations extracted from the BaPDEsx water simulations showed the bound water to be on this side of the epoxide group. Interesting comparisons can also be made between the BaPDEsx simulations and the BcPhDEisx simulations. The water simulations for both of these PAHDEs showed that the bound water molecules were not located on the side of the epoxide group nearer the saturated ring possibly due to the location and steric interference of the axial hydroxyl groups. Additionally, in the BcPhDEisx configurations, the water orientations were such that there could be an additional stabilizing force between the water oxygen and the H12 atom across the fjord region. The planarity of the BaPDE makes the equivalent hydrogen atom across the bay region less accessible to the water molecules, and instead the position of the water molecule in the most frequent BaPDEsx configuration was such that there could be additional interactions between the water oxygen atom and the H9 and H10 atoms on the DE. This position of the bound water molecule in the BaPDEsx configuration was such that the water was closer to the plane of the saturated ring than it was in any of the BcPhDEisx configurations.

Discussion The molecular dynamics simulations in this study have shown that there are preferred locations and orientations for water molecules around the PAHDE that depend on both PAH type as well as DE conformation. The occurrence of water molecules is influenced to a large degree by subtle changes in the three-dimensional molecular structure of the DE. These structural changes alter the frequency of both vicinal and hydrogen-bonded water molecules and modify the spatial relationship between the molecules in the hydrogen-bonded system. Previous studies have shown that there can be a rapid exchange of water molecules between the first hydration shell of a solute molecule and the bulk solvent, and residence times for individual water molecules have been measured to be on the order of 4 ps for regions of proteins that are easily accessible to the solvent molecules (17). The binding of water molecules to the epoxide oxygen is primarily electrostatic, and while there is some steric component, the water molecules are not as confined as the water molecules bound in inaccessible regions of proteins where there will be longer residence times. During the course of these simulations, the water molecules bound to the epoxide oxygen exchange at least 4 times (see Table 3), indicating that the length of the present simulations does, therefore, afford enough time for the individual water molecules to move about the


solvent system and/or bind to the DE. Additionally, the high temperature of these simulations does provide for a larger sampling of the possible positions of the water molecules. While we have identified a number of different configurations for water molecules bound to the epoxide oxygen atoms of DE, longer simulations would ensure that all possible orientations of bound water molecules have been described. Overall, the simulations show that the most stable anti DE configurations for both BaP and BcPh are much more likely to have a water molecule bonded to the epoxide oxygen than the other DE configurations studied. While the frequency of water molecules within 3 Å of the epoxide oxygen is large for these two molecular configurations, it is also true that the fraction of those vicinal water molecules that appears hydrogen bonded is much larger than that observed for the other DEs in this study. We can also generalize some of the differences between the water-bonding frequencies and orientations seen around the low-energy syn and anti conformations of the two different PAH DEs. As mentioned above, the BaPDEae and BcPhDEiae conformations had bound water molecules far more frequently than the BaPDEsx and BcPhDEise conformations. It was also more probable to find a second water molecule, bound to a hydroxyl group and close to the epoxide oxygen, in the BaPDEs than in the BcPhDEs. Additionally, in the BcPhDE water simulations, it was more probable to find the bound water over the saturated ring or over the epoxide oxygen. This may be due to the nonplanarity of the BcPhDEs which allows a water molecule over the saturated ring to also be close to the H12 across the fjord. In the BaPDE simulations where the water molecule was more commonly found on the side of the epoxide group away from the saturated ring, it was frequently close enough to the hydrogen atoms of the epoxide group (H1 and H2) such that an additional stabilizing force could be exerted by that interaction. The interaction potential used for the simulation contains steric and electrostatic contributions, and the hydroxyl groups have both steric and electrostatic effects on the approaching water molecules. For the BaPDE with equatorial hydroxyl groups, the atoms of the molecule are all nearly in the same plane, and the structure is open to the approach of water molecules with perhaps a small, steric restriction in the bay region whereas BcPhDE has a much more restricted bay region. For either type of PAHDE, axial hydroxyl groups on the same side of the saturated ring as the epoxide oxygen limit the region in space for the approach of the water molecule to the epoxide. In both PAHs, this accounts for the infrequent appearance of bound water molecules over the saturated ring of DEs that have axial hydroxyl groups. The dipolar hydroxyl groups near the epoxide also influence the electrostatic interaction between the water molecule and the epoxide. The electrostatic terms control the orientation of the water molecules near the epoxide oxygen. Contributions to the electrostatic potential from the epoxide atoms, the nearby hydroxyl group(s), and the proton across the bay region orient the water molecule although steric interactions limit the region about the epoxide (O) that is available to the approaching molecule. These steric and electrostatic influences are demonstrated in the simulations of the BcPh iae and ise DEs. Both of these DEs have equatorial hydroxyl groups leaving the area over the

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saturated ring sterically accessible, allowing for the water molecule to bind from this region, which it does in the simulations of both DEs. However, in the iae isomer simulations, the water molecule is never found in a hydrogen bond on the side of the epoxide toward the conjugated ring and is instead frequently found over the saturated ring such that its oxygen atom will then be within 3 Å of the axial hydrogen on C4. Conversely, for the ise isomer of BcPhDE, the configuration observed two-thirds of the time does have the hydrogen-bonded water molecule on the conjugated side of the epoxide (and still over the saturated ring). In the ise conformer, the axial hydrogen on C4 is anti to the epoxide oxygen (the hydroxyl group and epoxide oxygen are syn instead), making it inaccessible for a favorable interaction of the type seen in the iae simulations between the hydrogen and an epoxide-bound water. Instead, this ise configuration is stabilized by an excellent steric fit and an additional electrostatic interaction between the oxygen atom of the water molecule and the proton across the fjord region (H12). In our studies of the BcPhDEs, we performed simulations of the water molecules about two DE conformations that are higher energy local minima and therefore unlikely to be seen experimentally. For both of these configurations, the frequency of hydrogen-bonded water molecules and the ratio of hydrogen-bonded water molecules to vicinal water molecules are significantly smaller than for the corresponding lower energy structure. Two of the BcPhDE conformations examined, the in-antiquasi-diequatorial and the in-syn-quasi-diequatorial, were chosen because they are experimentally observed (7). These two conformations are also calculated to be the lowest energy anti and syn conformers, respectively (6). The two BaPDE structures in this simulation are the lowest energy syn and anti configurations from both experimental and computational studies. As mentioned above, the most dramatic differences in the water orientations around these different DEs are the large variations in the frequency of bound waters. Interestingly, we observe that the frequency at which water molecules hydrogen-bonded to the epoxide of DEs reflects the relative frequency of DNA adduct formation. For instance, the BcPhDEiae diol epoxide has a bound water 61% of the time during the simulations whereas the BcPhDEise has a bound water only 23% of the time. Similarly, the BaPDEae conformation has a bound water 49% of the time whereas the BaPDEsx conformation has a bound water in only 10.5% of the snapshots studied. These patterns reflect patterns seen in the relationship between type of PAH DE and amount of DNA adduct formation, where it has been shown that anti DEs form DNA adducts more often than the corresponding syn DEs (11, 18). Experimental evidence also shows that the BcPhDEs form DNA adducts more frequently than the BaPDEs (11). In analogy, we see that the BcPhDEs have water molecules more frequently bound than the BaPDEs. We can suggest two possible underlying reasons for this relationship between frequency of bound water molecules and adduct formation. First, the hydrogenbonded water molecules might act as proton donors to open the epoxide ring and facilitate adduct formation. Hence, DEs that more frequently bind water molecules will more frequently have the opportunity for the epoxide ring to be opened and subsequently react with DNA.

Brown et al.

Second, it is quite possible that the water molecules in these simulations act as probes of the potential about the DEs. Then the frequency of bound water molecules may reflect the frequency of the approach of the DE to dipolar sites in the DNA bases as well as the frequency of the approach to water molecules. If this were the case, the potential is not a scalar potential because it also contains information about relative orientation of the probe (water molecule) and the DE. If we consider the frequency of water molecules as a type of vector potential function where the water molecules are probes for exploring the potential interactions of other molecules, the number of responsible water molecules is an (inverse) measure of the slope of the walls of the potential well. For BcPhDEiae, there is a strong binding site with steep walls because the frequency of bound water molecules is great and the number of individual water molecules is small. For BcPhDEoae, the potential at the binding site is both shallower and more open. The ratio of the frequency of hydrogen-bonded water to the frequency of vicinal water is a measure of the orientational requirements for the binding of a dipolar molecule. This effect is stronger in the anti-quasidiequatorial DEs, which are also the most carcinogenic.

Concluding Remarks The results indicate that 10-60% of the time, depending strongly on the conformation and type of the DE, there is a water molecule within a good hydrogen-bonding distance and orientation of the epoxide oxygen. Overall, the most stable anti DE configurations for both the BaP and BcPh DEs are far more likely to have a water molecule bound to the epoxide oxygen. Interestingly, the patterns seen in the frequency at which a DE binds a water molecule reflect patterns seen in the experimental relationship between the type of PAH DE and amount of DNA adduct formation. Examination of the orientations and arrangements of the water molecules around the DEs during the simulations showed that the bound water molecules existed in several preferred configurations which are also dependent upon the PAH DE geometry. These studies were performed to determine water molecule orientations around various DEs as a first step in the examination of the energetics of the epoxide ring opening due to a water molecule. The configurations extracted from the simulations will be used as starting geometries in quantum mechanical studies of the ring opening for each DE conformation. Future simulations will also be performed with H3O+ molecules contained in the water box to simulate the initial steps in the acidcatalyzed epoxide ring openings that can occur around the DNA polyanion.

Acknowledgment. We thank Dr. S. Nesnow for helpful discussions during the course of this study. We also thank Drs. R. J. Preston and A. Richard for reviewing the manuscript. The information in this document has been funded wholly (or in part) by the U.S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement of recom-


mendation for use. K.W.B. was funded by the EPA/UNC Toxicology Research Program (training agreements CT902908 and CT 827206). Some of the calculations reported here were performed at the National Computer Center of the U.S. Environmental Protection Agency.

References (1) Dipple, A. (1995) DNA adducts of chemical carcinogens. Carcinogenesis 16, 437-441. (2) Jerina, D., and Lehr, R. (1977) The bay-region theory: A quantum mechanical approach to aromatic hydrocarbon-induced carcinogenicity. In Microsomes and Drug Oxidations (Proceedings of the 3rd International Symposium) (Ullrich, V., Roots, I., Hildebrandt, A., Estabrook, R., and Conney, A., Eds.) pp 709-720, Pergamon Press, Oxford, U.K. (3) Lehr, R., Kumar, S., Levin, W., Wood, A., Chang, R., Conney, A., Yagi, H., Sayer, J., and Jerina, D. (1985) The bay-region theory of polycyclic aromatic hydrocarbon carcinogenesis. In Polycyclic Hydrocarbons and Carcinogenesis (ACS Symposium Series No. 283) (Harvey, R. G., Ed.) pp 63-84, American Chemical Society, Washington, DC. (4) Whalen, D., Ross, A., Yagi, H., Karle, J., and Jerina, M. (1978) Stereoelectronic factors in the solvolysis of bay region diol epoxides of polycyclic aromatic hydrocarbons. J. Am. Chem. Soc. 100, 5218-5221. (5) Rabinowitz, J., Little, S., and Lewis-Bevan, L. (1996) The effect of crowding in the bay/fjord region on the structure and reactivities of polycyclic aromatic hydrocarbons and their metabolites: quantum mechanical studies. Polycyclic Aromat. Compd. 11, 237244. (6) Lewis-Bevan, L., Little, S., and Rabinowitz, J. (1995) Quantum mechanical studies of the structure and reactivities of the diol epoxides of benzo[c]phenanthrene. Chem. Res. Toxicol. 8, 499505. (7) Sayer, J., Yagi, H., Croisy-Delcey, M., and Jerina, D. (1981) Novel bay-region diol epoxides from benzo[c]phenanthrene. J. Am. Chem. Soc. 103, 4970-4972.

Chem. Res. Toxicol., Vol. 15, No. 8, 2002 1079 (8) Geacintov, N. (1986) Is intercalation a critical factor in the covalent binding of mutagenic and tumorigenic polycyclic aromatic diol epoxides to DNA? Carcinogenesis 7, 759-766. (9) Fernando, H., Huan, C.-R., Milliman, A., Shu, L., and LeBreton, P. (1996) Influence of Na+ on DNA reactions with aromatic epoxides and diol epoxides: evidence that DNA catalyzes the formation of benzo[a]pyrene and benz[a]anthracene adducts at intercalation sites. Chem. Res. Toxicol. 9, 1391-1402. (10) Lamm, G., Wong, L., and Pack, G. (1996) DNA-mediated acid catalysis: calculations of the rates of DNA-catalyzed hydrolyses of diol epoxides. J. Am. Chem. Soc. 118, 3325-3331. (11) Szeliga, J., and Dipple, A. (1998) DNA adduct formation by polycyclic aromatic hydrocarbon dihydrodiol epoxides. Chem. Res. Toxicol. 11, 1-11. (12) Dipple, A., Pigott, M., Agarwal, S., Yagi, H., Sayer, J., and Jerina, D. (1987) Optically active benzo[c]phenanthrene diol epoxides bind extensively to adenine in DNA. Nature 327, 535-536. (13) Agarwal, S., Sayer, J., Yeh, H., Panell, L., Hilton, B., Pigott, M., Dipple, A., Yagi, H., and Jerina, D. (1987) Chemical characterization of DNA adducts derived from the configurationally isomeric benzo[c]phenanthrene-3,4-diol 1,2-epoxides. J. Am. Chem. Soc. 109, 2497-2504. (14) HyperChem 5.11 (1996), HyperCube, Inc., Waterloo, Canada. (15) Jorgensen, W., and Tirado-Rives, J. (1988) The OPLS potential functions for proteins. Energy minimizations for crystals of cyclic peptides and crambin. J. Am. Chem. Soc. 110, 1657-1666. (16) Jorgensen, W., Chandrasekhar, J., and Madura, J. (1983) Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926-935. (17) Makarov, V., Andrews, B., Smith, P., and Pettitt, B. (2000) Residence times of water molecules in the hydration sites of myoglobin. Biophys. J. 79, 2966-2974. (18) Sayer, J., Chadha, A., Agarwal, S., Yeh, H., Yagi, H., and Jerina, D. (1991) Covalent nucleoside adducts of benzo[a]pyrene 7,8-diol 9,10-epoxides: Structural reinvestigation and characterization of a novel adenosine adduct on the ribose moiety. J. Org. Chem. 56, 20-29.

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