Chem. Res. Toxicol. 1994, 7, 110-119
110
Halide-Catalyzed cis Product Formation in the Hydrolysis of anti-Benzo[a]pyrene Diol Epoxide and Its Alkylation of Poly (A) Alan R. Wolfe and Thomas Meehan* Division of Toxicology and Department of Pharmacy, University of California, Sun Francisco, California 94143 Received September 8, 1993"
The carcinogen 7-r,8-t-dihydroxy-9-t,10-t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (antiBPDE) forms diastereomeric cis and trans products in its reactions with nucleic acids and water (adducts and tetrols, respectively). The effects of salts, buffers, and DNA on the hydrolysis product ratio were tested. Halide ions increase the cis-tetrolltrans-tetrol ratio, with the order of effectiveness being I > Br > C1 >> F. No cation effect was apparent. Non-halide salts of strong acids increase the ratio to a small degree. Buffers decrease the ratio, with phosphate being more effective than cacodylate. DNA also reduces the ratio, with denatured DNA being more potent than native DNA. Halide ions appear to catalyze cis-tetrol formation via trans halohydrin intermediates. At the lowest halide concentrations which significantly raise the product ratio, and a t all levels of chloride ion, the rate of anti-BPDE hydrolysis is not greatly increased, indicating that the halide ions are reacting primarily with the BPDE carbocation formed in the rate-determining step. At higher concentrations, iodide ion and, to a lesser degree, bromide ion significantly accelerate hydrolysis, indicating that BPDE undergoes s N 2 attack by these ions. Chloride ion was also found to increase the proportion of cis adducts formed between anti-BPDE and poly(A). The cis adductltrans adduct ratio was quadrupled by 0.5 M NaC1. This suggests that chlorohydrins can be intermediates in the alkylation of nucleic acids by epoxides of polycyclic aromatic hydrocarbons.
Introduction Benzo[a]pyrene, a widespread environment contaminant, is metabolized into electrophilic intermediates which alkylate DNA. Among the most important of these intermediates are the 7,8-diol 9,lO-epoxy derivatives derived from the monooxygenase system (1-6). All four possible diastereomers of the 7-r,8-t,g-t,lO-tisomer (antiBPDE)' and the 7-r,8-t,g-c,lO-cisomer (syn-BPDE) are formed in mammalian cells (7). The compound with the 7R,8S,9S,lOR configuration [(+)-anti-BPDEIhas the most potent carcinogenic and mutagenic activity (8, 9). The principal stable adduct formed results from alkylation of the exocyclic amino group of guanine by the C-10 position of the hydrocarbon (5, 6, 10-12). DNA alkylated by racemic anti-BPDE in vitro contains four guanine and four adenine adducts which result from cis and trans opening of the epoxide ring of both anti-BPDE enantiomers. The cis and trans adducts of guanine differ with respect to their conformation relative to the DNA helix. The trans adducts of guanine have been shown by NMR to be in a solvent-exposedenvironment in the minor groove, minimally disruptive of DNA structure (13,14),while the (+)-cisadduct is intercalated, displacingthe alkylated base (15). The (-)-cis adduct is believed on the basis of less direct evidence also to adopt an intercalative conformation (16). Correlative evidence suggests that trans adducts of BPDE and other activated polycyclic aromatic hydrocarAbstract published in Advance ACS Abstracts, January 1, 1994. 1 Abbreviations: (k)-anti-BPDE, racemic 7-r,8-t-dihydroxy-9-t,lO-tepoxy-7,8,9,10-tetrahydrobenzo[alpyrene;syn-BPDE,racemic 7-r,8-tdihydroxy-9-c,l0-c-epoxy-7,8,9,lO-tetrahydrobenzo[alpyrene; cis- and and (k)-7-r,S-t,9-t,lO-c-tetrahydroxytrans-tetrol, (k)-7-r,8-t,g-t,lO-t7,8,9,10-tetrahydrobenzo[alpyrene, respectively.
0893-228~/94/2707-0110$04.50/0
bons, which are probably less easily recognized by DNA repair systems, are generally more carcinogenic than cis adducts (16). BPDE hydrolyzes in aqueous solution to give a mixture of stereoisomeric 7,8,9,10-tetrahydroxy-7,8,9,lO-tetrahydrobenzo[alpyrenes (trans- or cis-tetrol, referring to the relative positions of the 9- and 10-hydroxy substituents). The hydrolysis reaction is catalyzed by hydronium ion and general acid catalysts, and it can also occur by a spontaneous, non-acid-catalyzed route (17-21). Only a small fraction of the anti-BPDE that interacts with DNA forms covalent adducts; most of it undergoes DNAcatalyzed hydrolysis (22-25). The exocyclic amino group of guanine has been implicated as having an important role in this catalysis (26). DNA-catalyzedhydrolysisoccurs primarily by a pathway that is general acid catalyzed, but a spontaneous pathway is again present (27). The catalysis of BPDE hydrolysis by nucleic acids is inhibited by cations (22, 23, 28). Since the tetrols are noncarcinogenic, the hydrolysis reaction can be viewed as a passive detoxification pathway. The reaction catalyzed by hydronium ion, buffers, and nucleic acids yields predominantly transtetrol. We report here that the presence of iodide, bromide, or chloride ions increases the proportion of cis-tetrol formed in the hydrolysis reaction and that chloride ion also increases the proportion of cis adducts formed in the alkylation of poly(A). The change in hydrolysis product ratio occurs at halide ion concentrations which have little effect on the reaction rate, indicating that under these conditions the halide ions act primarily in an s N 1 fashion on the C-10 anti-BPDE carbocation intermediate, after the rate-determining step. However, at high concentra-
0 1994 American Chemical Society
Halide Reactions with Benzo[a]pyrene
Diol Epoxide
Chem. Res. Toxicol., Vol. 7, No. 1, 1994 111
to 2.0 mL of solution. The reaction was allowed to proceed tions, iodide ion and, to a lesser extent, bromide ion do significantlycatalyze hydrolysis,indicatingthat direct S N ~ overnight in the dark a t room temperature. The reaction mixture was then extracted twice with 2 mL of water-saturated ethyl attack of these halide ions on anti-BPDE also occurs.
Experimental Procedures Chemicals. Racemic anti-BPDE was prepared as reported previously (29). Stock solutions were prepared in THF, DMSO, or ethanol and stored with desiccant at -80 "C. Racemic anti[l-SHIBPDE (specific activity 420 Ci/mol) was obtained from the NCI Radiochemical Carcinogen Repository (of the Cancer Research Program of the National Cancer Institute, Division of Cancer Cause and Prevention, Bethesda, MD) at Chemsyn Science Laboratories. Clsreverse-phase Sep-Pak cartridges were obtained from Waters Associates. Extinction coefficients (M-l cm-l) used were as follows: poly(A), €257nm = 10 400 (30);antiBPDE in THF, t345m = 46 600 (our measurement). Hydrolysis Kinetics. The kinetics of anti-BPDE hydrolysis was measured in 10 mM sodium cacodylate buffer (pH 7.0) containing 10% acetone (v/v). The reaction was monitored either fluorometrically or spectrophotometrically. The fluorometric assay takes advantage of tetrol's high quantum yield, while the spectrophotometric assay is based on the 1.5-nm blue shift in the UV A,, of tetrol relative to that of BPDE. The fluorometric assay is poorly suited for work a t high concentrations of iodide ion, which quenches tetrol emission, so the spectrophotometric assay was used at iodide concentrations above 7.5 mM. In this case, stock solutions of BPDE in DMSO or ethanol were used, because traces of peroxides in T H F react with iodide to form Is(Iz + I-), which interferes with the assay. The fluorometric assay was performed with a Spex Fluorolog 1680 0.22-m double spectrometer. Exciting radiation of 344 nm was used, and emission was measured a t 400 nm. The spectrophotometric assay was performed with a Hewlett Packard 8452A diode array spectrophotometer,measuring the difference between the average absorbances in the 338-344- and 346-352-nm ranges. In either assay, the reaction mixture was maintained at a temperature of 24 "C. The reaction was initiated by the addition of 4 pM (fluorometric assay) or 11pM (spectrophotometricassay) of (&)-anti-BPDEto 1.0 mL of reaction mixture (with a final T H F or DMSO concentration of 10.4%). The reaction was monitored for 50 min (always 13.5 half-lives of the BPDE) in the slowest cases, and for a variable, shorter interval (always 2 7 half-lives) in more rapid cases. For all assays, 51 or more data points (obtained at equal intervals) were fitted using nonlinear regression to the equation: where S ( t ) is the spectroscopic signal as a function of time, So and SFare the initial and final signals, respectively, and komis the observed pseudo-first-order rate constant for the disappearance of BPDE. Three or more measurements were made for each set of experimental conditions. Hydrolysis Product Ratios. The product ratio ofanti-BPDE hydrolysis was measured in 10 mM sodium cacodylate buffer (pH 7.0) containing 10% acetone (v/v) and, in some cases, additional salt, additional buffer (pH 7.0), or DNA (in a few instances, reactions were run without buffer). DNA was denatured by heating native calf thymus DNA in distilled water to 100 "C for 10 min, and then rapidly cooling in ice water. A control experiment established that nothing was leached from the glass vial during boiling that would alter the product ratio. Reactions for each set of experimental conditions were run in duplicate, except for reactions run with 10 mM buffer only, or without any buffer or salt, which were run in quadruplicate. At high concentrations of KF, 10 mM buffer is inadequate to maintain a constant pH. Each K F solution (containing 10 mM sodium cacodylate) was brought to neutrality by titration with a solution of the same concentration of H F (also containing 10 mM sodium cacodylate). Each reaction was initiated by the addition of 1020 pL of BPDE in T H F or DMSO (5-10 pM final concentration)
acetate. The two organic phases from a given sample were combined and evaporated, and the residue was dissolved in 1mL of methanol. Aliquots (320 nm using a longband-pass filter. The fluorescence quantum yields of trans- and cis-tetrol were compared by injecting a large sample and monitoring their elution simultaneously with emission and UV absorption detectors. The ratios of peak heights obtained by the two instruments were the same, indicating that the quantum yields are equal. The BPDE hydrolysis product ratio can thus be calculated on the basis of the relative peak areas in the emission chromatogram. Preparation of BPDE-ModifiedPoly(A). Adducts between BPDE and poly(A) were generated in samples containing 3 mL of 10 mM sodium cacodylate buffer (pH 7.2) containing 5 mM (in bases) of the polymer, 0.1 mM EDTA, 10% acetone (v/v),and O.O,O.l, or 0.5 M NaC1. [Higher salt concentrations precipitated the poly(A).] Racemic anti-[sH]BPDE (10 pM final concentration) was added to each sample. The samples were allowed to react at room temperature overnight in the dark. Each sample was extracted 4 times with 2 mL of water-saturated 1-butanol, and then twice with 2 mL of diethyl ether, to remove tetrol. To remove any remaining traces of tetrol, the samples were transferred to dialysis tubing (Spectrapor, 2000 molecular weight cutoff) and dialyzed for 36 h at 4 "C in 250 mL of 10 mM sodium cacodylate buffer (pH 7.2) containing 100 mM NaCl and 1mM EDTA (to inhibit ribonucleases by removing traces of Mgz+). The dialysate was changed five times in this interval (EDTA was omitted from the last change). Magnesium chloride was then added to give a final concentration of 10 mM, followed by the addition of 2.5 volumes of ethanol. The samples were then chilled to -20 "C, and the precipitated RNA was recovered by centrifugation (10 min a t 1OOOg). The pellets were dried and dissolved in 1.2 mL of HzO plus 0.3 mL of 1.5 M KOH. Hydrolysis of BPDE-Modified Poly(A). RNA hydrolysis was carried out by boiling modified poly(A) samples in 0.3 M KOH for 15 min; the pH of these samples was then lowered to 10 by the addition of Tris-HC1. Magnesium and zinc chlorides were then added to each sample to a final concentration of 1mM for each salt. Alkaline phosphatase was added in four equally spaced doses (4 X 40 units for each sample) over a period of 24 h in the dark at room temperature. Isolation of BPDE-Adenosine Adducts. Modified and unmodified nucleosides were separated with CIS Sep-Pak cartridges. Aqueous samples were loaded onto the cartridge, which was washed with water to remove unmodified nucleosides. The adducts were eluted with methanol, and the samples were concentrated with a rotary evaporator. Individual adducts were resolved by reverse-phase HPLC using a0.46 x 25 cm 5-pm Rainin Microsorb CIS column and a methanol/water mobile phase. Elution was at 0.8 mL/min using the following gradient: 50% MeOH/H20, 0-40 min; linear 50-55% MeOH ramp, 40-45 min; 55% MeOH after 45 min. Detection was by fluorescence and radioactivity. Fluorescence emission was measured with a cutoff filter at wavelengths >320 nm, using 245-nm exciting radiation. The tritium content of 1-min (or half-minute) fractions was measured by liquid scintillation. Because fluorescence yields of different adducts vary (31),adducts were quantitated by radioactivity. Adduct samples stored for more than a few weeks were kept a t -80 "C.
112 Chem. Res. Toxicol.,
Vol. 7,No.1, 1994
Wolfe and Meehan Table 1. The Effect of NaCl Concentration on the Distribution of Adducts Formed between (i)-anti-BPDE and P o l d A P NaCl concentration adduct ratio OM 0.1 M 0.5 M (+)-trans/total 0.300 0.210 0.167 (-)-cis/total 0.175 0.307 0.396 (-)-transltotal 0.346 0.244 0.167 (+)-cis/total 0.179 0.239 0.280 cis/ trans 0.55 1.20 2.08 Reaction conditions were as outlined under Experimental Procedures. ~~
c
4
0.1
10
1
100
1000
1 .o
t
0
[X-I, mM
Figure 1. The effects of halide salts on the product ratio of
anti-BPDEhydrolysis. Racemic BPDE was allowed to hydrolyze
in solutions containing 10 mM sodium cacodylate buffer (pH 7.0) or 10mM HCI (pH 2.0),10% acetone (v/v), and the indicated salt concentration. The ratio of cis-tetrol to trans-tetrol was determined by reverse-phase HPLC as described under Experimental Procedures. The curves through the data points (for pH 7 measurements) were obtained from eq A8 using nonlinear regression as described in the text. The mean value f SD is shown. 0.5
I
I
I
I I
I
I
20
30
40
50
retention
(min)
60
70
Figure 3. Reverse-phase HPLC elution profiles of (*)-anti[aH]BPDE-poly(A) adducts formed at high and low chloride concentration. The chromatograms for adducts formed at pH 7.0 in solutions containing 0 M (-0-) or 0.5 M (- -A--) NaCl were normalized to the same amount of total radioactivity, and the ordinate scale was adjusted to give a maximum peak height of unity. The adducts elute in the following order: trans-(+), cis-(-), trans-(-), cis-(+) (44).
0
100
500
[CI-1, mM
Figure 2. The effects of chloride salts on the product ratio of anti-BPDE hydrolysis. Product ratios in the presence of the indicated salt concentrations a t pH 7.0 were determined as described for Figure 1. The mean value f SD is shown.
Results and Discussion The dependence of the cis-tetrolltrans-tetrol product ratio of anti-BPDE hydrolysis on halide ion concentration is shown in Figure 1. It can be seen that iodide, bromide, chloride, and fluoride salts all substantially increase the product ratio at concentrations in the 500-1000 mM range. At concentrations in the 1-100 mM range, there is a large difference in the potency of the halide ions, in the order I > Br > C1 >> F. It is also apparent that the effects of NaCl and MgC12, when compared at the same chloride ion concentration, are nearly identical, indicating that the cation has little or no effect on the product ratio. In Figure 2, the effects of five different chloride salts on the product ratio are comparedand are found not to differ significantly, supporting the same conclusion. The relative proportions of the cis and trans poly(A) adducts formed from both enantiomers of (*)-anti-BPDE in solutions containing 0.0, 0.1, and 0.5M sodium chloride are given in Table 1. The reverse-phase HPLC chro-
matograms of the adducts formed in 0.0 and 0.5 M NaCl are depicted in Figure 3. The proportion of adducts that form with the cis configuration increases with salt concentration, from roughly one-third in the absence of salt to about two-thirds at 0.5 M NaC1. The effect of chloride ion on the cisltrans product ratio in the poly(A) alkylation reaction is thus similar to its effect in the hydrolysis reaction. The most notable difference between the two cases is that, at all levels of chloride (including zero), the ratio is 3-5 times higher in the alkylation reaction. There is a small selectivity for alkylation of poly(A) by the (-) enantiomer of anti-BPDE, which also increases with salt, reflecting the fact that the proportion of the (-) cis adduct is increased most by chloride ion. Poly(A) is singlestranded a t neutral pH (32). In duplex DNA, there is a much stronger selectivity for trans adduct formation in the absence of halide ions (33,34). The effects of bromide and iodide ions on adduct formation have not yet been tested, but their propensity for the S Nreaction ~ (see below) might be expected to reduce adduct yields. Chloride ion has been reported to have effects on both the kinetics and product ratio of the hydrolysis reactions of a number of other epoxides, and these effects have been interpreted as resulting from the formation of chlorohydrin intermediates (35-39). The susceptibility of different epoxides to nucleophilic attack by chloride varies widely; in one study (38),neither of the tetrahydrodiol epoxides of naphthalene and only one of the two tetrahydro epoxides of naphthalene were subject to such attack. The mech-
Chem. Res. Toxicol., VoZ. 7, No. 1, 1994 113
Halide Reactions with Benzo[a]pyrene Diol Epoxide
I
Scheme 1
x
6+Z
5
A
OH
OH
I
ko
‘Ho8+@
k5ax
k-5 !
Ho’8 6 OH
3
0.7
[-3
m
Na cacodylate
I
--
buffer only pH 10 buffer
0.6
-5
I
0.5
Na phosphate NaCl
04
NaCl + K phosphate
0.3
NaCI, pH 10
KI
0.2 I
0
KI
0.1
+K
phosphate
KI, pH 10
0 0
100
[additional
anion(s)],
500
mM
Figure 4. The effects of general acid catalysts on the product
ratio of anti-BPDE hydrolysis. Product ratios in the presence of the indicated buffer or salt concentrations were determined as described for Figure 1. The mean value f SD is shown. All samples except the pH 10 and “no buffer” samples contained 10 mM sodium cacodylate buffer (pH 7.0). Any additional buffer added was a t pH 7.0. If both a salt and buffer were added, both were present a t the indicated concentration. The pH 10samples contained 5 mM sodium carbonate.
Table 2. Effects of Halide Salts on anti-BPDE Hydrolysis Product Ratioa
anism of chlorohydrin formation changes with pH. In a number of cases where chloride directly attacks epoxides at neutral pH, chlorohydrin formation at acidic pH results from s N 1 reaction with the carbocation,due to the inability of chloride ion to compete with hydronium ion for reaction with the epoxide under these conditions (37, 39). The chloride effect on product distribution is usually larger in the former situation. A depiction of these possibilities is shown in Scheme 1. The rate constants for the formation of carbocation 4 from anti-BPDE [1,represented by the (+)enantiomer] by the hydronium ion-catalyzed and buffer-catalyzed routes are designated kH and kbuf, respectively (with UH and abuf representing the hydrogen ion and buffer activities). In this kinetic scheme, it is assumed that the carbocations produced by both routes are equivalent. The rate constant for spontaneous hydrolysis is designated ko. Whether this reaction proceeds through a carbocation intermediate is unclear. The use of trapping agents has provided evidence for the presence of a carbocation intermediate in the spontaneous hydrolysis of syn-BPDE, but not in the case of anti-BPDE (40). The rate constants for nucleophilic attack of halide ion on 1 and 4 are 122 and k d + kg, respectively; a, is the activity of the halide ion. The k2 reaction, being an S Ndisplacement, ~ would give a trans halohydrin. The relative rates of the k-4 and k5 reactions (the relative proportions of the trans and cis halohydrins derived from the carbocation) are difficult to predict. On the basis of the results obtained at 0.5 M sodium phosphate (see Figure 4), the tetrol formed via the acid-catalyzed pathway is almost entirely trans (kG/k7 > 97/3). The sN2 attack of water on trans and cis halohydrins (the ks and kg reactions) should yield cis- and trans-tetrols, respectively. The analogous s N 2 attack of the exocyclic amino group of a nucleic acid base on halohydrins should yield cis and trans adducts, respectively. Since intermediates 2-5 in Scheme 1 are unstable in aqueous solution at neutral pH, they should be present at low, steady-state concentrations during the hydrolysis of BPDE. The relative concentrations of 1 and 3-5 can be
KI KBr NaCl MgCl2 KF
0.84 0.14 0.021 0.018 0.001
0.630
0.585 0.524 0.525 NDb
a The BPDE hydrolysis product ratio versus halide ion concentration data of Figure 1were fitted to eq A8 using nonlinear regression. Equation A8 was derived using the assumption that rate constants k 4 and 125 in Scheme 1are zero. The estimates for the ratio of the halide-catalyzedto the non-halide-catalyzed hydrolysis rate constants (kzl k y ) , and the cis-tetrolltruns-tetrolproduct ratio for the halidecatalyzed reaction (rz), are shown. The experimental value for ry was 0.103. An accurate value for rz could not be determined from the data for KF.
calculated in terms of the rate constants and ion activities under the assumption that the concentrations of 2-5 are constant. This allows calculationof kobd and the expected product ratio. The expressionsare given in the Appendix. In a number of examples, the “chloride effect” on the kinetics of epoxide hydrolysis has been successfully analyzed using a model in which it is assumed that chloride ion does not interact with the carbocation (35-39). This model yields an equation (eq A8) in two unknowns that describes the expected relationship between the observed hydrolysis product ratio and the halide ion concentration. The non-halide-catalyzed and halide-catalyzed routes of hydrolysis should lead to different proportions of cis- and trans-tetrols; the cis-tetrolltrans-tetrol ratios characteristic of the two routes will be designated ry and rz. The former quantity is the observed product ratio in the absence of halide ion. Fitting the data of Figure 1to eq A8 using nonlinear regression and the experimental value of ry gives the curves in Figure 1 and the values of the unknowns listed in Table 2. According to these results, it is predicted that I-, Br-, C1-, and F- will catalyze antiBPDE hydrolysis at the relative rates 40:7:1:0.05, respectively, which is consistent with their relative nucleophilicities. Catalytic effects of bromide and chloride ions on the hydrolysis of 1,2-epoxy-1,2,3,4-tetrahydronaphthalene in a ratio about half that predicted above have been reported (38). In order to test the predictions about the halide effect on anti-BPDE hydrolysis made under the assumption that the halide ions interact only with BPDE, and not with its
114 Chem. Res. Toxicol., Vol. 7, No. 1, 1994
Wolfe and Meehan
Table 3. Effects of Salts on the Kinetics of anti-BPDE Hydrolysis. koM (10-3 9-1) salt OmM 7.5mM 100mM 500mM 1OOOmM KI 0.81 f 0.08 1.66 f 0.04 9.32 f 0.17 33.3 f 1.1 47.4 f 2.6 1.50 f 0.01 3.60 f 0.11 5.28 f 0.51 KBr 0.81 f 0.08 NDb NaCl 0.81 f 0.08 NDb NDb 1.43 f 0.08 1.64 f 0.01 LiClO4 0.81 f 0.08 0.84 f 0.06 0.96 f 0.13 1.18 f 0.06 1.39 f 0.04 Hydrolysis rates in 10 mM sodium cacodylate buffer (pH 7.0) containing 10% acetone (viv) were measured as described under Experimental Procedures. The mean value f SD is shown. Not determined. Table 4. The Role of Halide Ion Attack on the BPDE Carbocation in Halide Ion Catalysis of anti-BPDE Hydrolysis. MX KI
KBr NaCl
concn (mM) 7.5 100 500 1000 100 500 1000 500 1000
ko~(MX)lko~(LiC1Od measured predicted f H (lower limit) 1.97 9.7 28 34 1.57 3.1 3.8 1.21 1.18
7.3 84 418 836 15.3 72 144 11.5 22
0.42 0.04 0.01 0.09 0.57 0.31 0.26 0.70 0.81
The measured halide salt/LiC104 ratio of BPDE hydrolysis rate constants for a given salt concentration (from Table 3) and the ratio predicted under the assumption that k 4 = kg = 0, i.e., that the halide ion interacts only with BPDE itself, and not with its carbocation (from Table 2), are given. Also shown is the lower limit estimate for f ~ the , fraction of the hydrolysis products whose formation is attributable to halide ion attack on the carbocation (see eq A22).
carbocation, the effects of NaC1, KBr, and KI at various concentrations on the rate of the reaction were measured. In each case the measured rates were compared with rates obtained at the same concentration of LiC104, a nonnucleophilic salt used to control for ionic strength effects. The results are shown in Table 3, and the comparison of these results with the results predicted from the data in Table 1is given in Table 4. In all cases, the observed rate increases were 1157%of the predicted rate increases. Thus, the model represented by eq A8 is not valid. Much of the halohydrin formation is due to s N 1 attack on the carbocation. However, the good fit of the data to curves based on eq A8 (which is rationalized in the Appendix) suggests that the values of rz in Table 2 should be good estimates for the product ratios of the halide-catalyzed reactions. The fraction of tetrol whose formation is due to halide attack on the carbocation cannot be accurately determined from our data. However, a lower limit estimate for this quantity can be obtained; the method for doing so is given in the Appendix (eq A22). The results (Table 4) are generally consistent with the expectation that stronger nucleophilesand higher nucleophile concentrations should ~ relative to the S Npathway. ~ favor the S Npathway At high concentrations of iodide ion, the hydrolysis of anti-BPDE is greatly accelerated, and the process should be dominated by the halide-catalyzed pathways. Under these conditions, it is predicted that the rate of reaction should be linearly proportional to [I-] (eq A20). This expectation was not borne out by experiment (Table 3). A possible explanation for the discrepancy is that at high iodide ion concentration, the concentration of an intermediate builds up transiently to appreciable levels, violating the steady-state assumption on which eq A20 is
Table 5. Effects of General Acid Catalysts on the Kinetics of anti-BPDE Hydrolysis. kobd
catalyst native DNA denatured DNA 100 mM sodium cacodylate 500 mM sodium cacodylate 100 mM potassium phosphate 500 mM potassium phosphate
OmM NaCl 26.5 f 0.8 15.5 f 0.7 6.23 f 0.015 24.0 f 0.5 13.0 f 0.1 40.3 f 0.3
5-l)
100mM NaCl
500mM NaCl
5.00 f 0.08 2.58 f 0.12 5.36 f 0.06 2.79 f 0.04
NDb NDb
NDb NDb 14.5 f 0.1 NDb NDb 50.0 f 0.9
a Hydrolysis rates in pH 7.0 solutions containing 10% acetone (viv) and 10 mM sodium cacodylate in addition to the indicated buffer were measured as described under Experimental Procedures. Calf thymus DNA was used at a concentration (in bases) of 3 mM. The mean value f SD is shown. Not determined.
based. This idea is supported by the observation of transient changes in the UV spectrum (increased absorbance in the 350-380-nm region, and decreased absorbance in the 320-350-nm region) during hydrolysis in 1 M KI. In strongly acidic solutions of HzS04 or HC1 (pH 1 2 ) containing 10% acetone (v/v),the hydrolysisof anti-BPDE is, under most conditions, virtually instantaneous (tip < 1 s). This was true at all concentrations of chloride ion and bromide ion tested, as well as at concentrations of iodide ion less than 200 mM. However, at higher levels of iodide ion (1450 mM), the hydrolysis proceeded with a minimum t l j 2 of 2.5 s, regardless of the pH. Under these conditions, the same transient changes in the UV spectrum observed during hydrolysis in 1 M KI at neutral pH are again evident. The presence of 10 mM ascorbic acid did not alter these observations, indicating that they were not artifacts due to the presence of traces of iodine. It seems unlikely that the observed intermediate in the hydrolysis is the iodohydrin of BPDE, which should be less stable than the bromohydrin or chlorohydrin, which were not detected. At high concentrations of iodide ion the hydrolysis may proceed by a different pathway, involving an intermediate of greater stability. This interpretation is supported by product ratio measurements made in 10 mM HC1 (Figure l), which indicate that the degree of Icatalysis of hydrolysis changes little between 100 and 1000 mM KI. (The product ratio in 10 mM HC1 alone was 0.21 f 0.01.) The effects of several general acid catalysts, both alone and in combination with halide salts, on the anti-BPDE hydrolysisproduct ratio are shown in Figure 4. The effects of these catalysts on the kinetics of hydrolysis are shown in Table 5. At pH 10, in the absence of halide ions or high concentrationsof buffer, hydrolysis should proceed mostly by the spontaneous route, which is characterized by formation of a high proportion of cis-tetrol (18); the cis/ trans product ratio obtained under these conditions was 0.59. The “no buffer”, 10, 100, and 500 mM sodium cacodylate (pH 7.01, and 100 and 500 mM sodium phosphate (pH 7.0) samples have a progressively decreasing product ratio (0.28,0.103,0.076,0.071,0.043, and0.028, respectively), due to the increasing fractions of the hydrolysis occurring via specificand general acid catalysis, and the much lower cis1trans product ratio characterizing the acid-catalyzed route (20). Phosphate is more potent than cacodylate in lowering the product ratio. Phosphate is also a better general acid catalyst than cacodylate (Table 5). However, the lower product ratio obtained with phosphate is not simply a result of its greater catalytic activity. Hydrolysis is faster in 500 mM cacodylate than
Chem. Res. Toxicol., Vol. 7, No. 1, 1994 115
Halide Reactions with Benzo[a]pyrene Diol Epoxide I
I
‘
I
T
I I
I I
ES LiC104 ; ; 0.16
-
T
c
A
T
--
L
e2
0.12
L \
-2
0.08
CI
L
-$
0.04
0
0
100
500
mM NaCl
Figure 5. The combined effects of;DNA and sodium chloride on the product ratio of anti-BPDE hydrolysis. Product ratios in the presence of 10 mM sodium cacodylate buffer (pH 7.0) and the indicated sodium chloride concentrationswere determined aa described for Figure 1. Calf thymus DNA was used at a concentration (in bases) of 3 mM.
in 100mM phosphate, but the product ratio is lower in the latter. This suggests that different general acid catalysts produce slightly different ratios of anti-BPDE hydrolysis products. In solutions containing chloride or iodide ion, the cis/ trans hydrolysis ratio is lowered by addition of phosphate and raised by changing the pH from 7 to 10 (Figure 4). The pH effect is very similar for the two halide ions, while the phosphate effect is substantially greater with chloride. Since the chloride effect on the hydrolysis product ratio results primarily from trapping of the carbocation intermediate, the large drop in the product ratio due to addition of phosphate suggeststhat the BPDE carbocations formed by phosphate catalysis are less accessible to chloride than carbocations formed from hydronium ion or cacodylate catalysis. The catalytically active form of phosphate at neutral pH is HzP04- (20),and the HP0d2-remaining after proton transfer may repel chloride ions enough to inhibit the chloride-carbocation reaction. The phosphate effect on the reaction in the presence of iodide would be expected to be smaller because of iodide’s greater reactivity with the carbocation, and its greater ability to compete kinetically with phosphate for reaction with BPDE. Calf thymus DNA, in both native and denatured forms, acts as a general acid catalyst in the hydrolysis of antiBPDE and, as expected, also lowers the cisltrans hydrolysis product ratio (Figure 5). Like phosphate, DNA also partially counteracts the chloride effect on the hydrolysis product ratio. DNA at a concentration of 3 mM largely abolishes the effect of 100 mM chloride ion and reverses more than half the effect of 500 mM chloride. DNA at 3 mM is thus more effective than 100 or 500 mM phosphate in countering the chloride effect (Figures 4 and 5). This disparity is also unrelated to the rate of hydrolysis. At pH 7 in the absence of salt, 3 mM DNA catalyzes antiBPDE hydrolysis at a rate intermediate between those of 100and 500 mM phosphate. Salt strongly inhibits catalysis by DNA (281, but not catalysis by phosphate. In 100 mM NaC1,3 mM DNA at pH 7.0 is only one-third as effective a catalyst as 100 mM phosphate; at 500 mM NaC1,3 mM DNA is less than one-tenth as effective a catalyst as 500 mM phosphate (Table 5). The greater potency of DNA
0
100 [salt],
500 mM
Figure 6. The effecta of non-halidesalts of strong acids on the product ratio of anti-BPDE hydrolysis. Product ratios in the presence of the indicated salt concentrations were determined as described for Figure 1. The mean value SD is shown.
*
in reversing the chloride effect is probably due to the much greater ability of the polyanion to electrostatically repel chloride ion from the zone of general acid catalysis. The DNA effect is reduced at higher salt concentrations, probably in part due to the inhibition of DNA catalysis by salt, and in part due to a higher chloride concentration in the vicinity of the DNA. In the absence of salt, catalysis of anti-BPDE hydrolysis by denatured DNA produces a slightly lower product ratio than catalysis by native DNA (Figure 5). This is another example of the lack of correlation between the effects on hydrolysis rate and product ratio of general acid catalysts, because under these conditions native DNA is a significantly better catalyst (Table 5). The potency of denatured DNA in reversing the effect of 100 or 500 mM chloride ion on the product ratio is also somewhat greater than that of native DNA. We have previously shown that, at sodium ion concentrations above 35 mM, benzo [a]pyrene derivatives physically bind to denatured DNA with greater affinity than to native DNA (41);we have also found that denatured DNA is a better catalyst for anti-BPDE hydrolysis than native DNA at sodium ion concentrations above 20 mM [these measurements were carried out using 3 mM DNA in 2 mM sodium phosphate buffer (pH 7.2) containing 0.1 mM The effects of three non-halide salts of strong acids on the anti-BPDE hydrolysis product ratio are shown in Figure 6. The three anions involved vary widely in their position on the Hofmeister series, which is a ranking of the relative abilities of different anions to alter the properties of solutes in aqueous solutions. The anions used in this study can be ranked in order of increasing potency of their “lyotropic” effect as follows: F C PO4 < SO4 < C1< Br < NO3 < I < C104. The chaotropic anions on the right end of this series tend to increase the water solubility of hydrophobic solutes (“salting-in”), while the kosmotropic anions to the left have the opposite effect (42). It was found that 500 mM clod-, NOa-, or SO4” all had a similar and rather modest effect on the product ratio. This rules out any significant effect due to the lyotropic properties of anions. The effect of LiC104 on the hydrolysis rate was also small; 1 M LiC104 led to a
* A. R. Wolfe and T. Meehan, unpublished results.
116 Chem. Res. Toxicol., Vol. 7, No. I , 1994
70% increase (Table 3). Ionic strength does not appear to mediate the observed effects of these salts, since if this were the case, Na2.304 would have the largest effect.
Conclusion Chloride, bromide, and iodide ions catalyze cis product formation during the hydrolysis of anti-BPDE. This catalysis appears to result from the formation of trans halohydrin intermediates. The formation of the trans hydrolysis product from cis halohydrins also occurs, but to a lesser degree than in the non-halide-catalyzedreaction. In the case of bromide and iodide ions, the catalysis can occur both by s N 1 attack on the BPDE C-10 carbocation, which changes the product ratio but not the reaction rate, and by sN2 attack on BPDE itself, which accelerates the reaction. In the case of chloride ion, only the former process is significant. Chloride ion also catalyzes cis product formation in the alkylation of nucleic acids by anti-BPDE. The proportion of cis products resulting from reaction of BPDE chlorohydrins with nucleic acids appears to be much greater than in their reaction with water. The chloride effect has been observed not only with single-stranded poly(A) but also with both dGuo and dAdo targets in duplex DNA, in tetrastranded poly(G),and in nucleotides (34). Although the proportion of anti-BPDE adducts that are cis varies widely with the alkylation target, the secondary structure of the nucleic acid, and the BPDE enantiomer involved, in all cases this proportion is increased by addition of chloride ion. This is apparently the first indication that the alkylation of DNA by anti-BPDE can occur via a chlorohydrin intermediate, rather than through direct interaction of DNA with the BPDE carbocation (or with BPDE itself). It is likely that epoxide derivatives of other polycyclic aromatic hydrocarbons undergo analogous reactions with chloride. The intracellular chloride concentration is in the 5-15 mM range (43). In our experiments, 10 mM chloride ion raised the cisltrans hydrolysisproduct ratio 5095, so BPDE chlorohydrin formation must occur to at least a limited degree inside the cell. We have also found that 10 mM MgCl2 can significantly increase the proportion of cis adducts in DNA (34). Since it appears that chloride ion tends to be repelled from the immediate vicinity of the DNA molecule, the degree to which chloride perturbs the BPDE-DNA reaction may be influenced by the concentrations and types of DNA counterions present. Higher concentrations of chloride ion (-110 mM) occur extracellularly, and therefore, the proportion of cis products from any BPDE reactions occurring in this region should be larger. In addition to its in vivo significance,recognition of the existence of chloride-catalyzed reaction pathways may be important in the interpretation of in vitro experiments conducted in buffers containing chloride.
Acknowledgment. We thank Dr. George Negrete for helpful discussions. This work was supported in part by grants from the National Cancer Institute (CA 40598) and the Elsa U. Pardee Foundation.
Appendix The overall rate constant for the disappearance of 1 (Scheme 1) can be calculated in several ways. The expression in terms of the concentrations of intermediates
Wolfe and Meehan
is as follows:
This rate constant can also be expressed as the sum of the rate constants attributable to the spontaneous, acidcatalyzed, and halide-catalyzed reactions:
(Ah) where k A and kx are the rate constants that would be observed in the cases ko = k2 = 0 and ko = 121 = 0, respectively. An overall rate constant for the acidcatalyzed formation of carbocation from BPDE will be defined as 'obsd
= kO + 'A +
+ kb,
(A21 If halide ion interacts only with BPDE, and not with its carbocation, eq Ala can be rewritten: k1 E k H U ,
(restrictions on the applicability of equations presented in this paper will be listed in parentheses to the right of the equation). When k 4 and k5 are zero, the steady-state assumption can be used to derive the following:
_ [31 [I]
k2ax k-Z(k-3
+ k4 + k8)/k3aH + k4 + k8 (k4, k, = 0; steady state) (A4)
Assuming that k-3 combined to give
>>
124
+ k8, eqs A3 and A4 can be
(k4, k, = 0; k-, >> k4 + 128; steady state) (A5) where K, = k-3/k3 (35). This can be simplified further to
Itobsd = k y + kzax (k4, k, = 0; steady state) (A6) where k y and kz represent the rate constants for the nonhalide-catalyzed and halide-catalyzed routes of hydrolysis, respectively ( k y and kz are functions of buffer concentration and/or pH). Each of these routes should lead to a different ratio of cis- and trans-tetrols; the cis-tetroll trans-tetrol ratios characteristic of the two routes will be designated r y and rz. (The terms kx and rx defined in eqs A15 and A18 are equivalent to kz and rz when k 4 = k5 = 0.) The observed product ratio will be
- f Y f c Y + f z fcz robad
- fYftY + f z f t z
(k4,k5
= 0)
(-47)
where f y and f z = 1- f y are the fractions of tetrol produced by the non-halide-catalyzed and halide-catalyzed routes, f c y and f t y = 1 - f C y are the fractions of tetrol produced by the non-halide-catalyzed route that are cis and trans, and f C zand f aare defined similarlyfor the halide-catalyzed route. (It should be noted that it is being assumed that the product ratio and rate constant of the non-halidecatalyzed route are not affected by changes in salt concentration. It is shown in Figure 6 and Table 3 that this is not strictly true. However, the effects are small, and neglecting them here will not affect our conclusions.) Since f y = k y / ( k y + kzax), f t y = 1/(1+ r y ) , and f t z = 1/(1 + rz), eq A7 can be rewritten (assuming unitary activity
Chem. Res. Toxicol., Vol. 7, No. 1,1994 117
Halide Reactions with Benzo[a]pyrene Diol Epoxide
that would be observed if ko and k l were zero):
coefficients):
r o k d ( l + ro) + rAkA/(l ‘,bad
(k4, k5 = 0; steady state) (A8)
We turn now to the complete version of Scheme 1, with no rate constants assumed to be zero. The expressions for the concentrations of intermediates 3-5 relative to that of anti-BPDE obtained using the steady-state approximation are as follows:
where k =
k5ax
- 1+ k-,/k,
+ k,+
k,
and
[51 _
k,ax
141
111 k-5 + k, [ll
(steady state)
(A12)
The expressions for kA and kx, the portions of the observed hydrolysis rate constant attributable to attack of BPDE by acids and halide ions, respectively, are
=
+ rA) + r x k x / ( l + rx) k d ( 1 + TO) + kA/(1 + rA) + k x / ( 1 + rx) (A16b)
The product ratios for the acid-catalyzed and halidecatalyzedhydrolysispathways are given by the expressions: rA=
k,
+ k&x/kB
(steady state)
(A17)
kT-kI
and
It may be seen by comparing the expressions (using eqs A10 and A14) that the value of rx will be greater than or equal to that of rA under all conditions. The expressions in eqs A8, A17, and A18 can all be put in the form (ax + b)/(cx + d), which gives a rectangular hyperbola as a function of x (=[X-l). For positive values of the variable and coefficients, neither the first or second derivatives of the expression can change sign. Thus, ?‘A and rx will either increase or decrease monotonically with increasing ax, and plots of r A or r X versus [X-I will not have inflection points. The experimental data suggest that both quantities increase with ax. The plot of (ax + b)/(cx + d) versus log x has a single inflection point, at x = d/c. When the concentration of halide ion is sufficiently high, the hydrolysis will be dominated by halide-catalyzed reactions; the expressions for the observed product ratio and reaction rate then simplify to those for rx and k x , with kg and k, dropping out:
and where
and
(steady state) (A15) The general expression for the cis-tetrolltrans-tetrol ratio for Scheme 1 is r o k d ( l + ro) + ([41/[1l)k, robsd
=
kd(l
+ ([31/[ll)k8
+ ro) + ([41/[11)k, + ([51/Cll)k, (A16a)
where ro is the product ratio for the spontaneous reaction, and ro/(l + ro) and 1/(1+ ro) are the fractions of tetrol from this reaction that are cis and trans, respectively. The expression for robed can also be written in terms of kA, kx, PA, the product ratio for the acid-catalyzed hydrolysis pathway (which is the product ratio that would be observed if ko and kz were zero), and r x , the product ratio for the halide-catalyzed hydrolysis pathway (the product ratio
The latter expression implies that kobsd would be linearly proportional to ax under these limiting conditions. To obtain an estimate of the fraction of total tetrol produced that is derived from halohydrins formed by halide ion attack on the BPDE carbocation, we begin by noting that the tetrol can be divided into the fraction produced by direct halide attack on BPDE, fx, and the fraction that is not so produced, f~ = 1- fx. The latter quantity can be subdivided into fractions of tetrol that ) are not c f ~produced ) by halide attack on the are ( f ~and carbocation; Le., f~ + f~ = fN. Now, the fraction of total tetrol produced that is cis-tetrol, fc, is a function of the fractions of cis-tetrol produced by these three pathways: Solving for f~ then gives
The quantity fx can be estimated as [kobsd(MX) -
118 Chem. Res. Toxicol., Vol. 7, No. 1, 1994
Wolfe and Meehan
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kobad(LiC104)l/kobsd(MX) (see Table 2). Each of the quantities f c , f c H , f c J , and f c x may be obtained from the corresponding product ratios [e.g., f c = robsd/(l + robsd)l. The quantities robad and TJ (=robad for LiC104 at the same concentration as the halide salt) were measured. For the quantities rx and rH, the extrapolated value of robsd at infinite halide ion concentration (rz,Table 2) may be taken as an upper limit, and this will give a lower limit estimate for fH.
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Announcements Eighth International Congress of Pesticide Chemistry The International Union of Pure and Applied Chemistry in conjunction with the American Chemical Society will be hosting the Eighth International Congress of Pesticide Chemistry. The theme of the Congress is “Options 2000” to reflect the changes that will carry the field into the next millennium. All aspects of pesticide chemistry will be included. The Congress will be held July 4-9, 1994, in Washington, DC. For further information, please contact: American Chemical Society, Office of the Secretariat c / o Dianne Ruddy, Eighth International Congress of Pesticide Chemistry, 1155 Sixteenth Street, N.W., Washington, DC 20036. Phone: (202) 872-6286; FAX: (202) 872-6128.
International Symposium on Human Health and Environment: “Mechanisms of Toxicity and Biomarkers To Assess Adverse Effects of Chemicals” The Scientific Committee on Occupational Toxicology of the International Commission on Occupational Health, the Commission of the European Communities, and the University of Parma, in collaboration with the World Health Organization: International Agency for Research on Cancer, International Programme on Chemical Safety (ILO-UNEP-WHO), Office of Occupational Health (Headquarters), and Regional Office for Europe, are sponsoring the International Symposium on Human Health and Environment: “Mechanisms of Toxicity and Biomarkers To Assess Adverse Effects of Chemicals”. The Symposium will be held September 25-30,1994, in Salsomaggiore Terme (Parma), Italy. The Symposium program will consist of oral sessions and poster presentations. Keynote speakers will provide an overview of the state of the art on five main topics chosen by the scientific committee. Themes for oral presentations and posters are the following: Mechanistic Basis for Health Risk Assessment; Biomarkers for Exposure Assessment; Biomarkers of Individual Susceptibility; Biomarkers for Health Surveillanceand Effect Monitoring; and Biomarkers in Health Risk Assessment and Regulation. Deadline for registration is February 28, 1994. For further information, please contact: Dr. A. Mutti, Secretary of ICOH-SCOT, Laboratorio di Tossicologia Industriale, Universith degli Studi, Via Gramsci, 14,I-43100 Parma, PR, Italy (FAX: 39 521 291343).
