Chem. Res. Toxicol. 1995,8, 693-698
693
Sulfotransferase-MediatedActivation of 7,8,9,10-Tetrahydro-7-01, 7,8-Dihydrodiol, and 7,8,9,1O=TetraolDerivatives of Benzo[a]pyrenel Young-Joon Surht and Steven R. Tannenbaum*$f Department of Epidemiology and Public Health, Yale University School of Medicine and Yale Comprehensive Cancer Center, New Haven, Connecticut 06520-8034, and Department of Chemistry and Division of Toxicology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received February 21, 1995@
Some hydroxymethyl-substituted polycyclic aromatic hydrocarbons have been shown to be converted to electrophilic, mutagenic, or tumorigenic sulfuric acid ester metabolites by cytosolic sulfotransferase activity in rodent liver. Likewise, certain types of aromatic compounds with a secondary alcoholic functional group at the benzylic position undergo metabolic activation through sulfonation. Enzymatic oxidation of benzo[alpyrene produces such secondary alcohols as dihydrodiol and tetraol derivatives as primary metabolites. Sulfo conjugation of the benzylic hydroxy group of each of these metabolites is expected to generate an electrophilic sulfuric acid ester capable of covalently binding to DNA, which may contribute to mutagenesis and carcinogenesis by benzo[alpyrene. Although the model benzo-ring secondary benzyl alcohol, 7-hydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene, covalently bound to DNA and also exerted mutagenicity in the presence of rodent hepatic cytosols and 3’-phosphoadenosine 5’-phosphosulfate, no such sulfotransferase-dependent activation was observed with dihydrodiol or tetraol derivatives of benzo[alpyrene. Thus, it seems likely that appearance of the adjacent nonbenzylic hydroxy functional group(s) in latter metabolites hinders the benzylic sulfonation in these molecules.
Introduction The formation of sulfate conjugates is recognized as a primary Phase I1 metabolic reaction which plays a crucial role in the detoxification of lipophilic foreign compounds. However, sulfo conjugation can contribute to toxicity of certain classes of xenobiotics ( 1 , 2). Since the first discovery in the 1960s of 2-(acety1amino)fluorene Nsulfate as an electrophilic metabolite of N-hydroxy-2there has been accumulated (acety1amino)fluorene (3,4), evidence for the association between sulfuric acid esterification and induction of tumors by this carcinogen and others. The majority of reactive sulfuric acid esters previously investigated have been those of benzylic hydroxy or N-hydroxy derivatives of aromatic compounds, which are considered to yield extremely electrophilic carbocations or nitrenium ions, respectively. These reactive species are capable of covalently modifying cellular DNA, which may lead to mutations and neoplastic transformation. Recent studies from several laboratories have shown that certain polycyclic aromatic hydrocarbons (PAH)2with benzylic hydroxymethyl functionality are activated to electrophilic and mutagenic sulfuric acid esters by sulfotransferase activity in rodent liver cytosols +Yale University School of Medicine and Yale Comprehensive Cancer Center. Massachusetts Institute of Technology. @Abstractpublished in Advance ACS Abstracts, May 15, 1995. A portion of this work was presented at the 14th International Symposium on PolycylicAromatic Compounds held in Tan-Tar-A,Lake of Ozarks, MO, on September 8-11, 1993. Abbreviations: PAH, polycyclic aromatic hydrocarbon; PAPS, 3’phosphoadenosine 5’-phosphosulfate; BP, benzo[alpyrene; 7-HTBP, 7-hydroxy-7,8,9,lO-tetrahydrobenzo[a]pyrene;DHEA, dehydroepiandrosterone; DCNP, 2,6-dichloro-4-nitrophenol;6-HMBP, 64hydroxymethyl)benzo[alpyrene; 7-STBP, 7-(sulfooxy)-7,8,9,1O-tetrahydrobenzo[alpyrene; DMF, dimethylformamide; 10-HTBP, 10-hydroxy-
*
7,8,9,10-tetrahydrobenzo[alpyrene.
(5-10). Besides hydroxymethyl polyarenes, aromatic hydrocarbons possessing the secondary benzylic alcoholic functional group such as 4-hydroxy-3,4-dihydrocyclopenta[cdlpyrene (11, 12) and 4H-cyclopenta[deflchrysen-401 (13,141can also form highly reactive and mutagenic sulfuric acid ester metabolites when incubated with rodent liver cytosol and the sulfo group donor, 3’phosphoadenosine 5’-phosphosulfate (PAPS). These findings led us to explore the possibility of sulfotransferasemediated activation of other PAHs with a benzylic hydroxy group in a cyclic form. Compounds of particular interest to us include dihydrodiol and tetraol metabolites of benzo[alpyrene (BPI which possess the secondary benzylic alcoholic group(s) at C-7 and/or C-10atoms. If these primary metabolites of BP would form reactive esters via sulfo conjugation, this could constitute a potential metabolic activation pathway for BP alternative to the generally accepted “bay-region epoxidation”. This concept is schematically represented in Figure 1. With this idea in view, we have examined the DNA binding and mutagenicity of BP dihydrodiol and BP tetraol in the presence and absence of hepatic sulfotransferase activity. For comparison, 7-hydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (7-HTBP),a model benzo ring reduced secondary benzylic alcohol, was also included in this study.
Materials and Methods Caution. BP and its derivatives are potential carcinogens, and appropriate safety procedures should be followed when handling these compounds. The synthetic benzylic sulfuric acid esters used in this study are highly reactive and labile, especially in the presence of water, and unused portions must be kept dry in a deep freezer. They are relatively stable in dry DMSO i f stored at -70 “C. Chemicals. (i.)-trans-7,8-Dihydr0[1,3-~H]BP-7,8-diol and (~k)-cis-4,5-dihydro[ring-G-~H]BP-4,5-diol were supplied from
0893-228d95/2708-0693$09.00/0 0 1995 American Chemical Society
Surh and Tannenbaum
694 Chem. Res. Toxicol., Vol. 8, No. 5, 1995
..-
OH ha
-
m
OSOsH
HO.
A
1
OH
HO.
A
1
'APS 17
MUTAGENESIS, CARCINOGENESIS
OSOsH
OH
OH
Covalent DNA Modification
OH
or Ho
lo@ '0
m
H
OH
W
Figure 1. Hypothetical routes for metabolic reactivation of BP tetraol via benzylic sulfuric acid esterification. Asterisks denote benzylic carbon centers. Note that sulfonation of the C-10 hydroxyl group with subsequent solvolysis would afford the same carbocation as that derived from BP-7,8-dihydrodiol-9,lO-epoxide. Chemsyn Science Laboratory (Lenexa, KS). Nonradioactive BP derivatives were acquired from NCI Chemical Carcinogen Standard Repository. [14C]BP tetraol isomers were prepared by acid hydrolysis of anti-[14C]-7,8-dihydroxy-7,8-dihydro-9,10epoxy-BP followed by purification on CISreverse-phase HPLC. PAPS, dehydroepiandrosterone (DHEA), and calf thymus DNA were products of Sigma Chemical Co. (St. Louis, MO). 2,6Dichloro-4-nitrophenol (DCNP) was purchased from Fluka Chemical Co. (Ronkonkoma, NY). 6-(Hydroxymethyl)-BP (6HMBP) used as a positive control for sulfotransferase activation was synthesized by reduction of BP-6-carboxaldehyde(Chemsyn Science Laboratory) as described previously (15). 7-HTBP was prepared by sodium borohydride reduction of 9,lO-dihydro-BP7(8H)-one (Aldrich Chemical Co., Milwaukee, WI) according to the procedure reported in the literature (16).All other chemicals and solvents used were commercial products of analytical grade. Synthesis of 7-(Sulfooxy)-7,8,9,1O-tetrahydrobenzo[a]pyrene. For the synthesis of the sulfuric acid ester of 7-HTBP, the parent alcohol (0.2 mmol) in 0.5 mL of dry dimethylformamide (DMF) was added to a stirred solution of dicyclohexylcarbodiimide (1.0 mmol) in 1mL of DMF. Sulfuric acid (0.4 mmol) in 0.4 mL of ice-chilled DMF was added dropwise to the above mixture with vigorous stirring. After 30 min reaction a t 0 "C, the reaction mixture was centrifuged a t 3000g for 15 min. The supernatant was made slightly alkaline (-pH 8) with 1 M methanolic NaOH and centrifuged to remove insoluble salts. To the supernatant was added slowly 10 volumes of dry ethyl ether. 7-(Sulfooxy)-7,8,9,10-tetrahydrobenzo[a]pyrene (7-STBP) was precipitated as a white sodium salt, which was collected by centrifugation, rinsed repeatedly with ethyl ether, and dried under vaccum to afford the product a t the yield of 70%. Thinlayer chromatography on Whatman reverse-phase KC18 thinlayer plate using 90% methanol in water as eluent revealed the main product as a blue fluorescent spot with an Rf value of 0.9. Under these conditions, 7-HTBP showed an Rfvalue of 0.5. The hydrolysis of the above product produced a single ethyl acetate extractable material which had the same Rf value as that of the parent alcohol. UV ,A (EtOH) nm (log E ) : 238 (4.72), 247 (4.95), 256 (4.221, 267 (4.47), 278 (4.701, 314 (4.111, 328 (4.491, and 345 (4.66). IR (KBr): 3050,2930,1600,1450,1220,1180, and 1060 cm -I. Negative ion fast atom bombardment mass spectrometry exhibited a strong peak a t m / z 351 ([M - Nal-). Enzyme Preparation. Sprague-Dawley r a t liver postmitochondrial supernatant (S9 fractions; from Molecular Toxicology, Inc., Annapolis, MD) prepared from 25% homogenate in 0.15 M KC1 was centrifuged a t 105000g for 1 h a t 4 "C. The supernatant obtained was stored a t -70 "C until use. The protein content of cytosol was determined using the Coomassie
blue G-20 reagent (Pierce Chemical Co., Rockford, IL) with bovine serum albumin as the standard. Determination of Sulfotransferase-MediatedCovalent DNA Binding. The metabolic activation of benzylic hydroxy arenes to electrophilic sulfuric acid esters was determined by measuring the covalent binding of hydrocarbons to calf thymus DNA in the presence of hepatic cytosol and PAPS. The standard incubation mixture in a final volume of 2 mL of Tris-HC1buffer (50 mM; pH 7.4) contained MgClz (3 mM), PAPS (0.1 mM), calf thymus DNA (2 mg), hepatic cytosol from 21-day-old female Sprague-Dawley rats (equivalent to 2 mg of protein), and a radioactive hydrocarbon (5-25 pM). In some experiments, sulfotransferase inhibitors were added to the above incubation mixtures. After incubation a t 37 "C for 2 h, DNA was isolated after removal of cytosolic proteins by phenol extraction as described previously (15). DNA pellets were successively rinsed with 75% EtOH containing 1%NaCl, 75% EtOH, 100% EtOH, acetone, and ethyl ether. Dried DNA samples were dissolved in 15 mM NaC1-1.5 mM sodium citrate buffer (pH 7.0) and quantitated by their UV absorbance a t 260 nm. Aliquots of the DNA solutions were solubilized in 0.5-1 mL of Solvable (DupontNEN Research Products, Boston, MA) by incubation a t 37 "C for a t least 3 h and neutralized with glacial acetic acid. The radioactivity derived from hydrocarbon residues covalently bound to DNA were measured by liquid scintillation counting upon addition of Liquiscint (National Diagnotics, Manville, NJ) as a cocktail. Bacterial Mutagenicity Assays. Sulfotransferase-mediated mutagenicity of benzylic hydroxy hydrocarbons was determined in terms of 8-azaguanine resistance (forward mutation) in Salmonella typhimurium TM677 or his+ reversion (back mutation) in S. typhimurium TA98. The forward mutation assay was based on the procedure developed by Skopek et al. (17)except that liver cytosol and PAPS were used as a metabolic activation system. The direct mutagenicity of 7-STBP was assessed without metabolic activation. f i r 2 h preincubation a t 37 "C, cells were plated and further incubated for 2 days to allow the development of colonies. The experimental details have been described elsewhere (18). Mutagenicity in his- S. typhimurium tester strain TA98 was determined using a liquid preincubation modification of the Ames standard assay as described previously (19). Each test compound (50 nmol) dissolved in 10 pL of DMSO was added to a glass tube containing 0.5 mL of medium which consisted of MgClz (3 mM), PAPS (0.3 mM), hepatic cytosol (2 mg of cytosolic protein) from young adult male rats, bacterial suspension (6 x lo7 cells), and 50 mM phosphate buffer (pH 7.4). Incubations were conducted in triplicate. After incubation a t 37 "C for 1 h, the mixtures were diluted with soft agar preheated a t 45 "C and poured onto
Chem. Res. Toxicol., Vol. 8, No. 5, 1995 695
Benzylic Sulfonation of Benzo[alpyrene Metabolites Petri dishes containing minimal agar. After incubation for 2 days in the dark, the his+ revertant colonies were counted.
Results and Discussion Data from recent studies have indicated that not only polyarenemethanols which are primary benzyl alcohols but also PAHs with a secondary benzylic hydroxy functional group are subject to further metabolism by sulfotransferases to yield highly reactive sulfuric acid esters (1, 2 , 5-15). In light of the presence of two benzylic carbon centers in 7,8,9,10-tetrahydro-7,8,9,lO-tetrahydroxy-BP, uiz., C-7 and C-10, we speculated that sulfonation of one of its benzylic hydroxy functional groups would lead to the formation of an electrophilic intermediate capable of covalently interacting with cellular DNA (see Figure 1). BP tetraol has been regarded as an inactive end product formed as a consequence of hydrolysis of the bay-region diol epoxide, the ultimate electrophilic and carcinogenic metabolite of BP. However, little is known about the further metabolic fate of this relatively polar compound in vivo as well as in vitro. If BP tetraol would undergo sulfonation at either the C-7 or the 12-10 hydroxy group, this would yield an electrophilic sulfuric acid ester that can attack critical cellular nucleophiles, thereby causing deleterious effects. The majority of the remaining portion of the presumed sulfuric acid ester, in an aqueous environment of tissues, will hydrolyze back to the parent molecule, which may be reutilized for sulfo conjugation as above. In this regard, the benzylic sulfonation of BP tetraol may represent futile cycling as often observed with other sulfate conjugates (20). As part of our study directed toward testing the novel idea regarding the possible activation of BP tetraol via benzylic sulfuric acid esterification, we first investigated the sulfotransferase-mediated activation of the readily available BP derivative, 7-HTBP, which possesses a single hydroxy functional group linked to a benzylic carbon center of the saturated benzo ring. By utilizing this model monofunctional benzyl alcohol, we could manage to avoid any complications which might arise from competition for sulfonation between two benzylic hydroxy functional groups or between benzylic and nonbenzylic groups in BP tetraol in sulfotransferase-catalyzed reactions. Figure 2 illustrates the comparative bacterial mutagenicity of 7-HTBP and its benzylic sulfuric acid ester. 7-HTBP, when incubated with rat liver cytosol and PAPS, induced 8-azaguanine-resistant mutants in S. typhimurium TM677 in a dose-dependent manner. Without metabolic activation, 7-HTBP showed marginal mutagenicity while its synthetic sulfuric acid ester 7-STBP showed strong direct mutagenicity (Figure 2). 7-STBP was also more toxic to bacterial cells than the parent alcohol (data not shown). The sulfotransferase-mediated activation of 7-HTBP was also assessed in terms of covalent binding of this benzylic alcohol to DNA in the presence of rat liver cytosol and PAPS. As shown in Table 1, 7-HTBP with hepatic cytosol alone did not exhibit any significant covalent DNA interactions, but in the presence of both cytosol and the sulfo-group donor, the hydrocarbon formed about 18 pmol of covalently bound adductdmg of DNA (Table 1). The primary benzyl alcohol 6-HMBP, which was included as a positive control, formed DNA adducts to a much greater extent than did 7-HTBP. The cytosol- and PAPS-dependent
X
U
z 9
6U
20-
E /
0
--
-0
I
I
I
I
50
100
150
200
HYDROCARBON (pM) Figure 2. Comparative mutagenicities of 7-HTBP and its benzylic sulfuric acid ester in S. typhimurium TM677. Mutagenicity of the parent alcohol was determinedby preincubating the bacteria at 37 "C for 2 h with the hydrocarbon and hepatic cytosol (equivalent to 2 mg of protein) from female Spragueof Dawley rats (21-day-old) in the presence ( 0 )or absence (0) PAPS (0.25 mM). Intrinsic mutagenicity of 7-STBP was measured without metabolic activation (W. Table 1. Sulfotransferase-DependentCovalent DNA Binding of 7-HTBP and 6-HMBP covalent concn incubation DNA binding hydrocarbon @M) conditions (pmol of hydrocarbodmg of DNA) 7-HTBP
25
6-HMBP
25
Complete -PAPS +DHEA +DCNP complete -PAPS
18.0b (19.8, 16.2) 0.17 (0.17, 0.16) 4.4 (4.5, 4.3) 6.3 (6.7, 5.9)
835.7
0.36
Incubation conditions and other experimental details are described under Materials and Methods. Average of 2 values obtained from duplicate analysis; individual values are shown in parentheses.
DNA binding of this cyclic secondary benzyl alcohol was markedly attenuated by both DHEA and DCNP (Table 1). DHEA has been shown to competitively inhibit the hydroxysteroid sulfotransferase-mediated activation of hydroxymethyl PAHs such as 7-(hydroxymethyl)-12methylbenz[alanthracene and 6-HMBP (6,7,15).DCNP is a selective inhibitor of phenol sulfotransferase activity. Based on this result, the metabolic activation of 7-HTBP is likely to be catalyzed by both hydroxysteroid and phenol sulfotransferases. While our manuscript was in preparation, Glatt and his co-workers have reported the sulfotransferase-mediated activation of 7-HTBP to a mutagenic metabolite (21). An interesting feature of their study is that, in contrast to primary benzylic PAHs which are preferentially activated by hepatic cytosols from female rats (7-9, 151, no such notable gender difference is evident for 7-HTBP (22). Opposite patterns of age-related gender differences in rodent hepatic phenol and hydroxysteroid sulfotransferases have been reported (22-25). Thus, while the former enzyme is male-tropic, the expression of the latter enzyme has been found to be downregulated by androgens and hence predominant in adult females. These findings again suggest that metabolic activation of 7-HTBP is mediated by both types of sulfotransferases. 7,8,9,10-Tetrahydro-BP bears a benzylic carbon center at the C-10 as well as the C-7 position. Therefore, it is expected that the 10-hydroxy derivative of the benzo ring
6fM Chem. Res. Toxicol., Vol. 8, No. 5, 1995 reduced BP can be activated via sulfuric acid esterification as in the case of 7-HTBP. This presumption was recently tested by other investigators who determined the bacterial mutagenicity of the model benzylic alcohol, 10HTBP, in the presence of rodent hepatic cytosol and PAPS (21). However, compared to 7-HTBP, the 10hydroxy analogue elicited no pronounced sulfotransferase-dependent mutagenicity (211. The C-10 benzylic atom of BP has been known to represent a more reactive center than the C-7 position as determined by comparing electron delocalization energies ( h E d e l o J b ) of the corresponding carbonium ions (0.794, (2-10 vs 0.488, C-7) (26). Certain polyaromatic compounds, such as 3-hydroxy-3,4dihydrocyclopenta[cdlpyrene and 14hydroxymethyl)pyrene, bear a benzylic hydroxy functionality equivalent to that of 10-HTBP. In contrast to 10-HTBP,the former benzylic alcohols were found to be activated in the presence of hepatic cytosol and PAPS (7, 8, 1 1 , 13, 22). The sulfate esters of these compounds as well as that of 10-HTBP would yield benzylic carbocations that are stabilized by the same aromatic system in the pyrenyl moiety as does the (2-10 carbocations derived from the 7,8-dihydrodiol9,10-epoxide of BP. Moreover, the (2-10 acetic and propionic acid esters of BP tetraol retain intrinsic mutagenicity while the parent compound is i n a ~ t i v e .Therefore, ~ the lack of sulfotransferase-mediated mutagenicity of 10-HTBP seems more likely to be related to weak catalytic activity or affinity of sulfotransferase(s) for this benzylic alcohol. In this context, it is of interest to note that, among the phenolic isomers of BP tested, 10-hydroxy-BP showed the lowest rate of sulfate conjugation (27,281. However, inherent reactivity or stability of the presumed sulfuric acid ester metabolites formed from 7- and 10-HTBP might be another determinant of their relative mutagenicity in the presence of sulfotransferase activity. After confirming the presence of rat hepatic cytosolic sulfotransferase activity responsible for generation of an electrophilic and mutagenic sulfuric acid ester from 7-HTBP, we determined whether 7,8-dihydrodiol and 7,8,9,10-tetrahydrotetraolmetabolites of BP which possess the same benzylic hydroxy functional group at the C-7 position as 7-HTBP could also be activated via benzylic sulfate esters. In line with this idea, the 3,4dihydrodiol derivative of cyclopenta[cdlpyrene as well as its mono benzylic hydroxy analogues has been shown to be further activated through sulfuric acid esterification (11,12,21).However, neither 7,8,9,10-tetrahydro-7,8,9,10-tetrahydroxy- nor 7,8-dihydro-7,8-dihydroxy-BPexerted sulfotransferase-dependentmutagenicity as determined by 8-azaguanine resistance in s. typhimurium TM677 (Table 2 ) or histidine independence in the strain S. typhimurium TA98 (Table 3). Furthermore, incubation of the aforementioned hydrocarbons with calf thymus DNA and hepatic cytosol fortified with PAPS gave rise to no appreciable increases in DNA adduct formation (data not presented). By analogy, BP-4,B-dihydrodiol(Kregion) did not exhibit any enhanced DNA binding in the presence of sulfotransferase activity. Although results of our present study are indicative of lack of sulfotransferase-dependentformation of reactive benzylic sulfuric acid ester metabolites from dihydrodiol and tetraol derivatives of BP, they do not necessarily preclude the possibility of sulfonation at the adjacent non-benzylic hydroxy groups (vide infra). CulY.-J. Surh and B. Day, unpublished observation.
Surh and Tannenbaum Table 2. Comparative Mutagenicity and Cytotoxicity of BP-7,8-dihydrodiolsand 6-HMBPin the Absence and Presence of Sulfotransferase Activity concn mutant cell survival of control) (uM) PAPS fraction ( x lo5) i% 5.6 100 50 8.3 81 6.0 88 100 6.2 77 6.9 65 trans-BP-7,8-diol 50 7.0 80 6.0 75 100 8.9 85 6.8 95 6-HMBP 100 6.6 81 40.1 91
comDound DMSO cis-BP-7,B-diol
+ + + + +
Hydrocarbons were preincubated with S. typhimurium TM677 and 21-day-old female rat liver cytosol with and without PAPS under the conditions described in the legend for Figure 2.
Table 3. Comparison of Sulfotransferase-Dependent Mutagenicities of 7-HTBP, BP-dihydrodiol, and BP-tetraol in S. typhimurium TA98" compound
concn mutagenicity @M) PAPS (no. of His+ revertantdplate)
BP-tetraol
100
cis-BP-7,B-diol
100
trans-BP-7,8-diol
100
7-HTBP
100
-
+ + + +
35 f 0.8 42 f 2.9 73 f 5.1 52 & 3.1 65 & 5.0 67 i 9.2 36 3 4.6 935 i 27
a Mutagenicity of each compound was determined in the absence or presence of PAPS (0.3 mM) as described in Materials and Methods. The average number of background revertants in the DMSO control was 24 (- PAPS) or 37 (+ PAPS).
tured human colon cells have been reported to sulfate trans-7,8-dihydro-7,8-dihydroxy-BP (291. BP dihydrodi01s were also shown to be converted to sulfate conjugates in cell-free extracts of rat liver enriched with the PAPSgenerating system although their sulfation was much less efficient than that of BP phenols (27,28). However, the structural identity of metabolically formed sulfo conjugates was not elucidated in these studies. Since the sulfotransferase assay adopted in the above investigation relied on the incorporation of the radioactivity derived from 35S-labeledNa2S04,the formation of unstable benzylic sulfate esters, if any, may not have been accurately assessed due to probable loss of the radioactivity (35S)as a consequence of leaving group departure during solvolysis. Other studies also support the notion that BP dihydrodiols are poor substrates for sulfotransferases compared to the phenolic derivatives (30,31).A similar conclusion was made for glucuronide conjugation of monohydroxy and dihydrodiol metabolites of BP (31-34). Taken together, our results suggest that dihydrodiol and tetraol metabolites of BP are unlikely to be activated via benzylic sulfuric acid ester metabolites. The extra hydroxy functional group(s) at the C-8 andlor the C-9 positions of these molecules might compete with adjacent benzylic hydroxy groups for the binding to the sulfate moiety donated from PAPS. Alternatively, the enhanced polarity in dihydrodiol and tetraol derivatives of BP may render these molecules less accessible to the active sites of sulfotransferases. In fact, the lipophilicity of the acceptor substrates has been reported to be a critical factor in specificity and affinity of aryl sulfotransferase N which catalyzes sulfonation of a wide array of benzylic alcohols as well as phenolic substances (35). In addition
Benzylic Sulfonation of Benzo[alpyrene Metabolites
to lipophilicity, the stereochemical configuration and spatial arrangement of chiral benzylic alcohols are thought to play an important role in determining the catalytic activity of aryl sulfotransferase N for these molecules (36, 37). Although BP dihydrodiol or tetraol could bind to the active site(s) of a given sulfotransferase, their extra secondary alcoholic group(s) might direct the orientation of the presumed enzyme-substrate complex in such a way that the transfer of ,903from PAPS is hampered. Indeed, a recent study by Rao and Duffel has shown that both enantiomers of trans-dihydrodiol-BPdo not serve as substrates for arylsulfotransferase IV,but rather act as reversible inhibitors of this enzyme, which is consistent with the above supposition (37, 38). Although dihydrodiol and tetraol derivatives of BP do not appear to undergo sulfotransferase-mediated activation, the formation of an electrophilic and mutagenic sulfuric acid ester metabolite from the monofunctional secondary alcohol, 7-HTBP, might be relevant to metabolic activation and tumorigenicity of PAHs with an alicyclic moiety, such as 1,2,3,4-tetrahydro-7,12-dimethylbenz[alanthracene and 3-methylcholanthracene. Thus, cytochrome P450-mediated hydroxylation of the cyclohexa or cyclopenta ring of these hydrocarbons at a benzylic carbon with subsequent sulfo conjugation would afford the corresponding electrophilic sulfuric acid esters, which might play a role as ultimate electrophilic and carcinogenic metabolites of the parent arenes. This possibility has been recently addressed elsewhere (1, 7, 39, 401, but remains to be proven. Acknowledgment. This research was supported by NIH Grants 2P01 ES07168 and ES02109 (to S.R.T.) and Swebilius Cancer Research Award (to Y.-J.S.). The authors thank Walter Bishop for conducting forward mutation assays.
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