Dihydrodiol Dehydrogenases and Polycyclic ... - ACS Publications

JANUARY 1999 VOLUME 12, NUMBER 1 © Copyright 1999 by the American Chemical Society

Invited Review Dihydrodiol Dehydrogenases and Polycyclic Aromatic Hydrocarbon Activation: Generation of Reactive and Redox Active o-Quinones Trevor M. Penning,* Michael E. Burczynski, Chien-Fu Hung, Kirsten D. McCoull, Nisha T. Palackal, and Laurie S. Tsuruda† Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084 Received June 16, 1998

Contents 1. IntroductionsMetabolic Pathways of PAH Activation 1.1. Activation to Diol Epoxides 1.2. Activation to Radical Cations 1.3. Activation to o-Quinones 2. Dihydrodiol DehydrogenasesMembers of the Aldo-Keto Reductase (AKR) Gene Superfamily 2.1. Dihydrodiol Dehydrogenases 2.2. cDNA Cloning and Nomenclature 2.3. Steroids and Prostaglandins as Natural Substrates 2.4. X-ray Crystal Structures 3. trans-Dihydrodiol Specificity and Generation of Reactive and Redox Active o-Quinones 3.1. trans-Dihydrodiol Specificity of Rat and Human Dihydrodiol Dehydrogenases (AKR1C Subfamily) 3.2. Modeling B[a]P-diol Bound to Rat Dihydrodiol Dehydrogenase 3.3. Product Characterization and Generation of Reactive and Redox Active o-Quinones 3.4. Mechanism of Catechol Autoxidation

1 2 2 2 4

4 4 4 4 5

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4. o-Quinone Formation as a Pathway of PAH Metabolism 4.1. Metabolism Studies 4.2. Tissue Distribution of Dihydrodiol Dehydrogenases 5. Properties of PAH o-Quinones 5.1. Conjugation Reactions 5.2. DNA Adduct Formation 5.3. Redox Cycling Capability 5.4. Cytotoxic Properties 5.5. Mutagenic Properties 5.6. Disposition Studies 5.7. Chemical Nuclease Activity 6. Regulation of the Dihydrodiol Dehydrogenase (AKR1C) Genes 6.1. Rat Dihydrodiol Dehydrogenase (AKR1C9) Gene 6.2. Human Dihydrodiol Dehydrogenase (AKR1C1-AKR1C4) Genes 7. Future Directions

8 8 9 9 9 9 10 10 11 12 12 13 13 14 14

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1. IntroductionsMetabolic Pathways of PAH Activation Polycyclic aromatic hydrocarbons (PAHs)1 are ubiquitous environmental pollutants and may be causative

10.1021/tx980143n CCC: $18.00 © 1999 American Chemical Society Published on Web 12/10/1998

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Chem. Res. Toxicol., Vol. 12, No. 1, 1999

agents in human lung cancer. They are considered procarcinogens because they require metabolic activation to electrophiles to exert their deleterious effects (1-3). Three principal pathways of metabolic activation have been proposed. These involve the formation of diol epoxides catalyzed by several cytochrome P450s, where P450 1A1 can play a dominant role (2, 4-7), the formation of radical cations catalyzed by P450 peroxidases (8, 9), and the formation of reactive and redox active o-quinones catalyzed by dihydrodiol dehydrogenases (DD) (10, 11), which are members of the aldo-keto reductase (AKR) gene superfamily (Scheme 1). The third pathway is the subject of this review where it is placed in context with the other pathways of PAH activation. 1.1. Activation to Diol Epoxides. A widely accepted pathway of PAH activation involves formation of electrophilic diol epoxides. For benzo[a]pyrene (B[a]P), a representative PAH, initial epoxidation occurs on the terminal benzo ring via P450s to form an arene oxide, which is then hydrolyzed by epoxide hydrolase to form a trans-dihydrodiol (proximate carcinogen), (-)-trans-7,8dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-diol) (1, 3, 12). Secondary epoxidation by P450 1A1 produces the diol epoxide (ultimate carcinogen), (()-anti-7β,8R-dihydroxy9R,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (antiBPDE), which then forms N2-deoxyguanosine (dGuo) adducts within DNA (Scheme 2) (6, 13-15). (()-antiBPDE is the most mutagenic of the known metabolites of B[a]P in bacterial and mammalian cell assays (1618), and it is the most tumorigenic in causing pulmonary adenomas in mice (19-21). Furthermore, anti-BPDE will activate the c-H-ras-1 proto-oncogene to transform NIH/ 3T3 cells (22). This gain in function results from a single point mutation in the 12th codon of ras in which there is a G to T transversion. Reactions of (()-anti-BPDE with the p53 tumor suppressor gene lead to adduct formation in codons 157, 248, and 273. These hot spots are mutated in lung cancer patients by G to T transversions and result in p53 inactivation (23, 24). Other PAHs which are known carcinogens, e.g., chrysene (C) (25, 26), 5-methyl-C (27, 28), benz[a]anthracene (BA), and 7,12-dimethylbenz[a]anthracene (7,12-DMBA) (29-32), are similarly activated to diol epoxides. Despite the compelling evidence that anti-diol epoxide-DNA adducts lead to the mutagenic events observed in human cancer, many observations suggest that diol epoxides are not the sole electrophilic metabolites * To whom correspondence should be addressed. Phone: (215) 8989445. Fax: (215) 573-2236. E-mail: [email protected]. † Present address: Miravant Pharmaceuticals, 7408 Hollister Ave., Santa Barbara, CA 93117. 1Abbreviations: AKR, aldo-keto reductase; BA, benz[a]anthracene; BAQ, benz[a]anthracene-3,4-dione; B[a]P, benzo[a]pyrene; B[a]P-diol, (()-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene; anti-BPDE, (()anti-7β,8R-dihydroxy-9R,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BPQ, benzo[a]pyrene-7,8-dione; C, chrysene; dAde, deoxyadenosine; dGuo, deoxyguanosine; DD, dihydrodiol dehydrogenase, trans1,2-dihydrobenzene-1,2-diol dehydrogenase (EC 1.3.1.20); 7,12-DMBA, 7,12-dimethylbenz[a]anthracene; 7,12-DMBAQ, 7,12-dimethylbenz[a]anthracene-3,4-dione; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; EIMS, electron impact mass spectrometry; ESI/MS/MS, electrospray ionization tandem mass spectrometry; G, guanine; 3R-HSD, 3R-hydroxysteroid dehydrogenase (EC 1.1.1.213, A-face specific); 5-methyl-C, 5-methylchrysene; MDA, malondialdehyde; Npdiol, (()-trans-1,2-dihydroxy-1,2-dihydronaphthalene; NPQ, naphthalene-1,2-dione; PAH, polycyclic aromatic hydrocarbon; 1,2-PQ, phenanthrene-1,2-dione; 9,10-PQ, phenanthrene-9,10-dione; RP-HPLC, reversed-phase highperformance liquid chromatography; RT-PCR, reverse transcriptase polymerase chain reaction; SOD, superoxide dismutase; SQ, o-semiquinone anion radical; O2•-, superoxide anion radical.

Penning et al. Scheme 1. Metabolic Activation of Polycyclic Aromatic Hydrocarbons

responsible for PAH carcinogenesis. First, the diol epoxides are weaker tumorigens that the parent hydrocarbon in the initiation of mouse skin papillomas (33-36). Second, diol epoxides do not provide a mechanism by which oxidatively damaged bases (e.g., 8-hydroxy-dGuo and thymine glycol) are formed in rodent tissues and human mammary epithelial cells exposed to B[a]P (37, 38). Third, mechanisms must exist by which stable diol epoxide-DNA adducts lead to the G to T transversions responsible for the activation of ras and the inactivation of p53, yet these mechanisms are less straightforward than those involving apurinic sites or 8′-hydroxy-dGuo. Fourth, the diol epoxide pathway does not provide an easy route by which PAH can act as a complete carcinogen, i.e., tumor initiators and promoters. 1.2. Activation to Radical Cations. A second pathway of PAH activation involves the formation of radical cations catalyzed by elevated levels of P450 peroxidase in 3-methylcholanthrene-induced rat liver microsomes. The radical cations arise from one-electron oxidation at the most electrophilic carbon, e.g., C6 on benzo[a]pyrene (8, 39). Radical cations form predominantly depurinating adducts with the N7 and C8 positions of guanine (G) and the N7 position of adenine (A) (9, 39). Once apurinic sites are formed, an A is always introduced opposite this site (40). During replication, T is paired with A, thus giving a straightforward route to the G to T transversions observed in ras and p53. Following the acute application of B[a]P or 7,12-DMBA to mouse skin, the depurinating adducts derived from radical cations exceed the number of stable adducts derived from diol epoxides (9, 41). Once papillomas are formed, the tumors are characterized by G to T transversions in H-ras, suggesting a causal relationship between the formation of depurinating adducts and ras activation (42). The radical cation pathway also explains why compounds such as tetrahydrodimethylbenz[a]anthracene, which cannot form diol epoxides, are also potent tumorigens (43-45). This activation pathway has its own associated difficulties. First, the highly reactive radical cations may be too short-lived to react with DNA in the nucleus. Second, the level of depurinating adducts formed by the potent carcinogen dibenzo[a,l]pyrene has been disputed (46). Third, similar to the diol epoxide pathway, radical cations provide no mechanism for the formation of 8′hydroxy-dGuo or thymine glycol observed following B[a]P treatment. Fourth, the radical cation pathway provides no mechanism by which PAH can act as initiators and promoters. 1.3. Activation to o-Quinones. This review article is focused on a third mechanism of PAH activation which

Invited Review

Chem. Res. Toxicol., Vol. 12, No. 1, 1999 3

Scheme 2. Cytochrome P450 (P450) and Dihydrodiol Dehydrogenase (AKR) Isoforms Compete for PAH trans-Dihydrodiols

involves the formation of reactive and redox active o-quinones by DD (10, 11). Evidence will be provided that rat and human DD successfully compete with P450s for trans-dihydrodiol proximate carcinogens (7, 47). These diols undergo NADP+-dependent oxidation to form a ketol which spontaneously rearranges to form a catechol. The catechol is unstable and undergoes autoxidation in air (Scheme 2) (10). The first one-electron oxidation results in the formation of an o-semiquinone anion radical (SQ) and hydrogen peroxide. The second one-electron oxidation produces the fully oxidized o-quinone and superoxide anion (O2•-) (11). We also provide evidence that the resultant o-quinones are highly reactive Michael acceptors which can form both stable and depurinating DNA adducts (48, 49). The latter adducts like those derived from the radical cations have the potential to give rise to the G to T transversions observed in ras and p53. Importantly, the o-quinone can undergo either a twoelectron nonenzymatic reduction to re-form the catechol or a one-electron enzymatic reduction to re-form the SQ (50, 51). These events establish futile redox cycles in which the formation of reactive oxygen species (ROS) [hydrogen peroxide, hydroxyl radical (OH•), and O2•-] are amplified. Theoretically, only one molecule of PAH transdihydrodiol needs to be diverted down this pathway, since the resulting catechol will redox cycle and generate ROS until cellular reducing equivalents are depleted. As a consequence, the level of DD expression and the amount of catechol formed are not important issues for this pathway to impact redox state. The link between PAH activation and ROS formation is significant. First, ROS are the causative agents in radiation-induced carcinogenesis (52). Second, ROS can cause the formation of

oxidatively damaged bases, e.g., 8-hydroxy-dGuo (5355), which if unrepaired mismatches with A on the replicative strand (56). This leads to G to T transversions and provides yet another potential route to ras and p53 mutations. Third, ROS can lead to elevated levels of lipid peroxidation, including the formation of malondialdehyde (MDA), a suspected mutagen and carcinogen (57, 58). Fourth, OH• can cause DNA strand scission and illegitimate recombination (59-61). Fifth, the pro-oxidant state produced could cause ROS to act as a mitogen, cause initiated cells to expand (62, 63), and activate protein kinase C (64) to enhance tumor promotion. Thus, this pathway of PAH activation provides a route by which PAH could act as complete carcinogens. It is similar to that currently proposed for the metabolic activation of endogenous estrogens (65-67) and exogenous estrogens (dehydroequilenin, equilenin, and hexestrol) (68-71) via catechols. Catechol estrogens will also autoxidize to the corresponding o-quinones and generate ROS. For example, estrone-3,4-quinone will cause single-strand DNA breaks in MCF-7 cells (72), an event that may contribute to estrogen carcinogenesis. We proposed more than five years ago that the o-quinones produced by DD were the oxidized PAH equivalents of the catechol estrogens (73) and like the catechol estrogens are important potential carcinogens. It is highly likely that diol epoxides, radical cations, and o-quinones all contribute to PAH-induced carcinogenesis. The relative importance of each pathway may depend on the tumor site (mouse skin or lung and Sprague-Dawley mammary gland), the level of expression of the activation enzyme(s), and whether the tumor site is exposed to the parent PAH or trans-dihydrodiol. No

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systematic study exists in which the relative contribution of each pathway to PAH activation has been examined. Such studies are hampered by the fact that P450s and their associated peroxidase activity can often be induced by PAH, while dihydrodiol dehydrogenases are both constitutively expressed and subject to induction (see Section 6.0).

Penning et al. Scheme 3. Cluster Analysis of the AKR1C Subfamily

2. Dihydrodiol DehydrogenasessMembers of the Aldo-Keto Reductase (AKR) Gene Superfamily 2.1. Dihydrodiol Dehydrogenases. DD activity was first detected in acetone powders of rabbit liver where it was found to oxidize benzenedihydrodiol to catechol (74). The activity drew attention because it provided an enzymatic route for aromatization. Subsequently, DD was purified from rat liver cytosol using benzenedihydrodiol as the substrate (75, 76). The activity was found to copurifiy with 3R-hydroxysteroid dehydrogenase (3RHSD), where it represents 1% of the soluble protein and has been designated 3R-HSD/DD. Because of the perceived importance of this enzyme in oxidizing PAH transdihydrodiols to innocuous catechols, DD was subsequently purified from a variety of sources, including murine (77), guinea pig (78), rabbit (79), monkey (80), and human liver (81-84). In these instances, the enzymes were either purified on the basis of their ability to oxidize benzenedihydrodiol or worse still purified on the basis of their ability to oxidize 1-acenaphthenol (a shared substrate) with little attention as to whether these enzymes could really oxidize PAH trans-dihydrodiols to catechols. In most instances, these enzymes had similar properties, were monomeric cytosolic proteins (34-37 kDa) that utilized NADP+, often had associated 3R, 17β, or 20R-HSD activities (77-79, 81, 83-86), and were sensitive to inhibition by nonsteroidal anti-inflammatory drugs (76). The relationship of these various enzymes became apparent once their cDNAs were cloned. 2.2. cDNA Cloning and Nomenclature. Using a rabbit anti-rat 3R-HSD/DD antiserum, a full-length cDNA clone was first obtained for rat liver 3R-HSD/DD (87). Expression and purification of the recombinant protein from Escherichia coli established that a single polypeptide catalyzed the oxidation of benzenedihydrodiol and 3R-hydroxysteroids (88). It was found that the cDNA had high sequence identity with aldose and aldehyde reductase, as well as prostaglandin F2R synthase (>58%), and this assigned the cDNA to the AKR gene superfamily (89). This superfamily now contains more than 57 members, and cluster analysis shows that subfamilies are divided on the basis of function. Thus, the AKR1 family contains the aldehyde reductase subfamily (AKR1A), the aldose reductase subfamily (AKR1B), and the dihydrodiol/hydroxysteroid dehydrogenase subfamily (AKR1C) (89). On the basis of this cluster analysis, rat liver 3R-HSD/DD is formally AKR1C9. cDNA cloning of the human DDs indicated that they were also AKRs; however, similarities in sequence made it difficult to assign these cDNAs to their respective proteins until recombinant human DDs were available for characterization. It is now clear that human DD1 [20R(3R)-HSD] is AKR1C1, human DD2 (bile acid binding protein) is AKR1C2, human DD4 (type 1 3R-HSD) is AKR1C4, and human DDx (type 2 3R-HSD) is AKR1C3 (86, 90, 91). A cluster analysis for the AKR1C subfamily is shown in Scheme 3.

2.3. Steroids and Prostaglandins as Natural Substrates. Clearly, DD did not evolve to oxidize PAH transdihydrodiols. Examination of kcat/Km values for members of the AKR1C subfamily demonstrates a preference for the oxidation of hydroxysteroids, bile acids, and hydroxyprostaglandins (76, 92, 93). They often act as bidirectional catalysts and interconvert carbonyls and secondary alcohols using NAD(P)H or NAD(P)+ as a cofactor. By producing alcohols from a variety of aldehydes and ketones, they increase the availability of metabolites for conjugation reactions. On the basis of the broad and overlapping substrate specificity of AKRs, and their ability to work on endogenous substrates as well as exogenous substrates (aromatic aldehydes, ketones, and quinones), it is likely that the AKR superfamily, like the P450 superfamily, plays a central role in drug and carcinogen metabolism. 2.4. X-ray Crystal Structures. As 3R-HSD/DD (AKR1C9) is highly expressed in rat liver cytosol, milligram quantities of this protein were easily purified (76). This led to its crystallization and the determination of an apoenzyme structure by molecular replacement using the coordinates for human placenta aldose reductase as a search molecule (94). The apoenzyme (E) is characterized by an alternating arrangement of R-helixes and β-strands that repeats itself eight times forming an (R/ β)8-barrel (Figure 1). At the back of the structure, there are three large loops (A-C) which define the substrate binding pocket and specificity. The (R/β)8-barrel with its associated loops has also been observed in the structures of aldose and aldehyde reductase (95, 96) and is now considered a structural motif for AKR superfamily members (97). Shortly thereafter, the structure of the AKR1C9 binary complex (E‚NADP+) was determined at 2.7 Å (98). The cofactor was found to lie perpendicular to the barrel axis with the nicotinamide ring lying across the lip of the barrel and the adenine ring straddling the periphery of the structure. Most recently, the structure of the AKR1C9 ternary complex (E‚NADP+‚testosterone) was determined at 2.4 Å (99). Interestingly, the steroid lies perpendicular to the cofactor, the position of its C3 ketone identified a catalytic tetrad (Tyr55, Lys84, His117, and Asp50) at the base of a hydrophobic cleft. The participation of this tetrad in oxidoreduction has been verified by site-directed mutagenesis (100). The mature steroid binding site is made up of 10 residues from five loops; the loops that undergo the most movement upon binding steroid are loop B and loop C, which is located at the C terminus. In the latter case, the C-terminal aromatic amino acid residues stack over the A ring of the steroid. The position of testosterone in the structure provides a reference point for docking PAH trans-dihydrodiols into the active site (see Section 3.2.).

Invited Review


Figure 1. Properties of the AKR superfamily members. The (R,β)8-barrrel is shown (top right), and the loop structures at the back of the barrel are shown (bottom right).

3. trans-Dihydrodiol Specificity and Generation of Reactive and Redox Active o-Quinones 3.1. trans-Dihydrodiol Specificity of Rat and Human Dihydrodiol Dehydrogenases (AKR1C Subfamily). A structural series of PAH trans-dihydrodiols were examined as substrates to determine whether rat and human DDs displayed the correct regio- and stereospecificity to oxidize the major trans-dihydrodiols formed in vivo and whether there was a preference for bay-region methylated and fjord-region PAH trans-dihydrodiols (Scheme 4) (91, 101-103). These studies were important since others have shown that the in vivo metabolism of PAH favors formation of the non-K region (-)-(R,R)-trans-dihydrodiols and that trans-dihydrodiols of bay-region methylated and fjord-region PAH are among the most potent proximate carcinogens. It was found that homogeneous rat liver DD efficiently oxidized racemic trans-dihydrodiols in the benzene, naphthalene, phenanthrene, C, and B[a]P series. In PAH with three or more rings, the enzyme displayed strict regioselectivity in that only trans-dihydrodiols on terminal benzo rings (non-K region) were oxidized. Thus, the trans-9,10-dihydroxy-9,10-dihydrophenanthrene and the trans4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene were not substrates (Table 1). In the lower ring series, CD spectroscopy on the unreacted stereoisomers showed that the enzyme displayed a strict stereochemical preference for the minor isomers formed in vivo; i.e., the (+)-S,S-stereoisomer was preferentially oxidized. However, in the 5-methylchrysene series, there was an inversion of stereochemical

Scheme 4. PAH trans-Dihydrodiols Examined as Substrates for DD Isoforms

c

g

preference so that the (-)-R,R-isomer of 7,8-dihydroxy7,8-dihydro-5-methylchrysene was oxidized. In the next ring series, the enzyme oxidized both the (+)-S,S- and

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Penning et al.

Table 1. PAH trans-Dihydrodiol Specificity of Rat and Human DD Isoformsa Vmax/Kmb PAH trans-dihydrodiol

rat DD

human DD1

human DD2

human DD4

human DDx

benzenedihydrodiol 1,2-dihydroxy-1,2-dihydronaphthalene 1,2-dihydroxy-1,2-dihydrophenanthrene 1,2-dihydroxy-1,2-dihydrochrysene 1,2-dihydroxy-1,2-dihydro-5-methylchrysene 7,8-dihydroxy-7,8-dihydro-5-methylchrysene 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene 3,4-dihydroxy-3,4-dihydro-7-methylbenz[a]anthracene 3,4-dihydroxy-3,4-dihydro-12-methylbenz[a]anthracene 3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz[a]anthracene 3,4-dihydroxy-3,4-dihydrobenzo[c]phenanthrene 11,12-dihydroxy-11,12-dihydrobenzo[g]chrysene

1735c 51 160 114 1058 784 338 456 884 463 1877 ND ND

56 1704 ND ND 322 180 634 168

96 2088 ND 210 ND 871 244 286 1808 1240 278

512 600 ND 148 ND 1466 100 820 1300 1600 130 2214

104 152 ND ND 330 20 225 307 580 415

a Data taken from refs 91 and 101-103. b V max/Km ) nanomoles per minute per milligram/[S], where v/[S] ) Vmax/Km when Km . [S]. All reactions were performed in 50 mM glycine (pH 9) with 2.3 mM NADP+. The highest catalytic efficiences for human DD isoforms are in italics. ND means not determined; - means there was no measurable rate.

c

the (-)-R,R-stereoisomers of B[a]P-diol (101). These studies were extended to the benz[a]anthracene (BA) series in which there is an increasing level of methylation. In this series, the 7- and 12-monomethyl- and the 7,12-dimethylbenz[a]anthracenes (7,12-DMBA) are more carcinogenic than the parent hydrocarbon, due in part to the presence of the bay-region methyl group. It was found that the 3,4-trans-dihydrodiol of 7,12-DMBA, which possesses both non-bay- and bay-region methyl groups, was oxidized 30 times faster than BA. Using CD spectroscopy, it was found that the enzyme consumed both the (+)-S,S- and (-)-R,R-isomers of BA, but displayed strict preference for the (+)-S,S-3,4-dihydrodiol of 7,12DMBA (102). In vivo, BA is metabolically activated to the (-)-R,R-isomer, while the diol of 7,12-DMBA is metabolically activated in almost equal amounts to the (+)-S,S- and (-)-R,R-isomers (104). Thus, rat DD displays the desired regio- and stereoselectivity for oxidizing the major trans-dihydrodiol metabolites formed in vivo from the important PAH carcinogens, 5-methylchrysene, B[a]P, BA, and 7,12-DMBA. Similar studies have been performed with the recombinant human DD isoforms (91, 103). These enzymes were obtained by isoform selective RT-PCR using HepG2 mRNA as a template. The PCR fragments were inserted into pET-16b vectors and the enzymes overexpressed and purified to homogeneity from E. coli. On the basis of the cDNA sequence and substrate specificity, the human recombinant isoforms were identical to DD1 (AKR1C1), DD2 (AKR1C2), DD4 (AKR1C4), and DDx (AKR1C3). It was found that all four isoforms oxidized both the (+)and (-)-isomers of [3H]B[a]P-diol using a RP-HPLCbased assay. The rank order of B[a]P-diol oxidation catalyzed by the isoforms was as follows: DD2 > DD1 > DD4 > DDx. Examination of a structural series similar to that described for the rat enzyme showed several important trends (Table 1). K-Region trans-dihydrodiols were not substrates for any human DD isoform. Proximate carcinogen non-K-region trans-dihydrodiols were the preferred substrates. The fjord-region diol, trans-11,12-dihydroxy-11,12-dihydrobenzo[g]chrysene, was a substrate for all four human DD isoforms. DD2 and DD4 had the highest catalytic efficiency for the potent carcinogenic PAH-trans-dihydrodiols (B[a]P, 5-MC, 7-MBA, and 7,12-DMBA), and extent of methylation was a major determinant of increased catalytic efficiency. Our studies support the concept that multiple-human DDs (AKRs)

play a role in the further metabolism of proximate carcinogen PAH trans-dihydrodiols. 3.2. Modeling B[a]P-diol Bound to Rat Dihydrodiol Dehydrogenases. To understand the structural basis of why rat DD oxidizes both the (+)-S,S- and (-)-R,Risomers of B[a]P-diol, computer-generated structures of these diols were superimposed on the crystal structure of androsterone (a 3R-hydroxysteroid substrate). These studies showed that the diaxial conformers of both B[a]Pdiol isomers overlaid the steroid but the diequatorial conformers did not. Further, the (-)-(R,R)-trans-dihydrodiol gave better superimposition since there was greater ring overlap with the steroid (101). NMR studies show that the trans-diequatorial conformers of dihydrodiols predominate in solution (105). Since 3R-HSD/DD displays an absolute stereochemical preference for the 3R-axial alcohol of steroids, it is assumed that preferential oxidation of the trans-diaxial conformers of dihydrodiols occurs at the active site and shifts the solution equilibrium between the two dihydrodiol conformers. With the availability of the E‚NADP+‚testosterone complex, the (+)-S,S- and (-)-R,R-isomers were modeled into the active site of the enzyme using testosterone as a template (Figure 2). In the resultant models, the C8′axial alcohol of B[a]P-diol was superimposed over the C3 ketone of testosterone which is oriented toward the catalytic tyrosine (Y55). It was found that both enantiomers of B[a]P-diol fit equally well into the active site coincident with the enzymatic data which showed that both isomers were oxidized. Importantly, the dihedral angle that defines the trajectory of hydride transfer from the C8 position of B[a]P-diol to the C4 position of the oxidized cofactor was found to be a good predictor of trans-dihydrodiol stereoselectivity. Only those transdihydrodiol isomers giving dihedral angles in the range of -90 to -160° were found to be substrates.2 3.3. Product Characterization and Generation of Reactive and Redox Active o-Quinones. Initial atempts to isolate catechols as the products of transdihydrodiol oxidation catalyzed by the rat and human DDs failed due to the rapid appearance of multiple polar species that were later found to be buffer conjugates. This suggested that a reactive species was the initial enzyme product. Starting with either trans-1,2-dihydroxy-1,2dihydronaphthalene (NpDiol) or B[a]P-diol, the reactive 2J.

M. Jez and T. M. Penning, unpublished observations.

Invited Review


Figure 2. B[a]P-diol stereoisomers docked into the active site of 3R-HSD/DD (AKR1C9). Computer-generated structures of the (+)-S,S- and (-)-R,R-isomers of B[a]P-diol (pink and red) were docked into the ternary complex structure (E‚NADP+‚testosterone) by substituting the diols for testosterone. In each case, the C8′ position of the diol was placed in the position of the C3 ketone of testosterone and the diol positioned to achieve maximal ring overlap with the steroid. (+)-S,S-B[a]P-diol (top left) and (-)-R,RB[a]P-diol (top right) are shown docked into the active site to reveal the stereochemistry of hydride transfer from the C8′ position of the diol to the C4 position of the nicotinamide ring; the position of the catalytic tyrosine (Y55) is also indicated. (+)-S,S-B[a]P-diol (bottom left) and (-)-R,R-B[a]P-diol (bottom right) are shown docked into the active site to indicate how both isomers fit into the binding pocket.

products were trapped as thiol-ether conjugates of the corresponding o-quinones [naphthalene-1,2-dione (NPQ) or benzo[a]pyrene-7,8-dione (BPQ)] (10, 91). The structures of the thiol-ether conjugates were confirmed by establishing identity with authentic synthetic standards using high-field proton NMR and EIMS (10). The identification of the o-quinone products suggested that enzymatic oxidation of the trans-dihydrodiol initially produced a ketol which tautomerizes to the catechol and that subsequent air oxidation generated the fully oxidized o-quinone (Scheme 5). 3.4. Mechanism of Catechol Autoxidation. The process of catechol autoxidation was anticipated to yield ROS. This mechanism of autoxidation was further stud-

ied using both Npdiol and B[a]P-diol as substrates for rat DD (11). Addition of enzyme and NADP+ showed that the oxidation of these trans-dihydrodiols was accompanied by the consumption of molecular oxygen and the production of hydrogen peroxide. With both trans-dihydrodiols, oxygen consumption was stoichiometric with respect to hydrogen peroxide production, consistent with the reaction in eq 1.

QH2 + O2 ) H2O2 + Q

(1)

where QH2 is the catechol and Q is the quinone. Using either trans-dihydrodiol substrate, a burst of O2•- production that was catalyzed by DD was observed

8


Scheme 5. Mechanism of Diol Transformation into o-Quinones

which could be detected as the rate of cytochrome c reduction that was inhibited by superoxide dismutase (SOD). Using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent, secondary spin adducts corresponding to DMPO-CH3 were formed during the enzymatic oxidation of trans-dihydrodiols. The formation of the CH3• radical arises from the OH• attack of DMSO which was used as the cosolvent. These spin adducts were attenuated by SOD and catalase, implying that O2•- and hydrogen peroxide were obligatory precursors in the formation of OH• and the generation of DMPO-CH3 adducts. It was proposed that the OH• radical was formed by the Fenton reaction. Superoxide anion was found to be the initiating radical in the autoxidation event because hydrogen peroxide formation preceded oxygen consumption during the enzymatic oxidation of B[a]P-diol. In the proposed mechanism, O2•- acts as a base and removes a proton from the catechol to yield an anion and a hydroperoxy radical. The hydroperoxy radical then acts as an oxidant to produce SQ. Oxygen is consumed by SQ in the final step to produce the fully oxidized o-quinone and O2•-. The O2•- produced then propagates the next cycle of autoxidation (Scheme 6). The o-quinones produced by the DD reaction are redox active and in the presence of reducing cofactor can undergo nonenzymatic two-electron reduction back to the fully reduced catechol (50). The catechol in turn is autoxidized in air to the fully oxidized o-quinone. The establishment of these futile redox cycles provides a mechanism for the amplification of ROS at the expense of cellular reducing equivalents. The formation of ROS initiated by DD may have important consequences for tumor initiation and promotion.

4. o-Quinone Formation as a Pathway of PAH Metabolism 4.1. Metabolism Studies. Studies with the homogeneous enzymes substantiated the fact that B[a]P-diol was oxidized to BPQ by DD isoforms. However, it was unknown whether this reaction represented a bona fide pathway of metabolism in whole cells. Several approaches were taken to answer this question. First, it was found that an S9 fraction from an uninduced male SpragueDawley rat liver fortified with NADP+ converted (()[3H]B[a]P-diol to BPQ (7). The percentage of B[a]P-diol converted to BPQ was similar to the percentage of B[a]Pdiol converted to tetraols derived from the hydrolysis of (()-anti-BPDE. Second, B[a]P-diol metabolism was measured in suspensions of primary rat hepatocytes (47). It was found that BPQ was formed in the organic soluble metabolites of the cell media. The production of BPQ was verified by RP-HPLC by its cochromatography with a

Penning et al. Scheme 6. Mechanism of Catechol Autoxidation

synthetic standard and by its identity with the standard by diode-array spectrometry. The production of BPQ was further substantiated by EIMS which showed that the metabolite gave the same molecular ion and fragmentation pattern as the synthetic standard (47). In rat hepatocytes, similar amounts of BPQ and the tetraols from anti-BPDE were detected, suggesting that DD and P450s contribute equally to the further metabolism of PAH trans-dihydrodiols. In both the S9 fraction and rat hepatocytes, the formation of BPQ was attenuated by DD inhibitors, confirming that DD was responsible for its formation. These studies established that DD converts B[a]P-diol to BPQ in a whole cell context. In a third approach, the cDNA for rat 3R-HSD/DD was stably transfected into mammalian cells. By challenging the transfectants with B[a]P-diol, Tsuruda et al. (106) examined the formation of BPQ and the consequences of its formation. In this study, the cDNA for rat 3R-HSD/ DD was inserted into a eukaroytic expression vector under the control of a constitutive cytomegalovirus promoter (CMV) and stably transfected into PAHmetabolizing cells that were DD-deficient [human breast (MCF-7) carcinoma cells]. G418-selected MCF-7 cells were found to express DD at 1.0% of the level of soluble protein, similar to the levels found in rat liver cytosol. When these cells were exposed to B[a]P-diol, a significant amount of BPQ was formed over a background level of tetraols, whereas untransfected cells produced no BPQ.

Invited Review Scheme 7. PAH o-Quinones Studied for Their Chemical and Biological Properties

Chem. Res. Toxicol., Vol. 12, No. 1, 1999 9 Table 2. Second-Order Rate Constants for the Addition of Various Nucleophiles to PAH o-Quinonesa o-quinone NPQ NPQ NPQ

The identity of BPQ was confirmed by coelution with a synthetic standard on RP-HPLC and by diode-array spectrometry which showed that the unknown had the unique spectral characteristics of BPQ. The ability to detect unconjugated BPQ in the organic soluble metabolites of rat hepatocytes and transfected MCF-7 cells suggests that despite the fact that the o-quinones can be readily conjugated, these conjugation mechanisms are insufficient to remove the reactive BPQ. 4.2. Tissue Distribution of Dihydrodiol Dehydrogenases. The tissue distribution of DD isoforms will also determine whether they contribute to PAH activation. Northern analysis using a probe for the open-reading frame for rat liver DD (AKR1C9) identified tissue selective expression in the liver, small intestine, and lung of male rats and in the small intestine, lung, ovary, and uterus of female rats (107). This tissue specific expression correlates well with tissues (liver, lung, and small intestine) most actively involved in drug and xenobiotic metabolism. In humans, no definitive tissue distribution studies exist because the high sequence identity between the four DD isoforms precludes the use of Northern analysis. Nevertheless, DD4 appears to be liver specific, whereas DD2 is more widely expressed (90). Also, Northern analysis using either an open-reading frame probe for DD2 which recognizes all four DD transcripts or the 3′-untranslated region for DDx (type 2 3R-HSD) revealed transcripts in almost all tissues inspected with the highest levels of expression in liver, small intestine, colon, and lung (108). Thus, DD isoforms are distributed in rat and human tissues that are major sites of xenobiotic metabolism and are appropriately localized to activate PAH. Importantly, they are expressed in human lung, a suspected site of PAH-induced tumor formation.

5. Properties of PAH o-Quinones The chemical and biological properties of a structural series of PAH o-quinones derived from the DD reactions have been studied (Scheme 7). This structural series contains PAH o-quinones with increasing ring sizes and extents of methylation; none are commercially available, and all required synthesis. No studies have yet been conducted on the properties of fjord-region PAH oquinones. 5.1. Conjugation Reactions. The chemistry of quinone conjugation reactions has been extensively studied with NPQ and BPQ. Both PAH o-quinones readily form conjugates in phosphate and glycine buffers, and the second-order rate constants for these reactions are not

NPQ NPQ NPQ NPQ BPQ BPQ BAQ 7-methyl-BAQ 12-methyl-BAQ 7,12-DMBAQ a

nucleophile potassium phosphate (pH 7.0) glycine-NaOH (pH 9.0) 2-mercaptoethanol glutathione L-cysteine glutathione L-cysteine glutathione L-cysteine glutathione glutathione glutathione glutathione

rate constant (min-1 M-1)

conditions

0.003

aqueous

3.7

aqueous

1.1 × 104

aqueous

4.9 × 103 8.2 × 103 2.3 × 105 1.8 × 106 1.3 × 103 7.7 × 102 1.8 × 104 1.6 × 104 2.3 × 104 >2.0 × 106

aqueous aqueous phosphate buffer phosphate buffer phosphate buffer phosphate buffer phosphate buffer phosphate buffer phosphate buffer phosphate buffer

Data taken from ref 109.

insignificant (10, 109). The ability of these o-quinones to be scavenged by cellular electrophiles, e.g., L-cysteine and GSH, has also been measured, and the corresponding rate constants have been determined (Table 2). Although BPQ was less reactive than NPQ, benz[a]anthracene o-quinones which contain a bay region were found to be highly reactive. A concerted effort was also made to isolate L-cysteinyl, N-acetyl-L-cysteinyl, and glutathionyl conjugates of NPQ and BPQ and to characterize their structures using a combination of 1H, 13C, and twodimensional NMR (109, 110). In every instance, the major adduct was found to be derived by 1,4-Michael addition and was isolated at the level of the fully oxidized o-quinone. Michael addition would be expected to form a ketol, which would rearrange to the catechol conjugate. The detection of adducts that are fully oxidized to o-quinones implies that autoxidation of the catechol adduct occurs which in turn generates ROS. In fact, during the conjugation reactions, immediate and rapid consumption of molecular oxygen has been observed.3 The ability of these o-quinone conjugates to redox cycle has not yet been assessed. Monks has shown that GSH conjugates of benzoquinone are not simply water soluble conjugates but instead redox cycle to produce renal toxicity (111). Thus, the formation of PAH o-quinone conjugates may not be an innocuous event. Using NPQ as a model, the conjugates obtained were not always simple Michael addition products. For example, studies on the addition of L-cysteine to NPQ showed that both N- and S-attack were possible and that following N-attack, rearrangement to a p-iminoquinone occurred (Scheme 8) (109). This complex chemistry coupled with the ability of several intermediates to redox cycle provides a significant challenge to the isolation of PAH o-quinone conjugates and assessment of their biological properties. 5.2. DNA Adduct Formation. PAH o-quinones have the potential to produce a spectrum of DNA adducts (Figure 3). First, they could form stable bulky adducts via 1,4-Michael addition of the N2-amino group of dGuo or the N6-amino group of dAde. Second, they could form depurinating adducts via 1,4-Michael addition of the N7 of guanine or the N7 of adenine with subsequent hydrolysis of the glycosidic bond. Because it is known that 3T.

M. Penning, unpublished observations.

10 Chem. Res. Toxicol., Vol. 12, No. 1, 1999 Scheme 8. Formation of p-Iminoquinone Conjugates with NPQ and L-Cysteine

SQ radicals are also sources of single electrons, depurinating adducts with the C8 of guanine and the C8 and N3 of adenine are also possible. Third, by entering redox cycles, o-quinones can generate ROS which can result in either oxidatively damaged bases, e.g., 8-hydroxy-dGuo, or DNA strand scission. Evidence exists for many of these reactions. When [3H]BPQ was incubated with either calf thymus DNA or plasmid DNA, and the resultant DNA was degraded to its constituent deoxyribonucleosides, a single predominant stable adduct was observed. It was found that this adduct coeluted with the deoxyribonucleoside adduct obtained from the reaction of BPQ with oligodGuo (48). Quantitation of the level of adduct formation was performed using [3H]-(()-anti-BPDE as a positive control and [3H]-(()-B[a]P-diol as a negative control. It was found that the incidence of dGuo adducts oberved with [3H]BPQ and anti-BPDE were similar (two to four adducts per 103 nucleotides), whereas none were observed with B[a]P-diol. Although the structure of the stable BPQ-dGuo adduct has remained elusive, it is likely to result from 1,4-Michael addition of the N2-amino group of dGuo. The PAH o-quinones generated by DD resemble the estrogen o-quinones. Catechol estrogens can be chemically oxidized in situ to form estrogen o-quinones (112), and when they are mixed with deoxyribonucleosides or DNA, estrogen o-quinone-depurinating adducts are observed (67). This observation led us to examine whether PAH o-quinones could also form depurinating adducts with DNA. NPQ, 1,2-PQ, and BPQ were reacted with dGuo under acidic conditions. The order of reactivity was as follows: NPQ > 1,2-PQ > BPQ. In each instance, adduct formation was observed by RP-HPLC. Structures of the depurinating adducts were obtained by a combination of high-field 1H NMR and electrospray ionization tandem mass spectrometry (ESI/MS/MS). NMR of the 7-(naphthalene-1,2-dion-4-yl)guanine adduct showed the absence of the deoxyribose ring, the loss of the C4 proton of NPQ, and the retention of the C8 proton of guanine consistent with a N7 adduct. It is assumed that 1,2-PQ and BPQ also form N7 adducts. All adducts were further characterized by ESI/MS/MS, and in each case, the protonated molecular ions detected corresponded to the o-quinone-guanine adduct: 7-(naphthalene-1,2-dion-4yl)guanine (MH+, m/z 308), 7-(phenanthrene-1,2-dion-4yl)guanine (MH+, m/z 358), and 7-(benzo[a]pyrene-7,8-

Penning et al.

dion-10-yl)guanine (MH+, m/z 432) (Scheme 9). The fragment ions observed upon collision-induced dissociation of each MH+ ion supported the assigned structures (49). PAH catechol-depurinating adducts were not observed, suggesting that like the thiol-ether conjugates these adducts autoxidized and generated ROS. These PAH o-quinone-depurinating adducts will be invaluable as synthetic standards for their detection in cell culture, stable transfectants, and sites of PAH-induced tumor formation. 5.3. Redox Cycling Capability. All PAH o-quinones in Scheme 7 undergo nonenzymatic redox cycling with the concomitant production of O2•- radicals. In some instances, the reactions are extensive; e.g., 35% of NPQ was redox-cycled within the first minute of the reaction. Significantly, only NPQ was found to be a substrate for DT-diaphorase and undergo enzymatic two-electron reduction (50). It has been proposed that quinones that are substrates for DT-diaphorase have a greater propensity to be detoxified because they can be conjugated at the level of the hydroquinone (113, 114). This is not the case for the majority of the PAH o-quinones produced by DD which prefer to undergo enzymatic one-electron reduction. Thus, microsomal NADPH-cytochrome P450 reductase catalyzed the one-electron reduction of all the PAH o-quinones studied, with NPQ, 9,10-PQ, and 7,12DMBAQ yielding the largest rate of O2•- production. Microsomal NADH-cytochrome b reductase also catalyzed the one-electron reduction of all the PAH oquinones studied, but only 9,10-PQ produced a significant amount of O2•-. In contrast, mitochondrial NADH: ubiquinone oxidoreductase catalyzed the one-electron reduction of all the PAH o-quinones, but the largest rates of O2•- production were now observed with BPQ and 7,12DMBAQ. In every case, there was a significant uncoupling of the one-electron reduction of PAH o-quinones from the production of O2•-. This implies either that stable SQ radicals are produced or that SQ radicals use terminal electron acceptors other than molecular oxygen (50). 5.4. Cytotoxic Properties. PAH o-quinones were examined for their cytotoxicity in rat (H4IIe) and human (HepG2) hepatoma cells (51, 115). These quinones were found to be potent cytotoxins yielding IC50 values for cell survival in the range of 1-30 µM (51). On the basis of their cytotoxicity profiles, PAH o-quinones could be grouped into three classes. Group I contained o-quinones (NPQ and 7,12-DMBAQ) that reduced cell viability and survival. Group II contained o-quinones [benz[a]anthracene-3,4-dione (BAQ) and 5-methyl-C-1,2-dione] which reduced cell survival only, and Group III contained quinones (BPQ) which reduced cell viability only. In cell free extracts, Group I o-quinones produced the most total free radicals (i.e., O2•- and SQ), caused a change in cellular redox state [measured as a decrease in the amount of GSH-GSSG and an increase in the amount of NAD(P)+- NAD(P)H], and mediated cell death in a manner reminiscent of menadione. Menadione toxicity results from an increase in the amount of intracellular Ca2+ and membrane blebbing resulting in necrotic cell death (116-118). PAH o-quinones are thought to mediate these events by the changes shown in Scheme 10, which lead to a change in redox state in the mitochondria, and activation of a Ca2+-dependent ATPase. By contrast, Group II o-quinones produced the most SQ in cell free extracts without the concomitant production of O2•-. In

Invited Review


Figure 3. Spectrum of DNA adducts anticipated with PAH o-quinones. Stable and depurinating adducts are shown on the left, and DNA modifications resulting from the generation of reactive oxygen species by the redox active o-quinones are shown on the right.

Scheme 9. Structures of PAH o-Quinone Depurinating DNA Adducts

a

this instance, changes in the degree of cell survival are thought to be mediated by SQ-mediated macromolecule damage. Group III o-quinones did not produce a measurable change in the redox state but depleted GSH, which may result in cell death (51). That BPQ is cytotoxic is borne out by the stable transfection studies performed with MCF-7 cells. When transfectants containing the rat DD cDNA were exposed to B[a]P-diol, there was a concentration-dependent cell death, measured as the release of cellular lactate dehydrogenase, which could be attenuated by DD inhibitors (106). 5.5. Mutagenic Properties. PAH o-quinones were examined as direct-acting mutagens in Salmonella ty-

Scheme 10. Changes in Redox State Mediated by PAH o-Quinones

phimurium tester strains TA97a, TA98, TA100, TA102, and TA104. They were found to have a greater mutagenic efficiency than the test mutagens for each strain and were found to be predominantly frame-shift mutagens (51) (Table 3). However, BPQ was 10-5500 times less mutagenic than (()-anti-BPDE depending on the tester strain used (Table 3). The presence of an activating system (Aroclor-induced rat liver S9 with a NADPHgenerating system) did not increase the mutagenicity of o-quinones in tester strains that were sensitive to oxidative mutagens (TA102 and TA104). Under these conditions, the mutagenicity of PAH o-quinones appears to be independent of ROS formation and is related to the ability of the o-quinones to intercalate and/or covalently modify DNA (51). No mammalian mutagenicity assays have yet been performed with the PAH o-quinones.

12 Chem. Res. Toxicol., Vol. 12, No. 1, 1999

Penning et al.

Table 3. Comparison of the Mutagenic Efficiencies of BPQ vs (()-anti-BPDE in the Ames Testa tester strain

compound

concn (nmol/plate)

no. of revertantsb per plate

n-fold increase

mutagenic efficiencyc

mutation type

TA97a TA98 TA100 TA102 TA104 TA97a TA98 TA100 TA102 TA104

BPQ BPQ BPQ BPQ BPQ (()-anti-BPDE (()-anti-BPDE (()-anti-BPDE (()-anti-BPDE (()-anti-BPDE

70 70 70 35 35 0.1 0.1 0.1 16.5 3.3

463 ( 57 52 ( 5 192 ( 6 368 ( 12 553 ( 15 444 ( 27 426 ( 33 845 ( 48 2200 ( 120 1620 ( 85

4.1 1.8 1.6 1.5 1.6 4.0 14.7 7.0 9.1 4.6

5.9 2.6 2.3 4.3 4.6 4000 14700 7000 55 140

frame-shift frame-shift point oxidative oxidative frame-shift frame-shift point oxidative oxidative

a Taken from ref 119. b Mean ( SE (n ) 3). c [(Number of revertants divided by the number of spontaneous revertants)/nanomoles of mutagen] × 100.

5.6. Disposition Studies. [3H]BPQ was incubated with isolated rat hepatocytes to determine its disposition in whole cells (119). It was found that a significant portion of the radioactivity (30%) was sequestered into the cell pellet (RNA, DNA, and protein). DNA was isolated from the hepatocytes, and the radioactivity bound to the DNA was taken as a measure of covalent adduct formation. The number of stable adducts (30 ( 17 per 106 bp) was far fewer than the number observed with B[a]P-diol (204 ( 30 per 106 bp) (positive control). These data suggest that the stable adducts attributed to B[a]P-diol were unlikely to have arisen from BPQ. Digestion of the DNA to its constituent deoxyribonucleosides and subsequent RP-HPLC analysis indicated that the adducts derived from B[a]P-diol comigrated with those formed between anti-BPDE and dGuo. By contrast, the adducts observed with BPQ did not survive the digestion procedure and may correspond to depurinating adducts (119). With the recent availability of synthetic standards for PAH o-quinone-depurinating adducts, these studies need to be repeated. When isolated hepatocytes were incubated with BPQ, extensive fragmentation of the genomic DNA was observed. DNA from untreated cells had a large molecular size (>23 kb), whereas DNA obtained from hepatocytes treated with 20 µM BPQ for 30 min had undergone extensive fragmentation, yielding a species with an average size of 0.5 kb. Concurrently, BPQ caused a robust production of O2•- in isolated hepatocytes. In contrast, when hepatocytes were treated with 20 µM B[a]P-diol, about one-third of the genomic DNA was fragmented and the amount of O2•- produced was one-third of that observed with BPQ. Both the B[a]P-diol-mediated strand scission and O2•- production were attenuated by DD inhibitors, suggesting that ROS generated by the DD pathway were responsible for DNA fragmentation. The ability to detect ROS formation in hepatocytes treated with B[a]P-diol suggests that the extensive protective cellular mechanisms that exist to remove reactive oxygen (catalase, SOD, and glutathione peroxidase) are overwhelmed by the DD pathway. To investigate the mechanism of DNA strand scission, model studies were performed with ΦX174DNA. BPQ caused a concentration-dependent (0.05-10 µM) strand scission in the presence of 1 mM NADPH (which promoted redox cycling) provided that CuCl2 was present. Complete destruction of the ΦX174DNA was observed using 10 µM BPQ. Strand scission was inhibited by catalase and OH• radical scavengers. These data are consistent with ROS being responsible for the destruction of the DNA. Using (()-anti-BPDE as a control, only single

nicks in the DNA were observed at much higher concentrations. It was estimated that BPQ was 200 times more potent as a chemical nuclease than (()-anti-BPDE (119). 5.7. Chemical Nuclease Activity. The nuclease activity of PAH o-quinones was studied further with NPQ, DMBAQ, and BPQ using ΦX174DNA and poly(dG)‚poly(dC) as the target DNA. Complete strand scission of ΦX174DNA was observed with either 1 µM NPQ or 1 µM 7,12-DMBAQ and with 10 µM BPQ provided 1 mM NADPH and 10 µM CuCl2 were both present. No strand scission was observed if o-quinone, NADPH, or CuCl2 was excluded. The 10-fold difference in the potency of select o-quinones to act as nucleases was assigned to the properties of their respective o-semiquinone radicals. The o-seminaphthalene-1,2-dione radical yields an EPR signal with a greater amplitude which decays faster than that of the o-semibenzo[a]pyrene-7,8-dione radical, and therefore may propagate O2•- more readily. The inclusion of 10 µM CuCl2 is physiologically significant since this transition metal is found chelated to guanine bases in DNA (120, 121). Reactive species detected which could contribute to DNA strand scission included O2•-, H2O2, OH•, SQ radicals (measured by EPR), and Cu(I) (measured as a bathocuproine complex). No strand scission was observed in the absence of Cu(II), and significant attenuation was seen with bathocuproine, indicating a role for a Cu(II)/Cu(I) redox cycle consistent with the reactions in eqs 2 and 3.

H2O2 + Cu2+ ) Cu+ + O2•- + 2H+

(2)

H2O2 + Cu+ ) Cu2+ + OH• + OH-

(3)

These equations would predict that Cu(I) should cause strand scission because hydrogen peroxide is produced in the redox cycling of o-quinones. Since Cu(I) is without effect, Cu(II) must play a more critical role. It is proposed that Cu(II) redox cycles PAH catechols, accelerating the production of SQ radicals and OH• (Scheme 11). This role for Cu(II) has been described for strand scission observed with 1,4-hydroquinone and the catechol estrogens (122, 123). Hydroxyl radical scavengers (e.g., formate and mannitol) attenuated strand scission, consistent with this event being mediated by OH•. When PAH o-quinones were incubated with NADPH under strict anaerobic conditions, the EPR signals assigned to SQ were quenched by the addition of ΦX174DNA and poly(dG)‚poly(dC) without concomitant strand scission. Thus, SQ radicals do no act as nucleases, and the species responsible are the OH• they generate. When calf thymus DNA was incubated with PAH o-quinones in the presence of

Invited Review

Chem. Res. Toxicol., Vol. 12, No. 1, 1999 13 Scheme 11. Cu2+-Dependent PAH o-Quinone-Mediated DNA Strand Scission

Scheme 12. Creigee Rearrangement and DNA Strand Scission

NADPH and CuCl2, MDA was released by acid hydrolysis. The formation of MDA was attenuated by OH• radical scavengers. Since MDA arises from the hydrolysis of base propenals, this points to the mechanism of DNA strand scission by OH•. In this mechanism, OH• produced during the redox cycling of PAH o-quinones abstracts a hydrogen atom from the C4 position of 2-deoxyribose, a Criegee rearrangement occurs resulting in phosphodiester bond cleavage, and base propenals are released (Scheme 12) (56, 124). In summary, this section documents the fact that PAH o-quinones readily undergo Michael addition reactions with cellular nucleophiles (L-cysteine and GSH) and

cellular macromolecules (bases within DNA). The formation of these conjugates results in the further generation of ROS. With DNA, both stable and depurinating adducts may form, providing several routes to mutations. Although the PAH o-quinones are considerably less mutagenic than the anti-diol epoxides, they are potent chemical nucleases. Thus, diversion of PAH trans-dihydrodiols down the DD pathway reduces the level of production of mutagenic diol epoxides but generates quinones that promote DNA strand scission. The extent of strand scission observed with PAH o-quinones in all likelihood represents a toxicological end point that may not be reached in vivo, suggesting that less dramatic


oxidative damage may occur in situ, e.g., formation of oxidatively damaged bases such as 8′-hydroxy-dGuo. The oxidative DNA insult that quinones provide may contribute to their potential carcinogenicity.

6. Regulation of the Dihydrodiol Dehydrogenase (AKR1C) Genes DD members of the AKR superfamily activate PAH to reactive and redox active o-quinones, yet little is known about the structure and regulation of the AKR1C genes. This is to be contrasted with our knowledge of the regulation of the P450 1A1 gene that forms anti-diol epoxides. 6.1. Rat Dihydrodiol Dehydrogenase (AKR1C9) Gene. The rat 3R-HSD/DD gene (AKR1C9) consists of nine exons and eight introns spanning more than 30 kb. Analysis of the 5′-flanking region of the gene (-6 kb) showed that it contained a weak basal promoter, a weak proximal enhancer, a silencer, and a powerful distal enhancer (-4.6 kb) (125, 126). The silencer or negative response element binds Oct transcription factors, and we have proposed that Oct factors may be repressors of the gene (125). Interestingly, Oct factors are also repressors of the rat P450 1A1 gene (127, 128), suggesting that common transcription factors may downregulate genes involved in PAH activation (125, 129). Deletion mutagenesis on the AKR1C9 gene promoter identified a powerful distal enhancer which contains a novel triple repeat (1 unit ) GTGGAAAAAC) which is responsible for its high level of constitutive expression (126). The transcription factors that bind to this element have not been identified. It is unknown whether this element also governs the tissue selective expression of this gene. The AKR1C9 gene is also regulated by a steroid response unit which contains multiple steroid hormone response elements. In rat hepatocytes, the AKR1C9 gene is induced by dexamethasone via a mechanism which involves the binding of an occupied glucocorticoid receptor to a proximal GRE (129). Unlike P450 1A1, the AKR1C9 gene is not regulated by planar aromatics working through the aryl hydrocarbon receptor (130) at a xenobiotic response element (131, 132). Also it is not induced by monofunctional inducers (133-135) working at an antioxidant response element (136-139). 6.2. Human Dihydrodiol Dehydrogenase (AKR1C1-AKR1C4) Genes. The transcripts from the four human DD (AKR1C1-AKR1C4) genes are closely related in sequence, and therefore, Northern analysis cannot distinguish among them. The pooled DD transcripts are upregulated by planar aromatics or bifunctional inducers (3-methylcholanthrene, B[a]P, and β-naphthoflavone) as well as by monofunctional inducers (antioxidants, ethacrynic acid, and t-BHQ) and ROS in human hepatoma (HepG2) cells (140). The kinetics of DD induction by bifunctional inducers in these cells is delayed relative to P450 1A1 and is consistent with a requirement for their metabolism to an electrophilic species prior to induction. Furthermore, the human DD transcripts are not upregulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin, an unmetabolizable aryl-hydrocarbon receptor ligand. Therefore, regulation of these AKRs is likely mediated by an electrophilic or antioxidant response element rather than by a xenobiotic response element. Ribonuclease protection assays showed that of the four DD genes expressed in HepG2 cells, DD1

Penning et al.

(AKR1C1) was the form that was induced by monofunctional inducers and ROS. It will be recalled that DD1 has a high catalytic efficiency for the oxidation of B[a]Pdiol. These data suggest that a central issue will be to determine how two signals, an electrophilic signal and ROS, are sensed and transmitted to the nucleus to increase the degree of transcription of the AKR1C1 gene. By generating ROS, BPQ the product of the DD reaction, can also increase the degree of DD1 expression. Thus, our studies show that BPQ formation by DD1 results in both a chemical (redox cycling) and genetic (DD1 induction) amplification of ROS in PAH-exposed cells.

7. Future Directions Much work needs to be done to test the hypothesis that PAH o-quinones contribute to PAH carcinogenesis. The structural characterization of the stable and depurinating PAH o-quinones-DNA adducts is an important initiative since the availability of these synthetic standards will permit their detection by sensitive MS methods in sites of tumor formation. The ability to stably transfect the cDNAs for rat and human DDs into mammalian cells provides a means for determining the biological consequences of diverting B[a]P-diol to BPQ and the oxidative DNA modifications that result. NIH/3T3 cells stably transfected with the cDNAs for DDs could be challenged with B[a]P-diol to determine whether the formation of PAH o-quinones results in cell transformation. ras and p53 need to be incubated with PAH o-quinones to determine whether hot spots are targeted and whether change-in-function mutations result. PAH o-quinones also have the potential to act as tumor promoters; they need to be tested as ligands or activators of PKC. Concurrently, PAH o-quinones need to be evaluated as tumor initiators or promoters (or both) in whole animals.

Acknowledgment. These studies were supported by NIH Grants CA39504 and CA55711 awarded to T.M.P. We thank Dr. Kapila Ratnam and Mr. Haiching Ma for providing views of B[a]P-diol docked into the active site of rat liver 3R-HSD/DD (AKR1C9).

References (1) Gelboin, H. V. (1980) Benzo[a]pyrene metabolism, activation and carcinogenesis: Role and regulation of mixed function oxidases and related enzymes. Physiol. Rev. 60, 1107-1166. (2) Conney, A. H. (1982) Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons. G. H. A. Clowes Memorial Lecture. Cancer Res. 42, 4875-4917. (3) Harvey, R. G. (1991) in Polycyclic Aromatic Hydrocarbons: Chemistry and Carcinogenicity (Coombs, M., Ed.) Cambridge University Press, Cambridge, U.K. (4) Thakker, D. R., Yagi, H., Lu, A. Y. H., Levin, W., Conney, A. H., and Jerina, D. M. (1976) Metabolism of benzo[a]pyrene: Conversion of (()-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene to highly mutagenic 7,8-diol-9,10-epoxides. Proc. Natl. Acad. Sci. U.S.A. 73, 3381-3385. (5) Yang, S. K., McCourt, D. W., Roller, P. P., and Gelboin, H. V. (1976) Enzymatic conversion of benzo[a]pyrene leading predominantly to the diol-epoxide r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10tetrahydrobenzo[a]pyrene through a single enantiomer of r-7,t8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Proc. Natl. Acad. Sci. U.S.A. 73, 2594-2598. (6) Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Weinstein, I. B., Beland, F. A., Harvey, R. G., Kasai, H., Miura, I., and Nakanishi, K. (1976) Benzo[a]pyrene-nucleic acid derivative found in vivo: Structure of a benzo[a]pyrene-tetrahydrodiol epoxide-guanosine adduct. J. Am. Chem. Soc. 98, 5714-5715. (7) Shou, M., Harvey, R. G., and Penning, T. M. (1992) Contribution of dihydrodiol dehydrogenase to the metabolism of (()-trans-7,8-

Invited Review

(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

(24)

(25)

(26)

dihydroxy-7,8-dihydrobenzo[a]pyrene in fortified rat liver subcellular fractions. Carcinogenesis 13, 1575-1582. Cavalieri, E. L., and Rogan, E. G. (1995) Central role of radical cations in metabolic activation of polycyclic aromatic hydrocarbons. Xenobiotica 25, 677-688. Chen, L., Devanesan, P. D., Higginbotham, S., Ariese, F., Jankowiak, R., Small, G. J., Rogan, E. G., and Cavalieri, E. (1996) Expanded analysis of benzo[a]pyrene-DNA adducts formed in vitro and in mouse skin: their significance in tumor inititation. Chem. Res. Toxicol. 9, 897-903. Smithgall, T. E., Harvey, R. G., and Penning, T. M. (1988) Spectroscopic identification of ortho-quinones as the products of polycyclic aromatic trans-dihydrodiol oxidation catalyzed by dihydrodiol dehydrogenase. A potential route of proximate carcinogen metabolism. J. Biol. Chem. 263, 1814-1820. Penning, T. M., Ohnishi, S. T., Ohnishi, T., and Harvey, R. G. (1996) Generation of reactive oxygen species during the enzymatic oxidation of polycyclic aromatic hydrocarbon trans-dihydrodiols catalyzed by dihydrodiol dehydrogenase. Chem. Res. Toxicol. 9, 84-92. Yang, S. K., Roller, P. P., and Gelboin, H. V. (1977) Enzymatic mechanism of benzo[a]pyrene conversion to phenols and diols and an improved high-pressure liquid chromatographic separation of benzo[a]pyrene derivatives. Biochemistry 16, 3680-3687. Osborne, M. R., Beland, F. A., Harvey, R. G., and Brookes, P. (1976) The reaction of (()-7R,8β-dihydroxy-9β,10β-epoxy-7,8,9,10tetrahydrobenzo[a]pyrene with DNA. Int. J. Cancer 18, 362-368. Jennette, K. W., Jeffery, A. M., Blobstein, S. H., Beland, F. A., Harvey, R. G., and Weinstein, I. B. (1977) Nucleoside adducts from the in vitro reaction of benzo[a]pyrene-7,8-dihydrodiol-9,10oxide or benzo[a]pyrene-4,5-oxide with nucleic acids. Biochemistry 16, 932-938. Koreeda, M., Moore, P. D., Wislocki, P. G., Levin, W., Conney, A. H., Yagi, H., and Jerina, D. M. (1978) Binding of benzo[a]pyrene7,8-diol-9,10-epoxides to DNA, RNA and protein of mouse skin occurs with high stereoselectivity. Science 199, 778-781. Nagao, M., and Sugimura, T. (1978) Mutagenesis: microbial systems. In Polycyclic Hydrocarbons and Cancer (Ts’O, P. O. P., and Gelboin, H. V., Eds.) Vol. 1, pp 99-121, Academic Press, New York. Malaveille, C., Kuroki, T., Sims, P., Grover, P. L., and Bartsch, H. (1977) Mutagenicity of isomeric diol-epoxides of benzo[a]pyrene and benz[a]anthracene in S. typhimurium TA98 and TA100 and in V79 Chinese hamster cells. Mutat. Res. 44, 313-326. Newbold, R. F., and Brookes, P. (1976) Exceptional mutagenicity of a benzo[a]pyrene diol-epoxide in cultured mammalian cells. Nature 261, 52-54. Buening, M. K., Wilsocki, P. G., Levin, W., Yagi, H., Thakker, D. R., Akagi, H., Koreeda, M., Jerina, D. M., and Conney, A. H. (1978) Tumorigenicity of the optical enantiomers of the diastereomeric benzo[a]pyrene-7,8-diol-9,10-epoxides in newborn mice: Exceptional activity of (+)-7β,8R-dihydroxy-9R,10R-epoxy-7,8,9,10tetrahydrobenzo[a]pyrene. Proc. Natl. Acad. Sci. U.S.A. 75, 53585361. Kapitulnik, J., Wislocki, P. G., Levin, W., Yagi, H., Jerina, D. M., and Conney, A. H. (1978) Tumorigenicity studies with diolepoxides of benzo[a]pyrene which indicate that (()-trans-7β,8Rdihydroxy-9R,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene is an ultimate carcinogen in newborn mice. Cancer Res. 38, 354-358. Chang, R. L., Wood, A. W., Conney, A. H., Yagi, H., Sayer, J. M., Thakker, D. R., Jerina, D. M., and Levin, W. (1987) Role of diaxial versus diequatorial hydroxyl groups in the tumorigenic activity of benzo[a]pyrene bay-region diol-epoxide. Proc. Natl. Acad. Sci. U.S.A. 84, 8633-8636. Marshall, C. J., Vousden, K. H., and Phillips, D. H. (1984) Activation of c-Ha-ras-1 proto-oncogene by in vitro chemical modification with a chemical carcinogen, benzo[a]pyrene diolepoxide. Nature 310, 585-589. Puisieux, A., Lin, S., Groopman, J., and Ozturk, M. (1991) Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res. 51, 6185-6189. Denissenko, M. F., Pao, A., Tang, M.-S., and Pfieifer, G. P. (1996) Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in p53. Science 274, 430-432. Wood, A. W., Levin, W., Ryan, D., Thomas, P. E., Yagi, H., Mah, H. D., Thakker, D. R., Jerina, D. M., and Conney, A. H. (1977) High mutagenicity of metabolically activated chrysene-1,2-dihydrodiol. Evidence for bay region activation of chrysene. Biochem. Biophys. Res. Commun. 78, 847-854. Nordqvist, M., Thakker, D. R., Vyas, K. P., Yagi, H., Levin, W., Ryan, D. E., Thomas, P. E., Conney, A. H., and Jerina, D. M.


(27)

(28)

(29)

(30)

(31)

(32)

(33)

(34)

(35)

(36)

(37)

(38)

(39)

(40)

(41)

(42)

(43)

(44)

(45)

(46)

(1981) Metabolism of chrysene and phenanthrene to bay region diol-epoxides by rat liver enzymes. Mol. Pharmacol. 19, 168-178. Melikian, A. A., LaVoie, E. J., Hecht, S. S., and Hoffmann, D. (1982) Influence of a bay-region methyl group on formation of 5-methyl-chrysene dihydrodiol epoxide: DNA adducts in mouse skin. Cancer Res. 42, 1239-1242. Hecht, S. S., Radok, L., Amin, S., Huie, K., Melikian, A. A., Hoffmann, D., Pataki, J., and Harvey, R. G. (1985) Tumorigenicity of 5-methylchrysene dihydrodiols and dihydrodiol epoxides: DNA adducts in newborn mice and mouse skin. Cancer Res. 45, 14491452. Chouroulinkov, I., Gentil, A., Tierney, B., Grover, P., and Sims, P. (1977) The metabolic activation of 7-methylbenz[a]anthracene in mouse skin: High tumor initiating activity of the 3,4-dihydrodiol. Cancer Lett. 3, 247-253. Levin, W., Thakker, D. R., Wood, A. W., Chang, R. L., Lehr, R. E., Jerina, D. M., and Conney, A. H. (1978) Evidence that benz[a]anthracene-3,4-diol-1,2-epoxide is an ultimate carcinogen on mouse skin. Cancer Res. 38, 1705-1710. Slaga, T. J., Huberman, E., Selkirk, J. K., Harvey, R. G., and Bracken, W. M. (1978) Carcinogenicity and mutagenicity of benz[a]anthracene diols and diol epoxides. Cancer Res. 38, 1699-1704. Slaga, T. J., Gleason, G. L., DiGiovanni, J., Sukumaran, K. B., and Harvey, R. G. (1979) Potent tumor initiating activity of the 3,4-dihydrodiol of 7,12-dimethylbenz[a]anthracene in mouse skin. Cancer Res. 39, 1934-1936. Chouroulinkov, I., Gentil, A., Grover, P. L., and Sims, P. (1976) Tumor-initiating activities on mouse skin of dihydrodiols derived from benzo[a]pyrene. Br. J. Cancer 34, 523-532. Slaga, T. J., Bracken, W. M., Viaje, A., Levin, W., Yagi, H., Jerina, D. M., and Conney, A. H. (1977) Comparison of the tumor initiating activities of benzo[a]pyrene arene oxides and diolepoxides. Cancer Res. 37, 4130-4133. Slaga, T. J., Viaje, A., Berry, D. L., and Bracken, W. (1976) Skin tumor initiating ability of benzo[a]pyrene 4,5-, 7,8- and 7,8-diol9,10-epoxides and 7,8-diol. Cancer Lett. 2, 115-122. Levin, W., Wood, A. W., Chang, R. L., Slaga, T. J., Yagi, H., Jerina, D. M., and Conney, A. H. (1977) Marked differences in the tumor initiating activity of optically pure (+)- and (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene on mouse skin. Cancer Res. 37, 2721-2725. Kim, K. B., and Lee, B. M. (1997) Oxidative stress to DNA, protein, and antioxidant enzymes (superoxide dismutase and catalase) in rats treated with benzo[a]pyrene. Cancer Lett., 205212. Leadon, S. A., Stampfer, M. R., and Bartley, J. (1988) Production of oxidative DNA damage during the metabolic activation of benzo[a]pyrene in human mammary epithelial cells correlates with cell killing. Proc. Natl. Acad. Sci. U.S.A. 85, 4345-4368. Devanesan, P. D., RamaKrishna, N. V. S., Todorovic, R., Rogan, E. G., Cavalieri, E. L., Jeong, H., Jankowiak, R., and Small, G. J. (1992) Identification and quantitation of benzo[a]pyrene-DNA adducts formed by rat liver microsomes in vitro. Chem. Res. Toxicol. 5, 302-309. Sagher, D., and Strauss, B. (1983) Insertion of nucleotides opposite apurinic/apyrimidinic sites in deoxyribonucleic acid during in vitro synthesis. Uniqueness of adenine nucleotides. Biochemistry 22, 4518-4526. Devanesan, P. D., RamaKrishna, N. V. S., Padmavathi, N. S., Higginbotham, S., Rogan, E. G., Cavalieri, E. L., Marsch, G. A., Jankowiak, R., and Small, G. J. (1993) Identification and quantitation of 7,12-dimethylbenz[a]anthracene-DNA adducts formed in mouse skin. Chem. Res. Toxicol. 6, 364-371. Chakravarti, D., Pelling, J. C., Cavalieri, E. L., and Rogan, E. G. (1995) Relating aromatic hydrocarbon-induced DNA adducts and c-H-ras mutations in mouse skin papillomas: The role of apurinic sites. Proc. Natl. Acad. Sci. U.S.A. 92, 10422-10426. DiGiovanni, J., Diamond, L., Singer, J. M., Daniel, F. B., Witiak, D. T., and Slaga, T. J. (1982) Tumor initiating activity of 4-fluoro7,12-dimethylbenz[a]anthracene and 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene in female SENCAR mice. Carcinogenesis 3, 651-655. Cavalieri, E. L., Rogan, E. G., Higginbotham, S., Cremonesi, P., and Salmasi, S. (1990) Tumorigenicity of 7,12-dimethylbenz[a]anthracene, some of its fluorinated derivatives and 1,2,3,4tetrahydro-7,12-dimethylbenz[a]anthracene in mouse skin and rat mammary gland. Polycyclic Aromat. Compd. 1, 59-70. Nair, R. V., Walker, S. E., Sharma, P. K., Witiak, D. T., and DiGiovanni, J. (1992) Mouse skin tumor initiating activity of fluorination derivatives of 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene. Chem. Res. Toxicol. 5, 153-156. MelendezColon, V. J., Smith, C. A., Seidel, A., Luch, A., Platt, K. L., and Baird, W. M. (1997) Formation of stable adducts and


(47)

(48)

(49)

(50)

(51)

(52) (53)

(54)

(55) (56) (57)

(58)

(59)

(60)

(61) (62) (63) (64) (65) (66)

(67)

(68)

(69)

absence of depurinating DNA adducts in cells and DNA treated with the potent carcinogen dibenzo[a,l]pyrene or its diol-epoxides. Proc. Natl. Acad. Sci. U.S.A. 94, 13542-13547. Flowers-Geary, L., Harvey, R. G., and Penning, T. M. (1996) Identification of benzo[a]pyrene-7,8-dione as an authentic metabolite of (()-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in isolated rat hepatocytes. Carcinogenesis 16, 2707-2715. Shou, M., Harvey, R. G., and Penning, T. M. (1993) Reactivity of benzo[a]pyrene-7,8-dione with DNA. Evidence for the formation of deoxyguanosine adducts. Carcinogenesis 14, 475-482. McCoull, K. D., Rindgen, D., Blair, I. A., and Penning, T. M. (1998) Synthesis and characterization of depurinating adducts of polycyclic aromatic hydrocarbon o-quinones. Proc. Am. Assoc. Cancer Res. 39, 637. Flowers-Geary, L., Harvey, R., and Penning, T. M. (1992) Examination of polycyclic aromatic hydrocarbon o-quinones produced by dihydrodiol dehydrogenase as substrates for redoxcycling in rat liver. Biochem. (Life Sci.) 11, 49-58. Flowers-Geary, L., Bleczinski, W., Harvey, R. G., and Penning, T. M. (1996) Cytotoxicity and mutagenicity of polycyclic aromatic hydrocarbon o-quinones produced by dihydrodiol dehydrogenase. Chem.-Biol. Interact. 99, 55-72. Halliwell, B., and Gutteridge, J. M. C. (1989) Free radicals in biology and medicine, 2nd ed., Clarendon Press, Oxford, U.K. Kasai, H., and Nishimura, S. (1984) Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other reducing agents. Nucleic Acids Res. 12, 2137-2145. Kasai, H., Crain, P. F., Kuchino, Y., Nishimura, S., Ootsuyama, A., and Tanooka, H. (1986) Formation of 8-hydroxyguanine moiety in cellular DNA by agents producing oxygen radicals and evidence for its repair. Carcinogenesis 7, 1849-1851. Frenkel, K. (1992) Carcinogen-mediated oxidant formation and oxidative DNA damage. Pharmacol. Ther. 53, 127-166. Breen, A. P., and Murphy, J. P. (1995) Reactions of oxyl radicals with DNA. Free Radical Biol. Med. 18, 1033-1077. Chaudhary, A. K., Nokubo, M., Reddy, G. R., Yeola, S. N., Morrow, J. D., Blair, I. A., and Marnett, L. J. (1994) Detection of endogenous malondialdehyde-deoxyguanosine adducts in human liver. Science 265, 1580-1582. Benamira, M., Johnson, K., Chaudhary, A., Bruner, K., Tibbets, C., and Marnett, L. J. (1995) Induction of mutations by replication of malondialdehyde-modified M13 DNA in Escherichia coli: Determination of the extent of DNA-modification, genetic requirements for mutagenesis, and types of mutations induced. Carcinogenesis 16, 93-99. DeMarini, D. M., Brockman, H. E., deSerres, F. J., Evans, H. H., Stankowski, L. F., Jr., and Hsie, A. W. (1989) Specific locus mutations induced in eukaroytes (especially mammalian cells) by radiation and chemicals: a perspective. Mutat. Res. 220, 11-29. Emerit, I., Keck, M., Levy, A., Feingold, J., and Michelson, A. M. (1982) Activated oxygen species at the origin of chromosome breakage and sister-chromatid exchange. Mutat. Res. 103, 165172. Povrik, L. F., and Austin, M. J. F. (1991) Genotoxicity of bleomycin. Mutat. Res. 257, 127-143. Ames, B. N., and Gold, L. S. (1990) Too many rodent carcinogens: Mitogenesis increases mutagenesis. Science 227, 375-381. Cohen, S. M., and Ellwein, L. B. (1990) Cell proliferation in carcinogenesis. Science 249, 1007-1011. Cerutti, P. A. (1985) Prooxidant states and tumor promotion. Science 227, 375-381. Liehr, J. G. (1990) Genotoxic effects of estrogens. Mutat. Res. 238, 269-276. Dwivedy, I., Devanesan, P., Cremonesi, P., Rogan, E., and Cavalieri, E. (1992) Synthesis and characterization of estrogen2,3- and 3,4-quinones. Comparison of DNA adducts formed by the quinones versus horseradish peroxidase-activated catechol estrogens. Chem. Res. Toxicol. 5, 828-833. Stack, D. E., Byun, J., Gross, M. L., Rogan, E. G., and Cavalieri, E. L. (1996) Molecular characteristics of catechol estrogen quinones in reactions with deoxyribonucleosides. Chem. Res. Toxicol. 9, 851-859. Shen, L., Pisha, E., Huang, Z., Pezzuto, J. M., Krol, E., Alam, Z., van Breeman, R. B., and Bolton, J. (1997) Bioreductive activation of catechol estrogen-ortho-quinones: aromatization of the B-ring in 4-hydroxyequilenin markedly alters quinoid formation and reactivity. Carcinogenesis 18, 1093-1101. Shen, L., Qiu, S., van Breeman, R. B., Zhang, F., Chen, Y., and Bolton, J. L. (1997) Reaction of the premarin metabolite 4-hydroxyequilenin semiquinone radical with 2′-deoxyguanosine: Formation of unusual cyclic adducts. J. Am. Chem. Soc. 119, 1112611127.

Penning et al. (70) Shen, L., Qiu, S., Chen, Y., Zhang, F., van Breeman, R. B., Nikolic, D., and Bolton, J. L. (1998) Alkylation of 2′-deoxynucleosides and DNA by the Premarin metabolite 4-hydroxyequilenin semiquinone radical. Chem. Res. Toxicol. 11, 94-101. (71) Jan, S.-T., Devanesan, P. D., Stack, D., Ramamathan, R., Byun, J., Gross, M. L., Rogan, E. G., and Cavalieri, E. L. (1998) Metabolic activation and formation of DNA adducts of hexestrol, a synthetic nonsteroidal carcinogen estrogen. Chem. Res. Toxicol. 11, 412-419. (72) Nutter, L. M., Ngo, E., and Abul-Hajj, Y. J. (1991) Characterization of DNA damage induced by 3,4-estrone-o-quinone in human cells. J. Biol. Chem. 266, 16380-16386. (73) Penning, T. M. (1993) Dihydrodiol dehydrogenase and its role in polycyclic aromatic hydrocarbon metabolism. Chem.-Biol. Interact. 89, 1-34. (74) Ayengar, P. K., Hayaishi, O., Nakajima, M., and Tomida, I. (1959) Enzymic aromatization of 3,5-cyclohexadiene-1,2-diol. Biochim. Biophys. Acta 33, 111-119. (75) Vogel, K., Bentley, P., Platt, K.-L., and Oesch, F. (1980) Rat liver cytoplasmic dihydrodiol dehydrogenase. Purification to apparent homogeneity and properties. J. Biol. Chem. 255, 9621-9625. (76) Penning, T. M., Mukharji, I., Barrows, S., and Talalay, P. (1984) Purification and properties of a 3R-hydroxysteroid dehydrogenase of rat liver cytosol and its inhibition by anti-inflammatory drugs. Biochem. J. 222, 601-611. (77) Bolcsak, L. E., and Nerland, D. E. (1983) Purification of mouse liver benzene dihydrodiol dehydrogenase. J. Biol. Chem. 258, 7252-7255. (78) Hara, A., Hayashibara, M., Nakayama, T., Hasebe, K., Usui, S., and Sawada, H. (1985) Guinea-pig liver testosterone 17β-dehydrogenase (NADP+) and aldehyde reductase exhibit benzene dihydrodiol dehydrogenase activity. Biochem. J. 225, 177-181. (79) Klein, J., Thomas, H., Post, K., Worner, W., and Oesch, F. (1992) Dihydrodiol dehydrogenase activities of rabbit liver are associated with hydroxysteroid dehydrogenases and aldo-keto reductases. Eur. J. Biochem. 205, 1155-1162. (80) Nakagawa, M., Harada, T., Hara, A., Nakayama, T., and Sawada, H. (1989) Purification and properties of multiple forms of dihydrodiol dehydrogenase from monkey liver. Chem. Pharm. Bull. 37, 2852-2854. (81) Hara, A., Taniguchi, H., Nakayama, T., and Sawada, H. (1990) Purification and properties of multiple forms of dihydrodiol dehydrogenase from human liver. J. Biochem. (Tokyo) 108, 250254. (82) Takikawa, H., Stolz, A., Sugiyama, Y., Yoshida, H., Yamanaka, M., and Kaplowitz, N. (1990) Relationship between the newly identified bile acid binder and bile acid oxidoreductases in human liver. J. Biol. Chem. 265, 2132-2136. (83) Deyashiki, Y., Taniguchi, H., Amano, T., Nakayama, T., Hara, A., and Sawada, H. (1992) Structural and functional comparison of two human liver dihydrodiol dehydrogenases associated with 3R-hydroxysteroid dehydrogenase activity. Biochem. J. 282, 741746. (84) Deyashiki, Y., Tamada, Y., Miyabe, Y., Nakanishi, M., Matsuura, K., and Hara, A. (1995) Expression and kinetic properties of a recombinant 3R-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver. J. Biochem. 118, 285-290. (85) Deyashiki, Y., Ohshima, K., Nakanishi, M., Sato, K., Matsuura, K., and Hara, A. (1995) Molecular-cloning and characterization of mouse estradiol 17β-dehydrogenase (A-Specific), a member of the aldo-keto reductase family. J. Biol. Chem. 270, 10461-10467. (86) Hara, A., Matsurra, K., Tamada, Y., Sato, K., Miyabe, Y., Deyashiki, Y., and Ishida, T. (1996) Relationship of human liver dihydrodiol dehydrogenases to hepatic bile-acid-binding protein and an oxidoreductase of human colon cells. Biochem. J. 313, 373-376. (87) Pawlowski, J. E., Huizinga, M., and Penning, T. M. (1991) Cloning and sequencing of the cDNA for rat liver 3R-hydroxysteroid/ dihydrodiol dehydrogenase. J. Biol. Chem. 266, 8820-8825. (88) Pawlowski, J. E., and Penning, T. M. (1994) Overexpression and mutagenesis of the cDNA for rat liver 3R-hydroxysteroid/dihydrodiol dehydrogenase. Role of cysteines and tyrosines in catalysis. J. Biol. Chem. 269, 13502-13510. (89) Jez, J. M., Flynn, T. G., and Penning, T. M. (1997) A new nomenclature for the Aldo-Keto Reductase superfamily. Biochem. Pharmacol. 54, 639-647. (90) Khanna, M., Qin, K.-N., Wang, R. W., and Cheng, K.-C. (1995) Substrate specificity, gene structure and tissue specific distribution of multiple human 3R-hydroxysteroid dehydrogenases. J. Biol. Chem. 270, 20162-20168. (91) Burczynski, M. E., Harvey, R. G., and Penning, T. M. (1998) Expression and characterization of four recombinant human dihydrodiol dehydrogenase isoforms: Oxidation of trans-7,8-

Invited Review dihydroxy-7,8-dihydrobenzo[a]pyrene to the activated o-quinone metabolite benzo[a]pyrene-7,8-dione. Biochemistry 37, 67816790. (92) Stolz, A., Takikawa, H., Ookhtens, M., and Kaplowitz, N. (1989) The role of cytoplasmic proteins in hepatic bile acid transport. Annu. Rev. Physiol. 51, 161-176. (93) Ohara, H., Nakayama, T., Deyashiki, Y., Hara, A., Miyabe, Y., and Tsukada, F. (1994) Reduction of prostaglandin D2 to 9R,11βprostaglandin F2 by a human liver 3R-hydroxysteroid dihydrodiol dehydrogenase isozyme. Biochim. Biophys. Acta 1215, 59-65. (94) Hoog, S. S., Pawlowski, J. E., Alzari, P. M., Penning, T. M., and Lewis, M. (1994) Three-dimensional structure of rat liver 3Rhydroxysteroid/dihydrodiol dehydrogenase: A member of the aldoketo reductase superfamily. Proc. Natl. Acad. Sci. U.S.A. 91, 2517-2521. (95) Wilson, D. K., Bohren, K. M., Gabbay, K. H., and Quicoho, F. A. (1992) An unlikely sugar substrate site in the 1.65 Å structure of the human aldose reductase holoenzyme implicated in diabetic complications. Science 257, 81-84. (96) El-Kabbani, O., Judge, K., Ginell, S. L., Myles, D. A. A., DeLucas, L. J., and Flynn, T. G. (1995) Structure of procine aldehyde reductase holoenzyme. Nat. Struct. Biol. 2, 687-692. (97) Jez, J. M., Bennett, M. J., Schlegel, B. P., Lewis, M., and Penning, T. M. (1997) Comparitive anatomy of the aldo-keto reductase superfamily. Biochem. J. 326, 625-636. (98) Bennett, M. J., Schlegel, B. P., Jez, J. M., Penning, T. M., and Lewis, M. (1996) Structure of 3R-hydroxysteroid/dihydrodiol dehydrogenase complexed with NADP+. Biochemistry 35, 1070210711. (99) Bennett, M. J., Albert, R. H., Jez, J. M., Ma, H., Penning, T. M., and Lewis, M. (1997) Steroid recognition and regulation of hormone action: crystal structure of testosterone and NADP+ bound to 3R-hydroxysteroid dehydrogenase. Structure 5, 799812. (100) Schlegel, B. P., Jez, J. M., and Penning, T. M. (1998) Mutagenesis of 3R-hydroxysteroid dehydrogenase reveals a “push-pull” mechanism for proton transfer in aldo-keto reductases. Biochemistry 37, 3538-3548. (101) Smithgall, T. E., Harvey, R. G., and Penning, T. M. (1986) Regioand stereospecificity of homogeneous 3R-hydroxysteroid-dihydrodiol dehydrogenase for trans-dihydrodiol metabolites of polycyclic aromatic hydrocarbons. J. Biol. Chem. 261, 6184-6191. (102) Smithgall, T. E., Harvey, R. G., and Penning, T. M. (1988) Oxidation of the trans-3,4-dihydrodiol metabolites of the potent carcinogen 7,12-dimethylbenz[a]anthracene and other benz[a]anthracene derivatives by 3R-hydroxysteroid-dihydrodiol dehydrogenase: Effects of methyl substitution on velocity and stereochemical course of trans-dihydrodiol oxidation. Cancer Res. 48, 1227-1232. (103) Burczynski, M. E., Palackal, N. T., Harvey, R. G., and Penning, T. M. (1999) Polycyclic aromatic hydrocarbon trans-dihydrodiol specificity of four recombinant human dihydrodiol dehydrogenase isoforms. Polycyclic Aromat. Compd. (in press). (104) Yang, S. K., Mushtaq, M., and Chiu, P. L. (1985) Stereoselective metabolism and activation of polycyclic aromatic hydrocarbons. In Polycyclic Hydrocarbons and Carcinogenesis (Harvey, R. G., Ed.) ACS Monographs, Vol. 283, pp 19-34, Amercian Chemical Society, Washington, DC. (105) Zacharias, D. E., Glusker, J. P., Fu, P. P., and Harvey, R. G. (1979) Molecular structures of the dihydrodiols and diol epoxides of carcinogenic polycyclic aromatic hydrocarbons. X-ray crystallographic and NMR analysis. J. Am. Chem. Soc. 101, 4043-4051. (106) Tsuruda, L., Hou, Y.-T., and Penning, T. M. (1999) Stable transfection of dihydrodiol dehydrogenase in MCF7-breast carcinoma cells promotes polycyclic aromatic hydrocarbon o-quinone formation which leads to cell death. Polycyclic Aromat. Compd. (in press). (107) Hou, Y.-T., Xia, W., Pawlowski, J. E., and Penning, T. M. (1994) Rat dihydrodiol dehydrogenase: complexity of gene structure and tissue-specific and sexually dimorphic gene expression. Cancer Res. 54, 247-255. (108) Lin, H.-K., Jez, J. M., Schlegel, B. P., Peehl, D. M., Pachter, J. A., and Penning, T. M. (1997) Expression and characterization of recombinant type 2 3R-hydroxysteroid dehydrogenase (HSD) from human prostate: Demonstration of bifunctional 3R/17βHSD activity and cellular distribution. Mol. Endocrinol. 11, 1971-1984. (109) Murty, V. S., and Penning, T. M. (1992) Polycyclic aromatic hydrocarbon (PAH) ortho-quinone conjugate chemistry: kinetics of thiol addition to PAH ortho-quinones and structures of thiolether adducts of naphthalene-1,2-dione. Chem.-Biol. Interact. 84, 169-188.

Chem. Res. Toxicol., Vol. 12, No. 1, 1999 17 (110) Murty, V. S., and Penning, T. M. (1992) Characterization of mercapturic acid and glutathionyl conjugates of benzo[a]pyrene7,8-dione by two-dimensional NMR. Bioconjugate Chem. 3, 218224. (111) Monks, T. J., and Lau, S. S. (1997) Biological reactivity of polyphenolic-glutathione conjugates. Chem. Res. Toxicol. 10, 1296-1313. (112) Abul-Hajj, Y.-J. (1984) Synthesis of 3,4-estrogen o-quinones. J. Steroid Biochem. 21, 621-622. (113) Chesis, P. L., Levin, D. E., Smith, M. T., Ernster, L., and Ames, B. N. (1984) Mutagenicity of quinones: Pathways of metabolic activation and detoxification. Proc. Natl. Acad. Sci. U.S.A. 81, 1696-1700. (114) Lind, C., Hochstein, P., and Ernster, L. (1982) DT-diaphorase as a quinone reductase: A cellular control device against semiquinone and superoxide radical formation. Arch. Biochem. Biophys. 216, 178-185. (115) Flowers-Geary, L., Harvey, R. G., and Penning, T. M. (1993) Cytotoxicity of polycyclic aromatic hydrocarbon o-quinones in rat and human hepatoma cells. Chem. Res. Toxicol. 6, 252-260. (116) Di Monte, D., Ross, D., Bellomo, G., Eklow, L., and Orrenius, S. (1984) Alterations in intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes. Arch. Biochem. Biophys. 235, 334-342. (117) Reed, D. J. (1990) Review of the current status of calcium and thiols in cellular injury. Chem. Res. Toxicol. 3, 495-502. (118) O’Brien, P. J. (1991) Molecular mechanisms of quinone cytotoxicity. Chem.-Biol. Interact. 80, 1-41. (119) Flowers, L., Bleczinski, W. F., Burczynski, M. E., Harvey, R. G., and Penning, T. M. (1996) Disposition and biological activity of benzo[a]pyrene-7,8-dione. A genotoxic metabolite generated by dihydrodiol dehydrogenase. Biochemistry 35, 13664-13672. (120) Wacker, W. E. C., and Vallee, B. L. (1959) Nucleic acids and metals. J. Biol. Chem. 231, 3257-3262. (121) Prutz, W. A., Butler, J., and Land, E. J. (1990) Interaction of Cu(I) with nucleic acids. J. Radiat. Biol. 58, 215-234. (122) Li, Y., and Trush, M. A. (1993) Oxidation of hydroquinone by copper: chemical mechanism and biological effects. Arch. Biochem. Biophys. 300, 346-355. (123) Li, Y., and Trush, M. A. (1994) Reactive oxygen-dependent DNA damage resulting from the oxidation of phenolic compounds by a copper-redox cycle mechanism. Cancer Res. 54, 1895s-1898s. (124) Giloni, L., Takeshita, M., Johnson, F., Iden, C., and Grollman, A. P. (1981) Bleomycin-induced strand scission of DNA. J. Biol. Chem. 256, 8608-8615. (125) Lin, H.-K., and Penning, T. M. (1995) Cloning, sequencing, and functional analysis of the 5′-flanking region of the rat 3Rhydroxysteroid/dihydrodiol dehydrogenase gene. Cancer Res. 5, 4105-4113. (126) Hung, C.-F., and Penning, T. M. (1998) Characterization of a powerful distal enhancer on the rat 3R-hydroxysteroid/dihydrodiol dehydrogenase gene. Proc. Am. Assoc. Cancer Res. 39, 249. (127) Sterling, K., Weaver, J., Ho, K. L., Xu, L. C., and Bresnick, E. (1993) Rat CYP1A1 negative regulatory element: biological activity and interaction with a protein from liver and hepatoma cells. Mol. Pharmacol. 44, 560-568. (128) Bhat, R., Weaver, J. A., Sterling, K. M., and Bresnick, E. (1996) Nuclear transcription factor Oct-1 binds to the 5′-upstream region of CYP1A1 and negatively regulates its expression. Int. J. Biochem. 28, 217-227. (129) Hou, Y.-T., Lin, H.-K., and Penning, T. M. (1998) Dexamethasone regulation of the rat 3R-hydroxysteroid/dihydrodiol dehydrogenase gene. Mol. Pharmacol. 53, 459-466. (130) Nebert, D. W., Eisen, H. J., Negishi, M., Lang, M. A., Hjelmeland, L. M., and Okey, A. B. (1981) Genetic mechanisms controlling the induction of polysubstrate monoxygenase (P-450) activities. Annu. Rev. Pharmacol. Toxicol. 21, 431-462. (131) Denison, M. S., Fisher, J. M., and Whitlock, J. P., Jr. (1988) The DNA recognition site for the dioxin-Ah receptor complex. Nucleotide sequence and functional analysis. J. Biol. Chem. 263, 17221-17224. (132) Denison, M. S., Fisher, J. M., and Whitlock, J. P., Jr. (1989) Protein-DNA interactions at recognition sites for the dioxin-Ah receptor complex. J. Biol. Chem. 264, 16478-16482. (133) Prochaska, H. J., De Long, M. J., and Talalay, P. (1985) On the mechanisms of induction of cancer-protective enzymes: A unifying proposal. Proc. Natl. Acad. Sci. U.S.A. 82, 8232-8236. (134) De Long, M. J., Santamaria, A. B., and Talalay, P. (1987) Role of cytochrome P1-450 in the induction of NAD(P)H:quinone reductase in a murine hepatoma cell line and its mutants. Carcinogenesis 8, 1549-1553.

18 Chem. Res. Toxicol., Vol. 12, No. 1, 1999 (135) Prochaska, H. J., and Talalay, P. (1988) Regulatory mechanisms of monofunctional and bifunctional anti-carcinogenic enzyme inducers in murine liver. Cancer Res. 48, 4776-4782. (136) Rushmore, T. H., and Pickett, C. B. (1990) Transcriptional regulation of the rat glutathione S-transferase Ya subunit gene: Characterization of a xenobiotic-responsive element controlling inducible expression by phenolic antioxidants. J. Biol. Chem. 265, 14648-14653. (137) Rushmore, T. H., Morton, M. R., and Pickett, C. B. (1991) The antioxidant responsive element. Activation by oxidative stress and identification of the DNA consensus sequence required for functional activity. J. Biol. Chem. 266, 11632-11639. (138) Friling, R. S., Bensimon, A., Tichauer, Y., and Daniel, V. (1990) Xenobiotic-inducible expression of murine glutathione S-trans-

Penning et al. ferase Ya subunit gene is controlled by an electrophile-responsive element. Proc. Natl. Acad. Sci. U.S.A. 87, 6258-6262. (139) Li, Y., and Jaiswal, A. K. (1992) Regulation of human NAD(P)H:Quinone oxidoreductase gene. Role of AP-1 binding site contained within the human antioxidant response element. J. Biol. Chem. 267, 15097-15104. (140) Burczynski, M. E., Lin, M. K., and Penning, T. M. (1999) Isoformspecific induction of a human aldo-keto reductase by PAH, electrophiles and oxidative stress. Implications for the alternative pathway of PAH activation catalyzed by human dihydrodiol dehydrogenase. Cancer Res. (in press).

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