Aldo-Keto Reductases and PAH Ortho-quinones:An Alternative Pathway of PAH Activation
September 1, 2000
Aldo-Keto Reductases and PAH Ortho-quinones:An Alternative Pathway of PAH Activation
By Michael E. Burczynski, PhD, and Trevor M. Penning, PhD
Polycyclic aromatic hydrocarbons (pah) are known carcinogens. they are ubiquitous pollutants formed during organic combustion that are found at extremely high levels in coal, tar, diesel exhaust, and car emissions, and they impregnate the food supply. Benzo[a]pyrene (B[a]P) and other PAH are also present in tobacco smoke and are suspected causative agents for the development of lung cancer. Because of their prevalence in the environment, daily exposure to PAH is considered to pose a significant health risk to the human population today.
Background
PAH are actually procarcinogens; these lipophilic xenobiotics require metabolism to electrophilic species in order to exert their carcinogenic effects. The best-characterized route of PAH activation has been that catalyzed by the cytochrome P450s (CYPs). In this pathway, the concerted actions of CYPs and epoxide hydrolases initially convert PAH to their corresponding trans-dihydrodiol metabolites. (See Figure 1.) PAH trans-dihydrodiols (proximate carcinogens) are believed to be important, bonafide intermediates along the pathway of PAH activation because they are often more carcinogenic than parent PAH.
Research has focused on the subsequent CYP-catalyzed oxidation of PAH trans-dihydrodiols to anti- and syn- diol-epoxides. The diol-epoxides are potent electrophiles and are believed to represent ultimate carcinogens (i.e., the actual downstream metabolites of PAH responsible for their DNA lesions). For instance, anti-BPDE (the diol-epoxide metabolite derived from B[a]P) forms stable N2-deoxyguanosine adducts within DNA.1 These adducts give rise to G to T transversions either via translesional synthesis by DNA polymerase or by excision and mis-repair. Transversions on codons 12 of ras or on codons 245 and 248 of p53 can lead to activation of the proto-oncogene or inactivation of the tumor suppressor gene, respectively. anti-BPDE targets these mutational hotspots in the ras and p53 genes, and it is likely that this plays an important role in tumor initiation by B[a]P.2,3
An important question to ask is whether CYP-derived diol-epoxides are the only downstream metabolites responsible for the complete carcinogenicity of PAH. Although compelling evidence supports a role for anti-BPDE and other diol-epoxides in PAH carcinogenesis, several observations suggest that diol-epoxides may not be the sole electrophilic metabolites responsible for the spectrum of DNA damage caused by PAH. For instance, apurinic sites, as well as 8-hydroxy-2’-deoxyguanosine (8-OHdG) and other markers of oxidative damage, also are observed in DNA from rodent tissues and human mammary epithelial cells exposed to B[a]P.4,5 Such lesions are also promutagenic—if an apurinic site is left unrepaired, an A is always introduced opposite that site so that during replication, a G to T transversion results. Similarly, the mispairing of 8-OHdG with adenine and subsequent replication of the opposite strand provides yet another pathway for G to T transversions. Indeed, apurinic sites and 8’-OH-dG give more straightforward routes to these dominant mutations than anti-BPDE.
An Alternative Pathway of PAH Activation
What could be the source of these apurinic sites and oxidatively damaged bases? To understand the process of PAH carcinogenesis, and devise therapeutic strategies for prevention/intervention, it is important to characterize each metabolic pathway that can contribute to PAH activation. An alternative pathway of PAH activation is that catalyzed by the aldo-keto reductase (AKR) superfamily of enzymes. (For more information, see www.med.upenn.edu/akr). AKRs are NAD(P)H-dependent oxidoreductases that convert ketones to alcohols on a variety of endogenous substrates and xenobiotics for eventual conjugation and elimination.6 While physiological substrates include steroid hormones and prostaglandins, several AKR family members also use PAH trans-dihydrodiols.
The pathway of PAH activation catalyzed by members of the AKR1C subfamily was initially worked out for the rat liver 3a-hydroxysteroid/dihydrodiol dehydrogenase (AKR1C9). In this pathway, PAH trans-dihydrodiols are diverted from CYPs to form catechols.7 The catechols are air-sensitive and undergo two, 1 electron oxidations, and via the intermediacy of an o-semiquinone anion radical, form the fully oxidized PAH o-quinone.8 This air-oxidation is accompanied by the formation of reactive oxygen species (ROS; superoxide anion, hydrogen peroxide, and hydroxyl radical).9 The resultant PAH o-quinone is both a reactive electrophile and is redox active. (See Figure 1.) It will readily form Michael addition products with macromolecules, including DNA, and evidence in vitro has been obtained for the formation of stable N2-deoxyguanosine adducts as well as N7-guanine depurinating adducts.10,11 The latter observation is important since it could explain the presence of apurinic sites in PAH-exposed cells. In the presence of reducing equivalents, the PAH o-quinones generated by AKRs redox cycle back to the catechol. This establishes a futile redox-cycle in which ROS are amplified multiple times until the reducing equivalent is exhausted.
Under redox-cycling conditions, low nanomolar concentrations of PAH o-quinones cause strand-scission of pX174 phage DNA in vitro, and toxicologic concentrations of BPQ (~20 uM) cause extensive genomic DNA strand scission in primary rat hepatocytes.12 These observations are of great interest, since the production of ROS by redox-cyling o-quinones could explain the presence of oxidative damage in DNA from PAH-exposed cells.
PAH Activation Pathway in Humans
Until recently it was unknown whether this pathway of metabolism could occur in human tissues. Several human homologs of AKR1C9 are constitutively expressed: Studies demonstrated that four closely related human AKR isoforms also catalyzed the oxidation of B[a]P-diol to its corresponding o-quinone, benzo[a]pyrene-7,8-dione (BPQ).13 Thus, the pathway of PAH activation to redox-cycling o-quinones has the potential to occur in human cells.
One of these human isoforms, AKR1C1, is highly inducible in human cell lines by Michael acceptors and ROS.14,15 AKR1C1 appears to be included in the battery of phase II enzymes regulated by the antioxidant response element (ARE), although the presumptive ARE in the regulatory region of the AKR1C1 gene has not yet been characterized. The inclusion of AKR1C1 in this battery of genes may be detrimental with respect to PAH metabolism, since BPQ (a Michael acceptor and product of the AKR1C1 reaction) feedback stimulates AKR1C1 expression. Such a scenario would lead to both a chemical (redox-cycling) and genetic (AKR1C1 induction) amplification of ROS in AKR1C1-expressing cells exposed to proximate carcinogens. (See Figure 2.)
Summary
Recently, we have found that BPQ also is a potent inducer of CYP1A1.16 The oxidation of PAH trans-dihydrodiols to o-quinones by AKRs results in a restored planarity to these electrophilic metabolites. BPQ appears to mediate its induction of CYP1A1 by "hijacking" the aryl hydrocarbon receptor (AhR) signaling pathway. While this has important implications for inducing diol and diol-epoxide formation via the CYP pathway, it also will result in more diol being available for the constitutive AKRs. It also may provide a mechanism whereby genotoxic PAH o-quinones generated by cytosolic AKRs are "shuttled" into the nucleus by a specific cytosolic receptor. If this is the case, then the pathway of PAH trans-dihydrodiol metabolism catalyzed by AKRs could ultimately result in the import of genotoxic PAH o-quinones into the nucleus. The availability of AhR-deficient cell lines should allow further determination of the contribution of the AhR to the genotoxic effects of PAH o-quinones in living cells. (See Figure 3.)
The generation of PAH o-quinones by AKRs is a relatively new concept in the activation of PAH. The studies summarized in this article have laid a foundation for the serious investigation of PAH o-quinones as alternative ultimate carcinogens. Many of the questions that should now be asked are similar to those already answered for the diol-epoxides. Are PAH o-quinones mutagenic in mammalian cells? Do PAH o-quinones target mutational hotspots in p53, ras, or other target genes? Are PAH o-quinones carcinogenic in vivo? Do they act as tumor initiators, promoters, or both? The concept that PAH o-quinones could act as tumor promoters is an intriguing one since prooxidant states have been linked to tumor promotion. Should PAH o-quinones possess these undesirable properties and contribute to PAH carcinogenesis, there will be a great impetus to study the polymorphisms and pharmacogenetics of the human AKR isoforms responsible for their formation in the future. (Dr. Burczynski is Post-Doctoral Fellow, Drug Safety Evaluation, R.W. Johnson Pharmaceutical Research Institute, Johnson & Johnson, Raritan NJ; and Dr. Penning Associate Dean of Post-Doctoral Research Training and Professor of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia PA. )
References
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