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Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and procarcinogens that require activation by host metabolism. Metabolic activation of PAHs by aldo-keto reductases (AKRs) leads to formation of reactive and redox active o-quinones, which may cause oxidatively generated DNA damage. Spectrophotometric assays showed that NADPH caused PAH o-quinones to enter futile redox cycles, which result in the depletion of excess cofactor. Copper(II) amplified NADPH-dependent redox cycling of the o-quinones. Concurrent with NADPH oxidation, molecular oxygen was consumed, indicating the production of ROS. To determine whether PAH o-quinones can cause 8-oxo-dGuo formation in salmon testis DNA, three prerequisite experimental conditions were satisfied. Quantitative complete enzymatic hydrolysis of DNA was achieved, adventitious oxidation of dGuo was eliminated by the use of chelex and desferal, and basal levels of less than 2.0 8-oxo-dGuo/10⁵ dGuo were obtained. The HPLC−ECD analytical method was validated by spiking the DNA with standard 8-oxo-dGuo and demonstrating quantitative recovery. HPLC−ECD analysis revealed that in the presence of NADPH and Cu(II), submicromolar concentrations of PAH o-quinones generated >60.0 8-oxo-dGuo adducts/10⁵ dGuo. The rank order of 8-oxo-dGuo generated in isolated DNA was NP-1,2-dione > BA-3,4-dione > 7,12-DMBA-3,4-dione > BP-7,8-dione. The formation of 8-oxo-dGuo by PAH o-quinones was concentration-dependent. It was completely or partially inhibited when catalase, tiron, or a Cu(I) specific chelator, bathocuproine, was added, indicating the requirement for H2O2, O2^-, and Cu(I), respectively. Methional, which is a copper−hydroperoxo complex [Cu(I)OOH] scavenger, also suppressed 8-oxo-dGuo formation. By contrast, mannitol, sodium benzoate, and sodium formate, which act as hydroxyl radical scavengers, did not block its formation. Sodium azide, which can act as both a hydroxyl radical and a ¹O2 scavenger, abolished the formation of 8-oxo-dGuo. These data showed that the production of 8-oxo-dGuo was dependent on Cu(II)/Cu(I) catalyzed redox cycling of PAH o-quinones to produce ROS and that the immediate oxidant was not hydroxyl radical or Cu(I)OOH and that it is more likely ¹O2, which can produce a 4,8-endoperoxide-dGuo intermediate.

(±)-7,8-Dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol), a proximate carcinogen derived from benzo[a]pyrene (BP) requires further metabolic activation to exert its carcinogenic effects. Two principal pathways have been implicated, and these involve either the formation of (±)-trans-7,8-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) catalyzed by P450 1A1/P450 1B1 (NADPH-dependent monoxygenases) or the formation of benzo[a]pyrene-7,8-dione (BP-7,8-dione) catalyzed by human aldo−keto reductases AKR1A1 and AKR1C1−AKR1C4 [NAD(P)(H)-dependent oxidoreductases]. The relative contributions of the two pathways to PAH activation are unknown. In this study, BP-7,8-diol metabolism was studied in human bronchoalveolar H358 cell extracts. Parental H358 cells do not constitutively express P450 1A1/P450 1B1 or AKRs but were manipulated by induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to express P450 1A1/P450 1B1 or by stable transfection to express AKR1A1 (aldehyde reductase). TCDD induction of AKR1A1 transfectants provided a cell line that expressed both pathways. Extracts derived from parental H358 cells plus TCDD (P450 induction) produced electrophilic anti-BPDE, which hydrolyzed to benzo[a]pyrene tetrahydrotetrols (BP-tetrols), extracts derived from AKR1A1-transfected cells (AKR1A1 expression) produced reactive and redox-active BP-7,8-dione, which was trapped in situ as its mono(thioether) conjugate, and extracts derived from AKR1A1 transfectants plus TCDD (coexpression of P450 1A1/P450 1B1 and AKR1A1) produced both anti-BPDE and BP-7,8-dione. The competing activation of BP-7,8-diol by P450 1A1/P450 1B1 and AKR1A1 was studied with varied NADPH:NAD⁺ ratios. The system with a relatively higher concentration of NADPH favored formation of anti-BPDE via P450 1A1/P450 1B1, while the system with the higher concentration of NAD⁺ favored formation of BP-7,8-dione via AKR1A1. Under conditions that mimic the cellular redox state, 10 μM NADPH and 1 mM NAD⁺, equal amounts of BP-tetrols and BP-7,8-dione were formed. This suggests that P450 1A1/P450 1B1 and AKR1A1 play competing roles in the metabolic activation of BP-7,8-diol and that the dominant pathway of BP-7,8-diol activation depends on the redox state of the cells. These model systems provide a cellular context in which the dominant DNA adducts/lesions formed by either pathway may be compared.

Resveratrol (1) is a polyphenolic natural product, which functions as both a mechanism-based inactivator and a co-reductant of the COX-1 peroxidase. These functions are mediated through different moieties on the molecule, namely, the m-hydroquinone moiety (mechanism-based inactivator) and the phenol moiety (co-reductant). Implicit in this bifunctionality is the notion that resveratrol is oxidized at the peroxidase active site of COX-1, resulting in the formation of two hypothetical radical species. Oxidation of the m-hydroquinone moiety can generate a hypothetical m-semiquinone radical, which is unstabilized and leads to irreversible enzyme inactivation. Oxidation of the phenol moiety can generate a hypothetical phenoxy radical, which is stabilized and leads to co-reduction during peroxidase catalysis. These two radicals have been trapped as the resveratrol dimers, cis-ε-viniferin (4, trapped m-semiquinone radical) and trans-δ-viniferin (5, trapped phenoxy radical), and identified by liquid chromatography (LC), absorbance spectroscopy, and LC/tandem mass spectrometry (MSⁿ) methods. Methoxy-resveratrol analogues, in which either the m-hydroquinone or the phenol moiety were protected as methyl ethers, were used to confirm the proposed mechanism of viniferin production by COX-1.

Resveratrol (1) is a m-hydroquinone found in red wine, which has antiinflammatory, cardiovascular protective (antiplatelet), and cancer chemopreventive properties. It is a potent peroxidase-dependent mechanism-based inactivator of COX-1, a desired target for antiplatelet agents, and has no similar effect on COX-2. Much attention has focused on resveratrol (1) as being the sole agent responsible for the cardioprotective effects associated with red wine consumption (commonly known as the “French paradox”). In this study we show that other red wine constituents, namely, the catechins (2, 3) and epicatechins (4, 5), act as peroxidase mediated mechanism-based inactivators of COX-1 but not of COX-2. Structure−activity relationships identify these agents as being as effective as resveratrol with respect to their ability to specifically inactivate COX-1. We show that resorcinol (6) is the minimum structure necessary for mechanism-based inactivation of COX-1. These findings imply that resveratrol is not the sole agent responsible for the antiplatelet activity of red wine and suggest that all dietary m-hydroquinones should be examined for cardioprotective effects.

Leukotriene B4 (LTB4) is a potent, proinflammatory mediator involved in the pathogenesis of a number of diseases including inflammatory bowel disease, psoriasis, rheumatoid arthritis, and asthma. The enzyme LTA4 hydrolase represents an attractive target for pharmacological intervention in these disease states, since the action of this enzyme is the rate-limiting step in the production of LTB4. Our previous efforts focused on the exploration of a series of analogues related to screening hit SC-22716 (1, 1-[2-(4-phenylphenoxy)ethyl]pyrrolidine) and resulted in the identification of potent, orally active inhibitors such as 2. Additional structure−activity relationship studies around this structural class resulted in the identification of a series of α-, β-, and γ-amino acid analogues that are potent inhibitors of the LTA4 hydrolase enzyme and demonstrated good oral activity in a mouse ex vivo whole blood LTB4 production assay. The efforts leading to the identification of clinical candidate SC-57461A (8d, 3-[methyl[3-[4-(phenylmethyl)phenoxy]propyl]amino]propanoic acid) are described.

Polycyclic aromatic hydrocarbons (PAHs) in tobacco smoke may cause human lung cancer via metabolic activation to ultimate carcinogens. p53 is one of the most commonly mutated tumor suppressor genes in this disease. An analysis of the p53 mutational database shows that G to T transversions are a signature mutation of lung cancer. Aldo-keto reductases (AKRs) activate PAH trans-dihydrodiol proximate carcinogens to yield their corresponding reactive and redox-active o-quinones, e.g., benzo[a]pyrene-7,8-dione (BP-7,8-dione). We employed a yeast reporter system to determine whether PAH o-quinones or the ROS they generate cause change-in-function mutations in p53. N-Methyl-N-nitroso-N‘-nitro-guanidine, a standard alkylating mutagen was used as a positive control. MNNG caused a dose-dependent increase in mutant yeast colonies and at the highest concentrations 8−14% of the yeast colonies were mutated and were characterized by G:C to A:T transitions in the p53 DNA binding domain. Treatment of p53 cDNA with micromolar concentrations of (±)-anti-7,8-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene, (anti-BPDE, an ultimate carcinogen) or sub-micromolar concentrations of BP-7,8-dione in the presence of redox-cycling conditions (NADPH and CuCl2) also caused p53 mutations in a dose-dependent manner. We found that no mutants were observed with PAH o-quinones or NADPH alone. p53 mutagenesis by BP-7,8-dione was attenuated by ROS scavengers and completely abrogated by a combination of superoxide dismutase and catalase, indicating that both superoxide anion and hydroxyl radicals were the responsible mutagens. The bulk of the mutations detected were single-point mutations and were not random in occurrence. Over 46% of BP-7,8-dione-induced mutations were G:C to T:A transversions, consistent with the formation of 8-oxo-dGuo or its secondary oxidation products. In addition, 25% of these mutations were at hotspots in p53 which are known to be mutated in lung cancer. Together these data suggest that PAH o-quinones generate an endogenous mutagen (ROS) which leads to p53 inactivation. These observations provide an alternative route to G to T transversions that dominate in p53 in lung cancer.

Dihydrodiol dehydrogenase members of the aldo-keto reductase (AKR) superfamily have been implicated in the metabolic activation of PAH trans-dihydrodiols because they convert these proximate carcinogens to reactive and redox-active o-quinones. In this study, rat liver 3α-hydroxysteroid/dihydrodiol dehydrogenase (AKR1C9) was stably transfected into human breast carcinoma (MCF-7) cells, which represent a null-environment for AKR expression, to detect the formation of PAH o-quinones in a cellular context and the cellular consequences of o-quinone formation. The heterologous transfected cells expressed AKR1C9 mRNA and protein. Immunotitration of the enzyme activity indicated that the expressed protein constituted 1.0% of the soluble protein. The specific activity of the expressed enzyme was also comparable to that observed in rat liver cytosol. The transfectants were found to convert (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) to benzo[a]pyrene-7,8-dione (BPQ). The identity of this metabolite was confirmed by co-chromatography and by UV−Vis diode-array spectrometry. This conversion was not evident in mock-transfected cells. The cytotoxic consequences of BPQ formation was also examined. Transfectants exposed to 1 μM B[a]P-7,8-diol revealed that cytotoxicity, as measured by lactate dehydrogenase (LDH) release, occurred over the time course of o-quinone formation leading to 77% of the cellular LDH being released by 16 h. AKR1C9 inhibitors blocked the B[a]P-7,8-diol dependent cytotoxicity indicating that it was mediated by the enzymatically formed BPQ. These data indicate that high stable constitutive expression of AKR1C9 will result in B[a]P-7,8-diol mediated cytotoxicity due to the formation of unconjugated BPQ.

Quinones represent a class of toxicological intermediates which can create a variety of hazardous effects in vivo, including acute cytotoxicity, immunotoxicity, and carcinogenesis. The mechanisms by which quinones cause these effects can be quite complex. Quinones are Michael acceptors, and cellular damage can occur through alkylation of crucial cellular proteins and/or DNA. Alternatively, quinones are highly redox active molecules which can redox cycle with their semiquinone radicals, leading to formation of reactive oxygen species (ROS), including superoxide, hydrogen peroxide, and ultimately the hydroxyl radical. Production of ROS can cause severe oxidative stress within cells through the formation of oxidized cellular macromolecules, including lipids, proteins, and DNA. Formation of oxidatively damaged bases such as 8-oxodeoxyguanosine has been associated with aging and carcinogenesis. Furthermore, ROS can activate a number of signaling pathways, including protein kinase C and RAS. This review explores the varied cytotoxic effects of quinones using specific examples, including quinones produced from benzene, polycyclic aromatic hydrocarbons, estrogens, and catecholamines. The evidence strongly suggests that the numerous mechanisms of quinone toxicity (i.e., alkylation vs oxidative stress) can be correlated with the known pathology of the parent compound(s).

Leukotriene B4 (LTB4) is a pro-inflammatory mediator that has been implicated in the pathogenesis of a number of diseases including inflammatory bowel disease (IBD) and psoriasis. Since the action of LTA4 hydrolase is the rate-limiting step for LTB4 production, this enzyme represents an attractive pharmacological target for the suppression of LTB4 production. From an in-house screening program, SC-22716 (1, 1-[2-(4-phenylphenoxy)ethyl]pyrrolidine) was identified as a potent inhibitor of LTA4 hydrolase. Structure−activity relationship (SAR) studies around this structural class resulted in the identification of a number of novel, potent inhibitors of LTA4 hydrolase, several of which demonstrated good oral activity in a mouse ex vivo whole blood assay.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants which may cause cancer and require metabolic activation to exert their carcinogenic effects. One pathway of activation involves the dihydrodiol dehydrogenase-catalyzed oxidation of non-K region trans-dihydrodiols to yield catechols, which autoxidize to form reactive o-quinones. As a step toward identifying the spectrum of PAH o-quinone−DNA adducts that may form in biological systems, depurinating PAH o-quinone−guanine adducts were synthesized. Naphthalene-1,2-dione, phenanthrene-1,2-dione, and benzo[a]pyrene-7,8-dione were reacted with 5 equiv of 2‘-deoxyguanosine (dGuo) under acidic conditions (1:1 acetic acid/water). The products were purified by reversed-phase HPLC, characterized by a combination of UV spectroscopy, electrospray ionization/tandem mass spectrometry, and high-field proton nuclear magnetic resonance spectroscopy, and identified as 7-(naphthalene-1,2-dion-4-yl)guanine (MH⁺, m/z 308), 7-(phenanthrene-1,2-dion-4-yl)guanine (MH⁺, m/z 358), and 7-(benzo[a]pyrene-7,8-dion-10-yl)guanine (MH⁺, m/z 432), respectively. Reaction at N7 of dGuo leads to cleavage of the glycosidic bond, producing depurinating adducts. Reaction of phenanthrene-1,2-dione with calf thymus DNA led to the formation of the corresponding depurinating adduct. The loss of modified bases in DNA generates apurinic sites which, if unrepaired, can lead to mutations and thus cellular transformation. These synthesized PAH o-quinone−N7-guanine adducts can be used as standards to identify such adducts in vitro and in vivo.

Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) catalyzes the oxidation of polycyclic aromatic hydrocarbon (PAH) trans-dihydrodiols (proximate carcinogens) to catechols which rapidly autoxidize to yield o-quinones (Smithgall, T. E., Harvey, R. G., and Penning, T. M. (1988) J. Biol. Chem 263, 1814−1820). Although this pathway suppresses the formation of the PAH anti- and syn-diol epoxides (ultimate carcinogens), the process of autoxidation is anticipated to yield reactive oxygen species (ROS). We now show that the NADP⁺ dependent oxidation of (±)-trans-1,2-dihydroxy-1,2-dihydronaphthalene (Npdiol) and (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (Bpdiol) catalyzed by homogeneous DD is accompanied by the consumption of molecular oxygen and the production of H2O2. With both trans-dihydrodiol substrates, oxygen consumption was stoichiometric with H2O2 production consistent with the reaction:  QH2 + O2 = H2O2 + Q, where QH2 is the catechol and Q is the o-quinone. Using Npdiol or Bpdiol as substrates, a burst of superoxide anion production is catalyzed by DD which can be detected as the rate of cyt c reduction that is inhibited by superoxide dismutase. Using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as spin-trapping agent, secondary spin adducts corresponding to DMPO-CH3 were formed during the enzymatic oxidation of Npdiol and Bpdiol. The formation of the CH3^• radical arises from the OH^• attack of DMSO, which was used as cosolvent. These spin adducts were attenuated by superoxide dismutase and catalase, implying that O2^-• and H2O2 are obligatory for the formation of DMPO-CH3. It is proposed that O2^-• is the radical that propagates autoxidation and that the resultant H2O2 undergoes Fenton chemistry to produce the OH^• radical. Identical spin adducts were observed using a superoxide anion generating system (hypoxanthine/xanthine oxidase) and DMPO as spin-trapping agent in the presence of DMSO. The ability of DD to generate ROS during the oxidation of PAH trans-dihydrodiols (proximate carcinogens) may have important implications for tumor initiation and promotion.

A NAD(P)-linked 3 alpha-hydroxysteroid dehydrogenase [3 alpha-hydroxysteroid: NAD(P) oxidoreductase, EC 1.1.1.50], purified to homogeneity from male rat liver cytosol, accounts for most of the oxidative activity for 3 alpha-hydroxysteroids and for benzenedihydrodiol (trans-1,2-dihydroxy-3,5-cyclohexadiene) of this tissue. This enzyme, which also promotes the reduction of quinones and certain aromatic aldehydes and ketones, is powerfully inhibited by the major types of nonsteroidal and steroidal anti-inflammatory drugs. The IC50 values for indomethacin and for betamethasone are in the low micromolar range. The rank order of the inhibitory potencies of a series of these agents paralleled closely that reported for the inhibition of cyclooxygenase by nonsteroidal anti-inflammatory agents or the indirect inhibition of phospholipase A2 by anti-inflammatory steroids. A good correlation is found also between the logarithm of the concentrations of these drugs required to inhibit 3 alpha-hydroxysteroid dehydrogenase and the human anti-inflammatory dose. The suggestion that this enzyme may play a role in mediating inflammation is further strengthened by the observation that the dehydrogenase binds arachidonic acid and is also potentially inhibited by certain prostaglandins. This enzyme may be of use in the evaluation of anti-inflammatory activity.

The 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) of rat brain cytosol has been purified to apparent homogeneity. The purification procedure involves six successive steps, includes one affinity chromatography, and yields enzyme which displays a 1,550-fold enhancement in specific activity. The homogeneous enzyme has a Km of 8.0 microM for 5 alpha-dihydrotestosterone, a Vmax of 1.3 mumol of 3 alpha-androstanediol formed per h/mg of protein, and displays a preference for NADPH. It appears to be the major activity responsible for the reduction of 5 alpha-dihydrotestosterone in this tissue and may play a pivotal role in brain androgen metabolism. The homogeneous enzyme has several properties in common with the 3 alpha-hydroxysteroid dehydrogenase purified from rat liver cytosol (Penning, T. M., Mukharji, I., Barrows, S., and Talalay, P. (1984) Biochem. J. 222, 601-611). It is a monomer with a molecular weight of 31,000, it has a pI of 5.5, and it is potently inhibited by the nonsteroidal anti-inflammatory drugs (IC50 value for indomethacin = 2.0 microM). The potency of inhibition observed for the brain enzyme parallels that observed for cyclooxygenase: indomethacin greater than fenamates greater than l-methylpyrrole acetic acids greater than arylpropionic acids greater than salicylates greater than acetaminophen. Examination of a variety of steroidal contraceptives as modulators of the dehydrogenase indicates that ethinylestradiol is a very poor inhibitor (IC50 = 100 microM), while 6-medroxyprogesterone acetate (Provera) is an extremely potent inhibitor (IC50 = 0.2 microM). The possibility exists that brain androgen metabolism may be altered by the nonsteroidal anti-inflammatory drugs and synthetic progestins.

Examination of the product of affinity labeling of delta 5-3-ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni by the suicide substrate [7-3H]5,10-secoestr-5-yne-3,10,17-trione has demonstrated that the steroid becomes bound by an acid- and base-labile linkage to an active-site peptide representing residues 55-58 (H2N-Tyr-Ala-Asn-Ser-CO2H) of the primary structure (Penning, T. M., and Talalay, P. (1981) J. Biol. Chem. 256, 6851-6858). Upon release of the steroid by mild acid hydrolysis, the peptide is converted into a more basic structure while retaining its amino acid composition (as determined by conventional means). These findings were rationalized by postulating that the steroid is bound as an imido ester via the amide group of asparagine 57 and that the polypeptide backbone participates (via attack by nitrogen or oxygen) in the hydrolysis of this ester with the formation of a cyclic amidine or basic oxazine. By comparing the properties of the isolated tetrapeptide, from which the steroid has been removed, with those of synthetic H2N-Tyr-Ala-Asn-Ser-CO2H and H2N-Tyr-Ala-Asp-Ser-CO2H by electron impact and fast atom bombardment mass spectrometry, we now have evidence for the presence of an oxazine (5,6-dihydro-6-iminio-4H-1,3-oxazine) in the modified peptide. Our results draw attention to the hitherto unsuspected degree of nucleophilicity of the amide group of the side chain of asparagine and the participation of this group in the formation of an imido ester.

2 α-Cyanoprogesterone potently inhibits the conversion of [3H]pregnenolone into progesterone catalysed by bovine corpora lutea, bovine adrenal cortex and human term placenta microsomes (microsomal fractions), yielding IC50 (concentration causing 50% inhibition) values of 66 nM, 120 nM and 700 nM respectively. By contrast, it is an exceedingly poor inhibitor of the isomerization of pregn-5-ene-3,20-dione, yielding IC50 values between 50 and 70 microM. On this basis, 2 α-cyanoprogesterone would appear to be an extraordinarily selective inhibitor of the 3 β-hydroxysteroid dehydrogenase. Dixon plots indicate that it is a very-tight-binding competitive inhibitor of the corpus-luteum enzyme, yielding a Ki of 15 nM. In the bovine adrenal cortex and human placenta the steroid is less potent and inhibits the dehydrogenase non-competitively with Ki values of 150 nM and 1.0 microM respectively. Thus 2 α-cyanoprogesterone inhibits the corpus-luteum dehydrogenase with substantial selectivity. Because of its high affinity for the ovarian enzyme, the presence of low-micromolar concentrations of 2 α-cyanoprogesterone can promote a complete cessation of progesterone synthesis in corpora-lutea microsomes for several hours. Since this effect is observed in the presence of saturating concentrations of pregnenolone (50 microM), it is predicted that this inhibitor may be even more potent in vivo. 2 α-Cyanoprogesterone displays very low affinity for the human progesterone receptor, yielding a Kd of 600 nM as against a Kd of 1.6 nM for progesterone. It is suggested that 2 α-cyanoprogesterone may be a selective inhibitor of ovarian progesterone synthesis and may act as an effective anti-gestational agent in vivo.