The role of the Ah receptor and p38 in benzo[a]pyrene-7,8-dihydrodiol and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-induced apoptosis

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in the environment. Benzo[a]pyrene (B[a]P), a prototypical member of this class of chemicals, affects cellular signal transduction pathways and induces apoptosis. In this study, the proximate carcinogen of B[a]P metabolism, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-dihydrodiol) and the ultimate carcinogen, B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide(+/-) (BPDE-2) were found to induce apoptosis in human HepG2 cells. Apoptosis initiated by B[a]P-7,8-dihydrodiol was linked to activation of the Ah receptor and induction of CYP1A1, an event that can lead to the formation of BPDE-2. With both B[a]P-7,8-dihydrodiol and BPDE-2 treatment, changes in anti- and pro-apoptotic events in the Bcl-2 family of proteins correlated with the release of mitochondrial cytochrome c and caspase activation. The onset of apoptosis as monitored by caspase activation was linked to mitogen-activated protein (MAP) kinases. Utilizing mouse hepa1c1c7 cells and the Arnt-deficient BPRc1 cells, activation of MAP kinase p38 by B[a]P-7,8-dihydrodiol was shown to be Ah receptor-dependent, indicating that metabolic activation by CYP1A1 was required. This was in contrast to p38 activation by BPDE-2, an event that was independent of Ah receptor function. Confirmation that MAP kinases play a critical role in BPDE-2-induced apoptosis was shown by inhibiting caspase activation of poly(ADP-ribose)polymerase 1 (PARP-1) by chemical inhibitors of p38 and ERK1/2. Furthermore, mouse embryo p38-/- fibroblasts were shown to be resistant to the actions of BPDE-2-induced apoptosis as determined by annexin V analysis, cytochrome c release, and cleavage of PARP-1. These results confirm that the Ah receptor plays a critical role in B[a]P-7,8-dihydrodiol-induced apoptosis while p38 MAP kinase links the actions of an electrophilic metabolite like BPDE-2 to the regulation of programmed cell death.     

Vanadium redox cycling, lipid peroxidation and co-oxygenation of benzo(a)pyrene-7,8-dihydrodiol.

Mechanism of lipid peroxidation triggered by vanadium in human term placental microsomes was reinvestigated in vitro. Production of lipid peroxyl radicals was estimated from co-oxygenation of benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol. Vanadyl(IV), but not vanadate(V) caused a dose-dependent co-oxygenation. Vanadate(V) required the presence of reduced nicotinamide adenine dinucleotide phosphate to trigger co-oxygenation of benzo(a)pyrene-7,8-dihydrodiol. To determine the role of pre-formed lipid hydroperoxides, the results obtained with partially peroxidized linoleic acid were compared with those of fresh linoleate. Superoxide dismutase inhibited the co-oxygenation of reaction when fresh linoleic acid was used. To further characterize the role of superoxide anion-radical in the vanadium redox cycling, the increase of optical density of vanadate(V) dissolved in Tris buffer was measured at 328 nm during the addition of KO2. The rate of this reaction producing peroxy-vanadyl complex was decreased by superoxide dismutase, especially, in the presence of catalase. It is suggested that vanadium catalyzes two separate processes, both leading to enhanced lipid peroxidation: (i) initiation, dependent on superoxide and triggered by peroxy-vanadyl; (ii) propagation, dependent on pre-formed lipid hydroperoxide not sensitive to superoxide dismutase. It is postulated that the vanadium-triggered initiation of lipid peroxidation may be crucial for toxicity in organs with limited endogenous lipid peroxidation.     

The binding of benzo[a]pyrene-7,8-dihydrodiol to bacteriophage phi X174 DNA

A solute enhanced phase partition method is described for the measurement of the binding of drugs to nucleic acids. The inclusion of a solute which acts as a phase transfer reagent allows one to enhance the solubility of charged molecules in organic solvents by as much as three orders of magnitude. This development increases the utility of partition analysis as a general method for studying the interaction of small molecules with macromolecules. Solute enhanced partition analysis (SEPA) has been used to measure the DNA binding of positively charged drugs at very low levels of drug binding, where we have observed cooperative binding of daunorubicin to calf thymus DNA.     

Lipoxygenase-catalyzed epoxidation of benzo(a)pyrene-7,8-dihydrodiol

Metabolism of resolved radioactive stereoisomer, [14C](+)-benzo-(a)pyrene-trans-7,8-dihydrodiol by highly purified soybean lipoxygenase plus linoleic acid was investigated. Trans-anti-7,8,9,10-tetrahydrotetrol, the product of hydrolytic breakdown of ultimate mutagenic benzo(a)pyrene-anti-7,8-dihydrodiol,9,10-epoxide, was detected as a major metabolite. The epoxidation, depended on the enzyme concentration and was inhibited by nordihydroguaiaretic acid. This study provides evidence on the ability of lipoxygenase to catalyze the epoxidation of benzo(a)pyrene-7,8-dihydrodiol.     

The oxidation of benzo[a]pyrene mediated by lipid peroxidation in irradiated synthetic diets

The effect of gamma-irradiation (1000-4000 Gy) on the formation of lipid peroxides and on the oxidation of the environmental carcinogen benzo[a]pyrene (BP) has been studied in mixtures of starch/fat and BP which were used as models for natural foods. When mixtures containing polyunsaturated fats (mackerel oil and cod-liver oil which contain relatively large proportions of C20:5 and C22:6) were exposed to gamma-irradiation, large concentrations of lipid peroxide were formed and a concomitant oxidation of BP to mutagenic and toxic BP quinones took place. The rate of BP oxidation was closely related to the extent of peroxidation of the lipids in the starch mixtures and was dependent on the dose of gamma-irradiation and the presence of air. Mackerel oil also underwent peroxidation during the storage of both irradiated and unirradiated starch/mackerel oil/BP mixtures and this resulted in a significant oxidation of the BP present in these samples. Antioxidants such as vitamin E and BHA inhibited both lipid peroxidation and BP oxidation resulting from gamma-irradiation. These results demonstrate that the species generated during the peroxidation of unsaturated fats in foodstuffs can react with polycyclic aromatic hydrocarbons such as BP and convert them into active mutagenic and toxic products. This has important toxicological implications, particularly as the consumption of polyunsaturated fat in the Western world is increasing and gamma-irradiation may soon be widely used for food sterilization.     

Microsomal chemiluminescence of benzo[a]pyrene-7,8-dihydrodiol and its synthetic analogues trans- and cis-1-methoxyvinylpyrene

Low-density lipoproteins (LDL) have been shown to have a number of effects on the function of various cell types. To appreciate whether the in vitro effects of LDL have in vivo relevance, it is necessary to demonstrate that the biologic action described can be accounted for by native LDL and not by an alteration in the molecule or an addition to the preparation occurring during isolation. EDTA is frequently added to LDL during preparation to prevent oxidation. The effect of EDTA dialysis on LDL-mediated inhibition of lymphocyte responses was therefore examined. LDL alone did not inhibit mitogen-induced initial lymphocyte activation but rather suppressed lymphocyte DNA synthesis and subsequent proliferation in a transferrin-reversible manner. LDL dialysed with EDTA also inhibited lymphocyte responsiveness but the inhibition was not reversed by transferrin. Further experiments demonstrated that after dialysis EDTA in the LDL accounted for the change in its inhibitory effects. EDTA did not alter the lipoprotein but itself inhibited lymphocyte responses by chelating zinc necessary for DNA synthesis. These data indicate that LDL preparations may exhibit at least two separate effects on lymphocyte function. LDL is directly suppressive, while small amounts of contaminating EDTA can additionally be suppressive by chelating zinc. Thus, EDTA present in LDL preparations can alter their apparent biologic effects.     

Conformation of dinucleoside monophosphates modified with benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide as measured by circular dichroism

The conformational properties of GpU modified with the reactive derivative of benzo[a]pyrene, (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, has been investigated utilizing circular dichroism spectroscopy. Binding of this carcinogen to the N2 of G residues in GpU resulted in the formation of four compounds (I to IV) representing two pairs of diastereoisomers. The molar ellipticity values of the modified dimers were approximately twofold higher than those of the modified guanosine monomers. These values were decreased appreciably when the spectra of the dimers were obtained at 80 degrees C or in methanol rather than at 25 degrees C in water, suggesting that under the latter conditions there is a stacking interaction between the carcinogen and the neighboring uridine residue. Based on these results, a conformation is proposed for modified GpU. It includes insertion of the benzo[a]pyrene moiety, by rotation of the modified guanine residue about its glycoside bond, coplanar to the neighboring uridine and perpendicular to the phosphodiester backbone.     

Mutagenicity of benzo[a]pyrene 7,8-dihydrodiol and 7,12-dimethylbenz[a]anthracene 3,4-dihydrodiol in S. typhimurium mediated by microsomes from rat liver and mouse skin

The mutagenic activities of trans-7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene (BP 7,8-diol) and of trans-3,4-dihydroxy-7,12-dimethylbenz[a]-anthracene (DMBA 3,4-diol) towards S. typhimurium TA100 were measured in assays that were carried out on a micro-scale in liquid medium in the presence of microsomal fractions prepared from mouse skin or rat liver. In the presence of an NADPH-generating system, microsomal enzymes converted both diols into mutagens that were probably the respective 'bay-region' diol-epoxides. The rate of the enzyme-catalysed conversion of the BP 7,8-diol into mutagens by microsomal preparations from mouse epidermis was similar to that occurring with microsomes from rat liver. Pretreatment of mice by the topical application of benz[a]anthracene (BA) or 7,12-dimethylbenz[a]-anthracene (DMBA) increased the mutagenic activity of BP 7,8-diol mediated by mouse skin microsomal preparations by 2-fold and this was paralleled by a 4-fold increase in epidermal aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity. The results are discussed in relation to the high susceptibility of mouse skin to polycyclic aromatic hydrocarbon (PAH) carcinogenesis.     

A chemiluminescence assay specific for the microsomal metabolite, benzo[a]pyrene-7,8-dihydrodiol

It is possible to assay for trans-7,8-dihydroxy 7,8-dihydrobenzo[a]-pyrene (BP-7,8-dihydrodiol) in complex metabolite mixtures produced during microsomal metabolism of benzo[a]pyrene (BP) because only the BP-7,8-dihydrodiol metabolite will produce significant chemiluminescence (CL) in the NaOCl-H2O2 singlet oxygen-generating system. The limiting CL sensitivity is 30 pmol in a 1-ml CL reaction mixture. CL assays for BP-7,8-dihydrodiol in microsomal reaction solutions gave concentrations identical with those determined by calibrated high-performance liquid chromatography.     

Activation of benzo(a)pyrene-7,8-dihydrodiol in rat uterus: an in vitro study

Peroxidatic metabolism of benzo(a)pyrene-7,8-dihydrodiol by calcium containing extracts of rat uteri was investigated. Covalently bound and soluble metabolites of benzo(a)pyrene-7,8-dihydrodiol were quantitated by radiometry and high performance liquid chromatography, respectively. 1. Uterine extracts incubated with benzo(a)pyrene-7,8-dihydrodiol activated this proximate mutagen to protein binding metabolite(s). 2. Hydrogen peroxide increased the protein binding and yielded a substantial amount of benzo(a)pyrene-trans-anti-tetrahydrotetrol, suggesting the peroxyl-type free-radical epoxidation process. 3. The results indicate that rat uterine peroxidase is able to catalyze free-radical activation of benzo(a)pyrene-7,8-dihydrodiol by epoxidation to its 9,10-dihydrodiolepoxide, a known ultimate mutagen and carcinogen.     

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (?)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (?)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (?)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ～30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ～5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.     

Quinone formation from carcinogenic benzo[a]pyrene mediated by lipid peroxidation in phosphatidylcholine liposomes

The behavior of benzo[a]pyrene (B[a]P) during peroxidation of phosphatidylcholine (PC) liposomes initiated by an azo compound was investigated to examine the mechanism of quinone formation from carcinogenic B[a]P mediated by nonenzymatic lipid peroxidation occurring in vivo. B[a]P had a retarding effect on the peroxidation of polyunsaturated fatty acid moiety of PC. The major oxidation products which accumulated in the peroxidized liposomes were B[a]P 1,6-, 3,6-, and 6,12-quinone. Antioxidants acting as scavengers of chain-propagating lipid peroxy radicals effectively prevented not only lipid peroxidation but also B[a]P oxidation in the liposomal suspension. PC hydroperoxides, the primary products of PC oxidation, did not react with B[a]P in the absence of the azo compound, indicating that lipid peroxy radicals, not lipid hydroperoxides, are responsible for the formation of these quinones. The experiments using 18O2 gas and 18O-labeled methyl linoleate hydroperoxides demonstrated that B[a]P quinones are formed by incorporating molecular oxygen and their origin is partly due to the lipid peroxy radical. The mechanism proposed for the formation of B[a]P quinones mediated by peroxidation of membrane lipids involves a direct attack of the lipid peroxy radical on B[a]P and subsequent autocatalytic oxidation. Weak carcinogenic and noncarcinogenic pentacyclic aromatic hydrocarbons showed little reactivity to the lipid peroxy radical in the liposomes. Thus, the facility of the peroxidative attack on B[a]P may be related to the powerful carcinogenic activity of this substance.     

Ellagic acid toxicity and interaction with benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol in human bronchial epithelial cells

Ellagic acid, a plant phenol present in various foods consumed by humans, has been reported to have both anti-mutagenic and anti-carcinogenic potential. To evaluate the potential anti-carcinogenic property of ellagic acid, we tested its effects on the toxicity of ben-zo[a]pyrene and benzo[a]pyrene, 7,8-dihydrodiol and binding of benzo[a]yrene to DNA in cultured human bronchial epithelial cells. The toxicity of ellagic acid itself for human bronchial epithelial cells was also determined. Using a colony-forming efficiency assay, it was found that a nontoxic concentration of ellagic acid (5 g/ml) enhanced the toxicity of benzo[a]pyrene.7,8-dihydrodiol in human bronchial epithelial cells. In contrast, ellagic acid at concentrations of l.5 and 3.0 g/ml inhibited binding of benzo[a]pyrenemetabolites to DNA in these cells. An explanation for the potentiating effect of ellagic acid on the toxicity of benzo[a]pyrene, 7,8-dihydrodiol will require further investigation into the possible mechanisms of interaction between these two compounds.Abbreviations B[a]P benzo[a]pyrene - B[a]P 7,8-DHD (±)trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene - B[a]PDE-1 (±)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-2 (±) 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-1:dG N²-]10{7,8,9-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - B[a]PDE-2:dG NZ-{10-[7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - CFE colony forming efficiency - EA ellagic acid - HBE human bronchial epithelial     

Benzo[a]pyrene-7,8-quinone-3'-mononucleotide adduct standards for 32P postlabeling analyses: detection of benzo[a]pyrene-7,8-quinone-calf thymus DNA adducts

Benzo[a]pyrene-7,8-quinone (BPQ) is one of the reactive metabolites of the widely distributed archetypal polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P). The formation of BPQ from B[a]P through trans-7,8-dihydroxy-7,8-dihydroB[a]P by the mediation of aldo-keto reductases and its role in the genotoxicity and carcinogenesis of B[a]P currently are under extensive investigation. Toxicity pathways related to BPQ are believed to include both stable and unstable (depurinating) DNA adduct formation as well as reactive oxygen species. We previously reported the complete characterization of four novel stable BPQ-deoxyguanosine (dG) and two BPQ-deoxyadenosine (dA) adducts (Balu et al., Chem. Res. Toxicol. 17 (2004) 827-838). However, the identification of BPQ-DNA adducts by 32P postlabeling methods from in vitro and in vivo exposures required 3'-monophosphate derivatives of BPQ-dG, BPQ-dA, and BPQ-deoxycytidine (dC) as standards. Therefore, in the current study, BPQ adducts of dGMP(3'), dAMP(3'), and dCMP(3') were prepared. The syntheses of the BPQ-3'-mononucleotide standards were carried out in a manner similar to that reported previously for the nucleoside analogs. Reaction products were characterized by UV, LC/MS analyses, and one- and two-dimensional NMR techniques. The spectral studies indicated that all adducts existed as diastereomeric mixtures. Furthermore, the structural identities of the novel BPQ-dGMP, BPQ-dAMP, and BPQ-dCMP adducts were confirmed by acid phosphatase dephosphorylation of the BPQ-nucleotide adducts to the corresponding known BPQ-nucleoside adduct standards. The BPQ-dGMP, BPQ-dAMP, and BPQ-dCMP adduct standards were used in 32P postlabeling studies to identify BPQ adducts formed in vitro with calf thymus DNA and DNA homopolymers. 32P postlabeling analysis revealed the formation of 8 major and at least 10 minor calf thymus DNA adducts. Of these BPQ-DNA adducts, the following were identified: 1 BPQ-dGMP adduct, 2 BPQ-dAMP adducts, and 3 BPQ-dCMP adducts. This study represents the first reported example of the characterization of stable BPQ-DNA adducts in isolated mammalian DNA and is expected to contribute significantly to the future BPQ-DNA adduct studies in vivo and thereby to the contribution of BPQ in B[a]P carcinogenesis.     

Status of reduced glutathione in primary cultures of rat hepatocytes and the effect on conjugation of benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide

The intracellular level of reduced glutathione (GSH) and GSH conjugation have been investigated in primary cell cultures of hepatocytes isolated from control rats, phenobarbitone (PB) and 3-methylcholanthrene (MC) treated rats. The data demonstrate that in all cell cultures the GSH concentrations show a triphasic pattern: (i) within 1 h of culture an initial marked decrease to 50% of the levels found in fresh hepatocytes; (ii) recovery of GSH concentrations to above the levels observed in fresh cells. This occurs after 6 h in culture with control cells and after 10-24 h with cells from either PB or MC treated rats and was most prominent in cells from PB-treated rats. (iii) A slow decline to between 30 and 40 nmol GSH/mg protein from 24 to 96 h in culture. Synthesis of GSH was slower in cultured cells from PB treated rats and this was confirmed by the resynthesis rates when diethylmaleate (DEM) was used to deplete GSH. The formation of GSH conjugates with racemic 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) was measured in control cells in suspension and after 3 and 24 h in culture. Despite the decrease in GSH concentrations observed between 1 and 4 h after culture, the conjugation rates were not decreased.     

Epoxidation of benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 in reconstituted membranes. Effects of charge and nonbilayer phase propensity of the membrane.

Human cytochrome P4501A1 (CYP1A1) is one of the key enzymes in the bioactivation of environmental pollutants such as benzo[a]pyrene (B[a]P) and other polycyclic aromatic hydrocarbons. To evaluate the effect of membrane properties and distinct phospholipids on the activity of human CYP1A1 purified insect cell-expressed human CYP1A1 and of human NADPH-P450 reductase were reconstituted into phospholipid vesicle membranes. Conversion rates of up to 36 pmol x min(-1) x pmol(-1) CYP1A1 of the enantiomeric promutagens (-)- and (+)-trans-7,8-dihydroxy-7,8-dihydro-B[a]P (7,8-diol) to the genotoxic diolepoxides were achieved. The highest rates were obtained when negatively charged lipids such as phosphatidylserine and phosphatidylinositol and/or nonbilayer phospholipids such as phosphatidylethanolamine were present in the membrane together with neutral lipids. Both Vmax and Km values were changed. This suggests a rather complex mechanism of stimulation which might include altered substrate binding as well as more effective interaction between CYP1A1 and NADPH-P450 reductase. Furthermore, the ratio of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE2) to r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE1) formed from (-)-7,8-diol was significantly increased by the introduction of anionic lipids, but not by that of nonbilayer lipids. Thus, charged lipids affect the stereoselectivity of the epoxidation by leading to the formation of a larger amount of the ultimate mutagen DE2 than of DE1, which is far less carcinogenic. These data suggest that membrane properties such as negative charge and nonbilayer phase propensity are important for the efficiency and selectivity of enzymatic function of human CYP1A1.     

Characterization of mercapturic acid and glutathionyl conjugates of benzo[a]pyrene-7,8-dione by two-dimensional NMR.

Non-K-region polycyclic aromatic hydrocarbon (PAH) o-quinones represent alternative metabolites of PAH trans-dihydro diol proximate carcinogens. These PAH o-quinones react readily with glutathione and N-acetyl-L-cysteine, and these adducts may be responsible for their detoxication. Reactions between benzo[a]pyrene-7,8-dione and either N-acetyl-L-cysteine or glutathione gave three predominant products which were purified by semipreparative reverse-phase high-pressure liquid chromatography and characterized by homonuclear two-dimensional correlation spectroscopy (COSY). The first product corresponded to a Michael type, 1,4-addition product isolated at the level of quinone oxidation. The second product converted to the first and is a presumptive 1,4-addition product isolated at the level of hydroquinone oxidation. The third product was 7,8-dihydroxybenzo[a]pyrene (a hydroquinone) and was formed as a result of the reductive potential of the thiol. Additional proof for the catechol structure was obtained by its conversion to its diacetate and its identity with authentic 7,8-diacetoxybenzo[a]pyrene. The structures of these adducts and intermediates confirm that thiol addition involves formation of the ketol and rearrangement to give a catechol followed by oxidation to yield the quinone adduct. No evidence was obtained for the formation of either bisphenol or bisglutathionyl adducts. The COSY spectra provide the first complete structure of a benzo[a]pyrenyl-peptide conjugate.     

In vitro inhibition of the metabolism and mutagenicity of benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol by naphthazarin and other naphthol derivatives

Among naphthol derivatives tested in the Ames assay, 5,8-dihydroxy-1,4-naphthoquinone or naphthazarin was found to be the most effective inhibitor of benzo(a)pyrene mutagenicity. The inhibitory activity is due in part to the redox cycling of naphthazarin with the concommitant transfer of reducing equivalents from NADPH to molecular oxygen, thus diverting electrons from cytochrome P-450 enzymes. Metabolite separations showed a decrease in microsomal metabolism of benzo(a)pyrene and of benzo(a)pyrene-7,8-dihydrodoil upon addition of naphthazarin. Since both NADP and dicoumarol inhibited the naphthazarin-stimulated non-stoichiometric consumption of NADPH and oxygen then naphthazarin redox cycling probably involves both DT-diaphorase and NADPH cytochrome P-450 reductase.