Catechol-induced alterations in metabolic activation and binding of enantiomeric and racemic 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrenes to DNA in mouse skin

Catechol (1,2-dihydroxybenzene) is a potent co-carcinogen withbenzo[a]pyrene (BaP) and with (?)-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene(BaP-7, 8-diol) in mouse skin. The effects of catechol on themetabolic activation of (+)- and (–)- [³H]BaP-7,8-diolsand on epidermal DNA adduct formation of racemic and enantiomeric[³H]BaP-7, 8-diols were examined by applying the tritlated diolsto mouse skin. The major metabolite of the (+)- [³H]BaP-7, 8-diolswas the hydrolysis product of (–)- [³H]-7,8ß-diolsepoxy-9ß,10ß-epoxy-7,8,9, 10-tetrahydroheiizo[a]pyrene (anti-BPDE).This suggests that a peroxyl radical-mediated pathway Is predominantlyresponsible for the epoxidation of this diol. Formation of (–)-anti[³H]BPDEfrom (+)-[³H]BaP-7,8-diol was greater than that of (+)-anti-BPDEfrom (–)-[³H]BaP-7,8-diol Co-application of catechol with[³H]BaP-7,8-diols inhibited epoxidation of the (+) enantiomerto a greater extent than that of the (–) enantiomer. Catecholdecreased the total DNA-binding and the formation of the majoradduct with (+)-[³H]BaP-7, 8-diols metabolites but catecholhad no significant effect on the binding and formation of (+)-anti-[³H]BPDE-deoxyguanosine the major DNA adduct derived from (–)-[³H]BaP-7,8-diolsCo-administration of catechol with (?)-[³H]BaP-7,8-diols increasedthe ratio of (–)- to (+)-[³H]BaP-7, 8-diols major DNAadducts in mouse skin suggesting that catechol selectively inhibitscertain pathways of metabolic activation of (? )-[³H]BaP-7,8-diols Thus, catechol modifies the tumorigenic activity of(?)- BaP-7 ,8.-diol either by alteration of the relative proportionof various hydrocarbon:DNA adducts or by a totally differentas yet unexplored mechanism.     

Dosimetry of PAH skin carcinogenesis: covalent binding of benzo[a]pyrene to mouse epidermal DNA

Benzo[a]pyrene (BaP) is metabolized to the chemically reactiveanti and syn isomers of the 7,8-diol-9,10-epoxides of BaP (BPDE)which bind covalently to DNA to form DNA/BPDE complexes. Tetrolsliberated from the DNA/BPDE complex by acid hydrolysis are easilyquantified by h.p.l.c. using fluorescence detection. This approachallows femtomole amounts of BPDE associated with the DNA isolatedfrom a single mouse to be detected using conventional instrumentation.The usefulness of this technique to estimate the interactionof BaP with DNA of mouse skin, both in the intact animal andin organ culture, was investigated. With mouse skin in organculture it could be demonstrated that: (i) upon a single topicalapplication of 5 µg of BaP, binding to DNA occurred viaBPDE at a linear rate for up to 65 h, (ii) the amount of bindingwas dose dependent at concentrations of BaP <10 µg.     

Physical interactions of isomeric benzo[a]pyrene diol-epoxides with DNA

The physical interactions of two diol-epoxides derived frombenzo[a]pyrene (B[a]P), 7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydroB[a]P (BPDE) and 9,10-dihydroxy-7,8-oxy-7,8,9,10-tetrahydroB[a]P, (reverse BPDE) with DNA have been studied in a simple,in vitro system. The effects of DNA on the rates of hydrolysisof BPDE and reverse BPDE were studied by fluorescence spectroscopy.For both compounds, interaction with DNA was indicated by anincrease in the rate of hydrolysis in the presence of DNA. Thisincreased hydrolysis was more marked for BPDE than for reverseBPDE. Direct confirmation of physical binding was obtained byu.v. spectroscopy, where a 10 nm redshift in absorbance maximacharacteristic of polycyclic aromatic hydrocarbon DNA intercalationcomplexes was observed. Using absorbance changes to monitorbinding, association constants of 6580 L/mol and 5080 L/molwere determined for BPDE and reverse BPDE, respectively. Consistentwith the intercalation model, binding was inhibited by low concentrationsof Mg²⁺. The enhancement of hydrolytic rate by DNA for BPDEand reverse BPDE was also inhibited by low concentrations ofMg²⁺, suggesting involvement of intercalation complexes in themechanism of enhanced hydrolysis.     

Benzo[a]pyrene-DNA adduct formation in target and activator cells in a Wistar rat embryo cell-mediated V79 cell mutation assay

To determine the relationship of the benzo[a]pyrene (BaP)-DNAadducts formed in the activator cells of a cell-mediated mutationassay to the adducts formed in the target cells and to mutationinduction, irradiated second passage Wistar rat embryo (WRE)cells and V79 Chinese hamster lung cells were exposed to [³H]BaPfor 5, 24 and 48 h under the conditions of a cell-mediated mutationassay. The V79 target cells were separated from the WRE activatorcells by an immunoseparation procedure; the resulting V79 cellpellet contained <7% WRE cells. The percentage of the BaP-DNAadducts containing cis vicinal hydroxyl groups and the h.p.l.cprofile of individual adducts in the V79 target cells were similarto those of the WRE activator cells for each time point. Thetransfer of reactive BaP metabolites from the activator cellsto the target cell DNA was detectable after only 5 h of exposureto BaP, however, exposure for this length of time did not resultin significant mutation induction. The (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(anti-BaPDE)-deoxyguanosine (dGuo) adduct was essentially absentafter 5 h of exposure, but the amount of this adduct increasedat longer times of exposure as did the mutation frequency. Thecomplex mixture of BaP-DNA adducts formed in the WRE cells wasalso present in the V79 cells after all three times of exposureto BaP, a result which demonstrates the value of this cell-mediatedmutation assay for investigating the role of species-specificdifferences in the activation of BaP. The correlation betweenthe increase in mutation induction and the relative amount ofthe (+)-anti-BaPDE-dGuo adduct present in V79 cell DNA suggeststhe importance of this adduct in mutation induction by BaP.     

Effects of catechol on the induction of tumors in mouse skin by 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrenes

Catechol (1,2-dihydroxybenzene) is a major phenolic compound present in the co-carcinogenic fraction of cigarette tar. It has been shown to be a potent co-carcinogen with benzo[a]pyrene (BaP) in mouse skin. In this study we have examined the co-carcinogenic and co-initiating activities of catechol with racemic and enantiomeric 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrenes (BaP-7,8-diols) in mouse skin. Similar to enhancement of BaP carcinogenesis, repeated concurrent applications of catechol and (+/-)-BaP-7,8-diol to mouse skin strongly enhanced (+/-)-BaP-7,8-diol tumor multiplicity and tumor incidence, and decreased latency. Co-application of catechol with the racemic or either of the enantiomers of BaP-7,8-diol in a two-stage initiation--promotion protocol increased the tumor initiating activity of racemic BaP-7,8-diol, similar to that of BaP, by approximately 50%, but had no statistically significant effect on the tumor initiating activity of the (+)- or (-)-enantiomers in mouse skin. Thus, catechol is as potent a co-carcinogen with (+/-)-BaP-7,8-diol as it is with BaP. However, as tested here catechol is a weak co-initiator when applied with (+/-)-BaP-7,8-diol or BaP.     

Transport of DNA-adducting metabolites in mouse serum following benzo [a]pyrene administration

Metabolic activation of benzo[]pyrene (BaP) by cellular enzymesis required for DNA adduct formation. In vivo DNA adducts mightalso arise from BaP metabolites supplied via the systemic circulation,rather than from in situ activation. We determined whether electrophilicmetabolites could be detected in mouse serum 4 h after BaP dosing(i.p.) by trapping metabolites with salmon sperm DNA (ssDNA),followed by ³²P-postlabeling analysis for DNA adducts. In vitrostudies demonstrated that mouse serum sequesters BaP-7,8-diol-9,10-epoxide(BPDE) and protects it from hydrolysis. BPDE was rapidly transferredfrom serum to ssDNA or splenocytes, with adduct levels in ssDNA4- to 7-fold greater than in splenocytes. After BaP administration,mouse serum produced two adduct spots when incubated with ssDNA.The major adduct (spot 3) co-chromatographed with a BPDE adductstandard, while the minor adduct (spot 2) was unrelated to BPDE.A BPDE standard curve in control serum was developed to quantitateBPDE levels in dosed serum. These levels ranged from 13.1 to19.1 nM. Tissue DNA contained three adduct spots: spots 2 and3 appeared identical to the respective adducts arising fromdosed serum. BPDE-DNA adducts in tisues were highest in liver,lung and spleen, with kidney and stomach levels significantlylower. Levels of adduct 2 did not correlate with levels of adduct3, especially in spleen where the adduct 2/adduct 3 ratio wasvery low. In vitro studies in which splenocytes were presentedwith both adducting metabolites suggested that splenocytes preferentiallyform adduct 3. These results indicate that two of the threeBaP electrophilic metabolites responsible for cellular DNA damageare present in mouse serum. The levels of BPDE in serum maybe sufficient to account for a substantial portion of the tissueload of BPDE-DNA adducts.     

Oxidation and DNA binding of (+)-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene in mouse epidermis in vivo and effects of coadministration of catechol

Using a stereochemical probe as described by Marnett (Carcinogenesis (Lond.), 8: 1365-1373, 1987), we have investigated the mechanism of oxidation of (+)-[3H]BaP-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene [(+)-[3H]BaP-7,8-diol] in mouse epidermis in vivo. Groups of mice were topically treated with (+)-[3H]BaP-7,8-diol (60 nmol/mouse) and sacrificed at intervals from 1/2 to 8 h post treatment. (-)-Anti- and (+)-syn-7,8-[3H]dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) were formed as metabolites in a ratio of about 4 to 1, respectively, as determined by HPLC analysis of the hydrolysis products. Pretreatment of mice with indomethacin, an inhibitor of prostaglandin H synthase, did not alter the ratio of anti- to syn-BPDE-derived hydrolysis products. Pretreatment of mice with the cytochrome P-450 inducer, beta-naphthoflavone, yielded twice the level of syn-[3H]BPDE in mouse skin at the 1/2-h survival point. However, this enhancing effect diminished over time. Coadministration of 1,2-dihydroxybenzene (catechol) with (+)-[3H]BaP-7,8-diol decreased the formation of (-)-anti-[3H]BPDE and also decreased lipid peroxidation, as measured by the extent of formation of thiobarbituric acid-reactive material in mouse epidermis. Analysis of mouse epidermal DNA adducts 24 h after topical application (+)-[3H]BaP-7,8-diol indicated that the major adduct is not formed from the major metabolite (-)-anti-BPDE. Acid hydrolysis of the major adduct resulted in the formation of a small amount of r-7,c-9,c-10,t-8-tetrahydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene and two unidentified products different from 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydro-BaP. Coadministered catechol suppressed the formation of this adduct by 30%. The present observation suggests that a peroxyl radical-mediated epoxidation pathway is involved in the oxidation of (+)-[3H]BaP-7,8-diol in mouse skin in vivo.     

Sulfite-dependent mutagenicity of benzo[a]pyrene derivatives

Benzo[a]pyrene (BP) and sulfur dioxide (SO₂) are ubiquitousair pollutants and are also components of tobacco smoke. AlthoughSO₂ itself is not carcinogenic, concurrent administration withBP results in enhancement of respiratory tract tumorigenesis.In biological systems, SO₂ exists as its hydrated form, sulfite(SO₃^2–). Sulfite readily undergoes autoxidation, generatingpotent oxidant species. When 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(BP-7,8-diol) is included in sulfite autoxidation mixtures itis converted to more polar products, most notably 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes(BP tetraols). This implies the intermediacy of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetra-hydro-benzo[a]pyrenes (BPDE). We report herethe sulfite-dependent conversion of BP-7,8-diol to forms highlymutagenic to Salmonella typhimurium strain TA 98. This activationis observed at BP-7,8-diol concentrations of from 2 to 40 µMand at sulfite concentrations of from 0.5 to 10 mM. In the presenceof 10 µM BP-7,8-diol, half-maximal activation is observedat 1.6 mM sulfite. Sulfite itself is neither toxic nor mutagenicto the bacteria under these conditions. The time course of theactivation of BP-7,8-diol and its sensitivity to inhibitionby antioxidants indicate a requirement for sulfite autoxidation.These data further support the sulfite-dependent epoxidationof BP-7,8-diol. Not only does sulfite convert this promutagento its active mutagenic form, sulfite also enhances the mutagenicactivity of BP diolepoxides toward the tester strain. The reversionfrequency in response to 0.1–0.5 µM anti-EPDE isincreased by up to 33% in the presence of 1 mM sulfite, andby up to 270% with 10 mM sulfite. The mechanism of this enhancementof anti-BPDE activity is not known, but could be related toinhibition of the glutathione-S-transferase system which hasbeen previously reported for sulfite. These results are discussedin regard to the noted cocarcinogenicity of sulfur dioxide forBP.     

Reactivity of benzo[a]pyrene-7,8-dione with DNA. Evidence for the formation of deoxyguanosine adducts

Polycyclic aromatic hydrocarbon (PAH) o-quinones are productsof the dihydrodiol dehydrogenase-catalyzed oxidation of trans-dihydrodiolswhich are proximate carcinogens. The PAH o-quinones are highlyreactive molecules and have the potential to alkylate DNA. Inthis study, the reactivity of [³H](+/–)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene([³H](+/–)-anti-BPDE), [³H]benzo[a]pyrene-7,8-dione ([³H]BPQ)and [³H](+/–)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene ([³H](+/–)-B[a]P-diol) with DNA were compared.(+/–)-anti-BPDE reacted equally well with native, deproteinatedand deproteinated/sheared calf thymus DNA. In each case DNAadducts were formed which upon digestion to deoxyribonucleosidescomigrated on reverse-phase (RP)-HPLC with adducts synthesizedby reacting (+/–)-anti-BPDE with oligo-p(dG)₁₀. (+/–)-anti-BPDEalso reacted with plasmid (pGEM-3) DNA to yield multiple adductsone of which comigrated with the (+)-anti-BPDE-deoxyguanosineadduct. Under identical conditions [³H]BPQ reacted preferentiallywith native calf thymus DNA but displayed low reactivity withdeproteinated and deproteinated/sheared calf thymus DNA. RP-HPLCanalysis of deoxyribonucleoside—BPQ adducts indicatedthat the predominant adduct formed comigrated with a standardsynthesized by reacting BPQ with oligo-p(dG)₁₀. BPQ also reactedwith pGEM-3 DNA to yield multiple adducts one of which comigratedwith the BPQ—deoxyguanosine adduct. Reactions between[³H]BPQ and poly(dA), poly(dT), poly(dC) and oligo-p(dG)₁₀ indicatedthat BPQ preferentially formed deoxyguanosine adducts. In thisstudy, [³H]BPQ and [³H](+/–)-anti-BPDE covalently labelednative calf thymus DNA to an equal extent, however, less [³H]BPQwas recovered as deoxyguanosine adducts. By contrast, no covalentmodification of calf thymus DNA, pGEM-3 DNA or oligonucleotideswas observed with [³H](+/–)-B[a]P-diol. These studiesindicate that BPQ has the potential to be genotoxic in vitro;that reactivity is heightened in the presence of protein orcircular DNA and that the major adduct formed is a deoxyguanosineadduct.     

Benzo[e]pyrene-induced alterations in the binding of benzo[a]pyrene to DNA in hamster embryo cell cultures

Benzo[e]pyrene (B[e]P), a weakly carcinogenic polycyclic aromatichydrocarbon (PAH) modifies tumor induction in mouse skin andthe induction of mutation in mammalian cells by carcinogenicPAH. To determine how B[e]P alters the metabolic activationof the carcinogen benzo[a]pyrene (B[a]P), early passage Syrianhamster embryo cell cultures were exposed to [³H]B[a]P or [³H]trans-7,8-dihydro-7,8-dihydroxyB[a]P B[a]P-7,8-diol) in the presenceof various concentrations of B[e]P for 24 h. The DNA was isolated,degraded to deoxyribonucleosides and the B[a]P-deoxyribo-nucleosideadducts were analyzed by h.p.l.c. As the dose of B[e]P increased,the amount of B[a]P bound to DNA decreased and the ratio ofanti-B[a]P-7,8-diol-9, 10-epoxide B[a]PDE)-deoxyguanosine adductto syn-B[a]PDE-deoxy-guanosine adduct decreased. B[e]P treatmentinhibited the binding of B[a]P-7,8-diol to DNA to a greaterextent than it inhibited the binding of B[a]P and decreasedthe ratio of anti to syn-B[a]PDE-deoxyguanosine adducts formedfrom the 7,8-diol. These results indicate that B[e]P decreasesthe activation of B[a]P to DNA-binding intermediates in thesecells; especially the oxidation of B[a]P-7,8-diol to a diol-epoxide.The B[e]P-induced alterations in the ratio of DNA adducts formedfrom the syn- and anti-isomers of B[a]PDE suggest that B[e]Pselectively inhibited certain pathways of metabolic activationof B[a]P. Thus, B[e]P-induced modifications in the biologicalactivity of PAH may result from alteration in both the amountsand the relative proportions of various isomeric forms of theultimate carcinogenic metabolites formed from PAH.     

Binding of benzo[a]pyrene metabolites in the rat intestinal lumen by magnetic polyethyleneimine microcapsules following an intragastric dose of [14C]benzo[a]pyrene

Semi-permeable magnetic polyethyleneimine (PEI) microcapsuleshave been developed to trap carcinogens and their metabolitesin vivo and their time-dependent binding of a model carcinogen,[¹⁴C]benzo[a]pyrene ([¹⁴C]BaP), is studied within the intestinallumen. Overall, 0.5% of an intragastrk BaP dose was bound bythese microcapsules recovered from faeces with specific bindingof metabolites (nmol/10⁶ recovered microcapsules) being similarin the 0–24-h and 24–48-h periods, but 10-foldlower in the 48–72-h period. Successive extractions ofmicrocapsules with ammoniacal methanol, 2.5 N HCI, methanoland dimethylsulfoxide released 60% of bound radiolabel andthe unextracted radiolabel was presumed to have been bound covalently.By contrast, > 90% of bound radiolabel was extractable fromthe faeces of the treated animals and from microcapsules treatedin vitro with [¹⁴C]7,8-dihydroxy-9,10-epoxytetrahydrobenzo[a]-pyrene(BaPDE), indicating that the in vivo microcapsule- bound metaboliteswere not derived either from adsorbed faecal material or from[¹⁴C]BaPDE formed in situ. A time-dependent appearance of BaP3,6-dione was found. Also the qualitative and quantitative patternsof metabolites trapped by microcapsules, as assayed by h.p.l.c,were consistent only with a unique set of BaP metabolites beingbound within the intestinal lumen. Hence these carcinogen-bindingmicrocapsules can be used to investigate the in situ formationof carcinogen metabolites within the intestinal tract.     

Ellagic acid: a potent naturally occurring inhibitor of benzo[a]pyrene metabolism and its subsequent glucuronidation, sulfation and covalent binding to DNA in cultured BALB/C mouse keratinocytes

The metabolism of [³H]benzo[a]pyrene (BP) by cultured primarykeratinocytes prepared from BALB/C mouse epidermis was foundto be largely inhibited by the dietary plant phenol, ellagicacid. Varying concentrations of ellagic acid added to the keratinocytecultures resulted in a dosedependent inhibition of the cytochromeP-450-dependent monooxygenases aryl hydrocarbon hydroxylase(AHH) and 7-ethoxycoumarin-0-deethylase (ECD). The major organicsolvent-extractable metabolites found intracellularly in thecultured cells were trans-7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene(BP-7,8-diol) and 3-hydroxybenzo[a]pyrene (3-OH-BP), althoughsmall amounts of 9-hydroxybenzo[a]pyrene, quinones and trans-9,10-dihydro-9,10-dihydroxybenzo[a]-pyrene(BP-9,10-diol) were also present. The major organic solvent-extractablemetabolites found in the extracellular culture medium were BP-7,8-dioland BP-9,10-diol, with smaller quantities of unconjugated phenolsand quinones. The major intracellular and extracellular water-solublemetabolites of BP were conjugated with glucuronide (primarily3-OH-BP and several BP-quinones), and to a lesser extent withsulfate (primarily BP-7,8-diol). Both intracellular and extracellularmetabolism of organic solvent-extractable and water-solubleconjugates was significantly inhibited by ellagic acid in adose-dependent manner. The intracellular enzyme-mediated bindingof BP to mouse keratinocyte DNA was also largely inhibited ina dose-dependent fashion by ellagic acid. Our results indicatethat cultured primary mouse keratinocytes offer a useful modelsystem for studying factors affecting the metabolic activationand detoxification of polycyclic aromatic hydrocarbon carcinogensin the epidermis, and that polyphenolic compounds such as ellagicacid may prove useful in modulating the risk of cutaneous cancerthat results from exposure to these environmental chemicals.     

Quantitative significance of glutathione and glutathione-S-transferase in regulating benzo[a]pyrene anti diol-epoxide level in reconstituted C3H/10T1/2 cell lysates, and comparison to rat liver

Induced 10T1/2 cell microsomes were independently reconstitutedwith [³H]benzo[a]pyrene (BP) and glutathione (GSH) or purifiedGSH-transferases. Levels of the primary BP anti 7,8-dihydrodiol9,10-epoxide (r-7,t-8 dihydroxy-t-9,10-oxy-7,8,9,10 tetrahydrobenzo[a]pyrene)hydrolysis product, 7,10/8,9-tetrol, were measured in incubationextracts, enabling us to monitor the level of free anti diol-epoxidein incubations and to determine the independent effects of GSHor GSH-transferases upon it. GSH alone had no effect on antidiol-epoxide levels over the concentration range tested (0–4.0mM), however, the addition of purified GSH-transferase fromrat liver resulted in a dose-dependent conjugation of anti diol-epoxideas well as 9,10-epoxide and 7,8-epoxide with 50% conjugationoccurring at 0.036, 0.039 and 0.17 units GSH-transferase/ml,respectively. Free anti diol-epoxide was reduced by >95%when we reconstituted with the GSH-transferase concentrationwhich we measured in 10T1/2 cells (0.15–0.27 units/mlcell cytosol); this GSH-transferase concentration representsonly 6% of that found in rat liver. The results suggest thatin both 10T1/2 cells and rat hepatocytes GSH-transferase catalyzedGSH conjugation is quantitatively significant in determiningthe intracellular level of anti diol-epoxide.     

Persistence of benzo[a]pyrene and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene in Fischer 344 rats: time distribution of total metabolites in blood, urine and feces

A comparison of the rates of elimination of [³H]benzo[a] pyrene(BaP) and 7,8-dihydro-7,8-diol-[³H]benzo[a]pyrene (BPD), aftersubcutaneous injection into Fischer 344 rats, shows they areboth eliminated at about the same rates and with the same patternover at least 7 days post-exposure. The end-rate of combinedurinary and fecal excretion was {small tilde}40 nmol/day. About20% of the injected BaP and {small tilde}3% of the injectedBPD remained at the site of injection for at least 9 days. Theremainder was distributed throughout the animal. If the rateof excretion continued at the observed steady-state rates, theBaP and BPD could persist for up to 40 days for each milligramof injected substance. The concentration of excretion productswere highest during day 1 and day 2 following exposure, decreasedexponentially to a concentration of {small tilde}0.5 µM(mixed metabolites) by day 5 following exposure, and then continuedto be excreted at that rate. Feces contained the highest totalamounts of radioactivity, which were {small tilde}2- to 4-foldhigher than the amounts in urine and {small tilde}15- to 50-foldhigher than in total blood. The conversion of organic ³H to³H₂O during the experimental period indicates.that whole-bodyphenol(quinone) formation was significant for BaP metabolism,but was much less for BPD metabolism. When BaP was injected,both blood and urine contained water-soluble, volatile tritiumcounts (³H₂O). Injection of BPD resulted in volatile ³H₂O inurine but not in blood. The persistence of BaP and BPD metabolitesin skin, blood, urine and feces compartments indicates thereis a substantial reservoir of the chemical(s) that could beused to replenish repaired or discarded DNA adducts.     

Synthesis of putative oxidized metabolites of 8-methylbenzo[a]pyrene

Synthesis is described of trans-7,8-dihydroxy-7,8-dihydro-8-methylbenzo[a]pyreneand trans-7,8-dihydroxy-anti-9,10-epoxy-8-methyl-7,8,9,10-tetrahydrobenzo[a]pyrene,potential proximate and ultimate carcinogenic metabolites, respecively,of 8-methylbenzo[a]pyrene.     

Characterization of benzo[a]pyrene metabolites isolated from muscle, liver, and bile of a juvenile flatfish

Juvenile English sole (Parophrys vetulus) were force fed (³H)benzotaipyrene(BaP) and, after 24 h, metabolites were isolated from bile,liver, and muscle. The metabolites were analyzed by t.l.c. andh.p.l.c., and further characterized by u.v., fluorescence, orm.s. analyses. The identities of BaP 7,8-dihydrodiol, BaP 9,10-diydrodiol,1-hydroxy BaP, 3-hydroxy BaP, and 9-hydroxy BaP were confirmed.The amounts of BaP 7,8-dihydrodiol plus its conjugates (sulfatesand glucuronides) were greater than twice the amounts of BaP9,10-dihydrodiol plus its conjugates in bile. Liver also containedtwice as much BaP 7,8-dihydrodiol as BaP 9,10-dihydrodiol, demonstratingthat English sole produced larger amounts of the 7,8-isomer.T.l.c. analysis showed the presence of an unknown metaboliteX migrating between BaP 9,10- and 7,8-dihydrodiols. M.s. showedX to be a non-vicinal dihydroxy derivative of BaP. Spectralproperties of X were similar to those reported for a dihydroxyderivative, presumably the 3,9-dihydroxy BaP, formed from either3- or 9-hydroxy BaP in the presence of rat liver microsomes.Compared to the dihydrodiols, X was resistant to both glucuroni-dationand sulfation. The resistance to form water-soluble metabolitesmay be responsible for the deposition of relatively large amounts(0.11% of the administered dose) of X in muscle; liver containeda much smaller amount (0.01%) of X. Thus, English sole convertedBaP into metabolites known to be toxic to mammals, and thesemetabolites were found in the liver and edible tissue of thefish.     

Formation and tumorigenicity of benzo[b]fluoranthene metabolites in mouse epidermis

The metabolism in mouse epidermis of benzo[b]fluoranthene (BbF)was studied. [³H]BbF was applied topically, mice were killedat various intervals, and metabolites were extracted from theepidermis and analyzed by h.p.l.c The major metabolites wereidentified by comparisons to standards as 4-, 5-, and 6-hydroxyBbF.Sulfate and glucuronide conjugates of these hydroxyBbF werealso detected. Minor metabolites included 12-hydroxyBbF, BbF-1,2-diol,and BbF-11,12-diol. BbF-9,10-diol, the only known tumorigenicoxygenated derivative of BbF, was not detected. The furthermetabolism of BbF-9,10-diol was studied in vitro, using ratliver 9000 g supernatant. The major metabolites were identifiedby their spectral characteristics as 5-and 6-hydroxyBbF-9,10-diol.Little if any BbF-9,10,11,12-tetraol was detected. 5-and 6-HydroxyBbF-9,10-diolwere not detected as metabolites of [³H]BbF in mouse epidermis.Several known and potential BbF metabolites—BbF-1,2-diol,BbF-11,12-diol, BbF-9,10-diol, BbF-9,10-diol-11, 12-epoxide,5-and 6-hydroxyBbF-9,10-diol, 1-hydroxyBbF, 5-hydroxyBbF, and6-hydroxyBbF—were tested for tumor initiating activityon mouse skin. Among these, only BbF-9,10-diol showed high tumorigenicactivity, but no evidence has been obtained for its formationin vivo from BbF. These studies do not support the hypothesisthat BbF is metabolically activated through formation of thebay region diol epoxide, BbF-9,10-diol-11,12-epoxide.     

Separation and characterization of post-labeled DNA adducts of stereoisomers of benzo[a]pyrene-7,8-diol-9,10-epoxide by immobilized boronate chromatography and HPLC analysis

The carcinogenic polycydlic aromatic hydrocarbon (PAH) benzo[a]pyrene(BaP) is enzymatically activated in cells to an ultimate carcinogenicmetabolite, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BaPDE),which reacts with DNA to form covalent adducts involved in theinitiation of cancer. Previously, a post-labeling procedurethat uses adenosne-5'-O-(3'-[³⁵S]-thiotriphosphate) was developedto facilitate adduct analysis by HPLC. The much greater carcinogenicpotency of (+)-anti-BaPDE makes it essential to be able to separateand identify the adducts formed by all four BaPDE enantiomersin DNA of cells exposed to BaP. Reversed-phase HPLC (RPHPLC)resolved the major (+)-anti-BaPDE-N²-deoxyguanosine [(+)-anti-BaPDE-N²-dG]adduct from the (+)-syn-BaPDE-N²-dG adduct. However, anti-BaPDE-N²-dGadducts formed by (+)-and (–)-anti-BaPDE were not resolved.By using ion-pair RPHPLC (IP-RPHPLC) with tetrabutylammoniumphosphate, the [³⁵S]post-labeled (–)-anti-BaPDE-N²-dGadduct eluted 3 min prior to the [³⁵S]labeled (+)-anti-BaPDE-N²-dGadduct. In contrast, the major syn-BaPDE-N²-dG adducts wereresolved better by RPHPLC than by IP-RPHPLC. The differencein conditions required for optimal separation of anti- and syn-BaPDE-DNAadducts necessitated the development of an immobilized boronatechromatography technique for the separation of anti- from syn-BaPDE-DNAadducts prior to analytical HPLC analysis. At 4°C and withelution buffers containing high salt concentrations, the [³⁵S]post-labeledanti-BaPDE-DNA adducts were selectively retained by a boronatecolumn whereas the [³⁵S]labeled syn- BaPDE-DNA adducts werenot. Analysis of the multiple BaP-DNA adducts formed in BaP-treatedhamster embryo cells by these techniques gave results comparableto those obtained by other methods. The major BaP-DNA adductswere anti-BaPDE-N²-dG 14% from (–)- and 86% from (+)-anti-BaPDE.The ability of these techniques to detect low levels of PAH-DNAadducts because of the high specific radioactivity of ³⁵S andto separate the DNA adducts formed by stereolsomeric PAN diolepoxides adducts by boronate chromatography and HPLC will facilitatestudies of the role of individual PAH-DNA adducts in the inductionof biological effects such as toxicity and carcinogenesis.     

Benzo[a]pyrene--DNA adduct formation in target cells in a cell-mediated mutation assay

In order to analyze the adducts formed in V79 Chinese hamstertarget cells in a Syrian hamster embryo (HE) cell-mediated mutationassay, a procedure was developed in which the HE cells are differentiallykilled when the mixed cell suspension is treated with antiserumto Syrian HE cells and complement. The V79 cell suspension,separated from lysed HE cells by gradient centrifugation, is> 90% HE cell-free. When the carcinogen - DNA interactionproducts formed in these two cell types were analyzed afterexposure to [³H]benzo[a]pyrene (B[a]P) for 24 h under the conditionsof a cell-mediated mutation assay, the major B[a]P-DNA adductsin both cell types resulted from reaction of the anti and synisomers of B[a]P-7,8-diol-9,10-epoxide with DNA. The amountof B[a]P bound/mg DNA in the target cells was only 30% lessthan in the activator cells, and the relative proportions ofthe adducts of the syn and anti isomers were similar in thetwo cell types. The target cells, which do not metabolize B[a]P,were also unable to metabolically activate B[a]P-7,8-diol toDNA-binding metabolites. Thus, the transfer of activated B[a]Pmetabolites from activator cells to the DNA of target cellsis a relatively efficient process that appears to be independentof the relative reactivity of specific metabolites with cellularnucleophiles. The immunological cell separation procedure wedescribe can be adapted to the analysis of carcinogenl-cellinteraction products formed in cell-mediated assays in whichother types of activator and target cells are used to measureeither mutation or another biological endpoint.     

The enzymatic conjugation of glutathione with bay-region diol-epoxides of benzo[a]pyrene, benz[a]anthracene and chrysene

The kinetics of the enzymatic conjugation of glutathione (GSH)with the anti-diastereoisomers of trans-7, 8-dihydroxy-9, 10-epoxy-7,8, 9, 10-tetrahydrobenzo[a]pyrene (BPDE), trans-3, 4-dihydroxy-1,2-epoxy-1, 2, 3, 4-tetrahyd-robenz[a] anthracene (BADE) andtrans-1, 2-dihydroxy-3, 4-epoxy-1, 2, 3, 4-tetrahydrochrysene(CDE) catalyzed by transferase 4-4 from rat liver have beencompared. When the concentration of these diol-epoxides wasvaried (using 2mM GSH) the apparent V_max values were 560, 2100and 1500 nmol/ing/min for (?)-anti-BPDE, (?)-anti-BADE and (?)-anti-CDE,respectively, with corresponding apparent K_m values of 11, 125and 105 µM. The catalytic efficiency of transferase 4–4in the GSH conjugation of (?)-anti-BADE and (?)-anti-CDE isthus approximately one-third of (?)-anti-BPDE (0.014 and 0.012s^-1 µM^-1 respectively versus 0.042 s^-1µM^-1). Similarnon-linear Lineweaver-Burk plots were obtained with each diol-epoxidewhen the concentration of GSH was varied, and two apparent.K_m values of 0.02–0.04 and 0.4–0.9 mM GSH were estimated.The GSH-conjugates formed with the individual enantiomers ofthe recemic substrates used were resolved by h.p.l.c. The dataindicate that with each diol-epoxide transferase 4–4 ishighly selective(95%) towards the biologically most active (+)-enantiomer.