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.     

Metabolism of benzo[a]pyrene-7,8-dihydrodiol and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide to protein-binding products and glutathione conjugates in isolated rat hepatocytes

Isolated hepatocytes from 3-methylcholanthrene (MC)-treatedrats metabolized trans-7, 8-dihydroxy-7, 8-dihydrobenzo[a]pyrene(BP-7, 8-diol) and (± )-7ß, 8-dihydroxy-9,10-oxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene (anti-BPDE) to watersoluble conjugates including glutathione (GSH) conjugates. Underthe conditions employed 35% of total water soluble productsderived from BP-7, 8-diol could be accounted for by GSH conjugates.The corresponding figure for anti-BPDE was estimated to be >80%.Isolated hepatocytes metabolized BP-7, 8-diol and anti-BPDEto GSH conjugates at maximal rates of 0.5 and 9 nmol per 106cells per min, respectively. Thus, identifying the rate limitingstep in the reaction sequence as the metabolism of BP-7, 8-diolto the GSH conjugating intermediates. In addition to the directconjugation of anti-BPDE with GSH, anti-BPDE but not the correspondingBP-tetraols, was further metabolized to reactive intermediatesthat subsequently bound to cellular proteins or reacted withGSH forming water soluble conjugates. The identity or identitiesof these novel reactive intermediates is discussed.     

Inactivation of DNA-binding metabolites of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by glutathione and glutathione S-transferases

The binding to DNA of reactive metabolites of trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene(BP-7, 8-diol) was studied following the incubation of tritiatedbenzo[a]pyrene (BP) and BP-7, 8-diol with nuclei from liversof 3-methyl-cholanthrene-treated rats. Binding was inhibitedto a small extent by glutathione (GSH) alone and to a much greaterextent by GSH and cytosol or purified GSH-transferases B andE. In this respect GSH-transferases A and C were also active,but less so. Inhibition of binding of BP-7,8-diol metabolitesto DNA mediated by GSH-transferases was associated with theformation of GSH conjugates. The extent of inhibition of bindingwas similar in incubations of nuclei alone, nuclei and rat livermicrosomes, and calf thymus DNA and rat liver microsomes. Thisindicates that reactive metabolites of BP-7, 8-diol, formedeither by nuclei or microsomes, are readily accessible to solubleGSH-transferases. GSH and cytosol were also active in inhibitingDNA-binding of reactive metabolites from 9-hydroxybenzo[a]pyrene(9-OH-BP). Thus, in the rat hepatocyte GSH and GSH-transferasesmay be important in protecting DNA from electrophilic attackby reactive BP-7, 8-diol and 9-OH-BP species.     

Separation by ion-pair high-performance liquid chromatography of the glucuronide, sulfate and glutathione conjugates formed from benzo[a]pyrene in cell cultures from rodents, fish and humans

A large proportion of the metabolites formed from benzo[a]pyrene (BP) in cell cultures from rodents, fish and humans result from conjugation of an oxidized metabolite of BP with sulfate, glucuronic acid or glutathione (GSH). To improve the analysis of these metabolites, a reversed-phase ion-pair h.p.l.c. system using a step gradient of methanol:tetrabutyl-ammonium bromide in ammonium formate buffer has been developed for the separation of these three classes of conjugates. This system separated 3-hydroxy-BP glucuronide and sulfate conjugates and resolved them from GSH conjugates of BP 4,5-oxide, 7,8-oxide and 7,8-diol-9,10-epoxide. Cultures of early passage Syrian hamster, Wistar rat and Sencar mouse embryo cells, a bluegill fry (BF-2) cell line and a human hepatoma cell line (HepG2) were exposed to [3H]BP for 24 h. Medium samples from each were extracted with chloroform: methanol:water, and the water-soluble metabolites were analyzed by ion-pair h.p.l.c. The largest peak of metabolites in the media from cell cultures from rodents and the bluegill fry cell line co-eluted with the glucuronic acid conjugate of 3-hydroxy-BP. These phenol-glucuronides represented 48-62% of the total water-soluble metabolites in the fish and rodent cell cultures. Treatment of this material with beta-glucuronidase released 3-hydroxy-BP and 9-hydroxy-BP in ratios from 3:4 to 13.3:1 in various cultures. Media from the bluegill fry cell line and the mouse embryo cell cultures also contained a peak of BP-diol glucuronides; treatment of these peaks with beta-glucuronidase released mainly BP-7,8-diol. In HepG2 cells, 40% of the water-soluble metabolites were identified as sulfate conjugates of 3-hydroxy-BP and 9-hydroxy-BP. No glucuronic acid conjugates of BP metabolites were detected in HepG2 cells. Only small amounts of the water-soluble metabolites from these cell cultures eluted in the same volumes as the synthetic GSH conjugate of BP-4,5-oxide, BP-7,8-oxide and BP-7,8-diol-9,10-oxide. These studies indicate that conjugation with glucuronic acid represents a major pathway of formation of water-soluble metabolites from BP in cells derived from a number of species and demonstrate the value of this ion-pair h.p.l.c. system for the analysis of conjugates formed from BP.     

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.     

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.     

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.     

In vivo formation and persistence of DNA adducts in mouse and rat skin exposed to ({+/-})-trans-7, 8-dihydroxy-7, 8-dihydrobenzo[a]-pyrene and ({+/-})-7{beta}, 8{alpha}-dihydroxy-9{alpha}, 10{alpha}-epoxy-7, 8, 9, 10-tetrahydro-benzo(a)pyrene

The in vivo DNA adduct formation of (±)-trans-7, 8-dihydroxy-7,8-dihydrobenzo(a)pyrene (BPD) and (±)-7ß, 8-dihydroxy-9,10-epoxy-7, 8, 9, 10-tetrahydrobenzo(a)pyrene (anti-BPDE) werecompared and the persistence and disappearance of the adductsin both mouse and rat epidermis determined. BPD (100 nmol/mousein 150 µl acetone and 200 nmol/rat in 300µl acetone)and anti-BPDE (77 nmol/mouse in 150 µJ tetrahydrofuran)and 154 nmol/rat in 300 µ tetra-hydrofuran) were topicallyapplied to 50-day-old male Swiss mice and 35-day-old Wistarrats. To improve the identification of the DNA adducts formed,an acid hydrolysis technique was used to convert the BPD- andanti-BPDE- de-oxyribonucleoside adducts formed in mouse andrat skin to BP tetrols. The modified deoxyribonucleosides andBP tetrols obtained by hydrolysis of adducts were isolated byreverse-phase h.p.l.c. At approximately similar doses per unitarea of treated skin, the initial total binding of these compoundsto epidermal DNA and the level of modified deox-yribonucleosideswas 6-fold lower in rat skin epidermis than in mouse skin epidermis.Similar ratios of (±)-anti-BPDE-deoxyguanosine (dGuo)to (±)-syn-BPDE-dGuo adducts (5.7 and 6.1, determinedby h.p.l.c. analysis of BP tetrols obtained by hydrolysis ofmodified dGuo) were found in both mouse and rat epidermis ashort time (6 h)after topical application of (±)-trans-BPD.Three hours after topical application of (±)-anti-BPDE,the ratios of BP-7, 10/8, 9-tetrol to 7/8, 9, 10-tetrol were9: 1 in mouse epidermal DNA and 6: 1 in rat epidermal DNA. Oneand three weeks after application of these two compounds, only(+)-anti-BPDE-dGuo was detected in mouse epidermis; 2 and 0.2%of the initial (+)-anti-BPDE-dGuo level was found to persistin the epidermal DNA from BPD- and anti-BPDE-treated mice respectively.No DNA adducts were detected in rat epidermis 3 weeks afterBPD and anti-BPDE treatment. Thus, 3 weeks after topical applicationof BPD and anti-BPDE to mouse and rat skin, the DNA adductscompletely disappeared form rat epidermis while they persistedin mouse epidermis. The results suggest that: (i) the persistenceof (+)-anti-BPDE-dGuo may be related to carcinogenesis in mouseepidermis by BPD and anti-BPDE; (ii) the complete disappearanceof the anti-BPDE-dGuo adduct may also account in part for therelative resistance of tissue from this species to the carcinogenicaction of benzo(a) pyrene.     

Prostaglandin H synthase-dependent co-oxygenation of ({+/-})-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in hamster trachea and human bronchus explants

The role of prostaglandin H synthase (PHS) in the metabolismof 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) hasbeen examined in short-term explant cultures of hamster andhuman tracheobronchial tissues. Labeled BP-7,8-diol was incubatedwith the explants in the presence and absence of the PHS substratearachidonic acid (20:4) and the PHS inhibitor indomethadn. Theaddition of 10 µM to 200 µM 20:4 to incubationsof hamster trachea with 5 µM BP-7,8-diol caused significantincreases in the formation of 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(anti-BPDE). These increases were not seen when 1 µM or20 µM BP-7,8-diol was employed. The stimulation of anti-BPDEformation was observed after incubations of from 1 to 48 h.This stimulation was inhibited to the basal level by 20 µMindomethacin, supporting the role of PHS in the response. Noeffect of 20:4 was seen on the uptake of BP-7,8-diol by thetracheas or on the formation of water-soluble metabolites. Significantincreases in covalent binding of BP-7,8-diol metabolites toDNA of the tracheal epithelium were also elicited by the additionof 20:4, however these increases were not well correlated quantitativelywith the increases in anti-BPDE formation. H.p.l.c. profilesof deoxynucleoside adducts from basal and 20:4-stimulated incubationswere qualitatively identical. Far greater variability of metabolismwas seen in human bronchus explants, but 20:4-dependent increasesin anti-BPDE formation could be demonstrated in those tissuesas well. Inhibition of this stimulation by indomethacin waseither absent or incomplete. This variation in the effect ofindomethacin was explained by the examination of the productsof 20:4 metabolism by the two tissues. Hamster trachea producedalmost exclusively PHS metabolites whereas human bronchus yieldedpredominantly products of lipoxygenases, enzymes insensitiveto indomethacin. In conclusion, this study indicates that co-oxygenationof chemical carcinogens can occur in hamster and human tracheobronchialtissues. The concentration-dependence observed with BP-7,8-diol,however, suggests that this pathway is of minor importance inthe activation of BP in these tissues.     

Regioselective inhibition of benzo[a]pyrene metabolism by butylated hydroxyanisole

The effects of butylated hydroxyanisole (BHA) on the metabolismof benzo[a]pyrene (BP) were investigated with mouse hepaticmicrosomes. Microsomes from BHA-fed mice showed a higher activityin catalyzing the formation of most metabolites from BP buta much lower activity in the formation of 9-hydroxyBP than controlmicrosomes. Dietary BHA treatment enhanced the total metabolismof BP but decreased the microsomal metabolism of BP-7,8-diol,especially the formation of BP-trans-7,8-diol-anti-9,10-oxide.The altered metabolism of BP is believed to be due to the inductionof new cytochrome P-450 species by dietary BHA. Consistent withthis interpretation is the observation that the treatment alsodecreased the K_m and increased the V_max of ethoxycou-marin O-dealkylase.Short-term treatment (BHA administered p.o.) and in vitro addedBHA were shown to inhibit BP metabolism. Thus, BHA can affectBP metabolism by exerting its inhibitory effect directly andby altering the composition of microsomal monooxygenase enzymesafter a few days of exposure.     

Enhancement of benzo[a]pyrene diol epoxide mutagenicity by sulfite in a mammalian test system

Sulfur dioxide, a ubiquitous air pollutant, is a co-carcinogenfor benzo[a]pyrene (BP). We have demonstrated previously thatthe interaction between sulfite, the physiological form of sulfurdioxide, and (±)-7r, 8t-dihydroxy-9t, 10t-epoxy-7, 8,9, 10-tetrahydrobenzo[a]pyrene (anti-BPDE), the ultimate carcinogenicform of BP, results in an enhanced mutagenic effect in Salmonellatyphimurium strains TA98 and TA100. We report here that thissame co-mutagenic effect of sulfite occurs in a mammalian cellline. Treatment of Chinese hamster V79 cells with 50 nM anti-BPDE,a concentration on the linear portion of the dose-response,resulted in a four-fold increase in mutations at the hprt locusrelative to the spontaneous rate. When V79 cells were exposedto 1 or 10 mM sulfite immediately prior to the addition of anti-BPDE,the mutation rate increased by 73% and 210%, respectively, overthat elicited by anti-BPDE alone. Sulfite itself was moderatelycytotoxic, but caused no increase in mutation over the spontaneousrate. Characterization of the dose- and time-dependance of thisenhancement of diol epoxide mutagenicity by sulfite closelyresembled the effects seen previously in the bacterial system.In particular, enhancement by sulfite was evident when sulfitewas added to the cells between 60 min and 1 min prior to theaddition of the diol epoxide. Concurrent addition of sulfiteand the diol epoxide attenuated the enhancement, and the effectwas lost altogether when sulfite was added 10 min after thediol epoxide. The specificity of this effect of sulfite wasshown by comparison with sulfate, which at concentrations ofeither 1 or 10 mM exhibited modest cytotoxicity, but neitherwas directly mutagenic nor able to enhance the mutagenic effectof anti-BPDE. Binding studies with labeled anti-BPDE showedthat the addition of 10 mM sulfite increased binding of anti-BPDEto DNA by over 43%, corresponding to the observed increase inmutant frequency. Interestingly, this difference in level ofDNA modification was not apparent after 30 min to 2 h exposures,but only emerged at the 4 h time point. The 4 h point was routinelyused for all mutagenicity studies. Binding of anti-BPDE-derivedmaterials to cellular RNA was not altered by 10 mM sulfite.The emergence of increased DNA modification at the latest timepoint suggests either a more prolonged period of active DNAbinding than would occur with diol epoxide, or a differencein the ability to recognize and clear specific DNA adducts.Both possibilities are discussed in regard to the observed formationof 7r,8t,9t-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene- lOc-sulfonate(BPT-10-sulfonate) in those incubations. BPT-10-sulfonate isa relatively stable BP derivative which retains the abilityto covalently modify DNA. The role of this derivative in theenhancement of diol epoxide mutagenicity by sulfite is stronglysuggested by these data.     

Cell specific activation of benzo[a]pyrene by fibroblasts and hepatocytes

The cell specific activation of benzo[a]pyrene (BP) by embryonicfibroblasts and by mature hepatocytes to intermediates thatcan interact with DNA, or cause mutations in Chinese hamsterV79 cells has been investigated. At BP concentrations of upto 15 ;µM, BP was activated to mutagenic intermediatesfor the V79 cells by embryonic fibroblasts but not by hepatocytes.However, hepatocytes from rats that had been pretreated withan inducer of the mixed function ox-idases, 3-methylcholanthrene,did metabolize higher doses of BP (> 15 µM) to mutagenicintermediates. BP was extensively metabolized by both cell types,but the hepatocytes and fibroblasts showed differences bothin the profiles of BP metabolites and the nature of the BP-DNAadducts formed. Hepatocytes metabolized BP principally to 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene,phenols, and quinones, which underwent further metabolism towater-soluble metabolites. Metabolism of BP to 7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene(BP-7,8-diol) occurred but proceeded rapidly to the formationof triols and tetraols. Fibroblasts metabolized BP predominantlytoward the formation of BP-7,8-diol. The proportion of primarymetabolites undergoing further metabolism to conjugates wasless extensive than in the hepatocytes. Hepatocytes bound moreBP to their DNA than the fibroblasts. In the hepatocytes themajor DNA adducts formed were hydrophilic derivatives, and no[±]7ß,8-di-hydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE) adducts were detected even after treatment with BP-7,8-diol.In the fibroblasts, the major BP-DNA adduct was derived fromthe reaction of BPDE with deoxyguanosine. These results suggestthat the differences in the response of embryonic fibroblastsand mature hepatocytes in the activation of BP to a mutagenfor mammalian cells is determined at least in part by the overallbalance of oxidation and detoxification processes in the cellsand, hence, by the levels of critical oxidative intermediatesthat interact with DNA.     

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.     

Metabolism of ({+/-})-trans-7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene in mouse liver microsomes and the effect of 2(3)-tert-butyl-4-hydroxy-anisole

The metabolism of (±) trans-7,8-dihydroxy-7,8-dihydroben-zo[a]pyrene(BP-7,8-diol) was examined using liver microsomes from micemaintained either on a standard laboratory food diet or on amixture of ground food pellets and 2(3)-tert-butyl-4-hydroxyanisole(BHA, 7.5 g/kg food). Dietary BHA had a statistically significantinhibitory effect both on the formation of polar metabolitesof BP-7,8-diol and on the co-valent binding of reactive productsto calf thymus DNA. When BHA (20 µM) was added in vitroto the microsomal incubation system, both the metabolism ofBP-7,8-diol and the covalent binding of BP-7,8-diol metabolitesto DNA, was reduced by -50% using either type of microsomes.The binding of [¹⁴C]7ß,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to calf thymus DNA was not affected by thepresence of BHA. The reduced metabolism of BP-7,8-diol in microsomesfrom BHA-treated mice compared to control was not due to theeffect of residual BHA in the microsomal preparation. Theseresults show that BHA acts as a potent inhibitor of the activationof the proximate carcinogen BP-7,8-diol to reactive, DNA-bindingproducts both when administered as a dietary constituent andas an additive to microsomal incubation systems. Both of theseproperties may be of relevance to the inhibitory effect of BHAon benzo[a]-pyrene carcinogenesis.     

Comparison of the metabolism of benzo[a]pyrene and its activation to biologically active metabolites by low-passage hamster and rat embryo cells

The activity of benzo[a]pyrene (BP) in cell transformation andcell-mediated mutation assays in which the hydrocarbon was activatedby Wistar-Lewis rat embryo (RE) or hamster embryo (HE) cellswas compared, and the metabolism of BP was analyzed under theculture conditions of the assays. In the transformation assay,BP (0.1 µg/ml) decreased the cloning efficiency of HEtarget cells 74% with RE feeder cells, whereas with HE cellsas feeders, BP (10 µg/ml) produced little toxicity. Inthe cell-mediated mutation assay, BP (0.5 µg/ml) reducedthe cloning efficiency of V79 target cells 40% with either REor HE cells as activators. Under the high-cell density conditionsof the mutation assay, the major [³H]BP metabolites formed inboth cell types were dihydrodiols and phenol-glucuronides. REcells formed similar amounts of BP-9, 10-diol and BP-7, 8-diol;HE cells formed four times as much BP-9, 10-diol as BP-7, 8-diol.The ratio of the glucuronide conjugates of 3-hydroxy-BP to 9-hydroxy-BPwas 2:1 in RE cells and 1:2 in HE cells. Under the low-celldensity conditions of the transformation assay, the [³H]BP metabolitesreleased into the medium were dihydrodiols, phenol-glucuronidesand substantial amounts of free phenols, mainly 9-hydroxy-BPin HE cells and both 9-hydroxy-BP and 3-hydroxy-BP in RE cells.Differences in the ratios of specific free phenols and of thetwo dihydrodiols which are produced and released into the mediumby the two cell types may explain, in part, the differencesin BP-induccd toxicity under conditions of the transformationassay. In fact, under such conditions, 3-hydroxy-BP (1 µg/ml)reduced the cloning efficiency of HE target cells 50% with bothRE and HE feeder cells whereas 9-hydroxy-BP had no effect; BP-7,8-diol(0.1µg/ml) reduced the cloning efficiency 60% with REfeeder cells but had little effect with HE feeder cells. Theseexperiments indicate that differences in carcinogen metabolismat different cell densities should be considered in designingand interpreting short-term screening assays.     

Metabolic activation of benzo[a]pyrene-7,8-dihydrodiol and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide to protein-binding products and the inhibitory effect of glutathione and cysteine

Trans-7, 8-dihydroxy-7, 8-dihydrobenzo(a)pyrene (BP-7, 8-diol)and the anti-isomer of trans-7, 8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene(BPDE) were found to be activated by microsomes isolated from3-methylcholanthrene (MC)-treated rats to reactive intermediatesthat bound covalently to microsomal proteins. The extent ofbinding was markedly reduced by the presence of reduced glutathione(GSH) or cysteine. Fluorescence spectroscopic studies on theproducts derived from BP-7,8-diol and BPDE after microsomalactivation in presence of GSH or cysteine revealed the formationof a common reactive intermediate with unique fluorescence properties.The involvement of cytochrome P-448 in the activation of BP-7,8-dioland BPDE to protein-binding products was inferred by the requirementfor NADPH and almost complete inhibition by -naphtho-flavone.Furthermore, microsomes from MC-treated rats could be replacedby a reconstituted system containing purified cytochrome P-448,NADPH-cytochrome reductase and co-factors. The conjugation ofthe reactive intermediates from BP-7,8-diol and BPDE with GSHor cysteine did not require the presence of either microsomesor cyutosol, thus indicating a non-catalytic reaction. Theseresults emphasize the importance of cellular nucleophiles suchas GSH and cysteine in the deactivation of reactive benzo(a)pyrene(BP) intermediates and also provides evidence for the furtheractivation of the ultimate carcinogen BPDE to more reactivedectro-philes and may thus have relevance concerning the regulationof BP-induced 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.     

Synthesis and characterization of covalent adducts derived from the binding of benzo[a]pyrene diol epoxide to a -GGG- sequence in a deoxyoligonucleotide

Direct synthesis and purification procedures are described forthe preparation of adducts derived from the covalent bindingof 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene[(+)-ante-BPDE or (+)-BPDE 2] to each of the three guanine residues(trans-N² lesions) in the oligodeoxyribonucleotide d(CTATG¹G²G³TATC)The positions of the modified Gs are defined by Maxam-Gilbertsequencing techniques. Six different oligonucleotides with oneor two precisely positioned (+)-anti-BPDE residues are identified.The absorbance, circular dichroism and fluorescence characteristicsare changed upon formation of duplexes with the complementarystrands d(GATACCCATAG). In the doubly-modified oligonucleotides,a broad, excimer-like long wavelength fluorescence emissionband is observed with a maximum near 455 nm only if the two(+)-anti-BPDE-modified Gs are adjacent to one another. The covalentlyattached (+)-anti-BPDE residues decrease the thermodynamic stabilitiesof the duplexes; their melting points are markedly dependenton the position of the lesions, being highest with the (+)-anti-BPDEresidue at G¹ (T^m=40°C, only 2°C lower than in the caseof the unmodified oligonucleotide) and lowest when it is situatedat G³ (T^m=29°C). The implications of these and other physicalcharacteristics are discussed. The facile synthesis of theseor similar site-specific and stereochemically defined (+)-trans-anti-BPDE-N²-dGlesions in runs of contiguous guanines in oligodeoxyribonucleotidesof specified base sequence should be useful for the design ofsite-directed mutagenesis studies in vitro and in vivo.     

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.     

Prostaglandin synthetase and cytochrome P-450-dependent metabolism of (+/-)benzo(a)pyrene 7,8-dihydrodiol by enriched populations of rat Clara cells and alveolar type II cells

The metabolism of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol) by prostaglandin synthetase and cytochrome P-450-dependent monooxygenases was studied using enriched fractions of Clara cells and alveolar type II cells from rat lung. Arachidonic acid-fortified fractions enriched in Clara cells and alveolar type II cells metabolized BP-7,8-diol to the 7,10/8,9-tetrol of benzo(a)pyrene and the 7/8,9,10-tetrol of benzo(a)pyrene. These tetrols are formed upon solvolysis of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha- epoxy-7,8,9,10-tetrahydrobenzo(a)-pyrene (BP diol-epoxide I). Arachidonic acid-dependent metabolism of BP-7,8-diol to BP diol-epoxide I in enriched Clara cells and alveolar type II cells was completely inhibited by indomethacin, a classical inhibitor of prostaglandin synthetase. Enriched Clara cells and alveolar type II cells also metabolized BP-7,8-diol to BP diol-epoxide I in the presence of NADPH. Amounts of BP diol-epoxide I-derived tetrols formed from BP-7,8-diol by the prostaglandin synthetase-dependent and the cytochrome P-450-dependent pathways varied significantly between the two pulmonary cell fractions examined. In fractions enriched in Clara cells, cytochrome P-450-dependent BP-7,8-diol oxidation was higher than was prostaglandin synthetase-dependent BP-7,8-diol oxidation; while in fractions of alveolar type II cells, prostaglandin synthetase-dependent BP-7,8-diol oxidation to BP diol-epoxide I predominated. Pretreatment of rats with beta-naphthoflavone resulted in a 2- to 3-fold increase in BP diol-epoxide I formation by prostaglandin synthetase and cytochrome P-450-dependent monooxygenases in both enriched Clara cells and alveolar type II cells. These increases in BP-7,8-diol oxidation to BP diol-epoxide I appear to be due to induction of the two enzymatic pathways in both pulmonary cell types. No qualitative changes in the pattern of BP-7,8-diol metabolism by either enzymatic pathway in enriched Clara cells or alveolar type II cells were observed following beta-naphthoflavone treatment. The results suggest that pulmonary prostaglandin synthetase may serve as either an additional or an alternative bioactivating enzyme to the cytochrome P-450-dependent monooxygenases for the formation of reactive chemical carcinogens in the lung.