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.     

Secondary metabolites of benzo[a]pyrene: 3-hydroxy-trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, a biliary metabolite of 3-hydroxybenzo[a]pyrene in the rat

Rats administered 3-hydroxybenzo[a]pyrene (50 mg/kg, i.p.),excrete via the bile metabolites which, after treatment withß-glucuronidase and aryl sulphatase, yield, in additionto 3-hydroxybenzo[a]pyrene, 3-hydroxy-trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene(3-OH-BP-7, 8-diol) and a minor, highly labile, metabolite tentativelyidentified as 3,5-dihydroxybenzo[a]pyrene. These novel metabolitesare readily isolated in a pure state via preparative layer chromatography.The structure of the 3-OH-BP-7, 8-diol was revealed by its u.v.,proton magnetic resonance and mass spectral properties. Itshydroxyl functions are in a predominantly quasi-diequatorialconformation.     

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.     

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.     

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.     

Sodium nitrite-stimulated metabolic activation of benzo[a]pyrene 7,8-dihydrodiol in human polymorphonuclear leukocytes

Sodium nitrite was shown to enhance the metabolism of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) to 7/8,9,10- and 7,10/8,9-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (tetraols) in phorbol myristate acetate (PMA)-stimulated polymorphonuclear leukocytes (PMNs). The production of these tetraols implicates the intermediate formation of the corresponding trans-7,8-dihydroxy-9,10-epoxy-7,8-9,10-tetrahydrobenzo[a]pyrene (anti-BPDE). A 2- to 3-fold increase in the tetraol yield was observed in the presence of nitrite in excess of 1 mM. Sodium azide, an inhibitor of myeloperoxidase and catalase, reduced the nitrite-stimulated metabolism of BP-7,8-diol in PMA-activated leukocytes. Diphenylene iodonium sulphate, a NADPH-oxidase inhibitor, lowered the production of tetraols in PMA-stimulated leukocytes both in the absence and presence of nitrite. Additionally, nitrite markedly enhanced the covalent binding of metabolites derived from [3H](-)-BP-7,8-diol to leukocyte proteins as well as to DNA present extracellularly. The nitrite-stimulated covalent binding to both proteins and DNA was inhibited by the presence of sodium azide. The mechanism underlying the effect of nitrite on the metabolism of BP-7,8-diol to reactive intermediates in PMA-activated human polymorphonuclear leukocytes is not known. However, the results are compatible with a peroxidase-dependent mechanism although other possible pathways may contribute to the enhanced rate of metabolism.     

Epoxidation of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene via a hydroperoxide-dependent mechanism catalyzed by lipoxygenases

The lipoxygenase catalyzed epoxidation of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) was examined. Epoxidation of the BP-7,8-diol was catalyzed by 5- and 15-lipoxygenase in the presence of either arachidonic acid, gamma-linolenic acid, or 15-hydroperoxyeicosatetraenoic acid (15-HPETE). The anti-9,10-epoxy-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene isomer was formed in greater quantities than the syn isomer, indicative of peroxyl radical mediated epoxidation. Epoxidation was dependent on time, enzyme and fatty acid concentration. There was no difference in the time course of epoxidation with either arachidonic acid or 15-HPETE, although the initial rate of oxygen consumption was approximately 55-fold greater with arachidonic acid. The lipoxygenase inhibitor and anti-oxidant nordihydroguaiaretic acid inhibited epoxidation in a dose-dependent manner in incubations initiated with either arachidonic acid or 15-HPETE. The anti-oxidant butylated hydroxyanisole also inhibited the epoxidation. Incubations conducted under anaerobic conditions with 15-lipoxygenase and either arachidonic acid or 15-HPETE significantly decreased epoxidation. This suggests that the oxygen inserted into BP-7,8-diol is derived from the atmosphere. The epoxidizing peroxyl radicals could not be detected but their precursors, carbon-centered radicals, were detected by using the ESR spin trapping technique in incubations of 15-lipoxygenase with 15-HPETE. This radical, formed by reduction and rearrangement of the hydroperoxide, may trap oxygen to form a peroxyl radical. We propose that the epoxidizing species is a peroxyl radical derived from 15-HPETE rather than from arachidonic acid. This proposal is based on the similar amounts of epoxidation, but dissimilar amount of oxygen consumed with both fatty acids. Since lipoxygenases are widely distributed in vivo, especially in areas where tumors arise such as the pulmonary epithelium, peroxyl radical formation by these enzymes may have an important role in chemical carcinogenesis.     

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.     

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.     

Different patterns of benzo[a]pyrene metabolism of purified cytochromes P-450 from methylcholanthrene, {beta}-naphthoflavone and phenobarbital treated rats

An improved high-pressure liquid chromatography system was usedto analyze the amount of benzo[a]pyrene metabolites formed inreconstituted microsomal mixed-function oxidase systems containingdifferent cytochromes P-450. We separated twelve identifiedand seven unknown metabolites of BP which included three diols:the 9,10-, 4,5-, and 7,8-dihydrodiols; four phenols, 9-, 7-,1-, and 3-hydroxybenzo[a]pyrene (OH-BP); and three quinones:the 1,6-. 3,6-, and 6,12-quinones. Two additional peaks co-migratedwith synthetic 4-OH-BP and 5-OH-BP, respectively. The former,designated fraction 1, was shown by u.v. spectra to containprimarily the 4,5-epoxide with small amounts of 4-OH-BP. Thetotal metabolism of BP was found to be 20-fold greater withthe cytochrome P-450 from the 3-methylcholanthrene (P-450 3-MC)and ß-naphthoflavone (P-450 BNF) treated rats thanwith the phenobarbital preinduced cytochrome P-450 (P-450 PB).3-OH-BP and 9-OH-BP were the major phenolic products for bothP-450 3-MC and P-450 BNF whereas the 3-OH-BP and 1-OH-BP werethe major phenolic products for P-450 PB. The ratio of totalphenols to diols was found to be 3.34, 4.85 and 0.70 for P-4503-MC, P-450 BNF and P-450 PB. The major dihydrodiol generatedby P-450 3-MC and P-450 BNF was 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene,whereas the 9,10-diol was the major diol from P-450 PB. Theamount of 1,6- and 3,6-quinones produced was greater than the6,12-quinone with the P-450 3-MC and P-450 BNF but all threequinones were produced in low and equal amounts by the P-450PB. In respect to the percent metabolites formed at a givenregion of the BP, P-450 3-MC and P-450 BNF preferred oxidationat the 1, 3 positions, 6 position and the 7, 8 positions, whereasthe P-450 PB preferred oxidation at the 4, 5 position. Thisstudy demonstrates the unique positional specificity of differentforms of cytochrome P-450 which may regulate the balance betweenactivation and detoxification pathways of polycyclic aromatichydrocarbon metabolism.     

Prostaglandin endoperoxide synthetase-dependent cooxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene in C3H/10T 1/2 clone 8 cells

(+/-)trans-7,8-Dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol), the proximate form of the carcinogen benzo(a)pyrene, is cooxidized during the oxidation of arachidonic acid to prostaglandins by prostaglandin endoperoxide synthetase (PES). This enzyme can oxidize BP-7,8-diol to the reactive intermediate (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, which binds covalently to macromolecules, is mutagenic in bacterial test systems, and forms 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BP-tetrol) isomers. We have examined the cooxidation of BP-7,8-diol in an intact cell culture system of C3H/10T 1/2 clone 8 mouse embryo fibroblasts, in which both the mixed-function oxidase and PES systems are present. When BP-7,8-diol is incubated for 72 hr with approximately 10(6) confluent cells, high-performance liquid chromatography analysis of the organic extractable products reveals all four pairs of BP-tetrols, with those from (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene predominating. The addition of arachidonic acid (100 microM) produced a 2- to 3-fold increase in the formation of BP-tetrols from (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, while the metabolism of BP-tetrols from (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10 tetrahydrobenzo(a)pyrene was unchanged. The addition of the PES inhibitor indomethacin (100 microM) completely eliminated this stimulation. Cell transformation assays were carried out under the same conditions. The addition of arachidonic acid resulted in a 10-fold increase in foci formation, while indomethacin inhibited the increase in foci formation by 70%. These results suggest that cooxidation of BP-7,8-diol to reactive intermediates by PES can occur in an intact cell system if stimulated with arachidonic acid. In addition to mixed-function oxidase-dependent activation of carcinogens, the cooxidation of chemicals to reactive metabolites during prostaglandin biosynthesis may also play a role in carcinogenesis.     

Oxidation of (+)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene by mouse keratinocytes: evidence for peroxyl radical- and monoxygenase-dependent metabolism

The role of prostaglandin H (PGH) synthase and peroxyl radicals as well as cytochrome P-450 in the metabolism of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) was examined in fresh skin keratinocytes isolated from hairless mice. Labeled (+)-BP-7,8-diol was oxidized after incubation with the keratinocytes to syn- and anti-diolepoxides in greater than a 4:1 ratio as estimated by h.p.l.c. analysis of the stable hydrolysis products. Formation of diolepoxides was dependent on cell number and the concentration of BP-7,8-diol. Incubation in the presence of the PGH synthase substrate, 20:4 or the inhibitor, indomethacin did not alter the total formation or the ratio of diolepoxides. However, the addition of butylated hydroxyanisole (1 micron) an inhibitor of peroxyl radical dependent-metabolism significantly inhibited diolepoxide formation. The time course for the formation of the anti-diolepoxide and lipid peroxidation, measured as malondialdehyde was determined. The results suggest an excellent correlation between peroxyl radical and diolepoxide formation. Pretreatment of mice with the cytochrome P-450 inducer, beta-naphthoflavone greatly altered the metabolism of (+)-BP-7,8-diol by keratinocytes. The major metabolite was the syn-diolepoxide with significant formation of two unknown metabolites. Pretreatment of mice with BP-7,8-diol did not induce aryl hydrocarbon hydroxylase activity but did increase the yield of syn-diolepoxide formed from labeled (+)-BP-7,8-diol by 1.5-fold. Our results suggest that peroxyl radical-mediated metabolism is primarily responsible for the oxidation of (+)-BP-7,8-diol in control animals while the cytochrome P-450 system is primarily responsible for oxidation in animals pretreated with inducers.     

Peroxidase-catalyzed oxidation of (bi)sulfite: reaction of free radical metabolites of (bi)sulfite with ({+/-})-7,8-dihydroxy-7,8-di-hydrobenzo[a]pyrene

The peroxidase-catalyzed metabolism of (bi) sulfite (hydratedsulfur dioxide) in the presence of (±)-7, 8-dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7, 8-diol) was examined. Both horseradishperoxidase and prostaglandian peroxidase catalyze the one-electionoxidation of (bi)sulfite. This results in the formation of asulfurtrioxide radical anion which then reacts with molecularoxygen to form a peroxyl radical. This (bi)sulfite-derived peroxylradical then reacts with BP-7, 8-diol to form BP-7, 8-diol-9,10-epoxides, the ultimate carcinogenic form of benzo(a)pyrene(BP). Addition of (bi)sulfite to incubations containing BP-7,8-diol and an active peroxidase resulted in significantly increasedlevels of BP diol-epoxide formation. This result may, in part,explain the reported co-carcinogenic effect of sulfur dioxideon BP-induced tumors in the respiratory tracts of rats and hamsters.The sulfur trioxide radical anion also reacts directly withBP-7, 8-diol to form a sulfonate adduct. This reaction was particularlysignificant under conditions where molecular oxygen was depletedfrom the incubations. While the significance of this particularadduct is not known, its formation suggests that the sulfurtrioxide radical anion generated during the peroxidase-catalyzedoxidation of (bi)sulfite could react with a wide assortmentof compounds to form sulfonate adducts.     

Mutagenicity of benzo[a]pyrene bay-region sulfonates

The interaction between the sulfite anion and specific benzo[a]pyrene (B[a]P) derivatives produces a novel class of benzo[a]pyrene sulfonates. (+/-)-7,8,9-Trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10-sulfonate (B[a]PT-10-sulfonate) is formed in high yields in incubations containing (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyre ne (anti-BPDE) and sulfite, and sulfite strongly enhances the mutagenicity of the diolepoxide toward Salmonella typhimurium under those conditions. Although B[a]PT-10-sulfonate itself shows little direct mutagenicity over a 1-20 microM concentration range, this reactive bay-region intermediate does enhance the mutagenicity of anti-BPDE in strains TA98 and TA100 by up to 280%. No significant enhancement was seen when up to 20 microM B[a]PT-10-sulfonate was used in concert with another direct-acting mutagen, N-acetoxy-acetylaminofluorene (N-AcO-AAF). The isomeric product derived from sulfite and (+/-)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) is (+/-)-7,8,10-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-9-sulfonate (B[a]PT-9-sulfonate). Like B[a]PT-10-sulfonate, B[a]PT-9-sulfonate is not mutagenic to strains TA97, TA98 and TA100. This sulfonate exhibited little enhancing activity with anti-BPDE over a 1-20 microM concentration range, but did enhance the mutagenic response of strain TA98 to 0.2 microM N-Aco-AAF by up to 128%. Sulfite, anti-BPDE and B[a]PT-sulfonates were also examined for the ability to induce a forward mutation at the hgprt locus (8-azaguanine resistance) in strains of S.typhimurium. Sulfite caused a marked enhancement of forward mutation due to anti-BPDE in both TA98 and TA100. Surprisingly, concurrent administration of B[a]PT-10-sulfonate with anti-BPDE did not increase the number of mutant colonies. The extensive conversion of anti-BPDE to B[a]PT-10-sulfonate under conditions where sulfite enhances diolepoxide mutagenicity, when coupled with this enhancement of diolepoxide mutagenicity by B[a]PT-10-sulfonate in the reverse mutation assay, supports this novel B[a]P derivative as a mediator of the sulfite-dependent enhancement of B[a]P genotoxicity. Determining why this enhancing effect was not seen when selecting for mutation at the hgprt locus of S.typhimurium will require further study.     

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.     

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.     

Glutathione depletion suppresses conjugation of benzo[a]pyrene metabolites and ({+/-})-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites with glutathione but does not affect their binding to DNA in C3H/10T1/2 mouse fibroblasts

The study was aimed at determining the role of glutathione (GSH)conjugation in the binding of reactive benzo[a]pyrene (BaP)species to DNA of C3H/10T1/2 cells. In order to suppress GSHconjugation cells were depleted of GSH by treatment with buthioninesulfoximine for 18 h and 1-chloro-2,4-dinitrobenzene for 1 hprior to incubation with radiolabelled substrates. Under theseconditions GSH levels decreased to <1% of the control value.C3H/10T1/2 cells produced GSH conjugates with 7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydro-benzo[a]pyrene(BaPDE) comprising 6% of the total metabolites formed from BaPor (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(BaP-7,8-diol). In GSH-depteted cells formation of GSH conjugateswith metabolic products of BaP or BaP-7,8-diol was suppressedto 1% of total metabolites during an 8-h incubation period.Metabolic activation of BaP and BaP-7,8-diol by C3H/10T1/2 cellsresulted in the formation of DNA adducts which largely consistedof BaPDE:deoxyguanosine. Depletion of GSH altered neither thedegree of DNA binding nor the pattern of DNA adducts to anysignificant extent. When C3H/10T1/2 cells were co-incubatedwith microsomes from liver of 3-methylcholanthrene-treated ratsfor 1 h in order to activate BaP or BaP-7,8-diol extracellularly,the same pattern of GSH conjugates and DNA adducts was generatedas by intracellular metabolism of the polycyclic hydrocarbons.No GSH conjugates were detected following co-incubation of microsomeswith GSH-depleted C3H/10T1/2 cells. The formation of DNA adductsagain remained unaffected by the suppression of conjugation.C3H/10T1/2 cells are apparently capable of conjugating BaPDEwith GSH but are not capable of trapping by GSH conjugationthose BaPDE moieties which bind to DNA. The results are compatiblewith the notion that BaPDE is partially contained in a cellularcompartment—presumably the lipid environment of membranes—whereit is inaccessible to GSH transferases of C3H/10T1/2 cells.     

Differential effects of dietary BHA on hepatic enzyme activities and benzo[a]pyrene metabolism in male and female NMRI mice

The effect of dietary 2(3)-tert-butyl-4-hydroxy-anisole (BHA)alone and in combination with intraperitoneal injections of3-methylcholanthrene (MC), on hepatic enzyme activities andbenzo[a]pyrene (BP) metabolism was compared in male and femaleNMRI mice. In general, the characteristic induction patternfollowing dietary BHA administration in female mice could alsobe seen when male mice were used. The increase in epoxide hydrolaseand cytosolic glutathione S-transferase following BHA feedingwas however not as pronounced in males as in females. Also,MC treatment appeared to counteract the induction of GST activityby BHA in females but had no such effect in males. Liver microsomesfrom untreated male mice catalyzed the metabolism of BP lessefficiently than did microsomes from females. BHA treatmentincreased this activity in both sexes to a comparable extentand the overall activity was the same in males and females havingreceived MC. The pattern of BP metabolites was altered followingBHA treatment. In general, an increase in BP-4,5-diol and adecrease in 9-OH-BP was observed. This pattern was also noticedwhen microsomes from MC treated and MC + BHA treated animalswere compared. MC treatment alone increased the amount of BP-7,8-diol,the quinones and the phenols. The present report indicates thatseveral factors may contribute to the response to dietary BHAin mice. Whether this has any consequence in regard to thiscompound's anticarcinogenic effect remains to be elucidated.     

Effects of benzo[a]pyrene and ({+/-})-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene on mitosis in Chinese hamster V79 cells with stable expression of rat cytochrome P4501A1 or 1A2

The effect of bioactivation of benzo[a]pyrene (B[a]P) and (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(B[a]P-7,8-diol) on spindle disturbances and toxicity has beeninvestigated in V79 Chinese hamster cells genetically engineeredto express cytochrome P4501A1 (CYP1A1) and cytochrome P4501A2(CYP1A2). B[a]P induces spindle disturbances in native V79 Chinesehamster cells. This effect was enhanced by the expression ofCYP1A1 but not CYP1A2. The increased effect seen in the CYP1A1-expressingcell line could be brought back to the level seen in the nativecell line by     

Differential metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 variants

Cytochrome P450 1A1 (CYP1A1) plays a key role in the metabolism of carcinogens, such as benzo[a]pyrene (B[a]P) and metabolites to ultimate carcinogens. Three human allelic variants, namely wild-type (CYP1A1.1), CYP1A1.2 (I462V) and CYP1A1.4 (T461N), were coexpressed by coinfection of baculovirus-infected insect cells with human NADPH-P450 reductase. These recombinant enzymes (in microsomal membranes) were used to analyze whether CYP1A1 polymorphisms affect catalytic activities towards B[a]P and B[a]P-7,8-dihydrodiol. The complete spectrum of phase I metabolites, including the tetrahydrotetrols resulting from hydrolysis of the ultimate carcinogen, B[a]P-7,8-dihydrodiol-9,10-epoxide, was examined by HPLC. Wild-type enzyme showed the highest total metabolism of B[a]P, CYP1A1.2 was approximately 50%, and CYP1A1.4 approximately 70%. Km values for all metabolites with CYP1A1.2 were generally significantly lower than with wild-type enzyme (e.g. B[a]P-7,8-diol formation: 13.8 microM for wild-type, 3.5 microM for CYP1A1.2 and 7.7 microM for CYP1A1.4). Addition of epoxide hydrolase markedly increases the relative diol-to-phenol activities by all three variants. However, CYP1A1.4 exhibits the greatest efficiency to produce diol species. Each variant produced the diol epoxides from B[a]P-7,8-dihydrodiol. CYP1A1.1 exhibited with 10.4 pmol/min/pmol CYP1A1 the greatest total rate for 7,8-diol metabolites followed by CYP1A1.2 (7.2 pmol/min/pmol CYP1A1) and CYP1A1.4 (5.5 pmol/min/pmol CYP1A1). All enzyme variants produced about three times more diol epoxide 2-derived metabolites than diol epoxide 1-derived ones, whereby both rare allelic variants exhibited statistically significantly increased formation of diol epoxide 2. This study showed that the three CYP1A1 variants had different enzyme kinetics properties to produce both the diol metabolites from B[a]P and the ultimate mutagenic species diol epoxide 2 from B[a]P-7,8-dihydrodiol, which must be considered in the evaluation of individual susceptibility to cancer.