X-ray crystallographic analysis of ({+/-})-7{beta},8{alpha}-dihydroxy-9{beta},10{beta}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene: molecular structure of a 'syn' diol epoxide

X-ray crystallographic analysis has been used to define themolecular structure of the cis (syn) diol epoxide, (±)-7ß,8-dihydroxy-9ß,10ß-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.The two hydroxyl groups are oriented equatorially to the tetrahydrobenzenering, contrary to predictions and there is no intramolecularhydrogen bonding in the structure.     

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.     

Glutathione conjugation and DNA-binding of ({+/-})-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and ({+/-})-7{beta},8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in isolated rat hepatocytes

Isolated rat liver hepatocytes, previously depleted of glutathione(GSH) by treatment with diethylmaleate, were allowed to incorporate[³H]glycine into their GSH. Incubation of ³H-labelled cellswith ¹⁴C-labelled (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene((±)-BP-7,8-dihydrodiol) or (±)7ß,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene(()-BPDE) revealed the formation of double labelled products.This together with evidence from amino acid analysis indicatesformation of GSH-conjugates of the highly carcinogenic BP-derivatives.Incubation of hepatocytes isolated from 3-methylcholanthrene(MC) treated rats with ³H-labelled (±)-BP-7,8-dihydrodiolor (±)-BPDE resulted in binding of radioactivity to DNA.Reduction of the intracellular level of GSH to 40% of the normallevel resulted in an approximate 2-fold increase in the DNA-bindingof either substrate. In addition there was a concurrent decreasein the amount of GSH-conjugates formed. These data clearly demonstratethat GSH participates in conjugation reactions with carcinogenic(±)-BP-7,8-dihydrodiol and (±)-BPDE and that theintracelluilar level of GSH is important in preventing reactiveintermediates from reacting with the DNA in intact cells.     

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.     

Relation between benzo[a]pyrene-DNA adducts, cell proliferation and p53 expression in tracheal epithelium of hamsters fed a high {beta}-carotene diet

Vitamin A and ß-carotene protect against respiratorytract cancer by inhibiting the formation of DNA damage and controllingcellular proliferation and differentiation. Recently, it hasbeen shown that the p53 tumor-suppressor gene plays a crucialrole in the etiology of respiratory tract cancer. In the presentstudy, we investigated the relationship between benzo[a]pyrene(B[a]P)-DNA adducts, cell proliferation and p53 expression andthe possible effect of ß-carotene on such a relationshipin tracheal epithelium of hamsters given intratracheal instillationsof B[a]P-Fe₂O₃ particles suspended in saline. DNA-adducts werequantified by the ³²P-postlabeling assay, cell proliferationwas quantified by immunocytochemical detection of incorporatedBrdU during S-phase, and p53 protein was detected by immuno-histochemistrywith an antibody that recognized both the wild-type and themutated protein (BioGenex, Clone BP53–12–1). A clearrelationship appeared to exist between the extent of B[a]P-DNAadduct formation, the induction of cell proliferation and theexpression of p53 protein in hamster tracheal epithelium. Theseresults suggest that B[a]P induces cell proliferation in hamstertracheal epithelial cells most likely by the induction of mutationsin the p53 gene. Furthermore, ß-carotene was not foundto influence the formation of B[a]P-DNA adducts, which is probablydue to the high B[a]P dose. Moreover, ß-carotene didnot statistically significantly affect cell proliferation andp53-protein expression in hamster tracheal epithelial cells.     

Studies by fluorescence and n.m.r. spectroscopy of the major product formed after further metabolism of ({+/-})-7{beta}, 8{alpha}-dihydroxy-9{alpha}, 10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

Racemic 7ß, 8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene(anti-BPDE) is further metabolized by liver microsomes obtainedfrom 3-methylcholanthrene pretreated rats in the presence ofNADPH to at least four products as revealed by h.p.l.c. Dataobtained from measurements by fluorescence spectroscopy underneutral and alkaline conditions and high resolution two-dimensional¹H n.m.r. spectroscopy on the major metabolite derived fromanti-BPDE are consistent with aromatic hydroxylation at the3-position either directly or indirectly via transient epoxideintermediates.     

The molecular structure of ({+/-})- 7{alpha}, 8{beta}-dihydroxy-7,8-dihydrobenzo[a]pyrene, an early metabolite of benzo[a]pyrene

The molecular structure of (±)-7, 8ß-dihydroxy-7,8-dihydrobenzo[a]pyrene has been determined by X-ray crystallographicmethods. The analysis has shown that the two hydroxyl groupsare trans to each other and di-equatorial to the ring. The dihydrobenzenegroup adopts a distorted half-chair pucker. Trends in severalbond distances indicate reactive points in the molecule.     

Glutathione transferases in rat lung: the presence of transferase 7-7, highly efficient in the conjugation of glutathione with the carcinogenic (+)-7{beta},8{alpha}-dihydroxy-9{alpha},10{alpha}-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene

The enzyme-catalysed conjugation of (±)-7ß,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(±)-anti-BPDE] with glutathione(GSH) by cytosolic GSH transferases isolated primarily fromrat lung has been studied. GSH transferase 4-4 was active inthe GSH conjugation of anti-BPDE, whereas transferases 2-2 and3-3 showed little activity. GSH transferase 1-1 did not contributeto the activity since significant amounts were not detectedin the rat lung. Activity was also obtained with several acidicpulmonary GSH transferases and with a newly described form,transferase 7-7, also isolated from rat kidney and from hyperplasticliver nodules. The catalytic efficiency (k_cat/K_m) of transferase7-7 was seven times that of transferase 4-4, the most activerat transferase previously identified. When the GSH concentrationwas varied at constant (±)-anti-BPDE concentration inthe presence of transferases 4-4, 7-7 or the major acidic transferase,non-linear Lineweaver-Burk plots were obtained. Resolution ofthe GSH conjugates of the two enantiomers of (±)-anti-BPDEby h.p.l.c. showed that all isoenzymes with notable activitywere selective (97%) for the (+)-enantiomer of anti-BPDE, whichis generally considered to be the most carcinogenic form ofBPDE. The possibility that one enan-tiomer inhibits the conjugationof the other enantiomer with GSH cannot be excluded and mayquantitatively affect the results obtained.     

Interaction of ({+/-})-7r, 8t-dihydroxy-9t, 10t-oxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene with purified rat liver chromatin

Previous studies have shown that in addition to serving as atarget for covalent adduct formation, purified DNA catalyzesthe detoxification of (±)–7r, 8t-dihydroxy–9t,10t-oxy–7, 8, 9, 10-tetrahydrobenzo[a]pyrene (BPDE). Tobegin to relate these in vitro findings with the processes importantin carcino-genesis in vivo, we have prepared native chromatinfrom rat liver nuclei and analyzed its interactions with BPDE.Using several different methods to follow the hydrolysis ofBPDE, we find the ability of chromatin to catalyse this detoxificationis severely reduced relative to purified DNA. The rate of formationof covalent adducts is also reduced, although the final levelof modification is almost the same in chromatin and purifiedDNA. The difference in rates could be an important in vivo protectionmechanism, especially in the presence of competing nucleophiles,e.g.-SH compounds. In addition, non-covalent, physical bindingto chromatin is altered, both quantitatively and qualitatively,compared with purified DNA. The specificity of covalent bindingof BPDE to histone proteins in chromatin is identical to thespecificity found in intact nuclei.     

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.     

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.     

Characterization of DNA adducts at the bay region of dibenz[a,h]anthracene formed in vitro

Bay region diolepoxide—DNA adducts of dibenz[a,h]anthracene(DBA) formed in vitro were identified and their absolute stereochemistrywas assigned. After activation of [5,12-¹⁴C]DBA with liver microsomesobtained from Aroclor 1254 treated male Sprague—Dawleyrats in the presence of calf thymus DNA for 1 h, the amountof DNA adducts was found to be 9.9 ± 2.4 pmol/mg DNA,calculated on the basis of the portion of radioactivity elutedfrom the HPLC reversed-phase column with a water/acetonitrilegradient. Bay region diolepoxide—DNA adducts represented27.5% of radioactivity associated with DNA adducts. The absoluteconfiguration of the various adducts was determined from thereaction of the (+)- and (–)-3,4-dihydrodiol after metabolicactivation and the reaction of the anti- andsyn-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracenewith DNA or with the individual deoxyribonucleotides. The mainbay region adduct was identified as a deoxyguanosine adductof (anti)-3S,4R-dihydroxy-1R,2S-epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracene, a metabolite of (–)-3,4-dihydroxy-3,4-dihydrodi-benz[a,h]anthracene.Anti bay region diolepoxide-deoxyguanosine adducts of DBA contributedto 17.7% and syn diolepoxide-derived deoxyguanosine adductsto 5.8% of adduct-associated radioactivity. The amount of bayregion deoxyadenosine adducts was calculated to be 4%. For sixof probably eight different deoxyadenosine adducts absolutestereochemistry could be assigned. ₃₂P-Postlabelling experimentsrevealed a binding of 23 ± 6 pmol/mg DNA for (–)-3,4-dihydrodioland of 1.5 ± 0.4 pmol/mg DNA for (+)-3,4-dihydrodiolof DBA.     

Effects of the co-carcinogen catechol on benzo[a]pyrene metabolism and DNA adduct formation in mouse skin

We have studied the effects of the co-carcinogen catechol (1,2-dihydroxybenzene)on the metabolic activation of [³H] benzo[a]pyrene (BaP) inmouse skin, in vivo and on the binding of BaP metabolites toDNA and protein at intervals from 0.5–24 h. Upon topicalapplication of 0.015 mg [³H]BaP and 0.25 or 0.5 mg catecholper mouse, catechol had little effect on the total amount of[³H]BaP metabolized in mouse skin, but it affected the relativeproportions of [³H]BaP metabolites. Catechol (0.5 mg/mouse)decreased the proportion of watersoluble [³H]BaP metabolites,ethyl acetate-soluble polar metabolites and quinones, but doubledthe levels of unconjugated 3-hydroxy-BaP at all measured intervalsafter treatment. Catechol also caused a small increase in thelevels of trans-7,8-dihydroxy-7,8-dihydroBaP and trans-9,10-dihydroxy-9,10-dihydroBaP0.5 h after treatment. Two hours after treatment, the levelsof these metabolites subsided to those of the controls. Catecholdid not affect the levels of glutathione conjugates of BaP.However, it caused a decrease in glucuronide and sulphate conjugateformation from BaP. Catechol caused an 2-fold increase in theformation of anti-7, 8-dihydroxy-9, 10-epoxy-7, 8, 9, 10-tetrahydroBaP(BPDE) DNA adducts and elevated the ratio of anti-syn-BPDE-DNAadducts 1.6 to 2.9-fold. Catechol treatment increased the radioactivityassociated with epidermal proteins after [³H]BaP application.Because catechol increased levels of 3-hydroxyBaP, we consideredthe possibility that 3-hy-droxyBaP might enhance the tumor initiatingactivities of BaP or BPDE in mouse skin; a bioassay demonstratedthat this was not the case. The results of this study indicatethat one important effect of catechol related to its co-carcinogenicityis its ability to enhance formation of anti-BPDE-DNA adductsin mouse skin.     

Effect of ellagic acid and hydroxylated flavonoids on the tumorigenicity of benzo[a]pyrene and ({+/-})-7{beta}, 8{alpha}-dihydroxy-9{alpha}, 10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene on mouse skin and in the newborn mouse

Ellagic acid, quercetin and robinetin were tested for theirability to antagonize the tumor-initiating activity of benzo[a]pyrene(B[a]P) and (±)-7ß, 8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(B[a]P 7,8-diol-9,10-epoxide-2), the ultimate carcinogenic metaboliteof benzo[a]pyrene. Ellagic acid, robinetin or quercetin (2500nmol) had no tumor-initiating activity on mouse skin, but thetopical application of 2500 nmol of ellagic acid 5 min beforea tumor-initiating dose of 200 nmol of B[a]P 7,8-diol-9,10-epoxide-2caused a 59–66% inhibition in the number of skin tumorsper mouse that were observed after 15–20 weeks of promotionwith 12-O-tetradecanoylphorbol-13-acetate. Similar treatmentwith 2500 nmol of robinetin or quercetin caused a statisticallyinsignificant 16–24% inhibition in the tumor-initiatingactivity of 200 nmol of B[a]P 7,8-diol-9,10-epoxide-2 applied5 min later. Treatment of mice with 2500 nmol of ellagic acid5 min before the application of 50 nmol of B[a]P inhibited themean number of skin tumors per mouse by 28–33% after 15–20weeks of promotion, but these decreases were not statisticallysignificant. Robinetin and quercetin had little or no effecton the tumor-initiating activity of B[a]P on mouse skin. Treatmentof preweanling mice with 1/7, 2/7 and 4/7 of the total doseof ellagic acid (300 nmol), robinetin (1400 nmol), myricetin(1400 nmol) or quercetin (1400 nmol) i.p. on their first, eighthand fifteenth day of life, respectively, did not cause the formationof tumors in animals that were killed 9–11 months later.Similar treatment of preweanling mice with the above doses ofthe phenolic compounds 10 min before the i.p. injection of atotal dose of 30 nmol of B[a]P 7,8-diol-9,10-epoxide-2 duringthe animal's first 15 days of life caused a 44–75% inhibitionin the number of diol-epoxide-induced pulmonary tumors per mouse.Similar treatment with these plant phenols had little or noeffect on B[a]P-induced pulmonary tumors.     

The molecular structure of ({+/-})-8{alpha}, 9{beta}, 10{beta}, 11{alpha}-tetrahydroxy -8, 9, 10, 11 - tetrahydrobenz[a]anthracene; an X-ray crystallographic analysis

The molecular structure of a tetrahydrotetrol that was formedby the hydrolysis of 8, 9ß-dihydroxy-10ß,11ß-epoxy-8,9,10,11-tetrahydrobenz[a]anthracene hasbeen determined by using X-ray single crystal analysis, andrefined to a final discrepancy index R of 0.0684. The data showthat the relative arrangement of the four hydroxyl groups is8, 9ß, 10ß, 11 and that the tetra-hydrobenzenering has a half-chair pucker. Two of the hydroxyl groups areaxial (O8 and O9) and two are equatorial (O10 and O11).     

Mutagenic specificity of syn-benzo[g]chrysene 11,12-dihydrodiol 13,14-epoxide in the dihydrofolate reductase gene of Chinese hamster ovary cells

Polycyclic aromatic hydrocarbons (PAHs) with sterically hinderedfjord region diol epoxides are interesting with respect to theirpotency as carcinogens, interactions with DNA and mutagenicspecificities. Unlike the bay region PAH derivative, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydroxybenzo[a]pyrene(BPDE), reactive metabolites of two fjord region PAH, trans-3,4-dihydroxy-anti-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]-Phenanthrene[(±)-anti-BcPHDE] and trans-11,12-dihydroxy-syn-13,14-epoxy-11,12,13,14-tetrabenzo[g]chrysene[(±)-syn-BgCDE], react with DNA to yield high levelsof adenine adducts. We previously found that forward mutationsinduced by (±)-anti-BcPHDE in the dihydrofolate reductase(dhfr) gene of Chinese hamster ovary (CHO) cells preferentiallytargeted mRNA splice acceptor sites. (±)-anti-BcPHDEand (±)-syn-BgCDE are structurally similar; they differonly by the presence of an additional benzene ring. Thus weused (±)-syn-BgCDE to learn if the mutational targetbias reflects aspects of the mutagen structure or its capacityto efficiently modify deoxyadenosine (dA) in vivo. dhfr^–mutants were induced after treatment of hemizygous UA21 cellswith a 0.75 µM dose of (±)-syn-BgCDE. Cell survivalafter carcinogen exposure was 40%. The induced mutation frequencywas 9x10^–6, nearly 10-fold higher than the spontaneousone, but     

Reactions of stereoisomeric non-bay-region benz[a]anthracene diol epoxides with DNA and conformations of non-covalent complexes and covalent adducts

The reactions of the non-bay-region diol epoxides racemic trans-8,9-dihydroxy-anti-10,11-epoxy-8,9,10,11-tetrahydro-benz[a]anthracene(anti-BA-10,11-DE) and racemic trans-8,9-dihydroxy-syn-10,11-epoxy-8,9,10,11-tetrahydrobenz[a]-anthracene(syn-BA-10,11-DE) with native double-stranded DNA in aqueoussolutions (5 mM sodium cacodylate buffer, pH 7.0, 23°C)was investigated utilizing various spectroscopic techniques.The results of linear dichroism experiments suggest that bothdiastereomers form non-covalent, intercalative complexes withDNA prior to undergoing chemical reactions; the associationconstant for the anti stereoisomers is about twice as large(850 ± 100 M^–1) as that for the syn-diastereomers,thus qualitatively paralleling the behavior established previouslyfor the bay-region diol epoxides of benzo[a]pyrene and benz[a]anthracene.The reaction rates of both anti- and syn-BA-10,11-DE are significantlyaccelerated in the presence of DNA, and the fraction of diolepoxide molecules which bind covalently to DNA is 13 ±2% and 3 ± 1% respectively; these levels of covalentbinding are lower by factors of about two respectively, thanin the case of the bay-region diol epoxides of benz The phenanthrenylresidues in the covalent anti-BA-10,11-DE—DNA adductsare tilted with their long axes closer to the average orientationsof the normals to the DNA bases; in contrast, the adducts derivedfrom the binding of the syn diastereomers, appear to be characterizedby intercalative-type conformations; however, the overall degreesof orientations are weak in the cases of these non-bay-regiondiol epoxide-DNA adducts. Nevertheless, these adduct conformationsresemble those derived from the highly tumorigenic anti andthe less active syn diastereomers of benzo[a] and benz[a]anthracene,thus providing one additional example to the previously observedcorrelations between adduct structure and biological activity.     

Minor products from the reaction of (+) and (-) benzo[a]-pyrene-anti-diolepoxide with DNA

The reaction of trans-7, 8-dihydroxy-anti-9, 10-epoxy-7, 8,9, 10-tetrahydrobenzo[a]pyrene (BP-diolepoxide) with deoxyguanosinehas been studied. In addition to the expected N2-guanine derivativeminor products resulting from reaction at the O6 and 7-positionshave been identified. Reaction of racemic, (+) or (–)BP-diolepoxide with [¹⁴C] and [³H]purine labelled DNA allowedthese same products to be identified and their yields estimated.It was found that the O6 and 7-guanine products were derivedmainly from reaction of the (–)isomer. The 7-substitutedguanine derivative in DNA was unstable, undergoing either spontaneousrelease of the substituted guanine or imidazole ring opening.     

Persistence, gestation stage-dependent formation and interrelationship of benzo[a]pyrene-induced DNA adducts in mothers, placentae and fetuses of Erythrocebus patas monkeys

Since DNA adducts have been detected in the placentae of pregnantwomen who smoke cigarettes, the importance of these adductsas biomarkers of fetal exposure and risk has been evaluatedusing a non-human primate as a model. Pregnant Erythrocebuspatas monkeys on days 50, 100 or 150 of gestation (term = 160± 5 days) were treated once with 5–50 mg/kg benzo[a]pyrene(B[a]P), p.o. Fetuses were removed by Cesarean section 1–50days after treatment and analyzed for DNA adducts by the nucleaseP₁ version of the ³²P-postlabeling method. B[a]P induced highlevels of DNA adducts in all fetal organs, placentae and maternallivers in all three trimesters of gestation. DNA adduct levelswere higher in mid-gestation compared to early and late gestation.The major adduct detected was 10ß-(deoxyguanosin)-N²-yl-7ß,8     

Determination of stereospecificity of benzo[a]pyrene diolepoxide-DNA antisera with site-specifically modified oligonucleotides

Antisera developed against benzo[a]pyrene diolepoxide (BPDE)—DNAadducts are sensitive tools for detection of DNA adducts inhuman samples. All antisera currently used for biomonitoringstudies were produced against DNA or guanosine modified withracemic anti-BPDE. Using a non-competitive enzyme-linked immunosorbentassay (ELISA), Venkatachalam and Wani (Carcinogenesis, 15, 565–572,1994) recently tested polyclonal and monoclonal (5D2) antiserafor cross-reactivity against oligonucleotides containing (+)-and (–)-trans-anti-BPDE-N²-guanine or N⁶-adenine adductsand showed different stereospecificity for the two antisera.Because of the importance of antiserum specificity in humanbiomonitoring studies, we have tested several monoclonal (Mab5D11 and 5D2) and polyclonal (Pab #29) antisera developed againstracemic anti-BPDE-DNA adducts, and Mab 8E11 developed againstanti-BPDE-guanosine adducts. Stereoisomeric anti-BPDE-modifiedoligonucleotide adducts in the sequence 5'-d(CCAT-CG^*CTACC)-3'where G^* = anti-BPDE-N²-dG with (+ )-trans, (–)-trans,(+ )-cis and (–)-cis adduct stereochemistry at the C10position of anti-BPDE were tested by competitive ELISA. Twostructurally related 5-methylchrysene diolepoxide adducts withG^* = (+)- and (–)-trans-anti-5-MeCDE-N²-dG in the sameoligonucleotide were also tested. While Mab5D2 had the highestaffinity for the (–)-trans-anti-BPDE-modified oligomer,Mab 5D11 and 8E11 and Pab #29 recognized the (+ )-trans-anti-BPDE-modifiedoligomer better than the (–)-trans-anti-BPDE modifiedoligomer. Mab 5D11 and Pab #29 recognized racemic anti-BPDE-modifiedDNA adducts better than trans-anti-BPDE-modified oligonucleotides;however, Mab 8E11 showed similar sensitivity to racemic anti-BPDE-DNAadducts and (+ )-and (–)-trans-anti-BPDE-modified oligomers.All antisera exhibited lower reactivities with both 5-MeCDEmodified oligomers. Because of their sensitive detection of(+)-trans-anti-BPDE-dG adducts, the primary adduct producedin vivo, Mab 8E11 and 5D11 and Pab #29 are appropriate for measurementof most adducts formed in humans.