Inhibition of the mutagenicity of bay-region diol-epoxides of polycyclic aromatic hydrocarbons by phenolic plant flavonoids

Myricetin, robinetin and luteolin inhibited the mutagenic activityresulting from the metabolic activation of benzo[a]-pyrene and(±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]-pyrene byrat liver microsomes. These naturally occurring plant flavonoidsand seventeen additional flavonoids and related derivativeswith phenolic hydroxyl groups inhibited the mutagenic activityof (±)-7ß,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(B[a]P 7,8-diol-9,10-epoxide-2), which is an ultimate mutagenicand carcinogenic metabolite of benzo[a]pyrene. Several flavonoidswithout phenolic hydroxyl groups or with methylated phenolichydroxyl groups were inactive. The mutagenic activity of 0.05nmol of BP 7,8-diol-9,10-epoxide-2 towards strain TA 100 ofS. typhimurium was inhibited 50% by incubation of the bacteriaand the diol-epoxide with myricetin (2 nmol), robinetin (2.5nmol), luteolin (5 nmol), quercetin (5 nmol), 7-methoxyquercetin(5 nmol), rutin (5 nmol), quercitrin (5 nmol), delphinidin chloride(5 nmol), morin (10 nmol), myricitrin (10 nmol), kaempferol(10 nmol), diosmetin (10 nmol), fisetin (10 nmol), or apigenin(10 nmol). Considerably less antimutagenic activity was observedfor dihydroquercetin, naringenin, robinin, D-catechin, genistein,kaempferide and chrysin. Pentamethoxyquercetin, tangere-tin,nobiletin, 7,8-benzoflavone, 5,6-benzoflavone, and flavone,which lack free phenolic groups, were inactive. The antimutagenicactivity of hydroxylated flavonoids results from their directinteraction with B[a]P 7,8-diol-9,10-epoxide-2 since the rateof disappearance of the diol-epoxide from cell-free solutionsin 1:9 dioxane:water was markedly stimulated by myricetin, robinetinand quercetin. Myricetin was a highly potent inhibitor of themutagenic activity of bayregion diol-epoxides of benzo[a]pyrene,dibenzo[a,h]pyrene and dibenzo[a,i]pyrene, but higher concentrationsof myricetin were needed to inhibit the mutagenicity of thechemically less reactive benzo[a]pyrene 4,5-oxide and bay regiondiol-epoxides of benz[a]anthracene, chrysene and benzo[c]phenanthrene.     

Inhibition of the mutagenicity of bay-region diol-epoxides of polycyclic aromatic hydrocarbons by tannic acid, hydroxylated anthraquinones and hydroxylated cinnamic acid derivatives

Tannic acid and several hydroxylated anthraquinone and cinnamicacid derivatives inhibited the mutagenic activity of (±)-7ß,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P7,8-diol-9,10-epoxide-2), an ultimate mutagenic and carcinogenicmetabolite of benzo[a]pyrene. The mutagenic activity of 0.05nmol of B[a]P 7,8-diol-9, 10-epoxide-2 towards strain TA 100of Salmonella typhimurium was inhibited 50% by incubation ofthe bacteria and the diol-epoxide with tannic acid (0.5 nmol),anthraflavic acid (7 nmol), rufigallol (7 nmol), quinalizarin(10 nmol), alizarin (30 nmol), purpurin (60 nmol), and danthron(88 nmol). Dose-dependent, but weaker antimutagenic activitywas observed for quinizarin, and a number of hydroxylated cinnamicacid derivatives. Gallic acid and m-digallic acid, major componentsof tannic acid, possessed < 1% of the antimutagenic activityof tannic acid, although m-digallic acid was over 3 times moreactive than gallic acid. The antimutagenic activity of tannicacid was a result of its interaction with B[a]P 7,8-diol-9,10-epoxide-2 since the rate of disappearance of the diol-epoxidefrom cell-free solutions in 1: 9dioxane:water was markedly stimulatedby the polyphenol. Tannic acid was a highly potent inhibitorof the mutagenic activity of the bay-region diol-epoxides ofbenzo[a]pyrene, dibenzo[a,h]pyrene and dibenzo[a,i]pyrene, buthigher concentrations of tannic acid were needed to inhibitthe mutagenicity of the chemically less reactive benzo[a]-pyrene4,5-oxide and the bay-region diol-epoxides of benz[a]-anthracene,chrysene and benz[c]phenanthrene.     

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.     

Inhibitory effect of 3-hydroxybenzo(a)pyrene on the mutagenicity and tumorigenicity of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene

The 12 isomeric phenols of benzo(a)pyrene were tested for their ability to inhibit the mutagenic activity of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [B(a)P 7,8-diol-9,10-epoxide-2], an ultimate mutagenic and carcinogenic metabolite of benzo(a)pyrene. 3-Hydroxybenzo(a)pyrene [3-HO-B(a)P], a major metabolite of benzo(a)pyrene, was the most potent antagonist tested. Approximately 3 nmol of 3-HO-B(a)P, 14 nmol of 10-HO-B(a)P, and 5-8 nmol of 1-, 2-, 4-, 5-, 6-, 7-, 8-, 9-, 11-, and 12-HO-B(a)P inhibited the mutagenic activity of 0.05 nmol of B(a)P 7,8-diol-9,10-epoxide-2 by 50% in Salmonella typhimurium strain TA 100. The importance of the phenolic group for antimutagenic activity was indicated by the lack of antimutagenic activity of benzo(a)pyrene itself. 3-HO-B(a)P also inhibited the mutagenic activity resulting from the metabolic activation of benzo(a)pyrene and (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene by rat liver microsomes. This inhibition may have resulted from an effect of 3-HO-B(a)P on the metabolic activation of these carcinogens and/or from a direct effect on the action of B(a)P 7,8-diol-9,10-epoxide-2. In a mammalian cell culture system utilizing Chinese hamster V79 cells, 3-HO-B(a)P (8 microM) inhibited the mutagenicity of B(a)P 7,8-diol-9,10-epoxide-2 (0.2 microM) by 50%. Although 3-HO-B(a)P was a potent inhibitor of the mutagenic activity of bay-region diol epoxides of benzo(a)pyrene, dibenzo(a,h)pyrene, and dibenzo(a,i)pyrene in S. typhimurium strain TA 100, higher concentrations of 3-HO-B(a)P were needed to inhibit the mutagenicity of the chemically less reactive benzo(a)pyrene 4,5-oxide and the bay-region diol epoxides of benz(a)anthracene, chrysene, and benzo(c)phenanthrene. Both 3-HO-B(a)P and 10-HO-B(a)P accelerated the disappearance of B(a)P 7,8-diol-9,10-epoxide-2 from 1:9 dioxane-water solutions at pH 7 and 25 degrees C. 3-HO-B(a)P, the most effective antimutagen of the B(a)P phenols tested, was much more reactive with the diol epoxide than 10-HO-B(a)P, the least effective antimutagen. The rate constant for the reaction of 3-HO-B(a)P with the diol epoxide exhibited a nonlinear (greater than first-order) dependence on the concentration of the phenol. Evidence was obtained for covalent adduct formation between the diol epoxide and each of the two phenols.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Tumor-initiating activity of the bay-region dihydrodiols and diolepoxides of dibenz[a,j]anthracene and cholanthrene on mouse skin

The tumor-initiating activity of dibenz[a,j]anthracene, (±)dibenz[a,j]anthracene-trans-3,4-dioland (±)dibenz[a,j]anthracene-anti-3,4-diol-1,2-epoxidein mouse skin was examined and compared to that of cholanthrene,(±)cholanthrene-trans-9, 10-diol and (± )cholanthrene-anti-9,10-diol-7,8-epoxide.The tumor-initiating activity of these compounds dissolved inacetone or in tetrahydrofuran (THF) was also compared. In acetone,dibenz[a,j]anthracene was a weak tumor initiator with maximaltumor yields of 1.27 and 3.00 per mouse at 400 and 800 nmoldoses, respectively. At the 400 nmol dose, the diol of thiscompound was slightly more active than the parent compound whilethe tumorigenic activity of the diol-epoxide was significantlyhigher. The diol-epoxide was almost three times more activethan the parent compound as a tumor-initiator. Cholanthrenewas a moderate tumor-initiator with maximal tumor yields of6.90 and 8.86 tumors per mouse at 200 and 600 nmol doses, respectively,after 20 weeks of promotion. At comparable doses, (±)cholanthrene-trans-9,10-diolwas 50% as potent as cholanthrene as a tumor initiator whereasthe diol-epoxide was only minimally active. Replacing the acetonewith THF as solvent vehicle increased the tumor-initiating activityof cholanthrene-diol-epoxide; however, the parent compound stillretained higher tumor-initiating activity than its bay-regiondiol-epoxide. The low tumorigenic activity of cholanthrene-diol-epoxideis thought to reflect the high chemical reactivity and low stabilityof this derivative, which may prevent it from penetrating toepidermal targets. In contrast, the bay-region diol-epoxidederivative of dibenz-[a,j]anthracene appears to be stable enoughto exert greater biologic activity when applied to mouse skin.     

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.     

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).     

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.     

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.     

Instability of ({+/-})-7{beta}, 8{alpha}-dihydroxy-9{beta}, 10{beta}-epoxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene (syn-BaPDE)- DNA adducts formed in benzo[a]pyrene-treated Wistar rat embryo cell cultures

One of the peaks present in HPLC profiles of [³H]benzo[a]-pyrene(BaP)-deoxyribonucleosides prepared by enzymatic degradationof [³H)BaP-DNA isolated from Wistar rat embryo cell culturesexposed to [G-³H)BaP was found to be r-7, c-9, c-10 t-8-tetrahydroxy-7,8, 9, 10-tetrahydroBaP, a BaP-DNA adduct decomposition product(Pruess-Schwartz, D. and Baird, W.M., Cancer Res., 46, 545–552,1986). To investigate the stability of the hydrocarbon-deoxyribo-nucleosidelinkages in intact BaP-modified DNA, DNA was isolated from Wistarrat embryo cells that had been exposed to [G-³H]BaP- and incubatedin darkness at 37°C at a range of pH values from 5 to 11for 72 h or for 1– 150 h at pH 7. The rate of breakdownof (³H)BaP-DNA adducts (0.25%/h) was linear over 150 h. Theamounts of the two major BaP-DNA adduct decomposition products,I and II (present in a ratio of 1: 3), increased with lengthof time of incubation. Formation of I was not affected by pH.whereas, formation of II was highest at acidic and neutral pH.Analysis of the decomposition products by immobilized boronatechromatography and reverse-phase HPLC demonstrated that bothI and II contained cis-vicinal hydroxyl groups and decompositionproduct II cochromatographed with r-7, c-9, c-10, t-8-tetrahydroxy-7, 8, 9, 10-tetrahydroBaP, a (±)- 7ß,8-dihydroxy-9ß, 10ß-epoxy-7, 8, 9, 10-tetrahydroBaP(syn-BaPDE)-derivedtetraol. At neutral pH [³H](±)-syn-BaPDE-modified calfthymus DNA formed a decomposition product identical to II. Analysisof the BaP-DNA adducts that remained covalently bound to theDNA after the above incubations demonstrated that the amountsof both major syn-BaPDE-deoxyguanosine adducts decreased withlength of time of incubation. Thus, syn-BaPDE-deoxyribonucleosideadducts formed in the DNA of [³H)BaP-treated Wistar rat embryocells are unstable and breakdown spontaneously in the absenceof light to yield syn-BaPDE-tetraol decomposition products.     

Carcinogenic activity of benzo[a]pyrene and some of its synthetic derivatives by direct injection into the mouse fetus

We have studied the carcinogenic effects of direct injectionof benzo[a]pyrene (BP) and 6-methyIbenzo[a]pyrene (BP6M) intofetal Swiss mice at 15 days of intrauterine growth. To determinethe sensitivity of mouse fetuses to the action of ultimate carcinogens,benzo[a]pyrene-4,5-oxide (BPO) and a racemic mixture of 7ß,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE), respectively, a proximal metabolite and an ultimatecarcinogenic metabolite of BP, were also investigated in oursystem. The compounds were dissolved in acetone and trioctanoin(1:1) and injected at doses ranging from a minimum of 0.4 toa maximum of 19.8 nmol/fetus. Controls received 1µl/fetusof solvent. The experiment was terminated when the animals were12–15 weeks old. On the basis of the percentages of lungadenomas and the average number of nodules/mouse, BPDE appearsto be the most potent carcinogen of this group, causing 55%of tumors at a dose of 0.4 nmol and 73% at a dose of 4.0 nmol/fetus.At the dose of 0.4 nmol/fetus, neither BP nor BP6M induced tumors;injection of 4.0 or 19.8 nmol/fetus, caused incidences of 53%and 92%, respectively, in the case of BP6M and 24% and 39%,respectively, in the case of BP. BPO was not tumorigenic inthis system, even at doses as high as 15.7 nmol/fetus. Elevenpercent of the control animals had lung adenomas. The averagenumber of nodules/mouse varied with the compound and the doseinjected and closely followed the pattern of tumor incidence.     

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.     

Structures of covalent adducts derived from the reactions of the 9,10-epoxides of 7,8,9,10-tetrahydrobenzo [a] pyrene and 9,10,11,12-tetrahydrobenzo [e] pyrene with DNA

The reaction of a racemic mixture of 7ß, 8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE) and itsenantiomer with DNA is highly stereoselective. About 90% ofthe adducts are derived from the former enantiomer reactingwith the amino group of guanine residues. To investigate thisstereoselectively we compared the reactions of 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyreneand 9,10-epoxy-9,10,11,12-tetrahydrobenzo[e]pyrene with DNA.Most of the stereoselectivity seen with B[a]PDE is lost. Bothepoxides give mainly adducts on the N² group of guanine by bothcis and trans additions to the epoxide. Other adducts, tentativelyidentified as deoxyadenosine derivatives, were also detected.     

High selectivity of polyclonal antibodies against DNA modified by diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxides.

Polyclonal antibodies were developed in New Zealand White rabbits against DNA modified with diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxide (B[c]PhDE)-1 (4-hydroxyl and epoxide cis) and B[c]PhDE-2 (4-hydroxyl and epoxide trans). Antiserum developed against B[c]PhDE-2-DNA was stereoselective. In competitive ELISA assays using wells coated with 160 fmol B[c]PhDE-2-DNA adducts, B[c]PhDE-2-DNA gave 50% inhibition at 200 fmol adducts/well. B[c]PhDE-1-DNA required a 10-fold higher amount of adducts/well to give 50% inhibition. Benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA and 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA caused only a 30% inhibition even at the highest doses tested (greater than 4000 fmol adducts/well). For antiserum developed against B[c]PhDE-1-DNA, 50% inhibition required 570 fmol B[c]PhDE-1-DNA adducts in wells coated with 100 fmol B[c]PhDE-1-DNA adducts. 7,12-Dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA and B[c]PhDE-2-DNA were also effective competitors: they caused 50% inhibition at 1900 and 1800 fmol adducts/well respectively. In contrast, benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA gave no inhibition at the highest dose of competitor tested (4050 fmol adducts/well). Antisera from three rabbits immunized with B[c]PhDE-2-DNA demonstrated similar antigen specificities. The properties of these antisera differ from those reported previously for antibodies developed against benzo[a]pyrene-DNA in that they show selectivity for DNA modified by specific hydrocarbon diolepoxides, in one case for B[c]PhDE-2-DNA and in the other for B[c]PhDE-DNA or 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA. The specificity of these antisera will facilitate analysis of the modification of DNA by different polycyclic aromatic hydrocarbon diolepoxides.     

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

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

Metabolism of benzo[a]pyrene and (-)-trans-benzo[a]pyrene-7,8-dihydrodiol by freshly isolated hepatocytes from mirror carp

The metabolism of benzo[a]pyrene (B[a]P) and (-)-trans-benzo[a]pyrene-7,8-dihydrodiol [(-)-B[a]P-7,8-diol], a major putative proximate carcinogenic metabolite of B[a]P, was compared in freshly isolated hepatocytes from mirror carp, a strain of common carp (Cyprinus carpio, L.). Hepatocytes incubated with 40 microM [3H]B[a]P produced 1.22 nmol equivalents of B[a]P metabolites/mg dry wt of cells/h. Conjugated derivatives represented approximately 65% of all B[a]P metabolites and included glucuronides (38%), glutathione conjugates (21%) and sulfates (6%). About 14% of the total accumulated metabolites of B[a]P determined after 1 h incubations were identified as unconjugated derivatives, predominantly B[a]P-9,10-dihydrodiol and B[a]P-7,8-diol (7.4 and 3.1% of total metabolites respectively), with only traces of B[a]P tetrols (less than 1%). Hepatocytes incubated with 40 microM (-)-[14C]B[a]P-7,8-diol produced 4.78 nmol equivalents of metabolites/mg dry wt during a 1 h incubation, yielding an average rate of metabolism during this time period approximately 53% of that determined after a 5 min incubation. The profile of (-)-B[a]P-7,8-diol metabolites remained constant with incubation time (glucuronides, 30-33%; conjugates with glutathione, 43-46%; polyhydroxylated B[a]P derivatives plus sulfate conjugates, 22-24%). HPLC analysis revealed that polyhydroxylated metabolites amounted to 18% of the total metabolites; thus sulfate conjugates amounted to only 4% of the total metabolites. The trans-2 B[a]P-tetrol, which is the major hydrolysis product of (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (anti-BPDE), represented approximately 11% of the accumulated metabolites of (-)-B[a]P-7,8-diol. Despite the much larger amounts of BPDE formed from (-)-B[a]P-7,8-diol than from B[a]P, the amounts of B[a]P equivalents covalently bound to cellular DNA were the same following 1 h incubations with either substrate (247 +/- 42 or 212 +/- 42 pmol/mg DNA respectively). Thus biochemical and physiological factors other than the production of BPDE are critically involved in determining the level of DNA adducts in hepatocytes as well as the role of these adducts in hepatocarcinogenesis.     

Comparative dose-response tumorigenicity studies of dibenzo[alpha,l]pyrene versus 7,12-dimethylbenz[alpha]anthracene, benzo[alpha]pyrene and two dibenzo[alpha,l]pyrene dihydrodiols in mouse skin and rat mammary gland

Comparative studies were conducted of the tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland of dibenzo[a,l]pyrene (DB[a,l]P) versus 7,12-dimethyl-benz[a]anthracene (DMBA), the most potent recognized carcinogenic polycyclic aromatic hydrocarbon (PAH); benzo[a]pyrene (B[a]P), the most potent recognized carcinogenic environmental PAH; DB[a,l]P 8,9-dihydrodiol, the K-region dihydrodiol; and DB[a,l]P 11,12-dihydrodiol, precursor to the bay-region diolepoxide. The tumor-initiating activity of DB[a,l]P and B[a]P was compared in the skin of female SENCAR mice at doses of 300, 100 and 33.3 nmol. The mice were promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA) twice-weekly for 13 weeks. DB[a,l]P at all doses induced significantly more tumors than B[a]P at the corresponding dose, with a significantly shorter latency. Subsequently, the tumor-initiating activity of DB[a,l]P was compared in the skin of female SENCAR mice to that of DMBA, B[a]P, DB[a,l]P 8,9-dihydrodiol and DB[a,l]P 11,12-dihydrodiol at doses of 100, 20 and 4 nmol. The mice were promoted with TPA twice-weekly for 24 weeks. In addition, groups of mice were initiated with 100 nmol of DB[a,l]P, DMBA, B[a]P, DB[a,l]P 8,9-dihydrodiol or DB[a,l]P 11,12-dihydrodiol and kept without promotion. This experiment showed that in the mouse skin, DB[a,l]P and DB[a,l]P 11,12-dihydrodiol displayed similar tumor-initiating activity with a response inversely proportional to the dose, presumably due to the toxicity of the compounds. At the high dose they elicited tumors earlier than DMBA, though DMBA produced a much higher tumor multiplicity. At the low dose, DMBA, DB[a,l]P and DB[a,l]P 11,12-dihydrodiol exhibited similar tumorigenicities. DB[a,l]P 8,9-dihydrodiol was a marginal tumor initiator. Once again, DB[a,l]P was by far a much stronger tumor initiator than B[a]P. Female Sprague-Dawley rats were treated with 1.0 or 0.25 mumol of DB[a,l]P, DMBA or B[a]P by intramammillary injection at eight teats. DB[a,l]P at both doses was a more potent carcinogen than DMBA at the corresponding dose in the rat mammary gland. B[a]P was a marginal mammary carcinogen, eliciting only a few fibrosarcomas. Thus, these data suggest that DB[a,l]P is the strongest PAH carcinogen ever tested.     

Effects of glutathione transferase activity on benzo[a]pyrene 7,8-dihydrodiol metabolism and mutagenesis studied in a mammalian cell co-cultivation assay

This study deals with the role of glutathione transferase (GST)-mediatedconjugation of (+)-7ß,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE) in two mammalian cell lines, human mammary carcinomacells (MCF-7) and rat hepatoma cells (H4IIE), in relation tothen-capacity to metabolize (–)-trans-7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene[(–)-BP-7,8-diol] to products that induce mutations inco-cultivated V79 cells. Both MCF-7 and H4IIE cells metabolized(–)-BP-7,8-diol to BPDE, but mutations in co-cultivatedV79 cells were only detected with MCF-7 cells. However, depletionof glutathione (GSH) in H4HE cells increased the mutagenidtyof (– )-BP-7,8-diol to a similar level to that found withMCF-7 cells. Measurements of GST activity using GSH and post-microsomalsupernatants from H4IIE, V79 and MCF-7 cells indicated a substantialdifference in conjugation capacity. Although preparations fromall three cell-lines showed GST activity with l-chloro-2,4-dlnitro-benzeneas the substrate, GST activity towards BPDE could only be detectedin supernatants from H4HE cells. This is consistent with thepresence of GST 7-7 an isoenzyme highly efficient hi catalysingBPDE-GSH conjugation. The difference in GSH-conjugation activitytowards BPDE was confirmed using intact H4IIE and MCF-7 cellsin culture. These results indicate that GSH-conjugation playsa pivotal role in mutagenesis induced by polycyclk aromatichydrocarbons (PAH). Accordingly, a deficiency in GSH-conjugationcapacity may be regarded as one important factor in defininga target cell population with an increased risk for tumour initiationfollowing exposure to PAH.     

Anticarcinogenic and cocarcinogenic effects of benzo[e]pyrene and dibenz[a,c]anthracene on skin tumor initiation by polycyclic hydrocarbons

In the present study, we have examined the effects of benzo[e]pyrene(B[e]P) and dibenz[a,c]anthracene (DB[a,c]A) on the skin tumor-initiatingactivities of methylated and nonmethylated polycyclic aromatichydrocarbons (PAH). B[e]P, when applied 5 min prior to initiationwith seven different PAH skin carcinogens, effectively inhibitedthe tumorinitiating activities of 7,12-dimethylbenz[a]anthracene(DMBA) and dibenz[a,h]anthracene (DB[a,h]A) but had little orno effect on the tumor-initiating activities of 3-methylcholanthrene(MCA), 7-methylbenz[a]anthracene (7-MBA), 12-methylbenz[a]anthracene(12-MBA), and 5-methylchrysene (5-MeC). B[e]P potentiated thetumor-initiating activity of benzo[a]pyrene (B[a]P) by 30%.DB[a,c]A, when applied 5 min prior to initiation, inhibitedthe tumor-initiating activities of DMBA, MCA, and DB[a,h]A buthad little or no effect on the tumor-initiating activities ofB[a]P, 7-MBA, 12-MBA, and 5-MeC. DB[a,c]A, when applied 12,24, or 36 h prior to initiation with B[a]P, which allowed timefor induction of epidermal monooxygenase enzymes, inhibitedtumor initiation. The covalent binding of DMBA and B[a]P toepidermal DNA was examined under the influence of B[e]P. Dosesof 20 and 200 nmol B[e]P given 5 min prior to 10 nmol [³H]DMBAreduced binding to 47 and 22%, respectively, of the controlvalue. In contrast, doses of 200 or 2000 nmol B[e]P given 5min prior to 200 nmol [³H]B[a]P had little or no effect on totalbinding. The data indicate that one cannot predict anti andcocarcinogenic effects of B[e]P and DB[a,c]A on the basis ofa presence or absence of a methyl substituent. In addition,fundamental differences exist in the processing and metabolismof DMBA and B[a]P by mouse epidermal cells.