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.     

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.     

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.     

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.     

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 ({+/-})- 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.     

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).     

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.     

Application of fluorescence and linear dichroism techniques to the characterization of the covalent adducts derived from interaction of ({+/-})-trans-9,10-dihydroxy-anti-11,12-epoxy-9,10,11,12-tetrahydrobenzo[e]pyrene with DNA

Spectroscopic techniques including absorption, fluorescenceexcitation and emission spectra, fluorescence decay profiles(determined by single photon counting techniques), and electriclinear dichroism are applied to a study of the conformationof covalent adducts derived from a reaction of 9,10-dihydroxy-11,12-epoxy-9,10,11,12-tetrahydro[e]pyrene(B[e]PDE) with DNA. The characteristics of non-covalent adductsobtained from the intercalative binding of 9,10,11,12-tetrahydroxytetrahydrobenzo[e]pyrene(B[e]PT) (derived from the hydrolysis of B[e]PDE) with DNA arecompared to those of the covalent B[e]PDE–DNA adducts.It is shown that there are two types of binding sites in B[e]PDE–DNAadducts: (1) an exterior binding site similar to the one observedwith the isomeric 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(B[a]PDE)–DNA adducts, and (2) a quasi-intercalative typeof binding site in which the properties of the pyrene chromophoreare similar to those of an intercalated pyrene moiety, but inwhich the red shift in the absorption maximum, and fluorescencequenching are less pronounced. This latter conformation is notobserved in covalent B[a]PDE–DNA adducts. It is shownthat the DNA concentration is an important parameter in determiningthe relative number of pyrene chromophores at these two bindingsites. The extent of covalent binding of B[e]PDE is 4–8times less than the binding of B[a]PDE to DNA under the sameexperimental conditions. The reduced reactivity of B[e]PDE istentatively attributed to steric hindrance due to quasi-diaxialconformations of the two hydroxyl groups in one of the two bay-regionsof B[e]PDE.     

Quantification of (7S,8R)-dihydroxy-(9R,10S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene adducts in human serum albumin by laser-induced fluorescence: implications for the in vivo metabolism of benzo[a]pyrene.

The ubiquitous environmental carcinogen benzo[a]pyrene (BaP) is metabolized in vivo in humans to its ultimate carcinogenic form of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Mouse skin tumorigenicity studies indicate that the (7R,8S,9S,10R) enantiomer of BPDE, (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(7R,8S,9S,10R)-BPDE], is a potent tumor initiator, whereas the (7S,8R,9R,10S) enantiomer of BPDE, (7S,8R)-dihydroxy-(9R,10S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(7S,8R,9R,10S)-BPDE], may act as a tumor promoter. In vitro experiments have shown that human liver microsomes are capable of metabolizing BaP to both the (7R,8S,9S,10R) and (7S,8R,9R,10S) enantiomers of BPDE. However, the metabolism of BaP to (7S,8R,9R,10S)-BPDE has not been demonstrated in humans in vivo. The adducts formed between human serum albumin (HSA) and the (7S,8R,9R,10R) and (7R,8S,9S,10R) enantiomers of BPDE have been described previously. (7S,8R,9R,10S)-BPDE forms a stable adduct at histidine146 of HSA, whereas (7R,8S,9R,10R)-BPDE forms a relatively unstable ester adduct at aspartate187 or glutamate188 of HSA. Using high-performance liquid chromatography with laser-induced fluorescence (LIF) detector, we quantified the level of (7S,8R,9R,10S)-BPDE adducts at histidine146 in HSA isolated from 63 healthy males who were population control subjects for an ongoing case-control study of bladder cancer. By design, roughly half of the participants were lifelong nonsmokers (n = 35), whereas the remaining 28 participants were current smokers of varying intensities. HP-BPDE adducts were detected in 60 of the 63 samples (95%) by HPLC-LIF. Adduct levels ranged from undetectable (<0.04 fmol/mg HSA) to 0.77 fmol/mg HSA. The samples had a mean and median (7S,8R,9R,10S)-BPDE-HSA adduct level of 0.22 and 0.16 fmol of adduct/mg albumin, respectively. Mean adduct levels did not differ between smokers and nonsmokers (P = 0.72). Occupational exposure to polycyclic aromatic hydrocarbons was unrelated to adduct level (P = 0.62). Intake frequencies of two food items showed statistically significant associations with adduct levels. Consumption of sweet potatoes was negatively related to adduct level (P = 0.029), whereas intake of grapefruit juice was positively related to adduct level (P = 0.045). None of the three indices of residential ambient air pollution under study showed a statistically significant association with adduct levels.     

Molecular structure of (+/-) 7alpha,8beta-dihydroxy 9beta, 10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; an X-ray crystallographic study

The molecular structure of 7, 8ß-dihydroxy-9ß,10ß-epoxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene, anultimate carcinogenic form of benzo[a]pyrene has been determinedby X-ray crystallography. The tetra-hydrobenzene ring has diequatorialhydroxyl sub-stitutents and a C8 half-chair conformation. Theepoxide ring is symmetric and the plane of the epoxide ringis approximately at right angles to the plane of the aromaticsystem. A variety of other geometric parameters are reported.     

Contrasting disposition and metabolism of topically applied benzo(a)pyrene, 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 mouse epidermis in vivo

Whereas extensive evidence indicates that 7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BPDE) is a major ultimate carcinogen of benzo(a)pyrene (BaP) in mouse skin, tumorigenicity studies have consistently shown that anti-BPDE is less active then BaP in this model system. In order to investigate factors responsible for this apparent contradiction, we have compared the disposition, metabolism, and DNA binding of [3H]BaP, (+/-)-trans-7,8-[14C]dihydroxy-7,8-dihydrobenzo(a)pyrene [(+/-)-[14C]BaP-7,8-diol), and (+/-)-anti-[3H]BPDE in mouse epidermis in vivo. There were remarkable differences in the total radioactivity recovered in epidermis at various times after topical application of BaP, BaP-7,8-diol, and anti-BPDE. BaP and its metabolites were removed from epidermis gradually (t1/2 approximately equal to 2 h). However, 60-65% of anti-BPDE disappeared from mouse epidermis within 3 min of application, while a second slower phase of removal of radioactivity was observed between 8 min and 2 h. The kinetics of removal of BaP-7,8-diol and its metabolites were intermediate between those of BaP and anti-BPDE. The half-life of anti-BPDE in mouse epidermis was measured by trapping it with 2-mercaptoethanol. The initial half-life was about 6 min, similar to that observed in vitro. However, following the initial rapid penetration of anti-BPDE through epidermis most of the remaining material became immobilized in an epidermal binding site in which its half-life was greater than 2 h. Qualitatively, the metabolite patterns of BaP, BaP-7,8-diol, and anti-BPDE were similar to expectations based on in vitro studies. However, the kinetics of metabolite formation from BaP were different from those of BaP-7,8-diol or anti-BPDE. The extents of formation of anti-BPDE-DNA adducts 24 h after application of BaP, BaP-7,8-diol, or anti-BPDE to mouse skin were similar despite the fact that the levels of anti-BPDE present in epidermis were about 50 to 100 times greater after application of BaP-7,8-diol or anti-BPDE than after application of BaP. The results of this study demonstrate that the quantitative aspects of BaP-7,8-diol and anti-BPDE metabolism and disposition in mouse skin are different from those of BaP and indicate that the relatively low tumorigenicity of BaP-7,8-diol and anti-BPDE in mouse skin may be partially attributable to differences between the disposition of these metabolites when topically applied compared to when they are generated intracellularly from BaP.     

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)     

Studies on glutathione transferases belonging to class pi in cell lines with different capacities for conjugating (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The glutathione transferases (GST) belonging to class pi are primarily responsible for the intracellular detoxification of the highly mutagenic and carcinogenic compound (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). The aim of the present investigation was to study the nature and function of the GST pi gene in relation to the mutagenicity of BPDE in different cell lines. The studies were performed on three cell lines commonly used in toxicological studies, i.e. rat hepatoma cells (H4IIE), human mammary carcinoma cells (MCF-7) and Chinese hamster lung fibroblasts (V79). Western blotting with antisera against GST pi revealed a high level of reaction with cytosol from V79 and H4IIE cells. Furthermore, cytosol from the V79 cells demonstrated low levels of GSTs belonging to the alpha and mu classes, suggesting that a considerable portion of the total capacity of these cells to conjugate chlorodinitrobenzene (CDNB) was provided by GST pi. The level of mRNA for GST pi, as measured by Northern blots, was high in V79 and H4IIE and undetectable in the MCF-7 cell line. Analysis of the DNA fragment patterns using a series of restriction enzymes, revealed that all three cell lines have the pi class gene, although with different band patterns. The findings with H4IIE and MCF-7 cells with respect to their expression of the GST pi gene and their ability to conjugate BPDE were in agreement with the mutagenic effects of BPDE, produced by metabolic activation of (-)-7 beta, 8 alpha-dihydroxybenzo[a]-pyrene in the cells. In contrast, V79 cells although expressing high levels of GST pi, showed no ability to conjugate BPDE or to inhibit the mutagenicity of this compound. Based on these results, we suggest that V79 Chinese hamster lung cells contain a GST pi with a different substrate specificity from those of the human and rat GST pi enzymes.     

Inhibition of the skin tumorigenicity of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene by tannic acid, green tea polyphenols and quercetin in Sencar mice

The effect of pretreatment of skin of Sencar mice with topically applied tannic acid, quercetin and green tea polyphenols (GTP) on the skin tumor initiating activity of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-2) has been evaluated. The animals were pretreated with the plant phenols (tannic acid and quercetin (3000 nmol) or GTP 24 mg/mouse) for 7 days after which they received a single topical application of 200 nmol of BPDE-2 as the initiating agent. Beginning 7 days following initiation animals received twice weekly applications of 3.24 nmol of 12-O-tetradecanoyl phorbol-13-acetate (TPA). Tannic acid and GTP afforded significant protection against skin tumor induction. These inhibitory effects were verified both by prolongation of the latency period and subsequent development of tumors. Quercetin, on the other hand, afforded only moderate protection. Each phenolic compound was found to be highly effective in accelerating the disappearance of BPDE-2 from aqueous medium. Our results suggest that tannic acid and GTP have substantial potential for protecting against the skin tumorigenic response to BPDE-2 and the mechanism of inhibition may involve inactivation of the reactive carcinogenic moiety.     

The p53 tumor suppressor protein reduces point mutation frequency of a shuttle vector modified by the chemical mutagens (+/-)7, 8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, aflatoxin B1 and meta-chloroperoxybenzoic acid.

p53 has been postulated to be the guardian of the genome. However, results supporting the prediction that point mutation frequencies are elevated in p53-deficient cells either have not been forthcoming or have been equivocal. To analyse the effect of p53 on point mutation frequency, we used the supF gene of the pYZ289 shuttle vector as a mutagenic target. pYZ289 was treated in vitro by ultraviolet irradiation, aflatoxin B1, (+/-)7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and meta-chloroperoxybenzoic acid and then transfected into p53-deficient cells with or without a p53 expression vector. p53 reduced the mutant frequency up to fivefold when pYZ289 was treated with aflatoxin B1, (+/-)7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene or meta-chloroperoxybenzoic acid but not when it was ultraviolet-irradiated. The p53-dependent mutation frequency reduction was higher at a higher level of premutational lesions for aflatoxin B1 and (+/-)7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and at a lower level of lesions for meta-chloroperoxybenzoic acid. This suggests that the chemical mutagens produce, in a dose-dependent fashion, two kinds of DNA damage, one subject to p53-dependent mutation frequency reduction and the other not. These results indicate that p53 can reduce the point mutation frequency in a shuttle vector treated by chemical mutagens and suggest that p53 can act as guardian of the genome for at least some kinds of point mutations.     

Lack of a cell cycle-dependent strand bias for mutations induced in the HPRT gene by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene in excision repair-deficient human cells

We showed previously that in repair-proficient human cells the location of the premutagenic lesion induced by (+-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo(a)pyrene (BPDE), namely, the guanine in a G.C base substitution, in mutants derived from cells treated at the beginning of S phase just when the hypoxanthine (guanine) phosphoribosyltransferase gene is replicated, differs significantly from their location in cells treated 12 h prior to the beginning of S phase (early G1 phase) (R-H. Chen et al., Proc. Natl. Acad. Sci. USA, 87:8680-8684, 1990). This suggests that the cells preferentially remove BPDE adducts from the transcribed strand. We have now determined the kinds and location of independent mutations induced by BPDE in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene of synchronized repair-deficient xeroderma pigmentosum cells (XP12BE, complementation group A), treated at S or in G1. Nineteen of 25 mutants derived from S-treated cells and 23 of 28 mutants from G1-treated cells contained base substitutions. Eighty-nine percent of these involved a G.C base pair, primarily G.C----T.A transversions. This is similar to the kinds of mutations we saw in the repair-proficient cells. However, in contrast to our earlier results, there was no change in strand distribution of premutagenic BPDE lesions. In both populations, approximately 26% of the base substitutions involving G.C base pairs had the G located in the transcribed strand, 5 of 18 in the S phase mutants, and 5 of 21 in the G1 phase mutants. These results support the hypothesis that the strong strand bias of induced mutations observed in the repair-proficient cells results from preferential repair of BPDE-induced DNA damage from the transcribed strand.