p53Protein expression is correlated with benzo[a]pyrene-DNA adducts in carcinoma cell lines

p53 inhibits cell cycle progression and DNA damaging cytostaticsinduce p53 protein expression, indicating that p53 respondsto DNA damage. We have measured benzo[a]pyrene (BP)-inducedDNA damage in association with p53 expression. The most relevantDNA adducts for carcinogenesis, benzo[a]pyrene-7,8-diol-9,10-epoxide-DNAadducts, were measured by synchronous fluorescence spectrophotometryand p53 immunohistochemistry using polyclonal antibody CM1,which detects both wild-type and mutated forms of p53. Activationof BP in A-549 lung carcinoma and MCF-7 breast adenocarcinomacell lines containing wild-type p53 was followed by an increasein p53 protein expression.     

High survial rate of hamsters given intratracheal instillations of benzo[a]pyrene and ferric oxide and kept on a high {beta}-carotene diet

The study described in this paper was primarily conducted toidentify the cell types involved in the formation, progressionand regression of metaplastic changes in the respiratory tractepithelium of hamsters after intratracheal intubations withbenzo[a]pyrene Furthermore, the role of vitamin A and ß;-carotenein these processes was studied. In the course of the study aremarkable effect of dietary ß;-carotene on survivalof hamsters became a subject of investigation. Hamsters werefed diets with various levels of vitamin A or ß-caroteneand were treated intratracheally with a suspension of benzo[a]pyrenewith ferric oxide in saline. The tumour response of the respiratorytract was very low (2.8%) and hyper- and metaplasia of respiratoryepithelium were virtually absent. However, an interesting observationwas an exceptionally low mortality of only 2% after 69 weeksin the group of hamsters fed a high ß-carotene diet(1% w/w), whereas in the other groups mortality after 69 weeksamounted to 25%. Although the exact cause of death of most ofthe hamsters could not be established, a 40% reduction of lipidperoxidation in the livers was found in the high ß-carotenegroup. Moreover, In this group the degree and incidence of nephroslsand of focal mineralization of kidneys and heart were lowerthan in the other groups. These favourable effects of the highß-carotene diet may have contributed to the unusuallyhigh survival rate in hamsters fed this diet. Further studiesare planned to verify and study this observation.     

SHORT COMMUNICATION: TPA decreases the p53 response to benzo[a]pyrene-DNA adducts in vivo in mouse skin

TPA, a well-known tumor promoter, decreased the response ofnuclear P⁵³ immunoreactivity to benzo[a]pyrene-7, 8-diol-9,10-epoxide (BPDE)-DNA adducts in C57BL/6 mouse skin in vivo.A dose-dependent increase in both the level of BPDE-DNA adductsand nuclear p⁵³ immunoreactivity was found in mice treated topicallywith 50–750 µg benzo[a]pyrene. Such a positive correlationbetween the adducts and p53 positivity was suggested by an earlierstudy. Since p⁵³ probably functions in DNA damage control, interferenceby TPA with the p53 response could be a mechanism in TPA- inducedtumor promotion. Whether such a mechanism is more general intumor promotion deserves further study.     

Thapsigargin has similar effect on p53 protein response to benzo[a]pyrene-DNA adducts as TPA in mouse skin.

The level of p53 tumor suppressor protein increases in response to DNA damage caused by benzo[a]pyrene (B[a]P). The most used tumor promoter in the two step mouse skin carcinogenesis model, 12-O-tetradecanoylphorbol-13-acetate (TPA) decreases this response in mouse skin. In this study the effect of another promoter, thapsigargin was tested on B[a]P-induced p53 response using immunohistochemistry, western blotting and immunoelectron microscopy. We also studied the localization of p53 protein after treatments with BP and TPA or thapsigargin. Thapsigargin had a TPA-like effect on the acute induction of p53 protein related to benzo[a]pyrene-7, 8-diol-9,10-epoxide-DNA adducts in the skin of C57BL/6 mouse. After B[a]P treatment, there was slightly more putatively wild-type p53 protein in nuclei than in cytoplasm of the cells. Neither TPA nor thapsigargin affected the localization of p53 protein. Since both compounds increase the level of intracellular calcium, the inhibition of the p53 response may depend on the level of intracellular calcium. Inhibition of the putatively genome-protecting increase in p53 protein may be one of the critical effects of tumor promoters.     

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.     

Detection of benzo[a]pyrene-DNA adducts in cultured cells treated with benzo[a]pyrene diol-epoxide by quantitative immunofluorescence microscopy and 32P-postlabelling; immunofluorescence analysis of benzo[a]pyrene-DNA adducts in bronchial cells from smoking individuals

Monoclonal antibodies were raised against the reaction product of benzo[a]pyrene diol-epoxide (BPDE) and deoxyguanosine-5'-monophosphate. The antibodies were used for detection of DNA adducts in situ in BPDE-treated cultured human fibroblasts by immunofluorescence microscopy. Analogue-digital conversion of the fluorescence signal and further image processing allowed measurement of the immunospecific fluorescence in the nuclei of these cells. The results are compared with the adduct levels measured in isolated DNA by 32P-postlabelling. Preliminary results are shown of the application of the immunofluorescence method to the analysis of DNA adducts in bronchial cells obtained from smoking individuals.     

A comparison between the activation of benzo[a]pyrene in organ cultures and microsomes from the tracheal epithelium of rats and hamsters

Epidermoid cancers of tracheal origin produced in Syrian goldenhamsters and Fischer strain 344 rats are models for human bronchogeniccarcinomas. These two species differ, however, in the sensitivityof their tracheal epithelia to tumor induction elicited by intratrachealbenzo[a]pyrene (BP)-ferric oxide administration. The tracheasof hamsters are quite senistive to the carcinogenic effectsof BP-ferric oxide, but rat tracheas are apparently resistantto effects of comparable treatments by this route of administration.Rat tracheas are not completely resistant to polynuclear hydrocarboncarcinogenesis because in the heterotopic tracheal graft model,epidermoid carcinomas have been produced frequently. To determinewhether differences in BP metabolism could explain this differencebetween species, quantitative kinetic and chromatographic studiesof benzo[a]pyrene monooxygenase activity were carried out inepithelial microsomes and cells from organ cultures of rat andhamster tracheas. The V_max was 2-fold greater in hamster trachealcells than in rat tracheal cells whereas the K_m values wereidentical. H.p.l.c. profiles from microsomes of rat and hamstertracheal epithelial cells incubated with BP exhibited extremedifferences. Hamster tracheal microsomes produced large proportionsof BP-quinones, BP-phenols, and BP-diols but rat tracheal microsomesproduced mostly 3-OH BP. The total metabolic rate for BP inrat tracheal organ cultures was half that in cultures of hamstertracheas. The metabolites isolated in organ cultures of hamsterand rat tracheas well reflected secondary reactions of conjugationand recycling. When evaluated with respect to the amount oftissue used, the most striking difference between rat and hamstertracheal organ cultures was in the amount of products whichcochromatographed with bay-region BP-tetrols. The amount ofBP-tetrols produced by hamster tracheas was 0.22 pmol/mg tissue/24h, and by rat, 0.012 pmol/mg tissue/24 h. The level of BP-DNAbinding (in pmol/µg DNA/24 h) catalyzed by hamster trachealcells was 26.6 ± 11.4, and by rat tracheas     

Formation and persistence of benzo[a]pyrene-DNA adducts in mouse epidermis in vivo: importance of adduct conformation

The formation and repair of benzo[a]pyrene diol epoxide-N²-deoxyguanosineadducts (BPDE-N²-dG) in DNA isolated from the skin of mice treatedtopically with benzo[a]pyrene (BP) was studied by ³²P-postlabelingand by low-temperature fluorescence spectroscopy under low resolutionand under high resolution fluorescence line narrowing (FLN)conditions. In agreement with earlier studies, total BP-DNAbinding reached a maximum at 24 h after treatment (dose: 1 µmol/mouse),then declined rapidly until 4 days after treatment and muchmore slowly thereafter. An HPLC method was developed which resolvedthe ³²P-postlabeled (–)-trans- from (–)-cis-anti-BPDE-N²-dG,and (+)-trans- from (+)-cis-anti-BPDE-N² High performance liquidchromatography analysis of the major TLC adduct spot (containing>80% of the total adducts) obtained by postlabeling BP-modifiedmouse skin DNA showed that it consisted of a major componentthat coeluted with (–)-cis-/(+)-trans-anti-BPDE-N²-dGand a minor component that coeluted with (–)-trans-/(+)-cis-anti-BPDE-N²-dGand that the minor component was repaired at a slower rate thanthe major component. Low-temperature fluorescence spectroscopyof the intact DNA identified the major adduct as (+)-trans-anti-BPDE-N²-dGand the minor adduct fraction consisted mainly of (+)-cis-anti-BPDE-N²In agreement with the ³²P-postlabeling results it was observedby fluorescence spectroscopy that the (+)-cis-adducts were repairedmore slowly than most other adducts. Moreover, the (+)-trans-adductsexhibited a broad distribution of base-stacked, partially base-stackedand helix-external conformations. Mouse skin DNA samples obtainedat early timepoints (2–8 h) after treatment with BP containedsubstantially more of the ‘external’ adducts, whilesamples at later timepoints (24–48 h) contained relativelymore adducts in the base-stacked conformation, indicating alsothat the latter adducts are repaired less readily than the former.The possible biological significance of these novel observationsof conformation-dependent rates of DNA adduct repair and theirpossible dependence on DNA sequence, are discussed.     

p53-dependent global genomic repair of benzo[a]pyrene-7,8-diol-9,10-epoxide adducts in human cells

The global genomic repair of DNA adducts formed by the human carcinogen (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) has been studied by 32P-postlabeling in human fibroblasts in which p53 expression can be regulated. At low BPDE adduct levels (10-50 adducts/10(8) nucleotides), repair was rapid and essentially complete within 24 h in p53+ cells, whereas no repair was detected within 72 h in similarly treated p53- cells. At 10-fold higher BPDE adduct levels, repair under both conditions was rapid up to 8 h, after which a low level of adducts persisted only in p53- cells. These results demonstrate a dependence on p53 for the efficient repair of BPDE adducts at levels that are relevant to human environmental exposure and, thus, have significant implications for human carcinogenesis.     

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.     

Formation and repair of benzo[a]pyrene--DNA adducts in cultured hamster tracheal epithelium determined by 32P-postlabeling analysis and unscheduled DNA synthesis

Hamster tracheal organ cultures were used to investigate therelationship between DNA adduct formation measured directlyby the ³²P-postlabeling assay, and the DNA damage measured indirectlyby the unscheduled DNA synthesis (UDS) assay. Hamster tracheaswere treated with three concentrations of benzo[a]pyrene (B[a]P)for 2 days. Postlabeling and UDS assays were also carried outa few days after removal of the B[a]P. Furthermore, the typesof B[a]P—DNA adducts formed in the in vitro organ culturewere qualitatively compared with those formed in vivo afterintratracheal intubation of B[a]P attached to Fe₂O₃ particles.In vivo only one adduct was detected by ³²P-postlabeling. Thisadduct co-chromatographed with the trans-addition produce ofdG and (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE). In vitro, a clear B[a]P-DNA adduct pattern was alsofound with the ³²P-postlabeling assay. Four different adductswere found. The main adduct spot migrated to the same positionon the thin-layer chromatogram as the in vivo adduct. B[a]P-DNAadduct formation was both time-and dose-dependent. During thefirst day after removal of B[a]P the adduct levels still increased,thereafter they decreased at all B[a]P concentrations. A time-and dose-dependent increase in UDS was observed in the trachealepithelial cells treated with B[a]P in vitro. After removalof the B[a]P, UDS decreased immediately, in contrast to theformation of DNA adducts. The results of the present study showthat B[a]P induces time- and dose-dependently both DNA adductsand UDS in hamster tracheal organ culture. Moreover, the mainDNA adduct formed in vitro, dG-(+)-anti-BPDE, was the same asthat found in vivo.     

Highly sensitive chemiluminescence immunoassay for benzo[a]pyrene-DNA adducts: validation by comparison with other methods,and use in human biomonitoring

A chemiluminescence immunoassay (CIA) utilizing antiserum elicited against DNA modified with (+/-)-7beta, 8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]- pyrene (BPDE) has been developed and validated to study the formation of polycyclic aromatic hydrocarbon (PAH)-DNA adducts in human tissues. Advantages include a low limit of detection for 10b-(deoxyguanosin-N(2)-yl)-7beta,8alpha,9alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG, approximately 1.5 adducts/10(9) nucleotides using 20 micro g DNA) and a high signal-to-noise ratio (> or =100). The CIA BPDE-DNA standard curve gave 50% inhibition at 0.60 +/- 0.08 fmol BPdG (mean +/- SE, n = 30), which was a 10-fold increase in sensitivity compared with the dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). Calf thymus DNA modified with [1,3-(3)H]BPDE was assayed by radiolabeling, (32)P-postlabeling, DELFIA and CIA, and all assays gave similar values. Liver DNAs from mice exposed to 0.5 and 1.0 mg [7,8-(3)H]benzo[a]pyrene (BP) were assayed by the same four assays and a dose-response was obtained with all assays. The BPDE-DNA CIA was further validated in MCL-5 cells exposed to 4 micro M BP for 24 h, where nuclear and mitochondrial DNA adduct levels were associated with an increase in DNA tail length measured by the Comet assay. Human peripheral blood cell (buffy coat) DNA samples (n = 43) obtained from 25 individuals who were either colorectal adenocarcinoma patients or controls were assayed by BPDE-DNA CIA. Three samples (7%) were non-detectable, and the remaining 40 samples had values between 0.71 and 2.21 PAH-DNA adducts/10(8) nucleotides. The intra-assay coefficient of variation (CV), for four wells on the same microtiter plate, was 1.85%. Sufficient DNA for two assays, on separate plates, was available for 38 of the 43 samples, and the PAH-DNA adduct values obtained were highly correlated (r(2) = 0.95). Coded duplicate DNA samples from 15 individuals were assayed four times gave an inter-assay CV of 13.8%.     

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

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

Alterations in keratin expression in hamster tracheal epithelial cells exposed to benzo[a]pyrene.

Changes in keratin expression were documented in cultures of hamster tracheal epithelial (HTE) cells exposed to vitamin A (retinol 10(-6) M) and benzo[a]pyrene (B[a]P), two agents known to affect the differentiation of tracheal epithelial cells in vivo. Keratin protein patterns were determined after 10 days in culture in the presence and absence of B[a]P in order to determine whether the expression of these proteins was altered by this carcinogen. HTE cells maintained in the presence of vitamin A expressed four simple epithelial keratins (7,8,18 and 19) while vitamin A deficient HTE cells expressed four additional cytokeratins (5,6,14 and 17). No effect of B[a]P on keratin expression was observed in vitamin A treated cells. However, vitamin A deficient HTE cells exposed to B[a]P (0.05-10 micrograms/ml) demonstrated a decrease in the expression of the four differentiation-related keratins while the simple epithelial keratins appeared to be unaffected. These observations were verified at the RNA level employing Northern blot analysis using cDNA probes for human keratins 5,6 and 14. Results demonstrate that B[a]P alters the expression of differentiation-related genes, the cytokeratins, in cell types known to develop into tumors of the respiratory tract.     

Asbestos and benzo[a]pyrene act synergistically to induce squamous metaplasia and incorporation of [3H]thymidine in hamster tracheal epithelium

When exposed to either crocidolite asbestos (single 1-h exposureto 0.4 mg/ml medium) or the polycyclic aromatic hydrocarbon,benzo [a]pyrene (BaP) (2.5 µg/ml medium, 1x weekly for4 weeks), the epithelium of hamster tracheal explants exhibitsinsignificant amounts of squamous metaplasia, an atypical lesion,in comparison to amounts observed in untreated tissues. Incorporationof [³H]thymidine, an indication of DNA synthesis by epithelialcells, likewise is unchanged. However, the extent of squamousmetaplasia and numbers of labeled basal and suprabasal cellsare increased substantially when BaP and asbestos are addedin combination. These results suggest an important mechanismof co-carcinogenesis involving chemical and physical carcinogensand support epidemiologic observations documenting an increasedrisk of bronchogenic carcinoma in asbestos workers who smoke.     

The binding efficiency of polyclonal and monoclonal antibodies to DNA modified with benzo[a]pyrene diol epoxide is dependent on the level of modification. Implications for quantitation of benzo[a]pyrene-DNA adducts in vivo

A number of polyclonal antibodies specific for DNA modifiedwith (±)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE) were obtained from the sera of New Zealand white rabbitsimmunized with BPDE-DNA, complexed with methylated bovine serumalbumin (mBSA). Monoclonal antibodies were developed by fusionof mouse myeloma cells with spleen cells isolated from BALB/cmice immunized with the same complex of BPDE-DNA and mBSA. Theseantibodies have been characterized for specificity in a highlysensitive, enzyme-linked immunosorbent assay (ELISA). All antibodiesshowed a very high affinity for single-stranded BPDE-DNA, buthad lower affinity towards native BPDE-DNA. The affinity forthe free mononucleoside BPDE-dG was at least 100-fold lowerthan that for BPDE-DNA, and no affinity was detected for BPtetrols or DNA modified with N-acetoxy-N-acetyl-2-aminofluorene.A high cross reactivity was observed with DNA modified with(±)-trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydrochrysene.Using five different antibodies, monoclonal or polyclonal, weobserved that the antibody affinity for BPDE-DNA was dependenton the level of modification; in the competitive ELISA as littleas 4 fmol BPDE-DNA (50 pmol/µg) was sufficient for 50%inhibition with our best antisera, but 17 fmol of the adductwas required when [³H]BPDE-DNA of low modification (1–10fmol/µg) was used as inhibitor. When samples of [³H]BP-DNAisolated from the livers of mice, treated i.p. with differentdoses of [³H]BP were examined by competitive ELISA and calibratedwith [³H]BPDE-DNA of low modification (1–10 fmol/µg),binding values calculated from the immunoassay were in goodagreement with those obtained from radioactivity measurements.In contrast, when this DNA was quantitated in competitive ELISAusing highly modified BPDE-DNA as standards, values by ELISAwere 20–40% of those obtained by radioactivity. Theseresults indicate that the use of serially diluted BPDE-DNA ofhigh modification as standard competitor in the ELISA will leadto erroneous results in the measurement of adducts in DNAs modifiedto a low extent (biological samples). The property of antiseraspecific for BP-DNA, recognizing highly modified DNA more efficientlythan DNA modified to a low extent, may be common to all antiseraelicited against highly modified DNA immunogens. Therefore weconclude that antibody affinity must be tested also with DNAsamples of low modification, obtained either in vitro or invivo.     

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.     

Comparison of the morphological transforming activities of dibenzo[a,l]pyrene and benzo[a]pyrene in C3H10T1/2CL8 cells and characterization of the dibenzo[a,l]pyrene-DNA adducts

C3H10T1/2CL8 (C3H10T1/2) mouse embryo fibroblasts were used to study the in vitro carcinogenic activities of dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (B[a]P). The morphological transforming activities of these rodent carcinogens were compared using replicate concentration- response studies. In concentration ranges where both polycyclic aromatic hydrocarbons (PAHs) were active, DB[a,l]P proved to be four to 12 times as potent as B[a]P based on concentration. At lower concentrations DB[a,l]P was active at 0.10 and 0.20 microM, concentrations where B[a]P was inactive. This makes DB[a,l]P the most potent non-methylated PAH evaluated to date in C3H10T1/2 cells. DNA adducts of DB[a,l]P in C3H10T1/2 cells were analyzed by both TLC and TLC/HPLC 32P-postlabeling methods using mononucleotide 3'-phosphate adduct standards derived from the reactions of anti-DB[a,l]P-11,12-diol- 13,14-epoxide (anti-DB[a,l]PDE) and syn-DB[a,l]P-11,12-diol-13,14- epoxide (syn-DB[a,l]PDE) with deoxyadenosine 3'-monophosphate and deoxyguanosine 3'-monophosphate. All of the DNA adducts observed in C3H10T1/2 cells treated with DB[a,l]P were identified as being derived from the metabolism of DB[a,l]P to its fjord region diol epoxides through DB[a,l]P-11,12-diol. The predominant adduct was identified as an anti-DB[a,l]PDE-deoxyadenosine adduct. Other major adducts were anti- DB[a,l]PDE-deoxyguanosine and syn-DB[a,l]PDE-deoxyadenosine adducts with minor amounts of syn-DB[a,l]PDE-deoxyguanosine adducts. These DNA adduct data are consistent with similar findings of DB[a,l]PDE- deoxyadenosine adducts in mouse skin studies and human mammary cells in culture.