咸鱼提取液对小鼠C3H 10T 1／2CL 8细胞的毒性及转化作用研究

Epidemiological case-control study had shown that consumption of salted fish might be a causative factor for human nasopharyngeal carcinoma. In order to test this hypothesis, salted fish extract was studied by C3H 10T 1/2 CL8 cell transformation system. Neither cytotoxicity nor transformation effect was found in the solvent control, but a definite dose-response relation was obtained using relative transformation index when the cells were treated with 0.025-2.000 micrograms/ml 3-methylcholanthrene in the positive control. Current study showed that in the salted fish extract, dose-response relation in cytotoxicity was present in twelve fractions and transformation index significantly increased in five fractions. The maximum relative transformation indices in dichloromethane-methanol, hexane and acetone extract fractions from Murihura croaker were 15.38, 7.69 and 4.29, respectively; and those of dichloromethane-methanol and hexane extract fractions from Sudyoke croaker were 21.69 and 7.69, respectively. In addition, one type-III-focus of cell transformation was found in dichloromethane-methanol fraction and another in acetone fraction of Murihura croaker. In conclusion, there must be more than one carcinogen in the salted fish.     

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.     

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.     

Microsome-mediated bioactivation of dibenzo[a,l]pyrene and identification of DNA adducts by 32P-postlabeling

Dibenzo[a,l]pyrene (DBP) is one of the most potent bacterial mutagen and mammary carcinogens. When DBP (50 microM) was incubated with calf thymus DNA (300 microg/ml) in the presence of liver microsomes from beta-naphthoflavone (beta-NF)- or Aroclor 1254-treated rats, at least eight adduct spots were detected as analyzed by nuclease P1-enhanced 32P-postlabeling assay. DNA adduction was enhanced by nearly 20- and 60- fold with beta-NF- and Aroclor 1254-induced microsomes, respectively, as compared with uninduced microsomes, suggesting a possible involvement of CYP1A family in DBP activation. Inclusion of the selective P4501A1 inhibitor, alpha-naphthoflavone (50 microM) in the activation reaction almost completely (>98%) abolished adduct formation further supporting involvement of P4501A in DBP activation. Analysis of DNA and 2'-deoxynucleosides 3'-mononucleotide reacted with anti- and syn-DBP-11,12-diol-13,14-epoxides (DBPDEs) and co-chromatography analyses in multiple solvents showed that the microsomal DBP-DNA adducts were derived by interaction of both anti- and syn-DBPDEs with adenine and guanine in DNA in the following order: anti-DBPDE-dA approximately syn-DBPDE-dG >> anti-DBPDE-dG approximately syn-DBPDE-dA. It is concluded that (i) most or all DBP adducts were P4501A-mediated; (ii) both the anti- and syn-stereoisomers were involved in the DNA adduct formation; and (iii) both adenine and guanine in the DNA contributed equally to the formation of the major and minor adducts.      

Chemoprotection by natural chlorophylls in vivo: inhibition of dibenzo[a,l]pyrene-DNA adducts in rainbow trout liver

Naturally occurring chlorophylls (Chl) have shown anti-mutagenic activity but little is known about their chemoprotective properties in vivo. This study examined the effect of Chl on formation in vivo of DNA adducts by the potent environmental carcinogen dibenzo[a,l]pyrene (DBP), using rainbow trout as the animal model. Fingerling trout were fed diets containing 200 p.p.m. DBP alone or with one of the following preparations incorporated at 3000 p.p.m. total chlorins: purified pheophytin a (Phe a) (94%); semi-purified Chl a (77%, 23% Phe a), commercial Chl a (88%, 12% other Chl a-related compounds); crude spinach extract (53% Chl a, 19% Chl b, 14% Phe a, 9% carotenoids); commercial Cu-chlorophyllin (55% chlorins, 45% neutral salts), as a known inhibitory control. After 2 weeks dietary treatment, the animals were killed and organs were collected. Stable DBP-DNA adducts from liver were quantified after 33P-post-labeling and separation by reversed-phase HPLC. Total DBP-DNA adducts in the DBP-only group were 2.46 +/- 0.32 adducts/10(6) nucleotides. All chlorophyll treatment groups showed significantly lower adduct levels (P < 0.001, Tukey's HSD test), as follows: crude spinach extract, 0.64 +/- 0.14; semi-pure Chl a, 0.5 +/- 0.11; commercial Chl a, 1.26 +/- 0.17; Phe a, 0.95 +/- 0.01; chlorophyllin, 0.78 +/- 0.09. The various treatments suppressed DBP-DNA adducts essentially uniformly across the HPLC profile, which is consistent with complex formation and reduced carcinogen uptake as the predominant protective mechanism. The chlorophyll-mediated reduction in DBP-DNA adducts in vivo is the first demonstration of anti-genotoxic activity of these common dietary phytochemicals in any vertebrate animal model.      

8,9-dihydroxy-8,9-dihydrodibenzo[a,l]pyrene is a potent morphological cell-transforming agent in C3H10T(1)/(2)Cl8 mouse embryo fibroblasts in the absence of detectable stable covalent DNA adducts

The comparative genotoxic effects of racemic trans-8,9-dihydroxy-8, 9-dihydrodibenzo[a,l]pyrene (trans-DB[a,l]P-8,9-diol), the metabolic K-region dihydrodiol of dibenzo[a,l] pyrene (DB[a,l]P) (dibenzo[def, p]chrysene) and DB[a,l]P in transformable mouse embryo C3H10T(1)/(2)Cl8 (C3H10T(1)/(2)) fibroblasts was investigated. The C3H10T(1)/(2) mouse embryo morphological cell-transforming activities of these polycyclic aromatic hydrocarbons (PAHs) were assayed using concentration-response studies. At concentrations of 33 nM and above both trans-DB[a,l]P-8,9-diol and DB[a,l]P produced significant (and similar) numbers of type II and III foci per dish and numbers of dishes with type II and II foci. Concomitant cytotoxicity studies revealed a reduction in colony survival of approximately 25% up to 198 nM for both PAHs. DNA adducts of trans-DB[a,l]P-8,9-diol and DB[a,l]P in C3H10T(1)/(2) cells were analyzed by a (32)P-post-labeling TLC/HPLC method. No adducts were observed in the DNA of C3H10T(1)/(2) cells treated with trans-DB[a, l]P-8,9-diol at concentrations that induced morphological cell transformation. Under the same exposure and chromatographic conditions, DNA adducts of deoxyadenosine and deoxyguanosine derived from the fjord region anti-DB[a,l]P-11,12-diol-13,14-epoxide and syn-DB[a,l]P-11,12-diol-13,14-epoxide were observed in the DNA of DB[a,l]P-treated cells. These results indicate that trans-DB[a,l]P-8, 9-diol has intrinsic genotoxic activity equal to that of DB[a,l]P, based on morphological cell transformation of mouse embryo fibroblasts. The activity of trans-DB[a,l]P-8,9-diol is apparently not associated with the formation of observable stable covalent DNA adducts. These results suggest that under appropriate conditions, trans-DB[a,l]P-8,9-diol may serve as an intermediate in the genotoxicity of DB[a,l]P.     

Characterization of naphtho[1,2-a]pyrene and naphtho[1,2-e]pyrene DNA adducts in C3H10T1/2 fibroblasts

Polycyclic aromatic hydrocarbons (PAHs) are a class of carcinogenic chemicals that are ubiquitous in the environment. Fjord-region naphthopyrene isomers are structurally similar to the potent fjord-region PAH carcinogen dibenzo[a,l]pyrene and thus have the potential to be potent carcinogens. Naphtho[1,2-a]pyrene (N[1,2-a]P) exhibited similar bacterial mutagenicity and morphological cell transforming activity when compared to benzo[a]pyrene (B[a]P), whereas the structural isomer, naphtho[1,2-e]pyrene (N[1,2-e]P) was inactive is these bioassays. In this study, we examined the formation of DNA adducts in C3H10T1/2Cl8 (C3H10T1/2) mouse embryo fibroblasts exposed to N[1,2-a]P or N[1,2-e]P and their respective dihydrodiols. The DNA adducts were characterized by co-chromatography with reaction products from anti-N[1,2-a]P diol epoxide (DE) or anti-N[1,2-e]PDE and polydeoxyadenosine (dAdo) or oligodeoxyguanosine (dGuo). C3H10T1/2 fibroblasts exposed to N[1,2-a]P or N[1,2-a]P-9,10-diol produced both anti-N[1,2-a]P-DE-dAdo and -dGuo adducts with total DNA adduction levels of 22.2 to 33.3 pmol DNA adducts/mug DNA. C3H10T1/2 fibroblasts exposed to N[1,2-e]P produced 2 major and 1 minor adducts. C3H10T1/2 fibroblasts exposed to N[1,2-e]P-11,12-diol produced 2 major adducts. All of the identified adducts were anti-N[1,2-e]PDE-dGuo and -dAdo adducts. While the total DNA adduct level in N[1,2-e]P-11,12-diol-treated fibroblasts was extremely high, 105.9 pmol DNA adducts/mug DNA, the level in N[1,2-e]P-treated fibroblasts was 1.47 pmol DNA adducts/microg DNA. We conclude that lack of biological activity of N[1,2-e]P may be related to its inability to form sufficient amounts of N[1,2-e]P-11,12-diol, which would then be metabolized to sufficient amounts of anti-N[1,2-e]PDE needed to transform these fibroblasts.     

Mammary carcinogenicity in female CD rats of fjord region diol epoxides of benzo[c]phenanthrene, benzo[g]chrysene and dibenzo[a,l]pyrene

We compared the mammary carcinogenicity in female CD rats ofthree fjord region diol epoxides to test our hypothesis thatsuch sterically hindered molecules would be potent carcinogens.The diol epoxides tested were racemic anti-3,4-dihydroxy-l,2-epoxy-l,2,3,4-tetrahydrobenzo[c]phenanthrene(BcPDE), anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrobenzo[g]chry-sene(BgCDE) and anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene(DB[a,l]PDE). Each diol epoxide was dissolved in dimethylsulfoxide(DMSO) and injected under the six nipples on the left side ofthe rat, with DMSO only being injected under the nipples onthe right side. The total dose of each diol epoxide was 1.2µmol/rat and there were 20 rats/group. The experimentwas terminated 41 weeks after treatment. All three diol epoxideswere potent mammary carcinogens, with activity greater thanpreviously observed for a bay region diol epoxide, anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE). DB[a,l]PDE induced tumors most rapidly, followed byBcPDE and BgCDE. However, different types of tumors were induced.For induction of adenomas and adenocarcinomas, BcPDE and BgCDEhad comparable potency; both were more active than DB[a,l]PDE.In contrast, for induction of sarcomas, DB[a,l]PDE was significantlymore active than BcPDE and BgCDE. The results of this studysupport our hypothesis that sterically hindered fjord regiondiol epoxides are potent mammary carcinogens in the rat.     

Tumor-initiating activity and carcinogenicity of dibenzo[a,l]pyrene versus 7, 12-dimethylbenz[a]anthracene and benzo[a]pyrene at low doses in mouse skin

Dibenzo[a,l]pyrene (DB[a,l]P) is an extremely potent carcinogenthat may be present in environmental samples. Dose-responsestudies were conducted at low doses in mouse skin by initiation-promotionand repeated application to compare its activity to that of7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (B[a]P),DB[a,l]P-8,9-dihydrodiol and DB[a,l]P-11,12-dihydrodiol. FemaleSENCAR mice were initiated with 1 or 0.25 nmol of DB[a, l]P,DMBA, B[a]P or DB[a,l]P-11,12-dihydrodiol and promoted withphorbol ester acetate. At 1 nmol, DB[a, l]P induced 2.6 tumors/mouse,whereas DB[a,l]P-11,12-dihydrodiol and DMBA induced 0.17 and0.29 tumors/mouse respectively. At the low dose, DB[a,l]P induced0.79 tumors/mouse, but the other two compounds were virtuallyinactive. B[a]P, tested only at 1 nmol, was inactive. Thesethree compounds, as well as DB[a,l]P-8,9-dihydrodiol, were testedby repeated application twice weekly for 40 weeks at 1 and 4nmol per dose. In addition, DB[a,l]P, DMBA and B[a]P were alsotested at 8 nmol. At 8 and 4 nmol, DB[a,l]P induced malignanttumors in 91 and 70% of mice respectively. At 4 nmol DB[a, l]P-11,12-dihydrodiolelicited only benign tumors in 36% of mice. At 4 nmol DMBA inducedtwo carcinomas in one mouse and at 8 nmol it induced one papillomaand one sebaceous gland adenoma. B[a]P and DB[a,l]P-8,9-dihydrodiolwere inactive at all doses tested. These results demonstratethat DB[a, l]P is a much more potent carcinogen than DMBA, thearomatic hydrocarbon previously considered to be the most potent.Combination of these results with previous comparisons of DB[a,l]P,DB[a,l]P-11,12-dihydrodiol, DMBA and B[a]P at higher doses (E.L.Cavalieri et al. (1991) Carcinogenesis, 12, 1939–1944)shows clearly the interference of toxicity with the tumorigenicityof DB[a,l]P and its 11,12-dihydrodiol.     

Synthesis of fjord region tetraols and their use in hepatic biotransformation studies of dihydrodiols of benzo[c]chrysene, benzo[g]chrysene and dibenzo[a,l]pyrene

Metabolic activation of the racemic benzo[c]chrysene-trans-9,10-, benzo[g]chrysene-trans-11,12- and dibenzo[a,l]pyrene-trans-11,12- dihydrodiols to fjord region syn- and anti-dihydrodiol epoxides by microsomes of Aroclor 1254-treated Sprague-Dawley rats has been examined. Since the fjord region dihydrodiol epoxides were hydrolytically unstable under the experimental conditions, their enzymatic formation was determined by analyzing the tetraols as their products of acidic hydrolysis upon addition of perchloric acid. The various stereoisomeric tetraols formed were separated by HPLC and identified by co-chromatography with authentic tetraols, which had been prepared by acidic hydrolysis of synthetically available syn- and anti- dihydrodiol epoxides and characterized by NMR and UV spectroscopy. Under standardized conditions the acidic hydrolysis of syn-dihydrodiol epoxides of benzo[c]chrysene, benzo[g]chrysene and dibenzo[a,l]pyrene resulted in the formation of two tetraols with cis/trans ratios of 81:19, 77:23 and 80:20, respectively, whereas the anti-dihydrodiol epoxides underwent almost exclusively trans hydrolysis. The proportion of the stereoisomeric tetraols obtained from microsomal incubations indicates that all three dihydrodiols are predominantly oxidized at the adjacent olefinic double bond to the anti-diastereomers of the corresponding fjord region dihydrodiol epoxides accounting for 4-35% of the ethyl acetate-extractable metabolites. To allow quantitative assessment of the metabolites 3H-labeled trans-dihydrodiols were synthesized by reduction of the corresponding o-quinones with sodium borotritide. Metabolic conversion of benzo[c]chrysene-trans-9,10- and dibenzo[a,l]pyrene-trans-11,12-dihydrodiol by rat liver microsomes were in a similar low range during the first 10 min of incubation (6.2 +/- 1.2 and 3.4 +/- 1.0 nmol substrate/nmol cytochrome P450/10 min, respectively), whereas the conversion of benzo[g]chrysene-trans-11,12- dihydrodiol was much higher (20.6 +/- 2.2 nmol substrate/nmol cytochrome P450/10 min). Given the strong intrinsic mutagenic and carcinogenic activity of the fjord region dihydrodiol epoxides, our data indicate that their formation, even at a relatively low level, may contribute significantly to the biological activity of the parent hydrocarbons.      

Dibenzo[a,l]pyrene (dibenzo[def,p]chrysene): fjord-region distortions

The molecular dimensions of the potent chemical carcinogen dibenzo[def,p]chrysene, also known as dibenzo[a,l]pyrene, have been determined by X-ray diffraction methods. This analysis shows that the molecule is considerably distorted so that it is non-planar with an angle of 27.6 degrees between the outermost rings and a widening of C-C- C bond angles in the fjord region. The dimensions of the molecular distortion due to atomic overcrowding in the fjord region are presented. This polycyclic aromatic hydrocarbon is a more potent carcinogen than is benzo[a]pyrene or its 11-methyl derivative. Comparisons of the distortions in dibenzo[a,l]pyrene with the geometries of various other polycyclic aromatic hydrocarbons containing fjord- or bay-region methyl groups provide structural data on the ratio of angular to torsional distortion in such overcrowded molecules.      

Inhibition of morphological transformation of C3H10T1/2CL8 mouse embryo cells by multiple carcinogen treatments

C3H10T1/2CL8 cells treated on the first day after seeding with benzo[a]pyrene (B[a]P) and then treated again with B[a]P displayed an inhibited response of morphological transformation if the second treatment was administered from 14 days to 33 days after seeding. Under these conditions the cells exhibited up to 100% inhibition of morphological transformation, the extent of inhibition being related to the concentration of B[a]P administered in the second treatment. 3-Methylcholanthrene (3MC) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) also inhibited B[a]P-induced morphological transformation as a function of concentration when administered to cells 21 days after the initial B[a]P treatment. Delayed recovery of transformed foci was examined in cells treated with B[a]P on days 1 and 22 and scored 6-9 weeks after the first B[a]P treatment. No recovery of cell transformants was observed. Reconstruction experiments with normal and transformed C3H10T1/2CL8 cells suggested that selective cytotoxicity to incipient transformed cells could account for the inhibition by MNNG, but could not account for up to 50% of the inhibition induced by the second treatment of B[a]P or 3MC.     

Detection and identification of benzo[a]pyrene-DNA adducts by [35S]phosphorothioate labeling and HPLC

Two generally applicable [35S]phosphorothioate postlabeling procedures for the HPLC analysis of polycyclic aromatic hydrocarbon (PAH)-DNA adducts have been developed based upon [32P]phosphate postlabeling assays described by Gupta and Randerath et al. In one procedure, benzo[a]pyrene (B[a]P)-modified DNA was digested to nucleoside 3'-phosphates by micrococcal nuclease and spleen phosphodiesterase and the adducted nucleotides were extracted with 1-butanol. The adducted nucleoside-3'-phosphates were 5'-thiophosphorylated by T4 polynucleotide kinase (T4PNK) and adenosine 5'-O-(3-[35S]thiotriphosphate) to yield [35S]B[a]P-nucleoside-5'-phosphorothioate-3'-phosphate adducts. Although thiophosphorylation of B[a]P-DNA adducts was slower than the corresponding phosphorylation reaction, similar recoveries of the postlabeled adducts were achieved with longer incubation times and higher concentrations of T4PNK. A major advantage of this procedure over the 32P-postlabeling procedure is that the resistance of phosphorothioates to degradation by phosphatases allows selective removal of the unlabeled 3'-phosphate from the [35S]B[a]P-nucleoside-5'-phosphorothioate-3'-phosphate adducts by brief treatment with alkaline phosphatase. [35S]B[a]P-nucleoside-5'-phosphorothioate adducts were also prepared using a nuclease P1/prostatic acid phosphatase DNA degradation method. For anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-modified DNA, overall adduct recoveries were substantially higher with the nuclease P1/prostatic acid phosphatase method (48-51%) than with the micrococcal nuclease/spleen phosphodiesterase/alkaline phosphatase method (22-29%). There were no significant differences in the HPLC profiles of the [35S]phosphorothioate-postlabeled adducts obtained from these two procedures. HPLC analysis of B[a]P-DNA adducts formed in B[a]P-treated hamster embryo cell cultures demonstrated the formation of two major adducts, (+)syn-BPDE-deoxyguanosine-5'-phosphorothioate and (+)anti-BPDE-deoxyguanosine-5'-phosphorothioate, along with other minor adducts. Based upon an overall adduct recovery of 20% and 0.5 mol as the detection limit of this 35S-postlabeling/HPLC assay, the sensitivity of this assay is 1 adduct/10(8) nucleotides for a 60 micrograms DNA sample. This method offers the advantages of using 35S which has a longer half-life and lower radioactive decay energy than 32P and the ability to prepare PAH-DNA adducts at the monophosphorothioate level which greatly facilitates separation of individual 35S-postlabeled PAH-DNA adducts by HPLC.     

Morphological transformation and DNA adduct formation by dibenz[a,h]anthracene and its metabolites in C3H10T1/2CL8 cells

The major routes of metabolic activation of dibenz[a,h]-anthracene(DBA) have been studied in transformable C3H10T1/2CL8 (C3H10T1/2)mouse embryo fibroblasts in culture. The morphological transformingactivities of three potential intermediates formed by metabolismof DBA by C3H10T1/2 cells, trans-3,4-dihydroxy-3,4-dihydro-DBA-(DBA-3,4-diol),trans-dihydroxy-3,4-dihydro-DBA-anti-1,2-oxide (DBA-3,4-diol-1,2-oxide)and DBA-5,6-oxide were determined. DBA-3,4-diol-1,2-oxide wasa strong morphological transforming agent giving a mean of 73%dishes with Type II or III foci and 1.63 Type II and HI fociper dish at 0.5 µg/ml. DBA-3,4-diol produced a mean of42% dishes with Type II or III foci and 0.81 Type II and IIIfoci per dish at 2.5 µg/ml. DBA gave a mean of 24% disheswith Type II or III foci and 0.29 Type II and III foci per dishat 2.5 µg/ml. DBA-5,6-oxide was found to be inactive.DNA adducts of DBA, DBA-3,4-diol, DBA-3,4-diol-1,2-oxide, DBA-1,4/2,3-tetroland DBA-5,6-oxide in C3H10T1/2 cells were analyzed by ³²P-postlabelingmethod. DBA gave 11 adducts, nine of which were observed inthe DNA of cells treated with DBA-3,4-diol and seven from cellstreated with DBA-3,4-diol-1,2-oxide. Two of these adducts thatappear in each of the treatment groups have been identifiedas the product of the interaction of DBA-3,4-diol-1,2-oxidewith 2-deoxyguanosine. Furthermore, there is evidence for DBA-DNAadducts in cells treated with DBA, DBA-3,4-diol and DBA-3,4-diol-1,2-oxidearising from metabolism to (+,-)-trans,trans-3,4,10,11-tetrahydroxy-3,4,10,11-tetrahydro-DBA(DBA-3,4,10,11-bis-diol). These results are based on co-migrationof C3H10T1/2 DNA adducts with skin DNA adducts formed aftertopical treatment of mice with DBA-3,4,10,11-bis-diol. In C3H10T1/2cells, DBA is metabolically activated through DBA-3,4-diol,which is further activated via the DBA-3,4-diol-1,2-oxide andDBA-3,4,10,11-bis-diol pathways. No evidence is provided forthe metabolism of DBA by the Kregion pathway.     

Post-mortem stability of benzo[a]pyrene diolepoxide-DNA adducts in rat organs

In order to evaluate the stability of benzo[a]pyrene diolepoxide-DNAadducts two separate studies were carried out in rats, eithertreated i.p. with benzo[a]pyrene (100 mg/kg body wt) or sham-exposed.The measurement of DNA adducts in 155 samples of liver, lungor heart, each of them tested in duplicate, was performed bysynchronous fluorescence spectrophotometry. In the first studyfragments of rat liver or lung were stored for varying timesat varying temperatures. No decrease of adduct levels occurredat 4°C for at least 72 h, whereas a significant decreasewas recorded in both liver and lung after 48 h at 20°C or24 h at 37°C. In the second study liver, lungs and heartwere collected from rats either immediately after killing orafter storage of cadavers for 16 h at 20°C or 16 h at 20°Cplus 24 h at 4°C, thereby mimicking typical storage conditionsof human cadavers before autopsy. Under these conditions nosignificant variation of fluorescent adducts was observed inany organ. In conclusion, at least for this kind of adduct,the use of autopsy samples following proper storage of cadaversseems to be acceptable.     

Quantitation by electron microscopy of the binding of highly specific antibodies to benzo[a]pyrene-DNA adducts

Highly specific antibodies bound to carcinogen adducts in DNAmodified with (+ / - )7ß, 8-dihydroxy-9, 10-epoxy-7,8, 9, 10-tetrahydrobenzo[a]pyrene (BPDE I) were quantitatedby electron microscopy (EM) visualization and these observationswere compared with quantitation of adducts by enzyme-linkedimmunosorbent assay (ELISA). The antiserum, elicited in rabbitsfollowing inoculation with BPDE I-modified DNA, has been foundto be highly specific in its recognition of BPDE I-deoxyguanosinemoieties. Parallel DNA samples prepared for analysis by ELISAand EM quantitation were randomized, encoded, and analyzed todetermine extents of carcinogen modification in double-blindstudies. After levels of modification were determined by immunoassays,DNA samples were prepared for EM analysis by incubation withamounts of anti-BPdG-DNA serum in excess of that necessary forcomplete binding of antibody to antigenic sites. At equilibrium,samples were enzymatically digested with papain in order tocleave anti-BPdG-DNA IgG molecules into Fab fragments in situ.Following column exclusion chromatography, BPdG-DNA-Fab complexeswere incubated with ferritin-labeled Fab‘ fragments ofgoat [anti-rabbit F(ab’)2] IgG in amounts in excess ofthose necessary for complete binding. When DNA samples weremodified to between 0 and 40 fmol adduct/ µg DNA, excellentagreement was obtained between ELISA quantitation and visualizationby EM of antibodies bound to adducts.     

Tumorigenicity in newborn mice of fjord region and other sterically hindered diol epoxides of benzo[g]chrysene, dibenzo[a, l]pyrene (dibenzo[def, p]chrysene), 4H-cyclopenta[def]chrysene and fluoranthene

Diol epoxides of benzo[g]chrysene, dibenzo[a, l]pyrene (dibenzo[def,p]chrysene), 4H-cyclopenta[def]chrysene and fluoranthene weretested for tumorigenicity in newborn mice. The compounds testedwere racemic trans-11, 12-dihydroxy-anti-13,14-epoxy-11,12,13,14-tetrahydrobenzo[g]-chrysene(BgCDE), trans-11, 12-dihydroxy-anti-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a, l]PDE),trans-1,2-dihydroxy-anti-3,3a-epoxy-1,2,3,3a-tetrahydro-4H-cyclopenta[def]chrysene(C[def]C-1-3a-DE), trans-6,7-dihydroxy-anti-8,9-epoxy-6,7,8,9-tetrahydro-4H-cyclopenta-[def]chrysene(C[def]C-6-9-DE) and trans-2,3-dihydroxy-anti-1,10b-epoxy-10b,1,2,3-tetrahydrofluoranthene(FDE). BgCDE and DB[a,l]PDE are fjord region diol epoxides andtheir tumorigenic activities were compared to those of trans-3,4-dihydroxy-anti-1,2-epoxy-1,2,3,4-tetrahydrobenzo-[c]phenanthrene (BcPDE), a fjord region diolepoxide with known high tumorigenicity and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene(BPDE), a highly tumorigenic bay region diol epoxide. The protocolcalled for testing of each compound at a total dose of 25 nmolper mouse, administered on days 1, 7 and 15 of life, with killingat age 35 weeks. BgCDE had similar activity as BcPDE for inductionof lung tumors and was more active than BcPDE for inductionof liver tumors in male mice. Both compounds were significantlymore tumorigenic than BPDE. DB[a,l]PDE was highly toxic. Allmice died within 1 week of the first dose. It was then testedin a second study using total doses of 5 and 1 nmol per mouse.Only the first dose of the intended 5 nmol total dose was givendue to toxicity. The full course of doses with a total of 1nmol per mouse was administered; DB[a,l]PDE induced a significantincidence and multiplicity of lung tumors and, in male mice,liver tumors at both doses. These results demonstrate that fjordregion diol epoxides are highly active tumorigens in newbornmice, with activity greater than that of the most active unsubstitutedbay region diol epoxide, BPDE. C[def]C-1-3a-DE and C[def]-6-9-DEwere compared to trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydrochrysene(CDE), at a total dose of 500 nmol per mouse. FDE was also testedat this dose. The most active compound among the chrysene derivativeswas C[def]C-1-3a-DE, followed by C[def]C-6-9-DE and CDE. C[def]C-1-3a-DEhas a sterically constrained bay region, in which the benzyliccarbon of the tri-substituted epoxide ring is part of a fusedring system. This feature is also present in FDE, which hadconsiderable tumorigenic activity, greater than that of CDEin lung and greater than any of the chrysene derivatives inliver. These results demonstrate that the tumorigenicity ofdiol epoxides in newborn mice is enhanced by steric constraintspresent when the epoxide ring is part of a fjord region or isattached to a fused ring system. The tumorigenic activity ofFDE may be relevant to human risk since it is a stable metaboliteof the widely distributed environmental pollutant fluoranthene.     

Improved transformation of C3H10T1/2CL8 cells by direct- and indirect-acting carcinogens

Oncogenic transformation of C3H10T1/2CL8 cells was improvedby treating the cells 5 days after seeding. Benzo[a]pyrene-inducedtransformation was increased 3.5-fold by this method, comparedwith treating the cells 1 day after seeding. N-Methyl-N' -nitro-N-nitrosoguanidine,which does not transform asynchronous cultures of C3H10T1/2CL8cells when administered 1 day after seeding, produced an averageof 1 focus/dish, with 61% of the dishes exhibiting foci, whenadministered 5 days after seeding. Propane sultone and aflatoxinB₁ also produced marked transformation responses when administered5 days after seeding. However, 4-dimethylaminoazobenzene didnot induce transformation when administered either 1 day or5 days after seeding. With all chemicals examined, clonal cytotoxicitywas reduced when they were administered 5 days after seeding.These results indicate the utility of this new procedure forthe qualitative analysis of the transforming ability of chemicals.     

Modification of benzo[a]pyrene-induced transformation of C3H/10T12 cells by pregnenolone-16α-carbonitrile and dexamethasone

The compounds pregnenolone-16α-carbonitrile (3β-hydroxypregn-5-en-20-one-16α-carbonitrile (PCN)) and dexamethasone (9α-fluoro-16-methylprednisolone (DEX)), both known inducers of microsomal enzymes in vivo were tested for their effect on benzo[a]pyrene (B[a]P)-induced transformation of $\text{C3H/10T}\text{1}\text{2}$ cells. Our data indicate that even though PCN and DEX slightly increase aryl hydrocarbon hydroxylase (AHH) activity and metabolism of B[a]P, they have opposite effects on cellular transformation. While DEX inhibited transformation by as much as 65% when added 20 h prior to the carcinogen, PCN enhanced transformation by as much as 250% at equivalent doses. Possible mechanisms by which PCN and DEX could exert opposite effects in altering B[a]P-induced transformation of $\text{C3H/10T}\text{1}\text{2}$ cells are discussed.