Comparison of blood protein and target organ DNA and protein binding following topical application of benzo[a]pyrene and 7H-dibenzo[c,g]carbazole to mice

7H-Dibenzo[c,g]carbazole (DBC) induces skin and liver tumorsin mice following topical application, whereas benzo[a]pyrene(BP) induces only skin tumors. DBC also binds to liver DNA toa much greater extent than does BP. The present study examinedfactors that might account for the difference in DNA bindingactivity. [³H]DBC was applied topically to CD-1 mice at dosesof 15, 100 and 1000 nmol/mouse and tissues and blood sampleswere taken 24 h later. Absorption of DBC from skin into bloodand binding to blood proteins occurred linearly with dose. DBCbound to albumin at a 50-fold higher level than to globin andlevels of albumin adducts showed good correlation with levelsof DNA adducts in liver. Hepatic preference over skin in DNAbinding was found to be dose-dependent For comparison of [3H]BPand [3H]DBC binding, doses of 1000 nmol/mouse were used andthe mice were sacrificed at 12, 24 and 48 h. The rate of DBCuptake from skin was 70% higher than for BP over the first 24h, which was reflected in 40-50% higher plasma levels of DBCradiolabel. Skin protein and DNA binding were 2- to 5-fold higherfor BP than DBC. Conversely, total 3H radioactivity levels inliver were 2- to 3-fold higher and liver DNA and protein bindingwere 15- to 20-fold and 3- to 5-fold higher respectively forDBC. Blood protein adduct levels were similar for both chemicals,suggesting that DBC metabolites formed in the liver were tooreactive to re-enter the systemic circulation. Only minor amountsof the radiolabel in the liver were present as the parent compoundsby 12 h after dosing. These results indicate that more rapidabsorption from skin and selective accumulation in the livercontribute to the greater liver DNA binding seen with DBC, butthe types of liver metabolites appear to be the major factoraccounting for the binding difference.     

Tissue-specific DNA adduct formation in mice treated with the environmental carcinogen, 7H-dibenzo[c,g]carbazole

Covalent adduction of DNA by chemical agents is commonly thoughtto be an essential part of the initiation of chemical carcinogenesis.Until recently, assays of DNA damage by covalent binding ofchemicals have been restricted mostly to substances that areavailable in radiolabeled form, which excludes many environmentalcompounds with carcinogenic potential. In this paper, the bindingof non-radioactive 7H-dibenzo[c, g]carbazole (DBC), a knownenvironmental carcinogen, to DNA in female CD-1 mice after s.c.injection of 44 µmol/kg of the compound has been investigatedusing a ³²P-postlabeling assay. DBC showed strong hepatic specificitywith a mean total level of 107 adducts per 10⁷ nucleotides at24 h, while much lower levels of binding were seen in kidney,lung, spleen, skin and brain with 4.3, 2.1, 1.3, 0.4 and 0.04adducts, respectively, per 10⁷ nucleotides. Proportions of individualDBC adducts also varied considerably between tissues. The degreeof hepatic preference displayed by DBC is not seen with otherpolycyclic aromatic carcinogens such as benzo[a]pyrene and 2-acetylaminofluorene.The DNA-binding data, together with other hepatotoxic effectsof the compound, may be causally related to the known hepato-carcinogencityof DBC.     

Binding of 7H-dibenzo[c,g]carbazole to polynucleotides and DNA in vitro

The N-heterocyclic aromatic pollutant, 7H-dibenzo[c,g]- carbazole(DBC), is a potent carcinogen having both local and systemiceffects. The overall objective of this research was to investigatethe nature of the covalent binding of DBC with nucleic acidsin vitro. DBC was shown to bind to polynucleotides, RNA andDNA in an in vitro rat or hamster microsomal enzyme assay, exhibitinga preferential binding to polyguanylic acid (poly[G]). Benzo[a]pyrene(BaP) binding to these same nucleic acids was determined simultaneouslyand was 10-fold higher than DBC binding under identical experimentalconditions. DBC-nucleic acid binding was shown to be dependentupon the presence of a microsomal activating system, the resultsbeing similar for rat or hamster liver microsomes. This microsome-dependentbinding was unaffected by the addition of epoxide hydrase activitymodifiers but was almost completely inhibited by -naphthoflavone.The nature of DBC-nucleic acid binding was investigated usingfluorescence spectroscopy. Benzo-[c]carbazole and 5,5,6,6-tetrahydrodibenzo[c,g]carbazolewere synthesized as representatives of the effect of disruptionof the DBC -electron system on fluorescence excitation and emission.DBC-poly[G] adducts were isolated from binding assay mixturesand separated by HPLC. Results indicated that there are at leastthree different DBC-poly[G] adducts formed in vitro. The emissionspectra of isolated adducts were similar in shape to that ofDBC; however, the adduct spectra were shifted 5–10 nrntoward longer wavelengths. This suggests that the bound DBCspecies have intact -electron systems. Results are consistentwith binding through the nitrogen position as well as bindingthrough the 1,2,3,4-ring of the molecule.     

Comparative carcinogenic potencies of 7H-dibenzo[c,g]carbazole, dibenz[a,j]acridine and benzo[a]pyrene in mouse skin

The relative carcinogenic potencies of three combustion products of fossil fuels, 7H-dibenzo[c,g]carbazole (7H-DB[c,g]C), dibenz[a,j]acridine (DB[a,j]A) and benzo[a] pyrene (B[a]P) were compared using complete carcinogenicity C3H mouse skin bioassays. Both 7H-DB[c,g]C and B[a]P produced tumors in 48 of 50 mice with latency periods of 36.6 and 32.4 weeks, respectively. DB[a,j]A produced tumors in 25 of 50 mice with a latency period of 80 weeks. 7H-DB[c,g]C was found to be as potent a carcinogen as B[a]P when applied to mouse skin. These results have important implications in the determination of relative carcinogenic potencies of complex mixtures.     

32P-Postlabeling analysis of DNA adduction in mice by synthetic metabolites of the environmental carcinogen, 7H-dibenzo[c,g]carbazole: chromatographic evidence for 3-hydroxy-7H-dibenzo[c,g]carbazole being a proximate genotoxicant in liver but not skin

The DNA adduction by the environmental carcinogen 7H-dibenzo[c,g]carbazole(DBC) and chemically synthesized 2-OH, 3-OH, and 4-OH metabolitesof DBC was investigated in liver and skin of female CD-1 mice.After topical application to the skin of 37 µmol/kg ofDBC or the phenolic metabolites, DNA adducts were measured bya ³²P-post-labeling assay employing carrier-free [-³²P]ATP andATP-deficient conditions. In liver, DBC produced four majorand several minor chromatographically distinct adducts of asyet undetermined chemical structure. The adduct pattern elicitedby 3-OH-DBC was qualitatively similar to the DBC adduct pattern,while this was not the case for 2-OH-DBC and 4-OH-DBC. On thebasis of co-chromatography experiments under various conditions,the DBC and 3-OH-DBC adducts appeared identical, and the totallevel of adduction elicited by these compounds in liver wassubstantial. Similar results were observed when DBC or 3-OH-DBCwere administered i.p. As a major difference between the twocompounds, one 3-OH-DBC adduct (no. 3) was 4.4- and 7.0-foldlower than the corresponding DBC adduct after i.p. and topicaldosing, respectively. In skin, DBC produced two major adductfractions after topical application, one of which could be chromatographicallyresolved into three subcomponents. Prominent adducts producedin skin DNA by each of the three metabolites were differentfrom those elicited by DBC, and the level of adduction by themetabolites was significantly lower than that by DBC. Comparisonof the skin and liver DBC-DNA adduct patterns after topicalapplication of DBC showed that only one of the four major chromatographicallyresolved skin adducts corresponded to a major liver adduct (no.3), and that total adduction in liver was 13.5-fold higher thanin skin. These results suggested that (i) activation of DBCto DNA-binding compounds in liver occurs through at least twopathways with 3-OH-DBC being a proximate carcinogen involvedin the formation of most of the adducts; (ii) 3-OH-DBC and theother two phenolic metabolites investigated play a minor role,if any, in the formation of DBC-DNA adducts in skin; (iii) metabolicactivation of DBC to DNA-binding compounds in liver and skinappears to follow pathways that are different in terms of boththe chemical nature and the amount of the adducts formed; andiv) DBC and 3-OH-DBC exhibit a strong preference for liver versusskin DNA.     

Persistence of benzo(a)pyrene metabolite:DNA adducts in lung and liver of mice

The persistence of benzo(a)pyrene (BP) metabolite:DNA adducts has been studied in lung and liver of A/HeJ and C57BL/6J mice after a dose of BP (6 mg/mouse) which induces pulmonary adenomas in A/HeJ mice but not in C57BL/6J mice. BP is not a hepatic carcinogen in either strain. Following p.o. administration of [3H]BP, animals were killed at times ranging from 10 hr to 28 days, and BP metabolite:DNA adducts were analyzed by high-pressure liquid chromatography. The major adduct identified in each tissue was the (+)-7 beta-8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct. A 7 beta, 8 alpha-dihydroxy-9 beta,10 beta,epoxy-7,8,9, 10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct, a (-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct, and an unidentified adduct were also observed. The disappearance of (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydro-BP adduct in A/HeJ mice followed first-order kinetics over the time period examined, with a half-life of 18 and 9 days in lung and liver, respectively. The decay of this adduct in C57BL/6J mice was biphasic in both tissues. Our data on cell turnover suggest that there is active removal of adducts in liver, but that normal DNA turnover can account for the partial or possibly total observed disappearance of adducts in lung. These results suggest that the tissue specificity for BP-induced neoplasia in A/HeJ mice may be related to the relative persistence of adducts and high cell turnover rates in lung. In contrast, the results on formation and persistence of adducts and cell turnover do not provide an explanation for the strain difference in susceptibility to BP-induced pulmonary adenomas. It was also shown that the rates of removal of BP metabolite:DNA adducts in A/HeJ mice are not significantly different at a 500-fold lower BP dose.     

Human cell-mediated cytotoxicity, mutagenicity, and DNA adduct formation of 7H-dibenzo(c,g)carbazole and its N-methyl derivative in diploid human fibroblasts

The aromatic N-heterocyclic 7H-dibenzo(c,g)carbazole (DBC), like polynuclear aromatic hydrocarbons, is a potent inducer of local sarcomas, papillomas, and respiratory tumors, but unlike such compounds it also induces hepatomas. The N-methyl derivative of DBC, N-methyl-dibenzo(c,g)carbazole (MeDBC), also induces sarcomas, papillomas, and respiratory tumors but at a lower frequency than DBC. However, MeDBC lacks hepatocarcinogenic potential, suggesting that DBC undergoes metabolic activation at its carbon atoms and also at the nitrogen position and that the N-7 position plays a role in liver carcinogenesis by DBC. We compared the cytotoxic and mutagenic potential of DBC and MeDBC using a human epithelial cell-mediated activation assay. Repair-deficient, diploid human fibroblasts derived from a xeroderma pigmentosum (XP) patient were used as target cells, and a human hepatoma cell line, Hs703T, was used as a source of exogenous metabolism. Resistance to 6-thioguanine served as the genetic marker for mutations. The Hs703T cells, but not the target XP cells, activated DBC and MeDBC into forms capable of interacting with DNA. In the cell-mediated assay, both DBC and MeDBC induced cytotoxicity and mutations in the target XP cells in a dose-dependent manner. However, DBC was effective at 2-fold lower concentrations than MeDBC. DNA adduct analysis using a 32P-postlabeling assay showed that at biologically significant low doses DBC was 2.5 times more effective than MeDBC in covalent binding to DNA. At higher doses, the difference in ability to bind to DNA was 1.3-fold. In both XP and Hs703T cells, DBC produced three major adducts, while MeDBC produced two major adducts, one of which was the same as one detected in the DBC adduct pattern. The number of DBC- and MeDBC-induced DNA adducts corresponding to a particular level of cytotoxicity and mutagenicity in the XP cells was 10 times lower than that for (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene.     

Comparison of Ha-ras mutational spectra of N-methyldibenzo[c,g]carbazole and 7H-dibenzo[c,g]carbazole-induced mouse skin tumors.

Carcinogenic N-heterocyclic aromatic hydrocarbons are formed during the incomplete combustion of fossil fuels as well as cigarette smoke. N-Methyldibenzo[c,g]carbazole (NMeDBC) and 7H-dibenzo[c,g]carbazole (DBC) are members of this group. DBC induces mouse skin and liver tumors, whereas NMeDBC induces only mouse skin tumors. The objective of this study was to elucidate the mechanism of action of these compounds in skin by assessing the Ha-ras mutational spectra induced by a two-stage initiation-promotion protocol. NMeDBC (200 nmol) or DBC (200 nmol) was applied to the back skin of 24 female Hsd:ICR(Br) mice (12 per group) once. 12-O-tetradecanoylphorbol-13-acetate (TPA) (2 microg) was then applied twice weekly for 28 wk. Tumors were screened for Ha-ras mutations using enriched polymerase chain reaction and mutations defined by dideoxy sequencing. In DBC animals 58% produced papillomas, of which 71% had codon 61 mutations, 4% had codon 12 mutations, 4% had codon 13 mutations, and 21% had no Ha-ras mutations. In NMeDBC animals 92% produced papillomas, of which 73% had codon 61 mutations and 27% had no Ha-ras mutations. All of the codon 61 mutations, from both NMeDBC and DBC, were CAA-->CTA transversions. The DBC-induced tumors with the codon 12 mutation had a GGA-->GAA transition, and the codon 13 mutation was a GGC-->GTC transversion. These results suggest that NMeDBC is a more potent tumor inducer than DBC, but the resulting H-ras mutations in each group were predominantly in codon 61, and, therefore, mutation induction in skin by each chemical appears to proceed by a similar mechanism.     

Human and rat kidney cell metabolism of 2-acetylaminofluorene and benzo(a)pyrene

The metabolism and mutagenic activation of the model carcinogens benzo(a)pyrene [B(a)P] and 2-acetylaminofluorene (AAF) by human and rat kidney cells were measured. A slicing technique followed by enzyme digestion was utilized to obtain the kidney cells. Although levels of total metabolism of B(a)P by rat and human kidney cells were similar, analysis of specific metabolites of B(a)P indicated that species differences existed. Human kidney cells produced the organic-soluble metabolites B(a)P-9,10-diol, B(a)P-4,5-diol, B(a)P-7,8-diol, B(a)P-3,6-quinone, and B(a)P-9-phenol. Rat kidney cells produced organic-soluble B(a)P-pre-9,10-diols, B(a)P-9,10-diol, B(a)P-4,5-diol, and B(a)P-6,12-quinone. Both species produced sulfate and glucuronide conjugates of all products. For AAF, kidney cells from some human tissues produced up to four times the level of total metabolites compared to rat kidney cells. Organic-soluble metabolites were qualitatively similar between the species and consisted of 2-aminofluorene (AF), N-hydroxy-AAF and ring-hydroxylated products at the 1, 3, 5/9, 7, and 8 positions. Sulfate and glucuronide conjugates of these metabolites were also detected. Human interindividual variation with kidney cells was about 2.5-fold for total AAF metabolism and up to 6-fold for individual AAF metabolites. For B(a)P metabolism, human interindividual variation in total metabolism was low while for specific metabolites there was up to a 4-fold variation. Levels of AAF and AF cell-mediated Salmonella typhimurium mutagenesis were significantly higher with human cells as compared to rat kidney cells. It appears that the differences between human and rodent kidney cell metabolism of chemical carcinogens vary with the chemical class and understanding these differences will be necessary in the extrapolation of rodent carcinogenesis data to humans.     

7H-dibenzo[c,g]carbazole metabolism by the mouse and human CYP1 family of enzymes

Found in tobacco smoke, fossil fuel and other organic combustion products, 7H-dibenzo[c,g]carbazole (DBC) is a potent mouse lung carcinogen and potential human carcinogen. Although the first hydroxylation is critical for determining activation versus detoxication, the enzymes responsible for site-specific hydroxylation of DBC are not known. We found that DBC-DNA adduct levels are significantly higher in aromatic hydrocarbon receptor null Ahr(-/-) mice, suggesting that the induction of Aromatic hydrocarbon receptor (AHR)-regulated genes, such as those in the CYP1 family, decrease DBC genotoxicity. Using knockout mice for Cyp1a1, Cyp1a2 and Cyp1b1, we showed that the major CYP1 enzymes that metabolize DBC are CYP1A1 in beta-naphthoflavone (BNF)-induced liver, CYP1A2 in non-induced liver, CYP1B1 and CYP1A1 in induced lung and none in non-induced lung. DBC metabolism by the human CYP1 enzymes was examined in vitro using Supersomestrade mark. Each mouse CYP1, as well as each human CYP1, has a unique DBC metabolite profile. Comparison of the metabolite profile in BNF-induced mice suggested that CYP1A1 primarily generates 1-OH, 2-OH and (5 + 6)-OH-DBC, whereas CYP1A2 generates primarily (5 + 6)-OH-DBC and CYP1B1 primarily generates 4-OH-DBC. This was similar to that observed in the human CYP1 enzymes. Most importantly, lung CYP1B1 is associated with forming 4-OH-DBC, the most potent metabolite leading to DBC-DNA adducts. These studies suggest that for non-pulmonary routes of exposure (i.e. skin, gastric, i.p.), low hepatic expression of CYP1A2 and CYP1A1, together with high expression levels of lung CYP1B1 and CYP1A1, may define a phenotype for high susceptibility to carcinogens such as DBC.     

Carcinogenicity, DNA adduct formation and K-ras activation by 7H-dibenzo[c, g]carbazole in strain A/J mouse lung

N-Heterocyclic polynuclear aromatic hydrocarbons (NHA) are environmentalpollutants formed during the combustion of organic materials.7H-Dibenzo[c,g]carbazole (DBC) is a potent carcinogen in lung,liver and skin. We undertook these studies to determine whethertissue specificity for DBC lung carcinogenicity inthe strainA/J mouse is mirrored by formation of DBC-DNA adducts in lungtissue and whether these adducts are consistent with mutationpatterns in the K-ras gene. Strain A/J mice were given a singlei.p. injectionof DBC at doses of 0, 5, 10, 20 or 40 mg/kg andlevels of DNA adducts in the lung were monitored by ³²P-postlabelingon days 1,3,5,7,14 and 21. The remaining animals were sacrificed8 months after DBC treatment and lung tumor multiplicity andK-ras mutation patterns in the tumors were determined. The lungtumor response to DBC was dose related, with an average of 4.7± 1.2 tumors/mouse at 5 mg/kg and 48.1 ± 5.5 tumors/mouseat 40 mg/kg. As many as seven DBC-DNA adducts were observedin the lung. DNA binding levels in the lung were highest at40 mg/kg, with maximum binding at 5-7 days. At lower dose levelsthe maximum binding to DNA decreased and shifted to earliertime points. The DBC-DNA adduct in the lung with the highestlevel of binding at all dose levels was DBC-DNA adduct 3. Themajority of DBC-induced mutations in the K-ras gene in the lungwere A     

One-electron oxidation is not a major route of metabolic activation and DNA binding for the carcinogen 7H-dibenzo[c,g]carbazole in vitro and in mouse liver and lung

7H-Dibenzo[c,g]carbazole (DBC) is a potent multi-site, multi-species carcinogen present in a variety of complex mixtures derived from the incomplete combustion of organic matter. Like many carcinogens, DBC requires metabolic activation to an electrophilic species to exert its mutagenic and carcinogenic effects. One-electron oxidation, leading to the formation of radical cation intermediates, has been proposed as a mechanism of metabolic activation for DBC in vitro resulting in unstable DNA adducts. The purpose of this research was to determine whether one-electron oxidation is a mechanism of activation and DNA binding for DBC in vivo. Specific depurinating DBC-DNA adducts formed by one-electron oxidation were analyzed in mouse liver at 4 h following a single i.p. dose of 40 mg/kg of 11 microCi [(14)C]DBC. In addition to five previously published adduct standards, two newly identified adduct standards were characterized by mass spectrometry and NMR, namely DBC-6-N7-Ade and DBC-6-N1-Ade; however, neither was observed in mouse liver. Only the DBC-5-N7-Gua adduct was observed in mouse liver extracts at a level of 6.5 +/- 1. 8 adducts/10(6) nucleotides. In addition, the formation of AP sites and stable DBC-DNA adducts was analyzed in mouse liver and lung at 4, 12 and 24 h following a single i.p. dose of 0.4, 4 or 40 mg/kg DBC (n = 3/group). There was a distinct time- and dose-response of stable DBC-DNA adducts detected by (32)P-post-labeling. There was not a clear dose-response for formation of AP sites; however, a significant increase over control levels was observed at the 4 and 40 mg/kg dose groups at 4 and 12 h post dosing, respectively. Quantitative comparisons indicate that the depurinating DBC-5-N7-Gua adduct constitutes approximately 0.4% of total adducts measured whereas the stable adducts detected by (32)P-post-labeling constitute 99.6% of total adducts measured following a 40 mg/kg dose and a 4 h time-point. The data indicate that one-electron oxidation does occur in mouse liver in vivo. However, one-electron oxidation is a minor mechanism of activation in that the percentage of total adducts formed through this route constitutes a minor percentage of the total adducts formed.     

Effects of retinoids on metabolizing enzymes and on binding of benzo(a)pyrene to rat tissue DNA

Retinyl acetate, 13-cis-retinoic acid (13cisRA), and N-(4-hydroxyphenyl)-retinamide (4HPR) were assayed for their in vivo effects on hepatic levels of cytochrome P450, cytosolic glutathione-S-transferase, and quinone reductase. When given p.o. to Sprague-Dawley rats, all of the retinoids caused significant suppression in the levels of arylhydrocarbon hydroxylase, yet 13cisRA and 4HPR caused elevations in cytosolic levels of quinone reductase and glutathione-S-transferase, respectively. Scans of sodium dodecyl sulfate-polyacrylamide gels of microsomal proteins from the livers of retinoid-dosed animals showed changes in both the intensities and the number of stained bands. For microsomes from 13cisRA-dosed animals, there were additional changes in the absorption maximum of the carbon monoxide and octylamine difference spectra. There was, compared to controls, a 62% reduction in the NADPH-dependent binding of (+)-7S-trans-7,8-dihydro[7-14C]benzo(a)pyrene-7,8-diol to microsomal proteins from 13cisRA-dosed animals. Fluorography of the sodium dodecyl sulfate-polyacrylamide gels showed that the major reduction in metabolite binding occurred in the Mr 50,000 region of the gel. The reduction in the NADPH-dependent binding of (+)-7S-trans-7,8-dihydro[7-14C]benzo(a)pyrene-7,8-diol to microsomal proteins in vitro and the reduction in hepatic arylhydrocarbon hydroxylase levels correlated with a reduction in the in vivo binding of benzo(a)pyrene to rat liver DNA. Animals dosed for 7 days with 13cisRA, retinyl acetate, or 4HPR showed a 38, 27, and 40% reduction in binding of benzo(a)pyrene to liver DNA and a 29, 32, and 21% reduction in binding to stomach DNA, respectively, when the carcinogen was administered on the eighth day, and the tissues were harvested 24 h later. Binding to lung DNA was reduced by 23 and 11%, respectively, in the 13cisRA- and 4HPR-dosed rats. No differences were observed in binding to kidney. Thus, retinoids, by altering the metabolism of carcinogens, could influence the initiation stage of carcinogenesis.     

Metabolism and formation of DNA adducts of benzo(a)pyrene in human diploid fibroblasts.

Cultured human diploid skin fibroblasts incubated with [G-3H]benzo(a)pyrene yielded about 10 times more H2O-=soluble benzo(a)pyrene metabolites and DNA adducts of stationary growth phase than did proliferating cultures. This increased formation could be blocked by alpha-naphthoflavone. Trichloropropenoxide and cyclohexenoxide, inhibitors of the epoxide hydratase, inhibited predominantly the formation of DNA adducts. Cultures from older individuals formed significantly more benzo(a)pyrene metabolites and DNA adducts, but control cultures from patients with either lung cancer or melanoma did not. The age influence was not apparent when the ratio of DNA adducts to H2O-soluble metabolites was determined for each individual cell line. However, the proportion of DNA-bound material in the cells from patients with lung cancer was significantly increased compared to cells from melanoma patients or healthy individuals.     

Quantitation of protein adducts as a marker of genotoxic exposure: immunologic detection of benzo(a)pyrene -- globin adducts in mice

Immunologic methods have been developed for the determinationof benzo(a)pyrene (BP)-protein adducts and validated in animalstreated with (³H)BP. A previously developed antibody, 8E11,which recongnizes 7ß, 8-dihydroxy-9, 10-epoxy-7, 8,9, 10tetrahydrobenzo(a)pyrene (BPDE-I)-modified DNA or proteinas well as BPDE-I- tetraols, was used. The sensitivity of theassay was increased by enzymatic digestion of the modified proteinwith insoluble protease into peptides and amino acids beforeanalysis. In a competitive enzymelinked immunosorbent assay(ELISA) with digested BPDE-I-modified bovine serum albumin,50% inhibition occured at 400 fmol of adduct compared to 1450fmol for the nondigested albumin. Analysis of globin (Gb) isolatedfrom animals treated in vivo with 0.3–3 mg (³H)BP indicatedthat the ELISA could detect 90–100% of the adducts determinedby radioactivity. Levels of adducts in lung and liver DNA andserum albumin were correlated with the levels of Gb adducts.Of the total radioactivity associated with hemoglobin, only10% was from Gb while {small tilde}80% was from the heme fractionand the remainder from free BP metabolites. Significant cross-reactivityof antibody 8E11 was found with several BP-diols and phenols,suggesting that the immunoassay will not only be specific forBPDE-I adducts but will also detect adducts of other BP metabolitesas well as other aromatic hydrocarbon diol epoxides. An immunoaffinitycolumn of antibody 8E11 coupled to Sepharose 4B was used toisolate modified peptides from the digested Gb. About 65% ofthe applied radioactivity was retained on the column. Between1 and 2 mg of non-modified digested Gb could be added to thesample without interfering with binding of adducts. Proteindigestion and immunoaffinity chromatography should be usefulfor the measurement of protein adducts in biomonitoring studies.     

Analysis of benzo(a)pyrene:DNA adducts formed in cells in culture by immobilized boronate chromatography

A chromatographic procedure using boronic acid residues linked to a cellulose support [(N-(N'-[m-(dihydroxyboryl)-phenyl]succinamyl)amino]ethyl cellulose), used by Sawicki et al. (Cancer Res., 43: 3212-3218, 1983) for analysis of 7,12-dimethylbenz(a)anthracene:DNA adducts, was modified to allow the analysis of benzo(a)pyrene (BaP):DNA adducts formed in cells in culture. Adducts resulting from reaction of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BaPDE) contain cis-vicinal hydroxyl groups that complex with the boronic acid residues; adducts resulting from 7 beta, 8 alpha-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (syn-BaPDE) do not. A mixture of [3H]-syn-BaPDE:deoxyguanosine (dGuo) adduct and [14C]-anti-BaPDE:dGuo adduct was completely resolved on a column of boronate:cellulose. Early-passage cultures of Sencar mouse, Syrian hamster, and Wistar rat embryo cells and a culture of a human hepatoma cell line (Hep G2) were exposed to [3H]BaP, and the BaP:DNA adducts were resolved by boronate chromatography and high-performance liquid chromatography. The Hep G2 cells and mouse embryo cells contained two major adducts, a (+)-anti-BaPDE:dGuo adduct and a syn-BaPDE:dGuo adduct. Boronate chromatography permitted the resolution of an additional minor syn-BaPDE:deoxyribonucleoside adduct in the mouse embryo cells. The hamster and rat embryo cells contained a number of major BaP-DNA adducts that were resolved by boronate chromatography followed by high-performance liquid chromatography. The rat embryo cells contained three syn-BaPDE:deoxyribonucleoside adducts and approximately equal amounts of two adducts tentatively identified as dGuo adducts of the (+) and (-) enantiomers of anti-BaPDE. The boronate chromatography-high-performance liquid chromatography procedure improves the separation of the BaP:DNA adducts formed in biological systems and facilitates the identification of the BaP metabolite(s) responsible for the formation of these adducts.     

Species- and length of exposure-dependent differences in the benzo(a)pyrene:DNA adducts formed in embryo cell cultures from mice, rats, and hamsters

The activation of benzo(a)pyrene (BaP) to DNA-binding metabolites in early-passage embryo cell cultures prepared from various species of rodents was investigated by exposing cells from mice (BALB/c and Sencar), rats (Wistar and Fischer 344), and Syrian hamsters to [3H]BaP for various lengths of time. The BaP:DNA adducts containing cis-vicinal hydroxyl groups such as those formed from 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BaPDE) were separated from the other types of BaP:DNA adducts by immobilized boronate chromatography, and the individual adducts were analyzed by high-performance liquid chromatography. A number of BaP:DNA adducts were present in the DNA from the cultures from all three species after 5 h of BaP treatment. After a 24-h exposure to BaP, the mouse and hamster embryo cell DNA contained a large amount of the adduct formed by reaction of (+)-anti-BaPDE with the 2-amino group of deoxyguanosine (dGuo) and a small amount of a 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:dGuo adduct. A large number of BaP:DNA adducts derived from 7 beta, 8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene and other unidentified BaP metabolites were present in rat embryo cell cultures at all times. Neither the Fischer 344 nor the Wistar rat embryo cell cultures had a significant amount of (+)-anti-BaPDE:dGuo adduct after 5 h of BaP treatment, and in the Wistar rat cells larger amounts of other adducts were present even after a 96-h exposure to BaP. In cell cultures from all three species the proportion of (+)-anti-BaPDE:dGuo adduct increased as the length of time of exposure to BaP increased. There are major differences in the metabolic activation of BaP to DNA binding metabolites in embryo cells from various species of rodents. However, the variations between cell cultures from different strains of rats or mice are not as great as the variations between cell cultures from different species. The time-dependent alterations in the BaP:DNA adducts indicate that analysis after various lengths of time of exposure to BaP is essential to characterize accurately the pathways of metabolic activation of BaP in cells from various species and tissues.     

Immunohistochemical localization of DNA adducts in rat liver tissue and phenotypically altered foci during oral administration of 2-acetylaminofluorene

Histological studies using paired immunofluorescence stainingand peroxidase-anti-peroxidase staining were performed on sectionsof rat livers with an antiserum specific for the 2-acetylaminofluorene(AAF)-DNA adduct N-deoxyguanosin-(8-yl)-aminofluorene (dG-8-AF).This is the predominant adduct in rat liver DNA at 5 (80%) and28 (100%) days of AAF feeding. Nuclear staining was observedin livers of male Fischer rats fed 0.02% AAF for these timeperiods, and was not present in livers of animals fed controldiet or detected when specific antiserum, first absorbed withthe immunogen adduct, was utilized. In addition, nuclear stainingwas unchanged after incubation with RNase and abolished afterincubation with DNase. Adducts were not readily detectable whenwhole-liver adduct concentrations were less than an averageof 10⁵ adducts per cell (30–50 fmol/µg DNA). Theoverall pattern of adduct distribution in livers of AAF-fedanimals was distinctly non-uniform. A predominance of nuclearstaining was found in the periportal areas by both immunofluorescenceand immunoperoxidase procedures. In contrast, staining was veryweak in the centrilobular areas. When animals were fed AAF for28 days and control diet subsequently for 7, 14, 21 or 28 days,the overall intensity of the immunohistochemical staining decreasedwith time on control diet. However, the pattern of localizationremained the same as in livers of rats fed AAF for 28 days,with the predominance of adducts being in the periportal areas.In male rats fed 0.02% AAF for 8 weeks, foci positive for -glutamyltranspeptidase(GGT) became apparent, and the nuclei in these areas showedno immunofluorescence, indicating the absence of detectablelevels of the dG-8-AF adduct. Twenty adduct-negative areas inthe median lobes of three rat livers were positive for GGT,which suggests that loss of ability to form adducts in theseregions occurs concomitantly with early phenotypic changes.