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.     

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.     

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     

Frequent Ha-ras mutations in murine skin and liver tumors induced by 7H-dibenzo[c,g]carbazole.

7H-dibenzo[c,g]carbazole (DBC) is a potent liver and skin carcinogen when topically applied to the back skin of mice. This compound is found in complex mixtures produced during incomplete combustion of fossil fuels as well as in wood and tobacco smoke. The objective of this study was to elucidate the mechanism of action of this compound by assessing the Ha-ras mutational spectra of skin and liver tumors induced in a mouse model system. Low doses (50 nmol) and high doses (100 nmol) of DBC were applied topically to the backs of Hsd:ICR(Br) mice twice weekly. No treatment and solvent application were used as controls. After the mice were killed, the skin and liver tumors were removed, DNA was isolated, and tumor DNA was screened for Ha-ras codon 12, 13, and 61 mutations by using an enriched polymerase chain reaction method. Mutations were confirmed by reverse cyclic dideoxy sequencing. No mutations were found in codons 12 and 13 of DBC-induced tumors, whereas one acetone-control tumor had a codon 13 mutation. Sixty-seven percent of skin tumors and 45% of liver tumors induced by high doses of DBC and 67% of skin tumors induced by low doses of DBC contained codon 61 mutations, whereas liver tumors induced by low doses of DBC did not. The codon 61 mutations were exclusively A:T-->T:A transversions within the second base (CAA-->CTA). These results indicate that DBC is a unique polycyclic aromatic hydrocarbon in that it induces both skin and liver tumors upon topical application and that the mutational spectra are the same in tumors from two target organs, skin and liver, yet different from tumors from a third target organ, lung.     

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.     

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.     

The chemistry and biology of 7H-dibenzo[c,g]carbazole: synthesis and characterization of selected derivatives, metabolism in rat liver preparations and mutagenesis mediated by cultured rat hepatocytes

Authentic stable standards of 7H-dibenzo[c, g]carbazole (DBC),a potent environmental carcinogen, were synthesized in orderto study the compound's metabolism and mutagenesis in wholecell systems. Complete characterization of 2-OH-DBC, 3-OH-DBC,4-OH-DBC, 13c-OH-DBC and N-methyl-DBC was accomplished by UV,IR, fluorescence and high resolution NMR spectra, and by highresolution mass spectrometric procedures. Metabolites of DBCwere isolated and separated by HPLC from extracts of rat livermicrosomal incubations and the medium of primary cultures ofrat liver cells. Identification of metabolites was accomplishedby comparisons between the authentic standards and isolatedmetabolites by UV and fluorescence spectroscopy, mass spectralanalyses, and by co-chromatographic techniques. 2-OH-DBC and3-OH-DBC were found in all rat liver preparations as well asthree other unidentified phenols. 4-OH-DBC, 13c-OH-DBC or N-methyl-DBCwere not isolated under any conditions. The rates of appearanceof DBC metabolites in cultures of rat liver cells were comparedto those for benzo[a]pyrene (BaP) at 10, 25, 50 and 100 µMsubstrate. At 25 µM substrate or greater, DBC metabolitesappeared in the culture medium at significantly faster ratesthan those of BaP. At 100 µM substrate, DBC metabolitesappeared at a rate 4-times the rate observed for BaP. When themutagenk potential of DBC was compared to that of BaP underidentical conditions in a co-cultivation system of rat livercells and an epithelial cell line, DBC was found to producesignificantly higher rates of mutagenesis than BaP at concentrationsof 0.4, 4.0 and 40.0 µM in the culture medium. The mutagenicpotential of DBC was compared to that of several derivativesof the parent compound. 3-OH-DBC, 13c-OH-DBC and N-methyl-DBCwere found to be mutagenic in the co-cultivation system at 40µM, with mutation frequencies of 4.4 ± 0.8, 8.0±3.1 and 12.9 ± 5.4 mutants per 105 survivors,respectively. The parent compound induced 8.0 ± 2.8 mutantsper 10⁵ survivors at the same concentration. 2-OH-DBC and 4-OH-DBCwere not mutagenic under the same conditions. The studies haveshown that metabolism of 7H-DBC leads predominantly to phenolsin rat liver cells. The results of the mutagenesis experimentsindicate that, of the derivatives studied, those associatedby induction to the nitrogen are mutagenic. The latter studiessuggest that the nitrogen is involved in the activation of theparent compound through inductive mechanisms.     

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.     

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.     

N-methylation reduces the DNA-binding activity of 7H-dibenzo[c,g]carbazole ~300-fold in mouse liver but only ~2-fold in skin: possible correlation with carcinogenic activity

N-methyl-dibenzo[c,g]carbazole (MeDBC) lacks the potent hepatocarcinogenicactivity in mice characteristic for 7H-dibenzo[c,g]carbazole(DBC), while both compounds are local carcinogens, leading topapilloma and carcinoma formation in skin after topical application.Because DNA binding is considered an essential step in the initiationof chemical carcinogenesis, the DNA adduction by MeDBC was comparedwith that by DBC in mouse liver and skin via a ³²P-post-labelingtechnique. Both compounds elicited chromatographically similaradducts in liver; however, the extent of total DNA binding ofDBC was 343- and 265-fold greater than that of MeDBC 24 h aftertopical and i.p. administration, respectively, of a 37 µmol/kgdose. In skin, the adduct pattern elicited by either compoundafter topical application was dif ferent from that seen in liver,and three of four adducts derived from MeDBC were chromatographicallydistinct from those produced by DBC. Quantitative analysis revealedthat total adduction in skin by DBC was 2.3-fold higher thanby MeDBC. When the adduct levels were compared between liverand skin, topically applied MeDBC bound preferentially to skinversus liver DNA by a factor of 10, while the opposite was truefor DBC. These data are in agreement with the car cinogenicityreported for DBC and MeDBC and support the hypothesis that theextent of covalent DNA modification by these compounds is associatedwith their biological activity. We conclude that an unsubstitutednitrogen is essential for the genotoxic activity of DBC in liverbut not skin. The results also demonstrate the potential ofthe ³²P-postlabeling assay in predicting the organotropism ofclosely related carcinogens.     

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.     

Kinetics of DNA adduct formation and removal in mouse hepatocytes following in vivo exposure to 5,9-dimethyldibenzo[c,g]carbazole

5,9-Dimethyldibenzo[c,g]carbazole (DMDBC), a potent mouse hepatocarcinogen, has been shown to induce a non-linear increase in mutant frequency in the liver of the transgenic MutaMouse. To gain insight into the mechanisms underlying the mutagenicity of DMDBC in vivo, DNA damage formation and removal were monitored in mouse hepatocytes over 4-144 h after a single skin application of 10 or 90 mg/kg DMDBC. DNA adducts were measured by (32)P-post-labeling. DNA repair was assessed by: (i) the unscheduled DNA synthesis (UDS) assay, which measures [(3)H]thymidine incorporation into hepatocyte DNA undergoing excision repair; (ii) the Comet assay, which detects DNA strand breaks transiently produced between the incision and rejoining steps of the excision repair process. A plateau of approximately 400 DNA adducts/10(8) nucleotides was reached 24 h after treatment with 10 mg/kg and remained unchanged until 144 h. UDS activity was significantly induced at 15 and 24 h, while no DNA strand breaks were observed at any sampling time. These results suggest that DNA repair mechanisms were efficiently induced and the formation of a high degree of DNA damage was avoided at this dose level. Following exposure to 90 mg/kg DMDBC, the number of DNA adducts increased sharply to a maximum at 24 h ( approximately 8000/10(8) nucleotides) and then declined to approximately 500/10(8) nucleotides at 144 h. UDS activity was markedly induced from 15 to 72 h. Low levels of DNA strand breaks were observed at 24 and 48 h. The formation of large numbers of DNA adducts and the emergence of DNA strand breaks despite a strong initial induction of UDS activity suggested that DNA repair mechanisms were saturated at this dose level. This phenomenon could partly account for the non-linear induction of gene mutations previously reported in the liver of the transgenic MutaMouse.     

Differential metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 variants

Cytochrome P450 1A1 (CYP1A1) plays a key role in the metabolism of carcinogens, such as benzo[a]pyrene (B[a]P) and metabolites to ultimate carcinogens. Three human allelic variants, namely wild-type (CYP1A1.1), CYP1A1.2 (I462V) and CYP1A1.4 (T461N), were coexpressed by coinfection of baculovirus-infected insect cells with human NADPH-P450 reductase. These recombinant enzymes (in microsomal membranes) were used to analyze whether CYP1A1 polymorphisms affect catalytic activities towards B[a]P and B[a]P-7,8-dihydrodiol. The complete spectrum of phase I metabolites, including the tetrahydrotetrols resulting from hydrolysis of the ultimate carcinogen, B[a]P-7,8-dihydrodiol-9,10-epoxide, was examined by HPLC. Wild-type enzyme showed the highest total metabolism of B[a]P, CYP1A1.2 was approximately 50%, and CYP1A1.4 approximately 70%. Km values for all metabolites with CYP1A1.2 were generally significantly lower than with wild-type enzyme (e.g. B[a]P-7,8-diol formation: 13.8 microM for wild-type, 3.5 microM for CYP1A1.2 and 7.7 microM for CYP1A1.4). Addition of epoxide hydrolase markedly increases the relative diol-to-phenol activities by all three variants. However, CYP1A1.4 exhibits the greatest efficiency to produce diol species. Each variant produced the diol epoxides from B[a]P-7,8-dihydrodiol. CYP1A1.1 exhibited with 10.4 pmol/min/pmol CYP1A1 the greatest total rate for 7,8-diol metabolites followed by CYP1A1.2 (7.2 pmol/min/pmol CYP1A1) and CYP1A1.4 (5.5 pmol/min/pmol CYP1A1). All enzyme variants produced about three times more diol epoxide 2-derived metabolites than diol epoxide 1-derived ones, whereby both rare allelic variants exhibited statistically significantly increased formation of diol epoxide 2. This study showed that the three CYP1A1 variants had different enzyme kinetics properties to produce both the diol metabolites from B[a]P and the ultimate mutagenic species diol epoxide 2 from B[a]P-7,8-dihydrodiol, which must be considered in the evaluation of individual susceptibility to cancer.     

Comparative regioselective and stereoselective metabolism of 7-chlorobenz[a]anthracene and 7-bromobenz[a]anthracene by mouse and rat liver microsomes

Quantitative metabolism of 7-chlorobenz[a]anthracene (7-Cl-BA) and 7-bromobenz[a]anthracene (7-Br-BA) by liver microsomes of uninduced mice and rats was studied. Both enzymatic systems metabolize 7-Cl-BA preferentially at the C-8 and C-9 aromatic double bond region, approximately 42 and approximately 56% respectively, of the total metabolites. 7-Cl-BA and 7-Br-BA were metabolized considerably at C-3 and C-4, C-5 and C-6, C-8 and C-9, and C-10 and C-11. While 7-Cl-BA trans-3,4-dihydrodiol was formed in a 7-8% yield of the total metabolites in both enzymatic systems, 7-Br-BA trans-3,4-dihydrodiol was formed 16.0 and 9.9% respectively, from the mouse and rat liver microsomal metabolism. In mutagenicity assays with the Salmonella typhimurium tester strain TA100 in the presence of S9 activation enzymes, both of these trans-3,4-dihydrodiols exhibited higher mutagenicity than 7-Cl-BA and 7-Br-BA, while the other trans-dihydrodiol metabolites were either essentially inactive or weaker than the parent compounds. These results suggest that 7-Cl-BA trans-3,4-dihydrodiol and 7-Br-BA trans-3,4-dihydrodiol are the proximate metabolites of 7-Cl-BA and 7-Br-BA. Metabolism of 7-Cl-BA and 7-Br-BA by mouse liver microsomes was also in a stereoselective manner, preferentially giving trans-dihydrodiol metabolites an R, R stereochemistry.     

Effects of route of administration on tissue distribution of DNA adducts in mice: comparison of 7H-dibenzo(c,g)carbazole, benzo(a)pyrene, and 2-acetylaminofluorene

The environmental pollutant 7H-dibenzo(c,g)carbazole (DBC) has been shown to be a potent carcinogen in various mouse tissues, but displays an unusual degree of hepatocarcinogenicity. We have previously reported that in accord with this activity, mouse liver is the target organ for DBC-DNA binding, with total levels being up to 2700 times greater than in extrahepatic tissues after s.c. administration. To elaborate on this finding, we have directly compared the tissue distribution of DNA damage by three diverse aromatic carcinogens, DBC, benzo(a)pyrene (BP), and 2-acetylaminofluorene (AAF). Following a single topical, p.o., or s.c. administration of 80 mumol/kg of test compound to male BALB/c mice, a 32P-postlabeling assay showed the total number of DBC adducts in liver DNA to be 11-138 times that in kidney, lung, or skin DNA. The degree of hepatic adduction varied as a function of the route of administration, with the highest occurring after topical application and the lowest after s.c. injection. The tissue preference for AAF and BP adducts varied with the route of administration and was much less than for DBC adducts, except that topical application of BP gave DNA adduct levels in skin that were 91-218 times greater than in other tissues. For a given tissue and route of administration, DNA adduction by DBC was 1.7- to 950-fold greater than that by BP and AAF, except in skin where the level of DNA adducts from BP was 3 to 4 times that from DBC. We conclude that (a) DBC exhibits an exceptional and unique preference for liver DNA adduction after different routes of administration; (b) DBC is more potent overall than BP or AAF in causing tissue DNA damage; and (c) for each of the three carcinogens, the route of exposure is a much less important factor than the nature of the carcinogen in determining the tissue distribution of covalent DNA damage.     

The activation and DNA binding of 7-methylbenz[c]-acridine catalysed by mouse liver microsomes

The metabolism of the carcinogen, 7-methylbenz[c]acridine (7MBAC), in liver microsomes prepared from untreated, and phenobarbital sodium (PB) and 3-methylcholanthrene (MC) induced male C57BL/6 mice was examined by high pressure liquid chromatography (HPLC). The abilities of control and MC induced liver microsomes to catalyse covalent binding of the carcinogen to DNA were comparable (60 pmol 7MBAC bound/mg DNA), although this maximum binding level observed was achieved at different 7MBAC concentrations for control (100 microM) and MC induced (25 microM) preparations. In vivo binding of 7MBAC to liver DNA was greater than that observed for lung DNA of the same animals but over 21 h bound 7MBAC levels decreased to a greater extent in liver than in lung. One fraction of a digest of in vitro alkylated DNA gave a fluorescence emission spectrum very similar to that of 7-methyl-1,2,3,4-tetrahydrobenz[c]acridine. This result suggests that diol epoxides functionalised at the 1,2,3,4-positions may be ultimate carcinogens derived from 7MBAC.     

The effects of dietary corn oil on the metabolism and activation of benzo[a]pyrene by the benzo[a]pyrene metabolizing enzymes of the mouse

Male ICR Swiss mice, weighing 16 — 20 g, were fed ad libitumeither a fat-free diet or a diet containing 10% com oil. Afterthree weeks on these diets, the rates of benzo[a]pyrene (B[a]P)metabolite formation and metabolism to products which covalentlybind with macromolecules were compared using hepatic nucleiand microsomal preparations. The maximum activity of B[a]P hydroxylasehi microsomes from untreated animals was increased 50% by feedingthe com oil diet, however, B[a]P hydroxylase in microsomes from3-methylcholanthrene (3-MC)-treated mice was unaffected by diet.In animals treated with phenobarbital, B[a]P metabolism andB[a]P - DNA adduct formation were greater hi microsomes fromcorn oil fed mice compared to those fed the fat-free diet. Ata B[a]P concentration of 96 µM, microsomes from corn oilfed untreated mice produced 26% more extractable metabolitesand covalent binding to exogenous DNA was increased 46%. Atlower substrate concentrations (0.94–15.0 µM B[a]P),B[a]P-DNA and B[a]P -protein binding were 300 – 400% greaterwhen incubated with microsomes from corn oil fed mice than whenincubated with microsomes from mice fed fat-free diet. The apparentV_max's determined for the formation of each extractable metabolitewere increased 1.5 – 3.0 times by the corn oil diet. Hepaticnuclear B[a]P hydroxylase and nuclear activation of B[a]P toproducts which covalently bind to DNA in both non-induced and3-MC-pretreated animals fed the corn oil diet were greater thanthat observed in animals fed the fat-free diet. B[a]P hydroxylaseactivities in the lungs of these animals were unaltered by diet.