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Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), is a known human carcinogen. In non-smoking adults greater than 95% of BaP exposure is through diet. The carcinogenicity of BaP is utilized by the U.S. EPA to assess relative potency of complex PAH mixtures. PAH relative potency factors (RPFs, BaP = 1) are determined from high dose animal data. We employed accelerator mass spectrometry (AMS) to determine pharmacokinetics of [14C]-BaP in humans following dosing with 46 ng (an order of magnitude lower than human dietary daily exposure and million-fold lower than animal cancer models). To assess the impact of co-administration of food with a complex PAH mixture, humans were dosed with 46 ng of [14C]-BaP with or without smoked salmon. Subjects were asked to avoid high BaP-containing diets and a 3-day dietary questionnaire given to assess dietary exposure prior to dosing and three days post-dosing with [14C]-BaP. Co-administration of smoked salmon, containing a complex mixture of PAHs with an RPF of 460 ng BaPeq, reduced and delayed absorption. Administration of canned commercial salmon, containing very low amounts of PAHs, showed the impacts on pharmacokinetics were not due to high amounts of PAHs but rather a food matrix effect.

The cytochrome P450 (CYP) 1 family is active toward numerous environmental pollutants, including polycyclic aromatic hydrocarbons (PAHs). Utilizing a mouse model, null for Cyp1b1 and expressing human CYP1B1, we tested the hypothesis that hCYP1B1 is important for dibenzo[def,p]chrysene (DBC) transplacental carcinogenesis. Wild-type mCyp1b1, transgenic hCYP1B1 (mCyp1b1 null background), and mCyp1b1 null mice were assessed. Each litter had an equal number of siblings with Ahrb-1/d and Ahrd/d alleles. Pregnant mice were dosed (gavage) on gestation day 17 with 6.5 or 12 mg/kg of DBC or corn oil. At 10 months of age, mortality, general health, lymphoid disease and lung tumor incidence, and multiplicity were assessed. hCYP1B1 genotype did not impact lung tumor multiplicity, but tended to enhance incidence compared to Cyp1b1 wild-type mice (P = 0.07). As with Cyp1b1 in wild-type mice, constitutive hCYP1B1 protein is non-detectable in liver but was induced with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Wild-type mice were 59% more likely to succumb to T-cell Acute Lymphoblastic Leukemia (T-ALL). Unlike an earlier examination of the Ahr genotype in this model (Yu et al., Cancer Res, 2006;66:755-762), but in agreement with a more recent study (Shorey et al., Toxicol Appl Pharmacol, 2013;270:60-69), this genotype was not associated with lung tumor incidence, multiplicity, or mortality. Sex was not significant with respect to lung tumor incidence or mortality but males exhibited significantly greater multiplicity. Lung tumor incidence was greater in mCyp1b1 nulls compared to wild-type mice. To our knowledge, this is the first application of a humanized mouse model in transplacental carcinogenesis. © 2016 Wiley Periodicals, Inc.

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), is a known human carcinogen. In non-smoking adults greater than 95% of BaP exposure is through diet. The carcinogenicity of BaP is utilized by the U.S. EPA to assess relative potency of complex PAH mixtures. PAH relative potency factors (RPFs, BaP=1) are determined from high dose animal data. We employed accelerator mass spectrometry (AMS) to determine pharmacokinetics of [(14)C]-BaP in humans following dosing with 46 ng (an order of magnitude lower than human dietary daily exposure and million-fold lower than animal cancer models). To assess the impact of co-administration of food with a complex PAH mixture, humans were dosed with 46 ng of [(14)C]-BaP with or without smoked salmon. Subjects were asked to avoid high BaP-containing diets and a 3-day dietary questionnaire given to assess dietary exposure prior to dosing and three days post-dosing with [(14)C]-BaP. Co-administration of smoked salmon, containing a complex mixture of PAHs with an RPF of 460 ng BaP(eq), reduced and delayed absorption. Administration of canned commercial salmon, containing very low amounts of PAHs, showed the impacts on pharmacokinetics were not due to high amounts of PAHs but rather a food matrix effect.

Polycyclic aromatic hydrocarbons (PAHs) are a class of over 1500 chemicals formed as incomplete combustion products and released into the environment from both natural (e.g. forest fires) or anthropogenic (e.g. burning of fossil fuels, tobacco, charbroiled meats) sources. Several PAHs, particularly those with more than 4 rings such as benzo[a]pyrene (BaP), dibenzo[def,p]chrysene (DBC), have been designated as Class 1 known or Class 2A probable human carcinogens by the International Agency for Research on Cancer (IARC, 2010). While much of the research on PAH carcinogenicity focuses on individual PAHs and BaP, in particular, most human exposures to PAHs result from chemical mixtures through dietary, inhalation, or dermal routes of exposure. Primary sources of environmental exposure to these PAHs include wood smoke, creosote, and burning of fossil fuels and tobacco (IARC, 2010). Recently, diesel engine exhaust was added to the list of Class 1 known human carcinogens and certain other PAH-containing mixtures, including air pollution, have been designated as probable or possible Class2A/B carcinogens in humans (IARC, 2010, 2014). One of the most difficult challenges for risk assessment is the evaluation of health hazards from exposure to environmental chemical mixtures. Currently, significant data gaps exist for understanding carcinogenicity of PAH mixtures and complex environmental mixtures containing PAHs. Further, little is known about the mechanisms of tumorigenesis for PAH mixtures. Current assessment of cancer risk for PAHs involves testing compounds in the 2-year rodent bioassay, which is not practical for screening large numbers of compounds or mixtures due to expense and time. Therefore, alternative approaches are typically utilized for evaluating the carcinogenic potential of PAHs and PAH-containing mixtures. Currently, the primary method for assessing cancer risk of complex mixtures is the relative potency factor (RPF) approach in which complex mixtures are evaluated based on a subset of individual component PAHs compared with BaP as a surrogate or reference (US EPA, 2010). However, we and others have found this approach inadequate for predicting carcinogenicity of mixtures and certain individual PAHs, particularly those that function through alternate pathways or exhibit greater promotional capacity compared to BaP (Courter et al., 2008; Siddens et al., 2012). Significant challenges have also been identified in utilizing such reference-based approaches for estimating risk from exposure to PAHs in air pollution or waste sites. Complex environmental mixtures subjected to weathering and aging processes can contain many different PAHs, including alkyl-, N-, S-, and O-substituted forms, along with other unknown chemicals; however, only a limited number of unsubstituted PAHs have been characterized for use in RPF calculations. Mixture toxicity for risk assessment is calculated based on select individual components and assumes additivity through a common mechanism of action for PAHs compared to BaP as a standard. Therefore, the RPF approach does not take into consideration mechanistic information about the different pathways, cells, and tissues affected by PAHs during initiation and promotion. This approach is also insufficient for predicting carcinogenicity of complex real-world environmental mixtures of unknown composition. In this study, we propose an innovative model for determining carcinogenic risk of PAH mixtures using mechanistic approaches. We hypothesize that a chemical bioactivity profile measured after short-term exposure to individual and mixture PAHs from global transcriptional profiling can be used to discriminate future carcinogenic potential based on important mechanistic differences among exposures. The bioactivity profile acts as a unique fingerprint for genes and pathways activated by chemicals and mixtures postexposure and can be used for predicting long-term consequences such as cancer outcome. An important aspect of the bioactivity profile is that the gene signatures are linked to chemical mechanism of action and can also provide insight into alternate mechanisms of PAH carcinogenesis and related mechanisms for complex mixtures. Based on preliminary data, we demonstrate that long-term cancer outcome for PAHs and mixtures can be predicted from high-content genomic evaluation of bioactivity after short-term exposure.

Polycyclic aromatic hydrocarbons (PAHs) are present in the environment as complex mixtures with components that have diverse carcinogenic potencies and mostly unknown interactive effects. Non-additive PAH interactions have been observed in regulation of cytochrome P450 (CYP) gene expression in the CYP1 family. To better understand and predict biological effects of complex mixtures, such as environmental PAHs, an 11 gene input-1 gene output fuzzy neural network (FNN) was developed for predicting PAH-mediated perturbations of dermal Cyp1b1 transcription in mice. Input values were generalized using fuzzy logic into low, medium, and high fuzzy subsets, and sorted using k-means clustering to create Mamdani logic functions for predicting Cyp1b1 mRNA expression. Model testing was performed with data from microarray analysis of skin samples from FVB/N mice treated with toluene (vehicle control), dibenzo[ def,p ]chrysene (DBC), benzo[ a ]pyrene (BaP), or 1 of 3 combinations of diesel particulate extract (DPE), coal tar extract (CTE) and cigarette smoke condensate (CSC) using leave-one-out cross-validation. Predictions were within 1 log 2 fold change unit of microarray data, with the exception of the DBC treatment group, where the unexpected down-regulation of Cyp1b1 expression was predicted but did not reach statistical significance on the microarrays. Adding CTE to DPE was predicted to increase Cyp1b1 expression, whereas adding CSC to CTE and DPE was predicted to have no effect, in agreement with microarray results. The aryl hydrocarbon receptor repressor (Ahrr) was determined to be the most significant input variable for model predictions using back-propagation and normalization of FNN weights.

Dibenzo[def,p]chrysene (DBC) is a transplacental carcinogen in mice (15mg/kg; gestation day (GD) 17). To mimic residual exposure throughout pregnancy, dams received four smaller doses of DBC (3.75mg/kg) on GD 5, 9, 13 and 17. This regimen alleviated the previously established carcinogenic responses in the thymus, lung, and liver. However, there was a marked increase in ovarian tumors (females) and hyperplastic testes (males). [(14)C]-DBC (GD 17) dosing revealed transplacental distribution to fetal tissues at 10-fold lower concentrations than in paired maternal tissue and residual [(14)C] 3weeks post-dose. This study highlights the importance of developmental stage in susceptibility to environmental carcinogens.

The polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), was compared to dibenzo[def,p]chrysene (DBC) and combinations of three environmental PAH mixtures (coal tar, diesel particulate and cigarette smoke condensate) using a two stage, FVB/N mouse skin tumor model. DBC (4nmol) was most potent, reaching 100% tumor incidence with a shorter latency to tumor formation, less than 20 weeks of 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion compared to all other treatments. Multiplicity was 4 times greater than BaP (400 nmol). Both PAHs produced primarily papillomas followed by squamous cell carcinoma and carcinoma in situ. Diesel particulate extract (1 mg SRM 1650b; mix 1) did not differ from toluene controls and failed to elicit a carcinogenic response. Addition of coal tar extract (1 mg SRM 1597a; mix 2) produced a response similar to BaP. Further addition of 2 mg of cigarette smoke condensate (mix 3) did not alter the response with mix 2. PAH-DNA adducts measured in epidermis 12 h post initiation and analyzed by ³²P post-labeling, did not correlate with tumor incidence. PAH-dependent alteration in transcriptome of skin 12 h post initiation was assessed by microarray. Principal component analysis (sum of all treatments) of the 922 significantly altered genes (p

Many of the toxic and carcinogenic effects of urban air pollution have been linked to polycyclic aromatic hydrocarbons (PAHs) adsorbed to airborne particulate matter (PM). The carcinogenic properties of PAHs in complex organic mixtures derived from PM have been chiefly attributed to their mutagenicity. Nevertheless, PAHs are also potent activators of the aryl hydrocarbon receptor (AhR), which may contribute to their nongenotoxic effects, including tumor promotion. As the genotoxicity of carcinogenic PAHs in complex mixtures derived from urban PM is often inhibited by other mixture constituents, the AhR-mediated activity of urban PM extracts might significantly contribute to the carcinogenic activity of such mixtures. In the present study, we used an organic extract of the urban dust standard reference material, SRM1649a, as a model mixture to study a range of toxic effects related to DNA damage and AhR activation. Both the organic extract and its neutral aromatic fraction formed a low number of DNA adducts per nucleotide in the liver epithelial WB-F344 cells model, without inducing DNA damage response, such as tumor suppressor p53 activation and apoptosis. In contrast, we found that this extract, as well as its neutral and polar fractions, were potent inducers of a range of AhR-mediated responses, including induction of the AhR-mediated transcription, such as cytochrome P450 1A1/1B1 expression, and the AhR-dependent cell proliferation. Importantly, these toxic events occurred at doses one order of magnitude lower than DNA damage. The AhR-mediated activity of the neutral fraction was linked to PAHs and their derivatives, as polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls were only minor contributors to the overall AhR-mediated activity. Taken together, our data suggest that more attention should be paid to the AhR-dependent nongenotoxic events elicited by urban PM constituents, especially PAHs and their derivatives.

Assessment of human cancer risk from animal carcinogen studies is severely limited by inadequate experimental data at environmentally relevant exposures and by procedures requiring modeled extrapolations many orders of magnitude below observable data. We used rainbow trout, an animal model well-suited to ultralow-dose carcinogenesis research, to explore dose-response down to a targeted 10 excess liver tumors per 10000 animals (ED(001)). A total of 40800 trout were fed 0-225 ppm dibenzo[a,l]pyrene (DBP) for 4 weeks, sampled for biomarker analyses, and returned to control diet for 9 months prior to gross and histologic examination. Suspect tumors were confirmed by pathology, and resulting incidences were modeled and compared to the default EPA LED(10) linear extrapolation method. The study provided observed incidence data down to two above-background liver tumors per 10000 animals at the lowest dose (that is, an unmodeled ED(0002) measurement). Among nine statistical models explored, three were determined to fit the liver data well-linear probit, quadratic logit, and Ryzin-Rai. None of these fitted models is compatible with the LED(10) default assumption, and all fell increasingly below the default extrapolation with decreasing DBP dose. Low-dose tumor response was also not predictable from hepatic DBP-DNA adduct biomarkers, which accumulated as a power function of dose (adducts = 100 x DBP(1.31)). Two-order extrapolations below the modeled tumor data predicted DBP doses producing one excess cancer per million individuals (ED(10)(-6)) that were 500-1500-fold higher than that predicted by the five-order LED(10) extrapolation. These results are considered specific to the animal model, carcinogen, and protocol used. They provide the first experimental estimation in any model of the degree of conservatism that may exist for the EPA default linear assumption for a genotoxic carcinogen.

Polycyclic aromatic hydrocarbon (PAH) DNA adducts have been associated with carcinogenesis, which is accompanied by multiple alterations in gene expression. We used two-dimensional electrophoresis to distinguish protein expression changes induced in MCF-7 cells by individual PAH (B[a]P and DB[a,l]P) and PAH mixtures (coal tar extract [SRM 1597] and diesel exhaust extract [SRM 1975]). Spots of interest were identified by MALDI-TOF-TOF. Our results have shown alterations in the expression of heat-shock proteins, cytoskeletal proteins, DNA associated proteins, and glycolytic and mitochondrial proteins. The proteins that were universally altered in expression were actin cytoplasmic 1, tubulin alpha and myosin light chain alkali, cyclophilin B, and heterogeneous ribonucleoprotein B1 (a protein involved in access to telomerase and mRNA maturation). Additional proteins with altered expression include histone H2A.1, heat-shock protein 70-2, galectin-3, nucleoside diphosphate kinase, ATP synthase, and electron transfer flavoprotein. While sharing similarities, each PAH treatment exhibited a unique proteomic fingerprint.

Dibenzo(a,l)pyrene (DBP) is among the most potent carcinogenic polycyclic aromatic hydrocarbons. Previously, we showed that DBP administration to pregnant mice resulted in high mortality of offspring from an aggressive T-cell lymphoma. All mice that survive to 10 months of age exhibit lung tumors with high multiplicity. Recombinant cytochrome P450 (cyp) 1b1 from mice and the homologue 1B1 in humans exhibit high activity toward the metabolic activation of DBP. Targeted disruption of the cyp1b1 gene protects against most DBP-dependent cancers. Mice heterozygous for the disrupted cyp1b1 allele were used to examine the effect of cyp1b1 gene dosage on DBP transplacental carcinogenesis. Dams were treated with 1 or 15 mg/kg of DBP or 50 mg/kg of benzo(a)pyrene. Cyp1b1-null offspring did not develop lymphoma, whereas wild-type and heterozygous siblings, born to dams given the high dose of DBP, exhibited significant mortalities between 10 and 30 weeks of age. At 10 months, all groups had lung adenomas or carcinomas [9.5%, 40.3%, 25.6%, and 100% incidences for controls, benzo(a)pyrene, 1 and 15 mg/kg DBP, respectively]. Cyp1b1 status did not alter benzo(a)pyrene-dependent carcinogenesis. At 1 mg/kg DBP, cyp1b1 status altered the incidence of lung tumors (19.0, 27.8, and 28.6% for nulls, heterozygous, and wild-type, respectively). At 15 mg/kg, tumor multiplicities in cyp1b1 wild-type (9.3) and heterozygous (9.5) offspring were nearly twice that of cyp1b1-null siblings (5.0). These data confirm that cyp1b1 bioactivation of DBP occurs in fetal target tissues, following transplacental exposure, with the thymus and lung as primary and secondary targets, respectively.

The carcinogenic polycyclic aromatic hydrocarbon ns (PAHs) benzo[a]pyrene (B[a]P) and dibenzo[a,l]pyrene (DB[a,l]P) are widespread environmental pollutants, however their toxicological effects within a mixture is not established. We investigated the influence of diesel exhaust (DE) on B[a]P and DB[a,l]P-induced PAH-DNA adduct formation, metabolic activation, gene expression and 8-oxo-dG adduct levels in human breast epithelial cells (MCF-10A) in culture. Following 24 and 48 h, cells co-exposed to DE plus B[a]P exhibited a significant decrease in PAH-DNA adduct levels, compared with B[a]P alone, as determined by 33P-postlabeling combined with reversed-phase high performance liquid chromatography (HPLC). Cytochrome P450 (CYP) enzyme activity, as measured by the ethoxyresorufin O-deethylase (EROD) assay and CYP1B1 expression, significantly increased with co-exposure of DE plus DB[a,l]P, compared with DB[a,l]P alone. Aldo keto-reductase (AKR)1C1, AKR1C2,and AKR1C3 expression also significantly increased in cells exposed to DE plus PAH, compared with PAH exposure alone. Cell populations exhibiting 8-oxo-dG adducts significantly increased in response to exposure to B[a]P or DE plus B[a]P for 24 h, compared with vehicle control, as quantified by flow cytometry. These results suggest that complex mixtures may modify the carcinogenic potency of PAH by shifting the metabolic activation pathway from the production of PAH diol-epoxides to AKR pathway-derived metabolites.

Recent studies have shown urban dust particulate matter (UDPM) to decrease PAH-DNA adduct levels and cytochrome P450 (CYP) metabolic capacity in human mammary epithelial cell lines. The present study was designed to further elucidate the biochemical mechanisms involved in this inhibition process. We examined the effects of UDPM and its aromatic components on the metabolizing activities of human CYP1A1 and CYP1B1 enzymes as analyzed by the 7-ethoxyresorufin O-deethylation (EROD) assay. The data obtained indicate that the aromatic component of UDPM, derived from a neutral aromatic fraction of dichloromethane extract, inhibited the activity of CYP (no change in K M, decrease in k cat /K M ). Taken together, the decreases in the tumor initiating activity, PAH-DNA adduct formation and CYP1A1 and CYP1B1 activity by UDPM observed in vivo can be attributed to a noncompetitive inhibitory mechanism by the PAH component. Therefore, PAHs within a complex mixture could inhibit CYP metabolic capacity, thereby decreasin...

Persistent carbocations were generated from five A-ring mono- and di-substituted phenanthrenes [3-OMe; 4-OMe, 1,3-bis(OMe), 2,4-bis(OMe), and 1,3-bis(Me)]. In all cases protonation occurs in the A-ring, ortho/para relative to methoxy or methyl substituent(s). Complete NMR assignments of the resulting carbocations are reported and their charge delocalization modes are discussed. Mild nitration (with 20–50 % aqueous HNO3 at –10 °C or at room temp.) and bromination (NBS/MeCN/room temp.) of these substrates resulted in the synthesis of several novel mononitro-/dinitro- as well as monobromo/dibromo derivatives, including those with nitro or bromo substituent in the bay-region. Correspondence between the site of attack in low-temperature protonation study and nitro substitution in ambient mild nitrations are examined. Complete NMR assignments for the new derivatives are reported as well as X-ray structures for 2,4-dimethoxy-1-nitro- and 1,3-dimethyl-4-nitrophenanthrenes. A comparative DNA binding study with MCF cells on three of the synthesized mononitro and one dinitro derivative showed that 1,3-dimethyl-9-nitro- (nitro at the meso position), 3-methoxy-4-nitro- (nitro in bay-region), and 1,3-dimethoxy-4,9-dinitrophenanthrenes (nitro in both meso and bay-regions) formed DNA adducts. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Metabolic activation and DNA adduct formation of the carcinogenic aromatic hydrocarbon dibenzo[a,l]pyrene (DBP) was investigated in human mammary carcinoma MCF-7 cells and human cytochrome P450 (CYP) 1B1-expressing Chinese hamster V79 cells in culture. It has been shown that DBP is metabolically activated to DNA-binding diol epoxides both in vitro and in vivo. To further establish the role of human CYP1B1 in the activation of DBP, both cell lines were cotreated with DBP and a selective chemical inhibitor of CYP1B1, 2,4,3' ,5'-tetramethoxy-stilbene (TMS). Results from DBP-DNA adduct analyses revealed the complete inhibition of DNA binding when cells were cotreated with DBP and TMS in comparison to DBP alone. Inactivation of CYP1B1 by TMS was also demonstrated through a decrease in the 7-ethoxyresorufin O-deethylase (EROD) activity in microsomes isolated from these cells. Emodin, 3-methyl-1,6,8-trihydroxyanthraquinone, an active ingredient of an herb, has been recently shown of being able to induce CYP1 gene expression. Examination of human CYP1B1 induction and EROD activity confirmed an increase in protein levels upon cotreatment with emodin and DBP. Despite increases in protein levels and enzyme activity, there was no significant change in DBP-DNA binding levels at very low substrate concentrations (17 nM). The data obtained in this study emphasize the central role of CYP1B1 in the activation of DBP in human cells in culture.

The cytochrome P450 (CYP) 1 family is active toward numerous environmental pollutants, including polycyclic aromatic hydrocarbons (PAHs). Utilizing a mouse model, null for Cyp1b1 and expressing human CYP1B1, we tested the hypothesis that hCYP1B1 is important for dibenzo[def,p]chrysene (DBC) transplacental carcinogenesis. Wild-type mCyp1b1, transgenic hCYP1B1 (mCyp1b1 null background), and mCyp1b1 null mice were assessed. Each litter had an equal number of siblings with Ahrb-1/d and Ahrd/d alleles. Pregnant mice were dosed (gavage) on gestation day 17 with 6.5 or 12 mg/kg of DBC or corn oil. At 10 months of age, mortality, general health, lymphoid disease, and lung tumor incidence and multiplicity were assessed. hCYP1B1 genotype did not impact lung tumor multiplicity, but tended to enhance incidence compared to Cyp1b1 wild-type mice (p = 0.07). As with Cyp1b1 in wild-type mice, constitutive hCYP1B1 protein is non-detectable in liver but was induced with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Wild type mice were 59% more likely to succumb to T-cell Acute Lymphoblastic Leukemia (T-ALL). Unlike an earlier examination of the Ahr genotype in this model (Yu et al., Cancer Res., 2006), but in agreement with a more recent study (Shorey et al., Toxicol. Appl. Pharmacol., 2013), this genotype was not associated with lung tumor incidence, multiplicity, or mortality. Sex was not significant with respect to lung tumor incidence or mortality but males exhibited significantly greater multiplicity. Lung tumor incidence was greater in mCyp1b1 nulls compared to wild-type mice. To our knowledge this is the first application of a humanized mouse model in transplacental carcinogenesis.

FVB/N mice wild-type, heterozygous or null for Cyp 1b1 were used in a two-stage skin tumor study comparing PAH, benzo[a]pyrene (BaP), dibenzo[def,p]chrysene (DBC), and coal tar extract (CTE, SRM 1597a). Following 20 weeks of promotion with TPA the Cyp 1b1 null mice, initiated with DBC, exhibited reductions in incidence, multiplicity, and progression. None of these effects were observed with BaP or CTE. The mechanism of Cyp 1b1-dependent alteration of DBC skin carcinogenesis was further investigated by determining expression of select genes in skin from DBC-treated mice 2, 4 and 8 h post-initiation. A significant reduction in levels of Cyp 1a1, Nqo1 at 8 h and Akr 1c14 mRNA was observed in Cyp 1b1 null (but not wt or het) mice, whereas no impact was observed in Gst a1, Nqo 1 at 2 and 4 h or Akr 1c19 at any time point. Cyp 1b1 mRNA was not elevated by DBC. The major covalent DNA adducts, dibenzo[def,p]chrysene-(±)-11,12-dihydrodiol-cis and trans-13,14-epoxide-deoxyadenosine (DBCDE-dA) were quantified by UHPLC-MS/MS 8 h post-initiation. Loss of Cyp1 b1 expression reduced DBCDE-dA adducts in the skin but not to a statistically significant degree. The ratio of cis- to trans-DBCDE-dA adducts was higher in the skin than other target tissues such as the spleen, lung and liver (oral dosing). These results document that Cyp 1b1 plays a significant role in bioactivation and carcinogenesis of DBC in a two-stage mouse skin tumor model and that loss of Cyp 1b1 has little impact on tumor response with BaP or CTE as initiators.

The zebrafish (Danio rerio) is a sensitive non-mammalian model used for studying polycyclic aromatic hydrocarbon (PAH)-induced chemical carcinogenesis. The susceptibility of zebrafish to PAH-induced carcinogenesis may be related to the ability of the zebrafish P450s to bioactivate these procarcinogens. As a part of our overall effort to identify the various P450 enzymes that are involved in the activation and detoxification of PAHs in zebrafish, therefore, we have examined the ability of recombinant zebrafish CYP1A (zCYP1A) expressed in yeast to metabolize BaP in vitro. Comparison studies also were conducted with liver microsomes from beta-naphthoflavone (BNF)-treated rainbow trout (Oncorhynchus mykiss). Results demonstrated that the trout liver microsomes were almost twice as active as zCYP1A in oxidizing BaP, with Vmax values of 1.7 and 0.94 nmol/min/nmol P450 for trout and zebrafish preparations, respectively. Like trout CYP1A1, cDNA-expressed zCYP1A was found to oxidize BaP to phenols, quinones and diols (BaP-7,8-diol and BaP-9,10-diol) in the presence of exogenous human microsomal epoxide hydrolase (hEH). BaP-7,8-diol is the precursor of the ultimate carcinogen, BaP-7,8-diol-9,10-epoxide (BaPDE). The ability of zCYP1A to bioactivate BaP was confirmed by the formation of DNA adducts when calf thymus DNA was added to the incubation mixture. BaP-DNA binding was enhanced by the addition of hEH to the incubation mixture. HPLC analysis of the [33P]-postlabeled DNA adducts showed the formation of at least four adducts mediated by both zCYP1A and trout liver microsomes, and one of these adducts co-migrated with BaPDE-dG in HPLC analysis. The addition of hEH to the incubation mixture decreased the formation of BaPDE-dG by zCYP1A and by trout liver microsomes while increasing the formation of an unidentified DNA adduct in the case of zCYP1A. zCYP1A also mediated the binding of BaP to protein, providing further evidence that this enzyme is capable of oxidizing BaP to reactive metabolites that bind to macromolecules. It thus appears that zCYP1A may play an important role in BaP-induced carcinogenesis in the zebrafish model by catalyzing the sequential formation of the ultimate diol epoxide carcinogenic metabolite of BaP.

In this study, we investigated the effects of histone deacetylase (HDAC) inhibitors suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) on the metabolism of polycyclic aromatic hydrocarbons (PAH) in human mammary carcinoma derived MCF-7 cells in culture. Benzo[a]pyrene (B[a]P) induces cytochrome P450 (CYP) 1A1, CYP1B1 and other xenobiotic metabolizing enzymes. Results from our study indicated a significant increase in CYP activity in comparison to vehicle control in cells treated with SAHA or TSA as measured by ethoxyresorufin-O-deethylase assay. However, co-treatment with 1.0 microM SAHA and BP, reduced the mRNA levels of CYP1B1 relative to B[a]P alone. When co-treated with 1.0 microM TSA and BP, a reduction in the mRNA levels of both CYP1A1 and CYP1B1 was observed relative to BP alone. We further investigated to ascertain if the differential expression and activity of CYP1A1 and CYP1B1 influenced levels of B[a]P DNA adduct formation. MCF-7 cells co-treated with B[a]P and SAHA or TSA formed DNA adducts, although no significant differences in levels of DNA binding were revealed. These results suggest that while CYP enzyme activity and gene expression were affected by the HDAC inhibitors SAHA and TSA, they had no apparent influence on B[a]P DNA binding.

Seven selfcomplementary decamer oligodeoxyribonucleotides were reacted with (–)–anti–(11R,12S)–dihydrodiol (13S,14R)–epoxide of dibenzo[a,l]pyrene (DB[a,l]PDE). The oligodeoxyribonucleotides were postlabeled with 33P–ATP, digested to mononucleotides and the amount of adducts formed was determined by HPLC. We found that the nucleotide sequence affects the binding of DB[a,l]PDE to DNA. DB[a,l]PDE binds better to GC rich oligodeoxyribonucleotides than to AT rich oligodeoxyribonucleotides. The CGC sequence binds more DB[a,l]PDE than GGC, TGC, GAT or AGC sequences. The reactions were carried out on ice or at 37°C. There was no evidence of temperature effects on the amount of DB[a,l]PDE binding.

This article addresses the evidence for the mechanism of activation of polycyclic aromatic hydrocarbons (PAH) to “ultimate carcinogenic” DNA-binding metabolites in cells and describes how analysis of DNA adducts allowed the determination that the metabolites are dihydrodiol epoxides of PAH. Initially, the PAH-DNA adduct analysis techniques we developed allowed us to establish that the reactive form of PAH that bind to DNA in cells was not the K-region epoxide. Further development of PAH-DNA adduct analysis techniques allowed us to determine that in the case of the very potent carcinogen dibenzo[a,l]pyrene, the reactive metabolite was a diol epoxide with “fjord region” of the molecule. Collaborative studies of DNA adducts in cells from a mouse in which cytochrome P450 1B1 levels were knocked out demonstrated that DB[a,l]P activation to DNA binding intermediates was reduced to undetectable levels demonstrating the great importance of this enzyme in activating fjord region containing PAH.

Human exposure to polycyclic aromatic hydrocarbons (PAHs) occurs through complex mixtures. The National Institute of Standards and Technology has established standard reference materials (SRMs) for selected PAH mixtures that are composed of carcinogenic, noncarcinogenic, and weakly carcinogenic compounds, such as those derived from coal tar (SRM 1597), atmospheric particulate matter (SRM 1649), and diesel particulate matter (SRM 1650). To study the effects of PAHs with different carcinogenic potential in complex mixtures, and to investigate the metabolic activation of noncarcinogenic and weakly carcinogenic PAHs to DNA-binding derivatives, artificial mixtures (1597H, 1649H, and 1650H) were prepared in the laboratory. These artificial mixtures contained the same relative ratios of noncarcinogenic and weakly carcinogenic PAHs present in SRM 1597, SRM 1649, and SRM 1650. The human mammary carcinoma-derived cell line MCF-7 was treated with these artificial mixtures and analyzed for PAH-DNA adduct formation and the induction of cytochrome P450 (CYP) enzymes. We found that the artificial mixtures formed lower but detectable levels of DNA adducts 24 and 48 hr after treatment than benzo[a]pyrene. Induction of CYP enzyme activity was measured by the ethoxyresorufin-O-deethylase assay, and the expression of CYP1A1 and CYP1B1 was confirmed by immunoblots. Both noncarcinogenic and weakly carcinogenic PAHs present in the artificial mixtures have the ability to induce CYP1A1 and CYP1B1 in MCF-7 cells and contribute to DNA binding. Therefore, it is necessary to take into account the noncarcinogenic and weakly carcinogenic PAHs present in environmental mixtures in assessing the potential risk associated with human exposure. Environ. Mol. Mutagen. 44:99–107, 2004. © 2004 Wiley-Liss, Inc.

A stable ion study of a series of BaP derivatives is reported. 7,8-Dihydro-BaP 1 gives a persistent bay-region benzyliclike carbocation which shows extensive charge delocalization into the pyrene moiety. In contrast, a "benzylic" carbocation can not be generated from 9,10-dihydro-BaP 2. Introduction of bulky substituents at peri C-6 of 9,10-dihydro-BaP (as in 4 and 5) prevents side reactions (dimerization) to the extent that the initially formed carbocation undergoes rearrangement to generate the corresponding bay-region "benzylic" carbocation as a persistent species. Introduction of methoxy substituents into the 1- or 3-positions of 9,10-dihydro-BaP-7(8H)-one (6,7) increases its electrophilic reactivity to the extent that stable carboxonium-arenium dications are produced in FSO3H-SO2ClF. A detailed NMR study (at 500 MHz) of the resulting mono- and dications is reported, and charge delocalization mode (as well as conformational aspects) are addressed. Other oxidized derivatives of BaP such as the 7,8-dihydrodiol 9 and the 7,8-dihydrodibenzoate 8 are not suitable models for stable ion study because of competing O-protonation (and elimination). Energies for various possible arenium ions and regioisomeric "benzylic" cations were computed by the DFT method at the B3LYP/6-31G(d)//B3LYP/6-31G(d) level or by AM1 for comparison with the experimental results. These findings provide further evidence in support of the stability sequence: 1-pyrenyl4-pyrenyl2-pyrenyl in alpha-pyrene-substituted carbocations as models for the intermediates arising from BaP-epoxide ring opening. In an effort to provide a parallel, a series of alpha-pyrenylcarbinols were subjected to a DNA binding study using human MCF-7 cells. The results/trends are discussed and compared with the stable ion data.

Metabolic activation, DNA binding, and tumorigenicity of the carcinogenic polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P) catalyzed by murine cytochrome P450 (P450) enzymes were investigated. DNA binding of DB[a,l]P in human mammary carcinoma MCF-7 and human P450-expressing Chinese hamster V79 cell lines was previously shown to occur preferentially with metabolically generated fjord region DB[a,l]P-11,12-dihydrodiol 13,14-epoxides (DB[a,l]PDE). To elucidate different capabilities of murine P450 1A1 and 1B1 for metabolic activation of DB[a,l]P, V79 cell cultures stably expressing P450s 1A1 or 1B1 from mice were exposed to 10 or 100 nM DB[a,l]P. Both cell lines transformed DB[a,l]P to DNA binding intermediates. As with V79 cells expressing the corresponding human P450 enzyme [Luch et al. (1998) Chem. Res. Toxicol. 11, 686-695], murine P450 1B1-catalyzed metabolism and DNA binding proceeded exclusively through generation of fjord region DB[a,l]PDE. In addition, only DB[a,l]PDE-derived DNA adducts were found in V79 cells expressing P450 1A1 from mice. This is in contrast to our recent findings with V79 cells expressing P450 1A1 from humans or rats which catalyzed the formation of both highly polar DNA adducts as well as nonpolar DB[a,l]PDE-DNA adducts. To establish the role of P450 1B1 in DB[a,l]P-induced tumor formation in vivo, we treated P450 1B1-null and wild-type mice intragastrically and monitored survival rates and appearance of neoplasias in various organs. All wild-type mice (n = 17) used in this study developed at least one tumor at one site (tumor rate of 100%). In contrast, 5 of 13 P450 1B1-null mice were observed to be free from any tumor (tumor rate of 62%). The organ sites of tumor formation and the dignity of tumors were different between wild-type and P450 1B1-null mice. Wild-type mice were diagnosed with both benign and malignant tumors of the ovaries, lymphoid tissues, as well as with skin and endometrial hyperplasias, whereas P450 1B1-null mice developed only lung adenomas and endometrial hyperplasias. DNA binding studies using embryonic fibroblasts isolated from these animals provided further evidence that P450 1B1-catalyzed formation of fjord region DB[a,l]PDE-DNA adducts is the critical step in DB[a,l]P-mediated carcinogenesis in mice, and probably also in man.

Benzo[ a ]pyrene (B[ a ]P) and dibenzo[ a,l ]pyrene (DB[ a,l ]P) induce cytochrome P450 (CYP) CYP1A1 and CYP1B1, which metabolize these polycyclic aromatic hydrocarbons (PAHs) into DNA-binding species. In order to detail roles of CYP1A1 and CYP1B1 in activation of DB[ a,l ]P to the diol epoxide, we here report the inhibition of CYP1A1 in human MCF-7 cells with phosphorodiamidate morpholino antisense oligomers (morpholinos). PAH-DNA adduct formation was also determined after treatment with morpholinos and B[ a ]P or DB[ a,l ]P. p53 is involved in DNA repair, cell cycle arrest, and apoptosis. Cells with normal p53 protein arrest in the G1 phase of the cell cycle on exposure to DNA-damaging agents (presumably allowing the cell sufficient time to repair damaged DNA prior to replication). Previous studies in human MCF-7 cells indicate that cells with PAH-DNA adducts escape cell cycle arrest and accumulate in the S phase. In the present study the effect of PAH-DNA adducts on the cell cycle were observed in huma...

In human cells, the most carcinogenic polycyclic aromatic hydrocarbon dibenzo[ a,l ]pyrene (DB[ a,l ]P) forms high levels of DNA adducts through formation of the ( m )- anti -(11 R ,12 S )-diol (13 S ,14 R )-epoxide (DB[ a,l ]PDE) and its metabolic precursor, the ( m )-(11 R ,12 R )-diol. Generation of these adducts results from the catalytic activity of cytochrome P450 (P450) 1A1 and 1B1. Additional adducts such as (+)- syn -DB[ a,l ]PDE-DNA or more polar DNA adducts were detected only after increasing exposure doses of the parent compound or in cells that express P450 1A1. At low concentrations (·;100 nM) exclusively ( m )- anti -DB[ a,l ]PDE-DNA adducts were formed by P450 1B1, which is constitutively expressed in many mammalian tissues. Measurement of DNA binding and mutagenicity of DB[ a,l ]P in V79 cells expressing human P450 enzymes revealed a higher activity of P450 1B1 compared to 1A1 at low concentrations. Treatment of P450 1B1 knockout mice and DNA binding studies with fibroblasts isolated from...

Air quality has a direct impact on pulmonary and cardiovascular health. Short term exposures to high concentrations of fine particulate matter (PM2.5), coarse particulate matter (PM10) and ozone are associated with increased risk of asthma attacks [1]. Long term exposure to lower levels of these pollutants are associated with increased risk of developing asthma [2] and decreased respiratory function [3]. It is currently estimated that 147 million Americans (41%) are exposed to elevated levels of air pollutants [4]. The United States Environmental Protection Agency (U.S. EPA) has identified ground-level ozone and PM2.5 as the two air pollutants that pose the greatest threat to human health [5], yet the level of concern among the general public regarding these pollutants varies widely and is largely dependent upon a person's perception of risk [6]. Efforts to communicate air quality using terminology such as “PM10” and μg/m3” are perceived as too complex for the general public [7], and air quality information that is reported at national and regional levels is not always practical for an individual [8]. Smartphones have great potential to contribute to communicating personalized information about health risks including air pollution [5, 9]. There are an estimated one billion active smartphone users worldwide [10]. For a smartphone to facilitate communication and understanding of the health risks posed from air pollution the software (app) must 1) provide pollutant information that is representative of actual pollutant conditions at the person's current location and 2) be perceived as useful and easy to use by the general public [11]. The U.S. EPA released an “AirNow” app which identifies the user's current location and lists measured air pollutant concentrations as the air quality index (AQI),at the closest U.S. EPA air monitoring station. The AQI combines the concentration of ozone, particulate matter, carbon monoxide, sulfur dioxide, and nitrogen dioxide into a single index that describes the local air quality using a scale from 1-500 with 6 color coded categories ranging from good (green, index value 1-50) to hazardous (maroon, index value 300-500) [5]. This app provides useful personal air quality information but has limited utility for users who are not located near an air monitoring station or are uncertain of the nearest air monitoring station location. AirNow is also limited to the most recent measurements of pollutant information and requires focused attention from the user, making it difficult to evaluate personal air quality conditions over time and/or during health events of interest such as an asthma attack. To address these limitations, we developed the ParME (particulate matter estimator), app. This is a new software package that utilizes spatial pollutant distribution models to predict air pollutant levels at any smartphone location and send predicted pollutant level information to the smartphone device. The software accesses all the predicted pollutant information associated with the current location of the user's smartphone and lets users set personalized warning levels for predicted atmospheric pollutants. The software is consistent with the color warning categories employed by the the EPA AQI and presents this information in well-known formats such as Google maps and simple graphs. The complex sampling information such as predicted levels and latitude/longitude values is hidden from the user until that information is requested. ParME further minimizes user effort by collecting predicted air quality data and checking for warning levels while running in the background, which further empowers the user to collect personal information about air quality at their location.

The tumor suppressor protein p53 plays an important role in recognition of DNA damage and induction of subsequent cell cycle arrest. One of its target genes encodes the protein p21(WAF1), which is involved in mediation of growth arrest after DNA damage has occurred. Dibenzo[a,l]pyrene (DB[a,l]P) is a polycyclic aromatic hydrocarbon which is an exceptionally potent carcinogen. A reactive secondary metabolite of DB[a,l]P, the fjord region (-)-anti-11R,12S-dihydrodiol 13R,14S-epoxide [(-)-anti-DB[a,l]PDE] was used to investigate DNA damage via adduct formation and cell cycle arrest in human diploid fibroblast cell cultures (HDF). Synchronous HDF were exposed to increasing concentrations (0.014, 0.028 and 0.07 microM) of (-)-anti-DB[a,l]PDE and at 1, 12, 24 and 42 h after treatment cell pellets were analyzed for DNA adduct formation and cell cycle arrest. Exposure of HDF to 0.07 microM (-)-anti-DB[a,l]PDE caused a total DNA binding level of 113 pmol adducts/mg DNA (42 h after treatment). G(1) arrest was induced by this treatment, with 91% of the cells remaining in G(1) phase compared with the solvent-treated control cultures (50%) as analyzed by propidium iodide staining and flow cytometry. Further investigation of the percentage of cells in S phase by 5-bromo-2'-deoxyuridine incorporation confirmed the G(1) arrest in HDF treated with 0.07 microM (-)-anti-DB[a,l]PDE, with only 1.5% of the cells moving into S phase compared with 39% in the control 42 h after treatment. Induction of p53 and p21(WAF1) was demonstrated by western blot analysis.

1,4-Dimethylbenzo[c]phenanthrene (1,4-DMBcPh) is the dimethylated analogue of the benzo[c]phenanthrene (BcPh), where one of its two methyl groups resides in the highly congested fjord-region. A comparative X-ray crystallographic analysis, described herein, shows that BcPh is distorted out-of-plane so that there is an angle of 27° between the outermost rings. The additional methyl groups in 1,4-DMBcPh increase this nonplanarity to an angle of 37°. This methyl group-induced disruption of planarity results in P and M enantiomers of 1,4-DMBcPh, and this helicity is observed in a pronounced manner in its putative metabolites, the dihydrodiol and diol epoxide. Synthetically, photochemical cyclization offers convenient access to 1,4-DMBcPh as well as its metabolites. For this, Wittig reaction of 2,5-dimethylbenzyltriphenylphosphonium chloride and 2-naphthaldehyde provided a cis/trans mixture of alkenes which, when subjected to photolysis, provided 1,4-DMBcPh. Despite the high steric congestion in the fjord-regio...

The polycyclic aromatic hydrocarbon (PAH) dibenzo[a, l]pyrene (DB[a, l]P) has been found to be an environmental pollutant and, considering the available data from rodent bioassays, it represents the most carcinogenic member compound of the class of PAH yet discovered. To sort out the contribution of individual cytochromes P450 (P450) in the metabolic activation of this PAH, V79 cells stably expressing a single P450 isoform were treated with DB[a, l]P or enantiomeric DB[a, l]P-11,12-dihydrodiols (diols). Subsequent analysis of the DNA adducts formed revealed substantial differences in the adduct pattern and the total DNA binding depending on the cell line used. Human P450 1B1 effectively activated 0.1 μM DB[a, l]P (350 pmol adducts/mg DNA) and 0.05 μM (-)-(R,R)-diol (1010 pmol/mg DNA). Human P450 1A1 and rat P450 1A1 were less effective than human 1B1 in activating 0.1 μM DB[a, l]P (27 and 28 pmol/mg DNA, respectively), but significantly more active than human 3A4, human 2A6, and rat 1A2 (0.1, 0.2...

Incubation of DB[a, l]P with microsomes from TCDD-treated MCF-7 cells produced mainly (-) anti DB[a, l]P-11,12-diol 13,14-epoxide-dAdo adducts. Addition of antibodies against CYP1A1 and CYP1B1 inhibited the formation of DNA adducts up to 88% and 51%, respectively. The level of P450 1B1 protein was dramatically elevated, but P450 1A1 protein is not detectable by blottings in MCF-7 cells treated with 5 μM or 8 μM DB[a, l]P. MCF-7 cells treated with TCDD or B[a]P contained elevated P450 1A1 and P450 1B1. The current results demonstrate that both P450 1A1 and P450 1B1 are involved in metabolic activation of DB[a, l]P in MCF-7 cells treated with TCDD and suggest that P450 1B1 may be the major DB[a, l]P activating enzyme in MCF-7 cells treated with DB[a, l]P.

The repair of polycyclic aromatic hydrocarbon (PAH)-DNA adducts by nucleotide excision repair is a critical factor in the human response to exposure to these environmental carcinogens. In this study, we utilized a laser cleavage technique and Southern Blotting to measure the repair of benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) adducts in the transcribed and nontranscribed strands of a human cell line containing an amplified DHFR gene. Treatment of the cultures with 1.5 μM BPDE resulted in little adduct removal for 24 hours, whereas 1 μM BPDE-treated cells removed most of the adducts over a 24 hour period. Southern analyses of repair in individual strands of the human cell line revealed that adducts on the transcribed strand are repaired more rapidly than those on the non-transcribed strand during the first 8 hours after 1 μM BPDE treatment. These results suggest that human cells can repair BPDE-DNA adducts through a transcription-coupled repair process.

The tumor suppressor protein p53 plays an important role in recognition of DNA damage and induction of subsequent cell cycle arrest. One of its target genes encodes the p21 WAFI protein which is involved in the mediation of growth arrest after DNA damage has occured. The exceptionally potent carcino-genic polycyclic aromatic hydrocarbon (PAH) dibenzo[a, l]pyrene (DB[a, l]P) and its ultimate metabolites, the fjord region (+)-syn- and (-)-anti-11,12-diol 13,14-epoxides (DB[a, l]PDE), were used in order to investigate DNA damage via adduct formation, subsequent induction of p53 and p21 WAFI , and cell growth behavior in human mammary carcinoma MCF-7 cells. Exposure of MCF-7 cells to 0.005 μM DB[a, l]P caused a total DNA binding of 25 pmol adducts/mg DNA (48 hrs after treatment) and a significant increase in the level of p53 (12–72 hrs after treatment). 48 hrs after exposure an increased amount of the p21 WAFI protein was detected and its level remained elevated for the time measured (168 hrs). Treat...

Carcinogenic polycyclic aromatic hydrocarbons (PAH), such as benzo[a]pyrene (B[a]P), 7,12-dimethylbenz[a]anthracene (DMBA), and dibenzo[a,l]pyrene (DB[a,l]P), are metabolically activated to electrophilically reactive bay or fjord region diol epoxides that bind to the exocyclic amino groups of purine bases in DNA to form stable adducts. In addition, it has been reported that these PAH can be enzymatically oxidized to yield radical cations that form apurinic (AP) sites in DNA via depurinating adducts. The formation of stable adducts and AP sites in DNA of human cells exposed to PAH was examined in cytochrome P450 (P450)-expressing mammary carcinoma MCF-7 cells and in leukemia HL-60 cells, which display a high peroxidase but no P450-mediated activity, after exposure to these PAH. Stable DNA adducts were assessed by (33)P-postlabeling/HPLC analysis, and the induction of AP sites in DNA was analyzed by an aldehyde reactive probe (ARP) and a slot blot method. After exposure for 4 h, the levels of stable DNA adducts were comparable in MCF-7 cells treated with B[a]P and DMBA, but significantly lower than those observed in MCF-7 cells treated with the stronger carcinogen DB[a,l]P. While the levels of stable adducts increased more than 10-fold (B[a]P and DMBA) or 100-fold (DB[a,l]P) after exposure for 24 h, the levels of AP sites remained low after both treatment periods. Thus, the levels of stable adducts were approximately 5-fold higher than the levels of AP sites after treatment with B[a]P or DMBA and more than 100-fold higher in cells exposed to DB[a,l]P for 24 h. None of these carcinogenic PAH formed detectable levels of stable DNA adducts or AP sites in HL-60 cells. The results demonstrate that metabolic activation of B[a]P, DMBA, and DB[a,l]P is catalyzed by P450 enzymes leading to diol epoxides that form predominantly stable DNA adducts but only low levels of AP sites.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants with high carcinogenic potencies that have been linked to the etiology of human cancers through their presence in cigarette smoke and environmental mixtures. They are metabolically activated in cells by cytochrome P450 enzymes and/or peroxidases to reactive intermediates that damage DNA. One pathway of activation forms dihydrodiol epoxides that covalently bind to exocyclic amino groups of purines in DNA to form stable adducts. Another pathway involves formation of radical cations that bind to the N7 or C8 of purines to form unstable adducts that depurinate to leave apurinic (AP) sites in DNA. In the present study the proportions of stable DNA adducts and AP sites formed by the carcinogenic PAHs dibenzo[a,l]-pyrene (DB[a,l]P), 7,12-dimethylbenz[a]anthracene (DMBA), and benzo[a]pyrene (B[a]P) have been investigated in a target tissue for carcinogenesis, mouse epidermis. After topical application of the PAHs on the skin of female SENCAR mice epidermal DNA was isolated and the formation of stable DNA adducts was measured by (33)P-postlabeling and HPLC analysis. AP sites in DNA were measured with an aldehyde reactive probe in a slot-blot assay. At both 4 and 24 h after exposure, DB[a,l]P formed significantly higher amounts of stable DNA adducts than DMBA, and B[a]P exhibited the lowest level of binding. In contrast, the number of AP sites present in mice treated with these PAHs was in the order: DMBA > B[a]P >> DB[a,l]P. The level of AP sites was significantly lower than the level of stable adducts for each PAH. The most potent carcinogen, DB[a,l]P, induced the highest level of stable adducts and the lowest level of AP sites in epidermal DNA. These results indicate that stable DNA adducts rather than AP sites are responsible for tumor initiation by carcinogenic PAHs.

Cancer chemoprevention by dietary chlorophyllin (CHL) was investigated in a rainbow trout multi-organ tumor model. In study 1, duplicate groups of 130 juvenile trout were treated for 2 weeks with control diet, 500 p.p.m. dibenzo[a,l]pyrene (DB[a,l]P) or 500 p.p.m. DB[a,l]P + 2052 p.p.m. CHL, then returned to control diet. DB[a,l]P alone proved somewhat toxic but induced high tumor incidences in liver (61%), stomach (91%) and swimbladder (53%) 11 months after initiation. CHL co-feeding abrogated DB[a,l]P acute toxicity and reduced tumor incidences to 18% in liver, 34% in stomach and 3% in swimbladder (P

Polycyclic aromatic hydrocarbons (PAH) are widespread environmental contaminants, and some are potent carcinogens in rodents. Carcinogenic PAH are activated in cells to metabolites that react with DNA to form stable covalent DNA adducts. It has been proposed [Cavalieri, E. L. & Roger, E. G. (1995) Xenobiotica 25, 677–688] that unstable DNA adducts are also formed and that apurinic sites in the DNA resulting from unstable PAH adducts play a key role in the initiation of cancer. The potent carcinogen dibenzo[ a,l ]pyrene (DB[ a,l ]P) is activated in cells to (+)- syn - and (−)- anti -DB[ a,l ]P-11,12-diol-13,14-epoxide (DB[ a,l ]PDE), which have been shown to form stable adducts with DNA. To evaluate the importance of unstable PAH adducts, we compared stable adduct formation to apurinic site formation. Stable DB[ a,l ]PDE adducts were determined by 33 P-postlabeling and HPLC. To measure apurinic sites they were converted to strand breaks, and these were monitored by examining the integrity of a particular restriction fragment of the dihydrofolate reductase gene. The method easily detected apurinic sites resulting from methylation by treatment of cells or DNA with dimethyl sulfate or from reaction of DNA with DB[ a,l ]P in the presence of horseradish peroxidase. We estimate the method could detect 0.1 apurinic site in the 14-kb fragment examined. However, apurinic sites were below our limit of detection in DNA treated directly with (+)- syn - or (−)- anti -DB[ a,l ]PDE or in DNA from Chinese hamster ovary B11 cells so treated, although in these samples the frequency of stable adducts ranged from 3 to 10 per 14 kb. We also treated the human mammary carcinoma cell line MCF-7 with DB[ a,l ]P and again could not detect significant amounts of unstable adducts. These results indicate that the proportion of stable adducts formed by DB[ a,l ]P activated in cells and its diol epoxides is greater than 99% and suggest a predominant role for stable DNA adducts in the carcinogenic activity of DB[ a,l ]P.

Polyclonal antiserum prepared against DNA that was modified with racemic benzo[c]phenanthrene-3,4-diol-1,2-epoxide-2 (B[c]PhDE-2; benzylic hydroxyl and epoxide oxygen trans) was characterized for specificity of antigen recognition. Previous studies have demonstrated that the antisera stereoselectively recognized B[c]PhDE-2-DNA and failed to recognize DNA modified with racemic benzo[c]phenanthrene-3,4-diol-1,2-epoxide-1 (B[c]PhDE-1-DNA, benzylic hydroxyl and epoxide oxygen cis), benzo[a]pyrene-7,8-diol-9, 10-epoxide-2-DNA (B[a]PDE-2-DNA) and 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA (DMBADE-1-DNA). DNA samples modified by diol-epoxide-2 diastereomers of several hydrocarbons were tested in competitive ELISA assays utilizing B[c]PhDE-2-DNA (270 fmol adducts per well). DNA modified with racemic diol-epoxide-2 of various substituted chrysenes (including chrysene, benzo[g]chrysene (B[g]C), 6-methylchrysene (6-MeC), and 5-methychrysene (5-MeC), gave 50% inhibition of antisera binding at ...

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants generated during combustion. Dibenzo[def,p]chrysene (DBC) is a high molecular weight PAH classified as a 2B carcinogen by the International Agency for Research on Cancer. DBC crosses the placenta in exposed mice, causing carcinogenicity in offspring. We present pharmacokinetic data of DBC in pregnant and nonpregnant mice. Pregnant (gestational day 17) and nonpregnant female B6129SF1/J mice were exposed to 15mg/kg DBC by oral gavage. Subgroups of mice were sacrificed up to 48h postdosing, and blood, excreta, and tissues were analyzed for DBC and its major diol and tetrol metabolites. Elevated maximum concentrations and areas under the curve of DBC and its metabolites were observed in blood and tissues of pregnant animals compared with naive mice. Using a physiologically based pharmacokinetic (PBPK) model, we found observed differences in pharmacokinetics could not be attributed solely to changes in tissue volumes and blood flows that occur during pregnancy. Measurement of enzyme activity in naive and pregnant mice by activity-based protein profiling indicated a 2- to 10-fold reduction in activities of many of the enzymes relevant to PAH metabolism. Incorporating this reduction into the PBPK model improved model predictions. Concentrations of DBC in fetuses were one to two orders of magnitude below maternal blood concentrations, whereas metabolite concentrations closely resembled those observed in maternal blood.

Dibenzo[a,l]pyrene (DB[a,l]P) is an important polycyclic aromatic hydrocarbon because of possible human exposure and its exceptionally high carcinogenicity in rodents. We examined the metabolism of DB[a,l]P and the formation of DB[a,l]P-DNA adducts in the human mammary carcinoma cell line (MCF-7). Analysis of the DNA adducts by 33P-postlabeling, immobilized boronate chromatography, HPLC and TLC demonstrated that DB[a,l]P is stereoselectively metabolized to specific optical isomers of DB[a,l]P-11,12-diol-13,14-epoxide (DB[a,l]PDE). The major anti-DB[a,l]PDE adduct formed in DB[a,l]P-treated MCF-7 cells resulted from reaction of (-)-anti-DB[a,l]PDE with DNA whereas the two major syn-DB[a,l]PDE adducts resulted from (+)-syn-DB[a,l]PDE. All three major adducts were with deoxyadenosine, but small amounts of deoxyguanosine adducts were also detected. These results indicate that DB[a,l]P is metabolized to both the (+)- and (-)-11,12-dihydrodiols and that the (-)-dihydrodiol is stereoselectively activate...

The tumor suppressor p53 is involved in the recognition of DNA damage induced by radiation and chemicals. The effect of polycyclic hydrocarbons on p53 was investigated by treating MCF-7 cells with anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), an ultimate carcinogenic metabolite of benzo[a]pyrene. Western blotting of lysates with antibody mAb1801 showed that B[a]PDE doses of 0.1 to 0.5 μM caused detectable increases in p53 protein. In cells treated with 0.3 μM B[a]PDE, p53 protein levels increased by 2 hours after treatment, reached a maximum between 8 and 24 hours and returned to control value by 120 hours after treatment. Levels of p21WAF1 protein increased by 8 hours after treatment, reached a maximum by 48 hours and returned to control value by 168 hours after treatment. B[a]PDE-DNA adducts were quantitated by [γ-33P]ATP postlabeling and separation on reverse-phase HPLC. Adduct levels dropped rapidly between 2 and 24 hrs after treatment (48 to 15 pmol/mg DNA), and subsequently decreased...

7,12-Dimethylbenz[a]anthracene (DMBA), one of the most carcinogenic polycyclic aromatic hydrocarbons in rodent bioassays, is metabolically activated in many tissues to "bay-region" DMBA-3,4-diol-1,2-epoxides (DMBADE). Unlike benzo[a]pyrene, for which the high biological activity of the (7R,8S)-diol-(9S,10R)-epoxide has been established, the low chemical stability of anti-DMBADE has made it impossible to evaluate the role of specific stereoisomers in the biological activity of DMBA. In order to characterize the role of formation of DMBADE diastereomers in the induction of mutations, postlabeling assays using [35S]phosphorothioate with adduct separation by HPLC and immobilized boronate chromatography analyses were developed to allow separation and quantitation of DNA adducts formed from each stereoisomer of DMBADE. In DMBA-treated hamster embryo cell cultures, large quantities of three major adducts (anti-DMBADE-deoxyguanosine, anti-DMBADE-deoxyadenosine, and syn-DMBADE-deoxyadenosine) along with five minor adducts were completely resolved and quantitated. The DNA isolated from a human mammary carcinoma MCF-7 cell-mediated V79 cell mutation assay treated with increasing doses of racemic DMBA-3,4-dihydrodiol contained large amounts of two anti-DMBADE-DNA adducts. The anti-DMBADE adducts accounted for more than 90% of the total adducts at all doses. The number of 6-thioguanine-resistant mutants was proportional to the amount of anti-DMBADE-DNA adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Dibenzo[a,l]pyrene (DB[a,l]P) is an environmental contaminant and a very potent carcinogen. DB[a,l]P exceeds the carcinogenic potency of both benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in rodent bioassays. Previous studies demonstrated that DB[a,l]P is metabolized to DB[a,l]P-11,12-diol-13,14-epoxide (DB[a,l]PDE) in the human mammary carcinoma cell line MCF-7. In the present study the major DNA adducts formed in DB[a,l]P-treated MCF-7 cells have been identified through the use of 33P-postlabeling. TLC and HPLC. DB[a,l]P is metabolically activated in MCF-7 cells to form large amounts of three major DNA adducts and smaller amounts of three other adducts. The three major DNA adducts are with deoxyadenosine: two are formed by reaction of (+)-syn-DB[a,l]PDE (11S,12R,13S,14R), the third by reaction of (-)-anti-DB[a,l]PDE (11R,12S,13S,14R). The results demonstrate that DB[a,l] is stereoselectively metabolized in MCF-7 cells to form one enantiomer of each diol epoxide diastereomer; (+)-syn-DB[a,l]PDE and (-)-anti-DB[a,l]PDE. The high extent of binding of these diol epoxides to deoxyadenosine in DNA of MCF-7 cells may help to explain the very high carcinogenic potency of DB[a,l]P and suggests that DB[a,l]P could also pose a carcinogenic threat to humans.

Carcinogenic polycyclic aromatic hydrocarbons induce DNA damage through direct covalent interactions with nucleotides of the DNA in cells in which they are activated to 'ultimate carcinogenic metabolites'. To determine whether they also induce oxidative damage to DNA under the same circumstances, early passage Syrian hamster embryo and human mammary carcinoma cell line MCF-7 cultures were treated for 24 h with 0-5 micrograms/ml benzo[a]pyrene (BaP) or for 1 h with 0-100 microM methylene blue (as a positive control for oxidative damage). The cells were then exposed to fluorescent light for 1 or 4 h or retained in darkness. After cell harvest, DNA isolation and enzymatic digestion of the DNA to deoxyribonucleosides, the amounts of 8-hydroxy-2'deoxyguanosine (8-OH-dGuo) and unmodified deoxyguanosine present were determined by reverse-phase HPLC with electrochemical and UV detection respectively. Cultures treated with methylene blue for 1 h followed by light exposure for 1 h contained 5-fold (10 microM) and 8- to 28-fold (100 microM) higher 8-OH-dGuo levels than cells treated with methylene blue not exposed to light or untreated cells with methylene blue not exposed to light or untreated cells exposed to light. There was no significant change in 8-OH-dGuo levels in cultures treated with 1-5 micrograms/ml BaP for 24 h in the absence of light. However, both the human and hamster cell cultures treated with BaP and then exposed to fluorescent light for 4 h contained 3-fold (1 micrograms/ml) and 8- to 10-fold (5 micrograms/ml) higher 8-OH-dGuo levels than those not exposed to light or not treated with BaP. These results indicate that BaP treatment does not cause 8-OH-dGuo formation in DNA of cells maintained in darkness. Exposure of BaP-treated cells to fluorescent light causes formation of significant amounts of oxidative DNA damage as measured by 8-OH-dGuo formation. These findings suggest that oxidative damage of DNA could be involved in tumor induction by BaP in tissues, such as skin, in which exposure to BaP can occur in the presence of light.

The DNA adducts formed from the potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) in cultures of Sencar mouse embryo cells and a mouse keratinocyte cell line and in Sencar mouse epidermis in vivo were analyzed by 35S−phosphorothioate postlabeling, immobilized boronate chromatography and reverse-phase HPLC. The results demonstrate that the major DNA adducts result from the activation of DB[a,l]P to DB[a,l]P-11,12-diol-13,14-epoxide. Adducts from both the syn− and anti−isomers of this diol epoxide are formed in cells in culture, but in mouse epidermis only anti−isomer adducts are detected.

Diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxides (B[c]PhDE)-1 and -2 (with the benzylic hydroxyl and epoxide oxygen cis and trans, respectively) are nonplanar polycyclic aromatic hydrocarbons (PAH) with sterically congested fjord regions. Polyclonal antibodies developed against DNA modified with B[c]PhDE-1 or B[c]PhDE-2 exhibited selective recognition of B[c]PhDE-DNA adducts and no cross-reactivity with DNA modified with benzo[a]pyrene-7,8-diol-9,10-epoxide. To determine if these antisera recognize DNA adducts formed by other PAH diol epoxides with sterically congested bay regions, 5-6-dimethylchrysene-1,2-diol-3,4-epoxide (5,6-diMeCDE)-1 or -2 modified DNA were tested in competitive ELISA assays. 5,6-diMeCDE-2-DNA gave 50% inhibition at 270 fmol of adducts/well with B[c]PhDE-2-DNA (160 fmol adducts/well) and its antiserum, but 5,6-diMeCDE-1-DNA was not an effective competitor. Neither 5,6-diMeCDE-1-DNA nor 5,6-diMeCDE-2-DNA was an effective competitor for B[c]PhDE-1-DNA and its antiseru...

Polycyclic aromatic hydrocarbons (PAHs) are formed upon incomplete combustion of organic matter. Due to the abundant use of fossil energy sources, PAHs are readily detectable as ubiquitous contaminants in the environment. The great interest in this group of chemicals originated on the observation in animal tumor models that some member compounds possessed strong carcinogenic activity in skin, lung, breast, and other organs. Epidemiological meta-analyses confirmed that heavy exposures to mixtures of PAHs entail a substantial risk to develop cancer in certain organs. No matter what kind of source, humans are always exposed to mixtures of PAH with different degrees of biological activity. From all hydrocarbons detectable in the human environment the most intensively studied example benzo[a]pyrene (B[a]P) has been traditionally used as an indicator for carcinogenic PAHs. About four decades ago it was proposed that there is a significant positive correlation between the binding to DNA and the biological potency of carcinogenic PAHs. In more recent years it became clear that vicinal diol-epoxides of PAHs that contain the epoxy moiety in a sterically crowded bay or fjord region are the actual DNA-binding metabolites that mediate the biological effects associated with their parent structures. Hence, PAHs would not be carcinogenic if they were not stereoselectively metabolized by cytochrome P450-dependent monooxygenases (CYPs). The DNA adduct level at a given time point is an integrated product of PAH’s toxicokinetic and toxicodynamic behavior, including metabolic activation and detoxification prior to covalent binding, as well as the effectiveness of the repair of those DNA lesions that have been formed. Covalent PAH-DNA adducts are fixed as mutations if left to error-prone excision repair, misrepair, or replication errors during early S phase. If such somatic mutations occur in proto-oncogenes (e.g., K-Ras) and/or tumor suppressor genes (e.g., TP53), they can contribute to the aggravation of neoplastic growth through the processes of tumor promotion and progression. Given the chemical complexity of most environmental matrices, it seems difficult, if not impossible, to uncover causative relationships between certain forms of human cancer and the exposure to particular carcinogenic PAHs though. Nevertheless, molecular epidemiology has been able to point to the role of individual compounds and to extract their contribution from the overall biological response on environmental mixtures. One of the most well worked-out examples is the crucial role of B[a]P in the etiology of human lung cancer based on its presence in cigarette smoke. Its important role in tumor initiation is supported by several lines of evidence such as (1) increased levels of PAH activating CYP enzymes in lung cancer patients compared to controls, (2) a correlation between pulmonary levels of activating CYP enzymes and bulky B[a]P–DNA adduct levels in human lung tissue from cancer patients, (3) increased levels of B[a]P–DNA adducts in lung tissue of smokers compared to nonsmokers, and (4) the coincidence of mutational hotspots at certain codons of K-Ras or TP53 and B[a]P–DNA adduct hotspots as the preceding lesions found at the same sites.

Dasytrichone, A Novel Flavone With An Unusual A-Ring Substitution Pattern Was Isolated From The Stems And Leaves Of Dasymaschalon Trichophorum Merr. (Family Annonaceae). Its Structure Was Established By Spectroscopic Methods And Single Crystal X-Ray Crystallography. Dasytrichone Inhibits The Metabolism Of The Carcinogen Benzo[A]Pyrene By Hamster Embryo Cell Cultures, Suggesting That It Warrants Further Evaluation As A Potential Chemopreventive Agent.