ToxSci Advance Access originally published online on October 23, 2006
Toxicological Sciences 2007 95(1):63-73; doi:10.1093/toxsci/kfl137
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Urban Dust Particulate Matter Alters PAH-Induced Carcinogenesis by Inhibition of CYP1A1 and CYP1B1
* Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon 97331-7302
College of Veterinary Medicine, Oregon State University, Corvallis, Oregon 97331
Department of Statistics, Oregon State University, Corvallis, Oregon 97331
1 To whom correspondence should be addressed at Department of Environmental and Molecular Toxicology, 1007 Agriculture and Life Sciences Bldg., Corvallis, OR 97331-7302. Fax: (541) 737-0497. E-mail: william.baird{at}oregonstate.edu.
Received August 4, 2006; accepted October 16, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The polycyclic aromatic hydrocarbons (PAHs) benzo[a]pyrene (B[a]P) and dibenzo[a,l]pyrene (DB[a,l]P) are well-studied environmental carcinogens, however, their potency within a complex mixture is uncertain. We investigated the influence of urban dust particulate matter (UDPM) on the bioactivation and tumor initiation of B[a]P and DB[a,l]P in an initiation-promotion tumorigenesis model. SENCAR mice were treated topically with UDPM or in combination with B[a]P or DB[a,l]P, followed by weekly application of the promoter 12-O-tetradecanoylphorbol-13 acetate. UDPM exhibited weak tumor-initiating activity but significantly delayed the onset of B[a]P-induced tumor initiation by two-fold. When cotreated with UDPM, DB[a,l]P-treated animals displayed no significant difference in tumor-initiating activity, compared with DB[a,l]P alone. Tumor initiation correlated with PAH-DNA adducts, as detected by 33P-postlabeling and reversed-phase high-performance liquid chromatography. Induction of cytochrome P450 (CYP)1A1 and 1B1 proteins was also detected following UDPM treatment or cotreatment with B[a]P or DB[a,l]P, indicating PAH bioactivation. Further genotoxicity analyses by the comet assay revealed that cotreatment of UDPM plus B[a]P or DB[a,l]P resulted in increased DNA strand breaks, compared with PAH treatment alone. The metabolizing activities of CYP1A1 and CYP1B1, as measured by the 7-ethoxyresorufin O-deethylation (EROD) assay, revealed that UDPM noncompetitively inhibited CYP1A1 and CYP1B1 EROD activity in a dose-dependent manner. Overall, these data suggest that components within complex mixtures can alter PAH-induced carcinogenesis by inhibiting CYP bioactivation and influence other genotoxic effects, such as oxidative DNA damage. These data further suggest that in addition to the levels of potent PAH, the effects of other mixture components must be considered when predicting human cancer risk.
Key Words: polycyclic aromatic hydrocarbons; benzo[a]pyrene; dibenzo[a,l]pyrene; cytochrome P450; aldo-keto reductase; air pollution.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Air pollution affects millions worldwide and its adverse effects on human health are a serious concern. Previous studies report increased morbidity and mortality due to ambient air pollution (Dockery et al., 1993
; Samet et al., 2000), increased risk of lung cancer (Pope et al., 2002), genotoxicity in various tissues (Burgaz et al., 2002; Palli et al., 2001; Soares et al., 2003), and heritable mutations in mice (Somers et al., 2004). Polycyclic aromatic hydrocarbons (PAHs) are the products of the incomplete combustion of fossil fuels (Pershagen, 1990) and have been detected on suspended particulate matter (PM) in ambient air in urban areas (Menichini, 1992; Pleil et al., 2004).
Benzo[a]pyrene (B[a]P) and dibenzo[a,l]pyrene (DB[a,l]P) are well-studied PAH found in many environmental complex mixtures, including tobacco smoke, automobile exhaust, and air pollution (De Raat et al., 1987). The mutagenic effects of such PAH can be attributed to three distinct mechanistic pathways in the metabolic activation of PAHs, involving the (1) diol epoxide path (Conney, 1982; Jeffrey et al., 1977; Jennette et al., 1977; Sims et al., 1974; Wood et al., 1976), (2) the quinone path (Smithgall et al., 1986, 1988a,b), and (3) the radical cation path (Cavalieri and Rogan, 1995; Melendez-Colon et al., 1999). In the diol epoxide path, PAHs are oxidized by cytochrome P450 (CYP) enzymes to a trans-dihydrodiol which is then further oxidized to highly mutagenic, electrophilic diol epoxides. The bay region–containing PAH, B[a]P ((+)-anti-B[a]P-7,8-diol 9,10-epoxide) (B[a]PDE), preferentially binds to deoxyguanosine residues (Cheng et al., 1989), whereas the fjord region–containing PAH, DB[a,l]P ((+)-syn and (–)-anti-DB[a,l]P-11,12-dihydrodiol 13,14-epoxide) (DB[a,l]PDE) primarily binds to deoxyadenosine residues in DNA (Ralston et al., 1995). Covalent DNA adduct formation by these PAH-diol epoxides is correlated with tumor formation and linked to carcinogenic risk (Brookes and Lawley, 1964; Nesnow, 1990). The quinone path involves dehydrogenation of the dihydrodiol by aldo-keto reductase (AKR) enzymes to form a catechol that enters into a redox cycle with O2 to form a PAH o-quinone and reactive oxygen species (ROS). The one-electron oxidation pathway entails oxidation of PAH by peroxidase enzymes to form radical cations which react with DNA. It is suggested that all three pathways are involved in the initiation or promotion stages of carcinogenesis (Harvey et al., 2005; Jiang et al., 2006).
The carcinogenic risk associated with individual PAH exposure is well characterized (IARC, 1983), therefore concern exists about the regulation of PAH emissions and human risk assessment. Although PAHs are typically found in complex mixtures, much of the PAH research has focused on the effects of individual compounds. Because scientific evaluation is difficult, limited research has been done on binary or artificial mixtures (DiGiovanni et al., 1982; Hughes and Phillips, 1990; Mahadevan et al., 2004; Rice et al., 1984, 1988; Smolarek et al., 1987), and to a lesser extent complex mixtures (Mahadevan et al., 2005a,b; Marston et al., 2001). Results from these studies vary; in some in vitro and in vivo studies, PAH-DNA binding and/or tumor formation increased, while other studies have reported decreased activities. Many of these reports primarily focus on induction of CYPs (Mahadevan et al., 2004, 2005b; Marston et al., 2001); however, the mechanism by which the mixtures act on CYP metabolic activity has not been investigated. Because PAHs require CYP-mediated bioactivation, it is useful to investigate complex mixtures as a chemical inducer or inhibitor (Shimada and Guengerich, 2006). We employed the well-characterized two-stage SENCAR mouse model to determine the effect of urban dust particulate matter (UDPM) on PAH-DNA adduct formation and tumor initiation. Further, we investigated the effect of UDPM on the metabolic capacity of CYP1A1 and CYP1B1 through biochemical kinetic assays.
We tested the hypothesis that UDPM alters the carcinogenic potential of two potent PAHs, B[a]P and DB[a,l]P in SENCAR mice. Three specific aims were addressed: (1) to assess whether UDPM modifies B[a]P or DB[a,l]P tumor initiation, (2) to determine if UDPM alters PAH-DNA adduct formation and DNA strand breaks by B[a]P or DB[a,l]P, and (3) to determine whether UDPM acts as an inhibitor on CYP-mediated bioactivation to contribute to altered PAH-induced carcinogenesis. We report that the potency of carcinogenic PAH is influenced by other chemical components present in UDPM, in part, through inhibition of CYP1A1 and CYP1B1.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Caution: B[a]P and DB[a,l]P are potent carcinogens and should be handled according to National Cancer Institute (NCI) guidelines. Standard Reference Material (SRM) 1649a (UDPM) is a known mutagen and should be handled with the same precautions. Due to the instability of these compounds, PAHs and SRM 1649a were not prepared and used under direct light.
Chemicals and reagents.
B[a]P and DB[a,l]P were purchased from Chemsyn Science Laboratories (Lenexa, KS). Nuclease P1 (EC 3.1.30.1
[EC]
; from Penicillium citrinum), human prostatic acid phosphatase (EC 3.1.3.2
[EC]
; from human semen), apyrase (EC 3.6.1.5
[EC]
; from Solanum tuberosum), phosphodiesterase I (EC 3.1.4.1
[EC]
; from Crotalus atrox), and proteinase K (EC 3.4.21.64
[EC]
; from Tritirachium album) were purchased from Sigma (St Louis, MO). RNase T1 (EC 3.1.21.3
[EC]
; from Asperigillus oryzae) and RNase (DNase free, a heterogenous mixture of ribonucleases from bovine pancreas) were obtained from Boehringer Mannheim (Indianapolis, IN). Tris-equilibrated phenol and cloned T4 polynucleotide kinase were purchased from United States Biochemical (Cleveland, OH). [
-33P] Adenosine 5'-triphosphate (ATP) (1 mCi) was purchased from Perkin Elmer (Boston, MA). SRM 1649a was utilized in this study as UDPM. SRM 1649a was obtained from the National Institute of Standards and Technology (Gaithersberg, MD) and one bottle unit of SRM 1649a contained 2.5 g of atmospheric particulate material. The amount of B[a]P present in SRM 1649a was determined as 2.509 mg/kg and a detailed description of the composition of SRM 1649a is available at http://patapsco.nist.gov/srmcatalog/certificates/1649a.pdf. DB[a,l]P (47.2 µg/kg) has been recently detected in SRM 1649a (Schubert et al., 2003).
Tumor initiation in mouse epidermis.
This study used the SENCAR mouse two-stage skin tumorigenesis model. These mice are genetically susceptible to chemical carcinogens and thus sensitive to tumor initiation, and they generally respond more rapidly and uniformly to the induction of skin tumors compared with other available rodent strains or stocks. The initiation and promotion stages are clearly distinguished, thus facilitating the study of biochemical and molecular mechanisms involved in a particular stage of carcinogenesis (Slaga et al., 1996). During the resting phase of their hair-growth cycle (at 6–7 weeks of age), the dorsal area of the female SENCAR mice (NCI-Frederick Cancer Research and Development Center, Frederick, MD) was shaved 2 days prior to treatment with toluene (vehicle control) B[a]P or DB[a,l]P, UDPM, and UDPM plus B[a]P or DB[a,l]P. There were six treatment groups with six mice per group. Mice were housed in polycarbonate cages (three mice per cage) and fed Teklad rodent diet (no. 8604, Harlan) and water ad libitum. They were maintained at 72°F on a standard 12-h light/dark cycle with 40–60% relative humidity. Animals were housed and cared for in accordance with the Institute of Laboratory Animal Resources (ILAR, 1996), Commission of Life Sciences, National Research Council document entitled, Guide for the Care and Use of Laboratory Animals.
The concentrations of B[a]P and DB[a,l]P were chosen based on previous studies (Cavalieri et al., 1991; Gill et al., 1994; Higginbotham et al., 1993; Marston et al., 2001). Topical treatments with the PAH initiator were carried out 5 min after the UDPM treatment and dosed as follows: 10 mice with 200 µl of toluene, 35 mice with 50.4 µg B[a]P, 35 mice with 0.6 µg DB[a,l]P, 35 mice with 1 mg UDPM, 35 mice with 1 mg UDPM plus 50.4 µg B[a]P, and 35 mice with 1 mg UDPM plus 0.6 µg DB[a,l]P. Two weeks after initiation, 12-O-tetradecanoylphorbol-13 acetate (TPA) at 1 µg/200 µl acetone per mouse was administered for 25 weeks. The mice were examined weekly for skin papillomas. Following necropsy, tumors were confirmed by routine histopathology techniques by the Tissue Analysis Core and Pathology Facilities and Services Core of the Environmental Health Sciences Center (EHSC) at Oregon State University.
DNA isolation from PAH-treated mouse epidermis.
Following acclimatization, mice were shaved and treated with PAH alone or UDPM plus PAH. Mice were killed by cervical dislocation 24 and 48 h after exposure, and the epidermal cells were harvested by the method described by Slaga et al. (1974). Briefly, Nair depilatory cream was applied to the shaven dorsal area to remove residual fur, skin was rinsed off with cold water, harvested, and placed in water on ice. The skins were then submerged in a 58°C water bath for 30 s and placed in water on ice, followed by removal of the epidermal cells by scraping with a razor blade. The epidermal cells from six mice per treatment group were pooled, placed in 500 µl of 0.025M ethylenediaminetetraacetic acid (EDTA)/0.075M NaCl buffer, and stored in – 80°C for DNA isolation or used immediately for microsome preparation.
The DNA was isolated as described by Beach and Gupta (1992). Briefly, the pooled mouse epidermal samples from each treatment group were homogenized in a glass homogenizer containing EDTA, sodium dodecyl sulfate (SDS) buffer (10mM Tris, 1mM Na2EDTA, 1% [w/v] SDS, pH 8). Homogenates were treated with 50 U/ml RNase, DNase-free and 1000 U/ml RNase T1 at 37°C for 1 h, followed by treatment with 500 µg/ml proteinase K at 37°C for 1 h. DNA was extracted with 25:24:1 phenol:chloroform:isoamyl alcohol, precipitated with 100% ethanol, and dissolved in sterile distilled water.
33P-Postlabeling of DNA adducts.
Postlabeling was carried out as described previously (Marston et al., 2001). Briefly, 10 µg DNA was digested with nuclease P1 and prostatic acid phosphatase, postlabeled with [-33P]ATP (3000 Ci/mmol), cleaved to adducted mononucleotides with snake venom phosphodiesterase I, treated with apyrase, and prepurified with a Sep-Pak C18 cartridge (Waters, Milford, MA), as previously described (Lau and Baird, 1994). Subsequent separation by analytical high-performance liquid chromatography (HPLC-Varian system equipped with two pumps and an autosampler Varian Systems, Walnut Creek, CA) was carried out using a 5-µm Symmetry C18 reverse-phase column (4.6 x 250 mm; Waters, Milford, MA). Adducts were resolved by elution at 1 ml/min with 0.1M ammonium phosphate, pH 5.5 (solvent A) and 100% HPLC-grade methanol (solvent B). The elution gradient for B[a]P DNA adducts was 44–55% solvent B over 5 min, 55–60% solvent B over 20 min, and elution at 100% solvent B over 5 min. The DB[a,l]P-DNA adducts were resolved by elution at 1 ml/min with 0.1M ammonium phosphate, pH 5.5 (solvent A), and 90% HPLC-grade methanol/10% acetonitrile (solvent B). The elution gradient was as follows: 20–44% solvent B over 20 min, 44–60% solvent B over 40 min, 60–80% solvent B over 15 min, and 80–20% solvent B over 1 min. The radiolabeled nucleotides were detected by an on-line dry cell radioisotope flow-detector, BETA-RAM Model 3 (INUS, Tampa, FL), and the level of PAH-DNA binding was calculated based on the labeling efficiency of a [3H] B[a]PDE standard (Lau and Baird, 1991). Three independent sets of the postlabeling reaction were carried out for every sample treated.
Single-cell alkaline gel electrophoresis (comet assay).
Plain glass microscope slides were precoated by dipping in a solution of 1% normal melting point agarose (Gibco BRL, Invitrogen, Carlsbad, CA) in distilled water and dried. Following 24 h PAH or UDPM treatment, blood samples were collected from SENCAR mice, and 4 µl of whole blood was diluted in 70 µl of 0.5% low melting point agarose (LMPA) at 37°C. A diluted cell suspension was placed on precoated slides covered by a glass coverslip, and kept on ice for 30 min. The coverslip was then removed and another layer of 70 µl of 0.5% LMPA was added and maintained using a coverslip on ice for 10 min. After removal of the coverslip, the slides were immersed in cold lysing solution (2.5M NaCl, 0.1M Na2EDTA, 10mM Tris) brought to pH 10.0 with NaOH, and kept in 1% Triton X-100 and 10% dimethyl sulfoxide (DMSO) overnight. Slides were then placed in an electrophoresis tank and immersed in 0.3M NaOH, 1mM Na2EDTA for 30 min, before electrophoresis at 25 V (0.8 V/cm) for 30 min at 4°C. Following electrophoresis, slides were neutralized by dropwise addition of 0.4M Tris, pH 7.4, to neutralize the excess alkali. A Nikon E400 fluorescence microscope and the Comet Assay Image Analysis System were utilized at the Cancer and Chemoprotection Core Laboratory (Linus Pauling Institute, Oregon State University) to analyze and score comets after staining each slide with 60 µl of ethidium bromide. Three sets of 50 whole blood cell nuclei were randomly scored for each of the treatment groups.
Microsome preparation.
Microsomes were prepared as previously described (Otto et al., 1991), with minor modifications. The epidermal cells were harvested as similar to the protocol mentioned previously; however, the heat treatment was carried out at 52°C. Mouse epidermal samples were homogenized with a steel homogenizer containing microsomal homogenization buffer (0.25M K2PO4, 0.15M KCl, 10mM EDTA, and 0.25mM phenylmethlysulfonylfluoride [PMSF]) and centrifuged at 15,000 x g for 20 min at 4°C. The supernatant was centrifuged at 100,000 x g for 90 min at 4°C, and the pellet was resuspended in microsome dilution buffer (0.1M KPO4, 20% glycerol, 10mM EDTA, 0.1mM dithiotreitol, and 0.25mM PMSF). Protein concentrations were determined by the BCA protein assay (Pierce, Rockford, IL).
Western blot analysis.
Microsomal proteins were separated on a 7.5% Tris-HCl Ready Gel (BioRad, Hercules, CA) according to Laemmli (42). Briefly, microsomal proteins (50 µg) were diluted with 5x loading buffer (0.625M Tris-base [pH 6.8], 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.001% bromophenol blue). Proteins were denatured by boiling for 3 min followed by rapid cooling on ice. Following gel electrophoresis at 100 V for 1 h, the microsomal proteins were transferred overnight to a polyvinylidene fluoride membrane in a MiniBlotter apparatus (BioRad, Hercules, CA) at 30 V. The membrane was washed three times with phosphate-buffered saline (PBS)-T (PBS with 0.3% [w/v] Tween-20) for 5 min each and blocked with 1:3 Nap-Sure blocker (Geno Technology, St Louis, MO): PBS-T for 1 h on a shaker. After three further washes with Nap-Sure:PBS-T (1:7), the membrane was incubated with the primary antibody for 2 h and was washed again with Nap-Sure blocker: PBS-T (1:7). CYP1A1 was detected by a goat polyclonal CYP1A1/1A2 antibody (1:1000) prepared against purified recombinant rat CYP1A1 protein (BD Gentest, Woburn, MA). Dr C. Marcus (Department of Pharmacology and Toxicology, University of New Mexico, Albuquerque, NM) prepared and provided the human CYP1B1 rabbit polyclonal antibody (1:1000). The immunoreactive proteins were detected by peroxidase-conjugated anti-rabbit IgG (1:20,000) for 30 min. After three washes with Nap-Sure blocker:PBS-T (1:7), the immunoreactive proteins were observed by enhanced chemiluminescence detection method, as described by the manufacturer (Amersham Life Science, Arlington Heights, IL). Microsomal protein (2 µg) from Aroclor-induced rat liver (ICN Pharmaceuticals, Inc., Aurora, OH) was used as a positive control in the immunoblots.
Ethoxyresorufin O-deethylase assay.
Two hundred microliter 7-ethoxyresorufin O-deethylation (EROD) reactions consisted of 0.25pmol human CYP1A1 or 0.65pmol CYP1B1 (Sigma), 60µM nicotinamide adenine dinucleotide phosphate (reduced), 0.05–1.25µM 7-ethoxyresorufin (ERES), and 0–200 µg of UDPM, in 0.1M Tris-HCl, pH 7.8, buffer were aliquoted in a black 96-well plate (E&K Scientific, Campbell, CA), and the reaction was carried out for 10 min. Fluorescence was measured on Spectra MAX Gemini plate reader (Molecular Devices, Sunnyvale, CA) using 535 nm excitation and 585 emission filters. Experiments and reactions were assayed in triplicates and the amount of resorufin (RES) produced was calculated from the fluorescence of a known concentration of RES. The inhibitor (UDPM) was dissolved as a stock solution (1 mg/ml) in DMSO, and further diluted to working solutions.
Statistical analysis.
Mortality and final tumor incidences were compared between treatments with two-sided Fisher's exact tests (FET) utilizing the Exact statement in the SAS FREQ procedure. Tumors/tumor-bearing animal (TBA) were compared between treatments with the Wilcoxon rank test (W) utilizing the Exact statement in the SAS NPAR1WAY procedure. Time until tumor data were compared between treatments with survival analysis methods. The Kaplan-Meier log rank (LR) test was used to compare treatments nonparametrically utilizing the SAS LIFETEST procedure. Treatments were also compared semiparametrically with Cox proportional hazard regression utilizing the SAS PHREG procedure. For all of the analyses above, prior to treating the mouse as the individual independent unit, it was first confirmed that there was little evidence of any cage effects. In each case, this was done by comparing cages within each treatment with the analysis method appropriate for the response (FET, W, LR, or Cox regression). For the comet assay, analysis of tail moment and tail intensity data treatments were compared by analysis of variance utilizing the SAS MIXED procedure after averaging over cells within each mouse (as responses for cells within each mouse were not independent). Subsequent t-tests were used to make a priori comparisons between a carcinogen treatment (B[a]P or DB[a,l]P) and the SRM 1649a plus carcinogen treatment. Dunnett's procedure was used to adjust p values for all pair-wise comparisons to the toluene control. Residuals were graphically examined and revealed no problems with the model assumptions. All analyses were conducted within version 9.1 of the SAS System for Windows (SAS Institute, Inc., 2003, Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effect of UDPM on PAH Tumor Initiation
In order to determine the effects of UDPM on B[a]P and DB[a,l]P tumor-initiating activity we used experimental parameters such as time until tumor formation, tumor incidence, and the number of tumors per TBA. Table 1 shows the tumor-initiating activity by all PAH and PAH cotreatments after 25 weeks of promotion with TPA. The effect of UDPM on the tumor-initiating activity of B[a]P in mouse skin is illustrated in Figure 1A and Table 1. UDPM exhibited weak tumor-initiating activity, where only 5.7% of UDPM-treated animals exhibited tumors (Table 1). All treatments involving B[a]P or DB[a,l]P with or without the addition of UDPM significantly induced greater final tumor incidence than UDPM treatment alone (FET, p < 0.0001, all comparisons). Mice initiated with UDPM alone developed papillomas at week 16, compared to weeks 5, 6, 5, and 4 for mice initiated with B[a]P alone, UDPM plus B[a]P, DB[a,l]P, and UDPM plus DB[a,l]P, respectively (Fig. 1A, 1B).
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UDPM significantly delayed the onset of tumor incidence by B[a]P, compared with B[a]P treatment alone (LR, p = 0.0251) (Fig. 1A). Mice in the UDPM and B[a]P treatment group were estimated to have about half the hazard of developing tumors compared to mice receiving B[a]P alone (point estimate for hazard ratio = 0.499, 95% confidence interval [CI] [0.271, 0.920]). At 25 weeks, tumor incidence and tumors per TBA were reduced in the group receiving both UDPM and B[a]P (51% incidence and 2.5 tumors per TBA), compared to the group receiving B[a]P alone (71% and 3.3 tumors per TBA), but the reduction was not statistically significant (FET, p = 0.1431; W, p = 0.2431) (Table 1). Both papillomas and carcinomas developed in B[a]P and UDPM plus B[a]P treatments; however, no significant difference in papilloma (two sided p = 0.2321) and carcinoma occurrence was observed between groups (Table 1).
Table 1 and Figure 1B illustrate the effects of UDPM on DB[a,l]P tumor-initiating activity. In DB[a,l]P and DB[a,l]P plus UDPM cotreated animals, no significant difference in time until tumor incidence was observed (LR, p = 0.3946) (Fig. 1B). Mice receiving DB[a,l]P alone were estimated to have one fourth the hazard of developing tumors compared to those receiving both UDPM plus DB[a,l]P (point estimate for hazard ratio = 0.825, 95% CI [0.503, 1.354]) (Fig. 1B). At 25 weeks, tumor incidence and tumors per TBA were higher in the group receiving both UDPM and DB[a,l]P (88.6% tumor incidence, 4.97 tumors per TBA), compared to the group receiving DB[a,l]P alone (100% tumor incidence, 4.44 tumors per TBA) but the difference was not significant (FET, p = 0.2391, exact Monte Carlo estimate, p = 0.4651) (Table 1, Fig. 1B). No significant differences were observed between the numbers of papillomas and carcinomas developed in DB[a,l]P and UDPM plus DB[a,l]P treatments (Monte Carlo estimate, p = 0.2270, p = 0.5186, respectively) (Table 1).
PAH-DNA Adducts Correlate with Tumor Initiation
The effect of UDPM on PAH-DNA adducts in the SENCAR epidermis was investigated to determine if the observed tumorigenic activity correlated with PAH-DNA adduct formation. Mice were treated in an identical method as done in the initiation-promotion study (see Materials and Methods). PAH-DNA adduct detection at 24 and 48 h after PAH exposure indicated no DNA adduct formation in toluene (vehicle control)-treated mice (data not shown). Adduct profiles for all treatment groups were consistent between 24 and 48 h. Therefore, representative profiles of the general PAH-DNA–binding pattern in the mouse epidermis following 24 and 48 h exposure are shown in Figure 2.
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UDPM treatment exhibited low but detectable B[a]PDE-DNA adducts at both 24 and 48 h (Fig. 2, bottom left panel). Adducts detected in SENCAR epidermal DNA treated with UDPM, B[a]P, and UDPM plus B[a]P were primarily B[a]P diol epoxide (B[a]PDE)-DNA adducts (verified by a B[a]PDE standard, representing the (+)-anti-B[a]PDE adduct eluting at 7 min, not shown) (Fig. 2A and 2B, top panel). The peak representing the (+)-anti-B[a]PDE adduct is the stereoisomeric metabolite responsible for the majority of the tumor-initiating activity of B[a]P (Slaga et al., 1979
).
DB[a,l]P was actively metabolized to its fjord region diol epoxides. The four major DB[a,l]PDE-DNA adducts eluted at 57, 69, 73, and 82 min, in addition to a minor polar adduct eluting at 22 min. The DB[a,l]PDE-DNA adduct peaks labeled in Figure 2 (middle panel) are based on previously reported identification (Buters et al., 2002). Peak 4 represents the potent tumor-initiating stereoisomer of DB[a,l]PDE (–)-anti-diol epoxide bound to deoxyadenosine, and it was detected in DNA from mice treated with DB[a,l]P and UDPM plus DB[a,l]P. The detection of PAH-DNA adducts correlated with the observed tumor-initiating activity of the PAH and UDPM plus PAH.
DNA Strand Breaks by UDPM
In addition to analyzing PAH-DNA adduct formation and tumor initiation as events contributing to genotoxicity and carcinogenesis; we investigated the extent of DNA strand breaks in whole blood cells from treated mice by the comet assay. The tail intensity, or the mean percentage DNA (percent DNA) in the tail, is presented in Figure 3A. Each of the treatment groups exhibited a significant increase in the mean percent of DNA present in the comet tail (p < 0.05), compared to the control. Cotreatment of UDPM with B[a]P also exhibited a significant increase of percent DNA in comet tail, compared with B[a]P alone (p = 0.0019).
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Tail moment is the product of tail length and percentage of DNA in tail; thus, tail moment represents both the amount of DNA migrated into the tail and the distance DNA migrated. The tail moment is commonly reported as a valid marker of single-strand DNA breaks (Speit and Hartmann, 2005
). We observed a significant increase in the tail moment in cells from mice cotreated with UDPM and either B[a]P or DB[a,l]P, compared with PAH treatment alone (p = 0.0477 and p = 0.0084) (Fig. 3B). A significant increase in tail moment was also observed in mice treated with DB[a,l]P plus UDPM compared to the control (p = 0.0007).
CYP Expression
Immunoblots using antibodies against CYP1A1 and CYP1B1 were used to determine the effect of UDPM on CYP protein expression (Fig. 4A, 4B). Following 24 h PAH exposure, microsomes were isolated from SENCAR mouse epidermal cells. Although CYP1A1 protein expression was observed in all treatment groups, an additive increase in expression was detected in cotreatments with UDPM, compared with B[a]P or DB[a,l]P alone (Fig. 4A). CYP1B1 protein was expressed in mouse epidermis treated with UDPM and B[a]P, however, no CYP1B1 expression was observed in the UDPM plus B[a]P treatment group. Conversely, CYP1B1 increased in expression following cotreatment of UDPM plus DB[a,l]P, compared with DB[a,l]P treatment alone (Fig. 4B).
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UDPM Inhibits EROD Activity
To determine whether UDPM inhibits the metabolizing activity of CYP1A1 and CYP1B1, dealkylation of the substrate ERES to fluorescent RES by supersomes expressing human recombinant CYP1A1 and CYP1B1 was assessed. When all three concentrations of UDPM were added to the reaction mixture, kcat of CYP1A1 decreased in a dose-dependent manner (Fig. 5A, Table 2). The Michaelis constant (Km), or the substrate concentration for half-maximal enzyme activity, for CYP1A1 was (0.09µM), and the kcat was 8.09nmol RES/min/nmol of CYP1A1 protein. With the addition of 2 and 20 µg UDPM, there was no significant change in the Km (0.11 and 0.10µM, respectively) but a decrease in the kcat (7.11 and 5.44nmol RES/min/nmol CYP1A1, respectively) was observed. Addition of the highest UPDM concentration (200 µg) caused an increase in the Km (0.36µM), and no change in kcat (5.93nmol RES/min/ng CYP1A1) compared to kcat at 20 µg. The y-intercept of the double reciprocal Lineweaver-Burk (LWB) plot increased, yet the x-intercept remained relatively constant for concentrations up to 20 µg (Fig. 5B). However, addition of 200 µg UDPM increased the 1/[ERES] value.
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2 µg 
20 µg 
and 200 µg UDPM [---; x]. The EROD activity of both enzymes was measured with 0.05, 0.10, 0.20, 0.50, 0.75, and 1.25µM of the substrate ERES as described in the Materials and Methods. (A) Michaelis-Menten plot on the effect of various concentrations of UDPM on the EROD activity of CYP1A1. (B) The double reciprocal LWB plot of CYP1A1 kinetics. (C) Michaelis-Menten plot on the effect of UDPM on CYP1B1 EROD activity. (D) The LWB plot of CYP1B1 kinetics.
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The Km for CYP1B1 was 0.67µM and the kcat was 1.07nmol RES/min/nmol (Fig. 5C, Table 2). The Km of CYP1B1 following addition of 20 and 200 µg UDPM remained relatively constant (0.47 and 0.64nmol RES/min/nmol CYP1B1, respectively), compared to vehicle control; however, addition of the lowest UDPM concentration caused an increase in the Km (1.62µM) (Table 2). The kcat values decreased in a dose-dependent manner with the addition of 20 and 200 µg (0.69 and 0.17 nmol RES/min/nmol CYP1B1), compared with vehicle control (Table 2). In concordance with results obtained with CYP1A1, kinetics from CYP1B1 activity increased the y-intercept, but the x-intercept remained constant as the concentration of UDPM increased (Fig. 5D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The toxicological assessment of complex mixtures is difficult (Mauderly, 1993
), and although the characterization of individual environmental carcinogens and their mechanisms of genotoxicity are well established, limited toxicological information is available regarding carcinogens within a complex mixture. Toxicologic as well as genotoxic evaluations of complex mixtures are complicated by the presence of biologically active components at low concentrations, which may have additive, antagonistic, or synergistic interactions. Therefore, known concentrations of PAH within a mixture may not provide adequate representation of a complex mixture's carcinogenic potential. Here we report the effect of UDPM on the bioactivation, DNA adduct formation, DNA strand breaks, and tumor initiation of B[a]P (moderately potent PAH) and DB[a,l]P (extremely potent) to contribute to the understanding of how the carcinogenic potency of PAH can be altered within a complex mixture.
Although limited research has been done on complex mixtures and no standard approach or protocol is established, the fundamental concepts of evaluation are the same as those for single substances (Yang et al., 1989). Studies involving artificial or binary mixtures report PAH to alter tumor-initiating activity in mouse skin. For example, an additive effect was exhibited when B[a]P tumor initiation increased upon benzo[e]pyrene (B[e]P), pyrene and flouranthene coexposure (Van Duuren and Goldschmidt, 1976). It was also demonstrated that B[e]P increased B[a]P tumor-initiating activity but decreased tumor initiation by dimethyl benz[a]anthracene (DiGiovanni et al., 1982). Recently, it was shown that a coal tar–derived complex mixture significantly decreased DB[a,l]P tumor-initiating activity (Marston et al., 2001). In our study, UDPM exhibited weak tumor-initiating activity, however, it decreased the onset of tumor initiation by B[a]P (Table 1, Fig. 1A). This is in concordance with previous findings that components within a complex mixture can reduce the carcinogenic potential of PAH by having an antagonistic effect on PAH-induced tumor formation (DiGiovanni et al., 1982; Hughes and Phillips, 1990; Mahadevan et al., 2004). UDPM coexposure did not significantly modify DB[a,l]P-induced tumor initiation (Table 1, Fig. 1B). However, tumor initiation by DB[a,l]P and UDPM plus DB[a,l]P revealed a greater tumorigenic response than that of B[a]P and UDPM plus B[a]P (Table 1, Fig. 1). This can be attributed to the potent (–)-anti DB[a,l]PDE adducts detected, which have been shown to exhibit a greater tumor-initiating potential compared with B[a]P (Cavalieri et al., 1991; Higginbotham et al., 1993).
As seen in previous studies (Marston et al., 2001), we attribute the tumor initiation observed to PAH-DNA adducts. HPLC profiles from both B[a]P- and UDPM plus B[a]P–treated mice reveal the potent tumor-initiating (+)-anti-B[a]PDE-deoxyguanosine DNA adducts (Fig. 2). DB[a,l]P and UDPM plus DB[a,l]P also exhibited the potent tumor initiator (–)-anti-DB[a,l]PDE (Fig. 2). These results are consistent with the hypothesis that the formation of DNA adducts is essential for PAH-induced tumorigenesis (Brookes and Lawley, 1964). Recent in vitro studies have shown that other components within artificial mixtures can modify PAH-DNA adduct formation (Binkova and Sram, 2004; Mahadevan et al., 2004), which can contribute to altered tumorigenic response. Coexposure to an artificial mixture consisting of DB[a,l]P, dibenzo[a,e]pyrene (DB[a,e]P), and B[a]P resulted in decreased PAH-DNA adduct formation in mouse skin, compared with a single PAH treatment (Hughes and Phillips, 1990). However, a greater decrease in PAH-DNA adducts was found in mice treated with a binary mixture of DB[a,e]P and B[a]P. Marston et al. (2001) demonstrated that decreased tumor incidence in mice cotreated with coal tar and B[a]P or DB[a,l]P, compared with PAH treatment alone, was attributed to decreased PAH-DNA adduct formation.
Bioactivation of B[a]P and DB[a,l]P to its reactive diol epoxide metabolites can be associated to both CYP1A1 and CYP1B1 expression (Fig. 3). An increase in CYP1A1 protein expression was observed in mice cotreated with B[a]P plus UDPM, compared with B[a]P alone. However, we investigated whether the delay in the onset of tumor formation by B[a]P was attributed to enzyme inhibition by UDPM. With increasing concentrations of UDPM, CYP1A1, and CYP1B1 kcat values generally decreased while Km values remained relatively constant (Table 2). However, at 200- and 2-µg UDPM treatment CYP1A1 and CYP1B1 Km and kcat values increased compared to values resulting from other UDPM concentrations, respectively. Our data suggest that UDPM inhibits CYP metabolic capacity primarily through a noncompetitive inhibitory mechanism; thereby modifying PAH-DNA adduct formation and tumor initiation. Through such a mechanism, components within UDPM may not bind to the CYP active site; rather bind to sites distinct from substrate binding sites. Recently, Shimada and Guengerich (2006) reported that PAH, including B[a]P and DB[a,l]P, noncompetitively inhibited the metabolic activation of procarcinogenic PAH-diols by CYP1A1, 1A2, and 1B1. Therefore, we hypothesize that the PAH component within the mixture may contribute to the noncompetitive inhibition observed.
Bioactivation of PAHs by CYP also generate radical cations (Cavalieri and Rogan, 1995) and PAH o-quinones (Smithgall et al., 1986, 1988a). PAH o-quinones can stimulate the metabolic generation of DNA-damaging ROS (DiGiulio et al., 1989) which may produce DNA strand breaks (Eastman and Barry, 1992). Oxidative DNA lesions may lead to mutagenesis, and thus contribute to tumor initiation. We observed a significant increase in DNA strand breaks with UDPM plus B[a]P or DB[a,l]P treatment, compared with PAH treatment alone (Fig. 2). A previous study determined DNA strand breaks detected in human fibroblasts to be attributed to 8-oxo-2'-deoxyguanine formation by the PM portion of UDPM (Karlsson et al., 2004). Therefore, the significant increase in tail moment in mice cotreated with UDPM, compared to mice treated with B[a]P or DB[a,l]P alone (Fig. 3B), may indicate an increase in the production of ROS by PM. Mahadevan et al. (2005) reported an increase in the expression of AKR1C messenger RNA upon exposure to UDPM. In addition to metabolism by CYP, oxidation of PAH dihydrodiols by AKRs can result in PAH o-quinone formation and redox-active PAH metabolites (Jiang et al., 2005; Palackal et al., 2001). Furthermore, these quinones can form abasic sites by forming unstable lesions (Flowers et al., 1997; Park et al., 2005). The observed DNA strand breaks by oxidative damage may be the result of the PM component of the mixture, the production of PAH o-quinones, or ROS.
In summary, we demonstrate that UDPM altered the carcinogenic potential of B[a]P. Results from this study provide insight into the mechanisms by which complex mixtures can alter PAH carcinogenesis. Different mixture components of UDPM may compete at CYP, thereby altering PAH bioactivation to its DNA-binding metabolites. As a result of CYP inhibition, other PAH metabolism pathways may compensate bioactivation giving rise to metabolites causing other deleterious effects, such as oxidative DNA damage. Although B[a]P is currently used as a carcinogenic indicator of human risk to air pollution and other complex mixtures, it is necessary to take into consideration the toxicological and mechanistic effects of other components within a mixture.
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ACKNOWLEDGMENTS |
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This work was supported by NCI grant CA28825, Department of Health and Human Services (DHHS), and the National Institute of Environmental Health Science (NIEHS) training grant T32 ES007060 (L.A.C.). The authors thank W. M. Dashwood for assistance with the animal work, and T.-W. Yu of the Cancer Chemoprotection Core Laboratory at the Linus Pauling Institute for advice and assistance with the comet assay. We further acknowledge the EHSC at OSU for the Nucleic Acids and Proteins, Tissue Analysis and Pathology Facilities, and the Statistics Service Cores which are supported by the NIEHS Center, grant P30 ES00210. We also wish to show appreciation to Dr Brinda Mahadevan for critical review and helpful suggestions toward this manuscript.
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