Long-Term Effects of a Standardized Complex Mixture of Urban Dust Part
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《毒物学科学杂志》
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon 97331
Department of Statistics, Oregon State University, Corvallis, Oregon 97331
ABSTRACT
Humans are exposed to complex mixtures of polycyclic aromatic hydrocarbons in the atmosphere. We examined the long term effects of a standard reference material (SRM) 1649a over time on the metabolic activation and DNA adduct formation by two carcinogenic PAHs, benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) in the human mammary carcinoma derived cell line MCF-7. PAH-DNA adduct analysis, cytochrome P450 (CYP) enzyme activity, CYP1A1 and CYP1B1 protein expression were determined in cells treated with SRM 1649a alone or SRM 1649a with either BP or DBP for 24 to 120 h. Averaging over time, significantly higher levels of DNA adducts were observed in cells treated with BP or DBP alone than in co-treatments with SRM 1649a and BP or DBP. Ethoxyresorufin O-deethylase assay indicated a significant increase in activity in cells treated with BP alone and co-treated with SRM1649a in comparison to other treatment groups. Induction of CYP1A1 and CYP1B1 protein expression was observed by immunoblots in cells treated with BP alone or in co-treatments of SRM 1649a and BP or DBP. These data demonstrate the influence of the components of SRM 1649a in inhibiting the activation of BP or DBP by CYP enzymes in the formation of DNA adducts. It also suggests that the carcinogenic activity of PAH within a complex mixture may vary with composition and activation of the components present in the complex mixture.
Key Words: SRM 1649a; dibenzo[a,l]pyrene; benzo[a]pyrene; cytochrome P450; DNA adducts; PAH.
INTRODUCTION
Humans can be environmentally exposed to airborne pollutants resulting from industrial processes, residential heating, and motor vehicle exhausts (Pershagen, 1990). Air pollution might also contribute to increased cancer risk in humans (Hemminki and Pershagen, 1994). The incomplete combustion of organic matter in the environment results in the release of a complex mixture of airborne pyrolysis products, including polycyclic aromatic hydrocarbons (PAHs). PAHs constitute one of the major classes of airborne carcinogens (IARC, 1993) and can be metabolized to DNA binding derivatives that form DNA adducts (Pershagen, 1990). Studies have shown that the main contributors to genotoxicity are PAHs and their derivatives (Binkova et al., 1999; Cerna et al., 1999; Hsiao et al., 2000). Recently, it has been demonstrated that reduction of exposure to particulate air pollution lowers the risk of heritable mutations in mice suggesting health risks for future generations (Samet et al., 2004; Somers et al., 2004).
Chemical analysis of complex mixtures in ambient air is difficult since many chemically diverse and biologically active components occur at low concentrations. The National Institute of Standards and Technology (NIST) have developed a number of natural matrix standard reference materials (SRMs) since the early 1980s for the determination of organic contaminants in environmental matrices (Wise and Schantz, 1997). The first particle-based, environmental natural matrix SRM developed by NIST for organic contaminants was SRM 1649 (Urban dust/Organics) (May and Wise, 1984; Wise et al., 2000). Standard Reference Material 1649a is the same particulate material that was issued previously in 1982 as SRM 1649 (National Institute of Standards and Technology, 1982); it contains 44 or more PAHs, many polychlorbiphenyl (PCB) congeners, chlorinated pesticides, and inorganic constituents.
In our studies, we used SRM 1649a, in addition to representative examples of carcinogenic PAHs, benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP), to understand the metabolic activation and DNA binding of these PAHs within complex mixtures. Both BP and DBP have been demonstrated to form stable DNA adducts in MCF-7 cells in culture (Mauthe et al., 1995; Ralston et al., 1994). It has been shown that PAHs accumulate in breast tissue (Larsen et al., 1998) and that exposure to PAHs may lead to increased risk of breast cancer (Brody and Rudel, 2003; Jeffy et al.. 2002). In a recent study, the expression of cytochrome P450-1A1 (CYP1A1) and CYP1B1 genes was increased in MCF-7 cells, 24 h after treatment with BP alone or co-treatment with SRM 1649a (Mahadevan et al., 2005a). In the current work we studied the long-term effects of the complex environmental mixture SRM 1649a on the metabolic activation of BP and DBP by CYP1A1 and CYP1B1, and PAH-DNA adduct formation in MCF-7 cells in culture.
MATERIALS AND METHODS
Chemicals.
Benzo[a]pyrene and DBP 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 heterogeneous 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 ATP (1 mCi) was purchased from PerkinElmer (Boston, MA). Standrad Reference Material 1649a was obtained from the National Institute of Standards and Technology (Gaithersberg, MD) and had a unit of SRM in a bottle containing 2.5 g of atmospheric particulate material (National Institute of Standards and Technology 1982). The amount of BP present in SRM 1649a was determined as 2.509 mg/kg, and a detailed description of the chemical composition of SRM 1649a is available online at the NIST Web site (http://patapsco.nist.gov/srmcatalog/certificates/1649a.pdf). Schubert et al. (2003) have recently determined the presence of DBP (47.2 μg/kg) in SRM 1649a.
Cell culture and treatment.
The MCF-7 cells (obtained from the Karmanos Cancer Center, Detroit, MI) were cultured in a 75 cm2 flask (Corning, Corning, NY) in a 1:1 mixture of F-12 Nutrient Mixture and Dulbecco's Modified Eagle's Medium (DMEM; Gibco BRL, Grand Island, NY) at 37°C with 5% CO2. The medium was supplemented with 10% fetal bovine serum (FBS; Intergen, Purchase, NY) and 15 mM HEPES buffer. Cell cultures were subcultured at a ratio of 1:4 when the cells covered the entire surface of the flask.
All cell culture flasks, in which the cells covered approximately 80% of the surface, were replenished with fresh medium (20 ml) 24 h prior to treatment. The cells were treated in 20 ml media with DMSO (75 μl) as solvent control and with a final concentration of SRM 1649a alone (400 μg), BP (10 μg or 1.9 μM), SRM 1649a (400 μg) and BP (10 μg), DBP (0.1 μg or 0.0165 μM), or SRM 1649a (400 μg) and DBP (0.1μg). The cells were harvested after 24–120 h of continuous exposure and stored at –80°C until needed for DNA or microsome isolation.
The concentrations of BP and DBP used in this study were based on previous studies in our laboratory that indicated that these doses gave detectable levels of DNA binding without causing excessive toxicity to the cell (Mahadevan et al., 2005b). We used a dose of BP that was 100-fold higher in concentration than DBP based on their relative carcinogenic potency in mouse skin assay (Marston et al., 2001). Determination of cytotoxicity for 120 h by MTT cell proliferation assay indicated that co-treatment of SRM 1649a and BP or DBP was more toxic to MCF-7 cells compared to SRM 1649a and DBP alone (data not shown). Also, treatment with SRM 1649a alone was toxic to cells.
DNA isolation.
A standard DNA isolation protocol was used (Luch et al. 1998). Briefly, cell pellets from two T-75 flasks per treatment group were pooled and homogenized in cell lysis buffer [10 mM Tris, 1 mM Na2EDTA, 1% SDS, pH 8]. The homogenates were treated with RNase, DNase-free (50 U/ml) and RNase T1 (1000 U/ml) at 37°C for 1 h, followed by treatment with proteinase K (500 μg/ml) at 37°C for 1 h. The DNA was extracted in light phase gel lock tubes with equal volumes of Tris-equilibrated phenol followed by extraction with 1:1 volume of Tris-equilibrated phenol and chloroform:isoamyl alcohol (24:1) and then with equal volumes of chloroform:isoamyl alcohol (24:1). The aqueous layer was treated with 1/10 volume of 5 M NaCl and twice the volume of cold 100% ethanol to precipitate the DNA which was then dissolved in sterile double-distilled water; its concentration was determined by UV absorbance at 260 nm.
33P-postlabeling of DNA adducts and HPLC.
Post-labeling was carried out as described previously (Ralston et al., 1995). Briefly, 10 μg DNA isolated from MCF-7 cells after treatment were digested with nuclease P1 (0.4 U) and prostatic acid phosphatase (350 mU). It was then post-labeled with [-33P]ATP (3,000 Ci/mmol), cleaved to adducted mononucleotides with snake venom phosphodiesterase I (15 mU) and apyrase (100 mU) followed by pre-purification on a Sep-Pak C18 cartridge (Waters, Milford, MA). Subsequent separation by analytical high performance liquid chromatography (HPLC-Varian system equipped with two pumps and an autosampler; Varian Systems, CA) was carried out using a C18 reverse-phase column (5 μm Ultrasphere ODS, 4.6 x 250 mm). The BP-DNA adducts were resolved by elution at 1 ml/min with 0.1 M ammonium phosphate, pH 5.5 (solvent A) and 100% HPLC grade methanol (solvent B). The elution gradient for BP and SRM 1649a treated samples was as follows: 44–55% solvent B for 5 min, 55–60% solvent B over 5 min, 60–90% solvent B over 20 min and 90–100% solvent B over 5 min. The DBP DNA-adducts were resolved by elution at 1 ml/min with 0.1 M ammonium phosphate, pH 5.5 (solvent A) and 50% HPLC grade methanol/50% acetonitrile (solvent B). The elution gradient for DBP and SRM 1649a was as follows: 20–44% solvent B for 20 min, 44%–60% solvent B over 40 min, and 60–80% solvent B over 15 min. The radiolabeled nucleotides were detected by an on-line 400 μl dry cell -RAM Model 3 (IN/US Systems, Tampa, FL) radioisotope detector and the level of DNA binding was calculated based on the labeling efficiency of a [3H]B[a]P-7,8-dihydrodiol 9,10-epoxide (NCI Chemical Carcinogen Reference Stanadard Repository, Midwest Research Institute, Kansas City, MO) standard (Lau and Baird, 1991). Three independent sets of the postlabeling reactions were carried out for every sample treated, to determine the total PAH-DNA adduct levels.
Microsome isolation.
Microsomes were prepared as described previously (Otto et al., 1991) with minor modifications. Cell culture samples pooled from five T75 flasks for each treatment group were homogenized in a steel homogenizer with microsomal homogenization buffer [0.25 M K2HPO4, 0.15 M KCl, 10 mM EDTA, and 0.25 mM phenylmethylsulfonylfluoride (PMSF)] and were centrifuged at 15,000 x g for 20 min at 4°C in a Sorvall RC-5B refrigerated superspeed centrifuge (DuPont Instruments, Willmington, DE). The supernatant was centrifuged at 100,000 x g for 90 min at 4°C in a Beckman TL-100 Ultracentrifuge (Beckman Coulter, Fullerton, CA), and the pellet was resuspended in microsome dilution buffer (0.1 M KH2PO4, 20% glycerol, 10 mM EDTA, 0.1 mM DTT and 0.25 mM PMSF). The protein concentration was determined in a Turner UV-VIS spectrophotometer SP-890 (Barnstead International, Dubuque, IA) using the Bicinchoninic acid colorimetric assay (Pierce, Rockford, IL). Three independent sets of microsomes were prepared for all the treatment groups.
Ethoxyresorufin O-Deethylation (EROD) assay.
Fifty micrograms of microsomal protein were added to 1 μM 7-ethoxyresorufin (ERES) (Sigma) in 200 μl of 0.1 M Tris HCl (pH 7.8) buffer per well in black 96-well plate (E&K Scientific, Campbell, CA). NADPH (Sigma) was added to each well and the fluorescence was measured in a Spectra MAX Gemini plate reader (Molecular Devices, Sunnyvale, CA). The excitation and emission wavelength were 535 nm and 585 nm, respectively, and the kinetic assay was monitored over 10 min. The experiment was repeated three times and each sample was assayed in triplicate. The amount of resorufin produced was calculated from the fluorescence of a known concentration of resorufin.
Western blots.
Microsomal proteins (50 μg) were denatured by boiling for 3 min and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 8% acrylamide gels (Sigma). After electrophoresis, the proteins were transferred on to a PVDF membrane (Bio-Rad, Hercules, CA) in a Mini Blotter apparatus (Bio-Rad). The membrane was blocked with 1:3 Nap-Sure:PBS-T solution (Geno Technology, St. Louis, MO) and incubated with the primary antibody for 2 h. Human CYP1A1 was detected by a rabbit polyclonal CYP1A1 antibody (1:1500) prepared against purified recombinant human CYP1A1 protein. The CYP1A1 antibody was a gift from Dr. F. P. Guengerich (Center for Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN). Dr. C. Marcus (Department of Pharmacology and Toxicology, University of New Mexico, Albuquerque, NM) prepared and provided the human CYP1B1 rabbit polyclonal antibody (1:1,000). The immunoreactive proteins were detected by incubating the membrane with peroxidase-conjugated anti-rabbit IgG (1:20,000) for 30 min. After washes in PBS-T, the immunoreactive proteins were observed by enhanced chemiluminescence (ECL) detection method, as described by the manufacturer (Amersham Life Science, Arlington Heights, IL). -Actin (protein loading control) was detected by anti--actin primary antibody (1:1500) (Sigma) followed by anti-mouse HRP conjugated secondary antibody (1:20,000). Microsomal proteins (10 μg) from Chinese hamster ovary V79 cells expressing either human CYP1A1 or CYP1B1 were used as positive controls.
Statistical analysis.
DNA adduct data were averaged over one to three replicate reactions and the means were log transformed for analysis due to residuals on the original scale either having right skew or variation increasing with mean. Benzo[a]pyrene and DBP data were analyzed separately because of a few zero values in the DBP data. For log scale analysis of DBP data, four zero values were replaced with a small positive value (half of the smallest positive value observed 0.21/2 = 0.105). The same conclusions were obtained whether means were given equal weight or were weighted according to the number of replicate reactions; results from equally weighted analyses are presented herein. EROD data were averaged over triplicate assays and the means were log transformed for analysis as the variation increased with the response. For the log scale analysis only 3 means (out of 96) were zero, and they were replaced with small positive values (half of the smallest positive value observed within a complete set.
Linear mixed models were used for the analysis of both EROD and adduct data with MIXED procedures in SAS, version 9.1, 2003 (SAS Institute Inc. Cary, NC). Each assay included treatments from a single time point so that treatments could be compared within assays. This resulted in a split-plot type model with two error terms: (1) assays within time points for the main time effect and (2) the final residual for the treatment main effect and treatment-by-time interaction.
RESULTS
Effects of the Co-treatment with SRM 1649a and Carcinogenic PAH on DNA Adduct Formation
The effects of SRM 1649a on DNA adduct formation on co-treatment with carcinogenic PAH, BP, and DBP were determined by 33P-postlabeling and analyzed by HPLC. Representative HPLC elution profiles of the PAH-DNA adducts on co-treatment with BP or DBP after 48 and 120 h are depicted in Figures 1(A,B) and 2(A,B). The retention times vary between Figures 1 and 2A and B, panels 1 and 2, as two different elution gradients were used. MCF-7 cells treated with DMSO (vehicle control) or SRM 1649a alone did not exhibit any detectable levels of DNA adducts (Fig.1A, panel 3, and Fig. 1B, panel 3). The cells treated with SRM 1649a alone (400 μg) contained 1.0036 ng of BP and 18.88 pg of DBP. The presence of such low concentrations of carcinogenic PAHs explains the lack of detection of DNA adduct formation in cells treated with SRM 1649a alone.
The HPLC profile of BP-DNA adducts revealed one major peak eluting at a retention time of 19 min (Fig. 1A,B, panel 1). This major peak corresponded with the (+)-anti-BPDE-dG adduct peak of BP and was the significant adduct resolved in the SRM 1649a and BP co-treatment (Fig. 1A,B panel 2). A reduction in the peak area was observed over time in co-treatment of SRM 1649a and BP in comparison to BP alone (Fig. 1A,B, panels 1 and 2). MCF-7 cells treated with DBP exhibited four major DBPDE-DNA adducts with retention times of 50, 55, 57, and 62 min (Fig. 2A). Over time, a reduction in the peak area was observed in co-treatment of SRM 1649a and DBP (Fig. 2A,B, panels 1 and 2).
Reduction in Total DNA Binding on Co-treatment with SRM 1649a and Carcinogenic PAH Over Time
The level of PAH-DNA adducts formed in MCF-7 cells treated with BP, or in co-treatment with SRM 1649a are graphically represented in Figure 3A. The highest level of DNA binding was observed in cells treated with BP (79.2 pmol/mg of DNA) after 48 h of exposure (Fig. 3A). The mean adduct levels for the BP treatment group was higher in comparison to co-treatment with SRM 1649a. When averaged over time, a significant decrease (p < 0.01) was observed in the total BP-DNA adducts formed in co-treatment with SRM 1649a and BP. Analysis of log transformed data revealed that co-treatment with SRM 1649a and BP there was 2.8-fold less DNA adducts than BP alone. The maximum decrease in the level of DNA binding when co-treated with SRM 1649a and BP was identified at 120 h (3.6 pmol/mg of DNA) in comparison to BP alone (8.66 pmol/mg of DNA), although it was not significant (p = 0.36) (Fig. 3A).
The levels of PAH-DNA adducts formed in MCF-7 cells treated with DBP and co-treatment with SRM 1649a are graphically represented in Figure 3B. The level of DNA adducts formed when treated with DBP alone was relatively stable from 48 h to 120 h, with the highest level of DNA binding (34.2 pmol/mg) after 72 h of exposure (Fig. 3B). On co-treatment with SRM 1649a and DBP there was 18.7-fold less DBP-DNA adducts than with DBP alone. Therefore, a very significant decrease (p < 0.0001) in the total DBP-DNA adducts was observed over time in co-treatment with SRM 1649a and DBP (Fig. 3B). The maximum decrease in the level of DNA binding was identified at 120 h, when the total DNA adducts in cells treated with DBP alone was 19.5 pmol/mg of DNA in comparison to 0.3 pmol/mg of DNA on co-treatment with SRM 1649a plus DBP (Fig. 3B).
Cytochrome P450 Enzyme Activity and Induction
The effect of SRM 1649a in co-treatments with carcinogenic PAH on CYP enzyme activity as measured by EROD assay is illustrated in Figure 4. A significant difference (p < 0.05) in CYP enzyme activity between treatment groups was evident over time. Benzo[a]pyrene alone and co-treatment of SRM 1649a plus BP exhibited the highest EROD activity levels over time. Treatment groups SRM 1649a alone and SRM 1649a plus DBP indicated very low but similar EROD activity levels over time (Fig. 4). Dibenzo[a,l]pyrene alone and DMSO exhibited the lowest level of EROD activity in comparison to other treatment groups analyzed over time.
Western analyses were performed to examine the effect of SRM 1649a alone and in co-treatments with BP or DBP on CYP1A1 and CYP1B1 in MCF-7 cells 24 h, 48 h, and 120 h after exposure (Fig. 5). Treatment with DMSO, DBP or SRM 1649a alone did not induce CYP1A1 protein in the exposure times studied. However, basal levels of CYP1B1 were observed with DMSO, DBP, and SRM 1649a treatment groups 24 h and/or 48 h after exposure (Fig. 5A, 5B, 5A,B). An additive increase in the expression of CYP1A1 and CYP1B1 was observed with co-treatments of SRM 1649a and BP in comparison to BP alone (Fig. 5A,B), whereas in 1649a co-treatment with DBP only at 48 h, basal levels of CYP1B1 protein were observed (Fig. 5A,B). However, treatment with DBP alone revealed no induction of CYP1B1 except at 48 h. At 120 h CYP1A1 induction was observed in BP and co-treatment with SRM 1649a, whereas CYP 1B1 protein was detected in treatment groups BP, BP plus SRM 1649a, and DBP plus SRM 1649a (Fig. 5C).
DISCUSSION
Environmental pollutants from coal tar, diesel exhaust, and urban dust are known to cause DNA damage. The carcinogens present in complex mixtures of environmental pollutants can be metabolized by CYP enzymes producing metabolites that bind to DNA. Recently, in vitro and in vivo experiments have demonstrated the formation of DNA adducts after exposure to a complex mixture containing PAH from coal tar (Cizmas et al., 2004; Mahadevan et al., 2005a, 2005b; Marston et al., 2001), diesel exhaust particulate (Kuljukka-Rabb et al., 2001; Lazarova and Slamenova, 2004; Pohjola et al., 2003; Reliene et al., 2005) and urban dust (Mahadevan et al., 2005a). In an occupational cohort study by Peluso et al. (2001) where they examined the relationship between air pollution exposure and DNA adducts, the authors conclude that the association was significant for both heavily exposed industrial workers and less severely exposed urban workers. Also, DNA adduct levels in exposed workers were significantly correlated with levels of BP in the air (Peluso et al., 2001). In order to understand the possible link to breast cancer and long term effects potentially attributable to exposure to urban dust, we studied the effects of SRM 1649a on MCF-7 cells in culture up to 120 h. An epidemiological study on the short-term exposure (24 h) to SRM 1649a indicated increased nasal secretion levels of interleukins, supporting the observations that short-term increase of outdoor particulate matter concentration increases the frequency of upper respiratory diseases (Riechelmann et al., 2004). Recently, it was also demonstrated that short-term exposure (24 h) of MCF-7 cells to SRM 1649a altered gene expression patterns (Mahadevan et al., 2005a).
Here we discuss the first study involving an extended length of exposure (120 h) to SRM 1649a in vitro to determine if the components of SRM 1649a were competing with the metabolic activation of the carcinogenic PAH in co-treatment with BP or DBP. The level of BP-DNA adducts significantly decreased over time in the presence of SRM 1649a (Fig. 3A). SRM 1649a very significantly decreased the level of DBP-DNA adducts formed over time. This reduction was evident at each time point and was more pronounced at 96 h and 120 h of exposure (Fig. 3B). A similar phenomenon was reported by Binkova and Sram (2004) in human diploid lung fibroblasts where BP-DNA adduct levels were up to fivefold lower in an artificial carcinogenic PAH mixture and up to tenfold lower in an environmental mixture. In our study, it was not clear if the decrease occurred in response to treatment toxicity or removal of DNA adducts by DNA repair and replication. It is possible that the DNA adduct levels reached a saturation point after exposure to the complex mixture and carcinogenic PAH. As reported in a study by (Peluso et al., 2001), the relation between DNA adducts and BP in peripheral blood cells was linear at low doses and sublinear at high doses indicating that DNA adduct formation tends to reach a saturation point at higher levels of exposure to chemical mixtures (Peluso et al., 2001). Further experiments need to be conducted with varying levels of exposure to the urban dust mixture and carcinogenic PAH to test if this may have occurred with co-treatments of SRM 1649a and BP or DBP.
Previous studies on SRM 1649a (Mahadevan et al., 2004, 2005a), as well as this study, suggest that there may be a selective metabolism of certain PAH within complex mixtures, which may be influenced by different CYP enzymes. We hypothesized that the components within SRM 1649a may be affecting the metabolism of BP and DBP by CYP enzymes. Higher EROD activity levels corresponded with protein induction of CYP enzymes as analyzed by Western blots in SRM 1649a co-treatment with BP (Figs. 4 and 5A, B). However, co-treatment with SRM 1649a and DBP did not reveal a clear correlation with regard to EROD activity and CYP1A1 or CYP1B1 induction levels. These results suggest that the two CYP enzymes may have different roles in the metabolic activation of certain carcinogenic PAHs found in environmental mixtures (Diamond and Baird, 1977; Shimada and Fujii-Kuriyama, 2004). The difference in CYP1A1 enzyme activity and protein induction levels observed in co-treatments with SRM 1649a and BP or DBP could also occur because DBP is a poor inducer of CYP1A1-dependent proteins and enzyme activity, as the CYP1A1 induction levels observed were in accordance with induction equivalency factors reported for DBP (Machala et al., 2001).
The decrease in PAH-DNA adduct levels in co-treatment with SRM 1649a (Fig. 3) was similar to a study with complex mixture derived from coal tar (Mahadevan et al., 2005b). This decrease implies that an inhibitory effect of SRM 1649a on CYP enzymes may have resulted in the decreased metabolism of the carcinogenic PAH. Future biochemical experiments are planned with SRM 1649a to elucidate the mechanism of inhibition of CYP enzymes. On the other hand, although there was no decrease in the EROD activity in the BP and SRM 1649a co-treatment (Fig. 4) a decrease in the DNA adduct levels in comparison to BP was observed (Fig. 3A). There is a possibility that this decrease in DNA adduct levels may be due to detoxification of BP metabolites that form DNA adducts by the induced CYP1A1 as discussed in the in vivo studies by Uno et al. (2004). Another possible mechanism by which the components of SRM 1649a could affect the DNA is through the formation of quinones (for example BP-7,8-dione) that could cause oxidative damage to the DNA, as described by Harvey et al. (2004).
Overall, this study indicates that components of a complex mixture may interact with each other to produce synergistic, antagonistic, or additive effects. It also highlights the fact that complex mixture interactions can influence the toxicodynamic properties that may result in enhanced or reduced genotoxicity. Furthermore, the presence of carcinogenic PAH such as BP in complex mixtures is not sufficient to allow prediction of the human risk on exposure to mixtures. Our studies take into account the modifiers within the mixture that may alter the carcinogenic potency of the carcinogenic PAH present. Therefore, these studies are important and may assist in evaluating the carcinogenic potential or toxic equivalency (TE) of several complex PAH mixtures (Cizmas et al., 2004; Petry et al., 1996; Willett et al., 1997).
NOTES
2 These authors contributed equally to this work.
Caution: BP (benzo[a]pyrene) and DBP (dibenzo[a,l]pyrene) are potent carcinogenic agents and should be handled according to National Institutes of Health (NIH) guidelines for the use of carcinogens. SRM 1649a is a suspected human carcinogen and should be handled with the same precautions.
ACKNOWLEDGMENTS
This research was supported by grant CA 28825, DHHS (Department of Health and Human Services) from the National Cancer Institute, National Institutes of Health.
This publication was made possible, in part, by the Nucleic Acid and Protein Service Core, Cell Culture Core and Statistics Core of the Environmental Health Science Center, Oregon State University, funded by grant P30 ES00210 from the National Institute of Environmental Health Sciences. The authors thank Kathleen Yeager and Kendall Dutcher for assistance with DNA and microsome isolations. We also thank Dr. Louisa Hooven for isolating microsomes from V79 cells for use as positive controls; Dan Albershardt for the preparation of figures; Tony Rianprakaisang for organization of references; and Jack Giovanini for help in statistical analysis.
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Department of Statistics, Oregon State University, Corvallis, Oregon 97331
ABSTRACT
Humans are exposed to complex mixtures of polycyclic aromatic hydrocarbons in the atmosphere. We examined the long term effects of a standard reference material (SRM) 1649a over time on the metabolic activation and DNA adduct formation by two carcinogenic PAHs, benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) in the human mammary carcinoma derived cell line MCF-7. PAH-DNA adduct analysis, cytochrome P450 (CYP) enzyme activity, CYP1A1 and CYP1B1 protein expression were determined in cells treated with SRM 1649a alone or SRM 1649a with either BP or DBP for 24 to 120 h. Averaging over time, significantly higher levels of DNA adducts were observed in cells treated with BP or DBP alone than in co-treatments with SRM 1649a and BP or DBP. Ethoxyresorufin O-deethylase assay indicated a significant increase in activity in cells treated with BP alone and co-treated with SRM1649a in comparison to other treatment groups. Induction of CYP1A1 and CYP1B1 protein expression was observed by immunoblots in cells treated with BP alone or in co-treatments of SRM 1649a and BP or DBP. These data demonstrate the influence of the components of SRM 1649a in inhibiting the activation of BP or DBP by CYP enzymes in the formation of DNA adducts. It also suggests that the carcinogenic activity of PAH within a complex mixture may vary with composition and activation of the components present in the complex mixture.
Key Words: SRM 1649a; dibenzo[a,l]pyrene; benzo[a]pyrene; cytochrome P450; DNA adducts; PAH.
INTRODUCTION
Humans can be environmentally exposed to airborne pollutants resulting from industrial processes, residential heating, and motor vehicle exhausts (Pershagen, 1990). Air pollution might also contribute to increased cancer risk in humans (Hemminki and Pershagen, 1994). The incomplete combustion of organic matter in the environment results in the release of a complex mixture of airborne pyrolysis products, including polycyclic aromatic hydrocarbons (PAHs). PAHs constitute one of the major classes of airborne carcinogens (IARC, 1993) and can be metabolized to DNA binding derivatives that form DNA adducts (Pershagen, 1990). Studies have shown that the main contributors to genotoxicity are PAHs and their derivatives (Binkova et al., 1999; Cerna et al., 1999; Hsiao et al., 2000). Recently, it has been demonstrated that reduction of exposure to particulate air pollution lowers the risk of heritable mutations in mice suggesting health risks for future generations (Samet et al., 2004; Somers et al., 2004).
Chemical analysis of complex mixtures in ambient air is difficult since many chemically diverse and biologically active components occur at low concentrations. The National Institute of Standards and Technology (NIST) have developed a number of natural matrix standard reference materials (SRMs) since the early 1980s for the determination of organic contaminants in environmental matrices (Wise and Schantz, 1997). The first particle-based, environmental natural matrix SRM developed by NIST for organic contaminants was SRM 1649 (Urban dust/Organics) (May and Wise, 1984; Wise et al., 2000). Standard Reference Material 1649a is the same particulate material that was issued previously in 1982 as SRM 1649 (National Institute of Standards and Technology, 1982); it contains 44 or more PAHs, many polychlorbiphenyl (PCB) congeners, chlorinated pesticides, and inorganic constituents.
In our studies, we used SRM 1649a, in addition to representative examples of carcinogenic PAHs, benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP), to understand the metabolic activation and DNA binding of these PAHs within complex mixtures. Both BP and DBP have been demonstrated to form stable DNA adducts in MCF-7 cells in culture (Mauthe et al., 1995; Ralston et al., 1994). It has been shown that PAHs accumulate in breast tissue (Larsen et al., 1998) and that exposure to PAHs may lead to increased risk of breast cancer (Brody and Rudel, 2003; Jeffy et al.. 2002). In a recent study, the expression of cytochrome P450-1A1 (CYP1A1) and CYP1B1 genes was increased in MCF-7 cells, 24 h after treatment with BP alone or co-treatment with SRM 1649a (Mahadevan et al., 2005a). In the current work we studied the long-term effects of the complex environmental mixture SRM 1649a on the metabolic activation of BP and DBP by CYP1A1 and CYP1B1, and PAH-DNA adduct formation in MCF-7 cells in culture.
MATERIALS AND METHODS
Chemicals.
Benzo[a]pyrene and DBP 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 heterogeneous 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 ATP (1 mCi) was purchased from PerkinElmer (Boston, MA). Standrad Reference Material 1649a was obtained from the National Institute of Standards and Technology (Gaithersberg, MD) and had a unit of SRM in a bottle containing 2.5 g of atmospheric particulate material (National Institute of Standards and Technology 1982). The amount of BP present in SRM 1649a was determined as 2.509 mg/kg, and a detailed description of the chemical composition of SRM 1649a is available online at the NIST Web site (http://patapsco.nist.gov/srmcatalog/certificates/1649a.pdf). Schubert et al. (2003) have recently determined the presence of DBP (47.2 μg/kg) in SRM 1649a.
Cell culture and treatment.
The MCF-7 cells (obtained from the Karmanos Cancer Center, Detroit, MI) were cultured in a 75 cm2 flask (Corning, Corning, NY) in a 1:1 mixture of F-12 Nutrient Mixture and Dulbecco's Modified Eagle's Medium (DMEM; Gibco BRL, Grand Island, NY) at 37°C with 5% CO2. The medium was supplemented with 10% fetal bovine serum (FBS; Intergen, Purchase, NY) and 15 mM HEPES buffer. Cell cultures were subcultured at a ratio of 1:4 when the cells covered the entire surface of the flask.
All cell culture flasks, in which the cells covered approximately 80% of the surface, were replenished with fresh medium (20 ml) 24 h prior to treatment. The cells were treated in 20 ml media with DMSO (75 μl) as solvent control and with a final concentration of SRM 1649a alone (400 μg), BP (10 μg or 1.9 μM), SRM 1649a (400 μg) and BP (10 μg), DBP (0.1 μg or 0.0165 μM), or SRM 1649a (400 μg) and DBP (0.1μg). The cells were harvested after 24–120 h of continuous exposure and stored at –80°C until needed for DNA or microsome isolation.
The concentrations of BP and DBP used in this study were based on previous studies in our laboratory that indicated that these doses gave detectable levels of DNA binding without causing excessive toxicity to the cell (Mahadevan et al., 2005b). We used a dose of BP that was 100-fold higher in concentration than DBP based on their relative carcinogenic potency in mouse skin assay (Marston et al., 2001). Determination of cytotoxicity for 120 h by MTT cell proliferation assay indicated that co-treatment of SRM 1649a and BP or DBP was more toxic to MCF-7 cells compared to SRM 1649a and DBP alone (data not shown). Also, treatment with SRM 1649a alone was toxic to cells.
DNA isolation.
A standard DNA isolation protocol was used (Luch et al. 1998). Briefly, cell pellets from two T-75 flasks per treatment group were pooled and homogenized in cell lysis buffer [10 mM Tris, 1 mM Na2EDTA, 1% SDS, pH 8]. The homogenates were treated with RNase, DNase-free (50 U/ml) and RNase T1 (1000 U/ml) at 37°C for 1 h, followed by treatment with proteinase K (500 μg/ml) at 37°C for 1 h. The DNA was extracted in light phase gel lock tubes with equal volumes of Tris-equilibrated phenol followed by extraction with 1:1 volume of Tris-equilibrated phenol and chloroform:isoamyl alcohol (24:1) and then with equal volumes of chloroform:isoamyl alcohol (24:1). The aqueous layer was treated with 1/10 volume of 5 M NaCl and twice the volume of cold 100% ethanol to precipitate the DNA which was then dissolved in sterile double-distilled water; its concentration was determined by UV absorbance at 260 nm.
33P-postlabeling of DNA adducts and HPLC.
Post-labeling was carried out as described previously (Ralston et al., 1995). Briefly, 10 μg DNA isolated from MCF-7 cells after treatment were digested with nuclease P1 (0.4 U) and prostatic acid phosphatase (350 mU). It was then post-labeled with [-33P]ATP (3,000 Ci/mmol), cleaved to adducted mononucleotides with snake venom phosphodiesterase I (15 mU) and apyrase (100 mU) followed by pre-purification on a Sep-Pak C18 cartridge (Waters, Milford, MA). Subsequent separation by analytical high performance liquid chromatography (HPLC-Varian system equipped with two pumps and an autosampler; Varian Systems, CA) was carried out using a C18 reverse-phase column (5 μm Ultrasphere ODS, 4.6 x 250 mm). The BP-DNA adducts were resolved by elution at 1 ml/min with 0.1 M ammonium phosphate, pH 5.5 (solvent A) and 100% HPLC grade methanol (solvent B). The elution gradient for BP and SRM 1649a treated samples was as follows: 44–55% solvent B for 5 min, 55–60% solvent B over 5 min, 60–90% solvent B over 20 min and 90–100% solvent B over 5 min. The DBP DNA-adducts were resolved by elution at 1 ml/min with 0.1 M ammonium phosphate, pH 5.5 (solvent A) and 50% HPLC grade methanol/50% acetonitrile (solvent B). The elution gradient for DBP and SRM 1649a was as follows: 20–44% solvent B for 20 min, 44%–60% solvent B over 40 min, and 60–80% solvent B over 15 min. The radiolabeled nucleotides were detected by an on-line 400 μl dry cell -RAM Model 3 (IN/US Systems, Tampa, FL) radioisotope detector and the level of DNA binding was calculated based on the labeling efficiency of a [3H]B[a]P-7,8-dihydrodiol 9,10-epoxide (NCI Chemical Carcinogen Reference Stanadard Repository, Midwest Research Institute, Kansas City, MO) standard (Lau and Baird, 1991). Three independent sets of the postlabeling reactions were carried out for every sample treated, to determine the total PAH-DNA adduct levels.
Microsome isolation.
Microsomes were prepared as described previously (Otto et al., 1991) with minor modifications. Cell culture samples pooled from five T75 flasks for each treatment group were homogenized in a steel homogenizer with microsomal homogenization buffer [0.25 M K2HPO4, 0.15 M KCl, 10 mM EDTA, and 0.25 mM phenylmethylsulfonylfluoride (PMSF)] and were centrifuged at 15,000 x g for 20 min at 4°C in a Sorvall RC-5B refrigerated superspeed centrifuge (DuPont Instruments, Willmington, DE). The supernatant was centrifuged at 100,000 x g for 90 min at 4°C in a Beckman TL-100 Ultracentrifuge (Beckman Coulter, Fullerton, CA), and the pellet was resuspended in microsome dilution buffer (0.1 M KH2PO4, 20% glycerol, 10 mM EDTA, 0.1 mM DTT and 0.25 mM PMSF). The protein concentration was determined in a Turner UV-VIS spectrophotometer SP-890 (Barnstead International, Dubuque, IA) using the Bicinchoninic acid colorimetric assay (Pierce, Rockford, IL). Three independent sets of microsomes were prepared for all the treatment groups.
Ethoxyresorufin O-Deethylation (EROD) assay.
Fifty micrograms of microsomal protein were added to 1 μM 7-ethoxyresorufin (ERES) (Sigma) in 200 μl of 0.1 M Tris HCl (pH 7.8) buffer per well in black 96-well plate (E&K Scientific, Campbell, CA). NADPH (Sigma) was added to each well and the fluorescence was measured in a Spectra MAX Gemini plate reader (Molecular Devices, Sunnyvale, CA). The excitation and emission wavelength were 535 nm and 585 nm, respectively, and the kinetic assay was monitored over 10 min. The experiment was repeated three times and each sample was assayed in triplicate. The amount of resorufin produced was calculated from the fluorescence of a known concentration of resorufin.
Western blots.
Microsomal proteins (50 μg) were denatured by boiling for 3 min and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 8% acrylamide gels (Sigma). After electrophoresis, the proteins were transferred on to a PVDF membrane (Bio-Rad, Hercules, CA) in a Mini Blotter apparatus (Bio-Rad). The membrane was blocked with 1:3 Nap-Sure:PBS-T solution (Geno Technology, St. Louis, MO) and incubated with the primary antibody for 2 h. Human CYP1A1 was detected by a rabbit polyclonal CYP1A1 antibody (1:1500) prepared against purified recombinant human CYP1A1 protein. The CYP1A1 antibody was a gift from Dr. F. P. Guengerich (Center for Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN). Dr. C. Marcus (Department of Pharmacology and Toxicology, University of New Mexico, Albuquerque, NM) prepared and provided the human CYP1B1 rabbit polyclonal antibody (1:1,000). The immunoreactive proteins were detected by incubating the membrane with peroxidase-conjugated anti-rabbit IgG (1:20,000) for 30 min. After washes in PBS-T, the immunoreactive proteins were observed by enhanced chemiluminescence (ECL) detection method, as described by the manufacturer (Amersham Life Science, Arlington Heights, IL). -Actin (protein loading control) was detected by anti--actin primary antibody (1:1500) (Sigma) followed by anti-mouse HRP conjugated secondary antibody (1:20,000). Microsomal proteins (10 μg) from Chinese hamster ovary V79 cells expressing either human CYP1A1 or CYP1B1 were used as positive controls.
Statistical analysis.
DNA adduct data were averaged over one to three replicate reactions and the means were log transformed for analysis due to residuals on the original scale either having right skew or variation increasing with mean. Benzo[a]pyrene and DBP data were analyzed separately because of a few zero values in the DBP data. For log scale analysis of DBP data, four zero values were replaced with a small positive value (half of the smallest positive value observed 0.21/2 = 0.105). The same conclusions were obtained whether means were given equal weight or were weighted according to the number of replicate reactions; results from equally weighted analyses are presented herein. EROD data were averaged over triplicate assays and the means were log transformed for analysis as the variation increased with the response. For the log scale analysis only 3 means (out of 96) were zero, and they were replaced with small positive values (half of the smallest positive value observed within a complete set.
Linear mixed models were used for the analysis of both EROD and adduct data with MIXED procedures in SAS, version 9.1, 2003 (SAS Institute Inc. Cary, NC). Each assay included treatments from a single time point so that treatments could be compared within assays. This resulted in a split-plot type model with two error terms: (1) assays within time points for the main time effect and (2) the final residual for the treatment main effect and treatment-by-time interaction.
RESULTS
Effects of the Co-treatment with SRM 1649a and Carcinogenic PAH on DNA Adduct Formation
The effects of SRM 1649a on DNA adduct formation on co-treatment with carcinogenic PAH, BP, and DBP were determined by 33P-postlabeling and analyzed by HPLC. Representative HPLC elution profiles of the PAH-DNA adducts on co-treatment with BP or DBP after 48 and 120 h are depicted in Figures 1(A,B) and 2(A,B). The retention times vary between Figures 1 and 2A and B, panels 1 and 2, as two different elution gradients were used. MCF-7 cells treated with DMSO (vehicle control) or SRM 1649a alone did not exhibit any detectable levels of DNA adducts (Fig.1A, panel 3, and Fig. 1B, panel 3). The cells treated with SRM 1649a alone (400 μg) contained 1.0036 ng of BP and 18.88 pg of DBP. The presence of such low concentrations of carcinogenic PAHs explains the lack of detection of DNA adduct formation in cells treated with SRM 1649a alone.
The HPLC profile of BP-DNA adducts revealed one major peak eluting at a retention time of 19 min (Fig. 1A,B, panel 1). This major peak corresponded with the (+)-anti-BPDE-dG adduct peak of BP and was the significant adduct resolved in the SRM 1649a and BP co-treatment (Fig. 1A,B panel 2). A reduction in the peak area was observed over time in co-treatment of SRM 1649a and BP in comparison to BP alone (Fig. 1A,B, panels 1 and 2). MCF-7 cells treated with DBP exhibited four major DBPDE-DNA adducts with retention times of 50, 55, 57, and 62 min (Fig. 2A). Over time, a reduction in the peak area was observed in co-treatment of SRM 1649a and DBP (Fig. 2A,B, panels 1 and 2).
Reduction in Total DNA Binding on Co-treatment with SRM 1649a and Carcinogenic PAH Over Time
The level of PAH-DNA adducts formed in MCF-7 cells treated with BP, or in co-treatment with SRM 1649a are graphically represented in Figure 3A. The highest level of DNA binding was observed in cells treated with BP (79.2 pmol/mg of DNA) after 48 h of exposure (Fig. 3A). The mean adduct levels for the BP treatment group was higher in comparison to co-treatment with SRM 1649a. When averaged over time, a significant decrease (p < 0.01) was observed in the total BP-DNA adducts formed in co-treatment with SRM 1649a and BP. Analysis of log transformed data revealed that co-treatment with SRM 1649a and BP there was 2.8-fold less DNA adducts than BP alone. The maximum decrease in the level of DNA binding when co-treated with SRM 1649a and BP was identified at 120 h (3.6 pmol/mg of DNA) in comparison to BP alone (8.66 pmol/mg of DNA), although it was not significant (p = 0.36) (Fig. 3A).
The levels of PAH-DNA adducts formed in MCF-7 cells treated with DBP and co-treatment with SRM 1649a are graphically represented in Figure 3B. The level of DNA adducts formed when treated with DBP alone was relatively stable from 48 h to 120 h, with the highest level of DNA binding (34.2 pmol/mg) after 72 h of exposure (Fig. 3B). On co-treatment with SRM 1649a and DBP there was 18.7-fold less DBP-DNA adducts than with DBP alone. Therefore, a very significant decrease (p < 0.0001) in the total DBP-DNA adducts was observed over time in co-treatment with SRM 1649a and DBP (Fig. 3B). The maximum decrease in the level of DNA binding was identified at 120 h, when the total DNA adducts in cells treated with DBP alone was 19.5 pmol/mg of DNA in comparison to 0.3 pmol/mg of DNA on co-treatment with SRM 1649a plus DBP (Fig. 3B).
Cytochrome P450 Enzyme Activity and Induction
The effect of SRM 1649a in co-treatments with carcinogenic PAH on CYP enzyme activity as measured by EROD assay is illustrated in Figure 4. A significant difference (p < 0.05) in CYP enzyme activity between treatment groups was evident over time. Benzo[a]pyrene alone and co-treatment of SRM 1649a plus BP exhibited the highest EROD activity levels over time. Treatment groups SRM 1649a alone and SRM 1649a plus DBP indicated very low but similar EROD activity levels over time (Fig. 4). Dibenzo[a,l]pyrene alone and DMSO exhibited the lowest level of EROD activity in comparison to other treatment groups analyzed over time.
Western analyses were performed to examine the effect of SRM 1649a alone and in co-treatments with BP or DBP on CYP1A1 and CYP1B1 in MCF-7 cells 24 h, 48 h, and 120 h after exposure (Fig. 5). Treatment with DMSO, DBP or SRM 1649a alone did not induce CYP1A1 protein in the exposure times studied. However, basal levels of CYP1B1 were observed with DMSO, DBP, and SRM 1649a treatment groups 24 h and/or 48 h after exposure (Fig. 5A, 5B, 5A,B). An additive increase in the expression of CYP1A1 and CYP1B1 was observed with co-treatments of SRM 1649a and BP in comparison to BP alone (Fig. 5A,B), whereas in 1649a co-treatment with DBP only at 48 h, basal levels of CYP1B1 protein were observed (Fig. 5A,B). However, treatment with DBP alone revealed no induction of CYP1B1 except at 48 h. At 120 h CYP1A1 induction was observed in BP and co-treatment with SRM 1649a, whereas CYP 1B1 protein was detected in treatment groups BP, BP plus SRM 1649a, and DBP plus SRM 1649a (Fig. 5C).
DISCUSSION
Environmental pollutants from coal tar, diesel exhaust, and urban dust are known to cause DNA damage. The carcinogens present in complex mixtures of environmental pollutants can be metabolized by CYP enzymes producing metabolites that bind to DNA. Recently, in vitro and in vivo experiments have demonstrated the formation of DNA adducts after exposure to a complex mixture containing PAH from coal tar (Cizmas et al., 2004; Mahadevan et al., 2005a, 2005b; Marston et al., 2001), diesel exhaust particulate (Kuljukka-Rabb et al., 2001; Lazarova and Slamenova, 2004; Pohjola et al., 2003; Reliene et al., 2005) and urban dust (Mahadevan et al., 2005a). In an occupational cohort study by Peluso et al. (2001) where they examined the relationship between air pollution exposure and DNA adducts, the authors conclude that the association was significant for both heavily exposed industrial workers and less severely exposed urban workers. Also, DNA adduct levels in exposed workers were significantly correlated with levels of BP in the air (Peluso et al., 2001). In order to understand the possible link to breast cancer and long term effects potentially attributable to exposure to urban dust, we studied the effects of SRM 1649a on MCF-7 cells in culture up to 120 h. An epidemiological study on the short-term exposure (24 h) to SRM 1649a indicated increased nasal secretion levels of interleukins, supporting the observations that short-term increase of outdoor particulate matter concentration increases the frequency of upper respiratory diseases (Riechelmann et al., 2004). Recently, it was also demonstrated that short-term exposure (24 h) of MCF-7 cells to SRM 1649a altered gene expression patterns (Mahadevan et al., 2005a).
Here we discuss the first study involving an extended length of exposure (120 h) to SRM 1649a in vitro to determine if the components of SRM 1649a were competing with the metabolic activation of the carcinogenic PAH in co-treatment with BP or DBP. The level of BP-DNA adducts significantly decreased over time in the presence of SRM 1649a (Fig. 3A). SRM 1649a very significantly decreased the level of DBP-DNA adducts formed over time. This reduction was evident at each time point and was more pronounced at 96 h and 120 h of exposure (Fig. 3B). A similar phenomenon was reported by Binkova and Sram (2004) in human diploid lung fibroblasts where BP-DNA adduct levels were up to fivefold lower in an artificial carcinogenic PAH mixture and up to tenfold lower in an environmental mixture. In our study, it was not clear if the decrease occurred in response to treatment toxicity or removal of DNA adducts by DNA repair and replication. It is possible that the DNA adduct levels reached a saturation point after exposure to the complex mixture and carcinogenic PAH. As reported in a study by (Peluso et al., 2001), the relation between DNA adducts and BP in peripheral blood cells was linear at low doses and sublinear at high doses indicating that DNA adduct formation tends to reach a saturation point at higher levels of exposure to chemical mixtures (Peluso et al., 2001). Further experiments need to be conducted with varying levels of exposure to the urban dust mixture and carcinogenic PAH to test if this may have occurred with co-treatments of SRM 1649a and BP or DBP.
Previous studies on SRM 1649a (Mahadevan et al., 2004, 2005a), as well as this study, suggest that there may be a selective metabolism of certain PAH within complex mixtures, which may be influenced by different CYP enzymes. We hypothesized that the components within SRM 1649a may be affecting the metabolism of BP and DBP by CYP enzymes. Higher EROD activity levels corresponded with protein induction of CYP enzymes as analyzed by Western blots in SRM 1649a co-treatment with BP (Figs. 4 and 5A, B). However, co-treatment with SRM 1649a and DBP did not reveal a clear correlation with regard to EROD activity and CYP1A1 or CYP1B1 induction levels. These results suggest that the two CYP enzymes may have different roles in the metabolic activation of certain carcinogenic PAHs found in environmental mixtures (Diamond and Baird, 1977; Shimada and Fujii-Kuriyama, 2004). The difference in CYP1A1 enzyme activity and protein induction levels observed in co-treatments with SRM 1649a and BP or DBP could also occur because DBP is a poor inducer of CYP1A1-dependent proteins and enzyme activity, as the CYP1A1 induction levels observed were in accordance with induction equivalency factors reported for DBP (Machala et al., 2001).
The decrease in PAH-DNA adduct levels in co-treatment with SRM 1649a (Fig. 3) was similar to a study with complex mixture derived from coal tar (Mahadevan et al., 2005b). This decrease implies that an inhibitory effect of SRM 1649a on CYP enzymes may have resulted in the decreased metabolism of the carcinogenic PAH. Future biochemical experiments are planned with SRM 1649a to elucidate the mechanism of inhibition of CYP enzymes. On the other hand, although there was no decrease in the EROD activity in the BP and SRM 1649a co-treatment (Fig. 4) a decrease in the DNA adduct levels in comparison to BP was observed (Fig. 3A). There is a possibility that this decrease in DNA adduct levels may be due to detoxification of BP metabolites that form DNA adducts by the induced CYP1A1 as discussed in the in vivo studies by Uno et al. (2004). Another possible mechanism by which the components of SRM 1649a could affect the DNA is through the formation of quinones (for example BP-7,8-dione) that could cause oxidative damage to the DNA, as described by Harvey et al. (2004).
Overall, this study indicates that components of a complex mixture may interact with each other to produce synergistic, antagonistic, or additive effects. It also highlights the fact that complex mixture interactions can influence the toxicodynamic properties that may result in enhanced or reduced genotoxicity. Furthermore, the presence of carcinogenic PAH such as BP in complex mixtures is not sufficient to allow prediction of the human risk on exposure to mixtures. Our studies take into account the modifiers within the mixture that may alter the carcinogenic potency of the carcinogenic PAH present. Therefore, these studies are important and may assist in evaluating the carcinogenic potential or toxic equivalency (TE) of several complex PAH mixtures (Cizmas et al., 2004; Petry et al., 1996; Willett et al., 1997).
NOTES
2 These authors contributed equally to this work.
Caution: BP (benzo[a]pyrene) and DBP (dibenzo[a,l]pyrene) are potent carcinogenic agents and should be handled according to National Institutes of Health (NIH) guidelines for the use of carcinogens. SRM 1649a is a suspected human carcinogen and should be handled with the same precautions.
ACKNOWLEDGMENTS
This research was supported by grant CA 28825, DHHS (Department of Health and Human Services) from the National Cancer Institute, National Institutes of Health.
This publication was made possible, in part, by the Nucleic Acid and Protein Service Core, Cell Culture Core and Statistics Core of the Environmental Health Science Center, Oregon State University, funded by grant P30 ES00210 from the National Institute of Environmental Health Sciences. The authors thank Kathleen Yeager and Kendall Dutcher for assistance with DNA and microsome isolations. We also thank Dr. Louisa Hooven for isolating microsomes from V79 cells for use as positive controls; Dan Albershardt for the preparation of figures; Tony Rianprakaisang for organization of references; and Jack Giovanini for help in statistical analysis.
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