Mutagenesis, Vol. 16, No. 4, 353-358,
July 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Time- and dose-dependent DNA binding of PAHs derived from diesel particle extracts, benzo[a]pyrene and 5-methylchrysene in a human mammary carcinoma cell line (MCF-7)
FIOH, Topeliuksenkatu 41b, FIN-00250 Helsinki and 1 Fortum Oil and Gas Oy, Porvoo, Finland
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Abstract |
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Cultures of a human mammary carcinoma cell line (MCF-7) were exposed to the soluble organic fraction of diesel particle emissions, benzo[a]pyrene (B[a]P) and 5-methylchrysene (5-MeCHR) to study time- and dose-related PAH–DNA binding. The concentrations of 14 PAHs in three extracts were analyzed by HPLC and PAH–DNA adducts were measured by 32P post-labeling assay. Time-dependent DNA adducts formation of 2.5 µM B[a]P was lower than that of 2.5 µM 5-MeCHR. In comparison with B[a]P, 2-fold higher adduct formation by 5-MeCHR was observed at 12 h exposure, after which BPDE adducts decreased and 5-MeCHR continued to form adducts linearly during 48 h exposure. The data for these two PAH compounds demonstrate a large variation in adduct-forming potency, which should be taken into account when estimating DNA adducts formed by mixtures of unknown PAHs. A clear dose–response effect on formation of DNA adducts was obtained for B[a]P and a Standard Reference Material (SRM) of diesel particulate matter. The amount of B[a]P contributed more to total DNA adduct formation by SRM than by three diesel extracts. Thus, no conclusions can be drawn from diesel particle-derived B[a]P as to the adduct-forming potency of other carcinogenic PAHs. There was little change in adduct levels formed by three diesel extracts from 0 to 12 h exposure. Thereafter, the number of adducts formed by RD2 increased more rapidly than those formed by RD1 and EN97. The concentrations of 14 PAHs and adduct levels analyzed at 24 and 48 h did not change in the same proportion between the extracts. Neither could PAH–DNA adduct levels be explained by the sum of strong and weak adduct-forming PAHs analyzed in the extracts. This indicates that other PAHs in the extracts RD1, RD2 and EN97 contributed to adduct formation more than the carcinogenic adduct-forming PAHs analyzed in this study.
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Introduction |
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Diesel engine driven vehicles emit oxides of nitrogen (NOx) and particles, which cause a potential health risk. The International Agency for Research on Cancer (IARC) has considered diesel exhausts as a probable carcinogen to humans (International Agency for Research on Cancer, 1989
; Cox, 1997). Investigations have focused on both the chemical composition of exhausts and the size and amount of fine carbon particles to provide methods and models to better understand the possible effects of diesel particulates on human health (Health Effect Institute, 1995). To improve the investigation of particles and source identification, a new standard for fine particles, which have a diameter <2.5 µm (PM2.5), has recently been introduced in USA (Federal Register 1997).
Polycyclic aromatic hydrocarbons (PAHs) are products of incomplete combustion and are commonly found in diesel exhaust. Pollutants in diesel exhausts exist as particles or gas phase compounds and the contribution of nitrated, oxygenated and alkylated PAHs to the mutagenic and carcinogenic activity has been recognized (Scheepers and Bos, 1992; Durant et al., 1996; Rosengranz, 1996). Mutagenicity studies on diesel exhausts have shown that the activity is primarily due to direct acting compounds, such as mono- and dinitro-polyaromatic hydrocarbons (Rosengranz, 1982). When rats or mice were exposed to the soluble organic fraction of diesel exhaust particles the bioavailability of several compounds which are metabolically activated to reactive intermediates was demonstrated (Bond et al., 1990; Gallagher et al., 1994; Savela et al., 1995). However, some studies also focused on the mechanisms by which particles without organic pollutants cause carcinogenic effects (Heinrich et al., 1995; Nikula et al., 1995). Cell culture studies have shown that PAHs are bound to DNA after being activated by cytochrome P450 enzymes to electrophilic metabolites (Einolf et al., 1996; Luch et al., 1999a; Melendez-Colon et al., 2000).
This study investigates time- and dose-dependent DNA adduct formation by PAHs derived from three diesel particulate extracts, diesel particulate matter Standard Reference Material 1650 (SRM), benzo[a]pyrene (B[a]P) and 5-methylchrysene (5-MeCHR) in a human mammary carcinoma cell line (MCF-7).
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Materials and methods |
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Chemicals
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-32P]ATP (7000 Ci/mmol) was obtained from ICN Biochemicals (Costa Mesa, CA), micrococcal nuclease and nuclease P1 were from Sigma Chemical Co. (St Louis, MO), calf spleen phosphodiesterase was from Boehringer Mannheim (Indianapolis, IN) and T4 polynucleotide kinase was from US Biochemical (Cleveland, OH). Polyethyleneimine (PEI)–cellulose TLC plates were from Macherey-Nagel (Duren, Germany). RPMI 1640 medium was purchased from Gibco BRL (Gaithersburg, MD) and Qiagen genomic tips from Qiagen (Chatsworth, CA). SRM was from the National Institute of Standards and Technology (Gaithersburg, MD). All other chemicals were of analytical grade.
Test fuels, vehicle and emission test
Two diesel fuels of reformulated grade (RD1 and RD2) and a standard fuel (EN97) representing a European EN590 specification diesel fuel (sulfur 
0.05 wt%) were used. Details of the fuels, test vehicle and diesel exhaust sampling are reported elsewhere (Kuljukka et al., 1998). Fuel RD1 is a typical summer grade reformulated diesel fuel used in Finland, while fuel RD2 is the Swedish Class 1 specified diesel fuel. The test vehicle was a passenger car without a turbocharger or an oxidation catalyst having done 230 000 km. A modified emission test was carried out at 22°C using European transient exhaust emissions test procedures EU 91/441/EEC and 94/12/EC (Rantanen et al., 1993; Concawe Report, 1997). Regulated emissions, such as total hydrocarbons, carbon monoxide and nitrogen oxides, were analyzed according to directive EU 91/441/EEC.
Sampling and extraction of diesel particles
A dilution tunnel was used to collect diesel exhaust particles on 142 mm diameter Teflon-coated glassfiber filters with a constant flow of 150 l/min at a temperature <50°C. Sampling was performed six or seven times on each day to obtain enough particulate material for testing of each fuel. Filters were extracted with dichloromethane using a Soxhlet apparatus and the amount of soluble organic fraction (SOF) was calculated using the weight difference of the filters before and after extraction. The yield varied from 14 to 25 mg SOF depending on the fuel.
PAH analysis of the extracts
PAHs in SOF were analyzed using HPLC with fluorescence detection (NIOSH, 1998). Benzo[e]pyrene was added to the EPA 610 polynuclear aromatic mixture and was used as a PAH standard mixture. The amounts of the 14 PAHs (fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, B[a]P, dibenz[a,h]anthracene, benzo[ghi]perylene and indeno[1,2,3-cd]pyrene) in three different extracts are given in Table I. The compounds were divided into non-carcinogenic and weak and strong carcinogenic PAHs.
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Treatment of human mammary carcinoma cell line
The MCF-7 cell line, obtained from ABL-Basic Research Program (MD) was cultured according to the method of Agarwal et al. (1997). Approximately 4x106 cells were grown in 5 ml of RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin and 1 ml/100 ml L-glutamine at 37°C in a humidified atmosphere with 5% CO2. The average doubling time for MCF-7 cells was determined by counting the number of living cells using Trypan blue exclusion in two cultures on a day during the following 4 days. The volume of dimethylsulfoxide, in which all samples were diluted, did not exceed 1% of the total culture medium. Cells were exposed in fresh medium for 24 h, except in experiments in which time-dependent formation of adducts was studied. Time-dependent adduct formation of 2.5 µM B[a]P and 2.5 µM 5-MeCHR was estimated at time points of 0, 3, 8, 12, 24 and 48 h. In the dose–response effect study the amounts of B[a]P were 313 (0.25 µM), 1563 (1.25 µM), 3125 (2.5 µM), 3750 (3.0 µM), 4500 (3.6 µM), 5625 (4.5 µM), 6250 (5.0 µM) and 7500 ng (6.0 µM). When the dose–response effect on formation by SRM diesel particulate was studied the concentrations were 17, 33, 84, 100 and 167 µg SOF/ml medium, containing 0.1, 0.2, 0.5, 0.6 and 1.0 ng B[a]P, respectively. Doses of 1.1 mg (220 µg/ml) of each of three diesel extracts (SOF) were used to study time-dependent activation of particulate PAHs in MCF-7 cells.
DNA isolation and adduct analysis
DNA was isolated and purified from cells using Qiagen genomic tips according to the manufacturer's handbook. Aromatic adducts were analyzed by the 32P post-labeling method (Reddy and Randerath, 1986). DNA was first hydrolyzed with micrococcal nuclease and spleen phosphodiesterase and adducted nucleotides were then selected using the nuclease P1 enhancement procedure (Reddy and Randerath, 1986). The enriched adducts were next labeled using T4 polynucleotide kinase and [-32P]ATP. After chromatographic separation on thin layer plates adducts were visualized by autoradiography and quantified based on the specific activity of [-32P]ATP (Reddy and Randerath, 1986).
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Results |
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Autoradiograms resulting from the post-labeled DNA adducts formed in MCF-7 cells exposed to B[a]P, 5-MeCHR, SRM and the RD1, RD2 and EN97 diesel particulate extracts are shown in Figure 1A–F.
B[a]P- and 5-MeCHR-derived adducts eluted as single spots (Figure 1A and B), whereas SRM and all three diesel particulate extracts formed a diagonal radioactive zone of aromatic adducts (Figure 1C–F). The most intense area of the radioactive zone (indicated by an arrow in Figure 1D–F) migrated close to the B[a]P and 5-MeCHR adducts.
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HPLC analysis of the extracts showed that phenanthrene, fluoranthene and pyrene were the most abundant PAHs in all samples. About 90% of the compounds analyzed in SOF were PAHs which contained three or four rings. The amounts of PAHs present in the RD1, RD2 and EN97 extracts were 3907, 2565 and 3076 ng/mg SOF, respectively. The concentrations of B[a]P in the RD1, RD2 and EN97 extracts were 79.5, 57.4 and 48.8 ng/mg SOF, respectively. The sum of non-carcinogenic PAHs was ~80% of the total 14 PAHs, whereas the weak and strong carcinogenic PAHs were ~12 and 8%, respectively.
The formation of B[a]P and 5-MeCHR adducts by 2.5 µM solutions in MCF-7 cells during a 48 h treatment was considerably different (Figure 2A and B). B[a]P-related DNA adduct formation started 3 h after exposure and the number of adducts increased linearly during the first 12 h, after which the number of adducts started to decrease (Figure 2A). In contrast, adduct formation by 5-MeCHR was linear during 48 h exposure (Figure 2B). The maximum number of B[a]P-related adducts was 126/108 normal nucleotides (nt) at 12 h, whereas 5-MeCHR formed 260 adducts/108 nt at the same time point. The maximum number of 5-MeCHR–DNA adducts, 464 adducts/108 nt, was measured after 48 h exposure and at the same time point B[a]P–DNA adducts had already decreased by half from the highest number of adducts (Figure 2).
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Exposure of MCF-7 cells for 24 h to eight doses of B[a]P (313–7500 ng) resulted in a clear dose-related increase in adduct formation (Figure 3A
). A maximum of 311 and a minimum of 6 adducts/108 nt was obtained with 6250 and 313 ng B[a]P, respectively. Adduct levels went down to 130 adducts/108 nt with the highest B[a]P dose of 7500 ng. Dose-dependent formation of adducts was also obtained after exposure of MCF-7 cells to SRM (Figure 3B). Adduct levels of 7, 14 and 6/108 nt were obtained with the three extracts RD1, RD2 and EN97, containing 87.5, 63.1 and 53.5 ng B[a]P, respectively (Figure 3B). The adduct levels of 0.08, 0.22 and 0.11/ng B[a]P formed by the three diesel extracts RD1, RD2 and EN97, respectively, were lower than those for SRM (Figure 3B).
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The levels of strong and weak carcinogenic PAHs analyzed by HPLC and DNA adducts measured at 24 and 48 h exposure are shown in Table II
. The amount of strong carcinogenic PAHs in the extracts increased in the order of RD1 > EN97 
RD2, whereas the sum of strong and weak carcinogenic PAHs was highest in RD1 following by EN97 and RD2. The highest adduct levels were formed in MCF-7 cells exposed to RD2, followed by RD1 and EN97. The number of adducts was not associated with the concentrations of strong and weak carcinogenic PAHs found in the extracts.
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Time-dependent adduct formation by three diesel extracts was determined during 48 h treatment (Figure 4
). After the first 12 h adduct formation by all three extracts was low, but the adducts derived from the RD2 and RD1 extracts started to increase after 24 h and resulted in maximum levels of 18.4 and 11.8 adducts/108 nt, respectively, after 48 h exposure (Figure 4). DNA adducts formed by the diesel extract EN97 remained nearly constant (2.2–6.5 adducts/108 nt) during 24 h treatment and decreased to 4.0 adducts/108 nt after 48 h exposure. The numbers of DNA adducts derived from the diesel extracts RD2 and RD1 were about five and three times higher than adduct levels derived from EN97 after 48 h exposure. Adduct levels at this time point were significantly different between RD2 and EN97 and RD1 and EN97, with P values of 0.00006 and 0.002, respectively.
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Discussion |
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In this study we have analyzed the time- and dose-dependent formation of DNA adducts by PAHs derived from diesel exhaust particles, B[a]P and 5-MeCHR in human MCF-7 cells. Nitrated PAHs have been shown to play an important role in mutagenicity induced by PAHs in diesel particle extracts, but our goal was to study adduct formation by the `non-polar fraction' of the diesel particulates (Durant et al., 1996
; Enya et al., 1997; Fu and Herreno-Saenz, 1999). We deliberately used the nuclease P1 enhanced 32P post-labeling procedure, which excludes DNA adducts formed by nitro-PAHs. Studies carried out in several human cell lines have indicated that CYP1A1, CYP1A2 and CYP1B1 are induced by PAHs in generating DNA-binding metabolites (Christou et al., 1994; Einolf et al., 1997; Li et al., 1998; Spink et al., 1998; Luch et al., 1999b). In MCF-7 cells induction of both CYP1A1 and CYP1B1 has been shown with different levels of expression (Christou et al., 1994; Einolf et al., 1997; Spink et al., 1998). Various forms of cytochrome P450-dependent mixed function oxidases catalyze activation of PAHs, with subsequent intermediate hydrolysis by microsomal epoxide hydrolases (Gelboin, 1980). An additional metabolic pathway of PAHs has been suggested to occur via a one-electron oxidation process catalyzed by cytochrome P450 or peroxidase enzymes, leading to radical cation intermediates (Cavalieri and Rogan, 1995).
In our study B[a]P was converted to DNA adducts in MCF-7 cells with a lower intensity compared to 5-MeCHR. The level of adducts formed by B[a]P was 2- to 15-fold lower than that formed by 5-MeCHR (Figure 2
). The reason for the different levels of adduct formation could be the induction of cytochrome P450 enzymes that have a substrate-specific catalytic activity in MCF-7 cells (Spink et al., 1998). The high level of 5-MeCHR–DNA adducts obtained is in accordance with the data of Reardon et al. (1987), who showed that 32% of 5-MeCHR dihydrodiol epoxide was converted to adducts, but only ~6% of BPDE formed adducts in vitro. Furthermore, some investigators have suggested that methyl substitution in the bay region enhances adduct formation in general and, particularly, with adenine residues in DNA (Melikian et al., 1985; Reardon et al., 1987). Glutathione S-transferase isoenzymes (GSTs) (class hGSTP1-1) may play a different role in the detoxification pathway of diol epoxides of B[a]P and 5-MeCHR (Robertson et al., 1986; Hu et al., 1998). Although no data on the substrate specificity of GSTs on B[a]P and 5-MeCHR in MCF-7 cells is available, studies carried out with alkylating agents and BPDE have indicated that GSTs have substrate specificity and that expression of GST isoenzymes may vary (classes 
, µ and 
) (Swedmark et al., 1992; Chen and Waxman, 1994; Townsend et al., 1998). Not only activation and detoxification reactions but also repair of DNA adducts is an important cellular phenomenon (Sancar, 1994). When five PAH dihydrodiol epoxides were studied in a human cell-free extract nucleotide excision repair was shown to repair stable DNA adducts formed by carcinogens such as B[a]P, benzo[b]fluoranthene and benz[a]anthracene (Braithwaite et al., 1998). It was also demonstrated that BPDE adducts were more efficiently removed from the transcribed strand than from the non-transcribed strand (Chen et al., 1992).
DNA adducts were formed dose-dependently in MCF-7 cells exposed to B[a]P and the diesel particle extract SRM (Figure 3). The lower adduct level formed by 6 µM B[a]P than by 5 µM was not likely due to the cytotoxicity, because exposure of MCF-7 cells to 8 µM dibenzo[a,l]pyrene, a strong carcinogen, did not result in cytotoxicity (Ralston et al., 1997). An exposure time of 24 h was mostly applied in this study to avoid any cytotoxic effects of a long treatment time (Einolf et al., 1996). Estimation of the matrix effect on B[a]P-derived adduct formation is challenging due to a lack of methods to identify DNA adducts formed by a single PAH compound in a complex mixture. The number of adducts formed by SRM, if adjusted for the amount of B[a]P, was much higher (4 adducts/ng B[a]P) than the adduct levels formed by RD1, RD2 and EN97 (0.08, 0.22 and 0.11 adducts/ng B[a]P), suggesting that an induction process may play an important role in adduct formation. It should be noted that SRM was generated from a heavy duty diesel engine, while RD1, RD2 and EN97 were from a light duty vehicle. This difference may contribute to the PAH content of the extracts. Furthermore, the aromatic content of emissions was also affected by several other parameters, such as fuel, size and amount of particles, type of engine, driving style and weather conditions (Health Effect Institute, 1995). Due to the co-elution of several PAH–DNA adducts on TLC, single adducts are difficult to identify and quantify from the radioactive diagonal zone formed by complex mixtures of PAHs. In animal studies synergistic and inhibitory influences of PAHs were observed when complex mixtures and/or B[a]P along with dibenzopyrenes were applied to mouse skin (Springer et al., 1989; Hughes and Phillips, 1990; Culp et al., 2000). In coal tar fed mice dG-N2–BPDE levels were 6-fold higher than indicated by the B[a]P content of the coal tar (Culp et al., 2000). Binding of [3H]B[a]P to mouse epidermal DNA was studied by co-administration of five coal-derived complex mixtures, which showed decreased binding of B[a]P to DNA when applied together with a mixture of PAHs (Hughes and Phillips, 1990). Hughes and Phillips (1990) reported a synergistic interaction between DB[a,e]P and B[a]P, but an inhibitory effect between dibenzo[a,e]pyrene, dibenzo[a,l]pyrene and B[a]P.
The number of adducts was not related to the amounts of the 14 PAHs, neither were the PAH–DNA adduct levels associated with the sum of strong and weak PAHs analyzed in the extracts. This indicates that other PAHs may by more potent in forming adducts in the RD1, RD2 and EN97 extracts than those analyzed. RD2 formed more adducts than RD1 and EN97 at 24 and 48 h exposure. This finding was suprising, because RD2 contained the lowest amounts of strong and weak carcinogenic PAHs (Table II). This observation indicates that RD2 extract contains other DNA adduct-forming PAHs than those in RD1 and EN97 extracts. In addition, variations in the adduct levels formed by each diesel extract could be due to differences in the DNA damage induced by strong and weak carcinogenic PAHs. Hence, DNA adducts formed by diesel extract RD2 are induced by carcinogenic adduct-forming PAHs not analyzed in this study. Adducts formed by RD2 may also be chemically more stable and repaired by a slow nucleotide excision repair pathway (Braithwaite et al., 1998; Melendez-Colon et al., 2000). In contrast, RD1 and EN97 diesel extract could have formed DNA adducts by carcinogenic PAHs which are repaired more efficiently, by base excision repair (Braithwaite et al., 1998; Melendez-Colon et al., 2000).
In several in vitro and in vivo studies both direct and metabolically activated PAH compounds have been analyzed in diesel exhausts (Bond et al., 1990; Gallagher et al., 1991, 1994; Scheepers et al., 1992; Heinrich et al., 1995; Nikula et al., 1995; Savela et al., 1995; Rosengranz, 1996; Enya et al., 1997; Fu and Herreno-Saenz, 1999). We have previously shown that diesel extracts, when incubated in vitro with calf thymus DNA and nitroreductive xanthine oxidase, formed higher levels of adducts than those incubated with S9 rat liver microsomal activation mixture (Kuljukka et al., 1998). This study was conducted to investigate adduct formation by complex mixtures of PAHs derived from diesel particulate extracts, by diesel particulate matter SRM and by B[a]P and 5-MeCHR in MCF-7 cells. Furthermore, adduct-forming PAHs analyzed by HPLC and SRM were used to clarify adduct formation and to optimize the dose and exposure conditions. We were able to show time- and dose-related quantitative and qualitative differences in adduct formation in MCF-7 cells after exposure to PAHs derived from diesel particulate extracts. The 32P post-labeling assay was an applicable method to measure stable PAH–DNA adducts, but the method was unable to identify the specific adducts present in PAH mixtures. These results indicate that carcinogenic PAHs of diesel particle extracts lead to stable DNA adduct formation in MCF-7 cells and to potential risk for humans.
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Acknowledgments |
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The ABL-Basic Research Program, USA, is thanked for the MCF-7 cell line. Ms Nina Tamminen and the personnel of the Chemistry Laboratory in the Fortum Technology Centre are thanked for excellent assistance in several analyses. This work was supported by the Neste Oy Foundation and Technology Development Center of Finland.
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Notes |
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2 To whom correspondence should be addressed. Tel: +358 9 4747 2494; Fax: +358 9 4747 2114; Email: kirsti.savela{at}occuphealth.fi
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References |
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Received on December 18, 2000; accepted on April 6, 2001.
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