Identification of Benzo[a]pyrene 7,8-Diol 9,10-Epoxide N2

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Identification of Benzo[a]pyrene 7,8-Diol 9,10-Epoxide N2-Deoxyguanosine in Human Lung Adenocarcinoma Cells Exposed to Cooking Oil Fumes from Frying Fish under Domestic Conditions Sen-Chih Yang,† Shinn-Nen Jenq,† Zhi Chyang Kang,‡ and Huei Lee*,† Institute of Biochemistry & Toxicology, Chung Shan Medical & Dental College, Taichung, Taiwan, PRC, and Department of Health and Nutrition, Chia Nan University of Pharmacy and Science, Tainan, Taiwan, PRC Received February 22, 2000

Lung cancer is the most common cause of cancer death among women in Taiwan. Epidemiological studies of lung cancer in Chinese women indicate that factors other than cigarette smoking are related to lung cancer risk. One such factor may be exposure to carcinogens formed during the cooking of food. The carcinogenic compounds in oil smoke particulates from Chinese cooking practice have not yet been characterized. To reveal the relationship between the high mortality rate of lung cancer in Chinese women and exposure to cooking oil fumes (COF), DNA adduct formation, induced by COF collected from frying fish under domestic conditions, was assessed in human lung adenocarcinoma CL-3 cell lines using the 32P-postlabeling assay. DNA adduct levels were induced by COF in CL-3 cells in a dosedependent manner. DNA adducts with a diagonal radioactive zone (DRZ) were observed when CL-3 cells were treated with COF. Surprisingly, only one spot of the DNA adduct profile was in the DRZ. The DNA adduct was analyzed by HPLC coupled with an on-line radioactive detector. The retention time of the major DNA adduct corresponded to that of authentic benzo[a]pyrene 7,8-diol 9,10-epoxide N2-deoxyguanonsine (BPDE-N2-dG). Moreover, the mass spectrum of the major DNA adduct in CL-3 cells was confirmed to be BPDE-N2-dG by liquid chromatography/mass spectrometry. In conclusion, BPDE-N2-dG adduct formation in human lung cells supports epidemiological findings of an association between cooking fume exposure and lung cancer in Chinese women.

Introduction Lung cancer is the most common cause of cancer death among women in Taiwan (1). Most Chinese women are nonsmokers, and 60% of female lung cancer patients present with adenocarcinoma (2). Adenocarcinoma is the most common form of lung cancer in nonsmoking women (62.4% of cases) (3), in agreement with the percentage (64.5%) of nonsmoking cases reported in a summary of 16 studies from six countries (4). The aetiology of lung adenocarcinoma may differ from that of squamous cell lung carcinoma which has been demonstrated to be associated with cigarette smoking (4). Epidemiological studies of lung cancer in Chinese women indicate that factors other than cigarette smoking are related to lung cancer risk (5-7). A number of studies have tried to identify other potential risk factors, including passive smoking, incense burning, mosquito coil smoke, domestic coal fires, and cooking oil emissions (8-10). Multiple conditional logistic regression analyses of risk factors of lung cancer in Taiwan reveal that working as a cook is a significant risk factor for adenocarcinoma of the lung (10). * To whom correspondence should be addressed: Professor of Biochemistry, Institute of Toxicology, Chung Shan Medical & Dental College, No. 110, Sec. 1, Chien-Kuo N. Rd., Taichung 40203, Taiwan, ROC. Phone: 886-4-475-9400. Fax: 886-4-472-0407. E-mail: hl@ mercury.csmc.edu.tw. † Chung Shan Medical & Dental College. ‡ Chia Nan University of Pharmacy and Science.

A possible explanation for high lung cancer mortality in nonsmoking Chinese women is exposure to carcinogenic components formed during the cooking of food. The characterization of carcinogenic compounds in oil smoke particulates from Chinese cooking practices is still limited, although the mutagenicity of cooking smoke formed during pan-broiling and frying of lean pork has been demonstrated (11-13). Our recent report indicated that cooking fumes from stir-frying fish contain relatively high amounts of a rodent lung carcinogen, 2-methyl-3,8dimethylimidazo[4,5-f]qunoxaline (MeIQx)1 (14). In this study, we attempted to elucidate other possible carcinogens in cooking oil fumes (COF).

Experimental Procedures Collection and Preparation of COF from Fish Frying. Each pomfret (about 250 g) was fried with 15 mL of soybean oil in a stainless steel wok for 10 min. The fish was added to the wok at an oil temperature of 180 °C, and the temperature 1 Abbreviations: BPDE-N2-dG, benzo[a]pyrene 7,8-diol 9,10-epoxide N2-deoxyguanosine; [3H]BPDE, anti-7,8-diol 9,10-epoxide [3H]B[a]P; COF, cooking oil fumes; LC/MS, liquid chromatography/mass spectrometry; DRZ, diagonal radioactive zone; B[a]P, benzo[a]pyrene; B[b]F, benzo[b]fluranthene; B[g,h,i]P, benzo[g,h,i]perylene; B[a]A, benzo[a]anthracene; B[k]F, benzo[k]fluoranthene; DB[a,h]A, dibenzo[a,h]anthracene; PAH, polycyclic aromatic hydrocarbon; MeIQx, 2-amino3,8-dimethylimidazo[4,5-f]quinoxaline; FBS, fetal bovine serum; NEAA, nonessential amino acids.

10.1021/tx0000419 CCC: $19.00 © 2000 American Chemical Society Published on Web 09/22/2000

Identification of BPDE-N2-dG reached a maximum of 250 °C. The oil smog particulates from frying four pomfrets were collected with a high-volume air sampler through the hood at a flow rate of 1 m3/min. The particulate samples were filtered with high-purity glass filters (Whatman, EPM1000) and extracted with acetone in a shaker as described previously (15). The acetone extracts were weighed and redissolved in the original solvent and stored at -80 °C until assessed via HPLC analysis or 32P-postlabeling. Sample Cleanup and HPLC Analysis for PAH. Polycyclic aromatic hydrocarbon (PAH) fractions in COF were partially concentrated with a Baker-10 column (J. T. Baker Chemical Co., Phillipsburg, NJ), according to the manufacturer’s instructions. The eluant of COF purified through the Baker-10 column was called COF-B. The PAH content of COF-B was determined by HPLC as described previously (16). Cell Culture. Human lung adenocarcinoma CL-3 cells were kindly provided by P. C. Yang (Department of Internal Medicine, College of Medicine, National Taiwan University, Taiwan, ROC). The cells were routinely grown in Dulbecco’s Modified Eagle’s Medium with 10% (v/v) fetal bovine serum (FBS, Gibco), 2 mM glutamine, 10 mM NEAA, a 100 mM sodium pyruvate solution, 5.5% bicarbonate, 100 units of penicillin/mL, and 100 µg of streptomycin/mL. 32P-Postlabeling Assay. Cells were seeded at a density of 1.0 × 106 cells/100 mm dish 3 days prior to experimental manipulation. The cultures were treated with either various concentrations of COF or individual standard PAHs, including benzo[a]pyrene (B[a]P), benzo[a]anthracene (B[a]A), benzo[b]fluoranthene (B[b]F), pyrene, fluoranthene, and benzo[g,h,i]perylene (B[g,h,i]P) for 24 h. Then, the DNA of the cells was extracted using the chloroform/phenol method. 32P-Postlabeling was performed as described previously (17). Briefly, DNA samples (2 µg) were hydrolyzed to deoxyribonucleoside 3′monophosphates by incubation with spleen phosphodiesterase and micrococcal endonuclease at 37 °C for 4 h. The hydrolysates were treated with 6 µg of nuclease P1 at 37 °C for 1 h. The DNA adducts were labeled with [γ-32P]ATP (6000 Ci/mmol) and 4 units of T4 polynucleotide kinase, then separated by TLC on PEI-cellulose plates, and developed in three directions. Autoradiography was carried out on Kodak XAR-5 film. DNA was modified in vitro by anti-7,8-diol 9,10-epoxide [3H]B[a]P ([3H]BPDE), a standard for calibrating the 32P-postlabeling assay, kindly provided by P. P. Fu (National Center for Toxicological Research, Jeffersonville, AR). Adduct levels were calculated as described previously (17). HPLC Analysis of DNA Adducts. HPLC was performed with a Waters 600E multisolvent delivery system with a radioactive detector (β-RAM, Inus System Inc.). Gradient control and data processing were achieved with a Waters Datastation with Waters Baseline 810 software. 32P-Labeling adducts were applied on a PEI-cellulose plate and developed with 0.65 M sodium phosphate (pH 6.0) to remove the free nucleosides. The residual 32P-labeled adducts on the original spot of the PEI plate were scrapped and extracted with 500 µL of 4.0 M pyridinium formate (pH 4.5). The extracts were concentrated using a Speed Vac (Savant, NY), and the residues were dissolved in 500 mM NaH2PO4 (pH 2.5). The extracted 32P-labeled adducts were resolved on an RP-18 column (5 µm particle, 250 mm × 4.6 mm i.d., E. Merck, Darmstadt, Germany) with a linear gradient at a flow rate of 1 mL/min. The mobile phase conditions were the same as those described previously (18). LC/MS Analysis. The mass spectrum was performed by electrospray (VG platform, Fisons). The DNA samples were hydrolyzed with nuclease P1 and alkaline phosphatase and then filtered through a 0.2 µm filter membrane. The residues and authentic [3H]BPDE-N2-dG were injected onto a Nucleosil C18 column (5 µm particle, 250 mm × 2.1 mm i.d.) with a delivery system of a water/acetone/formic acid mixture (50/50/0.1) and a flow rate of 10 µL/min. This makeup flow was necessary to obtain stable ES conditions. The electrospray efficacy was the determining factor for ascertaining when the mobile phase reached the probe tip. Positive ES ionization (MH+) was

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Figure 1. Relationship of the dose response of DNA adduct levels induced by COF in CL-3 cells. Table 1. Amounts of PAHs in COFa PAHs

ng/mg of COF

% of total PAHs

benzo[b]fluoranthene fluoranthene benzo[a]pyrene benzo[g,h,i]perylene benzo[a]anthracene benzo[k]fluoranthene total PAHs

289 ( 70 259 ( 34 186 ( 1 212 ( 43 73 ( 8 59 ( 1 1413 ( 99

21 18 13 15 5 5

a Values are presented as means ( the SD of three independent experiments.

performed using an ionization voltage of -4.5 kV at the probe tip. The cone voltage was set at 30 V, and the source temperature was kept constant at 60 °C. The mass spectrum was acquired over a 600 amu range at 0.5 s/scan during peak elution.

Results 32

P-Postlabeling was used to examine whether COF had the capacity to form DNA adducts in CL-3 cells. As shown in Figure 1, a linear dose-dependent response of DNA adduct levels was observed when CL-3 cells were treated with COF concentrations ranging from 50 to 1000 µg/mL. We suspected that PAHs were responsible for DNA adduct formation; therefore, we analyzed the amounts of PAH in COF-B by HPLC. The largest amounts of PAH in COF were those of pyrene, B[b]F, fluoranthene, B[g,h,i]P, B[a]P, B[a]A, and benzo[k]fluoranthene (B[k]F) (Table 1). To elucidate which PAH was responsible for the DNA adduct of COF, the profile of DNA adducts in CL-3 cells treated with COF was evaluated by 32P-postlabeling and compared with the profiles of DNA adducts induced by each authentic PAH (B[a]P, B[a]A, B[b]F, pyrene, fluoranthene, and B[g,h,i]P). The DNA adduct levels induced by COF-B were 3-fold higher than those induced by COF (Table 2). This result indicated that the PAH fraction in COF may play an important role in DNA adduct formation in CL-3 cells. In addition, certain compound(s) in COF other than the PAH fraction may inhibit the formation of DNA adducts in CL-3 cells. Our finding was consistent with Binkova’s conclusion that the chemical fractionation procedure facilitates the assessment of the DNA adduct-forming ability of different chemical com-

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Table 2. DNA Adduct Levels Induced by COF or Authentic PAH Found in COF in Human Lung Adenocarcinoma CL-3 Cellsa COF or PAH

DNA adduct levels/ 108 nucleotides

OSF OSF-B benzo[b]fluoranthene benzo[a]pyrene benzo[a]anthracene pyrene fluoranthene benzo[g,h,i]perylene

29 ( 4 98 ( 3 30 ( 2 82 ( 1 15 ( 2 ND ND ND

a One hundred micrograms per milliliter of COF and COF-B (PAH fractions in OSF were partially purified through a Baker10 resin column) were used in this experiment. Each of six authentic PAHs (at 2 µM) was used for the treatment of CL-3 cells. Values are presented as means ( the SD of three independent experiments. B[k]F was not used to examine the formation of the DNA adduct in CL-3 cells because the content of B[k]F in total PAHs of COF was less than 5%.

pound classes (19). Table 2 shows that three of seven PAHs (B[a]P, B[b]F, and B[a]A) have the capacity to induce DNA adducts in CL-3 cells. The capacity of DNA adduct formation induced by B[a]P was greater than that induced by B[b]F and B[a]A (Table 2). The adduct profiles of COF and COF-B in CL-3 cells showed an obvious spot, including a diagonal radioactive zone (DRZ) (Figure 2A,B). The adduct profiles of COF were similar to those of B[b]F and B[a]P (Figure 2C,D). However, two adduct spots were revealed in calf thymus DNA treated with B[a]P in the presence of Aroclor 1254-induced rat liver S9 mix, showing that different metabolic activation enzyme systems between rats and humans may result in different B[a]P adduct formation. DNA adduct levels in the specific spot induced by COF and COF-B were about 56 and 68% of the level of total adducts, respectively (Table 2). According to the adduct profile and adduct levels, the major DNA adduct induced by COF in CL-3 cells may be B[a]P or B[b]F. To identify the specific DNA adduct, the 32P-labeled adducts from PEI plates were further analyzed by HPLC. Radiochromatograms of the authentic BPDE-N2-dG adduct, the adducts of B[b]F in calf thymus DNA in the presence of Aroclor 1254-induced rat liver S9, and the adducts of COF-B in CL-3 cells are shown in Figure 3. Two B[b]F-induced DNA adducts were detected with tRs of 29 and 50 min (Figure 3A). The DNA adduct of COF with a tR of 47 min corresponded to those of authentic BPDE-N2-dG adducts (Figure 3B,C). This was confirmed by an increase in the intensity of the peak when the combination of authentic BPDE-N2-dG and DNA adducts of COF was injected onto the HPLC column. The HPLC data showed that the DNA adducts of COF may be BPDE-N2-dG. The mass spectrum of the adduct of COF was further identified by LC/MS. The positive ion electrospray mass spectrum of [3H]BPDE-N2-dG standard, detected at m/z 573 [M + 1]+ and 595 [M + Na]+ and eluted from the HPLC column at 45.0 min, is shown in Figure 4A. A similar mass spectrum of the major DNA adduct of COF was observed (Figure 4B).

Discussion Wok frying whole fish with a small amount of soybean oil (∼15 mL) is common in Chinese domestic life. Extremely large amounts of COF are produced during

Figure 2. 32P-Postlabeling of DNA adduct profiles of COF, COF-B, and authentic PAH in human lung adenocarcinoma CL-3 cells: (A) 100 µg/mL COF, (B) COF-B, equivalent amount of 1 mg of COF in 10 mL of medium, (C) 2 µM B[b]F, (D) 2 µM B[a]P, (E) 2 µM B[a]A, (F) calf thymus DNA treated with B[a]P in the presence of Aroclor 1254-induced rat liver S9 mix, (G) 2 µM DB[a,h]A, and (H) solvent control, DMSO. These autoradiograms were recorded by electronic autoradiography (Packard Instrument Co.).

Chinese cooking practices. Taiwanese women spend about 1-2 h every day in the kitchen preparing meals. Exposure to cooking oil smoke has been demonstrated to be linked with the high incidence of lung cancer in Chinese women, including those in Taiwan, in several epidemiological studies (6-10). Alkylated three- and fourring PAHs in the smoky coal and wood combustion sample extract had been demonstrated to be a significant factor that can be linked to the high lung cancer mortality rates of nonsmoking women in Xuan Wei, China (20, 21). In this study, the COF were prepared from frying fish with a small amount of soybean oil on a wok heating by gas. Thus, we consider that most PAHs in COF come from frying fish, not from heating gas fuel. Li et al. (22) first showed that oil fumes from heated rapeseed oil and soybean oil contain relatively large amounts of carcinogenic PAHs, such as B[a]P and dibenzo[a,h]anthracene (DB[a,h]A). The concentration of DB[a,h]A is 5.7-22.8 times higher than that of B[a]P in oil fumes. However, we did not detect any DB[a,h]A in COF in this study. We suspect that this is due to using soybean oil only.

Identification of BPDE-N2-dG

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Figure 4. Mass spectra of authentic [3H]BPDE-N2-dG (A) and HPLC-purified BPDE-N2-dG (B) that were present in COF.

Figure 3. Radiochromatograms obtained from reversed-phase HPLC analysis of partially purified 32P-labeling DNA from CL-3 cells after treatment with (A) B[b]F, (B) B[a]P, (C) COF-B, or (D) B[a]P with COF-B.

Several lines of evidence show that B[a]P is a model carcinogen which is found ubiquitously in pollutants, such as coke oven and engine exhaust (23-25). Recent studies indicate that BPDE, a B[a]P ultimate metabolite, exhibits strong and selective adduct formation at N2 guanine positions (26, 27). The BPDE-N2-dG adduct has been shown to cause hot spots of p53 gene mutations, in codons 157, 248, and 273, frequently found in human lung cancer (28). In this study, we demonstrated that the DNA adduct of COF is BPDE-N2-dG. This study supports epidemiological findings of an association of lung cancer in Chinese women who are exposed to COF. The oil fumes from cooking fish contain large amounts of airborne particulates. However, fumes generated by heating oil predominately contain volatile organic compounds, such as 1,3-butadiene (29). Airborne particulates

from Chinese cooking processing collected by an air sampler with a glass filter predominately contained PAHs in COF. Few volatile organic compounds were collected under our experimental conditions. Therefore, PAHs in COF from Chinese cooking practices may play a role in the development of lung cancer in Chinese women. Our data showed seven types of PAHs detected in COF. Four of the seven were demonstrated to have the capability to induce DNA adduct formation in CL-3 cells. Surprisingly, only BPDE-N2-dG adducts were predominately detected when the CL-3 cells were treated with COF. Moreover, the DNA adduct levels induced by COF-B, in an equivalent amount of 1 mg of COF, were relatively higher than the observed B[a]P concentration of 2 mM. This result suggests that other PAHs contained in COF synergistically act to increase BPDE-N2-dG adduct formation. This observation is consistent with results found in other in vitro and in vivo studies of complex mixtures (19, 30, 31). Our recent data also demonstrated that B[g,h,i]P plays a synergistic role in increasing the level of B[a]P-DNA adduct formation in human HepG2 cells.2 Thus, chemical interactions in COF may be used to explain why COF has more potent DNA adduct formation than B[a]P alone. Our recent data from enzyme-linked immunosorbent assays using the polyclonal antibody of the BPDE-DNA adduct showed that B[a]P-DNA-like adduct levels in lung tissues from Taiwanese female nonsmokers, including lung cancer 2 Cherng, S.-H., Chen, Y.-M. A., Lin, P., Yang, J.-L., Hsu, S.-L., and Lee, H. (2000) Benzo[g,h,i]perylene synergistically transactivates benzo[a]pyrene-induced CYP1A1 gene expression by Ah receptor pathway (submitted for publication). 3 Cheng, Y.-W., Hsieh, L.-L., Lin, P., Chen, C.-P., Chen, C.-Y., Lin, T.-S., Wu, M.-H., and Lee, H. (2000) Gender difference in DNA adduct levels among non-smoking lung cancer patients (submitted for publication).

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patients and non-cancer controls, are significantly higher than in Taiwanese male nonsmokers.3 This result suggests that high B[a]P-DNA adduct levels in Taiwanese women may be linked to greater exposure to COF than males. Thus, exposure to COF in Taiwanese women appears to be associated with the high lung cancer mortality rate of Taiwanese female nonsmokers. In conclusion, BPDE-N2-dG adducts have been well demonstrated to cause p53 mutations. In this study, our data show that BPDE-N2-dG is predominately formed in human lung adenocarcinoma cells when exposed to COF under Chinese domestic cooking conditions. Our findings support epidemiological results showing the association between exposure to COF and lung cancer incidence in Chinese women, including those in Taiwan.

Acknowledgment. This work was supported by Grants DOH88-HR-611 and DOH88-TD-1050 from the National Health Research Institute, Department of Health, The Executive Yuan, ROC. We thank Ms. Cheryl Robbins for her editorial assistance.

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