Trans, Trans-2,4-Decadienal, a Product Found in Cooking Oil Fumes, Induces Cell Proliferation and Cytokine Production Due to Reactive Oxygen
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《毒物学科学杂志》
Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Kaoshiung, Taiwan, R.O.C.
Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung, Taiwan, R.O.C.
ABSTRACT
Dienaldehydes are by-products of peroxidation of polyunsaturated lipids and commonly found in many foods or food-products. Both National Cancer Institute (NCI) and NTP have expressed great concern on the potential genotoxicity and carcinogenicity of dienaldehydes. Trans, trans-2,4-decadienal (tt-DDE or 2,4-De), a specific type of dienaldehyde, is abundant in heated oils and has been associated with lung adenocarcinoma development in women due to their exposure to oil fumes during cooking. Cultured human bronchial epithelial cells (BEAS-2B cells) were exposed to 0.1 or 1.0 μM tt-DDE for 45 days, and oxidative stress, reactive oxygen species (ROS) production, GSH/GSSG ratio, cell proliferation, and expression of TNF and IL-1 were measured. The results show that tt-DDE induced oxidative stress, an increase in ROS production, and a decrease in GSH/GSSG ratio (glutathione status) in a dose-dependent manner. Treatment of BEAS-2B cells with 1.0 μM tt-DDE for 45 days increased cell proliferation and the expression and release of pro-inflammatory cytokines TNF and IL-1. Cotreatment of BEAS-2B cells with antioxidant N-acetylcysteine prevented tt-DDE-induced cell proliferation and release of cytokines. Therefore, these results suggest that tt-DDE-induced changes may be due to increased ROS production and enhanced oxidative stress. Since increased cell proliferation and the release of TNF- and IL-1 are believed to be involved in tumor promotion, our results suggest that tt-DDE may play a role in cancer promotion. Previous studies on dienaldehydes have focused on their genotoxic or carcinogenic effects in the gastrointestinal tract; the present study suggests a potential new role of tt-DDE as a tumor promoter in human lung epithelial cells.
Key Words: tt-DDE; dienaldehydes; cell proliferation; cytokines.
INTRODUCTION
Trans, trans-2,4-decadienal (tt-DDE or 2,4-De), a specific type of dienaldehyde, is a by-product of peroxidation of polyunsaturated lipids during storage (National Toxicology Program, 1993) or heated oils during cooking (Wu et al., 2001). Ingestion of these lipid peroxidation products and oxidized fats has been reported to induce cellular toxicity in liver and kidney (Hageman et al., 1991), as well as induction of cell proliferation in gastrointestinal epithelial cells (National Toxicology Program, 1993). Furthermore, tt-DDE is known to react with DNA (Carvalho et al., 2001; Loureiro et al., 2000). Indeed, there are increasing concerns for a potential link between the dienaldehydes and development of cancer in humans (Hageman et al., 1991; National Toxicology Program, 1993). Because of a potential role of tt-DDE in human carcinogenesis and its widespread presence in food products, these compounds are regarded as high priority compounds of concern by the National Cancer Institutes (NCI) and National Toxicology Program (NTP) at the National Institutes of Environmental Health Sciences (NIEHS).
It is well recognized that cigarette smoking is a major contributory factor in human lung cancer development (Doll and Peto, 1981). However, several recent epidemiological studies revealed that incidence of lung cancer in nonsmoking women in China, Taiwan, Hong Kong, and Singapore, is increasing at an alarming rate (Ko et al., 2000; Tan et al., 2003; Zhong et al., 1999b). Data from these epidemiological studies also provided strong association between incidence of female lung adenocarcinoma and cooking-oil fume (COF) exposure (Ko et al., 2000; Zhong et al., 1999a,b).
COF is a complex mixture of chemicals, and its precise composition and proportion of its chemical constituents varies with cooking conditions such as type of oil used, food being cooked, and cooking temperature (Shields et al., 1995; Zhu and Wang, 2003). However, regardless of the cooking conditions, upon heating, cooking oils, especially polyunsaturated fats, undergo thermal and oxidative decomposition to yield aldehydes (Warner, 1999). Among these aldehydes, tt-DDE is the major fatty acid decomposition product in the COF (Wu et al., 2001).
Because tt-DDE is commonly found in food products and because of its potential toxicity and carcinogenicity, most attention has been directed toward its effects on both the liver and the gastrointestinal tract (Hageman et al., 1991; National Toxicology Program, 1993). Due to current awareness about tt-DDE, its abundance in COF, and epidemiological evidence showing an association between COF and lung cancer in humans, more information about its biological effects is needed to elucidate its potential health implications.
Although, induction of oxidative stress and genotoxicity by tt-DDE in human lung carcinoma A549 cells has been reported (Wu and Yen, 2004), information about its effects on noncancerous human bronchial epithelial cells is still lacking. In the present study, immortalized human bronchial epithelial cells BEAS-2B were chronically (up to 45 days) exposed to noncytotoxic doses of tt-DDE, and reactive oxygen species (ROS) production, oxidative induction, cell proliferation, and cytokine expression were examined. We believe that present study will provide useful scientific information to further elucidate the toxicological as well as the potential carcinogenic roles of tt-DDE in human lung cells.
MATERIALS AND METHODS
Chemicals.
tt-DDE was purchased from Acros (Geel, Belgium). Dimethylsulfoxide (DMSO), dimethylthiazol-diphenyltetrazolium, 2'-7'-dichlorofluorescein diacetate, Tris, NaCl, dithiothreitol, glycerol, leupeptin, aprotinin, pepstatin A, phenylmethylsulfonyl fluoride, and N-acetylcysteine (NAC) were purchased from Sigma (St. Louis, MO).
Cell culture.
Human bronchial epithelial cell lines (BEAS-2B cells) immortalized with SV40 (American Type Culture Collection, Manassas, VA) were maintained in serum-free LHC-9 medium (BioSource International Inc., Nivelles, Belgium) in a 37°C incubator with a humidified mixture of 5% CO2 and 95% air. The medium was changed twice a week, and cells were passaged by trypsinization every week. The schedule of tt-DDE treatment and the following measurements are summarized in Figure 1.
Dose determination of tt-DDE for the study.
Cytotoxicity of tt-DDE was determined with dimethylthiazol-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 24-well plates and incubated either with 0.1% DMSO or different concentrations of tt-DDE for 48 h. Subsequently, 1 mg/ml MTT was added to medium, and cells were incubated for an additional 4 h. Precipitated formazan was dissolved in 0.5 ml DMSO, and the absorbance was measured at 535 nm. The data is presented as the percentage of controls.
ROS measurement.
Intracellular ROS were detected using 2'-7'-dichlorofluorescein diacetate (DCFDA) as previously described (Frenkel and Gleichauf, 1991). Briefly, after treatment with tt-DDE for 10 min or 30 days, cells were incubated with 50 μM DCFDA for 15 min at 37°C. A flow cytometer (Becton, Dickson and Company, San Jose, CA) was used to detect 2'-7'-dichlorofluorescein (DCF) formed by the reaction of DCFH with intracellular peroxides. Relative levels of intracellular ROS were determined by measuring the mean value of fluorescence per cell. The data is presented as the percentage of DMSO-treated control cells. Each data point indicates the average of four replicates.
Measurement of GSH/GSSG ratio.
BEAS-2B cells were seeded in 10-cm dishes. After tt-DDE treatment for 1 or 30 days, amounts of reduced (GSH) and oxidized (GSSG) glutathione were separately determined by colorimetric method using a glutathione assay kit (Cayman Chemical Company, Ann Arbor, MI), and GSH and GSSG ratio was calculated.
Determination of cell viability during long-term treatment with tt-DDE.
BEAS-2B cells (3 x 104) were seeded in 6-well dishes and treated either with 0.1% DMSO or different concentrations of tt-DDE on the second day. Culture medium was changed every 3 days, and the cells were passaged once a week by trypsinization. DMSO or tt-DDE was present in the media all the time from 2, 22, 32, or 45 days, and cell viability was determined with the MTT assay.
DNA synthesis determination.
Quantification of DNA synthesis was performed by bromodeoxyuridine (BrdU) incorporation using an ELISA kit (Roche Applied Science, Mannhein, Germany). BEAS-2B cells (1 x 104) were seeded in 96-well plates, and after 24 h media containing BrdU was added, and cells were incubated for 4 h. The incorporated BrdU was measured according to the manufacturer's instructions.
Gene expression assessment by cDNA microarray and RT-PCR.
cDNA microarray assay was performed with Human Cancer PathwayFinder Gene Array and Human Inflammatory Cytokines or Receptors Gene Array (SuperArray Bioscience Corp. Frederick, MD). Total cellular RNA was prepared by TriReagent (Life Technologies, Rockville, MD) and chloroform extraction. Synthesis of cDNA was done by RT-PCR using 3 μg of total RNA, M-MLV Reverse Transcriptase (Promega, Madison, WI), and 200 ng of oligo dT (New England BioLabs, Inc., Ipswich, MA). Quantitative PCR of tumor necrosis factor alpha (TNF-), interleukin-1 beta (IL-1), p53, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were performed using the TaqMan Universal PCR Master Mix (Perkin-Elmer Applied Biosystems, Foster City, CA) and analyzed on the ABI PRISM 7700 Sequence Detector System (Perkin-Elmer Applied Biosystem, Foster City, CA). TNF-, IL-1, and GAPDH primers and fluorogenic probes were designed with the assistance of computer program, Primer ExpressTM 1.5 (Perkin-Elmer Applied Biosystem, Foster City, CA) and are listed in Table 1. The p53 primers and probe were the Assay-on-DemandTM Gene Expression Assay Mix (Perkin-Elmer Applied Biosystems, Foster City, CA). Each data point was repeated two times. Quantitative values were obtained from the threshold PCR cycle number (CT) that allows the increase in signal associated with an exponential growth for PCR product starts to be detected. The TNF- and IL-1 expression levels in each sample were normalized to GAPDH expression.
Measurement of TNF- and IL-1 secretion.
BEAS-2B cells (5 x 105 cells/well) were seeded into 12-well dishes and treated with tt-DDE for 45 days. The culture medium was replaced with 0.5 ml serum-free medium per well, and conditioned medium was collected 72 h later. TNF- and IL-1 concentration was determined using the human TNF and IL-1 ELISA kits (Pierce Biotechnology Inc., Rockford, IL) according to manufacturer's instructions.
Treatment of cells with antioxidant N-acetylcysteine (NAC).
BEAS-2B cells were cotreated with 0.5 mM NAC and/or 1 μM tt-DDE for 7 days. Then NAC was removed, and cells were continually treated with either 0.1% DMSO or tt-DDE for a total of 45 days. Cell viability and cytokines expression were determined on day 45 as described above.
Statistics.
Comparisons of data between groups were done by Student's t-test. Comparison of data between NAC cotreated groups was done by one-way ANOVA.
RESULTS
Dose-Response of tt-DDE in BEAS-2B Cells
BEAS-2B cells were exposed to tt-DDE at doses of 0, 1, 2.5, 5, 10, 15, and 20 μM for 48 h. A significant cell loss was detectable at 5 μM dose, and a sharp decline in cell viability at dose levels greater than 10 μM (acute toxic dose) was observed (Fig. 2). Based on this information, a dose at 5 μM tt-DDE was considered to be high-dose exposure, and doses at 1/10 (1 μM) and 1/100 (0.1 μM) of the acute toxic dose level (10 μM) were selected as low-dose exposures.
Oxidative Stress Induced in BEAS-2B Cells by tt-DDE
The tt-DDE-induced oxidative stress was evaluated by two criteria: ROS production and a change in GSH/GSSG ratios. At low (0.1 and 1 μM) and high (5 μM) doses of short-termed treatments, a dose-dependent increase in ROS production was observed after short term of exposure of BEAS-2B cells to either 0.1, 1, or 5 μM tt-DDE. Increases of 110.7, 121.9, and 179.4% in ROS production over controls were observed 10 min after the addition of 0.1, 1, and 5 μM tt-DDE, respectively (Table 2). However, a dose-dependent decline in GSH/GSSG ratio was also evident at 24 h after either 0.1, 1, or 5 μM tt-DDE treatment (Table 2). While the GSH/GSSG ratio in the DMSO-treated control cells were 21.49, the ratios declined to 2.56, 1.24, and 0.16 in cells treated with 0.1, 1, and 5 μM tt-DDE, respectively (Table 2). This decline in GSH/GSSG ratios reflects glutathione status in the cells as a result of tt-DDE treatment.
To explore the low-dose and long-term effects of tt-DDE, BEAS-2B cells were treated with either 0.1 μM or 1 μM tt-DDE for up to 45 days. There was an apparent increase in the number of cells in the tt-DDE-treated cells after 30 days of treatment. In addition, there was also a dose-dependent increase in ROS production (120.6 and 246.4% over control) and decline in GSH/GSSG ratios (0.69 and 0.17) in cells treated with 0.1 μM and 1 μM of tt-DDE, respectively (Table 3). These data suggest that the increased oxidative stress induced by low tt-DDE dose exposure stimulated cell proliferation rather than cell death. However, cells treated with 5 μM tt-DDE could not survive after replating and could not be studied for the long-term effects.
Cell Viability under Low-Dose and Long-Term Treatment with tt-DDE
To further study the increase in cell number by tt-DDE, cells were treated with this compound for different time points (2, 22, 32, and 45 days). A time- and dose-dependent increase in cell growth was observed after 22 days and 32 days in cells treated with either 1 μM or 0.1 μM tt-DDE, respectively (Fig. 3A). By 45 days, highly significant increases in cell growth (approximately 145 and 160% of control) were observed in cells treated with 0.1 and 1 μM tt-DDE, respectively (Fig. 3A).
It was important to demonstrate that the observed tt-DDE-induced cell proliferation, especially at 45 days after the treatment, is accompanied by DNA synthesis. A significant increase in BrdU incorporation (DNA synthesis) into BEAS-2B cells chronically exposed to low levels (0.1 and 1 μM) of tt-DDE was observed (Fig. 3B).
Changes in TNF- and IL-1 Expression and Secretion by Chronic Exposure to Low Doses of tt-DDE
By cDNA microarray analysis, we screened for pro-inflammatory or cancer-related cytokine alterations. Our analysis revealed that TNF- and IL-1 mRNA levels were specifically elevated by exposure to 1 μM tt-DDE for 45 days. This elevation was further confirmed in a real-time RT-PCR study. The expression of TNF- (Fig. 4A) and IL-1 (Fig. 4B) was significantly elevated compared to controls after 45 days of 1 μM tt-DDE exposure. In addition, elevation in gene expression was accompanied by an increased secretion of TNF- and IL-1 into the media (Table 4). Interestingly, treatment of cells with 0.1 μM tt-DDE for 45 days, although it resulted in significant cell proliferation, produced no increase in cytokine expression. However, these results do not exclude the possibility of changes in cytokine expression if the tt-DDE exposure times were further prolonged.
Prevention of tt-DDE Effects by Antioxidant NAC Treatment
The data presented above suggest that tt-DDE-induced increase in oxidative stress (ROS generation and reduction of GSH/GSSG ratio) in the BEAS-2B cells may lead to cell proliferation and induction of TNF- and IL-1 expression and release. To confirm this conclusion, BEAS-2B cells were cotreated with NAC, a known antioxidant that increases cellular glutathione levels. There was a significant induction of cell proliferation, TNF-, and IL-1 expression in the BEAS-2B cells treated with 1 μM tt-DDE alone; however, cotreatment with 0.5 mM NAC effectively prevented all these changes (Figs. 5A, 5B, 5C). These observations affirm that all induction effects of tt-DDE on cell proliferation and cytokine expression and release may be the result of increased oxidative stress and oxidation of glutathione in the cells.
DISCUSSION
Our results show an increase in oxidative stress and ROS production and a decrease in GSH/GSSG ratio as a result of exposure of immortalized BEAS-2B cells to tt-DDE. This phenomenon was demonstrated in both short-term (48 h) acute exposure with high doses (1–5 μM) of tt-DDE as well as in chronic (up to 30 days) lower-dose (0.1–1 μM) exposures. Recently, Wu and Yen (2004) demonstrated that high doses (50–200 μM) of tt-DDE induced oxidative stress and oxidative DNA damages in A549 cells, a human lung cancer cell line. While this study was informative, the doses used were very high and cytotoxic. Our preliminary study has shown that BEAS-2B cells were much more vulnerable to tt-DDE toxicity than the human lung cancer cell lines, including A549. Thus, we believe that selecting BEAS-2B cells and exposing them to low toxic doses of tt-DDE represents a more physiological approach. Our finding, that chronic exposure of BEAS-2B cells to an extremely low level (0.1 μM) of tt-DDE still resulted in ROS accumulation and a significant suppression of GSH/GSSG ratio, is an important observation. This observation suggests that prolonged exposure to noncytotoxic low levels of tt-DDE can significantly compromise the glutathione status and induce oxidative stress in BEAS-2B cells. With sustained ROS production and elevated oxidative stress for a prolonged period, we believe risk of DNA damage and other potential pathological consequences, including carcinogenesis, would be increased.
Our results also show that an increase in oxidative stress was followed by a significant increase in cell proliferation and DNA synthesis. Enhancement of the expression and release of pro-inflammatory cytokines TNF- and IL-1 in tt-DDE-treated cells was also observed. It has been well documented that enhanced ROS production and accumulation could induce increases in cell proliferation and cytokine secretion, which in turn contribute to tumor promotion (Haddad et al., 2001; Klaunig et al., 1998; Nguyen-Ba and Vasseur, 1999; Updyke et al., 1989). Hanahan and Weinberg (2000) proposed that excessive cell growth capacity is acquired, via self-sufficiency in growth signals and/or insensitivity to anti-growth signals, in the early steps of carcinogenesis. Haddad et al. (2001) also indicated that accumulation of ROS in alveolar epithelial cell cultures was accompanied by an increased release of TNF- and IL-1. Since both TNF- and IL-1 are pro-inflammatory cytokines, they are commonly found during "inflammatory conditions." However, aside from inflammatory association, prolonged elevation of these cytokines is known to be involved in early stages of tumor promotion (Lappalainen et al., 2005; Moore et al., 1999). Indeed, TNF- has been closely associated with TPA-induced cellular hyperproliferation and tumor growth in mouse skin (Moore et al., 1999; Suganuma et al., 1999). IL-1 increased the number of transformed foci in v-Ha-ras-transfected BALA/3T3 cells (Suganuma et al., 1999). Thus both TNF- and IL-1, aside from their association with inflammatory response, are also considered to have tumor promotion activities (Moore et al., 1999; Suganuma et al., 1999; Updyke et al., 1989). Our present observations on elevated TNF- and IL-1 levels in the tt-DDE-exposed BEAS-2B cells provide the needed scientific platform to suggest that prolonged exposure to tt-DDE would increase the risk of lung cancer development. Future animal studies are needed to confirm this suggestion.
To confirm our hypothesis that oxidative stress (ROS production and decrease in GSH/GSSG ratio) was indeed involved in the induction of cell proliferation and cytokine release, cotreatment of tt-DDE-exposed cells with antioxidant (NAC) prevented the enhancement of cell proliferation as well as cytokine expression induced by tt-DDE. This observation indicated that, while tt-DDE induced oxidation of glutathione via ROS production reduces GSH/GSSG ratio, NAC protects the cells from oxidative stress by stimulating glutathione production. NAC is a precursor of glutathione and has been shown to prevent or reduce particulate-induced lung injury in vivo (Dick et al., 2003; Rhoden et al., 2004). Indeed, glutathione is a major antioxidant in the lung, and its status is considered to be critically important for the integrity of the lung tissues (Rahman et al., 1999). It is concluded that increased cell proliferation and release of pro-inflammatory cytokines are the consequence of induced oxidative stress in tt-DDE-treated lung epithelial cells.
It is worth mentioning that, although an increase in cell proliferation was observed in the BEAS-2B cells treated with low doses (0.1 and 1 μM) of tt-DDE for 45 days, cell transformation, as evaluated by anchorage-independent assay, was still not observed at this time point (data not shown). Nevertheless, tumor promoters are known to induce clonal selection and expansion which could carry endogenous and/or carcinogen-induced mutations (Rubin, 2002). Since tt-DDE has been demonstrated to be genotoxic and mutagenic (Wu and Yen, 2004; Wu et al., 2001), we believe that clonal selection and expansion in tt-DDE-treated BEAS-2B cells can occur under longer treatment conditions (60, 90, or 120 days). Attempts are currently being made in our laboratory to establish clonal selection and expansion carrying gene mutations. Further experiments are planned for genotoxicity investigations and to demonstrate cell transformation by tt-DDE.
In summary, the present investigation represents the first study focused on the effects of tt-DDE, a major type of dienaldehyde, on immortalized human lung epithelial cells. Our results provide clear evidences that tt-DDE induces ROS production and oxidative stress in human lung cells that may lead to induction of pro-inflammatory responses such as enhanced cell proliferation and increased cytokine TNF- and IL-1 release. Since enhancement of cell proliferation and release of these pro-inflammatory cytokines are known to be involved in early stages of tumor promotion (Lappalainen et al., 2005; Moore et al., 1999; Suganuma et al., 1999), our findings strongly suggest that tt-DDE, a component in COF, could be a tumor promoter in human lung epithelial cells and may play a potentially important role in the development of lung adenocarcinoma (Ko et al., 2000; Tan et al., 2003; Zhong et al., 1999a,b). While ingestion of tt-DDE was previously believed to be a major factor, we suggest that its inhalation is equally important, especially for personnel who operate deep frying facilities. In support of NTP and NCI's concerns, we strongly suggest that future investigations on the carcinogenic potential of tt-DDE and other dienaldehydes are warranted both in vitro and in vivo in human subjects.
ACKNOWLEDGMENTS
This work was supported by research grant, DOH94-TD-G-111-010, from the National Research Program for Genomic Medicine and Department of Health, and EO-093-PP-03 from Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Taiwan, ROC. The scientific content of this manuscript does not necessarily signify the view and policies of DOH and DEHOM/NHRI or condemn, endorse, or recommend for use on this issue presented. Conflict of interest: The scientific content of this manuscript does not necessarily signify the content reflects the view and policies of DOH and DEHOM/NHRI or condemnation or endorsement and recommendation for use on this issue presented.
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Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung, Taiwan, R.O.C.
ABSTRACT
Dienaldehydes are by-products of peroxidation of polyunsaturated lipids and commonly found in many foods or food-products. Both National Cancer Institute (NCI) and NTP have expressed great concern on the potential genotoxicity and carcinogenicity of dienaldehydes. Trans, trans-2,4-decadienal (tt-DDE or 2,4-De), a specific type of dienaldehyde, is abundant in heated oils and has been associated with lung adenocarcinoma development in women due to their exposure to oil fumes during cooking. Cultured human bronchial epithelial cells (BEAS-2B cells) were exposed to 0.1 or 1.0 μM tt-DDE for 45 days, and oxidative stress, reactive oxygen species (ROS) production, GSH/GSSG ratio, cell proliferation, and expression of TNF and IL-1 were measured. The results show that tt-DDE induced oxidative stress, an increase in ROS production, and a decrease in GSH/GSSG ratio (glutathione status) in a dose-dependent manner. Treatment of BEAS-2B cells with 1.0 μM tt-DDE for 45 days increased cell proliferation and the expression and release of pro-inflammatory cytokines TNF and IL-1. Cotreatment of BEAS-2B cells with antioxidant N-acetylcysteine prevented tt-DDE-induced cell proliferation and release of cytokines. Therefore, these results suggest that tt-DDE-induced changes may be due to increased ROS production and enhanced oxidative stress. Since increased cell proliferation and the release of TNF- and IL-1 are believed to be involved in tumor promotion, our results suggest that tt-DDE may play a role in cancer promotion. Previous studies on dienaldehydes have focused on their genotoxic or carcinogenic effects in the gastrointestinal tract; the present study suggests a potential new role of tt-DDE as a tumor promoter in human lung epithelial cells.
Key Words: tt-DDE; dienaldehydes; cell proliferation; cytokines.
INTRODUCTION
Trans, trans-2,4-decadienal (tt-DDE or 2,4-De), a specific type of dienaldehyde, is a by-product of peroxidation of polyunsaturated lipids during storage (National Toxicology Program, 1993) or heated oils during cooking (Wu et al., 2001). Ingestion of these lipid peroxidation products and oxidized fats has been reported to induce cellular toxicity in liver and kidney (Hageman et al., 1991), as well as induction of cell proliferation in gastrointestinal epithelial cells (National Toxicology Program, 1993). Furthermore, tt-DDE is known to react with DNA (Carvalho et al., 2001; Loureiro et al., 2000). Indeed, there are increasing concerns for a potential link between the dienaldehydes and development of cancer in humans (Hageman et al., 1991; National Toxicology Program, 1993). Because of a potential role of tt-DDE in human carcinogenesis and its widespread presence in food products, these compounds are regarded as high priority compounds of concern by the National Cancer Institutes (NCI) and National Toxicology Program (NTP) at the National Institutes of Environmental Health Sciences (NIEHS).
It is well recognized that cigarette smoking is a major contributory factor in human lung cancer development (Doll and Peto, 1981). However, several recent epidemiological studies revealed that incidence of lung cancer in nonsmoking women in China, Taiwan, Hong Kong, and Singapore, is increasing at an alarming rate (Ko et al., 2000; Tan et al., 2003; Zhong et al., 1999b). Data from these epidemiological studies also provided strong association between incidence of female lung adenocarcinoma and cooking-oil fume (COF) exposure (Ko et al., 2000; Zhong et al., 1999a,b).
COF is a complex mixture of chemicals, and its precise composition and proportion of its chemical constituents varies with cooking conditions such as type of oil used, food being cooked, and cooking temperature (Shields et al., 1995; Zhu and Wang, 2003). However, regardless of the cooking conditions, upon heating, cooking oils, especially polyunsaturated fats, undergo thermal and oxidative decomposition to yield aldehydes (Warner, 1999). Among these aldehydes, tt-DDE is the major fatty acid decomposition product in the COF (Wu et al., 2001).
Because tt-DDE is commonly found in food products and because of its potential toxicity and carcinogenicity, most attention has been directed toward its effects on both the liver and the gastrointestinal tract (Hageman et al., 1991; National Toxicology Program, 1993). Due to current awareness about tt-DDE, its abundance in COF, and epidemiological evidence showing an association between COF and lung cancer in humans, more information about its biological effects is needed to elucidate its potential health implications.
Although, induction of oxidative stress and genotoxicity by tt-DDE in human lung carcinoma A549 cells has been reported (Wu and Yen, 2004), information about its effects on noncancerous human bronchial epithelial cells is still lacking. In the present study, immortalized human bronchial epithelial cells BEAS-2B were chronically (up to 45 days) exposed to noncytotoxic doses of tt-DDE, and reactive oxygen species (ROS) production, oxidative induction, cell proliferation, and cytokine expression were examined. We believe that present study will provide useful scientific information to further elucidate the toxicological as well as the potential carcinogenic roles of tt-DDE in human lung cells.
MATERIALS AND METHODS
Chemicals.
tt-DDE was purchased from Acros (Geel, Belgium). Dimethylsulfoxide (DMSO), dimethylthiazol-diphenyltetrazolium, 2'-7'-dichlorofluorescein diacetate, Tris, NaCl, dithiothreitol, glycerol, leupeptin, aprotinin, pepstatin A, phenylmethylsulfonyl fluoride, and N-acetylcysteine (NAC) were purchased from Sigma (St. Louis, MO).
Cell culture.
Human bronchial epithelial cell lines (BEAS-2B cells) immortalized with SV40 (American Type Culture Collection, Manassas, VA) were maintained in serum-free LHC-9 medium (BioSource International Inc., Nivelles, Belgium) in a 37°C incubator with a humidified mixture of 5% CO2 and 95% air. The medium was changed twice a week, and cells were passaged by trypsinization every week. The schedule of tt-DDE treatment and the following measurements are summarized in Figure 1.
Dose determination of tt-DDE for the study.
Cytotoxicity of tt-DDE was determined with dimethylthiazol-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 24-well plates and incubated either with 0.1% DMSO or different concentrations of tt-DDE for 48 h. Subsequently, 1 mg/ml MTT was added to medium, and cells were incubated for an additional 4 h. Precipitated formazan was dissolved in 0.5 ml DMSO, and the absorbance was measured at 535 nm. The data is presented as the percentage of controls.
ROS measurement.
Intracellular ROS were detected using 2'-7'-dichlorofluorescein diacetate (DCFDA) as previously described (Frenkel and Gleichauf, 1991). Briefly, after treatment with tt-DDE for 10 min or 30 days, cells were incubated with 50 μM DCFDA for 15 min at 37°C. A flow cytometer (Becton, Dickson and Company, San Jose, CA) was used to detect 2'-7'-dichlorofluorescein (DCF) formed by the reaction of DCFH with intracellular peroxides. Relative levels of intracellular ROS were determined by measuring the mean value of fluorescence per cell. The data is presented as the percentage of DMSO-treated control cells. Each data point indicates the average of four replicates.
Measurement of GSH/GSSG ratio.
BEAS-2B cells were seeded in 10-cm dishes. After tt-DDE treatment for 1 or 30 days, amounts of reduced (GSH) and oxidized (GSSG) glutathione were separately determined by colorimetric method using a glutathione assay kit (Cayman Chemical Company, Ann Arbor, MI), and GSH and GSSG ratio was calculated.
Determination of cell viability during long-term treatment with tt-DDE.
BEAS-2B cells (3 x 104) were seeded in 6-well dishes and treated either with 0.1% DMSO or different concentrations of tt-DDE on the second day. Culture medium was changed every 3 days, and the cells were passaged once a week by trypsinization. DMSO or tt-DDE was present in the media all the time from 2, 22, 32, or 45 days, and cell viability was determined with the MTT assay.
DNA synthesis determination.
Quantification of DNA synthesis was performed by bromodeoxyuridine (BrdU) incorporation using an ELISA kit (Roche Applied Science, Mannhein, Germany). BEAS-2B cells (1 x 104) were seeded in 96-well plates, and after 24 h media containing BrdU was added, and cells were incubated for 4 h. The incorporated BrdU was measured according to the manufacturer's instructions.
Gene expression assessment by cDNA microarray and RT-PCR.
cDNA microarray assay was performed with Human Cancer PathwayFinder Gene Array and Human Inflammatory Cytokines or Receptors Gene Array (SuperArray Bioscience Corp. Frederick, MD). Total cellular RNA was prepared by TriReagent (Life Technologies, Rockville, MD) and chloroform extraction. Synthesis of cDNA was done by RT-PCR using 3 μg of total RNA, M-MLV Reverse Transcriptase (Promega, Madison, WI), and 200 ng of oligo dT (New England BioLabs, Inc., Ipswich, MA). Quantitative PCR of tumor necrosis factor alpha (TNF-), interleukin-1 beta (IL-1), p53, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were performed using the TaqMan Universal PCR Master Mix (Perkin-Elmer Applied Biosystems, Foster City, CA) and analyzed on the ABI PRISM 7700 Sequence Detector System (Perkin-Elmer Applied Biosystem, Foster City, CA). TNF-, IL-1, and GAPDH primers and fluorogenic probes were designed with the assistance of computer program, Primer ExpressTM 1.5 (Perkin-Elmer Applied Biosystem, Foster City, CA) and are listed in Table 1. The p53 primers and probe were the Assay-on-DemandTM Gene Expression Assay Mix (Perkin-Elmer Applied Biosystems, Foster City, CA). Each data point was repeated two times. Quantitative values were obtained from the threshold PCR cycle number (CT) that allows the increase in signal associated with an exponential growth for PCR product starts to be detected. The TNF- and IL-1 expression levels in each sample were normalized to GAPDH expression.
Measurement of TNF- and IL-1 secretion.
BEAS-2B cells (5 x 105 cells/well) were seeded into 12-well dishes and treated with tt-DDE for 45 days. The culture medium was replaced with 0.5 ml serum-free medium per well, and conditioned medium was collected 72 h later. TNF- and IL-1 concentration was determined using the human TNF and IL-1 ELISA kits (Pierce Biotechnology Inc., Rockford, IL) according to manufacturer's instructions.
Treatment of cells with antioxidant N-acetylcysteine (NAC).
BEAS-2B cells were cotreated with 0.5 mM NAC and/or 1 μM tt-DDE for 7 days. Then NAC was removed, and cells were continually treated with either 0.1% DMSO or tt-DDE for a total of 45 days. Cell viability and cytokines expression were determined on day 45 as described above.
Statistics.
Comparisons of data between groups were done by Student's t-test. Comparison of data between NAC cotreated groups was done by one-way ANOVA.
RESULTS
Dose-Response of tt-DDE in BEAS-2B Cells
BEAS-2B cells were exposed to tt-DDE at doses of 0, 1, 2.5, 5, 10, 15, and 20 μM for 48 h. A significant cell loss was detectable at 5 μM dose, and a sharp decline in cell viability at dose levels greater than 10 μM (acute toxic dose) was observed (Fig. 2). Based on this information, a dose at 5 μM tt-DDE was considered to be high-dose exposure, and doses at 1/10 (1 μM) and 1/100 (0.1 μM) of the acute toxic dose level (10 μM) were selected as low-dose exposures.
Oxidative Stress Induced in BEAS-2B Cells by tt-DDE
The tt-DDE-induced oxidative stress was evaluated by two criteria: ROS production and a change in GSH/GSSG ratios. At low (0.1 and 1 μM) and high (5 μM) doses of short-termed treatments, a dose-dependent increase in ROS production was observed after short term of exposure of BEAS-2B cells to either 0.1, 1, or 5 μM tt-DDE. Increases of 110.7, 121.9, and 179.4% in ROS production over controls were observed 10 min after the addition of 0.1, 1, and 5 μM tt-DDE, respectively (Table 2). However, a dose-dependent decline in GSH/GSSG ratio was also evident at 24 h after either 0.1, 1, or 5 μM tt-DDE treatment (Table 2). While the GSH/GSSG ratio in the DMSO-treated control cells were 21.49, the ratios declined to 2.56, 1.24, and 0.16 in cells treated with 0.1, 1, and 5 μM tt-DDE, respectively (Table 2). This decline in GSH/GSSG ratios reflects glutathione status in the cells as a result of tt-DDE treatment.
To explore the low-dose and long-term effects of tt-DDE, BEAS-2B cells were treated with either 0.1 μM or 1 μM tt-DDE for up to 45 days. There was an apparent increase in the number of cells in the tt-DDE-treated cells after 30 days of treatment. In addition, there was also a dose-dependent increase in ROS production (120.6 and 246.4% over control) and decline in GSH/GSSG ratios (0.69 and 0.17) in cells treated with 0.1 μM and 1 μM of tt-DDE, respectively (Table 3). These data suggest that the increased oxidative stress induced by low tt-DDE dose exposure stimulated cell proliferation rather than cell death. However, cells treated with 5 μM tt-DDE could not survive after replating and could not be studied for the long-term effects.
Cell Viability under Low-Dose and Long-Term Treatment with tt-DDE
To further study the increase in cell number by tt-DDE, cells were treated with this compound for different time points (2, 22, 32, and 45 days). A time- and dose-dependent increase in cell growth was observed after 22 days and 32 days in cells treated with either 1 μM or 0.1 μM tt-DDE, respectively (Fig. 3A). By 45 days, highly significant increases in cell growth (approximately 145 and 160% of control) were observed in cells treated with 0.1 and 1 μM tt-DDE, respectively (Fig. 3A).
It was important to demonstrate that the observed tt-DDE-induced cell proliferation, especially at 45 days after the treatment, is accompanied by DNA synthesis. A significant increase in BrdU incorporation (DNA synthesis) into BEAS-2B cells chronically exposed to low levels (0.1 and 1 μM) of tt-DDE was observed (Fig. 3B).
Changes in TNF- and IL-1 Expression and Secretion by Chronic Exposure to Low Doses of tt-DDE
By cDNA microarray analysis, we screened for pro-inflammatory or cancer-related cytokine alterations. Our analysis revealed that TNF- and IL-1 mRNA levels were specifically elevated by exposure to 1 μM tt-DDE for 45 days. This elevation was further confirmed in a real-time RT-PCR study. The expression of TNF- (Fig. 4A) and IL-1 (Fig. 4B) was significantly elevated compared to controls after 45 days of 1 μM tt-DDE exposure. In addition, elevation in gene expression was accompanied by an increased secretion of TNF- and IL-1 into the media (Table 4). Interestingly, treatment of cells with 0.1 μM tt-DDE for 45 days, although it resulted in significant cell proliferation, produced no increase in cytokine expression. However, these results do not exclude the possibility of changes in cytokine expression if the tt-DDE exposure times were further prolonged.
Prevention of tt-DDE Effects by Antioxidant NAC Treatment
The data presented above suggest that tt-DDE-induced increase in oxidative stress (ROS generation and reduction of GSH/GSSG ratio) in the BEAS-2B cells may lead to cell proliferation and induction of TNF- and IL-1 expression and release. To confirm this conclusion, BEAS-2B cells were cotreated with NAC, a known antioxidant that increases cellular glutathione levels. There was a significant induction of cell proliferation, TNF-, and IL-1 expression in the BEAS-2B cells treated with 1 μM tt-DDE alone; however, cotreatment with 0.5 mM NAC effectively prevented all these changes (Figs. 5A, 5B, 5C). These observations affirm that all induction effects of tt-DDE on cell proliferation and cytokine expression and release may be the result of increased oxidative stress and oxidation of glutathione in the cells.
DISCUSSION
Our results show an increase in oxidative stress and ROS production and a decrease in GSH/GSSG ratio as a result of exposure of immortalized BEAS-2B cells to tt-DDE. This phenomenon was demonstrated in both short-term (48 h) acute exposure with high doses (1–5 μM) of tt-DDE as well as in chronic (up to 30 days) lower-dose (0.1–1 μM) exposures. Recently, Wu and Yen (2004) demonstrated that high doses (50–200 μM) of tt-DDE induced oxidative stress and oxidative DNA damages in A549 cells, a human lung cancer cell line. While this study was informative, the doses used were very high and cytotoxic. Our preliminary study has shown that BEAS-2B cells were much more vulnerable to tt-DDE toxicity than the human lung cancer cell lines, including A549. Thus, we believe that selecting BEAS-2B cells and exposing them to low toxic doses of tt-DDE represents a more physiological approach. Our finding, that chronic exposure of BEAS-2B cells to an extremely low level (0.1 μM) of tt-DDE still resulted in ROS accumulation and a significant suppression of GSH/GSSG ratio, is an important observation. This observation suggests that prolonged exposure to noncytotoxic low levels of tt-DDE can significantly compromise the glutathione status and induce oxidative stress in BEAS-2B cells. With sustained ROS production and elevated oxidative stress for a prolonged period, we believe risk of DNA damage and other potential pathological consequences, including carcinogenesis, would be increased.
Our results also show that an increase in oxidative stress was followed by a significant increase in cell proliferation and DNA synthesis. Enhancement of the expression and release of pro-inflammatory cytokines TNF- and IL-1 in tt-DDE-treated cells was also observed. It has been well documented that enhanced ROS production and accumulation could induce increases in cell proliferation and cytokine secretion, which in turn contribute to tumor promotion (Haddad et al., 2001; Klaunig et al., 1998; Nguyen-Ba and Vasseur, 1999; Updyke et al., 1989). Hanahan and Weinberg (2000) proposed that excessive cell growth capacity is acquired, via self-sufficiency in growth signals and/or insensitivity to anti-growth signals, in the early steps of carcinogenesis. Haddad et al. (2001) also indicated that accumulation of ROS in alveolar epithelial cell cultures was accompanied by an increased release of TNF- and IL-1. Since both TNF- and IL-1 are pro-inflammatory cytokines, they are commonly found during "inflammatory conditions." However, aside from inflammatory association, prolonged elevation of these cytokines is known to be involved in early stages of tumor promotion (Lappalainen et al., 2005; Moore et al., 1999). Indeed, TNF- has been closely associated with TPA-induced cellular hyperproliferation and tumor growth in mouse skin (Moore et al., 1999; Suganuma et al., 1999). IL-1 increased the number of transformed foci in v-Ha-ras-transfected BALA/3T3 cells (Suganuma et al., 1999). Thus both TNF- and IL-1, aside from their association with inflammatory response, are also considered to have tumor promotion activities (Moore et al., 1999; Suganuma et al., 1999; Updyke et al., 1989). Our present observations on elevated TNF- and IL-1 levels in the tt-DDE-exposed BEAS-2B cells provide the needed scientific platform to suggest that prolonged exposure to tt-DDE would increase the risk of lung cancer development. Future animal studies are needed to confirm this suggestion.
To confirm our hypothesis that oxidative stress (ROS production and decrease in GSH/GSSG ratio) was indeed involved in the induction of cell proliferation and cytokine release, cotreatment of tt-DDE-exposed cells with antioxidant (NAC) prevented the enhancement of cell proliferation as well as cytokine expression induced by tt-DDE. This observation indicated that, while tt-DDE induced oxidation of glutathione via ROS production reduces GSH/GSSG ratio, NAC protects the cells from oxidative stress by stimulating glutathione production. NAC is a precursor of glutathione and has been shown to prevent or reduce particulate-induced lung injury in vivo (Dick et al., 2003; Rhoden et al., 2004). Indeed, glutathione is a major antioxidant in the lung, and its status is considered to be critically important for the integrity of the lung tissues (Rahman et al., 1999). It is concluded that increased cell proliferation and release of pro-inflammatory cytokines are the consequence of induced oxidative stress in tt-DDE-treated lung epithelial cells.
It is worth mentioning that, although an increase in cell proliferation was observed in the BEAS-2B cells treated with low doses (0.1 and 1 μM) of tt-DDE for 45 days, cell transformation, as evaluated by anchorage-independent assay, was still not observed at this time point (data not shown). Nevertheless, tumor promoters are known to induce clonal selection and expansion which could carry endogenous and/or carcinogen-induced mutations (Rubin, 2002). Since tt-DDE has been demonstrated to be genotoxic and mutagenic (Wu and Yen, 2004; Wu et al., 2001), we believe that clonal selection and expansion in tt-DDE-treated BEAS-2B cells can occur under longer treatment conditions (60, 90, or 120 days). Attempts are currently being made in our laboratory to establish clonal selection and expansion carrying gene mutations. Further experiments are planned for genotoxicity investigations and to demonstrate cell transformation by tt-DDE.
In summary, the present investigation represents the first study focused on the effects of tt-DDE, a major type of dienaldehyde, on immortalized human lung epithelial cells. Our results provide clear evidences that tt-DDE induces ROS production and oxidative stress in human lung cells that may lead to induction of pro-inflammatory responses such as enhanced cell proliferation and increased cytokine TNF- and IL-1 release. Since enhancement of cell proliferation and release of these pro-inflammatory cytokines are known to be involved in early stages of tumor promotion (Lappalainen et al., 2005; Moore et al., 1999; Suganuma et al., 1999), our findings strongly suggest that tt-DDE, a component in COF, could be a tumor promoter in human lung epithelial cells and may play a potentially important role in the development of lung adenocarcinoma (Ko et al., 2000; Tan et al., 2003; Zhong et al., 1999a,b). While ingestion of tt-DDE was previously believed to be a major factor, we suggest that its inhalation is equally important, especially for personnel who operate deep frying facilities. In support of NTP and NCI's concerns, we strongly suggest that future investigations on the carcinogenic potential of tt-DDE and other dienaldehydes are warranted both in vitro and in vivo in human subjects.
ACKNOWLEDGMENTS
This work was supported by research grant, DOH94-TD-G-111-010, from the National Research Program for Genomic Medicine and Department of Health, and EO-093-PP-03 from Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Taiwan, ROC. The scientific content of this manuscript does not necessarily signify the view and policies of DOH and DEHOM/NHRI or condemn, endorse, or recommend for use on this issue presented. Conflict of interest: The scientific content of this manuscript does not necessarily signify the content reflects the view and policies of DOH and DEHOM/NHRI or condemnation or endorsement and recommendation for use on this issue presented.
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