* Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Kaoshiung, Taiwan, R.O.C., and Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung, Taiwan, R.O.C.
1 To whom correspondence should be addressed. Fax: 886424751101. E-mail: ppl{at}csmu.edu.tw.
Received May 22, 2005; accepted July 5, 2005
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ABSTRACT |
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Key Words: tt-DDE; dienaldehydes; cell proliferation; cytokines.
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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.
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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-1ß), 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-1ß, 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-1ß expression levels in each sample were normalized to GAPDH expression.
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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.
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RESULTS |
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Changes in TNF- and IL-1ß 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-1ß 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-1ß (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-1ß 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.
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DISCUSSION |
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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-1ß 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-1ß. Since both TNF-
and IL-1ß 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-1ß increased the number of transformed foci in v-Ha-ras-transfected BALA/3T3 cells (Suganuma et al., 1999
). Thus both TNF-
and IL-1ß, 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-1ß 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-1ß 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.
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ACKNOWLEDGMENTS |
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