Berberine inhibits arylamine N-acetyltransferase activity and gene expression in mouse leukemia L 1210 cells.
N-acetyltransferases (NATs) are recognized to play a key role in the primary step of arylamine compounds metabolism. Polymorphic NAT is coded for rapid or slow acetylators, which are being thought to involve cancer risk related to environmental exposure. Berberine has been shown to induce apoptosis and affect NAT activity in human leukemia cells. The purpose of this study is to examine whether or not berberine could affect arylamine NAT activity and gene expression (NAT mRNA) and the levels of NAT protein in mouse leukemia cells (L 1210). N-acetylated and non-N-acetylated AF were determined and quantited by using high performance liquid chromatography. NAT mRNA was determined and quantited by using RT-PCR. The levels of NAT protein were examined by western blotting and determined by using flow cytometry. Berberine displayed a dose-dependent inhibition to cytosolic NAT activity and intact mice leukemia cells. Time-course experiments indicated that N-acetylation of AF measured from intact mice leukemia cells were inhibited by berberine for up to 24h. The NAT1 mRNA and NAT proteins in mouse leukemia cells were also inhibited by berberine. This report is the first demonstration, which showed berberine affect mice leukemia cells NAT activity, gene expression (NAT1 mRNA) and levels of NAT protein.
[c] 2005 Published by Elsevier GmbH.
Keywords: N-acetyltransferase; Berberine; Mouse leukemia cells; Arylamines (N-acetyl-2-aminofluorene and 2-aminofluorene)
The N-acetyltransferases (NATs) play an important role in the metabolism of arylamine chemicals and carcinogens that catalyze both detoxifying N-acetylation and O-acetylation that generate intermediate metabolites in electrophiles which then bind to DNA and form DNA adduct formation that finally generates cancer in specific target organs or tissues (Hein, 1988; Fretland et al., 2002). N-acetyltransferase-1 (NAT1) and N-acetyltransferase-2 (NAT2) are encoded by NAT1 and NAT2 in human chromosome 8 (Blum et al., 1990). The genetic determined the variants of both enzymes of individuals which then lead to the specific NAT activity for rapid or slow acetylation (Weber and Hein, 1985; Evans, 1989; Weber, 1987). Based on the NAT activity (N-acetylation of substrate) the individuals are classified into rapid and slow acetylators. From the epidemiological statistic analysis, it was demonstrated that slow acetylators have an increased bladder cancer risk after exposed to smoking-derived or occupational carcinogens (Risch et al., 1995; Brockmoller et al., 1996; Cartwright, 1982) but rapid acetylator with high intake of cooked meat containing carcinogenic heterocyclic amines could be a potential risk factor for colorectal cancer (Lang, 1986; Roberts-Thomson et al., 1996; Chen et al., 1998). Lenkemia usually occurs in animals and children. Usually, mutagen by oral administration was absorbed from the intestinal system, and then passed through the circulation system to the whole body. The in vivo studies of our research colleagues had been evaluated, and these mutagen and intermediates were deposited in the blood and had been accumulated in other tissues for a long time. Recently, comprehensive reviews on the molecular genetic analysis and epidemiology of the both NATs acetylation polymorphisms have been reported (Brockton et al., 2000; Cascorbi and Roots, 1999; Hein et al., 2000).
Berberine (5,6-Dihydro-9,10-dimethoxybenzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium), a benzodioxoloquinolozine alkaloid present in the plant genera Berberis and Coptis, and also in many other plants, has been widely used to treat gastroenteritis and diarrhea patients in the Chinese population for a long time (Tang and Eisenbrand, 1992). Berberine acts as an antimicrobial (Amin et al., 1969), antidiarrhea (Tai et al., 1981; Yamamoto et al., 1993), and antineoplastic agent (Hoshi et al., 1976; Zhang et al., 1990) and it also has a high binding affinity for mast cells (Berlin and Enerback, 1983) and influenced mast cell-mediated chloride secretion in rat colons (Taylor and Baird, 1993). Recently, it was also demonstrated that berberine could be used as an anti-inflammatory agent which may arise in part from the inhibition of DNA-synthesis in human activated peripheral lymphocytes (Weber and Hein, 1985; Ckless et al. 1995). In our laboratory, we also found out that berberine affected NAT activity of human colon (Chung et al., 1999) and bladder (Lin et al., 1999a, b) tumor cells. However, there is no available information to address berberine effects on NAT activity and gene expression in the mouse leukemia cells. Thus, the initial studies were focused on the effects of berberine on the NAT activity and gene expression of a mouse leukemia L 1210 cell line.
Materials and methods
Chemicals and reagents
Berberine, ethylenediaminetetraacetic acid (EDTA), 2-aminofluorene (AF), N-acetyl-2-aminofluorene (AAF), dithiothreitol (DTT), Tris, acetylcarnitine, leupeptin, bovine serum albumin (BSA), phenylmethylsulfonylfluoride (PMSF), dimethyl sulfoxide (DMSO), acetyl-coen-zyme A (Acetyl-CoA) and carnitine acetyltransferase were obtained from Sigma Chemical Co. (St. Louis, MO). All of the chemicals used were reagent grade.
Mouse leukemia cell line
Mouse lymphocytic leukemia cell line (L 1210) was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). The cells (1 X [10.sup.6]) were placed into 75 [cm.sup.3] tissue culture flasks and grown in Dulbecco's modified Eagle's Medium (DMEM) (Gibco BRL, Grand Island, NY) supplemented with 10% horse serum (Gibco BRL, Grand Island, NY), 2% penicillin-streptomycin (10 U/ml penicillin and 10 mg/ml streptomycin) in the incubator for 37[degrees]C, 10% C[O.sub.2].
Preparation of mouse leukemia cell cytosols
About 5 X [10.sup.7] cells were placed in 2 ml of the lysis buffer (20 mM Tris/HCl, pH 7.5, 1 mM DTT, 1 mM EDTA, 50 [micro]M PMSF, and 10 [micro]M leupeptin) previously described (Chung et al., 2000). The suspensions were centrifuged at 9000g for 1 min at 4[degrees]C in a model 3200 Eppendorf/Brinkman centrifuge, and the supernatant fraction was subsequently centrifuged at 10,000g for 60 min 4[degrees]C. The supernatant was kept on ice for NAT activity and protein determinations.
NAT activity determinations
The determination of Acetyl-CoA-dependent N-acet-ylation of AF was performed as previously described by Chung et al. (2000).
Determination of the levels of protein
Protein concentrations in the mouse leukemia cell cytosols were determined by the method of Bradford with BSA as the standard (Bradford, 1976). All of the samples were assayed in triplicate.
Intact cell NAT activity determination
Mouse leukemia cells (in 1 ml DMEM media with glutamine and 10% fetal calf serum) were incubated with arylamine substrate (AF) at 1 X [10.sup.6] cells/ml in individual wells of a 24-well cell culture plate with or without various concentrations of berberine co-treatment for 24h at 37[degrees]C in 95% air 5% C[O.sub.2]. At the conclusion of incubation, the cells and media were removed and centrifuged. The supernatant was immediately extracted with ethyl acetate/methanol (95:5), the solvent evaporated, and the residue redissolved in methanol and assayed for AAF and AF as described above.
Time-course effects of berberine on NAT activity in mouse leukemia intact cells
Mouse leukemia cells in DMEM media were incubated 22.5 [micro]M AF at 1 X [10.sup.6] cells/ml in individual wells of 24-well-cell culture plate with or without berberine co-treatment for the 6, 12, 18, and 24h incubation. At the conclusion of incubation, the acetylated AF (AAF) and unacetylated AF were determined by using HPLC as described above (Chen et al., 2003).
Preparation of recombinant proteins for immunization
Each primer pair, used in revise transcription-PCR, was designed to incorporation a restriction enzyme site (BamHI; as show in below section of PCR for NAT; DyNAzyme DNA polymerase. The sequence of primers as follows: B-MDIEA-NAT1, 5'-CACCCGGATCCGG-GATCATGGACATTGAAGC-3', NT 435-454, GenBank accession number X17059; VPKHGD-X-NAT1, 5'-GGTCCTCGAGTCAATCACCATGTTTGGGCAC-3', nt 1295-1278, GENBANK accession GGTTGCTGGCC-3', nt 79-98, GenBank accession number NM-00015; RP1-NAT2, 5'-TAACGTGAGGGTAGAGAGGA-3', nt 1073-1054, GenBank accession number NM-000015). The PCR product, treated with BamHI nd Xhol, was subcloned into the corresponding sites of the expression vector pET-29a + (Novagen, Inc., Madison, WI, USA). After transforming the recombinant plasmid into the expression (Escherichia coli strain BL21), the recombinant protein was introduced to overexpress after 3-h induction in the presence of 1 mM isopropyl-1 thio-beta-D-galactopyranoside. The isolated inclusion body was washed twice with distilled water and redissolved in 1% SDS overnight at 25[degrees]C. The prepared recombinant protein showed near homogeneity, as judged by SDS-PAGE, and was ready for immunization (Chung et al., 2001, 2003).
Preparation of polyclonal antibody
Six 6-week-old female BALB/c mice were immunized with the prepared recombinant protein (NAT). At first each mouse was injected with 0.5 ml of pristane. Then about 100 [micro]g of antigen, mixed with an equal volume of complete Freund's adjuvant, were applied by s.c, for 10-15 days. The antigen was emulsified with incomplete Freund's adjuvant, and injected by i.p. for 10-20 days then boosted again. Myeloma cells (1 X [10.sup.6]/ml) in PBS were then injected by i.p. into the mouse. The ascites fluids, which normally accumulated after 1 week, were collected daily for 5-8 days (Chung et al., 2001).
Detection of NAT protein by flow cytometry
The level of intracellular NAT of the examined cells was determined by flow cytometry (Becton Dickinson FACS Calibur), using the prepared polyclonal antibody mentioned above. Cells were co-treated with various concentrations (0.4, 4, 40, 80 and 160 [micro]M) of berberine for 24 hours to detect the intracellular NAT. The cells were washed twice, re-suspended in 100 [micro]l of ice-cold 1% formaldehyde for 5 min, and mixed with 100 [micro]l of ice-cold 99% methanol for 30 min. Then the cells were washed three times with 0.1% BSA in PBS and mixed with 100 [micro]l of 0.1% Triton X-100 in PBS with 0.1% sodium citrate on ice for 45 min. After being washed three times with the same buffer, the cells were incubated with polyclonal antibody at 4 [degrees]C for 2.5 h, and then washed three times with 0.1% BSA in PBS. The cells were then stained with FITC-labeled secondary antibody (goat antimouse IgG; Jackson ImmunoResearch; Laboratories, West Grove, PA, USA) at 4 [degrees]C for 35 min. Again, the cells were washed three times, resuspended in PBS, and analyzed by flow cytometry (Chung et al., 2001).
Detection of NAT protein by western blotting
About 5 X [10.sup.6] cells were placed in 6-well plate co-treated with or without various concentrations of berberine for 24 h. They were then harvested and centrifuged to remove and discard the medium, while the cells were washed with PBS. The cells were lysed in 100 [micro]l of triple detergent buffer (50 mM Tris-Cl [pH 8.0], 150 mM NaCl, 0.02% Na[N.sub.3], 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 100 [micro]g/ml phenylmethylsulfonyl fluoride, 1 [micro]g/ml aprotinin, 1 [micro]g/ml leupeptin, and 1[micro]g/ml pepstatin A). The samples were then sonicated, incubated on ice for 20 min and centrifuged at 12,000g for 10 min at 4 [degrees]C. The supernatants were collected and the Bradford assay was performed to determine the protein concentration. Proteins (50 [micro]g/lane) were separated on 12% SDS-polyacrylamide gels and blotted onto polyvinyldiene difluoride membranes. The membrane was incubated with 5% bovine serum albumin and primary antibody (anti-NAT) for overnight at 4 [degrees]C overnight. The blots were washed three times in PBS with 0.04% Tween-20 (PBST) for 5 min then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at 1:1000 dilution in PBST containing 5% fresh defatted milk for 2 h at room temperature. The membranes were washed with PBST and visualized using ECL detection system (Amersham, Piscataway, NJ) and quantitated by densitometry using ImageQuant image analysis.
Reverse transcriptase polymerase chain reaction (RT-PCR)
The total RNA was extracted from L 1210 cells by using Qiagen RNeasy Mini Kit at 24 h after with or without cotreatment of different concentrations of berberine. Total RNA (1.5 [micro]g), 0.5 [micro]g of oligo-dT primer and DEPC (diethyl pyrocarbonate)-treated water were combined into a microcentrifuge tube to a final volume of 12.5 [micro]l. The entire mixture was heated at 70 [degrees]C for 10 min and chilled on ice for at least 1 min. The subsequent procedures for conducting reverse transcription were exactly the same as those in the instruction manual (First-strand cDNA synthesis kit, Novagen). The reverse transcription products from total RNA served as a template for PCR. When amplifying target cDNA, components in 50 [micro]l of solution were as follows: 1.5 mM Mg[Cl.sub.2], 0.2 mM dNTP mix, 20 pmol of each primer (B-MDIEA-NAT1 & VPKHGD-X-NAT1 for NAT1, FP1-NAT2 & RP1-NAT1 for NAT2, Act b1 & Act b2 for beta-actin), cDNA template corresponding to the amount synthesized from 50 ng of total RNA and 2 units of DyNAzyme DNA polymerase. The sequence of primers as follows: B-MDIEA-NAT1, 5'-CACCCGGATCCGGGATCATGGACATTGAAGC-3', nt 435-454, GenBank accession number X17059; VPKHGD-X-NAT1, 5'-GGTCCTCGAGTCAATCACCATGTTTGGGCAC-3', nt 1295-1278, GenBank accession number X17059; FP1-NAT2, 5'-CTAGTTCCTGGTTGCTGGCC-3', nt 79-98, GenBank accession number NM-000015; RP1-NAT2, 5'-TAACGTGAGGGTAGAGAGGA-3', nt 1073-1054, GenBank accession number NM-000015; Act b1, 5'-GCTCGTCGTCGACAACGGCTC-3', nt 94-114, GenBank accession number NM-001101; Act2 b2, 5'-CAAACATGATCTGGGTCATCTTCTC-3', nt 446-422, GenBank accession number NM-001101 (Blum et al., 1990; Ebisawa and Deguchi, 1991; Ponte et al., 1984; Chiu et al., 2003) [30-32].
Statistical treatment of data
Statistical analysis of the data was performed with an unpaired Student's t-test.
Effects of various concentrations of berberine on mouse leukemia cells in cytosol and intact cells
The possible effects of berberine on NAT activity in mouse leukemia cells cytosols were examined by HPLC assessing the percentage of acetylation of AF. For the cytosolic examinations, AF was added to the cytosol for N-acetylation of AF then the determination of AF and AAF by HPLC. The means [+ or -] SD (standard deviation) of NAT activity co-treated with or without various concentrations of berberine with AF in cytosols is given in Table 1. The data indicated that there were decreased amounts of AAF associated with increased berberine in examined cytosols. In the presence of 0.4, 4, 40, 80, and 160 [micro]M berberine, the N-acetylation of AF were inhibited 13-84% for AF acetylation in L 1210 cell, respectively. For the intact cells' examination, the results indicated that after being cotreated with 0.4, 4, 40, 80, and 160 [micro]M berberine, the N-acetylation of AF were inhibited 4-88% for AF acetylation in L 1210 cell (Table 1).
Time course effects of berberine on NAT activity in intact mouse leukemia cells
Mouse leukemia cells were incubated with AF produced AAF in the culture media, whereas AF without cells and cells without AF did not lead to any detectable AAF in the media in all examined time (Fig. 1). Increased incubation time from culture cells led to increased AAF production upto 24 h. In the presence of 40 [micro]M of berberine, the amounts of AAF were decreased about 44-58% for L 1210 cells, respectively. Precentage of the viable cells by the treatment of 40 [micro]M of berberine was kept about 80% after 24 h periods.
Effects of berberine on the NAT protein in mouse leukemia cells
NAT protein was measured by the NAT antibody to form an antigen-antibody complex. The percentage of NAT-antibody complex from examined L 1210 cells co-treated with or without various concentrations of berberine for 24 h were measured by flow cytometry. The data indicated that berberine decreased the percentage of NAT-antibody complex in examined cells (Table 2). The effect of berberine on the total levels of NAT also were confirmed by western blotting as shown in Fig. 2. The NAT levels waere decreased in response to berberine concentrations.
[FIGURE 1 OMITTED]
Dose-dependent effects of berberine on NAT mRNA expression in intact leukemia cells
In order to examine dose-dependent effects of berberine on the NATs mRNA expression, the RT-PCR and PCR were carried out using various doses of berberine, which were co-treated with intact L 1210 cells. Data presented in Figs. 3 and 4 show that NAT1 mRNA level became decreased at the higher levels (40-160 [micro]M) of berberine. NAT1 mRNA was presented in L-1210 cells but NAT2 was not detected in these examined cells.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
Berberine had been demonstrated to inhibit DNA, RNA and protein synthesis in sarcoma S180 cells in vitro (Creasey, 1979). Berberine-treated HL-60 cells had an increased G2/M phase population (Kuo et al., 1995). Recently, it was also reported that berberine modulates expression of mdr1 gene product and the responses of digestive track cancer cells to paclitaxel (Lin et al., 1999a, b). However, there is no available information to address berberine affect NAT gene expression on mouse leukemia cells. Therefore, in the present study, we have examined berberine affect NAT activity (N-acetylation of AF), NAT gene expression and protein levels in mouse leukemia cells (L 1210). The results indicated that berberine did affect the N-acetylation of AF in cytosols and intact cells based on the following observations: (i) The amounts of AAF (acetylated AF) in mouse leukemia intact cells and cytosol were decreased and this effect has a dose-dependent manner and (ii) increased incubation times of cells with AF led to increased AAF production in all examined time peroids, berberine did decrease the amounts of AAF production. However from present results whether or not decreased NAT activity (N-acetylation of AF) could lead to inhibit leukemia development is not clear. But it had been reported that the attenuation of liver NAT activity is associated with several disease processes such as breast and bladder cancer  and increased levels of NAT activity are associated with increased sensitivity to the mutagenic effects of many arylamines (Einisto et al., 1991). NAT enzyme has been reported to play the role of some chemical carcinogenesis (Grant et al., 1991; Minchin et al., 1992). Our earlier studies already found out that mouse blood contains NAT activity (Chung et al., 1993) and mouse leukemia L 1210 cells also contain NAT activity (Li et al., 2001). Apparently, the in vivo studies for berberine affect leukemia in mouse are needed for future studies.
[FIGURE 4 OMITTED]
The data also indicated that berberine decreased the NAT kinetic constants (Km and Vmax) in the cytosols of L 1210 cells (data not shown). This inhibition of berberine for NAT is uncompetitive. The patterns in enzyme inhibitors were described in the textbook (Zubay et al., 1995). The nature and how the interaction between berberine and the NAT protein domains from L 1210 cells is remain unclear. Cytochrome P450 enzyme was involved in the metabolism of acetylated AF (AAF production) and cytochrome P450-dependent formation of N-hydroxy-AAF is recognized to be the initial rate-limiting step in the metabolism of AAF to mutagenic and potentially carcinogenic products (Thorgeirsson, 1983) and P4501A1 is particularly efficient for catalyzing conversion of AAF to 7-OH-AAF (Hayes et al., 1986). The susceptibility to the carcinogenic effects of aromatic and heterocyclic amines may depend on (i) the relative rates of N-acetylation and N-hydroxylation in the liver, (ii) the route of excretion of metabolites within the tissues or organs, and (iii) possibly the rates of glucuronide hydrolysis and NAT mediated activation in the target tissue (Smith, 1995). Therefore, there are at least three particular enzymes such as NAT, glucuronide hydrolysis and P4501A1 that are involved in the metabolism of arylamine (AF). Although the present studies did not involve whether or not berberine could affect two other enzymes but the berberine induce inhibition of NAT may be a useful additional tool to distinguish between different NAT enzymes based on the different inhibition. Based on the results (Figs. 2 and 3) from western blotting and flow cytometric analysis it is indicated that berberine did decrease the levels of NAT protein. Twenty-four hours was selected as the longest time point, since Fig. 2 indicates a further increase in activity, whereas Fig. 3 indicates strong decrease in the protein amount. Fig. 4 also show that berberine decreased gene expression (NAT1 mRNA) of L-1210 cells. But the NAT2 gene product was not found in these examined cells based on the RT-PCR and NAT PCR. The finding is in agreement with our earlier studies that show L 1210 cells did not acetylate sulfamethazine (data not shown). In this study, we did not detect any acetylated sulfamethazine in cytosol and intact cells. It is well known that sulfamethaze is one of the substrates of NAT2. It is already known that mouse blood cannot acetylate sulfamethaze and NAT2 is not present in these cells.
With distinction to be made between inhibition of DNA transcription, being that there was a decreased in protein amount and protein activity. Also, effects of cancer induction by NAT should be separated from cancer prevention by NAT-catalysis. The biological activity of berberine is considered to be associated in part with affect DNA, RNA and protein and also induce mdr1 gene expression and cell apoptosis (Lin et al., 1999a, b). Therefore, berberine decreased the levels of NAT activity and gene expression and NAT protein are associated with sensitivity to the mutagenic effects in mouse leukemia cells, but whether it could lead to decreased leukemia production is still not exactly known. In the other words, the role of arylamine NAT activity in the blood leukemia and any other tissues remain enigmatic. So far the exact role of NAT in the cancer production, is still controversial, and studies from liver tissues suggests that this NAT enzyme is involved in the detoxification of exogenous amines (Blum et al., 1990; Weber and Hein, 1985), significant NAT activity in these enzyme structure studies indicate that this NAT enzyme could be involved in N-acetylation of ocular drugs (Campbell et al., 1991). The other factors that may be concern for arylamine carcinogen induced carcinogenesis in target organs or tissues such as (1) conversion to mutagenic metabolites involves hydroxylation and acetylation, largely in the liver tissue, with additional acetylation of circulating hydroxyl metabolites may occur in the other tissues, (2) the metabolites of arylamine compounds from liver or other tissues may undergo blood circulation to transfer to other target tissues. In conclusion, the present results offer some information to show that berberine decreased the NAT activity and gene expression (NAT mRNA) in mouse leukemia cells' cytosols and intact cells. On the public health, maybe we should prevent the taking of more carcinogens, and suggest taking the relevance of NAT inhibitors for the public. What is helpful in combination of NAT inhibitors for prevention of cancer? The topic will be evaluated in the future, and many companies will be more interested in commercial products.
Table 1. Effects of berberine on mouse leukemia cells' NAT activity in cytosols Berberin AAF (nmol/min/ AAF (nmol/[10.sup.6] treatment mg protein) cells) Control 0.38[+ or -]0.10 9.0[+ or -]0.44 0.4 [micro]M 0.33[+ or -]0.12 8.8[+ or -]0.33 4 [micro]M 0.26[+ or -]0.09 7.5[+ or -]0.25 40 [micro]M 0.20[+ or -]0.06 (a) 4.5[+ or -]0.22 80 [micro]M 0.13[+ or -]0.04 (b) 2.8[+ or -]0.20 160 [micro]M 0.06[+ or -]0.02 (c) 1.0[+ or -]0.15 The cytosols of L-1210 cells were prepared as described in the section "Materials and Methods". The AcCoA and berberine concentrations were 0.1 mM and 0-160 [micro]M, respectively. Values are mean[+ or -]SD of activity (nmol/min/mg protein) n = 6. (a,b,c) Differs between berberine and control p < 0.05. Table 2. The percentage of mouse leukemia cells cells treated by berberine were stained by the NAT antibody Berberine treatment Percentage of cells stained by anti-NAT L 1210 0 (control) 46.2[+ or -]4.8 0.4 ([micro]M) 44.9[+ or -]5.2 4 ([micro]M) 36.5[+ or -]4.0 40 ([micro]M) 22.8[+ or -]3.2 (a) 80 ([micro]M) 14.9[+ or -]2.7 (b) 160 ([micro]M) 8.4[+ or -]0.6 (c) Values are mean [+ or -] S.D. n = 6. The L 1210 cells (1 X [10.sup.6] cells/ml) were co-treated with various concentrations of berberine. The zero concentration was defined as control. The percent of cells were stained by NAT antoibody, and the stained cells were determined by flow cytometry as described in the section "Materials and Methods". (a,b,c) Differs between berberine and control p < 0.05.
This work was supported by Grant NSC90-2745-P-039-001 from the National Science Council of Taiwan, ROC.
Received 5 August 2003; accepted 5 November 2003
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S.S. Lin (a), J.G. Chung (b,*), J.P. Lin (c), J.Y. Chuang (d), W.C. Chang (e), J.Y. Wu (f), Y.S. Tyan (g)
(a) Department of Radiological Technology, Chungtai Institute of Health and Technology, Taichung, Taiwan, ROC
(b) Department of Microbiology, China Medical University, 91 Hsueh-Shih Road, Taichung 404, Taiwan, ROC
(c) Chinese Medicine, China Medical University, Taichung, Taiwan, ROC
(d) Medical Technology, China Medical University, Taichung, Taiwan, ROC
(e) Physiology, China Medical University, Taichung, Taiwan, ROC
(f) Department of Chest Surgery, Armed Force Taichung General Hospital, Taichung, Taiwan, ROC
(g) Department of Radiology, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC
*Corresponding author. Tel.: + 88642205 3366; fax: + 88642205 3764.
E-mail address: email@example.com (J.G. Chung).