Correlation between Gene Expression of Aryl Hydrocarbon Receptor (AhR), Hydrocarbon Receptor Nuclear Translocator (Arnt), Cytochromes P4501A1 (CYP1A1) and 1B1 (CYP1B1), and Inducibility of CYP1A1 and CYP1B1 in Human Lymphocytes

Pinpin Lin*,1, Suh-Woan Hu{dagger} and Tzu-Hua Chang*

* Institute of Toxicology, Chung Shan Medical University, Taichung, Taiwan; and {dagger} Institute of Stomatology, Chung Shan Medical University, Taichung, Taiwan

Received July 12, 2002; accepted September 27, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The relationships between gene expression of aryl hydrocarbon receptor (AhR), aryl hydrocarbon receptor nuclear translocator (Arnt), cytochromes P4501A1 (CYP1A1), 1B1 (CYP1B1), CYP1A1, and the inducibility of CYP1A1 and CYP1B1 were determined in 32 cultivated human lymphocytes. Cytochrome P450 induction was performed by incubating lymphocytes with benzanthracene. The relative gene expression levels were determined by quantitative real-time RT-PCR assay. We found that gender is an important confounding factor for gene expression in cultivated lymphocytes. AhR, CYP1A1 and CYP1B1 levels in noninduced lymphocytes were significantly higher in female nonsmokers than in male nonsmokers (p < 0.05). Nevertheless, CYP1A1 and CYP1B1 inducibility was lower in female nonsmokers. CYP1A1 inducibility was higher in male smokers than in male nonsmokers (p < 0.05). After controlling for gender and cigarette smoking, AhR levels positively correlated with CYP1B1 levels and CYP1A1 inducibility (p < 0.01 and p = 0.03, respectively). Arnt levels also correlated with CYP1B1 levels in induced lymphocytes (p < 0.01). However, AhR levels were negatively correlated with CYP1B1 inducibility. These data indicate that AhR expression associates with individual variation of CYP1A1 inducibility and CYP1B1 expression in cultivated lymphocytes. Furthermore, gender and cigarette smoking are important confounding factors for gene expression levels in cultivated lymphocytes.

Key Words: aryl hydrocarbon receptor; hydrocarbon receptor nuclear translocator; cytochrome P4501A1; cytochrome P4501B1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several studies have indicated that drug-metabolizing enzyme activities are highly variable in the human population (Nebert, 1991Go). Some drug-metabolizing enzymes are responsible for metabolic activation of environmental carcinogens. Thus, individual variation in metabolic activation or detoxification of environmental carcinogens partially explains the host susceptibility to chemical-induced toxicity (Nebert, 1991Go; Perera, 1996Go).

The cytochrome P450-1 family is one of the major cytochrome P450 families involved in xenobiotic metabolism. One of the well known examples is cytochrome P4501A1 (CYP1A1), which has been shown to participate in metabolic activation of polycyclic aromatic hydrocarbons (PAHs) (Shimada et al., 1992Go). PAHs are some of the major carcinogens found in the environment as pollutants. Heavy exposure to PAH-contaminated air pollutants has been associated with the increased risk of lung cancer (Boffetta et al., 1997Go). It has been demonstrated that CYP1A1 activity and expression are inducible by PAHs through activation of aryl hydrocarbon receptor (AhR) (Whitlock, 1999Go). CYP1A1 inducibility measured in cultivated lymphocytes has correlated well with that measured in lung tissue explants (Jacquet et al., 1997Go). Therefore, it was suggested that CYP1A1 inducibility is a susceptibility marker for PAH-induced lung carcinogenesis (Kellermann et al., 1973Go; Kiyohara et al., 1998Go). Although the association between CYP1A1 inducibility and lung cancer risk is still controversial (Kellermann et al., 1973Go; Prasad et al., 1979Go; Stucker et al., 2000Go; Ward et al., 1978Go), CYP1A1 inducibility by PAHs is variable in the human population (Kellermann et al., 1973Go). Little information has been available on the molecular mechanism of variation in CYP1A1 inducibility. CYP1A1 genetic polymorphisms have been demonstrated to correlate with CYP1A1 inducibility (Kiyohara et al., 1996Go, 1998Go). However, the genotypes with high CYP1A1 inducibility are extremely rare in some ethnic groups (Hirvonen et al., 1992Go; Tefre et al., 1991Go; Xu et al., 1996Go), and other factors should contribute to individual variation in CYP1A1 inducibility (Smart and Daly, 2000Go).

Differential CYP1A1 inducibility was also reported in different strains of mice and was correlated with PAH-induced carcinogenesis in these mice (Nebert, 1989Go). The mice with high CYP1A1 inducibility had a higher AhR ligand-binding capacity. The PAHs are AhR ligands. The liganded AhR translocates from the cytosol to the nuclei, heterodimerizes with AhR nuclear translocator (Arnt), binds to the cognate enhancer sequence, and subsequently transactivates gene expression of CYP1A1 and cytochrome P4501B1 (CYP1B1) (Whitlock, 1999Go). CYP1B1 was also involved in metabolic activation of PAHs (Shimada et al., 1996Go). Constitutive expression and induction of CYP1B1 has been found in lung cancer cell lines and lymphocytes (Chang et al., 1999Go; Spencer et al., 1999Go). However, no research has been done to investigate the individual variation in CYP1B1 expression and inducibility in human population. It has been demonstrated that AhR activation is required for PAH-induced toxicity (Nebert, 1989Go; Shimizu et al., 2000Go). The study by Hayashi et al. (1994)Go demonstrated individual differences in the mRNA levels of AhR and Arnt in human liver and lung tissues. The mRNA levels of CYP1A1 from the blood of healthy subjects correlated with that of AhR and Arnt (Hayashi et al., 1994Go). Nevertheless, it is still uncertain whether the differential expression of AhR and Arnt contributes to the interindividual variation in CYP1A1 inducibility.

CYP1A1 inducibility by PAHs can be measured in cultivated lymphocytes. Aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD) assays were utilized to determine CYP1A1 activity in cultivated lymphocytes (Jacquet et al., 1997Go; Kellermann et al., 1973Go). However, a few problems exist in these assays. For example, the AHH assay is not specific to CYP1A1 activity and the EROD assay is not sensitive enough to detect basal levels of CYP1A1 activity (Jacquet et al., 1997Go). It is worthwhile to explore more accurate and specific quantitative assays for CYP1A1 induction in lymphocytes. Since it is well known that the PAHs induce CYP1A1 activity at the transcription level (Whitlock, 1999Go), quantification of CYP1A1 mRNA levels is a potential tool for measuring CYP1A1 inducibility in lymphocytes. In the present study, we developed the quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay to quantify the relative mRNA levels in cultivated lymphocytes. We propose that the expression levels of AhR and Arnt contribute to the differences in CYP1A1 and CYP1B1 inducibility in humans. Utilizing this technique, we investigated whether the expression levels of AhR and Arnt correlated with inducibility of CYP1A1 and CYP1B1 in cultivated peripheral lymphocytes isolated from 32 healthy subjects. Other factors, such as gender and cigarette smoking, were also considered. Information generated from this study will be helpful in the elucidation on the mechanism of interindividual variation in CYP1A1 and CYP1B1 inducibility and expression. These techniques can be further utilized to assess the roles of AhR, Arnt, CYP1A1, and CYP1B1 in lung carcinogenesis in the future.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Study subjects.
A total of 32 healthy subjects were recruited in this study, including 10 male nonsmokers, 12 male smokers, and 10 female nonsmokers. The average age was 24 ± 3 years. All of the donors were Taiwan citizens and were interviewed for cigarette smoking status. The Chung Shan Medical University Hospital Institutional Review Board approved this project. Samples were collected in the morning. Approximately 20 ml of venous blood was collected from each donor with heparinized tubes.

Lymphocytes isolation and culture.
Lymphocytes were isolated from fresh blood samples using Ficoll-Paque gradients according to the manufacturer’s instructions. Isolated lymphocytes were washed with phosphate-buffered saline and cell numbers were determined. Cell concentrations were adjusted to 106/ml in RPMI medium containing 5 µg/ml PHA, 10% fetal bovine serum, and 0.1% dimethylsulfoxide (DMSO) or 12 µM benzanthracene (BA). Cell cultures were maintained at 37°C, 5% CO2, and 95% relative humidity until harvest. After harvest, viable cell numbers in each culture were determined with trypan blue exclusion method. Total RNAs of cells and tissues were prepared using TRIZOL® Reagent (Life Technologies, Rockville, MD) and the phenol-chloroform extraction method. Isolated RNA was stored at –70°C until analysis.

Quantitative real-time RT-PCR assay.
Total RNA of cells and tissues were measured using the TRIZOL Reagent (Life Technologies, Rockville, MD) and the phenol-chloroform extraction method. Synthesis of cDNA was done with 2 µg total RNA, 1 µg oligo dT primer and 20 nmol deoxynucleotide triphosphates using M-MLV Reverse Transcriptase (Promega, Madison, WI). Quantitative PCR was performed using the QuantiTect SYBR Green PCR kit (Qiagen, Hilden, Germany) and analyzed on an ABI PRISM 7700 Sequence Detector System (Perkin-Elmer Applied Biosystems, Foster City, CA). Primers were chosen with the assistance of the computer program Primer Express (Perkin-Elmer Applied Biosystems). To avoid amplification of contaminating genomic DNA, one of the two primers was placed at the junction between two exons. The primer sequences and optimal concentrations of AhR, Arnt, CYP1A1, CYP1B1, and ß-actin are listed in Table 1Go. The thermal cycling comprised an initial step at 50°C for 2 min, followed by a denaturation step at 95°C for 10 min, 40 cycles at 95°C for 15 s, and 40 cycles at 60°C for 1 min. Varying lengths of oligonucleotides produce dissociation peaks at different melting temperatures. Consequently, at the end of the PCR cycles, the PCR products were analyzed using the heat dissociation protocol to confirm that one single PCR product was detected by SYBR Green dye. Each data point was repeated four times. Quantitative values were obtained from the threshold PCR cycle number (Ct) at which the increase in signal associated with an exponential growth for PCR product starts to be detected. The relative mRNA levels in each sample were normalized to its ß-actin content. The relative expression levels of the target gene: 2{Delta}Ct, {Delta}Ct = Cttarget gene - Ctß-actin.


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TABLE 1 Primer Sequences and Concentrations for Quantitative Real-time RT-PCR
 
Statistical analyses.
Data analysis started with descriptive statistics of all important variables. Arithmetic means and standard deviation of each gene expression level and frequency of categorical variables were calculated. In order to normalize the distributions, CYP1A1 and CYP1B1 expression levels were transformed into natural logarithmic scale. Comparisons of gene expression levels between males and females, between smokers and nonsmokers, and between BA treatment and DMSO treatment, respectively, were done by Student’s t-test. Pearson’s correlation coefficient was used to assess the correlation between gene expression levels. Finally, the multiple linear regression models were applied to assess the association between AhR and Arnt, respectively, with CYP1A1 and CYP1B1 controlling for gender and smoking status. Potential interaction between AhR or Arnt and gender or smoking status was evaluated by adding the interaction terms AhR*gender, AhR*smoking, Arnt*gender, and Arnt*smoking, respectively, into the linear regression models. An alpha level of 0.05 was used for all statistical tests.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The linear range of the quantitative real-time RT-PCR assay was determined by amplifying serial dilutions of cDNA converted from lung cancer cell lines NCI-H1355. The standard curve was examined by amplifying 4-fold serial dilutions of the cDNA using primers for AhR, Arnt, CYP1A1, CYP1B1, and ß-actin. The standard curve is plotted by the log of the template dilution fold versus the Ct (Fig. 1Go). The standard curve of AhR, Arnt, CYP1A1, and CYP1B1 were all parallel to that of ß-actin, which indicated that the amplification efficiency of ß-actin and target genes was equal. The calculation for the relative gene expression levels is described in Materials and Methods.



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FIG. 1. Validation of the quantitative real-time RT-PCR method. The cDNA from human lung adenocarcinoma cells NCI-H1355 cells was serially diluted, amplified with ß-actin primer and (A) CYP1A1, (B) CYP1B1, (C) AhR, or (D) Arnt primers, and detected by SYBR green with the quantitative real-time RT-PCR assay. The quantification data was presented with Ct. These data were fitted using linear regression analysis.

 
The time course of gene expression in cultivated lymphocytes was determined after exposure to 12 µM BA for 0 to 3 days. Lymphocytes were isolated from four donors: two male smokers, one male nonsmoker and one female nonsmoker. As shown in Figure 2Go, CYP1A1 levels were increased at day 1 and continued to increase within 3 days. Similarly, CYP1B1 levels started to increase at day 2 and then kept increasing to day 3. AhR and Arnt levels remained consistent after BA treatment. Lymphocytes isolated from 2 of 4 subjects died on day 4 after BA treatment (data not shown). Therefore, we decided to measure the induction of CYP1A1 and CYP1B1 expression on day 3 in the following study. However, it is unclear why the basal CYP1B1 expression, but not AhR, Arnt, or CYP1A1, kept decreasing during 3 days of cultivation. We also compared gene expression levels in uncultured lymphocytes and 3-day cultivated lymphocytes. We found that CYP1A1 levels were significantly lower in uncultivated lymphocytes than in cultivated lymphocytes (data not shown). However, AhR, Arnt, and CYP1B1 levels in uncultivated lymphocytes were not significantly different from those in cultivated lymphocytes (data not shown).



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FIG. 2. Time course of gene expression levels in DMSO- and BA-treated lymphocytes. Peripheral lymphocytes isolated from 4 donors were cultivated in the presence of 0.1% DMSO or 12 µM BA for 1, 2, and 3 days. The relative gene expression levels of (A) AhR, (B) Arnt, (C) CYP1A1, and (D) CYP1B1 were determined with the quantitative real-time RT-PCR assay. The relative gene expression levels were normalized with those of ß-actin and calculated from 2{Delta}Ct, as described in Materials and Methods.

 
Isolated peripheral lymphocytes were treated with 0.1% DMSO or 12 µM BA for 3 days and then harvested to quantify the relative gene-expression levels. Cell viability in DMSO- or BA-treated cells was, respectively, 85 or 75% (data not shown). The mRNAs of AhR, Arnt, CYP1A1, and CYP1B1 were all detectable in lymphocytes (Table 2Go). CYP1A1 and CYP1B1 levels in DMSO-treated cells varied by ~200- and 100-fold, respectively. Individual variations of AhR and Arnt levels in DMSO-treated cells were, respectively, 50- and 13-fold. CYP1A1 and CYP1B1 levels were significantly increased after BA treatment, but AhR and Arnt levels were not significantly changed. The average induction folds of CYP1A1 and CYP1B1 were 45.86 and 4.73, respectively.


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TABLE 2 Gene Expression Levels and Inducibility of AhR, Arnt, CYP1A1, and CYP1B1 in DMSO- and BA-treated Lymphocytes
 
The effects of smoking and gender on gene expression levels were evaluated by analyzing log-transformed data with a Student’s t-test. The nontransformed data was presented in Table 3Go. No significant difference in gene expression levels was found between male smokers and nonsmokers, but CYP1A1 inducibility was significantly higher in smokers than in nonsmokers. AhR, CYP1A1, and CYP1B1 levels in DMSO-treated cells from female nonsmokers were significantly higher than those from male nonsmokers. On the other hand, CYP1A1 and CYP1B1 inducibility was significantly higher in males than in females. However, Arnt levels did not differ by gender or smoking status. Nonparametric analysis (Mann-Whitney U-test) for untransformed data was consistent with the above results (data not shown).


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TABLE 3 Gene Expression Levels and Inducibility in Lymphocytes after Stratified by Gender and Cigarette Smoking Status
 
It is well known that AhR and Arnt regulate CYP1A1 and CYP1B1 gene expression. Therefore, we further investigated whether AhR and Arnt levels are associated with expression levels and the inducibility of CYP1A1 and CYP1B1. Log-transformed data were analyzed with the Pearson correlation. We found that CYP1B1 and Arnt levels positively correlated with AhR levels in DMSO-treated cells (Figs. 3A and 3BGo). In BA-treated cells, AhR and Arnt levels both correlated with CYP1B1 levels (Figs. 3C and 3DGo). CYP1B1 inducibility was negatively correlated with AhR levels in DMSO-treated cells (Fig. 3EGo). However, the correlation was not observed between CYP1A1, Arnt, and AhR (data not shown). When subjects were stratified according to smoking status, CYP1A1 inducibility positively correlated with AhR levels in DMSO-treated cells from smokers (Fig. 3FGo). The correlation between CYP1A1 inducibility and AhR levels was not observed in nonsmokers (data not shown).



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FIG. 3. Correlation of gene expression levels of AhR, Arnt, CYP1A1, CYP1B1, and inducibility of CYP1A1 and CYP1B1 in lymphocytes. Gene expression levels were normalized with those of ß-actin and calculated from 2{Delta}Ct as described in Materials and Methods. Inducibility was the fold change in gene expression levels of BA- versus DMSO-treated cells. These data were fitted using linear regression analysis. In DMSO-treated cells, AhR levels correlated with (A) CYP1B1 and (B) Arnt levels. In BA-treated cells, CYP1B1 levels correlated with (C) AhR and (D) Arnt levels. AhR levels in DMSO-treated cells correlated with (E) CYP1B1 and (F) CYP1A1 inducibility.

 
Since gene expression levels and inducibility differed by gender and smoking status (Table 3Go), the correlation between gene expression levels was further assessed by multiple linear regressions. The effects of gender and smoking status were controlled in this model. As shown in Table 4Go, AhR levels were positively correlated with CYP1B1 levels in DMSO and BA-treated cells, but negatively correlated with CYP1B1 inducibility. Arnt levels were also associated with CYP1B1 levels in BA-treated cells. During data analysis, we observed that cigarette smoking had significant interaction with AhR levels (Table 3Go). The interaction meant that smoking modified the association between AhR and CYP1A1 levels in DMSO-treated cells and CYP1A1 inducibility. Other interaction terms, such as AhR*gender, Arnt*gender, and Arnt*smoking, were not significant in all models (data not shown). After controlling for gender, smoking, and their interaction, AhR levels negatively correlated with CYP1A1 levels in the DMSO-treated cells, but positively correlated with CYP1A1 inducibility.


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TABLE 4 Associations between AhR and Arnt and CYP1A1 and CYP1B1 Gene Expression Levels and Inducibility
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CYP1A1 inducibility has been considered as a susceptibility factor for lung cancer and is usually determined by measuring enzyme activity, such as AHH or EROD (Kellermann et al., 1973Go; Kiyohara et al., 1998Go). Induction of CYP1A1 is dependent on AhR activation (Whitlock, 1999Go). CYP1A1 inducibility was highly variable in human population. Therefore, we hypothesized that AhR and Arnt expression might count for interindividual variation in CYP1A1 inducibility. Our study showed that gender and cigarette smoking in cultivated lymphocytes affected CYP1A1 inducibility. We also noticed that cigarette smoking had interaction with AhR levels and modified the association between AhR levels and CYP1A1 inducibility. After controlling for gender, cigarette smoking, and their interaction, CYP1A1 inducibility correlated with AhR levels in noninduced lymphocytes. These data partially support our hypothesis that the differences in AhR expression, but not in Arnt expression, associate with individual variation in CYP1A1 inducibility.

In DMSO-treated lymphocytes, CYP1B1 levels were higher than CYP1A1 levels and positively correlated with AhR levels. This was consistent with the results of a previous study, which showed that CYP1B1 was constitutively expressed in cultivated lymphocytes (Spencer et al., 1999Go). Shehin et al.(2000)Go have demonstrated that the dioxin response elements in the enhancer region of CYP1B1 were responsible for the constitutive expression of CYP1B1. These data suggest that the AhR signaling pathway regulates the constitutive expression of CYP1B1 in lymphocytes. Therefore, in the absence of exogenous AhR ligands, CYP1B1 should play a role in AhR-mediated biological and physiological functions. CYP1B1 expression was also induced by PAHs in cultivated lymphocytes, although the induction fold of CYP1B1 was much lower than that of CYP1A1. In BA-treated lymphocytes, CYP1B1 levels positively correlated with AhR and Arnt levels. However, AhR levels negatively correlated with CYP1B1 inducibility. It is possible that the AhR-dependent increase of constitutive CYP1B1 levels (in DMSO-treated cells) was greater than that of induced CYP1B1 levels (in BA-treated cells).

Several epidemiological studies have indicated that female smokers are at higher risk of lung cancer than male smokers (Engeland, 1996Go; Prescott et al., 1998Go; Zang and Wynder, 1996Go). Mollerup et al.(1999)Go reported that the levels of hydrophobic DNA adducts in the nontumor lung tissues of female smokers were higher than those of male smokers. Furthermore, CYP1A1 expression levels correlated with DNA adduct levels in nontumor lung tissues, and were significantly higher in female smokers than in male smokers (Mollerup et al., 1999Go). Consistent with these results, we observed that AhR, CYP1A1, and CYP1B1 levels in noninduced lymphocytes were significantly higher in female nonsmokers than in male nonsmokers. CYP1A1 and CYP1B1 participate in the metabolic activation of PAHs (Shimada et al., 1992Go, 1996Go). Many carcinogenic PAHs have been identified in tobacco smoke (Hecht, 1999Go). These data imply that higher AhR expression causes higher CYP1A1 and CYP1B1 expression in females.

Cigarette smoking is an important confounding factor for AHH inducibility (Kiyohara and Hirohata, 1997Go). We found that CYP1A1 inducibility was significantly higher in male smokers than that in male nonsmokers. Furthermore, the relationship between AhR, CYP1A1 inducibility, and CYP1A1 in noninduced lymphocytes differed by smoking habit. AhR levels positively correlated with CYP1A1 inducibility in male smokers. However, the correlation coefficient between AhR levels and CYP1A1 inducibility was negative in male nonsmokers, although it was not statistically significant (data not shown). The opposite relationship was also noticed between AhR and CYP1A1 levels in noninduced lymphocytes (data not shown). It is possible that cigarette smoking interfered with the mechanism of AhR-mediated CYP1A1 induction. More experiments are necessary to understand the interaction between cigarette smoking and the AhR signaling pathway.

CYP1A1 genetic polymorphisms were demonstrated to correlate with AHH and EROD inducibility in Japanese populations (Kiyohara et al., 1996Go, 1998Go). When we determined the m1 and m2 polymorphisms of CYP1A1 in our samples, these polymorphisms were not associated with CYP1A1 inducibility in our study (data not shown). This is probably because our sample size was too small.

In the present study, we demonstrated that AhR expression correlated with CYP1A1 inducibility in human lymphocytes and cigarette smoking was an important confounding factor for the relationship. On the other hand, AhR expression correlated with CYP1B1 expression, but not CYP1B1 inducibility. In subsequent studies, we will compare AhR and CYP1B1 expression in lung cancer patients and healthy individuals. These studies allow us to understand whether AhR and CYP1B1 expression levels can be used to assess the susceptibility to lung cancer.


    ACKNOWLEDGMENTS
 
This work was supported by the National Science Council of the R.O.C. under Grant Number NSC 90–2318-B-040-006-M51 and NSC 90–2320-B-040–018 and by Phillip Morris‘s External Research Program.


    NOTES
 
1 To whom correspondence should be addressed at Institute of Toxicology, Chung Shan Medical University, No. 110, Sec. 1, Chien-Kuo N. Rd., Taichung 402, Taiwan, R.O.C. Fax: 886–4–24751101. E-mail: ppl{at}csmu.edu.tw. Back


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 DISCUSSION
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