* Institute of Toxicology, Chung Shan Medical University, Taichung, Taiwan; and
Institute of Stomatology, Chung Shan Medical University, Taichung, Taiwan
Received July 12, 2002; accepted September 27, 2002
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ABSTRACT |
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Key Words: aryl hydrocarbon receptor; hydrocarbon receptor nuclear translocator; cytochrome P4501A1; cytochrome P4501B1.
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INTRODUCTION |
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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., 1992). 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., 1997
). It has been demonstrated that CYP1A1 activity and expression are inducible by PAHs through activation of aryl hydrocarbon receptor (AhR) (Whitlock, 1999
). CYP1A1 inducibility measured in cultivated lymphocytes has correlated well with that measured in lung tissue explants (Jacquet et al., 1997
). Therefore, it was suggested that CYP1A1 inducibility is a susceptibility marker for PAH-induced lung carcinogenesis (Kellermann et al., 1973
; Kiyohara et al., 1998
). Although the association between CYP1A1 inducibility and lung cancer risk is still controversial (Kellermann et al., 1973
; Prasad et al., 1979
; Stucker et al., 2000
; Ward et al., 1978
), CYP1A1 inducibility by PAHs is variable in the human population (Kellermann et al., 1973
). 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., 1996
, 1998
). However, the genotypes with high CYP1A1 inducibility are extremely rare in some ethnic groups (Hirvonen et al., 1992
; Tefre et al., 1991
; Xu et al., 1996
), and other factors should contribute to individual variation in CYP1A1 inducibility (Smart and Daly, 2000
).
Differential CYP1A1 inducibility was also reported in different strains of mice and was correlated with PAH-induced carcinogenesis in these mice (Nebert, 1989). 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, 1999
). CYP1B1 was also involved in metabolic activation of PAHs (Shimada et al., 1996
). Constitutive expression and induction of CYP1B1 has been found in lung cancer cell lines and lymphocytes (Chang et al., 1999
; Spencer et al., 1999
). 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, 1989
; Shimizu et al., 2000
). The study by Hayashi et al. (1994)
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., 1994
). 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., 1997; Kellermann et al., 1973
). 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., 1997
). 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, 1999
), 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.
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MATERIALS AND METHODS |
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Lymphocytes isolation and culture.
Lymphocytes were isolated from fresh blood samples using Ficoll-Paque gradients according to the manufacturers 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 1. 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
Ct,
Ct = Cttarget gene - Ctß-actin.
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RESULTS |
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DISCUSSION |
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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., 1999). Shehin et al.(2000)
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, 1996; Prescott et al., 1998
; Zang and Wynder, 1996
). Mollerup et al.(1999)
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., 1999
). 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., 1992
, 1996
). Many carcinogenic PAHs have been identified in tobacco smoke (Hecht, 1999
). 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, 1997). 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., 1996, 1998
). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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