Laboratoire de Toxicologie Moléculaire, U-490 INSERM, 45 rue des Saints-Pères, F-75270 Paris Cedex, France,
1 Laboratoire de Recherches Epidémiologiques et Statistiques sur l'Environnement et la Santé, U-170 INSERM, Villejuif, France and
2 Laboratoire de Chimie Médicale, Sart Tilman, CHU, Liège, Belgique
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Abstract |
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Abbreviations: AHH, aryl hydrocarbon hydroxylase; AhR, aryl hydrocarbon receptor; B[a]P, benzo[a]pyrene; CYP, cytochrome P-450; DGGE, denaturing gradient gel electrophoresis; OLA, oligonucleotide ligation assay; PAH, polycyclic aromatic hydrocarbons; XRE, xenobiotic responsive element.
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Introduction |
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The multigene family of cytochrome P-450 (CYP) (5) plays an important role in the biotransformation of many xenobiotics (4). These enzymes, particularly CYP1A1, are involved in the metabolic activation of benzo[a]pyrene (B[a]P), a widely distributed environmental carcinogen. B[a]P and pro-carcinogens are found in tobacco smoke, which is associated with lung cancer in humans (6,7). Cytochrome P450-dependent metabolic activation appears to be required for B[a]P activation and in vitro studies have showed that ultimate metabolite BPDE forms DNA adducts which were found also in human lung (8,9). The transcription factors, AhR, play critical roles in responding to xenobiotic compounds. There is a direct proof that the AhR plays a role in vivo as a mediator of carcinogenesis initiated with B[a]P (10) and that AhR signalling has a net potentiating effect on the genetic toxicity and, presumably, carcinogenicity of cigarette smoke (11).
The expression of the CYP1A1 gene is induced in a ligand-dependant fashion by the AhR and ARNT, which both contain basic helixloophelix (bHLH) and PAS domains (1216). The binding of various pro-carcinogens such as B[a]P to the AhR, is followed by the dissociation of Hsp90 from the receptor, leading to translocation of the ligand attached-AhR from the cytosol to the nucleus. In the nucleus, ARNT interacts with the ligand attached-AhR to form a heteromeric DNA-binding complex that can activate CYP1A1 gene transcription through binding to xenobiotic responsive element (XRE) (16).
The variability of CYP1A1 induction by various substances including PAH, may be due to genetic factors (17). It can be presumed that the AhR is involved in the variability of inducibility and, therefore, in the susceptibility to smoking-induced lung cancer. It constitutes an essential host factor regulating the level of the CYP1A1 protein in individuals (1821).
Polymorphisms in the AhR gene and their relation to levels of CYP1A1 activity and lung cancer susceptibility have been examined in a Japanese (22) and in a Caucasian population (23). A common polymorphism was found in both populations, in the coding region, which results in replacement of Arg 554 by Lys. No association was found between genotype, ability to induce CYP1A1 or lung cancer susceptibility in the Japanese population; however, this polymorphism appears to be a determinant of the level of CYP1A1 inducibility in the Caucasian population (23).
In this study, we explored the genetic polymorphisms of the AhR gene including the promoter. For each polymorphism that was found, its frequency was determined and the association between CYP1A1 inducibility and lung cancer susceptibility was examined in a case-control study of a French population.
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Materials and methods |
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Study population and DNA isolation
Study population and DNA isolation has been described previously by Stücker et al. (24,25). For technical or medical reasons, the DNA bank was established for only 552 of the 611 subjects recruited (89.5%) (24). In order to perform our study, we needed both lymphocytes (24) and DNA bank, this is the reason why we could study only 177 cases and 162 controls.
EROD activity assay
The procedures for the assay and optimization of the parameters influencing the results have been published previously (24,26). EROD measurements were carried out two to six times per subject on the same blood sample (mean = 2.55). The intra-individual variability was equal to 0.087, this corresponds to a small fraction (1/6) of the total variance of EROD measurements (s2 = 0.50). Therefore, the two to three measurements on the same blood sample show very good reproducibility.
The inducibility, which corresponds to the induced CYP1A1 activity, was defined as the ratio of the two enzyme activities EROD/Cyt.c red determined on the same cells after the benzanthracene treatment. Because some of the parameters could be cross-correlated, we looked at which factor(s) were significantly linked to the inducibility factor adjusted for the others (24). Therefore, statistical analysis used the log value of inducibility, so that the results would follow a Gaussian distribution.
Inducibility distribution
The classification of CYP1A1 inducibility in three classes (low, intermediate and high) was performed among controls (n = 162) according to Kellermann et al. (27). The cut-off was determined in the whole control population (185 controls): 45% low inducers (Ln: 3.6 to 0.75), 45% intermediate inducers (Ln: 0.75 to 0) and 10% high inducers (Ln: 0 to 1.2). As we used only 162 controls, this cut-off led us to observe a slightly different distribution, which did not exhibit a statistical difference with the initial one. Then the proportions were as follows: 48% low inducers, 41.5% intermediate inducers and 10.5% high inducers.
Samples used. In order to detect polymorphisms of the human AhR gene, we analysed 30 DNA samples. We have selected, randomly, 10 samples from each category of inducers defined as: (i) 10 `low inducers'; (ii) 10 `intermediate inducers'; and (iii) 10 `high inducers' defined in the `Inducibility distribution' chapter. When a polymorphism was detected, it was investigated in the whole DNA library (177 cases and 162 controls) by oligonucleotide ligation assay (OLA).
Primers used and PCR condition
Based on the nucleotide sequence information of human AhR cDNA (28) and the structure of human AhR gene (22), oligonucleotide primers for the PCR were designed to produce DNA fragments of 400600 base pairs (bp) as indicated in Figure 1. The primers used are shown in Table II
. PCR was performed with 100 ng of DNA in 25 µl PCR reaction containing 10 mM TrisHCl pH 8.3, 1.5 mM MgCl2, 200 µM dNTPs, 0.6 UI Taq polymerase and 0.3 µM of each primer. Amplifications were performed with 30 s denaturation step at 94°C, 30 s annealing at 60°C and 1 min extension at 74°C. The PCR products were detected and sized by agarose gel (2%).
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Direct DNA sequencing
Polymorphic regions of DNA fragments detected by DGGE analysis; exon 1 and the promoter region were screened by sequencing (because of the presence of GC-rich sequences which could not be easily analysed by DGGE). Primers used for amplification are indicated in Table II. The products were sequenced by the dideoxy chain termination method (30) using a sequencing Kit system in both directions, according to manufacturer instructions. The product of the sequencing reaction was analysed in the ABI PRISM 310 sequencer (Perkin-Elmer).
OLA
OLA (31) was used to screen the whole genomic library for 157(G/A) and 1721(G/A) polymorphisms. After PCR amplification, 2 µl of amplification product were added to 2 µl buffer 10x, 0.1 µl Taq ligase, 12.9 µl water + 1 µl (100 fmol) of each oligonucleotide (common 5'-phosphorylated, both labelled `mutated' or `wild-type') (Table III). The ligation was programmed as follows: initial denaturing 30 s at 94°C, hybridization and ligation 3 min at 45°C for 15 cycles, followed by a final step of denaturing at 98°C during 10 min. The ligation products were detected by fluorometric measure using the sequencer.
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All the estimations are given with the 95% confidence interval (CI = 95%). SigmaStat (Jandel Scientific Software) was used for all univariate analysis and SAS version 6.12 was used for the logistic regression.
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Results |
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Polymorphisms in the exon 1, the 5'-untranslated and promoter region of AhR were explored by direct sequencing of the 30 DNA controls. Five couples of primers were used to amplify the region between 1314 and 781 bp (Table II). One polymorphism was found in 11 out of 30 DNA samples in the 5'-untranslated region, it was a substitution of a G by an A at position 157: 157(G/A).
Polymorphism frequency in the 162 control subjects
From the genomic DNA library constituted by case-control study of lung cancer, we investigated the frequency of the three polymorphisms found in the 162 control subjects.
The allelic variant identified in the coding B region of exon 10 was investigated by OLA in the 162 controls. It was found in 25 of 162 controls tested; genotype distribution showed that 22 of 162 subjects were heterozygous and three of 162 were homozygous for the allelic variant. The allele distribution fitted the HardyWeinberg equation very well with an allele frequency of 0.92 for Arg- and 0.08 for Lys-coding alleles, respectively, with 95% CI 0.0430.129 (Table IV). The other allelic variant found was in exon 10 (E region); it was analysed in 100 subjects from the control population, no other subject presenting this allelic variant was found in this population. Because of the low frequency of the mutated subjects (1%, allele frequency 0.005, 95% CI 0.0030.018), we did not investigate it in all the 162 controls.
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Relationship between AhR polymorphisms and CYP1A1 inducibility
According to the three categories of inducers. The relationship between the most frequently observed allelic variants [1721(G/A) and 157(G/A)] and the CYP1A1 inducibility was investigated within the 162 control subjects studied. No association was found between CYP1A1 inducibility in the three classes of inducers and the frequency of 157(G/A) polymorphism found in the 5'-untranslated region (2 = 1.67, df = 2, P = 0.79). A similar result was observed for the 1721(G/A) allelic variant (
2 = 1.45, df = 2, P = 0.83).
According to CYP1A1 inducibility. The mean inducibility of the 1721(G/A) polymorphism was 0.677 ± 0.559, n = 137 for Arg/Arg versus 0.793 ± 0.722, n = 25 for Arg/Lys + Lys/Lys showing no statistical difference (P = 0.5) between genotypes. For the 157(G/A) the mean inducibility was 0.811 ± 0.716, n = 92 for GG versus 0.724 ± 0.677, n = 70 for GA + AA showing also no statistical significance (P = 0.3) between genotypes.
Relationship between the polymorphisms found
In order to test whether the polymorphisms found, 157(G/A) and 1721(G/A), are linked, a 2-test was performed. We did not observe any statistical association between these two allelic variants (
2 = 2.9, P = 0.6).
Relationship between the polymorphisms found and lung cancer
Within the 177 cases, two subjects were homozygous for the 1721(G/A) allelic variant and 28 were heterozygous (Table IV). The comparison between mutated allele frequency among already studied controls and cases did not show a statistical difference (0.086 for controls and 0.087 for cases). The odds ratio of lung cancer associated with the heterozygous genotype was 1.5 (95% CI 0.71.0) and 1.0 (95% CI 0.18.0) for the homozygous mutated. We adjusted this results for age, hospital, mean number of cigarette consumption, total duration of smoking and GSTM1 polymorphism (Table IV
). Results were unchanged.
As in the control population, this 157(G/A) allelic variant found in the 5'-untranslated region was more frequently observed than the polymorphism in the coding region, allele frequency: 0.25. Fifteen subjects out of 177 were homozygous for the allelic variant (8.5%) and 56 heterozygous (31.6%) (Table IV). The odds ratio of lung cancer associated with the heterozygous genotype was 0.8 (CI 0.51.4) and 1.2 (CI 0.43.4) for the homozygous mutated genotype. These results were non-significantly different from 1. Adjustments for age, hospital, mean number of cigarette consumption, total duration of smoking and GSTM1 polymorphism did modify this result.
Taking into account the histological type did not again alter these results. The distribution of mutated homozygous was identical within the different histological types (squamous cell carcinoma 7.4%; small cell carcinoma 10.7%; adenocarcinoma 8.7%).
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Discussion |
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The inter-individual heterogeneity of aryl hydrocarbon hydroxylase (AHH) activity, in lymphocytes treated with PAH was first demonstrated by Kellermann et al. (27). Moreover, a positive association was found between high CYP1A1 inducibility and lung cancer (24,33,34). In humans it has been suggested that most of the variability in CYP1A1 levels after induction might be due to a major gene such as the AhR gene (18). It has also been suggested that AhR polymorphism in the human population could be similar to that found in inbred mouse strains (35), but it is not yet well established if phenotypic variation is due to differences in the AhR locus in humans.
First, we systematically investigated the promoter and coding region of the AhR gene for new allelic variants in 30 controls either by DGGE and/or by sequencing. As these subjects were selected according to their CYP1A1 inducibility, the number of alleles screened was sufficient to find polymorphisms linked to the inducibility. Moreover, the systematic search of polymorphisms among 60 alleles should find any polymorphism with a frequency of 10%.
Three allelic variants were found, two located in exon 10 1721(G/A) and 2417(A/G) the other in the 5'-untranslated 157(G/A). Both 2417(A/G) and 157(G/A) allelic variants have never been described previously.
To determine the frequency of these polymorphisms and their relationship with CYP1A1 inducibility and lung cancer risk, a case-control study of lung cancer DNA library was screened.
The allelic variant 1721(G/A) detected had a 9% frequency; it has already been described in Japanese (22), Caucasian and African-American populations (23). The mutated allele frequency is quite similar between Japanese and African-American populations (respectively, 0.43 and 0.41) but much lower in the Caucasian populations [0.12 in Smart and Daly's study (23) and 0.09 in our study].
Our statistical analyses showed that the 1721(G/A) polymorphism did not affect the CYP1A1 inducibility. In this study, the absence of a relationship between the AhR polymorphism and CYP1A1 inducibility is in good agreement with the small change in amino acid (substitution of an Arg by a Lys) outside the ligand-binding domain which is localized in exons 78 of the protein. Moreover, this is in accordance with the results of Kawajiri et al. (22) who found no association between the 1721(G/A) polymorphism and CYP1A1 inducibility using EROD assay in treated lymphocytes. However, it disagrees with the results of Smart and Daly (23) who showed a significantly higher level of induced CYP1A1 activity in individuals with at least one copy of the 1721(G/A) variant allele (GA or AA) compared with individuals carrying (GG) allele.
The discrepancies between studies could be due to the way CYP1A1 inducibility was expressed and also to gender. In our study, we have measured the induced CYP1A1 activity using EROD activity/Cyt.c red; Smart and Daly measured EROD activity/mg cellular protein (23) and in the Kawajiri et al. study the inducibility level of AHH was measured (22). Concerning gender, only men were analysed in our study, whereas both genders were used in the Smart and Daly study (20 women and 10 men) (23). They found a significantly lower level of induced CYP1A1 activity in women compared with men, this observation appears to have also occurred in an earlier study performed by Thompson et al. in healthy volunteers (36). The other allelic variant 2417(A/G) detected in the coding region (exon 10, fragment E) with a 0.5% frequency leads to the replacement of Met by Val at codon 786. This allelic variant, which is not a polymorphism (frequency <1%), has never been described before in either Japanese or Caucasian populations probably because of its rareness. The allelic variant 2417(A/G) is located in the C-terminal region of AhR gene and could play an important role in the transcriptional activity. It was detected in one `high inducible phenotype' out of 100 controls studied. Considering the rareness of this allelic variant, the number of subjects included in this study could not allow us to link it with inducibility with a sufficient statistical power. However, it cannot be a significant contributor to the observed inter-individual variation in CYP1A1 inducibility in Caucasian population.
Other polymorphic sites in the coding region of the AhR gene were described in a Japanese population (22) and in an Amercican-African population (23). These polymorphisms were not detected in our study. Kawajiri described a substitution of T by C at codon 44 (ATT to AAC) in exon 2, but this did not lead to amino acid (Asn) replacement (22). Smart and Daly described a substitution of G by A at codon 1768 in exon 10, which results in amino acid replacement of Val 570 by Ile (23). This polymorphism was only found in African-American population at 0.05 allele frequency.
The allelic variant found in the 5'-untranslated region (between the initiation site of transcription and the ATG codon), has also not been described previously. It leads to the substitution of a G by an A in position 157 with an elevated frequency (0.25); no significant association has been established between this polymorphism and CYP1A1 inducibility. We assumed that a sequence modification in this region could influence the transcription of the gene or translation of the mRNA but this is apparently not the case since no relationship was found between this allelic variant and CYP1A1 inducibility.
We also looked for a relationship between the AhR polymorphisms and lung cancer. The 1721(G/A) and 157(G/A) polymorphisms were investigated in the lung cancer cases. Our results clearly showed that the OR is similar among wild-type and mutated genotypes for both allelic variants. Furthermore, the genotype distribution among the three categories of inducers is the same for patients and controls and variant alleles were identical within the different histological tissue types.
Finally, we investigated the relationship between AhR polymorphisms and GSTM1 in the lung cancer risk. A significant association between GSTM1 and lung cancer was shown previously (25). To our best knowledge, the association between both GSTM1, which is involved in the detoxification of polyaromatic hydrocarbon epoxides of B[a]P, and AhR polymorphisms has never been studied previously. Our results did not show any significant association between GSTM1 and AhR polymorphisms in lung cancer risk or in CYP1A1 inducibility.
In conclusion, two polymorphisms and one allelic variant in the AhR gene were found in a French population, none of them being involved in the inducible expression of CYP1A1 or in the susceptibility to develop a lung cancer. Thus, the heterogeneity of AHH activity could be due to polymorphisms that are either present in a different region of the AhR gene or in a different gene regulating CYP1A1 expression. Indeed, in mice, a novel mechanism of feedback inhibition of AhR by the Ahr repressor (AhRR) has been described (37). Activation of AhR by xenobiotics induces expression of the AhRR gene through binding to the XRE as a heterodimeric complex with ARNT. The induction of AhRR could inhibit, the AhR function by competing with it for ARNT- and XRE-binding activity. It could be interesting to investigate the genetic polymorphism of the AhRR gene to study the association between a putative polymorphism found with CYP1A1 inducibility activity and lung cancer.
Acknowledgements
We are grateful to the `Ligue Contre le Cancer', the `Région Ile de France; ARC and the `SESAME program' for supporting this work.
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Notes |
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References |
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