Mutagenesis Advance Access originally published online on December 7, 2007
Mutagenesis 2008 23(1):67-73; doi:10.1093/mutage/gem046
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Influence of TCDD and natural Ah receptor agonists on benzo[a]pyrene–DNA adduct formation in the Caco-2 human colon cell line
Department of Health Risk Analysis and Toxicology, Maastricht University, PO box 616, 6200 MD Maastricht, The Netherlands 1Toxicology and Effect Monitoring Group, RIKILT Institute of Food Safety, PO box 230, 6700 EA Wageningen, The Netherlands 2Division of Toxicology, Wageningen University, PO box 8000, 6700 EA Wageningen, The Netherlands
Several compounds originating from cruciferous vegetables and citrus fruits bind to and activate the aryl hydrocarbon receptor (AhR). This receptor plays an important role in the toxicity of the known tumour promoter and potent AhR-agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, vegetables and fruits are generally considered as healthy. Therefore, besides the AhR activation, the natural AhR agonists (NAhRAs) are assumed to show other health-concerning effects. AhR activation induces several cytochrome P450 phase I enzymes involved, e.g. in the bioactivation of carcinogenic polycyclic aromatic hydrocarbons, like benzo[a]pyrene (BaP), and may as such stimulate DNA adduct formation of those compounds. Therefore, the influence of TCDD, indolo[3,2-b]carbazole (ICZ, an NAhRA originating from cruciferous vegetables) and an NAhRA-containing extract of grapefruit juice (GJE) on BaP–DNA adduct formation in the human Caco-2 cell line was studied. Also, we investigated if different effects of TCDD, ICZ and GJE on adduct formation could be related to the modulation of transcription of biotransformation- and DNA-repair enzymes. Co-exposure to high AhR-activating concentrations of both TCDD and ICZ significantly reduced the amount of BaP–DNA adducts at 0.1 µM BaP, while at higher concentrations of BaP no influence was observed. In contrast, exposure to 0.1 µM BaP combined with GJE showed a significant increase in BaP–DNA adducts, and a significant decrease at 0.3 and 1 µM BaP. These differences could not be related to transcription of the phase I and II enzymes CYP1A1, CYP1B1, NQO1, GSTP1 and UGT1A6 or to transcription of the nucleotide excision repair enzymes ERCC1, XPA, XPC, XPF and XPG. We conclude that ICZ showed a similar effect on BaP–DNA adduct formation than TCDD, while GJE influenced the adduct formation in a different way. The difference in the influence on adduct formation may be due to effects at the level of enzyme activity, rather than gene expression.
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Introduction |
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Cruciferous vegetables and citrus fruits have been shown to contain significant amounts of compounds that can act as natural aryl hydrocarbon receptor (AhR) agonists (NAhRAs) (1
–3). Binding of an agonist to the AhR activates it as a transcription factor, which is considered the main pathway by which dioxins, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), exert their toxicity (4–6). Nevertheless, NAhRA-containing vegetables and fruits were reported to possess several health-promoting properties, including cancer-preventive potential (7–10), although not all epidemiological studies are consistent in this respect (8,11,12). Benzo[a]pyrene (BaP), a well-known carcinogenic compound originating from incomplete combustion of organic material, for instance by cigarette smoking, is also an AhR agonist. Phase I biotransformation enzymes like the P450 cytochromes CYP1A1 and CYP1B1 can convert BaP into reactive epoxide metabolites, and in the presence of epoxide hydrolase activity also dihydrodiol epoxides are formed. These electrophilic compounds can bind to the DNA and form BaP–DNA adducts, which can cause mutations, and finally may lead to cancer (13,14). Several detoxifying mechanisms in the cell may prevent these injurious effects; phase II biotransformation enzymes such as glutathione-S-transferase transform the electrophilic compounds into products which can be eliminated rapidly from the cell, and DNA-repair enzymes, like the nucleotide excision repair (NER) system, can remove the bulky BaP–DNA adducts from the DNA (13,15). Thus, there appears to be a dynamic balance between these possible metabolic routes of BaP. Since both TCDD and NAhRAs activate the AhR, they theoretically can stimulate the BaP–DNA adduct formation by induction of CYP1A1 and CYP1B1. It has indeed been reported that TCDD can promote the BaP adduct formation by AhR activation in rats (16) and that mice lacking the AhR did not suffer from BaP carcinogenicity (17).
In this study, we investigated the influence of NAhRAs on BaP–DNA adduct formation in a human colon cancer cell line, and compared it with the effects of TCDD. The Caco-2 cell line was chosen since it is generally used as a model system to study the effect and absorption of compounds in the small intestine, and it is known to show AhR-activity and BaP–DNA adduct formation (18). We examined the pure NAhRA indolo[3,2-b]carbazole (ICZ), a metabolite from cruciferous vegetables, and a hexane extract of grapefruit juice, containing the suspected NAhRAs bergamottin and certain polymethoxy flavones (19–21). Based on the above-mentioned tumour-promoting effects of TCDD on the one hand and the health-protective properties of vegetables and fruits on the other hand, we hypothesized that addition of TCDD to cells exposed to BaP will show an increase in BaP–DNA adduct formation, while addition of NAhRAs will result in a decrease in BaP–DNA adduct formation. The influence on adduct formation was subsequently related to the effects on bioactivation, elimination and DNA repair as examined at the transcription level by measuring mRNA of phase I and II enzymes and NER enzymes.
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Materials and methods |
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Chemicals
Methanol, ethanol, n-hexane, diethyl ether, dimethyl sulfoxide (DMSO), NaCl, EDTA, isoamylalcohol, sodium acetate, NaH2PO4, formic acid and LiOH were purchased from Merck (Darmstadt, Germany); phenol, Tris, endonuclease, spleen phosphodiesterase and urea from Sigma–Aldrich (Steinheim, Germany); foetal bovine serum, penicillin/streptomycin and trypsin from GIBCO (Paisley, UK); sodium dodecyl sulphate (SDS), nuclease P1, [
-32P]-ATP and LiCl from MP Biomedicals (Irvine, CA, USA); chloroform from Biosolve (Valkenswaard, The Netherlands) and T4 polynucleotide kinase from Fermentas (Hanover, MD, USA). ICZ (purity >95%) was synthesized as described by Bergman (22) and checked for quality by HPLC, NMR, IR and MS, using a reference sample which was a kind gift of Prof. J. Bergman, Department of Chemistry, Royal Institute of Technology, and Department of Biosciences at Novum, Huddinge, Sweden).
Preparation of the grapefruit juice extract
Ten ml of grapefruit juice, obtained from the local supermarket, was mixed with 20 ml of methanol/water 85/15, and extracted twice with 20 ml of n-hexane/diethyl ether 97/3 by mixing for 1 h. Hexane layers were collected, evaporated and the residue dissolved in 40 µl of DMSO for analysis. In a recent study (19), the grapefruit juice extract (GJE) was analysed by HPLC and found to contain a mixture of suspected NAhRAs, like polymethoxy flavones and a furocoumarin. The GJE contained the polymethoxy flavones tangeretin (48.5 µM), sinensetin (3.7 µM), tetramethylscutellarein, nobiletin and heptamethoxyflavone (all three < 0.1 µM), and the furocoumarin bergamottin (4150 µM). Because bergamottin showed the highest concentration, we express in this study the amount of extract as µM bergamottin. Also a blank extraction with 10 ml water has been performed.
Cell culture and exposure
The Caco-2 human colorectal adenocarcinoma cell line (American Type Culture Collection, Manassas, VA, USA) was grown in Dulbecco’s Modified Eagle’s Medium (Sigma, St-Louis, MO, USA), containing 10% foetal bovine serum and 1% penicillin/streptomycin, at 37°C, and 5% CO2. Cells were seeded 24 h before exposure in 6-well plates (5.105 cells/well) and exposed for 24 h to sample solutions in DMSO added to the culture medium. Concentration ranges were made of 1.5–1500 pM TCDD (purity 98%, Schmidt BV, Amsterdam, The Netherlands), 1–1000 nM ICZ, 0.008–25 µM BaP (purity >98%, Aldrich, Steinheim, Germany) and 0.03–6.9 µM bergamottin in GJE for the CYP1A1 mRNA induction experiments. For the adduct analysis, cells were exposed to 0.1 (duplicate), 0.3 or 1 µM BaP (triplicate) together with a very low, low or high AhR-activating concentration of TCDD, ICZ or GJE. In two additional experiments, 0.1 µM BaP was added 6 h (duplicate) or 0.3 µM BaP was added 15 h (triplicate) after treatment with TCDD, ICZ and GJE, to investigate if pre-treatment with a CYP1A1 inducer could result in a more pronounced effect. The final concentration of DMSO in all exposures was 0.5% (v/v). Cytotoxicity of the mixtures was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-colorimetric assay according to Mosmann (23), with modifications as described previously (24).The number of replicates was four.
RNA and DNA isolation
After exposure, total RNA was isolated from the cells using Trizol (Invitrogen, Breda, The Netherlands) according to the manufacturer's protocol. Purity and integrity of the RNA were checked by UV absorption and gel electrophoresis, respectively. For DNA isolation, cells were washed twice with ice-cold PBS, trypsinized and suspended in 1 ml medium. After centrifugation (1 min, 1500 r.p.m.), liquid was removed and cells were incubated with 400 µl Salt/EDTA/Tris/SDS (100 mM NaCl, 20 mM EDTA, 50 mM Tris, 0.5% SDS, pH 8.0) for 2 h at 37°C, followed by treatment with RNAse A/T1 and proteinase K (DNAse-free, Boehringer Mannheim). The DNA was purified by phenol/chloroform/isoamylalcohol (25:24:1) extraction and precipitation with 3 M sodium acetate, pH = 5.2, and 100% ethanol. After washing with 70% ethanol (v/v), the DNA was dried under a mild nitrogen flow and dissolved in 2 mM Tris (pH 7.4). Concentration and purity of the DNA were determined spectroscopically at 230, 260 and 280 nm.
Quantitative reverse transcription–polymerase chain reaction
One microgram of total RNA was reverse transcribed into cDNA using iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) in a final volume of 20 µl. Table I lists the primer sequences used. β-Actin was used to normalize for differences in RNA input and efficiency during the reverse transcription. Samples were diluted 15 times and duplicates were mixed with primers and iQTMSYBR®Green Supermix (Bio-Rad) and amplification took place in an iCycler PCR machine with an MyiQ Single Color Real-Time PCR Detector System (Bio-Rad, Veenendaal, The Netherlands). The specificity of the polymerase chain reaction (PCR) products was checked by melt curve peak analysis and by gel electrophoresis. Gene expression levels were calculated from the threshold cycle values and normalized by calculating the ratio with the β-actin expression level.
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32P-post-labelling of BaP–DNA adducts
The 32P-post-labelling assay for BaP–DNA adducts was performed as described earlier (25
). In short, 10 µg DNA was digested into deoxyribonucleoside 3'-monophosphates by incubation with micrococcal endonuclease and spleen phosphodiesterase for 4 h at 37°C. Seventy-five percent of the digest was treated with nuclease P1 for 30 min at 37°C and the reaction was terminated by adding 0.5 M Tris. The labeling was performed by using [-32P]-ATP (50 µCi) in the presence of T4 polynucleotide kinase for 30 min at 37°C, followed by two-dimensional chromatography using polyethylene-imine cellulose sheets (Macherey Nagel, Düren, Germany) using the following solvent systems: S1, 1 M NaH2PO4 (pH 6.5); S2, 5.3 M lithium formate, 8.5 M urea (pH 3.5); S3, 1.2 M lithium chloride, 0.5 M Tris, 8.5 M urea (pH 8.0); S4, 1.7 M NaH2PO4 (pH 6.0). For calibration, three standards of [3H]-BaP-diol-epoxide modified DNA with known modification levels (1 per 106, 107 and 108 nucleotides) were run in parallel in each experiment. Quantification was performed using a PhosphorImager (Raytest-Fujifilm FLA-3000, Düsseldorf, Germany) with a detection limit of 1 adduct/109 nucleotides. The other 25% of the digest was used for the determination of the amount of normal nucleotides by HPLC using UV detection.
Statistics
Differences in DNA adduct levels and gene expression levels were statistically evaluated using the Student's t-test.
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CYP1A1 and CYP1B1 mRNA induction
To estimate the onset and progress of the AhR response in Caco-2 cells, different exposure times and concentration ranges have been applied for TCDD, BaP, ICZ and GJE and the amount of mRNA of CYP1A1 was measured by use of quantitative reverse transcription (RT)–PCR (Figure 1). Exposure to TCDD resulted in increased gene expression in the low picomolar range and a maximum of about 400-fold change in mRNA amount was reached after 15 h of incubation with 1.5 nM. Six hours of exposure showed a considerable lower induction of CYP1A1 mRNA. BaP induced CYP1A1 in a comparable way as TCDD, however, at concentrations a thousand times higher (Figure 1A). The maximum induction of CYP1A1 by ICZ and GJE was about a 100-fold change, both after 15 and 24 h. The effective concentration of ICZ was in the same range as of BaP, and GJE, expressed as µM bergamottin, induced CYP1A1 to the same level in a concentration range about a factor 10 higher (Figure 1B). CYP1B1 mRNA determination resulted in similar induction properties of the samples, although the maximum levels were much lower (10- to 30-fold change, results not shown). The extraction procedure blank (water subjected to the extraction procedure applied to extract the grapefruit juice) did not induce CYP1A1 or CYP1B1 mRNA significantly (results not shown).
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The concentrations chosen for BaP, TCDD, ICZ and GJE in the following studies were based on the level of induction of CYP1A1 mRNA after 24 h of exposure, a marker for AhR activation. Based on Figure 1A, we estimated a low, medium and high effective concentration of BaP and a low and high effective concentration for TCDD, ICZ and GJE. Low is defined as around 10% of the maximum activation, and high is the maximum activation.
BaP–DNA adducts
In order to determine the effects of TCDD and NAhRAs on the DNA adduct formation by BaP, human Caco-2 cells were exposed to BaP in combination with TCDD or NAhRAs, and the adduct formation was measured by 32P-post-labelling (Figure 2). Exposure to 0.1, 0.3 and 1 µM BaP resulted in a dose-dependent formation of DNA adducts of respectively 3.7, 38 and 244 adducts per 108 nucleotides. Two concentration levels of TCDD and NAhRAs were tested, resulting in either a relatively high or low level of AhR activation, as judged from the CYP1A1 gene expression (Figure 1A). The concentration of TCDD producing a high level of CYP1A1 gene expression (500 pM) showed nearly a 4-fold decrease in BaP–DNA adduct formation (P = 0.11, n = 2), but only at the lowest concentration of BaP tested (0.1 µM) (Figure 2A). The low 15 pM TCDD concentration reduced the adduct formation significantly to 64% at 1 µM BaP, but had no significant effect at 0.1 and 0.3 µM BaP. Both the low and high concentrations of ICZ caused a strong reduction of BaP–DNA adduct levels (100 nM ICZ: P = 0.11, n = 2), down to one-third of the adducts induced by 0.1 µM BaP, but, like TCDD, ICZ showed no effect at the higher concentrations of BaP (Figure 2B), with the exception of a small but significant increase at 0.3 µM BaP. In contrast with TCDD and ICZ, both concentrations of GJE showed an approximately 2-fold increase in adduct formation at 0.1 µM BaP (Figure 2C). However, both at 0.3 and 1 µM BaP, the highest concentration of GJE decreased the amount of adducts significantly by five times (Figure 2C). TCDD, ICZ and GJE without co-exposure with BaP, just as the solvent DMSO, had no effect on DNA adduct formation (results not shown).
We also tested whether pre-treatment with a CYP1A1 inducer would produce a more pronounced effect on BaP–DNA adduct formation as compared to simultaneous exposure. Overall, both after 6 h (0.1 µM BaP) and 15 h (0.3 µM BaP) of pre-treatment of the cells with TCDD or NAhRAs, the adduct levels induced by BaP were not significantly different from those induced by simultaneous exposure to these compounds (results not shown). An only small, but statistically significant, difference was observed between simultaneous incubation with 0.3 µM BaP and 100 nM ICZ and 15 h pre-incubation with 100 nM ICZ followed by simultaneous incubation. Overall, this indicates that a 6- to 15-h earlier onset of AhR-induction does not produce a more pronounced effect on adduct formation.
For statistical purpose, and because the pre-incubation step was found to have mostly no significant effect at all, the results of the duplicate experiments with 0.1 µM BaP simultaneously exposed with (N)AhRAs, and with 6 h pre-incubation with (N)AhRAs, were pooled to obtain n = 4. In this way, the decrease in BaP–DNA adduct formation by 500 pM TCDD (P < 0.05), 3 nM ICZ (P < 0.05) and 100 nM ICZ (P < 0.01) and the increase of adduct formation by 0.15 µM GJE (P < 0.05) and 2.3 µM GJE (P < 0.01) were found significant (results not shown).
Gene expression modulation
To examine whether the effects of (N)AhRAs on BaP–DNA adduct formation could be related to modulation of the expression of biotransformation- or DNA-repair genes, the mRNA levels of several genes involved in these cellular processes have been measured by quantitative RT–PCR (Figure 3). Of the biotransformation enzymes, it is known that they are effected by AhR activation (1,4), for the DNA-repair enzymes it is not and therefore adding new information.
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CYP1A1. BaP increased the mRNA levels of the phase I biotransformation enzyme CYP1A1 by 12 (0.1 µM BaP) to 84 (1 µM BaP) times (Figure 3A). Addition of the relatively high (in terms of AhR activation) concentrations of TCDD, ICZ and GJE significantly increased the amount of CYP1A1 mRNA at all BaP concentrations, except for TCDD at 1 µM BaP.
CYP1B1. BaP increased the mRNA levels of the phase I biotransformation enzyme CYP1B1 by 2 (0.1 µM BaP) to 7 (1 µM BaP) times (Figure 3B). Addition of TCDD and GJE significantly increased the amount of CYP1B1 mRNA caused by BaP (Figure 3B). ICZ showed only a minor increase at 0.1 µM BaP. The induction by ICZ at 1 µM BaP was not determined.
NQO1. No up- or down-regulation of the phase II biotransformation enzyme NQO1 was observed after exposure to BaP, TCDD and NAhRAs (results not shown).
GSTP1. BaP caused a 2.5- to 3-fold increase of the phase II biotransformation enzyme GSTP1 but the response was not dose related. Although TCDD, ICZ and GJE alone showed significantly a 1.6- to 2.5-fold increase, they inhibited the GSTP1 induction by BaP at 0.1 and 0.3 µM BaP (except for GJE at 0.1 µM BaP), whereas they had no effect at 1 µM BaP (Figure 3C).
UGT1A6. BaP induced this phase II biotransformation enzyme mRNA significantly by about 2-fold. TCDD and the NAhRAs showed a similar induction, and did not have a significant effect in the mixture with 0.1 µM BaP (Figure 3D). The UGT1A6 mRNA induction at higher levels of BaP has not been determined.
ERCC1. BaP had no significant effect on the gene expression of ERCC1 (Figure 3E). ICZ down-regulated the ERCC1-mRNA expression 1.7 times. Co-exposure to TCDD, ICZ and GJE significantly decreased the ERCC1 expression levels found at 0.3 µM BaP by nearly 2-fold but had no or minimal effects at 0.1 and 1 µM BaP.
XPF (ERCC4). The NER enzyme XPF tended to be up-regulated 1.4–2.6 times by TCDD and the NAhRAs and 0.1 µM BaP, however, not significantly (Figure 3F). Simultaneous exposure to TCDD had no effect at 0.1 µM BaP, but ICZ and GJE decreased the induction at 0.1 µM BaP by 2-fold, but again not significantly. No influence was observed at 0.3 µM BaP. No determinations were carried out at 1 µM BaP, and for ICZ at 0.3 µM BaP.
XPG (ERCC5). TCDD and the NAhRAs had no significant influence on the induction of the NER enzyme XPG mRNA. BaP showed a 1.6-fold increase at 0.1 µM BaP, which was diminished by addition of TCDD significantly (P < 0.05). GJE decreased the XPG mRNA found at 0.3 µM BaP (results not shown). No analyses were performed at 1 µM BaP, and for ICZ at 0.3 µM BaP.
XPA and XPC. No significant effects of BaP alone and in combinations with TCDD or GJE on the NER enzymes XPA and XPC mRNA were observed at 0.3 µM BaP (results not shown). Therefore, no other concentrations of BaP were tested any more.
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Discussion |
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In this study, we investigated the influence of natural AhR-activating compounds in fruits and vegetables on BaP–DNA adduct formation in relation to the effect of the prototypical AhR agonist TCDD. Possible differences observed might be explained by differences between TCDD and NAhRAs with respect to the induction of biotransformation or DNA-repair activity. As a first step in this complicated process, we evaluated the differences between the effects of TCDD and NAhRAs on BaP–DNA adduct formation in relation to the modulation of expression of genes for several biotransformation and DNA-repair enzymes.
The influence of TCDD and NAhRAs on adduct formation appeared highly dependent on the concentration of BaP. Both TCDD and ICZ appear to reduce the amount of adducts considerably at 0.1 µM BaP, while they showed no clear effect at higher concentrations of BaP. This suggests that at low concentrations of BaP, which has a relatively low effect on, e.g. CYP1A1 and CYP1B1 mRNA levels by itself, the extra induction of CYP1A1 and CYP1B1 by the added AhR agonists can accelerate the elimination of the electrophilic BaP metabolites out of the cells; at higher concentrations of BaP, this effect already seems to be saturated by BaP itself. This detoxification process by TCDD was not hypothesized, but for ICZ it is in agreement with the findings of Ebert et al. (26), who found that the NAhRAs indole-3-carbinol (I3C, a precursor of ICZ) and flavone induced CYP1A1 in Caco-2 cells and that pre-treatment with these compounds accelerated metabolism and clearance of BaP. They also found that I3C, ICZ and also TCDD AhR dependently induced breast cancer resistance protein, which is involved in the transport of phase II metabolites of BaP (27). Arif et al. (28) showed that gavage treatment of I3C to rats exposed to cigarette smoke inhibited the DNA adduct formation in lung, trachea, bladder and heart, which is also indicative for I3C-stimulated BaP removal. Furthermore, the apparent absence of BaP metabolization by CYP1A1 and/or CYP1B1 and the subsequent lack of BaP clearance apparently resulting from it could explain the increase in BaP–DNA adducts in BaP-exposed CYP1A1/1B1-deficient mice (29,30). In contrast to these findings, the results from some other studies do not support the hypothesis that induction of CYP1A1 and CYP1B1 decreases the formation of reactive BaP metabolites. For instance, liver and lung tissues of rats, pre-treated with TCDD 48 h before sacrifice, showed a significant increase in BaP–DNA adducts after in vitro incubation with 10 µM BaP, which was related to a higher induction of CYP1A1 and CYP1B1 (16). Also Shimizu et al. (17) reported that BaP-exposed AhR–/– mice, which lacked the CYP1A1 induction, showed no BaP-related tumour formation. A potential other explanation may be that the induction of CYP1A1 and CYP1B1 results in the accelerated activation of BaP, initially resulting in higher DNA adduct levels, which are subsequently removed by DNA repair. As a result, the levels are lower due to the longer time available for the repair enzymes.
The GJE showed a completely different effect on BaP-DNA formation than TCDD and ICZ (an increase at 0.1 µM BaP instead of a decrease and a decrease at 0.3 and 1 µM BaP instead of no effect or a slight increase), although it also strongly induced CYP1A1 and CYP1B1 mRNA. GJE can inhibit the CYP1A1-related 7-ethoxyresorufin-O-deethylase activity (19) and it has been reported that constituents in grapefruit, e.g. bergamottin, inhibit the enzyme activity of CYP1A1 and CYP1B1 (21). Inhibition of these enzymes could hamper the rapid clearance of BaP, and thereby cause an increase in DNA adducts. This would be in agreement with the adduct-reducing effects at 0.1 µM BaP of TCDD and ICZ, which do not inhibit the CYP enzymes. Additionally, the formation of reactive BaP metabolites could be reduced by GJE-mediated inhibition of enzyme activity. This might explain why at higher levels of BaP, where the detoxification process seems also saturated according to our TCDD and ICZ results, GJE has a strong reducing effect on the BaP–DNA adduct formation.
Modulating effects on the transcription of the phase II enzymes NQO1, GSTP1 and UGT1A6 and the NER enzymes were moderate and could not be related to the different effects on adduct formation by the AhR agonists. Both the expression of GSTP1 and UGT1A6 was slightly induced by TCDD and NAhRAs, but the mixtures with BaP did not show an additive effect, as was seen for the induction of CYP1A1 and CYP1B1, but at 0.1 and 0.3 µM BaP even an inhibition of GSTP1 gene expression was shown. Although the influence on gene expression of the NER enzymes fluctuated considerably between the several BaP, TCDD and NAhRA exposures, presumably the opposite effects on adduct formation by GJE and the other compounds tested are not related to NER enzyme transcription.
We conclude that the influence of TCDD and NAhRAs on BaP–DNA adduct formation is not linearly related to AhR activation and that ICZ as a single NAhRA compound shows considerably more resemblance to TCDD, than GJE as a complex mixture containing NAhRAs, at high AhR-activating concentrations. In the lower and therefore more physiological BaP concentration range, TCDD and ICZ showed an apparent inhibiting effect on BaP–DNA adduct formation, while the GJE showed a stimulating effect. This seems to contradict our hypothesis based on the presumed toxic properties of TCDD and the healthy properties of citrus fruits. Differences between the effects of TCDD/ICZ and GJE on BaP genotoxicity could not be attributed to phase I and II enzymes and NER enzymes at the transcription level. Therefore, the influences on the dynamic balance between BaP elimination, bioactivation/DNA adduct formation and DNA repair should further also be examined at the level of enzyme activity. Our results emphasize the importance of testing mixtures at realistic concentrations for risk–benefit evaluations. In view of the complex mechanisms, our in vitro findings should be confirmed with in vivo studies.
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The Dutch Centre of Human Nutrigenomics.
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Acknowledgments |
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The authors thank Jennifer Collins and Stefan Camps kindly for their contribution to the cell culture, sample treatment and cytotoxicity test.
Conflict of interest statement: None declared.
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* To whom correspondence should be addressed. Tel: +31 43 3881091; Fax: +31 43 3884146; Email: t.dekok{at}grat.unimaas.nl
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Received on September 7, 2007; revised on November 9, 2007; accepted on November 11, 2007.
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