Sulforaphane and quercetin modulate PhIP–DNA adduct formation in human HepG2 cells and hepatocytes

James R. Bacon1, Gary Williamson1,4, R. Colin Garner2, Graham Lappin2, Sophie Langouët3 and Yongping Bao1,5

1 Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, UK, 2 Xceleron Ltd, York Biocentre, Innovation Way, Heslington, York YO10 5NY, UK and 3 INSERM U456, Faculté de Pharmacie, University de Rennes I, F-35043 Rennes, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The formation of DNA adducts in human HepG2 cells and human hepatocytes exposed to 14C-labelled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was examined using Accelerator Mass Spectrometry (AMS). PhIP generated DNA adducts in a linear dose-dependent manner between 100 pM and 20 µM. Co-treatment with the dietary isothiocyanate, sulforaphane (SFN, 1–10 µM), or the flavonoid, quercetin (5–20 µM), significantly reduced the level of PhIP–DNA adducts in a dose-dependent manner. The degree of protection was dependent on PhIP concentration, i.e. after 100 pM PhIP exposure, SFN or quercetin reduced adduct levels to below the limit of detection (0.15 amol PhIP/µg DNA) but at higher PhIP exposure (10 nM and 1 µM), the protection was 60 and 10%, respectively. The involvement of phase I, phase II and DNA repair enzymes in this protection against PhIP–DNA adduct formation was investigated using real-time RT–PCR and enzyme activity assays. In intact HepG2 cells, quercetin inhibited cytochrome P450 (CYP)1A2, the main phase I enzyme responsible for PhIP bioactivation. In contrast, SFN induced phase II detoxification enzymes, UDP-glucuronosyltransferase 1A1 and glutathione S-transferase A1 mRNA expression. SFN and quercetin showed no effect on DNA repair, neither in terms of the level of PhIP–DNA adducts, when cells were treated with phytochemicals after the carcinogen exposure, nor the regulation of mRNA expression of two DNA repair enzymes, apurinic endonuclease and DNA polymerase ß. This study indicates that dietary isothiocyanates and flavonoids modulate phase I and phase II enzyme expression, hence increasing the rate of detoxification of the dietary carcinogen PhIP in human HepG2 cells but do not affect the rate of PhIP–DNA adduct repair. The formation of PhIP–DNA adducts in human hepatocytes was also dose-dependent with PhIP-concentration and the levels of protection by SFN or quercetin were up to 60% after 10 nM PhIP treatment, but showed large inter-individual variation with no observed protection in some individuals.

Abbreviations: AMS, Accelerator Mass Spectrometry; APE, apurinic endonuclease; GST, glutathione S-transferase; HCAs, heterocyclic amines; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; Pol ß, DNA polymerase ß; SFN, sulforaphane; UGT, UDP-glucuronosyltransferase; RT–PCR, reverse transcription polymerase chain reaction


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Exposure to heterocyclic amines (HCAs) derived from cooked meat has been implicated in the aetiology of certain human cancers including colon, prostate and breast cancer (13). It has been estimated that the total intake of HCAs by an individual can be up to tens of micrograms per day (4). 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most mass abundant type of HCA formed in cooked meat and fish (57). In order to exert its genotoxicity, ingested PhIP must undergo metabolic activation via cytochrome P450 (CYP)1A2-mediated N-hydroxylation and followed by N-O-esterification catalyzed generally by N-acetyltransferases and sulfotransferases (8,9). N-Acetoxy-PhIP can covalently bind to DNA at the C8 of 2'-deoxyguanosine to form the DNA adduct, N-(deoxyguaunosin-8-yl)-2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (dG-C8-PhIP) (1012), generating predominantly G->T transversion mutations in mammalian cells (13,14). However, not all DNA adducts cause mutation because cells are equipped with defence systems to metabolize ingested PhIP and to repair adducted DNA. The activities of phase II enzymes, such as UDP-glucuronosyltransferase (UGT), are particularly important in the detoxification of PhIP, forming less toxic and more readily excretable conjugates such as N-OH-PhIP-N2-glucuronide (15,16).

Food is a complex matrix including nutrients, phytochemicals and possible toxic compounds such as HCAs. There are many reports of phytochemicals, including isothiocyanates and flavonoids, acting as chemopreventive agents against PhIP-induced carcinogenesis and PhIP–DNA adduct formation in rats (1721). Flavonoids such as quercetin, acting as blocking agents, can reduce the formation of PhIP–DNA adducts through the inhibition of PhIP bioactivation (21,22). Isothiocyanates can induce UGT and glutathione S-transferase (GST), which may increase the rate of metabolism and detoxification of PhIP (2326). The level of DNA adducts is an intermediate end-point biomarker, which can be used to measure the overall interactions and effects of PhIP and protective agents via activation, detoxification and repair (27,28). Isothiocyanates and flavonoids have been shown to protect against DNA damage and modulate DNA repair in rats and cultured cells (2932). It is not known, however, at what level PhIP treatments can be effectively modulated by the protective effects of phytochemicals. DNA damage produced by oxidative stress agents may induce DNA repair enzymes such as apurinic/apyrimidinic endonuclease (APE) and DNA polymerase ß (Pol ß) (33,34), which may also play a role in modulation of PhIP–DNA adduct levels. It is believed that PhIP causes bulky DNA damage and nucleotide excision repair (NER) may be involved in the repair (12,35). Pol ß has been shown to be involved in both base excision repair and NER in mammalian cells (36,37). The role of phytochemicals, however, in the repair of PhIP–DNA adducts has not been elucidated and the effects of sulforaphane (SFN) and quercetin on these mechanisms are largely unknown.

This study seeks to identify the effects of interactions and dose related changes between PhIP and phytochemicals on the level of PhIP–DNA adduct formation in HepG2 cells, a human hepatoma cell line that has been shown to express the principal enzymes (CYP1A2, N-acetyltransferase 2, sulfotransferase 1, UGT, GST, quinone reductase) necessary for the bioactivation and detoxification of HCAs (38) and in primary human hepatocytes using the most sensitive method yet developed for the determination of radioactivity in DNA samples; the nuclear physics technique of Accelerator Mass Spectrometry (AMS) (3941). Detection limits for quantitative assays are typically in the range of one adduct in 109 or 107 nucleotides (equivalent to 3 or 300 amol PhIP/µg DNA). However, AMS has a detection limit down to one adduct in 1012 nucleotides (or 3 zmol/µg DNA), depending upon background levels (40). This sensitivity allows in this study the investigation of the protection against DNA adduct formation by SFN and quercetin after treatment of cells with very low (pM) concentrations of 14C- PhIP and the identification of the threshold levels of PhIP below which phytochemicals may have beneficial effects. Combined with enzyme activity assays and real-time RT–PCR to determine mRNA levels of relevant phase II metabolizing and DNA repair enzymes, this will allow an understanding of the mechanisms by which phytochemicals may modify the cellular metabolic pathways of PhIP activation, detoxification and repair using PhIP–DNA adduct formation as a biomarker.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Unless otherwise stated, all chemicals were purchased from Sigma. Cell culture media and supplements were from Invitrogen (Gibco), (Paisley, UK). SFN (4-methylsulfinylbutyl isothiocyanate, purity 97%) was purchased from ICN Biomedicals, UK. PhIP (purity, 98%) and [14C]PhIP (purity, 98%, radiochemical purity, 17.5%) (370 Mbq/mmol) were purchased from Toronto Research Chemicals (Toronto, Canada). Human liver S9 fraction was purchased from Gentest (Cambridge Bioscience, UK). Primers were ordered from Sigma Genosys, (Cambridge, UK). TaqMan probes with 5'-FAM and 3'-TAMRA modification were ordered from Applied Biosystems (Warrington, UK).

Cell culture and treatment
Human hepatoma HepG2 cells were cultured in 8.5 cm dishes and maintained in supplemented Eagle's Minimum Essential Medium 12 ml, with FBS (10%), penicillin (100 U/ml) and streptomycin (100 µg/ml) under 5% CO2 in air at 37°C. Test compounds, dissolved in dimethyl sulfoxide (to give a maximum final concentration of 0.05%) were applied when cells reached ~80% confluence.

Human hepatocytes for PhIP–DNA adduct measurement by AMS and for mRNA expression studies were either purchased from a commercial supplier (TCS Cellworks/Biopredic, France) or prepared from liver biopsies, obtained from Norfolk and Norwich University Hospital, in an extension of a study on antioxidants on human hepatocytes under Norwich District Ethics Committee, NDEC 96/088, and from INSERM, University of Rennes, in agreement with French laws and the requirements of the local ethics committee, by a two-step perfusion with collagenase (42,43). Purchased cells were attached at 90–100% confluency in 24-well plates. On arrival, cells were placed in fresh media (Hams medium 1) and incubated at 37°C in the presence of 5% CO2. Cells harvested from biopsies were allowed to attach to cell culture plates for 24 h. The cells were then exposed to PhIP or phytochemicals (quercetin and SFN) for studies of DNA repair and phase II enzyme mRNA induction.

Preparation of DNA and analysis of PhIP–DNA adducts by AMS
Hepatocytes or HepG2 cells were treated with appropriate concentrations of [14C]PhIP together with co-treatment with protective compounds. Pooled human liver S9, where applied, was added in a 7:1 ratio of media to a premix containing 1.6 mg/ml S9 protein [with CYP1A2 activity – 180 pmol product/(mg x min)] in PBS with 1.3 mM NADPH, 3.3 mM MgCl2. At the end of the treatment time, DNA was isolated from cells using Qiagen Blood and Cell Culture DNA kits according to the manufacturer's instructions. The Qiagen genomic tip protocols have been used previously by other workers to prepare DNA for AMS analysis (44,45). The DNA was redissolved in water, the yield determined spectrophotometrically at 260 nm and purity determined from the A260/A280 ratio. Yields of DNA were typically 80–100 µg from an 8.5 cm dish of HepG2 (~9 x 106) cells.

Samples were shipped to Xceleron (York, UK) for determination of 14C content and hence DNA adducts, by AMS. Briefly, DNA was graphitized in a heat sealed tube with tributyrin (glycerol tributanoate) as a carbon carrier and pre-baked copper oxide wire. The graphite was then pressed into aluminium cathodes for loading into the AMS. AMS measures the carbon isotope ratios. The level of PhIP adduction may then be calculated from the specific activity, % labelling and the molecular weight of the compounds after subtraction of the background level of 14C in DNA and carrier.

To confirm that the DNA isolation procedure did not carry over any free or non-specifically bound [14C]PhIP into AMS analyses, cell nuclei were isolated from HepG2 cells and incubated with 10 pM [14C]PhIP (the residual concentration, estimated by liquid scintillation counting to be present in the nuclear fraction when cells had been treated with 10 nM PhIP) and then processed to isolate DNA as usual. The level of 14C found in this DNA was not distinguishable from baseline.

Preparation and quantification of RNA
Cells were lysed and homogenized on a Qiashredder column and total RNA was then isolated using a Qiagen RNeasy mini kit according to the manufacturer's instructions. The RNA was eluted from the binding column with 80 µl of RNase-free water. RNase inhibitor (Applied Biosystems) was added immediately (20 U/preparation) and the product stored at -70°C. Total RNA yield was determined by Molecular Probes Ribogreen RNA Quantitation Kit (Cambridge Bioscience, Cambridge, UK) against a standard curve of ribosomal RNA (16S and 23S rRNA from Escherichia coli).

TaqMan® real-time RT–PCR assays
Expression of mRNA for each target was determined by real-time RT–PCR using the ABI Prism 7700 Sequence Detection System (Applied Biosystems). Forward and reverse primers and the fluorogenic TaqMan® probe for target genes were designed using Primer Express Software. Sequence homology of selected oligomers was checked using a NCBI BLAST search to ensure that sequences were specific to target genes. For GSTA1 (Acc. No. M14777/M16594), forward primer is 5'-CAGCAAGTGCCAATGGTTGA, reverse primer 5'-TATTTGCTGGCAATGTAGTTGAGAA and probe 5'-TGGTCTGCACCAGCTTCATCCCATC. For UGT1A1 (Acc. No. M57899), forward primer 5'-GGTGACTGTCCAGGACCTATTGA, reverse primer 5'-TAGTGGATTTTGGTGAAGGCAGTT and probe 5'-ATTACCCTAGGCCCATCATGCCCAATATG. APE (Acc. No. X59764) forward primer 5'-GCATAGGCGATGAGGAGCAT, reverse primer 5'-TGTTACCAGCACAAACGAGTCAA and probe 5'-AGCCACAATCACCCGGCCTTCCT. Pol ß (Acc. No. D29013) forward primer 5'-GCGCCGCAGGAGACTCT, reverse primer 5'-GCTTGGCTCACGTTCTTCTCA and probe 5'-CCGACATGCTCACAGAACTCGCAAACTT.

RT–PCR reactions were carried out in a 96-well plate using TaqMan® one-step RT–PCR master mix Reagent kit (Applied Biosystems) in a total of 25 µl/well consisting of 100–200 nM probe, 200–400 nM primers and 20 ng of total RNA. TaqMan RT–PCR conditions: 48°C 30 min, 95°C 10 min then 40 cycles of 95°C for 15 s and 60°C for 1 min. Data were analysed with software provided with the instrument. Reactions were carried out in triplicate. Gene expression was quantified using relative standard curve method (Applied Biosystems Prism 7700 SDS User Bulletin #2; http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf).

Cytochrome P450 (CYP)1A2 activity in intact cells
CYP1A2 activity was determined in adherent HepG2 cells by an adaptation of the method of Donato et al. (46). Briefly, cells were cultured in 6-well plates and grown to 80% confluence. Cells in replicate wells were then treated with phytochemicals for time intervals up to 24 h. Media was then replaced with 1 ml media containing CYP1A2 substrate, methoxyresorufin (10 µM) and dicumarol (10 µM, a diaphorase inhibitor). After 30 min, 6 x 75 µl aliquots of this media were transferred from each well to a 96-well plate and processed according to the published method (46). Fluorescence was measured on a Molecular Devices Fmax plate reader (Filters, Ex = 544 nm, Em = 590 nm) relative to a standard curve of the fluorescent product, resorufin (0–500 nM) that has been processed as samples. Adherent cells were harvested from the 6-well plates using trypsin–EDTA and extracted by sonication in 0.1 M Tris–HCl pH 7.4, containing 0.1% digitonin, and 1 mM phenylmethylsulfonyl fluoride. Cytosolic protein was determined (Bradford dye-binding assay) using bovine serum albumin as standard (47).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Formation of PhIP–DNA adducts: reproducibility
HepG2 cells were treated with 10 nM [14C]PhIP for 24 h in six separate experiments at different cell passage numbers. The values obtained for the level of PhIP bound [mean = 14.2 ± 1.2 (SD) amol/µg DNA] give a measure of the consistency and reproducibility of the methodology. The relative cost of AMS measurements has, however, prohibited the analysis of multiple replicates of some treatments. The number of replicates is as indicated in the figure legends.

Formation of PhIP–DNA adducts in HepG2 cells: dose-dependence
A dose–response curve with respect to [14C]PhIP treatments of HepG2 cells is shown in Figure 1. Cells were treated for 24 h with a 2 x 107 fold dilution series of [14C]PhIP from 1 pM to 20 µM and data are shown as log/log plots of the levels of PhIP–adduct formation against PhIP concentration. A linear correlation between PhIP treatment and adduct level was observed and adducts were detected after treatment with 100 pM PhIP but were not discernible above background (i.e. <0.15 amol PhIP/µg DNA for HepG2 cells) after 1 pM PhIP treatment. HepG2 cells typically bound 280 amol PhIP/µg DNA following 24 h treatment with 1 µM PhIP.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1. [14C]PhIP DNA adduct formation in HepG2 cells (closed diamonds) and hepatocytes (open squares) after 24 h treatment with [14C]PhIP. Error bar (10 nM PhIP treatment, HepG2) indicates mean ± SD (14.2 ± 1.2) of six treatments at different passage numbers. *Below detection limits, <0.15 amol PhIP/µg DNA for HepG2 cells and 15 amol PhIP/µg DNA for hepatocytes.

 
Formation of PhIP–DNA adducts in hepatocytes: dose-dependence
A dose–response curve is shown (Figure 1) with respect to [14C]PhIP treatments of hepatocytes from one individual (Subject 3, see later). Cells were treated for 24 h with a 106-fold dilution series of [14C]PhIP from 1 pM to 1 µM. As noted in HepG2 cells, there was a linear correlation between PhIP treatment and adduct level and adducts were detected after treatment with 100 pM PhIP but were not discernible above background (i.e. <15 amol PhIP/µg DNA for hepatocytes) after 1 pM PhIP treatment. The differing detection limits for the two cell types were a function of cell number available and hence yield of DNA isolated and available for AMS analysis. Dose–response curves were also determined with hepatocytes from Subjects 4 and 5. However, for both these subjects, the levels of adducts formed were relatively low and could not be detected at an exposure level of 100 pM but were identified after 10 nM PhIP treatment, giving 155 and 33 amol PhIP/µg DNA respectively. Hepatocytes, whilst showing considerable inter-individual variation between cells from the five subjects tested, generated higher levels of adducts than HepG2 cells. For example, following treatment with 1 µM [14C]PhIP for 24 h, Subjects 2, 3, 4 and 5 bound 20 900, 13 000, 976 and 970 amol PhIP/µg DNA, respectively, and Subject 1 bound 8670 amol PhIP/µg DNA with 10 µM PhIP treatment. These results suggest there is at least a x20-fold inter-individual variation in the level of formation of PhIP–DNA adducts in human hepatocytes from different individuals. Whether this is directly due to variations between individuals or whether this reflects differences in the integrity of the hepatocytes is not known.

Effects of phytochemicals on the formation of PhIP–DNA adducts in HepG2 cells
The effects of co-treatment of cells with PhIP and SFN or quercetin were studied. SFN and quercetin decreased the level of PhIP–DNA adduct formation in HepG2 cells in a dose-dependent manner (Figure 2A and B). SFN is effective at concentrations of 1 µM and above and quercetin at doses of 5 µM and above. When the cells were treated with different levels of [14C]PhIP, quercetin and SFN exhibited different levels of protection (Figure 3). For example, at 1 µM PhIP, SFN (10 µM) and quercetin (20 µM) reduced the levels of PhIP–DNA adducts by 10 and 30%, respectively. At 10 nM PhIP, the percentage reduction was 40 and 60%, but at 100 pM PhIP, the level of adducts in co-treated cells, with either SFN or quercetin, were decreased to a level below detection limit (<0.15 amol PhIP/µg DNA). No morphological alterations as determined by microscopic observation were observed at any of the concentrations of phytochemicals tested here. Modification of hepatocytes was also not noted following treatment with higher concentrations of SFN (25 µM) (48).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2. Effects of SFN and quercetin on PhIP–DNA adduct formation in HepG2 cells. (A) Co-treatment with SFN (24 h); (B) co-treatment with quercetin (24 h). Error bars for control, 2 and 5 µM SFN treatments indicate range of two separate experiments. Control is the average of six replicates treated with 10 nM PhIP.

 


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3. Effect of SFN and quercetin on PhIP–DNA adduct formation in HepG2 cells at different [14C]PhIP doses. (Control at 10 nM PhIP is one of six replicates described in Figure 1.) *Below detection limit <0.15 amol PhIP/µg DNA.

 
Effects of phytochemicals on the formation of PhIP–DNA adducts in hepatocytes
Quercetin and SFN reduced the level of DNA adducts formed when hepatocytes were treated in combination with PhIP in three out of five subjects (Figure 4). However, one of the other individuals (subject 5), who showed no response to phytochemicals, generated relatively low levels of PhIP–DNA adducts in comparison with those individuals that showed a protective response. This preliminary experiment showed that the inter-individual response to PhIP and phytochemicals is huge and further studies on a larger number of individuals are required.



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 4. Effect of 24 h co-treatment of [14C]PhIP with SFN or quercetin on [14C]PhIP–DNA adduct formation in hepatocytes from five subjects. Data for Subjects 1–4 are from single treatments. Data for Subject 5 are from duplicate treatments—DNA from the SFN treatments were combined and adducts measured as one sample. Error bars indicate range of values obtained for replicate control and quercetin treatments.

 
Role of phytochemicals in the repair of PhIP–DNA adducts
To determine whether phytochemicals possess the ability to promote the repair of PhIP–DNA adducts and to separate the effects of DNA repair from PhIP bioactivation and detoxification, HepG2 cells were treated sequentially with 1 µM [14C]PhIP and after washing, with SFN or quercetin for 6 or 24 h prior to determination of levels of PhIP–DNA adducts, i.e. so that potential protective compounds were not present during the bioactivation of PhIP and formation of PhIP–DNA adducts. First, an estimate of the rate of repair of PhIP–DNA adducts in HepG2 cells was obtained by assaying the untreated control cells at a series of time points in this experiment (Figure 5). Over the first 3 h post-treatment, adduct levels fell sharply to 66% of the initial level and then decreased more gradually to 25% at 24 h. This finding suggests the overall genome repair rate appeared to consist of a faster and a slower repair phase; this is consistent with the report by Fan et al. using N-hydroxy-PhIP (28).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5. Time course of [14C]PhIP–DNA adduct removal/repair in HepG2 cells. Cells were treated with 1 µM [14C]PhIP for 24 h, followed by treatment with either SFN (10 µM) or quercetin (20 µM) at time points indicated. The yield of DNA at 24 h time point was only 20% more than that of controls at time 0 h, i.e. yields of DNA/dish of cells were: for time points up to 3 h, 161 ± 13 mg; for all time points taken at 6 h, 177 ± 9 mg; for all time points taken at 24 h, 194 ± 25 mg.

 
None of the compounds tested had a more potent effect on the rate of clearance of the PhIP–DNA adducts formed (Figure 5). Therefore, it can be concluded that SFN and quercetin have no influence on the rate of PhIP–DNA adduct repair and that the role of these compounds in PhIP metabolism is through their ability to modify phase I activation and phase II detoxification enzymes. This result also showed that human HepG2 cells possess efficient capacity to repair PhIP–DNA adducts.

Modulation of DNA repair enzyme mRNA by PhIP and phytochemicals
It is not clear how PhIP–DNA adducts are repaired in mammalian cells although it seems NER is likely to be involved in removing most of the PhIP-damaged DNA (12). There is no evidence of induction of APE or polß mRNA by PhIP, SFN or quercetin in HepG2 cells or hepatocytes (n = 7 subjects); results which are in accordance with levels of PhIP–DNA adduct repair. Major variations in basal levels (up to 10-fold) of both APE and polß mRNA in hepatocytes between subjects were noted.

Modulation of phase II enzymes by PhIP and phytochemjcals
SFN consistently induced levels of both UGT1A1 and GSTA1 mRNA in HepG2 cells (24 h) by ~12- and 3-fold, respectively (Figure 6). Results indicate, however, that PhIP, SFN and quercetin have little effect on the induction of expression of UGT1A1 or GSTA1 mRNA in hepatocytes (n = 6). Only one significant induction (x42-fold) of UGT1A1 in the presence of SFN was observed for hepatocytes from one subject but little effect has been seen with any other subjects and no effect was observed on GSTA1 mRNA expression. The subject that showed UGT1A1 induction, however, had a very low basal level, 130 ± 13 (mean ± SD) copies/ng total RNA. The variations in basal levels of UGT1A1 (130 ± 13 to 8010 ± 110 copies/ng total RNA) and GSTA1 mRNA (8606 ± 150 to 85 900 ± 1700 copies/ng total RNA) in hepatocytes from six subjects are highly significant. This is consistent with the report that individual difference in the rate of phase II glucuronidation of N-OH-PhIP in humans (8). GST mRNA expression has been shown, by other researchers (48), to be inducible in hepatocytes following prolonged treatment by SFN but no indication of inter-individual variation has been published.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 6. Effect of 24 h treatment with 10 µM SFN on the expression of GSTA1 and UGT1A1 mRNA in HepG2 cells. Mean ± SD of three replicate dishes of cells. Data normalized with mean level of expression in control cells = 1.

 
Modulation CYP1A2 activity by phytochemicals
An important factor determining levels of PhIP–DNA adduct formation is the level of PhIP bioactivation by CYP1A2 (49). HepG2 expresses CYP1A2 but at a reduced level (10–20%) relative to hepatocytes (50), subject to significant inter-individual variation. Levels of CYP1A2 activity observed in HepG2 cells (Figures 7 and 8) were comparable with published data (50). To increase the level of PhIP bioactivation in cell culture, HepG2 cells were co-treated for 24 h with a human liver S9 fraction, containing CYP1A2 activity with appropriate co-factors. This increased the level of PhIP–DNA adducts generated compared with cells treated with PhIP alone (5410 versus 1620 amol PhIP/µg DNA at 10 µM PhIP). Co-treatment with quercetin counteracted the effect of S9 (2700 vs 5410 amol PhIP/µg DNA at 10 µM PhIP) whereas SFN (5630 amol PhIP/µg DNA at 10 µM PhIP) had no effect (Figure 7). The activity of CYP1A2, the enzyme responsible for the first step in the bio-activation of PhIP, was estimated in intact cells. In this assay, HepG2 cells treated with 5 µM 3-methyl cholanthrene [a known CYP inducer (51)] showed a CYP1A2 activity x10-fold higher than the basal level after 24 h treatment. Time courses of up to 24 h HepG2 treatment with quercetin and SFN were performed (Figure 8). CYP1A2 activity was not detectable in HepG2 cells that had been treated with quercetin for 1 h, but the level gradually recovered over the following 23 h to a level slightly higher than the basal level. SFN induced CYP1A2 activity by 30% after 6 h treatment, but returned to the basal level at 24 h. Quercetin clearly inhibits CYP1A2 activity and also, in experiments reported previously (52), the addition of human liver fraction S9 (rich in CYP1A2) to HepG2 media increased the level of adduct formation and this increase was modulated by quercetin. SFN is a possible inhibitor of cytochrome P450 enzymes (53) but did not inhibit CYP1A2 activity in this assay. It also had no effect on PhIP–DNA adduct formation in S9 treated cells and therefore the most likely mode of action is through induction of phase II enzymes.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 7. Effects of human liver S9 fraction and 24 h co-treatment with 10 µM SFN or 20 µM quercetin on [14C]PhIP–DNA adduct formation in HepG2 cells. Data are from single treatments.

 


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 8. Effect of 20 µM quercetin (closed diamonds) or with 10 µM SFN (open squares) on cytochrome P450 (CYP)1A2 activity in intact HepG2 cells. Means are from six assays of duplicate wells.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
AMS is the most sensitive method (up to 1000 times more sensitive than 32P-postlabelling) so far developed for determining radioactivity levels in DNA samples, and allows estimation of PhIP–DNA adducts after treatment of cells with physiologically relevant concentrations of [14C]PhIP. Fan et al. treated mammary epithelial cells with 50 µM PhIP and could not detect any adducts by 32P-postlabelling (28). In both cultured HepG2 cells and human hepatocytes, PhIP can generate DNA adducts even at concentrations as low as 100 pM; however, no adducts were detected in either cell type following treatment with 1 pM PhIP. The variation in the level of PhIP–DNA adduct formation in hepatocytes among the five individuals tested exceeded 20-fold. In comparison, more than a 25-fold range of induction in EROD activities, mediated by CYP1 family, has been observed in human liver samples and in human primary hepatocytes (16,54,55) but the hepatocytes, whilst having vastly differing rates of PhIP metabolism, produced a similar spectrum of metabolites (16). There are no data available on the human plasma levels of PhIP resulting from the consumption of a portion of well-cooked meat and whilst it is possible to make estimates based on levels of consumption it remains difficult to relate data from our in vitro model to human risk. The detection of PhIP–DNA adducts using AMS has been reported in DNA extracted from the breast tissue of human subjects following the administration of a typical dietary dose of 20 µg (0.1 µmol) PhIP (44). In this study, PhIP was taken orally by human volunteers undergoing breast surgery. PhIP–DNA adducts were in the range 46–477 adducts/1012 nucleotides (equivalent to 0.14–1.44 amol PhIP/µg DNA), with no statistical difference between tumour and normal tissue. There is some evidence, however, that breast tissue is deficient in CYP1A2 (56).

Treatment of cells with dietary phytochemicals, such as SFN and quercetin at physiological relevant levels (57,58), significantly reduced the level of PhIP–DNA adducts formed. The levels of the protection depended on the level of PhIP exposure, i.e. at lower levels of exposure, quercetin and SFN exerted 100% protection, but at higher levels of PhIP exposure, the protection was limited to 10–60%. In another words, the threshold effect of PhIP could be modulated by the presence of SFN and quercetin. It is therefore critical to know the concentration of PhIP in vivo to be able predict the likely effects of SFN or quercetin on the threshold concentrations of PhIP required for the formation of DNA adducts.

The mechanisms by which levels of PhIP–DNA adducts are reduced by SFN and quercetin are different. CYP1A2 activation is one of the key steps in the bio-activation of PhIP that leads to DNA adduct formation. We confirmed that quercetin could inhibit CYP1A2 activity in intact HepG2 cells. Such a rapid response to quercetin indicates that the observed effect is probably a result of direct inhibition of the enzyme. The subsequent recovery is compatible with the known rate of metabolism of quercetin by HepG2 cells to its conjugates (quercetin is substantially metabolized by 10 h) (59). Conversely, SFN did not inhibit CYP1A2 activity in HepG2 cells although it inhibited adduct formation and SFN, unlike quercetin, showed no effect on the number of adducts formed with S9 treatment. Thus, the protective effect of SFN on PhIP–DNA adduct formation arises primarily from the induction of phase II enzymes such as UGT1A1 and GST A1-1 (24,26,60). SFN has also been shown to have no inhibitory effect on recombinant CYP enzymes even with pre-incubation (53) but inhibition of CYP1A2 activity (measured as EROD activity) had been observed previously in one sample of human hepatocytes, although no corresponding change in mRNA expression was observed (48). This may suggest that this inhibition of activity in hepatocytes may be related to the formation of an SFN metabolite, i.e. a conjugate or other biotransformed compound, which is not generated in HepG2 cells.

The two-step protocol we devised for measurement of PhIP–DNA adducts by AMS, separating the effects on DNA repair from those of bioactivation and detoxification, revealed that none of the compounds tested had any effect on levels of PhIP–DNA adducts when added after adduct formation. This indicates that they may not have a significant role in modifying PhIP–DNA adducts in HepG2 cells. The levels of expression of mRNA of two DNA repair enzymes (APE and Pol ß) were similarly not modified by the same compounds in either HepG2 cells or hepatocytes. APE is mainly involved in base excision repair and it is possible that PhIP–DNA adducts are repaired by other mechanisms such as nucleotide excision repair. Thus, modulation of expression and activity of other DNA repair enzymes involved in both these and other mechanisms by phytochemicals cannot be ruled out. There are reports that myricetin induces Pol ß in rat hepatocytes (31) and quercetin inhibits Pol ß activity from viruses (32) but no similar effects on the modulation of Pol ß by phytochemicals were identified in this study. Moreover, none of the compounds tested appeared to have a significant effect on the rate of removal of PhIP–DNA adducts formed in HepG2 cells.

The combination of data from PhIP–DNA adducts measurements with enzyme activity assay and real-time RT–PCR to determine mRNA levels of relevant PhIP metabolizing enzymes, allows an understanding of the mechanisms by which dietary phytochemicals may modify the cellular metabolic pathways of PhIP activation, detoxification and DNA adduct repair. SFN and quercetin inhibit the formation of PhIP–DNA adducts and hence may subsequently play a role in mutation and carcinogenesis. From the limited amount of data obtained in this study, it was not realistic to begin to make any correlations between basal levels of mRNA of phase II or other enzymes and levels of DNA adducts in hepatocytes and if such relationships exist they would need to be substantiated by a larger data set in a future study.

In conclusion, the determination of PhIP–DNA adducts by AMS, as an intermediate end-point biomarker that can be used to measure the overall effects of exposure, activation, detoxification and repair, has been validated. This model, combined with mechanistic studies, allows the investigation of the potential role of dietary phytochemicals and micronutrients on the metabolism and genotoxicity of food-borne mutagens in cultured cells. In view of the hypothesis that cancer cells exhibit a mutator phenotype (61), where genes encoding proteins involved in DNA polymerization and repair may be mutated leading to a further increase in DNA replication errors, phytochemicals decrease DNA adduct formation and may consequently reduce the rate of mutations, therefore the consumption of a diet rich in isothiocyanate SFN and flavonoid, quercetin may present an opportunity for cancer chemoprevention. More work is needed using primary human hepatocytes to correlate the phase I and phase II enzyme activities with the DNA adduct levels and to understand the molecular basis of the inter-individual variations in the formation of DNA adduct and repair.


    Notes
 
4 Present address: Nestlé Research Center, PO Box 44, CH-1000 Lausanne 26, Switzerland Back

5 To whom correspondence should be addressed Email: yongping.bao{at}bbsrc.ac.uk Back


    Acknowledgments
 
This study was funded by the Food Standards Agency, United Kingdom. The authors thank Dr C.S.M.Tahourdin and Professor G.Gibson for helpful discussions and Mr G.Plumb for culture of HepG2 cells.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Wakabayashi,K., Nagao,M., Esumi,H. and Sugimura,T. (1992) Food-derived mutagens and carcinogens. Cancer Res., 52, 2092–2098.
  2. Sinha,R, Gustafson,D.R., Kulldorff,M., Wen,W.Q., Cerhan,J.R. and Zheng,W. (2000) 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, a carcinogen in high-temperature-cooked meat and breast cancer risk. J. Natl Cancer Inst., 92, 1352–1354.[Free Full Text]
  3. Knize,M.G., Salmon,C.P., Mehta,S.S. and Felton,J.S. (1997) Analysis of cooked muscle meats for heterocyclic aromatic amine carcinogens. Mutat. Res., 376, 129–134.[ISI][Medline]
  4. Sugimura,T. (2000) Nutrition and dietary carcinogens. Carcinogenesis, 21, 387–395.[Abstract/Free Full Text]
  5. Sinha,R., Rothman,N., Brown,E.D., Salmon,C.P., Knize,M.G., Swanson,C.A., Rossi,S.C., Mark,S.D., Levander,O.A. and Felton,J.S. (1995) High-concentrations of the carcinogen 2-amino-1-methyl-6- phenylimidazo-[4,5-b] pyridine (PhIP) occur in chicken but are dependent on the cooking method. Cancer Res., 55, 4516–4519.[Abstract]
  6. Layton,D.W., Bogen,K.T., Knize,M.G., Hatch,F.T., Johnson,V.M. and Felton,J.S. (1995) Cancer risk of heterocyclic amines in cooked foods: an analysis and implications for research. Carcinogenesis, 16, 39–52.[Abstract]
  7. Skog,K. (2002) Problems associated with the determination of heterocyclic amines in cooked foods and human exposure. Food Chem. Toxicol., 40, 1197–1203.[CrossRef][ISI][Medline]
  8. Stillwell,W.G., Sinha,R. and Tannenbaum,S.R. (2002) Excretion of the N(2)-glucuronide conjugate of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine in urine and its relationship to CYP1A2 and NAT2 activity levels in humans. Carcinogenesis, 23, 831–838.[Abstract/Free Full Text]
  9. Frandsen,H. and Alexander,J. (2000) N-Acetyltransferase-dependent activation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine: formation of 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo [4,5-b]pyridine, a possible biomarker for the reactive dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Carcinogenesis, 21, 1197–1203.[Abstract/Free Full Text]
  10. Frandson,H. (1997) Excretion of DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, PhIP-dG, PhIP-DNA and DiMeIQx-DNA from the rat. Carcinogenesis, 18, 1555–1560.[Abstract]
  11. Lin,D. Kaderlik,K.R., Tureky,R.J., Miller,D.W., Lay,J.O. and Kadlubar,F.F. (1992) Identification of N-(deoxyguanosin-8-yl)-2- amino-1-methyl-6-phenylimidazo[4,5-b]pyridine as the major adduct formed by the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, with DNA. Chem. Res. Toxicol., 5, 691–697.[ISI][Medline]
  12. Brown,K., Hingerty,B.E., Guenther,E.A., Krishnan,V.V., Broyde,S., Turteltaub,K.W. and Cosman,M. (2001) Solution structure of the 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine C8-deoxyguanosine adduct in duplex DNA. Proc. Natl Acad. Sci. USA, 98, 8507–8512.[Abstract/Free Full Text]
  13. Shibutani,S., Fernandes,A., Suzuki,N., Zhou,L., Johnson,F. and Grollman,AP. (1999) Mutagenesis of the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine DNA adduct in mammalian cells. J. Biol. Chem., 274, 27433–27438.[Abstract/Free Full Text]
  14. Wu,R.W., Wu,E.M., Thompson,L.H. and Felton,J.S. (1995) Identification of aprt gene-mutations induced in repair-deficient and p450-expressing CHO cells by the food-related mutagen/carcinogen, PhIP. Carcinogenesis, 16, 1207–1213.[Abstract]
  15. Malfatti,M.A., Kulp,K.S., Knize,M.G. et al. (1999) The identification of [2-C-14]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine metabolites in humans. Carcinogenesis, 20, 705–713.[Abstract/Free Full Text]
  16. Langouet,S., Paehler,A., Welti,D.H., Kerriguy,N., Guillouzo,A. and Turesky,R.J. (2002) Differential metabolism of 2-amino-1-methyl- 6-phenylimidazo[4,5-b]pyridine in rat and human hepatocytes. Carcinogenesis, 23, 115–122.[Abstract/Free Full Text]
  17. He,Y.H., Friesen,M.D., Ruch,R.J. and Schut,H.A.J. (2000) Indole- 3-carbinol as a chemopreventive agent in 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP) carcinogenesis: inhibition of PhIP-DNA adduct formation, acceleration of PhIP metabolism and induction of cytochrome P450 in female F344 rats. Food Chem. Toxicol., 38, 15–23.[CrossRef][ISI][Medline]
  18. Hammons,G.J., Fletcher,J.V., Stepps,K.R., Smith,E.A., Balentine,D.A., Harbowy,M.E. and Kadlubar,F.F. (1999) Effects of chemoprotective agents on the metabolic activation of the carcinogenic arylamines PhIP and 4-aminobiphenyl in human and rat liver microsomes. Nutr. Cancer, 33, 46–52.[ISI][Medline]
  19. Hirose,M., Nishikawa,A., Shibutani,M., Imai,T. and Shirai,T. (2002) Chemoprevention of heterocyclic amine-induced mammary carcinogenesis in rats. Environ. Mol. Mutagen., 39, 271–278.[CrossRef][ISI][Medline]
  20. Schut,H.A.J. and Yao,R.S. (2000) Tea as a potential chemopreventive agent in PhIP carcinogenesis: effects of green tea and black tea on PhIP-DNA adduct formation in female F-344 rats. Nutr. Cancer, 36, 52–58.[ISI][Medline]
  21. Huber,W.W., McDaniel,L.P., Kaderlik,K.R., Teitel,C.H., Lang,N.P. and Kadlubar,F.F. (1997) Chemoprotection against the formation of colon DNA adducts from food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the rat. Mutat. Res., 376, 115–122.[ISI][Medline]
  22. Hirose,M., Takahashi,S., Ogawa,K., Futakuchi,M. and Shirai,T. (1999) Phenolics: blocking agents for heterocyclic amine-induced carcinogenesis. Food Chem. Toxicol., 37, 985–992.[CrossRef][ISI][Medline]
  23. Knize,M.G., Kulp,K.S., Salmon,C.P., Keating,G.A. and Felton,J.S. (2002) Factors affecting human heterocyclic amine intake and the metabolism of PhIP. Mutat. Res., 506–507, 153–162.[ISI]
  24. Basten,G.P., Bao,Y.P. and Williamson,G. (2002) Sulforaphane and its glutathione conjugate but not sulforaphane nitrile induce UDP- glucuronosyl transferase (UGT1A1) in HepG2 and HT29 cells. Carcinogenesis, 23, 1399–1404.[Abstract/Free Full Text]
  25. Murray,S., Lake,B.G., Gray,S., Edwards,A.J., Springall,C., Bowey,E.A., Williamson,G., Boobis,A.R. and Gooderham,N.J. (2001) Effect of cruciferous vegetable consumption on heterocyclic aromatic amine metabolism in man. Carcinogenesis, 22, 1413–1420.[Abstract/Free Full Text]
  26. Nowell,SA., Massengill,J.S., Williams,S. et al. (1999) Glucuronidation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine by human microsomal UDP-glucuronosyltransferases: identification of specific UGT1A family isoforms involved. Carcinogenesis, 20, 1107–1114.[Abstract/Free Full Text]
  27. Alexander,J., Reistad,R., Hegstad,S., Frandsen,H., Ingebrigtsen,K., Paulsen,J.E. and Becher,G. (2002) Biomarkers of exposure to heterocyclic amines: approaches to improve the exposure assessment. Food Chem. Toxicol., 40, 1131–1137.[CrossRef][ISI][Medline]
  28. Fan,L., Schut,H.A. and Snyderwine,E.G. (1995) Cytotoxicity, DNA adduct formation and DNA repair induced by 2-hydroxyamino-3-methylimidazo[4,5-f]quinoline and 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine in cultured human mammary epithelial cells. Carcinogenesis, 16, 775–779.[Abstract]
  29. Bonnesen,C., Eggleston,I.M. and Hayes,J.D. (2001) Dietary indoles and isothiocyanates that are generated from cruciferous vegetables can both stimulate apoptosis and confer protection against DNA damage in human colon cell lines. Cancer Res., 61, 6120–6130.[Abstract/Free Full Text]
  30. Webster,R.P., Gawde,M.D. and Bhattacharya,R.K. (1996) Protective effect of rutin, a flavonol glycoside, on the carcinogen-induced DNA damage and repair enzymes in rats. Cancer Lett., 109, 185–191.[CrossRef][ISI][Medline]
  31. Abalea,V., Cillard,J., Dubos,M.P., Sergent,O., Cillard,P. and Morel,I. (1999) Repair of iron-induced DNA oxidation by the flavonoid myricetin in primary rat hepatocyte cultures. Free Radic. Biol. Med., 26, 1457–1466.[CrossRef][ISI][Medline]
  32. Ono,K., Nakane,H., Fukushima,M., Chermann,J.C. and Barre-Sinoussi,F. (1990) Differential inhibitory effects of various flavonoids on the activities of reverse transcriptase and cellular DNA and RNA polymerases. Eur. J. Biochem., 190, 469–476.[Abstract]
  33. Grösch,S., Fritz,G. and Kaina,B. (1998) Apurinic endonuclease (Ref-l) is induced in mammalian cells by oxidative stress and involved in clastogenic adaptation. Cancer Res., 58, 4410–4416.[Abstract]
  34. Cabelof,D.C., Raffoul,J.J., Yanamadala,S., Guo,Z. and Heydari,A.R. (2002) Induction of DNA polymerase ß-dependent base excision repair in response to oxidative stress in vivo. Carcinogenesis, 23, 1419–1425.[Abstract/Free Full Text]
  35. Klein,J.C., Beems,R.B., Zwart,P.E., Hamzink,M., Zomer,G., van Steeg,H. and van Kreijl,C.F. (2001) Intestinal toxicity and carcinogenic potential of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in DNA repair deficient XPA (-/-) mice. Carcinogenesis, 22, 619–626.[Abstract/Free Full Text]
  36. Bennett,R.A., Wilson,D.M.,3rd, Wong,D. and Demple B. (1997) Inter-action of human apurinic endonuclease and DNA polymerase ß in the base excision repair pathway. Proc. Natl Acad. Sci. USA, 94, 7166–7169.[Abstract/Free Full Text]
  37. Canitrot,Y., Hoffmann,J.S., Calsou,P., Hayakawa,H., Salles,B. and Cazaux,C. (2000) Nucleotide excision repair DNA synthesis by excess DNA polymerase beta: a potential source of genetic instability in cancer cells. FASEB J., 14, 1765–1774.[Abstract/Free Full Text]
  38. Knasmuller,S., Schwab,C.E., Land,S.J., Wang,C.Y., Sanyal,R., Kundi,M., Parzefall,W. and Darroudi,F. (1999) Genotoxic effects of heterocyclic aromatic amines in human derived hepatoma (HepG2) cells. Mutagenesis, 14, 533–539.[Abstract/Free Full Text]
  39. Barker,J. and Garner,R.C. (1999) Biomedical applications of accelerator mass spectrometry isotope measurements at the level of the atom. Rapid Commun. Mass Spectr., 13, 285–293.[CrossRef][ISI]
  40. Poirier,M.C., Santella,R.M. and Weston,A. (2000) Carcinogen macromolecular adducts and their measurement. Carcinogenesis, 21, 353–359.[Abstract/Free Full Text]
  41. Lappin,G. and Garner,R.C. (2003) Ultra-sensitive detection of radiolabelled drugs and their metabolites using accelerator mass spectrometry. In Wilson,I.D. (ed.) Bioanalytical Separations, Handbook of Analytical Separations. Elsevier, Amsterdam, Vol. 4, pp. 331–349.
  42. Gomez-Lechon,M.J., Lopez,P., Donato,T., Montoya,A., Larrauri,A., Gimenez,P. Trullenque,R., Fabra,R. and Castell,J.V. (1990) Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo. In Vitro Cell Dev. Biol., 26, 67–74.[ISI][Medline]
  43. Guguenguillouzo,C., Campion,Jp., Brissot,P., Glaise,D., Launois,B., Bourel,M. and Guillouzo,A. (1982) High-yield preparation of isolated human adult hepatocytes by enzymatic perfusion of the liver. Cell Biol. Int. Rep., 6, 625–628.[ISI][Medline]
  44. Lightfoot,T.J., Coxhead,J.M., Cupid,B.C., Nicholson,S. and Garner,R.C. (2000) Analysis of DNA adducts by accelerator mass spectrometry in human breast tissue after administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and benzo[a]pyrene. Mutat. Res., 472, 119–127.[ISI][Medline]
  45. Dingley,K.H., Roberts,M.L., Velsko,C.A. and Turteltaub,K.W. (1998) Attomole detection of 3H in biological samples using accelerator mass spectrometry: application in low-dose, dual-isotope tracer studies in conjunction with 14C accelerator mass spectrometry. Chem. Res. Toxicol., 11, 1217–1222.[CrossRef][ISI][Medline]
  46. Donato,T.M., Gómez-Lechón,J.M. and Castell,J.V. (1993) A microassay for measuring cytochrome P450IA1 and P450IIB1 in intact human and rat hepatocytes cultured on 96 well plates. Anal. Biochem., 213, 29–33.[CrossRef][ISI][Medline]
  47. Bradford,M. (1976) A rapid and sensitive method for the quantition of microgram quantities of protein utilizing the principle of dye-binding. Analyt. Biochem., 72, 248–254.[CrossRef][ISI][Medline]
  48. Maheo,K., Morel,F., Langouet,S., Kramer,H., Le Ferrec,E., Ketterer,B. and Guillouzo, A. (1997) Inhibition of cytochromes P450 and induction of glutathione S-transferases by sulforaphane in primary human and rat hepatocytes. Cancer Res., 57, 3649–3652.[Abstract]
  49. Gooderham,N.J., Murray,S., Lynch,A.M., Yadollahi-Farsani,M., Zhao,K., Rich,K., Boobis,A.R. and Davies,D.S. (1997) Assessing human risk to heterocyclic amines. Mutat. Res., 376, 53–60.[ISI][Medline]
  50. Doostdar,H., Duthie,S.J., Burke,M.D., Melvin,W.T. and Grant,M.H. (1988) The influence of culture medium composition on drug metabolising enzyme activities of the human liver derived Hep G2 cell line. FEBS Lett., 241, 15–18.[CrossRef][ISI][Medline]
  51. Runge,D., Kohler,C., Kostrubsky,V.E. et al. (2000) Induction of cytochrome P450 (CYP)1A1, CYP1A2 and CYP3A4 but not of CYP2C9, CYP2C19, multidrug resistance (MDR-1) and multidrug resistance associated protein (MRP-1) by prototypical inducers in human hepatocytes. Biochem. Biophys. Res. Commun., 273, 333–341.[CrossRef][ISI][Medline]
  52. Reid,J.M., Kuffel,M.J., Miller,J.K. Rios,R. and Ames,M.M. (1999) Metabolic activation of dacarbazine by human cytochromes P450: the role of CYP1A1, CYP1A2 and CYP2E1. Clin. Cancer Res., 5, 2192–2197.[Abstract/Free Full Text]
  53. Langouet,S., Furge,L.L., Kerriguy,N., Nakamura,K., Guillouzo,A. and Guengerich,F.P. (2000) Inhibition of human cytochrome P450 enzymes by 1,2-dithiole-3-thione, oltipraz and its derivatives and sulforaphane. Chem. Res. Toxicol., 13, 245–252.[CrossRef][ISI][Medline]
  54. Turesky,R.J., Garner,R.C., Welti,D.H., Richoz,J., Leveson,S.H., Dingley,K.H., Turteltaub,K.W. and Fay,L.B. (1998) Metabolism of the food-borne mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in humans. Chem. Res. Toxicol., 11, 217–225.[CrossRef][ISI][Medline]
  55. Langouet,S., Welti,D.H., Kerriguy,N., Fay,L.B., Huynh-Ba.T., Markovic,J., Guengerich,F.P., Guillouzo,A. and Turesky,R.J. (2001) Metabolism of 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline in human hepatocytes: 2-Amino-3-methylimidazo [4,5-f]quinoxaline-8-carboxylic acid is a major detoxication pathway catalyzed by cytochrome P450 1A2. Chem. Res. Toxicol., 14, 211–221.[CrossRef][ISI][Medline]
  56. Ghoshal,A., Davis,C.D., Schut,H.A.J. and Snyderwine,E.G. (1995) Possible mechanisms for PhIP-DNA adduct formation in the mammary gland of female Sprague–Dawley rats. Carcinogenesis, 16, 2725–2731.[Abstract]
  57. Ye,L.X., Dinkova-Kostova,A.T., Wade,K.L., Zhang,Y.S., Shapiro,T.A. and Talalay,P. (2002) Quantitative determination of dithiocarbamates in human plasma, serum, erythrocytes and urine: pharmacokinetics of broccoli sprout isothiocyanates in humans. Clin. Chim. Acta, 316, 43–53.[CrossRef][ISI][Medline]
  58. Scalbert,A. and Williamson,G. (2000) Dietary intake and bioavailability of polyphenols. J. Nutr., 130, 2073S–2085S.[Abstract/Free Full Text]
  59. O'Leary,K.A., Day,A.J., Needs,P.W., Mellon,F.A., O'Brien,N.M. and Williamson,G. (2003) Metabolism of quercetin-7- and quercetin-3-glucuronides by an in vitro hepatic model: the role of human beta-glucuronidase, sulfotransferase, catechol-O-methyltransferase and multi-resistant protein 2 (MRP2) in flavonoid metabolism. Biochem. Pharmacol., 65, 479–491.[CrossRef][ISI][Medline]
  60. Alexander,J., Wallin,H., Rossland,O.J., Solberg,K.E., Holme,J.A., Becher,G., Andersson,R. and Grivas,S. (1991) Formation of a glutathione conjugate and a semistable transportable glucuronide conjugate of N2-oxidized species of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rat liver. Carcinogenesis, 12, 2239–2245.[Abstract]
  61. Loeb,L.A., Loeb,K.R. and Anderson,J.P. (2003) Multiple mutations and cancer. Proc. Natl Acad. Sci. USA, 100, 776–781.[Abstract/Free Full Text]
Received April 29, 2003; revised August 18, 2003; accepted August 19, 2003.