Sulforaphane and its glutathione conjugate but not sulforaphane nitrile induce UDP-glucuronosyl transferase (UGT1A1) and glutathione transferase (GSTA1) in cultured cells

Graham P. Basten, Yongping Bao and Gary Williamson,1

Institute of Food Research, Norwich Research Park, Conley Lane, Norwich, NR4 7UA, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Glucoraphanin in Brassica vegetables breaks down to either sulforaphane or sulforaphane nitrile depending on the conditions, and sulforaphane can be further conjugated with glutathione. Using a high-throughput microtitre plate assay and TaqMan real time quantitative RT-PCR to measure mRNA, we show that sulforaphane and its glutathione conjugate, but not the nitrile, increased significantly (P < 0.05) both UGT1A1 and GSTA1 mRNA levels in HepG2 and HT29 cells. These changes were accompanied by an increase in UGT1A1 protein, as assessed by immunoblotting, and a 2–8-fold increase in bilirubin glucuronidation. When treated together, the nitrile derivative did not affect sulforaphane induction. The induction of UGT1A1 and GSTA1 mRNA by sulforaphane was time and concentration dependent. The results show a functional induction of glucuronidation by sulforaphane but not sulforaphane nitrile, and show that the pathway of metabolism of glucosinolates in Brassica vegetables is important in determining the resulting biological and anticarcinogenic activities.

Abbreviations: ARE, antioxidant responsive element; Bis-Tris, bis(2-Hydroxyethyl)amino-tris(hydroxymethyl)methane; DMSO, Dimethyl sulfoxide; GST, glutathione-S-transferase; HRP, horseradish peroxidase; PBS, phosphate buffered saline; QR, quinone reductase; RT-PCR, real time polymerase chain reaction; SDS, sodium dodecyl sulfate; TBST, Tris-buffered saline with 0.05% Tween 20; UGT, UDP-glucuronosyltransferase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
UDP-Glucuronosyltransferases (UGTs, EC 2.4.1.17) are a family of proteins, localised to the endoplasmic reticulum, which catalyse the glucuronidation of a wide range of substrates (1–3). Glutathione transferases (GSTs, EC 2.5.1.18) are a family of soluble proteins which conjugate xenobiotics with glutathione (4). Metabolites after glucuronidation or glutathionylation are more hydrophilic, and thus biologically inactive; they are readily excreted in bile or urine, either as glucuronides or as N-acetylcysteine conjugates respectively (5). Human UGT1A1 (accession no. M57899 (6)) is a major isoform of the UGT1A family. The main substrate of UGT1A1 is bilirubin, which is conjugated with UDP-glucuronic acid to form water-soluble mono- and diglucuronides. Enhanced expression of UGT1A1 has implications for patients with Gilbert’s syndrome in which basal expression is decreased, leading to impaired bilirubin glucuronidation, reduced bilirubin clearance and increased toxicity (7,8). UGT1A1 is also capable of glucuronidating xenobiotics (9,10) and so induction may lead to enhanced clearance of carcinogens (11). The flavonoid chrysin induced UGT1A1 mRNA, protein levels and enzyme activity in human HepG2 cells (12), demonstrating the suitability of these cells as a model for studying UGT1A1 induction.

Glucoraphanin (4-methylsulfinyl-butyl glucosinolate) is found in Brassica vegetables, especially broccoli (13). It is deglycosylated on damage to the plant tissue, such as chewing or cooking, by myrosinase. The intermediate then breaks down to the isothiocyanate sulforaphane, or to the nitrile, depending on several factors such as pH and specific proteins (Scheme 1Go). The biological activity of the nitrile has received little attention, but is a poor inducer of quinone reductase in Hepa1c1c7 cells (14). Sulforaphane is a potent inducer of drug metabolising enzymes such as quinone reductase (15) and glutathione-S-transferase (16); this action is at least partly via an upstream ARE (17,18). UGT1A1 has an upstream ARE-like sequence (19) and thus may be inducible by sulforaphane. Sulforaphane is rapidly conjugated in the presence of glutathione (20), which is abundant in many biological tissues including the gut lumen (21). We therefore tested if metabolites of the glucosinolate glucoraphanin, namely sulforaphane, its glutathione conjugate and the nitrile derivative, could functionally induce glucuronidation and GSH conjugation in a liver and colon cell model, using an assay suitable for screening in 96-well microtitre plates.



View larger version (25K):
[in this window]
[in a new window]
 
Scheme. 1. Breakdown of glucoraphanin catalysed by the endogenous plant enzyme, myrosinase, leads to sulforaphane (isothiocyanate) or sulforaphane nitrile. Sulforaphane, but not the nitrile, can be further conjugated with glutathione.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Source of materials
Sulforaphane was purchased from ICN Biomedical, UK. The sulforaphane nitrile and sulforaphane glutathione conjugate were prepared as previously described (22,23). All other chemicals and solvents if not listed below were obtained from Sigma Chemicals, UK. The polyclonal antibody WB-UGT1A1, raised against human UGT1A1 (catalogue number 458411), and the secondary antibodies, HRP-conjugated goat anti-rabbit IgG and recombinant human UGT1A1 positive control, were from Gentest, USA. http://www.gentest.com/products/antibodies/anti_wes.shtm The antibody detects human UGT1A1 but does not detect UGT1A4, 1A6, 1A9, 1A10, 2B7, or 2B15. Pre-stained biotinylated molecular weight markers were purchased from New England Biolabs, USA. Electrophoresis and blotting supplies were from Bio-Rad Laboratories, UK, and Invitrogen, UK. Chemiluminescence chemicals were from National Diagnostics, USA, Hyperfilm MP was from Genetic Research Instrumentation, USA, and the Hypercassette RPN (18 x 24 cm) was from Amersham, UK.

Cell culture
Human colorectal adenocarcinoma HT29 cells and human hepatoma HepG2 cells (European Cell Culture Collection, Wiltshire, UK) were initially seeded in a 96-well plate (Costar, New York, USA) at either 2 or 4 x 104 cells for HepG2 or 15 x 104 cells for HT29. Preliminary experiments (results not shown) demonstrated that seeding densities above and below these values gave a lower response. Seeding was in Dulbecco’s medium (200 µl, Sigma, UK) supplemented with 5% charcoal-treated serum and 1% penicillin–streptomycin (GibcoBRL, UK), followed by incubation for 16 h at 37°C in 5% CO2. The medium was changed and the cells were then treated and subsequently lysed at the stated time point. Cells were treated with test compounds dissolved in 0.1% DMSO vehicle; controls were 0.1% DMSO only or medium only as appropriate.

Cell lysis and DNase treatment
Lysis of the cells was performed using Cells-to-cDNATM Kit and DNA-FreeTM (Ambion, Texas, USA). These provide DNAse treatment of genomic DNA and a novel non-heat inactivation of the DNAse enzymes, and this step is vital since the primers do not span an intron/exon junction. The medium was aspirated from the wells using a multi-channel pipette and the cells washed twice with phosphate buffered saline (GibcoBRL, UK). Cell lysis buffer (100 µl) was added to each well followed by incubation at 75°C for 5 min; 10 µl DNase buffer and 2 ml DNase I were added and incubation was at 37°C for 60 min, followed by transfer of 100 µl to a serocluster plate (Costar, UK) containing 10 µl DNase inactivation reagent. The plate was centrifuged for 2 min, 200 g, 4°C to pellet the inactivation reagent. When whole cell lysate was stored at –80°C for 2 weeks with one thaw cycle, comparable results to freshly analysed samples were obtained. Cell lysis efficiency was measured by seeding an extra set of wells and performing the lysis as above; the adherent cells were washed with 2% trypan blue, incubated at 37°C for 30 min, and lysis of the cell membrane estimated as a dark blue staining.

RNA quantification and normalization
To determine the amount of RNA present after lysis, a one-step fluorescence assay was used. This step was performed under subdued lighting using disposable, RNase free sterile plastic. RiboGreen dye (Molecular Probes, The Netherlands) was diluted 1:2000 in Tris–EDTA (0.2 M Tris–HCl, 20 mM EDTA, pH 7.5). A standard curve using 16 S/23 S rRNA at 100, 50, 25, 5 and 1 ng/µl in Tris-EDTA was prepared. Standard or cell lysate (100 ml, diluted 1:200 Tris–EDTA) was added to 100 µl of RiboGreen dye in a sterile 96-well plate. The plate was protected from light and incubated at room temperature for 5 min and read (Ex. 485 nm, Em. 510 nm) using a SPECTRAmaxTM Gemini XS fluorescent micro plate reader (Molecular Devices, Wokingham, UK). The absolute RNA concentration per well was calculated using SOFTmaxTM Pro 3.1 software (Molecular Devices, UK) and the original plate with remaining cell lysate was suitably diluted to ensure all wells contained the same amount of starting total RNA ({approx}7 ng/µl per original seeding of 4 x 104 HepG2 cells or 15 x 104 HT29 cells). `Pure’ total RNA refers to RNA harvested from either HepG2 or HT29 cells by Qiagen RNeasy (Qiagen, UK) and then quantified using RiboGreen and stored in TE (pH 8).

Primers and probes
The primers and probes were designed using ABI PRISMTM Primer ExpressTM (Applied Biosytems, CA, USA). UGT1A1 primers and probes were from the human cDNA sequence HumHugBr1, accession # M57899 (6). Sense primer; 5'-GGTGACTGTCCAGGACCTATTGA-3', antisense primer; 5'-TAGTGGATTTTGGTGAAGGCAGTT-3' and probe with 5'-FAM (6-carboxyfluoroscein) reporter and 3'-TAMRA (6-carboxytetramethylrhodamine) quencher dye modifications; 5'-ATTACCCTAGGCCCATCATGCCCAATATG-3' (MWG-Biotech, Germany) produced a single 130 bp PCR product on a 2% agarose gel (10 ml of final reaction mixture from each well). To confirm a single product, initial analysis of the PCR product from the wells was performed using a Gilson 715 HPLC with autoinjector, two pumps and a UV detector, on an analytical Shodex IEC DNApak column (6 x 50 mm) (Phenomenex, UK) with a linear gradient program for binary mobile phase as follows: buffer A; 0.1 M Tris–HCl and 1 mM EDTA (pH 8.0) and buffer B; buffer A + 1 M NaCl (pH 8.0). The mobile phase was linear from 50% to 75% B in 16 min, then to 50% B in 1 min, followed by re-equilibration for 5 min at 50% B. Flow rate was 1 ml/min, with detection at 260 nm (0.01 AUFS), using a column temperature 30°C and an injection volume of 20 µl.

GSTA1 mRNA assay was using primers and probe designed according to the homologous sequences between GST A1-1 (M14777) and GST A1-2 (M16594): sense primer 5'-CAGCAAGTGCCAATGGTTGA-3', antisense primer 5'-TATTTGCTGGCAATGTAGTTCAGAA-3' which together creates an 80 bp amplicon and the probe 5'-TGGTCTGCACCAGCTTCATCCCATC-3' (with 5'-FAM and 3'-TAMRA modifications).

Real time RT-PCR
One step RT-PCR was performed with 2 µl ({approx}14 ng total RNA) of original cell lysate in a final volume of 26 µl in the presence of 200 nM forward and reverse primers, 100 nM probe, 0.25 U/µl MultiScribeTM and TaqMan one step master mix (Applied Biosystems, UK) in a microamp optical 96-well plate with optical caps (Applied Biosystems, UK). Reverse transcription was for 30 min at 48°C, AmpliTaqTM gold activation for 10 min at 95°C, with 40 PCR cycles (reaction completion); denaturation was for 15 s at 95°C. Anneal/extend for 1 min at 60°C was performed using the ABI PRISMTM 7700 Sequence Detection System and data were processed with the 7700 Software Sequence Detector 1.6 (Applied Biosystems, UK), using either {Delta}Ct or the standard curve method (24). Each determination was performed in triplicate and statistical significance between control and treated cells was calculated by one-way ANOVA, using DMSO as the reference treatment group. There was no significant difference in results between standard curve or {Delta}Ct methods.

Additional controls were used as follows: no template control, in which water was substituted for the lysate to measure RNA or DNA contamination of reagents; 10 ng of `pure RNA’, to check quality of the RT-PCR reaction and to compare between assays; no multiscribe enzyme, to measure any DNA contamination. In addition, the normalised reporter dye emission ({Delta}Rn) was measured as a function of PCR cycle number for UGT1A1 mRNA expression in HepG2 cells, using 2 µl of whole lysate per 25 µl reaction. A plot of Ct from each dilution of lysate against starting quantity of total RNA gave a straight line for both UGT1A1 (R2 = 0.99, Slope = –2.99) and GSTA1 (R2 = 0.99, Slope = –3.10) showing equal amplification of GSTA1 and UGT1A1 products.

Bilirubin glucuronidation
Cells were seeded in 6-well dishes, at a density of 0.27 x 105 cells (HepG2) or 1 x 105 cells (HT29), and incubated for 16 h. Cells were then incubated in the presence of bilirubin (5 µM), and treated with either sulforaphane (30 µM) or the 0.1% DMSO vehicle. Cell media (2 ml) was removed at the stated time point, snap frozen and stored at –80°C. Bilirubin glucuronide formation was measured using a modification of a described method (25,26). Briefly, the media was incubated with a diazo reagent and incubated in darkness for 30 min, followed by rotary inversion with ascorbic acid and 2-pentanone to separate the azopigments. The organic solvent layer was then read at 530 nm and quantification was performed using the extinction coefficient of 4.07 x 104 mol–1 cm–1.

Preparation of samples for immunoblotting analysis of UGT1A1 protein
Cells were seeded in 100 mm dishes, at a density of 0.7 x 105 cells (HepG2) or 2.6 x 105 cells (HT29), and incubated for 16 h. Cells were then incubated for a further 16 h in the presence of either sulforaphane (30 µM) or the 0.1% DMSO vehicle. Cells were washed three times with PBS (4°C) before cell lysis. Protein was isolated using the TRI-ReagentTM method (27) and then concentrated using the Microcon-3 system from Amicon, dissolved in 1% SDS and quantified using the bicinchoninic acid assay (28).

Immunoblotting
Samples were diluted 1:4 with sample treatment buffer, reducing agent and antioxidant, agitated for 15 min at 70°C, and loaded on 10% Bis-Tris SDS-polyacrylamide gels. After electrophoresis (31), the proteins were transferred to nitrocellulose (30) and probed for UGT1A1. After 1 h blocking with 5% non-milk powder in TBST (milk-blot), the blots were hybridised with a 1:500 dilution of WB-UGT1A1 primary antibody in milk-blot for 1 h. Blots were washed three times with TBST and incubated for 1 h with 1:500 dilution of secondary antibody (HRP-conjugated goat anti-rabbit) in milk-blot. After thorough washing with TBST, the antibody binding was determined by chemiluminescence.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Measurement of UGT1A1 mRNA from cells in 96-well microtitre plates
UGT1A1 and UGT1A4 share 80% sequence homology with many extended runs of identical nucleotides (6), and a Blast search confirmed that the primers we designed were specific to UGT1A1 (National Library of Medicine, USA). Following real-time RT-PCR with test RNA, a single expected band of 130 bp was seen on a 2% agarose gel and a single peak was obtained by HPLC analysis. Multi-component analysis of the data showed an increase in fluorescence from FAM and a decrease from TAMRA dye demonstrating chemical cleavage of the probe.

The assay was initially tested with dilutions of pure total RNA in TE. The amplification rates were linear over the entire range for both GSTA1 (R2 = 0.99, Slope = –3.13) and UGT1A1 (R2 = 0.99, Slope = –3.21) when performed with one dye per tube, implying comparable amplification rates for each reaction >100-fold concentration range. Dilution of the lysate also produced linear amplification of GSTA1 and UGT1A1 over a hundred-fold range of total RNA.

Induction by sulforaphane is concentration- and time-dependent
Cells were treated with 15 µM sulforaphane, and significant increase in GSTA1 and UGT1A1 mRNA was rapidly observed and maintained for 18 h (Figure 1Go). Cells were treated with various concentrations (0.3–30 µM) of sulforaphane for 18 h, and significant induction was observed for both UGT1A1 and GSTA1 mRNA up to 30 µM with no cytotoxicity (Figure 2Go).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1. Effect of sulforaphane on mRNA levels of UGT1A1 (solid square), GSTA1 (solid triangle) in HepG2 cells and UGT1A1 (solid circle) in HT29 cells. Cells were seeded at 4 x 104 (HepG2) or 15 x 104 (HT29) and exposed to 15 µM sulforaphane over a total of 18 h, against a DMSO control. Significant induction in HepG2 cells (P < 0.005) and in HT29 cells (P < 0.05) was observed from 0.5 to 18 h. Results are shown as mean and standard deviation (n > 3).

 


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2. Effect of sulforaphane concentration on induction of UGT1A1 (solid square) (P < 0.05, 15–30 µM), GSTA1 (solid triangle) (P < 0.01, 3–30 µM) in HepG2 cells, and UGT1A1 (solid circle) (P < 0.01, 3–30 µM) mRNA levels in HT29 cells. Cells were seeded at 4 x 104 (HepG2) or 15 x 104 (HT29) and treated at various concentrations (0.3–30 µM) of sulforaphane and incubated for 18 h. Fold induction against DMSO controls is plotted. No measurable cytotoxicity was observed even up to 30 µM or with the DMSO control.

 
Bilirubin glucuronidation and UGT1A1 protein expression
HepG2 and HT29 cells glucuronidated bilirubin; media alone yielded no conjugates. Treatment with sulforaphane resulted in a significant increase (P < 0.01) of excreted bilirubin glucuronides compared with DMSO controls (Table IGo). As expected, the basal activity of glucuronidation in HT29 cells was lower than that in HepG2 cells.


View this table:
[in this window]
[in a new window]
 
Table I. Effect of sulforaphane treatment on bilirubin conjugation. HepG2 or HT29 cells were incubated with bilirubin (5 µM) in the presence of sulforaphane (15 µM) or DMSO control; values shown are the mean ± SD of three replicates with analysis by ANOVA. There was no detectable endogenous glucuronide formation in cell-free medium
 
Figure 3Go shows immunoblots using the UGT1A1 specific polyclonal antibody WB-UGT1A1. In control cells, almost no UGT1A1 protein can be detected. Treatment with sulforaphane produced a significant increase in UGT1A1 band intensity in both HepG2 and HT29 cells. These data confirm that the increased levels of glucuronidation (Table IGo) were at least partly due to increased UGT1A1 protein levels. These results demonstrate, for the first time, UGT1A1 protein levels and activity are inducible by sulforaphane in HT29 and HepG2 cells.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3. Measurement of UGT1A1 protein using immunoblotting. Cell-free extract from HepG2 cells, treated with 30 µM sulforaphane, 16 h: lane 1, sulforaphane-treated (20 µg protein loaded); lane 2, DMSO-treated control (20 µg); lane 3, sulforaphane-treated (40 µg); lane 4, DMSO-treated control (40 µg); lane 5, recombinant UGT1A1 positive control (1 µg). Lane 2 is below the limit of detection of the immunoblot.

 
Induction of UGT1A1 and GSTA1 in HepG2 cells by sulforaphane-glutathione conjugate and sulforaphane nitrile
Figure 4Go shows the effects of treating HepG2 cells with the alternative products of glucoraphanin, sulforaphane nitrile, and with a sulforaphane metabolite (glutathione conjugate). The latter induced GSTA1 with a similar potency to sulforaphane. When cells were treated with sulforaphane nitrile in addition to sulforaphane, no effect on induction of UGT1A1 compared to sulforaphane alone was observed. In contrast, the nitrile derivative did not induce significantly GSTA1 or UGT1A1.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4. Effect of sulforaphane metabolites on induction of UGT1A1 and GSTA1 in HepG2 cells. Cells were treated for 18 h with the indicated compound(s) at 15 µM and mRNA measured relative to solvent only controls.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Brassica vegetable consumption causes changes in cancer risk biomarkers, at least in part due to the content of isothiocyanates, derived from the parent glucosinolates (reviewed in ref. 32). Several reviews discuss the anti-carcinogenic properties of isothiocyanates (32–35), and this is at least in part through induction of phase II enzymes such as QR (15) and GST (16). For example, in humans, conjugation and excretion of the food-borne mutagen PhIP is increased after consumption of broccoli (36). Intact glucosinolates have low or no activity (37). Bioavailability of glucosinolates and isothiocyanates depend on the method of preparation of the vegetables (38) and on processing methods (39). Absorption of ITCs occurs in the small intestine in humans (38) and in rats (40) as well as from the colon (41), and isothiocyanates have been detected in plasma (38). ITC and nitriles are both found normally in broccoli and their amounts depend on storage and preparation conditions (42). As shown in Scheme 1Go, the isothiocyanate sulforaphane and sulforaphane nitrile are metabolites of the parent compound, 4-methylsulphinyl-butyl glucosinolate (glucoraphanin) (13,31). After formation of sulforaphane, but not the nitrile, conjugation with glutathione readily occurs both in vivo and in vitro. Metabolism of SFN to SFN-SG could occur in the plant (endogenous GSH) (43), in the gut lumen from GSH secreted in the bile (GSH ~0.25 mM) (44), or intracellularly in the small intestine epithelium, in hepatic cells or in other cells (45). In rats and humans, the main product in urine of SFN and other ITC metabolism is N-acetyl cysteine, formed via a GSH conjugate (38,46). In the current study, we show that sulforaphane is a potent inducer of the glucuronidating enzyme, UGT1A1, at the levels of mRNA, protein and bilirubin activity in two human carcinoma cell lines, used as models for the liver (47) and colon (48). Conjugation with glutathione to form a dithiocarbamate does not significantly modify the activity of SFN. In marked contrast, sulforaphane nitrile does not induce either UGT1A1 or GSTA1. UGT1A1 activity is induced by sulforaphane in rodents, or cells derived from rodents: murine hepatoma cells (Hepa 1c1c7), male Wistar rats (49) and female Sprague–Dawley rats. UGT1A1 is inducible by other compounds such as chrysin in HepG2 (12,50) and HT29 cells (51). Chrysin is high affinity substrate for UGT1A1 with Km = 350 nM, and induced its own glucuronidation (12,52). It is interesting to note that SFN induced glucuronidation, but there is no evidence for glucuronidation of SFN.

Other activities have been reported for isothiocyanates. Sulforaphane stimulated extracellular signal regulated protein kinase 2 (erk2) but not JNK1 in HepG2 and Hepa1c1c7 cells, activated mitogen activated protein kinase (MAPK) and also stimulated raf kinase activity (53). SFN down-regulated COX-2 expression in Raw 264.7 macrophages at the transcriptional level but did not interact with nitric oxide directly and did not induce iNOS activity. SFN also selectively reduced binding of NF-{kappa}B, but with no effect of LPS-induced degradation of I-{kappa}B nor with nuclear translocation of NF-{kappa}B (55). Isothiocyanates react readily with protein amino, sulfhydryl and tryptophan residues (56) and are taken up into cells by conjugation with glutathione (45). A two-fold induction of GST by sulforaphane in human hepatocytes has been reported (15), and sulforaphane may affect apoptosis in some systems (56). On the other hand, there is very little information on the biological activities of nitriles derived from glucosinolates. Nitriles derived from several glucosinolates are less effective at inhibiting cultured human K562 erythroleukemic cell growth than corresponding isothiocyanates (57). SFN nitrile was a poor inducer of quinone reductase in Hepa1c1c7 cells compared with SFN (14). The biological activities of dithiocarbamates have been reviewed, although not for glutathione conjugates of isothiocyanates from Brassica vegetables. Dithiocarbamates in general have numerous biological effects including increasing copper uptake into cells (58).

Induction of UGT1A1 increases conjugation of xenobiotics (9), which has the potential to reduce breast cancer risk (11) and of enhancing bilirubin clearance (8). A common genetic defect in the TATA box promoter of the UGT1A1 gene is associated with Gilbert’s syndrome causing mild hyperbilirubinaemia, and enhanced bilirubin clearance would be desirable in this condition. However, adverse effects of anticancer agents have been observed in Gilbert’s patients due to reduced drug or bilirubin glucuronidation (59) and so further human trials are needed to assess the role of drug–food interaction in a clinical environment. Decreased serum bilirubin levels have also been attributed to an increase in coronary heart risk, as bilirubin is believed to be an endogenous antioxidant preventing the formation of oxidised LDL and subsequent atherosclerosis (60). The results also suggest that sulforaphane treatment could be used to improve the potential of HepG2 cells as a model to study glucuronidation.


    Notes
 
1 To whom correspondence should be addressed at: Head of Metabolic and Genetic Regulation, Nestlé Research Center, PO Box 44, CH-1000 Lausanne 26, Switzerland Email: gary.williamson{at}rdls.nestle.com Back


    Acknowledgments
 
We thank the European Union (EFGLU; FAIR CT97 3029) and the Biotechnology and Biological Sciences Research Council for funding this work. We also thank Richard Bennett for the sulforaphane nitrile and glutathione conjugate.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Tukey,R.H. and Strassburg,C.P. (2000) Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Ann. Rev. Pharmacol. Toxicol., 40, 581–616.[ISI][Medline]
  2. Burchell,B. and Coughtrie,M.W. (1989) UDP-glucuronosyltransferases. Pharmacol. Ther., 43, 261–289.[ISI][Medline]
  3. Mackenzie,P.I., Owens,I.S., Burchell,B., et al. (1997) The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence. Pharmacogenetics, 7, 255–269.[ISI][Medline]
  4. Ketterer,B. (2001) A bird’s eye view of the glutathione transferase field. Chem. Biol. Interact., 138, 27–42.[ISI][Medline]
  5. Dutton,G.J. (1980) Glucuronidation of Drugs and Other Compounds. CRC Press, Boca Raton, Florida.
  6. Ritter,J.K., Crawford,J.M. and Owens,I.S. (1991) Cloning of two human liver bilirubin UDP-glucuronosyltransferase cDNAs with expression in COS-1 cells. J. Biol. Chem., 266, 1043–1047.[Abstract/Free Full Text]
  7. Chowdhury,J.R. and Chowdhury,N.R. (1983) Conjugation and excretion of bilirubin. Semin. Liver Dis., 3, 11–23.[ISI][Medline]
  8. Clarke,D.J., Moghrabi,N., Monaghan,G., Cassidy,A., Boxer,M., Hume,R. and Burchell,B. (1997) Genetic defects of the UDP-glucuronosyltransferase-1 (UGT1) gene that cause familial non-haemolytic unconjugated hyperbilirubinaemias. Clin. Chim. Acta, 266, 63–74.[ISI][Medline]
  9. Burchell,B., Brierley,C.H. and Rance,D. (1995) Specificity of human UDP-glucuronosyltransferases and xenobiotic glucuronidation. Life Sci., 57, 1819–1831.[ISI][Medline]
  10. Strassburg,C.P., Strassburg,A., Nguyen,N., Li,Q., Manns,M.P. and Tukey,R.H. (1999) Regulation and function of family 1 and family 2 UDP-glucuronosyltransferase genes (UGT1A, UGT2B) in human oesophagus. Biochem. J., 338, 489–498.[ISI][Medline]
  11. Guillemette,C., Millikan,R.C., Newman,B. and Housman,D.E. (2000) Genetic polymorphisms in uridine diphospho-glucuronosyltransferase 1A1 and association with breast cancer among African Americans. Cancer Res., 60, 950–956.[Abstract/Free Full Text]
  12. Walle,T., Otake,Y., Galijatovic,A., Ritter,J.K. and Walle,U.K. (2000) Induction of UDP-glucuronosyltransferase UGT1A1 by the flavonoid chrysin in the human hepatoma cell line HepG2. Drug Metab. Dispos., 28, 1077–1082.[Abstract/Free Full Text]
  13. Rosa,E.A., Heaney,R., Fenwick,G.R. and Portas,C.A.M. (1997) Glucosinolates in crop plants. In Janick,J. (ed.) Horticultural Reviews. John Wiley, Oxford, pp. 99–215.
  14. Matusheski,N.V. and Jeffery,E.H. (2001) Comparison of the bioactivity of two glucoraphanin hydrolysis products found in broccoli, sulforaphane and sulforaphane nitrile. J. Agric. Food Chem., 49, 5743–5749.[ISI][Medline]
  15. Zhang,Y.S., Talalay,P., Cho,C.G. and Posner,G.H. (1992) A major inducer of anticarcinogenic protective enzymes from broccoli – isolation and elucidation of structure. Proc. Natl Acad. Sci. USA, 89, 2399–2403.[Abstract/Free Full Text]
  16. Maheo,K., Morel,F., Langouet,S., Kramer,H., Le Ferrec,E., Ketterer,B. and Guillouzo,A. (1997) Inhibition of cytochromes P-450 and induction of glutathione S-transferases by sulforaphane in primary human and rat hepatocytes. Cancer Res., 57, 3649–3652.[Abstract]
  17. Favreau,L.V. and Pickett,C.B. (1995) The rat quinone reductase antioxidant response element – identification of the nucleotide sequence required for basal and inducible activity and detection of antioxidant response element-binding proteins in hepatoma and non-hepatoma cell lines. J. Biol. Chem., 270, 24468–24474.[Abstract/Free Full Text]
  18. Wang,B. and Williamson,G. (1996) Transcriptional regulation of the human NAD(P)H:quinone oxidoreductase (NQO1) gene by monofunctional inducers. Biochim. Biophys. Acta, 1307, 104–110.[ISI][Medline]
  19. Brierley,C.H., Senafi,S.B., Clarke,D., Hsu,M.H., Johnson,E.F. and Burchell,B. (1996) Regulation of the human bilirubin UDP-glucuronosyltransferase gene. Adv. Enzyme Regul., 36, 85–97.[ISI][Medline]
  20. Zhang,Y. (2000) Role of glutathione in the accumulation of anticarcinogenic isothiocyanates and their glutathione conjugates by murine hepatoma cells. Carcinogenesis, 21, 1175–1182.[Abstract/Free Full Text]
  21. Hagen,T.M., Wierzbicka,G.T., Bowman,B.B., Aw,T.Y. and Jones,D.P. (1990) Fate of dietary glutathione: disposition in the gastrointestinal tract. Am. J. Physiol., 259, G530–G535.[Abstract/Free Full Text]
  22. Kjaer,A., Larsen,I. and Gmelin,R. (1955) Isothiocyanates XIV. 5-Methylthiopentyl isothiocyanate, a new mustard oil present in nature as a glucoside (glucoberteroin). Acta Chem. Scand., 9, 1311–1316.[ISI]
  23. Kassahun,K., Davis,M., Hu,P., Martin,B. and Baillie,T. (1997) Biotransformation of the naturally occurring isothiocyanate sulforaphane in the rat: identification of phase I metabolites and glutathione conjugates. Chem. Res. Toxicol., 10, 1228–1233.[ISI][Medline]
  24. Bowen,W.P., Carey,J.E., Miah,A., McMurray,H.F., Munday,P.W., James,R.S., Coleman,R.A. and Brown,A.M. (2000) Measurement of cytochrome P450 gene induction in human hepatocytes using quantitative real-time reverse transcriptase-polymerase chain reaction. Drug Metab. Dispos., 28, 781–788.[Abstract/Free Full Text]
  25. Van Roy,F.P. and Heirwegh,K.P. (1968) Determination of bilirubin glucuronide and assay of glucuronyltransferase with bilirubin as acceptor. Biochem. J., 107, 507–518.[ISI][Medline]
  26. Cuff,R.J., Wade,L.T., Rychlik,B., Jedlitschky,G.A. and Burchell,B. (2001) Characterisation of glucuronidation and transport in V79 cells co-expressing UGT1A1 and MRP1. Toxicol Lett., 120, 43–49.[ISI][Medline]
  27. Chomczynski,P. (1993) A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques, 15, 532–537.[ISI][Medline]
  28. Carubelli,R., Graham,S.A. and McCay,P.B. (1992) Effect of dietary butylated hydroxytoluene on nuclear envelope cytochrome P-450 during the initiation and promotion stages of hepatocarcinogenesis. Nutr. Cancer, 18, 59–62.[ISI][Medline]
  29. Laemmli,U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.[ISI][Medline]
  30. Towbin,H., Staehelin,T. and Gordon,J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl Acad. Sci. USA, 76, 4350–4354.[Abstract]
  31. Fenwick,G.R., Heaney,R.K. and Mullin,W.J. (1983) Glucosinolates and their breakdown products in food and food plants. CRC Food Sci. Nutr., 18, 123–128.
  32. Verhoeven,D.T.H., Verhagen,H., Alexandra-Goldbohm,R., van der Brandt,P.A. and van Poppel,G. (1997) A review of mechanisms underlying anticarcinogenicity by Brassica vegetables. Chem. Biol. Interact., 103, 79–129.[ISI][Medline]
  33. Mithen,R.F., Dekker,M., Verkerk,R., Rabot,S. and Johnson,I.T. (2000) The nutritional significance, biosynthesis and bioavailability of glucosinolates in human foods. J. Sci. Food Agric., 80, 967–984.[ISI]
  34. Williamson,G., Faulkner,K. and Plumb,G.W. (1998) Glucosinolates and phenolics as antioxidants from plant foods. Eur. J. Cancer Prev., 7, 17–21.[ISI][Medline]
  35. Hecht,S.S. (1999) Chemoprevention of cancer by isothiocyanates, modifiers of carcinogen metabolism. J. Nutr., 129, 768s–774s.[ISI][Medline]
  36. Kulp,K.S., Knize,M.G. and Felton,J.S. (2000) Broccoli affects the metabolism of PhIP in humans. 7th International Conference on Mechanisms of Antimutagenesis and Anticarcinogenesis, Grand Rapids, Michigan.
  37. Tawfiq,N., Heaney,R.K., Plumb,J.A., Fenwick,G.R., Musk,S.R.R. and Williamson,G. (1995) Dietary glucosinolate as blocking agents against carcinogenesis: correlation between glucosinolate structure and induction of quinone reductase in murine hepa1c1c7 cells. Carcinogenesis, 16, 1191–1194.[Abstract]
  38. Conaway,C.C., Getahun,S.M., Liebes,L.L., Pusateri,D.J., Topham,D.K., Botero-Omary,M. and Chung,F.L. (2000) Disposition of glucosinolates and sulforaphane in humans after ingestion of steamed and fresh broccoli. Nutr. Cancer, 38, 168–178.[ISI][Medline]
  39. Shapiro,T.A., Fahey,J.W., Wade,K.L., Stephenson,K.K. and Talalay,P. (2001) Chemoprotective glucosinolates and isothiocyanates of broccoli sprouts: metabolism and excretion in humans. Cancer Epidemiol. Biomark. Prev., 10, 501–508.[Abstract/Free Full Text]
  40. Michaelsen,S., Otte,J. and Simonsen,L.O. (1993) Absorption and degradation of individual glucosinolates in the digestive track of rodents. Acta Agric Scand A: Anim. Sci., 44, 25–37.
  41. Elfoul,L., Rabot,S., Khelifa,N., Quinsac,A., Duguay,A. and Rimbault,A. (2001) Formation of allyl isothiocyanate from sinigrin in the digestive tract of rats monoassociated with a human colonic strain of bacteroides thetaiotaomicron. FEMS Microbiol. Lett., 197, 99–103.[ISI][Medline]
  42. Howard,L.A., Jeffrey,E.H., Wallig,M.A. and Klein,B.P. (1997) Retention of phytochemicals in fresh and processed broccoli. J. Food Sci., 62, 1098–1100.[ISI]
  43. Creissen,G., Firmin,J., Fryer,M., et al. (1999) Elevated glutathione biosynthetic capacity in the chloroplasts of transgenic tobacco plants paradoxically causes increased oxidative stress. Plant Cell, 11, 1277–1292.[Abstract/Free Full Text]
  44. Samiec,P.S., Dahm,L.J. and Jones,D.P. (2000) Glutathione S-transferase in mucus of rat small intestine. Toxicol. Sci., 54, 52–59.[Abstract/Free Full Text]
  45. Zhang,Y.S. and Talalay,P. (1998) Mechanism of differential potencies of isothiocyanates as inducers of anticarcinogenic phase 2 enzymes. Cancer Res., 58, 4632–4639.[Abstract]
  46. Kassahun,K., Davis,M., Hu,P., Martin,B. and Baillie,T. (1997) Biotransformation of the naturally occurring isothiocyanate sulforaphane in the rat: Identification of phase I metabolites and glutathione conjugates. Chem. Res. Toxicol., 10, 1228–1233.[ISI][Medline]
  47. Grant,M.H., Duthie,S.J., Gray,A.G. and Burke,M.D. (1988) Mixed function oxidase and UDP-glucuronyltransferase activities in the human HepG2 hepatoma cell line. Biochem. Pharmacol., 37, 4111–4116.[ISI][Medline]
  48. Britten,C.D., Hilsenbeck,S.G., Eckhardt,S.G., Marty,J., Mangold,G., MacDonald,J.R., Rowinsky,E.K., Von Hoff,D.D. and Weitman,S. (1999) Enhanced antitumor activity of 6-hydroxymethylacylfulvene in combination with irinotecan and 5-fluorouracil in the HT29 human colon tumor xenograft model. Cancer Res., 59, 1049–1053.[Abstract/Free Full Text]
  49. Kashi,K., Zhang,Y., Talalay,P. and Dannenberg,A.J. (1999) Anticarcinogenic organic isothiocyanates induce UDP-glucuronosyltransferase. Cancer Metab. Nutr., A868, 5033–5038.
  50. Knasmuller,S., Parzefall,W., Sanyal,R., et al. (1998) Use of metabolically active human hepatoma cells for the detection of mutagen and antimutagens. Mutat. Res., 402, 185–202.[ISI][Medline]
  51. Franklin,T.J., Jacobs,V., Jones,J.G., Ple,P. and Bruneau,P. (1996) Glucuronidation associated with intrinsic resistance to mycophenolic acid in human colorectal carcinoma cells. Cancer Res., 56, 984–987.[Abstract]
  52. Walle,T., Otake,Y., Galijatovic,A., Ritter,J.K. and Walle,U.K. Induction of UDP-glucuronosyltransferase UGT1A1 by the flavonoid chrysin in the human hepatoma cell line HepG2. (2000) Drug Metab. Disposit., 28, 1077–1082..[Abstract/Free Full Text]
  53. Yu,R., Lei,W., Mandlekar,S., Weber,M.J., Der,C.J., Wu,J. and Kong,A.N.T. (1999) Role of a mitogen-activated protein kinase pathway in the induction of phase II detoxifying enzymes by chemicals. J. Biol. Chem., 274, 27545–27552.[Abstract/Free Full Text]
  54. Heiss,E., Herhaus,C., Klimo,K., Bartsch,H. and Gerhauser,C. (2001) Nuclear factor kappa B is a molecular target for sulforaphane-mediated anti-inflammatory mechanisms. J. Biol. Chem., 276, 32008–32015.[Abstract/Free Full Text]
  55. Rawel,H., Kroll,J., Haebel,S. and Peter,M.G. (1998) Reactions of a glucosinolate breakdown product (benzyl isothiocyanate) with myoglobin. Phytochemistry, 48, 1305–1311.[ISI][Medline]
  56. Gamet-Payrastre,L., Li,P., Lumeau,S., Cassar,G., Dupont,M.A., Chevolleau,S., Gasc,N., Tulliez,J. and Terce,F. (2000) Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells. Cancer Res., 60, 1426–1433.[Abstract/Free Full Text]
  57. Nastruzzi, C., Cortesi, R., Esposito, E., Menegatti, E., Leoni, O., Iori, R. and Palmieri,S. (2000) In vitro antiproliferative activity of isothiocyanates and nitriles generated by myrosinase-mediated hydrolysis of glucosinolates from seeds of cruciferous vegetables. J. Agric. Food Chem., 48, 3572–3575.[ISI][Medline]
  58. Orrenius,S., Nobel,C.S.I., Van den Dobbelsteen,D.J., Burkitt,M.J. and Slater,A.F.G. (1996) Dithiocarbamates and the redox regulation of cell death. Biochem. Soc. Trans., 24, 1032–1038.[ISI][Medline]
  59. Burchell,B., Soars,M., Monaghan,G., Cassidy,A., Smith,D. and Ethell,B. (2000) Drug-mediated toxicity caused by genetic deficiency of UDP-glucuronosyltransferases. Toxicol. Lett., 112, 333–340.[ISI]
  60. Hunt,S.C., Kronenburg,F., Eckfeldt,J., Hopkins,P., Myers,R.H. and Heiss,G. (2001) Association of plasma bilirubin with coronary heart disease and segregation of bilirubin as a major gene trait: the NHLBI family heart study. Atherosclerosis, 15, 747–754.
Received December 27, 2001; revised May 3, 2002; accepted May 10, 2002.