Total intracellular accumulation levels of dietary isothiocyanates determine their activity in elevation of cellular glutathione and induction of Phase 2 detoxification enzymes

Lingxiang Ye and Yuesheng Zhang1,2

Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 and
1 Arizona Cancer Center and Department of Medicine, College of Medicine, University of Arizona, 1515 N. Campbell Avenue, PO Box 245024, Tucson,AZ 85724-5024, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 References
 
Many dietary isothiocyanates (ITCs) have shown cancer chemoprotective activity in animal models. Isothiocyanates rapidly accumulate in cells of various types as glutathione conjugates, and the total intracellular accumulation levels of ITCs (area under time–concentration curve; AUC) were critical for their Phase 2 enzyme inducer activities in murine hepatoma Hepa 1c1c7 cells. Induction of Phase 2 detoxification enzymes is recognized as a major cellular defense against carcinogens and other toxic agents. In order to further define the importance of intracellular AUC of ITCs in stimulating cellular detoxification functions, we have compared the intracellular AUCs and the inducer activities of four common dietary ITCs, allyl-ITC, benzyl-ITC, phenethyl-ITC and sulforaphane [1-isothiocyanato-(4R,S)-(methylsulfinyl)butane], in mouse skin papilloma (PE) cells. When PE cells were incubated with 5 µM of each ITC for 24 h, significant elevations of glutathione content (1.8–4.3-fold), quinone reductase activity (2.1–5.4-fold) and glutathione transferase activity (0.8–1.5-fold) were observed. These elevations were closely correlated with the AUCs of the ITCs. Increasing intracellular AUC of a weaker ITC by multiple dosing also increased its inducer activity. Further studies revealed that the AUC-dependent elevation of the above elements were mediated by the DNA regulatory element EpRE/ARE. In human HepG2 cells, which were stably transfected with a reporter construct under EpRE/ARE control, the intracellular AUC of the four ITCs closely correlated with the levels of reporter gene product (green fluorescent protein). These results showed that cellular accumulation levels of ITCs determine their activity in inducing cellular detoxification capacity and suggested that the intracellular AUC might be a valuable biomarker of the Phase 2 enzyme inducer activity of ITCs.

Abbreviations: allyl-ITC, allyl isothiocyanate; AUC, area under concentration–time curve; benzyl-ITC, benzyl isothiocyanate; DTC, dithiocarbamates; GFP, green fluorescence protein; GSH, glutathione; GSTs, glutathione transferases; phenethyl-ITC, phenethyl isothiocyanate; QR, quinone reductase; ITCs, isothiocyanates; SF, sulforaphane [-isothiocyanato-(4R,S)-(methylsulfinyl)butane].


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 References
 
It is widely accepted that induction of Phase 2 detoxification enzymes [e.g. quinone reductase (QR) and glutathione transferases (GSTs)] and elevation of glutathione (GSH) are a major strategy for protecting cells against a variety of endogenous and exogenous toxic components, such as reactive oxygen species and chemical carcinogens (1,2). Cellular Phase 2 enzymes and GSH levels can be elevated by an array of chemically diverse compounds. Studies from a number of laboratories including our own have shown that many isothiocyanates (ITCs) are effective inducers (3–5). These compounds and their precursor glucosinolates are broadly distributed in edible plants (6,7), and thus are of considerable interest as possible dietary protectors against carcinogens. Indeed, many ITCs have displayed potent activity in blocking chemical carcinogenesis in several animal models (4,8,9).

In a previous study, we showed that ITCs are rapidly accumulated in many cell lines, with intracellular levels reaching the millimolar range (10). We found that ITCs were accumulated as dithiocarbamates (DTC) derived from conjugation with intracellular GSH and that cellular GSH content appeared to be the factor determining the extent of accumulation (11,12). Of particular interest to us was that the total intracellular accumulation levels (area under time–concentration curve; AUC) of nine ITCs differing considerably in chemical structure, not the peak accumulation levels, correlated with their ability to induce Phase 2 enzymes (GST and QR) (10). Thus the possibility was raised that the intracellular AUCs of these compounds might provide a suitable marker for determining their inducer activity and anticarcinogenic potential. Because this phenomenon had only been shown in Hepa 1c1c7 cells, we needed to verify the utility and reliability of intracellular AUCs as markers of ITC inducer activity in other cell lines and to determine whether we could increase the inducer activity of a weaker ITC by increasing its intracellular AUC through multiple dosing of the compound. Much evidence indicates that transcriptional activation of Phase 2 enzyme genes is primarily mediated by antioxidant responsive element (ARE) or electrophile responsive element (EpRE) located on the upstream region of the genes (13–16). In order to determine whether ARE/EpRE also play a role in AUC-dependent induction of Phase 2 enzymes and elevation of GSH, human hepatoma HepG2 cells that were stably transfected with a reporter construct under regulatory control of EpRE were examined for the correlation of intracellular AUCs of ITCs with the induction of the reporter gene product.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 References
 
Chemicals
Allyl-ITC, benzyl-ITC and phenethyl-ITC were purchased from Aldrich (Milwaukee, WI) and sulforaphane (SF) was from LKT (St Paul, MN).

Cell culture
Mouse skin papilloma cells were cultured in Eagle MEM without Ca2+ (BioWhittaker, Walkersville, MD) plus 8% fetal bovine serum (FBS; Life Technologies, Rockville, MD) treated with Chelex 100 ion-exchange resin (Bio-Rad, Richmond, CA) (12). MCF-7 cells (human breast cancer cells) were cultured in DMEM (Life Technologies) with 5% FBS and Hepa 1c1c7 cells were cultured in {alpha}-MEM (Life Technologies) plus 10% FBS. Recombinant HepG2 cells that are stably transfected with an EpRE–TK–GFP construct (HepG2/EpRE–TK–GFP cells) or TK–GFP construct (HepG2/TK–GFP cells) were gifts of Drs M.Zhu and W.E.Fahl (McArdle Laboratory for Cancer Research, University of Wisconsin) (17). Briefly, for the EpRE–TK–GFP construct, four concatamerized 41 base pair (bp) EpREs were linked to a 123 bp thymidine kinase (TK) promoter and the entire fragment was cloned into the multiple cloning site of pEGFP (green fluorescent protein expression vector). For the TK–GFP construct, the EpRE sequences were excluded. HepG2 cells were cultured in DMEM with high glucose and glutamine (Life Technologies) containing 10% FBS. All cells were grown on 10 cm plates at 37°C in a humidified 5% CO2 incubator.

Measurement of intracellular accumulation of ITCs in PE and HepG2 cells
Intracellular accumulation of ITCs was measured by the cyclocondensation assay (reaction with 1,2-benzenedithiol) which detects both free ITCs and their dithiocarbamate (DTC) derivatives. Thus, the intracellular accumulation levels of ITCs reported in this paper refer to the total levels of both ITCs and dithiocarbamates formed in cells. The procedures for cell exposure to ITCs, cell harvest, preparation of cell lysates and quantitation of ITC/DTC in cell lysates by the cyclocondensation assay have been previously described (10). Briefly, 1x106 cells were cultured with 10 ml medium on each 10 cm plate for 3–5 days so that the cells were nearly confluent. The medium was then replaced with the same volume of fresh medium containing 5 µM ITC in 10 µl acetonitrile and incubation was continued. At 0.5, 1, 2, 3, 6, 12 and 24 h, cells in two plates were rapidly trypsinized and harvested. HepG2 cells were detached by incubation of each plate with 1 ml 0.05% trypsin for 5 min and 9 ml medium were then added to each plate. PE cells were more resistant to trypsin treatment. Consequently, 0.2 ml 10% trypsin was added to the existing medium (final concentration 0.2%) 25 min before the harvest time to detach cells. We found that trypsin did not interfere with ITC uptake. Each 10 ml suspension was then quickly layered on top of 2 ml 1:1 (v/v) dibutyl phthalate/diisononyl phthalate in a 15 ml conical tube and centrifuged at 3000 r.p.m. and 4°C for 2 min. Each cell pellet was resuspended in 200 µl distilled water and stored at –70°C. The cells were sonicated before use and the total content of ITC/DTC in each lysate was determined by the HPLC-based cyclocondensation assay (10,18). The ITC/DTC content in lysates was adjusted by protein levels in the lysates, which were measured by the BCA assay (19).

The intracellular accumulation levels of each ITC at all time points were then determined by an interactive computer program (LAGRAN method) to calculate the AUC (20).

Determination of cellular GSH levels, QR and GST activities, and GFP levels after exposure to ITCs
Cell pellets were prepared as described above. In experiments designed to assess the effects of multiple dosing of ITCs, the medium was replaced each time with fresh medium containing 5 µM ITC as indicated.

Cellular GSH levels were determined by the glutathione reductase-coupled 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) assay in 96-well microtiter plates (21). Briefly, each cell pellet was sonicated in ~200 µl Dulbecco's phosphate-buffered saline (DPBS) (PE cells) or lysed in 200 µl 0.08% digitonin solution (HepG2 cells, see QR assay below for details). GSH contents were very similar in lysates prepared by the two methods. Lysates (20 µl) were mixed with 80 µl ice-cold 2.5% metaphosphoric acid (MPA) on ice for 20 min and centrifuged at 14 000 r.p.m. for 2 min at 4°C. The supernatant fraction was diluted 20-fold with 100 mM sodium phosphate plus 5 mM EDTA (assay buffer), pH 7.5. In each well, 50 µl each diluted sample was mixed with 50 µl 1.26 mM DTNB in assay buffer and 50 µl 2.5 U/ml glutathione reductase (bakers yeast; Sigma, St Louis, MO) in assay buffer. After 5 min incubation at room temperature, 50 µl 0.72 mM NADPH in assay buffer were added to each well and the initial reaction rates were measured at 405 nm by a Spectra Max Plus microtiter plate reader (Molecular Devices, Sunnyvale, CA). Several concentrations of pure GSH were also assayed on the same plate to establish a calibration curve for calculation of GSH content in the samples.

To measure cytosolic QR activity, cell lysates were prepared by mixing each cell pellet with ~200 µl digitonin solution (0.8 g/l digitonin in 2 mM EDTA, pH 7.8) and incubating at 37°C for 10 min, followed by gentle shaking for 10 min at room temperature and centrifugation at 14 000 r.p.m. and 4°C for 3 min. The QR activity in the supernatant fractions was determined in a 300 µl assay in 96-well microtiter plates (5,22). Each fraction was diluted with 10 mM Tris–HCl buffer, pH 7.4 and 50 µl aliquots of the diluted solution were added to each well together with 50 µl phosphate buffer (5 mM potassium phosphate, pH 7.4 and 0.5% dimethyl sulfoxide). In order to exclude non-specific enzyme activity (dicumarol non-inhibitable), 50 µl phosphate buffer containing 0.3 mM dicumarol were added to parallel wells in place of the regular buffer. Then to each well was added 200 µl assay solution containing 25 mM Tris–HCl pH 7.4, 0.5% bovine serum albumin, 0.025% Tween 20, 5 µM FAD, 30 µM NADP, 1 µM glucose-6-phosphate, 2 U/ml glucose-6-phosphate dehydrogenase (bakers yeast; Sigma), 0.3 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and 25 µM menadione. The reaction rates were immediately measured at 610 nm for 5 min at room temperature by the microtiter plate reader. For each sample, the specific activity of QR was derived by subtracting the initial reaction rates in wells containing no dicumarol from those in wells containing dicumarol.

Cytosolic GST activity was assayed with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate (23) either in a 1 or 0.3 ml volume. The 1 ml assay was slightly modified from the published method and consisted of 100 mM sodium phosphate buffer (pH 7.4), 1 mM GSH, 1 mM CDNB, 2% (v/v) ethanol (CDNB was originally dissolved in ethanol) and 10 µl sample solution. Each cell pellet prepared as above was suspended in 200 µl 10 mM Tris–HCl (pH 7.4), sonicated for complete cell lysis and centrifuged at 14 000 r.p.m. for 3 min at 4°C. The supernatant fractions were used for the assay and the initial reaction rate was measured at 340 nm. We later found that GST activity could be more conveniently determined in microtiter plates by lysing the cells with digitonin (as described for QR) and that this procedure also resulted in higher recovery of GST activity than the sonication method. Thus, cell lysates were prepared as described for the QR assay. Each 0.3 ml assay contained 100 mM potassium phosphate (pH 6.5), 1 mM GSH, 1 mM CDNB, 2% (v/v) ethanol and 20 µl sample solution. The initial reaction rates were measured at 340 nm by the microtiter plate reader.

Cellular GFP content was determined by fluorescence emitted in samples derived from HepG2/EpRE–TK–GFP and HepG2/TK–GFP cells. Cell lysates were prepared by digitonin treatment as described above and the lysates were centrifuged at 14 000 r.p.m. for 3 min at 4°C. Supernatant fractions (usually 10 µl) were diluted to 2 ml with DPBS and the fluorescence intensity of each solution was read with a luminescence spectrometer (LS50; Perkin Elmer, Norwalk, CT) at excitation/emission wavelengths 485/530 nm (17).


    Results
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 Results
 References
 
GSH elevation by ITCs in different cell lines
In order to select a suitable cell line to examine whether intracellular ITC accumulation levels (AUCs) play a role in Phase 2 enzyme induction and GSH elevation, PE, MCF-7 and Hepa 1c1c7 cells were screened for the degree of GSH elevation by known Phase 2 enzyme inducers: allyl-ITC, benzyl-ITC and SF. Cellular GSH contents were measured after cells were incubated with each ITC at 5 µM for 24 h. As expected, basal intracellular GSH levels differed widely with cell types, but all these cell lines showed elevated GSH levels after ITC exposure. Table IGo shows that PE cells were the most responsive, with intracellular GSH increasing from 1.8-fold with allyl-ITC to 4.3-fold with SF. Smaller elevations were observed with MCF-7 cells and Hepa 1c1c7 cells. In PE cells the degree of GSH elevation differed considerably with each ITC inducer, suggesting that this cell line would be suitable for evaluating the relation between the intracellular AUC of ITCs and the degree of enzyme induction. We also examined the effect of ITC exposure time on elevation of GSH levels by incubating cells with each ITC at 5 µM for 12, 24, 48 or 72 h. We found that levels of GSH were highest after incubation with ITCs for 24 h (data not shown). Therefore, for subsequent studies, PE cells were routinely incubated with each ITC for 24 h, unless otherwise stated.


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Table I. Elevation of glutathione levels in three cell lines by isothiocyanates
 
Correlation of intracellular accumulation levels (AUCs) of ITCs with elevation of GSH and induction of QR and GST in PE cells
To determine whether the AUCs of intracellular accumulation of ITCs correlated with their ability to raise levels of GSH, and GST and QR activity, we incubated PE cells in monolayers with allyl-ITC, benzyl-ITC and SF, as well as phenethyl-ITC which is also a known Phase 2 enzyme inducer, each at 5 µM for 0.5, 1, 2, 3, 6, 12 and 24 h. At each time point the intracellular accumulation of each ITC was measured by the cyclocondensation assay as described above. The assay measures both free ITCs and their DTC derivatives in cell lysates, and the AUCs were calculated based on these measurements (our earlier study in Hepa 1c1c7 cells had shown that ITCs were accumulated mainly as DTCs derived from conjugation with GSH and that the DTC formed may be metabolized further) (11). We found that, similar to prior studies in Hepa 1c1c7 cells, (i) uptake was very rapid for all compounds; intracellular levels reached a peak around 0.5–1 h after initial exposure; (ii) the peak levels differed significantly among ITCs and (iii) the rates of decrease in intracellular ITC levels also differed considerably. Whereas intracellular SF levels decreased relatively slowly (half-life about 22 h) after the peak level was reached, levels of the other three ITCs decreased by 50% within 3–6 h (Figure 1Go, top; note also the different levels in HepG2/EpRE–TK–GFP cells in Figure 1Go, bottom, which are described later).



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Fig. 1. Time-course of intracellular accumulation of isothiocyanates in (top) PE cells and (bottom) HepG2/EpRE–TK–GFP cells. PE and HepG2/EpRE–TK–GFP cells were grown on 10 cm plates for 4 days to reach ~8x106 cells per plate and were then incubated with 10 ml fresh medium containing 5 µM allyl-ITC ({square}), benzyl-ITC (•), phenethyl-ITC ({circ}) or SF ({blacksquare}) for indicated times. Cells were then harvested and intracellular ITC contents were determined by the cyclocondensation assay. Each value is presented as mean ± SD (n = 4).

 
Cellular GSH content and QR and GST activities were measured at the end of the 24 h exposure. As shown in Figure 2Go, all four ITCs elevated cellular GSH level and QR activity, ranging from 1.8- to 5.4-fold, with SF the most potent inducer. GST was less responsive to ITCs and again SF was the best inducer. We then calculated the AUC of the intracellular accumulation levels within 24 h and compared these values with the ratios (treated over control) of specific enzyme activities and GSH levels. Figure 3Go shows that these AUCs correlated closely with the increases in GSH, QR and GST (correlation coefficients of linear regression range from 0.82 to 0.94), even though the increases by each ITC differed widely. In contrast, there was no clear correlation between the increases in GSH, QR and GST and the peak ITC accumulation levels. For example, the peak intracellular benzyl-ITC level was much higher than that of SF, but the latter compound was a much more potent inducer. Similarly, the peak accumulation of phenethyl-ITC was only slightly lower than that of SF, but the former compound was a much weaker inducer.



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Fig. 2. Induction of glutathione transferase and quinone reductase and elevation of glutathione in PE cells by isothiocyanates. PE cells were grown on 10 cm plates with 10 ml medium for 4 days to reach ~5x106 cells per plate. Cells on each plate were then incubated with 10 ml fresh medium containing 5 µM allyl-ITC, benzyl-ITC, phenethyl-ITC or SF for 24 h. Cells were harvested by trypsin treatment and the activities of both GST ({square}) and QR ({blacksquare}), and GSH levels ( ) were determined. GST activity was determined in a 1 ml assay and cell lysates were prepared by sonication. The ratios were calculated from values each of which was a mean of three, four or six measurements (standard errors are generally <than 4% of the means). The specific activities of GST and QR in control cells were 11.92 and 11.86 nmol/min/mg, respectively, and the GSH level was 9.4 nmol/mg protein.

 


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Fig. 3. Correlation of total intracellular accumulation of isothiocyanate (AUC) within 24 h with its inducer activity in PE cells. The total intracellular AUC of each ITC in PE cells (exposed to 5 µM ITC for 24 h) was calculated from the data shown in Figure 1Go (top) by the LAGRAN computer program (20). The specific GST and QR activities, and GSH levels were determined in PE cells treated with or without 5 µM ITC for 24 h and the ratios of the specific enzyme activities and GSH levels (treated cells/control cells) were calculated (see Figure 2Go). The intracellular AUCs of ITCs were then plotted against the ratios of specific enzyme activities and GSH levels: a, allyl-ITC; b, phenethyl-ITC; c, benzyl-ITC; d, SF.

 
Effect of multiple dosing with ITCs on QR and GST activities in PE cells
On the basis of the above experiments showing that induction of Phase 2 enzymes and elevation of GSH closely correlated with the degree of accumulation of ITCs, we questioned whether increasing the intracellular accumulation of ITCs by multiple dosing would enhance induction. Accordingly, PE cells were incubated with either 5 µM phenethyl-ITC or SF for 24 h, but the medium was replaced with fresh medium containing the same concentration of ITC every 8 h. As shown in Figure 2Go, phenethyl-ITC was a much weaker inducer than was SF, but Table IIGo shows that treatment of the cells three times with fresh medium containing 5 µM phenethyl-ITC substantially increased induction of QR and GST compared with one treatment. In contrast, treatment with SF three times did not increase induction, suggesting that both GST and QR activity may have been maximally induced by the single SF incubation for 24 h. This notion is supported by the finding that increases in GST and QR activities in cells treated three times with phenethyl-ITC were very similar to those in cells treated with SF either once or three times. Accordingly, we calculated that the AUC of intracellular accumulation of an ITC required for maximal elevation of GSH, GST and QR in PE cells within 24 h is ~260 (nmol/mg)xh. The effect of multiple dosing on intracellular GSH content was not clear-cut, because replacement with medium alone also resulted in a significant increase in intracellular GSH content, possibly due to cystine or cysteine present in the medium.


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Table II. Effect of multiple dosing of isothiocyanates on induction of glutathione transferase and quinone reductase in mouse skin papilloma cells
 
Correlation of cellular accumulation levels (AUCs) of ITCs with both GFP and GSH elevation levels in HepG2/EpRE–TK–GFP cells and HepG2/TK–GFP cells
It is now widely recognized that Phase 2 enzyme induction and GSH elevation are mediated by a small regulatory DNA element, namely ARE or EpRE, which resides in the upstream region of many Phase 2 enzyme genes. In order to determine whether the intracellular AUC-dependent induction of Phase 2 enzymes by ITCs is mediated by EpRE/ARE, we examined induction of GFP in both HepG2/EpRE–TK–GFP and HepG2/TK–GFP cells. The GFP gene was stably introduced into HepG2 cells through an expression vector with (the former cells) or without (the latter cells) four concatamarized EpREs in the upstream region of the gene by Zhu and Fahl (17). They have shown that incubation of HepG2/EpRE–TK–GFP cells with various known Phase 2 enzyme inducers including ITC resulted in significant induction of GFP, whereas little increase of GFP level was detected in HepG2/TK–GFP cells, indicating that the transcriptional activation of GFP gene is strictly under EpRE control.

Incubation of HepG2/EpRE–TK–GFP cells with allyl-ITC, benzyl-ITC, phenethyl-ITC or SF at 5 µM for 24 h resulted in 1.83-, 1.70-, 1.36- and 2.49-fold increases in GFP levels, respectively, whereas there were no detectable changes in GFP levels when HepG2/TK–GFP cells were incubated with the same ITCs under the same conditions (Table IIIGo), indicating again that the EpREs were essential in transcriptional activation of GFP by ITCs.


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Table III. Effect of isothiocyanates on green fluorescence protein and glutathione contents in recombinant HepG2 cells
 
We then measured the intracellular AUC of each ITC at 5 µM in HepG2/EpRE–TK–GFP cells after incubation for different times up to 24 h, as described in Materials and methods. The accumulation kinetics of the four ITCs differed significantly from each other (Figure 1Go, bottom). When the AUCs of intracellular accumulations of each ITC within 24 h were compared with induction of GFP (-fold increases), however, a very close correlation was seen (correlation coefficient of linear regression 0.98) (Figure 4Go), indicating that GFP induction is dependent on overall intracellular AUC of ITCs and such dependence is mediated by EpRE. This finding therefore suggests that the intracellular AUC-dependent induction of Phase 2 enzyme and elevation of GSH in PE cells and other cells is likely mediated by EpRE/ARE. Moreover, although induction of GST and QR by the ITCs in HepG2/EpRE–TK–GFP cells did not reach significance, intracellular GSH levels were raised by as much as 2.2-fold and these levels again closely correlated with the intracellular AUCs (correlation coefficient of linear regression 0.95) (Figure 4Go).



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Fig. 4. Correlation of total intracellular accumulation of isothiocyanate (AUC) within 24 h with its inducer activity in HepG2/EpRE–TK–GFP cells. The intracellular AUC of each ITC in HepG2/EpRE–TK–GFP cells (exposed to 5 µM ITC for 24 h) was calculated from the data shown in Figure 1Go (bottom) by the LAGRAN computer program (20). The specific GFP and GSH levels in cells treated with or without 5 µM ITC for 24 h were determined and the ratios of the specific GSH and GFP levels (treated cells/control cells) were calculated (see Table IIIGo). The intracellular AUCs of ITCs were then plotted against the ratios of the specific GFP and GSH levels: a, phenethyl-ITC; b, benzyl-ITC; c, allyl-ITC; d, SF.

 
Of note, the level of accumulation of ITCs in HepG2/EpRE–TK–GFP cells were lower than those found in PE cells and the peak levels were reached later, 0.5–3 h after the start of exposure compared with 0.5–1 h in PE cells. Moreover, the intracellular AUC of allyl-ITC was higher than those of benzyl-ITC and phenethyl-ITC in HepG2/EpRE–TK–GFP cells, in contrast to PE cells where intracellular AUC of allyl-ITC was lower than those of benzyl-ITC and phenethyl-ITC in PE cells.

Effect of multiple dosing of ITCs on elevation of both GFP and GSH in HepG2/EpRE–TK–GFP cells
Since multiple dosing of phenethyl-ITC enhanced the elevation of both GST and QR in PE cells, whereas multiple dosing with SF did not, we examined the effect of three doses of these ITCs on elevation of GFP and GSH in HepG2/EpRE–TK–GFP cells. Thus, the cell monolayers were incubated with either 5 µM phenethyl-ITC or SF and the medium was replaced with fresh medium containing the same ITC at the same concentration every 8 h for a total of 24 h (three times). As shown in Table IIIGo, we found that three doses of phenethyl-ITC increased levels of both GFP and GSH in the cells, but three doses of SF in 24 h did not, indicating that, similar to the effects in PE cells, induction by some ITCs with initially low activity may be enhanced by multiple dosing in a fixed time period. The above results also allowed us to calculate the intracellular AUC of ITCs within 24 h incubation required for maximal elevation of GFP and GSH at 155 (nmol/mg)xh, which is less than the value we measured in PE cells, suggesting that the AUC value required for maximal inducer activity of ITCs may differ among cell types.

Discussion

Many ITCs simultaneously elevate cellular GSH and several Phase 2 enzymes and thus have potential for significant protection of cells against a variety of toxic compounds. In the present study, GST, QR and GSH were coordinately elevated by the four common dietary ITCs, allyl-ITC, benzyl-ITC, phenethyl-ITC and SF, in PE cells. Similar results have also been reported with other ITCs as well as other types of inducers in a variety of cell lines and animal tissues. The degree of enzyme induction and GSH elevation, however, varies with cell line. For example, GST and QR were not significantly induced by ITCs in HepG2/EpRE–TK–GFP cells. The four ITCs studied, which have very different chemical structures, also have very different accumulation kinetics in PE cells. We found that elevation of GSH, GST and QR by the four ITCs in PE cells was closely dependent on their overall intracellular accumulation levels (AUC). In contrast, the maximal peak accumulation levels of ITCs did not determine their inducer activities. Similar correlations between the intracellular AUCs of the four ITCs and their activities in elevating GSH in the recombinant HepG2 cells (HepG2/EpRE–TK–GFP) were also found, although neither GST nor QR were significantly induced by the ITCs in these cells.

The correlation between the inducer activity of ITCs and their overall intracellular accumulation levels, AUCs, was further illustrated by phenethyl-ITC in a multiple dosing experiment. Phenethyl-ITC accumulated at a much lower level than SF, and the accumulated compound was also removed more rapidly than SF in both PE and HepG2/EpRE–TK–GFP cells. The induction of GST and QR in PE cells and GSH in HepG2/EpRE–TK–GFP cells by phenethyl-ITC was very low when the cells were incubated with 5 µM for 24 h. Induction was dramatically increased, with levels very close to those induced by SF, when the cells were incubated with fresh medium containing 5 µM phenethyl-ITC three times during the 24 h period. Similar replacement with SF, however, did not result in enhanced induction. It seems likely, therefore, that maximal induction of GST, QR and GSH might have been attained when the cells were treated with 5 µM SF for 24 h. Sulforaphane was previously found to be the most potent inducer among several dozen of ITCs tested (3,5).

The intracellular accumulation levels of ITCs in the present study were measured by the cyclocondensation assay, which detects both free ITCs and DTCs that result from conjugation of ITCs with sulfhydryl agents such as GSH. Hence the accumulated ITCs are likely to be a mixture of several chemical entities. As previously pointed out, however, both free ITCs and their DTC derivatives may be the ultimate inducer forms (12). Inducer activities of nearly all Phase 2 enzyme inducers are related to their chemical reactivity with sulfhydryl groups (24–26). It is known that both ITCs and their DTC derivatives can undergo reaction with sulfhydryl groups.

Whatever is the ultimate inducer form, much evidence indicates that transcriptional activation of Phase 2 enzymes by inducers are regulated by the ARE/EpRE located on the upstream region of the cognate genes and that such a mechanism operates across cell types (13,16,27). In the present study, by utilizing cells that were stably transfected with an EpRE driven reporter construct (EpRE–TK–GFP), we showed that the intracellular AUC-dependent elevation of Phase 2 enzymes and GSH by ITCs may be mediated by EpRE/ARE. This, together with the findings in PE cells and our previous finding in Hepa 1c1c7 cells (10), strongly suggests that the intracellular AUC-dependent induction of Phase 2 enzymes and elevation of GSH by ITCs is a common mechanism shared by a variety of cell types. We therefore propose that the overall intracellular accumulation (AUCs) levels of ITCs may serve as useful markers of their activity in elevating cellular detoxification capacity. If confirmed in vivo, these markers may provide valuable guidance in developing ITCs as cancer chemoprotective agents in humans. In fact, Conaway et al. (28) recently reported that higher levels of phenylhexyl-ITC in target organs in F344 rats, compared with that of phenethyl-ITC, correlated with its greater potency as a cancer chemopreventive agent. In conclusion, it appears that understanding of the intracellular ITC/DTC accumulation kinetics, as measured by the AUCs, is the key to developing rational dosing regimens for chemoprotection of humans by induction of Phase 2 enzymes and elevation of GSH.


    Notes
 
2 To whom correspondence should be addressed Email: yzhang{at}azcc.arizona.edu Back


    Acknowledgments
 
We would like to thank Drs Paul Talalay and Xiangqun Gao for their insightful comments on this work. We also wish to thank Dr Pamela Talalay for much editorial assistance in preparing this manuscript. These studies were supported by an RO1 grant (CA 80962) and a Program-Project grant (PO1 CA44530) from the National Cancer Institute, Department of Health and Human Services.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 References
 

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Received April 19, 2001; revised July 10, 2001; accepted July 20, 2001.