NUTRITION AND CANCER, 55(2), 210–223 Copyright © 2006, Lawrence Erlbaum Associates, Inc.
Induction of Phase II Enzyme Activity by Various Selenium Compounds Hang Xiao and Kirk L. Parkin
Abstract: Twenty-seven selenium compounds and sixteen structurally related organosulfur compounds were tested for quinone reductase (QR) and glutathione-S-transferase (GST) inducing activity in murine hepatoma (Hepa 1c1c7) cells. Sixteen selenium compounds were able to double QR activity, and seven of them also doubled GST activity. The nine most potent compounds, dimethyl diselenide, 2,5-diphenyl-selenophene, dibenzyl diselenide, methylseleninic acid, diphenyl diselenide, benzeneseleninic acid, benzene selenol, triphenylselenonium chloride, and ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), doubled QR-specific activity at levels lower than 7 mM. The concentration-dependence of QR induction and cell growth inhibition were linearly correlated (P < 0.001, r2 = 0.96) among the group of organoselenium compounds with putative selenol-generating potential, implying that both responses of Hepa 1c1c7 cells were based on these selenol metabolites.
Introduction Selenium compounds have been shown to inhibit the development of chemically induced carcinogenesis in animal models (1,2) and reduce total cancer incidence and mortality in a human dietary intervention trial (3,4). Selenium supplementation was found to significantly decrease cancer incidence of lung and prostate among individuals with low baseline selenium level (4,5). Selenium-enriched garlic (Segarlic) treatment effectively inhibited both initiation and post-initiation stages of dimethylbenz(a)anthracene (DMBA)-mediated mammary carcinogenesis in rats (6). Subsequent studies further revealed that prior administration of Se-garlic resulted in a consistent reduction of DMBA-DNA adducts detected after DMBA treatment in both mammary gland and liver (7,8) indicating that Se-garlic might diminish genotoxic effects by modulating in vivo DMBA metabolism. Se-Garlic treatment at 1, 2, or 3 ppm Se did not affect activities of general Phase I P450 enzymes but did enhance two major Phase II enzymes, GST and UDP-glucuronosyltransferase, by up to 2- to 2.5-fold in liver
and kidney in a dose-dependent fashion in rats (8). Selenium in Se-garlic exists principally as Se-methylselenocysteine (MSC) or γ-glutamyl-Se-methylselenocysteine, depending on degree of Se-enrichment (9). The candidate component(s) responsible for the induction of Phase II enzyme activities include MSC and/or γ-glutamyl-Se-methylselenocysteine, or metabolically transformed derivatives of these compounds, possibly enhanced by other chemicals in Se-garlic. Phase II enzyme induction has emerged as an important strategy for cancer chemoprevention (10). Structure-activity studies revealed that a wide array of compounds are capable of coordinately inducing Phase II enzymes, and they can be classified into nine categories (11): 1) diphenols, phenylenediamines, and quinones; 2) various Michael reaction acceptors; 3) isothiocyanates, dithiocarbamates, and related sulfur compounds; 4) 1,2-dithiole-3-thiones, oxathiolene oxides, and alk(en)yl (poly)sulfides; 5) hydroperoxides; 6) trivalent arsenicals; 7) heavy metals; 8) vicinal dimercaptans; and 9) carotenoids and related polyenes. A limited number of studies have focused on Phase II enzyme induction by (organo)selenium compounds. Recently, 1,4-phenylenebis(methylene)selenocyanate (p-XSC) and related selenium compounds were found capable of inducing Phase II enzyme activity, and GST in particular, in rodents (12–14). A subsequent study using cDNA microarrays further documented the inducing effect of p-XSC for different GST isoforms (15). Another organoselenium compound, ebselen, was examined in both rat liver epithelial (RL34) cells and the mouse skin model (16), and it exhibited a strong inducing capacity toward QR and GST. Moreover, the ability of ebselen to induce QR in RL34 cells was at a magnitude equivalent to that of ter-butylhydroquinone (tBHQ), a well-known inducer. Several selenocysteine (SeCys) Se-conjugates increased mRNA levels of GST α isoforms and GST Pi, but not of GST Mu isoforms in cultured primary rat hepatocytes and H35 Reuber rat hepatoma cells (17). Se-Allyl-L-selenocysteine was the most active GST inducer among the SeCys-conjugates tested. Both experimental studies in animal models and epidemiological studies have suggested protective roles of Allium
Both authors were affiliated with the Department of Food Science, University of Wisconsin-Madison, Madison WI 53706. H. Xiao is currently affiliated with the Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854.
vegetables, such as garlic and onions, against carcinogenesis (18,19), and it has been shown that organosulfur compounds (OSCs) are responsible for some of the chemoprotective effects of these vegetables (20–22). The ability of these OSCs to induce a battery of Phase II enzyme activities has been proposed as one mechanism of cancer chemoprevention (23,24). OSCs have been studied mainly in rodents, and the most studied OSCs are diallyl sulfide (DAS), diallyl disulfide (DADS), dipropyl sulfide (DPS), and dipropyl disulfide (DPDS) (25). The objective of this study was to systematically investigate Phase II enzyme activity induction (as one chemopreventive mechanism) by various selenium and related sulfur compounds (including those derived from Allium vegetables) with the intent of surveying for possible structure-function relationships. A total of 43 compounds were studied including well-known chemopreventive agents such as MSC, methylseleninic acid (MSeA), sodium selenite, selenomethionine, DAS, and DADS, as well as other structurally related compounds.
Experimental Procedures Materials Dimethyl diselenide (DMDSe), diphenyl diselenide, dibenzyl diselenide, MSeA, benzeneseleninic acid, sodium selenite, 2,1,3-benzoselenadiazole, benzeneselenol, allyl methyl sulfide, 1,1-dimethyl-2-selenourea, DADS, DMDS, propyl methyl disulfide, DAS, DPS, DPDS, propyl methyl sulfide, dimethyl trisulfide, benzyl trisulfide were purchased from Aldrich Chemical Co. (Milwaukee, WI). Sodium selenate, 2,5-diphenyl-selenophene, 4,4-dimethyl-2-(2(methylseleno)phenyl)-2-oxazoline, N,N′-diphenylselenourea, benzyl selenide, ebselen, allyl phenyl sulfide, allyl 4-methoxyphenyl sulfide, allyl ethyl sulfide, allyl 4-bromophenyl sulfide, S-methyl-cysteine, S-ethyl-cysteine, MSC, seleno-L-methionine, seleno-DL-methionine, selenocystamine and 3,3′-diselenodipropionic acid were from Sigma Chemical Co. (St. Louis, MO). Allyl phenyl selenide and Se-phenyl-L-selenocysteine were from Fluka (Milwaukee, WI). S-Allyl-L-cysteine was from TCI America (Portland, OR). Dimethyl selenide, diethyl selenide, diallyl selenide, diphenyl selenide, and triphenylselenonium chloride were from Organometallics, Inc. (East Hampstead, NH). α-Minimum essential medium (with L-glutamine, without ribonucleosides and deoxyribonucleoside; MEM), trypsinEDTA (0.25% trypsin with EDTA-4Na), fetal bovine serum (certified), and penicillin-streptomycin were from Gibco (Grand Island, NY).
Cell Culture Hepa 1c1c7 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA) for use in the Vol. 55, No. 2
“Prochaska” bioassay (11,26). Cells were cultured in 96-well plates at a density of 5,000 cells/well in 200 µl of α-MEM containing 10% fetal bovine serum (FBS), 100 U/ml of penicillin G, and 100 µg/ml of streptomycin. FBS was treated with activated charcoal (60°C, 90 min) to remove traces of endogenous QR inducers prior to use. After 24 h incubation at 37°C in a humidified atmosphere at 5% CO2 in air, the medium was replaced with 150 µl of fresh medium supplemented with test compounds, along with compound-free (non-induced) controls and cell-free controls. The plates were then incubated for another 48 h before the cells were lysed by replacing medium with 50 µl of saturated digitonin aqueous solution (in 2mM EDTA, pH 7.8) and then incubating at 37°C for 20 min with gentle shaking prior to measuring enzyme activity. For test compounds of limited aqueous solubility, the culture medium was supplemented with 0.1% of hydroxylpropyl β-cyclodextrin to facilitate dispersion. Also, some samples required dissolution in DMSO before adding to the medium, and in this case, final concentration of DMSO in medium was ≤0.5%. QR Activity Assay QR activity was measured using the menadione-coupled reduction of tetrazolium dye assay as modified from Prochaska et al. (26). A stock solution was prepared by mixing together 100 mg of BSA, 3.75 ml of 1 M Tris-Cl, 1 ml of 1.5% (wt/vol) Tween-20, 1 ml of 0.75 M FAD, 39 mg of D-glucose-6-phosphate, 3.5 mg of NADP, and 300 units of glucose-6-phosphate dehydrogenase and then brought to a final volume of 140 ml with H2O. Immediately prior to assay, 21 ml of the stock solution was mixed with 1.5 ml of 0.46% (wt/vol) 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 22.5 µl of 0.0861% (wt/vol) menadione (in acetonitrile). Two hundred microliters of this complete stock solution was added to each well containing cell lysates, and then the absorbance of the reduced tetrazolium dye was measured over a 10-min period using an optical microtiter plate scanner (SPECTRA MAX plus, Molecular Devices, Sunnyvale, CA) set at 490 nm. Glutathione S-Transferase Activity Assay GST activity was measured by a common method (27) adapted to 96-well plates. One hundred fifty microliters of stock solution containing 1.33 mM of 1-chloro-2, 4-dinitrobezene (CDNB), 1.33 mM of glutathione (reduced), and 100 mM PBS (pH 6.4) was added to each well containing cell lysates. The absorbance of the conjugate formed was recorded over a 10-min period using an optical microtiter plate scanner at 340 nm. Cell Viability Assessment A replicate plate prepared in the same manner as those for enzyme activity assays was used to evaluate the effect of test samples on cellular viability as quantified by cellular protein 211
levels that also allowed determination of enzyme-specific activity (26). At the end of the 48-h induction period, culture medium was decanted gently and 100 µl of 0.2% crystal violet (in 2% ethanol) was added to each well. After 10 min incubation at room temperature, the plate was rinsed thoroughly with lukewarm tap water to remove residual dye. The bound dye was then solublized by incubation (with agitation) at 37°C for 1 h with 150 µl of 0.5% (wt/vol) SDS in 50% (vol/vol) ethanol. The plate then was scanned at 610 nm using an optical microtiter plate scanner. Enzyme Activity and Cell Growth Inhibition Enzyme-specific activity was calculated by taking the ratio of enzyme activity and cell density (protein) derived from crystal violet staining assay. Inducing potency was determined by comparing enzyme-specific activity of cells subjected to the test compounds with that of the non-induced controls, the latter taken as 1.0. The concentration required for doubling the specific enzyme activity (CD value) was used as an indicator of inducer potency. To compare effects of test compounds on loss of cellular viability, the dose required to reduce cellular protein by 50% was calculated as the inhibitory concentration (IC50) as an indicator of cell growth inhibitory potency. Statistical Analysis Data are shown as means ± SD from at least three replicate experiments, each with replicated samples. The differences among treatment means were evaluated by a two-tailed Student’s t-test.
Results and Discussion Among 43 compounds tested (Fig. 1 and Table 1), twenty-four of them significantly elevated both QR- and GST-specific activity in Hepa 1c1c7 cells, and another ten compounds increased only QR-specific activity. Sixteen compounds were found capable of doubling QR-specific activity, and seven of them also doubled GST-specific activity. Nine compounds exhibited strong inducing potency toward QR with CD values ranging from 0.16 to 6.72 µM. All of them were organoselenium compounds: DMDSe, 2,5-diphenyl-selenophene, dibenzyl diselenide, MSeA, diphenyl diselenide, benzeneseleninic acid, benzene selenol, triphenylselenonium chloride, and ebselen. Induction by Methylated Selenium Compounds DMDSe was the most potent inducer among the compounds tested, and it doubled QR in Hepa 1c1c7 cells at 0.16 µM, a value that compares favorably to that reported for sulforaphane, one of the most potent naturally occurring inducers, at 0.2-0.8 µM (28). DMDSe also doubled GST at 212
0.39 µM, and QR and GST were elevated by DMDSe up to 5.33- and 2.41-fold, respectively, at levels near cell growth inhibitory dose of 0.57 µM (IC50 of 0.83 µM). In contrast, a sulfur analog of DMDSe, dimethyl disulfide (DMDS), and a mono-selenide version of DMDSe, dimethyl selenide, were inactive at inducing either QR or GST at concentrations up to 1,000 and 600 µM, respectively (Fig. 2). There is compelling evidence that a monomethylated selenium metabolite (CH3SeH, methylselenol) is responsible for the cancer chemopreventive action of selenium, rather than the element per se (29). As shown in Fig. 3, two of the most studied selenium compounds, sodium selenite and selenomethionine, are metabolized via different pathways to yield H2Se, which in turn can be transformed to CH3SeH by the action of methyl transferase (30). MSC, a lower homolog of selenomethionine, can be converted to CH3SeH by the action of cysteine conjugate β-lyases. MSeA can undergo facile reduction to CH3SeH by enzymatic and non-enzymatic pathways involving glutathione (GSH) and NADPH (31). In the presence of GSH and other thiols, DMDSe can be reduced to CH3SeH via the transient formation of selenenylsulfide intermediates (32, 33). The results of the present study on Phase II enzyme inducing patterns of methylated selenium compounds (Fig. 2) are consistent with the scheme shown in Fig. 3, and we propose that monomethylated metabolite (CH3SeH) plays a key role in Phase II induction by related methylated selenium compounds such as DMDSe, MSeA, MSC, selenomethionine, and dimethyl selenide. Once taken up by cells, both DMDSe and MSeA may be readily transformed into CH3SeH because of the availability of reducing equivalents from GSH, NADPH, and other thiols (31–33). DMDSe exhibited lower CD values for both QR and GST induction than MSeA, which may be due to the fact that two equivalents of CH3SeH can be theoretically generated from DMDSe, while only one equivalent can be produced from MSeA. The reason why inducing power of MSC was much less than DMDSe and MSeA in Hepa 1c1c7 cells may be that the rate of β–lyase dependent production of CH3SeH from MSC may be limited. An endogenous β–lyase from rat kidney has a fairly high Km value for MSC (29). There may also be limited β–lyase activity in cultured Hepa 1c1c7 cells compared to animal tissues, as the β–lyase-catalyzed β–elimination rates of several selenocysteine Se-conjugates, including MSC, in H35 Reuber rat hepatoma cells were found to be two- to sixfold lower than in cultured primary rat hepatocytes (17). Also, Ip et al. (34) indicated that MSeA produced more robust inhibition of cell accumulation and induction of apoptosis in TM12 and TM2H mouse mammary hyperplastic epithelial cells at one-tenth of the concentration of MSC. However, MSeA and MSC exhibit similar chemoprevention efficacies in rat mammary tumor models because animals have an ample capacity to metabolize MSC to CH3SeH. Thus, MSC may be expected to behave similarly as MSeA if β–lyase is not a limiting factor in processes yielding CH3SeH. It would be of interest to test whether using in vivo animal Nutrition and Cancer 2006
Figure 1. Structures of selenium and sulfur compounds tested for Phase II enzyme activity induction.
models would diminish the difference in extents of Phase II enzyme induction between MSC (β–lyase-dependent) and MSeA (β–lyase-independent). In Hepa 1c1c7 cells, both seleno-L-methionine and seleno-DL-methionine induced QR activity marginally and had no effect on GST activity, which indicated weak inducing abilities relative to that of the lower homolog, MSC. This may be associated with 1) inefficiency of Hepa 1c1c7 cells in Vol. 55, No. 2
carrying out multistep metabolism of selenomethionine to yield CH3SeH (Fig. 3) and 2) possible nonspecific channeling of selenomethionine into the protein pool. However, in vivo, selenium-enriched yeast (primarily as selenomethionine) can increase hepatic GST and QR-specific activity in Sprague-Dawley rats (35), possibly indicating a greater ability to transform selenomethionine into CH3SeH than in cultured Hepatoma cells. 213
Figure 1. (Continued)
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Figure 1. (Continued)
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216 Table 1. Quinone Reductase (QR) and Glutathione-S-Transferase (GST) Inducing Potency of Selenium and Sulfur Compoundsa Quinone Reductase
Test Compounds Dimethyl selenide Diethyl selenide Diallyl selenide Diphenyl selenide Allyl phenyl selenide Benzyl selenide Dimethyl diselenide Dibenzyl diselenide Diphenyl diselenide Selenocystamine 3,3’-Diselenodipropionic acid Sodium selenite Sodium selenate N,N’-Diphenyl selenourea Methylseleninic acid Benzeneseleninic acid Ebselen 2,5-Diphenyl-selenophene 2,1,3-Benzoselenadiazole 4,4-Dimethyl-2-(2-(methyseleno)phenyl)-2 oxazoline 1,1-Dimethyl-2-selenourea
Dose Range (µM) 1.19–611 0.61–310 0.176–90 2.32–1,187 2.33–1,192 1.08–555 0.072–38.3 0.06–32 0.21–108 0.4–200 0.4–200 0.055–28.3 6.51–3,334 1.05–536 0.02–9.59 0.35–180 0.71–365 0.12–60 2.43–1,243 1.09–559 2.42–1,238
IC50 (µM) >611 >310 67 ± 4 276 ± 15 516 ± 2 160 ± 20 0.83 ± 0.10 5.6 ± 0.11 33.3 ± 4.4 9.39 ± 0.25 67.4 ± 5 6.03 ± 0.77 334 ± 23 49 ± 3 3.6 ± 0.39 37.3 ± 5.7 155 ± 22 47 ± 3.6 690 ± 59 193 ± 3 261 ± 30
Imax Mean ± SD NS NS NS NS 2.32 ± 0.18 1.98 ± 0.34 5.33 ± 0.23 5.29 ± 0.48 4.53 ± 0.61 1.23 ± 0.15 2.02 ± 0.3 NS 1.48 ± 0.12 2.57 ± 0.13 4.67 ± 0.44 4.87 ± 0.15 4.32 ± 0.85 8.8 ± 1.4 2.76 ± 0.03 6.24 ± 0.25 2.33 ± 0.38
CImax (µM) NS NS NS NS 298 139 0.57 4.04 27 6.25 50 NS 208 33 2.40 22 45.6 31 621 140 155
Glutathione S-Transferase CD (µM) Mean ± SD
Imax Mean ± SD
CImax (µM)
CD (µM) Mean ± SD
NA NA NA NA 246 ± 36 NA 0.16 ± 0.007 0.57 ± 0.04 1.50 ± 0.16 NA 50 NA NA 22 ± 0.6 0.79 ± 0.07 2.33 ± 0.23 6.72 ± 0.43 0.36 ± 0.09 492 ± 28 41 ± 1.9 109 ± 17
NS NS NS NS 1.30 ± 0.06 1.70 ± 0.01 2.41 ± 0.18 2.61 ± 0.35 2.25 ± 0.43 1.79 ± 0.27 NS NS NS 1.57 ±0.03 2.23 ± 0.03 1.95 ± 0.38 1.59 ± 0.07 2.05 ± 0.39 3.31 ± 0.20 2.91 ± 0.05 1.43 ± 0.08
NS NS NS NS 298 139 0.57 4.04 6.74 6.25 NS NS NS 33 2.40 11.2 23 15.45 621 140 155
NA NA NA NA NA NA 0.39 ± 0.075 2.02 ± 0.25 3.37 ± 0.57 NA NA NA NA NA 1.97 ± 0.37 NA NA 7.73 ± 1.06 310 ± 12 69 ± 3 NA
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Vol. 55, No. 2 Benzene selenol Triphenylselenonium chloride Se-Phenyl-L-selenocysteine Seleno-D,L-methionine Seleno-L-methionine Se-Methyl-selenocysteine Allyl-4-bromophenyl sulfide Allyl 4-methoxyphenyl sulfide Allyl phenyl sulfide Diallyl disulfide Allyl ethyl sulfide Allyl methyl sulfide Dimethyl trisulfide Benzyl trisulfide Diallyl sulfide Propyl methyl disulfide Dipropyl disulfide Dimethyl disulfide Propyl methyl sulfide S-Methyl-cysteine S-Ethyl-cysteine S-Allyl-cysteine
0.6–300 0.54–279 2.56–1,311 6.08–3,110 5.98–3,069 7.06–3,616 1.79–916 2.17–1,109 2.60–1,331 6.14–1,572 4.2–2,153 3.32–1,701 6.03–3,089 1.75–898 2.22–1,138 2–1,000 2–1,000 2–1,000 2–1,000 50–25,000 50–25,000 50–25,000
64 ± 8.7 7.7 ± 0.5 >1,311 139 ± 14 91 ± 8 705 ± 68 576 ± 21 404 ± 10 649 ± 92 150 ± 26 >2,153 364 ± 14 44 ± 1 2.56 ± 0.24 66 ± 10 >1,000 386 ± 27 >1,000 >1,000 22,833 ± 644 5,257 ± 297 1,388 ± 182
4.0 ± 0.71 2.06 ± 0.09 4.71 ± 0.01 1.16 ± 0.06 1.11 ± 0.03 2.73 ± 0.35 1.76 ± 0.52 1.40 ± 0.06 1.92 ± 0.44 1.83 ± 0.27 NS 1.58 ± 0.37 NS 1.24 ± 0.28 1.35 ± 0.22 NS 1.34 ± 0.03 1.33 ± 0.08 NS 1.59 ± 0.16 1.41 ± 0.14 1.88 ± 0.03
37 4.35 1,311 97 48 452 229 277 665 98 NS 106 NS 1.75 35 NS 125 125 NS 1,2204 3,142 1,314
3.35 ± 0.53 4.13 ± 0.23 256 ± 34 NA NA 310 ± 30 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA
1.87 ± 0.42 1.57 ± 0.02 1.70 ± 0.02 NS NS 1.92 ± 0.35 1.49 ± 0.04 1.42 ± 0.28 1.56 ± 0.04 1.77 ± 0.55 NS NS NS NS NS NS 1.21 ± 0.03 1.14 ± 0.08 NS NS NS NS
9.33 4.35 655 NS NS 452 57 277 333 98 NS NS NS NS NS NS 125 125 NS NS NS NS
NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA
a: IC50, concentration where cell survival (protein) is reduced by ³ 50%; Imax, maximum induction ratio; CImax, concentration for maximum induction; CD, concentration required to double enzyme specific activity; CImax, and CD are reported as means ± standard deviation from at least three replicates. All values were significantly different (P < 0.01) from the controls by a two-tailed Student’s t-test. NS, not statistically different from non-induced control; NA, not appropriate; underlined compounds are potent inducers with CD values lower than 7 µM.
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cells, and this may account for differences in behavior observed in the present study. Induction by Aromatic Selenium Compounds
Figure 2. Quinone reductase (QR) induction by methylated selenium compounds. Culture and assay conditions are specified in Experimental Procedures. The results represent the mean values ± SD from at least three replicates. Dimethyl disulfide was included for comparison. MSC, Se-methylselenocysteine.
Both dimethyl selenide and sodium selenite were not capable of QR and GST induction in Hepa 1c1c7 cells. Dimethyl selenide is the methylation product of CH3SeH and may not be subject to the reverse transformation to yield the putative bioactive derivative in Hepa cells. Production of CH3SeH from sodium selenite, although possible in vivo by methylation (Fig. 3), may not take place sufficiently enough to produce enzyme-inducing effects in Hepa cells. In prostate cancer, leukemia, mammary cancer, and vascular endothelial cells, sodium selenite induces different biochemical and cellular responses relative to more immediate precursors of CH3SeH, such as MeSeA (36). It was suggested that CH3SeH precursors and sodium selenite comprise different selenium metabolite pools in cultured
As shown in Fig. 4, among five phenylated selenium compounds, diphenyl diselenide, benzeneseleninic acid, and benzene selenol were potent inducers of QR activity, with CD values of 1.50, 2.33, and 3.35 µM, respectively (Table 1). Se-Phenyl-L-cysteine doubled QR-specific activity at 256 µM and elevated QR-specific activity up to 4.71-fold at about 1,300 µM, indicating that Se-phenyl-L-cysteine was a weaker inducer than diphenyl diselenide, benzeneseleninic acid, and benzene selenol by about two orders of magnitude. Diphenyl selenide was inactive as an inducer at concentrations up to its IC50 (276 µM). From our results in Hepa 1c1c7 cells, the structure-activity relationship of these five phenylated selenium compounds in Phase II enzyme induction appears similar to that of the methylated selenium compounds discussed in the previous section, where the selenol derivative seems to play an important role in Phase II enzyme induction. Diselenides can oxidatively mediate the transformation of GSH to glutathione disulfide and produce two equivalents of selenol (32). Similarly, diphenyl diselenide can readily form benzene selenolate (ionized form of benzene selenol) in the presence of excess of GSH (37). In this study, diphenyl diselenide, which can yield two molar equivalents of selenol, was the strongest inducer among these five phenylated selenium compounds, with a CD value 1.6-fold (P < 0.001) lower than that of benzeneseleninic acid, which can only generate one equivalent of selenol by reduction. As a selenol, benzene selenol had a CD value comparable to that of benzeneseleninic acid. A series of Se-substituted
Figure 3. Scheme of methylselenol formation (compiled from Refs. 28–31).
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Figure 4. Quinone reductase (QR) induction by phenylated selenium compounds. Culture and assay conditions are specified in Experimental Procedures. The results represent the mean values ± SD from at least three replicates.
Figure 5. Quinone reductase (QR) induction by phenmethylated selenium compounds. Culture and assay conditions are specified in Experimental Procedures. The results represent the mean values ± SD from at least three replicates.
selenocysteine conjugates, including Se-phenyl-L-cysteine, were found to be substrates for β-lyases from H35 Reuber rat hepatoma cells, primary rat hepatocytes (17) and rat renal cytosol (38), and such biotransformation gives rise to the corresponding selenols. In Hepa 1c1c7 cells, Se-phenyl-Lcysteine exhibited a CD value (for QR) close to that of MSC (256 vs. 310 µM), which is consistent with the observation that Se-phenyl-L-cysteine had a similar or higher reaction rate with β-lyases than MSC (17,38). Similar to the case for MSC and related methylated selenium compounds discussed in the previous section, the lesser inducing potency of Se-phenyl-L-cysteine, compared to other β-lyase-independent selenol-generating phenylated selenium compounds such as diphenyl diselenide and benzeneseleninic acid, was likely associated with a lower capacity of Hepa 1c1c7 cells to transform this compound to the respective selenol. Vol. 55, No. 2
Another pair of selenium compounds, dibenzyl diselenide and benzyl selenide, were compared for their QR-inducing potencies (Fig. 5). Not surprisingly, the potential selenol-generating dibenzyl diselenide was the superior inducer with a CD value as low as 0.79 µM, whereas benzyl selenide had no QR-inducing effect at concentrations ranging from 1–70 µM. Several other selenium compounds were also tested for Phase II enzyme induction (Fig. 6). Ebselen, triphenylselenonium chloride, and 2,5-diphenylselenophene exhibited potent QR-inducing activities with CD values lower than 7 µM, while N,N′-diphenyl selenourea, 4,4-dimethyl-2-(2-(methyseleno)phenyl)-2 oxazoline 1,1-dimethyl-2-selenourea, and 2,1,3-benzoselenadiazole were found to double QR-specific activity at greater concentrations of 17, 27, 109, and 310 µM, respectively. Ebselen is known as an antioxidant (16), and it was investigated as a neuroprotective agent in a clinical trial (39). In Hepa 1c1c7 cells, ebselen doubled QR-specific activity at 6.72 µM and had an IC50 of 155 µM, which is consistent with the previous finding that ebselen was able to double QR in rat liver epithelial RL34 cells at concentration between 5–25 µM (16). Ebselen can react with glutathione to yield an ebselen selenenylsulfide, which, in turn, is converted to ebselen selenol and ebselen diselenide in the presence of excess glutathione (40,41). Recently, ebselen selenol was shown to be generated from ebselen via reductions by mammalian thioredoxin reductase (TrxR) and thioredoxin (Trx) (42). Further reaction between ebselen and its selenol gives rise to ebselen diselenide that is also a substrate of TrxR to form ebselen selenol (43). Triphenylselenonium chloride is a cation with three benzene rings directly bonded to a selenium atom (Fig. 1), and several studies have demonstrated its chemopreventive effects in rat mammary tumor models (44–46). Triphenylselenonium is resistant to metabolism (34), and there is no indication of metabolic generation of selenol from this compound. Triphenylselenonium significantly induced both QR- and GST-specific activity in Hepa 1c1c7 cells, and QR-specific activity was doubled at 4.35 µM where only about 60% cell viability was maintained (IC50 of 7.7 µM). Ip et al. (46) indicated that the maximum tolerable doses (added to the diet of rats) for diphenyl selenide and triphenylselenonium were 30 and >200 ppm Se, respectively. In contrast, the present results showed less growth inhibition by diphenyl selenide (IC50: 276 µM) in Hepa 1c1c7 cells than triphenylselenonium (IC50: 7.7 µM). This discrepancy may be explained by the fact that in the animal model, triphenylselenonium has poor intestinal absorption and most of it is excreted through feces while a large proportion of diphenyl selenide is absorbed (45), while both compounds may be quite available for cell uptake in the cell culture model. Relative to diphenyl selenide, triphenylselenonium is both lipophilic and amphiphilic. The combined structural elements of lipophilicity and cationic Se are viewed as being important factors to afford tissue uptake and chemopreventive activitity (44–46). Chemopreventive effects of triphenylselenonium appear to be mechanistically different 219
Figure 6. Quinone reductase (QR) induction by other selenium compounds. Culture and assay conditions are specified in Experimental Procedures. The results represent the mean values ± SD from at least three replicates.
than other organoselenium compounds, including selenol generators, in part because Se in triphenylselenonium is not bioavailable. Another potent Phase II enzyme inducer was found to be 2,5-diphenyl-selenophene that was capable of inducing QRand GST-pecific activity up to 8.8- and 2.05-fold, respectively. The QR-inducing capacity of 2,5-diphenyl-selenophene was of similar potency as sulforaphane, dibenzyl diselenide, and DMDSe, but it was less growth inhibitory to Hepa 1c1c7 cells. The IC50 of 2,5-diphenyl-selenophene was 4.7-, 8.4-, and 57-times greater than that of sulforaphane, dibenzyl diselenide, and DMDSe, respectively (47 vs. 10, 5.6, and 0.82 µM). These findings suggest that 2,5-diphenyl-selenophene may be a good candidate as a chemopreventive agent, having a broad margin between the effective dose range and the growth inhibitory dose range (IC50/CD = 131) based on the Hepa cellular bioassay. To our knowledge, there is no report on the biological activity of 2,5-diphenyl-selenophene, and it would be of interest to investigate possible mechanisms for Phase II enzyme induction and other potentially related biological activities of this compound.
Induction by Organosulfur Compounds Sixteen organosulfur compounds were tested in Hepa 1c1c7 cells for their inducing potencies, and they included mono-, di-, and tri-sulfides (both aliphatic and aromatic), as well as three cysteine derivatives: S-methyl-, S-ethyl-, and S-allyl-cysteine (Table 1). None of these organosulfur compounds was able to double QR activity at concentration ranges up to their respective IC50 values. In contrast, more than 20 in vivo studies have suggested that some Allium-derived sulfides, including DAS and DADS tested in this study, are potent Phase II enzyme inducers in rodents (25). However, most of those experiments employed dosing levels above what could be considered plausible for common human intakes and may be of limited relevance to human dietary exposures. The inductive activity of organosulfides in 220
vivo may be conferred through specific metabolic transformations that are not incumbent to Hepa 1c1c7 cells. In vivo studies on monosulfides with rats indicated metabolic conversion of DAS (47) and DPS (48) to the sulfoxide and sulfone. For disulfides, DADS was found to be reduced to allyl mercaptan (allyl-SH) in an isolated perfused rat liver (49) and oxidized to diallyl thiosulfinate (allicin) in rat or human liver microsomes (50). DPDS is oxidized to dipropyl thiosulfinate in rat liver microsomes, whereas it is transformed to propylglutathione sulfide and propyl mercaptan by liver cytosol, and to propyl mercaptan, propylglutathione sulfide, methylpropyl sulfide, and methylpropyl sulfone in an isolated perfused rat liver (51). Benzyl trisulfide and dimethyl trisulfide were the two most cell growth inhibitory agents to Hepa 1c1c7 cells among all sulfides tested. Alkyl trisulfides are known to generate reactive oxygen species (ROS) and cause oxidative damage to erythrocytes in vitro to a greater extent than diand mono-sulfides (52). In contrast to DMDSe, a potent Phase II enzyme inducer with a CD value of 0.16 µM, DMDS failed to double QR-specific activity at concentrations up to 1,000 µM, although the disulfide is believed to be capable of generating the respective mercaptan (RSH), the sulfur analog of selenol, by thiol-disulfide exchange with GSH (53). This may reinforce the key and specific role of the Se atom in selenol structures for their various biological activities. S-Methyl-, S-ethyl-, and S-allyl-cysteine induced QR activity weakly at near IC50 doses, and relative cell growth inhibitory potency was S-allyl- > S-ethyl- > S-methyl-cysteine, based on the ratio of IC50 values of 1:3.8:16.5, respectively. It was shown that SeCys conjugates, such as Se-methyl-Lselenocysteine and Se-phenyl-L-selenocysteine, were better substrates for rat renal cytosolic β-lyases than their sulfur analogs, such as S-ethyl-L-cysteine and S-phenyl-L-cysteine, the latter which were such poor substrates that no appropriate enzyme kinetic parameters were experimentally obtained (38). This finding suggests that differences between Se and S atoms may dictate the fate and bioactivities of cysteine conjugates in biological milieu. Correlation Between CD and IC50 Values Among Putative Selenol-Generating Selenium Compounds Among the nine potent Phase II enzyme inducers capable of doubling QR-specific activity at concentrations lower than 7 µM, seven of them are selenol or potential selenol-generating compounds, including DMDSe, MSeA, diphenyl diselenide, benzeneseleninic acid, benzeneselenol, dibenzyl diselenide, and ebselen. A correlation was found (r = 0.98, P < 0.001) between the Phase II enzyme inducing potency (quantified by CD value) and cell growth inhibitory potency (quantified by IC50) of these compounds (Fig. 7). Inclusion of the other two potent inducers, triphenylselenonium chloride and 2,5-diphenyl-selenophene, in the correlation plot resulted in a substantial decrease in correlation coefficient (from 0.98 to 0.72), which suggested triphenylselenonium chloride and Nutrition and Cancer 2006
more than four-times stronger QR-inducing and cell growth inhibitory activity than did ebselen in the Hepa 1c1c7 cell line. We also tested Phase II enzyme induction activity of two other diselenides, selenocystamine and 3,3′-diselenodipropionic acid, which showed weaker QR-inducing activity relative to dimethyl, dibenzyl, and diphenyl diselenides (Table 1), although both of the former compounds have been reported to undergo reaction with GSH and produce selenol (32,33). These results may indicate that structural differences among potential selenol-generating compounds (e.g., different R groups in diselenides, RSeSeR) profoundly influence their intracellular metabolism and transformation as well as reactivity of the respective selenols generated. This, in turn, may modulate their relative biological activities as Phase II enzyme inducers and cell growth inhibitors in cells. Figure 7. Correlation between the concentration required for doubling the specific enzyme activity (CD) and the dose required to reduce cellular protein by 50% (IC50) of selenol-generating selenium compounds. CD values for quinone reductase (QR) induction by seven putative selenol-generating compounds were correlated with their respective IC50s. Correlation coefficient was 0.98 (P < 0.001). Two potent inducers, triphenylselenonium chloride and 2,5-diphenyl selenophene, are included for comparison.
2,5-diphenyl-selenophene may not adhere to the same relationship between Phase II enzyme induction and cell growth inhibition as did the putative selenol-generating compounds. This correlation between CD and IC50 may imply that there is/are commonly shared mechanism(s) underlying the mode of actions of these compounds as Phase II enzyme inducers and growth inhibitors in Hepa 1c1c7 cells, and that a single active agent is responsible for both activities. The present results support the premise that Phase II enzyme induction and cell growth inhibition of selenium compounds are attributable to selenol-generating processes in Hepa 1c1c7 cells. Methylselenol (CH3SeH), derived from direct reduction of DMDSe, is highly reactive and cannot be isolated (29), while other selenols, such as benzeneselenol and ebselen selenol, are sufficiently stable to permit their isolation. DMDSe was the most potent Phase II enzyme inducer and cell growth inhibitor in this study. Sinha et al. (54) showed that DMDSe and MSeA (but not DMDS or dimethyl selenoxide, neither of which give rise to CH3SeH) similarly inhibited growth of synchronized mouse mammary epithelial tumor cells at 5 µM Se concentration (DMDSe:MSeA = 1:2), which provided experimental support for the hypothesis that CH3SeH is a critical affector molecule in Se-mediated growth inhibition in vitro. Cotgreave et al. (41) and Engman et al. (37) studied selenol-generation in the presence of excess of GSH from ebselen and diphenyl diselenide, respectively. Comparison of their results revealed that under similar conditions the initial rate of formation of ebselen selenol was about half that of benzeneselenol. Moreover, only 10–20% ebselen was transformed to ebselen selenol while 96% of diphenyl diselenide was reduced to benzeneselenol by GSH. This may explain, in part, why diphenyl diselenide showed Vol. 55, No. 2
Conclusions Potent inductive activity toward Phase II enzymes by the selenium compounds studied herein suggests a new category of compounds, in addition to those nine categories summarized by Dinkova-Kostova et al. (11), as Phase II enzyme inducers. In addition, further experimental support was provided for cancer chemopreventive roles of (organo)selenium compounds. It seems evident that selenol derivatives of these compounds are the putative active agents that induce Phase II enzymes. Metabolic formation rate and substituent structures of the respective selenols may account for differences in inducing potency of parent selenium compounds. The correlation between Phase II enzyme induction potency and cell growth inhibitory potency of putative selenol-generating compounds implied possible shared common mechanism(s) for both activities. The exact molecular mechanism involved in the induction of Phase II enzymes by these selenium compounds remains to be explored. It is important to understand the fate of structurally diverse selenols under physiological conditions and their reactivity toward potential molecular targets associated with Phase II enzyme induction to account for their bioactive effects.
Acknowledgments and Notes This work was supported by the College of Agricultural and Life Sciences of the University of Wisconsin–Madison and by a grant from the United States Department of Agriculture (NRI-CGP #2004–35503–14131). Address correspondence to K.L. Parkin, Department of Food Science, University of Wisconsin, 1605 Linden Drive, Madison, WI 53702. Phone: 608–263–2011, FAX: (608)-262–6872. E-mail:
[email protected]. Submitted 27 October 2005; accepted in final form 8 May 2006.
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