Selective expression of glutathione S-transferase genes in the murine gastrointestinal tract in response to dietary organosulfur compounds
John H. Andorfer,
Tatyana Tchaikovskaya and
Irving Listowsky1
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA
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Abstract
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A short-term feeding regimen was designed to analyze the effects of compounds such as diallyl disulfide (DADS), diallylthiosulfinate (allicin) from garlic and butylated hydroxyanisole (BHA) on glutathione S-transferase (GST) expression in the gastrointestinal tract and liver of male mice. After animals were force-fed these compounds, tissue GSTs were purified and individual subunits resolved by HPLC and identified on the basis of mass spectrometry (ESI MS) and immunoreactivity data. The effects of DADS and allicin on GST expression were especially prominent in stomach and small intestine, where there were major coordinate changes in GST subunit profiles. In particular, the transcripts of the mGSTM1 and mGSTM4 genes, which share large segments of common 5'-flanking sequences, and their corresponding subunits were selectively induced. Levels of
class subunits also increased, whereas mGSTM3 and mGSTP1 were not affected. The inducible mGSTA5 and non-responsive mGSTM3 subunits had not been identified previously. Liver and colon GSTs were also affected to a lesser extent, but this short-term feeding regimen had no effect on GST subunit patterns from other organs, including heart, brain and testis. Real-time PCR (TaqMan) methods were used for quantitative estimations of relative amounts of the mRNAs encoding the GSTs. Effects on the transcripts generally paralleled changes at the protein level, for the most part, however, the greatest relative increases were observed for those mRNAs that were expressed at low abundance constituitively. Mechanisms by which the organosulfur compounds operate to affect GST transcription could involve reversible modification of certain protein sulfhydryl groups, shifts in reduced glutathione/oxidized glutathione ratios and resultant changes in cellular redox status.
Abbreviations: ARE, antioxidant-responsive element; BHA, butylated hydoxyanisole; CDNB, 1-chloro 2,4-dinitrobenzene; DADS, diallyl disulfide; GAPDH, glyceraldehyde phosphate dehydrogenase; GSH, glutathione; GST, glutathione S-transferase; MALDI, matrix-assisted laser desorption ionization spectrometry; TFA, trifluoroacetic acid
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Introduction
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Some naturally occurring dietary agents, particularly non-nutrient phytochemicals, have been shown to exhibit chemopreventive/chemoprotective effects against development of cancer (14). In that regard, several lines of evidence have linked consumption of organosulfur compounds from garlic with chemoprotection against cancer and certain degenerative diseases (5,6). It has been suggested that organosulfur compounds such as allicin and diallyl disulfide (DADS) modify signal transduction pathways, induce apoptosis and suppress tumor cell growth (715). Antioxidants such as butylated hydroxyanisole (BHA) have also been shown to have anticarcinogenic effects (16). These types of compounds are known to activate transcription of phase II drug metabolizing enzymes, including glutathione S-transferases (GSTs) (24,17).
Mammalian GSTs are products of gene superfamilies that are expressed in a tissue-specific manner (1821). They have been subdivided into at least seven categories based on sequence homologies, dimeric subunit assembly patterns and other common properties, of which the
, µ,
and
forms are usually the most abundant (20). GSTs allow cells to adapt to various types of noxious agents by catalyzing nucleophilic addition or substitution reactions between glutathione (GSH) and reactive electrophilic compounds and by reduction of organic hydroperoxides generated from reactive oxygen species (18,20,2224). GSTs are also intracellular stoichiometric binding proteins for various non-substrate ligands (25). The multiple forms are characterized by discrete differences in substrate specificities and binding properties (20). For instance, stable transfection of the mouse GSTA3 subunit to cells confers protection against aflatoxin B1 mutagenicity (26). The mGSTA4 subunit exhibits selective activities against toxic products of lipid peroxidation, including 4-hydroxy-nonenal (27).
Some classes of GST-inducing compounds activate transcription of GST genes directly and some require further metabolic activation in order to function. The inducing agents are considered to be monofunctional if they increase expression of only phase II drug-metabolizing enzymes and NAD(P)H-quinone oxidoreductase but not cytochrome P450 s, and others are bifunctional in that they also induce phase I mixed function oxygenases (cytochrome P450 s) (28). Because GSTs are considered to act as cellular chemoprotective proteins by functioning in the direct detoxification of reactive substances, including some carcinogens, some strategies for dietary intervention for the chemoprevention of various diseases include manipulation of GST systems (2932). In the present study, differential effects of dietary organosulfur compounds on the family of GSTs in mouse tissues are analyzed and molecular mechanisms by which they function are considered.
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Materials and methods
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Materials
DADS was obtained from LKT Laboratories (St Paul, MN), BHA from ICN (Costa Mesa, CA), 1-chloro 2,4-dinitrobenzene (CDNB) and epoxy-GSH affinity resins from Sigma-Aldrich (St Louis, MO) and other reagents were of high purity. Allicin was a gift from Drs Meir Wilchek and Talia Miron (Weizmann Institute of Science, Rehovot, Israel). Peptide sequence-specific antisera prepared in our laboratories were used to distinguish among different subclasses of GST (3335).
Dietary regimen
Cohorts of 67-week-old C57BL/6 male mice (Charles River Laboratories, Wilmington, MA) were fed a laboratory chow diet (505A; Purina Mills, St Louis, MO) ad libitum. Groups of at least three mice were force-fed either DADS (20 µmol/animal) or BHA (30 µmol) by gavage. The DADS was mixed with corn oil for delivery as a 200 µl bolus. The animals were given a second dose 24 h later. Some control animals were given corn oil alone, but no changes in GST patterns were observed between those animals as compared with animals fed laboratory chow. These pharmacological doses were well tolerated by the mice, whereas toxicity was observed after administration of 100 and 200 µmol DADS, some animals not surviving at the higher doses. For comparison, mice were maintained on a semi-purified diet that contained no antioxidants or other preservatives (AIN-76; Purina Mills), for 4 weeks prior to treatment. However, animals maintained on laboratory chow diets tolerated the force-feeding procedure and higher doses of DADS without the serious adverse effects experienced by some animals on the AIN-76 diet. Accordingly, laboratory chow was the diet chosen for comprehensive studies to determine the effects of DADS and other dietary supplements on GST expression. Another group of older animals (4 males, 20 weeks old) maintained on laboratory chow were force-fed two doses of allicin (2.2 µmol) in citrate buffer pH 5.0. All animals were killed by CO2 asphyxiation 24 h after the experimental group received the second dose of compound; the major organs were removed, rinsed in ice-cold 1xphosphate-buffered saline, snap frozen on dry ice and stored at -70°C.
GST purification
Homogenates were prepared by disrupting the tissue with a rotor-stator homogenizer in loading buffer (10 mM TrisHCl pH 7.9, 10 mM NaCl, 1 mM DTT) at 4°C. The homogenates were centrifuged at 35 000 g for 1 h at 4°C and the cytosolic fractions were used for GST purification. Protein concentrations were determined by a modification of the Bradford assay (Cytoskeleton Inc., Denver, CO) and GST enzymatic activities were determined using CDNB as substrate (36). Cytosolic GSTs were purified using epoxy-linked GSHagarose (Sigma-Aldrich, St Louis, MO) affinity chromatography. The affinity columns (2 ml gel volume) were loaded with cytosolic extracts and washed with
50 column volumes of loading buffer. The GSTs were eluted with 30 ml of elution buffer (50 mM TrisHCl pH 9.6, 10 mM GSH, 0.1 mM dithiothreitol). The eluted GSTs were concentrated to 1 ml using 10K MWCO Ultrafree Centrifugal Filter devices (Millipore, Bedford, MA). The GSH concentration was reduced by dilution and refiltration in elution buffer (minus GSH).
Gel electrophoresis
Tissue cytosolic extracts (40 µg protein) were mixed with 2x SDS loading buffer (10% SDS, 5% ß-mercaptoethanol) and resolved in 12% Trisglycine SDSPAGE minigels (Invitrogen, Carlsbad, CA). Duplicate gels were obtained for staining and for immunoblots. Proteins were transferred to nitrocellulose membranes (Invitrogen) and the membranes probed with peptide sequence-specific antisera raised against various GSTs (33,34). The GSTs were detected using horseradish peroxidase-conjugated anti-rabbit secondary antibody (Sigma). Loading amounts were normalized using ß-actin as a control (Stressgen, San Diego, CA), with a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Southern Biotechnology Associates, Birmingham, AL).
HPLC
Purified GSTs (150 µg) obtained from GSHagarose affinity columns for each treatment were resolved by HPLC on an analytical C4 (0.46 x 25 cm) reversed phase column (Grace Vydac, Hesperia, CA) using an HP-1090 instrument (Hewlett Packard, Palo Alto, CA). Proteins were eluted with linear gradients of buffer B [0.1% trifluoroacetic acid (TFA) in acetonitrile] at a flow rate of 0.7 ml/min in two phases (2%/min, 010 min; 0.5%/min, 1050 min). Fractions were collected and peaks detected by absorbance at 214 nm were classified by peptide-specific antibody recognition and identified by electrospray ionization mass spectrometry (ESI MS) using a quadrapole ion-trap mass spectrometer (LCQ-Finnigan Corp., San Jose, CA) (35). Each subunit was quantitated by measuring the area under its peak relative to the total area and known amounts of loaded protein.
Real-time PCR
RNA was purified from 2030 mg of the different tissues (RNeasy Mini Kit; Qiagen, Valencia, CA) and concentrations were estimated from absorption values at 260 nm. The isolated total RNA (1.5 µg/20 µl reaction volume) was used for first strand cDNA synthesis (SuperScript II; Invitrogen). Relative mRNA levels of the various GSTs and differences between control and treated animals were measured using TaqMan® chemistry (ABI Prism 7000 Sequence Detection System; Applied Biosystems, Foster City, CA). Each TaqMan® assay was performed in triplicate for each gene, tissue and treatment combination. Primers (Invitrogen) and TaqMan probes (Qiagen Operon) designed using Primer Express software (Applied Biosystems) or those previously published (37) were used to amplify and detect the mRNAs that are shown in Table I. mRNA levels were normalized to that of GAPDH using rodent-specific primers and probes (Applied Biosystems). GST gene-specific probes were fluorescently labeled at the 5'-end with 6-carboxy-fluorescein phosphoramide (FAM, reporter dye) and at the 3'-end with 6-carboxy-tetramethyl-rhodamine (TAMRA, quencher dye). The GAPDH probe was 5'-labeled with VICTM (Applied Biosystems), enabling both the gene-specific and GAPDH probes to be used in the same well with each replicate cDNA sample. Relative changes in gene expression as measured by real-time PCR were calculated by comparing treated samples with untreated controls. Data are presented as the fold change in gene expression normalized to mGAPDH and relative to the control using the 2-
CT method (38).
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Table I. Primer and probe sequences used for quantitation of mRNA levels by real-time PCR using Taqman® chemistry
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RTPCR
To substantiate results obtained by real-time PCR, an independent DADS feeding experiment was carried out according to the procedures outline above and data for the mGSTA3 transcript analyzed by reverse transcription PCR methods. Thus, total RNA was isolated from five treated and five control mice and first strand cDNA was synthesized using SuperScript II RNase H- Reverse Transcriptase according to the manufacturer's suggestions (Invitrogen). After digestion of RNADNA hybrids with RNase H (Invitrogen), the reverse transcriptase reaction was diluted 100x with Ultra-Pure H2O (Millipore) and 5 µl used directly in a PCR reaction using Platinum Pfx DNA Polymerse (Invitrogen). Forward and reverse primers (GeneLink) were designed that contained 3'-ends specific for mGSTA3 (5'-GTTGTGGACAACTTCCCTCTC-3' and 5'-CTGACTCAACACATTTTGCGTCATC-3', unique mGSTA3 nucleotides shown in bold). Temperature cycling was carried out for 31 cycles and the reaction products loaded onto a 1.5% agarose gel containing ethidium bromide. The gel was digitally imaged using an Image Station 2000R (Eastman Kodak, Rochester, NY) and bands were analyzed using Kodak 1D Image Analysis software.
Peptide mass fingerprinting
Unidentified mGST HPLC fractions from DADS-treated intestine were subjected to peptide mass fingerprinting analysis. Briefly, the HPLC fraction was collected and the organic solvent (acetonitrile or TFA) removed by vacuum evaporation. Ammonium bicarbonate was added to a final concentration of 100 mM and digestion was conducted with 8 µg trypsin (Promega, Madison, WI) at 25°C for 16 h. The resulting peptides were resolved by loading the digest onto a Vyadac reversed phase C18 column (1 mm x 15 cm) connected to an HP-1100 (Hewlett Packard) HPLC apparatus. Peptides were eluted with increasing concentrations (0.2%/min) of buffer B (0.1% TFA in acetonitrile) at a flow rate of 50 µl/min. Peptides were subjected to analysis by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS) using a Voyager-DE mass analyzer (PerSeptive Biosystems, Framingham, MA). A portion of each fraction was mixed 1:1 with MALDI matrix (
-cyano-4-hydroxy-cinnamic acid; Sigma) in 50% acetonitrile and 0.1% TFA and spotted on a MALDI target before analysis. The peptide masses obtained were compared to theoretical tryptic digests of known mouse GSTs. Additionally, sequences were determined for a few abundant peptides using Edman chemistry on a Procise-494 sequencer (Applied Biosystems).
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Results
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Dietary protocols using short-term feeding regimens of naturally occurring organosulfur compounds were established for studies on induction of GSTs in the gastrointestinal tract and liver of mice. Cytosolic extracts from those tissues were initially screened for overall GST enzymatic activities and probed by immunoblots with specific antisera to distinguish among the major
, µ and
class GSTs. Two courses of 20 µmol DADS resulted in almost 3-fold increases in GST enzymatic activities of stomach and small intestine (Table II). There were smaller changes in GST activities of liver and colon (Table II).
Immunoblots of cytosolic proteins from control and DADS-treated mice showed relatively small variations in GST
levels after DADS administration, however, substantial increases in the µ and
class GSTs were observed in both liver and small intestine of DADS-treated animals (Figure 1). To refine the analysis and identify GSTs affected by the dietary organosulfur compounds, HPLC methods were developed to resolve seven µ, five
and two
class GST subunits. Subunit peaks were classified on the basis of immunoreactivity and individual subunits within each subclass identified by comparison of molecular masses determined by ESI MS with molecular masses based on deduced sequences from the database.

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Fig. 1. Immunoblots of cytosolic proteins from intestine (columns 1 and 2) and liver (columns 3 and 4). Homogenates were prepared according to the methods described in Materials and methods. Columns 1 and 3 show data obtained for untreated control mice and columns 2 and 4 show the effect of DADS treatment on the indicated classes of GSTs.
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Basis for GST nomenclature
It has been pointed out that many studies on regulation of expression of mouse GSTs failed to distinguish among individual subunits which were either incompletely resolved or not identified (37). Accordingly, retention times of each subunit, molecular masses determined by ESI MS, accession numbers and nomenclature adopted in this report for
, µ and
class GSTs are summarized in Table III. Most of the subunits are named according to recent literature consensus designations, however, some have been renamed for this study. For instance, mGSTM1 and mGSTM2 are related to their rat counterparts (rGSTM1 and rGSTM2), however, the protein subunit corresponding to mGSTM3 of Table III had not been reported prior to this study. The identity of mGSTM3 was deduced from a putative GST sequence in the genomic database that exactly corresponds to the experimental molecular mass of the HPLC peak of Table III. The rationale for this assignment is based on its sequence homology to rat rGSTM3 (39) and the unique characteristic pentapeptide sequence (ERNQV) at residues 163167 present only in these subunits. Moreover, the tissue distribution of the mGSTM3 subunit is similar to that of rat rGSTM3 (39). Likewise, mGSTM4 was previously designated M3 (40) (before the mGSTM3 subunit cited above was identified), but it is more homologous to rGSTM4, and both contain the characteristic Cys115 residue in their active site pocket (40). The mGSTM7 sequence is identical to that of the mGSTM7 described by Guo et al. (41) and mGSTM4 (rGSTYb5) of Hayes and co-workers (37,42). The mGSTA5 subunit is a previously unknown inducible form. Peptide mapping data indicate that the sequence of this subunit is very similar to that of mGSTA1. This previously uncharacterized mGSTA5 subunit undergoes an S-glutathiolation reaction (see Figure 2).
Differential induction of GSTs by DADS
Typical HPLC profiles of GST subunits isolated from small intestines of control and DADS-treated animals are shown in Figure 2A. mGSTP1 is the predominant subunit in the small intestine and usually comprises >60% of the total GST content of that organ. After acute feeding of DADS, however, sharp increases in the mGSTM1 and mGSTM4 subunits were observed. Consequently, in response to DADS administration mGSTM1 subunit levels exceeded those of mGSTP1 and mGSTM4 was elevated to levels comparable with those of mGSTP1. The
class subunits, mGSTA1, mGSTA4 and mGSTA5 also increased after feeding of DADS.
class GSTs were shown to be restricted mainly to the epithelial cell layer of the villi and crypts of the small intestine (43). In addition, the low abundance mGSTM6, mGSTM7 and mGSTA2 subunits, which were not detected in control small intestines, were clearly discernible in small intestines of DADS-treated animals. On the other hand, no substantial changes were observed in mGSTP1, mGSTM2 and mGSTM3 levels. A consistent pattern of induction of GSTs in small intestine (as represented in Figure 2A) was observed for three different animals.
In mouse stomach, the major GST subunits are mGSTM1 (
25% of total GST), mGSTP1 (
30%) and mGSTA5 and mGSTA4 (
10% each). In contrast to the small intestine, the stomach has extremely low levels of mGSTM4 in untreated animals. Effects of direct force-feeding of DADS into the stomach yielded a typical pattern of GST induction as shown in Figure 2B, with little variation among animals. Similar to findings in the small intestine, the greatest increases caused by DADS in the stomach were in the mGSTM1 and mGSTM4 subunits. The levels of mGSTA4 and mGSTA5 also increased, as did the minor subunits mGSTA1 and mGSTA2. Subunits mGSTM2, mGSTM3 and mGSTA3 did not undergo substantial changes.
In colon of chow-fed animals, subunits mGSTM1 and mGSTP1 together comprise >85% of the GST composition (Figure 3A). Although the effects of DADS were less pronounced than in stomach and small intestine, increases in the mGSTM1 and mGSTM4 subunits were also observed in the colon and the relative proportions of minor subunits mGSTA5 and mGSTA1 increased substantially (Figure 3A).

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Fig. 3. HPLC profiles of affinity-purified GST subunits from the indicated tissues. The GST subunit patterns are for colon (A), liver (B), brain (C), heart (D) and testis (E) with DADS treatment indicated by black lines and those of corresponding control animals indicated by grey lines. Selected GST subunits of particular interest are labeled in (A) according to a system of nomenclature outlined in Table III. A detailed description of methods used to prepare and purify GSTs is given in Materials and methods.
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Mouse liver GSTs consist primarily of three subunits, mGSTM1, mGSTA3 and, mostly, mGSTP1 (
65% of the total GST in livers of male mice) (Figure 3B). mGSTM4 subunits were not detected in control livers. The greatest changes in livers of animals after administration of DADS were in the
class GSTs. In particular, mGSTA5 and mGSTA1, which were present at very low levels in livers of control mice (Figure 3B, inset), showed the greatest percentage increase in DADS-treated animals.
GSTs from other organs, such as heart, brain and testis, were not substantially influenced by the short-term DADS feeding regimen (Figure 3CE). The data in Figures 2 and 3 illustrate the discrete tissue-specific GST expression patterns in mice and Figure 3CE underscores the small degree of experimental variability in the tissue GST subunit patterns among different mice. Testicular and brain GSTs are characterized by a relatively high level of mGSTM5 subunits, which are present at low or below detectable levels in most other tissues. Testicular GSTs are also notable for the absence of mGSTM4 subunits and for greater expression of the mGSTM3 and mGSTM6 subunits as compared with their expression in most other tissues.
Transcriptional activation profiles
Gene-specific primers were designed to distinguish among closely related genes encoding for the GST subclasses (Table I) and for quantitative estimation of their RNA levels by real-time PCR (TaqMan) methods. In general, DADS treatment enhanced transcription of individual GST genes (Figure 4), which paralleled the patterns observed for the corresponding protein subunits (Figures 2 and 3). Thus, increased transcripts of mGSTM1 were observed in stomach, small intestine, liver and colon of DADS-fed animals. Expression of mGSTM4 and some
class GSTs also increased in stomach and small intestine after DADS feeding. Neither mGSTP1 (a major
class GST transcript) nor mGSTP2 (expressed at lower levels and which encodes for a subunit that is catalytically deficient) (44,45), was substantially affected by DADS. The mGSTA4 transcript increased in small intestine, with smaller changes in that transcript in stomach. This feeding regimen caused little change in mGSTM2 transcripts and those of mGSTM3 were not affected.

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Fig. 4. Effects of DADS on GST mRNA expression. The indicated mRNA levels were determined by real-time PCR methods. Total RNA was extracted from stomach, liver, small intestine and colon of both DADS-treated and age-matched control animals. First strand cDNA synthesis was performed using oligo(dT) as primer before relative mRNA levels of the indicated GSTs were measured using TaqMan® chemistry. The sequences of the gene-specific primers and probes employed for real-time PCR are shown in Table I. Levels of mGST mRNA for DADS-treated samples are reported relative to corresponding mRNA levels from untreated control animals. The inset is an image of an ethidium bromide stained agarose gel showing the results of RT--PCR reactions using RNA purified from small intestine, for mGSTA3 and adenylate kinase 3 (AK3) as a control gene. Band intensity was analyzed by densitometry for five control mice (lanes 15) and 5 DADS treated mice (lanes 610) using Kodak 1D Image Analysis Software.
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From a quantitative perspective, however, the apparent increases in mRNA and protein levels were sometimes divergent; thus, the greatest increases were often observed for the low abundance mRNAs. For instance, the constitutive levels of mGSTM4 mRNA expression are much lower in stomach (normalized average Ct = 34) and liver (Ct = 32) as compared with those of small intestine (Ct = 25). Accordingly, greater proportional increases in its mRNA were observed in stomach and liver after DADS feeding (Figure 4). Large changes in mGSTM4 mRNA levels (>80-fold) were also observed in stomach after feeding allicin (data not shown). In small intestine, the more abundant mGSTM1 subunit exhibited the greatest increase in absolute amount at the protein level (Figure 2A), but increases in terms of fold increase of its mRNA was not proportionally so great as that of mGSTM4 and those that occurred for the less abundant GST mRNAs (Figure 4). The selective increase in transcription of mGSTA3 in small intestine was unexpected (Figure 4) in view of the limited increase in protein level (Figure 2). Real-time PCR data were therefore reproduced for eight different sets of treated animals. In addition, semi-quantitative RTPCR followed by densitometric analysis of agarose gel electrophoretic bands was carried out on five separate control and five DADS-treated animals from independent feeding experiments (Figure 4, inset).The 9-fold increase in mGSTA3 transcripts for animals treated with DADS thereby confirmed the results obtained by real-time PCR (Figure 4).
Effects of allicin and BHA
HPLC patterns of GSTs from small intestine and stomach of mice force-fed with allicin or BHA are shown in Figure 5. Although some quantitative differences were evident between the effects of these compounds and the effects of DADS, certain general features of induction were consistent for the different substances and for animals of different ages. For instance, the three compounds selectively induced expression of mGSTM1 and mGSTM4 subunits to approximately the same extent in small intestine and stomach. The
class subunits, in addition to mGSTM6 and mGSTM7 that are induced by DADS, were also increased by administration of allicin or BHA. In liver, BHA, which is a prototypic inducer of GSTs (43,4649), appeared to be more effective than the organosulfur compounds for induction of mGSTM1 and the low abundance mGSTM4 subunits.

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Fig. 5. Effects of allicin and BHA on HPLC profiles of affinity-purified mouse GST subunits. The pattern of GST subunits for small intestine (A and C) and stomach (B and D) of treated (indicated by black lines) and untreated (indicated by grey lines) animals are shown. Treatments consisted of two oral doses of allicin (A and B) or BHA (C and D) administered by gavage. The allicin-treated mice and their untreated paired controls were 6 months old when gavaged (which is different from the mean age of 7 weeks for both BHA- and DADS-treated animals used in all other experiments). Refer to Figure 2 for subunit identification.
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Discussion
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The short-term feeding regimen of organosulfur compounds or BHA to mice resulted in substantial and coordinate increases in some GST subunits in the stomach and small intestine. The rapid induction of GSTs by DADS and allicin documented in this study could in fact be an immediate response required to counteract noxious dietary substances encountered by organs that are directly exposed to them. Moreover, a reduced risk of gastric cancer due to garlic consumption has been reported (50,51). Liver and colon GSTs were also induced, but to a lesser extent. Each tissue is marked by characteristic cell type-specific patterns of expression of the multiple GSTs, with mGSTM1 and mGSTP1 usually being the most abundant and widely distributed mouse subunits (52). The mGSTM1, mGSTM4 and some
class GSTs are considerably induced by the treatments (Figures 2 and 3).
Induction of a GST subunit by these compounds (Figures 2 and 3) is likely to occur by transcriptional activation of its corresponding gene (Figure 4). However, some apparent inconsistencies between changes in amount of a particular subunit (Figures 2 and 3) relative to the change in its corresponding mRNA (Figure 4) are noteworthy. For instance, although mGSTM4 became a major GST subunit in small intestine after induction by DADS (Figure 2), the increase in level of its mRNA (5-fold, Figure 4) was much less than corresponding DADS-induced increases that occurred in stomach (>100-fold). Yet, the stomach is an organ in which the mGSTM4 protein subunit remained a minor component even after induction. Thus, in stomach and liver, mRNAs encoding for mGSTM4, which are of relatively low abundance, exhibited greater proportional increases after administration of DADS. Moreover, the redox-active mGSTM4 subunit, which has an active site Cys115 residue (40), although expressed at very low levels in most tissues, increased considerably in response to these inducers (Figures 2
4). Likewise, the low abundance small intestinal mRNA for mGSTA3 increased >300-fold after feeding of DADS (Figure 4), while significant changes of this transcript did not occur in liver, an organ in which its constitutive expression is much greater. It is noteworthy that the mGSTA3 subunit has been shown to exhibit selective activity against certain substrates, such as aflatoxin B1 (26). Conversely, very large increases were observed for the low abundance mRNA encoding mGSTM4 in liver (>30-fold), whereas the relative amount of this protein subunit remained relatively low even after induction (Figure 3B). Although there are few studies on GST protein turnover in tissues, the results obtained here may be related, in part, to differences in translational efficiencies of the mRNAs and the relatively long half-lives observed for some GST proteins (53).
Some components involved in the transcriptional activation process of various redox-sensitive gene products, including GSTs, have recently been linked to antioxidant-responsive element (ARE) motifs in the 5'-flanking regions of their genes (5458). However, with the possible exception of the mGSTA1 gene (which contains tandem repeat ARE elements, also designated an electrophile response element, EpRE) (54), no other mouse GST genes are known to contain functional AREs, even though ARE-like sequences are present in some of their 5'-flanking regions. In that connection it is noteworthy that the DADS-responsive mGSTM1 and mGSTM4 genes have extensive sequence homologies in their 5'-flanking regions that include a consensus ARE element (Figure 6), whereas the non-responsive mGSTM2 and mGSTM3 genes have 5'-flanking sequences that are distinct from those of mGSTM1 and mGSTM4. However, there is no direct evidence that these ARE elements are functional.

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Fig. 6. Sequence alignment of the 5'-flanking region of the mGSTM1 and mGSTM4 genes. Portions of the 5'-flanking region of mGSTM1 (5000 bp) and mGSTM4 (9000 bp) obtained from the mouse genome database were aligned using CLUSTAL W (v.1.8). A region of high homology is shown and the upstream nucleotides are numbered relative to the start of exon 1 of both genes. There are additional homologous regions in the 5'-flanking region of these genes that are not shown. The 5'-flanking segments of mGSTM1 and mGSTM4 were scanned for the presence of antioxidant-responsive elements (AREs) using the TEIRESIAS algorithm (IBM Bioinformatics Group). The perfect AREs are indicated by bold type and the consensus core regions are underlined. The AREs are located at -1653 bp and -2324 bp for mGSTM1 and mGSTM4, respectively.
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A common mechanism by which many of the structurally diverse chemoprotective compounds could affect transcription and function of GSTs is by modification of cellular redox status and chemical stress. Many of the known inducers of GSTs are electrophilic, including alkylating agents, free radical-generating compounds (59,60) or, in the case of DADS and allicin used in this study, compounds that can undergo sulfhydryl/ disulfide exchange reactions. In the presence of excess GSH, conditions that are likely to exist in the tissues (10,61), both DADS and allicin can form mixed disulfides to yield allylmercaptoglutathione (62). Many of the compounds that induce GSTs can thus generate changes in the intracellular reduced glutathione/oxidized glutathione ratio (63). Moreover, some naturally occurring organosulfur compounds (DADS or allicin) can also form mixed disulfides with reactive and accessible cysteine residues of proteins. S-thiolated species of certain proteins would thus be formed and, by thioldisulfide exchange reactions with the abundant GSH, S-glutathiolated proteins could be produced. Likewise, inducing compounds such as isothiocyanates (64) can react with sulfhydryl groups to form dithiocarbamate intermediates, which, in turn, can undergo an exchange reaction with GSH to yield S-glutathiolated proteins. Redox-active compounds, such as quinones produced by oxidation of BHA (and phenolic compounds) or nitric oxide, can lead to the formation of glutathiyl radicals, sulfenic acids or S-nitrosothiols. All of those intermediates also can react with GSH to produce S-glutathiolated proteins (6570). Hence, S-thiolation of proteins may lead to induction of chemoprotective proteins and other biological consequences.
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Notes
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1 To whom correspondence should be addressed. Email: irving{at}aecom.yu.edu 
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Acknowledgments
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The authors thank Drs John D.Hayes, Sam Seifter and John A.Milner for their valuable comments about this manuscript. This work was supported by grant CA42448 from the National Cancer Institute of the National Institutes of Health. J.A. was supported by Hepatology Training Grant T32DK07218 from the National Institutes of Health.
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Received June 15, 2003;
revised September 3, 2003;
accepted October 19, 2003.