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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

In Vivo Pharmacokinetics and Regulation of Gene Expression Profiles by Isothiocyanate Sulforaphane in the Rat

Rong Hu, Vidya Hebbar, Bok-Ryang Kim, Chi Chen, Bozena Winnik, Brian Buckley, Patricia Soteropoulos, Peter Tolias, Ronald P. Hart and A.-N. Tony Kong
Journal of Pharmacology and Experimental Therapeutics July 2004, 310 (1) 263-271; DOI: https://doi.org/10.1124/jpet.103.064261

Abstract

Sulforaphane (SUL) is one member of the isothiocyanate class of cancer chemopreventive compounds that has been shown to be effective in blocking initiation and progression of carcinogenesis. Previously, many studies have shown that SUL can potently induce phase II detoxifying enzymes, which contributes to its chemopreventive functions. In this study, we used 4967 oligonucleotides microarray to assess the genes that are modulated by SUL in in vivo rat livers, as well as time course of expression of these genes. The pharmacokinetics of SUL was assessed after oral dose of 50 μmol of SUL. The plasma concentration occurred at 1 h and peaked around 20 μM at 4 h after dosing and declined with a half-life of about 2.2 h. Analysis of the gene expression data found various clusters of genes that are important in cellular defense mechanisms and cell cycle regulation. The most robust cluster of genes is the metallothionein-like genes (MT-1/2 and MT-1a), which are increased up to 10-fold by 2 to 4 h after SUL dosing. The second cluster of genes is the glutathione S-transferase-A3-like genes, which include aflatoxin B1 aldehyde reductase and aldehyde oxidase. These genes are increased slightly by 4 h and peaked at 12 h. Real-time polymerase chain reaction was performed to authenticate the mRNA expression of some of these genes. In summary, this in vivo study of SUL provides the first clue as to the plasma concentrations of SUL, in vivo mitogen-activated protein kinase activations in rat livers, as well as what other genes are modulated in addition to phase II detoxifying genes. The results from this study may yield better insights for its chemopreventive functions.

Sulforaphane (SUL) belongs to the isothiocyanate (ITC) class of chemopreventive compounds and is found abundantly in many cruciferous vegetables such as broccoli and cauliflower (Zhang et al., 1992). This class of compounds has been shown to be effective in blocking initiation as well as progression of various chemically induced carcinogenesis models in animals (Hecht, 1995, 1999; Fahey et al., 1997). Previously, many studies have shown that SUL and other ITCs can induce phase II drug-metabolizing enzymes or detoxifying enzymes in in vitro cell lines as well as in animals (Guo et al., 1992; Zhang et al., 1992, 1994; Prestera and Talalay, 1995; Thimmulappa et al., 2002). The induction of phase II detoxifying enzymes includes glutathione S-transferases (GSTs) and NADPH:quinone oxidoreductase as well as cellular defensive enzymes such as heme-oxygenase-1) by ITC, commonly occurred via the activation of a basic leucine zipper Nrf2 transcription factor (Venugopal and Jaiswal, 1996; Itoh et al., 1997) acting on the antioxidant response element (ARE) or electrophile response element located in the 5′-flanking region of these genes (Nguyen et al., 1994, 2000; Kensler, 1997). The induction of these detoxifying enzymes would result in the detoxification and clearance of potential carcinogens as well as endogenous reactive oxygen species, consequently leading to protection of these cells against DNA or other cellular damage and thereby blocking the initiation of carcinogenesis. It seems that in general, many of the naturally occurring chemopreventive compounds possess this mechanism of action via the induction of detoxifying enzymes and cellular defensive enzymes (Kensler, 1997). In addition, the ITCs can also induce apoptotic cell death (Kirlin et al., 1999; Gamet-Payrastre et al., 2000) either via the caspase pathway (Yu et al., 1998) or the p53-dependent pathway (Huang et al., 1998). Furthermore, the ITC can also induce cell cycle arrest (Chiao et al., 2000; Gamet-Payrastre et al., 2000) and/or potentially induce cell death genes, leading to apoptosis.

To further understand the in vivo mechanisms of cancer preventive action of ITC such as SUL, we have conducted a study in the F344 rats to elucidate its in vivo pharmacokinetics and to ascertain what other genes are modulated in normal rat livers after oral administration of SUL. An additional objective was to examine the in vivo activation of the mitogen-activated protein kinase (MAPK) in rat liver after SUL administration. Because previously we as well as others have found that the ITC, including SUL, can modulate MAPKs in different mammalian cell lines, and one of the biological consequences of modulation of MAPKs would lead to changes in gene expression (Patten and DeLong, 1999; Yu et al., 1999). In this study, we also investigated the time course (kinetics) of the gene expression profiles elicited by SUL using the rat oligonucleotide-based DNA microarray. The dose chosen has been shown to be effective against azoxymethane-induced rat intestinal cancer model (Chung et al., 2000). We found that in this in vivo study, it provided the first clue as to the plasma concentrations of SUL, in vivo MAPK activations in rat livers, as well as what other genes are modulated in addition to phase II detoxifying genes by SUL. The results from this study would yield better insights for the chemopreventive functions of SUL and other ITCs.

Materials and Methods

Animals and Drug Treatments. Male Fisher F344 rats (120–150 g) were purchased from Hilltop Laboratories (Wilmington, DE) and kept ad libitum with food and water and housed in the Rutgers University Animal Facilities. After acclimatization for 1 week, the animals were put on the AIN76A diet (without any antioxidants) for another 2 weeks. The animals were dosed by oral gavage with 50 μmol of SUL (purchased from LKT Laboratories, St. Paul, MN, and the purity was checked by LC/MS) suspended in about 0.5 ml of corn oil at 0 h and again at 24 h. At 0, 1, 2, 4, 8, 12, 16, 24, 36, and 48 h after SUL administration, three animals per group were sacrificed; plasma and liver samples were obtained and frozen in liquid nitrogen immediately and then stored in -80°C until further analysis.

LC/MS Assay. Plasma drug concentrations (Cp) were determined using LC/MS (Thermo Finnigan, San Jose, CA). The plasma samples were passed through Centricon filters at 4°C (hydrophilic membrane, 3000-mol. wt pore size; Millipore Corporation, Bedford, MA) to remove molecules with molecular masses larger than 3000 Da. One volume of acetonitrile was added to each filtrate. The LC/MS system is composed of a Thermo Finnigan series of binary pumps, a degasser, a cooled autosampler, and a system controller (Thermo Finnigan). An analytical C18 column (Shimadzu, Kyoto, Japan) was equilibrated with solvent composed of 45:55 (v/v) A/B, where A was 0.1% (v/v) formic acid in water and B was 0.1% formic acid in acetonitrile, with a flow rate of 0.2 ml/min. After loading of a 20-μl sample, the column was eluted with the equilibrium solvent isocratically for 5 min and then eluted in a linear gradient mode up to 100% solvent B over 15 min. The eluant was directed to a mass spectrometer (LCQ DECA; Thermo Finnigan) equipped with its Turbo Ionspray heated at 45°C using the nebulizer gas and nitrogen as the auxiliary gas. The Ionspray needle was maintained at 5.5 kV under positive mode to generate sulforaphane ion (M + H)+ (Kassahun et al., 1997). The ion optics was adjusted to operate at unit mass resolution. Optimization and calibration was achieved with Xcalibur (Thermo Finnigan) software to obtain a state file for mass spectrometer.

Pharmacokinetics Analysis. The Cp values of SUL from three animals per time point (0–24-h samples) were averaged, and the resultant concentrations were fitted to a one-compartmental model with first-order absorption kinetics using WinNonlin (Pharsight, Mountain View, CA) as described in our previous publication (Kong and Jusko, 1991). The area under the curve and the absorption rate constant (ka) and elimination rate constant (kel) as well as the elimination half-life (t1/2; = 0.693/kel) were generated using the same software.

MAPK Assay. The rat liver samples were crushed into powder with a mortar and pestle. After evaporation of most of the nitrogen, powdered tissues were lysed with lysis buffer (10 mM Tris-HCl, pH 7.4, 50 mM sodium chloride, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 100 mM sodium orthovanadate, 2 mM iodoacetic acid, 5 mM ZnCl2, 1 mM phenylmethylsulfonyl fluoride, and 0.5% Triton X-100). The lysate was homogenized by passing through a 23-gauge needle three times or sonicating 10 s, and kept in ice for 30 min. The homogenate was centrifuged at 13,000 rpm for 15 min at 4°C. The supernatant was transferred into a clean tube and stored at -80°C. The protein concentration of whole lysates was determined by protein assay kit (Bio-Rad, Hercules, CA). Equal amount of protein was mixed with 4× loading buffer and preheated at 95°C for 3 min. The samples were then loaded on a 10% mini SDS-polyacrylamide gel and run at 200 V. The proteins were transferred onto a polyvinylidene difluoride membrane for 1.5 h using semi-dry transfer system (Fisher Scientific Co., Pittsburgh, PA). The membrane was blocked in 5% bovine serum albumin solution for 1 h at room temperature and then incubated overnight at 4°C with three antiphospho-MAPK primary antibodies (1:1000 dilution; New England Biolabs, Beverly, MA), which specifically recognized phosphorylated ERK1 (Thr202/Tyr204), JNK (Thr183/Tyr185), and p38 (Thr180/Tyr182), respectively. After hybridization with primary antibody, membrane was washed with Tris buffered saline/Tween 20) three times, and then incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 45 min at room temperature and washed with Tris buffered/saline Tween 20 three times. Final detection was performed with enhanced chemiluminescence Western blotting reagents (Amersham Biosciences Inc., Piscataway, NJ).

Microarray Design and Construction. A collection of 4854 oligonucleotides was purchased from Compugen, Inc. (Jamesburg, NJ), and a set of 113 oligos was designed and synthesized by MWG-Biotech AG (Ebersberg, Germany) based on a set of GenBank accession numbers provided by us. The combined set of oligos constituted nearly every identified rat UniGene group known at the time of design. The oligos, 65 to 70 nucleotides in length, are standardized for melting temperature and minimal homology. All bioinformatics for the oligonucleotides are provided on our searchable Web site (www.ngelab.org). Microarrays were printed on poly-l-lysine-coated glass slides using an OmniGrid microarrayer (GeneMachines, San Carlos, CA) and quill-type printing pins (Telichem, Sunnyvale, CA) following standard procedures described in detail previously (Carmel et al., 2004).

Hybridization. RNA was prepared from rat livers that had been frozen in liquid nitrogen and crushed into a powder with a mortar and pestle. After evaporation of most of the nitrogen, powdered tissues were homogenized in TRIzol (Invitrogen, Carlsbad, CA) and then extracted with chloroform by vortexing. A small volume (0.5 ml) of aqueous phase after chloroform extraction of the TRIzol homogenate was adjusted to 35% ethanol and loaded onto an RNeasy column (QIAGEN, Valencia, CA). The column was washed and RNA was eluted following the manufacturer's recommendations. Fluorescent probes were prepared from 2 μg of total cellular RNA using the Genisphere 3DNA dendrimer system (Genisphere, Inc., Montvale, NJ). Three separate microarrays, one for each rat at each time point, were conduced. Briefly, the control untreated RNA was labeled with Cy3 and the treated RNA was labeled with Cy5 in a parallel reaction, mixed, and hybridized to the chips. Automated microarray hybridization and washing was performed using a Ventana Discovery System (Ventana Medical Systems, Tucson, AZ) following the protocol described previously (Carmel et al., 2004). In brief, the sequence-tagged target was hybridized for 12 h at 58°C, and microarrays were washed twice in 2× SSC for 10 min at 55°C and once in 0.1× SSC for 2 min at 42°C. Florescent dendrimer was then applied and incubated at 55°C for 2 h. The microarrays were then washed with 2× SSC and spin-dried, followed by scanning on an Axon GenePix 4000B (Axon Instruments, Union City, CA).

Data Analysis. Image files were processed using Axon GenePix 4.0 software, resulting in text files containing median fluorescence intensities as well as median local backgrounds. The text files were loaded into the public domain MIAME-compliant database BASE (Saal et al., 2002) and normalized using Lowess (Yang et al., 2001, 2002a; Yang and Speed, 2002). Normalized data were imported along with spot flag information into GeneSpring (Silicon Genetics, Redwood City, CA). Here, the ratios were calculated with a minimum net control intensity of 10 (of 65,536) fluorescence units only if the spot was not flagged. The average of three control untreated RNA was used as reference for the experiment.

Quantitative Real-Time PCR. We confirmed selected microarray results by comparison with mRNA levels obtained by quantitative reverse transcription PCR using selected gene-specific primer pairs (Table 2). Total RNA was isolated, DNase I-treated, and first-strand cDNA was synthesized using the Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) with oligo-dT primers. The PCR reactions were carried out using 10 ng of cDNA, 50 nM of each primer, and SYBR Green master mix (Applied Biosystems, Foster City, CA) in 10-μl reactions. Levels of quantitative reverse transcription product were measured using SYBR Green fluorescence (Ririe et al., 1997; Wittwer et al., 1997; Schmittgen et al., 2000) collected during real-time PCR on an Applied Biosystems PRISM 7900HT sequence detection system. A control cDNA dilution series was created for each gene to establish a standard curve. Each reaction was subjected to melting point analysis to confirm single amplified products. The data generated from each PCR were analyzed using SDS 2.0 software (Applied Biosystems).

View this table:
TABLE 2

Oligonucleotide primers used for real-time PCR The nucleotide sequences of the respective up- and downstream primers are indicated.

Results

Treatment with SUL Results in Substantial Plasma Concentrations of SUL. Previous studies have shown that administration of radioactive 6-phenylhexyl isothiocyanate and phenethyl isothiocyanate resulted in substantial amounts of the radioactive compound in various tissues, including in the lung and plasma (Conaway et al., 1999). In this study, we selected oral dosing of SUL because it may simulate the natural absorption pathway after consumption of cruciferous vegetables. The average Cp of SUL after oral administration of 50 μmol of SUL are illustrated in Fig. 1. The Cp of SUL increased very rapidly, detectable at 1 h and then peaked around 20.8 μM at 4 h after dosing. The pharmacokinetic parameters generated from fitting the Cp of SUL to a one-compartment model with first-order absorption using WinNonlin yielded ka of 0.313 h-1, kel of 0.311 h-1, and volume of distribution divided by bioavailability of 0.903 liter. SUL displayed a fairly rapid absorption, which peaked around 4 h and an elimination half-life of 2.23 h (0.693/kel).

Fig. 1.
Fig. 1.

Plasma concentrations of SUL versus time after a 50-μmol SUL oral dose in rats with three animals per time point. Results are mean ± S.D. The curve is the fitted plasma concentrations to a one-compartmental model with first-order absorption using WinNonlin.

SUL Induces MAPK Activation in Rat Liver in Vivo. MAPKs characterized as proline-directed serine/threonine [ProXSer/ThrPro; [Gonzalez, 1991 #935; Alvarez, 1991 #936]] kinases (Marshall, 1994) are important cellular signaling components, which convert various extracellular signals into intracellular responses through serial phosphorylation cascades (Cobb and Goldsmith, 1995). Several laboratories, including ours, have found that other chemopreventive agents such as tea polyphenols can modulate MAPK signaling pathways (Dong et al., 1997; Yu et al., 1997). In terms of the ITCs, we and others have also shown that phenethyl isothiocyanate, SUL, and benzylisothiocyanate can activate MAPK, resulting in gene expression and apoptosis in many mammalian cell lines (Yu et al., 1996, 1998; Samaha et al., 1997; Huang et al., 1998; Kirlin et al., 1999; Patten and DeLong, 1999; Chiao et al., 2000; Gamet-Payrastre et al., 2000; Xu and Thornalley, 2000; Yang et al., 2002b). However, it is not clear regarding the roles of MAPK in the in vivo situation. As shown in Fig. 2, the maximum activation of ERK1/2 occurred at 2 h (and again at 36 and 48 h; due to second dose); however, very little JNK or p38 MAPK activation was observed. This result suggests that in the in vivo rat liver, the ERK pathway may play an important role in the early signal transduction event leading to the transcriptional activation of ARE-mediated genes expression, analogous to the in vitro cell lines situation (Yu et al., 1999).

Fig. 2.
Fig. 2.

In vivo kinetics of MAPK (ERK, JNK, and p38) activation in rat livers after SUL administered orally. The data were obtained from the combination of three liver samples per time point.

Gene Expression Profiles Induced by SUL in Rat Liver in Vivo. DNA microarrays are a powerful tool to assess simultaneously the expression profile of tens or thousands of genes in one experiment. The data were obtained after two oral doses of 50 μmol of SUL given at 0 and 24 h. Three animals per time point were obtained at 0, 2, 4, 12, 24, and 48 h to show the kinetics profile of gene expression. The data were normalized and the average of three animals per time point and the standard errors were obtained. The Student's t test, p < 0.05, filter was used to select data reproducibly different from control. From the Genespring analysis, 562 genes were found to be either 2-fold up- or down-regulated in at least one of the five time points compared with control. The genes represented in Table 1 have been generated from these 562 genes with specific importance in the regulation of the stress response, homeostasis, cell cycle, phase I and II drug metabolizing enzymes, transcription factors, kinases, phosphatases, as well as others. As shown in Table 1, very robust induction of certain genes, up to 30-fold as early of 2 to 4 h after SUL administration, was observed. Similarly, inhibition of certain genes down to 0.1-fold of the controls was observed as early as 2 to 4 h. Figure 3 shows the up-regulation of metallothionein (MT-2/1 and -1a). MT-2/1 was induced as early as 2 h up to 37-fold, decreased slightly at 4 h, and then peaked at 24 h up to 43-fold induction over control RNA. Similarly pattern was observed for MT-1a, although with less induction.

View this table:
TABLE 1

Microarray analysis of differentially expressed genes in SUL-treated rats

Fig. 3.
Fig. 3.

Changes in the MT cluster of genes (MT-I/II and -1a) as a function of time in rat livers after SUL administered orally. Values are the average fold of changes over the vehicle-treated controls (n = 3).

Figure 4 shows a second cluster of genes that is induced by SUL. These include the GST-A3, aflatoxin B1 aldehyde reductase (ABAR), aldehyde oxidase (AO), and UDP-glucuronosyltransferase-phenobarbital (pb)-inducible form (UGT-pb). ABAR and GST-A3 shows similar kinetics, which were not induced by 4 h, but then increased up to 7- and 2.5-fold at 12 h, decreased at 24 h, and then increased slightly at 48 h with 4.6- and 2.8-fold over control, respectively. AO (female form) and UGT-pb were induced in these male rat livers with similar kinetics as GST-A3, increased up to 2.3-, 2.5-, and 2.1-fold and 2.8-, 2.7-, and 2.3-fold at 12, 24, and 48 h, respectively.

Fig. 4.
Fig. 4.

Changes in the GST cluster of genes as a function of time in rat livers after SUL administered orally. Values are the average fold of changes over the vehicle-treated controls (n = 3).

Figure 5 shows the regulation of the cytochrome P450 (P450) genes. Most of this cluster of genes is up-regulated and these include P450 2b19, 3A3, 3A9, 1b1, 2b19, 4b1, and b-type b-clone HCB1, as well as the phenobarbital CAR receptor. There are two down-regulated genes and these include P450 2c29 and Cyp1a1.

Fig. 5.
Fig. 5.

Changes in the cytochrome P450 cluster of genes as a function of time in rat livers after SUL administered orally. Values are the average fold of changes over the vehicle-treated controls (n = 3).

Figure 6 shows the down-regulated gene, which includes mitochrondrial genes, cytochrome c oxidase subunits II and III; cell cycle-related genes, cyclin D1 and check point kinase Bub1; protein tyrosine phosphatase, phosphodiesterase gene (CaM-PDE); as well as transcription factors, NF-A1, NF-X1, and HNF-3-β.

Fig. 6.
Fig. 6.

Down regulation of cell cycle-related cluster of genes as a function of time in rat livers after SUL administered orally. Values are the average fold of changes over the vehicle-treated controls (n = 3).

Real-time RT-PCR Confirms the Microarray Results. To further validate the microarray results, we used quantitative real-time RT-PCR to examine the effect of SUL on individual genes. Seven genes (five up-regulated and two down-regulated genes) as relate to our interest were selected as targets for real-time RT-PCR (Table 2). RNA samples from each time points were reverse-transcribed into cDNA and then quantitative PCR was performed as described under Materials and Methods. Values for each gene were normalized to values obtained for β-actin and then a ratio of treated/control was calculated. Table 3 shows the averages of three separate experiments with the standard deviations. In all cases, differential expression could be qualitatively confirmed. The Spearman correlation was done, and it showed that the data obtained by microarray analysis and real-time PCR were highly correlated (r2 = 0.889; Fig. 7).

View this table:
TABLE 3

Real-time PCR results shown in fold changes over control

Fig. 7.
Fig. 7.

Correlation of microarray and real-time PCR data. Log10-transformed fold of changes of transcripts measured by quantitative real-time PCR at various time points were plotted against the corresponding log10-transformed values obtained by microarray analysis. Data obtained from the two techniques were highly correlated (r2 = 0.889).

Discussion

SUL is one of the ITC class of chemopreventive compounds, which is found in abundance in many cruciferous vegetables such as broccoli and cauliflower. This class of compounds has been shown to be effective in blocking initiation as well as progression of various chemically induced carcinogenesis models in animals. However, limited information is available regarding their in vivo pharmacokinetics, MAPK activation, as well as induction of different genes in rat liver. Previously, our laboratory as well as other laboratories have studied the signal transduction mechanism and the induction of phase II drug metabolizing enzymes elicited by ITC-chemopreventive compounds, including SUL. Thimmulappa et al. (2002) have generated a transcriptional profile of small intestine of wild-type (nrf2 +/+) and knock out (nrf2 -/-) mice treated with vehicle or SUL using microarray and identified genes regulated by nrf2. In this study, we used DNA microarray to assess the battery of genes that are modulated by SUL in in vivo rat liver. In addition, we investigated the kinetics (time course) of gene expression profiles in these animals.

To ascertain the concentrations of SUL in the blood of the animals after oral administration, the pharmacokinetics of SUL was assessed after oral dose of 50 μmol of SUL. The Cp occurred at 1 h and increased to a peak of around 20 μM at 4 h after dosing. The peak in vivo concentrations of 20 μM SUL offers clear relevance for numerous in vitro cell culture studies, where between 1 and 30 μM SUL was typically used for various signal transduction studies as well as phase II gene induction studies.

Analysis of the gene expression data found various clusters of genes that are up-regulated as well as some that are down-regulated. The most robust cluster of genes is the metallothionein-like genes (MT-1/2 and MT-1a), which increased up to 10-fold by 2 to 4 h after SUL dosing. Role of MT as an antioxidant has been of great interest in addition to its major biological function of regulation of zinc and copper homeostasis. Studies using cell-free system have demonstrated the ability of MT as a free radical scavenger (Thornalley and Vasak, 1985; Abel and de Ruiter, 1989). The biosynthesis and induction of MTs in vivo and in vitro is enhanced by various stimuli, including metals, hormones, cytokines, oxidants, stress, and irradiations (De et al., 1990; Schroeder and Cousins, 1990; Bauman et al., 1991; Palmiter, 1994; Andrews, 2000). In vertebrate, MT gene promoters contain metal (Buhler and Kagi, 1974) and glucocorticoid response elements (Kelly et al., 1997), and also ARE (Dalton et al., 1994). Previous gene arrays studies with cadmium chloride but not benzo[a]pyrene nor ticholoroethylene induced high expression of MT-1 and MT-II as well as several heat shock/stress response genes and early response genes (Bartosiewicz et al., 2000, 2001). However, for SUL, the latter genes were not found to be modulated. The second cluster of genes that is induced by SUL is the GST-A3-like genes, which include ABAR, aldehyde oxidase, and UGT-pb. These genes increased slightly by 4 h and peaked at 12 h. It has been shown that GST and NADPH:quinone oxidoreductase were induced by SUL in mouse liver (Zhang et al., 1992). These genes are most likely modulated by the ARE/Nrf2 signaling pathway. There is a slight delay in their induction (the plasma concentrations peaked at 4 h; however, the liver concentrations were not examined, but presumably they would be similar to the plasma levels or higher due to potential sequestration; Zhang and Callaway, 2002), suggesting that SUL may have the first effect on possibly increasing the protein level of Nrf2 transcription factor, either by transcription (Kwak et al., 2001) or via blocking the degradation of Nrf2 (Nguyen et al., 2003) followed by the release of Nrf2 from its cytosolic partner Keap1 (Itoh et al., 1999) with the subsequent nuclear translocation and binding to its partners (small Mafs) to exert its transcriptional activity on the ARE of these genes (Venugopal and Jaiswal, 1996; Itoh et al., 1997; Nguyen et al., 2000).

In terms of the down-regulated genes, they include mitochrondrial genes, cytochrome c oxidase subunits II and III; cell cycle-related genes, cyclin D1 and check point kinase Bub1; protein tyrosine phosphatase, phosphodiesterase gene (CaM-PDE); as well as transcription factors, NF-A1, NF-X1, and HNF-3-β. The biological functions as well as the mechanisms of down-regulation are not clear at this time; however, these could potentially contribute to SUL's toxicity, especially under chronic dosing. On the other hand, if these occur in preinitiated or tumor cells, it may have beneficial biological effects as seen in human cancer cell lines (Kelloff et al., 1994; Kirlin et al., 1999; Gamet-Payrastre et al., 2000; Hu et al., 2003). Future studies with higher doses under chronic administration in animal tumor models will be needed to elucidate these differential effects.

In summary, this microarray study of SUL in in vivo rat livers provided the first clue as to what genes are modulated in addition to phase II detoxifying genes such as GST and UGT. The induction of MT genes by SUL is somewhat surprising and has not been reported before. The down modulation of cell cycle and cell death genes suggest that SUL may have biological effects in preinitiated or tumor cells, and future studies would be needed to confirm this phenomenon. The elucidation of this global gene expression profile elicited by isothiocyanates such as SUL may yield further insights for their chemopreventive functions. Future studies focusing on potential cell signaling and biological significance of isothiocyanate-induced gene expression would greatly extend our understanding of the mechanisms of chemopreventive functions of isothiocyanates.

Acknowledgments

We thank Jessica Lam for technical assistance.

Footnotes

  • This work was supported by the National Institutes of Health Grant R01-CA73674.

  • DOI: 10.1124/jpet.103.064261.

  • ABBREVIATIONS: SUL, sulforaphane; ITC, isothiocyanate; GST, glutathione S-transferase; ARE, antioxidant response element; MAPK, mitogen-activated protein kinase; LC/MS, liquid chromatography/mass spectrometry; Cp, plasma drug concentration(s); ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; SSC, standard saline citrate; PCR, polymerase chain reaction; MT, metallothionein; ABAR, aflatoxin B1 aldehyde reductase; pb, phenobarbital; RT-PCR, reverse transcription-polymerase chain reaction; ARE, antioxidant response element; UGT, uridine diphosphate glucuronosyltransferase.

  • ↵1 These authors contributed equally to this work.

    • Received December 12, 2003.
    • Accepted February 19, 2004.

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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

In Vivo Pharmacokinetics and Regulation of Gene Expression Profiles by Isothiocyanate Sulforaphane in the Rat

Rong Hu, Vidya Hebbar, Bok-Ryang Kim, Chi Chen, Bozena Winnik, Brian Buckley, Patricia Soteropoulos, Peter Tolias, Ronald P. Hart and A.-N. Tony Kong
Journal of Pharmacology and Experimental Therapeutics July 1, 2004, 310 (1) 263-271; DOI: https://doi.org/10.1124/jpet.103.064261
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