Sulforaphane Pretreatment Research Articles

DSS-induced acute colitis in C57BL/6 mice is mitigated by sulforaphane pre-treatment

The Brassica-derived isothiocyanate sulforaphane (SFN) is known to induce factor erythroid 2-related factor 2 (Nrf2), a transcription factor centrally involved in chemoprevention. Furthermore, SFN exhibits anti-inflammatory properties in vitro and in vivo. However, little is known regarding the anti-inflammatory properties of SFN in severe inflammatory phenotypes. In the present study, we tested if pre-treatment with SFN protects mice from dextran sodium sulphate (DSS)-induced colitis.C57BL/6 mice received either phosphate-buffered saline (control) or 25 mg/kg body weight (BW) SFN per os for 7 days. Subsequently, acute colitis was induced by administering 4% DSS via drinking water for 5 days and BWs, stool consistency and faecal blood loss were recorded. Following endoscopic colonoscopy, mice were sacrificed, the organs excised and spleen weights and colon lengths measured. For histopathological analysis, distal colon samples were fixed in 4% para-formaldehyde, sectioned and stained with hematoxylin/eosin. Inflammatory biomarkers were also measured in distal colon.Treatment with SFN prior to colitis induction significantly minimised both BW loss and the disease activity index compared to control mice. Furthermore, colon lengths in SFN pre-treated mice were significantly longer than in control mice. Both macroscopic and microscopic analysis of the colon revealed attenuated inflammation in SFN pre-treated animals. mRNA analysis of distal colon samples confirmed reduced expression of inflammatory markers and increased expression of Nrf2-dependent genes in SFN pre-treated mice.Our results indicate that pre-treating mice with SFN confers protection from DSS-induced colitis. These protective effects were corroborated macroscopically, microscopically and at the molecular level.

Sulforaphane preconditioning of the Nrf2/HO-1 defense pathway protects the cerebral vasculature against blood–brain barrier disruption and neurological deficits in stroke

Disruption of the blood–brain barrier (BBB) and cerebral edema are the major pathogenic mechanisms leading to neurological dysfunction and death after ischemic stroke. The brain protects itself against infarction via activation of endogenous antioxidant defense mechanisms, and we here report the first evidence that sulforaphane-mediated preactivation of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target heme oxygenase-1 (HO-1) in the cerebral vasculature protects the brain against stroke. To induce ischemic stroke, Sprague-Dawley rats were subjected to 70min middle cerebral artery occlusion (MCAo) followed by 4, 24, or 72h reperfusion. Nrf2 and HO-1 protein expression was upregulated in cerebral microvessels of peri-infarct regions after 4–72h, with HO-1 preferentially associated with perivascular astrocytes rather than the cerebrovascular endothelium. In naïve rats, treatment with sulforaphane increased Nrf2 expression in cerebral microvessels after 24h. Upregulation of Nrf2 by sulforaphane treatment prior to transient MCAo (1h) was associated with increased HO-1 expression in perivascular astrocytes in peri-infarct regions and cerebral endothelium in the infarct core. BBB disruption, lesion progression, as analyzed by MRI, and neurological deficits were reduced by sulforaphane pretreatment. As sulforaphane pretreatment led to a moderate increase in peroxynitrite generation, we suggest that hormetic preconditioning underlies sulforaphane-mediated protection against stroke. In conclusion, we propose that pharmacological or dietary interventions aimed to precondition the brain via activation of the Nrf2 defense pathway in the cerebral microvasculature provide a novel therapeutic approach for preventing BBB breakdown and neurological dysfunction in stroke.

Isothiocyanates protect against oxidized LDL-induced endothelial dysfunction by upregulating Nrf2-dependent antioxidation and suppressing NFκB activation

Oxidative stress plays a pivotal role in the pathophysiology of cardiovascular diseases. Oxidized low-density lipoprotein (oxLDL) is a key contributor to atherogenesis through multiple mechanisms. In this study, we investigated the protection by three structurally related isothiocyanates, i.e., sulforaphane (SFN), benzyl isothiocyanate (BITC), and phenethyl isocyanate (PEITC), against oxLDL-induced leukocyte adhesion to vascular endothelium and the mechanism involved. The protection against oxLDL-induced endothelial dysfunction by isothiocyanates was studied in human umbilical vein endothelial cells (HUVECs). oxLDL increased reactive oxygen species (ROS) production, stimulated nuclear factor-kappaB (NFκB) activation, and enhanced intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin expression in HUVECs, which led to promotion of monocyte adhesion to HUVECs. Treatment with SFN, BITC, and PEITC (0-10 μM) dose-dependently induced heme oxygenase (HO)-1, glutamate cysteine ligase (GCL) catalytic and modifier subunit expression, intracellular glutathione content, and antioxidant response element (ARE)-luciferase reporter activity. SFN, BITC, and PEITC pretreatment reversed oxLDL-induced ROS production, NFκB nuclear translocation, κB-reporter activity, ICAM-1, VCAM-1, and E-selectin expression, and monocyte adhesion to endothelial cells. Both heme oxygenase 1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) knockdown attenuated the isothiocyanate inhibition of oxLDL-induced ROS production, κB-reporter activity, and adhesion molecule expression. SFN, BITC, and PEITC protect against oxLDL-induced endothelial damage by upregulating Nrf2-dependent HO-1 and GCL expression, which leads to inhibition of NFκB activation and ICAM-1, VCAM-1, and E-selectin expression.

Temporal and spatial distribution of Nrf2 in rat brain following stroke: quantification of nuclear to cytoplasmic Nrf2 content using a novel immunohistochemical technique

Activation of the redox-sensitive transcription factor NF-E2 related factor 2 (Nrf2) affords protection against cerebral ischaemia-reperfusion injury via the upregulation of antioxidant defence genes. We have quantified for the first time Nrf2 content in brains from rats subjected to stroke and from cultured bEnd.3 brain endothelial cells using a novel immunohistochemical technique. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion for 70 min followed by reperfusion for 4, 24 or 72 h. Coronal brain sections were incubated with anti-Nrf2 primary and biotinylated-horseradish peroxidase-conjugated secondary antibody, after which sections were reacted with 3,3-diaminobenzidine (DAB) in the presence of hydrogen peroxide. The initial rates of DAB polymer formation were directly proportional to the Nrf2 protein concentration. Image processing was used to determine the temporal and spatial distribution of Nrf2 in nuclear and cytoplasmic compartments in stroke-affected and contralateral hemispheres. Nuclear to cytoplasmic Nrf2 ratios were increased in the stroke region after 24 h reperfusion and declined after 72 h reperfusion. Pretreatment with the Nrf2 inducer sulforaphane reduced total cellular Nrf2 levels in peri-infarct and core regions of the stroke hemisphere after 24 h reperfusion. Treatment of cultured murine brain endothelial cells with sulforaphane (2.5 μm) increased nuclear accumulation of Nrf2 over 1-4 h. We report the first quantitative measurements of spatial and temporal nuclear Nrf2 expression in rat brains following stroke, and show that sulforaphane pretreatment affects Nrf2 distribution in the brain of naïve rats and animals subjected to cerebral ischaemia. Our findings provide novel insights for targeting endogenous redox-sensitive antioxidant pathways to ameliorate the damaging consequences of stroke.

Attenuation ofβ-Amyloid-Induced Oxidative Cell Death by Sulforaphane via Activation of NF-E2-Related Factor 2

β-amyloid peptide (Aβ), a major component of senile plaques, plays important roles in neuropathology of Alzheimer's disease (AD). An array of in vitro and in vivo data indicates that Aβ-induced neuronal death is mediated by oxidative stress. In this study, we aimed to investigate effects of sulforaphane (SUL), an isothiocyanate in cruciferous vegetables, on Aβ-induced oxidative cell death in SH-SY5Y cells. Cells treated with Aβ 25–35 exhibited decreased cell viability and underwent apoptosis as determined by MTT assay and TUNEL, respectively. Aβ 25–35-induced cytotoxicity and apoptotic characteristics such as activation of c-JNK, dissipation of mitochondrial membrane potential, altered expression of Bcl-2 family proteins, and DNA fragmentation were effectively attenuated by SUL pretreatment. The antiapoptotic activity of SUL seemed to be mediated by inhibition of intracellular accumulation of reactive oxygen species and oxidative damages. SUL exerted antioxidant potential by upregulating expression of antioxidant enzymes including γ-glutamylcysteine ligase, NAD(P)H:quinone oxidoreductase-1, and heme oxygenase-1 via activation of NF-E2-related factor 2(Nrf2). The protective effect of SUL against Aβ 25–35-induced apoptotic cell death was abolished by siRNA of Nrf2. Taken together, the results suggest that pharmacologic activation of Nrf2 signaling pathway by SUL might be a practical prevention and/or protective treatment for the management of AD.

Protective effect of sulforaphane pretreatment against cisplatin-induced liver and mitochondrial oxidant damage in rats

In the present work was analyzed whether sulforaphane (SFN) may protect against cisplatin (CIS)-induced hepatic damage, oxidant stress and mitochondrial dysfunction. Four groups of male Wistar rats were studied: control, CIS, CIS + SFN and SFN. SFN was given i.p. (500 μg/kg/d × 3 days) before CIS administration (single i.p. injection, 10 mg/kg). Rats were sacrificed 3 days after CIS injection to evaluate hepatic damage (histological analysis, liver/body weight ratio and serum activity of aspartate aminotransferase and alanine aminotransferase), oxidant stress (lipid peroxidation and protein carbonyl and glutathione content), antioxidant enzymes (catalase, glutathione reductase, glutathione peroxidase, glutathione-S-transferase and superoxide dismutase) in liver homogenates and isolated mitochondria and mitochondrial function (oxygen consumption using either malate/glutamate or succinate as substrates and the activity of mitochondrial complex I, II, II–III, IV and V). Furthermore it was evaluated if SFN is able to scavenge some reactive oxygen species in vitro. It was found that SFN prevents CIS-induced (a) hepatic damage, (b) oxidant stress and decreased activity of antioxidant enzymes in liver and mitochondria and (c) mitochondrial alterations in oxygen consumption and decreased activity of mitochondrial complex I. It was also found that the scavenging ability of SFN for peroxynitrite anion, superoxide anion, singlet oxygen, peroxyl radicals, hydrogen peroxide and hydroxyl radicals was very low or negligible. The hepatoprotective effect of SFN was associated to the preservation of mitochondrial function, antioxidant enzymes and prevention of liver and mitochondrial oxidant stress.

Sulforaphane protects human chondrocytes against cell death induced by various stimuli

Chondrocyte cell death can contribute to cartilage degeneration in articular diseases, such as osteoarthritis (OA). Sulforaphane (SFN), a natural compound derived from cruciferous aliment, is well known as an anti-carcinogen, but according to recent evidence it also shows cytoprotective effects on a variety of non-tumoral cells. Therefore we have tested the ability of SFN to protect chondrocytes from cell death in vitro. Treatment of growing monolayer cultures of human C-28/I2 chondrocytes with SFN in the low micro-molecular range for a few days, reduced cell growth without affecting cell survival or inducing apoptosis. However it decreased cell death in C-28/I2 chondrocytes exposed to stimuli previously reported to promptly trigger apoptosis, that is, the cytokine tumor necrosis factor-α (TNF) plus cycloheximide (CHX) or the polyamine analogue N(1),N(11)-diethylnorspermine (DENSPM) plus CHX. In particular pre-treatment with SFN reduced effector and initiator caspase activities and the associated activation of JNK kinases. SFN exerted a cytoprotective action even versus H(2)O(2) , which differently from the previous stimuli induced cell death without producing an evident caspase activation. SFN pre-treatment also prevented caspase activation in three-dimensional micromass cultures of OA chondrocytes stimulated with growth-related oncogene α (GROα), a pro-apoptotic chemokine. The suppression of caspase activation in micromasses appeared to be related to the inhibition of p38 MAPK phosphorylation. In conclusion, the present work shows that low micro-molecular SFN concentrations exert pro-survival and anti-apoptotic actions and influence signaling pathways in a variety of experimental conditions employing chondrocyte cell lines and OA chondrocytes treated with a range of death stimuli.

Sulforaphane protects brains against hypoxic–ischemic injury through induction of Nrf2-dependent phase 2 enzyme

Neonatal hypoxia–ischemia (HI) brain injury involves reactive oxygen species (ROS) and inflammatory responses. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, has cytoprotective effects against oxidative stress and its effect was mediated by NF-E2-related factor-2 (Nrf2), a transcription factor, and heme oxygenase 1 (HO-1) which is one of Nrf2 downstream target genes. This study was undertaken to investigate the neuroprotective mechanisms of SFN in a neonatal HI rat model. Seven-day-old rat pups were subjected to left common carotid artery ligation and hypoxia (8% oxygen at 37 °C) for 90 min. SFN (5 mg/kg) was systemically administered 30 min before HI insult. Brain injury was assessed by 2,3,5-triphenyltetrazoliumchloride (TTC), Nissl, TUNEL staining, malondialdehyde (MDA), 8OH-dG level, and caspase-3 activity in the cortex and hippocampus. SFN pretreatment increased the expression of Nrf2 and HO-1 in the brain and reduced infarct ratio at 24 h after HI. The number of TUNEL-positive neurons as well as activated macroglia and the amount of 8OH-dG, were markedly reduced after SFN treatment, accompanied by suppressed caspase-3 activity and reduced lipid peroxidation (MDA) level. These results demonstrated that SFN could exert neuroprotective effects through increasing Nrf2 and HO-1 expression.

Sulforaphane protects liver injury induced by intestinal ischemia reperfusion through Nrf2-ARE pathway

To investigate the effect of sulforaphane (SFN) on regulation of NF-E2-related factor-2 (Nrf2)-antioxidant response element (ARE) pathway in liver injury induced by intestinal ischemia/reperfusion (I/R). Rats were divided randomly into four experimental groups: control, SFN control, intestinal I/R and SFN pretreatment groups (n = 8 in each group). The intestinal I/R model was established by clamping the superior mesenteric artery for 1 h and 2 h reperfusion. In the SFN pretreatment group, surgery was performed as in the intestinal I/R group, with intraperitoneal administration of 3 mg/kg SFN 1 h before the operation. Intestine and liver histology was investigated. Serum levels of aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured. Liver tissue superoxide dismutase (SOD), myeloperoxidase (MPO), glutathione (GSH) and glutathione peroxidase (GSH-Px) activity were assayed. The liver transcription factor Nrf2 and heme oxygenase-1 (HO-1) were determined by immunohistochemical analysis and Western blotting analysis. Intestinal I/R induced intestinal and liver injury, characterized by histological changes as well as a significant increase in serum AST and ALT levels (AST: 260.13 +/- 40.17 U/L vs 186.00 +/- 24.21 U/L, P < 0.01; ALT: 139.63 +/- 11.35 U/L vs 48.38 +/- 10.73 U/L, P < 0.01), all of which were reduced by pretreatment with SFN, respectively (AST: 260.13 +/- 40.17 U/L vs 216.63 +/- 22.65 U/L, P < 0.05; ALT: 139.63 +/- 11.35 U/L vs 97.63 +/- 15.56 U/L, P < 0.01). The activity of SOD in the liver tissue decreased after intestinal I/R (P < 0.01), which was enhanced by SFN pretreatment (P < 0.05). In addition, compared with the control group, SFN markedly reduced liver tissue MPO activity (P < 0.05) and elevated liver tissue GSH and GSH-Px activity (P < 0.05, P < 0.05), which was in parallel with the increased level of liver Nrf2 and HO-1 expression. SFN pretreatment attenuates liver injury induced by intestinal I/R in rats, attributable to the antioxidant effect through Nrf2-ARE pathway.

Inhibition of Activator Protein-1 by Sulforaphane Involves Interaction with Cysteine in the cFos DNA-Binding Domain: Implications for Chemoprevention of UVB-Induced Skin Cancer

Sulforaphane is an isothiocyanate derived from cruciferous vegetables that has been linked to decreased risk of certain cancers. Although the role of sulforaphane in the induction of the transcription factor Nrf2 has been studied extensively, there is also evidence that inhibition of the transcription factor activator protein-1 (AP-1) may contribute to the chemopreventive properties of this compound. In this study, we show for the first time that sulforaphane is effective at reducing the multiplicity and tumor burden of UVB-induced squamous cell carcinoma in a mouse model using cotreatment with the compound and the carcinogen. We also show that sulforaphane pretreatment is able to reduce the activity of AP-1 luciferase in the skin of transgenic mice after UVB. Chromatin immunoprecipitation analysis verified that a main constituent of the AP-1 dimer, cFos, is inhibited from binding to the AP-1 DNA binding site by sulforaphane. Electrophoretic mobility shift assay analysis of nuclear proteins also shows that sulforaphane and diamide, both known to react with cysteine amino acids, are effective at inhibiting AP-1 from binding to its response element. Using truncated recombinant cFos and cJun, we show that mutation of critical cysteines in the DNA-binding domain of these proteins (Cys(154) in cFos and Cys(272) in cJun) results in loss of sensitivity to both sulforaphane and diamide in electrophoretic mobility shift assay analysis. Together, these data indicate that inhibition of AP-1 activity may be an important molecular mechanism in chemoprevention of squamous cell carcinoma by sulforaphane.

Chemopreventive agent sulforaphane enhances radiosensitivity in human tumor cells

Sulforaphane (SFN), an isothiocyanate derived from broccoli and other cruciferous vegetables, is a positive regulator of phase II detoxification enzymes and is highly effective in protection against chemically induced cancers by inducing apoptosis and cell cycle arrest. Here, we report that SFN also enhances radiosensitivity in human tumor cells. Cell survival in HeLa human cervix carcinoma cells pretreated with SFN was significantly lower than in cells treated with radiation only. Constant-field gel electrophoresis and a gamma-H2AX foci assay showed marked inhibition of DSB repair in irradiated cells treated with SFN, while little inhibition was observed in cells with DMSO (control). In addition, immunofluorescence experiments revealed a significant delay in Rad51 (a key protein for homologous recombination repair) foci formation and disappearance in irradiated cells treated with SFN when compared to the cells with X-irradiation alone. The dephosphorylation of DNA-PKcs (a critical nonhomologous end joining protein) was also markedly delayed by SFN pretreatment in irradiated cells. These DSB repair inhibition data partially support the high apoptotic frequency of irradiated cells pretreated with SFN. Furthermore, the combined treatment of X-rays and SFN (i.p. 300 micromol/kg) in the xenograft model with HeLa cells showed efficient inhibition of in vivo tumor growth. To the best of our knowledge, our study is the first report showing SFN-enhanced radiosensitivity of tumor cells in vitro and in vivo, which opens the door for a multitude of clinical applications for chemoradiotherapy using SFN.

Transcription factor Nrf2 mediates an adaptive response to sulforaphane that protects fibroblasts in vitro against the cytotoxic effects of electrophiles, peroxides and redox-cycling agents

Sulforaphane can stimulate cellular adaptation to redox stressors through transcription factor Nrf2. Using mouse embryonic fibroblasts (MEFs) as a model, we show herein that the normal homeostatic level of glutathione in Nrf2 −/− MEFs was only 20% of that in their wild-type counterparts. Furthermore, the rate of glutathione synthesis following its acute depletion upon treatment with 3 µmol/l sulforaphane was very substantially lower in Nrf2 −/− MEFs than in wild-type cells, and the rebound leading to a ∼ 1.9-fold increase in glutathione that occurred 12–24 h after Nrf2 +/+ MEFs were treated with sulforaphane was not observed in Nrf2 −/− fibroblasts. Wild-type MEFs that had been pre-treated for 24 h with 3 µmol/l sulforaphane exhibited between 1.4- and 3.2-fold resistance against thiol-reactive electrophiles, including isothiocyanates, α,β-unsaturated carbonyl compounds (e.g. acrolein), aryl halides and alkene epoxides. Pre-treatment of Nrf2 +/+ MEFs with sulforaphane also protected against hydroperoxides (e.g. cumene hydroperoxide, CuOOH), free radical-generating compounds (e.g. menadione), and genotoxic electrophiles (e.g. chlorambucil). By contrast, Nrf2 −/− MEFs were typically ∼ 50% less tolerant of these agents than wild-type fibroblasts, and sulforaphane pre-treatment did not protect the mutant cells against xenobiotics. To test whether Nrf2-mediated up-regulation of glutathione represents the major cytoprotective mechanism stimulated by sulforaphane, 5 µmol/l buthionine sulfoximine (BSO) was used to inhibit glutathione synthesis. In Nrf2 +/+ MEFs pre-treated with sulforaphane, BSO diminished intrinsic resistance and abolished inducible resistance to acrolein, CuOOH and chlorambucil, but not menadione. Thus Nrf2-dependent up-regulation of GSH is the principal mechanism by which sulforaphane pre-treatment induced resistance to acrolein, CuOOH and chlorambucil, but not menadione.

Sulforaphane protects astrocytes against oxidative stress and delayed death caused by oxygen and glucose deprivation

Oxidative stress is an important molecular mechanism of astrocyte injury and death following ischemia/reperfusion and may be an effective target of intervention. One therapeutic strategy for detoxifying the many different reactive oxygen and nitrogen species that are produced under these conditions is induction of the Phase II gene response by the use of chemicals or conditions that promote the translocation of the transcriptional activating factor NRF2 from the cytosol to the nucleus, where it binds to genomic antioxidant response elements. This study tested the hypothesis that pre- or post-treatment of cultured cortical astrocytes with sulforaphane, an alkylating agent known to activate the NRF2 pathway of gene expression protects against death of astrocytes caused by transient exposure to O(2) and glucose deprivation (OGD). Rat cortical astrocytes were exposed to 5 muM sulforaphane either 48 h prior to, or for 48 h after a 4-h period of OGD. Both pre- and post-treatments significantly reduced cell death at 48 h after OGD. Immunostaining for 8-hydroxy-2-deoxyguanosine, a marker of DNA/RNA oxidation, was reduced at 4 h reoxygenation with sulforaphane pretreatment. Sulforaphane exposure was followed by an increase in cellular and nuclear NRF2 immunoreactivity. Moreover, sulforaphane also increased the mRNA, protein level, and enzyme activity of NAD(P)H/Quinone Oxidoreductase1, a known target of NRF2 transcriptional activation. We conclude that sulforaphane stimulates the NRF2 pathway of antioxidant gene expression in astrocytes and protects them from cell death in an in vitro model of ischemia/reperfusion.

Potent Induction of Total Cellular and Mitochondrial Antioxidants and Phase 2 Enzymes by Cruciferous Sulforaphane in Rat Aortic Smooth Muscle Cells: Cytoprotection Against Oxidative and Electrophilic Stress

Sulforaphane, a cruciferous isothiocyanate compound, upregulates cytoprotective genes in liver, but its effects on antioxidants and phase 2 defenses in vascular cells are unknown. Here we report that incubation of rat aortic smooth muscle A10 cells with sulforaphane (0.25-5 microM) resulted in concentration-dependent induction of a spectrum of important cellular antioxidants and phase 2 enzymes, including superoxide dismutase (SOD), catalase, the reduced form of glutathione (GSH), glutathione peroxidase, glutathione reductase (GR), glutathione S-transferase (GST), and NAD(P)H:quinone oxidoreductase 1 (NQO1). Sulforaphane also increased levels/activities of SOD, catalase, GSH and GST in isolated mitochondria of aortic smooth muscle cells. Time-dependent sulforaphane-induced increases in the mRNA levels for MnSOD, catalase, the catalytic subunit of gamma-glutamylcysteine ligase, GR, GST-A1, GST-P1, and NQO1 were observed. Pretreatment with sulforaphane (0.5, 1, and 5 microM) protected aortic smooth muscle cells from oxidative and electrophilic cytotoxicity induced by xanthine oxidase (XO)/xanthine, H2O2, SIN-1-derived peroxynitrite, 4-hydroxy-2-nonenal, and acrolein. Furthermore, sulforaphane pretreatment prevented intracellular accumulation of reactive oxygen species (ROS) after exposure of the cells to XO/xanthine, H2O2, or SIN-1. Taken together, this study demonstrates that in the aortic smooth muscle cells sulforaphane at physiologically relevant concentrations potently induces a series of total cellular as well as mitochondrial antioxidants and phase 2 enzymes, which is accompanied by dramatically increased resistance of these vascular cells to oxidative and electrophilic stress.

Protective Effect of Sulforaphane against Dopaminergic Cell Death

Parkinson's disease (PD) is a progressive neurodegenerative disorder with a selective loss of dopaminergic neurons in the substantia nigra. Evidence suggests oxidation of dopamine (DA) to DA quinone and consequent oxidative stress as a major factor contributing to this vulnerability. We have previously observed that exposure to or induction of NAD(P)H:quinone reductase (QR1), the enzyme that catalyzes the reduction of quinone, effectively protects DA cells. Sulforaphane (SF) is a drug identified as a potent inducer of QR1 in various non-neuronal cells. In the present study, we show that SF protects against compounds known to induce DA quinone production (6-hydroxydopamine and tetrahydrobiopterin) in DAergic cell lines CATH.a and SK-N-BE(2)C as well as in mesencephalic DAergic neurons. SF leads to attenuation of the increase in protein-bound quinone in tetrahydrobiopterin-treated cells, but this does not occur in cells that have been depleted of DA, suggesting involvement of DA quinone. SF pretreatment prevents membrane damage, DNA fragmentation, and accumulation of reactive oxygen species. SF causes increases in mRNA levels and enzymatic activity of QR1 in a dose-dependent manner. Taken together, these results indicate that SF causes induction of QR1 gene expression, removal of intracellular DA quinone, and protection against toxicity in DAergic cells. Thus, this major isothiocyanate found in cruciferous vegetables may serve as a potential candidate for development of treatment and/or prevention of PD.

Inhibition of 7,12-Dimethylbenz(a)anthracene-Induced Skin Tumorigenesis in C57BL/6 Mice by Sulforaphane Is Mediated by Nuclear Factor E2–Related Factor 2

Sulforaphane, a dietary isothiocyanate, possesses potent chemopreventive effects through the induction of cellular detoxifying/antioxidant enzymes via the transcription factor nuclear factor E2-related factor 2 (Nrf2). To investigate carcinogenesis mechanisms related to the regulation of Nrf2, we examined the tumor incidence and tumor numbers per mouse in Nrf2 wild-type (+/+) and Nrf2 knockout (-/-) mice. 7,12-Dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate treatments resulted in an increase in the incidence of skin tumors and tumor numbers per mouse in both genotypes; however, both indices were markedly higher in Nrf2(-/-) mice as compared with Nrf2(+/+) mice. Western blot analysis revealed that Nrf2 as well as heme oxygenase-1, a protein regulated by Nrf2 were not expressed in skin tumors from mice of either genotype, whereas expression of heme oxygenase-1 in Nrf2(+/+) mice was much higher than that in Nrf2(-/-) mice in nontumor skin samples. Next, we examined the chemopreventive efficacy of sulforaphane in mice with both genotypes. Topical application of 100 nmol of sulforaphane once a day for 14 days prior to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate applications decreased the incidence of skin tumor in the Nrf2(+/+) mice when compared with the vehicle-treated group. Importantly, there was no chemoprotective effect elicited by sulforaphane pretreatment in the Nrf2(-/-) mice group. Taken together, our results show for the first time that Nrf2(-/-) mice are more susceptible to skin tumorigenesis and that the chemopreventive effects of sulforaphane are mediated, at least in part, through Nrf2.

Sulforaphane-stimulated phase II enzyme induction inhibits cytokine production by airway epithelial cells stimulated with diesel extract

Airborne particulate pollutants, such as diesel exhaust particles, are thought to exacerbate lung and cardiovascular diseases through induction of oxidative stress. Sulforaphane, derived from cruciferous vegetables, is the most potent known inducer of phase II enzymes involved in the detoxification of xenobiotics. We postulated that sulforaphane may be able to ameliorate the adverse effects of pollutants by upregulating expression of endogenous antioxidant enzymes. Stimulation of bronchial epithelial cells with the chemical constituents of diesel particles result in the production of proinflammatory cytokines. We first demonstrated a role for phase II enzymes in regulating diesel effects by transfecting the airway epithelial cell line (BEAS-2B) with the sentinel phase II enzyme NAD(P)H: quinine oxidoreductase 1 (NQO1). IL-8 production in response to diesel extract was significantly reduced in these compared with untransfected cells. We then examined whether sulforaphane would stimulate phase II induction and whether this would thereby ablate the effect of diesel extracts on cytokine production. We verified that sulforaphane significantly augmented expression of the phase II enzyme genes GSTM1 and NQO1 and confirmed that sulforaphane treatment increased glutathione S-transferase activity in epithelial cells without inducing cell death or apoptosis. Sulforaphane pretreatment inhibited IL-8 production by BEAS-2B cells upon stimulation with diesel extract. Similarly, whereas diesel extract stimulated production of IL-8, granulocyte-macrophage colony-stimulating factor, and IL-1beta from primary human bronchial epithelial cells, sulforaphane pretreatment inhibited diesel-induced production of all of these cytokines. Our studies show that sulforaphane can mitigate the effect of diesel in respiratory epithelial cells and demonstrate the chemopreventative potential of phase II enzyme enhancement.