Epigenetic Control of NRF2-Directed Cellular Antioxidant Status in Dictating Life-Death Decisions.

In this issue of Molecular Cell, Chen et al. (2017) demonstrate that the tumor suppressor protein ARF sensitizes cancer cells to programmed death through a surprising mechanism: ARF physically interacts with and antagonizes activation by acetylation of the master redox regulator NRF2, providing an unusual mode of posttranslational NRF2 regulation.

Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.

NOX4-derived ROS are neuroprotective by balancing intracellular calcium stores.

Hyperexcitability is associated with neuronal dysfunction, cellular death, and consequently neurodegeneration. Redox disbalance can contribute to hyperexcitation and increased reactive oxygen species (ROS) levels are observed in various neurological diseases. NOX4 is an NADPH oxidase known to produce ROS and might have a regulating function during oxidative stress. We, therefore, aimed to determine the role of NOX4 on neuronal firing, hyperexcitability, and hyperexcitability-induced changes in neural network function. Using a multidimensional approach of an in vivo model of hyperexcitability, proteomic analysis, and cellular function analysis of ROS, mitochondrial integrity, and calcium levels, we demonstrate that NOX4 is neuroprotective by regulating ROS and calcium homeostasis and thereby preventing hyperexcitability and consequently neuronal death. These results implicate NOX4 as a potential redox regulator that is beneficial in hyperexcitability and thereby might have an important role in neurodegeneration.

Chronic hyperglycaemia increases the vulnerability of the hippocampus to oxidative damage induced during post-hypoglycaemic hyperglycaemia in a mouse model of chemically induced type 1 diabetes.

AIMS/HYPOTHESIS: Chronic hyperglycaemia and recurrent hypoglycaemia are independently associated with accelerated cognitive decline in type 1 diabetes. Recurrent hypoglycaemia in rodent models of chemically induced (streptozotocin [STZ]) diabetes leads to cognitive impairment in memory-related tasks associated with hippocampal oxidative damage. This study examined the hypothesis that post-hypoglycaemic hyperglycaemia in STZ-diabetes exacerbates hippocampal oxidative stress and explored potential contributory mechanisms. METHODS: The hyperinsulinaemic glucose clamp technique was used to induce equivalent hypoglycaemia and to control post-hypoglycaemic glucose levels in mice with and without STZ-diabetes and Nrf2-/- mice (lacking Nrf2 [also known as Nfe2l2]). Subsequently, quantitative proteomics based on stable isotope labelling by amino acids in cell culture and biochemical approaches were used to assess oxidative damage and explore contributory pathways. RESULTS: Evidence of hippocampal oxidative damage was most marked in mice with STZ-diabetes exposed to post-hypoglycaemic hyperglycaemia; these mice also showed induction of Nrf2 and the Nrf2 transcriptional targets Sod2 and Hmox-1. In this group, hypoglycaemia induced a significant upregulation of proteins involved in alternative fuel provision, reductive biosynthesis and degradation of damaged proteins, and a significant downregulation of proteins mediating the stress response. Key differences emerged between mice with and without STZ-diabetes following recovery from hypoglycaemia in proteins mediating the stress response and reductive biosynthesis. CONCLUSIONS/INTERPRETATION: There is a disruption of the cellular response to a hypoglycaemic challenge in mice with STZ-induced diabetes that is not seen in wild-type non-diabetic animals. The chronic hyperglycaemia of diabetes and post-hypoglycaemic hyperglycaemia act synergistically to induce oxidative stress and damage in the hippocampus, possibly leading to irreversible damage/modification to proteins or synapses between cells. In conclusion, recurrent hypoglycaemia in sub-optimally controlled diabetes may contribute, at least in part, to accelerated cognitive decline through amplifying oxidative damage in key brain regions, such as the hippocampus. DATA AVAILABILITY: The datasets generated during and/or analysed during the current study are available in ProteomeXchange, accession no. 1-20220824-173727 ( www.proteomexchange.org ). Additional datasets generated during and/or analysed during the present study are available from the corresponding author upon reasonable request.

The Triterpenoid Nrf2 Activator, CDDO-Me, Decreases Neutrophil Senescence in a Murine Model of Joint Damage.

The synthetic 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me) is a potent activator of the erythroid 2-p45-derived factor 2, Nrf2, a leucine-zipper regulator of the antioxidant response. Herein, we investigated the effect of CDDO-Me on neutrophil function in a murine model of joint damage. Collagenase-induced osteoarthritis (CIOA) was initiated by the intra-articular injection of collagenase in the knee-joint cavity of Balb/c mice. CDDO-Me was administrated intra-articularly twice a week starting at day 7 post-CIOA, and its effect was evaluated at day 14. Neutrophils in blood and bone marrow (BM), cell apoptosis, necrosis, expression of C-X-C chemokine receptor 4 (CXCR4), beta-galactosidase (ß-Gal), and Nrf2 levels were measured by flow cytometry. In vitro, CDDO-Me promoted cell survival, reduced cell necrosis, and increased Nrf2 levels by 1.6 times. It decreased surface CXCR4 expression and reduced the frequency of senescent ß-Gal+CXCR4+ neutrophils by three times. In vivo, the degree of knee-joint damage in CIOA was correlated with upregulated CXCR4 on CD11b+ neutrophils. CDDO-Me improved the disease histological score, increased the levels of Nrf2, and downregulated surface CXCR4 on mature BM cells. Our data suggest that CDDO-Me may act as a potent regulator of neutrophil senescence during the progression of knee-joint damage.

Nrf2 is activated by disruption of mitochondrial thiol homeostasis but not by enhanced mitochondrial superoxide production.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the expression of genes involved in antioxidant defenses to modulate fundamental cellular processes such as mitochondrial function and GSH metabolism. Previous reports proposed that mitochondrial reactive oxygen species production and disruption of the GSH pool activate the Nrf2 pathway, suggesting that Nrf2 senses mitochondrial redox signals and/or oxidative damage and signals to the nucleus to respond appropriately. However, until now, it has not been possible to disentangle the overlapping effects of mitochondrial superoxide/hydrogen peroxide production as a redox signal from changes to mitochondrial thiol homeostasis on Nrf2. Recently, we developed mitochondria-targeted reagents that can independently induce mitochondrial superoxide and hydrogen peroxide production mitoParaquat (MitoPQ) or selectively disrupt mitochondrial thiol homeostasis MitoChlorodinitrobenzoic acid (MitoCDNB). Using these reagents, here we have determined how enhanced generation of mitochondrial superoxide and hydrogen peroxide or disruption of mitochondrial thiol homeostasis affects activation of the Nrf2 system in cells, which was assessed by the Nrf2 protein level, nuclear translocation, and expression of its target genes. We found that selective disruption of the mitochondrial GSH pool and inhibition of its thioredoxin system by MitoCDNB led to Nrf2 activation, whereas using MitoPQ to enhance the production of mitochondrial superoxide and hydrogen peroxide alone did not. We further showed that Nrf2 activation by MitoCDNB requires cysteine sensors of Kelch-like ECH-associated protein 1 (Keap1). These findings provide important information on how disruption to mitochondrial redox homeostasis is sensed in the cytoplasm and signaled to the nucleus.

Anti-Inflammatory Effects of Auranamide and Patriscabratine-Mechanisms and In Silico Studies.

Auranamide and patriscabratine are amides from Melastoma malabathricum (L.) Smith. Their anti-inflammatory activity and nuclear factor erythroid 2-related factor 2 (NRF2) activation ability were evaluated using Escherichia coli lipopolysaccharide (LPSEc)-stimulated murine macrophages (RAW264.7) and murine hepatoma (Hepa-1c1c7) cells, respectively. The cytotoxicity of the compounds was assessed using a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. The anti-inflammatory activity was determined by measuring the nitric oxide (NO) production and pro-inflammatory cytokines (Interleukin (IL)-1ß, Interferon (IFN)-γ, tumour necrosis factor (TNF)-α, and IL-6) and mediators (NF-κB and COX-2). NRF2 activation was determined by measuring the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) quinone oxidoreductase 1 (NQO1), nuclear NRF2 and hemeoxygenase (HO)-1. In vitro metabolic stability was assessed using the mouse, rat, and human liver microsomes. The compounds were non-toxic to the cells at 10 µM. Both compounds showed dose-dependent effects in downregulating NO production and pro-inflammatory cytokines and mediators. The compounds also showed upregulation of NQO1 activity and nuclear NRF2 and HO-1 levels. The compounds were metabolically stable in mouse, rat and human liver microsomes. The possible molecular targets of NRF2 activation by these two compounds were predicted using molecular docking studies and it was found that the compounds might inhibit the Kelch domain of KEAP1 and GSK-3ß activity. The physicochemical and drug-like properties of the test compounds were predicted using Schrodinger small molecule drug discovery suite (v.2022-2).

Activation of transcription factor Nrf2 to counteract mitochondrial dysfunction in Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder, for which no disease-modifying therapies are available to date. Although understanding of the precise aetiology of PD is incomplete, it is clear that age, genetic predisposition and environmental stressors increase the risk. At the cellular level, oxidative stress, chronic neuroinflammation, mitochondrial dysfunction and aberrant protein aggregation have been implicated as contributing factors. These detrimental processes are counteracted by elaborate networks of cellular defence mechanisms, one of which is orchestrated by transcription factor nuclear factor-erythroid 2 p45-related factor 2 (Nrf2; gene name NFE2L2). A wealth of preclinical evidence suggests that Nrf2 activation is beneficial in cellular and animal models of PD. In this review, we summarise the current understanding of mitochondrial dysfunction in PD, the role of Nrf2 in mitochondrial function and explore the potential of Nrf2 as a therapeutic target for mitochondrial dysfunction in PD.

Novel iodinated quinazolinones bearing sulfonamide as new scaffold targeting radiation induced oxidative stress.

Reactive oxygen species (ROS) play an integral role in the pathogenesis of most diseases. This work presents the design and synthesis of fourteen new diiodoquinazolinone derivatives bearing benzenesulfonamide moiety with variable acetamide tail and evaluation of their ability to activate nuclear factor erythroid 2-related factor 2 (Nrf2) using its classical target NAD(P)H: quinone oxidoreductase 1 (NQO1) in Hepa1c1c7 murine hepatoma cells. The N-(2-chloropyridin-3-yl)-2-((6,8-diiodo-4-oxo-3-(4-sulfamoylphenyl)-3,4-dihydroquinazolin-2-yl)thio) acetamide 17 was the most potent NQO1 inducer (CD = 25 µM) with free radical scavenging activity (IC50 = 28 µM) and in vivo median lethal dose (LD50) of 500 mg/Kg. The possible radioprotective activity of compound 17 was evaluated in (7 Gy) irradiated mice. Compound 17 showed a reduction in radiation induced oxidative stress as evidenced by the lower levels of ROS, malondialdehyde (MDA) and NQO1 in liver tissues. Moreover, compound 17 showed improvement in the complete blood count (CBC) of irradiated mice and decreased mortality over 30 days following irradiation. Additionally, docking studies inside the Nrf2-binding site of Kelch-like ECH associated protein 1 (Keap1), the main negative regulator of Nrf2, confirmed that 17 revealed the same interactions with the key amino acids as those of the co-crystallized ligand. This study identifies 17 as a novel antioxidant that protects against the harmful effect of radiation.

Obesity and NRF2-mediated cytoprotection: Where is the missing link?

The expanding dimensions of the global health crisis of overweight population has defined the term "globesity". Among the most common pathological conditions connected with excessive adiposity are hyperglycemia, insulin resistance, dyslipidemia and hypertension which result in chronic non-communicable diseases (NCD) such as metabolic syndrome (MetS), type 2 diabetes (T2D), and nonalchoholic steatohepatitis (NASH). The contribution of inflammatory-immune reactions in obesity and its related co-morbidities is unequivocal. Increased levels of free fatty acids (FFA), reactive oxygen species (ROS) and reactive nitrogen species (RNS) overloads the homeostatic system resulting in pro-inflammatory adipokines secretion, immune-activation and chronic inflammation in obesity. The cellular mechanisms of defense against oxidative stress are orchestrated by the transcription factor nuclear factor erythroid 2 p45-related factor 2 (NRF2). Excessive oxidative stress in the cell activates NRF2 which upregulates genes encoding major cytoprotective enzymes such as NAD(P)H:quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO1), and glutathione S-transferases (GST). The present review aims to clarify the interconnections between chronic inflammation, oxidative overload and NRF2-mediated cytoprotection as potential therapeutic approach in obesity.

KEAP1-modifying small molecule reveals muted NRF2 signaling responses in neural stem cells from Huntington's disease patients.

The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntington's disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.

Measuring the Interaction of Transcription Factor Nrf2 with Its Negative Regulator Keap1 in Single Live Cells by an Improved FRET/FLIM Analysis.

Transcription factor NF-E2 p45-related factor 2 (Nrf2) and its principal negative regulator, Kelch-like ECH-associated protein 1 (Keap1), comprise a molecular effector and sensor system that robustly responds to perturbations of the cellular redox homeostasis by orchestrating a comprehensive cytoprotective program. Under homeostatic conditions, Nrf2 is a short-lived protein, which is targeted for ubiquitination and proteasomal degradation. Upon encounter of electrophiles, oxidants, or pro-inflammatory stimuli, the cysteine sensors in Keap1 are chemically modified, rendering Keap1 unable to target Nrf2 for degradation, and consequently leading to accumulation of the transcription factor and enhanced transcription of cytoprotective genes. A detailed understanding of the protein-protein interactions between Nrf2 and Keap1 has been achieved by use of various in vitro systems, but few assays are available to assess these interactions in the context of the living cell. We previously developed an imaging-based FLIM/FRET methodology to visualize and measure the interaction between Nrf2 and Keap1 in single cells. Here, our goal was to improve this methodology in order to increase throughput and precision, and decrease cell-to-cell variability. To eliminate the possibility of orientation bias, we incorporated a flexible linker between Keap1 and the FRET acceptor fluorescent protein tag. To ensure the correct image capture of Nrf2 fused to the FRET donor fluorescent protein tag, we matched the maturation time of the fluorescent tag to the half-life of the endogenous Nrf2, by using sfGFP as the FRET donor. Using a global binning approach increased the assay throughput, whereas including the measured instrument response function in the analysis improved precision. The application of this methodology revealed a strong covariation of the results with the expression level of the acceptor. Taking the acceptor level into account circumvented cell-to-cell variability and enhanced sensitivity of the measurements of the Keap1-Nrf2 interaction in live cells.

KEAP1 inhibition is neuroprotective and suppresses the development of epilepsy.

Hippocampal sclerosis is a common acquired disease that is a major cause of drug-resistant epilepsy. A mechanism that has been proposed to lead from brain insult to hippocampal sclerosis is the excessive generation of reactive oxygen species, and consequent mitochondrial failure. Here we use a novel strategy to increase endogenous antioxidant defences using RTA 408, which we show activates nuclear factor erythroid 2-related factor 2 (Nrf2, encoded by NFE2L2) through inhibition of kelch like ECH associated protein 1 (KEAP1) through its primary sensor C151. Activation of Nrf2 with RTA 408 inhibited reactive oxygen species production, mitochondrial depolarization and cell death in an in vitro model of seizure-like activity. RTA 408 given after status epilepticus in vivo increased ATP, prevented neuronal death, and dramatically reduced (by 94%) the frequency of late spontaneous seizures for at least 4 months following status epilepticus. Thus, acute KEAP1 inhibition following status epilepticus exerts a neuroprotective and disease-modifying effect, supporting the hypothesis that reactive oxygen species generation is a key event in the development of epilepsy.

Oxidative stress management in the hair follicle: Could targeting NRF2 counter age-related hair disorders and beyond?

Widespread expression of the transcription factor, nuclear factor (erythroid-derived 2)-like 2 (NRF2), which maintains redox homeostasis, has recently been identified in the hair follicle (HF). Small molecule activators of NRF2 may therefore be useful in the management of HF pathologies associated with redox imbalance, ranging from HF greying and HF ageing via androgenetic alopecia and alopecia areata to chemotherapy-induced hair loss. Indeed, NRF2 activation has been shown to prevent peroxide-induced hair growth inhibition. Multiple parameters can increase the levels of reactive oxygen species in the HF, for example melanogenesis, depilation-induced trauma, neurogenic and autoimmune inflammation, toxic drugs, environmental stressors such as UV irradiation, genetic defects and aging-associated mitochondrial dysfunction. In this review, the potential mechanisms whereby NRF2 activation could prove beneficial in treatment of redox-associated HF disorders are therefore discussed.

Broccoli or Sulforaphane: Is It the Source or Dose That Matters?

There is robust epidemiological evidence for the beneficial effects of broccoli consumption on health, many of them clearly mediated by the isothiocyanate sulforaphane. Present in the plant as its precursor, glucoraphanin, sulforaphane is formed through the actions of myrosinase, a ß-thioglucosidase present in either the plant tissue or the mammalian microbiome. Since first isolated from broccoli and demonstrated to have cancer chemoprotective properties in rats in the early 1990s, over 3000 publications have described its efficacy in rodent disease models, underlying mechanisms of action or, to date, over 50 clinical trials examining pharmacokinetics, pharmacodynamics and disease mitigation. This review evaluates the current state of knowledge regarding the relationships between formulation (e.g., plants, sprouts, beverages, supplements), bioavailability and efficacy, and the doses of glucoraphanin and/or sulforaphane that have been used in pre-clinical and clinical studies. We pay special attention to the challenges for better integration of animal model and clinical studies, particularly with regard to selection of dose and route of administration. More effort is required to elucidate underlying mechanisms of action and to develop and validate biomarkers of pharmacodynamic action in humans. A sobering lesson is that changes in approach will be required to implement a public health paradigm for dispensing benefit across all spectrums of the global population.

The Nrf2 regulatory network provides an interface between redox and intermediary metabolism.

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2, also called Nfe2l2) is a transcription factor that regulates the cellular redox status. Nrf2 is controlled through a complex transcriptional/epigenetic and post-translational network that ensures its activity increases during redox perturbation, inflammation, growth factor stimulation and nutrient/energy fluxes, thereby enabling the factor to orchestrate adaptive responses to diverse forms of stress. Besides mediating stress-stimulated induction of antioxidant and detoxification genes, Nrf2 contributes to adaptation by upregulating the repair and degradation of damaged macromolecules, and by modulating intermediary metabolism. In the latter case, Nrf2 inhibits lipogenesis, supports ß-oxidation of fatty acids, facilitates flux through the pentose phosphate pathway, and increases NADPH regeneration and purine biosynthesis; these observations suggest Nrf2 directs metabolic reprogramming during stress.

Activation of Nrf2 Signaling Augments Vesicular Stomatitis Virus Oncolysis via Autophagy-Driven Suppression of Antiviral Immunity.

Oncolytic viruses (OVs) offer a promising therapeutic approach to treat multiple types of cancer. In this study, we show that the manipulation of the antioxidant network via transcription factor Nrf2 augments vesicular stomatitis virus Δ51 (VSVΔ51) replication and sensitizes cancer cells to viral oncolysis. Activation of Nrf2 signaling by the antioxidant compound sulforaphane (SFN) leads to enhanced VSVΔ51 spread in OV-resistant cancer cells and improves the therapeutic outcome in different murine syngeneic and xenograft tumor models. Chemoresistant A549 lung cancer cells that display constitutive dominant hyperactivation of Nrf2 signaling are particularly vulnerable to VSVΔ51 oncolysis. Mechanistically, enhanced Nrf2 signaling stimulated viral replication in cancer cells and disrupted the type I IFN response via increased autophagy. This study reveals a previously unappreciated role for Nrf2 in the regulation of autophagy and the innate antiviral response that complements the therapeutic potential of VSV-directed oncolysis against multiple types of OV-resistant or chemoresistant cancer.

Keap1, the cysteine-based mammalian intracellular sensor for electrophiles and oxidants.

The Kelch-like ECH associated protein 1 (Keap1) is a component of a Cullin3-based Cullin-RING E3 ubiquitin ligase (CRL) multisubunit protein complex. Within the CRL, homodimeric Keap1 functions as the Cullin3 adaptor, and importantly, it is also the critical component of the E3 ligase that performs the substrate recognition. The best-characterized substrate of Keap1 is transcription factor NF-E2 p45-related factor 2 (Nrf2), which orchestrates an elaborate transcriptional program in response to environmental challenges caused by oxidants, electrophiles and pro-inflammatory agents, allowing adaptation and survival under stress conditions. Keap1 is equipped with reactive cysteine residues that act as sensors for endogenously produced and exogenously encountered small molecules (termed inducers), which have a characteristic chemical signature, reactivity with sulfhydryl groups. Inducers modify the cysteine sensors of Keap1 and impair its ability to target Nrf2 for ubiquitination and degradation. Consequently, Nrf2 accumulates, enters the nucleus and drives the transcription of its target genes, which encode a large network of cytoprotective proteins. Here we summarize the early studies leading to the prediction of the existence of Keap1, followed by the discovery of Keap1 as the main negative regulator of Nrf2. We then describe the available structural information on Keap1, its assembly with Cullin3, and its interaction with Nrf2. We also discuss the multiple cysteine sensors of Keap1 that allow for detection of a wide range of endogenous and environmental inducers, and provide fine-tuning and tight control of the Keap1/Nrf2 stress-sensing response.

KEAP1 and Done? Targeting the NRF2 Pathway with Sulforaphane.

BACKGROUND: Since the re-discovery of sulforaphane in 1992 and the recognition of the bioactivity of this phytochemical, many studies have examined its mode of action in cells, animals and humans. Broccoli, especially as young sprouts, is a rich source of sulforaphane and broccoli-based preparations are now used in clinical studies probing efficacy in health preservation and disease mitigation. Many putative cellular targets are affected by sulforaphane although only one, KEAP1-NRF2 signaling, can be considered a validated target at this time. The transcription factor NRF2 is a master regulator of cell survival responses to endogenous and exogenous stressors. SCOPE AND APPROACH: This review summarizes the chemical biology of sulforaphane as an inducer of NRF2 signaling and efficacy as an inhibitor of carcinogenesis. It also provides a summary of the current findings from clinical trials using a suite of broccoli sprout preparations on a series of short-term endpoints reflecting a diversity of molecular actions. KEY FINDINGS AND CONCLUSIONS: Sulforaphane, as a pure chemical, protects against chemical-induced skin, oral, stomach, colon, lung and bladder carcinogenesis and in genetic models of colon and prostate carcinogenesis. In many of these settings the antitumorigenic efficacy of sulforaphane is dampened in Nrf2-disrupted animals. Broccoli preparations rich in glucoraphanin or sulforaphane exert demonstrable pharmacodynamic action in over a score of clinical trials. Measures of NRF2 pathway response and function are serving as guideposts for the optimization of dose, schedule and formulation as clinical trials with broccoli-based preparations become more commonplace and more rigorous in design and implementation.