Albena T. Dinkova-Kostova

Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants

Coordinate induction of phase 2 proteins and elevation of glutathione protect cells against the toxic and carcinogenic effects of electrophiles and oxidants. All inducers react covalently with thiols at rates that are closely related to their potencies. Inducers disrupt the cytoplasmic complex between the actin-bound protein Keap1 and the transcription factor Nrf2, thereby releasing Nrf2 to migrate to the nucleus where it activates the antioxidant response element (ARE) of phase 2 genes and accelerates their transcription. We cloned, overexpressed, and purified murine Keap1 and demonstrated on native gels the formation of complexes of Keap1 with the Neh2 domain of Nrf2 and their concentration-dependent disruption by inducers such as sulforaphane and bis(2-hydroxybenzylidene)acetone. The kinetics, stoichiometry, and order of reactivities of the most reactive of the 25 cysteine thiol groups of Keap1 have been determined by tritium incorporation from [3H]dexamethasone mesylate (an inducer and irreversible modifier of thiols) and by UV spectroscopy with sulforaphane, 2,2′-dipyridyl disulfide and 4,4′-dipyridyl disulfide (titrants of thiol groups), and two closely related Michael reaction acceptors [bis(2- and 4-hydroxybenzylidene)acetones] that differ 100-fold in inducer potency and the UV spectra of which are bleached by thiol addition. With large excesses of these reagents nearly all thiols of Keap1 react, but sequential reaction with three successive single equivalents (per cysteine residue) of dipyridyl disulfides revealed excellent agreement with pseudo-first order kinetics, rapid successive declines in reaction velocity, and the stoichiometric formation of two equivalents of thiopyridone per reacted cysteine. This finding suggests that reaction of cysteine thiols is followed by rapid formation of protein disulfide linkages. The most reactive residues of Keap1 (C257, C273, C288, and C297) were identified by mapping the dexamethasone-modified cysteines by mass spectrometry of tryptic peptides. These residues are located in the intervening region between BTB and Kelch repeat domains of Keap1 and probably are the direct sensors of inducers of the phase 2 system.

Protection against electrophile and oxidant stress by induction of the phase 2 response: Fate of cysteines of the Keap1 sensor modified by inducers

Induction of a family of phase 2 genes encoding for proteins that protect against the damage of electrophiles and reactive oxygen intermediates is potentially a major strategy for reducing the risk of cancer and chronic degenerative diseases. Many phase 2 genes are regulated by upstream antioxidant response elements (ARE) that are targets of the leucine zipper transcription factor Nrf2. Under basal conditions, Nrf2 resides mainly in the cytoplasm bound to its cysteine-rich, Kelch domain-containing partner Keap1, which is itself anchored to the actin cytoskeleton and represses Nrf2 activity. Inducers disrupt the Keap1-Nrf2 complex by modifying two (C273 and C288) of the 25 cysteine residues of Keap1. The critical role of C273 and C288 was established by (i) their high reactivity when purified recombinant Keap1 was treated with dexamethasone mesylate and the dexamethasone-modified tryptic peptides were analyzed by mass spectrometry, and (ii) transfection of keap1 and nrf2 gene-deficient mouse embryonic fibroblasts with constructs expressing cysteine to alanine mutants of Keap1, and measurement of the ability of cotransfected Nrf2 to repress an ARE-luciferase reporter. Reaction of Keap1 with inducers results in formation of intermolecular disulfide bridges, probably between C273 of one Keap1 molecule and C288 of a second. Evidence for formation of such dimers was obtained by 2D PAGE of extracts of cells treated with inducers, and by the demonstration that whereas C273A and C288A mutants of Keap1 alone could not repress Nrf2 activation of the ARE-luciferase reporter, an equal mixture of these mutant constructs restored repressor activity.

Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups

Induction of phase 2 enzymes and elevations of glutathione are major and sufficient strategies for protecting mammals and their cells against the toxic and carcinogenic effects of electrophiles and reactive forms of oxygen. Inducers belong to nine chemical classes and have few common properties except for their ability to modify sulfhydryl groups by oxidation, reduction, or alkylation. Much evidence suggests that the cellular “sensor” molecule that recognizes the inducers and signals the enhanced transcription of phase 2 genes does so by virtue of unique and highly reactive sulfhydryl functions that recognize and covalently react with the inducers. Benzylidene-alkanones and -cycloalkanones are Michael reaction acceptors whose inducer potency is profoundly increased by the presence of ortho- (but not other) hydroxyl substituent(s) on the aromatic ring(s). This enhancement correlates with more rapid reactivity of the ortho-hydroxylated derivatives with model sulfhydryl compounds. Proton NMR spectroscopy provides no evidence for increased electrophilicity of the β-vinyl carbons (the presumed site of nucleophilic attack) on the hydroxylated inducers. Surprisingly, these ortho-hydroxyl groups display a propensity for extensive intermolecular hydrogen bond formation, which may raise the reactivity and facilitate addition of mercaptans, thereby raising inducer potencies.

The cytoprotective role of the Keap1–Nrf2 pathway

An elaborate network of highly inducible proteins protects aerobic cells against the cumulative damaging effects of reactive oxygen intermediates and toxic electrophiles, which are the major causes of neoplastic and chronic degenerative diseases. These cytoprotective proteins share common transcriptional regulation, through the Keap1–Nrf2 pathway, which can be activated by various exogenous and endogenous small molecules (inducers). Inducers chemically react with critical cysteine residues of the sensor protein Keap1, leading to stabilisation and nuclear translocation of transcription factor Nrf2, and ultimately to coordinate enhanced expression of genes coding for cytoprotective proteins. In addition, inducers inhibit pro-inflammatory responses, and there is a linear correlation spanning more than six orders of magnitude of concentrations between inducer and anti-inflammatory activity. Genetic deletion of transcription factor Nrf2 renders cells and animals much more sensitive to the damaging effects of electrophiles, oxidants and inflammatory agents in comparison with their wild-type counterparts. Conversely, activation of the Keap1–Nrf2 pathway allows survival and adaptation under various conditions of stress and has protective effects in many animal models. Cross-talks with other signalling pathways broadens the role of the Keap1–Nrf2 pathway in determining the fate of the cell, impacting fundamental biological processes such as proliferation, apoptosis, angiogenesis and metastasis.

Direct and indirect antioxidant properties of inducers of cytoprotective proteins

Cellular protection against oxidative and electrophile toxicities is provided by two types of small-molecule antioxidants: (i) direct antioxidants, which are redox active, short-lived, are sacrificed in the process of their antioxidant actions and need to be replenished or regenerated, and may evoke pro-oxidant effects; and (ii) indirect antioxidants, that may or may not be redox active. Indirect antioxidants activate the Keap1/Nrf2/ARE pathway resulting in transcriptional induction of a battery of cytoprotective proteins (also known as phase 2 enzymes) that act catalytically, are not consumed, have long half-lives, and are unlikely to evoke pro-oxidant effects. These protective systems are involved in a complex functional interplay, such that many cytoprotective proteins participate in the synthesis and/or regeneration of direct antioxidants, whereas some direct antioxidants are required for the catalytic functions of cytoprotective proteins. Importantly, many inducers of cytoprotective proteins have been isolated from edible plants, e. g., sulforaphane from broccoli and curcumin from turmeric. Both are pleiotropic agents with multiple biological activities that could collectively contribute to their protective effects in various animal studies, including models of carcinogenesis, hypertension, neuronal and retinal damage. In addition to inducing cytoprotective proteins, molecules like curcumin which contain Michael acceptor functionalities (olefins or acetylenes conjugated to electron withdrawing groups) and phenolic hydroxyl groups can scavenge directly and potently oxygen- and nitrogen-centered reactive intermediates. Such bifunctional antioxidants can play a dual protective role by: (i) scavenging hazardous oxidants directly and instantaneously, and (ii) inducing cytoprotective enzymes that in turn function to resolve the consequences of hazardous processes that are already in progress, and to ensure long-term protection against subsequent challenges.

Quantitative determination of dithiocarbamates in human plasma, serum, erythrocytes and urine: pharmacokinetics of broccoli sprout isothiocyanates in humans

BACKGROUND Humans are exposed to substantial quantities of isothiocyanates and glucosinolates from vegetables. Since dietary isothiocyanates are widely regarded as potentially important chemoprotectors against cancer, reliable methods for measuring the plasma and tissue pharmacokinetics of isothiocyanates and their dithiocarbamate metabolites are essential for defining dosing regimens. METHODS Isothiocyanates (ITC) and dithiocarbamates (DTC) react quantitatively with 1,2-benzenedithiol to produce 1,3-benzodithiole-2-thione that can be quantified spectroscopically. Although this cyclocondensation reaction has been highly useful for analyzing plant material and urine samples, the determination of DTC/ITC (the total quantity of DTC and ITC components in a sample that react in the cyclocondensation reaction) in blood and tissues has been hampered by their low levels and the high concentrations of proteins that interfere with the cyclocondensation reaction. The protein content of blood and tissues was reduced by the precipitation with polyethylene glycol (PEG) or ultrafiltration, and the sensitivity of the method was increased substantially by the solid phase extraction of the cyclocondensation product. RESULTS Pharmacokinetic measurements were made in four human volunteers who received single doses of about 200 micromol of broccoli sprout isothiocyanates (largely sulforaphane, with lesser amounts of iberin and erucin). Isothiocyanates were absorbed rapidly, reached peak concentrations of 0.943-2.27 micromol/l in plasma, serum and erythrocytes at 1 h after feeding and declined with first-order kinetics (half-life of 1.77+/-0.13 h). The cumulative excretion at 8 h was 58.3+/-2.8% of the dose. Clearance was 369+/-53 ml/min, indicating active renal tubular secretion. CONCLUSION A sensitive and specific method for quantifying DTC levels in human plasma, serum, and erythrocytes has been devised. Determinations of ITC/DTC levels are important because: (i) dietary isothiocyanates are of potential value in reducing the risk of cancer, and (ii) humans are extensively exposed to DTC as fungicides, insecticides, pesticides and rubber vulcanization accelerators.

Glucosinolates and isothiocyanates in health and disease

Glucosinolates and isothiocyanates have both been objects of research for more than half a century. Interest in these unique phytochemicals escalated following the discovery that sulforaphane, an isothiocyanate from broccoli, potently induces mammalian cytoprotective proteins through the Keap1-Nrf2-ARE pathway. In parallel with the advances in understanding the molecular regulation of this pathway and its critical role in protection against electrophiles and oxidants, there have been increased efforts toward translating this knowledge to improve human health and combat disease. This review focuses on the animal studies demonstrating the beneficial effects of glucosinolates and isothiocyanates in models of carcinogenesis, and cardiovascular and neurological diseases, as well as on the intervention studies of their safety, pharmacokinetics, and efficacy in humans.

Cellular Stress Responses, The Hormesis Paradigm, and Vitagenes: Novel Targets for Therapeutic Intervention in Neurodegenerative Disorders

Despite the capacity of chaperones and other homeostatic components to restore folding equilibrium, cells appear poorly adapted for chronic oxidative stress that increases in cancer and in metabolic and neurodegenerative diseases. Modulation of endogenous cellular defense mechanisms represents an innovative approach to therapeutic intervention in diseases causing chronic tissue damage, such as in neurodegeneration. This article introduces the concept of hormesis and its applications to the field of neuroprotection. It is argued that the hormetic dose response provides the central underpinning of neuroprotective responses, providing a framework for explaining the common quantitative features of their dose-response relationships, their mechanistic foundations, and their relationship to the concept of biological plasticity, as well as providing a key insight for improving the accuracy of the therapeutic dose of pharmaceutical agents within the highly heterogeneous human population. This article describes in mechanistic detail how hormetic dose responses are mediated for endogenous cellular defense pathways, including sirtuin and Nrf2 and related pathways that integrate adaptive stress responses in the prevention of neurodegenerative diseases. Particular attention is given to the emerging role of nitric oxide, carbon monoxide, and hydrogen sulfide gases in hormetic-based neuroprotection and their relationship to membrane radical dynamics and mitochondrial redox signaling.

Cellular stress responses, hormetic phytochemicals and vitagenes in aging and longevity.

Modulation of endogenous cellular defense mechanisms represents an innovative approach to therapeutic intervention in diseases causing chronic tissue damage, such as in neurodegeneration. This paper introduces the emerging role of exogenous molecules in hormetic-based neuroprotection and the mitochondrial redox signaling concept of hormesis and its applications to the field of neuroprotection and longevity. Maintenance of optimal long-term health conditions is accomplished by a complex network of longevity assurance processes that are controlled by vitagenes, a group of genes involved in preserving cellular homeostasis during stressful conditions. Vitagenes encode for heat shock proteins (Hsp) Hsp32, Hsp70, the thioredoxin and the sirtuin protein systems. Dietary antioxidants, such as polyphenols and L-carnitine/acetyl-L-carnitine, have recently been demonstrated to be neuroprotective through the activation of hormetic pathways, including vitagenes. Hormesis provides the central underpinning of neuroprotective responses, providing a framework for explaining the common quantitative features of their dose response relationships, their mechanistic foundations, their relationship to the concept of biological plasticity as well as providing a key insight for improving the accuracy of the therapeutic dose of pharmaceutical agents within the highly heterogeneous human population. This paper describes in mechanistic detail how hormetic dose responses are mediated for endogenous cellular defense pathways including sirtuin, Nrfs and related pathways that integrate adaptive stress responses in the prevention of neurodegenerative diseases. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.

Safety, tolerance, and metabolism of broccoli sprout glucosinolates and isothiocyanates: a clinical phase I study.

Abstract: Broccoli sprouts are widely consumed in many parts of the world. There have been no reported concerns with respect to their tolerance and safety in humans. A formal phase I study of safety, tolerance, and pharmacokinetics appeared justified because these sprouts are being used as vehicles for the delivery of the glucosinolate glucoraphanin and its cognate isothiocyanate sulforaphane [1-isothiocyanato-(4R)-(methylsulfinyl)butane] in clinical trials. Such trials have been designed to evaluate protective efficacy against development of neoplastic and other diseases. A placebo-controlled, double-blind, randomized clinical study of sprout extracts containing either glucosinolates (principally glucoraphanin, the precursor of sulforaphane) or isothiocyanates (principally sulforaphane) was conducted on healthy volunteers who were in-patients on our clinical research unit. The subjects were studied in three cohorts, each comprising three treated individuals and one placebo recipient. Following a 5-day acclimatization period on a crucifer-free diet, the broccoli sprout extracts were administered orally at 8-h intervals for 7 days (21 doses), and the subjects were monitored during this period and for 3 days after the last treatment. Doses were 25 μmol of glucosinolate (cohort A), 100 μmol of glucosinolate (cohort B), or 25 μmol of isothiocyanate (cohort C). The mean cumulative excretion of dithiocarbamates as a fraction of dose was very similar in cohorts A and B (17.8 ± 8.6% and 19.6 ± 11.7% of dose, respectively) and very much higher and more consistent in cohort C (70.6 ± 2.0% of dose). Thirty-two types of hematology or chemistry tests were done before, during, and after the treatment period. Indicators of liver (transaminases) and thyroid [thyroid-stimulating hormone, total triiodothyronine (T3), and free thyroxine (T4)] function were examined in detail. No significant or consistent subjective or objective abnormal events (toxicities) associated with any of the sprout extract ingestions were observed.