Biomarker Exploration in Human Peripheral Blood Mononuclear Cells for Monitoring Sulforaphane Treatment Responses in Autism Spectrum Disorder

Autism Spectrum Disorder (ASD) is one of the most common neurodevelopmental disorders with no drugs treating the core symptoms and no validated biomarkers for clinical use. The multi-functional phytochemical sulforaphane affects many of the biochemical abnormalities associated with ASD. We investigated potential molecular markers from three ASD-associated physiological pathways that can be affected by sulforaphane: redox metabolism/oxidative stress; heat shock response; and immune dysregulation/inflammation, in peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with ASD. We first analyzed the mRNA levels of selected molecular markers in response to sulforaphane ex vivo treatment in PBMCs from healthy donors by real-time quantitative PCR. All of the tested markers showed quantifiability, accuracy and reproducibility. We then compared the expression levels of those markers in PBMCs taken from ASD patients in response to orally-delivered sulforaphane. The mRNA levels of cytoprotective enzymes (NQO1, HO-1, AKR1C1), and heat shock proteins (HSP27 and HSP70), increased. Conversely, mRNA levels of pro-inflammatory markers (IL-6, IL-1beta, COX-2 and TNF-alpha) decreased. Individually none is sufficiently specific or sensitive, but when grouped by function as two panels, these biomarkers show promise for monitoring pharmacodynamic responses to sulforaphane in both healthy and autistic humans, and providing guidance for biomedical interventions

Characterization of theThreshold for NAD(P)H:quinone Oxidoreductase Activity in Intact Sulforaphane-treated Pulmonary Arterial Endothelial Cells

Treatment of bovine pulmonary arterial endothelial cells in culture with the phase II enzyme inducer sulforaphane (5 μM, 24 h; sulf-treated) increased cell-lysate NAD(P)H:quinone oxidoreductase (NQO1) activity by 5.7 ± 0.6 (mean ± SEM)-fold, but intact-cell NQO1 activity by only 2.8 ± 0.1-fold compared to control cells. To evaluate the hypothesis that the threshold for sulforaphane-induced intact-cell NQO1 activity reflects a limitation in the capacity to supply NADPH at a sufficient rate to drive all the induced NQO1 to its maximum activity, total KOH-extractable pyridine nucleotides were measured in cells treated with duroquinone to stimulate maximal NQO1 activity. NQO1 activation increased NADP+ in control and sulf-treated cells, with the effect more pronounced in the sulf-treated cells, in which the NADPH was also decreased. Glucose-6-phosphate dehydrogenase (G-6-PDH) inhibition partially blocked NQO1 activity in control and sulf-treated cells, but G-6-PDH overexpression via transient transfection with the human cDNA alleviated neither the restriction on intact sulf-treated cell NQO1 activity nor the impact on the NADPH/NADP+ ratios. Intracellular ATP levels were not affected by NQO1 activation in control or sulf-treated cells. An increased dependence on extracellular glucose and a rightward shift in the Km for extracellular glucose were observed in NQO1-stimulated sulf-treated vs control cells. The data suggest that glucose transport in the sulf-treated cells may be insufficient to support the increased metabolic demand for pentose phosphate pathway-generated NADPH as an explanation for the NQO1 threshold

Effect of stevia supplementation of kale juice spheres on their quality changes during refrigerated shelf life

Kale is a vegetable with high contents of health–promoting compounds although its consumption as a beverage is highly limited due to its bitter flavour. Nonetheless, the bitter flavour of Brassicas may be masked by sweetening. Then, the effects of different stevia extracts (CTRL, S0.5 (g L–1), S1.25 and S2.5), added to an innovative kale beverage presentation, were studied on the kale juice spheres quality during 7 days at 5ºC. Kale juice spheres were produced with a double spherification technique, which allowed to obtain hydrogel spheres with high mechanical resistance.The authors express their gratitude to CNPq (Council for Scientific and Technological Development, Brazil) for a doctoral grant (232758/2014−0) made to Tâmmila Venzke Klug. The authorsare grateful to S.G. Hasa for her skillful technical assistance. The authors are also grateful to Sakata Seeds Ibérica SLU and to Sacoje SCL for providing the plant material

Benefits and risks of the hormetic effects of dietary isothiocyanates on cancer prevention

The isothiocyanate (ITC) sulforaphane (SFN) was shown at low levels (1-5 µM) to promote cell proliferation to 120-143% of the controls in a number of human cell lines, whilst at high levels (10-40 µM) it inhibited such cell proliferation. Similar dose responses were observed for cell migration, i.e. SFN at 2.5 µM increased cell migration in bladder cancer T24 cells to 128% whilst high levels inhibited cell migration. This hormetic action was also found in an angiogenesis assay where SFN at 2.5 µM promoted endothelial tube formation (118% of the control), whereas at 10-20 µM it caused significant inhibition. The precise mechanism by which SFN influences promotion of cell growth and migration is not known, but probably involves activation of autophagy since an autophagy inhibitor, 3-methyladenine, abolished the effect of SFN on cell migration. Moreover, low doses of SFN offered a protective effect against free-radical mediated cell death, an effect that was enhanced by co-treatment with selenium. These results suggest that SFN may either prevent or promote tumour cell growth depending on the dose and the nature of the target cells. In normal cells, the promotion of cell growth may be of benefit, but in transformed or cancer cells it may be an undesirable risk factor. In summary, ITCs have a biphasic effect on cell growth and migration. The benefits and risks of ITCs are not only determined by the doses, but are affected by interactions with Se and the measured endpoint

Nutrigenomics: Using Sulforaphane Consumption as a Mechanism to Prevent Cardiovascular Disease through Epigenetic Regulation

Cardiovascular disease is the leading cause of death in the United States. Diet composition and reduced expression of the transcription factor Nrf2 are both possible factors contributing to cardiovascular disease. As vitamin supplementation grows in scope and popularity, it is becoming common to replace vegetable consumption with multivitamins. The purpose of this research was to investigate how sulforaphane, an isothiocyanate found in its greatest quantities in broccoli, prevents cardiovascular disease through epigenetic regulation in order to promote the understanding that vitamin supplementation does not adequately replace the health benefits of phytonutrients found in vegetables. In order to investigate sulforaphane’s ability to prevent cardiovascular disease through epigenetic regulation, I studied scholarly journal articles that focused on experiments involving sulforaphane-induced activation of Nrf2 and the effects of Nrf2 activation such as up-regulation of antioxidant genes and phase II enzymes. Additionally, I studied articles examining sulforaphane-induced reductions in blood pressure and elimination of cardiac dysfunctions such as cardiac hypertrophy and decreased fractional shortening with the goal of identifying Nrf2 activation as the underlying mechanism. The results showed that up-regulation of antioxidant genes, signaling of phase II enzymes, lowered blood pressure, and elimination of cardiac dysfunctions were all a result of sulforaphane-induced activation of Nrf2. These results indicate that people who may be at risk for cardiovascular disease could benefit from including broccoli in their diet rather than using vitamin and mineral supplementation to replace vegetables that provide valuable phytonutrients.https://scholarscompass.vcu.edu/uresposters/1284/thumbnail.jp

Targeting crosstalk between Nrf-2, NF-κB and androgen receptor signaling in prostate cancer

Oxidative stress, inflammation and androgen receptor (AR) signaling play a pivotal role in the initiation, development and progression of prostate cancer (PCa). Numerous papers in the literature have documented the interconnection between oxidative stress and inflammation; and how antioxidants can combat the inflammation. It has been shown in the literature that both oxidative stress and inflammation regulate AR, the key receptor involved in the transition of PCa to castration resistant prostate cancer (CRPC). In this review, we discuss about the importance of targeting Nrf-2-antioxidant signaling, NF-κB inflammatory response and AR signaling in PCa. Finally, we discuss about the crosstalk between these three critical pathways as well as how the anti-inflammatory antioxidant phytochemicals like sulforaphane (SFN) and curcumin (CUR), which can also target AR, can be ideal candidates in the chemoprevention of PCa

The emerging role of Nrf2 in mitochondrial function

The transcription factor NF-E2 p45-related factor 2 (Nrf2; gene name NFE2L2) allows adaptation and survival under conditions of stress by regulating the gene expression of diverse networks of cytoprotective proteins, including antioxidant, anti-inflammatory, and detoxification enzymes as well as proteins that assist in the repair or removal of damaged macromolecules. Nrf2 has a crucial role in the maintenance of cellular redox homeostasis by regulating the biosynthesis, utilization, and regeneration of glutathione, thioredoxin, and NADPH and by controlling the production of reactive oxygen species by mitochondria and NADPH oxidase. Under homeostatic conditions, Nrf2 affects the mitochondrial membrane potential, fatty acid oxidation, availability of substrates (NADH and FADH2/succinate) for respiration, and ATP synthesis. Under conditions of stress or growth factor stimulation, activation of Nrf2 counteracts the increased reactive oxygen species production in mitochondria via transcriptional upregulation of uncoupling protein 3 and influences mitochondrial biogenesis by maintaining the levels of nuclear respiratory factor 1 and peroxisome proliferator-activated receptor γ coactivator 1α, as well as by promoting purine nucleotide biosynthesis. Pharmacological Nrf2 activators, such as the naturally occurring isothiocyanate sulforaphane, inhibit oxidant-mediated opening of the mitochondrial permeability transition pore and mitochondrial swelling. Curiously, a synthetic 1,4-diphenyl-1,2,3-triazole compound, originally designed as an Nrf2 activator, was found to promote mitophagy, thereby contributing to the overall mitochondrial homeostasis. Thus, Nrf2 is a prominent player in supporting the structural and functional integrity of the mitochondria, and this role is particularly crucial under conditions of stress

Monitoring Keap1-Nrf2 interactions in single live cells

AbstractThe transcription factor NF-E2 p45-related factor 2 (Nrf2) and its negative regulator Kelch-like ECH associated protein 1 (Keap1) control the expression of nearly 500 genes with diverse cytoprotective functions. Keap1, a substrate adaptor protein for Cullin3/Rbx1 ubiquitin ligase, normally continuously targets Nrf2 for degradation, but loses this ability in response to electrophiles and oxidants (termed inducers). Consequently, Nrf2 accumulates and activates transcription of its downstream target genes. Many inducers are phytochemicals, and cruciferous vegetables represent one of the richest sources of inducer activity among the most commonly used edible plants. Here we summarize the discovery of the isothiocyanate sulforaphane as a potent inducer which reacts with cysteine sensors of Keap1, leading to activation of Nrf2. We then describe the development of a quantitative Förster resonance energy transfer (FRET)-based methodology combined with multiphoton fluorescence lifetime imaging microscopy (FLIM) to investigate the interactions between Keap1 and Nrf2 in single live cells, and the effect of sulforaphane, and other cysteine-reactive inducers, on the dynamics of the Keap1–Nrf2 protein complex. We present the experimental evidence for the “cyclic sequential attachment and regeneration” or “conformation cycling” model of Keap1-mediated Nrf2 degradation. Finally, we discuss the implications of this mode of regulation of Nrf2 for achieving a fine balance under normal physiological conditions, and the consequences and mechanisms of disrupting this balance for tumor biology

PMI: A Delta Psi(m) Independent Pharmacological Regulator of Mitophagy

Mitophagy is central to mitochondrial and cellular homeostasis and operates via the PINK1/Parkin pathway targeting mitochondria devoid of membrane potential (ΔΨm) to autophagosomes. Although mitophagy is recognized as a fundamental cellular process, selective pharmacologic modulators of mitophagy are almost nonexistent. We developed a compound that increases the expression and signaling of the autophagic adaptor molecule P62/SQSTM1 and forces mitochondria into autophagy. The compound, P62-mediated mitophagy inducer (PMI), activates mitophagy without recruiting Parkin or collapsing ΔΨm and retains activity in cells devoid of a fully functional PINK1/Parkin pathway. PMI drives mitochondria to a process of quality control without compromising the bio-energetic competence of the whole network while exposing just those organelles to be recycled. Thus, PMI circumvents the toxicity and some of the nonspecific effects associated with the abrupt dissipation of ΔΨm by ionophores routinely used to induce mitophagy and represents a prototype pharmacological tool to investigate the molecular mechanisms of mitophagy

Genetic regulation of glucoraphanin accumulation in Beneforté® broccoli

Diets rich in broccoli (Brassica oleracea var italica) have been associated with maintenance of cardiovascular health and reduction in risk of cancer. These health benefits have been attributed to glucoraphanin that specifically accumulates in broccoli. The development of broccoli with enhanced concentrations of glucoraphanin may deliver greater health benefits. Three high-glucoraphanin F1 broccoli hybrids were developed in independent programmes through genome introgression from the wild species Brassica villosa. Glucoraphanin and other metabolites were quantified in experimental field trials. Global SNP analyses quantified the differential extent of B. villosa introgression The high-glucoraphanin broccoli hybrids contained 2.5–3 times the glucoraphanin content of standard hybrids due to enhanced sulphate assimilation and modifications in sulphur partitioning between sulphur-containing metabolites. All of the high-glucoraphanin hybrids possessed an introgressed B. villosa segment which contained a B. villosa Myb28 allele. Myb28 expression was increased in all of the high-glucoraphanin hybrids. Two high-glucoraphanin hybrids have been commercialised as Beneforte broccoli. The study illustrates the translation of research on glucosinolate genetics from Arabidopsis to broccoli, the use of wild Brassica species to develop cultivars with potential consumer benefits, and the development of cultivars with contrasting concentrations of glucoraphanin for use in blinded human intervention studie