Hydroxytyrosol Research Articles

Tyrosol: Repercussion of the Lack of a Hydroxyl-Group in the Response of MCF-7 Cells to Hypoxia.

In solid tumors, such as breast cancer, hypoxic microenvironment worsens patient prognoses. We have previously reported in MCF-7 breast cancer cells that, under hypoxic conditions, hydroxytyrosol (HT) downregulates the level of reactive oxygen species, reduces the expression of hypoxia inducible factor-1α (HIF-1α), and, at high concentrations, can bind to the aryl hydrocarbon receptor (AhR). With this background, the present study investigated whether the most abundant extra virgin olive oil (EVOO) phenolic compound tyrosol (TYR), with a chemical structure similar to HT but with only one hydroxyl group, exerts comparable effects. Our results revealed that, although TYR did not show any antioxidant activity in hypoxic MCF-7 cells, it inhibited the PI3K/Akt/mTOR/S6 kinase (S6K) pathway and reduced the expression of HIF-1α and some of its target genes. Besides, TYR showed a lower binding affinity with the cytosolic transcription factor AhR, and even reduced its transcriptional activity. Some of these results are positive to control tumor progression in a hypoxic environment; however, they are observed at doses unachievable with diet intake or nutraceutical presentations. Considering that EVOO phenols can have synergistic effects, a mixture of low doses of TYR and other phenols could be useful to achieve these beneficial effects.

EVOO Polyphenols Exert Anti-Inflammatory Effects on the Microglia Cell through TREM2 Signaling Pathway.

In Alzheimer's disease (AD), microglia, brain resident immune cells, become chronically inflammatory and neurotoxic. In recent years, neuroinflammation has attracted particular interest in the scientific community. The genetic variants of molecules associated with ''microgliopathies'', including the triggering receptor expressed in myeloid cells-2 (TREM2), result in increased risk of developing AD and cognitive decline. We performed a set of in vitro assays using human neuronal (SH-SY5Y) and microglial (BV2 and C13NJ) cell models. Cells were differentially treated with extra virgin olive oil (EVOO) polyphenols, oleuropein aglycone (OleA) and hydroxytyrosol (HT) before adding LPS. We evaluated the protective effects of these EVOO products by a set of biochemical and cell biology assays, including ELISA, MTT, ROS detection, Western blotting and immunofluorescence. Our results provide an integrated understanding of the neuroprotection exerted by polyphenols in terms of: (i) reduction of pro-inflammatory cytokines release (IL-6, IL-8, IP-10 and RANTES); (ii) activation of the TREM2-dependent anti-inflammatory pathway; (iii) enhancement of protective microglial activity favoring the M2 polarization phenotype. Such findings provide new and important insights into the mechanisms by which the dietary olive polyphenols exert beneficial properties against neuroinflammation and neuronal impairment.

Production of hydroxytyrosol through whole-cell bioconversion from L-DOPA using engineered Escherichia coli

Hydroxytyrosol (HT), a polyphenolic molecule of high value, is used in the nutraceutical, cosmetic, food, and livestock nutrition industries. As a natural product, HT is chemically manufactured or extracted from olives; nevertheless, the increasing demand mandates the exploration and development of alternative sources, such as heterologous production by recombinant bacteria. In order to achieve this purpose, we have molecularly modified Escherichia coli to carry two plasmids. For conversion of L-DOPA (Levodopa) into HT efficiently, it is necessary to enhance the expression of DODC (DOPA decarboxylase), ADH (alcohol dehydrogenases), MAO (Monoamine oxidase) and GDH (glucose dehydrogenases). The step that significantly affects the rate of ht biosynthesis is likely to be associated with the reaction facilitated by DODC enzymatic activity, as suggested by the result of in vitro catalytic experiment and HPLC. Then Pseudomonas putida, Sus scrofa, Homo sapiens and Levilactobacillus brevis DODC were taken into comparsion. The DODC from H. sapiens is superior to that of P. putida, S. scrofa or L. brevis for HT production. Seven promoters were introduced to increase the expression levels of catalase (CAT) to remove the byproduct H2O2 and optimized coexpression strains were obtained after screening. After the 10-hour operation, the optimized whole-cell biocatalyst produced HT at a maximum titer of 4.84 g/L with over 77.5% molar substrate conversion rate.

Enzymatic Modification of Pomace Olive Oil with Natural Antioxidants: Effect on Oxidative Stability.

Enzymatic lipophilization has been proposed as a cost-effective strategy to produce new liposoluble antioxidant compounds. In this study, modified oils rich in structured phenolipids were prepared via one-pot enzymatic acylation of hydroxytyrosol (HTYR), vanillyl alcohol (VA) and homovanillyl alcohol (HVA) with pomace olive oil (POO) in solvent-free conditions using immobilized lipase on biogenic nanoparticles. The effect of temperature (30-70 °C) and enzyme concentration (0.1-1%, w/w) on the efficiency of the bioprocess as well as the reusability of the nanobiocatalyst were thoroughly investigated. The modified oils exhibited increased antioxidant activity compared to the control oil according to DPPH and CUPRAC assays (p < 0.05). The oxidative stability of pomace olive oil was also significantly enhanced after modification, as depicted by the K232 values and TBARS contents under accelerated oxidation at 60 °C (p < 0.05). Moreover, a fortified mayonnaise containing modified oil with HTYR was prepared that was noticeably stable compared to the control mayonnaise at 28 °C for 5 months (p < 0.05). Enzymatically modified oils have great potential for application in the nutraceutical and food industry, encouraging the exploitation of immobilized lipases as effective and green catalytic tools.

Production of hydroxytyrosol from tyrosol via controllable oxidation with immobilized tyrosinase: A comparative study

Hydroxytyrosol (HT) is a novel functional pharmaceutical or food ingredient that can be prepared using tyrosinase (TYR) as the catalyst. In this study, cross-linked agarose resin (AG-NTA-Ni2+) was selected as the carrier for the one-step immobilization and purification of the enzyme via specific adsorption of the His-tag on the protein and metal ions on the carrier particles. The immobilized TYR exhibited excellent thermal and pH stabilities and retained 82.3% of its relative activity after seven cycles. Using different reduction methods, immobilized TYR was used as a catalyst to synthesize HT via the controlled oxidation of tyrosol. The enzymatic cascade reaction of co-immobilized TYR and glucose dehydrogenase (GDH) as a biocatalyst served as a bioreduction method with an HT yield of 88%. For the chemical reduction using ascorbic acid (AA) as the reducing agent, an 80% HT yield was achieved. These results indicate that the immobilization method, which relies on the specific coordination between transition metal ions and certain protein amino acids, is a potential strategy for the effective co-immobilization of TYR and GDH. The proposed affinity co-immobilization technique is among the potential strategies for the efficient production of HT.

Unveiling How Hydroxytyrosol Destabilizes α-Syn Oligomers Using Molecular Simulations.

The etiology of Parkinson's disease (PD) is mainly linked to the α-synuclein (α-Syn) fibrillogenesis. Hydroxytyrosol (HT), also known as 3,4-dihydroxyphenylethanol, is a naturally occurring polyphenol, found in extra virgin olive oil, and has been shown to have cardioprotective, anticancer, antiobesity, and antidiabetic properties. HT has neuroprotective benefits in neurodegenerative diseases and lessens the severity of PD by reducing the aggregation of α-Syn and destabilizing the preformed toxic α-Syn oligomers. However, the molecular mechanism by which HT destabilizes α-Syn oligomers and alleviates the accompanying cytotoxicity remains unexplored. The impact of HT on the α-Syn oligomer structure and its potential binding mechanism was examined in this work by employing molecular dynamics (MD) simulations. The secondary structure analysis depicted that HT significantly reduces the β-sheet and concomitantly increases the coil content of α-Syn trimer. Visualization of representative conformations from the clustering analysis depicted the hydrogen bond interactions of the hydroxyl groups in HT with the N-terminal and nonamyloid-β component (NAC) region residues of α-Syn trimer, which, in turn, leads to the weakening of interchain interactions in α-Syn trimer and resulted in the disruption of the α-Syn oligomer. The binding free energy calculations depict that HT binds favorably to α-Syn trimer (ΔGbinding = -23.25 ± 7.86 kcal/mol) and a notable reduction in the interchain binding affinity of α-Syn trimer on the incorporation of HT, which, in turn, highlights its potential to disrupt α-Syn oligomers. The current research provided mechanistic insights into the destabilization of α-Syn trimer by HT, which, in turn, will provide new clues for developing therapeutics against PD.

Redox self-sufficient production of hydroxytyrosol from dopamine by enzymatic cascade catalysis

Due to its powerful antioxidant capacity, hydroxytyrosol (HT) has a long history of application in the cosmetics and food industries. Owing to the fact that current isolation procedures for HT, both natural and synthetic, are not optimal, the efficient acceleration of HT synthesis is a critical problem in biocatalysis and metabolic engineering. The purpose of this study is to establish an Escherichia coli (E. coli) whole cell system capable of synthesizing HT from dopamine by using chosen enzymes to construct a custom biosynthetic pathway. The modified strain E. coli HT3 generated 4.43 g/L of HT under ideal conditions, with a yield and productivity of 0.44 g/L/h in shaking tube. Using alanine dehydrogenase (AlaDH), a “bridge” between the amine and its equivalent alcohol was created in the system. Specifically, ω-transaminase (ω-TA) were identified as the rate-limiting enzymes in the formation of HT using fast investigations employing cell-free systems. Subsequently, overexpression of ω-TA, AlaDH, and weakly overexpressed alcohol dehydrogenase (ADH), efficiently boosted HT synthesis based on a whole-cell biocatalyst system. Furthermore, this establishes a biocatalytic pathway for the synthesis of new building blocks for industrially high-performance HT.

Approaching Study on the Relationship Between Saccharomyces cerevisiae Production of Tyrosol, Hydroxytyrosol, and Melatonin with Volatile Compounds in Fermented Must

Yeasts are feasible and effective bioreactors and, therefore, there is a great interest in their industrial employment for the production of a wide range of molecules. In this study, the production by Saccharomyces cerevisiae of bioactive compounds such as hydroxytyrosol (HT), tyrosol (TYR) and melatonin (MEL) vs. volatile compounds in fermented must was studied. The concentration of the bioactive compounds HT and MEL in fermented must employing different yeast strains revealed that the higher the concentrations, the lower the amount of volatile compounds determined. This inverse correlation was especially remarkable with respect to the production of higher alcohols, especially 2-phenylethanol (2-PE) and esters. Furthermore, the employment of a modified Aro4pK229LS. cerevisiae QA23 yeast strain which overproduces HT, gave rise to fermented must also higher in 2-PE and their corresponding esters but with an outstanding less presence of other important esters such as ethyl hexanoate and ethyl octanoate. Both premises could point out that S. cerevisiae might have different approaches to handling cell stress/toxicity due to their nitrogen metabolism. One detoxifying pathway could be through the production of higher alcohols and these in turn to esters and the other be more related to synthesizing antioxidant molecules such as MEL and HT.

Hydroxytyrosol and Arginine as Antioxidant, Anti-Inflammatory and Immunostimulant Dietary Supplements for COVID-19 and Long COVID.

Phytochemicals from plant extracts are becoming increasingly popular in the world of food science and technology because they have positive effects on human health. In particular, several bioactive foods and dietary supplements are being investigated as potential treatments for chronic COVID. Hydroxytyrosol (HXT) is a natural antioxidant, found in olive oil, with antioxidant anti-inflammatory properties that has been consumed by humans for centuries without reported adverse effects. Its use was approved by the European Food Safety Authority as a protective agent for the cardiovascular system. Similarly, arginine is a natural amino acid with anti-inflammatory properties that can modulate the activity of immune cells, reducing the production of pro-inflammatory cytokines such as IL-6 and TNF-α. The properties of both substances may be particularly beneficial in the context of COVID-19 and long COVID, which are characterised by inflammation and oxidative stress. While l-arginine promotes the formation of •NO, HXT prevents oxidative stress and inflammation in infected cells. This combination could prevent the formation of harmful peroxynitrite, a potent pro-inflammatory substance implicated in pneumonia and COVID-19-associated organ dysfunction, as well as reduce inflammation, improve immune function, protect against free radical damage and prevent blood vessel injury. Further research is needed to fully understand the potential benefits of HXT and arginine in the context of COVID-19.

Metabolomics Insights into the Differential Response of Breast Cancer Cells to the Phenolic Compounds Hydroxytyrosol and Luteolin.

The aim of this study was to investigate the effects of two phenolic compounds found in extra virgin olive oil, hydroxytyrosol (HT) and luteolin (LUT), on the metabolism of breast cancer (BC) cells of different molecular subtypes. An untargeted metabolomics approach was used to characterize the metabolic responses of both triple-negative MDA-MB-231 cells and hormone-responsive MCF-7 cells to treatment with these phenols. Notably, while some effects were common across both cell types, others were dependent on the cell type, highlighting the importance of cellular metabolic phenotype. Common effects included stimulation of mitochondrial metabolism, acetate production, and formate overflow. On the other hand, glucose metabolism and lactate production were differentially modulated. HT and LUT appeared to inhibit glycolysis and promote the hexosamine biosynthetic pathway in MDA-MB-231 cells, while MCF-7 cells exhibited higher glycolytic flux when treated with phenolic compounds. Another significant difference was observed in lipid metabolism. Treated MDA-MB-231 cells displayed increased levels of neutral lipids (likely stored in cytosolic droplets), whereas treatment of MCF-7 cells with HT led to a decrease in triacylglycerols. Additionally, glutathione levels increased in MDA-MB-231 cells treated with HT or LUT, as well as in MCF-7 cells treated with LUT. In contrast, in HT-treated MCF-7 cells, glutathione levels decreased, indicating different modulation of cellular redox status. Overall, this work provides new insights into the metabolic impact of HT and LUT on different BC cell subtypes, paving the way for a better understanding of the nutritional relevance of these phenolic compounds in the context of BC prevention and management.

Dietary Vitamin E and/or Hydroxytyrosol Supplementation to Sows during Late Pregnancy and Lactation Modifies the Lipid Composition of Colostrum and Milk.

Modifying the composition of a sow's milk could be a strategy to improve the intestinal health and growth of her piglet during the first weeks of life. This study evaluated how dietary supplementation of vitamin E (VE), hydroxytyrosol (HXT) or VE+HXT given to Iberian sows from late gestation affected the colostrum and milk composition, lipid stability and their relationship with the piglet's oxidative status. Colostrum from VE-supplemented sows had greater C18:1n-7 than non-supplemented sows, whereas HXT increased polyunsaturated (∑PUFAs), ∑n-6 and ∑n-3 fatty acids. In 7-day milk, the main effects were induced by VE supplementation that decreased ∑PUFAs, ∑n-6 and ∑n-3 and increased the Δ-6-desaturase activity. The VE+HXT supplementation resulted in lower desaturase capacity in 20-day milk. Positive correlations were observed between the estimated mean milk energy output and the desaturation capacity of sows. The lowest concentration of malondialdehyde (MDA) in milk was observed in VE-supplemented groups, whereas HXT supplementation increased oxidation. Milk lipid oxidation was negatively correlated with the sow's plasma oxidative status and to a great extent with the oxidative status of piglets after weaning. Maternal VE supplementation produced a more beneficial milk composition to improve the oxidative status of piglets, which could promote gut health and piglet growth during the first weeks, but more research is needed to clarify this.

Efficient production of hydroxytyrosol by directed evolution of HpaB in Escherichia coli

Hydroxytyrosol (HT) is an olive-derived phenolic phytochemical that has gained increasing commercial interest due to its natural antioxidant properties. It is widely used in the field of food supplement and medicine. It is reported that 4-hydroxyphenylacetate 3-hydroxylase (EcHpaB) and flavin reductase (EcHpaC) from E. coli BL21(DE3) can successfully express and catalyze the production of HT from tyrosol. In this study, the tyrosol production strain YMG5∗R as chassis cells, and a random mutant library of EcHpaB was established using error-prone PCR to improve the ability of EcHpaB to convert tyrosol to HT. Finally, a highly efficient HT synthetic mutant strainYMG5∗R-HpaBTLEHC with high transformation efficiency was screened by directed evolution. The YMG5∗R-HpaBTLEHC strain efficiently converted 50 mM tyrosol, with a yield of hydroxytyrosol reaching 48.2 mM (7.43 g/L) and a space-time yield reached 0.62 g/L·h. Overall, our study demonstrates the successful development of a highly efficient synthetic enzyme mutant for the production of HT, which has the potential to significantly improve the commercial viability of this natural antioxidant.

Rapid pretreatment-free evaluation of antioxidant capacity in extra virgin olive oil using a laser-nanodecorated electrochemical lab-on-strip

An electroanalytical lab-on-a-strip device for the direct extra-virgin olive oil (EVOO) antioxidant capacity evaluation is proposed. The lab-made device is composed of a CO2 laser nanodecorated sensor combined with a cutter-plotter molded paper-strip designed for EVOOs sampling and extraction. Satisfactory performance towards the most representative o-diphenols of EVOOs i.e., hydroxytyrosol (HY) and oleuropein (OL) were achieved; good sensitivity (LODHY = 2 µM; LODOL = 0.6 µM), extended linear ranges (HY: 10–250 µM; OL: 2.5–50 µM) and outstanding reproducibility (RSD < 5%, n = 3) were obtained in rectified oil. The device was challenged for the extraction-free analysis of 15 different EVOO samples, with satisfactory recoveries (90–94%; RSD < 5%, n = 3) and correlation with classical photometric assays (r ≥ 0.91). The proposed device includes all analysis steps, needs 4 µL of sample, and returns reliable results in 2 min, resulting portable and usable with a smartphone.

Dual effects of extra virgin olive oil in acute wounds.

Extra virgin olive oil (EVOO) has proved beneficial effects in skin wound healing of chronic lesions; however, the effects of EVOO in acute wounds are not completely understood. This study investigated the effects of short-term and long-term administration of a diet rich in EVOO on acute wound healing. To check this, mice were fed with a diet rich in EVOO for 1 week (short term), 1 month, or 3 months (long term). The control group received a standard diet. Mouse macrophages were treated in vitro with EVOO or hydroxytyrosol (HT), which is the main EVOO polyphenol. Short-term administration of an EVOO rich diet in vivo increased lipid peroxidation and mRNA levels of pro-inflammatory cytokine levels and impaired acute wound closure. In contrast, long-term administration of an EVOO rich diet resulted in increased mRNA levels of anti-inflammatory cytokines and enhanced acute wound closure. In both in vivo and in vitro assays, the administration of EVOO or HT resulted in a predominantly anti-inflammatory macrophage phenotype. In conclusion, a diet rich in EVOO has a positive effect on acute wound healing that is dependent on the duration of EVOO administration. Short-term EVOO diet supplementation increases oxidative damage and pro-inflammatory responses, which impaired acute wound closure. On the other hand, long-term EVOO supplementation reduces oxidative damage and enhances anti-inflammatory responses, which improved acute wound closure. The effects of EVOO on oxidation and inflammation in acute wounds are linked to the EVOO polyphenol HT.

Optimization of low thermal treatments to increase hydrophilic phenols in the Alperujo liquid fraction

Hydrophilic phenols are the main bioactive compounds in alperujo. Among them, 3,4-Dihydroxyphenylglycol (DHPG), Hydroxytyrosol (HT) and Tyrosol (Ty), are the most relevant and deeply studied. These compounds exhibit high antioxidant capacity and a wide range of health benefits as well as technologically promising properties. Given that, their recovery represents an attractive opportunity to valorize this by-product. In this work low thermal treatments were applied to alperujo in order to obtain phenol-enriched liquid fractions. Optimization assays combining different levels of temperature (30 to 90 ºC), time (60 to 180 min) and water content (70 to 90%), followed by response surface methodologies were performed. The results indicated that by applying optimal conditions, is possible to obtain theoretical yields of Total phenols, DHPG, HT and Ty of 2.4, 957.8, 3.4 and 6.4 times greater, respectively, than raw dry alperujo. Interestingly, all the evaluated conditions can be reproduced with low investment in a standard olive oil industry.

Hydroxytyrosol attenuates ethanol-induced liver injury by ameliorating steatosis, oxidative stress and hepatic inflammation by interfering STAT3/iNOS pathway

ABSTRACTObjective: Hydroxytyrosol (HT) is a polyphenol with a wide range of biological activities. Excessive drinking can lead to oxidative stress and inflammation in the liver, which usually develop into alcohol liver disease (ALD). At present, there is no specific drug to treat ALD. In this paper, the protection effect of HT on ALD and the underline mechanism were studied.Methods: HepG2 cells were exposed to ethanol in vitro and C57BL/6J mice were fed with a Lieber-DeCarli ethanol liquid diet in vivo.Results: triglyceride (TG) level in serum and the expression of fatty acid synthase (FASN) were reduced significantly by the treatment with HT The acetaldehyde dehydrogenase (ALDH) activity was increased, the serum level of malondialdehyde (MDA) was decreased, catalase (CAT) and glutathione (GSH) were increased, suggesting that HT may reduce its oxidative damage to the body by promoting alcohol metabolism. Furthermore, according to the mRNA levels of tnf-α, il-6 and il-1β, HT inhibited ethanol-induced inflammation significantly. The anti-inflammatory mechanism of HT may be related to suppress the STAT3/iNOS pathway.Dissussion: Our study showed that HT could ameliorate ethanol-induced hepatic steatosis, oxidative stress and inflammation and provide a new candidate for the prevention and treatment of ALD.

NOTCH1: A Novel Player in the Molecular Crosstalk Underlying Articular Chondrocyte Protection by Oleuropein and Hydroxytyrosol.

Osteoarthritis (OA) is the most common joint disease, but no effective and safe disease-modifying treatment is available. Risk factors such as age, sex, genetics, injuries and obesity can concur to the onset of the disease, variably triggering the loss of maturational arrest of chondrocytes further sustained by oxidative stress, inflammation and catabolism. Different types of nutraceuticals have been studied for their anti-oxidative and anti-inflammatory properties. Olive-derived polyphenols draw particular interest due to their ability to dampen the activation of pivotal signaling pathways in OA. Our study aims to investigate the effects of oleuropein (OE) and hydroxytyrosol (HT) in in vitro OA models and elucidate their possible effects on NOTCH1, a novel therapeutic target for OA. Chondrocytes were cultured and exposed to lipopolysaccharide (LPS). Detailed analysis was carried out about the OE/HT mitigating effects on the release of ROS (DCHF-DA), the increased gene expression of catabolic and inflammatory markers (real time RT-PCR), the release of MMP-13 (ELISA and Western blot) and the activation of underlying signaling pathways (Western blot). Our findings show that HT/OE efficiently attenuates LPS-induced effects by firstly reducing the activation of JNK and of the NOTCH1 pathway downstream. In conclusion, our study provides molecular bases supporting the dietary supplementation of olive-derived polyphenols to revert/delay the progression of OA.

The Effect of Hydroxytyrosol in Type II Epithelial-Mesenchymal Transition in Human Skin Wound Healing.

Skin wound healing is a multiphase physiological process that involves the activation of numerous types of cells and is characterized by four phases, namely haemostasis, inflammatory, proliferative, and remodeling. However, on some occasions this healing becomes pathological, resulting in fibrosis. Epithelial mesenchymal transition (EMT) is an important process in which epithelial cells acquire mesenchymal fibroblast-like characteristics. Hydroxytyrosol (HT) is a phenolic compound extracted from olive oil and has been proven to have several health benefits. The aim of this study was to determine the effect of HT in type II EMT in human skin wound healing via cell viability, proliferation, migration, and proteins expression. Human dermal fibroblasts (HDF) isolated from skin samples were cultured in different concentrations of HT and EMT model, induced by adding 5 ng/mL of transforming growth factor-beta (TGF-β) to the cells. HT concentrations were determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells' migrations were evaluated using scratch and transwell migration assay. Protein expressions were evaluated via immunocytochemistry. The result showed that HT at 0.2% and 0.4% significantly increased the proliferation rate of HDF (p < 0.05) compared to control. Scratch assay after 24 h showed increased cell migration in cells treated with 0.4% HT (p < 0.05) compared to the other groups. After 48 h, both concentrations of HT showed increased cell migration (p < 0.05) compared to the TGF-β group. Transwell migration revealed that HT enhanced the migration capacity of cells significantly (p < 0.05) as compared to TGF-β and the control group. In addition, HT supplemented cells upregulate the expression of epithelial marker E-cadherin while downregulating the expression of mesenchymal marker vimentin in comparison to TGF-β group and control group. This study showed that HT has the ability to inhibit EMT, which has potential in the inhibition of fibrosis and persistent inflammation related to skin wound healing.

Efficacy of Hydroxytyrosol-Rich Food Supplements on Reducing Lipid Oxidation in Humans.

In the present study we report the efficacy of two food supplements derived from olives in reducing lipid oxidation. To this end, 12 healthy volunteers received a single dose (25 mL) of olive phenolics, mainly hydroxytyrosol (HT), provided as a liquid dietary supplement (30.6 or 61.5 mg HT), followed by an investigation of two reliable markers of oxidative stress. Blood and urine samples were collected at baseline and at 0.5, 1, 1.5, 2, 4, and 12 h post-intake. Plasma-oxidized low-density lipoprotein (oxLDL) cholesterol levels were measured with ELISA using a monoclonal antibody, while F2-isoprostanes (F2-IsoPs) were quantified in urine with UHPLC-DAD-MS/MS. Despite the great variability observed between individuals, a tendency to reduce lipoxidation reactions was observed in the blood in response to a single intake of the food supplements. In addition, the subgroup of individuals with the highest baseline oxLDL level showed a significant (p < 0.05) decrease in F2-IsoPs at 0.5 and 12 h post-intervention. These promising results suggest that HT supplementation could be a useful aid in preventing lipoxidation. Additionally, people with a redox imbalance could benefit even more from supplementing with bioavailable HT.

Abstract P2-07-01: Hydroxytyrosol, a Component of Olive Oil for Breast Cancer Prevention in Women at High Risk

Abstract Background Breast cancer is the most frequently diagnosed cancer in women in developed countries with increased incidence in women at high risk such as those with strong family history, BRCA mutations, atypical hyperplasia etc. Chemoprevention with drugs like tamoxifen and aromatase inhibitors come with challenges of intolerance and limited efficacy in estrogen receptor negative breast cancers. The purpose of our study was to evaluate the effects of hydroxytyrosol (HT), a component of olive oil on mammographic breast density reduction, which is a validated surrogate biomarker for breast cancer prevention as well as to explore on-target effects on Wingless-related integration site (Wnt) pathway, and alterations in key pathways of proliferation, DNA damage repair and stem cell function. Methods This study was conducted using 25mg/day oral dose of HT for 12 months in both pre- and post-menopausal women who are at increased risk of breast cancer with Gail 5-year risk score ≥ 1.67 and/or &amp;gt; 10% probability of BRCA mutation and had declined standard chemoprevention. These women underwent annual mammograms as well as had the option to have a biopsy of normal breast tissue before and after HT. Maximum volumetric breast density (Max VBD%) was assessed quantitatively using Volpara software (VIS 3.2) and the annualized percent decrease in max VBD% between baseline (BL) and end of treatment (EOT) with HT was analyzed in the study. RNA was extracted from the breast biopsies and RNA sequencing (RNA-Seq) and multiplex analysis of 28 proteins using NanoString nCounter analysis was performed to compare the BL with EOT samples. Results A total of 61 women were screened for the trial, 51 were enrolled and 41 patients completed the study. Median age for the study population was 54 years (range 35-76 years). There were 26 patients with paired BL and EOT mammograms. There were 14 patients with paired BL and EOT breast biopsies and seven patients with only BL and seven with only EOT biopsies. Mammographic density as measured by max VBD% showed a non-significant change of -0.038%, p = 0.49. However, on subgroup analysis in women 60-years or older, the mean decrease in max VBD% was - 3.7% (p = 0.0391), especially in those with high baseline mammographic density (&amp;gt; 10% baseline max VBD%). RNAseq data was generated from 15 BL and 17 EOT samples and pathways were confirmed by NanoString. Our bioinformatics pipeline identified 190 transcripts that were upregulated and 90 that were downregulated in EOT vs. BL by at least 1.5 fold. Reactome pathway R-HSA-195721 (Signaling by WNT) is identified as the top pathway enriched with EOT downregulated genes. On further review of the expression profiles, 82 of 261 member genes had p-value &amp;lt; 0.05 for EOT vs. BL, among which only 4 were upregulated and 78 were downregulated in EOT samples. These 82 genes were highly interactive with each other, with 379 high confidence protein-protein interactions (PPIs) (confidence score &amp;gt; 0.7 from STRING database) involving 81 out of 82 genes. In addition, mitotic telophase/cytokinesis (p = 0.004), ATM signaling (p = 0.007) were some pathways that were upregulated whereas, NOTCH signaling (p = 0.007), oxidative stress induced senescence (p = 0.0001) pathways were downregulated in EOT vs. BL. Conclusions Hydroxytyrosol, a component of olive oil did not show a decrease in breast density in the intention to treat population but showed some reduction in breast density in women 60-years of age and above, especially in those with high baseline breast density. HT showed on target effects by affecting Wnt signaling pathway, as well as decrease in proliferation. This study lays the foundation for future larger studies in exploring a natural compound such as HT for chemoprevention of breast cancer. Citation Format: Akshjot Puri, Zheng Yin, Sergio Granados-Principal, Joe Ensor, Liliana Guzman, Roberto Rosato, Hong Zhao, Stephen Wong, Tejal Patel, Jenny Chang. Hydroxytyrosol, a Component of Olive Oil for Breast Cancer Prevention in Women at High Risk [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-07-01.