The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol prevents TNF-α-mediated impairment of mineralization in mouse osteoblastic MC3T3-E1 cells: Impact of macrophage activation.

The phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), found in the Pacific oyster Crassostrea gigas, is a superior peroxyl radical scavenger compared to other materials, including Trolox. DHMBA may play an important role in the prevention of health disorders. This study elucidates whether DHMBA prevents the impairment of mineralization of mouse osteoblastic MC3T3-E1 cells under inflammatory conditions by using mouse macrophage RAW264.7 cells in vitro. Culturing with DHMBA (1-100 µM) did not affect the proliferation and death of MC3T3-E1 cells. DHMBA stimulated osteoblastic mineralization. DHMBA blocked the decrease in mineralization of MC3T3-E1 cells caused by culture with the inflammatory cytokine TNF-α. DHMBA inhibited the production of TNF-α by stimulation with lipopolysaccharide (LPS) in RAW264.7 cells. The growth of MC3T3-E1 cells was suppressed by coculture with macrophages under LPS stimulation through the crosstalk of both cells. Interestingly, the growth of MC3T3-E1 cells was suppressed by culturing with the conditioned medium obtained by culturing macrophages with LPS. The effect of the conditioned medium was blocked by the presence of DHMBA or Bay 11-7082, an inhibitor of the TNF-α pathway. The blocking effect of DHMBA was not further enhanced in the presence of Bay 11-7082. Mechanistically, DHMBA was found to decrease the levels of NF-κB p65 and the activity of NF-κB reporter expression in MC3T3-E1 cells. DHMBA was shown to prevent the impairment of osteoblastic mineralization via TNF-α signaling involved in macrophage activation in the bone marrow microenvironment. This study may provide a novel strategy for the therapy of osteoblastic impairment.

A Pacific Oyster-Derived Antioxidant, DHMBA, Protects Renal Tubular HK-2 Cells against Oxidative Stress via Reduction of Mitochondrial ROS Production and Fragmentation.

The kidney contains numerous mitochondria in proximal tubular cells that provide energy for tubular secretion and reabsorption. Mitochondrial injury and consequent excessive reactive oxygen species (ROS) production can cause tubular damage and play a major role in the pathogenesis of kidney diseases, including diabetic nephropathy. Accordingly, bioactive compounds that protect the renal tubular mitochondria from ROS are desirable. Here, we aimed to report 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), isolated from the Pacific oyster (Crassostrea gigas) as a potentially useful compound. In human renal tubular HK-2 cells, DHMBA significantly mitigated the cytotoxicity induced by the ROS inducer L-buthionine-(S, R)-sulfoximine (BSO). DHMBA reduced the mitochondrial ROS production and subsequently regulated mitochondrial homeostasis, including mitochondrial biogenesis, fusion/fission balance, and mitophagy; DHMBA also enhanced mitochondrial respiration in BSO-treated cells. These findings highlight the potential of DHMBA to protect renal tubular mitochondrial function against oxidative stress.

The Marine Factor 3,5-dihydroxy-4-methoxybenzyl Alcohol Suppresses Cell Growth, Inflammatory Cytokine Production, and NF-κB Signaling-enhanced Osteoclastogenesis in In Vitro Mouse Macrophages RAW264.7 Cells.

BACKGROUND AND OBJECTIVE: The novel marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) was originally identified in the Pacific oyster Crassostrea Gigas. DHMBA has been shown to prevent oxidative stress by scavenging radicals and enhance the production of antioxidant proteins. However, the pharmacologic role of DHMBA has been poorly understood. Inflammation is implicated in the pathogenesis of many diseases. Inflammatory cytokines are produced in macrophages with stimulation of lipopolysaccharide (LPS) and are used as biomarkers that cause diverse disease conditions. Therefore, this study has been undertaken to elucidate whether DHMBA expresses anti-inflammatory effects in in vitro mouse macrophage RAW264.7 cells. METHODS: Mouse macrophage RAW264.7 cells were cultured in a medium containing 10% fetal bovine serum (FBS) with or without DHMBA (1-1000 µM). RESULTS: Culturing with DHMBA (1-1000 µM) suppressed the growth and stimulated the death of RAW264.7 cells in vitro, leading to a decrease in cell number. Treatment with DHMBA reduced the levels of Ras, PI3K, Akt, MAPK, phospho-MAPK, and mTOR, which are signalling factors to promote cell proliferation, and it raised the levels of p53, p21, Rb, and regucalcin, which are cell growth suppressors. DHMBA treatment elevated caspase-3 and cleaved caspase-3 levels. Interestingly, DHMBA treatment repressed the production of inflammatory cytokines, including tumor necrosis factor-α, interleukin-6, interleukin-1ß, or prostaglandin E2, which were enhanced by LPS stimulation. Notably, the levels of NF-κB p65 increased by LPS treatment, and this augmentation was repressed by DHMBA treatment. Moreover, LPS treatment stimulated osteoclastogenesis of RAW264.7 cells. This stimulation was blocked by DHMBA treatment, and this effect was not caused by the presence of an NF-κB signalling inhibitor. CONCLUSION: DHMBA was found to potentially suppress the activity of inflammatory macrophages in vitro, suggesting its therapeutic usefulness in inflammatory conditions.

The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol suppresses growth, migration and invasion and stimulates death of metastatic human prostate cancer cells: targeting diverse signaling processes.

Prostate cancer is metastatic cancer and is the second leading cause of cancer-related death in men. It is needed to develop more effective treatment for metastatic prostate cancer. The present study investigates whether the novel factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), which was isolated from marine oyster, suppresses the activity of metastatic human prostate cancer PC-3 or DU-145 cells. Culture of DHMBA (1 or 10 µM) suppressed colony formation and growth of PC-3 or DU-145 cells in vitro. Suppressive effects of DHMBA on cell proliferation were not occurred by culturing with intracellular signaling inhibitors. Mechanistically, DHMBA (10 µM) reduced the levels of key proteins linked to promotion of cell growth, including Ras, PI3K, Akt, MAPK, and mTOR in PC-3 cells. Interestingly, DHMBA increased the levels of cancer suppressor p53, p21, Rb, and regucalcin. Moreover, culture of DHMBA simulated the death of PC-3 and DU-145 cells. This effect was implicated to caspase-3 activation in cells. Interestingly, the effects of DHMBA on cell proliferation and death were blocked by culturing with an inhibitor of aryl hydrocarbon receptor linked to transcriptional regulation. Furthermore, culture of DHMBA inhibited production of reactive oxygen species in PC-3 or DU-145 cells. Of note, DHMBA blocked migration and invasion by diminishing their related protein levels, including NF-κB 65, caveolin-1 and integrin ß1. The novel marine factor DHMBA was demonstrated to suppress metastatic prostate cancer cells via targeting diverse signaling pathways. This study may provide a new strategy for prostate cancer therapy with DHMBA.

The Phenolic Antioxidant 3,5-dihydroxy-4-methoxybenzyl Alcohol (DHMBA) Prevents Enterocyte Cell Death under Oxygen-Dissolving Cold Conditions through Polyphyletic Antioxidant Actions.

Cold preservation in University of Wisconsin (UW) solution is not enough to maintain the viability of the small intestine, due to the oxidative stress. The novel phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) has dual properties to reduce oxidative stress, radical scavenging, and antioxidant protein induction, in other cells. This study was designed to determine whether DHMBA reduces cold preservation injury of enterocytes, and to identify the effector site. Enterocytes were subjected to 48-h cold preservation under atmosphere in UW solution (±DHMBA), and then returned to normal culture to replicate reperfusion of the small intestine after cold preservation. At the end of cold preservation (ECP) and at 1, 3, 6, and 72 h after rewarming (R1h, R3h, R6h, and R72h), we evaluated cell function and the injury mechanism. The results showed that DHMBA protected mitochondrial function mainly during cold preservation, and suppressed cell death after rewarming, as shown by the MTT, ATP, mitochondrial membrane potential, LDH, and lipid peroxidation assays, together with enhanced survival signals (PI3K, Akt, p70S6K) and induction of antioxidant proteins (HO-1, NQO-1, TRX-1). We found that DHMBA mitigates the cold-induced injury of enterocytes by protecting the mitochondria through direct and indirect antioxidative activities.

Flazin as a Promising Nrf2 Pathway Activator.

Flazin is a ß-carboline-derived alkaloid found in Japanese fermented foods. Here, the potential of flazin as an antioxidant food was studied with particular reference to its effect on the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) system in human hepatocytes (C3A). Flazin and flazin analogues including the decarboxylated derivative perlolyrine were chemically synthesized and compared with each other and with chlorogenic acid and curcumin. Among these compounds, flazin showed the lowest cytotoxicity (IC50 < 500 µM) and the highest capacity to activate the Keap1-Nrf2 system. It provided the largest (>3-fold of the control) cytoprotection ability against a pro-oxidant, although its radical absorbance capacity was relatively low. Flazin increased the expressions of Nrf2-dependent phase II enzyme genes and their products (NQO1, GSTP, and GSH proteins). The strong cytoprotection ability of flazin associated with low log P (0-3) is shared by sulforaphane and 3,5-dihydroxy-4-methoxybenzyl alcohol, suggesting the potential value of flazin and flazin-rich foods for the prevention of oxidation-related health disorders.

Preventive Effect of 3,5-dihydroxy-4-methoxybenzyl Alcohol (DHMBA) and Zinc, Components of the Pacific Oyster Crassostrea gigas, on Glutamatergic Neuron Activity in the Hippocampus.

The effects of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), and zinc--both components of the Pacific oyster Crassostrea gigas--were examined by glutamatergic neuron activity in rats in an in vivo microdialysis experiment and an in vitro brain slice experiment. The basal concentration of extracellular glutamate in the hippocampus was decreased under hippocampal perfusion with DHMBA (1 mmol l(-1)) or ZnCl2 (µmol l(-1)), indicating that DHMBA and Zn(2+) suppress glutamatergic neuron activity under basal (static) conditions. To assess the preventive effect of DHMBA and Zn(2+) on glutamate release from neuron terminals, brain slices were pretreated with DHMBA (1 mmol l(-1)) or ZnCl2 (100 nmol l(-1)) for 1 h, then stimulated with high K(+). A high, K(+)-induced increase in extracellular Zn(2+) level, an index of glutamate release, was suppressed with pretreatment with DHMBA or zinc. A high, K(+)-induced increase in intracellular Ca(2+) level was also suppressed with pretreatment with DHMBA or Zn(2+). These results suggest that DHMBA and Zn(2+), previously taken up in the hippocampal cells, suppress high, K(+)-induced glutamate release in the hippocampus, probably via presynaptic suppression of intracellular Ca(2+) signaling. It is likely that Zn(2+) and DHMBA play a preventive role in suppressing excess glutamatergic neuron activity in rats and mice.

Mass Spectrometric Quantification of Amphipathic, Polyphenolic Antioxidant of the Pacific Oyster (Crassostrea gigas).

A novel amphipathic phenolic compound, 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), that can be isolated from the Pacific oyster (Crassostrea gigas) has been found to protect human hepatocytes against oxidative stress. This study aims to establish a method for the measurement of DHMBA for industrial application. Liquid chromatography-tandem mass spectrometry using deuterated DHMBA as an internal standard and a polar end-capped ODS (Hypersil GOLD aQ) as the solid phase was validated. The limit of detection was 0.04 pmol (S/N = 5), and the limit of quantitation was 0.1 pmol (S/N = 10). The calibration curve was linear throughout the range of 0.1 - 16 pmol (r(2) = 0.9995). This method successfully quantified DHMBA in oysters from 11 sea areas in Japan. The results showed that the yield of DHMBA was variable from 9.8 to 58.8 µg g(-1) whole oyster meat wet weight but not affected by the seawater temperature. The proposed LC-MS/MS method is useful in quantitative studies for DHMBA and potentially for other amphipathic substances.

Anti-apoptotic effects of novel phenolic antioxidant isolated from the Pacific oyster (Crassostrea gigas) on cultured human hepatocytes under oxidative stress.

The antioxidant, and hepatoprotective properties of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), a natural phenolic antioxidant isolated from the Pacific oyster, were defined using cultured human hepatocyte-derived cells (C3A). DHMBA showed no cytotoxicity at 62.5-500µM, as well as chlorogenic acid (CGA), vitamin C, and vitamin E. However, butylated hydroxytoluene, eicosapentaenoic acid, docosahexaenoic acid and catechin reduced cell viability. In the presence of the prooxidant 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), DHMBA at 125-500µM improved cell viability, whereas CGA had no effect. DNA ladder formation and flow-cytometric studies indicated that DHMBA inhibited AAPH-induced apoptosis and necrosis. CGA was ineffective. Thus, DHMBA is a novel, potent antioxidant, effectively protecting cultured hepatocytes from apoptosis and necrosis caused by oxidative stress. Additionally, the concentration of DHMBA was determined by mass spectrometry to be 24.4µmol/kg wet oyster meat, and three polyphenols (gentisic acid, daidzein, and matairesinol) were newly identified in the oyster extracts.

Isolation and characterization of a phenolic antioxidant from the Pacific oyster (Crassostrea gigas).

Using an oxygen radical absorbance capacity (ORAC) assay, antioxidant activity was detected in the ethanol extract of the Pacific oyster, which was purified by sequential extraction with organic solvents. The ethyl acetate fraction showed the strongest antioxidant activity and was further purified, yielding a single compound [as assessed by thin-layer chromatography (TLC) and reverse-phase high-performance liquid chromatography (HPLC)]. This compound was identified as 3,5-dihydroxy-4-methoxybenzyl alcohol on the basis of (1)H and (13)C nuclear magnetic resonance (NMR), heteronuclear multiple-bond correlation (HMBC), and electrospray ionization-mass spectrometry (ESI-MS) spectral analyses, a conclusion that was confirmed by chemical synthesis. The concentration of the compound was 6.7 mg/100 g of whole oyster meat wet weight. This amphiphilic antioxidant retarded the copper-mediated oxidation of low-density lipoproteins (LDLs) and the generation of thiobarbituric acid reactive substances. Furthermore, the compound showed substantial antioxidant activity using the ORAC and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays compared to natural antioxidants. Although the same compound was previously found in brown algae, its presence in other organisms and antioxidant activity are reported here for the first time.

Significance of serum glucocorticoid and chelatable zinc in depression and cognition in zinc deficiency.

Dietary zinc deficiency elicits neuropsychological symptoms and cognitive dysfunction. To pursue the mechanisms of these symptoms, in the present study, the relationship among serum glucocorticoid, chelatable zinc in the synaptic cleft and brain function based on behavior was examined in young rats fed a zinc-deficient diet for 4 weeks. Serum glucocorticoid level was significantly increased in zinc-deficient rats. However, the induction of in vivo dentate gyrus LTP and object recognition memory were not affected in zinc-deficient rats. Chelatable zinc levels were decreased in the stratum lucidum of the hippocampal CA3, but not in the molecular layer of the dentate gyrus. It is reported that dentate gyrus LTP and object recognition memory are affected in clioquinol (30mg/kg)-administered rats, in which chelatable zinc is significantly decreased in the molecular layer of the dentate gyrus. Thus, the significant decrease in chelatable zinc in the molecular layer of the dentate gyrus may be required for object recognition memory deficit in zinc deficiency. On the other hand, the time of grooming in the open-field test was decreased in zinc-deficient rats. Immobility time in the forced swim test was increased in zinc-deficient rats, but not in clioquinol-administered rats, in which chelatable zinc was more markedly decreased than in zinc-deficient rats, suggesting that the lack of chelatable zinc does not increase depression-like behavior. These results suggest that the chronic increase in serum glucocorticoid level is involved in the increase in depression-like behavior rather than the decrease in chelatable zinc after 4-week zinc deficiency.

A phenolic antioxidant from the Pacific oyster (Crassostrea gigas) inhibits oxidation of cultured human hepatocytes mediated by diphenyl-1-pyrenylphosphine.

3,5-Dihydroxy-4-methoxybenzyl alcohol (DHMBA), an antioxidant isolated from the Pacific oyster (Crassostrea gigas), was studied in a cell-based fluorometric antioxidant assay using human hepatocyte-derived cells (C3A) and diphenyl-1-pyrenylphosphine (DPPP) as a fluorescent probe. In comparison with two hydrophilic antioxidants, DHMBA showed the stronger inhibition of DPPP-mediated fluorescence than chlorogenic acid and l-ascorbic acid: at a concentration of 320 µM of DPPP, the inhibition was 26.4±2.6%, 11.1±1.2%, and 0±2.0% for DHMBA, chlorogenic acid, and l-ascorbic acid, respectively (mean±SD, n=4). Their relative oxygen radical absorbance capacities (ORAC) were dissociated with their cell-based antioxidant activities: 1.47±0.40, 4.57±0.30, and 0.53±0.13 µmol TE/µmol for DHMBA, chlorogenic acid, and l-ascorbic acid, respectively (mean±SD, n=4). The amphiphilicity of DHMBA was better than chlorogenic acid and l-ascorbic acid might underlie this dissociation. Since the C3A cells are human hepatoma-derived cells, DHMBA might be useful in the prevention and treatment of liver diseases by involving an oxidation process.

Susceptibility to stress in young rats after 2-week zinc deprivation.

Dietary zinc deficiency elicits abnormal behavior in stressful environment. It is possible that abnormal corticosterone secretion in zinc deficiency is linked to abnormal behavior. To understand the increase in depression-like behavior in zinc deficiency, in the present study, serum corticosterone concentration was checked in young rats fed a zinc-deficient diet for 2 weeks after exposure to acute stress. Serum corticosterone concentration was higher in zinc-deficient rats after exposure to water-immersed and forced swim stress. Immobility time in the forced swim test was significantly increased in zinc-deficient rats, but not in pair-fed rats, suggesting that the increase in depression-like behavior is due to zinc deficiency rather than decreased food intake. The increase in immobility time in zinc deficiency was restored to the control level by feeding of the control diet. In dexamethasone suppression test, serum corticosterone concentration was markedly decreased in both the control and zinc-deficient rats. These results suggest that excessive corticosterone secretion after exposure to stress is linked to the increase in depression-like behavior in zinc deficiency. It has been reported that exposure to stress and glucocorticoids facilitates the increase in extracellular glutamate in the hippocampus. When the hippocampus was stimulated with 100mM KCl, the concentration of extracellular glutamate was more increased in zinc-deficient rats. In hippocampal slices from zinc-deficient rats, the decrease in FM4-64 fluorescence (exocytosis) was more facilitated. It is likely that zinc deficiency excessively excites glutamatergic neurons in the hippocampus after exposure to acute stress. This excessive excitation seems to contribute to susceptibility to stress after 2-week zinc deprivation and its related behavior such as the increase in depression-like behavior.