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Abstract

The aim of the present study was to assess the effects of l-buthionine-(S,R)-sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor, and GBR 12909, a dopamine reuptake inhibitor, administered alone or in combination to Sprague-Dawley rats during early postnatal development (p5–p16), on the levels of reactive oxygen species (ROS), lipid peroxidation (LP) and the activities of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione disulfide reductase (GR) in peripheral tissues (liver, kidney) and selected brain structures (prefrontal cortex, PFC; hippocampus, HIP; and striatum, STR) of 16-day-old rats. The studied parameters were analyzed with reference to the content of GSH and sulfur amino acids, methionine (Met) and cysteine (Cys) described in our previous study. This analysis showed that treatment with a BSO + GBR 12909 combination caused significant decreases in the lipid peroxidation levels in the PFC and HIP, in spite of there being no changes in ROS. The reduction of lipid peroxidation indicates a weakening of the oxidative power of the cells, and a shift in balance in favor of reducing processes. Such changes in cellular redox signaling in the PFC and HIP during early postnatal development may result in functional changes in adulthood.

2.1. Animals and Treatment

To create the neurodevelopmental model of schizophrenia, pregnant Sprague-Dawley females were delivered to our laboratory by the Charles River Company (Sulzfeld, Germany). They were kept in individual cages, with free access to standard laboratory food and tap water under standard laboratory conditions i.e., at room temperature (22 °C) and under an artificial light/dark cycle (12/12 h). On the day of delivery, the sex of pups was determined. Only male pups which were left with their mothers were treated on the postnatal days p5–p16, with the selective inhibitor of GCL, compound l-buthionine-(S,R)-sulfoximine (BSO) and the DA reuptake inhibitor GBR 12909, alone or in combination. BSO at a dose of 3.8 mmol/kg s.c., was administered once daily, while GBR 12909 at a dose of 5 mg/kg s.c., every second day. Both model compounds were dissolved in 0.9% NaCl. Under the combined treatment regimen, BSO administration proceeded GBR 12909 injection. Control rats instead of the model compounds received a 0.9% NaCl once daily. Every day, pups were weighed and the injected volumes of the studied model compounds were adjusted accordingly with the actual body weight. On postnatal day p16, the rats were killed by decapitation 4 h after the last doses of model compounds, and their liver, kidney and selected brain structures (PFC, HIP, STR) were dissected, immediately frozen on dry ice and stored at −80 °C until further biochemical analysis.

Abstract

Bone remodeling and repair require osteogenic cells to reach the sites that need to be rebuilt, indicating that stimulation of osteoblast migration could be a promising osteoanabolic strategy. We showed that purified δ-tocotrienol (δ-TT, 10 μg/mL), isolated from commercial palm oil (Elaeis guineensis) fraction, stimulates the migration of both MC3T3-E1 osteoblast-like cells and primary human bone marrow mesenchymal stem cells (BMSC) as detected by wound healing assay or Boyden chamber assay respectively. The ability of δ-TT to promote MC3T3-E1 cells migration is dependent on Akt phosphorylation detected by Western blotting and involves Wnt/β-catenin signalling pathway activation. In fact, δ-TT increased β-catenin transcriptional activity, measured using a Nano luciferase assay and pretreatment with procaine (2 µM), an inhibitor of the Wnt/β-catenin signalling pathway, reducing the wound healing activity of δ-TT on MC3T3-E1 cells. Moreover, δ-TT treatment increased the expression of β-catenin specific target genes, such as Osteocalcin and Bone Morphogenetic Protein-2, involved in osteoblast differentiation and migration, and increased alkaline phosphatase and collagen content, osteoblast differentiation markers. The ability of δ-TT to enhance the recruitment of BMSC, and to promote MC3T3-E1 differentiation and migratory behavior, indicates that δ-TT could be considered a promising natural anabolic compound.

2.2. Effect of δ-TT on MC3T3-E1 and BMSC Cell Migration

We then studied the effects of δ-TT on cell migration, performing a scratch assay. MC3T3-E1 cells were exposed to δ-TT 24 h before and 24 h after the wound gap has been applied. As shown in , MC3T3-E1 cells treated with δTT exhibited a faster wound healing rate compared to that of control untreated cells at 18 and 24 h, reaching a statistical significance at 24 h.

2.2. Effect of δ-TT on MC3T3-E1 and BMSC Cell Migration

Lastly, we examined the effects of δ-TT on BMSC cell migration, performing a micro-chemotaxis assay in a Boyden chamber. BMCS exposed to δ-TT 24 h before and during micro-chemotaxis assay, showed a significant increase in cell migration (170%) in comparison to the control-treated cells ().

Abstract

Domoic acid (DA), the main toxin responsible for Amnesic Shellfish Poisoning, frequently affects the marine resources of Chile and other countries across the South Pacific, thus becoming a risk for human health. One of the affected resources is the scallop Argopecten purpuratus. Even though this species has a high commercial importance in Northern Chile and Peru, the characteristics of its DA depuration are not known. In this work, the DA depuration was studied by means of two experiments: one in controlled (laboratory) and another in natural conditions. All organs of A. purpuratus depurated the toxin very quickly in both experiments. In some organs, an increase or a very small decrease of toxin was detected in the early depuration steps. Several models were used to describe this kinetics. The one that included toxin transfer between organs and independent depuration from each organ was the model that best fit the data. It seems, therefore, that the DA in this species is quickly transferred from the digestive gland to all other organs, which release it into the environment. Physiological differences in the two experiments have been shown to have some effect on the depuration from each organ but the actual reasons are still unknown.

2. Results

Two DA depuration experiments were carried out using scallops which had fed on a naturally occurring bloom of toxic Pseudo-nitzschia australis. In the first experiment, scallops were removed from the field, held in the laboratory, and fed non-toxic Isochrysis galbana (Haptophyta). Changes in body weight and tissue-specific DA concentration were then measured for 12 days. In the second, experiment the scallops were maintained in the field and allowed to feed naturally on a post-bloom phytoplankton population containing only a relatively small population of Pseudo-nitzschia australis.

2.3.1. Depuration of Domoic Acid in the Natural Environment

Two types of models were fitted to the experimental data of the toxin burden. The first one was first-order exponential decrease of each organ, without toxin transfer between organs, and the second included toxin transfers from DG to all other organs, which depurated in the same way than in the first type of model. When fitted to the data from the experiment carried out in natural conditions (the complete dataset) the model without transfers of DA from the digestive gland to other organs was unable to correctly describe the depuration, especially because of the increase of toxin burden in the second day of the experiment in some organs. When the transfer between organs was included, the model fits the observed data much better (). The simplest model of this type, with only a common transfer rate to all organs and a common depuration rate, described the general shape of the kinetics but did not fit well the levels of DA in several organs, especially in DG (with levels noticeably underestimated).

2.3.2. Depuration of Domoic Acid in Laboratory

The same model fitted to the data obtained in the laboratory gave different estimates, but the amount of useful observations was very low because of the steep toxin burden descent (). The model estimates some transfer of toxin, but the organs to which the toxin transfer was estimated to be higher did not coincide with those in the experiment carried out in natural conditions (). Some of the observed differences could be explained if the depuration of the different organs does take place totally or partially through the kidney; unfortunately, it was not separated from the adductor muscle in any of the experiments. This may account for the increase of DA in Mu (adductor muscle plus kidney) during the initial steps of the laboratory experiment, in which the depuration rate was very high.

3.2. Domoic Acid Depuration

There were important differences in the apparent depuration rate between the two experiments. Scallops in laboratory depurated faster than in natural conditions (0.91 vs. 0.27 day−1, respectively). Most of the difference is probably due to some degree of reintoxication in natural conditions, because the toxic algae populations had not completely disappeared (). The two experiments also differed at least in temperature and food availability, and both could have contributed to the observed difference. The difference in the temperature at which the experiments were developed (16 °C in the laboratory and 13–14 °C at natural conditions) could have contributed to the difference in depuration rate, since temperature seems to accelerate the DA depuration in other species, like the king scallop Pecten maximus [] or the detoxification of paralytic shellfish toxins in the visceral mass of the surf clam Spisula solidissima []. The magnitude of the recorded difference seems, nevertheless, too high to be the only factor, as the estimated Q10 (temperature coefficient) is near 6, while, for most physiological rates in bivalves it is between 2 and 3 [,]. Food availability would not seem to be primarily responsible because in sight of its likely negative effect on depuration in the king scallop, Pecten maximus []. Nevertheless, its effect cannot be ruled out because the physiological differences between the two species could be important.

Abstract

This review considers the results of recent studies on marine excitatory amino acids, including kainic acid, domoic acid, dysiherbaine, and neodysiherbaine A, known as potent agonists of one of subtypes of glutamate receptors, the so-called kainate receptors. Novel information, particularly concerning biosynthesis, environmental roles, biological action, and syntheses of these marine metabolites, obtained mainly in last 10–15 years, is summarized. The goal of the review was not only to discuss recently obtained data, but also to provide a brief introduction to the field of marine excitatory amino acid research.

1. Introduction

Throughout the 55-year history of the G.B. Elyakov Pacific Institute of Bioorganic Chemistry (PIBOC), Far Eastern Branch, Russian Academy of Science, a vast variety of new marine-derived natural compounds including different glycosides, polar steroids, alkaloids, lipids, and other metabolites have been isolated from echinoderms and sponges, ascidians, algae, fungi, and marine bacteria and thoroughly studied. Many of the discovered new natural compounds proved to have a protective action and were repeatedly mentioned in well-known series of review articles published in Natural Products Reports []. To protect themselves against predators and pathogens, marine organisms use a variety of structural groups of toxins. The latter are synthesized by lower plants, including microalgae [,,], and then accumulated by some invertebrates that transfer them up to higher trophic levels. Marine excitatory amino acids (MEAAs) that belong to this category and play an important role in marine communities have found application in pharmacology. To date, there are only three known groups of marine-derived excitatory amino acids: (1) kainic acid and related compounds, (2) domoic acid and some its derivatives, (3) sponge-derived dysiherbaine and neodysiherbaine A, which specifically bind and activate the kainate-type of glutamate receptors (GluRs).

Abstract

A liquid chromatography–high resolution mass spectrometry (LC-HRMS) method for the direct determination of various saturated hydroxy fatty acids (HFAs) in milk was developed for the first time. The method involves mild sample preparation conditions, avoids time-consuming derivatization procedures, and permits the simultaneous determination of 19 free HFAs in a single 10-min run. This method was validated and applied in 17 cow milk and 12 goat milk samples. This work revealed the existence of various previously unrecognized hydroxylated positional isomers of palmitic acid and stearic acid in both cow and goat milk, expanding our knowledge on the lipidome of milk. The most abundant free HFAs in cow milk were proven to be 7-hydroxystearic acid (7HSA) and 10-hydroxystearic acid (10HSA) (mean content values of 175.1 ± 3.4 µg/mL and 72.4 ± 6.1 µg/mL in fresh milk, respectively). The contents of 7HSA in cow milk seem to be substantially higher than those in goat milk.

2.1. LC-ESI-MS Data

Nineteen SHFAs were used in the present study. Eight of them were regio-isomers of hydroxy palmitic acid [2-hydroxypalmitic acid (2HPA), 3-hydroxypalmitic acid (3HPA), 7-hydroxypalmitic acid (7HPA), 8-hydroxypalmitic acid (8HPA), 9-hydroxypalmitic acid (9HPA), 10-hydroxypalmitic acid (10HPA), 11-hydroxypalmitic acid (11HPA), 16-hydroxypalmitic acid (16HPA)], and seven were regio-isomers of stearic acid [2-hydroxystearic acid (2HSA), 3-hydroxystearic acid (3HSA), 7-hydroxystearic acid (7HSA), 8-hydroxystearic acid (8HSA), 9-hydroxystearic acid (9HSA), 10-hydroxystearic acid (10HSA), 12-hydroxystearic acid (12HSA)]. In addition, four 3-hydroxy FAs [3-hydroxycapric acid (3HCA), 3-hydroxylauric acid (3HLA), 3-hydroxymyristic acid (3HMA), 3-hydroxypentadecanoic acid (3HPDA)] were included in the set of compounds tested. The high-resolution mass spectra of these HFAs were recorded in electrospray ionization (ESI) negative mode. The structures of the SHFAs used along with the exact masses of the deprotonated molecules and the retention times observed for each one of them in the chromatographic method are presented in . The MS/MS spectra of all the 19 analytes in negative ESI mode are also presented in the .

2.3. Analysis of Samples

Seventeen cow milk samples and 12 goat milk samples were purchased from the local market and analyzed. The extracted ion chromatograms (EICs) of a cow milk sample (A) and a goat milk (B) sample are shown in . The simultaneous determination of 19 SHFAs was achieved in a single 10-min run. The contents of the 19 analytes in milk samples (in triplicate) are summarized in , and they are expressed as the µg of HFA per mL of milk. The integration of the peak areas was performed manually using MultiQuant 3.0.2. As Hill et al. discussed [], in particular cases a manual baseline may be employed for the quantification of the peak areas. The same integration parameters were used in all cases.

Abstract

Thiamin deficiency, or beriberi, is an increasingly re-recognized cause of morbidity and mortality in the developing world. Thiamin status has traditionally been measured through the erythrocyte activation assay (ETKA) or basal transketolase activity (ETK), which indirectly measure thiamin diphosphate (TDP). Thiamin diphosphate can also be measured directly by high-performance liquid chromatography (HPLC), which may allow a more precise estimation of thiamin status. We compared the direct measurement of TDP by HPLC with basal ETK activity and ETKA in 230 patients with Plasmodium falciparum malaria in rural southern Laos without overt clinical beriberi, as part of a trial of thiamin supplementation. Admission thiamin status measured by basal ETK activity and ETKA (α) were compared with thiamin status assessed by the measurement of TDP by HPLC. 55% of 230 included patients were male, and the median age was 10 (range 0.5–73) years. Using α ≥ 25% as the gold standard of thiamin deficiency, the sensitivity of TDP < 275 ng/gHb as a measure of thiamin deficiency was 68.5% (95% CI: 54.4–80.5%), with specificity of 60.8 (95% CI: 53.2–68.1%). There was a significant inverse correlation between the results of the two tests (Kendall’s tau = −0.212, P < 0.001). Basal ETK activity was also significantly positively correlated with TDP levels (Kendall’s tau = 0.576, P < 0.001). Thiamin diphosphate measurement may have a role in measuring thiamin levels in clinical settings. Further studies evaluating TDP concentration in erythrocytes with basal ETK activity and ETKA (α) in beriberi patients would help establish comparative values of these assays.

RESULTS

Using α ≥ 25% as the gold standard of biochemical thiamin deficiency, the sensitivity of erythrocyte TDP < 275 ng/gHb as a measure of thiamin deficiency was 68.5% (95% CI: 54.4–80.5%) with specificity of 60.8 (95% CI: 53.2–68.1%). There was a significant inverse correlation between the tests (Kendall’s tau = −0.212, P < 0.001). Inverse correlation was also observed between the two assays when adults () (78 patients, Kendall’s tau = −0.227, P = 0.003) and children () (152 patients, Kendall’s tau = −0.176, P = 0.001) were analyzed separately.

DISCUSSION

Limitations of this study include the fact that no patients in the cohort manifested clinical signs of thiamin deficiency, so it is not possible to correlate results with clinical thiamin deficiency. It is possible that some patients had subclinical deficiency. Determining a clinical endpoint for thiamin deficiency is challenging, as beriberi seems to occur not only when thiamin deficiency exists but also when additional exacerbating factors such as infections are present. The distinction between those who do and do not show clinical symptoms of beriberi at different biochemical assay levels may also be confounded by other factors. Importantly, these data cannot be used to inform biochemical diagnosis of infantile beriberi as children aged < 6 months were not included.

Abstract

Cytochrome P450 monooxygenases (CYPs/P450s) are well known for their role in organisms’ primary and secondary metabolism. Among 20 P450s of the tuberculosis-causing Mycobacterium tuberculosis H37Rv, CYP128A1 is particularly important owing to its involvement in synthesizing electron transport molecules such as menaquinone-9 (MK9). This study employs different in silico approaches to understand CYP128 P450 family’s distribution and structural aspects. Genome data-mining of 4250 mycobacterial species has revealed the presence of 2674 CYP128 P450s in 2646 mycobacterial species belonging to six different categories. Contrast features were observed in the CYP128 gene distribution, subfamily patterns, and characteristics of the secondary metabolite biosynthetic gene cluster (BGCs) between M. tuberculosis complex (MTBC) and other mycobacterial category species. In all MTBC species (except one) CYP128 P450s belong to subfamily A, whereas subfamily B is predominant in another four mycobacterial category species. Of CYP128 P450s, 78% was a part of BGCs with CYP124A1, or together with CYP124A1 and CYP121A1. The CYP128 family ranked fifth in the conservation ranking. Unique amino acid patterns are present at the EXXR and CXG motifs. Molecular dynamic simulation studies indicate that the CYP128A1 bind to MK9 with the highest affinity compared to the azole drugs analyzed. This study provides comprehensive comparative analysis and structural insights of CYP128A1 in M. tuberculosis.

2.6. CYP128A1 Protein Model Has High Affinity to Menaquinone-9

In P450 enzyme systems, substrates and azole drugs are known to coordinate with the ferric core of the heme group. In the case of MK9, coordination takes place at the end of its hydrophobic tail, consistent with omega hydroxylation of the molecule [,]. For azole drugs, coordination occurs via the imidazole ring. Importantly, docking of MK9 and azole drugs was performed in a non-covalent way in the present paper. Therefore, no coordination was predicted for the tested ligands, which would require more thorough investigation, especially considering changes in electronic density. However, since non-covalent binding is a critical step in the establishment of covalent interactions, we believe the present results still provide a good indication of how azoles drugs can interact with CYP128A1 and how they rank in terms of affinity.

3.11. Redocking of Compounds

MK9 and the azoles compounds were re-docked on their corresponding set of equilibrated structures using the MOE’s dock (six structures for MK9, 10 structures for each of the azole drugs). The same options as in the first docking step were used (see ).

Introduction

The community composition and toxigenicity of the diatom Pseudo-nitzschia in the open South Atlantic Ocean were characterized during the austral spring of 2007 by Guannel et al. (). Multiple morphological types of Pseudo-nitzschia were detected in coastal and in open-ocean waters. The toxin produced by Pseudo-nitzschia, DA, was present in at least 10 species in the South Atlantic (Gayoso, ; Reguera, ). Even though Pseudo-nitzschia sp. blooms occur in different environmental conditions, Marchetti et al. () and Wells et al., ) showed a positive correlation between these blooming events and high levels of Fe(III) and NO3-, and low levels of PO43- and SiO44-. Even more, differences in temperature, salinity, pH, high irradiance, and long-term photoperiod can affect the formation of Pseudo-nitzschia blooms (Lelong et al., ). Woods () suggested a possible photo-oxidative stress regulation on DA's higher production under high irradiance. Cellular stress produced by these conditions may favor the production of toxins during HAB events. The main objective of this opinion article is to briefly report the toxicological implications of the harmful marine phycotoxin DA and its intrinsic properties. Special focus will be made on the reported oxidative stress status in marine algae in relation to the exposure to DA. Moreover, because Fe presence is known to be implicated in oxidative stress generation, and its occurrence seems to be a fundamental factor in the production of DA, the oxidative effects of the biotoxin will be discussed in relation to its capacity to bind Fe.

Discussion

On the other hand, DA production starts in late EXP and ST phase of the Pseudo-nitzschia growth cycle (Lelong et al., ). In the late ST phase, cells are physiologically impaired by injury in the structure of the wall and in the membrane (Pan et al., ). Therefore, DA tends to be released into the culture medium, along with ROS generated by the cells (as shown by Cabrera et al., ,). Because of the short life span of ROS, this increased production could not be very effective in damaging other organisms in the aquatic community. However, the release of DA could allopathically affect (protect or injure) other species of non–toxin-producing photosynthetic microalgae. Using a highly sensitive adsorptive cathodic stripping voltammetric technique, Rue and Bruland () showed that DA forms Fe chelates with a conditional stability constant of KcondFeDA,Fe(III) = 108.7±0.5 M−1. The degree to which the DA acts as a trace metal chelator reveals that it has the potential to modify the availability of these metals in the seawater. Thus, the physiological role of DA for toxigenic Pseudo-nitzschia species may be tied closely to the acquisition of Fe in coastal waters. Among several other postulations, the formation of a Fe–DA complex could be a very attractive hypothesis to explain the increased generation of ROS both in producing and non–phycotoxin-generating species. Chemical aspects of the Fe chelators, such as Fe affinity, Fe selectivity, molecular weight, and lipophilicity, in addition to stability and redox properties of the resultant Fe complex, drastically change the ability of the Fe complex to catalyze radical generation. The presence of a catalytically active Fe–DA complex could favor lipid peroxidation and radical generation in a significative amount, by affecting the composition of the labile Fe content within the cells. This is a novel point of view that should be further explored. The capacity of the antioxidant defense in each species will be the main factor to determine the hormetic (beneficial) or the damaging effect of the triggering of the burst of oxidative species.

Discussion

However, other alternatives could be considered under changing environmental conditions that can modify DA-dependent active species generation in aquatic organisms. Hypoxia is a common condition in aquatic environments, and exposure to hypoxia followed by reoxygenation is often believed to induce oxidative stress and activation of relevant signaling molecules such as the hypoxia-inducible factor 1α. This transcription factor is a potent coordinator of acclimation processes in various stress conditions. Eutrophication and extensive algal blooms caused by anthropogenic activity (input of nutrients, fertilizers, and human waste) deplete the ocean and lake bottoms of O2, i.e., make them hypoxic. In the seas, the large hypoxic areas are also known as hypoxic dead zones, and they are expanding quickly (Conley et al., ). Hypoxic bottoms release phosphorus from the aquatic sediments, which feed the algae even further. Hypoxia has an acute effect on the benthic fauna, and if the hypoxic environments are allowed to expand further, the whole ecosystem will be disturbed (Conley et al., ; Conley, ). Recently, Borowiec and Scott () reported that hypoxia acclimation of killifish leads to adjustments in ROS homeostasis and oxidative status that do not reflect oxidative stress, but may instead be part of the suite of responses used to cope with chronic hypoxia. Thus, in situ studies could show the alterations due to other transitory changes in the environment that may occur during the HABs. Overall, the specific cellular response to the increased oxidative stress triggered by DA will be one of the important factors to allow survival of each organism that contribute to determine how the composition of the community will be affected by an increasing magnitude of the HAB season produced by the climate global change.

Abstract

Mushroom irradiation has been considered a sustainable process to generate high amounts of vitamin D2 due to the role of this vitamin for human health and of the global concerns regarding its deficient or inadequate intake. Mushrooms are also receiving increasing interest due to their nutritional and medicinal properties. However, there is still a knowledge gap regarding the effect of UV irradiation on mushroom bioactive compounds. In this study, two of the most cultivated mushroom species worldwide, Agaricus bisporus and Pleurotus ostreatus, were irradiated with UV-B, and the effect of processing was investigated on the contents of vitamin D2 as well as on antioxidant and antiglycation activities. UV irradiation increased vitamin D2 up to 57 µg/g d.w, which is an adequate level for the fortification of a number of target foods. UV irradiation decreased the antioxidant activity when measured by the Folin–Ciocalteu reagent, the 2,2-diphenyl-1-(2,4,6 trinitrophenyl) hydrazyl radical assay and the ferric ion-reducing antioxidant power assay, but did not decrease the mushroom’s ability to inhibit glycation of a target protein. These results open up a new area of investigation aimed at selecting mushroom species with high nutraceutical benefits for irradiation in order to maintain their potential properties to inhibit oxidative and glycation processes responsible for human diseases.

2.2. Mushrooms

P. ostreatus and A. bisporus were purchased from the market. The irradiation treatment was performed as described previously []. In brief, about 10 kg of body fruits were used for each mushroom type, cut into 4 mm slices, spread on an oven rack in a single layer and air-dried at 37 °C for 48 h. During the first 24 h of air-drying, the mushrooms were irradiated using two fluorescent lamps installed at 30 cm above the mushroom layer. In accordance to most of the previous studies so far performed [,], a UV-B source was chosen for the irradiation. The lamps delivered a UV-B (280–315 nm) irradiance of 0.4 W/m2. At the end of the process, about 1 kg of dried mushroom was obtained from each mushroom type and the product was grinded to a fine powder using a Thermomix TM 31 (Vorwerk Contempora S.r.l., Milan, Italy). Control P. ostreatus and A. bisporus powders were obtained by processing mushrooms with the same drying and grinding procedure without UV irradiation.

3.1. The Effect of Mushroom Irradiation on Vitamin D2 Content

Initial content of vitamin D2 was low in both commercial mushrooms considered. This result is consistent with previous studies and could be associated with the conditions used for industrial cultivation of mushrooms that generally occurs with low exposure to sunlight [,]. UV irradiation led to an increase in vitamin D2 content from 3.1 to 37 µg/g d.w. and from 0.3 to 57 µg/g d.w. in P. ostreatus and A. bisporus, respectively (). The initial contents of ergosterol were 3600 and 5700 µg/g d.w. in P. ostreatus and A. bisporus, respectively (). These values fall in the range previously reported [] and remained high after the generation of significant amounts of vitamin D2. This result may suggest that upon UV irradiation. the residual amount of ergosterol is still relevant for drug development and could contribute to the health-promoting effects of mushrooms [].

Abstract

Chronic low-grade inflammation is a primary characteristic of obesity and can lead to other metabolic complications including insulin resistance and type 2 diabetes (T2D). Several anti-inflammatory dietary bioactives decrease inflammation that accompanies metabolic diseases. We are specifically interested in delta-tocotrienol, (DT3) an isomer of vitamin E, and tart cherry anthocyanins (TCA), both of which possess individual anti-inflammatory properties. We have previously demonstrated that DT3 and TCA, individually, reduced systemic and adipose tissue inflammation in rodent models of obesity. However, whether these compounds have combinatorial effects has not been determined yet. Hence, we hypothesize that a combined treatment of DT3 and TCA will have great effects in reducing inflammation in adipocytes, and that these effects are mediated via the nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), a major inflammatory transcription factor. We used 3T3-L1 adipocytes and treated them with 1–5 µM doses of DT3 along with tart cherry containing 18–36 µg anthocyanin/mL, to assess effects on inflammation. Neither DT3 nor TCA, nor their combinations had toxic effects on adipocytes. Furthermore, pro-inflammatory markers interleukin-6 (IL-6) and p-65 (subunit of NFkB) were reduced at the protein level in media collected from adipocytes with both individual and combined treatments. Additionally, other downstream targets of NFkB including macrophage inflammatory protein 2 (Mip2), and Cyclooxygenase-2 (Cox2) were also significantly downregulated (p ≤ 0.05) when treated with individual and combined doses of DT3 and TCA with no additional combinatorial effects. In summary, DT3 and TCA individually, are beneficial in reducing inflammation with no additional combinatorial effects.