Induction of ferroptosis in human keratinocyte HaCaT cells by squalene hydroperoxide: Possible prevention of skin ferroptosis by botanical extracts.

Ever since the proposal of ferroptosis, it has been studied as a nonapoptotic cell death caused by iron ion-dependent phospholipid (PL) peroxidation. We previously showed that treatment of human hepatoma cell line HepG2 with prepared PL hydroperoxide (PLOOH) resulted in ferroptosis. However, in human sebum, the major hydroperoxide is not PLOOH but squalene hydroperoxide (SQOOH), and to our knowledge, it is not established yet whether SQOOH induces ferroptosis in the skin. In this study, we synthesized SQOOH and treated human keratinocyte HaCaT cells with SQOOH. The results showed that SQOOH induces ferroptosis in HaCaT cells in the same way that PLOOH causes ferroptosis in HepG2 cells. Some natural antioxidants (botanical extracts) could inhibit the ferroptosis in both the cell types. Consequently, future research focus would revolve around the involvement of SQOOH-induced ferroptosis in skin pathologies as well as the prevention and treatment of skin diseases through inhibition of ferroptosis by botanical extracts.

Expression and functional analyses for estrogen receptor and estrogen related receptor of Yesso scallop, Patinopecten yessoensis.

Estrogen receptors (ERs) were known as estrogen-activated transcription factors and function as major reproduction regulators in vertebrates. The presence of er genes had been reported in Molluscan cephalopods and gastropods. However, they were considered as constitutive activators with unknown biological functions since reporter assays for these ERs did not show a specific response to estrogens. In this study, we tried characterization of ER orthologues from the Yesso scallop, Patinopecten yessoensis, in which estrogens had been proven to be produced in the gonads and involved in the spermatogenesis and vitellogenesis. Identified ER and estrogen related receptor (ERR) of Yesso scallops, designated as py-ER and py-ERR, conserved specific domain structures for a nuclear receptor. Their DNA binding domains showed high similarities to those of vertebrate ER orthologues, while ligand binding domains had low similarities with them. Both the py-er and py-err expression levels decreased in the ovary at the mature stage while py-vitellogenin expression increased in the ovary by quantitative real-time RT-PCR. Also, the py-er and py-err showed higher expressions in the testis than ovary during the developing and mature period, suggesting both genes might function in the spermatogenesis and testis development. The py-ER showed binding affinities to vertebrate estradiol-17ß (E2). However, the intensity was weaker than the vertebrate ER, indicating scallops might exist endogenous estrogens with a different structure. On the other hand, the binding property of py-ERR to E2 was not confirmed in this assay, speculating that py-ERR was a constitutive activator as other vertebrate ERRs. Further, the py-er was localized in the spermatogonia in the testis and in the auxiliary cells in the ovary by in situ hybridization, indicating its potential roles in promoting spermatogenesis and vitellogenesis. Taken together, the present study demonstrated that py-ER was an authentic E2 receptor in the Yesso scallop and might have functions for the spermatogonia proliferation and vitellogenesis, while py-ERR was involved in the reproduction by undiscovered manners.

Elucidation of Olive Oil Oxidation Mechanisms by Analysis of Triacylglycerol Hydroperoxide Isomers Using LC-MS/MS.

Despite the importance of the insight about the oxidation mechanisms (i.e., radical and singlet oxygen (1O2) oxidation) in extra virgin olive oil (EVOO), the elucidation has been difficult due to its various triacylglycerol molecular species and complex matrix. This study tried to evaluate the mechanisms responsible for EVOO oxidation in our daily use by quantitative determination of triacylglycerol hydroperoxide (TGOOH) isomers using LC-MS/MS. The standards of dioleoyl-(hydroperoxy octadecadienoyl)-triacylglycerol and dioleoyl-(hydroperoxy octadecamonoenoyl)-triacylglycerol, which are the predominant TGOOHs contained in EVOO, were prepared. Subsequently, fresh, thermal-, and photo-oxidized EVOO were analyzed. The obtained results mostly agreed with the previously reported characteristics of the radical and 1O2 oxidation of linoleic acid and oleic acid. This suggests that the methods described in this paper should be valuable in understanding how different factors that determine the quality of EVOO (e.g., olive species, cultivation area, cultivation timing, and extraction methods) contribute to its oxidative stability.

Cellular concentrations of plasmalogen species containing a polyunsaturated fatty acid significantly increase under hypoxia in human colorectal cancer, Caco2 cells.

Plasmalogen localized in the raft of mammalian cell membranes plays a role in the storage of polyunsaturated fatty acid (PUFA), and exists to a higher extent in malignant cells that survive, and even grow in hypoxic conditions. The biosynthesis of plasmalogen in mammalian cells has been reported to depend on aerobic conditions. Using liquid chromatography-tandem mass spectrometry, we found that the intracellular concentration of plasmalogen species containing a PUFA at the sn-2-position did not change for two days from the start of hypoxic culture in human colorectal cancer-derived Caco2 cells. At the third day of hypoxia, Caco2 cells showed the average increase rate of 2.6 times in ethanolamine plasmalogen and 2.9 times in choline plasmalogen depending on the molecular species compared with those in the second day of hypoxia. In normoxic culture, there was little quantitative change in any species of both ethanolamine and choline plasmalogens for three days. The up-regulations of mRNA of Ca2+-independent phospholipase A2ß and cytoplasmic phospholipase A2γ as well as the down-regulation of lysoplasmalogenase observed in hypoxia were suggested to be responsible for the increase of plasmalogen in Caco2 cells under hypoxia.

Lipidomics and Redox Lipidomics Indicate Early Stage Alcohol-Induced Liver Damage.

Alcoholic fatty liver disease (AFLD) is characterized by lipid accumulation and inflammation and can progress to cirrhosis and cancer in the liver. AFLD diagnosis currently relies on histological analysis of liver biopsies. Early detection permits interventions that would prevent progression to cirrhosis or later stages of the disease. Herein, we have conducted the first comprehensive time-course study of lipids using novel state-of-the art lipidomics methods in plasma and liver in the early stages of a mouse model of AFLD, i.e., Lieber-DeCarli diet model. In ethanol-treated mice, changes in liver tissue included up-regulation of triglycerides (TGs) and oxidized TGs and down-regulation of phosphatidylcholine, lysophosphatidylcholine, and 20-22-carbon-containing lipid-mediator precursors. An increase in oxidized TGs preceded histological signs of early AFLD, i.e., steatosis, with these changes observed in both the liver and plasma. The major lipid classes dysregulated by ethanol play important roles in hepatic inflammation, steatosis, and oxidative damage. Conclusion: Alcohol consumption alters the liver lipidome before overt histological markers of early AFLD. This introduces the exciting possibility that specific lipids may serve as earlier biomarkers of AFLD than those currently being used.

Dietary Ethanolamine Plasmalogen Alleviates DSS-Induced Colitis by Enhancing Colon Mucosa Integrity, Antioxidative Stress, and Anti-inflammatory Responses via Increased Ethanolamine Plasmalogen Molecular Species: Protective Role of Vinyl Ether Linkages.

Dietary ethanolamine plasmalogen (PlsEtn) has been reported to have several health benefits; however, its functional role during colon pathophysiology remains elusive. The present study investigated the anticolitis effect of dietary ethanolamine glycerophospholipids (EtnGpls) with high PlsEtn from ascidian muscle (86.2 mol %) and low PlsEtn from porcine liver (7.7 mol %) in dextran sulfate sodium (DSS)-induced colitis in mice. Dietary EtnGpls lowered myeloperoxidase activity, thiobarbituric acid-reactive substances, proinflammatory cytokines and proapoptosis-related protein levels in colon mucosa after 16 days of DSS treatment, with ascidian muscle (0.1% EtnGpl in diet) showing higher suppression than porcine liver (0.1% EtnGpl in diet). Moreover, dietary EtnGpls suppressed DSS symptoms after 38 days of DSS treatment as evidenced by increased body weight, colon length, and ameliorated colon mucosa integrity. Additionally, dietary EtnGpls elevated short-chain fatty acid production in DSS-treated mice. Altogether, these results indicate the potential of utilizing diets with abundant PlsEtn for the prevention of colon inflammation-related disorders.

Ethanolamine Plasmalogen Suppresses Apoptosis in Human Intestinal Tract Cells in Vitro by Attenuating Induced Inflammatory Stress.

Ethanolamine plasmalogen (PlsEtn) is a subtype of ethanolamine glycerophospholipids (EtnGpl). Recently, PlsEtn has attracted increasing research interest due to its beneficial effects in health and disease; however, its functional role in colonic health has not been well established. This study was conducted to determine the mechanism underlying the antiapoptotic effect of PlsEtn in human intestinal tract cells under induced inflammatory stress. Lipopolysaccharide induced apoptosis of differentiated Caco-2 cells, which was suppressed by EtnGpl in a dose-dependent manner. Cells treated with ascidian muscle EtnGpl containing high levels of PlsEtn demonstrated a lower degree of apoptosis, and downregulated TNF-α and apoptosis-related proteins compared to those treated with porcine liver EtnGpl containing low PlsEtn. This indicates that PlsEtn exerted the observed effects, which provided protection against induced inflammatory stress. Overall, our results suggest that PlsEtn with abundant vinyl ether linkages is potentially beneficial in preventing the initiation of inflammatory bowel disease and colon cancer.

Dietary PlsEtn Ameliorates Colon Mucosa Inflammatory Stress and ACF in DMH-Induced Colon Carcinogenesis Mice: Protective Role of Vinyl Ether Linkage.

Ethanolamine plasmalogen (PlsEtn), a sub-class of ethanolamine glycerophospholipids (EtnGpl), is a universal phospholipid in mammalian membranes. Several researchers are interested in the relationship between colon carcinogenesis and colon PlsEtn levels. Here, we evaluated the functional role of dietary purified EtnGpl from the ascidian muscle (87.3 mol% PlsEtn in EtnGpl) and porcine liver (7.2 mol% PlsEtn in EtnGpl) in 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) in vivo, and elucidated the possible underlying mechanisms behind it. Dietary EtnGpl-suppressed DMH-induced aberrant crypt with one foci (AC1) and total ACF formation (P < 0.05). ACF suppression by dietary ascidian muscle EtnGpl was higher compared with dietary porcine liver EtnGpl. Additionally, dietary EtnGpl decreased DMH-induced oxidative damage, overproduction of TNF-α, and expression of apoptosis-related proteins in the colon mucosa. The effect of dietary ascidian muscle EtnGpl showed superiority compared with dietary porcine liver EtnGpl. Our results demonstrate the mechanisms by which dietary PlsEtn suppress ACF formation and apoptosis. Dietary PlsEtn attained this suppression by reducing colon inflammation and oxidative stress hence a reduction in DMH-induced intestinal impairment. These findings provide new insights about the functional role of dietary PlsEtn during colon carcinogenesis.

Revealing the thermal oxidation stability and its mechanism of rice bran oil.

Although the stability of rice bran oil (RBO) has been showed on several studies, the factors which make it capable on maintaining its stability under thermal oxidation has not been sure yet. We hypothesized that its fatty acid composition [high composition of oleic acid (OA), lower composition of linoleic acid (LA) and α-linolenic acid (LnA)] and/or its antioxidant agents [γ-oryzanol (OZ)] and vitamin E [tocopherol (Toc), tocotrienol (T3)] might be the biggest factor. To prove the hypothesis, we thermally oxidized RBO under 40 °C for 17 days to mimic the harsh daily storage condition, and compared it with soybean oil (SO) and rapeseed oil (RPO) then monitoring their primary oxidation products [triacylglycerol hydroperoxide (TGOOH)] from easily oxidized fatty acid contained in triacylglycerol (TG) and the amount loss of antioxidant agents. As a result, RBO showed the lowest TGOOH/TG ratio, followed by RPO and SO. The superior stability RPO compared SO might occur due to because of the influence of the fatty acid profile (higher OA and lower LA). For RBO's case, besides its fatty acid profile, the existence of OZ and the synergistic effect of OZ and vitamin E might have a greater contribution in maintaining its stability under thermal oxidation.

Modulation of Telomerase Activity in Cancer Cells by Dietary Compounds: A Review.

Telomerase is expressed in ~90% of human cancer cell lines and tumor specimens, whereas its enzymatic activity is not detectable in most human somatic cells, suggesting that telomerase represents a highly attractive target for selective cancer treatment. Accordingly, various classes of telomerase inhibitors have been screened and developed in recent years. We and other researchers have successfully found that some dietary compounds can modulate telomerase activity in cancer cells. Telomerase inhibitors derived from food are subdivided into two groups: one group directly blocks the enzymatic activity of telomerase (e.g., catechin and sulfoquinovosyldiacylglycerol), and the other downregulates the expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase, via signal transduction pathways (e.g., retinoic acid and tocotrienol). In contrast, a few dietary components, including genistein and glycated lipid, induce cellular telomerase activity in several types of cancer cells, suggesting that they may be involved in tumor progression. This review summarizes the current knowledge about the effects of dietary factors on telomerase regulation in cancer cells and discusses their molecular mechanisms of action.

Determination of triacylglycerol oxidation mechanisms in canola oil using liquid chromatography-tandem mass spectrometry.

Triacylglycerol (TG), the main component of edible oil, is oxidized by thermal- or photo- oxidation to form TG hydroperoxide (TGOOH) as the primary oxidation product. Since TGOOH and its subsequent oxidation products cause not only the deterioration of oil quality but also various toxicities, preventing the oxidation of edible oils is essential. Therefore understanding oxidation mechanisms that cause the formation of TGOOH is necessary. Since isomeric information of lipid hydroperoxide provides insights about oil oxidation mechanisms, we focused on dioleoyl-(hydroperoxy octadecadienoyl)-TG (OO-HpODE-TG) isomers, which are the primary oxidation products of the most abundant TG molecular species (dioleoyl-linoleoyl-TG) in canola oil. To secure highly selective and sensitive analysis, authentic OO-HpODE-TG isomer references (i.e., hydroperoxide positional/geometrical isomers) were synthesized and analyzed with HPLC-MS/MS. With the use of the method, photo- or thermal- oxidized edible oils were analyzed. While dioleoyl-(10-hydroperoxy-8E,12Z-octadecadienoyl)-TG (OO-(10-HpODE)-TG) and dioleoyl-(12-hydroperoxy-9Z,13E-octadecadienoyl)-TG (OO-(12-HpODE)-TG) were characteristically detected in photo-oxidized oils, dioleoyl-(9-hydroperoxy-10E,12E-octadecadienoyl)-TG and dioleoyl-(13-hydroperoxy-9E,11E-octadecadienoyl)-TG were found to increase depending on temperature in thermal-oxidized oils. These results prove that our methods not only evaluate oil oxidation in levels that are unquantifiable with peroxide value, but also allows for the determination of oil oxidation mechanisms. From the analysis of marketed canola oils, photo-oxidized products (i.e., OO-(10-HpODE)-TG and OO-(12-HpODE)-TG) were characteristically accumulated compared to the oil analyzed immediately after production. The method described in this paper is valuable in the understanding of oil and food oxidation mechanisms, and may be applied to the development of preventive methods against food deterioration.

Analysis of Plasmalogen Species in Foodstuffs.

Ethanolamine plasmalogen (PlsEtn), which is present at high levels in brains, is believed to be involved in neuronal protection. The present study was performed to search for PlsEtn resources in foodstuffs. The foodstuffs examined showed a wide range of PlsEtn contents from 5 to 549 µmol/100 g wet wt. The marine invertebrates, blue mussel, and ascidian had high PlsEtn contents (over 200 µmol/100 g wet wt). Profiling of the molecular species showed that the predominant fatty acids of PlsEtn species were 20:5 (EPA) and 22:6 (DHA) at the sn-2 position of the glycerol moiety in marine foodstuffs, whereas major PlsEtn species in land foodstuffs were 20:4. Following quantitative analysis by multiple reaction monitoring, the ascidian viscera were shown to contain the highest levels of 18:0/20:5-PlsEtn and 18:0/22:6-PlsEtn (86 and 68 µmol/100 g wet wt, respectively). In order to evaluate a neuronal antiapoptotic effect of these PlsEtn species, human neuroblastoma SH-SY5Y cells were treated with ethanolamine glycerophospholipid (EtnGpl), purified from the ascidian viscera, under serum starvation conditions. Extrinsic EtnGpl from ascidian viscera showed stronger suppression of cell death induced by serum starvation than with bovine brain EtnGpl. The EtnGpl from ascidian viscera strongly suppressed the activation of caspase 3. These results suggest that PlsEtn, especially that containing EPA and DHA, from marine foodstuffs is potentially useful for a therapeutic dietary supplement preventing neurodegenerative diseases, such as Alzheimer's disease (AD).

Molecular characterization of an estrogen receptor and estrogen-related receptor and their autoregulatory capabilities in two Mytilus species.

Vertebrate-like sex steroid hormones have been widely detected in mollusks, and numerous experiments have shown the importance of steroids in gonad development. Nevertheless, their signaling pathways in invertebrates have not been uncovered yet. Steroid receptors are an ancient class of transcription factors with multiple roles in not only vertebrates but also invertebrates. Estrogen signaling is thought to have major roles in mollusk physiology, but the full repertoire of estrogen receptors is unknown. We presented the successful cloning of two novel forms of estrogen receptor-like genes. These receptors are present in two closely related species of Mytilus: Mytilus edulis and Mytilus galloprovincialis, commonly known and widely distributed sentinel species. Our phylogenetic analysis revealed that one of these receptors is an estrogen receptor (ER) and the other one is an estrogen-related receptor (ERR). Studies of expression analysis showed that both receptor mRNAs were localized in the oocytes and follicle cells in contact with developing oocytes in the ovary and Sertoli cells in the testis, and in the ciliated cells of the gill. In addition, we have evidence that one (ER) of these may have a capacity to autoregulate its own expression in the gonadal cells by estrogen (E2) and that this gene is responsive to estrogenic compounds.