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.

Elucidation of decomposition pathways of linoleic acid hydroperoxide isomers by GC-MS and LC-MS/MS.

Food lipid oxidation provides various volatile compounds involved in food flavor via the decomposition of lipid hydroperoxide (LOOH). This study predicted the pathways which can coherently explain LOOH decomposition focusing on hydroperoxy octadecadienoic acid (HpODE) isomers (9-EZ-HpODE, 9-EE-HpODE, 10-HpODE, 12-HpODE, 13-ZE-HpODE, and 13-EE-HpODE) which are the major LOOH contained in edible oils. Each standard was first prepared and thermally decomposed. Generated volatile and non-volatile compounds were analyzed by GC-MS and LC-MS/MS. The results showed that all HpODE decomposition was based on the factors such as favorable scission, radical delocalization, and cyclization. Interestingly, the formation of 8-HpODE and 14-HpODE were demonstrated during HpODE decomposition. The insights obtained in this study would explain the generation pathways of flavor involved in food quality.

TRPV2 Promotes Cell Migration and Invasion in Gastric Cancer via the Transforming Growth Factor-ß Signaling Pathway.

BACKGROUND: Transient receptor potential vanilloid 2 (TRPV2) is a highly Ca2+-permeable ion channel that is involved in a number of cellular processes. It is expressed in various human cancers; however, the role of TRPV2 in gastric cancer (GC) remains poorly understood. METHODS: TRPV2 gene expression was knocked down in GC cell lines by small-interfering RNA (siRNA), and the biological roles of TRPV2 in the proliferation, migration, and invasion of GC cells were then investigated. The gene expression profile of GC was elucidated using a microarray analysis. TRPV2 expression in tumor tissue sections was analyzed by immunohistochemistry. RESULTS: The migration and invasion abilities of GC cells were inhibited by the knockdown of TRPV2. Moreover, the microarray assay revealed that TRPV2 was associated with the transforming growth factor (TGF)-ß signaling pathway. Immunohistochemical staining showed that the strong expression of TRPV2 correlated with lymphatic invasion, venous invasion, pathological T (pT), pathological N (pN), and a poor prognosis in GC patients. CONCLUSIONS: TRPV2 appeared to promote tumor migration and invasion via the TGF-ß signaling pathway, and the strong expression of TRPV2 was associated with a worse prognosis in GC patients.

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.

[Gastrectomy in Patients Aged<85 Years Who Had Gastric Cancer-A Single-Institutional Experience].

BACKGROUND: This study examined the treatment outcomes of gastrectomy in patients aged<85 years who had gastric cancer(GC). METHODS: The postoperative short- and long-term outcomes of 27 patients aged<85 years who underwent gastrectomy for GC at our institute were retrospectively investigated. RESULTS: The median age was 87 years(range: 85-94 years), and 17 patients(63%)had comorbidities. Total, distal, and proximal gastrectomies were performed for 12, 14, and 1 patient, respectively. Only 13 patients(48%)underwent standard lymph lymphadenectomy(LND), while R0, R1, and R2 were performed for 23, 2, and 2 patients, respectively. The overall, surgical, and non-surgical complication rates were 59%, 26%, and 44%, respectively, even though the incidence of GradeBⅢa complications was only 4%, and there was no mortality. The 1-, 2-, and 3-year overall survival rates(OSR)were 91.7%, 79.4%, and 63.2%, respectively. The 3-year OSRs of the patients who underwent R0, R1, and R2 were 76.2%, 35.4%, and 0%, respectively. The 3-year OSR was significantly higher in the patients who underwent the standard LND(100%)than in those who underwent limited LND(36.6%). CONCLUSION: The standard LND and R0 might also be useful for patients aged<85 years who had GC, although care should be taken for the high incidence of complications.

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.

[A Case of Laparoscopic Hartmann's Operation for Perforation of Rectal Cancer in Superior Elderly People].

We report a case where home discharge was possible after laparoscopic Hartmann's operation for superior elderly perforation of rectal cancer. The patient was 91-year-old, a woman. She was delivered to the emergency room complaining of weakness. We diagnosed rectal perforation and started emergency laparoscopic surgery. Rectal cancer perforation was observed during surgery and laparoscopic Hartmann's operation plus D2 lymph node dissection was performed. The operation time was 3 hours 21 minutes, the blood loss was 10 g. She resumed her meal intake from the postoperative day(POD)5 and became ready for discharge on POD 20 postoperatively. She moved to a comprehensive care ward and she was discharged to her house on POD 89. On POD 120, she visited the hospital complaining of anorexia and anal bleeding, and was diagnosed as local recurrence in the pelvis, multiple liver metastases, and cancerous peritonitis. She was admitted to palliative care unit on POD 132 and died on POD 141. It was suggested that laparoscopic surgery will be minimally invasive even at superior elderly patients and that they will be able to discharge from their homes.

A novel chiral stationary phase HPLC-MS/MS method to discriminate between enzymatic oxidation and auto-oxidation of phosphatidylcholine.

To elucidate the role of enzymatic lipid peroxidation in disease pathogenesis and in food deterioration, we recently achieved stereoselective analysis of phosphatidylcholine hydroperoxide (PCOOH) possessing 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-9Z,11E-HPODE) using HPLC-MS/MS with a CHIRALPAK OP (+) column. Because enzymatic oxidation progresses concurrently with auto-oxidation, we need to distinguish them further. Here, we attempted such an analysis. First, we used lipoxygenase, linoleic acid, and lysophosphatidylcholine (LPC) to synthesize the enzymatic oxidation product 13(S)-9Z,11E-HPODE PC, and the auto-oxidation products 13(RS)-9Z,11E-HPODE PC and 13(RS)-9E,11E-HPODE PC, which were used as standards to test the ability of various columns to separate the enzymatic oxidation product from auto-oxidation products. Separation was achieved by connecting in series two columns with different properties: CHIRALPAK OP (+) and CHIRALPAK IB-3. The CHIRALPAK OP (+) column separated 13(R)-9Z,11E-HPODE PC and 13(S)-9Z,11E-HPODE PC, whereas CHIRALPAK IB-3 enabled separation of 13(S)-9Z,11E-HPODE PC and 13(RS)-9E,11E-HPODE PC. The results for the analysis of both enzymatically oxidized and auto-oxidized lecithin (an important phospholipid mixture in vivo and in food) indicate that our method would be useful for distinguishing enzymatic oxidation and auto-oxidation reactions. Such information will be invaluable for elucidating the involvement of PCOOH in disease pathogenesis and in food deterioration.

[Long-Term Survival of a Patient with Stage IV Breast Cancer and An Intrathoracic Space-Occupying Metastatic Lesion].

A 50-year old woman noticed left axillary lymph node swelling and presented at our hospital. CNB showed adenocarcinoma( pap-tub, ER+, PgR+, HER2 3+). CT revealed a right lung metastatic nodule and swollen lymph nodes above the left collarbone and left axilla. However, no breast tumor was found at that time. We diagnosed occult breast cancer, TxN3bM1 (lung), Stage IV . FEC(100), paclitaxel, letrozole, anastrozole, exemestane plus trastuzumab, tegafur/uracil plus trastuzumab, and lapatinib plus capecitabine were sequentially administered. Five years and 9 months after the treatment started, CT revealed a right intrathoracic lesion that had gradually increased in size. Subsequently, trastuzumab plus pertuzumab plus docetaxel, bevacizumab plus paclitaxel, trastuzumab emtansine, trastuzumab plus fulvestrant, and doxifluridine plus medroxyprogesterone acetate plus cyclophosphamide(DMpC therapy)were sequentially administered. At this time, 8 years after the treatment started, trastuzumab plus pertuzumab plus vinorelbine were also administered. An intrathoracic space-occupying lesion due to breast cancer is rare, and anti-HER2 chemotherapy was effective for this patient.

Tandem mass spectrometry analysis of linoleic and arachidonic acid hydroperoxides via promotion of alkali metal adduct formation.

Recently, we demonstrated that tandem mass spectrometry (MS/MS) analysis in the presence of sodium ions was useful for identification of the position of the hydroperoxy group in phosphatidylcholine hydroperoxide (PCOOH). Likewise, MS/MS may enable identification of the hydroperoxy group position in various lipid hydroperoxides (LOOHs). To this end, we prepared major LOOHs, namely hydroperoxyoctadecadienoic acid (HPODE) and hydroperoxyeicosatetraenoic acid (HPETE), and analyzed them by quadrupole-time-of-flight MS/MS in both the absence and presence of alkali metals. Photo-oxidation (singlet oxygen-induced oxidation) of linoleic acid (LA) was used to prepare 9-10E,12Z-HPODE, 9-10E,12E-HPODE, 10-8E,12Z-HPODE, 12-9Z,13E-HPODE, 13-9Z,11E-HPODE, and 13-9E,11E-HPODE. Each isomer was analyzed under various MS/MS conditions (e.g., absence and presence of sodium). We found that in the presence of alkali metals, especially sodium, collision-induced dissociation (CID) of all HPODE isomers yielded structure-diagnostic fragment ions that were highly useful in identifying the position of the hydroperoxy group. For instance, CID spectra of sodiated 13-9Z,11E-HPODE revealed a neutral loss of 88 Da arising from fragmentation of the hydroperoxy group. Similar results were observed for HPETE isomers. Following oxidation of LA (or arachidonic acid) by lipoxygenase, the hydroperoxy group position of the resultant HPODE (or HPETE) was easily identified using this method, without any chromatographic separation processes. As information on the position of the hydroperoxy group provides insight into the processes that initiate lipid peroxidation (e.g., enzymatic oxidation, auto-oxidation and singlet oxygen-induced oxidation), the proposed method may be useful in elucidating the involvement and mechanism of lipid peroxidation in food deterioration and pathophysiological processes.

Metabolism and cytotoxic effects of phosphatidylcholine hydroperoxide in human hepatoma HepG2 cells.

In this study, we investigated cellular uptake and metabolism of phosphatidylcholine hydroperoxide (PCOOH) in human hepatoma HepG2 cells by high performance liquid chromatography-tandem mass spectrometry, and then evaluated whether PCOOH or its metabolites cause pathophysiological effects such as cytotoxicity and apoptosis. Although we found that most PCOOH was reduced to PC hydroxide in HepG2 cells, the remaining PCOOH caused cytotoxic effects that may be mediated through an unusual apoptosis pathway. These results will enhance our fundamental understanding of how PCOOH, which is present in oxidized low density lipoproteins, is involved in the development of atherosclerosis.

Liquid chromatography-tandem mass spectrometry determination of human plasma 1-palmitoyl-2-hydroperoxyoctadecadienoyl-phosphatidylcholine isomers via promotion of sodium adduct formation.

Accumulation of phosphatidylcholine hydroperoxide (PCOOH), a primary oxidation product of phosphatidylcholine, in blood plasma has been observed in various pathological conditions, including atherosclerosis. In this study, we investigated the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to develop a method for accurate quantification of PCOOH (1-palmitoyl-2-hydroperoxyoctadecadienoyl-sn-glycero-3-phosphocholine, 16:0/HpODE PC), focusing on isomers such as 16:0/13-HpODE PC and 16:0/9-HpODE PC. Sodiated PCOOH ([M+Na](+), m/z 812) provided not only a known product ion (m/z 147) but also characteristic product ions (m/z 541 for 16:0/13-HpODE PC and m/z 388 for 16:0/9-HpODE PC). Thus, three multiple reaction monitorings (MRMs) could be performed. MRM (812/147) enabled determination of 16:0/HpODE PC, and MRM (812/541) and MRM (812/388) allowed specific measurement of 16:0/13-HpODE PC and 16:0/9-HpODE PC, respectively. By using this method, we could determine plasma PCOOH concentrations in healthy subjects and patients with angiographically significant stenosis. In healthy subject and patient plasma, the concentration of 16:0/HpODE PC was close to the sum of the concentrations of 16:0/13-HpODE PC and 16:0/9-HpODE PC. This finding shows that radical and/or enzymatic oxidation, rather than singlet oxygen oxidation, is recognized to cause peroxidation of PC. The newly developed LC-MS/MS method appears to be a powerful tool for developing a better understanding of in vivo lipid peroxidation and its involvement in human diseases.

Corrigendum to "Preparation of saturated fatty acid hydroperoxide isomers and determination of their thermal decomposition products - 2-alkanones and lactones" [Food Chem.: X 21 (2024) 101074].

[This corrects the article DOI: 10.1016/j.fochx.2023.101074.].

LC-MS/MS analysis of milk triacylglycerol hydroperoxide isomers which are generated corresponding to the photo- and thermal-oxidation.

Milk is a rich source of essential nutrients such as lipids. However, lipid oxidation can be considered a crucial factor in determining the initial stage of milk deterioration. Therefore, it is essential to identify the mechanisms of lipid oxidation, such as photo-oxidation or thermal oxidation, to efficiently prevent it by selecting proper antioxidants. In this study, the oxidation mechanisms of long-life (LL) milk were investigated, and triacylglycerol hydroperoxide isomers generated corresponding to the oxidation mechanisms were analyzed by LC-MS/MS. This study first prepared the standard of TG 4:0_16:0_18:1;OOH isomers, which are the appropriate target for evaluating LL milk's oxidation mechanism. The authentic standards provided the robust analysis of TG 4:0_16:0_18:1;OOH isomers and suggested that LL milk was susceptible to photo-oxidation rather than thermal-oxidation. Furthermore, it was discovered that radicals play a role in the oxidation of LL milk during photo-oxidation. This information could be valuable in effectively preventing photo-oxidation in LL milk. It is important to note that milk is contained in a variety of food products. Hence, these findings would be applicable not only to milk but also to various milk-containing food products.

Preparation of saturated fatty acid hydroperoxide isomers and determination of their thermal decomposition products - 2-alkanones and lactones.

As known for quite a long time now, even saturated fatty acids can be oxidized at high temperatures to produce unique aroma compounds, such as 2-alkanones and lactones. Hydroperoxide positional isomers with a hydroperoxy group at the 2-, 3-, 4-, or 5-position are hypothesized to be responsible for the formation of these aroma components, but this hypothesis has not been verified. For the first time, this study successfully prepared a series of glyceryl trioctanoate hydroperoxide (C8TG;OOH) isomers. The isomers were thermally decomposed, proving that 2-heptanone was selectively formed from C8TG;3-OOH, and γ- and δ-octalactones were mainly formed from C8TG;4- and 5-OOH, respectively. C8TG;2-OOH was also involved in lactone formation, whereas C8TG;6- and 7-OOH were not. This proves the long-standing hypothesis. The mechanism revealed in this work is expected to be useful to create favorable aromas (i.e., 2-alkanones and lactones) from saturated fatty acids.

Comparison of the Catabolic Rates of Linoleic and Oleic Acid Hydroperoxides Using 13CO2 Expired from Mice.

Unsaturated fatty acids, such as oleic and linoleic acids, are easily oxidized by exposure to temperature and light in the presence of air to form unsaturated fatty acid hydroperoxides as primary oxidation products. However, the catabolic rates of unsaturated fatty acid hydroperoxides in the human body remain unknown. In this study, ethyl esters of 13C-labeled linoleic acid (*C18:2-EE) and oleic acid (*C18:1-EE) and their hydroperoxides (*C18:2-EE-OOH and *C18:1-EE-OOH, respectively) prepared by the photo-oxidation of *C18:2-EE and *C18:1-EE, respectively, were administered to mice and their catabolic rates were determined by measuring the expired 13CO2 levels. *C18:2-EE-OOH and *C18:1-EE-OOH were ß-oxidized faster than *C18:2-EE and *C18:1-EE, respectively. Notably, rapid ß-oxidation of *C18:2-EE-OOH and *C18:1-EE-OOH was similar to that of medium-chain fatty acids, such as octanoic acid. Then, degradation products of C18:2-EE-OOH and C18:1-EE-OOH were analyzed under gastric conditions by gas chromatography/mass spectrometry. Major decomposition products of C18:2-EE-OOH and C18:1-EE-OOH were medium-chain compounds, such as octanoic acid ethyl ester, 9-oxo-nonanoic acid ethyl ester, and 10-oxo-8-decenoic acid ethyl esters, indicating that C18:2-EE-OOH and C18:1-EE-OOH isomers formed during photo-oxidation were decomposed under acidic conditions. These findings support previous reports that dietary lipid hydroperoxides are not absorbed into the intestine as lipid hydroperoxides but as degradation products. This is the first study to suggest that dietary lipid hydroperoxides decompose during gastric digestion to form medium-chain compounds that are directly absorbed into the liver via the portal vein and rapidly catabolized via ß-oxidation.

Oxidative stress during development of alcoholic fatty liver: therapeutic potential of cacao polyphenol.

The lipid and antioxidative/oxidative profiles of livers from rats fed an ethanol liquid diet for 8 weeks provided evidence for an involvement of oxidative stress (e.g., phospholipid peroxidation) in the development of alcoholic fatty liver (AFL), possibly in an early stage. Cacao polyphenol supplementation produced an ameliorating effect, and may help in AFL prevention.

Analysis of docosahexaenoic acid hydroperoxide isomers in mackerel using liquid chromatography-mass spectrometry.

Docosahexaenoic acid (DHA) is mostly esterified in food and is easily oxidized by exposure to heat or light. Hydroperoxide positions of DHA mono-hydroperoxide (DHA;OOH) provide information on oxidation mechanisms (e.g., radical- or singlet oxygen oxidation), yet direct identification of esterified DHA;OOH isomers has not been achieved. We previously accomplished the direct analysis of free DHA;OOH isomers with liquid chromatography-mass spectrometry (LC-MS/MS). In this study, we developed an LC-MS/MS method for direct analysis of esterified DHA;OOH based on our previous study. The developed method was capable of distinguishing esterified DHA;OOH isomers in raw- and oxidized mackerel. The result suggested that radical oxidation of esterified DHA can progress even in refrigeration. Different transitions were observed depending on the oxidation mechanism and lipid class. The analytical method and insights obtained in this study would be valuable to further understand and effectively prevent DHA oxidation in food products.

Analysis of oxidized glucosylceramide and its effects on altering gene expressions of inflammation induced by LPS in intestinal tract cell models.

Glucosylceramide (GlcCer) belongs to sphingolipids and is found naturally in plant foods and other sources that humans consume daily. Our previous studies demonstrated that GlcCer prevents inflammatory bowel disease both in vitro and in vivo, whose patients are increasing alarmingly. Although some lipids are vulnerable to oxidation which changes their structure and activities, it is unknown whether oxidative modification of GlcCer affects its activity. In this research, we oxidized GlcCer in the presence of a photosensitizer, analyzed the oxide by mass spectrometric techniques, and examined its anti-inflammatory activity in lipopolysaccharide (LPS)-treated differentiated Caco-2 cells as in vitro model of intestinal inflammation. The results showed that GlcCer is indeed oxidized, producing GlcCer hydroperoxide (GlcCerOOH) as a primary oxidation product. We also found that oxidized GlcCer preserves beneficial functions of GlcCer, suppressing inflammatory-related gene expressions. These findings suggested that GlcCerOOH may perform as an LPS recognition antagonist to discourage inflammation rather than induce inflammation.