INTL SYMP. ON OXIDATIVE STRESS FOR SUSTAINABLE DEVELOPMENT OF HUMAN BEINGS

Reactive Oxygen Species (ROS) are closely related to various diseases of the brain. Twendee X (TWX) is an antioxidant composition consisting of Vitamin C, L-Glutamine, L-Cystine or L-Cysteine, Riboflavin, Succinic acid, Fumaric acid, Coenzyme Q10, and Niacin. TWX was invented from the basic experiments of alcohol, glucose, and fat metabolisms (1-4). TWX strongly reduces ROS (Patent: WIPO WO2013/072441 A1, COMPOSITION FOR PROTECTION AGAINST CELL-DAMAGING EFFECTS). Lysozyme radiation experiments showed that the antioxidant effects of TWX are 6-7 times higher than Vitamin C (Data from Dr. Helmut Durschschlarg, Regensburg Univ. Germany). Oxidative stress and mitochondrial expert ICDD (France) experiments using hepatic cancer cell line HepG2 showed TWX decrease of 63% of mitochondrial ROS and an increase of 147% mitochondrial SOD. ICDD confirmed that TWX is safe and has most strong anti-ROS and protection effects from ROS. TWX increase Neogenesis nerves cell numbers of the hippocampal dentate gyrus of the 56 weeks mouse. The same occurs within 6 weeks by TWX administration. The mitochondrial metabolism of the mouse’s hippocampal dentate gyrus was activated by TWX administration by the metabolome analysis.
The model of the brain of the mouse for ischemia and Alzheimer's disease were used to evaluate the effects of TWX. The double blind clinical trial of the Japanese Association for Prevention of Dementia from autumn 2017 to March 2019 for mild cognitive impairment was performed to analyze TWX prevention effects on Alzheimer's disease.
TWX decreased volume of brain ischemia to 2/3, and inflammation and oxidative stress in the brain was also reduced in the mouse ischemia model. TWX inhibited deposition of amyloid beta. Inflammation and oxidative stress in the brain was also reduced in the mouse model of Alzheimer. Double blind clinical research resulted in the TWX group significantly increasing the score of the Mini Mental State Examination compared to the placebo group at 6 months after the start of TWX administration. Thus, TWX is effective to prevent Alzheimer's disease and vascular dementia.

Reactive Oxygen/Nitrogen Species (ROS/RNS) produced by macrophages inside their phagolysosomes are closely related to immunity and inflammation by being involved in the removal of pathogens, altered cells, etc. The existence of a homeostatic mechanism regulating the ROS/RNS amounts inside phagolyso¬somes has been invoked to account for the efficiency of this crucial process, but this could never be unambiguously documented. Here, intracellular electrochemical analysis with platinized nanowires electrodes (Pt-NWEs) allowed monitoring of ROS/RNS effluxes with sub-millisecond resolution from individual phagolysosomes. These randomly impacted onto the electrode and were inserted inside a living macrophage. This evidenced for the first time that the consumption of ROS/RNS by their oxidation at the nanoelectrode surface stimulates the production of significant ROS/RNS amounts inside phagolysosomes. These results established the existence of the long-time postulated ROS/RNS homeostasis and allowed to quantify its kinetics and efficiency. ROS/RNS concentrations may then be maintained at sufficiently high levels for sustaining proper pathogen digestion rates without endangering the macrophage internal structures.

The exposome can be defined as the totality of exposures (diet, lifestyle, occupational, and environmental factors) received by an individual throughout the entire lifetime. It is unclear how these exposures are related to health. There is an increasing trend to intake food additives due to food mileage extension and maintenance of a healthy lifestyle. Therefore, we investigated the effect of aspartame (APM), an artificial sweetener, and ortho-phenyl phenol (OPP), a broad-spectrum fungicide and antibacterial agent, on oxidative stress in rats. APM (250 mg/kg/day; 1/16 NOAEL) or OPP (1 &#u181;g/kg/day; 1/10⁵ NOAEL) were administered to rats for &#~8776;eight weeks, through their mixed diet and drinking water, respectively. Administration of both exposomes for two weeks led to a significant reduction in reactive oxygen specie (ROS)-scavenging ability for hydroxyl radicals in plasma. In epididymis and hippocampus, both exposome treatments increased the 4-hydroxy-2-nonenal (HNE) levels, an index of lipid peroxidation. APM treatment for two weeks increased ROS generation in sperms without motility changes. Moreover, APM treatment for eight weeks slightly decreased sperm motility. Furthermore, amyloid beta (Abeta) levels were significantly increased in both epididymis and hippocampus after two weeks of OPP administration, that may be related to male infertility and dementia. Pretreatment with the antioxidant Twendee X (20 mg/kg/day; TIMA Japan) inhibited the Abeta level increase in both tissues. In conclusion, our data indicate that some exposomes systemically induce oxidative stress and increase Abeta levels in both epididymis and hippocampus. These results suggested that effective antioxidants such as Twendee X can inhibit exposome-induced oxidative stress in male infertility and neurodegenerative diseases.

It is widely known that oxidative stress causes many diseases. Free radical species triggering these results from transition metals, ischemia/reperfusion, drugs, inflammation, and environmental factors such as iron, and it causes the pathologies. Materials and mechanisms that are essential to the survival of many organisms including cells, protein and DNA is ruined by a free radical, causing many diseases, and these diseases were proposed that we called these oxidation stress diseases (free radical diseases) as a general term. A lot of specialized international journals about these are proud of the impact factor which is high in each. Although the disorder developmental mechanics and protective efficacy are studied in vitro and in animal experiments in detail, there is yet few antioxidative prevention or drugs to treat for those diseases in clinical studies using human.
Drug RADICUT (cut a free radical according to the name. Generic name “Edaravone”) used for brain stroke treatment widely or vitamin E effective in non-alcoholic lipid hepatitis (NASH) are the representative examples. These and other antioxidants cannot be an effective means for treatment and prevention of many other oxidative stress diseases probably because of the characteristics of the drugs. Appearance of a versatile antioxidant material which expresses an effect of prevention and treatment for many oxidation stress diseases without showing any side effects has been waited for. A supplement named Twendee X which was the vitamins and amino acids complex showing strong antioxidant effect finally came up recently. Ingredients and composition ratio of Twendee X was developed to maximize its antioxidant effects. The randomized, double-blind placebo control study of dementia prevention using Twendee X for human with mild cognitive impairment (MCI) was conducted by Professor Koji Abe of the Okayama University, Department of Neurology. Surprisingly, the result showed its preventive effect of MCI progression with a significant difference [1], and, as for this antioxidant material, it has been suggested to be effective in the prevention and treatment of many oxidative stress diseases, and more results are expected in the future. Here, based on these results, I will discuss about possibility of prevention and treatment by antioxidant materials.

Molecular targeting drugs were referred to as a paradigm shift in cancer treatment; it could not be beyond cytotoxic chemotherapy in terms of side effects and drug resistance. Only three treatments, namely surgery, chemotherapy and radiation have been the treatments for cancer for a long time. Because removal of cancer is the essence of these treatments, patients have been forced to carry a heavy burden. Various immunotherapies have been developed based on the theory, but almost all have failed. Immune checkpoint inhibitors (ICIs), however, which were the subjects of the Nobel Prize in Medicine and Physiology in 2018, have changed that aspect. Nivolumab, an immune checkpoint inhibitor, has dramatically improved the prognosis of melanoma which is one of the most refractory and poor-prone tumors. Melanoma is now followed by lung cancer, renal cancer, gastric cancer, and so on. ICIs make immune therapy the 4th cancer treatment. The most important thing is that immune therapy with ICIs is essentially different from the conventional three cancer treatments. ICIs work by adjusting the host side environment whereas the latter are is "artificially" eliminating cancer. ICIs only elicit the ability given by God to eliminate the cancer.
As antioxidants also have effects on the environment of the host’s side. These effects have been used in combination with chemotherapy and radiotherapy. Antioxidants have two main effects. One is the prevention from adverse effects of chemotherapy and radiotherapy. Another is the anti-tumor effects. As for the former, the preventing effects from mucinous inflammation and neuropathy caused by chemotherapy and radiotherapy have been reported. The control of these symptoms is important because these adverse effects are the main reason for the discontinuation of the treatments. Many studies have shown that antioxidants have the effects of suppressing the activation of NF-kappaB which leads to cancer growth and apoptosis suppression. According to some reports of chemotherapy, radiotherapy and molecular targeted therapies may have more host-mediated pro-tumorigenic and pro-metastatic effects than anti-tumor effects. Such therapy induced host effects are said to have angiogenesis, metastasis, and tumor-cell repopulation properties which are resistant to therapy. Preliminary in vivo mouse experiments with anti-oxidant composition showed elimination of the pro-metastatic effects of chemotherapy. Tumor growth was observed with short term exposure of 5-FU. Although anti-oxidant composition alone had no anti-tumor effect, tumor growth with 5-FU was completely reversed by anti-oxidant composition. The modulation of host side factors may have the same effects as the treatment itself and antioxidants are considered to be able to become a main player of cancer treatments.

Intestines are said to be involved in 60% of human immunity. More than 100 species, or 100 trillions of bacteria (intestinal bacteria) inhabit in the intestines and those bacteria can be divided into bad bacteria that trigger diseases, good bacteria that are considered to benefit health, and opportunistic bacteria. Regarding 20% of good, 10% of bad, and 70% of opportunistic bacteria as an ideal balance of gut bacteria, this balance can be disturbed by diarrhea, constipation and various diseases. In other words, oxidative stress can reduce diversity of gut bacteria. In that case, how does reducing oxidative stress alter gut microflora, and how does reducing oxidative stress affect daily defecation? Although administering antiseptic agent OPP to rats for 2 weeks increased their blood oxidative stress by 5%, there were no significant changes of bacterial gut diversity. Rats, however, were given a strong antioxidant composition, Twendee X(TWX), from 1 week before OPP decreased their oxidative stress by 13.3% against control, and increased bacterial diversity by 1.5 times. Also, observation of the defecating condition and gut microflora of 57 healthy persons showed increased diversity index and improvement of the defecating condition after TWX administration. These results suggest that, even if exposed to toxic substances which results in oxidative stress, daily effort of decreasing oxidative stress can improve/prevent their host's diseases by increasing gut bacterial diversity.

Oxidative stress is characterized by an excessive production of reactive oxygen species (ROS) and decreased antioxidant availability. It is considered as a major effector mechanism responsible for extensive cellular and molecular damage, perpetuating chronic inflammation and increased tissue destruction. Serum free thiols (R-SH, sulfhydryl groups) are considered as a robust and powerful read-out of the systemic in vivo reduction-oxidation (redox) status. Systemic oxidative stress is associated with decreased levels of reduced free thiols, since thiols are rapidly oxidized by circulating reactive species. Blood proteins, mainly albumin, harbor the largest amount of redox-active thiol groups (approximately 75% of the total thiol pool). Typically, high extracellular concentrations of reduced free thiols are representative of a healthy redox status.
We have examined systemic free thiol levels in a variety of (inflammatory) disease conditions, including pregnancy related, cardiovascular, renal, metabolic and rheumatological diseases. All these studies show a positive association between free thiol levels and favorable disease outcomes. Inversely, decreased free thiols have been linked to cardiovascular risk factors, such as ageing, obesity and alcohol consumption. In patient with Crohn’s disease in clinical remission systemic free thiols were still markedly reduced levels indicating the sensitivity of the Redox system as biomarker for (latent) disease. We evaluated free thiols in patients with inflammatory bowel disease and found them to be a strongly discriminating biomarker for endoscopic disease activity in IBD, even superiorly to classical fecal calprotectin levels. In a prospective clinical study that evaluated effects of dietary intake of the anti-oxidant riboflavin (vitamin B2) in Crohn’s disease, we observed significant improvement of systemic redox status after 3 weeks of riboflavin supplementation. This study highlights the potential significance of free thiols as therapeutic target of the redox signaling network in disease conditions. Finally, In a large population-based cohort (n=6000), the redox status was inversely associated with the risk of cardiovascular events and all-cause mortality (10 year follow up).
All these data highlight the potential significance and clinical relevance of the Redox status as modifiable biomarker in both health and disease.

The larynx and pharynx have important roles in maintaining voice and swallowing functions. Age-related deterioration of vocal and swallowing functions has become a significant problem for the elderly which can lead to aspiration pneumonia in the worst cases. Age-related histological and functional deficit of the pharyngolaryngeal system is the main issue. The vocal fold, the producer of the voice, becomes atrophied and weak which causes glottic insufficiency with reduced mucosal vibration. The voice becomes harsh and weak. Glottic insufficiency also causes mis-swallowing. Laryngeal elevation and pharyngeal constriction are weakened with age which can lead to aspiration. We have confirmed that oxidative stress increases with age in the vocal fold mucosa and muscle, as well as the pharyngeal muscles. Reactive oxygen species (ROS) damage the tissue and hamper function. In the case of singers or other professional voice users, overuse of the vocal fold also causes damage to the function of the vocal fold via ROS.
Anti-oxidants are the promising material that can maintain or improve the voice and the swallowing ability. We have revealed that anti-oxidants can maintain the vocal fold during excessive vocal loading tasks, avoiding inflammation or exhaustion of the vocal fold. Twendee X (TWX) has proven the strongest anti-oxidant, and our preliminary study demonstrated that an opera singer's voice was well maintained with the 1-month use of TWX. Another aspect of TWX is its capability to reduce allergic inflammation in the upper airway which is also important for protection of the pharyngolarynx.
Anti-oxidants should be effective, useful, and promising for maintaining the pharyngolarynx which secures voicing and swallowing functions.

Ginseng has been reported about for its immunostimulation and its effects on various diseases such as obesity, diabetes and cancer. In this study, from the viewpoint of antioxidant and oxidative stress, the influence of ginseng on humans was examined. By using the MULTIple free-radical Scavenging (MULTIS method), we measured the scavenging activity of the reactive oxygen species: hydroxyl radical (・OH), superoxide radical (O₂^・-), alkyloxy radical (RO・), alkyl peroxy radical (ROO・), methyl radical (・CH₃), and singlet oxygen (¹O₂). We also evaluated the antioxidant capacity multifaceted.
On the first day, subjects’ samples (plasma and saliva) were taken before ingesting ginseng tablets (n=5). After that, the samples were taken again, 4 hours after the administration of 18 tablets. They continued taking 18 tablets per day from the second day to the sixth day. On the seventh day, the samples were collected. The antioxidant capacity of the samples was examined using the MULTIS method.
By ingestion of ginseng tablets, the ・OH scavenging activity of samples increased after 1 week. Also, plasma ・CH₃, and saliva O₂^・- scavenging activity increased. It was suggested that ginseng improved the antioxidant capacity of human.

Traffic of the lipid-enclosed compartments (vesicles, endosomes, phagosomes etc.) within the cell is extremely important for sustaining cell life and allowing the cell to perform its function. These lipid bilayer bound organelles deliver their cargo through bilayer fusion with the other organelles or with the plasma membrane. In the case when the release of the cargo molecule is controlled only by diffusion, we have shown earlier for the case of vesicular exocytosis that emptying of such organelles occurs exponentially and the rate of the exponential decay is controlled by the size of the fusion pore [1]. The generality of the assumptions made for model derivation, as well as for laws underlying this model, suggest that the assumptions can be applied to a vast variety of cases. This can be done independently if organelles fuse with another part like the cellular membrane or electrode (like in vesicle impact electrochemical cytometry), etc. Moreover, being able to describe mass transport of the cargo molecules inside or out of the organelle allow one to characterize various physicochemical processes occurring within the organelle [2].
In particular, relevantly adapted models were applied to the case of the detection with the platinized carbon nanoelectrode of the reactive oxygen/nitrogen species (ROS/RNS) produced by macrophages inside their phagolysosomes (more on the experiment will be presented in Prof. Wei-Hua Huang's talk) [3]. Modelling the oxidation and mass transport of the ROS/RNS towards the nanoelectrode and comparison with experimental data evidenced for the first time that the consumption of ROS/RNS by their oxidation at the nanoelectrode surface stimulates the production of significant ROS/RNS amounts inside phagolysosomes, i.e., proved the very existence of a ROS/RNS homeostasis during phagocytosis. The homeostatic production rates of ROS/RNS inside individual phagolysosomes were quantified by employing the developed theory [3]. These results allowed measuring the long-time postulated ROS/RNS homeostasis within the phagolysosome, its kinetics and its efficiency. ROS/RNS concentrations may then be maintained at sufficiently high levels to sustain proper pathogen digestion rates without endangering the macrophage internal structures [3].

Oxidative stress is an essential metabolic outcome in aerobic organisms due to the activity of the mitochondria in providing the basic energy of cells or during the operation of several enzymatic pools. It also serves to regulate the size and shape of organs or restructure them during foetal development by apoptosis. Oxidative stress is also indispensable to the immune system by allowing macrophages to eliminate virus, bacteria and impaired or dead cells through phagocytosis [1]. In fact, no aerobic organism could live without oxidative stress: a fact that explains why evolution maintained such unsafe mechanisms in aerobic organisms. They are, however, associated to highly negative issues.
Indeed, oxidative stress mechanisms provide a variety of life-harmful radicals and species called generically Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) whose fluxes need to be finely controlled to avoid the destruction of most organic molecules (e.g., lipids in cell membranes, enzymes, etc.) and biological molecules (DNA, proteins, etc.) in cells. Thus, under normal conditions, a panoply of antioxidants and enzymatic systems ensures a fine homeostatic balance. Rupture of this delicate balance, however, is frequent and may provoke severe damages leading to human pathologies (aging, cancers, AIDS, hearth and brain strokes, Parkinson and Alzheimer’ diseases, etc.).
Using platinized carbon fiber ultramicroelectrodes, we could establish the composition of primary oxidative stress in macrophages [1, 2] and characterize the nature of functional hyperemia in the brain [3]. This led us to formulate an alternative hypothesis about the onset of Alzheimer disease when Amyloid-β and ascorbate molecules are present [4, 5].