The Antioxidant PAPLAL Protects against Allergic Contact Dermatitis in Experimental Models.

PAPLAL, a mixture of platinum (nPt) and palladium (nPd) nanoparticles, is widely used as a topical agent because of its strong antioxidant activity. Allergic contact dermatitis (ACD) is one of the most common occupational skin diseases worldwide. However, the role of oxidative stress in ACD remains unclear. In the present study, we investigated the protective effects of topical PAPLAL treatment on 2,4-dinitrofluorobenzene (DNFB)-induced ACD. DNFB treatment increased 8-isoprostane content; upregulated Xdh, Nox2, and Nox4, pro-oxidant genes; and downregulated Sod1, an antioxidant gene, indicating oxidative damage in the ear skin. PAPLAL therapy significantly reduced ear thickness associated with the downregulation of inflammatory cytokine-related genes. PAPLAL also significantly increased the expression of the stress-response-related genes Ahr and Nrf2, as well as their target genes, but failed to alter the expression of redox-related genes. Furthermore, Sod1 loss worsened ACD pathologies in the ear. These results strongly suggest that PAPLAL protects against ACD through its antioxidant activity and activation of the AHR and NRF2 axes. The antioxidant PAPLAL can be used as a novel topical therapy for ACD that targets oxidative stress.

Natural Compounds That Enhance Motor Function in a Mouse Model of Muscle Fatigue.

Musculoskeletal disease can be a serious condition associated with aging that may lead to fractures and a bedridden state due to decreased motor function. In addition to exercise training to increase muscle mass, increasing muscle function with the intake of functional foods is an effective treatment strategy for musculoskeletal disease. Muscle-specific SOD2-deficient mice (muscle-Sod2-/-) show a severe disturbance in exercise in association with increased mitochondrial reactive oxygen species, as well as mitochondrial dysfunction and muscle damage. In the present study, to develop a therapeutic strategy for musculoskeletal disease, we searched for substances that enhanced motor function among functional compounds by in vivo screening using muscle-Sod2-/- mice as a muscle fatigue model. We administered 96 compounds, including antioxidants, to muscle-Sod2-/- mice and assessed their effects on treadmill performance. Among the administered compounds, gossypin, genistein, kaempferol, taxifolin, fumaric acid, ß-hydroxy-ß-methylbutyrate Ca, and astaxanthin, which are dietary functional food factors, increased forced running time in muscle-Sod2-/- mice. In addition, troglitazone, tempol, trolox, and MnTE-2-PyP, which are antioxidants, also significantly increased the running ability of muscle-Sod2-/- mice. These results suggest that the intake of functional foods with antioxidant activity can improve motor function. Muscle-Sod2-/- mice, as a muscle fatigue model, are suitable for the in vivo screening of functional substances that promote improvements in exercise and muscle performance.

Xanthine Oxidoreductase-Mediated Superoxide Production Is Not Involved in the Age-Related Pathologies in Sod1-Deficient Mice.

Reactive oxygen species (ROS) metabolism is regulated by the oxygen-mediated enzyme reaction and antioxidant mechanism within cells under physiological conditions. Xanthine oxidoreductase (XOR) exhibits two inter-convertible forms (xanthine oxidase (XO) and xanthine dehydrogenase (XDH)), depending on the substrates. XO uses oxygen as a substrate and generates superoxide (O2•-) in the catalytic pathway of hypoxanthine. We previously showed that superoxide dismutase 1 (SOD1) loss induced various aging-like pathologies via oxidative damage due to the accumulation of O2•- in mice. However, the pathological contribution of XO-derived O2•- production to aging-like tissue damage induced by SOD1 loss remains unclear. To investigate the pathological significance of O2•- derived from XOR in Sod1-/- mice, we generated Sod1-null and XO-type- or XDH-type-knock-in (KI) double-mutant mice. Neither XO-type- nor XDH-type KI mutants altered aging-like phenotypes, such as anemia, fatty liver, muscle atrophy, and bone loss, in Sod1-/- mice. Furthermore, allopurinol, an XO inhibitor, or apocynin, a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor, failed to improve aging-like tissue degeneration and ROS accumulation in Sod1-/- mice. These results showed that XOR-mediated O2•- production is relatively uninvolved in the age-related pathologies in Sod1-/- mice.

Pathological Relationship between Intracellular Superoxide Metabolism and p53 Signaling in Mice.

Intracellular superoxide dismutases (SODs) maintain tissue homeostasis via superoxide metabolism. We previously reported that intracellular reactive oxygen species (ROS), including superoxide accumulation caused by cytoplasmic SOD (SOD1) or mitochondrial SOD (SOD2) insufficiency, induced p53 activation in cells. SOD1 loss also induced several age-related pathological changes associated with increased oxidative molecules in mice. To evaluate the contribution of p53 activation for SOD1 knockout (KO) (Sod1-/-) mice, we generated SOD1 and p53 KO (double-knockout (DKO)) mice. DKO fibroblasts showed increased cell viability with decreased apoptosis compared with Sod1-/- fibroblasts. In vivo experiments revealed that p53 insufficiency was not a great contributor to aging-like tissue changes but accelerated tumorigenesis in Sod1-/- mice. Furthermore, p53 loss failed to improve dilated cardiomyopathy or the survival in heart-specific SOD2 conditional KO mice. These data indicated that p53 regulated ROS-mediated apoptotic cell death and tumorigenesis but not ROS-mediated tissue degeneration in SOD-deficient models.

Acai Extract Transiently Upregulates Erythropoietin by Inducing a Renal Hypoxic Condition in Mice.

Acai (Euterpe oleracea Mart. Palmae, Arecaceae) is a palm plant native to the Brazilian Amazon. It contains many nutrients, such as polyphenols, iron, vitamin E, and unsaturated fatty acids, so in recent years, many of the antioxidant and anti-inflammatory effects of acai have been reported. However, the effects of acai on hematopoiesis have not been investigated yet. In the present study, we administered acai extract to mice and evaluated its hematopoietic effects. Acai treatment significantly increased the erythrocytes, hemoglobin, and hematocrit contents compared to controls for four days. Then, we examined the hematopoietic-related markers following a single injection. Acai administration significantly increased the levels of the hematopoietic-related hormone erythropoietin in blood compared to controls and also transiently upregulated the gene expression of Epo in the kidney. Furthermore, in the mice treated with acai extract, the kidneys were positively stained with the hypoxic probe pimonidazole in comparison to the controls. These results demonstrated that acai increases the erythropoietin expression via hypoxic action in the kidney. Acai can be expected to improve motility through hematopoiesis.

SOD1 deficiency alters gastrointestinal microbiota and metabolites in mice.

Redox imbalance induces oxidative damage and causes age-related pathologies. Mice lacking the antioxidant enzyme SOD1 (Sod1-/-) exhibit various aging-like phenotypes throughout the body and are used as aging model mice. Recent reports suggested that age-related changes in the intestinal environment are involved in various diseases. We investigated cecal microbiota profiles and gastrointestinal metabolites in wild-type (Sod1+/+) and Sod1-/- mice. Firmicutes and Bacteroidetes were dominant in Sod1+/+ mice, and most of the detected bacterial species belong to these two phyla. Meanwhile, the Sod1-/- mice had an altered Firmicutes and Bacteroidetes ratio compared to Sod1+/+ mice. Among the identified genera, Paraprevotella, Prevotella, Ruminococcus, and Bacteroides were significantly increased, but Lactobacillus was significantly decreased in Sod1-/- mice compared to Sod1+/+ mice. The correlation analyses between cecal microbiota and liver metabolites showed that Bacteroides and Prevotella spp. were grouped into the same cluster, and Paraprevotella and Ruminococcus spp. were also grouped as another cluster. These four genera showed a positive and a negative correlation with increased and decreased liver metabolites in Sod1-/- mice, respectively. In contrast, Lactobacillus spp. showed a negative correlation with increased liver metabolites and a positive correlation with decreased liver metabolites in Sod1-/- mice. These results suggest that the redox imbalance induced by Sod1 loss alters gastrointestinal microflora and metabolites.

Platinum and palladium nanoparticle-containing mixture, PAPLAL, does not induce palladium allergy.

Palladium (Pd) is a common metal found in jewellery and dental appliances, but it has been shown to be likely to cause metal allergy. We previously reported that platinum (nPt) and palladium (nPd) nanoparticle-containing mixture (PAPLAL) has both superoxide dismutase and catalase activities and that the topical application of PAPLAL improved skin atrophy induced by chronic oxidative damage in an ageing mouse model. However, the safety of PAPLAL for preventing Pd allergy remains unclear. In the present study, we investigated whether or not PAPLAL induces Pd allergy. We found that PAPLAL treatment caused no skin inflammation, while nPd administration caused only slight skin inflammation compared to the palladium chloride-induced severe reaction in an experimental metal allergy model. A gene expression analysis revealed that PAPLAL treatment significantly suppressed the expression of Inf-γ, Il-1ß and Tnfα genes. Even in human clinical trials using patches containing metal nanoparticles, nPd and PAPLAL failed to induce significant skin inflammation. These results suggest that mixing with nPt in PAPLAL suppresses the inflammation response of nPd. PAPLAL can be expected to be applied to various skin treatments as a safe topical substance.

Syringaresinol Reverses Age-Related Skin Atrophy by Suppressing FoxO3a-Mediated Matrix Metalloproteinase-2 Activation in Copper/Zinc Superoxide Dismutase-Deficient Mice.

Aging is characterized by accumulation of chronic and irreversible oxidative damage, chronic inflammation, and organ dysfunction. Superoxide dismutase (SOD) serves as a major enzyme for cellular superoxide radical metabolism and physiologically regulates cellular redox balance throughout the body. Copper/zinc superoxide dismutase-deficient (SOD1-/-) mice showed diverse phenotypes associated with enhanced oxidative damage in whole organs. Here, we found that oral treatment with syringaresinol (also known as lirioresinol B), which is the active component in the berries of Korean ginseng (Panax ginseng C.A. Meyer), attenuated the age-related changes in Sod1-/- skin. Interestingly, syringaresinol morphologically normalized skin atrophy in Sod1-/- mice and promoted fibroblast outgrowth from Sod1-/- skin in vitro. These protective effects were mediated by the suppression of matrix metalloproteinase-2 overproduction in Sod1-/- skin, but not by increased collagen expression. Syringaresinol also decreased the oxidative damage and the phosphorylation of FoxO3a protein, which was a transcriptional factor of matrix metalloproteinase-2, in Sod1-/- skin. These results strongly suggest that syringaresinol regulates the FoxO3-matrix metalloproteinase-2 axis in oxidative damaged skin and exhibits beneficial effects on age-related skin involution in Sod1-/- mice.

Heterosis extends the reproductive ability in aged female mice†.

Heterosis is the beneficial effect of genetical heterogeneity in animals and plants. Although heterosis induces changes in the cells and individual abilities, few reports have described the effect of heterosis on the female reproductive ability during aging. In this study, we investigated the reproductive capability of genetically heterogeneous (HET) mice established by the four-way crossing of C57BL/6N, BALB/c, C3H/He, and DBA/2. We found the HET females naturally and repeatedly produced offspring, even in old age (14-18 months of age). We also found that HET females showed a significantly enlarged body and organ sizes in both youth and old age. In histological analyses, the numbers of primordial follicles, primary follicles, secondary follicles, and corpora lutea were significantly increased in the old ovaries of HET females compared with those in inbred C57BL/6 mice of the same age. In vitro fertilization experiments revealed that aged HET oocytes showed identical rates of fertilization, early development, and birth compared to those of young and old C57BL/6 oocytes. We further found the significantly increased expression of sirtuin genes concomitant with the up-regulation of R-spondin2 in old HET ovaries. These results confirm the novel phenotype, characterized by fertility extension and follicular retention due to heterosis, in old HET females. The HET female will be a valuable model for clarifying the mechanism underlying the effect of heterosis in the field of reproduction.

Topical Application of Trisodium Ascorbyl 6-Palmitate 2-Phosphate Actively Supplies Ascorbate to Skin Cells in an Ascorbate Transporter-Independent Manner.

Ascorbic acid (AA) possesses multiple beneficial functions, such as regulating collagen biosynthesis and redox balance in the skin. AA derivatives have been developed to overcome this compound's high fragility and to assist with AA supplementation to the skin. However, how AA derivatives are transferred into cells and converted to AA in the skin remains unclear. In the present study, we showed that AA treatment failed to increase the cellular AA level in the presence of AA transporter inhibitors, indicating an AA transporter-dependent action. In contrast, torisodium ascorbyl 6-palmitate 2-phosphate (APPS) treatment significantly enhanced the cellular AA level in skin cells despite the presence of inhibitors. In ex vivo experiments, APPS treatment also increased the AA content in a human epidermis model. Interestingly, APPS was readily metabolized and converted to AA in keratinocyte lysates via an intrinsic mechanism. Furthermore, APPS markedly repressed the intracellular superoxide generation and promoted viability associated with an enhanced AA level in Sod1-deficient skin cells. These findings indicate that APPS effectively restores the AA level and normalizes the redox balance in skin cells in an AA transporter-independent manner. Topical treatment of APPS is a beneficial strategy for supplying AA and improving the physiology of damaged skin.

Resveratrol Derivative-Rich Melinjo Seed Extract Attenuates Skin Atrophy in Sod1-Deficient Mice.

The oxidative damages induced by a redox imbalance cause age-related changes in cells and tissues. Superoxide dismutase (SOD) enzymes play a pivotal role in the antioxidant system and they also catalyze superoxide radicals. Since the loss of cytoplasmic SOD (SOD1) resulted in aging-like phenotypes in several types of murine tissue, SOD1 is essential for the maintenance of tissue homeostasis. Melinjo (Gnetum gnemon Linn) seed extract (MSE) contains trans-resveratrol (RSV) and resveratrol derivatives, including gnetin C, gnemonoside A, and gnemonoside D. MSE intake also exerts no adverse events in human study. In the present studies, we investigated protective effects of MSE on age-related skin pathologies in mice. Orally MSE and RSV treatment reversed the skin thinning associated with increased oxidative damage in the Sod1 (-/-) mice. Furthermore, MSE and RSV normalized gene expression of Col1a1 and p53 and upregulated gene expression of Sirt1 in skin tissues. In vitro experiments revealed that RSV significantly promoted the viability of Sod1 (-/-) fibroblasts. These finding demonstrated that RSV in MSE stably suppressed an intrinsic superoxide generation in vivo and in vitro leading to protecting skin damages. RSV derivative-rich MSE may be a powerful food of treatment for age-related skin diseases caused by oxidative damages.

Palladium and platinum nanoparticles attenuate aging-like skin atrophy via antioxidant activity in mice.

Cu-Zn superoxide dismutase (Sod1) loss causes a redox imbalance as it leads to excess superoxide generation, which results in the appearance of various aging-related phenotypes, including skin atrophy. Noble metal nanoparticles, such as palladium (Pd) and platinum (Pt) nanoparticles, are considered to function as antioxidants due to their strong catalytic activity. In Japan, a mixture of Pd and Pt nanoparticles called PAPLAL has been used to treat chronic diseases over the past 60 years. In the present study, we investigated the protective effects of PAPLAL against aging-related skin pathologies in mice. Transdermal PAPLAL treatment reversed skin thinning associated with increased lipid peroxidation in Sod1-/- mice. Furthermore, PAPLAL normalized the gene expression levels of Col1a1, Mmp2, Has2, Tnf-α, Il-6, and p53 in the skin of the Sod1-/- mice. Pt nanoparticles exhibited marked SOD and catalase activity, while Pd nanoparticles only displayed weak SOD and catalase activity in vitro. Although the SOD and catalase activity of the Pt nanoparticles significantly declined after they had been oxidized in air, a mixture of Pd and Pt nanoparticles continued to exhibit SOD and catalase activity after oxidation. Importantly, a mixture of Pd and Pt nanoparticles with a molar ratio of 3 or 4 to 1 continued to exhibit SOD and catalase activity after oxidation, indicating that Pd nanoparticles prevent the oxidative deterioration of Pt nanoparticles. These findings indicate that PAPLAL stably suppresses intrinsic superoxide generation both in vivo and in vitro via SOD and catalase activity. PAPLAL is a potentially powerful tool for the treatment of aging-related skin diseases caused by oxidative damage.

Superoxide dismutase 1 loss disturbs intracellular redox signaling, resulting in global age-related pathological changes.

Aging is characterized by increased oxidative stress, chronic inflammation, and organ dysfunction, which occur in a progressive and irreversible manner. Superoxide dismutase (SOD) serves as a major antioxidant and neutralizes superoxide radicals throughout the body. In vivo studies have demonstrated that copper/zinc superoxide dismutase-deficient (Sod1(-/-)) mice show various aging-like pathologies, accompanied by augmentation of oxidative damage in organs. We found that antioxidant treatment significantly attenuated the age-related tissue changes and oxidative damage-associated p53 upregulation in Sod1(-/-) mice. This review will focus on various age-related pathologies caused by the loss of Sod1 and will discuss the molecular mechanisms underlying the pathogenesis in Sod1(-/-) mice.

Collagen peptide and vitamin C additively attenuate age-related skin atrophy in Sod1-deficient mice.

Age-related skin thinning is correlated with a decrease in the content of collagen in the skin. Accumulating evidence suggests that collagen peptide (CP) and vitamin C (VC) transcriptionally upregulate type I collagen in vivo. However, the additive effects of CP and VC on age-related skin changes remain unclear. We herein demonstrate that CP and a VC derivative additively corrected age-related skin thinning via reduced oxidative damage in superoxide dismutase 1 (Sod1)-deficient mice. Co-treatment with these compounds significantly normalized the altered gene expression of Col1a1, Has2, and Ci1, a proton-coupled oligopeptide transporter, in Sod1(-/-) skin. The in vitro analyses further revealed that collagen oligopeptide, a digestive product of ingested CP, significantly promoted the bioactivity of the VC derivative with respect to the migration and proliferation of Sod1(-/-) fibroblasts. These findings suggest that combined treatment with CP and VC is effective in cases of age-related skin pathology.

Sod1 loss induces intrinsic superoxide accumulation leading to p53-mediated growth arrest and apoptosis.

Oxidative damages induced by a redox imbalance cause age-related changes in cells and tissues. Superoxide dismutase (SOD) enzymes play a major role in the antioxidant system and they also catalyze superoxide radicals (O2·-). Since the loss of cytoplasmic SOD (SOD1) resulted in aging-like phenotypes in several types of mouse tissue, SOD1 is essential for the maintenance of tissue homeostasis. To clarify the cellular function of SOD1, we investigated the cellular phenotypes of Sod1-deficient fibroblasts. We demonstrated that Sod1 deficiency impaired proliferation and induced apoptosis associated with O2·- accumulation in the cytoplasm and mitochondria in fibroblasts. Sod1 loss also decreased the mitochondrial membrane potential and led to DNA damage-mediated p53 activation. Antioxidant treatments effectively improved the cellular phenotypes through suppression of both intracellular O2·- accumulation and p53 activation in Sod1-deficient fibroblasts. In vivo experiments revealed that transdermal treatment with a vitamin C derivative significantly reversed the skin thinning commonly associated with the upregulated p53 action in the skin. Our findings revealed that intrinsic O2·- accumulation promoted p53-mediated growth arrest and apoptosis as well as mitochondrial disfunction in the fibroblasts.

Revised culture-based system for identification of Malassezia species.

Forty-six strains of Malassezia spp. with atypical biochemical features were isolated from 366 fresh clinical isolates from human subjects and dogs. Isolates obtained in this study included 2 (4.7%) lipid-dependent M. pachydermatis isolates; 1 (2.4%) precipitate-producing and 6 (14.6%) non-polyethoxylated castor oil (Cremophor EL)-assimilating M. furfur isolates; and 37 (34.3%) M. slooffiae isolates that were esculin hydrolyzing, 17 (15.7%) that were non-tolerant of growth at 40 degrees C, and 2 (1.9%) that assimilated polyethoxylated castor oil. Although their colony morphologies and sizes were characteristic on CHROMagar Malassezia medium (CHROM), all strains of M. furfur developed large pale pink and wrinkled colonies, and all strains of M. slooffiae developed small (<1 mm) pale pink colonies on CHROM. These atypical strains were distinguishable by the appearance of their colonies grown on CHROM. Three clinically important Malassezia species, M. globosa, M. restricta, and M. furfur, were correctly identified by their biochemical characteristics and colony morphologies. The results presented here indicate that our proposed identification system will be useful as a routine tool for the identification of clinically important Malassezia species in clinical laboratories.