Edward Sharman

Hydroxytyrosol protects against oxidative damage by simultaneous activation of mitochondrial biogenesis and phase II detoxifying enzyme systems in retinal pigment epithelial cells.

Studies in this laboratory have previously shown that hydroxytyrosol, the major antioxidant polyphenol in olives, protects ARPE-19 human retinal pigment epithelial cells from oxidative damage induced by acrolein, an environmental toxin and endogenous end product of lipid oxidation, that occurs at increased levels in age-related macular degeneration lesions. A proposed mechanism for this is that protection by hydroxytyrosol against oxidative stress is conferred by the simultaneous activation of two critically important pathways, viz., induction of phase II detoxifying enzymes and stimulation of mitochondrial biogenesis. Cultured ARPE-19 cells were pretreated with hydroxytyrosol and challenged with acrolein. The protective effects of hydroxytyrosol on key factors of mitochondrial biogenesis and phase II detoxifying enzyme systems were examined. Hydroxytyrosol treatment simultaneously protected against acrolein-induced inhibition of nuclear factor-E2-related factor 2 (Nrf2) and peroxisome proliferator-activated receptor coactivator 1 alpha (PPARGC1α) in ARPE-19 cells. The activation of Nrf2 led to activation of phase II detoxifying enzymes, including γ-glutamyl-cysteinyl-ligase, NADPH (nicotinamide adenine dinucleotide phosphate)-quinone-oxidoreductase 1, heme-oxygenase-1, superoxide dismutase, peroxiredoxin and thioredoxin as well as other antioxidant enzymes, while the activation of PPARGC1α led to increased protein expression of mitochondrial transcription factor A, uncoupling protein 2 and mitochondrial complexes. These results suggest that hydroxytyrosol is a potent inducer of phase II detoxifying enzymes and an enhancer of mitochondrial biogenesis. Dietary supplementation of hydroxytyrosol may contribute to eye health by preventing the degeneration of retinal pigment epithelial cells induced by oxidative stress.

Dietary supplementation with melatonin reduces levels of amyloid beta-peptides in the murine cerebral cortex

Abstract:  Melatonin levels decrease with aging in mice. Dietary supplementation with melatonin has recently been shown to result in a significant rise in levels of endogenous melatonin in the serum and all other tissue samples tested. Herein, the effects of dietary melatonin on brain levels of nitric oxide synthase, synaptic proteins and amyloid beta‐peptides (Aβ) were determined in mice. Melatonin supplementation did not significantly change cerebral cortical levels of nitric oxide synthase or synaptic proteins such as synaptophysin and SNAP‐25. Increased brain melatonin concentrations however, led to a significant reduction in levels of toxic cortical Aβ of both short and long forms which are involved in amyloid depositions and plaque formation in Alzheimers diseases. Thus, melatonin supplementation may retard neurodegenerative changes associated with brain aging. Depletion of melatonin in the brain of aging mice may in part account for this adverse change.

Age-related changes in serum melatonin in mice: higher levels of combined melatonin and 6-hydroxymelatonin sulfate in the cerebral cortex than serum, heart, liver and kidney tissues

Abstract:  Age‐related changes in levels of melatonin and 6‐hydroxymelatonin sulfate and effects of dietary melatonin on their levels in different tissues were determined in mice. Levels of melatonin were highest in the serum followed by liver, kidney, cerebral cortex and heart as measured by a quantitative and sensitive enzyme‐labeled immunosorbent assay (ELISA). Serum melatonin levels decreased with age, and were reduced by 80% in 27‐month old mice relative to 12‐month old mice. Levels of 6‐hydroxymelatonin sulfate were measured independently in various tissues. Levels of the melatonin metabolite, 6‐hydroxymelatonin sulfate were significantly higher than free melatonin in all tissues tested. Levels of 6‐hydroxymelatonin sulfate were highest in the cerebral cortex followed by the serum, heart, kidney, and liver. In 12‐month old mice 6‐hydroxymelatonin sulfate concentration was approximately 1000‐fold greater than that of melatonin in the cerebral cortex, it was only 3‐fold greater than melatonin levels in the serum. Thus only 0.1% of total melatonin in the brain was present in the free and unconjugated form but the corresponding value for serum was 27.4%. The cerebral cortex had the highest levels of combined melatonin and 6‐hydroxymelatonin sulfate than other tissue tested in control mice. There was no significant change in 6‐hydroxymelatonin sulfate levels between young and old mice. There was also no age‐dependent change in levels of serotonin or cortisol in the serum samples. Dietary supplementation with melatonin resulted in a significant increase in levels of melatonin in the serum and all other tissue samples tested. Thus, any age‐related decline of tissue melatonin can be reversed by supplementation with dietary melatonin.

Effects of age and dietary antioxidants on cerebral electron transport chain activity.

Aging is a pleiotropic process involving genetic and environmental factors. Recently it has been demonstrated that dietary constituents may affect senescence. In the present study, adult (3 month-old) mice were fed diets supplemented with ubiquinone (coenzyme Q(10)), alpha-lipoic acid, melatonin or alpha-tocopherol for a six-month period to determine if antioxidants may reverse or inhibit the progression of certain age-associated changes in cerebral mitochondrial electron transport chain (ETS) enzyme activities. The control consisted of a group of mice maintained on a basal diet for the same period. The activity of cytochrome c oxidase (Complex IV) increased with age but melatonin supplementation restored the activity to levels of 3 month-old animals. The activity of succinate dehydrogenase (Complex II) showed no age-related changes. However, this enzyme complex was elevated, in animals supplemented with coenzyme Q(10), alpha-lipoic acid and alpha-tocopherol, above corresponding values obtained with basal diet. NADH-ubiquinone oxidoreductase (Complex I) and ubiquinol:ferricytochrome-c oxidoreductase (Complex III) activities remained unchanged.

Mitochondrial nutrients improve immune dysfunction in the type 2 diabetic Goto-Kakizaki rats.

The development of type 2 diabetes is accompanied by decreased immune function and the mechanisms are unclear. We hypothesize that oxidative damage and mitochondrial dysfunction may play an important role in the immune dysfunction in diabetes. In the present study, we investigated this hypothesis in diabetic Goto‐Kakizaki rats by treatment with a combination of four mitochondrial‐targeting nutrients, namely, R‐α‐lipoic acid, acetyl‐L‐carnitine, nicotinamide and biotin. We first studied the effects of the combination of these four nutrients on immune function by examining cell proliferation in immune organs (spleen and thymus) and immunomodulating factors in the plasma. We then examined, in the plasma and thymus, oxidative damage biomarkers, including lipid peroxidation, protein oxidation, reactive oxygen species, calcium and antioxidant defence systems, mitochondrial potential and apoptosis‐inducing factors (caspase 3, p53 and p21). We found that immune dysfunction in these animals is associated with increased oxidative damage and mitochondrial dysfunction and that the nutrient treatment effectively elevated immune function, decreased oxidative damage, enhanced mitochondrial function and inhibited the elevation of apoptosis factors. These effects are comparable to, or greater than, those of the anti‐diabetic drug pioglitazone. These data suggest that a rational combination of mitochondrial‐targeting nutrients may be effective in improving immune function in type 2 diabetes through enhancement of mitochondrial function, decreased oxidative damage, and delayed cell death in the immune organs and blood.

Dietary melatonin selectively reverses age-related changes in cortical cytokine mRNA levels, and their responses to an inflammatory stimulus

The basal levels of expression of mRNA of cytokines, interleukin-6 (IL-6) and tumor necrosis factor (TNF-alpha), in the cerebral cortex of 5 and 26 month-old male B6C3F1 mice have been compared. In addition, the responsivity of animals of differing age to an inflammatory stimulus (lipopolysaccharide, LPS) has been studied. Basal levels of both of these cytokine mRNAs were elevated in aged animals relative to the younger group. However LPS administration led to a robust increase in cytokine mRNA levels in the younger animals but in aged mice, there was either an unchanged (IL-6) or a depressed (TNF-alpha) response. Administration of dietary melatonin (200 ppm) to aged mice for 6 weeks prior to sacrifice, resulted in reduction of basal levels of cytokine mRNA to values found in the younger animals. Furthermore, following administration of LPS to melatonin fed animals, cerebral cytokine mRNA levels were significantly elevated rather than being unchanged or depressed. Taken together these findings reflect a trend in the cortices of melatonin-treated aged mice, to more closely approximate the status of younger mice. For comparative purposes, parallel studies were carried out using an immunologically active organ (spleen) and a non-neural organ with a low rate of cell turnover (heart muscle). In both these tissues, basal levels of cytokine mRNAs of animals of either age were very low, and there was a marked positive response to LPS. Dietary melatonin had no effect on the responses of TNF-alpha mRNA to LPS but attenuated the reaction of splenic IL-6 mRNA, thus bringing the response closer to that of the younger mice.

Age‐related alterations of gene expression patterns in human CD8+ T cells

Aging is associated with progressive T‐cell deficiency and increased incidence of infections, cancer and autoimmunity. In this comprehensive study, we have compared the gene expression profiles in CD8+ T cells from aged and young healthy subjects using Affymetrix microarray Human Genome U133A‐2 GeneChips. A total of 5.2% (754) of the genes analyzed had known functions and displayed statistically significant age‐associated expression changes. These genes were involved in a broad array of complex biological processes, mainly in nucleic acid and protein metabolism. Functional groups, in which down‐regulated genes were overrepresented, were the following: RNA transcription regulation, RNA and DNA metabolism, intracellular (Golgi, endoplasmic reticulum and nuclear) transportation, signaling transduction pathways (T‐cell receptor, Ras/MAPK, JNK/Stat, PI3/AKT, Wnt, TGFβ, insulin‐like growth factor and insulin), and the ubiquitin cycle. In contrast, the following functional groups contained more up‐regulated genes than expected: response to oxidative stress and cytokines, apoptosis, and the MAPKK signaling cascade. These age‐associated gene expression changes may be responsible for impaired DNA replication, RNA transcription, and signal transduction, possibly resulting in instability of cellular and genomic integrity, and alterations of growth, differentiation, apoptosis and anergy in human aged CD8+ T cells.

Age‐related changes in murine CNS mRNA gene expression are modulated by dietary melatonin

Abstract:  Brain cellular functions decline with normal aging, accompanied by a changing profile of gene expression. Gene array analysis was used to quantitatively estimate messenger RNA (mRNA) expression levels in the cerebral cortex of both young (4‐month) and old (27‐month) B6C3F1 male mice. A stringent degree of significance was obtained by using multiple gene chips. Out of 12,423 mRNA levels, only 25 changed significantly with age. Nine of these genes coded for inflammatory proteins, all of which were elevated in aged, relative to younger mice. Melatonin (200 p.p.m.) included in the diet of aged animals for 8 wk elevated serum and cortical melatonin and reversed 13 of the 25 genes altered with age. In no case did melatonin potentiate age‐related changes in gene expression. The restoration of a more youthful gene profile to brains of aged animals by melatonin, to a large extent, involves reversal of age‐induced elevation of basal inflammatory parameters.

Melatonin and the aging brain

The events associated with brain aging are enumerated with emphasis on increased oxidative and inflammatory processes and on mitochondrial dysfunction. Several of these factors are further increased in a wide range of overt age-related neurological diseases. This generality has given impetus to concepts concerning similar therapeutic approaches common to a series of neurodegenerative disorders. Animal and cell culture models of several such disorders have benefited from the application of melatonin. The mechanisms underlying the neuroprotective properties of melatonin are likely to involve activation of specific melatonin receptors. This can lead to modulation of transcription factors and consequent altered gene expression, resulting in enhancement of antioxidant enzymes and downregulation of basal levels of inflammation. Melatonin has potential utility both in slowing normal brain aging and in treatment of neurodegenerative conditions. This is reinforced by the low cost of melatonin and its very low toxic hazard.

Reversal of biochemical and behavioral parameters of brain aging by melatonin and acetyl L-carnitine

The potential utility of dietary supplementation in order to prevent some of the oxidative and inflammatory changes occurring in the brain with age, has been studied. The cerebral cortex of 27-month-old male B6C3F1 mice had elevated levels of nitric oxide synthase 1 (EC 1.14.13.39) (nNOS) and peptide nitrotyrosine relative to cortices of younger (4-month-old) animals. After 25-month-old mice received basal diet together with 300 mg/l acetyl L-carnitine in the drinking water for 8 weeks, these levels were fully restored to those found in younger animals. A partial restoration was found when old animals received basal diet supplemented with 200 ppm melatonin in the diet. Levels of mRNA (messenger RNA) for nNOS were unchanged following these treatments implying translational regulation of nNOS activity. Behavioral indices indicative of exploratory behavior were also depressed in aged animals. Dietary supplementation with melatonin or acetyl L-carnitine partially reversed these changes. These findings suggest that dietary supplementation cannot merely arrest but indeed reverse some age-related increases in markers of oxidative and inflammatory events occurring with the cortex.