Matrine and Oxymatrine: evaluating the gene mutation potential using in silico tools and the bacterial reverse mutation assay (Ames test).

The quinolizidine alkaloids matrine and its N-oxide oxymatrine occur in plants of the genus Sophora. Recently, matrine was sporadically detected in liquorice products. Morphological similarity of the liquorice plant Glycyrrhiza glabra with Sophora species and resulting confusion during harvesting may explain this contamination, but use of matrine as pesticide has also been reported. The detection of matrine in liquorice products raised concern as some studies suggested a genotoxic activity of matrine and oxymatrine. However, these studies are fraught with uncertainties, putting the reliability and robustness into question. Another issue was that Sophora root extracts were usually tested instead of pure matrine and oxymatrine. The aim of this work was therefore to determine whether matrine and oxymatrine have potential for causing gene mutations. In a first step and to support a weight-of-evidence analysis, in silico predictions were performed to improve the database using expert and statistical systems by VEGA, Leadscope (Instem®), and Nexus (Lhasa Limited). Unfortunately, the confidence levels of the predictions were insufficient to either identify or exclude a mutagenic potential. Thus, in order to obtain reliable results, the bacterial reverse mutation assay (Ames test) was carried out in accordance with OECD Test Guideline 471. The test set included the plate incorporation and the preincubation assay. It was performed with five different bacterial strains in the presence or absence of metabolic activation. Neither matrine nor oxymatrine induced a significant increase in the number of revertants under any of the selected experimental conditions. Overall, it can be concluded that matrine and oxymatrine are unlikely to have a gene mutation potential. Any positive findings with Sophora extracts in the Ames test may be related to other components. Notably, the results also indicated a need to extend the application domain of respective (Q)SAR tools to secondary plant metabolites.

Sub-Retinal Injection of Human Lipofuscin in the Mouse - A Model of "Dry" Age-Related Macular Degeneration?

Lipofuscin (LF) accumulates during lifetime in the retinal pigment epithelium (RPE) and is thought to play a crucial role in intermediate and late age-related macular degeneration (AMD). In an attemt to simulate aged retina and to study response of retinal microglia and RPE cells to LF, we injected a suspension of LF into the subretinal space of adult mice. LF suspension was obtained from human donor eyes. Subretinal injection of PBS or sham injection served as a control. Eyes were inspected by autofluorescence and optical coherence tomography, by electroretinography and on histological and ultrastructural levels. Levels of cytokine mRNA were determined by quantitative PCR separately in the RPE/choroid complex and in the retina. After injection of LF, microglial cells migrated quickly into the subretinal space to close proximity to RPE cells and phagocytosed LF particles. Retinal function was affected only slightly by LF within the first two weeks. After longer time, RPE cells showed clear signs of melanin loss and degradation. Levels of mRNA of inflammatory cytokines increased sharply after injection of both PBS and LF and were higher in the RPE/choroid complex than in the retina and were slightly higher after LF injection. In conclusion, subretinal injection of LF causes an activation of microglial cells and their migration into subretinal space, enhanced expression of inflammatory cytokines and a gradual degradation of RPE cells. These features are found also in an aging retina, and subretinal injection of LF could be a model for intermediate and late AMD.

Sub-Retinal Injection of Human Lipofuscin in the Mouse - A Model of "Dry" Age-Related Macular Degeneration?

Lipofuscin (LF) accumulates during lifetime in the retinal pigment epithelium (RPE) and is thought to play a crucial role in intermediate and late age-related macular degeneration (AMD). In an attemt to simulate aged retina and to study response of retinal microglia and RPE cells to LF, we injected a suspension of LF into the subretinal space of adult mice. LF suspension was obtained from human donor eyes. Subretinal injection of PBS or sham injection served as a control. Eyes were inspected by autofluorescence and optical coherence tomography, by electroretinography and on histological and ultrastructural levels. Levels of cytokine mRNA were determined by quantitative PCR separately in the RPE/choroid complex and in the retina. After injection of LF, microglial cells migrated quickly into the subretinal space to close proximity to RPE cells and phagocytosed LF particles. Retinal function was affected only slightly by LF within the first two weeks. After longer time, RPE cells showed clear signs of melanin loss and degradation. Levels of mRNA of inflammatory cytokines increased sharply after injection of both PBS and LF and were higher in the RPE/choroid complex than in the retina and were slightly higher after LF injection. In conclusion, subretinal injection of LF causes an activation of microglial cells and their migration into subretinal space, enhanced expression of inflammatory cytokines and a gradual degradation of RPE cells. These features are found also in an aging retina, and subretinal injection of LF could be a model for intermediate and late AMD.

Redox homeostasis and cell cycle activation mediate beta-cell mass expansion in aged, diabetes-prone mice under metabolic stress conditions: Role of thioredoxin-interacting protein (TXNIP).

Overnutrition contributes to insulin resistance, obesity and metabolic stress, initiating a loss of functional beta-cells and diabetes development. Whether these damaging effects are amplified in advanced age is barely investigated. Therefore, New Zealand Obese (NZO) mice, a well-established model for the investigation of human obesity-associated type 2 diabetes, were fed a metabolically challenging diet with a high-fat, carbohydrate restricted period followed by a carbohydrate intervention in young as well as advanced age. Interestingly, while young NZO mice developed massive hyperglycemia in response to carbohydrate feeding, leading to beta-cell dysfunction and cell death, aged counterparts compensated the increased insulin demand by persistent beta-cell function and beta-cell mass expansion. Beta-cell loss in young NZO islets was linked to increased expression of thioredoxin-interacting protein (TXNIP), presumably initiating an apoptosis-signaling cascade via caspase-3 activation. In contrast, islets of aged NZOs exhibited a sustained redox balance without changes in TXNIP expression, associated with higher proliferative potential by cell cycle activation. These findings support the relevance of a maintained proliferative potential and redox homeostasis for preserving islet functionality under metabolic stress, with the peculiarity that this adaptive response emerged with advanced age in diabetes-prone NZO mice.

Assessing autophagy in murine skeletal muscle: current findings to modulate and quantify the autophagic flux.

PURPOSE OF REVIEW: In addition to the currently available lysosomotropic drugs and autophagy whole-body knockout mouse models, we provide alternative methods that enable the modulation and detection of autophagic flux in vivo, discussing advantages and disadvantages of each method. RECENT FINDINGS: With the autophagosome-lysosome fusion inhibitor colchicine in skeletal muscle and temporal downregulation of autophagy using a novel Autophagy related 5-short hairpin RNA (Atg5-shRNA) mouse model we mention two models that directly modulate autophagy flux in vivo. Furthermore, methods to quantify autophagy flux, such as mitophagy transgenic reporters, in situ immunofluorescent staining and multispectral imaging flow cytometry, in mature skeletal muscle and cells are addressed. SUMMARY: To achieve clinical benefit, less toxic, temporary and cell-type-specific modulation of autophagy should be pursued further. A temporary knockdown as described for the Atg5-shRNA mice could provide a first insight into possible implications of autophagy inhibition. However, it is also important to take a closer look into the methods to evaluate autophagy after harvesting the tissue. In particular caution is required when experimental conditions can influence the final measurement and this should be pretested carefully.

Lipofuscin-dependent stimulation of microglial cells.

PURPOSE: To examine the reaction of microglial cells (MG) when incubated with lipofuscin (LP) in vitro with emphasis on the immunological reaction of the MG toward LP and the suppression of this reaction by immunomodulatory agents. MG are involved in the pathogenesis of degenerative eye disorders such as age-related macular degeneration (AMD). LP is a heterogeneous waste material that accumulates in the retinal pigment epithelium (RPE) cells with advancing age. LP is known to have toxic effects on RPE cells and therefore an elevated LP-derived fundus autofluorescence is a risk factor for AMD development. MG in the subretinal space have been reported in eyes affected by AMD. Moreover, in senescent mice, subretinal MG were found, which display an autofluorescence that may be derived from LP uptake. METHODS: In this study, we incubated MG (BV-2 cell line and primary cells from murine brain) in vitro with LP isolated from the human RPE. We observed phagocytosis, studied cell morphologies, and analyzed the cell culture supernatants. We also investigated the effect of the immunomodulatory agents hydrocortisone (HC), minocycline, and the tripeptide TKP. RESULTS: The MG phagocytosed the LP quickly and completely. We detected highly elevated levels of pro-inflammatory cytokines (especially of IL-6, IL-23p19, TNF-α, KC, RANTES, and IL-1α) in the cell culture supernatants. Furthermore, levels of vascular endothelial growth factor (VEGF) were raised in BV-2 cells. Anti-inflammatory agents added to the cell cultures inhibited the inflammatory reaction, in particular hydrocortisone (HC). Minocycline and TKP had less impact on the cytokine release. CONCLUSION: The interaction of MG and LP could play a role in the development of retinal degeneration by triggering an inflammatory reaction and angiogenesis.

Protein aggregates and proteostasis in aging: Amylin and ß-cell function.

The ubiquitin-proteasomal-system (UPS) and the autophagy-lysosomal-system (ALS) are both highly susceptible for disturbances leading to the accumulation of cellular damage. A decline of protein degradation during aging results in the formation of oxidatively damaged and aggregated proteins finally resulting in failure of cellular functionality. Besides protein aggregation in response to oxidative damage, amyloids are a different type of protein aggregates able to distract proteostasis and interfere with cellular functionality. Amyloids are clearly linked to the pathogenesis of age-related degenerative diseases such as Alzheimer's disease. Human amylin is one of the peptides forming fibrils in ß-sheet conformation finally leading to amyloid formation. In contrast to rodent amylin, human amylin is prone to form amyloidogenic aggregates, proposed to play a role in the pathogenesis of Type 2 Diabetes by impairing ß-cell functionality. Since aggregates such as lipofuscin and ß-amyloid are known to impair proteostasis, it is likely to assume similar effects for human amylin. In this review, we focus on the effects of IAPP on UPS and ALS and their role in amylin degradation, since both systems play a crucial role in maintaining proteome balance thereby influencing, at least in part, cellular fate and aging.

Soybean Oil-Derived Poly-Unsaturated Fatty Acids Enhance Liver Damage in NAFLD Induced by Dietary Cholesterol.

While the impact of dietary cholesterol on the progression of atherosclerosis has probably been overestimated, increasing evidence suggests that dietary cholesterol might favor the transition from blunt steatosis to non-alcoholic steatohepatitis (NASH), especially in combination with high fat diets. It is poorly understood how cholesterol alone or in combination with other dietary lipid components contributes to the development of lipotoxicity. The current study demonstrated that liver damage caused by dietary cholesterol in mice was strongly enhanced by a high fat diet containing soybean oil-derived ω6-poly-unsaturated fatty acids (ω6-PUFA), but not by a lard-based high fat diet containing mainly saturated fatty acids. In contrast to the lard-based diet the soybean oil-based diet augmented cholesterol accumulation in hepatocytes, presumably by impairing cholesterol-eliminating pathways. The soybean oil-based diet enhanced cholesterol-induced mitochondrial damage and amplified the ensuing oxidative stress, probably by peroxidation of poly-unsaturated fatty acids. This resulted in hepatocyte death, recruitment of inflammatory cells, and fibrosis, and caused a transition from steatosis to NASH, doubling the NASH activity score. Thus, the recommendation to reduce cholesterol intake, in particular in diets rich in ω6-PUFA, although not necessary to reduce the risk of atherosclerosis, might be sensible for patients suffering from non-alcoholic fatty liver disease.

Age-related oxidative changes in pancreatic islets are predominantly located in the vascular system.

Aged tissues usually show a decreased regenerative capacity accompanied by a decline in functionality. During aging pancreatic islets also undergo several morphological and metabolic changes. Besides proliferative and regenerative limitations, endocrine cells lose their secretory capacity, contributing to a decline in functional islet mass and a deregulated glucose homeostasis. This is linked to several features of aging, such as induction of cellular senescence or the formation of modified proteins, such as advanced glycation end products (AGEs) - the latter mainly examined in relation to hyperglycemia and in disease models. However, age-related changes of endocrine islets under normoglycemic and non-pathologic conditions are poorly investigated. Therefore, a characterization of pancreatic tissue sections as wells as plasma samples of wild-type mice (C57BL/6J) at various age groups (2.5, 5, 10, 15, 21 months) was performed. Our findings reveal that mice at older age are able to secret sufficient amounts of insulin to maintain normoglycemia. During aging the pancreatic islet area increased and the islet size doubled in 21 months old mice when compared to 2.5 months old mice, whereas the islet number was unchanged. This was accompanied by an age-dependent decrease in Ki-67 levels and pancreatic duodenal homeobox-1 (PDX-1), indicating a decline in proliferative and regenerative capacity of pancreatic islets with advancing age. In contrast, the number of p16Ink4a-positive nuclei within the islets was elevated starting from 10 months of age. Interestingly, AGEs accumulated exclusively in the islet blood vessels of old mice associated with increased amounts of inflammatory markers, such as the inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine (3-NT). In summary, the age-related increase in islet size and area was associated with the induction of senescence, accompanied by an accumulation of non-enzymatically modified proteins in the islet vascular system.

Mitochondrial contribution to lipofuscin formation.

Mitochondria have been in the focus of oxidative stress and aging research for decades due to their permanent production of ROS during the oxidative phosphorylation. The hypothesis exists that mitochondria are involved in the formation of lipofuscin, an autofluorescent protein aggregate that accumulates progressively over time in lysosomes of post-mitotic and senescent cells. To investigate the influence and involvement of mitochondria in lipofuscinogenesis, we analyzed lipofuscin amounts as well as the mitochondrial function in young and senescent cells. In addition we used an aging model and Lon protease deficient HeLa cells to investigate the influence of mitochondrial degradation processes on lipofuscin formation. We were able to show that mitophagy is impaired in senescent cells resulting in an increased mitochondrial mass and superoxide formation. In addition, the inhibition of mitochondrial fission leads to increased lipofuscin formation. Moreover, we observed that Lon protease downregulation is linked to a higher lipofuscinogenesis whereas the application of the mitochondrial-targeted antioxidant mitoTEMPO is able to prevent the accumulation of this protein aggregate.

Happily (n)ever after: Aging in the context of oxidative stress, proteostasis loss and cellular senescence.

Aging is a complex phenomenon and its impact is becoming more relevant due to the rising life expectancy and because aging itself is the basis for the development of age-related diseases such as cancer, neurodegenerative diseases and type 2 diabetes. Recent years of scientific research have brought up different theories that attempt to explain the aging process. So far, there is no single theory that fully explains all facets of aging. The damage accumulation theory is one of the most accepted theories due to the large body of evidence found over the years. Damage accumulation is thought to be driven, among others, by oxidative stress. This condition results in an excess attack of oxidants on biomolecules, which lead to damage accumulation over time and contribute to the functional involution of cells, tissues and organisms. If oxidative stress persists, cellular senescence is a likely outcome and an important hallmark of aging. Therefore, it becomes crucial to understand how senescent cells function and how they contribute to the aging process. This review will cover cellular senescence features related to the protein pool such as morphological and molecular hallmarks, how oxidative stress promotes protein modifications, how senescent cells cope with them by proteostasis mechanisms, including antioxidant enzymes and proteolytic systems. We will also highlight the nutritional status of senescent cells and aged organisms (including human clinical studies) by exploring trace elements and micronutrients and on their importance to develop strategies that might increase both, life and health span and postpone aging onset.

Macroautophagy is impaired in old murine brain tissue as well as in senescent human fibroblasts.

The overall decrease in proteolytic activity in aging can promote and accelerate protein accumulation and metabolic disturbances. To specifically analyze changes in macroautophagy (MA) we quantified different autophagy-related proteins (ATGs) in young, adult and old murine tissue as well as in young and senescent human fibroblasts. Thus, we revealed significantly reduced levels of ATG5-ATG12, LC3-II/LC3-I ratio, Beclin-1 and p62 in old brain tissue and senescent human fibroblasts. To investigate the role of mTOR, the protein itself and its target proteins p70S6 kinase and 4E-BP1 were quantified. Significant increased mTOR protein levels were determined in old tissue and cells. Determination of phosphorylated and basal amount of both proteins suggested higher mTOR activity in old murine tissue and senescent human fibroblasts. Besides the reduced levels of ATGs, mTOR can additionally reduce MA, promoting further acceleration of protein accumulation and metabolic disturbances during aging.

Reduced autophagy leads to an impaired ferritin turnover in senescent fibroblasts.

Changes in the two main intracellular degradation systems, the Ubiquitin-Proteasome System and the Autophagy-Lysosome pathway (ALP) are widely discussed as a hallmark of the aging process. To follow the age-related behavior of both degradation systems we examined their impact on ferritin, known to be degradable by both. Ferritin H was analyzed in young and senescent human fibroblasts, revealing a higher steady-state level in the senescent cells. By blocking both proteolytic systems, we confirmed that particularly the ALP plays a crucial role in ferritin H turnover. However, an unexpected increase in lysosomal activity in the senescent cells, suggests a dysregulation in the autophagy pathway. To further investigate the impaired ferritin H turnover, confocal microscopic colocalization studies of ferritin H with lysosomal-associated membrane protein 2a (Lamp2a) and monodansylcadaverine (MDC) were performed and clearly revealed the degradation of ferritin by macroautophagy. By induction of autophagy via inhibition of mTOR using rapamycin an increase of ferritin H turnover was obtained in senescent cells, demonstrating a mTOR dependent reduction of autophagy in senescent human fibroblasts.

Metabolic Syndrome, Redox State, and the Proteasomal System.

SIGNIFICANCE: Since the metabolic syndrome (MS) and pathologies associated with/resulting from metabolic dysregulations became a worldwide spreading and growing problem, the mechanisms mediating the according cellular changes got into a focus of interest. The ubiquitin-proteasomal system (UPS) is the main regulator of both the functional and dysfunctional protein pool of (not only) mammalian cells-thus, it is obvious that an impact on this system may also affect cellular functionality that directly depends on permanent regulation/adaption of the cell's proteostasis. However, the according research is still at the beginning. Recent Advances: It was also recently shown that maintaining a highly functional UPS positively correlates with increased health or even life span, thus modulation or restoration of UPS function may be an effective approach alleviating or even preventing MS detrimental consequences. CRITICAL ISSUES: Even if many consequences of metabolic dysregulation such as a slight but chronic redox shift to a more oxidative state (i.e., a low-grade systemic inflammation that increases reactive oxygen species formation, lipid peroxidation, protein oxidation, formation of advanced glycation end products, glycosylation, S-glutathionylation, redox shifts, endoplasmic reticulum stress, unfolded protein response, expression of transcription factors, and release of cytokines) are already known to affect the highly redox-regulated UPS, experimental data about UPS changes that are directly mediated by glucotoxic and/or lipotoxic stress are still rarely published. FUTURE DIRECTIONS: It may be taken into account that many MS-related pathologic changes result from UPS dysfunction or dysregulation. In this review, the main interface between MS effects and their impact on the UPS are highlighted since they may direct to new therapeutic approaches. Antioxid. Redox Signal. 25, 902-917.

Quantification of age-related changes of α-tocopherol in lysosomal membranes in murine tissues and human fibroblasts.

Considering the biological function of α-tocopherol (α-Toc) as a potent protective factor against oxidative stress, this antioxidant is in the focus of aging research. To understand the role of α-Toc during aging we investigated α-Toc concentrations in young and aged primary human fibroblasts after supplementation with RRR-α-Toc. Additionally, α-Toc contents were determined in brain, kidney, and liver tissue of 10 week-, 18 month-, and 24 month-old mice, which were fed a standard diet containing 100 mg/kg dl-α-tocopheryl acetate. α-Toc concentrations in isolated lysosomes and the expression of the α-Toc transport proteins Niemann Pick C1 (NPC1), Niemann Pick C2 (NPC2), and lipoprotein lipase were also analyzed. Obtained data show a significant age-related increase of α-Toc in murine liver, kidney, and brain tissue as well as in human dermal fibroblasts. Also liver and kidney lysosomes are marked by elevated α-Toc contents with aging. NPC1 and NPC2 protein amounts are significantly decreased in adult and aged murine kidney tissue. Also aged human dermal fibroblasts show decreased NPC1 amounts. Supplementation of young and aged fibroblasts led also to decreased NPC1 amounts, suggesting a direct role of this protein in α-Toc distribution. Our results indicate an age-dependent increase of α-Toc in different murine tissues as well as in human fibroblasts. Furthermore saturation and intracellular distribution of α-Toc seem to be strongly dependent on the availability of this vitamin as well as on the presence of the lysosomal protein NPC1. © 2016 BioFactors, 42(3):307-315, 2016.

Protein oxidation in aging and the removal of oxidized proteins.

Reactive oxygen species (ROS) are generated constantly within cells at low concentrations even under physiological conditions. During aging the levels of ROS can increase due to a limited capacity of antioxidant systems and repair mechanisms. Proteins are among the main targets for oxidants due to their high rate constants for several reactions with ROS and their abundance in biological systems. Protein damage has an important influence on cellular viability since most protein damage is non-repairable, and has deleterious consequences on protein structure and function. In addition, damaged and modified proteins can form cross-links and provide a basis for many senescence-associated alterations and may contribute to a range of human pathologies. Two proteolytic systems are responsible to ensure the maintenance of cellular functions: the proteasomal (UPS) and the lysosomal system. Those degrading systems provide a last line of antioxidative protection, removing irreversible damaged proteins and recycling amino acids for the continuous protein synthesis. But during aging, both systems are affected and their proteolytic activity declines significantly. Here we highlight the recent advantages in the understanding of protein oxidation and the fate of these damaged proteins during aging. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.