Could melatonin unbalance the equilibrium between autophagy and invasive processes?

The Syrian hamster Harderian gland (HG) is a juxtaorbital organ exhibiting marked gender-associated morphological differences. Regarding contents of porphyrins, this gland is a good model for studying physiological oxidative stress effects, since both sexes present strong (in females) and moderate (in males) levels of this stress in normal conditions. We have recently showed that autophagic processes are in the Syrian hamster HG as the first result of an elevated porphyrin metabolism observed in both sexes. In this case, autophagy is not a cell death mechanism per se but a constant renovation system which allows to continuing with the normal gland activity. Moreover, we have also reported that this gland presents invasive processes, resembling to tumoral progression, and are, additionally, a consequence of a strong oxidative stress environment that is mainly observed in female Syrian hamster HG and in minor proportion in male HG. Here, we present additional data and discuss a model of melatonin action on these cited processes by which melatonin would be able to destroy the equilibrium between both detoxifying actions. We postulate that melatonin reduces oxidative stress level into HG as direct antioxidant. This decrease of free radicals produces the autophagy inhibition due to outbreak signal disappearance in HG. Under these events and regarding the huge contents of porphyrins that this gland supports, the invasive process triggers.     

Autophagic Cell Death and its Importance for Fungal Developmental Biology and Pathogenesis

The Syrian hamster Harderian gland (HG) is a juxtaorbital organ exhibiting marked gender-associatedmorphological differences. Regarding contents of porphyrins, this gland is a good model for studyingphysiological oxidative stress effects, since both sexes present strong (in females) and moderate (inmales) levels of this stress in normal conditions. We have recently showed that autophagic processes arein the Syrian hamster HG as the first result of an elevated porphyrin metabolism observed in both sexes.In this case, autophagy is not a cell death mechanism per se but a constant renovation system whichallows to continuing with the normal gland activity. Moreover, we have also reported that this glandpresents invasive processes, resembling to tumoral progression, and are, additionally, a consequence of astrong oxidative stress environment that is mainly observed in female Syrian hamster HG and in minorproportion in male HG. Here, we present additional data and discuss a model of melatonin action on thesecited processes by which melatonin would be able to destroy the equilibrium between both detoxifyingactions. We postulate that melatonin reduces oxidative stress level into HG as direct antioxidant. Thisdecrease of free radicals produces the autophagy inhibition due to outbreak signal disappearance in HG.Under these events and regarding the huge contents of porphyrins that this gland supports, the invasiveprocess triggers.     

Antioxidant activity in Spalax ehrenbergi: a possible adaptation to underground stress

The blind subterranean mole rat Spalax ehrenbergi superspecies has evolved adaptive strategies to cope with underground stress. Hypoxia is known to stimulate reactive oxygen species generation; however, mechanisms by which Spalax counteracts oxidative damage have not been investigated before. We studied in Spalax the oxidative status of the Harderian gland (HG), an organ which is particularly vulnerable to oxidative stress in many rodents. With regard to the sexual dimorphism found in this gland, differences between males and females were determined and compared to the surface-dwelling Syrian hamster. Our results show, for the first time, that Spalax exhibits remarkably low biomolecular damage, which implies the existence of physiological strategies to avoid oxidative damage under fluctuating O₂ and CO₂ levels existing in the mole rat’s subterranean niche. Correspondingly, main antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione reductase (GR), exhibited high activities in both genders; in particular, remarkably high levels were measured in SOD. SOD and GR activities showed statistically significant differences between sexes. Melatonin, an important circadian agent is also a very important antioxidant molecule and is synthesized in the Harderian glands (HGs) of Spalax. Therefore, the possible interaction between antioxidant enzymes and melatonin is suggested.Joint senior authorship: Aaron Avivi and Ana Coto-Montes     

Effects of the circadian mutation 'tau' on the Harderian glands of Syrian hamsters

The Syrian hamster Harderian gland (HG) is an organ continually exposed to oxidative stress caused by high concentrations of porphyric metabolites. According to previous studies, melatonin, which is rhythmically secreted by the pineal gland and tonically produced by the HG, antagonizes the oxidative damage. HGs exhibit a strong gender-dependent correlation between porphyrins, melatonin, and histological appearance. In HGs of both sexes, we have investigated effects of a single gene defect in the circadian clock system (tau mutation) causing a shortened free-running period and an advanced maximum of circulating melatonin. Comparisons were made with wild-type animals, one group of which received daily pharmacological injections of melatonin in late photophase. Changes were observed in histological characteristics, porphyrin content, antioxidant enzyme activities, and damage of proteins and lipids. HGs of tau hamsters showed morphological changes which can be partially interpreted in terms of increased damage. Additionally, tau females exhibited a many-fold augmentation in the percentage of so-called type II cells, which are otherwise typical for the male glands. In tau hamsters of both sexes, major antioxidative enzyme activities (superoxide dismutase, glutathione reductase, and catalase) were markedly enhanced, a presumably compensatory response to increased oxidative stress. Higher oxidative damage in tau HGs was directly demonstrable by a many-fold increase in protein carbonyl. Rises in antioxidative enzymes were also observed upon injections of melatonin; this was, however, not accompanied by changes in protein carbonyl, so that enzyme inductions by the hormone should be understood as protective actions. Our data are not only in accordance with findings on protective effects by melatonin, but also with our earlier observation made in Drosophila that perturbations in the circadian system lead to increased oxidative stress.     

Castration Increases Cell Damage Induced by Porphyrins in the Harderian Gland of Male Syrian Hamster. Necrosis and Not Apoptosis Mediates the Subsequent Cell Death

It is known that the Harderian gland of male Syrian hamster synthesizes a much smaller amount of porphyrins than the gland of the female and that castration greatly increases this synthesis. We have studied in this experimental model the behavior of the different classes of secretory cells and their role in the synthesis of porphyrins, attempting to clarify the participation of these compounds in the cell damage leading to the formation of clear cells previously described in the gland of females. We have also investigated the mechanism underlying the death of these secretory cells after porphyrin accumulation (necrosis vs apoptosis). To achieve this, we have utilized the following techniques: (a) morphometrical; (b) ultrastructural; (c) biochemical (fluorescence spectrophotometry); and (d) molecular (DNA nick-end labeling in methacrylate sections and dot blot analysis). The glands from male hamsters (serving as control) present a very low rate of damaged cells that progressively rises after castration. This rise runs parallel to that of porphyrin synthesis, porphyrin deposits, and the decrease of Type II secretory cells. The damage and subsequent death of the secretory cells in the gland is produced by the deposit of porphyrins in the mitochondrial membrane. This porphyrin accumulation leads to a complete mitochondrial destruction that finally results in cell death and its secretion into the lumen. We finally conclude that this event is not a physiological cell death (apoptosis) but the consequence of the toxic accumulation of porphyrins (necrosis).     

Porphyric enzymes in hamster Harderian gland, a model of damage by porphyrins and their precursors. A chronobiological study on the role of sex differences

The Syrian hamster Harderian gland (HG), representing a highly porphyrogenic organ, was used as a model system for studying physiologically occurring damage of biomolecules by porphyrins and their precursors, phenomena associated with from the pathological situation of porphyrias. The species used exhibits the peculiarity of much higher porphyrogenesis in females than in males, offering possibilities for comparison of effects by different porphyrin levels in one species. Since concentrations of free, and therefore, radical-generating porphyric metabolites are difficult to determine in the presence of high amounts of secreted and crystallizing porphyrins, which are, moreover, mainly surface-reactive, and since indications existed for temporal changes in the oxidative stress caused by these molecules, the following approach was chosen: in HGs of both females and males, activities of the relevant porphyric enzymes, delta-aminolevulinate synthase (ALA-S), delta-aminolevulinate dehydratase (ALA-D) and porphobilinogen deaminase (PBG-D), were determined throughout the circadian cycle. Results were compared with the temporal patterns of lipid peroxidation and protein damage in the same glands. In females, a strong correspondence was observed between protein carbonyl and lipid peroxidation, peaking at the end of both photophase and scotophase; maximal activities of the three porphyric enzymes ALA-S, ALA-D, and PBG-D either coincided or slightly preceded the peaks of oxidative damage. In males, lower enzyme activities, especially in PBG-D, were associated with weakly expressed rhythmicity. Correspondingly, lipid peroxidation was lower and exhibited a smaller rhythm amplitude; protein carbonyl of males showed a temporal pattern differing from that of females, with regard to amplitude and phasing. These data are in agreement with morphological observations demonstrating particularly severe cell damage in the female HG under normal conditions.     

Effects of human chorionic gonadotropin and progesterone administration on porphyrin biosynthesis and histology of the Harderian glands in male and female Syrian hamsters.

We investigated the influence of hCG and progesterone on the control of porphyrin biosynthesis and histology in the Syrian hamster Harderian glands. Castration of male hamsters caused a marked elevation in porphyrin biosynthesis as revealed by the concentrations of porphyrins and the mRNA levels of the porphyrin pathway rate-limiting enzyme, 5-aminolevulinate synthase (ALV-S). Injection of hCG into castrated male hamsters also resulted in a significant increase in both porphyrin concentrations and levels of ALV-S mRNA compared with those in saline-injected castrated hamsters. Type II cells, which are filled with large lipid vacuoles and are characteristic of male phenotype, disappeared after castration, but administration of hCG partially prevented this change. On the other hand, neither administration of hCG nor progesterone implants could increase the very high porphyrin concentrations and ALV-S mRNA levels characteristic of female Syrian hamsters. As in the case of castrated male hamsters, injections of 20 IU hCG to female Syrian hamsters increased the relative number of Type II cells per square millimeter, whereas progesterone administration did not modify the relative number of Type II cells. These results indicate that hCG can modify Harderian gland morphology in both male and female hamsters and can exert a positive control in the expression of ALV-S gene in castrated male hamsters.     

L-carnitine protects C2C12 cells against mitochondrial superoxide overproduction and cell death

AIM To identify and characterize the protective effect that L-carnitine exerted against an oxidative stress in C2C12 cells.METHODS Myoblastic C2C12 cells were treated with menadione, a vitamin K analog that engenders oxidative stress, and the protective effect of L-carnitine(a nutrient involved in fatty acid metabolism and the control of the oxidative process), was assessed by monitoring various parameters related to the oxidative stress, autophagy and cell death. RESULTS Associated with its physiological function, a muscle cell metabolism is highly dependent on oxygen and may produce reactive oxygen species(ROS), especially under pathological conditions. High levels of ROS are known to induce injuries in cell structure as they interact at many levels in cell function. In C2C12 cells, a treatment with menadione induced a loss of transmembrane mitochondrial potential, an increase in mitochondrial production of ROS; it also induces autophagy and was able to provoke cell death. Pre-treatment of the cells with L-carnitine reduced ROS production, diminished autophagy and protected C2C12 cells against menadione-induced deleterious effects. CONCLUSION In conclusion, L-carnitine limits the oxidative stress in these cells and prevents cell death.     

Chloroquine inhibits glutamate-induced death of a neuronal cell line by reducing reactive oxygen species through sigma-1 receptor

Chloroquine, a widely used anti-malarial and anti-rheumatoid agent, has been reported to induce apoptotic and non-apoptotic cell death. Accumulating evidence now suggests that chloroquine can sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis by inhibiting autophagy. However, chloroquine is reported to induce GM1 ganglioside accumulation in cultured cells at low μM concentrations and prevent damage to the blood brain barrier in mice. It remains unknown whether chloroquine has neuroprotective properties at concentrations below its reported ability to inhibit lysosomal enzymes and autophagy. In the present study, we demonstrated that chloroquine protected mouse hippocampal HT22 cells from glutamate-induced oxidative stress by attenuating production of excess reactive oxygen species. The concentration of chloroquine required to rescue HT22 cells from oxidative stress was much lower than that sufficient enough to induce cell death and inhibit autophagy. Chloroquine increased GM1 level in HT22 cells at low μM concentrations but glutamate-induced cell death occurred before GM1 accumulation, suggesting that GM1 induction is not related to the protective effect of chloroquine against glutamate-induced cell death. Interestingly, BD1047 and NE-100, sigma-1 receptor antagonists, abrogated the protective effect of chloroquine against glutamate-induced cell death and reactive oxygen species production. In addition, cutamesine (SA4503), a sigma-1 receptor agonist, prevented both glutamate-induced cell death and reactive oxygen species production. These findings indicate that chloroquine at concentrations below its ability to inhibit autophagy and induce cell death is able to rescue HT22 cells from glutamate-induced cell death by reducing excessive production of reactive oxygen species through sigma-1 receptors. These results suggest potential use of chloroquine, an established anti-malarial agent, as a neuroprotectant against oxidative stress, which occurs in a variety of neurodegenerative diseases.     

Asymmetric learning to avoid heterospecific males in Mesocricetus hamsters

If a female mates with a male of a closely related species, her fitness is likely to decline. Consequently, females may develop behavioral mechanisms to avoid mating with heterospecific males. In some species, one such mechanism is for adult females to learn to discriminate against heterospecific males after exposure to such males. We have previously shown that adult, female Syrian hamsters (Mesocricetus auratus) learn to discriminate against male Turkish hamsters (Mesocricetus brandti) after exposure to a single heterospecific male during 8 days across a wire-mesh barrier. Here we repeated that experiment but this time we exposed female Turkish hamsters to a male Syrian hamster for 8 days and then measured sexual and aggressive behaviors towards that heterospecific male and towards a conspecific male. In contrast to female Syrian hamsters, female Turkish hamsters did not differ in their latency to go into lordosis or in any measure of aggression towards either type of male. Female Turkish hamsters spent less time in lordosis with the heterospecific male, but the percentage of trials in which females copulated with conspecific and heterospecific males did not differ. When comparing females from both species that had been exposed to a heterospecific male for 8days, female Syrian hamsters copulated less and were more aggressive towards the heterospecific male compared to the behavior of female Turkish hamsters. We discuss how this asymmetric response between females of the two species may be due to the much larger geographical range of Turkish hamsters compared to Syrian hamsters.     

Potential role of autophagy in behavioral changes of the flank organ

The flank organ of the Syrian hamster, which shows a biodynamic response to androgen stimulation, is considered a good model for studying the androgen effect on sebaceous gland and hair. This organ is susceptible to programmed cell death (PCD), a prominent feature associated with sexual organ adjustment. We have recently shown the first direct evidence of a relationship between autophagy and morphological changes in androgen-dependent organs. In this work, animals were stabulated (housed) for two months under control conditions and, after sacrifice, autophagy markers, Beclin 1 and LC3-II, were evaluated. The results revealed autophagic processes in the flank organ of both sexes, especially in females, and no detectable caspase-3 activity. Therefore, these data indicated that macroautophagy, and not apoptosis, was the main mechanism by which the flank organ responds to androgen. Here, we present additional data on the flank organ, showing that the intensity of autophagy between genders depends on the length of stabulation. These data could indicate a role for autophagy in response to behavioral influences. A possible explanation and its implication in Syrian hamster social defeat are discussed in this addendum.     

Dimorphic expression of uncoupling protein-3 in golden hamster harderian gland: effects of castration and testosterone administration

Hamster (Mesocricetus auratus) harderian gland (HG) is a dimorphic orbital gland producing a copious lipid secretion. Two cell-types are present in hamster HG, type I in both sexes, type II only in males. In hamster HGs, we found a marked sexual dichotomy in the expression of uncoupling protein-3 (UCP3), a mitochondrial protein carrier, that probably exports fatty acid anions and fatty acid peroxides from the mitochondrial matrix. Following castration and/or testosterone treatment: (1) UCP3 levels correlated with the type II-cell percentage, not with testosterone levels, (2) in male HGs, UCP3 was comparable to female levels at 30 days post-castration (when the type II-cell percentage had fallen from 50 to 5%), although testosterone was already near zero at 15 days (when neither the type II-cell percentage nor the UCP3 level had fallen), and testosterone-replacement therapy prevented these changes. Testosterone-treated females possessed type II cells and a UCP3 level about twofold higher than in control females. Males displayed more intense UCP3 immunohistochemical positivity in type I HG cells than females. Hence, testosterone may indirectly control UCP3 expression by regulating the gland's morphological and lipid dimorphism. Straight-chain fatty acids [found in alkyl diacylglycerols (ADGs) in males] are oxidized predominantly in mitochondria, branched-chain fatty acids (abundant in ADGs in females) predominantly in peroxisomes, so we speculate that the higher UCP3 expression in males reflects greater fatty acid flux in HG mitochondria. This is supported by our finding that in female (not male) HGs, the peroxisome-rich fraction contained alpha-methylacyl-CoA racemase (AMACR), an enzyme important in the beta-oxidation of branched-chain fatty acids.     

Autophagic and proteolytic processes in the Harderian gland are modulated during the estrous cycle

The Syrian hamster Harderian gland (HG) is an organ that undergoes physiological autophagy in response to oxidative stress induced by porphyrin production. Porphyrin production in the HG has marked sex differences and is closely linked to reproductive function. In the present study, we observed that the estrous cycle and associated estrogen variations may affect oxidative-stress-induced proteolytic processes. In particular, significant changes in autophagic activity were detected during the estrous cycle. Notably, increased activation of macroautophagy as well as chaperone-mediated autophagy in the estrus phase coincided with a minimal antioxidant capability and the highest protein damage levels. By contrast, autophagic machinery was found to be blocked in the diestrus phase, likely due to mammalian target of rapamycin activation, which could be corroborated by the subsequent pS6K activation. Analogous results were observed regarding proteasome activity, which also showed maximal activity in the estrus phase. Interestingly, all these mechanisms were associated with important morphological changes in the HG during the estrous cycle. We observed statistically significant increases in Type II cells, which may be related to extensive autophagy in the estrus phase. Physiologically, this would result in a significant release of porphyrins specifically when females are more receptive. These data support the role of porphyrins as pheromones, as other authors have previously suggested, thus making the HG a scent organ. In addition, these results suggest a porphyrin-based approach to the treatment of porphyria during pregnancy, a condition for which no treatment is currently known.     

Gender differences and time course of castration-induced changes in porphyrins, indoles, and proteins in the Harderian glands of the Syrian hamster.

Sexual differences and the effects of orchidectomy were determined for porphyrin and melatonin concentrations and for the activities of the enzymes N-acetyltransferase and hydroxyindole-O-methyltransferase, which synthesize melatonin from serotonin, in the Harderian glands of the Syrian hamster. Porphyrin concentrations in intact males were about 1/400th those of intact females. Castration for 1 week increased male Harderian porphyrin concentrations 10-fold; by 3 weeks, castrated male porphyrin levels were 140 times those of control values. N-Acetyltransferase activity in intact male Harderian glands was about 4 times that of females. Castration led to a drop in N-acetyltransferase activity to female levels within 2 weeks. Hydroxyindole-O-methyltransferase activity was 7 times higher in females than in males and castration had no effect on male Harderian hydroxyindole-O-methyltransferase activity. Neither gender nor castration influenced Harderian melatonin concentrations. Soluble proteins in Harderian glands from male and female hamsters and from male hamsters castrated for 1 and 4 weeks were examined by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. The gel profiles revealed several differences among the protein distribution in male and female gland lysates. Orchidectomy led to a female protein pattern within 4 weeks.     

Effects of delta-aminolevulinic acid and melatonin in the harderian gland of female Syrian hamsters

Effects of delta-aminolevulinic acid (ALA) and melatonin were investigated in the female Syrian hamster Harderian gland. This is an organ physiologically exposed to strong oxidative stress due to the highest porphyrinogenic rates known in nature. Enzyme activities of porphyrin biosynthesis and of antioxidative protection, oxidative protein modification, and histological integrity were studied. In the porphyrin biosynthetic pathway, ALA and melatonin acted synergistically by downregulating ALA synthase (ALA-S) and stimulating product formation from ALA; the combination of ALA and melatonin suppressed ALA-S activity, down to about 1% of that in controls. While ALA effects on porphyrinogenesis can be interpreted in terms of homeostasis, melatonin's actions may be seen in relation to seasonality and/or reduction of oxidative stress. Among antioxidant enzymes, superoxide dismutase (SOD) and glutathione reductase (GR) activities were diminished by ALA, presumably due to the vulnerability of their active centers to free radicals, whereas melatonin moderately increased SOD. Both ALA and melatonin strongly stimulated catalase (CAT), thereby counteracting the oxidative stress induced by ALA and its metabolites. Nevertheless, exogenous ALA caused a strong net rise in protein carbonyl and considerable damage of tissue. When given together with ALA, melatonin antagonized these effects and largely protected the integrity of glandular structures.     

Autophagy,programmed cell death and reactive oxygen species in sexual reproduction in plants

Autophagy is one of the major cellular processes of recycling of proteins, metabolites and intracellular organelles, and plays crucial roles in the regulation of innate immunity, stress responses and programmed cell death (PCD) in many eukaryotes. It is also essential in development and sexual reproduction in many animals. In plants, although autophagy-deficient mutants of Arabidopsis thaliana show phenotypes in abiotic and biotic stress responses, their life cycle seems normal and thus little had been known until recently about the roles of autophagy in development and reproduction. Rice mutants defective in autophagy show sporophytic male sterility and immature pollens, indicating crucial roles of autophagy during pollen maturation. Enzymatic production of reactive oxygen species (ROS) by respiratory burst oxidase homologues (Rbohs) play multiple roles in regulating anther development, pollen tube elongation and fertilization. Significance of autophagy and ROS in the regulation of PCD of transient cells during plant sexual reproduction is discussed in comparison with animals.     

Autophagy induction by SIRT6 is involved in oxidative stress-induced neuronal damage

SIRT6 is a NAD+-dependent histone deacetylase and has been implicated in the regulation of genomic stability, DNA repair, metabolic homeostasis and several diseases. The effect of SIRT6 in cerebral ischemia and oxygen/glucose deprivation (OGD) has been reported, however the role of SIRT6 in oxidative stress damage remains unclear. Here we used SH-SY5Y neuronal cells and found that overexpression of SIRT6 led to decreased cell viability and increased necrotic cell death and reactive oxygen species (ROS) production under oxidative stress. Mechanistic study revealed that SIRT6 induced autophagy via attenuation of AKT signaling and treatment with autophagy inhibitor 3-MA or knockdown of autophagy-related protein Atg5 rescued H2O2-induced neuronal injury. Conversely, SIRT6 inhibition suppressed autophagy and reduced oxidative stressinduced neuronal damage. These results suggest that SIRT6 might be a potential therapeutic target for neuroprotection.     

Male reproductive senescence: the price of immune-induced oxidative damage on sexual attractiveness in the blue-footed booby

In animals, male reproduction is commonly a function of sexual attractiveness, based on the expression of sexually dimorphic traits that advertise genuinely the male's quality. Male performance may decline with age because physiological functions underlying sexual attractiveness may be affected by senescence. Here we show that a sexual signal (foot colour) declines with age, due probably to the deleterious effects of oxidative damage. We found that in the blue-footed booby Sula nebouxii foot colour during courtship was less attractive in senescent than in middle-aged males. In addition, we increased reactive oxygen species experimentally by immunizing males with lipopolysaccharide, a bacterial cell wall component that induces marked oxidative stress in animals. The immune system activation induced greater lipid peroxidation and invoked changes on colour expression (less attractive), particularly in senescent males. These results support the idea that oxidative stress affects reproductive senescence, and suggest that oxidative damage might be a proximal mechanism underlying age-reproductive patterns in long-lived animals.     

RAGE regulates autophagy and apoptosis following oxidative injury

The receptor for advanced glycation end products (RAGE) plays a crucial role in several disease processes including diabetes, inflammation, and cancer. Compared with apoptosis ("programmed cell death"), autophagy is a genetically programmed, evolutionarily conserved cell survival process that degrades long-lived cellular proteins and organelles ("programmed cell survival"). Recently we reported that RAGE is an important regulator of oxidative stress in pancreatic cancer cells. Upregulation of RAGE expression by the nuclear factor (NF)-κB pathway decreases reactive oxygen species (ROS)-induced oxidative injury. In contrast, suppression of RAGE expression increases pancreatic tumor cell sensitivity to oxidative injury. Furthermore, RAGE is a positive regulator of autophagy, and negative regulator of apoptosis during oxidative stress. These findings provide insight into how crosstalk between apoptosis and autophagy is mediated via ROS signaling with a process involving RAGE.