Sexual dimorphism of autophagy in Syrian hamster Harderian gland culminates in a holocrine secretion in female glands.

The Syrian hamster Harderian gland (HG) has a large porphyrin metabolism with a sexual dimorphism, showing male HGs much lower porphyrin concentrations than female glands. Damage derived from this production of porphyrins, displayed by reactive oxygen species, forces the gland to develop morphological changes that must have a physiological significance. Thus, oxidative stress is present in two states: mild oxidative stress in male HGs and extreme oxidative stress in female HGs. Cathepsins data gave indirect indications about the presence of programmed cell death affecting the lysosomal pathway, especially in female HGs, which showed an accumulation of autophagic bodies. Our results showed different degrees of autophagy in Syrian hamster HGs depending on sex and probably controlled by the redox-sensitive transcription factors: NFkappaB and p53. The discovery of these sexual dimorphisms in redox signaling and in autophagy corroborates previous findings and underlines the key role of reactive oxygen species in the regulation of autophagy. In addition, in this paper we propose a physiological significance for these phenomena: male HGs develop a survival autophagy, while in female HGs, autophagy culminates in a detachment-derived cell death that plays a central role in its secretory activity, leading to a massive glandular secretion.

Autophagy upregulation and loss of NF-kappaB in oxidative stress-related immunodeficient SAMP8 mice.

Aged spleens from senescence-accelerated prone mice 8 (SAMP8) and senescence-accelerated resistant mice 1 (SAMR1) were examined to determine whether sex or melatonin had an effect on oxidative stress-related immune impairments. We observed that the immunosenescence of SAMP8 mice was associated with a redox imbalance, leading to an age-related increase in oxidative damage, resulting from a decrease in antioxidant defense and protease activity. Moreover, increased apoptotic cell death, a decrease in proliferative activity and the loss of NF-kappaB activation were also related to the immunodeficiency seen in SAMP8 compared to SAMR1 mice. Females demonstrated higher oxidative stress-related alterations in the immune response, and subsequent, melatonin treatment provided the best protective effects. Pathways involved in autophagy were upregulated in SAMP8 as an adaptive response to oxidative stress, in an attempt to rescue the cell from increased apoptosis and age-related immunodeficiency. However, the NF-kappaB signaling and autophagic processes were unaffected by treatment with melatonin. Therefore, we propose a key role for NF-kappaB signaling and autophagy in the oxidative stress-related immunosenescent spleens of SAMP8 mice.

Melatonin alters cell death processes in response to age-related oxidative stress in the brain of senescence-accelerated mice.

We studied the effect of age and melatonin on cell death processes in brain aging. Senescence-accelerated prone mice 8 (SAMP8) and senescence-accelerated resistant mice (SAMR1) at 5 and 10 months of age were used as models of the study. Melatonin (10 mg/kg) or its vehicle (ethanol at 0.066%) was administered in the drinking water from 1 to 9 months of age. Neurodegeneration, previously shown in the aged brain of SAMP8 and SAMR1 at 10 months of age, may be due to a drop in age-related proteolytic activities (cathepsin D, calpains, and caspase-3). Likewise, lack of apoptotic and macroautophagic processes were found, without apparent modification by melatonin. However, the caspase-independent cell death, owing to high p53 and apoptosis-inducing factor (AIF) levels, might be an alternative pathway of cell death in the aged brain. The main effects of melatonin treatment were observed in the aged SAMR1 mice; in this strain we observed a marked increase in antioxidant activity (catalase and superoxide dismutase). Likewise, a key antioxidant role of apoptosis-related proteins, Bcl-2 and AIF, was suggested in the aged brain of SAM mice, which was clearly influenced by melatonin. Moreover, the age-related increase of lysosomal activity of cathepsin B and a lysosomal membrane-associated protein 2 supports the possibility of the maintenance of lysosomal viability in addition to age-related impairments of the proteolytic or macroautophagic activities. The effectiveness of melatonin against the oxidative stress-related impairments and apoptosis during the aging process is, once more, corroborated in this article.

Sexual autophagic differences in the androgen-dependent flank organ of Syrian hamsters.

The flank organ of the Syrian hamster shows a biodynamic response to androgenic stimulation and is, therefore, a suitable model for the study of androgenic effects on hair and sebaceous glands. This organ is susceptible to programmed cell death (PCD), a prominent feature associated with sexual organ adjustment. In the present report, the type of PCD (apoptosis or autophagy) exhibited by this organ was evaluated. Caspase-3 activity, indicative of apoptosis, was not detectable in flank organ homogenates. Furthermore, cytokeratins, which are normally degraded during apoptosis, remained intact. On the other hand, Western blotting of Beclin 1 and light chain 3-II, both important autophagy markers, revealed autophagic processes in the flank organ in both sexes, especially in females. Cathepsin D activity, higher in males than in females, and procathepsin D expression were also consistent with autophagy and not apoptosis. Taken together, these data indicate that macroautophagy, and not apoptosis, is the main mechanism by which the flank organ responds to androgen. This is the first direct evidence establishing the relationship between autophagy and morphological changes in androgen-dependent organs.

Favorable effects of a prolonged treatment with melatonin on the level of oxidative damage and neurodegeneration in senescence-accelerated mice.

Senescence-accelerated mice (SAMP8) and senescence-accelerated resistant mice (SAMR1) were studied at 5 and 10 months of age, respectively. In the animals, neurodegenerative processes and how they were influenced by melatonin were examined. Melatonin (10 mg/kg) or vehicle (ethanol at 0.066%) treatments were administrated from the age of 1 to 9 months in the drinking water. Differences in the neurodegenerative markers examined were found between the two strains with a more damaged protein, phosphorylated Tau at Ser392, increased neurofibrillary tangles (NT) and higher alpha-synuclein expression in SAMP8 versus SAMR1 mice overall, when the mice were 10 months of age. Changes in density of receptors and oxidative stress-related signaling with age were found in the brains of SAM strains at 10 months as shown by a marked decrease in the level of MT-1 melatonin receptor and retinoic acid receptor-related orphan receptor (ROR)-alpha1. This diminution was earlier and more pronounced in SAMP8 mice. Likewise, the levels of nuclear factor-kappa B (NF-kB) transcriptional factor were higher in SAMP8 mice compared with SAMR1 mice regardless of age confirming the direct role of oxidative stress in the aging process. Treatment with melatonin in SAMP8 and SAMR1 mice reduced the neurodegenerative changes with an increase of ROR-alpha1 levels without an apparent influence in the levels of MT-1 receptor. However, different melatonin effects on NF-kB signaling were observed suggesting that NF-kB could trigger inflammatory processes in a different way, being SAM strain-dependent and associated with age-related oxidative stress levels. The effectiveness of melatonin in improving age-related neural impairments is corroborated.

Oxidative damage in the livers of senescence-accelerated mice: a gender-related response.

The prevalence of liver diseases emphasizes the need of animal models to research on the mechanism of disease pathogenesis. Furthermore, most of the liver pathologies have the oxidative stress as an important component. The senescence-accelerated mouse strain SAMP8 was proposed as a valuable animal model for the study of liver diseases. To gain a better understanding of the mechanisms underlying degenerative processes in SAMP8 mice livers, we studied the oxidative-induced damage in 5-month-old SAMP8 mice and SAMR1, senescence-accelerated-resistant mice. We found profound differences in the antioxidant response to aging between sexes, with males displaying lowest levels of main antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) in SAMP8; whereas females had no difference in their activities, except for GR, when compared with their SAMR1 controls. The results obtained show the binomial SOD/CAT as an important factor for counteracting reactive oxygen species-dependent damage. There were not pathological differences at the morphological level between both strains, although the decay in protection against free radicals had an immediate response by increasing lipid and protein oxidative damage in SAMP8 mice liver. At 5 months, both male and female SAMP8 mice confront the oxidative stress challenge to different extents. Indeed, proteins seem to be the most vulnerable biomolecule in SAMP8 male mice.

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 O(2) and CO(2) 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.

Survival mechanisms in a physiological oxidative stress model.

The Syrian hamster Harderian gland has as the remarkable feature of an extraordinary rate of porphyrin production, even higher than the liver. The low activity of the last enzyme of the route gives rise to the accumulation of the uncomplex porphyrins in the female glands. Moreover, due to the localization of the Harderian gland, porphyrins exposed to light produce reactive oxygen species and, thus, the gland presents a physiological oxidative stress, with a great number of sings of degeneration, but without compromising the gland integrity. The appearance of abnormal features in this gland was largely described in the past, but the significance is interpreted for the first time in this study. We have found that autophagic processes are the first result of an elevated porphyrin metabolism, as it is observed in both sexes. This mechanism is considered, in this case, as a constant renovation system that allows the normal gland activity to be sustained. Furthermore, there is a second procedure, invasive processes toward connective tissue, which even occasionally reach blood vessels with intravasation of damaged gland components into the bloodstream. This effect is a consequence of a strong oxidative stress environment that is mainly observed in the female gland, resembling to tumoral progression. Both mechanisms, autophagy and invasive processes, have to be implied in the maintenance of the gland integrity.

Melatonin neutralizes neurotoxicity induced by quinolinic acid in brain tissue culture.

Quinolinic acid is a well-known excitotoxin that induces oxidative stress and damage. In the present study, oxidative damage to biomolecules was followed by measuring lipid peroxidation and protein carbonyl formation in rat brain tissue culture over a period of 24 hr of exposure to this prooxidant agent at a concentration of 0.5 mm. Quinolinic acid enhanced lipid peroxidation in an early stage of tissue culture, and protein carbonyl at a later stage. These data confirm and extend previous studies demonstrating that quinolinic acid can induce significant oxidative damage. Melatonin, an antioxidant and neuroprotective agent with multiple actions as a radical scavenger and signaling molecule, completely prevented these prooxidant actions of quinolinic acid at a concentration of 1 mm. Morphological lesions and neurotoxicity induced by quinolinic acid were evaluated by light microscopy. Quinolinic acid produced extensive apoptosis/necrosis which was significantly attenuated by melatonin. Cotreatment with melatonin exerted a profound protective effect antagonizing the neurotoxicity induced by quinolinic acid. Glutathione reductase and catalase activities were increased by quinolinic acid and these effects were antagonized by melatonin. Furthermore, melatonin induced superoxide dismutase activity. Quinolinic acid and melatonin acted independently and by different mechanisms in modulating antioxidant enzyme activities. Our findings using quinolinic acid and melatonin clearly demonstrate that such changes should always be seen in the context of oxidative neurotoxicity and antioxidant neuroprotection.

Coexpression of MT1 and RORalpha1 melatonin receptors in the Syrian hamster Harderian gland.

Melatonin acts through several specific receptors, including membrane receptors (MT(1) and MT(2)) and members of the RZR/ROR nuclear receptors family, which have been identified in a large variety of mammalian and nonmammalian cells types. Both membrane and nuclear melatonin receptors have been partially characterized in Harderian gland of the Syrian hamster. Nevertheless, the identities of these receptors were unknown until this study, where the coexistence of MT(1) and RORalpha(1) in this gland was determined by nested RT-PCR followed by amplicon sequencing and Western-blot. Furthermore, the cellular localization of both receptors was determined by immunohistochemistry. Thus, MT(1) receptor was localized exclusively at the basal side of the cell acini, supporting the hypothesis that this receptor is activated by the pineal-synthesized melatonin. On the contrary, although a RORalpha(1)-immunoreactivity was observed in nuclei of epithelial cells of both sexes, an extranuclear specific staining, which was more frequently among those cells of males, was also seen. The implication of this possible nuclear exclusion of RORalpha(1) on the role of this indoleamine against oxidative stress is discussed.

Specific binding of melatonin to purified cell nuclei from mammary gland of swiss mice: day-night variations and effect of continuous light.

Melatonin binding sites were characterized in the nuclei of mouse mammary glands. The specific binding of 2-125I-iodomelatonin by homogenates of purified mammary gland cell nuclei was found to be rapid, reversible and saturable. Binding of 125I-melatonin exhibited day-night variations with the highest binding affinity observed during the dark period and the lowest affinity at midday. However, when the animals were maintained under continuous light exposure, binding of 125I-melatonin to cell nuclei showed a higher affinity than the daytime values found in animals maintained in normal photoperiod. Scatchard analysis of the binding data revealed the existence of a significant night-day variation in the binding kinetics, compatible with the existence of two classes of binding sites: a high-affinity binding site expressed during the night, with KD = 185 +/- 36 pm and a binding capacity of 6.24 +/- 0.4 fmol/mg protein, and a low-affinity site with KD = 562 +/- 57 nm and Bmax = 7.56 +/- 0.49 pmol/mg protein during the day. Interestingly, after 2 wk of continuous exposure to light, the animals killed at an equivalent midday time showed a significant increase in binding affinity, with KD = 1.43 +/- 0.2 nm and Bmax = 24.75 +/- 3.5 fmol/mg protein. Displacement experiments show an IC50 value compatible with the affinity constants obtained in the saturation experiments. These results indicate that the low-affinity binding site may be saturated by the high levels of melatonin found in the mouse mammary gland, and sustain the hypothesis of a circadian regulation of these melatonin binding sites by the photoperiod.

Characterization of melatonin high-affinity binding sites in purified cell nuclei of the hamster (Mesocricetus auratus) harderian gland.

In the present paper, binding of melatonin to purified cell nuclei from harderian glands of male and female hamsters was assessed. Binding of 125I-melatonin to cell nuclei fulfills the criteria for binding to a receptor site. Binding kinetics exhibit properties such as dependence on time and temperature as well as reversibility, saturability, high affinity and specificity. The dissociation constants (K(D)) and the number of binding sites (B(max)) for the binding of 125I-melatonin to harderian gland nuclei were 260 +/- 56 pm and 12.2 +/- 0.8 fmol/mg protein in male glands, and 280 +/- 43 pm and 9.8 +/- 0.6 fmol/mg protein in female glands, respectively. Competition experiments showed IC50 values for melatonin of 250 +/- 45 pm and 290 +/- 68 pm in male and female glands, respectively. Other indoleamines such as N-acetylserotonine and 5-metoxytryptamine showed IC50 values in the micromolar range, suggesting that the binding sites are specific for melatonin. Hill analyses of the data show nH values of 0.96-0.98, suggesting the existence of a single class of binding sites. These data indicate that specific 125I-melatonin binding sites exist in the cell nuclei of Harderian glands in male as well as in female hamsters, without significant differences between them. The K(D) and B(max) values obtained from the binding in both sexes correlates well with the concentration of melatonin described in these respective Harderian glands. It is hypothesized that the nuclear binding sites of melatonin here described could be a physiological melatonin receptor, which may be involved in the genomic-dependent antioxidant effects of melatonin on hamster Harderian glands elsewhere reported.

Effects of continuous light exposure on antioxidant enzymes, porphyric enzymes and cellular damage in the Harderian gland of the Syrian hamster.

The Syrian hamster Harderian gland (HG), an organ present in the male two secretory cell types (type-I and type-II cells), is physiologically exposed to high oxidative stress because of high concentrations of porphyrins and their precursor, 5-aminolevulinic acid. Because of its juxtaorbital location, the HG is accessible to light, and subject to phototoxic effects of these substances. After having previously demonstrated circadian rhythms in antioxidant enzymes, porphyric enzymes and oxidative damage of proteins and lipids, as well as influences of melatonin on these parameters, we have now studied the effects of continuous light (LL), which suppresses melatonin secretion by the pineal gland. Measurements were performed in two different circadian phases, in order to detect the presence or absence of day/night differences. In LL, no differences between circadian phases of subjective day and subjective night were demonstrable for 5-aminolevulinate synthase, 5-aminolevulinate dehydratase, porphobilinogen deaminase, or superoxide dismutase; temporal differences in glutathione reductase and catalase were markedly diminished, whereas all these parameters showed marked day/night differences in the rats exposed to a light/dark cycle of 14:10. In LL, oxidative damage to lipids was minimally effected, while protein damage was enhanced. LL also caused a reduction in the percentage of type-II cells. Therefore, cell differentiation in the HG does not seem to be controlled only by the androgen, but, unexpectedly, also by melatonin.

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.

Melatonin protects against delta-aminolevulinic acid-induced oxidative damage in male Syrian hamster Harderian glands.

Effects of the prooxidant delta-aminolevulinic acid (ALA) and the antioxidant melatonin (MEL) were investigated in the male Syrian hamster Harderian gland (HG). Rodent Harderian glands are highly porphyrogenic organs, which may be used as model systems for studying damage by delta-aminolevulinic acid and its metabolites, as occurring in porphyrias. Chronic administration of delta-aminolevulinic acid (2 weeks) markedly decreased activities of the porphyrogenic enzymes delta-aminolevulinate synthase (ALA-S) and delta-aminolevulinate dehydratase (ALA-D) and of the antioxidant enzymes superoxide dismutase (SOD), glutathione reductase (GR) and catalase (CAT), whereas porphobilinogen deaminase (PBG-D) remained unaffected. This treatment led to increased lipid peroxidation (LPO) and oxidatively modified protein (protein carbonyl) as well as to morphologically apparent tissue damage. Melatonin also caused decreases in delta-aminolevulinate synthase, delta-aminolevulinate dehydratase, superoxide dismutase, glutathione reductase and catalase. Despite lower activities of antioxidant enzymes, lipid peroxidation and protein carbonyl were markedly diminished. The combination of delta-aminolevulinic acid and melatonin led to approximately normal levels of delta-aminolevulinate dehydratase, glutathione reductase, catalase and protein carbonyl, and to rises in superoxide dismutase and porphobilinogen deaminase activities; lipid peroxidation remained even lower than in controls and the appearance of the tissue revealed a protective influence of melatonin. These results suggest that melatonin may have profound effects on the oxidant status of the Harderian gland.