Armando Luna-López

Senescence associated secretory phenotype profile from primary lung mice fibroblasts depends on the senescence induction stimuli

Cellular senescence is a multifactorial phenomenon of growth arrest and distorted function, which has been recognized as an important feature during tumor suppression mechanisms and a contributor to aging. Senescent cells have an altered secretion pattern called Senescence-Associated Secretory Phenotype (SASP) that comprises a complex mix of factors including cytokines, growth factors, chemokines, and matrix metalloproteinases. SASP has been related with local inflammation that leads to cellular transformation and neurodegenerative diseases. Various pathways for senescence induction have been proposed; the most studied is replicative senescence due to telomere attrition called replicative senescence (RS). However, senescence can be prematurely achieved when cells are exposed to diverse stimuli such as oxidative stress (stress-induced premature senescence, SIPS) or proteasome inhibition (proteasome inhibition-induced premature senescence, PIIPS). SASP has been characterized in RS and SIPS but not in PIIPS. Hence, our aim was to determine SASP components in primary lung fibroblasts obtained from CD-1 mice induced to senescence by PIIPS and compare them to RS and SIPS. Our results showed important variations in the 62 cytokines analyzed, while SIPS and RS showed an increase in the secretion of most cytokines, and in PIIPS only 13 were incremented. Variations in glutathione-redox balance were also observed in SIPS and RS, and not in PIIPS. All senescence types SASP displayed a pro-inflammatory profile and increased proliferation in L929 mice fibroblasts exposed to SASP. However, the behavior observed was not exactly the same, suggesting that the senescence induction pathway might encompass dissimilar responses in adjacent cells and promote different outcomes.

Protective Effect of Tert-Butylhydroquinone on the Quinolinic-Acid-Induced Toxicity in Rat Striatal Slices: Role of the Nrf2-Antioxidant Response Element Pathway

Tert-butylhydroquinone (tBHQ) is a xenobiotic with reported antioxidant properties. tBHQ has been shown to induce nuclear translocation of the transcription factor NF-E2-related factor 2 (Nrf2) to further activate the antioxidant response element (ARE). In turn, the Nrf2/ARE pathway is responsible for the induction of phase 2 antioxidant enzymes that detoxify oxidant promoters from different toxic insults. In this work, the antioxidant and protective actions of tBHQ were explored for the first time on different biomarkers of the neurotoxic model produced by the excitotoxic and pro-oxidant molecule quinolinic acid (QUIN) in rat striatal slices. For comparison purposes, 3-nitropropionic acid was used as reference model. Our results show that tBHQ (25 µM) prevented the QUIN-induced lipid peroxidation and mitochondrial dysfunction. In addition, tBHQ enhanced glutathione-S-transferase activity, partially recovering its depletion induced by QUIN treatment. Our results also demonstrated that tBHQ was able to induce nuclear accumulation of Nrf2 and further antioxidant protection: while QUIN alone decreased the nuclear Nrf2, a treatment with tBHQ preserved the nuclear levels Nrf2 in the presence of QUIN. Therefore, the tBHQ-mediated Nrf2/ARE induction constitutes a signaling-mediated antioxidant strategy and therapeutic tool to be tested in different neurotoxic models.

A noncanonical NF-κB pathway through the p50 subunit regulates Bcl-2 overexpression during an oxidative-conditioning hormesis response

Cells can respond to damage and stress by activating various repair and survival pathways. One of these responses can be induced by preconditioning the cells with sublethal stress to provoke a prosurvival response that will prevent damage and death, and which is known as hormesis. Bcl-2, an antiapoptotic protein recognized by its antioxidant and prosurvival functions, has been documented to play an important role during oxidative-conditioning hormesis. Using an oxidative-hormetic model, which was previously established in the L929 cell line by subjecting the cells to a mild oxidative stress of 50 μM H₂O₂ for 9 h, we identified two different transductional mechanisms that participate in the regulation of Bcl-2 expression during the hormetic response. These mechanisms converge in activating the nuclear transcription factor NF-κB. Interestingly, the noncanonical p50 subunit of the NF-κB family is apparently the subunit that participates during the oxidative-hormetic response.

Primary cultured astrocytes from old rats are capable to activate the Nrf2 response against MPP+ toxicity after tBHQ pretreatment

Astrocytes are key players for brain physiology, protecting neurons by releasing antioxidant enzymes; however, they are also susceptible to damage by neurotoxins. Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a central regulator of the antioxidant response, and therefore, pharmacologic inducers are often used to activate this transcription factor to induce cellular protection. To date, it still remains unknown if cells from aged animals are capable of developing this response. Therefore, the purpose of this work was to determine if cortical astrocytes derived from old rats are able to respond to tertbuthyl-hydroquinene (tBHQ) pretreatment and stimulate the Nrf2-antioxidant response pathway to induce an antioxidant strategy against MPP+ toxicity, one of the most used molecules to model Parkinsons disease. Our results show that, although astrocytes from adult and old rats were more susceptible to MPP+ toxicity than astrocytes from newborn rats, when pretreated with tertbuthyl-hydroquinene, they were able to transactivate Nrf2, increasing antioxidant enzymes and developing cellular protection. These results are discussed in terms of the doses used to create protective responses.

Bcl-2 sustains hormetic response by inducing Nrf-2 nuclear translocation in L929 mouse fibroblasts

Hormesis is the process whereby exposure to a low dose of a chemical agent induces an adaptive effect on the cell or organism. This response evokes the expression of cytoprotective and antioxidant proteins, allowing pro-oxidants to emerge as important hormetic agents. The antiapoptotic protein Bcl-2 is known to protect cells against death induced by oxidants; it has been suggested that Bcl-2 might also modulate steady-state reactive oxygen species levels. The aim of this work was to find out if Bcl-2 might play a role during the hormetic response and in Nrf-2 activation. We have established a model to study the oxidative conditioning hormesis response (OCH) by conditioning the cell line L929 with 50muM H(2)O(2) for 9h. This condition did not induce oxidative damage nor oxidative imbalance, and OCH cells maintained a 70-80% survival rate after severe H(2)O(2) treatment compared to nonconditioned cells. When cells were pretreated with the Bcl-2 inhibitor HA14-1 or were silenced with Bcl-2-siRNA, both the hormetic effect and the Nrf-2 nuclear translocation previously observed were abrogated. Our results suggest a sequence of causal events related to increase in Bcl-2 expression, induction of Nrf-2 activation, and sustained expression of cytoprotective proteins such as GST and gammaGCS.

DNA MMR systems, microsatellite instability and antioxidant activity variations in two species of wild bats: Myotis velifer and Desmodus rotundus, as possible factors associated with longevity

The accumulation of oxidative damage to biomolecules, such as DNA, is known to induce alterations in the cells mechanisms and structure that might lead to the aging process. DNA mismatch repair system (MMR) corrects base mismatches generated during DNA replication that have escaped the proofreading process. In addition, antioxidant enzymes can reduce reactive oxygen species effects in order to protect cells from oxidizing damage. In order to determine the importance of these associated factors during the aging process, in this study, levels of MMR proteins MSH2 and MLH1, as well as microsatellite markers, were compared in liver, lung, and brain of juvenile, adult, and old, both female and male, individuals from two species of wild bats: the short-lived Myotis velifer and the longer lived Desmodus rotundus. Catalase, glutathione peroxidase, and superoxide dismutase were also analyzed to determine if the antioxidant protection correlates negatively with DNA damage. Antioxidant activities were higher in the longer lived D. rotundus than in M. velifer. Furthermore, old M. velifer but not old D. rotundus bats had reduced MMR levels and increased microsatellite instability. Therefore, although our results correlate the reduced MMR efficiency, the deficient antioxidant activity, and the increase in DNA damage with the aging process, this is not always true for all living organisms.

Msh2 promoter region hypermethylation as a marker of aging-related deterioration in old retired female breeder mice

Aging is a process where individuals decrease the performance of their physiological systems and cellular stress response, making them more susceptible to disease and death. The increase in DNA damage associated with age might be recognized as the accumulation of physiological and environmentally induced mutations accompanied with a decline in DNA repair. DNA mismatch repair (MMR) is the main postreplicative correction pathway, which is known to decrease with age. However, since infrequent occurrence of direct DNA damage contrasts with the extensive cell and tissue dysfunction seen in older individuals, the withdrawing of DNA-repairing systems might be also related to epigenetic changes, such as DNA methylation. It has been reported that the physiological stress related to breeding might accelerate the acquisition of aging-related markers; therefore, the aim of this work was to link age with epigenetic modifications in this animal population. Hence, the correlation of Msh2 gene silencing with the deterioration of breeding female mice associated to aging was determined. Combined bisulfite restriction analysis assay was used to compare methylation on DNA isolated from twelve-month-old retired breeders against nulliparous female mice aged-matched, and two-month-old young adults. Our experiments clearly reveal Msh2 promoter hypermethylation associated to the aging process. A higher degree methylation was additionally observed in breeding females DNA. Nevertheless, this additional methylation did not correlate with a further decrease Msh2 mRNA, suggesting that the increase in methylation in old retired breeder might account for further epigenetic changes that could additionally promote the aging process.

Comparing the effects of two neurotoxins in cortical astrocytes obtained from rats of different ages: involvement of oxidative damage

Oxidative stress has been recognized as a potential mediator of cell death. Astrocytes play an active role in brain physiology responding to harmful stimuli by activating astrogliosis, which in turn has been associated either with survival or degenerative events. The characterization of the mechanistic actions exerted by different toxins in astrocytes is essential to understand the brain function and pathology. As age plays a critical role in degenerative processes, the aim of this study was to determine whether the administration of equimolar concentrations of two neurotoxins evoking different toxic patterns can induce differential effects on primary astrocytes obtained either from newborn or adult rats, with particular emphasis on those events linked to oxidative stress as a potential source of damage. Primary cortical astrocyte cultures derived from rat brains were exposed to 1‐methyl‐4‐phenylpyridinium (MPP+) or beta‐amyloid peptide (β‐amyloid). Mitochondrial functionality and cell viability were determined as physiological parameters, whereas lipid and protein oxidation were used as markers of oxidative damage. The results of these experiments pointed towards a higher vulnerability to MPP + over β‐amyloid, on most of the tested markers. Hence, in order to allow a comprehensive evaluation of astrocytic responses against MPP + intoxication, a third astrocyte group was included for dose‐response experiments: astrocytes derived from aged rats. The present data indicate that the differences associated with age were mainly found in astrocytes exposed to MPP + (25 and 50 μM) at 1‐h treatment. Results are discussed in terms of the differential mechanisms involved in each model. Copyright

Telomerase activity in response to mild oxidative stress.

We have analysed telomerase activity to determine whether it can be modified when BCL‐2 is endogenously overexpressed in response to a mild oxidative stress treatment as part of a survival mechanism, in contrast with an exogenous bcl‐2 overexpression due to a retroviral infection. Endogenous bcl‐2 overexpression was induced after a low oxidative insult of H2O2 in mice primary lung fibroblasts and L929 cell, whereas bcl‐2 exogenous overexpression was performed using a retroviral infection in L929 cells. Telomerase activity was quantified in Bcl‐2 overexpressing cells by the TRAP assay. When the cells were treated with different H2O2 concentrations, only those exposed to 50 μM showed increased telomerase activity. This correlates with BCL‐2 expression as part of the endogenous response to mild oxidative stress. Oxidative stress generated during the toxic mechanism of chemotherapeutic drugs might induce BCL‐2 increment, enhancing telomerase activity and reactivating the oncogenic process. Clinical trials should take into consideration the possibility of telomerase activation following increased BCL‐2 expression when treating patients with ROS (reactive oxygen species) generation by anti‐cancer drugs.

Hepatocyte Growth Factor Reduces Free Cholesterol-Mediated Lipotoxicity in Primary Hepatocytes by Countering Oxidative Stress.

Cholesterol overload in the liver has shown toxic effects by inducing the aggravation of nonalcoholic fatty liver disease to steatohepatitis and sensitizing to damage. Although the mechanism of damage is complex, it has been demonstrated that oxidative stress plays a prominent role in the process. In addition, we have proved that hepatocyte growth factor induces an antioxidant response in hepatic cells; in the present work we aimed to figure out the protective effect of this growth factor in hepatocytes overloaded with free cholesterol. Hepatocytes from mice fed with a high-cholesterol diet were treated or not with HGF, reactive oxygen species present in cholesterol overloaded hepatocytes significantly decreased, and this effect was particularly associated with the increase in glutathione and related enzymes, such as γ-gamma glutamyl cysteine synthetase, GSH peroxidase, and GSH-S-transferase. Our data clearly indicate that HGF displays an antioxidant response by inducing the glutathione-related protection system.