Mimicking Parkinson's Disease in a Dish: Merits and Pitfalls of the Most Commonly used Dopaminergic In Vitro Models.

Parkinson's disease (PD) is the second most common neurodegenerative disorder and has both unknown etiology and non-curative therapeutic options. Patients begin to present the classic motor symptoms of PD-tremor at rest, bradykinesia and rigidity-once 50-70% of the dopaminergic neurons of the nigrostriatal pathway have degenerated. As a consequence of this, it is difficult to investigate the early-stage events of disease pathogenesis. In vitro experimental models are used extensively in PD research because they present a controlled environment that enables the direct investigation of the early molecular mechanisms that are potentially involved with dopaminergic degeneration, as well as for the screening of potential therapeutic drugs. However, the establishment of PD in vitro models is a controversial issue for neuroscience research not only because it is challenging to mimic, in isolated cell systems, the physiological neuronal environment, but also the pathophysiological conditions experienced by human dopaminergic cells in vivo during the progression of the disease. Since no previous work has attempted to systematically review the literature regarding the establishment of an optimal in vitro model, and/or the features presented by available models used in the PD field, this review aims to summarize the merits and limitations of the most widely used dopaminergic in vitro models in PD research, which may help the PD researcher to choose the most appropriate model for studies directed at the elucidation of the early-stage molecular events underlying PD onset and progression.

RA Differentiation Enhances Dopaminergic Features, Changes Redox Parameters, and Increases Dopamine Transporter Dependency in 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Cells.

Research on Parkinson's disease (PD) and drug development is hampered by the lack of suitable human in vitro models that simply and accurately recreate the disease conditions. To counteract this, many attempts to differentiate cell lines, such as the human SH-SY5Y neuroblastoma, into dopaminergic neurons have been undertaken since they are easier to cultivate when compared with other cellular models. Here, we characterized neuronal features discriminating undifferentiated and retinoic acid (RA)-differentiated SH-SYSY cells and described significant differences between these cell models in 6-hydroxydopamine (6-OHDA) cytotoxicity. In contrast to undifferentiated cells, RA-differentiated SH-SY5Y cells demonstrated low proliferative rate and a pronounced neuronal morphology with high expression of genes related to synapse vesicle cycle, dopamine synthesis/degradation, and of dopamine transporter (DAT). Significant differences between undifferentiated and RA-differentiated SH-SY5Y cells in the overall capacity of antioxidant defenses were found; although RA-differentiated SH-SY5Y cells presented a higher basal antioxidant capacity with high resistance against H2O2 insult, they were twofold more sensitive to 6-OHDA. DAT inhibition by 3α-bis-4-fluorophenyl-methoxytropane and dithiothreitol (a cell-permeable thiol-reducing agent) protected RA-differentiated, but not undifferentiated, SH-SY5Y cells from oxidative damage and cell death caused by 6-OHDA. Here, we demonstrate that undifferentiated and RA-differentiated SH-SY5Y cells are two unique phenotypes and also have dissimilar mechanisms in 6-OHDA cytotoxicity. Hence, our data support the use of RA-differentiated SH-SY5Y cells as an in vitro model of PD. This study may impact our understanding of the pathological mechanisms of PD and the development of new therapies and drugs for the management of the disease.

Sulforaphane ameliorates the insulin responsiveness and the lipid profile but does not alter the antioxidant response in diabetic rats.

Diabetes is one of the most prevalent chronic non-communicable diseases and is characterized by hyperglycemia and increased oxidative stress. These two alterations are also responsible for the main diabetic complications: cardiovascular disease, retinopathy, nephropathy and peripheral neuropathy. Diabetes progression is governed by pancreatic ß-cell failure, and recent studies showed that sulforaphane (SFN) might be able to prevent this change, preserving insulin production. Consequently, our goal was to test the effects of SFN on metabolic parameters related to diabetic complications and antioxidant defenses (superoxide dismutase, catalase and sulfhydryl groups) in the pancreas, liver and kidney of non-diabetic and diabetic rats. Male Wistar rats were treated with water or 0.5 mg kg(-1) SFN i.p. for 21 days after diabetes induction. In diabetic animals treated with SFN, the serum levels of total cholesterol, non-HDL cholesterol and triacylglycerols were similar to those of non-diabetic animals, and the insulin responsiveness was higher than that of the diabetic animals that did not receive the compound. No effect of SFN on the superoxide dismutase and catalase activity or sulfhydryl groups was observed in the pancreas, liver or kidney of the treated animals. We conclude that SFN ameliorates some features of clinical diabetic complications particularly the lipid profile and insulin responsiveness, but it does not modulate the antioxidant response induced by superoxide dismutase, catalase and sulfhydryl groups in the evaluated organs.

In vitro evaluation of antitumoral efficacy of catalase in combination with traditional chemotherapeutic drugs against human lung adenocarcinoma cells.

Lung cancer is the most lethal cancer-related disease worldwide. Since survival rates remain poor, there is an urgent need for more effective therapies that could increase the overall survival of lung cancer patients. Lung tumors exhibit increased levels of oxidative markers with altered levels of antioxidant defenses, and previous studies demonstrated that the overexpression of the antioxidant enzyme catalase (CAT) might control tumor proliferation and aggressiveness. Herein, we evaluated the effect of CAT treatment on the sensitivity of A549 human lung adenocarcinoma cells toward various anticancer treatments, aiming to establish the best drug combination for further therapeutic management of this disease. Exponentially growing A549 cells were treated with CAT alone or in combination with chemotherapeutic drugs (cisplatin, 5-fluorouracil, paclitaxel, daunorubicin, and hydroxyurea). CalcuSyn(®) software was used to assess CAT/drug interactions (synergism or antagonism). Growth inhibition, NFκB activation status, and redox parameters were also evaluated in CAT-treated A549 cells. CAT treatment caused a cytostatic effect, decreased NFκB activation, and modulated the redox parameters evaluated. CAT treatment exhibited a synergistic effect among most of the anticancer drugs tested, which is significantly correlated with an increased H2O2 production. Moreover, CAT combination caused an antagonism in paclitaxel anticancer effect. These data suggest that combining CAT (or CAT analogs) with traditional chemotherapeutic drugs, especially cisplatin, is a promising therapeutic strategy for the treatment of lung cancer.

Oxidative stress associates with aggressiveness in lung large-cell carcinoma.

Oxidative stress is involved in many cancer-related processes; however, current therapeutics are unable to benefit from this approach. The lungs have a very exquisite redox environment that may contribute to the frequent and deadly nature of lung cancer. Very few studies specifically address lung large-cell carcinoma (LCC), even though this is one of the major subtypes. Using bioinformatic (in silico) tools, we demonstrated that a more aggressive lung LCC cell line (HOP-92) has an overall increase activity of the human antioxidant gene (HAG) network (P = 0.0046) when compared to the less aggressive cell line H-460. Gene set enrichment analysis (GSEA) showed that the expression of metallothioneins (MT), glutathione peroxidase 1 (GPx-1), and catalase (CAT) are responsible for this difference in gene signature. This was validated in vitro, where HOP-92 showed a pro-oxidative imbalance, presenting higher antioxidant enzymes (superoxide dismutase (SOD), CAT, and GPx) activities, lower reduced sulfhydryl groups and antioxidant potential, and higher lipoperoxidation and reactive species production. Also, HAG network is upregulated in lung LCC patients with worst outcome. Finally, the prognostic value of genes enriched in the most aggressive cell line was assessed in this cohort. Isoforms of metallothioneins are associated with bad prognosis, while the thioredoxin-interacting protein (TXNIP) is associated with good prognosis. Thus, redox metabolism can be an important aspect in lung LCC aggressiveness and a possible therapeutic target.

Increased cerebral oxidative damage and decreased antioxidant defenses in ovariectomized and sham-operated rats supplemented with vitamin A.

Previous studies have linked oxidative stress with aging and aging-related processes, including menopause. Abnormalities in the redox state similar to those observed in menopausal women can be modeled experimentally with rat ovariectomy. The aim of the present study was to investigate the effects of vitamin A (retinol palmitate) supplementation (500 or 1,500 IU kg(-1) day(-1) for 30 days) on behavioral parameters and brain redox profile in ovariectomized (OVX) and sham-operated rats. Ovariectomy caused pronounced uterine atrophy and decreased locomotor/exploratory activity. Moreover, we found increased hypothalamic and frontal cortex superoxide dismutase/catalase (SOD/CAT) ratio and decreased hippocampal thiol content, accompanied by increased frontal cortex lipid oxidative damage (TBARS) in OVX rats. Vitamin A at 1,500 IUkg(-1) day(-1) decreased exploratory behavior and decreased total hippocampal thiol content in sham-operated rats, increased hippocampal SOD/CAT ratio and decreased total antioxidant potential in the hippocampus of both sham and OVX groups, and increased cortical TBARS levels in OVX rats. Thus, vitamin A may induce a pro-oxidant state in discrete brain regions of sham-operated and OVX rats. These results suggest some caution regarding the use of high doses of vitamin A supplementation during menopause.

Evaluation of the neurotoxic/neuroprotective role of organoselenides using differentiated human neuroblastoma SH-SY5Y cell line challenged with 6-hydroxydopamine.

It is well established that oxidative stress plays a major role in several neurodegenerative conditions, like Parkinson disease (PD). Hence, there is an enormous effort for the development of new antioxidants compounds with therapeutic potential for the management of PD, such as synthetic organoselenides molecules. In this study, we selected between nine different synthetic organoselenides the most eligible ones for further neuroprotection assays, using the differentiated human neuroblastoma SH-SY5Y cell line as in vitro model. Neuronal differentiation of exponentially growing human neuroblastoma SH-SY5Y cells was triggered by cultivating cells with DMEM/F12 medium with 1% of fetal bovine serum (FBS) with the combination of 10 µM retinoic acid for 7 days. Differentiated cells were further incubated with different concentrations of nine organoselenides (0.1, 0.3, 3, 10, and 30 µM) for 24 h and cell viability, neurites densities and the immunocontent of neuronal markers were evaluated. Peroxyl radical scavenging potential of each compound was determined with TRAP assay. Three organoselenides tested presented low cytotoxicity and high antioxidant properties. Pre-treatment of cells with those compounds for 24 h lead to a significantly neuroprotection against 6-hydroxydopamine (6-OHDA) toxicity, which were directly related to their antioxidant properties. Neuroprotective activity of all three organoselenides was compared to diphenyl diselenide (PhSe)2, the simplest of the diaryl diselenides tested. Our results demonstrate that differentiated human SH-SY5Y cells are suitable cellular model to evaluate neuroprotective/neurotoxic role of compounds, and support further evaluation of selected organoselenium molecules as potential pharmacological and therapeutic drugs in the treatment of PD.

Peripheral oxidative damage in early-stage mood disorders: a nested population-based case-control study.

Systemic toxicity is a relevant dimension of pathophysiology in bipolar disorder, and oxidative damage is one potential link between central and peripheral pathology. Although there is mounting evidence that chronic bipolar disorder is associated with oxidative stress, studies in the early stages of bipolar disorder are scarce, and heavily reliant on clinical in lieu of population studies. The objective of this study was to confirm leading hypotheses about the role of oxidative damage in bipolar disorder. To that end, we nested a case-control study in a population-based study of young adults aged 18-24 yr. After an initial psychopathology screen, all people with a lifetime history of (hypo)mania and matched controls underwent a structured diagnostic interview. This yielded a sample of 231 participants, in whom we measured serum protein carbonyl content (PCC) and thiobarbituric acid reactive substances (TBARS). People with bipolar disorder had higher PCC levels than healthy subjects. Those with major depression were not different from control subjects in either PCC or TBARS levels. Both bipolar disorder and major depression were associated with higher PCC levels in the a priori regression model controlling for possible confounders. These findings indicate that protein oxidative damage is present from early stages and can be seen as a sign of early illness activity in mood disorders.

Modulation in reproductive tissue redox profile in sexually receptive female rats after short-term exposure to male chemical cues.

It is well known that antioxidants play an important role in sperm fertility, but there is no data on the literature regarding the effect of male chemical cues in the antioxidant defenses of the female reproductive tract. Here, we evaluated oxidative parameters in ovaries and uterus of virgin female rats isolated from contact to males and exposed only to male-soiled bedding (MSB). Four-month-old Wistar (regular 4-day cyclic) virgin female rats were utilized from proestrus to estrus phase of the reproductive cycle for experimental exposure. In an isolated room, female rats were exposed for 90 min to MSB. For biochemical assays, female rats were killed by decapitation at 30, 90, 180, and 240 min after the end of exposure, and the ovaries and uterus were removed for further analysis. Antioxidant enzyme activities (superoxide dismutase, catalase, and glutathione peroxidase), the nonenzymatic antioxidant potential (total radical-trapping antioxidant parameter), and the oxidative damage parameters (thiobarbituric acid-reactive species and carbonyl content) were analyzed. We observed an increase in the nonenzymatic antioxidant potential and diminished free radical oxidative damage in uterine tissue, 30 and 90 min after exposure. Furthermore, in ovaries, enzymatic defenses were modulated distinctly along the 240 min after exposure. MSB exposure modulates the antioxidant profile in ovaries and uterus of receptive female rats. It is possible that the modifications in the oxidative profile of the female genital tract may have important implications in the process of fertilization.

Vitamin A supplementation for different periods alters oxidative parameters in lungs of rats.

Lungs require an adequate supply of vitamin A (retinol) for normal embryonic development, postnatal maturation, and maintenance and repair during adult life. However, recent intervention studies revealed that supplementation with retinoids resulted in higher incidence of lung cancer, although the mechanisms underlying this effect are still unknown. Here, we studied the effect of vitamin A supplementation on oxidative stress parameters in lungs of Wistar rats. Vitamin A supplementation at either therapeutic (1,000 and 2,500 IU/kg) or excessive (4,500 and 9,000 IU/kg) doses for 3, 7, or 28 days induced lipid peroxidation, protein carbonylation, and oxidation of protein thiol groups, as well as change in catalase and superoxide dismutase activity. Together, these results suggest that vitamin A supplementation causes significant changes in redox balance, which are frequently associated with severe lung dysfunction.

Decreased anxiety-like behavior and locomotor/exploratory activity, and modulation in hypothalamus, hippocampus, and frontal cortex redox profile in sexually receptive female rats after short-term exposure to male chemical cues.

Chemical cues are widely used for intraspecific social communication in a vast majority of living organisms ranging from bacteria to mammals. As an example, mammals release olfactory cues with urine that promote neuroendocrine modulations with changes in behavior and physiology in the receiver. In this work, four-month-old Wistar (regular 4-day cyclic) virgin female rats were utilized in the proestrus-to-estrus phase of the reproductive cycle for experimental exposure. In an isolated room, female rats were exposed for 90 min to male-soiled bedding (MSB). Elevated plus-maze assay, open field test, and light/dark box task were performed to analyze behavioral alterations on females after exposure. For biochemical assays, female rats were killed and the hypothalamus, hippocampus, and frontal cortex were isolated for further analysis. Antioxidant enzyme activities (superoxide dismutase, catalase and glutathione peroxidase), non-enzymatic antioxidant defense measurements (TRAP and TAR), and the oxidative damage parameters (TBARS, Carbonyl and SH content) were analyzed. In behavioral analyses we observe that female rats show decreased anxiety and locomotory/exploratory activities after MSB exposure. In biochemical assays we observed an increase in both enzymatic and non-enzymatic antioxidant defenses in different central nervous system (CNS) structures analyzed 30 and 90 min after MSB exposure. Furthermore, hippocampus and frontal cortex showed diminished free radical oxidative damage at 180 and 240 min after exposure. These results provide the first evidence that oxidative profile of female CNS structures are altered by chemical cues present in the MSB, thus suggesting that pheromonal communication is able to modulate radical oxygen species production and/or clearance in the female brain.