Krisztian Stadler

Diesel Exhaust Activates and Primes Microglia: Air Pollution, Neuroinflammation, and Regulation of Dopaminergic Neurotoxicity

Background: Air pollution is linked to central nervous system disease, but the mechanisms responsible are poorly understood. Objectives: Here, we sought to address the brain-region–specific effects of diesel exhaust (DE) and key cellular mechanisms underlying DE-induced microglia activation, neuroinflammation, and dopaminergic (DA) neurotoxicity. Methods: Rats were exposed to DE (2.0, 0.5, and 0 mg/m3) by inhalation over 4 weeks or as a single intratracheal administration of DE particles (DEP; 20 mg/kg). Primary neuron–glia cultures and the HAPI (highly aggressively proliferating immortalized) microglial cell line were used to explore cellular mechanisms. Results: Rats exposed to DE by inhalation demonstrated elevated levels of whole-brain IL-6 (interleukin-6) protein, nitrated proteins, and IBA-1 (ionized calcium-binding adaptor molecule 1) protein (microglial marker), indicating generalized neuroinflammation. Analysis by brain region revealed that DE increased TNFα (tumor necrosis factor-α), IL-1β, IL-6, MIP-1α (macrophage inflammatory protein-1α) RAGE (receptor for advanced glycation end products), fractalkine, and the IBA-1 microglial marker in most regions tested, with the midbrain showing the greatest DE response. Intratracheal administration of DEP increased microglial IBA-1 staining in the substantia nigra and elevated both serum and whole-brain TNFα at 6 hr posttreatment. Although DEP alone failed to cause the production of cytokines and chemokines, DEP (5 μg/mL) pretreatment followed by lipopolysaccharide (2.5 ng/mL) in vitro synergistically amplified nitric oxide production, TNFα release, and DA neurotoxicity. Pretreatment with fractalkine (50 pg/mL) in vitro ameliorated DEP (50 μg/mL)-induced microglial hydrogen peroxide production and DA neurotoxicity. Conclusions: Together, these findings reveal complex, interacting mechanisms responsible for how air pollution may cause neuroinflammation and DA neurotoxicity.

Increased nitric oxide levels as an early sign of premature aging in diabetes

The levels of different reactive species, especially those of nitric oxide and peroxynitrite, were determined in streptozotocin-induced diabetic rat tissues, before the development of histopathological damages. Significantly higher steady state free radical concentrations were found in the liver 3 weeks after the onset of diabetes compared to age-matched control groups. Increased nitric oxide levels in diabetic vasculature and kidney decreased the production of detectable reactive oxygen species. High peroxynitrite generation suggested the onset of processes characteristic to premature aging of the endothelium. According to the histopathological results, there were no signs of late complications in the tissues up to 7 weeks after induction of diabetes. These results support the idea that oxidative stress is increased at a very early stage of diabetes and, in particular, that high levels of nitric oxide and peroxynitrite could play a decisive role in the development of late complications in the diabetic vasculature and kidney.

The effect of exercise and nettle supplementation on oxidative stress markers in the rat brain

Chronic swimming training and phytotherapeutic supplementation are assumed to alleviate oxidative damage, and support cell survival in the brain. The effect of forced, chronic swimming training, and enriched lab chow containing 1% (w/w) dried nettle (Urtica dioica) leaf were investigated for oxidative stress, inflammation and neurotrophic markers in Wistar rat brains. The rats were divided into groups subjected to swimming training (6 weeks) or to nettle supplementation (8 weeks) or to a combination of these two treatments. The level of oxidative stress was measured by electron spin resonance (EPR), and by the concentration of carbonylated proteins. Nettle supplementation resulted in a decreased concentration of free radicals in both cerebellum and frontal lobe. Swimming, however, did not influence significantly the oxidative damage nor was it reflected in the carbonyl content. The protein content of nerve growth factor (NGF), and brain-derived neurotrophic factors (BDNF) was evaluated by E-Max ImmunoAssay in the cerebellum. No changes occurred either with exercise or nettle diet treatments. On the other hand, nuclear factor kappa B (NF-kappaB) binding activity to DNA increased with the combined effect of swimming training and nettle diet, while the activator protein1 (AP-1) DNA binding activity showed a more profound elevation in the nettle treated animals. The amount of c-Jun decreased by swimming training. In conclusion, the results suggest that both exercise and nettle influenced physiological brain functions. Nettle supplementation reduces the free radical concentration and increases the DNA binding of AP-1 in the brain. Nettle was found to be an effective antioxidant and possible antiapoptotic supplement promoting cell survival in the brain. Exercise, as a downregulator of c-Jun and in combined group as an upregulator of NF-kappaB, may play also a role in antiapoptotic processes, which is important after brain injury.

Involvement of inducible nitric oxide synthase in hydroxyl radical-mediated lipid peroxidation in streptozotocin-induced diabetes.

Free radical production is implicated in the pathogenesis of diabetes mellitus, where several pathways and different mechanisms were suggested in the pathophysiology of the complications. In this study, we used electron paramagnetic resonance (EPR) spectroscopy combined with in vivo spin-trapping techniques to investigate the sources and mechanisms of free radical formation in streptozotocin-induced diabetic rats. Free radical production was directly detected in the diabetic bile, which correlated with lipid peroxidation in the liver and kidney. EPR spectra showed the trapping of a lipid-derived radical. Such radicals were demonstrated to be induced by hydroxyl radical through isotope-labeling experiments. Multiple enzymes and metabolic pathways were examined as the potential source of the hydroxyl radicals using specific inhibitors. No xanthine oxidase, cytochrome P450s, the Fenton reaction, or macrophage activation were required for the production of radical adducts. Interestingly, inducible nitric oxide synthase (iNOS) (apparently uncoupled) was identified as the major source of radical generation. The specific iNOS inhibitor 1400W as well as L-arginine pretreatment reduced the EPR signals to baseline levels, implicating peroxynitrite as the source of hydroxyl radical production. Applying immunological techniques, we localized iNOS overexpression in the liver and kidney of diabetic animals, which was closely correlated with the lipid radical generation and 4-hydroxynonenal-adducted protein formation, indicating lipid peroxidation. In addition, protein tyrosine nitration occurred in the diabetic target organs. Taken together, our studies support inducible nitric oxide synthase as a significant source of EPR-detectable reactive intermediates, which leads to lipid peroxidation and may contribute to disease progression as well.

High-fat diet induces an initial adaptation of mitochondrial bioenergetics in the kidney despite evident oxidative stress and mitochondrial ROS production

Obesity and metabolic syndrome are associated with an increased risk for several diabetic complications, including diabetic nephropathy and chronic kidney diseases. Oxidative stress and mitochondrial dysfunction are often proposed mechanisms in various organs in obesity models, but limited data are available on the kidney. Here, we fed a lard-based high-fat diet to mice to investigate structural changes, cellular and subcellular oxidative stress and redox status, and mitochondrial biogenesis and function in the kidney. The diet induced characteristic changes, including glomerular hypertrophy, fibrosis, and interstitial scarring, which were accompanied by a proinflammatory transition. We demonstrate evidence for oxidative stress in the kidney through 3-nitrotyrosine and protein radical formation on high-fat diet with a contribution from iNOS and NOX-4 as well as increased generation of mitochondrial oxidants on carbohydrate- and lipid-based substrates. The increased H(2)O(2) emission in the mitochondria suggests altered redox balance and mitochondrial ROS generation, contributing to the overall oxidative stress. No major derailments were observed in respiratory function or biogenesis, indicating preserved and initially improved bioenergetic parameters and energy production. We suggest that, regardless of the oxidative stress events, the kidney developed an adaptation to maintain normal respiratory function as a possible response to an increased lipid overload. These findings provide new insights into the complex role of oxidative stress and mitochondrial redox status in the pathogenesis of the kidney in obesity and indicate that early oxidative stress-related changes, but not mitochondrial bioenergetic dysfunction, may contribute to the pathogenesis and development of obesity-linked chronic kidney diseases.

Oxidative Stress in Diabetes

Oxidative stress and diabetes, both Type 1 and Type 2 as well as their related conditions have been extensively studied. As diabetes, obesity and metabolic syndrome have reached at epidemic levels, there is a huge need and effort to understand the detailed molecular mechanisms of the possible redox imbalance, underlying the cause of pathology and progression of the disease. These studies provide new insights at cellular and subcellular levels to design effective clinical interventions. This chapter is intended to emphasize the latest knowledge and current evidence on the role of oxidative stress in diabetes as well as to discuss some key questions that are currently under discussion.

Ascorbyl free radical and dehydroascorbate formation in rat liver endoplasmic reticulum

The mechanism of ascorbate oxidation was studied in rat liver microsomes. A continuous consumption of the added ascorbate was observed, which was accompanied with a prompt appearance of ascorbyl free radical and dehydroascorbate. Microsomes sustained steady-state level of ascorbyl free radical and dehydroascorbate till ascorbate was present in the medium. Ascorbyl free radical formation was diminished when microsomes had been pretreated with heat or trypsine. It was also decreased by addition of quercetin, econazole or metal chelators, including the copper specific neocuproine. Enzymatic (superoxide dismutase, catalase) and nonenzymatic (dimethyl sulfoxide, mannitol) antioxidants did not modify the microsomal production of ascorbyl free radical. Investigation of the subcellular distribution of ascorbate oxidation showed that the microsomal fraction of liver had the highest activity. The decrease of ascorbate oxidation after protease treatment and the negligible increase upon permeabilization of microsomal vesicles showed that a membrane protein is responsible for the activity, which is exposed to the outer surface of the endoplasmic reticulum. The results indicate the presence of a primary enzymatic ascorbate oxidation in rat liver endoplasmic reticulum which is able to generate dehydroascorbate, an important source of the oxidizing environment in the endoplasmic reticulum.

The Evolving Understanding of the Contribution of Lipid Metabolism to Diabetic Kidney Disease

Although diabetes is mainly diagnosed based on elevated glucose levels, dyslipidemia is also observed in these patients. Chronic kidney disease (CKD), a frequent occurrence in patients with diabetes, is associated with major abnormalities in circulating lipoproteins and renal lipid metabolism. At baseline, most renal epithelial cells rely on fatty acids as their energy source. CKD, including that which occurs in diabetes, is characterized by tubule epithelial lipid accumulation. Whether this is due to increased uptake or greater local fatty acid synthesis is unknown. We have recently shown that CKD also leads to decreased fatty acid oxidation, which might be an additional mechanism leading to lipid accumulation. Defective fatty acid utilization causes energy depletion resulting in increased apoptosis and dedifferentiation, which in turn contributes to fibrosis and CKD progression. Enhanced fatty acid oxidation in the kidney induced by fenofibrate, a peroxisomal proliferator-activated receptor (PPAR)-α agonist, showed benefit in mouse models of CKD. Fenofibrate treatment also reduced albuminuria in patients with diabetes in multiple clinical trials. Taken together, these findings suggest that further understanding of lipid metabolism in diabetic kidney disease may lead to novel therapeutic approaches.

The peroxidase activity of mitochondrial superoxide dismutase

Manganese superoxide dismutase (MnSOD) is an integral mitochondrial protein known as a first-line antioxidant defense against superoxide radical anions produced as by-products of the electron transport chain. Recent studies have shaped the idea that by regulating the mitochondrial redox status and H(2)O(2) outflow, MnSOD acts as a fundamental regulator of cellular proliferation, metabolism, and apoptosis, thereby assuming roles that extend far beyond its proposed antioxidant functions. Accordingly, allelic variations of MnSOD that have been shown to augment levels of MnSOD in mitochondria result in a 10-fold increase in prostate cancer risk. In addition, epidemiologic studies indicate that reduced glutathione peroxidase activity along with increases in H(2)O(2) further increase cancer risk in the face of MnSOD overexpression. These facts led us to hypothesize that, like its Cu,ZnSOD counterpart, MnSOD may work as a peroxidase, utilizing H(2)O(2) to promote mitochondrial damage, a known cancer risk factor. Here we report that MnSOD indeed possesses peroxidase activity that manifests in mitochondria when the enzyme is overexpressed.

The beneficial effects of nettle supplementation and exercise on brain lesion and memory in rat

Regular swimming and phytotherapeutic supplementation are assumed to alleviate the severity of neurodegeneration leading to dementia. The effect of swimming training and that of enriched lab chow containing 1% (w/w) dried nettle (Urtica dioica) leaf on the prevention of severity of brain injury caused by N-methyl-d-aspartate (NMDA) lesion in Wistar rats were investigated. Nettle supplementation and regular swimming exercise seem to improve the adverse effect of brain injury caused by NMDA lesion assessed by passive avoidance test and open-field test. Nettle supplementation decreases the level of reactive oxygen species, measured by electron paramagnetic resonance, and the DNA-binding activity of NF-kappaB. The data reveal that nettle supplementation has an effective antioxidant role, down-regulates the inflammatory transcription factors and could also promote learning performance in the brain. Regular swimming increases the concentration of reactive species in the cerebellum and alters the activity of transcription factors toward inflammation. The additive effect of the two treatments was more profound in the down-regulation of inflammatory transcription processes in NMDA lesion.