Oxidative Stress Agents Research Articles

Aberrant Insulin Receptor Signaling and Amino Acid Homeostasis as a Major Cause of Oxidative Stress in Aging

The mechanisms leading to the increase in free radical-derived oxidative stress in "normal aging" remains obscure. Here we present our perspective on studies from different fields that reveal a previously unnoticed vicious cycle of oxidative stress. The plasma cysteine concentrations during starvation in the night and early morning hours (the postabsorptive state) decreases with age. This decrease is associated with a decrease in tissue concentrations of the cysteine derivative and quantitatively important antioxidant glutathione. The decrease in cysteine reflects changes in the autophagic protein catabolism that normally ensures free amino acid homeostasis during starvation. Autophagy is negatively regulated by the insulin receptor signaling cascade that is enhanced by oxidative stress in the absence of insulin. This synopsis of seemingly unrelated processes reveals a novel mechanism of progressive oxidative stress in which decreasing antioxidant concentrations and increasing basal (postabsorptive) insulin receptor signaling activity compromise not only the autophagic protein catabolism but also the activity of FOXO transcription factors (i.e., two functions that were found to have an impact on lifespan in several animal models of aging). In addition, the aging-related decrease in glutathione levels is likely to facilitate certain "secondary" disease-related mechanisms of oxidative stress. Studies on cysteine supplementation show therapeutic promise.

Amyloid-Degrading Enzymes as Therapeutic Targets in Alzheimers Disease

The steady state concentration of the Alzheimer's amyloid-beta peptide in the brain represents a balance between its biosynthesis from the transmembrane amyloid precursor protein (APP), its oligomerisation into neurotoxic and stable species and its degradation by a variety of amyloid-degrading enzymes, principally metallopeptidases. These include, among others, neprilysin (NEP) and its homologue endothelin-converting enzyme (ECE), insulysin (IDE), angiotensin-converting enzyme (ACE) and matrix metalloproteinase-9 (MMP-9). In addition, the serine proteinase, plasmin, may participate in extracellular metabolism of the amyloid peptide under regulation of the plasminogen-activator inhibitor. These various amyloid-degrading enzymes have distinct subcellular localizations, and differential responses to aging, oxidative stress and pharmacological agents and their upregulation may provide a novel and viable therapeutic strategy for prevention and treatment of Alzheimer's disease. Potential approaches to manipulate expression levels of the key amyloid-degrading enzymes are highlighted.

The primary pathogenetic role of vascular hypoperfusion, mitochondria failure and oxidative stress in aging and Alzheimer disease

We studied the cellular and subcellular features of vascular lesions, immunoreactivity of vasoactive substances, and mitochondria in brain vascular wall cells and hippocampal neurons from human Alzheimer disease (AD) and animal models that mimic AD. Lesioned vessels with immunopositive staining for AbPP and iNOS, and large, lipid‐laden vacuoles in the cytoplasm of endothelial cells were observed. Mitochondrial abnormalities and lesions were pronounced in microvessels in human AD, YAC, ApoE4 and C57B6/SJL Tg (+) mice, 2‐vessel occluded and non‐occluded aged rats, with and without selective mitochondrial antioxidant (lipoic acid and ALCAR) treatment. In situ hybridization revealed damaged mitochondria in vascular endothelium and in perivascular cells of lesioned microvessels in human AD and rodent models proximal to regions of large amyloid deposition. mtDNA deletions were associated with increased amounts of immunoreactive AbPP, 8OHG and COX but not eNOS; these deletions and expression of oxidative stress markers indicate that energy deficiency and oxidative stress in AD selectively affect the brain vascular tree and vulnerable neurons, which can be ameliorated by selective mitochondrial treatment.

Peripheral biomarkers of oxidative stress in aging and Alzheimer's disease.

Aging is associated with a greatly increased incidence of a number of neurodegenerative disorders, including Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS). These conditions are associated with chronic inflammation, which generates oxygen reactive species, ultimately responsible for a process known as oxidative stress. It is well established that this process is the culprit of neurodegeneration, and there are also mounting evidences that it is not restricted to the central nervous system. Indeed, several studies, including some by our group, have demonstrated that increased peripheral oxidative stress markers are associated to aging and, more specifically, to AD. Therefore, it is very instigating to regard aging and AD as systemic conditions that might be determined by studying peripheral markers of oxidative stress.

Carbon source-dependent variation of acquired mutagen resistance of Moniliophthora perniciosa: Similarities in natural and artificial systems

The basidiomycete Moniliophthora perniciosa causes Witches’ Broom disease in Theobroma cacao. We studied the influence of carbon source on conditioning hyphae to oxidative stress agents (H 2O 2, paraquat, 4NQO) and to UVC, toward the goal of assessing the ability of this pathogen to avoid plant defenses involving ROS. Cells exhibited increased resistance to H 2O 2 when shifted from glucose to glycerol and from glycerol to glycerol. When exposed to paraquat, cells grown in fresh medium were always more resistant. Apparently glycerol and/or fresh media, but not old glucose media, up-regulate oxidative stress defenses in this fungus. For the mutagens UVC and 4NQO, whose prime action on DNA is not via ROS, change of carbon source did not elicit a clear change in sensitivity/resistance. These results correlate with expression of fungal genes that protect against ROS and with biochemical changes observed in infected cacao tissues, where glycerol and high amounts of ROS have been detected in green brooms.

Urinary 8-hydroxy-2'-deoxyguanosine: a biomarker of environmental oxidative stress?

The oxidative stress plays an important role in certain pathologies, notably in carcinogenesis. Indeed, reactive oxygen species (ROS) can induce a variety of damage to DNA, including oxidized bases which will then be repaired and eliminated in urine. The 8-hydroxy-2'-deoxyguanosine (8-OHdG), the most frequent member of these oxidized bases, can be measured in DNA or in urine by various methods. The urinary measurement was used in several studies among subjects with occupational or environmental exposure. Diverse chemical or physical agents can indeed contribute to the increase of oxidative stress, although the latter has several origins. Our objective is to analyze through these studies the interest, the limits and the implementation of this biomarker. The majority of the studies reveal an increase of the concentration of 8-OHdG in urine in exposed subjects (polycyclic aromatic hydrocarbons, metals, ionizing radiation and other agents) compared with controls or according to exposure levels. The urinary concentration of 8-OHdG is subject to important inter and intra-individual variations. The biomonitoring studies have to take into account diverse confounding factors, which may have conflicting effects. Moreover the results strongly depend on the analytical method used. Thus other investigations are necessary to validate this biomarker and better know its sources of variability, its biological significance, its dose-response relationship and its kinetics after exposure to oxidative stress agents.

Isoorientin induces Nrf2 pathway-driven antioxidant response through phosphatidylinositol 3-kinase signaling

Because oxidative stress is involved in the pathogenesis of various chronic diseases and the aging process, antioxidants that can increase the intrinsic antioxidant potency are proposed as desirable therapeutic agents to counteract oxidative stress-related diseases. NF-E2-related factor-2 (Nrf2) is a transcription factor that regulates important antioxidant and phase II detoxification genes, and therefore, the molecule that regulates nuclear translocation of Nrf2 and the induction of antioxidative proteins is thought to be a promising candidate as a cytoprotective agent for oxidative stress. In the present study, we show that isoorientin (luteolin 6-C-beta-D-glucoside) obtained from the leaves of Sasa borealis upregulates and activates Nrf2, and has protective ability against oxidative damage caused by reactive oxygen intermediates in HepG2 cells. Isoorientin induces increase in the level of antioxidant enzyme proteins, especially NQO1, and the cytoprotective and antioxidative effects of isoorientin are PI3K/Akt pathway-dependent. Together with direct radical scavenging activity, the novel effect of isoorientin on the regulation of antioxidative gene expression provides attractive strategy to prevent diseases associated with oxidative stress and attenuate the progress of the diseases.

Oxidative stress in aging

Oxidative stress in aging

The marC Gene of Escherichia coli Is Not Involved in Multiple Antibiotic Resistance

The marC gene of Escherichia coli is divergently transcribed from the marRAB operon involved in resistance to multiple antibiotics (5, 8), oxidative stress agents (2), and organic solvents (3, 16). Previous data from our laboratory had suggested a role for marC in intrinsic multiple antibiotic resistance (4, 9, 16), and the gene has been so annotated in most databases. Because that earlier work had suggested that marC was regulated by the repressor MarR and induced by tetracycline, we sought the transcriptional start site for marC to see if the marC promoter might overlap the MarR binding sites within the marRAB promoter (5′ rapid amplification of cDNA end [RACE] system of Gibco/BRL Life Technologies, cells grown with 2 μg/ml tetracycline to increase the amount of mRNA). Transcription of marC started 30 nucleotides upstream from the putative ATG initiation codon of MarC (bp 1266 of Cohen et al. [4]). Therefore, the marC promoter does not contain the MarR binding sites. Moreover, Northern blot analysis of AG100 and its isogenic marR mutant AG112 (in which MarR is inactive [8, 12]) showed no differences in levels of marC mRNA between the two strains (data not shown), nor was expression of marC induced by salicylate, which inactivates the repressor MarR (1) (Fig. ​(Fig.1).1). We conclude that marC is not regulated by MarR. Since chloramphenicol does not bind to MarR (1), the apparent up-regulation by tetracycline and chloramphenicol (Fig. ​(Fig.1)1) (4) likely reflects stabilization of mRNA rather than true induction (11, 13, 14). FIG. 1. Northern blot analysis of marC from E. coli. RNA was isolated from mid-exponential-phase cells grown in LB broth at 30°C and treated for 1 h with the specified compounds. Separate cultures of E. coli AG100 following exposure to 5 mM salicylate ... We replaced the marC locus in E. coli AG100 with a kanamycin cassette as described previously (10) and looked for any increase in susceptibilities to antimicrobials. MICs were determined on LB agar by use of Etest strips (AB Biodisk, Solna, Sweden) with about three dozen different agents, including beta lactams, tetracyclines, fluoroquinolones, cephalosporins, imipenem, macrolides, aminoglycosides, chloramphenicol, fusidic acid, trimethoprim, and rifampin. Gradient plates (6) were used for oxidative stress agents (plumbagin, paraquat, phenylmethylsulfonate, dinitrophenol, and menadione) and for ethidium bromide. No differences in susceptibilities were seen for the marC::kan deletion mutant relative to the wild type. We then replaced the genes for the E. coli MarC paralogs YchE and YhgN in the marC::kan strain by use of spectinomycin and gentamicin cassettes, respectively, to create a triple knockout mutant, but again no differences in susceptibilities were seen. We also used three plasmid constructs designed to overexpress marC via the araBAD, T7, or native marC promoter. Plasmid pHA-1::marC (obtained from D. Daley) specifies membrane-bound MarC-PhoA regulated by Salmonella enterica serovar Typhimurium pBAD/AraC (7). We constructed pETmarC11 (T7 promoter/lac operator with lac repressor; specifying MarC-6H) and pACmarC1 (marC promoter starting 58 bp upstream of the transcriptional start site; specifying native MarC) by cloning PCR-amplified DNA into vectors pET21b (Novagen) and pACYC184, respectively. None of these three plasmids led to a change in susceptibility of cells to a variety of antibiotics and oxidative stress agents. We suggest that MarC no longer be classified as a multiple antibiotic resistance protein. However, we do not advise a name change until a function is found.

Should Depressive Syndromes Be Reclassified as “Metabolic Syndrome Type II”?

A nascent explanatory theory regarding the pathophysiology of major depressive disorder posits that alterations in metabolic networks (e.g., insulin and glucocorticoid signaling) mediate allostasis. We conducted a PubMed search of all English-language articles published between January 1966 and September 2006. The search terms were: neurobiology, cognition, neuroprotection, inflammation, oxidative stress, glucocorticoids, metabolic syndrome, diabetes mellitus, insulin, and antidiabetic agents, cross-referenced with the individual names of DSM-III-R/IV/-TR-defined mood disorders. The search was augmented with a manual review of article reference lists; articles selected for review were determined by author consensus. Disturbances in metabolic networks: e.g., insulin-glucose homeostasis, immuno-inflammatory processes, adipokine synthesis and secretion, intra-cellular signaling cascades, and mitochondrial respiration are implicated in the pathophysiology, brain volumetric changes, symptomatic expression (e.g., neurocognitive decline), and medical comorbidity in depressive disorders. The central nervous system, like the pancreas, is a critical modulator of the metabolic milieu and is endangered by chronic abnormalities in metabolic processes. We propose the notion of "metabolic syndrome type II" as a neuropsychiatric syndrome in which alterations in metabolic networks are a defining pathophysiological component. A comprehensive management approach for depressive disorders should routinely include opportunistic screening and primary prevention strategies targeting metabolically mediated comorbidity (e.g., cardiovascular disease). Innovative treatments for mood disorders, which primarily target aberrant metabolic networks, may constitute potentially novel, and disease-modifying, treatment avenues.

Alternative splicing of CTP:phosphoethanolamine cytidylyltransferase produces two isoforms that differ in catalytic properties

CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) catalyzes the rate-controlling reaction of the CDP-ethanolamine (Kennedy) pathway. We have previously established that Pcyt2 is encoded by a single gene that can be alternatively spliced from an internal exon into two transcripts, designated Pcyt2alpha and Pcyt2beta. Little is currently known about the regulation of Pcyt2. Here, we functionally express both murine Pcyt2 (mPcyt2) transcripts and investigate the roles of the two proteins in the regulation of mPcyt2 activity. We demonstrate that the tagged and purified alpha and beta proteins differ significantly in their kinetic properties. The K(m) of mPcyt2alpha for phosphoethanolamine was 318.4 microM, compared with 140.3 microM for mPcyt2beta. The maximal velocities of the alpha and beta isoforms at saturating conditions for both substrates were 138.0 and 114.4 nmol/min/mumol enzyme, respectively. When phosphoethanolamine was used at a fixed concentration of 1 mM, the K(m) of mPcyt2alpha for CTP was 102.0 microM and that of mPcyt2beta was 84.09 microM. Using a combination of nondenaturing PAGE, gel filtration chromatography, and immunoprecipitation, we provide evidence that mPcyt2alpha and mPcyt2beta proteins can form both homodimeric and heterodimeric complexes. We show that alternative splicing of the mPcyt2 transcript is ubiquitous but could also be regulated in a tissue-specific manner, producing a variable ratio of mPcyt2alpha/mPcyt2beta mRNAs. The expression of two distinct protein isoforms maybe an important mechanism by which Pcyt2 activity is regulated.

The Nuclear Matrix Protein, NRP/B, Enhances Nrf2-Mediated Oxidative Stress Responses in Breast Cancer Cells

The transcription factor NF-E2-related factor 2 (Nrf2) translocates into the nucleus and activates phase II genes encoding detoxification enzymes and antioxidant proteins, resulting in the protection of cells from oxidative insults. However, the involvement of Nrf2-mediated oxidative stress responses in breast cancer cells is largely unknown. Notably, during our study of the Nrf2 pathway in breast cancer cells, we observed that the nuclear matrix protein NRP/B was expressed and colocalized with Nrf2 in these cells, suggesting that NRP/B is involved in Nrf2-mediated oxidative stress responses. The expression level of NRP/B was variable in different breast cancer cells and breast cancer tissues, and was found to be localized in the nucleus. NRP/B expression was increased after exposure to the oxidative stress agent, hydrogen peroxide (H(2)O(2)), particularly in the highly aggressive MDA-MB-231 breast cancer cells. Association of NRP/B with Nrf2 in vitro and in vivo was observed in MDA-MB-231 breast cancer cells, and this association was up-regulated upon exposure to H(2)O(2), but not to sodium nitroprusside, SIN-1, and DETA-NO. NRP/B also enhanced Nrf2-mediated NAD(P)H:quinine oxidoreductase 1 promoter activity. Thus, this study reveals that NRP/B enhances oxidative stress responses in breast cancer cells via the Nrf2 pathway, identifying a novel role of nuclear matrix protein(s) in oxidative stress responses.

Relationship between glutathione-S-transferase P1 Ile105Val polymorphism and docetaxel neurosensory toxicity

2527 Background: Glutathione-S-transferases (GST) regulate the cellular response to oxidative stress and various anticancer agents. We recently underlined the importance of oxidative stress in the side effects of taxanes [Alexandre et al,J Natl Cancer Inst 2006;98:236–44], and investigated the relationship between the GST isoforms M1, T1 and P1 genes polymorphisms and docetaxel-induced peripheral neuropathy. Methods: We retrospectively analysed GST polymorphisms in a cohort of cancer patients (pts) treated with docetaxel and entered in a clinical trial database. A clinical neurologic evaluation (according to the NCI-CTC v2.0) was performed at baseline and at each treatment cycle. The GST M1 (null genotype), GST T1 (null genotype), and GST P1 (Ile105Val and Ala114Val) polymorphisms were determined using PCR, followed by either sequencing or RFLP techniques. The relationship between GST polymorphisms and grade = 2 neurosenrory toxicity (NST) as primary endpoint was studied, using the Chi2-square (with Yates correction) and the Fischer’s two-tailed exact test. Results: Fifty-eight pts were included : F/M: 29/29; median age: 61 years (range: 47–75). Primary tumor: 27.6% breast, 27.6% prostate, 24.1% lung and 20.7% other cancers. Pts received docetaxel 75–100 mg/m2 given as single-agent. A total of 261 cycles were administered (median/pt: 4, range 2–12). Ten pts developed grade = 2 NST. Twenty-seven pts (47%) were homozygous for the GST P1 105Ile allele, and 31 pts were either homozygous or heterozygous for the GST P1 105Val allele. Grade = 2 NST was significantly more common in pts with GST P1 105Ile/105Ile genotype (8/27 pts, 30%) compared with patients with 105Ile/105Val or 105Val/105Val GSTP1 genotype (2/31 pts, 6,5%; P = 0.047). Pts who were genotyped as GST P1 105Ile/105Ile had a higher risk of developing a grade = 2 NST than did those with other GSTP1 genotypes (OR 6.11; 95% CI, 1.17–31.94; P < 0.05). Conclusions: We found a significant correlation between GST P1 105Ile homozygous genotype and the development of a docetaxel-induced peripheral neuropathy. No significant financial relationships to disclose.

Aging impairs neurogenic contraction in guinea pig urinary bladder: role of oxidative stress and melatonin

The incidence of urinary bladder disturbances increases with age, and free radical accumulation has been proposed as a causal factor. Here we investigated the association between changes in bladder neuromuscular function and oxidative stress in aging and the possible benefits of melatonin treatment. Neuromuscular function was assessed by electrical field stimulation (EFS) of isolated guinea pig detrusor strips from adult and aged female guinea pigs. A group of adult and aged animals were treated with 2.5 mg x kg(-1) x day(-1) melatonin for 28 days. Neurotransmitter blockers were used to dissect pharmacologically the EFS-elicited contractile response. EFS induced a neurogenic and frequency-dependent contraction that was impaired by aging. This impairment is in part related to a decrease in detrusor myogenic contractility. Age also decreased the sensitivity of the contraction to pharmacological blockade of purinergic and sensitive fibers but increased the effect of blockade of nitrergic and adrenergic nerves. The density of cholinergic and nitrergic nerves remained unaltered, but aging modified afferent fibers. These changes were associated with an increased level of markers for oxidative stress. Melatonin treatment normalized oxidative levels and counteracted the aging-associated changes in bladder neuromuscular function. In conclusion, these results show that aging modifies neurogenic contraction and the functional profile of the urinary bladder plexus and simultaneously increases the oxidative damage to the organ. Melatonin reduces oxidative stress and improves the age-induced changes in bladder neuromuscular function, which could be of importance in reducing the impact of age-related bladder disorders.

Consequences of expressing mutants of the hemochromatosis gene ( HFE ) into a human neuronal cell line lacking endogenous HFE

HFE mutations have traditionally been associated with the iron overload disorder known as hemochromatosis. Recently, it has become clear that the two most common mutations in the HFE gene, H63D and C282Y, may be genetic modifiers for risk of neurodegenerative disorders and cancer, respectively. We developed human neuroblastoma stable cell lines that express either wild-type (WT) or mutant HFE to determine the cellular consequences of the mutant forms of HFE. The presence of the C282Y mutation was associated with relatively higher labile iron pool and iron regulatory protein activity than WT or H63D HFE. Targeted gene arrays revealed that the signal transduction pathway was up-regulated in the C282Y cells. H63D cells had higher levels of lipid peroxidation, protein oxidation, and lower mitochondrial membrane potential, suggesting higher baseline stress. This cell line was also more vulnerable to exposure to oxidative stress agents and more responsive to iron chelation than the C282Y cells. These data demonstrate that the different mutations in the HFE gene have unique effects on the cells and provide insights into how the different mutations may have different clinical consequences. The results also raise multiple novel questions for future study about the function of the HFE protein.

Molecular mechanisms of calorie restriction's protection against age-related sclerosis

The current knowledge on the molecular mechanisms of the protective effect of calorie restriction (CR) against age-related fibrosclerosis is tentatively reviewed with specific reference to the role of oxidative stress in aging. The effects of oxidative stress are often mediated by its own final products. Of these, 4-hydroxy-2,3-nonenal (HNE) induces the expression and synthesis of transforming growth factor beta1 (TGFbeta1) and activates nuclear binding of transcription factor activator protein 1 (AP-1) thus stimulating fibrogenesis. Several studies have shown that, as well as extending mean and maximum life span in a variety of species, CR delays the onset and slows the progression of a variety of age-associated diseases, including diabetes, cardiovascular diseases and neoplasia. However, the anti-aging mechanisms of CR are still not clearly understood. Of the numerous hypotheses put forward, one that still remains popular is protection against the age-associated increase of oxidative stress and consequent cell damage. CR protects the rat aorta from the age-related increase of both oxidative damage and fibrosis; as regards the possible mechanism/s of CR's protection against fibrosclerosis, it is conceivable that, by decreasing oxidative stress, CR reduces HNE levels and consequently TGFbeta1 expression and collagen deposition, likely by down-regulating the activation of Jun-N terminal kinase and of AP-1. Through the modulation of reactive oxygen species and oxidative stress CR may also attenuate the age-associated increase in the inflammatory milieu, thus preserving vascular functional integrity by suppressing the age-associated increase in inflammatory enzyme activities and prostanoids.

MarA, SoxS and Rob function as virulence factors in an Escherichia coli murine model of ascending pyelonephritis

MarA, SoxS and Rob are transcription factors belonging to the AraC family. While these proteins have been associated historically with control of multiple antibiotic resistance, and tolerance to oxidative stress agents and organic solvents, only a paucity of experimental data support a role in regulating virulence. Clinical Escherichia coli isolates, and isogenic strains lacking marA, soxS and rob, were studied in a murine model of ascending pyelonephritis, which is a clinically relevant model of urinary tract infection. Organisms lacking all three transcription factors (triple knockouts) were significantly less virulent than parental strains, and complementation studies demonstrated that the addition of marA, soxS and rob individually restored wild-type virulence in the triple-knockout strain. Deletion of soxS or rob alone was more detrimental than the removal of marA. Thus, all three proteins contribute to virulence in vivo.

17β-Estradiol Protects Cortical Neurons Against Oxidative Stress-Induced Cell Death through Reduction in the Activity of Mitogen-Activated Protein Kinase and in the Accumulation of Intracellular Calcium

Although many studies have suggested that estrogen acts as a neuroprotective agent in oxidative stress, the underlying mechanism has not been fully elucidated. In the present study, we examined the effect of 17beta-estradiol (17beta-E2) on H(2)O(2)-induced death signaling in cultured cortical neurons. Exposure of the cortical neurons to H(2)O(2) triggered a series of events, including overactivation of p44/42 MAPK and intracellular Ca(2+) accumulation via voltage-gated Ca(2+) channels and ionotropic glutamate receptors, resulting in apoptotic-like cell death. The MAPK pathway might work as death signaling in our system, because the MAPK pathway inhibitor, U0126, blocked H(2)O(2)-induced MAPK activation, Ca(2+) overload, and cell death. Interestingly, a similar inhibitory effect on H(2)O(2)-triggered MAPK activation, Ca(2+) accumulation, and cell death was observed in cultures incubated with 17beta-E2 for 24 h before exposure to H(2)O(2), suggesting that the protective effect of 17beta-E2 is induced via attenuating overactivation of the MAPK pathway. Furthermore, we found that ionotropic glutamate receptor subunits, including NR2A and GluR2/3, but not NR2B and GluR1, were down-regulated in the 17beta-E2-treated cultures. The down-regulation of these glutamate receptor subunits was also observed after chronic treatment with U0126. Therefore, it is possible that 17beta-E2 down-regulates the expression of the ionotropic glutamate receptors by reducing activity of the MAPK pathway, which might be important for the protective effect of 17beta-E2 against oxidative stress-induced toxicity.

Response of human endothelial cells to oxidative stress on Ti6Al4V alloy

Titanium and its alloys are amongst the most frequently used materials in bone and dental implantology. The good biocompatibility of titanium(-alloys) is attributed to the formation of a titanium oxide layer on the implant surface. However, implant failures do occur and this appears to be due to titanium corrosion. Thus, cells participating in the wound healing processes around an implanted material, among them endothelial cells, might be subjected to reactive oxygen species (ROS) formed by electrochemical processes during titanium corrosion. Therefore, we studied the response of endothelial cells grown on Ti6Al4V alloy to H 2O 2 and compared this with the response of endothelial cells grown on cell culture polystyrene (PS). We could show that although the cell number was the same on both surfaces, metabolic activity of endothelial cells grown on Ti6Al4V alloy was reduced compared to the cells on PS and further decreased following prototypic oxidative stress (H 2O 2-treatment). The analysis of H 2O 2-induced oxidative stress showed a higher ROS formation in endothelial cells on Ti6Al4V than on PS. This correlated with the depletion of reduced glutathione (GSH) in endothelial cells grown on Ti6Al4V surfaces and indicated permanent oxidative stress. Thus, endothelial cells in direct contact with Ti6Al4V showed signs of oxidative stress and higher impairment of cell vitality after an additional oxidative stress. However, the exact nature of the agent of oxidative stress generated from Ti6Al4V remains unclear and requires further investigation.

GABA-synthesizing enzyme, GAD67, from dermal fibroblasts: Evidence for a new skin function

Glutamate decarboxylase (GAD) catalyzes the synthesis of γ-aminobutyric acid (GABA), an inhibitory neurotransmitter, from glutamate. An expression of GAD protein has been reported for brain and pancreas, but not for skin. In this study, we present evidence that GAD67 mRNA and protein are expressed in mouse skin and in human dermal fibroblasts. The expression of GAD67 gene is weaker in aged mouse than the young one. To further explore the function of GAD in skin, we have examined a potential role(s) of GABA in human dermal fibroblasts. We have observed that GABA stimulates the synthesis of hyaluronic acid (HA) and enhances the survival rate of the dermal fibroblasts when fibroblasts are exposed to H 2O 2 an oxidative stress agent. Also observed were lowering the levels of HA and collagen in the embryonic skin from GAD67 deficient mouse as compared to those from the wild-type (WT) mouse. In this study, we have presented the evidences that GAD67 is localized in the dermis and is potentially involved in variety of skin activities.