Nowdays there is a plethora of evidence for H2O2-mediated oxidative stress in the epidermis as well as in the system in patients with vitiligo (for review see (Schallreuter, Bahadoran et al. 2008). Xanthine dehydrogenase / xanthine oxidase (XDH / XO) catalyses the oxidative hydroxylation of hypoxanthine to xanthine followed by xanthine to uric acid, the last two steps in purine degradation pathway. Under oxidative conditions, XDH is converted to XO. The reactions catalysed by this enzyme generate H2O2 and O2 ¿- , yielding in the presence of ROS accumulation, allantoin from uric acid. Therefore XO has been considered a major biologic source of oxygen-derived free radicals in many organs. The presence of XO in the human epidermis has not been shown so far. In this study several techniques were utilised to nail the presence and activity of XO in epidermal melanocytes and keratinocytes. The enzyme is regulated by H2O2 in a concentration dependent manner, where concentrations of 10-6M upregulate activity. Importantly, the results showed that the activity of XO is little affected by H2O2 in the mM range. H2O2-mediated oxidation of tryptophan and methionine residues in the sequence of XO yields only subtle alterations in the enzyme active site. These findings are in agreement with enzyme kinetics in the presence of 10-3M H2O2. Since uric acid is the end product of XO activity and this can be oxidised to allantoin by H2O2, we wanted to know whether allantoin is formed in the epidermis of patients with vitiligo. In order to address this issue, we utilised HPLC/mass spectrometry analysis. Analysis of epidermal cell extracts from suction blister tissue identified the presence of allantoin in patients with acute vitiligo, while this product was absent in healthy controls. In conclusion, our results provide evidence for functioning epidermal XO in the human epidermis which 4 can be a major source for the production of H2O2 contributing to oxidative stress in vitiligo. In addition, this thesis also demonstrates for the first time the presence of XO in melanosomes, and we showed that both 7BH4 and 7-biopterin inhibit XO activity in a concentration dependent manner. Moreover, XO has the potential to bind to 6/7BH4 and 6/7-biopterin from the pterin/tyrosinase inhibitor complex. This discovery adds another receptor independent mechanism for regulation of tyrosinase within the melanocyte similar to ¿/ß-MSH as shown earlier (Moore, Wood et al. 1999; Spencer, Chavan et al. 2005). Since the entire epidermis of patients with vitiligo is under H2O2-mediated oxidative stress, oxidative DNA damage would be highly expected. This thesis shows for the first time that epidermal 8-oxoG levels as well as plasma level of this oxidised DNA base are significantly increased in patients compared to healthy controls. We have shown that epidermal cells from patients with vitiligo respond to oxidative DNA damage via the overexpression of p21 and Gadd45¿ leading to a functioning increased short-patch base-excision repair (BER), while increased apoptosis can be ruled out due to lower caspase 3 and cytochrome c response compared to healthy controls. Our results show that patients develop effective DNA repair machinery via hOgg1, APE1 and DNA polymeraseß. Taking into consideration that these patients do not have an increased prevalence for solar-induced skin cancers, our data suggest that BER is a major player in the hierarchy to combat H2O2-mediated oxidative stress preventing ROS-induced tumourigenesis in the epidermis of these patients.

Patients with classical vitiligo lose partially their protecting inherited pigment. The cause of the disease is still unknown. Despite massive epidermal oxidative / nitrative stress and signs for DNA-damage in the skin and in the plasma, these patients have no higher prevalence for sun induced non-melanoma skin cancer and increased photo-damage. Protection and DNA-repair have been attributed to a functioning up-regulated wild type p53 / p21 cascade in association with up-regulated p76 MDM2. As some patients with cutaneous melanoma develop depigmentations away from their primary tumour site post surgical excision, it became of our interest, whether this melanoma associated leucoderma (MAL) is the same as classical vitiligo. The purpose of this thesis was two-fold. In part I, we wanted to further substantiate the reasons behind the constantly up-regulated wild-type functioning p53 / p21 cascade in classical vitiligo utilising a panel of proteins with direct and / or indirect action on p53 regulation, including p21, p76MDM2, MDM4/MDM4phospho, SPARC, VEGF-A and TGF-β1. In part II, we wanted to characterize MAL and compare this peculiar leucoderma with classical vitiligo using the same protein panel and methodologies. To achieve our goals, we used in vivo FT-Raman spectroscopy, in vitro cell cultures, in vitro and in situ immuno-fluorescence labelling, Western blot, dot blot and computer modelling techniques. Our data showed distinct differences between classical vitiligo and MAL. Our results in MAL exhibited a concentration dependent protein expression gradient between the basal / suprabasl layers and the upper layers of the epidermal compartment using catalase, ONOO-, p53, p21, MDM4, p76MDM2, TGF-β1 and VEGF-A expression gradient. Moreover, we document for the first time the presence of a nitrated non-fuctional SPARC protein in classical vitiligo which is absent in MAL. Although we show in vivo considerable ROS / RNS- mediated stress in MAL and classical vitiligo documented by FT-Raman spectroscopy, Western blot and in situ immuno-fluorescence, our results prove that MAL and classical vitiligo are two distinct entities.

Cancer is one of the leading causes of death across the globe which needs appropriate and cost-effective treatment. Several recent studies have suggested that dietary intake of various flavonoids such as myricetin have a protective effect against different types of cancers and cardiovascular diseases. The present study was conducted to investigate the genoprotective and genotoxic effects of myricetin nano and bulk forms on the lymphocytes from pre-cancerous and multiple myeloma cancer patients compared to those from healthy individuals. Also, to investigate the protective potential of myricetin bulk and nano against the oxidative stress produced in vitro by 2- amino-1-methyl-6 phenylimidazo [4, 5-b] pyridine and reactive oxygen species- induced DNA damage using the Comet assay, micronucleus assay, cellular reactive oxygen species and glutathione detection assay, Western blotting, real-time polymerase chain reaction and immunofluorescence. Lymphocytes from the patient groups showed significantly higher levels of basal DNA damage compared to the lymphocytes from healthy individuals which was observed throughout the in vitro treatment. Myricetin in both forms has not induced any significant DNA damage in all of the investigative groups at selective lower concentrations; in fact, the results demonstrate a reduction in DNA damage upon treating with myricetin nano in lymphocytes from pre-cancerous patients demonstrated by significant reduction in micronuclei formation in mononucleated cells. DNA repair capacity of myricetin bulk and nano was determined by co-treating the drugs with hydrogen peroxide. Myricetin significantly reduced the oxidative stress related damage caused by hydrogen peroxide, where myricetin nano seemed to be more effective employing the Comet assay. In the presence of myricetin bulk and nano, the damaging effects of 2- amino-1-methyl-6 phenylimidazo [4,5-b] pyridine were considerably decreased, where myricetin nano was more effective. This could be because nanoparticles have a larger surface area which could improve their reactivity and also the reduction in size of the particles could improve the anti-cancer properties of this compound. Myricetin has shown genoprotective and anti-oxidant effects by demonstrating the potential to reduce DNA damage caused by over-production of reactive oxygen species and oxidative stress. It has also shown anti-cancer potential in the lymphocytes from multiple myeloma patients by regulating the apoptosis related proteins, dependent on oxidative stress. Therefore, this study suggests that myricetin supplementation in our regular diet with enhanced bioavailability could have potential health beneficial effects and possibly protect against various diseases including cancer.

No description available.

No / Nonsegmental vitiligo (NSV) is characterized by loss of inherited skin color. The cause of the disease is still unknown despite accumulating in vivo and in vitro evidence of massive epidermal oxidative stress via H2O2 and peroxynitrite (ONOO−) in affected individuals. The most favored hypothesis is based on autoimmune mechanisms. Strictly segmental vitiligo (SSV) with dermatomal distribution is a rare entity, often associated with stable outcome. Recently, it was documented that this form can be associated with NSV (mixed vitiligo). We here asked the question whether ROS and possibly ONOO− could be players in the pathogenesis of SSV. Our in situ results demonstrate for the first time epidermal biopterin accumulation together with significantly decreased epidermal catalase, thioredoxin/thioreoxin reductase, and MSRA/MSRB expression. Moreover, we show epidermal ONOO− accumulation. In vivo FT-Raman spectroscopy reveals the presence of H2O2, methionine sulfoxide, and tryptophan metabolites; i.e., N-formylkynurenine and kynurenine, implying Fenton chemistry in the cascade (n=10). Validation of the basic data stems from successful repigmentation of skin and eyelashes in affected individuals, regardless of SSV or segmental vitiligo in association with NSV after reduction of epidermal H2O2 (n=5). Taken together, our contribution strongly supports H2O2/ONOO-mediated stress in the pathogenesis of SSV. Our findings offer new treatment intervention for lost skin and hair color.—Schallreuter, K. U., Salem, M. A. E. L., Holtz, S., Panske, A. Basic evidence for epidermal H2O2/ONOO−-mediated oxidation/nitration in segmental vitiligo is supported by repigmentation of skin and eyelashes after reduction of epidermal H2O2 with topical NB-UVB-activated pseudocatalase PC-KUS.

No / Vitiligo is characterized by a mostly progressive loss of the inherited skin color. The cause of the disease is still unknown, despite accumulating in vivo and in vitro evidence of massive oxidative stress via hydrogen peroxide (H2O2) and peroxynitrite (ONOO−) in the skin of affected individuals. The most favored hypothesis is based on autoimmune mechanisms. Since depletion of the essential amino acid l-tryptophan (Trp) severely affects various immune responses, we here looked at Trp metabolism and signaling in these patients. Our in vivo and in vitro data revealed total absence of epidermal Trp hydroxylase activities and the presence of H2O2/ONOO− deactivated indoleamine 2,3-dioxygenase. Aryl hydrocarbon receptor signaling is severely impaired despite the ligand (Trp dimer) being formed, as shown by mass spectrometry. Loss of this signal is supported by the absence of downstream signals (COX-2 and CYP1A1) as well as regulatory T-lymphocytes and by computer modeling. In vivo Fourier transform Raman spectroscopy confirmed the presence of Trp metabolites together with H2O2 supporting deprivation of the epidermal Trp pool by Fenton chemistry. Taken together, our data support a long-expressed role for in loco redox balance and a distinct immune response. These insights could open novel treatment strategies for this disease.—Schallreuter, K. U., Salem, M. A. E. L., Gibbons, N. C. J., Maitland, D. J., Marsch, E., Elwary, S., Healey, A. R. Blunted epidermal l-tryptophan metabolism in vitiligo affects immune response and ROS scavenging by Fenton chemistry, part 2: epidermal H2O2/ONOO−-mediated stress in vitiligo hampers indoleamine 2,3-dioxygenase and aryl hydrocarbon receptor-mediated immune response signaling.

No / The biochemistry of hair pigmentation is a complex field involving a plethora of protein and peptide mechanisms. The in loco factory for melanin formation is the hair follicle melanocyte, but it is common knowledge that melanogenesis results from a fine tuned concerted interaction between the cells of the entire dermal papilla in the anagen hair follicle. The key enzyme is tyrosinase to initiate the active pigmentation machinery. Hence, an intricate understanding from transcription of mRNA to enzyme activity, including enzyme kinetics, substrate supply, optimal pH, cAMP signaling, is a must. Moreover, the role of reactive oxygen species on enzyme regulation and functionality needs to be taken into account. So far our knowledge on the entire hair cycle relies on the murine model of the C57BL/6 mouse. Whether this data can be translated into humans still needs to be shown. This article aims to focus on the effect of H2O2-redox homeostasis on hair follicle pigmentation via tyrosinase, its substrate supply and signal transduction as well as the role of methionine sulfoxide repair via methionine sulfoxide reductases A and B (MSRA and B).

The synthesis of pure tris(6-hydroxymethyl-2-pyridylmethyl)amine (H₃L₁₁) is reported for the first time. New complexes of H₃L₁₁ with copper(II), manganese(II) and iron(III) have been characterised by X-ray crystallography. Linear [Fe₃(L₁₁)₂](ClO₄)₃ reveals the tightest Fe-O-Fe angle (87.6°) and shortest Fe...Fe distance (2.834 Å) presently found for a weakly antiferromagnetically-coupled high spin alkoxide-bridged polyiron(III) system. H₃L₁₁ provides a route to various hydrophobic peralkylated TPA ligand derivatives for creating a hydrophobic pocket for the assembly of iron catalysts for the novel 1-hydroxylation of n-alkanes. New 6-py substituted TPA ligands containing methyl (L₁₅) and n-octyl (L₁₆) ether linkages were synthesised via alkylation. Two further novel 6-py substituted ligands were synthesized incorporating n-hexyl substituents on one (L₂₁) and two (L₂₂) of the py moieties. Here a urea spacer group was used to promote hydrogen–bond assisted heterolytic O-O cleavage (generation of the potent FeV=O oxidant) within the hydroxoperoxoiron(III) precursor. High spin [FeII(L)(CH₃CN)[subscript(x)]](CF₃SO₃)₂ complexes (x = 0–2, L = L₁₅,₁₆,₂₁,₂₂) were characterised in solution by ¹H NMR. The structure of [Fe(L₂₂)](CF₃SO₃)₂ reveals a distorted iron(II) centre bound to four N atoms and two urea carbonyls. Iron(II) complexes of H₃L₁₁, L₁₅,₁₆,₂₁,₂₂ and tris(6-Br)-TPA (L₂₄), were investigated for catalysis of the oxygenation of cyclohexane by H₂O₂. Reaction of the iron(II) complexes with H₂O₂ and [superscript(t)]BuOOH was followed by time-resolved EPR and UV-VIS spectrophotometry. A correlation between the observed catalytic activity and the nature of the FeIII(L)-OOR intermediates generated is apparent. A convenient ‘one-pot’ synthesis of benzene-1,3,5-triamido-tris(l-histidine methyl ester) is reported along with attempts at preparing N,N’-bis(pyridylmethyl)-1,3- diaminopropane-2-carboxylic acid (L₂₅), a new water soluble pyridine-amine ligand. The final demetallation step resulted in ligand hydrolysis to the novel amino acid; 1,3-diaminopropane- 2-carboxylic acid which was characterised as its HCl salt by X-ray crystallography.

In the present investigation peripheral blood lymphocytes from patients with inflammatory bowel disease (IBD) and different cancer states were treated with various agents and compared with lymphocytes from healthy control individuals (HCI) treated in the same way and measured in the Comet assay. For inflammatory bowel disease, patient's responses in IBD patients treated with H2O2 were higher than in HCI and Crohn's patients (CD) were found to have higher responses than Ulcerative colitis (UC) patients. The responses for all IBD and HCI were all reduced in the presence of chaga mushroom extract which behaved in an antioxidant manner. A second group of IBD patients were treated with the heterocyclic amine (food mutagen), IQ and H2O2 and responses were reduced in the presence of the flavonoids, quercetin and epicatechin and compared with HCI similarity treated. In all cells responses were reduced with flavonoids and again CD had higher responses than the UC patients and IBD patients higher than HCI. The responses with CD and UC were that confirmed in two independent studies with IBD, one with chaga mushroom extract and the other with flavonoids. Peripheral lymphocytes from malignant melanoma and suspected melanoma patients and colon cancer and polyposis patients were compared to the lymphocytes from HCI and treated with UVA. There were differential sensitivities when measured in the micronucleus and Comet assays. The cancer patients had higher responses than those in the precancerous states and they in turn were higher than responses in HCI. In all the studies, untreated baseline DNA damage values were also higher in IBD and cancer patients and pre-cancerous patients than HCIs. This would suggest that baseline frequencies of different diseases compared to controls could be an important biomarker in the diagnosis of pre-cancers and early stage cancers. Also peripheral lymphocytes are a useful surrogate for cancers and pre-cancerous disease states since, blood is present in all organs and tissues and DNA is basically the same in all cells.

Bisphenol A (BPA) is widely used in the production of plastics, epoxy resins and polycarbonates. It is a toxic, endocrine disruptor compound. Different studies have shown the presence of BPA in several environmental systems, classifying it as a worldwide persistent pollutant which may act synergistically with other pollutants. In this context, advanced oxidation processes (AOP) have received great attention due to their ability to degrade pollutants with such characteristics, through their transformation into less hazardous compounds or even their mineralization. Although there are investigations on the use of AOPs for BPA degradation, systematic studies on the effects of process variables, coupled with the statistical interpretation of the results are virtually non-existent. Furthermore, to the best of our knowledge, a rigorous kinetic model has not yet been proposed for the degradation of this pollutant by the H2O2/UV process. The objective of this work was to evaluate BPA degradation by the H2O2/UV process, investigating the effects of the initial H2O2 concentration and the specific rate of photons emission (EP,0) by means of a Doehlert experimental design, combined with the response surface methodology. The experiments were performed in a photochemical tubular reactor equipped with a 254-nm UV lamp, for [H2O2]0 and EP,0 in the ranges 1.6-9.6 mmol L-1 and 0.87 × 1018-3.6 × 1018 photons L-1 s-1, respectively. Total BPA degradation was achieved after 60 min of irradiation in all experiments. The best conditions were [H2O2]0 = 7.6 mmol L-1 and EP,0 = 3.6 × 1018 photons L-1 s-1, for which the best performance was obtained regarding the BPA degradation rate, BPA degradation after 15 min, and the second highest TOC removal after 180 min. However, in most experiments less than 75% TOC removal was observed, with 95% mineralization obtained only for the superior [H2O2]0 and EP,0. A mathematical model was developed, considering the reactor characteristics and the radiation field, based on the line source with parallel emission (LSPP) approach, in combination with the radiative transfer equation (RTE), mass balances, and a detailed kinetic model of the H2O2/UV process. The steady-state approximation was applied for all radical species. In the estimation of unknown kinetic constants, the non-linear least squares method was employed. The model was able to satisfactorily fit experimental BPA and H2O2 concentrations as a function of time. This work shows that the H2O2/UV process is a good alternative for BPA removal from aqueous streams, with total degradation of the target compound and adequate percent mineralization under optimal operating conditions. Such conditions may serve as first guidelines for pilot-plant and industrial processes operation. In addition, simulations using the proposed kinetic model may provide useful information for the design and scale-up of pre- or post-treatment of effluents containing this pollutant. / O bisfenol A (BPA) é amplamente utilizado na fabricação de plásticos, resinas epóxi e policarbonatos. Trata-se de um composto tóxico e um desregulador endócrino. Diferentes estudos evidenciam a presença do BPA em diversos compartimentos ambientais em todo planeta, identificando-o como um poluente persistente e resistente à degradação biológica, que apresenta efeitos sinergéticos com outros poluentes. Nesse contexto, os processos oxidativos avançados (POA) têm recebido atenção devido a sua capacidade em degradar poluentes com tais características, transformando-os em compostos menos perigosos ou até mesmo mineralizando-os totalmente. Apesar de haver trabalhos na literatura acerca da utilização de POA para degradação de BPA, estudos sistemáticos dos efeitos de variáveis de processo junto com a interpretação estatística dos resultados são virtualmente inexistentes. Além disso, até onde se sabe um modelo cinético rigoroso ainda não foi proposto para a degradação desse poluente por meio do processo H2O2/UV. Este trabalho teve por objetivo avaliar a degradação do BPA pelo processo H2O2/UV, investigando os efeitos da concentração inicial de H2O2 e da taxa específica de emissão de fótons (EP,0) por meio de um projeto experimental Doehlert, combinado com a análise de superfície de resposta. Os experimentos foram realizados em um reator tubular fotoquímico equipado com uma lâmpada UV de 254 nm, para [H2O2]0 e EP,0 entre 1,6-9,6 mmol L-1 e 0,87 × 1018 - 3,6 × 1018 fótons L-1 s-1, respectivamente. Todos os experimentos sob H2O2/UV resultaram em total degradação do BPA após 60 min de irradiação. Nesse caso, as melhores condições foram [H2O2]0 = 7,6 mmol L-1 e EP,0 = 3,6 × 1018 fótons L-1 s-1, para as quais se obteve o melhor desempenho quanto à taxa de degradação de BPA e à remoção após 15 min, e a segunda maior remoção de COT após 180 min. Entretanto, na maioria dos experimentos menos de 75% de remoção de COT foram observados, com 95% de mineralização obtida apenas para os maiores [H2O2]0 e EP,0. Elaborou-se um modelo matemático que considera as características do reator utilizado e o campo de radiação, baseado no modelo de fonte linear de emissão em planos paralelos (LSPP), combinado à equação de transferência radiativa (RTE), aos balanços materiais e a um modelo cinético detalhado do processo H2O2/UV. Foi empregada a aproximação de estado estacionário para todas as espécies radicalares. Na estimativa das constantes cinéticas desconhecidas, utilizou-se o método de mínimos quadrados não linear. Esse modelo foi capaz de ajustar satisfatoriamente as concentrações experimentais de BPA e de H2O2 em função do tempo. Este trabalho mostra que o processo H2O2/UV constitui uma alternativa conveniente para a degradação de BPA em matrizes aquosas, com total degradação do composto alvo e porcentagem de mineralização adequada nas condições ótimas de operação. Tais condições podem servir como diretrizes iniciais de processamento em escalas piloto e industrial. Por sua vez, simulações empregando o modelo matemático proposto permitem gerar informações úteis para projeto e aumento de escala de processos de pré- ou pós-tratamento de efluentes contendo esse poluente.