1,4-Diamino-2-butanone, a wide-spectrum microbicide, yields reactive species by metal-catalyzed oxidation

The alpha-aminoketone 1,4-diamino-2-butanone (DAB), a putrescine analogue, is highly toxic to various microorganisms, including Trypanosoma cruzi. However, little is known about the molecular mechanisms underlying DAB's cytotoxic properties. We report here that DAB (pK(a) 7.5 and 9.5) undergoes aerobic oxidation in phosphate buffer, pH 7.4, at 37 degrees C, catalyzed by Fe(II) and Cu(II) ions yielding NH(4)(+) ion, H(2)O(2), and 4-amino-2-oxobutanal (oxoDAB). OxoDAB, like methylglyoxal and other alpha-oxoaldehydes, is expected to cause protein aggregation and nucleobase lesions. Propagation of DAB oxidation by superoxide radical was confirmed by the inhibitory effect of added SOD (50 U ml(-1)) and stimulatory effect of xanthine/xanthine oxidase, a source of superoxide radical. EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) revealed an adduct attributable to DMPO-HO(center dot), and those with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone or 3,5-dibromo-4-nitrosobenzenesulfonic acid, a six-line adduct assignable to a DAB(center dot) resonant enoyl radical adduct. Added horse spleen ferritin (HoSF) and bovine apo-transferrin underwent oxidative changes in tryptophan residues in the presence of 1.0-10 mM DAB. Iron release from HoSF was observed as well. Assays performed with fluorescein-encapsulated liposomes of cardiolipin and phosphatidylcholine (20:80) incubated with DAB resulted in extensive lipid peroxidation and consequent vesicle permeabilization. DAB (0-10 mM) administration to cultured LLC-MK2 epithelial cells caused a decline in cell viability, which was inhibited by preaddition of either catalase (4.5 mu M) or aminoguanidine (25 mM). Our findings support the hypothesis that DAB toxicity to several pathogenic microorganisms previously described may involve not only reported inhibition of polyamine metabolism but also DAB pro-oxidant activity. (C) 2011 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)INCT Processos Redox em Biomedicina-RedoxomaUniv São Paulo, Inst Quim, Dept Bioquim, BR-05508900 São Paulo, BrazilUniversidade Federal de São Paulo, Inst Ciencias Ambientais Quim & Farmaceut, Dept Ciencias Exatas & Terra, Diadema, SP, BrazilUniversidade Federal de São Paulo, Inst Ciencias Ambientais Quim & Farmaceut, Dept Ciencias Exatas & Terra, Diadema, SP, BrazilWeb of Scienc

Neurotoxicity and aggressiveness triggered by low-level lead in children: a review

Lead-induced neurotoxicity acquired by low-level long-term exposure has special relevance for children. A plethora of recent reports has demonstrated a direct link between low-level lead exposure and deficits in the neurobehavioral-cognitive performance manifested from childhood through adolescence. In many studies, aggressiveness and delinquency have also been suggested as symptoms of lead poisoning. Several environmental, occupational and domestic sources of contaminant lead and consequent health risks are largely identified and understood, but the occurrences of lead poisoning remain numerous. There is an urgent need for public health policies to prevent lead poisoning so as to reduce individual and societal damages and losses. In this paper we describe unsuspected sources of contaminant lead, discuss the economic losses and urban violence possibly associated with lead contamination and review the molecular basis of lead-induced neurotoxicity, emphasizing its effects on the social behavior, delinquency and IQ of children and adolescent

Potential diagnostic of Branched-Chain Ketoaciduria by HPLC-DAD

A system of high performance liquid chromatography (HPLC) was used for the development and validation of efficient method for quantitative determination of three aminoacids involved in the inherited metabolic disease Branched-Chain Ketoaciduria (BCK), also called maple syrup urine disease. The analytical conditions were selected in order to obtain baseline separation profiles of the amino acids known to be altered in blood plasma of BCK patients, namely L-valine, L-isoleucine, and L-leucine. Most accurate data were obtained using HPLC/diode detector. As the analytes do not have chromophore groups, they were pre-derivatized with o-phthaldialdehyde (OPA), yielding an unsaturated adduct, making thus possible the detection of amino acids. The validation was conducted according to National Health Surveillance Agency (ANVISA) and Guidance for Industry (Bioanalytical Method Validation) United States Food and Drug Administration (U.S. FDA). The results were satisfactory, with high sensitivity, good linearity, precision and accuracy, limit of detection and quantification, all within the established parameters for bioanalytical methods, showing its applicability and low cost compared to other existing techniques such as sequential mass spectrometry. For the three amino acids, L-valine, L-isoleucine and L-leucine, the detection limits (LOD) found were: 1.61, 1.84 and 1.88 mmol L- 1 and the quantification limits (LOQ) 4.37, 6.13 and 6.27 mmol L- 1, respectively.Neste trabalho, um sistema de cromatografia líquida de alta eficiência (HPLC) foi usado para desenvolver e validar um eficiente método para determinar quantitativamente aminoácidos envolvidos na desordem rara conhecida como cetonúria de aminoácidos de cadeia ramificada ou doença do xarope de bordo. As condições analíticas foram desenvolvidas para obter os perfis dos aminoácidos de L-valina, L-isoleucina e L-leucina, sabidamente alterados no plasma sanguíneo dos pacientes. Empregou-se HPLC provido de um detector com arranjo de diodo. Os analitos não possuem grupos cromóforos e, por isso, foram pré-derivatizados com o-ftalaldeído (OPA) para tornar possível sua detecção. A validação foi conduzida de acordo com as normas da Agência Nacional de Vigilância Sanitária (ANVISA) (RDC No. 27, de 17 de maio de 2012) e secção de validação de bioanalítica da United States Food and Drug Administration (U.S. FDA). Os resultados foram satisfatórios, apresentando alta sensibilidade, boa linearidade, precisão e exatidão, limite de detecção e quantificação, todos parâmetros estabelecidos para métodos bioanalíticos, demonstrando a aplicabilidade e baixo custo do método comparado com outras técnicas como espectrometria de massas. Para os três aminoácidos, L-valina, L-isoleucina e L-leucina, os limites de detecção encontrados foram: 1,61, 1,84 e 1,88 mmol L 1 e limites de quantificação 4,37, 6,13 e 6,27 mmol L 1, respectivamente.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)INCT Redoxoma (National Institute of Science and Technology of Redox Processes in Biomedicine)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo (UNIFESP) Instituto de Ciências Ambientais, Química e FarmacêuticasUniversidade de São Paulo Instituto de Química Departamento de Química FundamentalUNIFESP, Instituto de Ciências Ambientais, Química e Farmacêuticas2006/60245-3 e 2007/59039-2 e 2012/02514-9SciEL

Bioanalytical studies of porphyric disorders using HPLC with fluorescence detection

We describe here the development, validation, quantification and application of a method for determination of heme porphyrin precursors in the urine of porphyric patients. The isomers coproporphyrinogen I and III (COPRO I and III), uroporphyrinogen I (URO I), heptacarboxylporphyrinogen I (HEPTA I), pentacarboxylporphyrinogen (PENTA I), and hexacarboxylporphyrinogen I (HEXA I) were analyzed. These six urinary heme precursors were determined in urine samples collected from 24 patients by high-performance liquid chromatography (HPLC) equipped with a fluorescence detector. The inter- and intra-day precision (coefficient of variation < 5%) and accuracy (95-99%) were evaluated. The limits of detection and of quantification of the porphyrins, expressed in nmol L-1, were as follows: URO I, 0.62 and 2.05; HEPTA I, 0.59 and 1.96; HEXA I, 0.54 and 1.81; PENTA I, 0.52 and 1.73; COPRO I, 2.03 and 6.77; and COPRO III, 0.43 and 1.44. The method described here satisfactorily results in an acceptable cost-benefit ratio, precision and speed for determining the concentrations of heme precursors in the urine of latent or symptomatic acute intermittent porphyria individuals or porphyria cutanea tarda carriers. Since it was analytically validated, this method may be used for accurate and reliable diagnostic reports to follow-up the onset of acute crisis in porphyria carriers to adopt preventive pharmacological treatment.Neste artigo, desenvolvemos, validamos e aplicamos um método para separação e quantificação de porfirinas precursoras do grupo heme na urina de portadores de porfirias. Os isômeros coproporfirinogenio I e III (COPRO I e III), uroporfirinogenio I (URO I), heptacarboxilporfirinogenio I (HEPTA I), pentacarboxilporfirinogenio I (PENTA I) e hexacarboxilporfirinogenio (HEXA I) foram determinados em amostras coletadas de 24 pacientes de porfiria aguda intermitente e de porfiria cutânea tarda. Utilizou-se cromatografia líquida de alta eficiência (HPLC) e detector de fluorescência. As concentrações de porfirinas foram determinadas com precisão inter e intra-dias (< 5%) e exatidão dentro da faixa 95-99%. Os limites de detecção e quantificação das porfirinas, expressos em nmol L-1, foram os seguintes: URO I, 0,62 e 2,05; HEPTA I, 0,59 e 1,96; HEXA I, 0,54 e 1,81; PENTA I, 0,52 e 1,73; COPRO I, 2,03 e 6,77; e COPRO III, 0,43 e 1,44. O método descrito aqui obedece a parâmetros analíticos satisfatórios, com excelente relação custo-benefício, e foi aplicado a amostras de urina de portadores assintomáticos e pacientes de porfirias. Este método foi validado analiticamente e mostrou potencial para diagnóstico de portadores de diferentes tipos de porfirias, imediatamente antes ou durante crises, e até mesmo para monitorar um tratamento farmacológico.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)INCT Redoxoma Redox Processes in BiomedicineFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo (UNIFESP) Instituto de Ciências Ambientais, Químicas e Farmacêuticas Departamento de Ciências Exatas e da TerraUniversidade de São Paulo Instituto de Química Departamento de Química FundamentalUNIFESP, Instituto de Ciências Ambientais, Químicas e Farmacêuticas Depto. de Ciências Exatas e da Terra2006/60245-3 e 2006/56530-4SciEL

Astaxanthin Restrains Nitrative-Oxidative Peroxidation in Mitochondrial-Mimetic Liposomes: A Pre-Apoptosis Model

Astaxanthin (ASTA) is a ketocarotenoid found in many marine organisms and that affords many benefits to human health. ASTA is particularly effective against radical-mediated lipid peroxidation, and recent findings hypothesize a "mitochondrial-targeted" action of ASTA in cells. Therefore, we examined the protective effects of ASTA against lipid peroxidation in zwitterionic phosphatidylcholine liposomes (PCLs) and anionic phosphatidylcholine: phosphatidylglycerol liposomes (PCPGLs), at different pHs (6.2 to 8.0), which were challenged by oxidizing/nitrating conditions that mimic the regular and preapoptotic redox environment of active mitochondria. Pre-apoptotic conditions were created by oxidized/nitr(osyl) ated cytochrome c and resulted in the highest levels of lipoperoxidation in both PCL and PCPGLs (pH 7.4). ASTA was less protective at acidic conditions, especially in anionic PCPGLs. Our data demonstrated the ability of ASTA to hamper oxidative and nitrative events that lead to cytochrome c-peroxidase apoptosis and lipid peroxidation, although its efficiency changes with pH and lipid composition of membranes.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESPBPE fellowship)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (Bolsa Produtividade em Pesquisa, Nivel 2, CNPq, Brazil)Programa Iberoamericano de Ciencia y Tecnologia para el Desarollo (CYTEDRed iberoamericana para el estudio de nuevos carotenoides bioactivos como ingredientes de alimentos, Spain)Univ Sao Paulo IQUSP, Inst Quim, Dept Bioquim, BR-05508000 Sao Paulo, SP, BrazilUniv Cruzeiro Sul, ICAFE, BR-01506000 Sao Paulo, SP, BrazilSuperintendencia Policia Tecn Cient, BR-05507060 Sao Paulo, SP, BrazilLychnoflora Pesquisa & Dev Prod Nat LTDA, BR-14030090 Ribeirao Preto, SP, BrazilGrp Fleury, BR-04344070 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Ciencias Exatas & Terra, UNIFESP, BR-09972270 Diadema, SP, BrazilUniv Sao Paulo IQUSP, Inst Quim, Dept Quim Fundamental, BR-05508000 Sao Paulo, SP, BrazilCSIC, IATA, Dept Ciencia Alimentos, Calle Catedrat Agustin Escardino 7, Paterna 46980, SpainUniv Fed Sao Paulo, Dept Ciencias Exatas & Terra, UNIFESP, BR-09972270 Diadema, SP, BrazilFAPESP: 017/06032-2CNPq: 304663/2015-8RIENCBI: 112RT0445Web of Scienc

Association of dental enamel lead levels with risk factors for environmental exposure

OBJECTIVE: To analyze household risk factors associated with high lead levels in surface dental enamel. METHODS: A cross-sectional study was conducted with 160 Brazilian adolescents aged 14-18 years living in poor neighborhoods in the city of Bauru, southeastern Brazil, from August to December 2008. Body lead concentrations were assessed in surface dental enamel acid-etch microbiopsies. Dental enamel lead levels were measured by graphite furnace atomic absorption spectrometry and phosphorus levels were measured by inductively coupled plasma optical emission spectrometry. The parents answered a questionnaire about their children's potential early (05 years old) exposure to well-known lead sources. Logistic regression was used to identify associations between dental enamel lead levels and each environmental risk factor studied. Social and familial covariables were included in the models. RESULTS: The results suggest that the adolescents studied were exposed to lead sources during their first years of life. Risk factors associated with high dental enamel lead levels were living in or close to a contaminated area (OR = 4.49; 95% CI: 1.69;11.97); and member of the household worked in the manufacturing of paints, paint pigments, ceramics or batteries (OR = 3.43; 95% CI: 1.31;9.00). Home-based use of lead-glazed ceramics, low-quality pirated toys, anticorrosive paint on gates and/or sale of used car batteries (OR = 1.31; 95% CI: 0.56;3.03) and smoking (OR = 1.66; 95% CI: 0.52;5.28) were not found to be associated with high dental enamel lead levels. CONCLUSIONS: Surface dental enamel can be used as a marker of past environmental exposure to lead and lead concentrations detected are associated to well-known sources of lead contamination.OBJETIVO: Analizar factores de riesgo en el ambiente domiciliar asociados con altos niveles de plomo en el esmalte dental superficial. MÉTODOS: Se realizó estudio transversal con 160 adolescentes brasileros (14 a 18 años), residentes en urbanizaciones pobres del municipio de Bauru, SP, de agosto a diciembre de 2008. La concentración de plomo en el esmalte dental fue evaluada por micro biopsias ácidas del esmalte dental superficial, cuantificada por espectrometría de absorción atómica con horno de grafito y la concentración de fósforo fue medida por espectrometría de absorción óptica con plasma inductivamente acoplado. Los padres de los adolescentes respondieron a cuestionario sobre posible exposición previa (5 primeros años de vida del adolescente) al plomo producto de fuentes de contaminación bien conocidas. Se usó regresión logística para identificar asociaciones entre concentración de plomo en el esmalte y factores de riesgo ambientales. Covariables familiares y sociales fueron incluidas en los modelos. RESULTADOS: Los resultados sugieren que los jóvenes evaluados fueron expuestos a fuentes de plomo durante sus primeros años de vida. Los factores de riesgo asociados con el resultado fueron residir en área contaminada por plomo o en sus proximidades (OR=4,49; IC 95%: 1,69;11,97) y haber convivido, en el mismo domicilio, con persona que trabajaba en fábrica de tintas, pigmentos, cerámicas o baterías (OR= 3,43; IC 95%: 1,31;9,00). Haber usado, en casa, cerámica vitrificada, juegos de baja calidad o piratas, haber aplicado zircón en portones de hierro sin cobertura esmaltada o almacenar baterías de carro usadas en la residencia (OR= 1,31; IC 95%: 0,56-3,03) y hábito de fumar no fueron asociados con altas concentraciones de plomo en el esmalte dental (OR=1,66; IC 95%: 0,52;5,28). CONCLUSIONES: El esmalte dental superficial puede ser utilizado como marcador de exposición ambiental pasada al plomo y las concentraciones encontradas de dicho metal están relacionadas con fuentes bien conocidas de contaminación por plomo.OBJETIVO: Analisar fatores de risco no ambiente domiciliar associados com altos níveis de chumbo no esmalte dentário superficial. MÉTODOS: Estudo transversal conduzido com 160 adolescentes brasileiros (14 a 18 anos), residentes em bairros pobres do município de Bauru, SP, de agosto a dezembro de 2008. A concentração de chumbo no esmalte dentário foi avaliada por microbiópsias ácidas do esmalte dentário superficial, quantificada por espectrometria de absorção atômica com forno de grafite e a concentração de fósforo foi medida por espectrometria de absorção óptica com plasma indutivamente acoplado. Os pais dos adolescentes responderam a questionário sobre possível exposição prévia (cinco primeiros anos de vida do adolescente) a chumbo decorrente de fontes de contaminação bem conhecidas. Usou-se regressão logística para identificar associações entre concentração de chumbo no esmalte e fatores de risco ambientais. Covariáveis familiares e sociais foram incluídas nos modelos. RESULTADOS: Os resultados sugerem que os jovens avaliados foram expostos a fontes de chumbo durante seus primeiros anos de vida. Os fatores de risco associados com o desfecho foram residir em área contaminada por chumbo ou nas suas proximidades (OR = 4,49; IC 95%: 1,69;11,97) e ter convivido, no mesmo domicílio, com pessoa que trabalhava em fábrica de tintas, pigmentos, cerâmicas ou baterias (OR = 3,43; IC 95%: 1,31;9,00). Ter usado, em casa, cerâmica vitrificada, brinquedos de baixa qualidade ou piratas, ter aplicado zarcão em portões de ferro sem cobertura esmaltada ou armazenar baterias de carro usadas na residência (OR = 1,31; IC 95%: 0,56;3,03) e hábito de fumar não foram associados com altas concentrações de chumbo no esmalte dentário (OR = 1,66; IC 95%: 0,52;5,28). CONCLUSÕES: O esmalte dentário superficial pode ser utilizado como marcador de exposição ambiental passada ao chumbo e as concentrações encontradas desse metal estão ligadas a fontes bem conhecidas de contaminação por chumbo

Ferricytochrome c Directly Oxidizes Aminoacetone to Methylglyoxal, a Catabolite Accumulated in Carbonyl Stress

Age-related diseases are associated with increased production of reactive oxygen and carbonyl species such as methylglyoxal. Aminoacetone, a putative threonine catabolite, is reportedly known to undergo metal-catalyzed oxidation to methylglyoxal, NH4+ ion, and H2O2 coupled with (i) permeabilization of rat liver mitochondria, and (ii) apoptosis of insulin-producing cells. Oxidation of aminoacetone to methylglyoxal is now shown to be accelerated by ferricytochrome c, a reaction initiated by one-electron reduction of ferricytochrome c by aminoacetone without amino acid modifications. the participation of O-2(center dot-) and HO center dot radical intermediates is demonstrated by the inhibitory effect of added superoxide dismutase and Electron Paramagnetic Resonance spin-trapping experiments with 5,5'-dimethyl-1-pyrroline-N-oxide. We hypothesize that two consecutive one-electron transfers from aminoacetone (E-0 values = -0.51 and -1.0 V) to ferricytochrome c (E-0 = 0.26 V) may lead to aminoacetone enoyl radical and, subsequently, imine aminoacetone, whose hydrolysis yields methylglyoxal and NH4+ ion. in the presence of oxygen, aminoacetone enoyl and O-2(center dot-) radicals propagate aminoacetone oxidation to methylglyoxal and H2O2. These data endorse the hypothesis that aminoacetone, putatively accumulated in diabetes, may directly reduce ferricyt c yielding methylglyoxal and free radicals, thereby triggering redox imbalance and adverse mitochondrial responses.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)INCT Processos Redox em Biomedicina (Brazil)Univ São Paulo, Dept Bioquim, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim & Biol Mol, São Paulo, BrazilUniversidade Federal de São Paulo, Inst Ciencias Ambientais Quim & Farmaceut, São Paulo, BrazilUniv São Paulo, Dept Fis & Informat, São Paulo, BrazilUniv Fed ABC, Ctr Ciencias Nat & Humanas, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim & Biol Mol, São Paulo, BrazilUniversidade Federal de São Paulo, Inst Ciencias Ambientais Quim & Farmaceut, São Paulo, BrazilWeb of Scienc

Increased chemical acetylation of peptides and proteins in rats after daily ingestion of diacetyl analyzed by Nano-LC-MS/MS

Background. Acetylation alters several protein properties including molecular weight, stability, enzymatic activity, protein protein interactions, and other biological functions. Our previous findings demonstrating that diacetyl/peroxynitrite can acetylate L-lysine, L-histidine, and albumin in vitro led us to investigate whether diacetyl-treated rats suffer protein acetylation as well. Methods. Wistar rats were administered diacetyl daily for four weeks, after which they were sacrificed, and their lung proteins were extracted to be analysed by Nano-LC-MS/MS (Q-TOF). A C18 reversed-phase colurnn and gradient elution with formic acid/acetonitrile solutions from 2 to 50% over 150 min were used to separate the proteins. Protein detection was performed using a microTOE-Q II (QTOF) equipped with captive source and an electrospray-ionization source. The data frommass spectrometry were processed using a Compass 1.7 and analyzed using Protein Scape, software that uses Mascot algorithms to perform protein searches. Results. A set of 3,162 acetylated peptides derived from 351 acetylated proteins in the diacetyl-treated group was identified. Among them, 23 targeted proteins were significantly more acetylated in the diacetyl-treated group than in the PBS control. Protein acetylation of the group treated with 540 mg/kg/day of diacetyl was corroborated by Western blotting analysis. Conclusions. These data support our hypothesis that diacetyl exposure in animals may lead to the generation of acetyl radicals, compounds that attach to proteins, affecting their functions and triggering adverse health problems.Sao Paulo Research Foundation (FAPESP)Brazilian Innovation Agency (FINEP)Univ Fed Sao Paulo, Inst Environm Chem & Pharmaceut Sci, Diadema, SP, BrazilUniv Fed Sul & Sudeste Para, Inst Studies Hlth & Biol, Collect Hlth, Maraba, PA, BrazilFundacao Univ Fed Rondonia, Dept Chem, Porto Velho, RO, BrazilUniv Sao Paulo, Sao Carlos Inst Chem, Sao Carlos, SP, BrazilUniv Sao Paulo, Inst Chem, Dept Fundamental Chem, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Inst Environm Chem & Pharmaceut Sci, Diadema, SP, BrazilFAPESP: 2012/02514-9FAPESP: 2013/07763-0FAPESP: 2010/01404-0Web of Scienc