Direct toxicity of nonsteroidal antiinflammatory drugs for renal medullary cells

Antipyretic analgesics, taken in large doses over a prolonged period, cause a specific form of kidney disease, characterized by papillary necrosis and interstitial scarring. Epidemiological evidence incriminated mixtures of drugs including aspirin (ASA), phenacetin, and caffeine. The mechanism of toxicity is unclear. We tested the effects of ASA, acetaminophen (APAF, the active metabolite of phenacetin), caffeine, and other related drugs individually and in combination on mouse inner medullary collecting duct cells (mIMCD3). The number of rapidly proliferating cells was reduced by ≈50% by 0.5 mM ASA, salicylic acid, or APAF. The drugs had less effect on confluent cells, which proliferate slowly. Thus, the slow in vivo turnover of IMCD cells could explain why clinical toxicity requires very high doses of these drugs over a very long period. Caffeine greatly potentiated the effect of acetaminophen, pointing to a potential danger of the mixture. Cyclooxygenase (COX) inhibitors, indomethacin and NS-398, did not reduce cell number except at concentrations greatly in excess of those that inhibit COX. Therefore, COX inhibition alone is not toxic. APAF arrests most cells in late G1 and S and produces a mixed form of cell death with both oncosis (swollen cells and nuclei) and apoptosis. APAF is known to inhibit the synthesis of DNA and cause chromosomal aberrations due to inhibition of ribonucleotide reductase. Such effects of APAF might account for renal medullary cell death in vivo and development of uroepithelial tumors from surviving cells that have chromosomal aberrations.

Three-dimensional reconstruction of the recombinant type 3 ryanodine receptor and localization of its amino terminus

Recombinant type 3 ryanodine receptor (RyR3) has been purified in quantities sufficient for structural characterization by cryoelectron microscopy and three-dimensional (3D) reconstruction. Two cDNAs were prepared and expressed in HEK293 cells, one encoding the wild-type RyR3 and the other encoding RyR3 containing glutathione S-transferase (GST) fused to its amino terminus (GST-RyR3). RyR3 was purified from detergent-solubilized transfected cells by affinity chromatography using 12.6-kDa FK506-binding protein in the form of a GST fusion as the affinity ligand. Purification of GST-RyR3 was achieved by affinity chromatography by using glutathione-Sepharose. Purified recombinant RyR3 and GST-RyR3 proteins exhibited high-affinity [3H]ryanodine binding that was sensitive to activation by Ca2+ and caffeine and to inhibition by Mg2+. 3D reconstructions of both recombinant RyR3 and GST-RyR3 appeared very similar to that of the native RyR3 purified from bovine diaphragm. Comparison of the 3D reconstructions of RyR3 and GST-RyR3 revealed that the GST domains and, hence, the amino termini of the RyR3 subunits are located in the “clamp” structures that form the corners of the square-shaped cytoplasmic region of homotetrameric RyR3. This study describes the 3D reconstruction of a recombinant ryanodine receptor and it demonstrates the potential of this technology for characterizing functional and structural perturbations introduced by site-directed mutagenesis.

Fluorescent light-induced chromatid breaks distinguish Alzheimer disease cells from normal cells in tissue culture.

The neurodegeneration and amyloid deposition of sporadic Alzheimer disease (AD) also occur in familial AD and in all trisomy-21 Down syndrome (DS) patients, suggesting a common pathogenetic mechanism. We investigated whether defective processing of damaged DNA might be that mechanism, as postulated for the neurodegeneration in xeroderma pigmentosum, a disease with defective repair not only of UV radiation-induced, but also of some oxygen free radical-induced, DNA lesions. We irradiated AD and DS skin fibroblasts or blood lymphocytes with fluorescent light, which is known to cause free radical-induced DNA damage. The cells were then treated with either beta-cytosine arabinoside (araC) or caffeine, and chromatid breaks were quantified. At least 28 of 31 normal donors and 10 of 11 donors with nonamyloid neurodegenerations gave normal test results. All 12 DS, 11 sporadic AD, and 16 familial AD patients tested had abnormal araC and caffeine tests, as did XP-A cells. In one of our four AD families, an abnormal caffeine test was found in all 10 afflicted individuals (including 3 asymptomatic when their skin biopsies were obtained) and in 8 of 11 offspring at a 50% risk for AD. Our tests could prove useful in predicting inheritance of familial AD and in supporting, or rendering unlikely, the diagnosis of sporadic AD in patients suspected of having the disease.

Calmodulin is a selective mediator of Ca(2+)-induced Ca2+ release via the ryanodine receptor-like Ca2+ channel triggered by cyclic ADP-ribose.

The ryanodine receptor-like Ca2+ channel (RyRLC) is responsible for Ca2+ wave propagation and Ca2+ oscillations in certain nonmuscle cells by a Ca(2+)-induced Ca2+ release (CICR) mechanism. Cyclic ADP-ribose (cADPR), an enzymatic product derived from NAD+, is the only known endogenous metabolite that acts as an agonist on the RyRLC. However, the mode of action of cADPR is not clear. We have identified calmodulin as a functional mediator of cADPR-triggered CICR through the RyRLC in sea urchin eggs. cADPR-induced Ca2+ release consisted of two phases, an initial rapid release phase and a subsequent slower release. The second phase was selectively potentiated by calmodulin which, in turn, was activated by Ca2+ released during the initial phase. Caffeine enhanced the action of calmodulin. Calmodulin did not play a role in inositol 1,4,5-trisphosphate-induced Ca2+ release. These findings offer insights into the multiple pathways that regulate intracellular Ca2+ signaling.

Modulation of Sarco/Endoplasmic Reticulum Ca2+-ATPase 2 (SERCA2) function by acetylation following the treatment with histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)

Background: Acetylation and deacetylation at specific lysine (K) residues is mediated by histone acetylases (HATs) and deacetylases (HDACs), respectively. HATs and HDACs act on both histone and non-histone proteins, regulating various processes, including cardiac impulse propagation. Aim of the present work was to establish whether the function of the Ca2+ ATPase SERCA2, one of the major players in Ca2+ reuptake during excitation-contraction coupling in cardiac myocytes (CMs), could be modulated by direct K acetylation. Materials and methods: HL-1 atrial mouse cells (donated by Prof. Claycomb), zebrafish and Streptozotocin-induced diabetic rat CMs were treated with the pan-inhibitor of class I and II HDACs suberanilohydroxamic acid (SAHA) for 1.5 hour. Evaluation of SERCA2 acetylation was analyzed by co-immunoprecipitation. SERCA2 activity was measured on microsomes by pyruvate/NADH coupled reaction assay. SERCA2 mutants were obtained after cloning wild-type and mutated sequences into the pCDNA3 vector and transfected into HEK cells. Ca2+ transients in CMs (loading with Fluo3-AM, field stimulation, 0.5 Hz) and in transfected HEK cells (loading with FLUO-4, caffeine pulse) were recorded. Results: Co-Immunoprecipitation experiments performed on HL-1 cells demonstrated a significant increase in the acetylation of SERCA2 after SAHA-treatment (2.5 µM, n=3). This was associated with an increase in SERCA2 activity in microsomes obtained from HL-1 cells, after SAHA exposure (n=5). Accordingly, SAHA-treatment significantly shortened the Ca2+ reuptake time of adult zebrafish CMs. Further, SAHA 2.5 nM restored to control values the recovery time of Ca2+ transients decay in diabetic rat CMs. HDAC inhibition also improved contraction parameters, such as fraction of shortening, and increased pump activity in microsomes isolated from diabetic CMs (n=4). Notably, the K464, identified by bioinformatic tools as the most probable acetylation site on human SERCA2a, was mutated into Glutamine (Q) or Arginine (R) mimicking acetylation and deacetylation respectively. Measurements of Ca2+ transients in HEK cells revealed that the substitution of K464 with R significantly delayed the transient recovery time, thus indicating that deacetylation has a negative impact on SERCA2 function. Conclusions: Our results indicate that SERCA2 function can be improved by pro-acetylation interventions and that this mechanism of regulation is conserved among species. Therefore, the present work provides the basis to open the search for novel pharmacological tools able to specifically improve SERCA2 activity in diseases where its expression and/or function is impaired, such as diabetic cardiomyopathy.

Consumo alimentar e estilo de vida: um estudo longitudinal com estudantes universitários

A universidade é um local de aprendizagem no qual conhecimento acadêmico, social e cultural permeia a relação entre os estudantes, onde estes terão a oportunidade de vivenciar diferentes situações durante o curso. A maior preocupação é como esta experiência influenciará o estado nutricional com a possível mudança dos hábitos cotidianos, como abuso de bebidas alcoólicas, uso irrestrito de suplementos vitamínicos e alimentares e a alimentação inadequada. O consumo alimentar de universitários foi o foco desta pesquisa que teve como objetivo geral identificar as principais mudanças do consumo de alimentos/ bebidas e estilos de vida por meio de estudo de coorte, envolvendo estudantes ingressantes nos cursos de graduação da Escola Superior de Agricultura \"Luiz de Queiroz\" - ESALQ/USP, e como objetivos específicos desenvolver, validar e aplicar um instrumento com a finalidade de identificar medidas comportamentais relacionadas aos hábitos de consumo alimentar, atividade física, situação socioeconômica e relacionar as informações obtidas ao estado nutricional do estudante; descrever o consumo dentro e fora do domicílio. Participaram da pesquisa estudantes com idade entre 18 e 30 anos. Um questionário foi aplicado juntamente com a avaliação antropométrica para mensuração do peso, altura e classificação do estado nutricional por meio do Índice de Massa Corporal (IMC). Este protocolo foi repetido após 8 meses de curso para que fosse identificada a situação do estado nutricional de cada indivíduo relacionada às mudanças do comportamento alimentar. Os dados coletados foram armazenados em base de dados no Microsoft Excel, sendo analisados por meio do Statistical Analysis System. Os dados quantitativos foram expressos em média e desvio-padrão (DP) com cálculos de intervalos de confiança de 95%. O teste do qui-quadrado foi utilizado para comparar a distribuição da prevalência de sobrepeso e obesidade quanto à variável sexo, associando-se ao IMC. Foi utilizado o coeficiente de correlação intraclasse de Pearson para verificação de concordância entre peso e altura aferidos e referidos. Foi realizada análise de regressão múltipla para identificação da mudança de consumo entre as fases, assim como para peso corporal. Utilizou-se o nível de significância de 5%. Observaram-se entre as duas fases quantidades preocupantes de nutrientes ingeridos aquém ou além dos limites preconizados para ambos os sexos; destaque para o elevado consumo de sódio, e insatisfatório de carotenóides. No caso dos carotenóides, houve crescimento significativo da contribuição da categoria 3 para as alunas. O consumo de cafeína na segunda fase foi maior, predominando o fornecimento pelos alimentos ultraprocessados. De forma geral houve aumento do consumo de alimentos processados e ultraprocessados. Observou-se aumento (significativo a 5%) no consumo de lipídios. Constatou-se diminuição na prática de exercícios físicos e aumento na ingestão de bebidas alcoólicas, e destas com energéticos e no tabagismo. Concluiu-se que o ingresso na universidade contribui para a mudança nos hábitos alimentares e estilos de vida de maneira negativa, sendo necessária intervenção adequada visando a promoção da saúde dos estudantes.

A Hydrodynamic Method For Measuring Aqueous Nanoparticle Surface Interactions

The objectives of this research dissertation were to develop and present novel analytical methods for the quantification of surface binding interactions between aqueous nanoparticles and water-soluble organic solutes. Quantification of nanoparticle surface interactions are presented in this work as association constants where the solutes have interacted with the surface of the nanoparticles. By understanding these nanoparticle-solute interactions, in part through association constants, the scientific community will better understand how organic drugs and nanomaterials interact in the environment, as well as to understand their eventual environmental fate. The biological community, pharmaceutical, and consumer product industries also have vested interests in nanoparticle-drug interactions for nanoparticle toxicity research and in using nanomaterials as drug delivery vesicles. The presented novel analytical methods, applied to nanoparticle surface association chemistry, may prove to be useful in assisting the scientific community to understand the risks, benefits, and opportunities of nanoparticles. The development of the analytical methods presented uses a model nanoparticle, Laponite-RD (LRD). LRD was the proposed nanoparticle used to model the system and technique because of its size, 25 nm in diameter. The solutes selected to model for these studies were chosen because they are also environmentally important. Caffeine, oxytetracycline (OTC), and quinine were selected to use as models because of their environmental importance and chemical properties that can be exploited in the system. All of these chemicals are found in the environment; thus, how they interact with nanoparticles and are transported through the environment is important. The analytical methods developed utilize and a wide-bore hydrodynamic chromatography to induce a partial hydrodynamic separation between nanoparticles and dissolved solutes. Then, using deconvolution techniques, two separate elution profiles for the nanoparticle and organic solute can be obtained. Followed by a mass balance approach, association constants between LRD, our model nanoparticle, and organic solutes are calculated. These findings are the first of their kind for LRD and nanoclays in dilute dispersions.

Spontaneous Abortions and Shift Work in a Cohort of Nurses in Norway

Background: An association between spontaneous abortions and shift work has been suggested, but present research results are conflicting. The aim of the study is to evaluate the relationship between spontaneous abortions among nurses, shift schedules, and nights worked. Methods: This is a longitudinal study where we identified 914 females from a cohort of nurses in Norway who had worked the same type of shift schedule 2008-2010; either permanent day shift, three-shift rotation or permanent night shift. Information on age, work and life-style factors, as well as spontaneous abortions during lifetime and the past three years (2008-2010) was obtained by annual questionnaires. Results: A higher prevalence of experienced spontaneous abortions before study start (2008) was found among nurses working permanent night shift compared to other nurses. In a linear regression analysis, a risk of 1.3 was found for experienced spontaneous abortions before study start among permanent night shift nurses, with day shift as reference, when adjusting for age, smoking, caffeine and job strain, but the finding was not statistical significant (95 per cent confidence interval 0.8-2.1). Permanent night shift workers had a risk of 1.5 experiencing spontaneous abortions in 2008-2010 compared to day shift nurses, although not statistical significant (95 per cent confidence interval 0.7-3.5). The number of night shifts the past three years was not associated with experiencing spontaneous abortions 2008-2010, but associated with a reduced risk of experiencing spontaneous abortions during lifetime. The results must be interpreted in the light of a possible selection bias; both selections into the occupation of nursing and into the different shift types of the more healthy persons may have occurred in this population. Conclusion: No significant increased risk of spontaneous abortion among permanent night shift nurses compared to day-time nurses was found in this study, and no association was found between spontaneous abortions and the number of worked night shifts.

The EBNA- 3 gene family proteins disrupt the G2/M checkpoint

The Epstein - Barr nuclear antigens (EBNA), EBNA-3, -4 and - 6, have previously been shown to act as transcriptional regulators, however, this study identifies another function for these proteins, disruption of the G2/M checkpoint. Lymphoblastoid cell lines (LCLs) treated with a G2/M initiating drug azelaic bishydroxamine ( ABHA) did not show a G2/M checkpoint response, but rather they display an increase in cell death, a characteristic of sensitivity to the cytotoxic effects of the drug. Cell cycle analysis demonstrated that the individual expression of EBNA-3, - 4 or - 6 are capable of disrupting the G2/M checkpoint response induced by ABHA resulting in increased toxicity, whereas EBNA-2, and - 5 were not. EBNA-3 gene family protein expression also disrupted the G2/M checkpoint initiated in response to the genotoxin etoposide and the S phase inhibitor hydroxyurea. The G2 arrest in response to these drugs were sensitive to caffeine, suggesting that ATM/ATR signalling in these checkpoint responses may be blocked by the EBNA-3 family proteins. The function of EBNA-3, - 4 and - 6 proteins appears to be more complex than anticipated and these data suggest a role for these proteins in disrupting the host cell cycle machinery.

The measurement of nicotine in human plasma by high-performance liquid chromatography-electrospray-tandem mass spectrometry

The authors describe a reverse-phase high-performance liquid chromatography-electrospray-tandem mass spectrometry method for the measurement of nicotine in human plasma. Samples (500 muL) with added deuterium-labeled d(3)-nicotine as an internal standard (IS) were treated with a 2-step process of ether extraction (6 mL) followed by back-extraction into 0.1% formic acid (50 muL). Chromatography was performed on a phenyl Novapak column with a mobile phase consisting of 50% 10 mM ammonium fortriate (pH 3.3) and acetonitrile (50:50, vol/vol). A flow rate of 0.2 mL/min resulted in a total analysis time of 5 minutes per sample. Mass spectrometric detection was by selected reactant monitoring (nicotine m/z 163.2 --> 130.2; IS m/z 166.2 --> 87.2). The assay was linear from 0.5 to 100 mug/L (r > 0.993, n = 9). The accuracy and imprecision of the method for quality control sampleswere 87.5% to 113% and < 10.2%, respectively. Interday accuracy and imprecision at the limit of quantification (0.5 mug/L) was 113% and 7.2% (n = 4). The process efficiency for nicotine in plasma was > 75%. The method described has good process efficiency, stabilized nicotine, avoided concentration steps, and most importantly minimized potential contamination. Further, we have established that water-based standards and controls are interchangeable with plasma-based samples. This method was used successfully to measure the pharmacokinetic profiles of subjects involved in the development of an aerosol inhalation drug delivery system.

HPLC analyses of flavanols and phenolic acids in the fresh young shoots of tea (Camellia sinensis) grown in Australia

As part of a 4-year project to study phenolic compounds in tea shoots over the growing seasons and during black tea processing in Australia, an HPLC method was developed and optimised for the identification and quantification of phenolic compounds, mainly flavanols and phenolic acids, in fresh tea shoots. Methanol proved to be the most suitable solvent for extracting the phenolic compounds, compared with chloroform, ethyl acetate and water. Immediate analysis, by HPLC, of the methanol extract showed higher separation efficiency than analyses after being dried and redissolved. This method exhibited good repeatability (CV 3-9%) and recovery rate (88-116%). Epigallocatechin gallate alone constituted up to 115 mg/g, on a dry basis, in the single sample of Australian fresh tea shoots examined. Four catechins (catechin, gallocatechin, epicatechin and epigallocatechin) and six catechin gallates (epigallocatechin gallate, catechin gallate, epicatechin gallate, gallocatechin gallate, epicatechin digallate and epigallocatechin digallate) have been identified and quantified by this HPLC method. In addition, two major tea alkaloids, caffeine and theobromine, have been quantified, while five flavonol glycosides and six phenolic acids, including quinic acids and esters, were identified and quantified. (C) 2003 Elsevier Ltd. All rights reserved.

Tooth wear: diet analysis and advice

Diet analysis and advice for patients with tooth wear is potentially the most logical intervention to arrest attrition, erosion and abrasion. It is saliva that protects the teeth against corrosion by the acids which soften enamel and make it susceptible to wear. Thus the lifestyles and diet of patients at risk need to be analysed for sources of acid and reasons for lost salivary protection. Medical conditions which put patients at risk of tooth wear are principally: asthma, bulimia nervosa, caffeine addiction, diabetes mellitus, exercise dehydration, functional depression, gastroesophageal reflux in alcoholism, hypertension and syndromes with salivary hypofunction. The sources of acid are various, but loss of salivary protection is the common theme. In healthy young Australians, soft drinks are the main source of acid, and exercise dehydration the main reason for loss of salivary protection. In the medically compromised, diet acids and gastroesophageal reflux are the sources, but medications are the main reasons for lost salivary protection. Diet advice for patients with tooth wear must: promote a healthy lifestyle and diet strategy that conserves the teeth by natural means of salivary stimulation; and address the specific needs of the patients' oral and medical conditions. Individualised, patient-empowering erosion WATCH strategies; on Water, Acid, Taste, Calcium and Health, are urgently required to combat the emerging epidemic of tooth wear currently being experienced in westernised societies.

Intravenous anaesthetics inhibit nicotinic acetylcholine receptor-mediated currents and Ca2+ transients in rat intracardiac ganglion neurons

1 The effects of intravenous (i.v.) anaesthetics on nicotinic acetylcholine receptor (nAChR)-induced transients in intracellular free Ca2+ concentration ([Ca2+](i)) and membrane currents were investigated in neonatal rat intracardiac neurons. 2 In fura-2-loaded neurons, nAChR activation evoked a transient increase in [Ca2+](i), which was inhibited reversibly and selectively by clinically relevant concentrations of thiopental. The half-maximal concentration for thiopental inhibition of nAChR-induced [Ca2+](i) transients was 28 muM, close to the estimated clinical EC50 (clinically relevant (half-maximal) effective concentration) of thiopental. 3 In fura-2-loaded neurons, voltage clamped at -60mV to eliminate any contribution of voltage-gated Ca2+ channels, thiopental (25 muM) simultaneously inhibited nAChR-induced increases in [Ca2+](i) and peak current amplitudes. Thiopental inhibited nAChR-induced peak current amplitudes in dialysed whole-cell recordings by - 40% at - 120, -80 and -40 mV holding potential, indicating that the inhibition is voltage independent. 4 The barbiturate, pentobarbital and the dissociative anaesthetic, ketamine, used at clinical EC50 were also shown to inhibit nAChR-induced increases in [Ca2+](i) by similar to40%. 5 Thiopental (25 muM) did not inhibit caffeine-, muscarine- or ATP-evoked increases in [Ca2+](i), indicating that inhibition of Ca2+ release from internal stores via either ryanodine receptor or inositol-1,4,5-trisphosphate receptor channels is unlikely. 6 Depolarization-activated Ca2+ channel currents were unaffected in the presence of thiopental (25 muM), pentobarbital (50 muM) and ketamine (10 muM). 7 In conclusion, i.v. anaesthetics inhibit nAChR-induced currents and [Ca2+](i) transients in intracardiac neurons by binding to nAChRs and thereby may contribute to changes in heart rate and cardiac output under clinical conditions.

Phenolic compounds in tea from Australian supermarkets

Phenolic compounds constitute 50-70% of tea water extract and are the main quality parameters for teas. Theaflavins (TF), thearubigins (TR) and theabrownins (TB) are the major polyphenols that determine the quality of black tea. These compounds were measured in 56 leaf teas and teabags sampled from Australian supermarkets in Queensland. The various quantities of TF, ranging from 0.29% to 1.25%, indicate a quality difference that exists among the teas studied. Low TF content in black tea may be due to over-fermenting and/or long periods of storage. The solubility of TR and TB from teabags ranged from 82% to 92%, indicating that the permeability of teabags was variable. Variable quantities of TF in Australian teas show instability and a tendency of TF to oxidize during storage. Total polyphenols in green teas ranged from 14% to 34%, indicating a large variation, which was not reflected in price. The solubility of total polyphenols from teabags has been proposed as a useful quality index of the filtering paper used for the teabags. This chemical analysis of phenolic compounds in commercial teas may be a potential tool for the quality control of Australian manufactured and imported teas in Australian markets. (C) 2005 Elsevier Ltd. All rights reserved.

Compositional analysis of teas from Australian supermarkets

Caffeine, total amino acids, water extract and moisture content are considered to be quality indicators for leaf teas and teabags. These analyses were examined in 20 leaf teas and 36 teabags sampled from Australian supermarkets. About 70% of the analysed samples showed a moisture content higher than Vie maximum accepted level, 6.5%, for tea storage and marketing by the tea industries and traders. Water appropriate extract of 15 samples out of 36 teabags was lower than that of the teas without teabags, which indicates that the quality of the paper used for teabags needs to be evaluated. Moreover, one of the black leaf tea samples was found to have a water extract below the lower limit of international standards. Four green and black teas of the same brand, claimed to contain less than 3% caffeine, were found to have 3-4%, the same as the other samples analysed in this study. The mean total contents of amino acids were 2.50% and 1.76% in black leaf teas and the teabags, respectively, whereas they were 3.44% and 2.28% in green leaf teas and the teabags, respectively. Furthermore, the weights of 28 teabags out of 36 samples were found to lie outside of the proposed +/- 2% variation accepted by the tea industries and traders, and 4 samples showed even larger variation, 10% being out of the proposed weights. This investigation also showed that the solubility of caffeine and water extract was affected by the permeability of teabags, whereas total amino acids were very variable. These results suggest that an efficient and practical quality control system for both imported and Australian-made teas in the Australian supermarkets should be developed, implemented and enforced. Chemical analysis should be a part of the system for establishing an objective assessment for the quality control. (c) 2004 Elsevier Ltd. All rights reserved.