Human enteric viruses in a wastewater treatment plant: evaluation of activated sludge combined with UV disinfection process reveals different removal performances for viruses with different features.

This study assess the quality of wastewater through the detection and quantification of important viruses causing gastroenteritis at different stages of the wastewater treatment process in an activated-sludge wastewater treatment plant with ultraviolet disinfection. Ten sampling events were carried out in a campaign along a period of 18 months collecting wastewater samples from the influent, after the activated-sludge treatment, and after the final disinfection with UV radiation. Samples were concentrated through ultracentrifugation and analysed using retro-transcription, PCR and real time quantitative PCR protocols, for detection and quantification of Group A Rotavirus (RVA), Human Astrovirus (HAstV), Norovirus Genogroup II (NoV GII) and Human Adenovirus (HAdV). HAdV (100%), NoV GII (90%), RVA (70%) and HAstV (60%) were detected in influent samples with concentration from 1·4 (NoV GII) to 8·0 (RVA) log10  gc l-1 . Activated-sludge treatment reached well quality effluents with low organic material concentration, although nonstatistical significant differences were registered among influent and postactivated sludge treatment samples, regarding the presence and concentration for most viruses. All post-UV samples were negative for NoV GII and HAstV, although RVA and HAdV were detected in 38% and 63% of those samples respectively, with concentration ranging from 2·2 to 5·5 and 3·1 to 3·4 log10  gc l-1 . SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that an activated-sludge wastewater treatment plant with UV disinfection reduces to levels below the detection limit those single-stranded RNA viruses as noroviruses and astroviruses and reach significant lower levels of rotaviruses and adenoviruses after the complete treatment process.

Intracellular spin-trapping of oxygen-centered radicals generated by human neutrophils.

Phagocytosing neutrophils secrete superoxide into a vacuole generally inaccessible for direct study. However, the spin-trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) enters the cytoplasm of several cell types where it can report free radical species including superoxide and hydroxyl radical. In the present study we employed a variety of experimental conditions to eliminate extracellular ESR signals and/or free radicals generated by stimulated neutrophils so that DMPO adducts reported events inside the cell. We identified a concentration of poly(ethylene glycol)-modified superoxide dismutase that permitted measurement of intracellular superoxide as determined by several criteria. It seems likely that poly(ethylene glycol)-modified superoxide dismutase is too large to enter the neutrophil phagosome. Under these conditions no hydroxyl radical was detected, as would be predicted from earlier studies with spin-trapping. Use of poly(ethylene glycol)-modified superoxide dismutase should allow on-line measurement of phagosomal events, thereby improving our understanding of microbicidal and inflammatory processes.

Does peroxynitrite generate hydroxyl radical?

Nitric oxide reacts with superoxide at a diffusion controlled rate to form peroxynitrite. Some studies have indicated that peroxynitrite underwent homolytic cleavage to give the highly toxic hydroxyl radical, while others have suggested that the decomposition of peroxynitrite did not generate hydroxyl radical. Because of this controversy, the fate of peroxynitrite decomposition was investigated using electron spin resonance (EPR) spectroscopy in combination with spin trapping technique. Utilizing 4-pyridyl 1-oxide N-tert-butylnitrone (4-POBN)/ethanol as a spin trapping system, we found that peroxynitrite, at physiological pH in either the absence or the presence of chelated iron, will produce hydroxyl radical. Further quantification experiments indicated that the yield of hydroxyl radical formation was only about 1-4%. Since the concentration of hydroxyl radical produced is so low, the cytotoxicity mediated by peroxynitrite might not be due to the formation of this free radical.

Involvement of the perferryl complex of nitric oxide synthase in the catalysis of secondary free radical formation.

Neuronal nitric oxide synthase (NOS I) has been shown to generate nitric oxide (NO*) and superoxide (O(2)* during enzymatic cycling, and the ratio of each free radical is dependent upon the concentration of L-arginine. Using spin trapping and electron paramagnetic resonance spectroscopy, we detected alpha-hydroxyethyl radical (CH(3)*CHOH), produced during the NOS I metabolism of ethanol (EtOH). The generation of CH(3)*CHOH by NOS I was found to be Ca(2+)/calmodulin dependent. Superoxide dismutase prevented CH(3)*CHOH formation in the absence of L-arginine. However, in the presence of L-arginine, the production of CH(3)*CHOH was independent of O(2)* but dependent upon the concentration of L-arginine. Formation of CH(3)*CHOH was inhibited by substituting D-arginine for L-arginine, or inclusion of the NOS inhibitors N(G)-nitro-L-arginine methyl ester, N(G)-monomethyl-L-arginine and the heme blocker, sodium cyanide. The addition of potassium hydrogen persulfate to NOS I, generating the perferryl complex (NOS-[Fe(5+)=O](3+)) in the absence of oxygen and Ca(2+)/calmodulin, and EtOH resulted in the formation of CH(3)*CHOH. NOS I was found to produce the corresponding alpha-hydroxyalkyl radical from 1-propanol and 2-propanol, but not from 2-methyl-2-propanol. Data demonstrated that the perferryl complex of NOS I in the presence of L-arginine was responsible for catalyses of these secondary reactions.

Can nitric oxide be spin trapped by nitrone and nitroso compounds?

Increasing interest in the study of nitric oxide (NO.) in many facets of biological research necessitates a search for accurate techniques to directly identify the free radical. One recently employed strategy for NO. detection is the method of electron spin resonance (ESR) used in combination with nitrone and nitroso spin traps. Applying this technique to our studies with nitric oxide synthase (NOS), we found that NO. generated directly from the enzyme system could not be detected. Further investigation revealed that 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) inhibited NO. generation by NOS at concentrations used for spin trapping. Reexamining the ability of various nitrones and DBNBS to spin trap authentic NO. dissolved in buffer, we obtained ESR spectra similar to those previously reported for the spin trap DBNBS. However, continuing our studies with 15NO. and N-hydroxylamine, we found these spectra to be artifactual. Our results emphasize the need to synthesize new spin traps, since currently available compounds are not capable of spin trapping NO. generated by NOS.

Spin trapping of nitric oxide by ferro-chelates: kinetic and in vivo pharmacokinetic studies.

Biologically generated nitric oxide appears to play a pivotal role in the control of a diverse series of physiologic functions. Iron-chelates and low-frequency EPR spectroscopy have been used to verify in vivo production of nitric oxide. The interpretation of in vivo identification of nitric oxide localized at the site of evolution in real time is complicated by the varied kinetics of secretion. The quantitative efficiency of the spectroscopic measurement, so important in understanding the physiology of nitric oxide, remains elusive. The development of a more stable iron-chelate will help better define nitric oxide physiology. In this report, we present data comparing the commonly used ferro-di(N-methyl-D-glucamine-dithiocarbamate) (Fe2+(MGD)2) and the novel chelate ferro-di(N-(dithiocarboxy)sarcosine) (Fe2+(DTCS)2) quantifying the in vitro and in vivo stability of the corresponding spin trapped adducts, NO-Fe(MGD)2 and NO-Fe(DTCS)2. Finally, very low frequency EPR spectroscopy has been used to evaluate the pharmacokinetics of NO-Fe(MGD)2 and NO-Fe(DTCS)2 in mice in real time.

Effect of trifluoromethyl ketone-based elastase inhibitors on neutrophil function in vitro.

Neutrophils release elastase, which is known secondarily to cause tissue damage. However, it is rapidly inactivated by the endogenous alpha1-proteinase inhibitor (alpha1Pi). Nevertheless, under pathological conditions, alpha1i is inactivated by oxidants released from neutrophils, resulting in an excess of elastase at the site of inflammation. This elastase/alpha1Pi imbalance has been implicated as a pathogenic factor in cystic fibrosis, acute respiratory distress syndrome, and emphysema. Elastase inhibitors, which do not interfere with the microbicidal activity of neutrophils and are resistant to neutrophil-released oxidants, would undoubtedly represent an important advance in the management of neutrophil-mediated tissue injury. We report that a new family of elastase inhibitors ICI200355 and ZD0892 was found to be resistant toward superoxide, hypochlorous acid, hydrogen peroxide, hydroxyl radical, and peroxynitrite mediated degradation as well as having no effect on the formation of these oxidants by activated neutrophils. More importantly, we found that these inhibitors did not interfere with the ability of human neutrophils to phagocytose and to kill Staphylococcus aureus. In conclusion, a new potent class of elastase inhibitors, while blocking the effects of neutrophil elastase, was found not to impede various physiological functions of human neutrophils, in particular the ability of these phagocytic cells to phagocytose and kill bacteria.

Hematogenous photosensitization. A mechanism for the development of age-related macular degeneration.

Age-related macular degeneration (ARMD) is one of the leading causes of severe visual loss in the United States. Numerous risk factors have been investigated, but the pathogenesis of ARMD has remained elusive. The authors propose that ARMD develops as a direct result of photosensitization of the vascular endothelium of the choriocapillaris, Bruch's membrane, and the retinal pigment epithelium (RPE) by superoxide anion and singlet oxygen generated by photoactive compounds in blood. Using electron-spin resonance spectrometry, the free-radical trap, 5,5-dimethyl-1-pyrroline-N-oxide, and the singlet-oxygen trap, 2-(9,10-dimethoxyanthracentyl)-t-butylhydroxylamine, the authors demonstrate that the photoactive compound, protoporphyrin IX (PP IX), a naturally occurring precursor molecule of hemoglobin found in erythrocytes and plasma, generates superoxide anion and singlet oxygen. The amount of reactive-oxygen species produced by this system is dependent on the concentration of PP IX and the intensity and wavelength of the light delivered. Furthermore, the production of these photooxidants is significantly reduced by filtering the excitatory wavelengths of PP IX. These photogenerated oxidants could damage the vascular endothelium of the choriocapillaris, Bruch's membrane, and the RPE, necessitating a reparative process. This could result in features characteristically seen in ARMD such as a thickened Bruch's membrane, RPE atrophy, and hyperplasia. Prevention of phototoxic damage by this mechanism could involve enhancing protective enzymes, increasing scavenger substances, or supplying appropriate filters to eliminate the exciting wavelengths of light.

Effect of anti-inflammatory drugs on myeloperoxidase-dependent hydroxyl radical generation by human neutrophils.

Neutrophils comprise a group of leukocytes that play a pivotal role in inflammation and vascular diseases like ischemia/reperfusion. These activated phagocytic cells are drawn to the site of injury, secreting superoxide and other oxidants derived from the formation of this free radical. This series of events frequently results in localized tissue damage. Surprisingly, free radical scavengers frequently offer only minimal relief. Why this is so may be due, in part, to our limited understanding of mechanisms that govern generation of free radicals in these settings. Although the metal ion-catalyzed Haber-Weiss reaction is considered the classical pathway for neutrophil-derived hydroxyl radical, an alternative mechanism, such as the myeloperoxidase-dependent pathway, may undoubtedly contribute to the formation of this free radical by stimulated neutrophils. In this study, we explored this possibility by investigating the role of different classes of anti-inflammatory drugs to ameliorate hydroxyl radical generation via the myeloperoxidase-dependent pathway. In this paper, we report that meclofenamic acid inhibited myeloperoxidase-dependent hydroxyl radical generation through scavenging of hypochlorous acid and not by direct inhibition of myeloperoxidase. The importance of these results with regard to the clinical efficacy of this anti-inflammatory compound remains to be determined as studies into the significance of myeloperoxidase-dependent hydroxyl radical formation in inflammatory tissue injury continue.

Effect of NPC 15669, an inhibitor of neutrophil recruitment and neutrophil-mediated inflammation, on neutrophil function in vitro.

The new anti-inflammatory agent N-[9H-(2,7-dimethylfluorenyl-9-methoxy)carbonyl]-L-leucine (NPC 15669) inhibits inflammation in several animal models dependent upon neutrophil activation and recruitment into the inflammatory lesion. NPC 15669 appears to elicit its pharmacological action by inhibiting the cell surface expression of CD11b/CD18 (Mac-1) on the neutrophil and subsequent adhesion of the neutrophil to the vascular endothelium. The current study sought to further characterize the action of NPC 15669 on neutrophil function. In the range of 1-100 microM, this fluorene enhanced superoxide production in a concentration-dependent fashion. Using spin trapping/ESR spectroscopy, NPC 15669 was found to inhibit myeloperoxidase (MPO)-dependent hydroxyl radical primarily by scavenging hypochlorous acid, and secondarily by inhibiting agonist-stimulated degranulation as assessed by MPO and elastase release. These studies demonstrated that NPC 15669, in addition to inhibiting adhesion, alters other neutrophil functions. Whether the pharmacological activities described for NPC 15669 resulted directly from changes in Mac-1 expression or through some other mechanism is currently under investigation.

The single-dose pharmacokinetics of midazolam and its primary metabolite in pediatric patients after oral and intravenous administration.

The first-dose pharmacokinetics of midazolam and its primary alpha-hydroxymetabolite were studied after single-dose administration. Eligible study patients were enrolled into one of three study arms: Arm I (midazolam/metabolite pharmacokinetic evaluation after oral administration of a syrup formulation), Arm II (the absolute bioavailability of midazolam syrup), and Arm III (midazolam and metabolite pharmacokinetics after IV administration). Complete blood sampling for pharmacokinetic analysis was available in 87 subjects. Midazolam absorption after administration of the oral syrupformulation was rapid, with adolescents absorbing the drug at approximately half the rate observed in younger children (ages 2 to < 12 years). Furthermore, midazolam t 1/2 was prolonged and CL/F reducedin adolescents as compared with younger children. Although the midazolam Vd/F appeared larger in the youngest age group after oral administration, this observation was not apparent after IV dosing, suggesting subject differences in bioavailability rather than distribution. Like midazolam, the disposition characteristics for a-hydroxymidazolam were also highly variable, with the greatest formation of metabolite (reflected by the AUC ratio) observed in children ages 2 to < 12 years. The A UC ratios of alpha-hydroxymidazolam to midazolam after IV dosing were similar across all age groups and were smaller than corresponding values following oral administration. The absolute bioavailability of midazolam averaged 36% with a very broad range (9%-71%). No relationship between midazolam bioavailability and age was observed. Overall, the disposition characteristics of midazolam and its a-hydroxy metabolite were highly variable, appeared independent of age and dose administered, and were linear over the dose range studied (0.25 to 1 mg/kg). These data suggest that an initial oral dose of 0.2 to 0.3 mg/kg should be adequateforsuccessful sedation of most pediatric patients. The inherent variability in midazolam bioavailability and metabolism underscores the importance of titrating midazolam dose to desired effect.

Potentiation of quinolinate-induced hippocampal lesions by inhibition of NO synthesis.

Low doses of quinolinic acid (QUIN) administered intracerebroventricularly (ICV) to rats produced either no damage or mild to moderate damage in the pyramidal cell layer of the hippocampus and resulted in mild, limbic seizures in the majority of animals treated. The same dose of QUIN following ICV pretreatment with the nitric oxide synthase inhibitor N-nitro-L-arginine (NARG), produced extensive hippocampal lesions with complete loss of the pyramidal layer in 50% of the animals, and moderate damage with total neuronal loss in areas CA1 and CA3 in the remainder of the group. Animals treated with both NARG and QUIN also exhibited a greater incidence of severe convulsive behavior (9/11) and 3 deaths. Pretreatment with the nitric oxide-generating drug molsidomine attenuated the enhanced toxicity observed with combined NARG-QUIN treatment, resulting primarily in no detectable hippocampal damages and mild seizures resembling those produced by QUIN alone. Administration of NARG alone produced neither seizure activity nor histological evidence of neurotoxicity. We conclude that inhibition of nitric oxide production with NARG potentiates the neurotoxicity of quinolinic acid in the rat hippocampus.

Use of nitroxides as NMR contrast enhancing agents for joints.

NMR imaging is a well-established technology for obtaining cross-sectional anatomic pictures of organs and tissues. In addition, NMR can provide valuable information about the physiologic state of organs and tissues, especially, as a consequence of cellular injury. With this in mind, NMR in combination with gadolinium-based contrast enhancing agents has been used to assist in the detection of abnormalities to joints as well as to evaluate the status of damage resulting from an injury to this site. We describe the synthesis of a new nitroxide, which is bioresistant to the one-electron reduction mediated by superoxide in the presence of cysteine. This model mimics the reduction of nitroxides by extracellular secretion of superoxide by PMA-stimulated neutrophils. With this nitroxide, we found, in the range from 15 to 17.5 mumoles, enhancement of an NMR image in the knee joint of rabbits. Of interest is the finding that the contrast image remained for at least 90 minutes. These results demonstrate the utility of nitroxides as contrast enhancing agents for NMR imaging of joints.

Light-dependent generation of superoxide from human erythrocytes.

Using the cytochrome c reduction method, we investigated light-dependent erythrocytic superoxide production. After 4 h light and dark exposure of erythrocytes from eight healthy human subjects, an average of 18.6% more superoxide was generated by erythrocytes exposed to light. Pretreatment of erythrocytes with the superoxide dismutase inhibitor N,N-diethyldithiocarbamate increased detection of superoxide while pretreatment with the anion channel blocker 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene decreased detection. These findings indicate that substantially more spontaneously generated superoxide is produced and escapes from normal erythrocytes at ambient oxygen tensions on exposure to light. This excess generation and escape of superoxide from erythrocytes exposed to light may result in tissue photosensitization, especially in the retina of the eye, where high oxygen tension, blood and chronic light exposure occur simultaneously.

Isotopically sensitive regioselectivity in the oxidative deamination of a homologous series of diamines catalyzed by diamine oxidase.

The equivalence of aminomethylene groups in selected diamine substrates of diamine oxidase was exploited for the determination of intramolecular isotope effects. In the series of substrates, [1,1-2H2]-1,3-diaminopropane, [1,1-2H2]-1,5-diaminopentane, [1,1-2H2]-1,6-diaminohexane, [1,1-2H2]-1,7-diaminoheptane and [alpha,alpha-2H2]-4-(aminomethyl)benzylamine, the preference of the enzyme for reaction at the unlabeled methylene was found to vary from 1.45 to 10.5-fold. The observed partitioning ratios go through a minimum value with 1,5-diaminopentane, the best substrate of diamine oxidase of the compounds tested. The results suggest that fast substrates have less opportunity to reorient into alternate binding conformations while bound to the active site of the enzyme. On the other hand, diamine substrates tested that cannot exist in energetically favorable conformations with internitrogen distances of about 7-8 A showed larger intramolecular isotope effects.

Nitroxide-doped liposomes containing entrapped oxidant: an approach to the "reduction problem" of nitroxides as MRI contrast agents.

The purpose of this investigation is to test the feasibility of a nitroxide regeneration system involving liposomes as an approach toward solving the "reduction problem" when nitroxides are used as contrast enhancing agents in MRI applications. It is shown that the inclusion of an entrapped oxidant (K3Fe(CN)6) in the aqueous compartment of nitroxide-doped liposomes causes a 4-5-fold increase in the duration of the nitroxide ESR signal in the presence of the external reductant sodium ascorbate. Confirmation was obtained by monitoring the concentration of the internalized Fe(CN)6(3-) ion versus time by visible spectroscopy at 410 nm. Trans bilayer (flip-flop) motion of the long chain nitroxide ester is the likely mechanism of this nitroxide regeneration system.

Unique pharmacological actions of atypical neuroleptic quetiapine: possible role in cell cycle/fate control.

Quetiapine is an atypical neuroleptic with a pharmacological profile distinct from classic neuroleptics that function primarily via blockade of dopamine D2 receptors. In the United States, quetiapine is currently approved for treating patients with schizophrenia, major depression and bipolar I disorder. Despite its widespread use, its cellular effects remain elusive. To address possible mechanisms, we chronically treated mice with quetiapine, haloperidol or vehicle and examined quetiapine-specific gene expression change in the frontal cortex. Through microarray analysis, we observed that several groups of genes were differentially expressed upon exposure to quetiapine compared with haloperidol or vehicle; among them, Cdkn1a, the gene encoding p21, exhibited the greatest fold change relative to haloperidol. The quetiapine-induced downregulation of p21/Cdkn1a was confirmed by real-time polymerase chain reaction and in situ hybridization. Consistent with single gene-level analyses, functional group analyses also indicated that gene sets associated with cell cycle/fate were differentially regulated in the quetiapine-treated group. In cortical cell cultures treated with quetiapine, p21/Cdkn1a was significantly downregulated in oligodendrocyte precursor cells and neurons, but not in astrocytes. We propose that cell cycle-associated intervention by quetiapine in the frontal cortex may underlie a unique efficacy of quetiapine compared with typical neuroleptics.