Oral administration of diphenylarsinic acid, a degradation product of chemical warfare agents, induces oxidative and nitrosative stress in cerebellar Purkinje cells.

A new clinical syndrome with prominent cerebellar symptoms in patients living in Kamisu City, Ibaraki Prefecture, Japan, is described. Since the patients ingested drinking water containing diphenylarsinic acid (DPA), a stable degradation product of both diphenylcyanoarsine and diphenylchloroarsine, which were developed for use as chemical weapons and cause severe vomiting and sneezing, DPA was suspected of being responsible for the clinical syndrome. The purpose of the present study was to elucidate prominent cerebellar symptoms due to DPA. The aim of the study was to determine if single (15 mg/kg) or continuous (5 mg/kg/day for 5 weeks) oral administration of DPA to ICR-strain mice induced oxidative and/or nitrosative stress in their brain. Significantly positive staining with malondialdehyde (MDA) and 3-nitrotyrosine (3-NT) was observed in the cerebellar Purkinje cells by repeated administration (5 mg/kg/day) with DPA for 5 weeks that led to the cerebellar symptoms from a behavioral pharmacology standpoint and by single administration of DPA (15 mg/kg). Furthermore, it is possible that the production of 3-NT was not caused by peroxynitrite formation. The present results suggest the possibility that arsenic-associated novel active species may be a factor underlying the oxidative and nitrosative stress in Purkinje cells due to exposure to DPA, and that the damage may lead to the cerebellar symptoms.

Specific induction of oxidative stress in terminal bronchiolar Clara cells during dimethylarsenic-induced lung tumor promoting process in mice.

The induction of oxidative stress in pulmonary cells during the process of lung tumor promotion by dimethylarsinic acid (DMA), a main metabolite of inorganic arsenics in mammals, was examined by immunohistochemical analysis using a specific antibody against 4-hydroxy-2-nonenal (4HNE) adducts, which are major aldehydic metabolites of lipid peroxidation. We demonstrated that 4HNE-modified proteins existed specifically in the secretory granules in terminal bronchiolar Clara cells. Furthermore, the degree of positive staining increased with the duration of DMA administration. Transmission electron microscopy revealed morphological changes in the Clara cells of DMA-treated mice. These results suggest that Clara cells are the major target cell for DMA-induced oxidative stress and that the cells may play an important role in the lung tumor promotion process in mice.

The role of trivalent dimethylated arsenic in dimethylarsinic acid-promoted skin and lung tumorigenesis in mice: tumor-promoting action through the induction of oxidative stress.

We investigated the relationship between lung- and skin-tumor promotion and oxidative stress caused by administration of dimethylarsinic acid (DMA(V)) in mice. The incidence of lung tumors induced by lung tumor initiator (4NQO) and DMA(V) were, as well as 8-oxo-2'-deoxyguanosine (8-oxodG), suppressed by cotreatment with (-)epigallocatechin gallate (EGCG). When mice were topically treated with trivalent dimethylated arsenic (DMA(III)), a further reductive metabolite of DMA(V), not only an increase in skin tumors but also an elevation of 8-oxodG in epidermis were observed. These results suggest that tumor promotion due to DMA(V) administration is mediated by DMA(III) through the induction of oxidative stress.

Evaluation of some halogen biocides using a microbial biofilm system.

A simple method for the formation of microbial biofilms of three species, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Klebsiella pneumoniae, on a small glass slide was established, and its suitability for evaluation of disinfectant efficacy was examined. The biofilms formed were observed in situ by confocal laser scanning microscopy (CLSM). Using the biofilms established, biocidal efficacy of several halogen biocides, such as hypochlorite (HOCl), bromochlorodimethylhydantoin (Br, Cl-DMH), ammonia monochloramine (NH2Cl), a stabilized hypobromite biocide named STABREX, and a mixed solution of NH4Br and HOCl, was evaluated. The formation of NHBrCl in the mixed solution was indicated by UV spectra analysis. Biofilm cells were more resistant to these biocides than planktonic cells and the extent of resistance varied with the biocide tested. Among the biocides tested, the biocidal potency of HOCl was the most susceptible to the change brought about by biofilm formation. By CLSM observation, differences in biofilm conformation were revealed between the microbial species. The efficacy of the biocide tested varied with the structure of biofilms formed. The assay method developed in the present study would be useful for further investigation on biofilm disinfection.

Immunohistochemical analysis of oxidative DNA damage in arsenic-related human skin samples from arsenic-contaminated area of China.

The appearance of 8-oxo-2'-deoxyguanosine (8-oxodG) was examined immunohistochemically using an 8-oxodG-monoclonal antibody in 28 cases of arsenic-related human skin tumors and in 20 cases of arsenic-unrelated human skin cancer to determine if the induction of oxidative stress participates in skin tumorigenesis caused by arsenics. The rate of 8-oxodG-positive was significantly higher in arsenic-related human skin cancer (28 of 28, 100%) than in arsenic-unrelated human skin cancer (3 of 20, 15%, P<0.01 by Chi2 test). Moreover, in all the arsenic-related skin samples, 8-oxodG was detected not only in tumor tissues but also in keratosis and normal tissues. These results suggest that the induction of oxidative stress may play an important role in arsenic carcinogenesis.

Oxidative DNA damage following exposure to dimethylarsinous iodide: the formation of cis-thymine glycol.

The purpose of the present study was to elucidate the genotoxic mechanism of trivalent dimethylated arsenic, particularly the induction mechanism of oxidative stress in nuclear bases. Cis-thymine glycol was used as a biomarker of DNA oxidation damage. The treatment of thymine with dimethylarsinous iodide (DMI), a model compound of dimethylarsinous acid, induced the formation of cis-thymine glycol. This oxidative damage was induced via the production of dimethylated arsenic peroxide, but not via the production of superoxide anion or hydrogen peroxide. Trivalent dimethylated arsenic may thus play an important role in arsenic carcinogenesis through the induction of oxidative base damage.

Development of a rabbit model of tear film instability and evaluation of viscosity of artificial tear preparations.

OBJECTIVE: The purposes of this study were to establish a quantitative method for evaluating rabbit tear film status and investigate the efficacy of artificial tear preparations through ocular surface bathing or eye drop application. METHODS: The rabbit tear film was evaluated using a noninvasive specular reflection video recording system. The appearance of a tear break area (TBA) on the tear film images (7.4 mm2/mm) after 30 seconds of eye opening was quantified by image analysis. To induce disruption of the rabbit tear film, the ocular surface was challenged for 60 minutes with 1 ppm hypochloric acid (HOCl). Immediately after irrigation, artificial tear preparations composed of viscosity agents sodium hyaluronate (SH), hydroxypropylmethycellulose (HPMC), hydroxyethylcellulose (HEC), or chondroitin sulfate (CS) were applied to the rabbit eye through ocular surface bathing or eye drop application, and the recovery of the disrupted tear film was compared for each preparation. RESULTS: A dramatic increase in TBA was observed immediately after the ocular surface was challenged with HOCl, and it returned to the initial level after 6 hours. Immediately after ocular surface bathing and eye drop application, a dramatic recovery of TBA was observed in all the test solution-treated eyes. One hour after treatments, prolonged amelioration of the tear film instability was observed after ocular surface bathing, but not by eye drop application, with the artificial tear preparations composed of HPMC or SH. CONCLUSION: Ocular surface bathing with artificial tear preparations composed of a suitable viscosity agents could be useful in managing tear film instability.

Active arsenic species produced by GSH-dependent reduction of dimethylarsinic acid cause micronuclei formation in peripheral reticulocytes of mice.

Dimethylarsine and trivalent dimethylated arsenic, metabolites of inorganic arsenics, have received considerable attention in current research because of their biological activities. We attempted to determine the appearance of micronucleated reticulocytes (MNRETs) in mouse peripheral blood following intraperitoneal administration of dimethylarsinous iodide (DMI) and trimethylarsine (TMA), model compounds of trivalent dimethylated arsenic and dimethylarsine, respectively. A significant increase in the number of MNRETs was observed with TMA, but not with DMI. Furthermore, MNRETs only appeared with 10.6 mg/kg of dimethylarsinic acid (DMA) following its co-injection with reduced glutathione (GSH). These results suggest that micronucleus formation may need further metabolic reduction of trivalent dimethylated arsenic, i.e. the production of dimethylarsine, by an excess amount of GSH. Meanwhile, the increase in MNRETs by administration of arsenite at 7.6 mg/kg, an equivalent dose to DMA as As, was remarkably diminished by co-administration with GSH. These results indicate that GSH plays an important role in the genotoxic process of arsenics, particularly by dimethylated arsenic.

In vivo genotoxicity evaluation of dimethylarsinic acid in MutaMouse.

Dimethylarsinic acid (DMA) induces DNA damage in the lung by formation of various peroxyl radical species. The present study was conducted to evaluate whether arsenite or its metabolite, DMA, could initiate carcinogenesis via mutagenic DNA lesions in vivo that can be attributed to oxidative damage. A transgenic mouse model, MutaMouse, was used in this study and mutations in the lacZ transgene and in the endogenous cII gene were assessed. When DMA was intraperitoneally injected into MutaMice at a dose of 10.6 mg/kg per day for 5 consecutive days, it caused only a weak increase in the mutant frequency (MF) of the lacZ gene in the lung, which was at most 1.3-fold higher than in the untreated control animals. DMA did not appreciably raise the MF in the bladder or bone marrow. Further analysis of the cII gene in the lung, the organ in which DMA induced the DNA damage, revealed only a marginal increase in the MF. Following DMA administration, no change in the cII mutation spectra was observed, except for a slight increase in the G:C to T:A transversion. Administration of arsenic trioxide (arsenite) at a dose of 7.6 mg/kg per day did not result in any increase in the MF of the lacZ gene in the lung, kidney, bone marrow, or bladder. Micronucleus formation was also evaluated in peripheral blood reticulocytes (RETs). The assay for micronuclei gave marginally positive results with arsenite, but not with DMA. These results suggest that the mutagenicity of DMA and arsenite might be too low to be detected in the MutaMouse.

Dimethylarsine likely acts as a mouse-pulmonary tumor initiator via the production of dimethylarsine radical and/or its peroxy radical.

AIMS: Recent animal experiments have indicated that dimethylarsinic acid (DMA), a main metabolite of inorganic arsenic, is a complete carcinogen in the lung of mice and the urinary bladder of rats, nevertheless, no ultimate-active metabolite from DMA has been identified thus far. We have proposed that dimethylarsine ((CH3)2AsH), an ultimate reductive metabolite of DMA, is excreted in the expired air of mice administered DMA, and furthermore, was easily converted into dimethylarsine radical ((CH3)2As•) and dimethylarsine peroxy radical ((CH3)2AsOO•) by its reaction with O2. The aim of the present study was to elucidate the possible mode of the tumorigenic action by dimethylated arsenic. MAIN METHODS: In vitro experiments using GSH reductase as a two-electron donor of dimethylarsenic-glutathione conjugate ((CH3)2As-SG) and DNA adduct assay via a photochemical approach were performed. A lung tumorigenicity assay of (CH3)2AsH suspended in argon-atmospheric olive oil for 5 days was also conducted in mice. KEY FINDINGS: The results indicated that (CH3)2AsH was easily produced enzymatically from (CH3)2As-SG and showed tumor-initiating action in mouse lung via the production of (CH3)2As• and (CH3)2AsOO• by its reaction with O2, and that these radicals have the ability to form DNA adducts. SIGNIFICANCE: The carcinogenicity of DMA, at least in mouse lung, could be explained based on the proposal that oral administration of DMA induces pulmonary tumors in mice, and arises from the arsine radicals produced through (CH3)2AsH, which was enzymatically reduced from (CH3)2As-SG.

Dimethylarsine likely acts as a mouse-pulmonary tumor initiator via the production of dimethylarsine radical and/or its peroxy radical.

AIMS: Recent animal experiments have indicated that dimethylarsinic acid (DMA), a main metabolite of inorganic arsenic, is a complete carcinogen in the lung of mice and the urinary bladder of rats, nevertheless, no ultimate-active metabolite from DMA has been identified thus far. We have proposed that dimethylarsine ((CH(3))(2)AsH), an ultimate reductive metabolite of DMA, is excreted in the expired air of mice administered DMA, and furthermore, was easily converted into dimethylarsine radical ((CH(3))(2)As*) and dimethylarsine peroxy radical ((CH(3))(2)AsOO*) by its reaction with O(2). The aim of the present study was to elucidate the possible mode of the tumorigenic action by dimethylated arsenic. MAIN METHODS: In vitro experiments using GSH reductase as a two-electron donor of dimethylarsenic-glutathione conjugate ((CH(3))(2)As-SG) and DNA adduct assay via a photochemical approach were performed. A lung tumorigenicity assay of (CH(3))(2)AsH suspended in argon-atmospheric olive oil for 5 days was also conducted in mice. KEY FINDINGS: The results indicated that (CH(3))(2)AsH was easily produced enzymatically from (CH(3))(2)As-SG and showed tumor-initiating action in mouse lung via the production of (CH(3))(2)As* and (CH(3))(2)AsOO* by its reaction with O(2), and that these radicals have the ability to form DNA adducts. SIGNIFICANCE: The carcinogenicity of DMA, at least in mouse lung, could be explained based on the proposal that oral administration of DMA induces pulmonary tumors in mice, and arises from the arsine radicals produced through (CH(3))(2)AsH, which was enzymatically reduced from (CH(3))(2)As-SG.

Effects of cyclosporine A in hyperzincaemia and hypercalprotectinaemia.

INTRODUCTION: Hyperzincaemia and hypercalprotectinaemia with systemic inflammation, recurrent infections, hepatosplenomegaly, arthritis, anemia, cutaneous inflammation, and failure to thrive is an extremely rare disease and no therapy is reported. AIM: To evaluated the effects of cyclosporine A in hyperzincaemia and hypercalprotectinaemia in terms of serum cytokine level changes before and after treatment. METHODS: A 10-year-old girl was admitted suffering from pyoderma gangrenosum, hepatosplenomegaly, anemia that was unresponsive to iron supplementation, persistent inflammation, arthritis, and increased serum zinc. The level of serum calprotectin was extremely high; therefore, we diagnosed hyperzincaemia and hypercalprotectinaemia and started cyclosporine A treatment. Twelve cytokines in serum were measured before and one year after treatment. RESULTS: Cyclosporine A was very effective. Her skin lesion and joint pain were alleviated and quality of life was markedly improved. C-reactive protein had decreased and anemia had improved. While zinc levels had fallen, calprotectin remained at an extremely high level. Of the cytokines examined, interleukin -6 serum levels had fallen and interleukin -8 showed a marked reduction after treatment. CONCLUSION: Cyclosporine A is effective for hyperzincaemia and hypercalprotectinaemia. Serum interleukin -8 may be useful in assessing the therapeutic effects of cyclosporine A in hyperzincaemia and hypercalprotectinaemia.

Deuterium-resistant algal cell line for D labeling of heterotrophs expresses enhanced level of Hsp60 in D2O medium.

Fully deuterated components from autotrophic cell lysate are useful materials for labeling of heterotrophs with deuterium. To facilitate the faster production of deuterated algal lysate, we selected a mutant Chlorella strain that grows faster in heavy water than the wild type. The mutant DR-17 was found to have a higher level of Hsp60 and an elevated level of protein synthesis. We previously isolated a deuterium-resistant yeast cell line that was also found to express elevated level of Hsp70 (K. Unno, T. Kishido, M. Morioka, S. Okada, and N. Oku, Biol. Pharm. Bull. 26:799-802, 2003). This suggests that the overexpression of heat shock proteins is required to compensate for the deuterium isotope effect.

Increased expression of Hsp70 for resistance to deuterium oxide in a yeast mutant cell line.

Labeling with stable isotopes, typically deuterium (D), is powerful tool for studying the functional structure of biomolecules by NMR. Biosynthesis of certain deuterated proteins in microorganisms cultured in deuterium oxide (D(2)O) is an attractive strategy. However, the growth of almost all microorganisms is inhibited at high concentrations of D(2)O. We isolated a mutant of yeast that grows well in D(2)O. The expression of Hsp70 was enhanced in the mutant. The increased expression also endowed the yeast with cold-resistance. The mutant might be useful for biosynthesis of D-labeled biomolecules.

Stress response caused by chronic alcohol intake in aged rat brain.

BACKGROUND: Chronic alcohol consumption may act as a cellular stressor for brain cells, as has been found for aging. In this study we examined one component of the cellular stress response (heat shock proteins) as a function of age and alcohol exposure. We have found that the level of constitutively expressed heat shock protein 70 (heat shock cognate 70, or Hsc70) increases in the aged rat brain. Among many heat shock proteins and molecular chaperones, Hsc70 might be important not only for the normal protein folding pathway but also for refolding of denatured proteins produced by mild and chronic stress. METHODS: Male Wistar rats that were 5.5 to 28.5 months old were fed a liquid diet that contained 5% (w/v) alcohol or a control diet for 6 weeks. The effects of alcohol consumption and aging on the expression of Hsc70 in the brain were investigated. The cytosol proteins in the 12,000 x g supernatant fraction were heat-treated at 42 degrees C for 1 hr. After the heat treatment, proteins that transferred from the soluble to insoluble aggregated fraction were estimated as heat-unstable proteins. RESULTS: In the 24- and 30-month-old rat brain, chronic consumption of alcohol increased levels of Hsc70 and heat-unstable proteins. On the other hand, those changes were not detected in the younger rat brain. CONCLUSION: Chronic alcohol intake causes a stress response in the aged rat brain. It is thought that the increased level of Hsc70 is brought about by an increase of denatured proteins.

The role of active arsenic species produced by metabolic reduction of dimethylarsinic acid in genotoxicity and tumorigenesis.

In recent research of arsenic carcinogenesis, many researchers have directed their attention to methylated metabolites of inorganic arsenics. Because of its high cytotoxicity and genotoxicity, trivalent dimethylated arsenic, which can be produced by the metabolic reduction of dimethylarsinic acid (DMA), has attracted considerable attention from the standpoint of arsenic carcinogenesis. In the present paper, we examined trivalent dimethylated arsenic and its further metabolites for their chemical properties and biological behavior such as genotoxicity and tumorigenicity. Our in vitro and in vivo experiments suggested that the formation of cis-thymine glycol in DNA was induced via the production of dimethylated arsenic peroxide by the reaction of trivalent dimethylated arsenic with molecular oxygen, but not via the production of common reactive oxygen species (ROS; superoxide, hydrogen peroxide, hydroxyl radical, etc.). Thus, dimethylated arsenic peroxide may be the main species responsible for the tumor promotion in skin tumorigenesis induced by exposure to DMA. Free radical species, such as dimethylarsenic radical [(CH(3))(2)As.] and dimethylarsenic peroxy radical [(CH(3))(2)AsOO.], that are produced by the reaction of molecular oxygen and dimethylarsine [(CH(3))(2)AsH], which is probably a further reductive metabolite of trivalent dimethylated arsenic, may be main agents for initiation in mouse lung tumorigenesis.