Sodium arsenite and dimethylarsenic acid induces apoptosis in OC3 oral cavity cancer cells.

Although arsenic is an environmental toxicant, arsenic trioxide (ATO) is used to treat acute promyelocytic leukemia (APL) with anticancer effects. Studies have demonstrated oral cancer is in the top 10 cancers in Taiwan. High rate of oral cancers is linked to various behaviors, such as excessive alcohol consumption and tobacco use. Similarly, betel chewing is a strong risk factor in oral cancer. In the present study, oral squamous carcinoma OC3 cells were investigated with the treatments of sodium arsenite (NaAsO2) and dimethylarsenic acid (DMA), respectively, to examine if arsenic compounds have anti­cancer efforts. It was found that 1 µM NaAsO2 and 1 mM DMA for 24 h induced rounded contours with membrane blebbing phenomena in OC3 cells, revealing cell apoptotic characteristics. In addition, NaAsO2 (10­100 µM) and DMA (1­100 mM) significantly decreased OC3 cell survival. In cell cycle regulation detected by flow cytometry, NaAsO2 and DMA significantly augmented percentage of subG1 and G2/M phases in OC3 cells, respectively. Annexin V/PI double staining assay was further used to confirm NaAsO2 and DMA did induce OC3 cell apoptosis. In mechanism investigation, western blotting assay was applied and the results showed that NaAsO2 and DMA significantly induced phosphorylation of JNK, ERK1/2 and p38 and then the cleavages of caspase­8, ­9, ­3 and poly ADP­ribose polymerase (PARP) in OC3 cells, dynamically. In conclusion, NaAsO2 and DMA activated MAPK pathways and then apoptotic pathways to induce OC3 oral cancer cell apoptosis.

[Effects of arsenic and its main metabolites on A549 cell apoptosis and the expression of pro-apoptotic genes Bad and Bik].

Objective: To investigate the effect of arsenic and its main metabolites on the apoptosis of human lung adenocarcinoma cell line A549 and the expression of pro-apoptotic genes Bad and Bik. Methods: In October 2020, A549 cells were recovered and cultured, and the cell viability was detected by the cell counting reagent CCK-8 to determine the concentration and time of sodium arsenite exposure to A549. The study was divided into NaAsO(2) exposure groups and metobol: le expoure groups: the metabolite comparison groups were subdivided into the control group, the monomethylarsinic acid exposure group (60 µmol/L) , and the dimethylarsinic acid exposure group (60 µmol/L) ; sodium arsenite dose groups were subdivided into 4 groups: control group (0) , 20, 40, 60 µmol/L sodium arsenite NaAsO(2). Hoechst 33342/propidium iodide double staining (Ho/PI) was used to observe cell apoptosis and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of Bad and Bik mRNA in cells after exposure. Western blotting was used to detect the protein expressions of Bad, P-Bad-S112, Bik, cleaved Bik and downstream proteins poly ADP-ribose polymerase PARP1 and cytochrome C (Cyt-C) , using spectrophotometry to detect the activity changes of caspase 3, 6, 8, 9. Results: Compared with the control group, the proportion of apoptotic cells in the 20, 40, and 60 µmol/L NaAsO(2) dose groups increased significantly (P<0.01) , and the expression levels of Bad, Bik mRNA, the protein expression levels of Bad, P-Bad-S112, Bik, cleaved Bik, PARP1, Cyt-C were increased (all P<0.05) , and the activities of Caspase 3, 6, 8, and 9 were significantly increased with significantly differences (P<0.05) . Compared with the control group, the expression level of Bad mRNA in the DMA exposure group (1.439±0.173) was increased with a significant difference (P=0.024) , but there was no significant difference in the expression level of Bik mRNA (P=0.788) . There was no significant differences in the expression levels of Bad and Bik mRNA in the poison groups (P=0.085, 0.063) . Compared with the control group, the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to MMA were 0.696±0.023, 0.707±0.014, 0.907±0.031, 1.032±0.016, and there was no significant difference between the two groups (P=0.469, 0.669, 0.859, 0.771) ; the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to DMA were 0.698±0.030, 0.705±0.022, 0.908±0.015, 1.029±0.010, and there was no difference between the two groups (P=0.479, 0.636, 0.803, 0.984) . Conclusion: Sodium arsenite induces the overexpression of Bad and Bik proteins, initiates the negative feedback regulation of phosphorylated Bad and the degradation of Bik, activates the downstream proteins PARP1, Cyt-C and Caspase pathways, and mediates the apoptosis of A549 cells.

Ionomic responses of rice plants to the stresses of different arsenic species in hydroponics.

Different ionomic profiles of plants are associated with different external stresses to which they are exposed. Investigation of ionomic variation is necessary for understanding the migration and detoxification of toxic elements in plants. In the current study, rice plants were treated with arsenite, arsenate, monomethylarsonic acid and dimethylarsinic acid in hydroponics. The ionomic responses of the rice plants to different arsenic (As) species stresses were measured and analyzed. The multielement approach is more sensitive at detecting significant variations from external environmental stresses than the consideration of several individual elements. The pairs of significant correlations between elements varied based on the rice tissues and As species used in treatment, resulting in specific correlation networks. However, some pairs of correlations existed regardless of As species treatment used in this study. Positive correlations between P and Fe were observed in rice roots treated with any of the As species, implying that P and Fe share similar biological processes. The heatmap from hierarchical cluster analysis (HCA) agreed with the principal component analysis (PCA) results in ionomic differentiation between roots and shoots. Furthermore, ionomic differences between rice plants treated with different As species were identified through PCA. This study revealed that the ionomic profiles in rice plants are sufficient to detect responses to environmental perturbations. Association studies between ionomics and genomics are necessary to further understand the potential mechanisms that promote uptake or exclusion of elements in plants.

Dimethylarsinic acid modulates the aryl hydrocarbon receptor-regulated genes in C57BL/6 mice: in vivo study.

1. Dimethylarsinic acid (DMA(V)) is the major metabolite of inorganic arsenic in human body. Thus we investigated the effect of DMA(V) on the alteration of phase I (typified by Cyp1a) and phase II (typified by Nqo1) AhR-regulated genes in vivo. C57BL/6 mice received DMA(V) (13.3 mg/kg, i.p.) with or without TCDD (15 µg/kg, i.p.), thereafter the liver, lung, and kidney were harvested at 6 and 24 h post-treatment. 2. Results demonstrated that DMA(V) has no significant effect on Cyp1a mRNA and protein expression or catalytic activity in the liver. On the other hand, DMA(V) significantly potentiated the TCDD-mediated induction of Cyp1a mRNA and protein expression, with a subsequent potentiation of catalytic activity in the lung. Moreover, DMA(V) significantly inhibited the TCDD-mediated induction of Cyp1a mRNA and protein expression with subsequent inhibition of catalytic activity in the kidney. 3. Regarding to phase II AhR-regulated genes, DMA(V) has no significant effect on Nqo1 mRNA and protein expression, or activity neither in the liver, lung, or kidney. 4. In conclusion, the present work demonstrates for the first time that DMA(V) modulates AhR-regulated genes in a tissue- and enzyme-specific manner. This modulation may play a crucial role in arsenic-induced toxicity and carcinogenicity.

Dimethylarsenate (DMA) exposure influences germination rates, arsenic uptake and arsenic species formation in wheat.

The contamination of cereals with arsenic (As) is a global health and agronomic concern. This study compared the physiological response, As uptake and As speciation in the grains and above ground tissues of 20 wheat cultivars exposed to 5 mg As kg-1 soil as either arsenate (AsV) or dimethylarsenate (DMA) under glasshouse conditions. Germination rates for the majority of cultivars exceeded 80% for the majority of cultivars when exposed to AsV, but fell significantly to 20-40% when exposed to DMA. For a number of cultivars, grain yields were 20-50% lower when plants were exposed to DMA compared to AsV. Grain As concentrations were between 0.6 and 1.6 µg As g-1 grain across the twenty cultivars when exposed to AsV, whereas grain As concentrations were much higher (2.2-4.6 µg As g-1 grain) when exposed to DMA. When plants were exposed to AsV, 100% of the As present in the grain was found as inorganic As while in plants exposed to DMA, 70-90% of As was present as DMA with the remainder found as inorganic As. DMA is believed to be incorporated by plants via silica (Si) acid channels and assessment of grain Si concentrations demonstrated that up to 40% less Si was accumulated in grains when plants were exposed to DMA. The decreased germination rates and grain yields in the presence of DMA is similar to the symptoms described for straight head disease in rice, which has been linked to DMA exposure. The results presented here indicate some analogous processes occur in wheat to those described in rice. We hypothesise that exposure to DMA may have inhibited Si-metabolism and translocation which resulted in both developmental impairment and possibly an increased susceptibility to soil pathogens.

DMA(V) in Drinking Water Activated NF-κB Signal Pathway and Increased TGF-ß and IL-1ß Expressions in Bladder Epithelial Cells of Rats.

Dimethylarsinic acid (DMA(V)) is the main product of arsenic methylation metabolism in vivo and is rat bladder carcinogen and tumor promoting agent. In this study, we measured the expressions of mRNA and proteins of NF-κB pathway members, IKKα, IKKß, p65, and p50 in rat bladder epithelium by qRT-PCR and immunohistochemical analysis after rats received drinking water containing 100 and 200 ppm DMA(V) for 10 weeks. Transforming growth factor-ß (TGF-ß) immunoexpression in rat bladder epithelium and urine level of IL-1ß also were determined. We found that DMA(V) dramatically increased the mRNA levels of NF-κB p50 and IKKα in the bladder epithelium of rats compared to the control group. Immunohistochemical examinations showed that DMA(V) increased immunoreactivities of IKKα, IKKß, and phospho-NF-κB p50 in the cytoplasm and phospho-NF-κB p50 and p65 in nucleus of rat urothelial cells. In addition, DMA(V) treated rats exhibited significantly increased inflammatory factor TGF-ß immunoreactivity in bladder epithelium and IL-1ß secretion in urine. These data suggest that DMA(V) could activate NF-κB signal pathway and increase TGF-ß and IL-1ß expressions in bladder epithelial cells of rats.

Methylated arsenic metabolites bind to PML protein but do not induce cellular differentiation and PML-RARα protein degradation.

Arsenic trioxide (As2O3) is one of the most effective therapeutic agents used for patients with acute promyelocytic leukemia (APL). The probable explanation for As2O3-induced cell differentiation is the direct targeting of PML-RARα oncoprotein by As2O3, which results in initiation of PML-RARα degradation. However, after injection, As2O3 is rapidly methylated in body to different intermediate metabolites such as trivalent monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)), therefore, it remains unknown that which arsenic specie is actually responsible for the therapeutic effects against APL. Here we have shown the role of As2O3 (as iAs(III)) and its intermediate metabolites (i.e., MMA(III)/DMA(III)) in NB4 cells. Inorganic iAs(III) predominantly showed induction of cell differentiation, while MMA(III) and DMA(III) specifically showed to induce mitochondria and endoplasmic reticulum-mediated apoptosis, respectively. On the other hand, in contrast to iAs(III), MMA(III) showed stronger binding affinity for ring domain of PML recombinant protein, however, could not induce PML protein SUMOylation and ubiquitin/proteasome degradation. In summary, our results suggest that the binding of arsenicals to the ring domain of PML proteins is not associated with the degradation of PML-RARα fusion protein. Moreover, methylated arsenicals can efficiently lead to cellular apoptosis, however, they are incapable of inducing NB4 cell differentiation.

Autocuidado do cuidador familiar de adultos ou idosos dependentes após a alta hospitalar / Self-care in family caregiver of dependent adults or elderly persons after hospital discharge / El autocuidado del cuidador familiar de adultos o ancianos dependientes tras el alta hospitalaria

Este artigo apresenta uma compreensão sobre o autocuidado do cuidador familiar segundo a teoria de Dorothea Orem. Resulta de uma pesquisa qualitativa com aporte da Teoria Fundamentada nos Dados, utilizando-se das técnicas de visita domiciliar, registro de notas de campo e entrevista semiestruturada com 11 cuidadores, após a internação de familiar em um Hospital Universitário de Minas Gerais. Foram obtidas quatro categorias, destacando uma categoria central, em torno da qual se analisaram as facilidades, dificuldades e estratégias para o autocuidado do cuidador. Entre as dificuldades, foram evidenciadas: tempo insuficiente para os cuidados com a saúde e, entre as facilidades, o apoio de outros familiares. As principais estratégias foram: apoio na fé; revezamento nos cuidados e recursos na comunidade. Concluiu-se que orientações no momento da alta e o acompanhamento de enfermagem após a alta contribuem para o autocuidado do cuidador, atuando sobre suas dificuldades e estimulando suas potencialidades.

This article presents an understanding concerning self-care in family caregivers according to Dorothea Orem's theory. Resulting from a qualitative research based on Grounded Theory, this work uses the techniques of home visiting, field notes and semistructured interviews with 11 caregivers after the hospitalization of a family member in a teaching Hospital located in Minas Gerais. Four categories were found and among them a central category is highlighted from which some facilities, difficulties and strategies for selfcare in caregiver were analyzed. Considering the difficulties, insufficient time for healthcare was noticed whereas the support from other family members appeared as a facility. The main strategies were: faith as a support; shift work in healthcare and community resources. This study demonstrated that hospital discharge guidelines and nursing follow-up after discharge were responsible for positive contributions to self-care in caregivers helping them to overcome their difficulties and enhancing their potentialities.

El artículo presenta una comprensión sobre el autocuidado del cuidador familiar, según la teoría de Dorothea Orem. Resulta de investigación cualitativa con aporte de la Teoría Fundamentada en los Datos, utilizándo se de las técnicas de visitas a domicilio; registro de apuntes de campo y entrevista semiestructurada, tras la internación de un familiar en un hospital universitario de Minas Gerais. Se llegó a cuatro categorías, señalando una categoría central, alrededor de la cual se analizaron las facilidades, dificultades y estrategias para el autocuidado del cuidador. Se evidenció, entre las dificultades, tiempo insuficiente para los cuidados con la salud y, entre las facilidades, el apoyo de otros familiares. Las principales estrategias fueron: apoyo en la fe, revezo en los cuidados y recursos en la comunidad. Se concluyó que orientaciones el momento del alta y el acompañamiento de enfermería tras el alta contribuyen para el autocuidado del cuidador, actuando sobre sus dificultades y estimulando sus potencialidades.

Trivalent methylated arsenic metabolites induce apoptosis in human myeloid leukemic HL-60 cells through generation of reactive oxygen species.

Arsenic trioxide (As2O3) has remarkable therapeutic efficacy against leukemia. However, after As2O3 biotransformation, the role of arsenic metabolites in the clinical efficacy against leukemia still needs to be elucidated. Therefore, to explore the contribution of trivalent methylated arsenicals in the therapeutic effects, we investigated and compared the effects of arsenite (iAs(III)), monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)) on HL-60 cells. Methylated arsenic species MMA(III) and DMA(III) showed potentially reduced cell survival with IC50 values of 3 and 2 µM, respectively. We found that methylated metabolites caused apoptosis through oxidative stress and loss of mitochondrial membrane potential. Furthermore, we found that the caspase-9 and -3 were markedly activated by exposure to methylated metabolites, with cleavage of poly-ADP ribose polymerase (PARP). Conversely, cellular apoptosis, generation of ROS, activation of caspase-3, -9 as well as PARP cleavage were significantly attenuated by pretreatment with an antioxidant, N-acetylcysteine (NAC). DNA damage was also markedly observed in HL-60 cells exposed to either MMA(III) or DMA(III), while iAs(III) did not show any relevant effects in HL-60 cells. Likewise, phosphorylation of the histone H2A variant (γ-H2AX), a biomarker of DNA damage, significantly occurred in cellular nuclei following exposure to two methylated species, which was reduced in the presence of NAC, suggesting that the induction of DNA damage was predominantly caused by the two metabolites via oxidative stress. In conclusion, we suggest that arsenic intermediate metabolites; MMA(III) and DMA(III) might prove to be of clinical relevance in future as such approaches may help in the treatment of leukemia and other types of cancers.

Release of apoptotic cytochrome C from mitochondria by dimethylarsinous acid occurs through interaction with voltage-dependent anion channel in vitro.

Arsenic is known to be a human carcinogen as well as one of the most effective drugs for treatment of patients with acute promyelocytic leukemia. The intermediate metabolites of arsenic, monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)), are formed by methylation reactions, and they are more reactive and toxic than the inorganic precursor arsenite (iAs(III)); however, the detailed mechanism of toxicity is poorly understood. Here, we studied the effects of three arsenic compounds (i.e., iAs(III), MMA(III), and DMA(III)) on mitochondrial permeability transition pore (mPTP) and release of apoptotic cytochrome c (Cyt c) after incubating with rat liver mitochondria. Inorganic iAs(III) had no effect on mitochondrial swelling even at higher concentrations ranging up to 100 µM, but swelling was significantly induced in the presence of Ca(2+). Additionally, mitochondrial swelling was strongly induced by exposure to the methylated forms of MMA(III) and DMA(III) in a dose-dependent manner in the absence of Ca(2+), suggesting that the methylated forms may have potent effects on cellular mitochondria. Although mitochondrial swelling was completely inhibited in the presence of cyclosporin-A (an inhibitor of mitochondrial permeability transition) or ruthenium red (an inhibitor of Ca(2+) uniporter) following exposure to methylated arsenicals, the release of apoptotic Cyt c from mitochondria was not inhibited, indicating that release of Cyt c is probably not dependent on mPTP opening. In addition, inhibitors of Bax (e.g., Bax-inhibiting peptide) did not reduce the release of Cyt c from the mitochondria by formation of Bax-voltage-dependent anion channel (VDAC) complex, whereas the recombinant Bcl-x(L )proteins significantly reduced the release of Cyt c after exposure to DMA(III), suggesting that dimethylated DMA(III) directly interacted with the VDAC in mitochondria and caused the release of Cyt c from mitochondria.

The rice aquaporin Lsi1 mediates uptake of methylated arsenic species.

Pentavalent methylated arsenic (As) species such as monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)] are used as herbicides or pesticides, and can also be synthesized by soil microorganisms or algae through As methylation. The mechanism of MMA(V) and DMA(V) uptake remains unknown. Recent studies have shown that arsenite is taken up by rice (Oryza sativa) roots through two silicon transporters, Lsi1 (the aquaporin NIP2;1) and Lsi2 (an efflux carrier). Here we investigated whether these two transporters also mediate the uptake of MMA(V) and DMA(V). MMA(V) was partly reduced to trivalent MMA(III) in rice roots, but only MMA(V) was translocated to shoots. DMA(V) was stable in plants. The rice lsi1 mutant lost about 80% and 50% of the uptake capacity for MMA(V) and DMA(V), respectively, compared with the wild-type rice, whereas Lsi2 mutation had little effect. The short-term uptake kinetics of MMA(V) can be described by a Michaelis-Menten plus linear model, with the wild type having 3.5-fold higher V(max) than the lsi1 mutant. The uptake kinetics of DMA(V) were linear with the slope being 2.8-fold higher in the wild type than the lsi1 mutant. Heterologous expression of Lsi1 in Xenopus laevis oocytes significantly increased the uptake of MMA(V) but not DMA(V), possibly because of a very limited uptake of the latter. Uptake of MMA(V) and DMA(V) by wild-type rice was increased as the pH of the medium decreased, consistent with an increasing proportion of the undissociated species. The results demonstrate that Lsi1 mediates the uptake of undissociated methylated As in rice roots.

Structure of SRP14 from the Schizosaccharomyces pombe signal recognition particle.

The signal recognition particle (SRP) Alu domain has been implicated in translation elongation arrest in yeasts and mammals. Fission yeast SRP RNA is similar to that of mammals, but has a minimal Alu-domain RNA lacking two stem-loops. The mammalian Alu-domain proteins SRP9 and SRP14 bind their cognate Alu RNA as a heterodimer. However, in yeasts, notably Saccharomyces cerevisiae, SRP14 is thought to bind Alu RNA as a homodimer, the SRP9 protein being replaced by SRP21, the function of which is not yet clear. Structural characterization of the Schizosaccharomyces pombe Alu domain may thus help to identify the critical features required for elongation arrest. Here, the crystal structure of the SRP14 subunit of S. pombe SRP (SpSRP14) which crystallizes as a homodimer, is presented. Comparison of the SpSRP14 homodimer with the known structure of human SRP9/14 in complex with Alu RNA suggests that many of the protein-RNA contacts centred on the conserved U-turn motif are likely to be conserved in fission yeast. Initial attempts to solve the structure using traditional selenomethionine SAD labelling failed. However, two As atoms originating from the cacodylate buffer were found to make cysteine adducts and strongly contributed to the anomalous substructure. These adducts were highly radiation-sensitive and this property was exploited using the RIP (radiation-damage-induced phasing) method. The combination of SAD and RIP phases yielded an interpretable electron-density map. This example will be of general interest to crystallographers attempting de novo phasing from crystals grown in cacodylate buffer.

Comparison of sensitivity to arsenic compounds between a Bhas 42 cell transformation assay and a BALB/c 3T3 cell transformation assay.

A short-term cell transformation assay has recently been developed, using Bhas 42 cells which were established from BALB/c 3T3 cells transfected by v-Ha-ras gene and postulated to be initiated in the two-stage carcinogenesis theory. The Bhas 42 cell transformation assay has been reported to be capable of detecting initiating and promoting activities of chemical carcinogens, according to the different protocols, initiation assay and promotion assay, respectively. The assay is superior to classical transformation assays in cost and labor performance. The present study was carried out to compare its sensitivity with that of a classical BALB/c 3T3 cell system. We performed the Bhas 42 cell transformation assay with inorganic arsenic compounds which are potent environmental carcinogens in human but not mutagens in bacteria or weak mutagens in mammalian cells in vitro. Sodium arsenite, disodium arsenate, and their metabolites, monomethylarsonic acid and dimethylarsinic acid (DMAA) were included in the study. Sodium arsenite was positive in the initiation assay and all compounds except for DMAA were positive in the promotion assay. These results were compared with reported data in a two-stage BALB/c 3T3 cell transformation assay. The sensitivity of Bhas 42 cell transformation assay was found to be similar to that of the conventional BALB/c 3T3 cell transformation assay for the detection of initiating activities of arsenic compounds. For the detection of promoting activities, its sensitivity was equivalent to that of the two-stage BALB/c 3T3 cell transformation assay where the target cells were initiated with sub-threshold dose of 3-methylcholanthrene, confirming that Bhas 42 cells behave as initiated cells in the transformation assay.

Arsenite and its mono- and dimethylated trivalent metabolites enhance the formation of benzo[a]pyrene diol epoxide-DNA adducts in Xeroderma pigmentosum complementation group A cells.

Recently, inorganic arsenite (iAs(III)) and its mono- and dimethylated metabolites have been examined for their interference with the formation and repair of benzo[a]pyrene diol epoxide (BPDE)-induced DNA adducts in human cells (Schwerdtle, ., Walter, I., and Hartwig, A. (2003) DNA Repair 2, 1449 - 1463). iAs(III) and monomethylarsonous acid (MMA(III)) were found to be able to enhance the formation of BPDE-DNA adducts, whereas dimethylarsinous acid (DMA(III)) had no enhancing effect at all. The anomaly manifested by DMA(III) prompted us to further investigate the effects of the three trivalent arsenic species on the formation of BPDE-DNA adducts. Use of a nucleotide excision repair (NER)-deficient Xeroderma pigmentosum complementation group A cell line (GM04312C) allowed us to dissect DNA damage induction from DNA repair and to examine the effects of arsenic on the formation of BPDE-DNA adducts only. At concentrations comparable to those used in the study by Schwerdtle et al., we found that each of the three trivalent arsenic species was able to enhance the formation of BPDE-DNA adducts with the potency in a descending order of MMA(III) > DMA(III) > iAs(III), which correlates well with their cytotoxicities. Similar to iAs(III), DMA(III) modulation of reduced glutathione (GSH) or total glutathione S-transferase (GST) activity could not account for its enhancing effect on DNA adduct formation. Additionally, the enhancing effects elicited by the trivalent arsenic species were demonstrated to be highly time-dependent. Thus, although our study made use of short-term assays with relatively high doses, our data may have meaningful implications for carcinogenesis induced by chronic exposure to arsenic at low doses encountered environmentally.

Difference of toxicity and accumulation of methylated and inorganic arsenic in arsenic-hyperaccumulating and -hypertolerant plants.

The arsenic (As) hyperaccumulators, Pteris vittata and Pteris cretica and an As-tolerant plant Boehmeria nivea, were selected to compare the toxicity, uptake, and transportation of inorganic arsenate (As(V)) and its methylated counterpart dimethylarsinic acid (DMA). The XANES method was used to elucidate the effect of As species transformation on As toxicity and accumulation characteristics. Significantly higher toxicity and lower accumulation of DMAthan inorganic As(V) was shown in the As hyperaccumulators and the As-tolerant plant. Reduction of As(V) was commonly found in the plants. Arsenic complexation with thiols, which have less mobility in plants and usually occur in As-tolerant plants, was also found in rhizoids of P. cretica. Plants with greater ability to form As-thiolate have lower ability for upward transport of As. Demethylation of DMA occurred in the three plants. The DMA component decreased from the rhizoids to the fronds in both hyperaccumulators, while this tendency is reverse in B. nivea.

Inositol hexakisphosphate kinase products contain diphosphate and triphosphate groups.

Eukaryotic cells produce a family of diverse inositol polyphosphates (IPs) containing pyrophosphate bonds. Inositol pyrophosphates have been linked to a wide range of cellular functions, and there is growing evidence that they act as second messengers. Inositol hexakisphosphate kinase (IP6K) is able to convert the natural substrates inositol pentakisphosphate (IP 5) and inositol hexakisphosphate (IP 6) to several products with an increasing number of phospho-anhydride bonds. In this study, we structurally analyzed IPs synthesized by three mammalian isoforms of IP6K from IP 5 and IP 6. The NMR and mass analyses showed a number of products with diverse, yet specific, stereochemistry, defined by the architecture of IP6K's active site. We now report that IP6K synthesizes both pyrophosphate (diphospho) as well as triphospho groups on the inositol ring. All three IP6K isoforms share the same activities both in vitro and in vivo.

Down-regulation of glutaminase C in human hepatocarcinoma cell by diphenylarsinic acid, a degradation product of chemical warfare agents.

In a poisonous incident in Kamisu, Japan, it is understood that diphenylarsinic acid (DPAA) was a critical contaminant of ground water. Most patients showed dysfunction of the central nervous system. To understand the overall mechanism of DPAA toxicity and to gain some insight into the application of a remedy specific for intoxication, the molecular target must be clarified. As an approach, a high throughput analysis of cell proteins in cultured human hepatocarcinoma HpG2 exposed to DPAA was performed by two-dimensional electrophoresis (2-DE). Four proteins, which were up- and down-regulated by exposure of cultured HepG2 cells to DPAA, were identified. They were chaperonin containing TCP-1 (CCT) beta subunit, aldehyde dehydrogenase 1 (ALDH1), ribosomal protein P0 and glutaminase C (GAC). Of these, GAC was the only protein that was down-regulated by DPAA exposure, and cellular expression levels were reduced by DPAA in a concentration- and time-dependent manner. Decrease in cellular GAC levels was accompanied by decreased activity of the enzyme, phosphate-activated glutaminase (PAG). Decreased expression of GAC by DPAA was also observed in human cervical carcinoma HeLa and neuroblastoma SH-SY5Y cells. By contrast, no significant changes in GAC protein expression were observed when cells were incubated with arsenite [iAs (III)] and trivalent dimethylarsinous acid [DMA (III)]. In the central nervous system, GAC plays a role in the production of the neurotransmitter glutamic acid. Selective inhibition of GAC expression by DPAA may be a cause of dysfunction of glutamatergic neuronal transmission and the resultant neurological impairments.

Role of the alkali labile sites, reactive oxygen species and antioxidants in DNA damage induced by methylated trivalent metabolites of inorganic arsenic.

In the last decade arsenic metabolism has become an important matter of discussion. Methylation of inorganic arsenic (iAs) to monomethylarsonic acid (MMA(V)) and dimethylarsinic acid (DMA(V)) is considered to decrease arsenic toxicity. However, in addition to these pentavalent metabolites, the trivalent metabolites monomethylarsonous (MMA(III)) and dimethylarsinous acid (DMA(III)) have been identified recently as intermediates in the metabolic pathway of arsenic in cultured human cells. To examine the role of oxidative damage in the generation of DNA strand breaks by methylated trivalent arsenic metabolites, we treated human lymphocytes with both metabolites at non-cytotoxic concentrations. We further tested whether these effects are sensitive to modulation by the antioxidants ascorbate (Vitamin C) and selenomethionine (Se-Met). Both trivalent metabolites produced oxidative stress related DNA damage, consisting of single strand breaks and alkali-labile sites, with MMA(III) being more potent at low concentrations than DMA(III). Neither MMA(III) nor DMA(III) induced DNA-double strand breaks. The oxidative stress response profiles of the metabolites were parallel as determined by lipid peroxidation induction. MMA(III) induced peroxidation from the lowest concentration tested, while effects of DMA(III) were apparent only at concentrations above 10 muM. The antioxidant Se-Met exhibited a more pronounced inhibition of trivalent arsenic metabolite-induced oxidative-DNA damage than did vitamin C. The present findings suggest that DNA damage by methylated trivalent metabolites at non-cytotoxic concentrations may be mediated by a mix of reactive oxygen and nitrogen oxidized species.

DNA-protein cross-links and sister chromatid exchanges induced by dimethylarsinic acid in human fibroblasts cells.

Biotransformation of inorganic arsenic to form both methylarsinic acid (MA) and dimethylarsinic acid (DMA) has traditionally been considered as a mechanism to facilitate the detoxification and excretion of arsenic. However, the methylation of inorganic arsenic as a detoxification mechanism has been questioned due to recent studies revealing an important role of organic arsenic in the induction of genetic damage. In a previous report a reduction of DNA migration after treatment of cells with DMA was described. In order to further evaluate the possible induction of protein-DNA adducts, an experiment was performed taking into account other parameters and modifications of the standard alkaline comet assay. In addition, the results obtained with the comet assay were compared with those obtained by analyzing the induction of sister chromatid exchanges (SCEs). SCE frequencies were significantly increased in treated cells in relation to controls (p<0.001). Furthermore, in the standard alkaline comet assay, as well as in the control assay for proteinase K treatment, a significant dose-dependent reduction in tail moment was observed. Nevertheless, the post-treatment with proteinase K induced the release of proteins joined to the DNA and consequently, a dose-dependent increment in DNA migration was observed (p<0.001). These results suggest that DNA-protein cross-links may be an important genotoxic effect induced by dimethylarsinic acid in human MRC-5 cells.

An integrated pharmacokinetic and pharmacodynamic study of arsenite action 2. Heme oxygenase induction in mice.

Heme oxygenase (HO) is the rate-limiting enzyme in heme degradation and its activity has a significant impact on intracellular heme pools. Rat studies indicate that HO induction is a sensitive, dose-dependent response to arsenite (As(III)) exposure in both liver and kidney. The objective of this study was to evaluate the relationship of HO induction to administered As(III) dose, and concentrations of inorganic arsenic (iAs) in tissues and urine. Levels of iAs, mono- (MMA) and dimethylated arsenic (DMA) as well as HO activity were determined in liver, lung and kidney over time in female B6C3F1 mice given a single oral dose of 0, 1, 10, 30 or 100 micromol/kg As(III). Increased HO activity was a time and dose-dependent response in liver and kidney, but not in lung. Activity peaked in the 4-6 h time range in liver and kidney with the responsiveness in liver being approximately 2- to 3-fold greater than kidney. The lowest observed effect levels (LOELs) in this study for HO induction are 30 and 100 micromol/kg, respectively, in liver and kidney. The predominant form of arsenic (As) was iAs in liver at all doses, whereas DMA was the predominant form of As in kidney at all doses. Three- to four-fold higher levels of iAs were achieved in liver compared to kidney. MMA was the least abundant form of As in liver and kidney, never exceeding more than 20% of the total As present. The concentration of iAs in tissue or urine demonstrated the strongest correlation with HO activity in both liver and kidney. Results of this study suggest that HO induction is a biomarker of effect that is specific for tissue iAs because a high, but nontoxic, acute dose of DMA (5220 micromol/kg) did not induce HO in mice. Thus, HO induction has potential for use as a biomarker of effect for inorganic arsenic exposure and may be used as an indicator response to further the development of a biologically-based dose response model for As.