Seishiro Hirano

Metabolism of arsenic and its toxicological relevance

Arsenic is a worldwide environmental pollutant and a human carcinogen. It is well recognized that the toxicity of arsenicals largely depends on the oxidoreduction states (trivalent or pentavalent) and methylation levels (monomethyl, dimethyl, and trimethyl) that are present during the process of metabolism in mammals. However, presently, the specifics of the metabolic pathway of inorganic arsenicals have yet to be confirmed. In mammals, there are two possible mechanisms that have been proposed for the metabolic pathway of inorganic arsenicals, oxidative methylation, and glutathione conjugation. Oxidative methylation, which was originally proposed in fungi, is based on findings that arsenite (iAsIII) is sequentially converted to monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV) in both humans and in laboratory animals such as mice and rats. However, recent in vitro observations have demonstrated that arsenic is only methylated in the presence of glutathione (GSH) or other thiol compounds, which strongly suggests that arsenic is methylated in trivalent forms. The glutathione conjugation mechanism is supported by findings that have shown that most intracellular arsenicals are trivalent and excreted from cells as GSH conjugates. Since non-conjugated trivalent arsenicals are highly reactive with thiol compounds and are easily converted to less toxic corresponding pentavalent arsenicals, the arsenic–glutathione conjugate stability may be the most important factor for determining the toxicity of arsenicals. In addition, “being a non-anionic form” also appears to be a determinant of the toxicity of oxo-arsenicals or thioarsenicals. The present review discusses both the metabolism of arsenic and the toxicity of arsenic metabolites.

Difference in the toxicity mechanism between ion and nanoparticle forms of silver in the mouse lung and in macrophages.

The health effects of silver nanoparticles (AgNPs) have not been well investigated, despite AgNPs now being widely used in consumer products. We investigated the metabolic behavior and toxicity of AgNPs in comparison to silver nitrate (AgNO3) both in vivo and in vitro. AgNPs (20 nm diameter) suspended in 1% albumin solution or AgNO3 solution was injected into the mouse lung. Less than 1% of the initial dose of AgNPs and more than 7% of the initial dose of AgNO3 was recovered in the liver 4h after administration, suggesting that the ionic form of silver was absorbed by the lung tissue and entered the systemic circulation more efficiently than AgNPs. The pro-inflammatory cytokine, IL-1β, and neutrophils in bronchoalveolar lavage fluid (BALF) increased following intratracheal instillation of AgNPs or AgNO3. AgNO3 recruited more neutrophils in the alveolar space than did AgNPs. In the in vitro study, AgNO3 was more cytotoxic than 20, 60, or 100 nm diameter AgNPs in a mouse macrophage cell line (J774.1). To investigate the intracellular distribution of Ag in detail, J774.1 cells were exposed to AgNO3 or 20 nm AgNPs and the distribution of Ag to cytosolic proteins was investigated using HPLC-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS). Ag was mainly distributed to metallothioneins (MT) and to high molecular weight proteins in AgNO3- and AgNPs-exposed cells, respectively. Confocal laser microscopic examination of LysoTracker(®)-labeled cells indicated that AgNPs were colocalized with lysosomes in J774.1 cells. These results suggest that AgNPs were transported to lysosomes and only gradually dissolved in the macrophages, causing milder inflammatory stimulation in the mouse lung compared to AgNO3.

Identification of arsenite-and arsenic diglutathione-binding proteins in human hepatocarcinoma cells

It is generally accepted that trivalent arsenicals are more toxic than the corresponding pentavalent arsenicals, since trivalent arsenicals bind the thiol groups of biomolecules, leading to a deterioration in cellular functions. In the present study, we prepared three different arsenic-bound sepharoses and investigated the binding of hepatic cytosolic proteins to pentavalent, trivalent, and glutathione-conjugated trivalent arsenicals. SDS-PAGE showed no proteins bound to pentavalent arsenic specifically. In contrast, we found a number of proteins that have specific and high affinity for trivalent arsenic. Two of those proteins were identified: protein disulfide isomerase-related protein 5 (PDSIRP5) and peroxiredoxin 1/enhancer protein (PRX1/EP). These proteins have vicinal cysteines, as previously reported. In contrast, one of the prominent proteins that did not bind to trivalent arsenic was identified as calreticulin precursor. Although there are 3 cysteines in calreticulin precursor, two of the cysteines are spaced more than 25 amino acids apart. Five synthetic peptides containing 2 vicinal cysteines were prepared to study whether they would inhibit the binding of PDSIRP5, PRX1/EP, and other arsenic-binding proteins to trivalent arsenicals. Only two of the five peptides effectively inhibited binding, suggesting that other amino acids besides the 2 vicinal cysteines may modulate the affinity of cysteine-rich proteins for trivalent arsenicals. We further investigated hepatic cytosolic proteins that bound specifically to glutathione-conjugated trivalent arsenic, which is the most abundant form of arsenical in bile fluid. Four proteins that bound specifically to glutathione-conjugated trivalent arsenic were identified; interestingly, these proteins were different from the trivalent arsenic-binding proteins. These results suggest that although glutathione-conjugation is an important process in the metabolism, excretion, and detoxification of arsenicals, glutathione-conjugated arsenicals can still react with some proteins in hepatic cells.

Mitochondrial electron transport is inhibited by disappearance of metallothionein in human bronchial epithelial cells following exposure to silver nitrate

Silver (Ag) possesses antibacterial activity and has been used in wound dressings and deodorant powders worldwide. However, the metabolic behavior and biological roles of Ag in mammals have not been well characterized. In the present study, we exposed human bronchial epithelial cells (BEAS-2B) to AgNO3 and investigated uptake and intracellular distribution of Ag, expression of metallothionein (MT), generation of reactive oxygen species (ROS), and changes in mitochondrial respiration. The culture medium concentration of Ag decreased with time and stabilized at 12h. The concentration of both Ag and MT in the soluble cellular fraction increased up to 3h and then decreased, indicating that cytosolic Ag relocated to the insoluble fraction of the cells. The levels of mRNAs for the major human MT isoforms MT-I and MT-II paralleled with the protein levels of Ag-MT. The intensity of fluorescence derived from ROS was elevated in the mitochondrial region at 24h. Ag decreased mitochondrial oxygen consumption in a dose-dependent manner and the activity of mitochondrial complex I-IV enzymes was significantly inhibited following exposure to Ag. In a separate experiment, we found that hydrogen peroxide (H2O2) at concentrations as low as 0.001% (equivalent to the concentration of H2O2 in Ag-exposed cells) removed Ag from MT. These results suggest MT was decomposed by cytosolic H2O2, and then Ag released from MT relocated to insoluble cellular fractions and inhibited electron chain transfer of mitochondrial complexes, which eventually led to cell damage.

Developmental Subchronic Exposure to Diphenylarsinic Acid Induced Increased Exploratory Behavior, Impaired Learning Behavior, and Decreased Cerebellar Glutathione Concentration in Rats

In Japan, people using water from the well contaminated with high-level arsenic developed neurological, mostly cerebellar, symptoms, where diphenylarsinic acid (DPAA) was a major compound. Here, we investigated the adverse effects of developmental exposure to 20mg/l DPAA in drinking water (early period [0–6 weeks of age] and/or late period [7–12]) on behavior and cerebellar development in male rats. In the open field test at 6 weeks of age, early exposure to DPAA significantly increased exploratory behaviors. At 12 weeks of age, late exposure to DPAA similarly increased exploratory behavior independent of the early exposure although a 6-week recovery from DPAA could reverse that change. In the passive avoidance test at 6 weeks of age, early exposure to DPAA significantly decreased the avoidance performance. Even at 12 weeks of age, early exposure to DPAA significantly decreased the test performance, which was independent of the late exposure to DPAA. These results suggest that the DPAA-induced increase in exploratory behavior is transient, whereas the DPAA-induced impairment of passive avoidance is long lasting. At 6 weeks of age, early exposure to DPAA significantly reduced the concentration of cerebellar total glutathione. At 12 weeks of age, late, but not early, exposure to DPAA also significantly reduced the concentration of cerebellar glutathione, which might be a primary cause of oxidative stress. Early exposure to DPAA induced late-onset suppressed expression of NMDAR1 and PSD95 protein at 12 weeks of age, indicating impaired glutamatergic system in the cerebellum of rats developmentally exposed to DPAA.

Western Blot Analysis

Electrophoresis and the following western blot analysis are indispensable to investigate biochemical changes in cells and tissues exposed to nanoparticles or nanomaterials. Proteins should be extracted from the cells and tissues using a proper method, especially when phosphorylated proteins are to be detected. It is important to select a good blocking agent and an appropriate pair of primary and peroxidase-tagged secondary antibodies to obtain good results in western blot analysis. One thing that may be specific to nanomaterials, and that you should keep in mind, is that some proteins may be adsorbed on the surface of particulate nanomaterials. In this chapter the whole process of western blot analysis, from sample preparation to quantitative measurement of target proteins, is described.

Effects of arsenic on modification of promyelocytic leukemia (PML): PML responds to low levels of arsenite

Inorganic arsenite (iAs(3+)) is a two-edged sword. iAs(3+) is a well-known human carcinogen; nevertheless, it has been used as a therapeutic drug for acute promyelocytic leukemia (APL), which is caused by a fusion protein comprising retinoic acid receptor-α and promyelocytic leukemia (PML). PML, a nuclear transcription factor, has a RING finger domain with densely positioned cysteine residues. To examine PML-modulated cellular responses to iAs(3+), CHO-K1 and HEK293 cells were each used to establish cell lines that expressed ectopic human PML. Overexpression of PML increased susceptibility to iAs(3+) in CHO-K1 cells, but not in HEK293 cells. Exposure of PML-transfected cells to iAs(3+) caused PML to change from a soluble form to less soluble forms, and this modification of PML was observable even with just 0.1 μM iAs(3+) (7.5 ppb). Western blot and immunofluorescent microscopic analyses revealed that the biochemical changes of PML were caused at least in part by conjugation with small ubiquitin-like modifier proteins (SUMOylation). A luciferase reporter gene was used to investigate whether modification of PML was caused by oxidative stress or activation of antioxidant response element (ARE) in CHO-K1 cells. Modification of PML protein occurred faster than activation of the ARE in response to iAs(3+), suggesting that PML was not modified as a consequence of oxidative stress-induced ARE activation.

Pharmacodynamics of S-dimethylarsino-glutathione, a putative metabolic intermediate of inorganic arsenic, in mice

Abstract Inorganic arsenicals are well‐known carcinogens, whereas arsenite (iAsIII) compounds are now recognized as potent therapeutic agents for several leukemias, and arsenic trioxide has been used for the treatment of recurrent acute promyelocytic leukemia (APL). However, recent clinical trials revealed that arsenite is not always effective for non‐APL malignancies. Another arsenical, S‐dimethylarsino‐glutathione ([DMAIII(GS)]), which is a putative metabolic intermediate in the hepatic metabolism of iAsIII, shows promise for treating several types of lymphoma. However, the metabolism of [DMAIII(GS)] has not been well investigated, probably because [DMAIII(GS)] is not stable in biological fluids where the concentration of glutathione is low. In the present study, we injected [DMAIII(GS)] intravenously into mice and compared the tissue distribution and metabolic dynamics of [DMAIII(GS)] with those of sodium arsenite (NaAsO2). We found a unique organ preference for the distribution of [DMAIII(GS)] to the lung and brain in comparison to NaAsO2. Furthermore, [DMAIII(GS)] appeared to bind to serum albumin by exchanging its glutathione moiety quickly after administration, providing novel insights into the longer retention of [DMAIII(GS)] in plasma.

Health Effects of Silver Nanoparticles and Silver Ions

The health effects of silver nanoparticles (AgNPs) have not been well investigated, despite AgNPs now being widely used in consumer products. We introduce living environment, analysis, metabolic behavior, toxicity, and human health effect of AgNPs in comparison to silver nitrate (AgNO3). The American Conference of Governmental Industrial Hygienists (ACGIH) has established separate threshold limit values (TLV) for metallic silver (0.1 mg/m3) and soluble compounds of silver (0.01 mg/m3). Argyria and argyrosis are chronic disorders of skin microvessels and eyes in humans, and these disorders reportedly develop following extended oral and inhalational exposure to Ag. In mammals, AgNO3 and AgNPs increased the number of the total cells, neutrophils, and pro-inflammatory cytokine production “IL-1β,” and these were distributed in the lung, kidney, and liver. The amount of Ag in the metallothionein (MT)-bound form was related in cellular behavior and toxicity of AgNPs and AgNO3. The cytotoxic effect of AgNPs is a simple function of neither the number nor total surface area. Although the effect may vary among the cell types and the culture conditions, AgNPs were transported to lysosomes and only gradually dissolved in mammals, causing milder inflammatory stimulation.

Solubility shift and SUMOylaltion of promyelocytic leukemia (PML) protein in response to arsenic(III) and fate of the SUMOylated PML.

Promyelocytic leukemia (PML), which is a tumor suppressor protein that nevertheless plays an important role in the maintenance of leukemia initiating cells, is known to be biochemically modified by As(3+). We recently developed a simple method to evaluate the modification of PML by As(3+) resulting in a change in solubility and the covalent binding of small ubiquitin-like modifier (SUMO). Here we semi-quantitatively investigated the SUMOylation of PML using HEK293 cells which were stably transfected with PML-VI (HEK-PML). Western blot analyses indicated that PML became insoluble in cold RadioImmunoPrecipitation Assay (RIPA) lysis buffer and was SUMOylated by both SUMO2/3 and SUMO1 by As(3+). Surprisingly SUMO1 monomers were completely utilized for the SUMOylation of PML. Antimony (Sb(3+)) but not bismuth (Bi(3+)), Cu(2+), or Cd(2+) biochemically modified PML similarly. SUMOylated PML decreased after removal of As(3+) from the culture medium. However, unSUMOylated PML was still recovered in the RIPA-insoluble fraction, suggesting that SUMOylation is not requisite for changing the RIPA-soluble PML into the RIPA-insoluble form. Immunofluorescence staining of As(3+)-exposed cells indicated that SUMO2/3 was co-localized with PML in the nuclear bodies. However, some PML protein was present in peri-nuclear regions without SUMO2/3. Functional Really Interesting New Gene (RING)-deleted mutant PML neither formed PML nuclear bodies nor was biochemically modified by As(3+). Conjugation with intracellular glutathione may explain the accessibility of As(3+) and Sb(3+) to PML in the nuclear region evading chelation and entrapping by cytoplasmic proteins such as metallothioneins.