Search

Diphenylarsinic acid (DPAA) was detected as the primary compound responsible for the arsenic poisoning that occurred in Kamisu, Ibaraki, Japan, where people using water from a well that was contaminated with a high level of arsenic developed neurological (mostly cerebellar) symptoms and dysregulation of regional cerebral blood flow. To understand the underlying molecular mechanism of DPAA-induced cerebellar symptoms, we focused on astrocytes, which have a brain-protective function. Incubation with 10 µM DPAA for 96 h promoted cell proliferation, increased the expression of antioxidative stress proteins (heme oxygenase-1 and heat shock protein 70), and induced the release of cytokines (MCP-1, adrenomedullin, FGF2, CXCL1, and IL-6). Furthermore, DPAA overpoweringly increased the phosphorylation of three major mitogen-activated protein kinases (MAPKs) (ERK1/2, p38MAPK, and SAPK/JNK), which indicated MAPK activation, and subsequently induced expression and/or phosphorylation of transcription factors (Nrf2, CREB, c-Jun, and c-Fos) in cultured rat cerebellar astrocytes. Structure-activity relationship analyses of DPAA and other related pentavalent organic arsenicals revealed that DPAA at 10 µM activated astrocytes most effective among organic arsenicals tested at the same dose. These results suggest that in a cerebellum exposed to DPAA, abnormal activation of the MAPK-transcription factor pathway and irregular secretion of these neuroactive, glioactive, and/or vasoactive cytokines in astrocytes can be the direct/indirect cause of functional abnormalities in surrounding neurons, glial cells, and vascular cells: This in turn might lead to the onset of cerebellar symptoms and disruption of cerebral blood flow.

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.

Although inorganic arsenate (iAsV) and arsenite (iAsIII) are metabolized in liver and excreted into bile and urine, the metabolites in the bile after the oral intake of iAs remain unclear. Male Sprague-Dawley rats were orally (po) or intravenously (iv) exposed to iAs and methylated arsenics, and the arsenic speciation in the urine and bile was analyzed by high performance liquid chromatography-inductively coupled argon plasma mass spectrometry. Arsenic caused induction of multidrug resistance-associated protein 2 (MRP2), and changes of glutathione (GSH) levels in the liver and bile were also determined. The metabolic speciation studies revealed that arsenic was excreted into bile in the methylarsenic-diglutathione (MADG) and/or dimethylarsenic acid (DMAV) forms in iAsIII- or iAsV-po rats, but that MADG and arsenic-triglutathione (ATG) are the main forms excreted into bile both in iAsIII- and iAsV-iv rats. In MADG-po rats, the MADG was excreted into bile in the MADG and DMAV forms. Monomethylarsonic acid (MMAV)- and DMAV-iv rats did not excrete significant amounts of either MMAV or DMAV into bile and mostly excreted into urine in the unchanged chemical forms. Taken together, the DMAV detected in the bile is mostly supposed to be the dissociation of dimethylarsenic-glutathione (DMAG). Urinary arsenic speciation showed that arsenic metabolized to 43% methylated DMAV, 47% unmethylated iAsIII, and 10% iAsV in iAsIII-iv rats, whereas only 3% methylated DMAV, 87% unmethylated iAsV, and 10% iAsIII were detected in iAsV-iv rats. Arsenic was accumulated dose dependently, and arsenic concentration was significantly higher in the iAsIII-po rat liver than in the iAsV-po rat liver. GSH levels in the bile were decreased by relatively higher doses of iAsV-po, but significantly increased by iAsIII- or iAsV-iv. iAs-exposure increased the expression of MRP2 in the liver. Pretreatment with buthionine sulfoximine predominantly inhibited arsenic excretion into bile in iAs-iv rats. In conclusion, our data demonstrated that biliary and urinary arsenic excretion and speciation are affected by the route, dose, and chemical forms of arsenical administration, and GSH plays a key role in arsenic metabolism. We are also first to show that DMAV that probably originated from DMAG is excreted into the bile in iAs-po rats.

Diesel exhaust particles (DEP) induce pulmonary diseases including asthma and chronic bronchitis. Comprehensive evaluation is required to know the effects of pollutants including DEP on these and other lung diseases. Alveolar macrophages (AM) and epithelial cells are important cellular targets for pollutants such as DEP in the lung. Alveolar macrophages encounter and phagocytose DEP in the alveolar space, and their biological responses have been implicated in DEP-induced pulmonary diseases. Expression profiles of genes induced by DEP in AM will lead to better understanding of the mechanisms involved in pulmonary diseases. To characterize the effect of the DEP extract on AM systematically, we analyzed the gene expression in AM exposed to DEP extract using the Atlas Rat Toxicology Array II. The finding in cDNA microarray was further confirmed by Northern blot analysis. AM were exposed to 10 microg/ml of DEP extract for 6 h in order to elucidate early response to DEP extract in AM. Early response to DEP extract in AM may affect the alteration of gene expression in subsequent responses so that it is important to identify the alteration in early response. In this study, the transcription of 6 genes in the cDNA microarray was significantly elevated by exposure of the AM to DEP extract. These genes were heme oxygenase (HO)-1 and -2, thioredoxin peroxidase 2 (TDPX-2), glutathione S-transferase P subunit (GST-P), NAD(P)H dehydrogenase, and proliferating cell nuclear antigen (PCNA). The antioxidative enzymes such as HO, TDPX-2, GST-P, and NAD(P)H dehydrogenase may play a role in the pulmonary defense against oxidative stress caused by various pollutants including DEP. PCNA may have contributed to the repair of DNA damage and to cell proliferation caused by exposure to these pollutants. Our results suggest that cDNA microarray analysis is a useful tool to investigate the biological responses to pulmonary toxicants.

Diphenylarsinic acid (DPAA) was a major compound found in the arsenic poisoning incident that occurred in Kamisu, Ibaraki, Japan in 2003. People exposed to DPAA via contaminated well water suffered from several neurological disorders, including cerebellar symptoms. We previously reported that DPAA induces cellular activation in cultured rat cerebellar astrocytes, dose-dependent promotion of cell growth (low DPAA), cell death (high DPAA), and increased phosphorylation of mitogen-activated protein (MAP) kinases (p38MAPK, SAPK/JNK, and ERK1/2). Moreover, DPAA induces up-regulation of oxidative stress-counteracting proteins, activation of CREB phosphorylation, increased protein expression of c-Jun and c-Fos, and aberrant secretion of brain-active cytokines (MCP-1, adrenomedullin, FGF2, CXCL1, and IL-6). Here, we explored the role of MAP kinases in DPAA-induced activation of astrocytes using specific MAP kinase signaling inhibitors [SB203580 (p38MAPK), SP600125 (SAPK/JNK), SCH772984 (ERK1/2), and U0126 (MEK1/2, a kinase for ERK1/2)]. DPAA-induced activation of MAP kinases had little contribution to DPAA-induced cell growth and death. On the other hand, a power relationship among MAP kinases was also observed, in which p38MAPK suppressed DPAA-induced SAPK/JNK and ERK1/2 activation, whereas ERK1/2 and MEK1/2 facilitated p38MAPK and SAPK/JNK activation. In addition, SAPK/JNK had minimal effects on the activation of other MAP kinases. DPAA-induced activation of transcription factors and secretion of brain-active cytokines were submissively but intricately dominated by MAP kinases. Collectively, our results indicate that DPAA-induced activation of MAP kinases is neither a cell growth-promoting response nor a cytoprotective one but leads to transcriptional disruption and aberrant secretion of brain-active cytokines in cerebellar astrocytes.