Yasuyuki Fujiwara

Lithium diffusion in LLNbO single crystals is directly visualized and quantitatively analyzed using isotope exchange and TOF-SIMS.

Methylmercury (MeHg) is a well-known environmental neurotoxicant that preferentially affects sensory neurons in the peripheral nervous system. While sensory-dominant neuropathy has long been described in Minamata disease, the temporal dynamics of dorsal root ganglion (DRG) injury and recovery remain incompletely understood. In this study, Wistar rats were exposed to MeHg for five consecutive days, followed by a two-day treatment-free period; this regimen was repeated once. The DRG and peripheral sensory fibers were analyzed up to 70 days after exposure. Histological and immunohistochemical analyses, DNA microarrays, and mercury quantification and distribution mapping were performed. The A-fiber density was significantly reduced at Day 14 but recovered by Day 70, whereas C-fibers showed no significant change. The total number of DRG neurons remained stable. Immunohistochemical analyses demonstrated that subtype marker-selected neurons (NF, TrkA, FAM19A1, TAC1, SST) decreased at Day 14 and gradually recovered thereafter. DNA microarray analysis at Day 14 revealed a broad downregulation of DRG neuronal subtype marker genes. The mercury concentration in the DRG peaked at Day 14 and declined to the control level by Day 70, with in situ imaging confirming preferential accumulation in DRG neurons. These data suggest that the short-term MeHg exposure caused a transient functional suppression of DRG neurons without widespread neuronal loss. The selective and reversible downregulation of neuronal phenotypes, coupled with preferential Hg accumulation in DRG neurons, underlies the sensory-dominant and potentially reversible features of MeHg neurotoxicity.

Sensory disturbances and central nervous system symptoms are important in patients with Minamata disease. In the peripheral nervous system of these patients, motor nerves are not strongly injured, whereas sensory nerves are predominantly affected. In this study, we investigated the mechanisms underlying the sensory-predominant impairment of the peripheral nervous system caused by methylmercury. We found that the types of cell death in rat dorsal root ganglion (DRG) neurons caused by methylmercury included apoptosis, necrosis, and necroptosis. Methylmercury induced apoptosis in cultured rat DRG neurons but not in anterior horn neurons or Schwann cells. Additionally, methylmercury activated both caspase 8 and caspase 3 in DRG neurons. It increased the expression of tumor necrosis factor (TNF) receptor-1 and the phosphorylation of receptor-interacting protein kinase 3 (RIP3) and mixed-lineage kinase domain-like pseudokinase (MLKL). The expression of TNF-α was increased in macrophage-like RAW264.7 cells by methylmercury. The increase was suggested to be mediated by the NF-κB pathway. Moreover, methylmercury induced neurological symptoms, evaluated by a hindlimb extension response, were significantly less severe in TNF-α knockout mice. Based on these results and our previous studies, we propose the following hypothesis regarding the pathogenesis of sensory nerve-predominant damage by methylmercury: First, methylmercury accumulates within sensory nerve neurons and initiates cell death mechanisms, such as apoptosis, on a small scale. Second, cell death triggers the infiltration of macrophages into the sensory fibers. Third, the macrophages are stimulated by methylmercury and secrete TNF-α through the NF-κB pathway. Fourth, TNF-α induces cell death mechanisms, including necrosis, apoptosis through the caspase 8/3 pathway, and necroptosis through the TNFR1-RIP1-RIP3-MLKL pathway, activated by methylmercury in sensory neurons. Consequently, methylmercury exhibits potent cytotoxicity specific to the DRG/sensory nerve cells in the peripheral nervous system. This chain of events caused by methylmercury may contribute to sensory disturbances in patients with Minamata disease.

Methylmercury is an environmental polluting organometallic compound that exhibits neurotoxicity, as observed in Minamata disease patients. Methylmercury damages peripheral nerves in Minamata patients, causing more damage to sensory nerves than motor nerves. Peripheral nerves are composed of three cell types: dorsal root ganglion (DRG) cells, anterior horn cells (AHCs), and Schwann cells. In this study, we compared cultured these three cell types derived from the rat for susceptibility to methylmercury cytotoxicity, intracellular accumulation of mercury, expression of L-type amino acid transporter 1 (LAT1), which transports methylmercury into cells, and expression of multidrug resistance-associated protein 2 (MRP2), which transports methylmercury-glutathione conjugates into the extracellular space. Of the cells examined, we found that DRG cells were the most susceptible to methylmercury with markedly higher intracellular accumulation of mercury. The constitutive level of LAT1 was higher and that of MRP2 lower in DRG cells compared with those in AHC and Schwann cells. Additionally, decreased cell viability caused by methylmercury was significantly reduced by either the LAT1 inhibitor, JPH203, or siRNA-mediated knockdown of LAT1. On the other hand, an MRP2 inhibitor, MK571, significantly intensified the decrease in the cell viability caused by methylmercury. Our results provide a cellular basis for sensory neve predominant injury in the peripheral nerves of Minamata disease patients.

Cadmium (Cd) is an environmental pollutant that damages various tissues. Cd may cause a depletion of iron stores and subsequently an iron deficiency state in the liver. However, the molecular mechanism of decreased iron accumulation in the liver induced by long-term exposure to Cd is unknown. In this study, we investigated the hepatic accumulation of iron and the proximal duodenal expression of the genes involved in iron transport using mice chronically exposed to Cd. Five-week-old female C57BL/6J mice were fed a diet containing 300 ppm Cd for 12, 15, 19 and 21 months. The iron concentration in the liver was markedly decreased by Cd. Among iron-transport-related genes in the proximal duodenum, the gene expression of HCP1 and Cybrd1 was significantly decreased by Cd. HCP1 is an influx transporter of heme iron. Cybrd1 is a reductase that allows non-heme iron to enter cells. The expression of iron-transport-related genes on the duodenal basolateral membrane side was hardly altered by Cd. These results suggest that long-term exposure to Cd suppresses the expression of HCP1 and Cybrd1 in the proximal duodenum, resulting in reduced iron absorption and iron accumulation in the liver.

Vascular endothelial cells synthesize and secrete perlecan, a large heparan sulfate proteoglycan that increases the anticoagulant activity of vascular endothelium by inducing antithrombin III and intensifying fibroblast growth factor (FGF)-2 activity to promote migration and proliferation in the repair process of damaged endothelium during the progression of atherosclerosis. However, the exact regulatory mechanisms of endothelial perlecan expression remain unclear. Since organic–inorganic hybrid molecules are being developed rapidly as tools to analyze biological systems, we searched for a molecular probe to analyze these mechanisms using a library of organoantimony compounds and found that the Sb-phenyl-N-methyl-5,6,7,12-tetrahydrodibenz[c,f][1,5]azastibocine (PMTAS) molecule promotes the expression of perlecan core protein gene without exhibiting cytotoxicity in vascular endothelial cells. In the present study, we characterized proteoglycans synthesized by cultured bovine aortic endothelial cells using biochemical techniques. The results indicated that PMTAS selectively induced perlecan core protein synthesis, without affecting the formation of its heparan sulfate chain, in vascular endothelial cells. The results also implied that this process is independent of the endothelial cell density, whereas in vascular smooth muscle cells, it occurred only at high cell density. Thus, PMTAS would be a useful tool for further studies on the mechanisms underlying perlecan core protein synthesis in vascular cells, which is critical in the progression of vascular lesions, such as those during atherosclerosis.

Vascular endothelial cell growth is essential for the repair of intimal injury. Perlecan, a large heparan sulfate proteoglycan, intensifies fibroblast growth factor-2 (FGF-2) signaling as a co-receptor for FGF-2 and its receptor, and promotes the proliferation of vascular endothelial cells. Previously, we reported that 2 µM of lead, a toxic heavy metal, downregulated perlecan core protein expression and then suppressed the growth of vascular endothelial cells. However, since the mechanisms involved in the repression of perlecan by lead remains unclear, we analyzed its detailed signaling pathway using cultured bovine aortic endothelial cells. Our findings indicate that 2 µM of lead inhibited protein tyrosine phosphatase (PTP) activity and induced cyclooxygenase-2 (COX-2) via phosphorylation of the epidermal growth factor receptor (EGFR) and its downstream extracellular signal-regulated kinases (ERK1/2). In addition, among the prostanoids regulated by COX-2, prostaglandin I2 (PGI2) specifically contributes to the downregulation of perlecan expression by lead. This study revealed an intracellular pathway—the EGFR-ERK1/2-COX-2-PGI2 pathway activated by inhibition of PTP by lead—as a pathway that downregulates endothelial perlecan synthesis. The pathway is suggested to serve as a mechanism for the repression of perlecan expression, which leads to a delay in cell proliferation by lead.

5-Aminolevulinic acid (5-ALA) is an amino acid derivative and a precursor of protoporphyrin IX (PpIX). The photophysical feature of PpIX is clinically used in photodynamic diagnosis (PDD) and photodynamic therapy (PDT). These clinical applications are potentially based on in vitro cell culture experiments. Thus, conducting a systematic review and meta-analysis of in vitro 5-ALA PDT experiments is meaningful and may provide opportunities to consider future perspectives in this field. We conducted a systematic literature search in PubMed to summarize the in vitro 5-ALA PDT experiments and calculated the effectiveness of 5-ALA PDT for several cancer cell types. In total, 412 articles were identified, and 77 were extracted based on our inclusion criteria. The calculated effectiveness of 5-ALA PDT was statistically analyzed, which revealed a tendency of cancer-classification-dependent sensitivity to 5-ALA PDT, and stomach cancer was significantly more sensitive to 5-ALA PDT compared with cancers of different origins. Based on our analysis, we suggest a standardized in vitro experimental protocol for 5-ALA PDT.

Chronic arsenic exposure is known to be related to the progression of atherosclerosis. However, the pathogenic mechanisms of arsenic-induced atherosclerosis have not been fully elucidated. Because disruption of the blood coagulation/fibrinolytic system is involved in the development of arteriosclerosis, we investigated the effect of arsenite on fibrinolytic activity in human vascular endothelial EA.hy926 cells in the present study. Fibrinolysis depends on the balance between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) secreted from vascular endothelial cells. We found that arsenite reduced fibrinolytic t-PA activity by inhibiting its synthesis without affecting PAI-1 production. The inhibitory effect of arsenite on t-PA expression was partially recovered by the reactive oxygen species (ROS) scavenger Trolox. The nuclear factor erythroid 2 related factor 2 (NRF2) pathway is known to be activated by arsenite via ROS production. We confirmed that arsenite activated the NRF2 pathway, and arsenite-induced inhibition of fibrinolytic t-PA activity was abrogated in NRF2-knockdown EA.hy926 cells. These results suggest that arsenite inhibits the fibrinolytic activity of t-PA by selectively suppressing its synthesis via activation of the NRF2 pathway in vascular endothelial cells.

Methylmercury (MeHg), the causal substrate in Minamata disease, can lead to severe and chronic neurological disorders. The main symptom of Minamata disease is sensory impairment in the four extremities; however, the sensitivity of individual sensory modalities to MeHg has not been investigated extensively. In the present study, we performed stimulus-response behavioral experiments in MeHg-exposed rats to compare the sensitivities to pain, heat, cold, and mechanical sensations. MeHg (6.7 mg/kg/day) was orally administered to 9-week-old Wistar rats for 5 days and discontinued for 2 days, then administered daily for another 5 days. The four behavioral experiments were performed daily on each rat from the beginning of MeHg treatment for 68 days. The pain sensation decreased significantly from day 11 onwards, but recovered to control levels on day 48. Other sensory modalities were not affected by MeHg exposure. These findings suggest that the pain sensation is the sensory modality most susceptive to MeHg toxicity and that this sensitivity is reversible following discontinuation of the exposure.