Manabu Kunimoto

Transcriptional regulation of thioredoxin reductase 1 expression by cadmium in vascular endothelial cells: Role of NF-E2-related factor-2†

Thioredoxin reductase (TrxR) is a selenoprotein that catalyzes the reduction of the active site disulfide of thioredoxin (Trx), which regulates the redox status of the cells. In the present study, we found that TrxR1, one of the three TrxR isozymes, was induced by cadmium as well as tumor necrosis factor α (TNFα) in bovine arterial endothelial cells (BAEC), and investigated the mechanism of cadmium‐induced TrxR1 expression. We here showed that cadmium, differently from TNFα, enhanced the promoter activity of the 5′‐flanking region of human TrxR1 gene (nucleotides −1692 to +49). Deletion and site‐directed mutation of antioxidant responsive element (ARE) (nucleotides −62 to −48) in this region abolished the response to cadmium. Overexpression of NF‐E2‐related factor‐2 (Nrf2) augmented the TrxR1 promoter activity. In contrast, overexpression of the dominant negative mutant of Nrf2 suppressed cadmium‐induced activation of TrxR1 promoter through the ARE. Chromatin immunoprecipitation (ChIP) assays showed that anti‐Nrf2 antibody precipitated ARE from the chromatin of the cadmium‐treated cells. These results indicated that cadmium‐induced TrxR1 gene expression is mediated by the activation of Nrf2 transcription factor and its binding to ARE in the TrxR1 gene promoter. We further found that in addition to cadmium, the activators of Nrf2, such as diethyl maleate (DEM) and arsenite, induced both TrxR1 and Trx gene expression in BAEC. Nrf2 might play an important role in the regulation of the cellular Trx system consisting of Trx and TrxR.

Bisphenol A causes hyperactivity in the rat concomitantly with impairment of tyrosine hydroxylase immunoreactivity

We examined the effects of bisphenol A, an endocrine disruptor, on rat behavioral and cellular responses. Single intracisternal administration of bisphenol A (0.2‐20 μg) into 5‐day‐old male Wistar rats caused significant hyperactivity at 4–5 weeks of age. Rats were about 1.6‐fold more active in the nocturnal phase after administration of both 2 and 20 μg of bisphenol A than were control rats. The response was dose‐dependent. Based on DNA macroarray analyses of the midbrain, bisphenol A decreased by more than twofold gene expression levels of the dopamine D4 receptor at 4 weeks of age and the dopamine transporter at 8 weeks of age. Furthermore, bisphenol A decreased by more than twofold gene expression levels of the dopamine D4 receptor at 4 weeks of age and the dopamine transporter at 8 weeks of age. We conclude that bisphenol A affected central dopaminergic system activity, resulting in hyperactivity due most likely to a large reduction of tyrosine hydroxylase activity in the midbrain.

Overexpression of thioredoxin reductase 1 regulates NF-κB activation

Thioredoxin reductase (TrxR) is a flavoprotein that contains a C‐terminal penultimate selenocysteine (Sec) and has an ability to reduce thioredoxin (Trx), which regulates the activity of NF‐κB. To date, three TrxR isozymes, TrxR1, TrxR2, and TrxR3, have been identified. In the present study, we found that among these isozymes only TrxR1 was induced by tumor necrosis factor‐α (TNFα) in vascular endothelial cells. Furthermore, the overexpression of TrxR1 enhanced TNFα‐induced DNA‐binding activity of NF‐κB and NF‐κB‐dependent gene expression. The catalytic Sec residue of TrxR1, which is essential for reducing Trx, was required for this NF‐κB activation, and aurothiomalate, an inhibitor of TrxR, suppressed TNFα‐induced activation of NF‐κB and the expression of NF‐κB‐targeted proinflammatory genes such as E‐selectin and cyclooxygenase‐2. These results suggest that TrxR1 may act as a positive regulator of NF‐κB and may play an important role in the cellular inflammatory response.

Influence of LIF and BMP-2 on differentiation and development of glial cells in primary cultures of embryonic rat cerebral hemisphere.

Cells prepared from the cerebral hemisphere of embryonic Day 18 rats were maintained for 2 days in serum‐free modified Bottenstein‐Sato (mBS) medium containing thyroid hormone (TH), with or without leukemia inhibitory factor (LIF) or bone morphogenetic protein (BMP)‐2, and these influences on the differentiation and development of glial cells were investigated using the cells maintained in mBS medium containing TH as controls. The levels of glial fibrillary acidic protein (GFAP) expression and the number of GFAP‐positive astrocytes increased markedly with the addition of LIF or BMP‐2, and were enhanced further with the addition of both LIF and BMP‐2. The number of O1‐positive oligodendrocytes increased with the addition of LIF, whereas it decreased with the addition of BMP‐2. The number did not change with the addition of both cytokines. Using antibody against platelet‐derived growth factor (PDGF), we then excluded indirect effects of these cytokines through PDGF, which would increase by accelerated astrocyte development. When PDGF was neutralized, the number of oligodendrocytes increased under all conditions examined. As a result of the neutralization, the effect of BMP‐2 on oligodendrocyte differentiation was eliminated, although LIF remained effective. These results suggest that the differentiation of oligodendrocytes was delayed partially by PDGF even in control cultures. It is also suggested that LIF and BMP‐2, each of which accelerates the differentiation and development of astrocytes, would seem to have different effects on oligodendrocyte differentiation, i.e., LIF would directly affect oligodendrocyte differentiation, whereas BMP‐2 would affect it mainly through PDGF.

Selective disappearance of an axonal protein, 440‐kDa ankyrinB, associated with neuronal degeneration induced by methylmercury

The 440‐kDa isoform of brain ankyrin, 440‐kDa ankyrinB, is a neuron‐specific protein and is confined to axons. Cerebellum is one of the areas characteristically altered by methylmercury intoxication both in the adult and during development. When rat cerebellar neurons matured for 7 days in vitro were exposed to methylmercury at 0.03 μM for 48 hr, viability of the cells was unaffected. However, the immunocytochemical staining of 440‐kDa ankyrinB diminished drastically, whereas that of microtubule‐associated protein‐2, which is localized in dendrites and cell bodies, and of glial fibrillary acidic protein (GFAP), a marker for astroglial cells coexisting in the culture, remained unchanged. To confirm these observations, a simplified dot blot assay was established to determine 440‐kDa ankyrinB and several other marker proteins in cultured cell samples. With this assay, we found that methylmercury at a submicromolar range induced a decrease of 440‐kDa ankyrinB and an increase of GFAP in a dose‐dependent manner in cerebellar cells in primary culture. Surprisingly, another axonal protein, tau, remained mostly in its intact molecular sizes even in the presence of 0.3–1.0 μM methylmercury, though its immunocytochemical localization was substantially altered. These results indicate that selective loss of the axonal protein 440‐kDa ankyrinB is associated with the early stage of degeneration of cerebellar neurons induced by methylmercury. Therefore, 440‐kDa ankyrinB should be useful as a specific and sensitive marker for the neurotoxicity of methylmercury.

β-Naphthoflavone disturbs astrocytic differentiation of C6 glioma cells by inhibiting autocrine interleukin-6

Regulation of astrocyte differentiation is a key process in the development of the central nervous system (CNS), and disturbance of the differentiation can lead to brain system dysfunction. Here we show that β‐naphthoflavone (βNF), an agonist of the aryl hydrocarbon receptor (AhR), disturbed the cAMP‐induced astrocytic differentiation of C6 glioma by inhibiting autocrine interleukin‐6 (IL‐6). Treatment of cells with βNF reduced the induction of an astrocyte marker glial fibrillary acidic protein (GFAP). This was caused by the inactivation of its upstream transcription factor signal transducer and activator of transcription 3 (STAT3) by βNF. In addition, βNF attenuated the induction of the IL‐6 gene, which leads to the activation of STAT3. Most importantly, the inhibitory effect of βNF on GFAP promoter activity was recovered by the addition of recombinant IL‐6. Taken together, these results indicate that the inhibitory effect of βNF on IL‐6 induction suppresses STAT3 activation. These processes subsequently lead to the attenuation of GFAP induction.

Influence of cell density and thyroid hormone on glial cell development in primary cultures of embryonic rat cerebral hemisphere.

The influence of cell density and thyroid hormone (TH) on the development of astrocytes and oligodendrocytes was investigated in primary cultures prepared from rat cerebral hemisphere on embryonic day (E)18. At the beginning of the culture, most of the cells were microtubule‐associated protein 2 (MAP2)‐positive neurons, whereas O1‐positive oligodendrocytes and glial fibrillary acidic protein (GFAP)‐positive astrocytes were rarely observed. After the cells were maintained in serum‐free defined medium, astrocytes developed at high cell density but rarely at a low one. When leukemia inhibitory factor (LIF) was supplemented in low‐density cultures, the levels of GFAP expression markedly increased to almost the same extent as in high‐density culture without TH. This suggests that, in low‐density cultures, astrocyte progenitors could not differentiate because of insufficient astrocyte‐inducing factors. Interestingly, the addition of TH increased GFAP expression levels only at high density. The number of oligodendrocytes increased with TH addition at both cell densities, although the effects were more remarkable at high density. These results suggest that cell density and TH are pivotal factors in the development of both astrocytes and oligodendrocytes. It is also suggested that the effects of TH on glial cell development could be accelerated via cell–cell communications.

Application of Biotests for Toxicity of Leachate and Evaluation Monitoring

廃棄物最終処分場が周辺水環境に与える影響の評価を行うため, 本研究では3種のバイオアッセイ (遺伝子毒性試験, ヒト乳癌由来細胞 (MCF-7) 増殖活性試験, 培養細胞毒性試験) を用い, 6ヶ月間にわたって廃棄物処分場試料, 周辺環境水の毒性監視を行った。また, 遺伝子毒性試験umu試験について環境監視に適した試料前処理法と新たな毒性単位を検討, 提唱した。その結果, 各試料の遺伝子毒性に季節特異的な変動は見られず, 浸出水に検出された毒性は生物処理によって周辺環境水程度に低減していた。浸出水のMCF-7増殖活性の平均値は低く, 放流先河川への影響はほとんどないと考えられた。細胞毒性試験において濃縮試料も細胞の生存率を50％以上低下させる毒性を示さず, 濃縮試料で検出された細胞毒性も, その由来は浸透圧であると推測された。この処理後放流水の細胞毒性の最大値は放流先上流の約9倍であり, 放流先河川に全く影響を与えないためには, 10倍の希釈が必要であった。