NIMD Forum 2011
Methylmercury Toxicity:
Up-to-date Research on Mechanisms, Toxicology
and Pathology

January 27-28, 2011

Venue: Minamata Disease Archives, Conference Hall
55-10 Myojin, Minamata City, Kumamoto 867-0055, Japan0Address
It is a great pleasure for me to be with you here and to have an opportunity to say a few
words at the occasion of opening of NIMD forum 2011. First of all, on behalf of
National Institute for Minamata Disease, I would like to extend our warmest welcome toall of you here to attend NIMD forum 2011. And also I would like to express my sincere
gratitude to invited researchers from abroad who came all the way to Minamata here.
Our institute was established in October 1978 and has more than 32 year’s history.
NIMD is a small but a unique institute; its purpose is to conduct comprehensive
research on single chemical “mercury”, especially “methylmercury” which caused
Minamata Disease. Comprehensive means that we are conducting medical,
environmental and social researches on Minamata disease and mercury. Therefore wecover very broad areas such as effects on human health, air and soil contamination,
biogeochemistry and epidemiology.
NIMD forum has been held almost every year since 1997. The forums have been indeed
excellent opportunities to bring many of active and experienced researchers from allover the world to Minamata and to share and to discuss the latest research findings on
mercury. This is the twelfth NIMD forum. This year’s forum focuses on methylmercury
toxicity with special attention to up-to-date research on mechanisms, toxicology andpathology. We have fifteen presenters, seven from abroad, five from universities inJapan and three from NIMD. They are all leading scientists in this field. I hope that all
participants will exchange ideas and views frankly so that everyone may be benefited
from this forum. Last but not least, I sincere hope that two days meeting will be usefuland fruitful for better understanding of current status of methylmercury toxicity
researches. I want to once again extend our warmest welcome to all of you.
January 27, 2011
Koji Okamoto, M.D., M.P.H.
Director General
National Institute for Minamata Disease1

Organizing Committee
Fusako Usuki, M.D., Ph.D.
Chief in Rehabilitation Section, Department of Clinical Medicine,National Institute for Minamata Disease

Masatake Fujimura, Ph.D.
Chief in Pathology Section, Department of Basic Medical Sciences,
National Institute for Minamata Disease

Isamu Tsuji
Officer in International Affairs and Information Section, Department of International
Affairs and Environmental Sciences,
National Institute for Minamata Disease

Keisuke Mori, Ph.D.
Chief in Survey Section, Department of Epidemiology,
National Institute for Minamata Disease

Takehiro Tsuchiya
Chief Officer in Finance and Accounting Section, Administration Division,
National Institute for Minamata Disease

Tomoyuki Nonomura
Officer in Finance and Accounting Section, Administration Division,
National Institute for Minamata Disease

Hiroyuki Watanabe
Officer in International Affairs and Information Section, Department of International
Affairs and Environmental Sciences,
National Institute for Minamata Disease2

Invited Speakers and Chairpersons
Dr. William Rostene (Inserm, France)
Dr. Chris Newland (Auburn University, USA)
Dr. Sandra Ceccatelli (Karolinska Institute, Sweden)
Dr. Michael Aschner (Vanderbilt University Medical Center, USA)
Dr. Yoshito Kumagai (Tsukuba University, Japan)
Dr. Jean-Paul Bourdineaud (Bordeaux University, France)
Dr. Shuji Izumo (Kagoshima University, Japan)
Dr. Roshan Tofighi (Karolinska Institute, Sweden)
Dr. Ebany J. Martinez-Finley (Vanderbilt University Medical Center,
USA)
Dr. Gi-Wook Hwang (Tohoku University, Japan)
Dr. Takashi Hirooka (Tokyo University of Science, Japan)
Dr. Takashi Toyama (Tsukuba University, Japan)

Dr. Akira Naganuma (Tohoku University, Japan)
Dr. Toshiyuki Kaji (Tokyo University of Science, Japan)

3

Program
January 27
9:20-9:25

Opening address

Koji Okamoto (NIMD, Japan)

9:25-9:35

Brief introduction

Fusako Usuki (NIMD, Japan)

9:35-10:55

Session 1

1-1.

Chair: Akira Yasutake (NIMD, Japan)
William Rostene (Inserm, France)

Chris Newland (Auburn University, USA)
Neuroprotection and Methylmercury: Selenium, DHA, andNimodipine (魚由来成分のセレンや DHA およびカルシウム遮断薬である
ニモジピンのメチル水銀毒性防御効果)

Follow-up remark: Mineshi Sakamoto (NIMD, Japan)
Protective effects of selenomethionine against methylmercury-induced
neuronal degeneration in developing rat brain (メチル水銀により惹起されるラット発達期脳の神経変性に対するセレノメチオニンの抑制効果)

1-2.

Sandra Ceccatelli (Karolinska Institute, Sweden)
Effects of methylmercury on survival and differentiation of neuralstem cells (神経幹細胞の生存と分化に及ぼすメチル水銀の影響)

10:55-11:15

Coffee break

11:15-12:25

Session 2

2-1.

Chair: Masaaki Nakamura (NIMD, Japan)
Michael Aschner(Vanderbilt University Medical Center, USA)

Takashi Toyama (Tsukuba University, Japan)
Keap1/Nrf2 system regulates cellular accumulation of methylmercury,
thereby blocking its toxicity(メチル水銀蓄積に対する Keap1/Nrf2 シス
テムの制御)

2-2.

Yoshito Kumagai (Tsukuba University, Japan)
A unique protein mediating cellular protection against
4

Methylmercury
12:25-13:40(メチル水銀毒性防御に関与する特異な蛋白分子)

Lunch

13:40-14:50 Session 3
Chair: Toshiyuki Kaji (Tokyo University of Science, Japan)
Jean-Paul Bourdineaud (Bordeaux University, France)
3-1.

Gi-Wook Hwang (Tohoku University, Japan)
Involvement of the post-translational modification of proteins inmethylmercury toxicity(メチル水銀毒性における蛋白質の翻訳後修飾
の関与)

3-2.

Fusako Usuki (NIMD, Japan)
Posttranscriptional defects of antioxidant selenoenzymes cause
oxidative stress under methylmercury exposure (メチル水銀曝露下における転写後変化と酸化ストレス)14:50-16:20

4-1.

Session 4

Chair: Yoshito Kumagai (Tsukuba University, Japan)
Megumi Yamamoto (NIMD, Japan)

William Rostene (Inserm, France)
Protection by the chemokine CCL2/MCP1 of methylmercury
neurotoxicity (メチル水銀神経毒性におけるケモカインの作用)

15:25-15:45 Coffee break
4-2.

Takashi Hirooka (Tokyo University of Science, Japan)
The microvascular cells are a target of methylmercury toxicity(メチル水銀の標的としての微小血管)

16:20-17:30 Session 5

5-1.

Chair: Gi-Wook Hwang (Tohoku University, Japan)
Chris Newland (Auburn University, USA)

Ebany J. Martinez-Finley (Vanderbilt University Medical Center, USA)
Methylmercury neurotoxicity in a C. elegans model system(線虫モデルシステムにおけるメチル水銀毒性)
5

5-2.

Jean-Paul Bourdineaud (Bordeaux University, France)
Zebrafish as a model of the mercurial contamination of the aquatic
food web: histological, bioenergetical, and transcriptional issues(メチル水銀毒性モデルとしてのゼブラフィッシュ)19:00-20:30 Reception

January 28
9:00-10:10 Session 6
6-1.

Chair: Akira Naganuma (Tohoku University, Japan)

Shuji Izumo (Kagoshima University, Japan)
Early changes of astrocytes in the molecular layer of cerebellar cortex
of rats with methyl mercury intoxication(メチル水銀毒性におけるア
ストログリアの初期変化)

6-2.

Michael Aschner (Vanderbilt University Medical Center, USA)The role of glia in modulating methylmercury neurotoxicity(メチル水銀神経毒性におけるグリア細胞の役割)

10:10-10:30 Coffee break
10:30-11:40 Session 7

Chair: Sandra Ceccatelli (Karolinska Institute, Sweden)
Shuji Izumo (Kagoshima University, Japan)

7-1.

Masatake Fujimura (NIMD, Japan)

7-2.

Neuritic degeneration contributes to MeHg-induced neuronal cell
death (メチル水銀による神経細胞死への神経軸索変性の寄与)
Roshan Tofighi (Karolinska Institute, Sweden)
Methylmercury activates multiple cell death pathways in neuronaland glial cells (メチル水銀曝露下での神経細胞及びグリア細胞における神経細胞死経路)

11:40-11:50 Brief summary

Fusako Usuki (NIMD, Japan)

11:50-11:55 Closing address

Koji Okamoto (NIMD, Japan)6

1-1

Neuroprotection and Methylmercury: Selenium, DHA, andNimodipine
Christopher Newland
Auburn University, Auburn, AL.

It has been suggested that fish nutrients like DHA and selenium might confer
protection against the neurotoxicty of methylmercury. Other hypotheses about
neuroprotection have derived from mechanisms by which methylmercury may exert its
neurotoxicity, like disruption of calcium homeostasis. We have investigated thesehypotheses by combining methylmercury exposure with fish nutrients or, in ongoingstudies, with the calcium channel blocker, nimodipine. Neuroprotection by fish
nutrients was examined both in developmental and adult-onset methylmercury
exposures. Neither DHA nor selenium protected against methylmercury’s
developmental neurotoxicity, which was found to occur in rats at very low daily intakesof 40 ug/kg/day. Selenium, however, significantly postponed the onset ofmethylmercury’s neurotoxicity when exposure began in adulthood. Whether this wasdue to actions in the brain or to peripheral actions is uncertain. In ongoing studies using
mice, dietary nimodipine delays the appearance of neuromotor deficits, including
performance on a running wheel. Nimodipine also delayed the appearance of cognitive
deficits that we detected using an “incremental repeated acquisition” procedure. This
exposure produced no detectable nimodipine in blood but did produce a large number ofdetectable metabolites. Interestingly, no such protection was seen when nimodipine
exposure occurred through subcutaneous implants, which produced detectable
nimodipinne but fewer metabolites. One hypotheses to test would be that the protection
came from a metabolite, rather than from nimodipine.
Key Words: selenium, DHA, nimodipine, neuroprotection, neuro-behavioral effects
References: 1) Heath JC et al., Neurotoxicology, 31, 169-179, 2010.
2) Reed MN et al., Neurotoxicology, 27, 721-732, 2006.
3) Newland MC et al., Animal Models of Cognitive Impairment. pp 101146, CRC Press, 2006.

7

[紹介]
演者の Newland 先生は、「アメリカ合衆国・オーバーン大学」において研究を行っておられます。今回の発表は、「魚由来成分のセレンや DHA およびカルシウム遮断薬であるニモジ
ピンのメチル水銀毒性防御効果」についてです。ラットを用いた実験で、 DHA やセレンは発達期のメチル水銀毒性には効果がありませんでしたが、成人期の曝露ではセレンはその毒性発現を遅延させました。また、食餌性のニモジピンが、マウスの運動障害や認知障害などの毒性発現を遅延させることが明らかになりました。

Selenium: セレン
DHA: ドコサヘキサエン酸
Nimodipine: ニモジピン
Neuroprotection: 神経保護作用
Neuro-behavioral effects: 神経行動効果

8

Follow-up remark
Protective effects of selenomethionine against methylmercury-induced
neuronal degeneration in developing rat brain
Mineshi Sakamoto1, Akira Yasutake1, Megumi Yamamoto1, Hing Man Chan2,Masae Ryufuku3, Akiyoshi Kakita3, Sanae Oumi4, Chiho Watanabe41National Institute for Minamata Disease, Japan
2
University of Northern British Columbia, Canada
3

Brain Research Institute, Niigata University, Niigata, Japan
4
University of Tokyo, Japan

Although a protective effect of selenium (Se) against the toxicity of methylmercury(MeHg) has been confirmed in some animal studies, its direct effects in the brain has
not been reported. Substantiation of the direct protective effects of selenomethionine(SeMet), one chemical form of Se in food, against MeHg-induced neurotoxicity would
provide important information for the risk evaluation of MeHg on seafood. Our goal inthis study is to demonstrate the direct protective effects of Se against the neuronaldegeneration caused by MeHg in developing brains which is vulnerable to MeHg
exposure. We used a model rat of fetal-type Minamata disease which causes widespreadneuronal degeneration in the brain at a high dose of MeHg. Male Wistar rats ofpostnatal days 14 (PD14) were maintained by their dam. They were orally administered
MeHg, either MeHg (8 mg Hg/kg/day in a solution containing L-cysteine andcondensed milk), SeMet (2 mg/kg/day) alone or MeHg and SeMet co-exposure (MeHg
was administered 30 min after SeMet) for 10 consecutive days. Twenty-four hours after
the final administration, the effects of different treatment were investigated byhistopathological and chemical analyses. Decreases in body and liver weights wereobserved in MeHg and SeMet groups, but were negated by co-exposure. Neuronaldegeneration in the cerebral neocortex, decreased GSH-Px activity and increasedhydroperoxide were observed in MeHg group, and co-exposure to SeMet prevented theeffects. On the other hand, SeMet decreased GSH and GSH-Px activity and increased TBil, which indicate liver damage by SeMet, but the effect was negated by co-exposure
to MeHg. Co-exposure to SeMet increased the total mercury concentrations in the
cerebral cortex and cerebellum. Co-exposure to SeMet also increased the percentage of
inorganic Hg in all organ tissues, which indicates increased demethylation by SeMet.
9This is, to our knowledge, the first report which shows the direct protective effect of Se
against MeHg-induced neuronal degeneration in the brain. We consider theneuroprotection effect will be derived by a MeHg and Se complex such as bismethylmercury selenide, which was formed by co-exposure to MeHg and SeMet. Thismay be also explained by the fact that SeMet-induced liver damage was prevented by
MeHg.
Key Words: methylmercury, developing rat brain, selenomethionine, neuronaldegeneration, neuroprotective effects
References: 1) Sakamoto M et al., Brain Res., 784, 351-354, 1998.
2) Sakamoto M et al., Brain Res., 949, 51-59, 2002.
3) Sakamoto M et al., Dev. Brain Res., 152, 171-176, 2004.

[紹介]
演者の坂本は、「国立水俣病総合研究センター」において研究を行っています。今回は、Newland 先生の発表に対する追加発言として「メチル水銀により惹起されるラット発達期脳の神経変性に対するセレノメチオニンの抑制効果」について発表を行います。メチル水銀はラットの発達期脳の神経に傷害をあたえますが、セレノメチオニンの同時投与によってその変化は抑制されました。

Methylmercury: メチル水銀
Developing rat brain: 発達期ラット脳Selenomethionine:
セレノメチオニン (セレンを含むアミノ酸で、食物などに含まれるセレンの摂取源)

Neuronal degeneration: 神経変性
Neuroprotection effects: 神経保護作用

10

1-2

Effects of methylmercury on survival and differentiation ofneural stem cells
Sandra Ceccatelli
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.

Neural stem cells (NSCs) play an essential role in both the developing and adultnervous system. In the last few years, we have studied the mechanisms by which NSCs
undergo cell death in response to neurotoxicants 1), such as methylmercury (MeHg) 2).
The mouse NSC line C17.2 and rat primary embryonic cortical NSCs were used as
experimental models to investigate the effects of MeHg on survival and differentiation.
The results show that NSCs are more vulnerable to MeHg, as compared to differentiatedcells. MeHg induces apoptosis via Bax-activation, cytochrome c translocation andcaspase activation with subsequent fodrin cleavage and formation of the 120 kD
fragment. In addition, there is a significant increase of the 150 kD fodrin breakdown
product indicating that calpains are also activated. The caspase and calpain proteases areconcomitantly activated in NSCs undergoing apoptosis, as proven by the partial
protection exerted by the caspases (zVAD-fmk) or calpains (E64d) inhibitor alone andthe full protective effect of the two inhibitors together. Remarkably, exposure to MeHg
at concentrations relevant to human exposure inhibits spontaneous neuronaldifferentiation of NSCs via Notch signaling 3). Conversely, other neurotoxic foodcontaminants, such as non-dioxin like PCBs, promote neuronal differentiation 4). The
evaluation of NSC differentiation offers sensitive endpoints to identify substances with
developmental neurotoxic potential and further points to NSCs as a relevant in vitro
model for neurotoxicity studies.
Key words: Apoptosis, caspases, calpains, spontaneous differentiation, developmental
neurotoxicity.
References: 1) Ceccatelli S. et al., Toxicol. Lett., 149, 59-66, 2004.
2) Tamm C. et al., J. Neurochem., 97, 69-78, 2006.
3) Tamm C. et al., Neuroreport, 19, 339-43, 2008.
4) Tofighi R. et al., Toxicol. Sci., submitted.

11

[紹介]
演者の Ceccatelli 先生は、「スウェーデン・カロリンスカ研究所」において研究を行っておられます。今回の発表は、「神経幹細胞の生存と分化に及ぼすメチル水銀の影響」についてです。神経幹細胞 (Neural stem cells) は、発生期および成体内に存在していて神経やグリア細胞に分化する重要な細胞です。培養細胞を用いた実験によって、神経幹細胞は分化した細胞よりもメチル水銀感受性が高く、アポトーシスをおこしやすいことが明らかになりました。
さらに、低濃度のメチル水銀は神経幹細胞の分化を抑制することも明らかになりました。

Apoptosis: アポトーシス (プログラムされた“自殺性”の細胞死)Caspases: カスパーゼ (アポトーシス誘導に関与する蛋白質分解酵素)
Calpains: カルパイン (カルシウム依存性の蛋白質分解酵素)
Spontaneous differentiation: 自然分化
Developmental neurotoxicity: 発達神経毒性

12

2-1

Keap1/Nrf2 system regulates cellular accumulation of methylmercury,
thereby blocking its toxicity

Takashi Toyama
Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan.
Research Fellow of the Japan Society for the Promotion of Science, Japan.
Methylmercury (MeHg), a naturally occurring environmental electrophile, exhibits
neurotoxicity through accumulation in the brain. We reported previously that MeHg
activates nuclear factor-erythroid 2-related factor 2 (Nrf2), a basic-leucine zipper
transcription factor, thereby up-regulating its downstream gene products and that Nrf2
plays an important role in reducing the cellular accumulation of MeHg1). In our current
study, we investigated the protective effect of isothiocyanates, which are known toactivate Nrf2, on the accumulation of mercury and its toxicity following exposure ofmice to MeHg in vitro and in vivo. Isothiocyanates 6-methylsulfinylhexyl
isothiocyanate (6-HITC) and sulforaphane (SFN) activated Nrf2 and upregulated
downstream proteins associated with MeHg excretion such as glutamate-cysteine ligase,glutathione S-transferase, and multidrug resistance–associated protein in primary mouse
hepatocytes. Under these conditions, intracellular glutathione levels increased in wildtype but not Nrf2-deficient primary mouse hepatocytes. Pretreatment with theseisothiocyanates before MeHg exposure suppressed cellular accumulation of mercury
and cytotoxicity in wild-type but not Nrf2-deficient primary mouse hepatocytes.
Compared with that in wild-type mice, oral administration of MeHg (5 mg/kg/day) to
Nrf2-deficient mice for 8 days resulted in increased sensitivity to mercury concomitantwith an increase in mercury accumulation in the brain and liver. Injection of SFN before
administration of MeHg resulted in a decrease in mercury accumulation in the brain andliver of wild-type, but not Nrf2-deficient, mice. These data suggesting that 6-HITC andSFN can suppress mercury accumulation and intoxication caused by MeHg intake
through activation of Nrf2.
Key words: Nrf2, isothiocyanate, chemoprevention.
References: 1) Toyama T et al., Biochem. Biophys. Res. Commun., 363, 645-650, 2007.

13

[紹介]
演者の外山先生は、「筑波大学」において研究を行っておられます。今回の発表は、「メチル水銀蓄積に対する Keap1/Nrf2 システムの制御」についてです。転写因子である Nrf2 はメ
チル水銀により活性化され、その結果、下流の遺伝子発現が増加してメチル水銀の細胞内蓄積を減尐させます。Nrf2 を活性化することが知られているイソチオシアン酸塩を細胞または実験動物に処置することによって、メチル水銀の細胞内蓄積および細胞死を抑制できることが明らかになりました。

Nrf2: (DNA に特異的に結合して遺伝子転写を制御する転写因子の一つ)
Isothiocyanate: イソチオシアン酸塩
Chemoprevention: 化学的予防

14

2-2

A unique protein mediating cellular protection against
methylmercury

Yoshito Kumagai
Doctoral Programs in Medical Sciences, Graduate School of Comprehensive Human
Sciences, University of Tsukuba.
Methylmercury (MeHg) is an environmental electrophile that reacts readily withprotein thiols to form a covalent bond formation, thereby causing the functional loss andalteration in the structure. Some of unbound MeHg is also covalently bound to low
molecule nucleophiles such as glutathione (GSH), by nonenzymatic and possibly
enzymatic processes by the action of GSH S-transferases (GSTs), to form a MeHg–
GSH adduct. This adduct is transported out to extracellular space by multidrug
resistance-associated proteins (MRPs). These detoxification processes can substantiallyreduce intracellular MeHg. Nuclear factor-erythroid 2-related factor 2 (Nrf2), a basicleucine zipper transcription factor, is found to coordinately regulate gene expression of
glutamate cysteine ligase (GCL) catalyzing GSH synthesis, GSTs and MRPs. We havefound that Nrf2 plays a role in the initial response through modification of Keap1, thenegative regulator of Nrf2, leading to activation of Nrf2 and cellular protection throughup-regulation of GCL, GSTs and MRPs during exposure to MeHg in vitro and in vivo1, 2,
3). Hydrogen sulfide (H2S) is found to be a biological signaling molecule involved in
vascular and nervous system functions. Most interestingly, H2S is extensively
dissociated into a nucleophile HS- because of its pKa value = 6.88 while pKa value of
GSH is 8.66, implying that most of GSH exists as its protonated form at physiologicalconditions. However, role of H2S in chemical modification of cellular proteins andcytotoxicity during exposure to MeHg is poorly understood. In this symposium, I will
introduce current our findings that not only GSH but also enzymatically produced H2S
are key nucleophiles that protect against electrophilic attack of MeHg, resulting in
reduction of the cell damage.
Key words: hydrogen sulfide, Keap1/Nrf2 system, covalent modification, detoxification,
thiol adduct.
References: 1) Toyama T et al., Biochem. Biophys. Res. Commun., 363, 645-650, 2007.
2) Toyama T et al., Toxicol. Appl. Pharmacol., 249, 86-90, 2010.
3) Toyama T et al., Environ. Health Perspect., submitted.
15

[紹介]
演者の熊谷先生は、「筑波大学」において研究を行っておられます。今回の発表は、「メチル水銀毒性防御に関与する特異な蛋白分子」についてです。メチル水銀は、蛋白質のチオー
ル (Thiol) 基と容易にイオウ原子を介して共有結合する親電子性環境物質です。イオウ原子を含む硫化水素は、血管系や神経系における生物学的なシグナル分子であることが最近知られてきました。メチル水銀曝露下での蛋白質の化学修飾や細胞毒性における硫化水素の役割について検討し、硫化水素はメチル水銀の親電子物質としての攻撃を防御し、細胞障害を減弱させることが明らかになりました。

Hydrogen sulfide: 硫化水素
Keap1/Nrf2 system: Keap1/Nrf2システム
Covalent modification: 共有結合性の修飾
Detoxification: 解毒
Thiol adduct: チオール付加化合物

16

3-1

Involvement of the post-translational modification of proteinsin methylmercury toxicity
Gi-Wook HWANG
Graduate School of Pharmaceutical Sciences, Tohoku University, Japan.

Methylmercury (MeHg) is a toxic environmental pollutant, causing serious
neurological and developmental effects in humans. However, the mechanism underlying
the MeHg toxicity is not fully understood. We used yeast cells (S. cerevisiae) to
elucidate the defense mechanisms against MeHg toxicity, and search for novel genes
involved in MeHg resistance because S. cerevisiae is a eukaryotic organism having
many gene products that are functionally similar to those of mammals such as humans.
We have previously reported that Cdc34, Hrt3, Ylr224w and Rad23, proteins related toubiquitin-proteasome system (UP system), confer MeHg resistance to yeast cells. It
seems likely that certain proteins that have not yet been identified but exist in cells,
increase MeHg toxicity. The toxicity of MeHg might be reduced by enhanced
degradation of these proteins through the UP system which is strongly conserved fromyeast to human cells. We also found that the UP system plays an important role inlending protection against MeHg toxicity not only in yeast cells and but also in humancells. Hrt3 and Ylr224w are F-box proteins which are known to recognize and bind to
substrate proteins that are then degraded by the UP system. We recently identified Dld3and Eno2 as proteins that specifically bind to these F-box proteins. We also found thatboth proteins involved in reinforcement of MeHg toxicity. Dld3 and Eno2 are known tobe proteins involved in synthesis of pyruvate, and overexpression of each protein might
induce increase in interacellular levels of pyruvate. We found that deletion of Yil006w,
a transports pyruvate into mitochondria, confers resistance to MeHg in yeast cells.
These results suggest that promotion of pyruvate inflow into the mitochondria mightenhance MeHg toxicity. In the future, detailed studies on the role of the pyruvate in
MeHg toxicity will help elucidate the mechanism of MeHg toxicity and thecorresponding biological defense mechanism.
Key words: ubiquitin, proteolysis, pyruvate, mitochondria.
References: 1) Hwang G.W. et al., FASEB J., 16, 709-711, 2002.
2) Hwang G.W. et al., FEBS Lett., 580, 6813-6818, 2006.
3) Hwang G.W. et al., J. Toxicol. Sci., 34, 413-416, 2009.
17

[紹介]
演者の黄先生は、「東北大学」において研究を行っておられます。今回の発表は、「メチル水銀毒性における蛋白質の翻訳後修飾の関与」についてです。蛋白質の翻訳後修飾には様々なものが知られています。その中で、細胞内で不要となった蛋白質にユビキチンで"印"を付け、これをアミノ酸へと分解してリサイクルするユビキチンープロテアソームシステム (UPシステム) (Ubiquitin-proteasome system, UP system) が、メチル水銀毒性の防御メカニズ
ムに重要な役割をもつことが明らかになりました。UP システムによって分解され、メチル水銀
毒性を増強させる蛋白質について検討したところ、新たな蛋白質が同定されました。この蛋白質はピルビン酸生成に関係しており、ミトコンドリアへのピルビン酸の流入がメチル水銀毒性に関与していることが明らかになりました。

Ubiquitin: ユビキチン (細胞内で不要となったタンパク質の"印"の役割をするタン パク質)
Proteolysis: 蛋白質分解
Pyruvate: ピルビン酸 (エネルギー代謝経路酵素の一種)
Mitochondria: ミトコンドリア (細胞内に存在する酸素呼吸を行う小器官)

18

3-2

Posttranscriptional defects of antioxidant selenoenzymes cause
oxidative stress under methylmercury exposure

Fusako Usuki
Department of Clinical Medicine, National Institute for Minamata Disease, Japan.
The critical role of oxidative stress in the pathogenesis of methylmercury (MeHg)
cytotoxicity has been clarified, but the molecular mechanisms underlying MeHgmediated oxidative stress remain to be elucidated. Here I demonstrate aposttranscriptional effect of MeHg on antioxidant selenoenzymes by using a myogenic
model cell line1) showing apoptosis by exposure to MeHg. Quantitative real-time PCR
analysis showed down-regulation of the major antioxidant selenoenzyme, glutathione
peroxidase 1 (GPx1) mRNA in spite of up-regulation of other antioxidant enzyme
mRNAs. MeHg-induced decrease in GPx1 mRNA was not suppressed by the coaddition with antioxidant Trolox but by the pretreatment with sodium selenite,suggesting that the decrease in GPx1 mRNA was due to MeHg-induced relative
intracellular selenium deficiency. This notion was supported by the inhibition study ofnonsense-mediated mRNA decay (NMD)2), which recognizes a UGA codon forselenocysteine on GPx1 as a nonsense codon and degrades GPx1 mRNA in seleniumdeficient condition. In contrast, thioredoxin reductase 1 (TrxR1), another antioxidant
selenoenzyme of the thioredoxin system, was likely skipped by NMD because of a UGA
codon in the last exon. However, TrxR1 activity was decreased despite mRNA
upregulation, which was probably due to the synthesis of aberrant TrxR1 protein
without selenocysteine. Changes in selenoenzyme GPx1 and TrxR1 mRNAs wereobserved earlier than was the incidence of oxidative stress and upregulation of otherantioxidant enzyme mRNAs. Results indicated that the MeHg-induced relative
selenium-deficient condition affects the major antioxidant selenoenzymes GPx1 andTrxR1 through a posttranscriptional effect, resulting in the disturbance of cellular redox
systems and the incidence of oxidative stress3). Treatment with ebselen, a seleno-organic
compound, effectively suppressed oxidative stress and protected cells against MeHginduced relative selenium deficiency and cytotoxicity.
Key words: oxidative stress, antioxidant selenoenzyme, nonsense-mediated mRNA
decay (NMD), posttranscriptional defect, ebselen
References: 1) Usuki F et al., Neurotoxicology, 29, 22-30, 2008.
192) Yamashita A et al., Biochim. Biophys. Acta., 1754, 305-315, 2005.
3) Usuki F et al., J. Biol. Chem., 2010 Nov 24 [Epub. Ahead of print].

[紹介]
演者の臼杵は、「国立水俣病総合研究センター」において研究を行っています。今回の発表は、「メチル水銀曝露下における転写後変化と酸化ストレス」についてです。メチル水銀の細胞毒性には酸化ストレス傷害が重要な役割をもちますが、なぜ、メチル水銀が酸化ストレスを起こすかはよくわかっていませんでした。培養細胞を用いた実験によって、メチル水銀がセレン動態への影響を介してセレン含有抗酸化酵素の遺伝子発現を制御し、酸化ストレスを引き起こすことが明らかになりました。また、セレノ有機化合物であるエブセレンが、メチル水
銀による酸化ストレスと細胞毒性を抑制することも明らかになりました。

Oxidative stress: 酸化ストレス
Antioxidant selenoenzyme: セレン含有抗酸化酵素
Nonsense-mediated mRNA decay (NMD):
遺伝子配列の途中に生じたナンセンスコドンを含む変異 mRNA を排除する mRNA 監視機構

Posttranscriptional defect: 転写後の欠陥
Ebselen: エブセレン (セレノ有機化合物)

20

4-1

Protection by the chemokine CCL2/MCP-1 of the neurotoxic
effect of methylmercury

David Godefroy1, Romain-Daniel Gosselin2, Akira Yasutake3, Masatake Fujimura3,
Régine Maury-Brachet4, Muriel Laclau4, Jean-Paul Bourdineaud4, William Rostène1
1
INSERM, UPMC Université Paris 06, UMRS 968, Institut de la Vision, 75012 Paris,
France.
2
University Hospital Center and University of Lausanne, Lausanne, Switzerland.
3
Dpt of Basic Med Sci NIMD, Minamata City, Kumamoto, Japan.
4

Université de Bordeaux 1, CNRS, UMR 5805, Station Marine d'Arcachon, 33120,
France.

Industrial pollution due to heavy metals such as mercury is a major concern for theenvironment and public health. Mercury, in particular methylmercury (MeHg),
primarily affects brain development and activity, resulting in neurotoxic effects. As
chemokines are considered to be involved in neuroinflammatory and neurodegenerative
diseases 1), we tested the possibility that one of these chemokines, CCL2/MCP-1, can be
implicated in the neurotoxic effects of MeHg. Using in vitro experiments on rat pure
cortical neurons in culture and in vivo by means of a fish-based diet to mice 2), we
demonstrate that the chemokine CCL2/MCP-1, via its cognate receptor CCR2, is able toprotect cortical neurons from MeHg-induced cell death through a mechanism involving
the glutathione pathway. Furthermore, MeHg induced a decrease in mice brain
CCL2/MCP-1 concentrations and a significant cortical microglial recruitment. Finally,
the protective effect of CCL2/MCP-1 was confirmed in CCL2 KO mice which showeda more pronounced neuronal cell death than wild-type mice in specific layers of thecortex in response to MeHg. We suggest that during the early phase of MeHg
contamination, an increased release of CCL2/MCP-1 by neurons acting as an autocrine
feedback loop and by glial cells such as microglia, could partially protect from neuronal
apoptotic cell death. The dying neurons thus inform the microenvironment in order toproduce more CCL2/MCP-1. When the contamination lasts, CCL2 cannot anymore
prevent the neurotoxic effect of MeHg and the neuronal cell death spreeds up. These
original findings open new avenues on the implication of the chemokine systems inbrain deficits due to MeHg intoxication.
Key words: chemokines, CCL2/MCP-1, neuronal cell death, microglia, methylmercury.
21References: 1) Rostene W et al., Nature Rev. Neurosci., 8, 895-903, 2007.
2) Bourdineaud JP et al., Environ. Health, 7, 53-66, 2008.

[紹介]
演者の Rostène 先生は、「フランス・インサーム」において研究を行っておられます。今回の発表は、「メチル水銀神経毒性におけるケモカインの作用」についてです。培養神経細胞およびケモカインを遺伝子操作で喪失させたケモカインノックアウトマウスを用いた実験によって、生体内のケモカインがメチル水銀毒性に対して抑制的に働いていることが明らかになりま
した。

Chemokines: ケモカイン (神経炎症や神経変性に関係する蛋白質)
CCL2/MCP-1: (ケモカインの一種)
Neuronal cell death: 神経細胞死
Microglia: ミクログリア (グリア細胞の一種、食作用がある)
Methylmercury: メチル水銀

22

4-2

The microvascular cells are a target of methylmercury toxicity
Takashi Hirooka
Faculty of Pharmaceutical Sciences, Tokyo University of Science, Japan.

Methylmercury is a toxic metal compound that induces severe neuropathy in thebrain of exposed humans and animals, as is observed in patients with Minamata disease.
The neuropathological lesions are localized in the cerebral cortices along deep sulci andfissures including the calcarine, and the granular cell layers in cerebellum. Although themechanisms of the selective vulnerability have been incompletely understood, it appears
to be possible that vascular toxicity of methylmercury is important for the selective
vulnerability in the brain. We have investigated the cytotoxic effects of methylmercury
on human brain microvascular pericytes and endothelial cells in a culture system. The
pericytes at low cell densities were highly susceptible to methylmercury cytotoxicity.
This susceptibility was due to the not only constitutively high but also methylmercuryinduced expression of LAT-1, which transports methylmercury into cells1). On the other
hand, the endothelial cells were resistant to methylmercury, which was due to higher
expression of glutathione and metallothionein2). Their proliferation was inhibited bysuppression of FGF-2 expression3). Vascular permeability is regulated by the VEGF
system, which is consisted of VEGF family proteins and receptors. In endothelial cells,
methylmercury induced the expression of VEGFR-1 and VEGFR-2; in addition,
methylmercury increased in the secretion of PlGF, a VEGF family protein that enhances
the binding of VEGF-A to VEGFR-2 by preventing the binding of VEGF-A to VEGFR1. In pericytes, methylmercury increased the VEGF-A165 secretion. Furthermore,methylmercury promoted the synthesis of hyaluronan in both endothelial cells andpericytes. These results suggest that methylmercury induces the vasogenic edema bystimulation the autocrine/paracrine signaling of VEGF and hyaluronan secretion in
microvessels. We propose the possible mechanisms of the selective localization of thecerebral lesion caused by methylmercury as follows: 1) Methylmercury induces thedamage of perocyes. 2) methylmercury inhibits the repair of damaged endothelial cell
layers. 3) methylmercury increases the permeability of brain microvascular tissue by
activation of the VEGF system. 4) methylmercury increases hyaluronan that maintains
water in the extracellular matrix of brain microvascular tissue.
Key words: brain microvascular cells, VEGF system, vasogenic edema.
23

References: 1) Hirooka T et al., Toxicol. In Vitro, 24, 835-41, 2010.
2) Hirooka T et al., J. Toxicol. Sci., 35, 287-94, 2010.
3) Hirooka T et al., J. Toxicol. Sci., 34, 433-39, 2010.
[紹介]
演者の廣岡先生は、「東京理科大学」において研究を行っておられます。今回の発表は、「メチル水銀の標的としての微小血管」についてです。ヒト脳の微小血管細胞を用いた実験によって、メチル水銀が血管周囲細胞の障害や血管内皮細胞の再生障害、血管内皮細胞増殖因子システムの活性化による血管透過性の増大、および血管構造の水分量を調整するヒア
ルロナン(Hyaluronan) 量の増大を引き起こすことが明らかになり、メチル水銀が血管性浮腫を誘発する可能性が示唆されました。

Brain microvascular cells: 脳微小血管細胞
VEGF system: 血管内皮細胞増殖因子システム
Vasogenic edema: 血管性浮腫

24

5-1

Role of Skn-1 in methylmercury-induced dopaminergic
neurotoxicity in Caenorhabditis elegans
Ebany Martinez-Finley and Michael Aschner

Division of Pediatric Toxicology and Center in Molecular Toxicology,
Vanderbilt University Medical Center, Nashville, TN.
Mercury (Hg) is a persistent environmental contaminant that exerts its toxic effects
on the nervous system through molecular mechanisms that remain unknown.
Epidemiological studies have pointed to the contribution of methylmercury (MeHg) to
dopamine neuron vulnerability and the predisposition to Parkinson’s disease (PD). Nrf2,
a phase II antioxidant transcription factor, has been shown to be involved in MeHg
neurotoxicity. Overexpression of Nrf2 inhibits MeHg-mediated cell death and deficits in
Nrf2 leaves cells vulnerable to MeHg. We hypothesize that skn-1, the Caenorhabditis
elegans (C. elegans) orthologue of mammalian Nrf2, is an important factor in MeHginduced dopaminergic neurodegeneration. We knocked down skn-1 (skn-1 KO) in C.
elegans and exposed both N2 (control) and skn-1 KO worms to 0, 10, 20 and 30µM
MeHgCl for 30 minutes or 4 hours following synchronization. Our data suggests thatskn-1 KO nematodes (LC50=16µM MeHg) are more sensitive to MeHg than N2 controls(LC50=25µM MeHg). Dopaminergic neuronal morphology was subsequently observed
via fluorescent analysis at L1, L4 and adult life stages. Presence of puncta was observed
at 1µM MeHg. Longevity and brood size were also assessed. Decreased longevity wasobserved at high doses in both N2 and skn-1KO nematodes. MeHg, at 20µM, had asignificant effect on brood size, and decreased nematode size immediately following
exposure indicates a developmental delay. We are currently testing the vulnerability ofparkin, a juvenile onset PD gene, to MeHg to delineate the contribution of MeHg to PD.
Our data suggest that C. elegans is a valuable model for studying the effects of MeHg
on the nervous system, delineating molecular mechanisms of toxicity and determining
genetic susceptibility. Acknowledgements: ES R01 07331.
Key Words: C. elegans, skn-1, Parkinson’s Disease.

25

[紹介]
演者の Martinez-Finley 先生は、「アメリカ合衆国・バンダービルド大学」で研究を行っておられます。今回の発表は、「線虫モデルシステムにおけるメチル水銀毒性」についてです。線
虫のシー・エレガンスは全遺伝子の解析が終了しており、遺伝子操作 (特定の遺伝子の欠損や付加) が容易で、毒性メカニズム研究に有用です。哺乳類における転写因子 Nrf2 に相当する線虫の Skn-1 を遺伝子操作で欠損させたシー・エレガンスでは、メチル水銀毒性に対する感受性が野生株よりも高くなることが明らかになりました。シー・エレガンスは、メチル水銀の神経系への影響を研究し、毒性や遺伝子感受性を明らかにするのに有用な実験モデルです。現在、メチル水銀とパーキンソン病への寄与について明らかにするために、若年性パーキンソン病の原因遺伝子であるパーキン (Parkin) のメチル水銀に対する脆弱性について検討しています。

C. elegans: シー・エレガンス (線虫の一種, モデル生物として広く利用されている)
Skn-1: (哺乳類における転写因子、Nrf2 に相当)
Parkinson’s Disease: パーキンソン病

26

5-2 Zebrafish as a model of the mercurial contamination of theaquatic food web: histological, bioenergetical, and transcriptional
issues.
Jean-Paul Bourdineaud
Bordeaux University, CNRS, Arcachon Marine Station, France.
Methylmercury (MeHg) is a potent neurotoxic involved in the Minamata tragedy. In
the Amazonian basin, gold mining leads to MeHg biomagnification all along the foodweb, culminating in piscivorous fish, ultimately responsible for contamination of humanbeings through fish consumption. In order to assess the biological impact of dietary
MeHg on fish, we contaminated zebrafish with MeHgHg/g of food, an environmentally relevant dose). A genome scale analysis of gene
expression was conducted on the skeletal muscle because this tissue does not perform
MeHg demethylation, and among 5280 different transcripts, 60 appeared up-regulated
and 15 down-regulated by more than 2 times. A net impact of MeHg was noticed on 14
ribosomal protein genes, indicating a perturbation of protein synthesis. Several genes
involved in mitochondrial metabolism, the electron transport chain, endoplasmic
reticulum (ER) function, detoxification, and general stress responses were differentiallyregulated, suggesting an onset of oxidative stress and ER stress. Several other genes forwhich expression varied with MeHg contamination could be clustered in various
compartments of the cell’s life, such as lipid metabolism, calcium homeostasis, ironmetabolism, muscle contraction, and cell cycle regulation. Mitochondria fromcontaminated zebrafish muscles presented structural abnormalities under electron
microscopy observation such as cristae disorganization. In permeabilized muscle fibers,
we observed a strong inhibition of state 3 mitochondrial respiration, a decrease ofcytochrome c oxidase activity and in the rate of ATP release after 49 days of exposure.
Contrarily to muscles, brain mitochondrial respiration was not modified by MeHg
exposure. GABA transaminase, glial fibrillary acidic protein, and glutathione Stransferase genes (abat, gfap, and gst) were up regulated, whereas the glutathione
peroxidase gene gpx1 was repressed, suggesting an impact of MeHg on GABA
concentration, and the onset of an oxidative stress and inflammation. A transmission
electron microscopic observation revealed a disorganization of the optical tectum.

27Key words: mitochondrial impairment, transcriptional response, zebrafish, muscle ATPdepletion, optical tectum.
References: 1) Cambier S et al., Environ. Sci. Technol., 44, 469-475, 2010.
2) Cambier S et al., Int. J. Biochem. Cell Biol., 41, 791-799, 2009.
3) Oliveira Ribeiro CA et al., Environ. Toxicol. Pharmacol., 25, 304-309,
2008.
4) Gonzalez P et al., Environ. Sci. Technol., 39, 3972-3980, 2005.

[紹介]
演者の Bourdineaud 先生は、「フランス・ボルドー大学」において研究を行っておられます。
今回の発表は、「メチル水銀毒性モデルとしてのゼブラフィッシュ」についてです。ゼブラフィッ
シュは全遺伝子の解析が終了していて、毒性研究に有用なモデルです。ゼブラフィッシュにメ
チル水銀を含む餌を与え、筋肉の 5280 種類の遺伝子発現解析の結果から、酸化ストレスや小胞体ストレスが誘導されていることが示唆されました。筋肉ではミトコンドリアの構造変化と機能異常が明らかになりましたが、脳ではミトコンドリア呼吸の変化は認められませんでした。
また、電子顕微鏡による検討で、視覚に関係する視蓋の異常が認められました。

Mitochondrial impairment: ミトコンドリア障害
Transcriptional response: 転写応答
Zebrafish: ゼブラフィッシュ (熱帯淡水魚の一種)
Muscle ATP depletion: 筋肉 ATP 枯渇
Optical tectum: 視蓋

28

6-1

Early changes of atrocities in the molecular layer of cerebellar
cortex of rats with methylmercury intoxication.
Shuji Izumo
Division of Molecular Pathology, Center for Chronic Viral Diseases,
Kagoshima University, Japan.

Selective involvement of granule neurons in the cerebellar cortex is one of thecharacteristic histopathology of methylmercury (MeHg) intoxication. However, its
pathogenic process is still unclear and little has been described about changes ofmolecular layer of the cerebellar cortex. Recent studies have described roles ofastrocytes in pathogenesis of the neuronal damage in MeHg intoxication, most of themwere done as in vitro studies and histopathologic changes of astrocytes are not reported.
In this study we performed histopathologic analysis of cerebellar lesions of MeHgpoisoned rats in order to clarify early phase of pathogenic process of cerebellar lesions
in MeHg intoxication. We administered MeHg to adult rats by exposure of drink-watercontaining 20 ppm of MeHg for 21 or 28 days, and sacrificed on 2-4 days aftertermination of MeHg exposure. Cerebellar vermis were processed for histopathologic
examination and performed routine neuropathological, immunohistochemical, andelectron microscopic studies. Sagittal sections of cerebellar vermis showed somepyknotic neurons in the granule layer indicating presence of cerebellar lesions. In themolecular layer, increase of Iba1-positive activated microglia and intense staining of
GFAP immunohistochemistry in Bergmann’s glia were observed. In addition, manysmall vacuoles were noticed in the neuropil of the molecular layer. Purkinje cells were
well preserved in number. EM study revealed swollen astrocytic processes in which
formation of vacuoles and accumulation of dense lamellar bodies were observed.
Surrounding structures such as Purkinje cell dendrites and pre- and post synapses did
not show any morphological abnormality. These glial cell changes seen in the molecularlayer of the cerebellum suggest that primary involvement of astrocytes may occur inacute phase of MeHg intoxication.
Key words: methylmercury intoxication, cerebellar cortex, astrocytes, neuropathology
electron microscopy

29

[紹介]
演者の出雲先生は、「鹿児島大学」において神経病理学的研究を行っておられます。今回の発表は、「メチル水銀毒性におけるアストロサイトの初期変化」についてです。メチル水銀
障害の病理像の特徴として小脳皮質の顆粒細胞が選択的に障害されることが知られていますが、その病理過程および分子層の病理変化についてはなお明らかではありません。今回、メチル水銀中毒モデルラットの小脳を用いた神経病理学的研究で、ミクログリアの活性化やアストロサイトの空胞変性が明らかになりました。プルキンエ細胞の樹状突起やニューロンシ
ナプス構造の形態変化は認められないことから、グリアの初期障害が示唆されました。

Methylmercury intoxication: メチル水銀中毒
Cerebellar cortex: 小脳皮質
Astrocytes アストロサイト
Neuropathology: 神経病理Electron microscopy 電子顕微鏡

30

6-2

Comparative Study on the Response of Rat Primary Astrocytes
and Microglia to Methylmercury Toxicity

Mingwei Ni1, Michael Aschner1,2,3
1
Department of Pharmacology, 2Neuroscience Graduate Program and 3Department ofPediatrics, Vanderbilt University Medical Center, Nashville, TN, USA.
As two major glial cell types in the brain, astrocytes and microglia play pivotal andyet different roles in maintaining optimal brain function. Even though both cell types
have been implicated as major targets of methylmercury (MeHg), their uniquesensitivities and adaptive responses to this metal can vary given their distinctive
properties and physiological functions. This study was carried out to compare therespective responses of neonatal rat primary cortical astrocytes and microglia following
treatment with MeHg, addressing the effects of MeHg on cell viability, reactive oxygen
species (ROS) generation and glutathione (GSH) levels, as well as mercury (Hg) uptake
and the expression of NF-E2-related factor 2 (Nrf2) (a major regulator of intracellularantioxidant response). Results showed that microglia are more sensitive to MeHg thanastrocytes, consistent with their higher Hg uptake and lower basal GSH levels.
Microglia also demonstrated higher ROS generation upon MeHg treatment compared toastrocytes. Nrf2 and its downstream genes were upregulated in both cell types, but withdifferent kinetics (much faster in microglia). In summary, microglia and astrocytes eachexhibit a distinct sensitivity to MeHg, resulting in their differential temporal adaptive
responses. These unique sensitivities appear to be dependent upon the cellular thiol
status of the particular cell type. Collectively, these studies suggest that microglia andastrocytes assume different roles on a protracted time-scale, with microglia representing
the early responders and astrocytes taking on a similar role at a later stage following
MeHg treatment (This work was supported by grant from the National Institute ofEnvironmental Health Sciences R01 ES07331).
Keywords: methylmercury, microglia, astrocytes, reactive oxygen species, glutathione,
Nrf2.

31

[紹介]
演者の Aschner 先生は、「アメリカ合衆国・バンダービルド大学」において研究を行っておられます。今回の発表は、「メチル水銀神経毒性におけるグリア細胞の役割」についてです。脳神経系に存在する 2 種類のグリア細胞、すなわちミクログリアとアストロサイトは脳機能を維持する上で重要な細胞です。培養細胞を用いた実験によって、アストロサイトよりもミクログリ
アの方がよりメチル水銀に感受性が高いこと、ミクログリアはより水銀の取り込みが多くグル
タチオン含量が低いこと、さらに活性酸素種産生が多いことが明らかになりました。以上のことから、メチル水銀毒性におけるミクログリアの初期応答の関与が示唆されました。

Methylmercury: メチル水銀
Microglia: ミクログリア (グリア細胞の一種)
Astrocytes: アストロサイト (グリア細胞の一種)
Reactive oxygen species: 活性酸素種
Glutathione: グルタチオン (生体内に存在する抗酸化物質の一種)
Nrf2: (DNA に特異的に結合して遺伝子転写を制御する転写因子の一種)

32

7-1

Neuritic degeneration contributes to methylmercury-induced
neuronal cell death
Masatake Fujimura
National Institute for Minamata Disease, Japan.

Methylmercury (MeHg) is an environmental neurotoxicant which induces
neuropathological changes in both central nervous and peripheral sensory nervous
systems. The molecular mechanism underlying MeHg-induced neuronal cell death wasinvestigated using cultured cortical neuronal cells. We demonstrated that neuritic
degeneration precedes MeHg-induced apoptotic death in neurons exposed to 100 nM
MeHg. Immunocytochemical and ELISA analyses for neurite-specific proteins namely,
tau and MAP2, showed that injury to tau-positive axons was first induced followed bydamage to the dendrites and cellular bodies. To further investigate the factors
responsible for neuronal death, we investigated the expression levels of Rho-family
proteins (Rac1, Cdc42, and RhoA), which regulate neuritic functions and apoptosis inneurons. Western blot analysis demonstrated that MeHg downregulated the expression
levels of Rac1 and Cdc42 but did not affect RhoA. The results indicate that neuritic
degeneration, in particular axonal degeneration triggered by the downregulation of Rac1
expression, contributes to MeHg-induced apoptotic cell death in cultured cerebrocorticalneurons1). So we hypothesized that MeHg-induced axonal degeneration might be causedby neuritic extension/retraction incoordination. This idea brought our attention to the
Rho/ROCK pathway because it has been known to be associated with the developmentof axon and apoptotic neuronal cell death. Next we show that inhibition of Rho/ROCK
pathway prevents MeHg-intoxication in cultured cortical neuronal cells. A Rho inhibitor,
C3 toxin, and two ROCK inhibitors, Fasudil and Y-27632, significantly protected
MeHg-induced axonal degeneration and apoptotic neuronal cell death in cultured
cortical neuronal cells exposed to 100 nM MeHg. The results suggest that inhibition of
Rho/ROCK pathway rescues MeHg-mediated neuritic extension/retraction
incoordination and is effective for the prevention of MeHg-induced axonal degeneration
and apoptotic neuronal cell death2).
Key words: neuritic degeneration, neuronal cell death, Rac1, ROCK inhibitor.
References: 1) Fujimura M et al., Neurotoxicology, 30, 16-22, 2009.
2) Fujimura M et al., Toxicol. Appl. Pharmacol., 250, 1-9, 2011.
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[紹介]
演者の藤村は、「国立水俣病総合研究センター」において研究を行っています。今回の発表は、「メチル水銀神経細胞死への神経軸索変性の寄与」についてです。神経細胞は細胞体、軸索、樹状突起から形成されているのですが、その中でも神経軸索は神経間の情報伝達に重要な役割を果たしています。培養神経細胞を用いた実験で、メチル水銀が最初に Rac1 という神経軸索を伸展させる蛋白質の発現を抑制することによって神経軸索を変性させた後、神経細胞死を引き起こすことが明らかになりました。また、メチル水銀による軸索変性と神経細胞死は薬剤 (ROCK 阻害剤等) によって防御できることも明らかになりました。

Neuritic degeneration: 神経軸索変性
Neuronal cell death: 神経細胞死
Rac1: (神経軸索を伸展させる蛋白質)
ROCK inhibitor: ROCK 阻害剤

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7-2

Methylmercury activates multiple cell death pathways
in neuronal and glial cells

Roshan Tofighi
Department of Neuroscience, Karolinska Institutet, S-17177 Stockholm, Sweden.
The identification of the mechanisms that drive methylmercury (MeHg)-induced
neurotoxicity has been a main research topic in neurotoxicology. In the nervous system
MeHg can induce necrotic or apoptotic cell death depending on the level and duration of
exposure. While necrosis is an ATP-independent process characterized by cell andorganelle swelling, loss of plasma membrane integrity and lysis, apoptosis is ATPdriven with hallmarks including cell shrinkage, chromatin condensation, plasma
membrane blebbing, activation of specific proteases (e.g. caspases and calpains), andDNA fragmentation at specific sites. We have investigated the signaling cascades
leading to cell death induced by MeHg in different cell types including primary rat
cerebellar granule cells (CGC), the mouse HT22 hippocampal neuron cell line, thehuman D384 astrocytoma cell line as well as the rodent pituitary cell lines AtT20 and
GH31,2). As a general feature, all these cells when exposed to MeHg undergo apoptosis
in a caspase-independent manner. Instead, the Ca2+-activated proteases calpains areactivated1,2). Also, translocation of apoptosis-inducing factor (AIF) from themitochondria to the nucleus with subsequent chromatin condensation and large-scale
DNA fragmentation occurs in CGC1). The Lysosomal proteases as executor factors in
MeHg-induced cell death appears to be critical in the astrocytoma, hippocampal andpituitary cell lines. Pretreatment with antioxidants prevent MeHg-induced apoptosis
pointing to oxidative stress playing a major role in MeHg-induced toxicity in our
experimental models1,2). Interesting, the plasma membrane voltage-dependent anion
channels (VDAC) that play a role in the redox homeostasis of normal cells, are open
and active in MeHg- exposed HT22 cells, suggesting that these channels also play a rolein anion efflux in apoptotic cells3). In conclusion, it is evident that the response to MeHg
depends on the cell type and that multiple cell death pathways can be activatedconcomitantly. Multiple cell models should be used for in vitro studies aimed at
identifying neurotoxic effects and their underlying molecular mechanisms.
Key words: apoptosis, necrosis, caspases, calpains, lysosomes, oxidative stress, VDAC.
References: 1) Ceccatelli S. et al., Chem. Biol. Interact., 188, 301-308, 2010. Review.
352) Tofighi R. et al., Neurotox. Res., 19, 183-194, 2011.
3) Elinder F. et al., Cell Death Differ., 12, 1134-1140, 2005.

[紹介]
演者の Tofighi 先生は、「スウェーデン・カロリンスカ研究所」において研究を行っておられます。今回の発表は、「メチル水銀曝露下での神経細胞及びグリア細胞における神経細胞死経路」についてです。メチル水銀は脳神経系において曝露量や曝露期間に応じてアポトーシ
スあるいはネクローシスという異なる細胞死を引き起こします。5 種類の培養細胞を用いた実験によって、メチル水銀に対する応答は細胞種によって異なり、様々な細胞死経路が活性化されることが明らかになりました。

Apoptosis: アポトーシス（プログラムされた“自殺性”の細胞死)
Necrosis: ネクローシス (壊死、“他殺性”の細胞死)Caspases: カスパーゼ (アポトーシス誘導に関与する蛋白質分解酵素)
Calpains: カルパイン (カルシウム依存性の蛋白質分解酵素)
Lysosomes: リソゾーム (細胞内小器官)
Oxidative stress: 酸化ストレス
VDAC (voltage-dependent anion channels): 電位依存性陰イオンチャネル

36SummaryIn this FORUM, we had seven topics on MeHg toxicity: up-to-date research onmechanisms, toxicology, and pathology.

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The first topic was MeHg-toxicity and selenium. Dr. Newland and Dr. Sakamoto
showed the protective effect of selenium component against MeHg cytotoxicity,although their reagents, rat models and assessment methods were different. Dr. Newland
showed that sodium selenite was useful to delay the onset of MeHg-related signs inchronic adult-onset MeHg exposure. And Dr. Sakamoto showed that selenomethionine
was useful to protect brain cells against MeHg-induced neuronal degeneration in fetaltype Minamata disease rat model.

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The second topic was MeHg-toxicity in the central nervous system in vitro and in vivo.
Dr. Ceccatelli mentioned the effect of MeHg on survival and differentiation of mouseneural stem cell line and rat primary embryonic cortical neural stem cells. Both neuralstem cells were highly susceptible to MeHg and showed apoptosis. In addition, at theconcentrations that were not cytotoxic but still were relevant for human exposure,
impairment of neuronal differentiation via Notch signaling was recognized in rat
primary cortical neural stem cells.

39Dr. Fujimura showed the process of MeHg-induced cerebrocortical neuronal cell death.
MeHg suppressed the expression of Rac1, which is known to promote neuritic extension,
during the early stage of MeHg-induced cytotoxicity, resulting in axonal degeneration
and ultimately neuronal apoptosis. Inhibition of the Rho/ROCK pathway protected cells
against MeHg-induced axonal degeneration and apoptotic neuronal cell death.

40Dr. Tofighi showed various signaling cascade leading to cell death under MeHg
exposure in 5 kinds of neuronal and glia cells. In these cells oxidative stress played acritical role in the onset of MeHg toxicity and apoptosis was induced via mitochondrial/
caspase-dependent and -independent pathways. The results suggest that cross-talkbetween the various pathways were activated concomitantly under MeHg exposure.

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The important roles of glia cells in MeHg-neurotoxicity were demonstrated by Dr.
Izumo and Dr. Aschner. Dr. Izumo showed neuropathological findings on molecularlayer of the cerebellum in MeHg-intoxicated model rat. Electron microscopy
demonstrated morphological changes in astrocyte processes with normal Purkinje cell
dendrites, and normal pre- and postsynapses. The results suggest that astrocytes areprimarily involved in acute phase of MeHg intoxication.

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Dr. Aschner showed comparative studies of the effect of MeHg on microglia andastrocytes. Microglia showed a lower basal glutathione pool and a significantly greater
mercury accumulation than astrocytes. Microglia was more susceptible to MeHg thanastrocytes. The results suggest the possibility that microglia is the first line of cellulardefense against MeHg toxicity in the central nervous system.

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The third topic was talked by two speakers. Dr. Hwang described the search for novel
genes involved in methylmercury resistance using yeast cells. Many of the identified
gene products were related to ubiquitin-proteasome systems. Cdc34 was one of them.
He also identified other proteins which enhanced methylmercury toxicity. They are
decreased by ubiquitin-proteasome system. The results suggest that ubiquitinproteasome system may play an important role as defense mechanisms against
methylmercury toxicity.

44Dr. Usuki showed the role of post-transcriptional defect of antioxidant selenoenzymes
in the incidence of MeHg-mediated oxidative stress. MeHg-induced relative seleniumdeficient condition affects the major antioxidant selenoenzymes through a
posttranscriptional effect, resulting in the disturbance of cellular redox systems and theincidence of oxidative stress. Treatment with ebselen protected cells against MeHginduced relative selenium deficiency and cytotoxicity.

45

The fourth topic on an approach from chemical aspects of MeHg was talked by two
speakers. Dr. Toyama showed the protective role of Keap1/Nrf2 system in MeHgcytotoxicity. The transcription factor Nrf2 is activated through the modification ofcysteines of Keap1 by electrophiles and oxidative stress. Nrf2 activators,
isothiocyanates, protect cells against MeHg-cytotoxicity. It was interesting that Wasabi
and broccoli sprout contain components of Nrf2 activator, isothiocyanate.

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MeHg is an electrophile and the conjugation of MeHg by nucleophiles is essential for its
detoxification. Recently hydrogen sulfide is found to be a biological signaling molecule.
CBS and CSE are known to be hydrogen sulfide producing enzymes. From the resultsof the experiments on the treatment with materials to generate hydrogen sulfide,
overexpression or knockdown of CBS, and CSE-knockout mice, Dr Kumagai showed
the possible protective role of hydrogen sulfide in MeHg-toxicity.

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The fifth topic was “Study on the MeHg-toxicity using small animal models”. Dr.
Martinez-Finley showed that C. elegans is a useful animal model system to investigate
MeHg-toxicity. C. elegans has the protective proteins against MeHg-toxicity such as
glutathione, heat shock protein, Skn-1 (an orthologue of mammalian Nrf2), andmetallothioneine, which are the same as mammals. And c. elegans allows us to visualize
individual neurons in vivo.

48

Zebrafish is another useful animal model for scientific research. Dr. Bourdineaud
showed the result of his research on the effect of dietary MeHg exposure atenvironmentally relevant doses. MeHg exposure mediated the modification of gene
expression pattern related to oxidative stress, ER stress, mitochondrial damage, anddetoxification. Morphological changes in muscle mitochondria and optical tectum inbrain were also observed. In addition, decreases in hatching and viability rates of theeggs were observed under MeHg exposure.

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The sixth topic on the effect of MeHg on microvascular systems was talked by Dr.
Hirooka. Study on cultured pericytes and endothelial cells showed that MeHg exposure
upregulated VEGF system proteins in these cells and increased the secretion of
hyaluronan from both cells. The results suggest hyperpermeability in these cells and thepossible induction of water accumulation in the extracellular matrix of vascular tissue.

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The seventh topic was MeHg-toxicity and chemokine. Chemokine CCL2 and its
receptor CCR2 are produced by neurons and glial cells. Dr. Rostene showed theprotective effect of CCL2 against MeHg-neurotoxicity using neuronal cell culture andCCL2 knock-out mice. MeHg increased CCL2 release in cortical neuronal cell culture.
Block of CCL2 and CCR2 in cortical neuronal cell culture and CCL2 knock-out micecaused significant increase in neuronal cell death under MeHg exposure.

51

Lastly, in this FORUM, materials listed here were shown to protect cells against MeHgtoxicity. We hope that continued investigation and discussion on MeHg research willlead to the development of useful drugs to treat with MeHg-intoxication.

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Photographs

Meeting Place (1)Meeting Place (2)Meeting Place (3)Meeting Place (4)Meeting Place (5)Group Photograph

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NIMD Forum 2011

February 21, 2011
平成 23 年 2 月 21 日

Published by National Institute for Minamata Disease, Ministry of the Environment,Japan
発行 環境省 国立水俣病総合研究センター

4058-18, Hama, Minamata, Kumamaoto 867-0008, Japan
Tel: +81-966-63-3111, Fax: +81-966-61-1145, http://www.nimd.go.jp〒867-0008 熊本県水俣市浜 4058-18

Inquiry: Department of International Affairs and Environmental Sciences
mda@nimd.go.jp
問い合わせ先：国際・総合研究部

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