生体微量金属研究における興味と手法

proteome

金属化合物の新規化学形態分析法の構築と毒性発現機構解明における応用

genomemetallome

小椋康光

千葉大学大学院薬学研究院
予防薬学研究室2

生体微量元素の化学形態分析（スペシエーション、speciation）
分離部

selenium

検出部

Σελήνη, Selene‐ essential for animals but not plants
HPLC
システム

紫外-可視検出器

蛍光検出器

ICP-MS

LC‐ICP‐MS
分離部には、LCの他、GC、CE、AF4等も利用可能であるが、生体試料との相性から、LCが多用される。

‐ 25 genes encoding selenoproteins in human (ex. glutathione peroxidase,thioredoxin reductase, etc.)‐ ambivalent effects (essential but poisonous)

ICP‐MS以前は、原子吸光光度計やICP‐発光分光計も使われていた。

Se is transformed on its metabolic pathway with forming organic Secompounds.
“Identification of Se metabolite” is synonymous with “elucidation of Se
metabolism.”some advantages of cultured cells‐ animal welfare‐ cost‐ easier gene modification
etc....

From experimental animals
to cultured cells

Experimental design
intact cells‐ Human hepatocellular carcinoma cells, HepG2, were used.
‐ HepG2 cells were exposed with 10 µM selenite for 24 h.
‐ The supernatant was obtained by ultracentrifugation, andsubjected to an LC‐ICP‐MS.

cell‐free‐ The homogenate of HepG2 was prepared.
‐ The homogenate was mixed with 100 µM selenite and 10 mM GSH
for 1 h at 37˚C.
‐ The supernatant was obtained by ultracentrifugation, andsubjected to an LC‐ICP‐MS and LC‐ESI‐MS‐MS.

However, limited information about
the Se metabolism in cultured cells areavailable......

HepG2

Elution profile of Se in the supernatant of HepG2 cells
obtained by LC‐ICP‐MS
intact cells

cell-free

82Se intensity (CPS)?

Elution profile of Se in the several supernatants of cells
obtained by LC‐ICP‐MS?

GSSeH?

?
Retention time (min)Retention time (min)The unknown Se metabolite was also detected in the supernatant of humanembryonic kidney cells (HEK293), pheochromocytoma cells of the rat adrenal
medulla (PC12), mouse hepatoma cells (hepa1‐6), rat liver cell (RL34) andfibroblast‐like cells from a monkey kidney (COS7) although an amount of theunknown Se metabolite varied among these cell lines.

?

?

?

?

Hyphenated techniques for the identification of
unknown metallometabolome

ESI‐QMS spectrum of the supernatant of HepG2 cells incubated with sodium
selenite and glutathione
monomerdetection
techniques

100
Relative abundance (%)

separation
technique
HPLC

LC‐ICP‐MS
ICP‐MS

105.9Se

trimer
315.8
317.9
313.9

10050

311.8

103.8

7476 77 78

80820102.6
101.8

determination

319.9

310 315 320 325

107.6

0100

120

140

160

180200220240260

280300320m/z
LC‐ESI‐MS‐MS conditions (Waters Quattro Micro)
Column

LC‐ESI‐MS/MS

ESI‐Q‐TOF‐MS

identification

GS-320A-2E (multi-mode size exclusion)

Buffer

10 mM ammonium acetate pH 6.5

Flow rate

0.04 mL/min

Injection volume5 µL

Detection mode

negative ion modeSeCN‐ std

ESI‐Q‐TOF‐MS spectrum of the supernatant of HepG2 cells incubated withsodium selenite and glutathione

LC‐ICP‐MS conditions (Agilent 7700)
Columnobserved mass containing 80Se

105.92055

Count (x103)

3

Δ9.06 ppm
2105.92055

theoretical mass of 80SeCN‐

1105.91959

GS-520 HQ (multi-mode size exclusion)

Buffer

50 mM Tris-HCl, pH 7.4

Flow rate

0.6 mL/min

Injection volume

20 µL

unspiked

80Se: 79.91652: 12.00000
14N: 14.00307
12C0607080

90100 110 120 130 140 150 160 170

spiked with SeCN‐SeCN‐m/z

ESI‐Q‐TOF MS conditions (Agilent 6450)
BufferElution profiles of Se in the supernatantof HepG2 cells incubated with sodium
selenite and glutathione

0.3 % ammonia

Flow rate

0.1 mL/min

Injection volume

5 µL

Detection mode

negative ion mode

Retention time (min)SeCN‐ is made fromendogenous CN‐.

GSSeH
SeCN‐

rhodanese (‐)

SeCN‐

rhodanese (+)

SeCN‐ is non‐enzymatically synthesized.

S O 2‐

2.5E+07

SeCN‐

200000S2O232‐ 3

2.0E+07

SeCN‐

1500001.5E+07
10000034S Intensity (CPS)

GSSeHreaction mixture‐ thosulfate / GSSeH/(selenosulfate)‐ cyanide‐ cofactors‐ buffer‐ rodanese (‐) / (+)(b) + CN‐(a) no addition

Effect of rhodanese on the production of selenocyanate

1.0E+07
5.0E+06
0.0E+00
010203040

2.5E+07

82Se Intensity (CPS)

Elution profiles of Se in the supernatant of HepG2 cells incubated with sodium selenite,glutathione and cyanide

50000
00102030

40010203040

2000002.0E+07

1500001.5E+07

1000001.0E+07

Retention time (min)Retention time (min)50000

5.0E+06
0.0E+0000The addition of cyanide (CN‐) increased the amount of selenocyanate (SeCN‐).

10203040Retention Time (min)Why and
WhyHow is endogenous cyanide generated?

Why andHowHow is endogenous cyanide generated?

‐ To reduce the toxicity of selenite‐ Proposed mechanisms underlying the cyanide generation

Cytotoxicity of selenite (SeO32‐), cyanide (CN‐) and selenocyanate (SeCN‐) on
HepG2 cells

Cell viability (% of control)

120(A) N‐chlorination by myeloperoxidase

glycine10080

SeO32‐

60CN‐(B) acidic dismutation of N‐chloroglycine
H++SeCN‐

40(C) Decomposition to nitrile and cyanide

20HCN + CO200101001000

10000

Concentration (µM)
Cell viability was determined by MTT assay. Values are represented as means ± SD, n = 4-2HCl
Cipollone and Visca, IUBMB Life 59, 187‐189, 2007

Bioavailability, metabolism and toxicological effect of SeCN‐
Exogenous CN‐+ thiosulfate

rhodanese

thiocyanate (SCN‐)

Excess amount of Se (‐II) + Endogenous CN‐

Animal experiment I

Reactive
CyanogenSpecies(RCNS)Se deficient rat？

(GS-SeH, HSe-)enzymaticallyutilization for selenoprotein synthesis,
excretion into urine

non‐enzymatically

SeCN‐selenocyanate (SeCN‐)
detoxification of excess Se

Y. Anan, M. Kimura, M. Hayashi, R. Koike and Y. Ogra: Chem. Res. Toxicol. (2015) 28, 1803–1814
10.1021/acs.chemrestox.5b00254

Experimental design

60000

normal diet 22 days

12 h

12 heGPxuu, sselenite (i.v.),
0.5 mg Se/kg bw
Se‐deficient diet 22 days

12 h

12 hu
SeCN‐ (i.v.),
0.5 mg Se/kg bw

SeCN‐ gr.

Se‐deficient diet 22 days

12 hu, sSe CPS

40000

30000

20000

10000

10000
0051060000

15 20 25 30
Retention time (min)35

40

05selenite

50000

50000

40000

40000

30000

35

40

3540

30000

20000

20000

10000

10000
00u, s15 20 25 30
Retention time (min)

SeCN‐

510

15 20 25 30
Retention time (min)

35

40

0

5Retention time (min)

u10

60000

012 h

30000

20000

0Se CPS

selenite gr.

Sel P

Se CPS

12 h

Se intensity (cps)

Se‐deficient diet 22 days

urine
serum
12 h

Se‐def.

50000

40000

urine

Se‐def. gr.

60000

control

50000

Se CPS

control gr.

acclimation
5 daysElution profile of Se in the serum obtained by LC‐ICP‐MSsaline (i.v.)

1015 20 25 30
Retention time (min)Elution profile of Se in the urine (12‐24 h) obtained by LC‐ICP‐MS

GPx activity in the serum

120000120000control

100000100000

80000

80000

Se‐def.

Se CPS30

60000

Se intensity (cps)

40000

bb
10

c

40000

selenosugar

20000

000

120000510

15 20 25 30
Retention time (min)35400
120000selenite

100000100000

80000

80000
Se CPS

2060000

20000

Se CPSGPx activity (U/mg protein)aSe CPS

4060000
400000control

Se‐def.

SeCN‐1015 20 25 30
Retention time (min)

20000

SeCN‐
TMSe

60000

CH320000

51015 20 25 30
Retention time (min)354005Retention time (min)Bars marked by the different letters (a, b or c) are significantly different among groups.

Experimental design

Animal experiment II

acclimation
7 dayssaline (s.c.)1015 20
25 30
Retention time (min)1h？

SeCN‐ (i.v.), 1.0 mg Se/kg bw

Hg
Hg+SeO32‐
Hg+SeCN‐

354024 hSeO32‐

SeCN‐

CH3saline (i.v.)

selenite (i.v.), 1.0 mg Se/kg bw

SeCN‐

Se+00control

4040000

0HgCl235H3C

selenite

Bioavailability, metabolism and toxicological effect of SeCN‐

5HgCl2 (s.c), 2.5 mg Hg/kg bwurine, dissect.

Hepato‐ and nephrotoxicity

Effects of selenium compounds on AST and BUN.
Data are presented as means ± SD (n=4 ‐ 6) . *p＜0.05 vs. thecontrol group , #p＜0.05 vs. the Hg2+ group.
AST: asparate aminotrasferase
BUN: blood urea nitrogen

結論
亜セレン酸, セレノアミノ酸など
栄養源となる

Hg

HSe- / GSSeH

排泄

重要な代謝中間体
セレンタンパク質としての利用

セレンタンパク質

排泄CN‐
解毒あるいは貯蔵OHCH2OHOOHSeCH3NHCOCH3(CH3)3Se+

SeCN‐‐‐‐

細胞内の過剰なセレンは、一時的に解毒・貯蔵されるが、その際にセレノシアン酸に変換される。
セレノシアン酸の生合成過程は、非酵素的で“活性シアン種”の存在を想定させるものである。
化学形態分析により活性シアン種の想定に辿り着いたが、この毒性学的意義について、今後さらに研究を続ける必要がある。

Concentrations of Se and Hg in the liver and urine.
Data are presented as means ± SD (n=4 ‐ 6) . *p＜0.05 vs. SeO32‐ or SeCN‐ alonegroup , #p＜0.05 vs. the Hg2+ group. nd:not detected