Mercury and selenium in seafood. Michiaki Yamashita. Selenoneine 2-selenyl-N,N,N-trimethyl-L-histidine

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1 Mercury and selenium in seafood Michiaki Yamashita Selenoneine 2-selenyl-N,N,N-trimethyl-L-histidine

2 不老不死の薬

3 Risk and benefit analysis neurotoxicity MeHg Molecular mechanism of metabolism, accumulation and toxicity risk assessment considering beneficial effects by animal trials and epidermiological studies Improved risk analysis

4 Ganther et al., Science 175, , 1972

5 Major epidemiological studies on methylmercury The Faroe islands study ( ) Neurobehavioral development tests were conducted at ages of 7 and 14 years of ages. Significant relationships between prenatal methylmercury exposure and several neurophysiological and neuropsychological endpoints were observed. The people eat pilot whale meat. The Seychelles child development study ( ) Neurodevelopmental tests were conducted at 6.5, 19, 29, and 66 months, and 9 years of ages. No effects of methylmercury exposure on nerves, cognition, and behavior of children were observed at any ages. Importance of selenium intake by fish consumption Se/Hg ratio Faroe islands 0.29 Seychelles 1.12 (National Institute of Minamata Disease, 2006)

6 A novel Se-containing imidazol antioxidant selenoneine reduced form Ergothioneine Firstly isolated from ergot (Tanret, 1909) Distributed in liver, red blood cell, testis Used as an antioxidant in food and cosmetics Selenoneine (oxidized form dimer) Yamashita and Yamashita, JBC 285, (2010)

7 Biological significance of selenium Group 16, the oxygen family (O,S,Se, Te) Essential trace element Dietary reference intake: µg/day Biological chemical form Selenocysteine residues in selenoproteins Low molecular wt, selenoneine Polypeptide chain NH CH CH 2 SeH Selenium deficiency diseases Keshan disease, Kashin-Beck disease, Myocardial necrosis Physiological effects Detoxification of heavy metals Thyroxine conversion Anti cancer Immune response Anti aging Insoluble inorganic Hg-Se in marine mammal liver CO

8 Selenium contents in bluefin tuna Tissues bluefin tuna blood kidney spleen dark muscle heart gill brain muscle Pacific mackerel muscle Pacific cod muscle Beef chicken muscle wheat Selenium content (mg Se/kg)

9 Speciation analysis of selenium compounds by HPLC-ICP-MS GPx selenite selenocystine selenomethionine standards selenoneine Retention time (min) GPC column:waters Ultrahydrogel 120 (7.8 x 300 mm) solvent:0.1 M formate ammonium Flow rate: 1 ml/min Observed element: Se-82

10 Se speciation analysis by HPLC-ICP-MS Bluefin tuna (body wt ~40 Kg) Selenoneine content in RBC 51.6 mg Se/kg Bluefin tuna dark muscle 3.04 mg Se/kg Bluefin tuna white muscle mg Se/kg Yamashita and Yamashita JBC 285, (2010)

11 Selenoneine and total Se contents in fish muscles pilot whale muscle bluefin tuna dark muacle swordfish muscle bigeye tuna muscle bluefin tuna muscle albacore muscle yellowfin tuna muscle skipjack muscle Pacific mackerel muscle red seabream muscle saury muscle Chum salmon muscle Japanese anchovy muscle beef selenoneine (mgse/kg) total Se (mgse/kg) Se (mg/kg)

12 Se speciation analysis of human RBC in the isolated island region of Kagoshima, Japan intencity (10 3 cps) intencity (10 3 cps) intencity (10 3 cps) #3 #4 #2 Se-protein * 5 10 * * selenoneine 5 10 retention time (min) total Se: mg Se/kg Selenoneine: mg Se/kg Selenoneine content 1.84 mg Se/kg 0.67 mg Se/kg mg Se/kg Selenoneine is a biomarker for fish eating and Se redox status

13 DPPH radical scavenging activity product RS 50 (µm) Trolox * 880 L ergothioneine 1700 selenoneine 1.9 * The water-soluble vitamin E-like antioxidant

14 Anti-oxidant activity of selenoneine normal human umbilical vein endothelial cell (HUVEC) Selenoeine was easily taken into the cells from culture medium No cytotoxicity against animal cells (~400 ppm) control Cell growth (%) control control 5µM selenoneine 5 µm H 2 O 2 25 µm H 2 O 2 5µM H 2 O 2 5 µm selenoneine 25µM H 2 O 2 5 µm selenoneine 100 nm selenoneine Selenoneine was detected in the cells

15 Effect on the cell growth Cell No. Zebrafish ZE 25 nm Cell No. Yellowtail YT 100 nm Cell No. Rainbow trout RTG nm Human endotherial HUVEC 100 nm Cell No. Human ataxia telanogiectasia mutated ATM Cell No. 25 nm Cell No. Human KIG3S 5 nm

16 GPx1 nm GPx4

17 zgpx1a-luc promoter assay in zebrafish ZE cells GPx1 promoter GPx1 promoter Selenoneine induced the GPx1 gene at a transcriptional level.

18 Selenoneine blocked metmb and ROS formation in vivo metmb in RM ROS in RM control Se-injected control Se-injected Red muscle ROS (AU) 0.07 Selenoneine vs. ROS in the red muscle 0.06 r2= Red muscle selenoneine (nmol/g) 1) Selenoneine was injected in blood vessel (12 nmol/kg). 2) After 18h of the injection, fish were killed and a fillet was prepared and stored for 72 h. Selenoneine-injected 3) The meat slice was stored for 4 C for 24 h.

19 Proposed function of selenoneine in redox cycle

20 Tamai et al.,1999 organic cations/carnitine transporter OCTN1 acts to selenoneine and MeHg excretion.

21 OCTN1 is a transporter of selenoneine OCTN1 transporter SLC22A4 The transporter expressed in blood cells. Gerhard et al., Blood 106: (2005) Selenoneine in zebrafish erythrocyte Km = 9.5 µm Selenoneine in OCTN1-expressed HEK293 cells Km = 13 µm Zebrafish erythrocyte Incorporated Se (cps) (min) (cps) Human kidney HEK293 cells transfected with hoctn1 (poctn1)

22 Abnormal morphogenesis and apoptosis induction by MeHg exposure anti-active caspase-3 staining of apoptotic cells

23 Protective effect of selenoneine against MeHg accumulation in zebrafish embryos MeHg exposure Se depletion ROS production Stress induced apoptosis Abnormal morphogenesis MeHg levels was reduced and inorganic Hg was produced by selenoneine exposure. Selenium levels were reduced by MeHg exposure. Selenoneine blocked MeHg accumulation and enhanced MeHg demethylation.

24 Possible metabolic pathway of selenoneine OCTN1 transporter