Selenium Biofortification and
Antioxidant Function:
Complexities and Interactions
John Finley
USDA-ARS
Se: A brief history
• Originally studied for
toxicity
• Shown to be essential in
1957
• Present DRI of 55ug/d
Se as an Antioxidant: A Rush to
Judgement?
• Exudative diathesis in chickens – indicative of tissue damage:
•Free Radicals?
Se as an Antioxidant
Glutathione Peroxidase: the first known Selenoprotein
Selenocysteine at active site
Reaction:
2GSH + ...
Se as an Antioxidant
Reaction of GPX fit within hypothesized scheme of antioxidant
protection
Se as an Antioxidant
• Selenium deficiency = signs of oxidative stress
• First selenoprotein = activity to reduce hydrogen...
Selenium: A trace mineral with
antioxidant properties, selenium may
be useful in preventing arthritis and
other conditions...
Selenium as an Antioxidant:
Current Thinking
• Other ideas as to GPX function:
• Cell signaling
• Se storage protein
• No ...
Selenium BioFortifcation:
Complexities
Selenium variation in
foods from different
geographical
areas and/or different
suppliers (Finley et
al. 1996)
Food Se (mg/...
Selenium in pasta
USDA Nutrient Database
Food Selenium/serving
Spaghetti 20.2 µg Se
Whole-wheat spaghetti 23.7 µg Se
Macar...
Selenium in pasta
Italian pasta
Brand ug Se/100g
Venecia, angel hair 6.7±0.1
Davinci, fusilli 7.1±0.2
Venecia, linguini 5....
Se Distribution in U.S. Soils
= Low Se
::::::::
:::: = Variable Se
::::::::
::::
… = Se toxicity
Kubota and
Allaway, 1972
...
Meeting Se DRI with Se-enriched
Beef Amount of Se supplied by a 100g serving of
beef
0
50
100
150
200
250
Se,ug
Men Women ...
Se Accumulation in Plants
• Wheat = Accumulates Se in direct relationship
to amount in soil
•Total conc. 0.01 -15 pp
• Se ...
NH3
COOH
WHEAT
CH-CH2-CH2-Se-CH3
Selenomethionine
Transulfuration
pathway
NH3
COOH
CH-CH2-SeH
Selenocysteine
General prote...
Selenium from broccoli does not activate rat
TR mRNA (A) or accumulate in muscle (B)
as readily as other food forms of Se....
250 kDa 
98 kDa 
64 kDa 
50 kDa 
30 kDa 
16 kDa 
6 kDa 
MW Meat Broc Meat BrocMeat Broc Meat Broc
Liver Kidney Test...
Selenium from
broccoli does not
accumulate in the
plasma of healthy
men as well as Se
salts
Form matters – but does it in
real life?
• Western diet = mixed foods, mixed origin
Selenium BioFortification:
Interactions
Broccoli Extract SF
(mM)
Se
(mM)
0 SM 239  2 c 1.3  0.2 a
100 SM 157  3 b 35.4  1.3 b
1000 SM 41  3 a 354.0  5.6 c
S...
Selenium fertilization alters
phenolic acid profile in broccoli
0 ppm Se
1000 ppm Se
1,2'-disinapoyl-2-
feruloyl gentiobio...
0
5
10
15
20
TRActivity
mU/mgprot/min
* *
TRT 6
Cntrl 20% broc 3.2 umol Sf/d 52 umol Sf/d 20% B +1.6 Sf 20% B + 50 Sf
Thio...
0
100
200
300
400
500
600
700
800
900
GSH-PxActivity
mU/mgprot/min
*
*
Cntrl 20% broc 3.2 umol Sf/d 52 umol Sf/d 20% B +1....
1 2 3 4 5 6 7 8 9 10 11 12
Lanes 1-2- AIN 76 Diet
Lanes 3-4- 20% Broccoli Diet
Lanes 5-6- 0.32 mMol SF/kg diet
Lanes 7-8 5...
Concerted regulation of multiple
Antioxidant proteins
Consensus ARE Sequence (GST-A2)
5’-GTGACAAAGCA-3’
(-57) 5’-ATGACAAAGCA-3’(-46)
TR Promoter
Putative Antioxidant Response E...
Effect of Sulforaphane on TR or QR
Transcription (luciferase promoter
construct)
0
10
20
30
40
50
Firefly/renillaratio
5.0...
Effect of Sulforaphane on TR or QR
Transcription (luciferase promoter
construct)
0
2
4
6
8
10
12
14
16
firefly/renillarati...
Broccoli Extract SF
(mM)
Se
(mM)
0 SM 239  2 c 1.3  0.2 a
100 SM 157  3 b 35.4  1.3 b
1000 SM 41  3 a 354.0  5.6 c
S...
ANOVA
Treatment P value
Effect of selenite 0.0009
Effect of SF 0.29
Interaction Se*SF 0.33
0
20
40
60
80
100
120GSH-PxActi...
Se and SF synergistically
upregulate thioredoxin reductase
activity
0
10
20
30
40
50
60
TRActivity(mU/mgprot./min)
A
B B
C...
ANOVA
Treatment P value
Effect of selenite 0.69
Effect of SF <0.0001
Interaction Se*SF 0.14
0
100
200
300
400
500
600
QRAc...
Se fertilization affects the
ability of broccoli
extracts to induce TR and
QR activity
Summary
• TR is transcriptionally regulated by an
ARE
• Transcriptional regulation increases TR
activity beyond the max in...
Summary – Lessons learned
1. The real story is often more complex than
the theory
Summary – Lessons learned
1. The real story is often more complex than
the theory
2. Understanding nutritional chemistry i...
Summary – Lessons learned
1. The real story is often more complex than
the theory
2. Understanding nutritional chemistry i...
Summary – Lessons learned
1. The real story is often more complex than
the theory
2. Understanding nutritional chemistry i...
Collaborators and partners
• Cindy Davis, NCI
• Joel Caton, NDSU
• Mark Roberge, UND
• Korry Hintze, NDSU
• Anna Keck, U. ...
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Finley developed world

  1. 1. Selenium Biofortification and Antioxidant Function: Complexities and Interactions John Finley USDA-ARS
  2. 2. Se: A brief history • Originally studied for toxicity • Shown to be essential in 1957 • Present DRI of 55ug/d
  3. 3. Se as an Antioxidant: A Rush to Judgement? • Exudative diathesis in chickens – indicative of tissue damage: •Free Radicals?
  4. 4. Se as an Antioxidant Glutathione Peroxidase: the first known Selenoprotein Selenocysteine at active site Reaction: 2GSH + H2O2 → GS–SG + 2H2O
  5. 5. Se as an Antioxidant Reaction of GPX fit within hypothesized scheme of antioxidant protection
  6. 6. Se as an Antioxidant • Selenium deficiency = signs of oxidative stress • First selenoprotein = activity to reduce hydrogen peroxides • Subsequent selenoproteins = have same reactive site: (selenocysteine) Selenium = Powerful Antioxidant Need to fortify low and deficient popuulations
  7. 7. Selenium: A trace mineral with antioxidant properties, selenium may be useful in preventing arthritis and other conditions, including age-related blindness, cancers, cardiovascular disease, cataracts and kidney disease. You’ll get selenium from whole-grain wheat products and shellfish, such as oysters and crab. A recent study raised the possibility that taking selenium supplements might increase the risk of developing diabetes, so talk to your doctor before taking extra selenium. Getting more selenium in your diet appears safe.
  8. 8. Selenium as an Antioxidant: Current Thinking • Other ideas as to GPX function: • Cell signaling • Se storage protein • No protein positively associated with Se- deficiency diseases • No positive health benefits in Westernized nations that have fortified
  9. 9. Selenium BioFortifcation: Complexities
  10. 10. Selenium variation in foods from different geographical areas and/or different suppliers (Finley et al. 1996) Food Se (mg/100g)Food Se (mg/100g) Corn masa mix, Maseca 4.7±0.6 Corn masa mix, Masa Harina 49.8±0.8 Flour white all-purpose, Gold Medal 15.9±0.9 Flour, white all-purpose, Pillsbury 39.9±1.3 Ground beef - North Dakota 33.6±0.7 Ground beef - SW Missouri 6.0±1.2
  11. 11. Selenium in pasta USDA Nutrient Database Food Selenium/serving Spaghetti 20.2 µg Se Whole-wheat spaghetti 23.7 µg Se Macaroni 20.2 µg Average selenium content of pasta analyzed at Grand Forks Human Nutrition Center 20.5 µg/serving
  12. 12. Selenium in pasta Italian pasta Brand ug Se/100g Venecia, angel hair 6.7±0.1 Davinci, fusilli 7.1±0.2 Venecia, linguini 5.7±1.0 Avg Italian brands 6.2 American pasta Brand ug Se/100g Market Pantry, spaghetti 139±7.0 Creamette, shells 102±11 Leonardo, shells 100±10 Avg American brands 57.4
  13. 13. Se Distribution in U.S. Soils = Low Se :::::::: :::: = Variable Se :::::::: :::: … = Se toxicity Kubota and Allaway, 1972 Volcanic soils, low pH = Se deficient/low bioavailability Jurrasic Shale = high Se Weathering = Se released to soil
  14. 14. Meeting Se DRI with Se-enriched Beef Amount of Se supplied by a 100g serving of beef 0 50 100 150 200 250 Se,ug Men Women U.S. avg ND grass-fed SD feedlot-fed DRI 100 g Beef
  15. 15. Se Accumulation in Plants • Wheat = Accumulates Se in direct relationship to amount in soil •Total conc. 0.01 -15 pp • Se Accumulator = Accumulates Se well beyond availability from soil • Broccoli = up to 1500 ppm; • Astragalus = up to 10,000 ppm
  16. 16. NH3 COOH WHEAT CH-CH2-CH2-Se-CH3 Selenomethionine Transulfuration pathway NH3 COOH CH-CH2-SeH Selenocysteine General proteins Lyase SeH2SeO4 SeO3 GS-Se-SG Lyase GSH GSH NH3 COOH CH-CH2-Se-CH3 Se-methyl-selenocysteine H-Se-CH3 Lyase CH3-Se-CH3 BREATH tRNA-C-C-COH O tRNA-serine tRNA-C-C-C-SeH O Lyase CH3-Se-CH3 tRNA-selenocystein Selenoprotein incorporation CH3 URINE MEAT MEAT BROCCOLI GARLIC SELENATE SELENITE GSH-Px (5) deIodinase (3) Selenoprotein p Selenoprotein W TR Selenophosphate synthase 2 15kDa selenoprotein 18kDa selenoprotein Form Matters – understand the chemistry
  17. 17. Selenium from broccoli does not activate rat TR mRNA (A) or accumulate in muscle (B) as readily as other food forms of Se. 0.0 0.5 1.0 1.5 0 0.5 1 1.5 2 Dietary Selenium (ug/g) C 0 0.5 1 1.5 2 C1 C2 C3 C4 1 1.5 2 2.5 3 3.5 4 0 0.5 1 1.5 2 Dietary Selenium (ug/g) BA
  18. 18. 250 kDa  98 kDa  64 kDa  50 kDa  30 kDa  16 kDa  6 kDa  MW Meat Broc Meat BrocMeat Broc Meat Broc Liver Kidney Testis Plasma Incorporation of 75Se from broccoli or meat into selenoproteins Selenium from meat incorporates into selenoproteins But Se from broccoli does not
  19. 19. Selenium from broccoli does not accumulate in the plasma of healthy men as well as Se salts
  20. 20. Form matters – but does it in real life? • Western diet = mixed foods, mixed origin
  21. 21. Selenium BioFortification: Interactions
  22. 22. Broccoli Extract SF (mM) Se (mM) 0 SM 239  2 c 1.3  0.2 a 100 SM 157  3 b 35.4  1.3 b 1000 SM 41  3 a 354.0  5.6 c Selenium fertilization decreases sulforaphane content of broccoli Robbins, Banuelos, Finley and Keck, 2004
  23. 23. Selenium fertilization alters phenolic acid profile in broccoli 0 ppm Se 1000 ppm Se 1,2'-disinapoyl-2- feruloyl gentiobiose (11), 1-sinapoyl-2- feruloyl gentiobiose (8), 1,2,2'-trisinapoyl gentiobiose (10) and 1,2-disinapoyl gentiobiose (7), 1,2- diferuloylgentiobiose (9), and 3-O-caffeoyl- quinic (neochlorogenic acid) (2).
  24. 24. 0 5 10 15 20 TRActivity mU/mgprot/min * * TRT 6 Cntrl 20% broc 3.2 umol Sf/d 52 umol Sf/d 20% B +1.6 Sf 20% B + 50 Sf Thioredoxin reductase in livers of rats fed broccoli or SF
  25. 25. 0 100 200 300 400 500 600 700 800 900 GSH-PxActivity mU/mgprot/min * * Cntrl 20% broc 3.2 umol Sf/d 52 umol Sf/d 20% B +1.6 Sf 20% B + 50 Sf Glutathione peroxidase in livers of rats fed broccoli or SF
  26. 26. 1 2 3 4 5 6 7 8 9 10 11 12 Lanes 1-2- AIN 76 Diet Lanes 3-4- 20% Broccoli Diet Lanes 5-6- 0.32 mMol SF/kg diet Lanes 7-8 5.16 mMol SF/kg diet Lanes 9-10 20% Broccoli Diet + 0.16 mMol SF/kg diet Lanes 11-12 20% Broccoli Diet + 5 mMol SF/kg diet GSH-Px TR Effect of Sulforaphane from broccoli on Rat Hepatic GSH-Px and TR Protein
  27. 27. Concerted regulation of multiple Antioxidant proteins
  28. 28. Consensus ARE Sequence (GST-A2) 5’-GTGACAAAGCA-3’ (-57) 5’-ATGACAAAGCA-3’(-46) TR Promoter Putative Antioxidant Response Element (ARE) in TR Promoter
  29. 29. Effect of Sulforaphane on TR or QR Transcription (luciferase promoter construct) 0 10 20 30 40 50 Firefly/renillaratio 5.0 uM SF 2.0 uM SF 0.5 uM SF 0 uM SF TR
  30. 30. Effect of Sulforaphane on TR or QR Transcription (luciferase promoter construct) 0 2 4 6 8 10 12 14 16 firefly/renillaratio 5.0 uM SF 2.0 uM SF 0.5 uM SF 0 uM SF 0 10 20 30 40 50 Firefly/renillaratio 5.0 uM SF 2.0 uM SF 0.5 uM SF 0 uM SF TR QR
  31. 31. Broccoli Extract SF (mM) Se (mM) 0 SM 239  2 c 1.3  0.2 a 100 SM 157  3 b 35.4  1.3 b 1000 SM 41  3 a 354.0  5.6 c Selenium fertilization decreases sulforaphane content of broccoli Robbins, Banuelos, Finley and Keck, 2004
  32. 32. ANOVA Treatment P value Effect of selenite 0.0009 Effect of SF 0.29 Interaction Se*SF 0.33 0 20 40 60 80 100 120GSH-PxActivity mU/mgprot./min C SF Se SF+Se GPx activity in Hepa 1c1c7 cells
  33. 33. Se and SF synergistically upregulate thioredoxin reductase activity 0 10 20 30 40 50 60 TRActivity(mU/mgprot./min) A B B C Control 2uM Sel 2uM SF 2uM Se and 2uM SF
  34. 34. ANOVA Treatment P value Effect of selenite 0.69 Effect of SF <0.0001 Interaction Se*SF 0.14 0 100 200 300 400 500 600 QRActivity nmolDPIP/mgprot./min C SF Se SF+Se Quinone reductase in Hepa 1c1c7 cells
  35. 35. Se fertilization affects the ability of broccoli extracts to induce TR and QR activity
  36. 36. Summary • TR is transcriptionally regulated by an ARE • Transcriptional regulation increases TR activity beyond the max induced by Se • Sf from broccoli regulates TR activity • Sf + Se = additive induction of TR • Se antagonizes Sf accumulation in broccoli and vice versa
  37. 37. Summary – Lessons learned 1. The real story is often more complex than the theory
  38. 38. Summary – Lessons learned 1. The real story is often more complex than the theory 2. Understanding nutritional chemistry is essential
  39. 39. Summary – Lessons learned 1. The real story is often more complex than the theory 2. Understanding nutritional chemistry is essential 3. Understanding the soil/plant interaction is essential
  40. 40. Summary – Lessons learned 1. The real story is often more complex than the theory 2. Understanding nutritional chemistry is essential 3. Understanding the soil/plant interaction is essential 4. Enrichment of one nutrient may result in unexpected interactions, both in the plant and the animal that consumes it
  41. 41. Collaborators and partners • Cindy Davis, NCI • Joel Caton, NDSU • Mark Roberge, UND • Korry Hintze, NDSU • Anna Keck, U. Ill. • Allan Hovland, SD • Phil Whanger, OSU • Gary Banuelos, USDA • Mike Grusak, USDA • Karl Wald, GFHNRC • Eliz. Jeffery, U. Ill. • Yi Feng, U. Louis.
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