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V9 mexico 2012


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Palestra de Los Cabos, Mexico, sobre antioxidantes e tolerancia a anoxia

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V9 mexico 2012

  1. 1. Los Cabos, MX, 2012 ANIMAL RESPONSES TO WILDFLUCTUATIONS IN OXYGEN AVAILABILITY Revisiting the concept of “preparation for oxidative stress”.Marcelo Hermes-LimaUniversidade de Brasília, Brasília, BrazilIn collaboration with Daniel Carneiro andDrs. Elida Campos and Alexis Welker
  2. 2. • It is known for folks gathered here, in Los Cabos, that a large number of animal species (aquatic or not) are able to withstand hours to months of exposure to conditions where oxygen availability can be quite limited.• These natural conditions include hypoxia, anoxia, aerial exposure, dehydration, freezing and, possibly, estivation/hibernation. Global ischemia (10 min) plus• Twenty years ago it was already well-known that reoxygenation (15 min) in a ROS can be overproduced following post-ischemic rat; effects in heart (Singal’s reperfusion in mammalian tissues. Lab, 1993)• Due to wild variations in oxygen availability, these animals may be under ischemic/reperfusion-like conditions, which could set a harmful state of oxidative stress.
  3. 3. • The big question, 20 years ago, was: what these animals do to survive a putative excess in ROS formation in reoxygenation. What are their secrets?• Do they have enormous amounts of endogenous antioxidants ? In 1986, Dr. Evaldo Reischl, from Brazil, showed the presence of SH-rich hemoglobins in a freshwater turtle (Phrynops hilarri); it winters underwater for months. He proposed that those SH groups could be a defense against reoxygenation-induced ROS formation. Anoxic-tolerant turtle Ascorbate is a relevant defense in brain (Margaret Rice lab, 1991)
  4. 4. Our first observations were presented in a cryobiology meeting, 20 years ago: anoxia exposure in garter snakes 1992
  5. 5. 1993anoxia freezing
  6. 6. Hermes-Lima and Storey 1993This is when we first proposedthe “preparation” hypothesis
  7. 7. 1998“(…) This pattern of preparation for oxidativeinsult while under a state where oxyradicalformation should be diminished [talking aboutestivation] is similar to what we have observedearlier with other stress-tolerant animals. (…)”
  8. 8. Several studies corroborated our “preparation” proposal 1996 Muscle Heart Brain Anoxia (30 h at 5 degrees) Catalase: up in muscle and heart Se-GPX: up in heart and brain GST: up in brain
  9. 9. 2001
  10. 10. Liver catalase in hatchling turtles under anoxia and freezing/thawing 2005 Dinkelacker et al., J. Comp. Physiol. B 14 to 50 days of anoxiaOther enzymes werenot studied…
  11. 11. 2005GPX in mantle GPX was also “up” in gill and hepatopancreas (maximum at 24 days)HSP70 in hepatopancreas
  12. 12. 201020 days in Hepatopancreas: CuZn-SOD,hypoxia Peroxiredoxin 5, GST, ferritin
  13. 13. 2009Nauplius Adults 24 h hypoxia or anoxia
  14. 14. 2009 2 h of air exposuregills hepatopancreas High Shore Low Shore
  15. 15. 2009South Korea study
  16. 16. 2011 xxxx 3 h air-exposureGPX C. danae C. ornatusCAT Ipanema Beach
  17. 17. 2012
  18. 18. Hypoxia There is a very large list of studies showing increase in expression or activity of endogenous antioxidants in animals under hypoxic/anoxic (or hypometabolic ) conditions . This is not limited to animalsHibernation from aquatic environments. However, in some studies there is clear evidence for oxidative stress under hypoxia or anoxia. This became a hard problem to deal with. But not quite anymore! Hypoxic-sensitive vertebrates
  19. 19. The first “problem” happened in our own frog study, from 1996 !! Hermes-Lima and Storey 1996 Controls, 10h anoxia, 30 h anoxia and two reoxygenation groups
  20. 20. 1998 (from Ken Storey lab)Anoxia (6 days)and recovery inLittorina littorea Legend: control (open bar) ; 6 days anoxia (light bar); recovery for 30 min, 1 h, 5 h and 12 h (other bars).
  21. 21. Hypoxia for 5 h
  22. 22. hepatopancreas2005
  23. 23. 20054 h air-exposure (hepatopancreas)Almeida and Bainy 2006
  24. 24. 2007 Hours under aerial exposure
  25. 25. Oxidative stress during air exposure (0 to 24 h) Carbonyl protein * * * *Peroxidation (CHP-eq) * * *
  26. 26. Hypoxia in Perccottus glenii (Lushchak and Bagnyukova 2007) SOD Carbonyl protein Lipid peroxides
  27. 27. 2010 Hypoxia for 5 or 9 days
  28. 28. 2011 paper Hypoxia for 30 days Hypoxia HypoxiaComet assay - erythrocytes
  29. 29. 2012
  30. 30. Outdated reasoning based on our initial idea! This is from a 2010 paper !Initial evidences for increasedROS formation in hypoxia datesback from the late 90s !
  31. 31. PNAS 1998 !!
  32. 32. 2005 “Cells express the FRET sensor following the transfer of a cDNA vector. This sensor is comprised of a redox sensitive regulatory domain, HSP-33, from the bacteria E. coli, to which have been attached cyan (CFP) and yellow (YFP) fluorescent proteins” Fearon and Ebselen Stephen (2009).
  33. 33. Guzy et al. 2005RISP 5K: with iRNA to inhibitexpression of the Rieske protein
  34. 34. 2007 1 h anoxia 2007 ROS determination in brain of turtle exposed to anoxia
  35. 35. Our current proposal activation/stabilization of: HIF-1 Nrf2 P53 NF-kappaB oxidative damage induction of expression of antioxidant enzymes Preparation for oxidative stress
  36. 36. Now it is tequila time !Alexis Daniel Élida