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

1. Los Cabos, MX, 2012
ANIMAL RESPONSES TO WILD
FLUCTUATIONS IN OXYGEN AVAILABILITY
Revisiting the concept of
“preparation for oxidative stress”.
Marcelo Hermes-Lima
Universidade de Brasília, Brasília, Brazil
In collaboration with Daniel Carneiro and
Drs. Elida Campos and Alexis Welker

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. • 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. Our first observations were presented in
a cryobiology meeting, 20 years ago:
anoxia exposure in garter snakes
1992

5. 1993
anoxia freezing

6. Hermes-Lima and Storey 1993
This is when we first proposed
the “preparation” hypothesis

7. 1998
“(…) This pattern of preparation for oxidative
insult while under a state where oxyradical
formation should be diminished [talking about
estivation] is similar to what we have observed
earlier with other stress-tolerant animals. (…)”

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. 2001

10. Liver catalase in hatchling turtles under
anoxia and freezing/thawing 2005
Dinkelacker et al., J. Comp. Physiol. B
14 to 50 days of anoxia
Other enzymes were
not studied…

11. 2005
GPX in mantle
GPX was also “up” in
gill and hepatopancreas
(maximum at 24 days)
HSP70 in hepatopancreas

12. 2010
20 days in Hepatopancreas: CuZn-SOD,
hypoxia Peroxiredoxin 5,
GST, ferritin

13. 2009
Nauplius Adults 24 h hypoxia or anoxia

14. 2009
2 h of air exposure
gills hepatopancreas
High Shore
Low Shore

15. 2009
South Korea study

16. 2011
xxxx 3 h air-exposure
GPX C. danae
C. ornatus
CAT
Ipanema Beach

17. 2012

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 animals
Hibernation 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. 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. 1998 (from Ken Storey lab)
Anoxia (6 days)
and recovery in
Littorina littorea
Legend: control (open bar) ; 6 days anoxia (light bar); recovery for 30 min, 1 h, 5 h and 12 h (other bars).

21. Hypoxia for 5 h

22. hepatopancreas
2005

23. 2005
4 h air-exposure (hepatopancreas)
Almeida and Bainy 2006

24. 2007
Hours under aerial exposure

25. Oxidative stress during air exposure (0 to 24 h)
Carbonyl protein
* * * *
Peroxidation (CHP-eq)
* * *

26. Hypoxia in Perccottus glenii
(Lushchak and Bagnyukova 2007)
SOD
Carbonyl protein Lipid peroxides

27. 2010
Hypoxia for 5 or 9 days

28. 2011 paper
Hypoxia for 30 days
Hypoxia
Hypoxia
Comet assay - erythrocytes

29. 2012

30. Outdated reasoning based on our initial idea!
This is from a 2010 paper !
Initial evidences for increased
ROS formation in hypoxia dates
back from the late 90s !

31. PNAS 1998 !!

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. Guzy et al. 2005
RISP 5K: with iRNA to inhibit
expression of the Rieske protein

34. 2007
1 h anoxia
2007
http://jap.physiology.org/content/102/6/2379.full.pdf
ROS determination in brain of turtle exposed to anoxia

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. Now it is tequila time !
Alexis Daniel
Élida