v9 mexico 2012
DESCRIPTION
Palestra de Los Cabos, Mexico, sobre antioxidantes e tolerancia a anoxiaTRANSCRIPT
ANIMAL RESPONSES TO WILD FLUCTUATIONS IN OXYGEN AVAILABILITY
Revisiting the concept of “preparation for oxidative stress”.
Marcelo Hermes-LimaUniversidade de Brasília, Brasília, Brazil
In collaboration with Daniel Carneiro and Drs. Elida Campos and Alexis Welker
Los Cabos, MX, 2012
• 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.
• Twenty years ago it was already well-known that ROS can be overproduced following post-ischemic reperfusion in mammalian tissues.
• 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.
G lo b a l is c h e m ia ( 1 0 m in ) p lu s
r e o x y g e n a t io n ( 1 5 m in ) in a
r a t ; e f f e c t s in h e a r t ( S in g a l ’s
L a b , 1 9 9 3 )
Global ischemia (10 min) plus reoxygenation (15 min) in a rat; effects in heart (Singal’s Lab, 1993)
• 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 ?
Anoxic-tolerant turtle Ascorbate is a relevant defense in brain (Margaret Rice lab, 1991)
Anoxic-tolerant turtle
Ascorbate is a relevant defense in brain (Margaret Rice lab, 1991)
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.
Our first observations were presented in a cryobiology meeting, 20 years ago:
anoxia exposure in garter snakes
1992
1993
anoxia freezing
Hermes-Lima and Storey 1993
This is when we first proposed the “preparation” hypothesis
“(…) This pattern of preparation for oxidativeinsult 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. (…)”
1998
Anoxia (30 h at 5 degrees)Catalase: up in muscle and heartSe-GPX: up in heart and brainGST: up in brain
Heart Brain
Several studies corroborated our “preparation” proposal
1996
Muscle
2001
Liver catalase in hatchling turtles under anoxia and freezing/thawing
Dinkelacker et al., J. Comp. Physiol. B
Other enzymes were not studied…
Other enzymes were not studied…
2005
14 to 50 days of anoxia
GPX in mantle
HSP70 in hepatopancreas
2005
GPX was also “up” in gill and hepatopancreas (maximum at 24 days)
2010
Hepatopancreas: CuZn-SOD, Peroxiredoxin 5, GST, ferritin
20 days in hypoxia
2009
24 h hypoxia or anoxiaAdultsNauplius
hepatopancreasgills
High Shore
Low Shore
2009
2 h of air exposure
South Korea study
2009
2011
C. danae
C. ornatus
GPX
Ipanema Beach
CAT
xxxx 3 h air-exposure
2012
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 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!
Hypoxia
Hibernation
Hypoxic-sensitive vertebrates
The first “problem” happened in our own frog study, from 1996 !!
Controls, 10h anoxia, 30 h anoxia and two reoxygenation groups
Hermes-Lima and Storey 1996
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).
1998 (from Ken Storey lab)
Hypoxia for 5 h
2005
hepatopancreas
4 h air-exposure (hepatopancreas)
Almeida and Bainy 2006
2005
2007
Hours under aerial exposure
Oxidative stress during air exposure (0 to 24 h)
* **
* * *
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Carbonyl proteinCarbonyl protein
Peroxidation (CHP-eq)Peroxidation (CHP-eq)
Hypoxia in Perccottus glenii (Lushchak and Bagnyukova 2007)
Carbonyl protein Lipid peroxides
SOD
2010
Hypoxia for 5 or 9 days
Comet assay - erythrocytes
Hypoxia for 30 days
2011 paper
Hypoxia
Hypoxia
2012
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 !
PNAS 1998 !!
2005
Ebselen
“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 Stephen (2009).
RISP 5K: with iRNA to inhibit expression of the Rieske protein
Guzy et al. 2005
2007
http://jap.physiology.org/content/102/6/2379.full.pdf
1 h anoxia
2007
ROS determination in brain of turtle exposed to anoxia
Our current proposalactivation/stabilization of:
Nrf2
P53
HIF-1
NF-kappaB
induction of expression ofantioxidant enzymes
Preparation for oxidative stress
oxidative damage
Now it is tequila time !
DanielAlexis Élida