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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* * *

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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