Hypoxia and oxidation levels of DNA and lipids in humans and animal experimental models

Critical Review

Hypoxia and Oxidation Levels of DNA and Lipids in Humansand Animal Experimental Models

Peter Møller1,* Lotte Risom1, Carsten Lundby2, Lone Mikkelsen1 and Steffen Loft11Institute of Public Health, University of Copenhagen, Copenhagen K, Denmark2The Copenhagen Muscle Research Centre, Rigshospitalet section 7652, Copenhagen N, Denmark

Summary

The objective of this review was to evaluate the associationbetween hypoxia and oxidative damage to DNA and lipids.Evaluation criteria encompassed specificity and validation statusof the biomarkers, study design, strength of the association,dose-response relationship, biological plausibility, analogousexposures, and effect modification by intervention. The collec-tive interpretation indicates persuasive evidence from the stud-ies in humans for an association between hypoxia and elevatedlevels of oxidative damage to DNA and lipids. The levels of oxi-datively generated DNA lesions and lipid peroxidation productsdepend on both the duration and severity of the exposure to hy-poxia. Largest effects are observed with exposure to hypoxia athigh altitude, but other factors, including ultraviolet light, exer-cise, exertion, and low intake of antioxidants, might contributeto the effect observed in subjects at high altitude. Most of theanimal experimental models should be interpreted with cautionbecause the assays for assessment of lipid peroxidation productshave suboptimal validity. � 2008 IUBMB

IUBMB Life, 60(11): 707–723, 2008

Keywords comet assay; oxidative DNA damage; lipid peroxidation;

oxidative stress.

Abbreviations 8-oxodG, 8-oxo-7,8-dihydro-20-deoxyguanosine; CI, con-fidence interval; ENDOIII, endonuclease III; FOX assay,

ferrous oxidation-xylenol orange assay; FPG, formamido-

pyrimidine DNA glycosylase; LH, lipid hydroperoxides;

MNBC, mononuclear blood cells; ROS, reactive oxygen

species; SB, strand breaks; SL, sea level; TBARS, thio-

barbituric acid reactive substances.

INTRODUCTION

Hypoxia may be defined as conditions of inadequate oxygen

supply to tissues, and is commonly experienced in humans

when lowlanders are exposed to high altitude. Hypoxia may

also be observed in patients with sleep apnea or other critical

conditions. In addition, there is more than 140 million people

worldwide living at altitudes above 2,500 m above sea level

(SL) (1). Short stay in high mountains is associated with alti-

tude illness, encompassing acute mountain sickness as well as

the potentially fatal conditions of high altitude pulmonary

edema or high altitude cerebral edema (1, 2). The risk of devel-

oping altitude illnesses increases with the level of altitude and

the speed of ascending. With exposure to acute and chronic hy-

poxia several defense mechanisms are initiated with the aim to

restore oxygenation levels.

In recent years, it has been hypothesized that exposure to

hypoxia may paradoxically be associated with elevated levels

of reactive oxygen species (ROS) and oxidative stress (3). It

is easier to assess the consequences of ROS generation by

measuring the level of oxidatively altered biomolecules,

including oxidation products of DNA and lipids, than assess-

ing the balance between oxidants and antioxidants. Oxidized

DNA lesions encompass a wide range of damages, including

strand breaks (SB) and base damage such as 8-oxo-7,8-hydro-

20deoxyguanosine (8-oxodG) (4). Similarly, there exist a

range of assays for the detection of lipid peroxidation prod-

ucts. However, it should be emphasized that many of the bio-

markers of oxidative stress are not reliable for in vivo tests

because they are unspecific or the analysis introduces spuri-

ous oxidation. In addition, the design of studies is of outmost

importance for the weight of evidence ascribed to various

investigations. For example, the effect of acute hypoxia can

be studied in controlled settings, but longer exposure periods

require special facilities and are mainly studied in mountains

as high altitude hypoxia. These studies typically have sequen-

tial designs that are weak because they do not control for pe-

riod effects (5). Stronger study designs encompassing parallel

groups of subjects exposed either to hypoxia or normal air

are difficult because subjects cannot wear oxygen masks

in mountains for longer times and long stay in chambers is

Address correspondence to: Peter Møller, Institute of Public Health,

University of Copenhagen, Øster Farimagsgade 5A, Postbox 2099, DK-

1014 Copenhagen K, Denmark. Tel: 14535327654.

E-mail: [email protected]

Received 13 March 2008; accepted 7 May 2008

ISSN 1521-6543 print/ISSN 1521-6551 online

DOI: 10.1002/iub.109

IUBMB Life, 60(11): 707–723, November 2008

associated with other difficulties such as altered cerebral

functions.

There are numerous studies on the association between expo-

sure to hypoxia and oxidation products of biomolecules in sam-

ples obtained from humans and animals. The aim of this survey

was to consider the strength of evidence that exposure to hy-

poxia is associated with oxidative stress with resulting damage

to DNA and lipids. To this end, we have used a molecular epi-

demiological approach and have developed a set of criteria to

assess the association between hypoxia and oxidative stress.

The survey focuses on biomarkers of DNA and lipid oxidation

products in animal experimental models and humans. These bio-

markers are intuitively comparable because both are damaged

biomolecules and can be regarded to reflect detrimental events.

IDENTIFICATION AND ANALYSIS OF STUDIES

The studies were identified by searches in the PubMed,

EMBASE, and Web of Science databases for endpoints of oxi-

dation products of DNA and lipids. We identified 40 investiga-

tions containing relevant data. Tables 1 and 2 outline detailed

summaries of the studies in humans in terms of DNA damage

and lipid peroxidation, respectively. Table 3 provides a sum-

mary of the investigations of oxidized DNA and lipid peroxida-

tion products in animal experimental models.

The analysis of the association between hypoxia and oxida-

tive stress is based on eight criteria encompassing issues related

to the biomarker, study design, plausibility, and magnitude of

exposure-effect relationship (Table 4). The criteria resemble

those of association (or sometimes referred to as the criteria of

causality or Bradford-Hill criteria) used in epidemiology (6).

However, it should be emphasized that we use these criteria in

the context of biomarker-based molecular epidemiology and

toxicology and they might have a different implication than in

epidemiology. The association criteria were originally discussed

as issues that would increase the confidence in a factor being

associated to a disease, whereas they nowadays sometimes are

misused as a checklist of causality where a lack of evidence

may be misinterpreted as a lack of causal relationship (7). The

criteria outlined in this survey should be viewed in a similar

way as epidemiological association criteria; the confidence in

an association is proportional to the number of fulfilled criteria.

A formal meta-analysis of the studies encompassed in the

survey is impossible because of differences in study design, ex-

posure length, and altitudes. The effect most likely depends on

both the duration and level of hypoxia. For practical purposes,

altitudes can be divided into low altitude (\2,500 m), high alti-

tude (2,500–3,500 m), very high altitude (3,500–5,800 m), and

extreme altitude ([5,800 m) (2). We stratified the studies into

these four classes of altitude. To resolve the time-window of

effect, the studies were categorized into four groups with dura-

tion of exposure as follows: period � 1 h, 1 h\ period � 24 h,

24 h\ period\ 8 weeks, period ‡ 8 weeks. For the analysis of

the effect of altitude level and time period, we calculated the

fold-difference in biomarkers relative to baseline data or the con-

trol groups. In case that the studies included data with more than

one biomarker per time point, or more than one time point per

biomarker, we calculated the geometric mean of the biomarkers.

This means that each study contributed with no more than one

data point for each group of classification. The dose-response

effect of hypoxia in different studies on DNA and lipid oxidation

markers was analyzed by one-factor ANOVA test on logarithmic

transformed data in order to achieve homogeneity of variance

between groups (Levene’s test). Trends of the dose-response rela-

tions were analyzed by categorized linear regressions. The analy-

sis of parametric tests was carried out in Statistica 5.5 for Win-

dows, StatSoft, (1997), Tulsa, OK, USA.

ASSESSMENT OF THE ASSOCIATION BETWEENHYPOXIA AND OXIDATIVE STRESS

Specificity of Biomarkers

The specificity of the biomarker reflects both methodological

properties and the biomarker’s ability to detect events in biolog-

ical or toxicological mechanisms. Oxidation products of lipids

and DNA can be considered as rough biomarkers of the internal

dose of oxidative stress, which depend on the intensity of oxida-

tive damage and the cellular ability of repairing or removing

those lesions or compounds. Therefore, the specificity refers to

the ability of these biomarkers to adequately assess the magni-

tude of oxidative stress.

Biomarkers of oxidative damage to DNA encompass meth-

ods that measure breaks in the DNA strand and base oxidation

products. Although exposure to oxidizing agents generates SB,

the assessment of SB in tissues and mononuclear blood cells

(MNBC) is usually carried out by methods that measure overall

genotoxicity rather than SB. The single cell electrophoresis

(comet) assay has become a very popular and sensitive assay

for the detection of SB in MNBC of humans exposed to differ-

ent occupational and environmental agents, but it is not a spe-

cific endpoint of oxidatively damaged DNA (8). The comet

assay provides a more reliable measurement of oxidative stress

when it is coupled to enzymatic digestion of DNA with endonu-

clease III (ENDOIII) or formamidopyrimidine DNA glycosylase

(FPG) from Escherichia coli that mainly detects oxidized purine

and pyrimidine lesions, respectively. Alternatively, DNA base

oxidation products in cells, tissues, and urine can be measured

by chromatographic methods. Urinary excretion products of oxi-

dized DNA are considered as measurements of whole-body ex-

posure representing repaired DNA lesions, sanitation of the nu-

cleotide pool, and apoptosis (9). The chromatographically mea-

surement of 8-oxodG in urine is considered to be more specific

than the antibody-based ELISA method where insufficient spec-

ificity of the antibodies is a problem (10, 11). A discrepancy

between these methods can be discerned in studies where the

subjects have been exposed to high altitude hypoxia for more

than 24 h; there is increased urinary excretion of 8-oxodG

detected by the antibody-based methods (12–14), whereas the

708 MØLLER ET AL.

Table

1

Oxidized

DNA

intissues,mononuclearbloodcells,andurineofhumansexposedto

hypoxia

Subjectsa

Ageb

Exposure

Biomarker

Baselinec

Effectd

Ref

Men

(7)

24(1)

Sim

ulatedaltitude(4,100m)

inahypobaric

cham

ber

withasingle

(2h)exposure

or2hexposureson14

differentdays(biopsies

wereobtained

afterthelast

exposure)e

Oxidized

DNA

(muscle)

SB(0.396

0.09

lesions/106bp),

ENDOIIIsites

(0.106

0.05

lesions/106bp),

andFPG

sites

(0.436

0.23

lesions/106bp)

Increasedlevel

ofSB(1.52-fold),but

notENDOIII(1.31-fold)andFPG

(0.91-fold)sitesafter2hhypoxia.

UnalteredlevelsofSB(0.98-fold),

ENDOIII(1.53-fold),andFPG

(0.83-fold)sitesafter14daysof2h

hypoxia

(42)

Men

and

women

(8)

22–31

Sim

ulatedexposure

(4,100m)

inmasksornorm

oxic

air

for1hwhilebicyclingwith

atan

intensity

of50%

of

VO2-m

axf

Oxidized

DNA

(MNBC)

SB(0.016

0.01

lesions/106bp)and

FPG

(0.106

0.09

lesions/106bp)

ThelevelsofSB(2.10-fold)andFPG

sites(1.09-fold)werenotstatistically

afterbicyclingin

hypoxia

(57)

Men

and

women

(25)

28(8)

Sim

ulatedaltitude(5,500m)

byinhalation10%

oxygen

inmasksfor2h.

Unexposedgroupinhaled

norm

alair.

Oxidized

DNA

(MNBC)

SB(0.076

0.05

lesions/106bp)and

FPG

sites(0.566

0.35lesions/106

bp)

Increasedlevel

ofSB(2.14-fold)and

FPG

sites(1.50-fold)

(47)

Men

and

women

(12)

26(5)

Bloodsamplestaken

at

baseline(D

enmark),and

after19h,2daysand3

daysstay

at4,559m

(Mt.

Rosa,Italy).Urinesamples

representbaseline

(Denmark),first19hand

subsequent24hat

high

altitude

Oxidized

DNA

(MNBC)

and

urinary8-

oxodG

excretion

(24h,

HPLC)

SB(0.156

0.14

lesions/106bp),

ENDOIIIsites

(0.066

0.05

lesions/106bp),

FPG

sites(1.076

0.45lesions/106

bp),andurinary

8-oxodG

(15.8

66.9

lesions/24h)

Thelevel

ofSBwas

increasedafter

19hhypoxia

(2.63-fold),2days

(2.76-fold),and3days(2.52-fold).

ENDOIIIsiteswereunalteredafter

19h(1.22-fold),whereasthey

were

increasedat

the2nd(1.94-fold)and

3rd

(2.88-fold)day

ofhypoxia.The

level

ofFPG

siteswas

unalteredafter

19h(1.14-fold),2ndday

(1.09-fold),

and3rd

day

(1.08-fold).Theurinary

excretionof8-oxodG

was

increased

duringthefirst19h(1.32-fold)and

unalteredfollowingthesubsequent

24h(1.03-fold)

(16)

Men

(18)

supplemented

with

antioxidants

orplacebo

26(5)

Sam

plestaken

atSLand

duringa2-w

eekstay

at

4,300m

(Pike’speak)

8-oxodG

(24h

urine,

HPLC)

3.4

60.7

ng/m

g

creatinine

Significantlylower

excretionof8-

oxodG

atthe2nd(0.91-fold),4th

(0.86-fold),9th

(0.79-fold),and12th

(0.83-fold)day

ofhypoxia,andno

effect

ofsupplementation

(15)

Table

1

(Continued)

Subjectsa

Ageb

Exposure

Biomarker

Baselinec

Effectd

Ref

Men

(40)

supplemented

withan

antioxidant

mixture

or

placebo

23(5)

Soldiers

participated

ina

wintermountain

operation

trainingat

2,053–2,804m

for24daysg

8-oxodG

(overnight

urine,

antibody-based)

7.5

66.1

lmol/

mgcreatinine

Increasedafterthe12th

(1.32-fold)

and24th

(1.70-fold)daysof

hypoxia.

(13)

Soldiers

(58)

supplemented

with

antioxidants

or

placebo

24(6)

MarineCorpsvolunteers

participatingin

awinter

fieldtraining(14day)

program

at2,546–2,804m

(cam

pground)andwith

daily

activities3,000m

aboveSL

8-oxodG

(spoturine,

antibody-based)

23.0

611.6

lMIncreasedafter14days(1.33-fold)

(12)

Soldiers

(30)

supplemented

with

antioxidants

orplacebo

22(3)

MarineCorpsvolunteers

participatingin

awinter

fieldtraining(14day)

program

2,053m

(training

center,presamples)

with

field-trainingat

2,743m

8-oxodG

(24hurine,

antibody-based)

156.5

6116ng/l/

kgfat-free

mass

Increasedurinaryexcretionof

8-oxodG

(day

7:2.02-fold,

day

14:2.13-fold)

(14)

Men

and

women

(7)

25(3)

Sam

plesobtained

atSL

(Denmark)andafter2and

8weeksstay

at4,100m

(Bolivia)

Oxidized

DNA

(muscle)

SB(0.446

0.20

lesions/106bp),

ENDOIIIsites

(0.076

0.07

lesions/106bp),

andFPG

sites

(0.236

0.13

lesions/106bp)

IncreasedlevelsofSB(1.37-fold)

andENDOIIIsites(4.0-fold)

after

2weeksofhypoxia,whereasno

effect

was

observed

after8weeks

(0.99-fold

and1.14-fold,

respectively).Thelevel

ofFPG

siteswas

unalteredat

both

time

points(1.0-fold

and1.09-fold,

respectively)

(40)

aThenumber

ofsubjectsisindicated

inbrackets.

bMeanandSD

(orrange).

cValues

representingthecontrolgroup(unexposedorsamplesobtained

atlow

altitude).Thedataarethemean6

SD.

dEffectis

outlined

asthefold-increase

relativeto

thecontrol(baseline)

values.Dataindicated

initalic

textreferto

statisticalsignificanteffect

reported

intheoriginal

publications.

eThemainstudy,includingdescriptionofthedesign,has

beendescribed

elsewhere(42).Thedataonoxidized

DNA

inmuscle

tissuehavenotbeenpublished

previously.Thelevel

ofoxidized

DNA

was

measuredin

muscle

biopsies

bythecomet

assayas

reported

previously(40).Thelevel

ofSB

was

increasedafter2hhypoxia

relativeto

thepre-hypoxia

samples(P

\0.05,repeatedmeasurement

ANOVA),whereasthelevelsofENDOIIIandFPG

siteswerenotstatisticallysignificant.

f Themainstudy,includingdescriptionofthedesign,has

beendescribed

elsewhere(57).Thestudyincluded

threeparts

asfollows:

(a)bicycling1hin

norm

oxia

at50%

VO2-m

ax,(b)bicyclingin

hy-

poxia

atan

intensity

equivalentto

50%

oftheVO2-m

axin

norm

oxia,(c)bicyclingin

hypoxia

atan

intensity

equivalentto

50%

oftheVO2-m

axin

hypoxia.ThelevelsofSBandFPG

siteswerestatistically

non-significantin

alloftheparts

(P\

0.05,repeatedmeasurementANOVA).ThelevelsofSB

andFPG

sitesbefore

andafterexercise

innorm

oxia

were0.036

0.02(SB,before),0.036

0.02(SB,

after),0.076

0.06(FPG,before),and0.056

0.04(FPG,after)lesions/106bp.

gTherewas

nobeneficial

effect

interm

softheoutlined

biomarkersoftheantioxidantintervention.Thedatarepresentthepooleddatafrom

theplaceboandinterventiongroup.

Table

2

Lipid

peroxidationproductsin

tissues,exhaled

air,andurineofhumansexposedto

hypoxia

Subjectsa

Ageb

Exposure

Biomarker

Baselinec

Effectd

Ref

Men

(14)

31(7)

Sam

plesobtained

after4min

hypoxia

(FiO

25

14%)and

afteramaxim

alexercise

test

TBARS(plasm

a;

spectrophotometry)

1.646

0.40lM

Noeffect

ofhypoxia

(1.03-

fold).TBARSincreased

afterexercise

inhypoxia

(1.37-fold),butnotin

norm

oxia

(43)

Men

(8)

30(3)

Sam

plesobtained

after10min

stable

hypoxem

iabyinhalation

of15%

O2

TBARS(plasm

ae)

343.2

623ng/m

L

(norm

oxia)

Unalteredlevel

ofTBARS

(1.05-fold)

(44)

Men

(18)

22(3)

Sam

plesobtained

before

and

afteran

increm

entalcycling

testin

norm

oxia

(FiO

25

21%)

andhypoxia

(FiO

25

16%)for

30min

f

LH

(serum)and

TBARS(plasm

a;

HPLC)

LH

(1.1

60.3

lM)

andTBARS(0.49

60.21lM

)

IncreasedLH

(1.08-fold)and

TBARS(1.16-fold)after

hypoxia

(48)

Men

andwomen

(15)

25(4)

Sam

plesobtained

after1h

inhalationof13.6%

O2

LH

(serum

g),TBARS

(spoturineat

1–2h

and24hafter

hypoxia;HPLC)

LH

(1.466

0.32

lM)andTBARS

(0.946

0.45

lmol/mg

creatinine)

Increasedlevelsofserum

LH

(1.10-fold),whereasthe

urinaryexcretionofTBARS

after24hwas

unaltered

(0.95-fold;1–2hresultsof

sampleswerenotreported)

(45)

Men

(8)

33(19–

53)

Inhalationof11%

O2or

norm

oxic

airfor1or2hin

a

paralleldesign

8-iso

PGF2a(plasm

a;

ELISA)

36.8

614.1

pg/m

L

(1h)and41.9

617.3

pg/m

L(2

h)

Unalteredlevelsafter1h

(0.95-fold)and2h(0.98-

fold)hypoxia

(49)

Men

(6)

36(2)

Sim

ulatedhypobaric

hypoxia

(5,500m)withsamplestaken

atSL(pre-sam

ple),1hand4

hofhypoxia,andim

mediately

afterreturn

toSL

TBARS(plasm

a;

spectrophotometry,

absorbance

at540nm

afterHPLC

purification)

7.876

3.18lM

Unalteredlevelsafter1h

hypoxia

(1.36-fold),

whereasincreasedafter4h

(1.50-fold).Unalteredafter

return

toSL(1.28-fold)

(46)

Men

(30)

21(1)

Sam

plesobtained

before

and

after2hcyclingwithdifferent

workload

asfollows:1)55%

ofVO2-m

axin

norm

oxia

and

exercise

innorm

oxia,FiO

25

21%;2)55%

ofVO2-m

axin

norm

oxia

andexercise

in

hypoxia,FiO

25

16%;3)55%

VO2-m

axin

hypoxia

(FiO

25

16%)andexercise

inhypoxia

(FiO

25

16%)

LH

(serum;FOX

assay)

TBARS(serum;

HPLCwith

fluorometric

detection)

LH

(0.596

0.06lM

(part1),0.626

0.16lM

(part2),

0.576

0.07lM

(part3))and

TBARS(0.486

0.1

lM(part1),

0.566

0.2

lM

(part2),0.656

0.2

lM(part3))

Increasedlipid

peroxidation

byhypoxia

interm

sofLH

(1.08-fold)andnoeffect

in

term

sofTBARS(1.02-

fold).

(51)

Table

2

(Continued)

Subjectsa

Ageb

Exposure

Biomarker

Baselinec

Effectd

Ref

Men

andwomen

(20)

24(2)

Cross-over

studywithsamples

obtained

atbaselineandafter

8hand15hofhypoxia

(FiO

25

12%)

LH

(serum,FOX

assay)

0.826

0.16lm

ol

Increasedlevel

ofLH

after8h

(1.25-fold)and15h(1.65-fold)

hypoxia.Noeffect

inthe

norm

oxia

treatm

ent.

(50)

Men

(28)

37(23–

58)

Sam

plesobtained

atSLand

after48hrin

highaltitude

(4,300m)

TBARS(plasm

a,

spectrophotometry,

absorbance

at532

nm)and8-iso

PGF2a

(spoturine)

TBARS(59.7

636

pmol/mgprotein)

and8-iso

PGF2a:

(1.316

0.8

g/lg

creatinine)

UnalteredlevelsofTBARS(1.07-

fold),whereastheurinary

excretionof8-iso

PGF2awas

increased(1.64-fold)

(41)

Men

(18)

supplemented

with

antioxidants

or

placebo

26(5)

Sam

plestaken

atSLand

duringa2-w

eekstay

at

4,300m

(Pike’speak)

LHg(plasm

a)5.4

60.9

lM

IncreasedLH

atthe3th

(1.14-fold),

5th

(1.27-fold),and10th

(1.43-

fold)day,andnoeffect

of

supplementation.

(15)

Sportsm

en

participatingin

asportscamp

(20)

24(3)

Sam

plestaken

atlow

altitude

(elevationnotreported)and

low

altitude(2,000m;time

notspecified).Low

altitude

sampleswereobtained

before

andafterexercise

atdays4,

10and18ofthestay.

TBARS(plasm

aand

erythrocytes;

spectrophotometry,

absorbance

at

532nm)

Plasm

a(0.506

0.17

lM)and

erythrocytes(18.0

68.4

nmol/g

hem

oglobin)

Unalteredplasm

aTBARSat

high

altitude(1.22-fold),butincreased

before

trainingat

the4th

(2.52-

fold),10th

(1.98-fold),and18th

(2.44-fold)day

ofhypoxia.

UnalteredTBARSin

erythrocytes

athighaltitude(1.28-fold),but

increasedbefore

trainingat

the

4th

(3.15-fold),10th

(1.98-fold),

and18th

(1.88-fold)day

of

hypoxia.

(35)

Men

(40)

supplemented

withan

antioxidant

mixture

or

placebo

23(5)

Soldiers

participated

inawinter

mountain

operationtraining

at2,053–2,804m

for24

daysh

Breathpentane,

LHg

(serum,kit),TBARS

(spectrophotometry;

overnighturine)

Breathpentane(0.19

nmol/l),serum

LH

(5.0

lmol/l),

urinaryTBARS

(6.0

ng/m

g

creatinine)

Increasedlevelsofbreathpentane

(2.54-fold),serum

LH

(1.16-fold)

after24daysofhypoxia.Urinary

TBARSwas

unaltered(1.18after

both

12after24daysofhypoxia).

(13)

Athletes(9)

24(19–

32)

Sam

plesobtained

atSLand

after8–15daysoflow

altitudehypoxia

(1,650m).

Sam

plingbefore

andafteran

exercise

test

atSL(20

biathlonrace)andhigh

altitude(30km

cross

country

skiing)

Conjugated

dienes

(serum)

16,8506

4,179

DAbs/l

Noeffect

afterhypoxia

(1.25-fold),

butincreasedafterexercise

(1.30-

fold

versusbaselineat

SL).

(36)

Table

2

(Continued)

Subjectsa

Ageb

Exposure

Biomarker

Baselinec

Effectd

Ref

Soldiers

(75)

supplemented

with

antioxidants

or

placebo

24(6)

MarineCorpsvolunteersparticipatingin

a

winterfieldtraining(14day)program

at

2,546–2,804m

(cam

pground)andwith

daily

activities3,000m

aboveSL

Breathpentane,

LH

(plasm

agand

serum

i ),urinary

malondialdehydej

andLHi

Breathpentane(0.1

60.08nM,n5

52),

plasm

aLH

(5.1

62.9

lM,n5

65),serum

LH

(datanotshown),

urinary

malondialdehyde

(0.306

0.34lM,

n5

69),urinary

LH

(4.9

62.7lM

,

n5

69)

Increasedbreathpentane(6th

day:4.3-fold

and14th

day:

16.9-fold),plasm

aLH

(1.08-fold),urinary

malondialdehyde(3.37-

fold),urinaryLH

(1.29-

fold).Unalteredlevelsof

LH

inserum

(resultsnot

shown).

(12)

Soldiers

(30)

supplemented

with

antioxidants

or

placebo

22(3)

MarineCorpsvolunteersparticipatingin

a

winterfieldtraining(14day)program

2,053m

(trainingcenter,presamples)

withfield-trainingat

2,743m

LH

(plasm

ag)and24

hurinaryexcretion

ofTBARSand

4-hydroxynonenal

Plasm

aLH

(116

5.6

lM),urinary

excretionofTBARS

(79.1

641ng/l/kg

fat-free

mass),and

4-hydroxynonenal

(1636

76ng/l/kg

fat-free

mass)

Increasedlevel

ofplasm

aLH

(1.30-fold,day

14)in

the

placeboandunalteredlevels

intheactivegroup.

Increasedurinaryexcretion

ofTBARS(day

7:2.03-

fold,day

14:2.59-fold),and

4-hydroxynonenal

(day

7:

2.07-fold,

day

14:2.30-

fold),withhigher

excretion

ofoxidationproductsin

the

supplementedgroup

(14)

Soldiers

(118-

148)

9(6)

Sam

pleswereobtained

after3wkor13

monthsin

highaltitude(4,000–4,500m)

TBARS(w

hole

bloode)

13.0

67.8

lMIncreasedlevel

ofTBARS

after3weeks(1.66-fold)

andunalteredafter13

months(1.05-fold)

(39)

Male

mountaineers

31(2)

Sam

plesobtained

before

andafterarrival

inPortugal

followingaclim

bingto

the

summitofMtPumori(7,161m).Three

wkabove5,250m.Post-sam

pleswere

obtained

afterreturningto

Portugal

(from

Nepal)4day

afterbeingat

5,250m.

TBARS(plasm

a,

spectrophotometry,

absorbance

at540

nm

afterHPLC

purification)

6.986

0.34lM

IncreasedTBARSafterhigh

altitudehypoxia

(1.48-fold)

(38)

Men

(10)

38(12)

Sam

plesobtained

atSL1(U

K),after

trekkingtoPangPem

abasecamp(5,100

m;206

5daysjourney

and76

5days

stay

atbasecamp),afterdescenttoSL2

(Kathmandu;day

39)

LH

(serum;FOX

assay)TBARS

(plasm

a;HPLC

withfluorometric

detection)

LH

(1.4

60.5

lM,

SL1)andTBARS(0.7

60.3

lM,SL1)

Increasedlevel

ofLH

(1.4-

fold;versusSL1andSL2),

whereasthelevel

of

TBARSwas

unaltered

(0.89-fold)

(20)

Table

2

(Continued)

Subjectsa

Ageb

Exposure

Biomarker

Baselinec

Effectd

Ref

Malesoldiers

(10)

29(4)

Sam

plesobtained

before

(Santiago;670m),during(day

3,2,500m;day

9,3,000m),

andafterascendingto

the

summitofSan

Pedro

ySan

Pablo

volcano(day

11,

6,125m)

TBARSandLH

(exhaled

breath

condensate;HPLC

andFOX

assay,

respectively)

TBARS(716

51

nM)andLH

(0.30

60.38lM

)

TBARSincreasedat

the3rd

(1.34),9th

(1.80-fold),and

11th

(1.82-fold)ofhypoxia.

LH

increasedat

the3rd

(2.81-fold),9th

(2.83-fold),

and11th

(3.80-fold)day

of

hypoxia.

(34)

Subjects(12)

given

vitam

inE

(2x200mg/d)

orplacebo

25–57

Sam

plestaken

before

(2,500m)

andafter4weeksat

5,000m.

Breathpentane

0.82ppm

(median)

Increasedlevel

ofbreath

pentanein

theplacebo

group(1.48-fold),and

unalteredlevelsin

the

vitam

inEsupplemented

group(1.05-fold)

(23)

Men

(8)

27(12)

Sim

ulatedhypobaric

hypoxia

withsamplestakes

atSL(pre-

samples).Successiveincreased

load

ofhypoxia

was

achieved

followingseven-day

stay

at

Vallotobservatory

(4,350m)

andthen

by6-daysstaysin

a

cham

ber

correspondingto

5,000m,6,000m,7,000m.

Final

exposuresrepresented

8,000m

(6h)and8,848m

(3h)

TBARS(plasm

a)e,k

246

0.97lg

/100

mL(SL)

Increasedlevel

ofTBARSat

6,000m

(1.2-fold),8,000m

(1.78-fold),and8,848m

(1.89-fold)

(37)

Mountaineers

(6)

Sam

plestaken

before

andafter

anexpeditionto

theHim

alayas

(8–10weeksabove5,000m)

Lipofuscin

(muscle;

electronmicroscopy)

0.02146

0.0106

percentoffiber

volume

Increasedlipofuscin

afterthe

expedition(3.35-fold).

(24)

aThenumber

ofsubjectsisindicated

inbrackets.

bMeanandSD

(orrange).

cValues

representingthecontrolgroup(unexposedorsamplesobtained

atlow

altitude).Thedataarethemean6

SD.

dEffectis

outlined

asthefold-increase

relativeto

thecontrol(baseline)

values.Dataindicated

initalic

textrefers

tostatisticalsignificanteffect

reported

intheoriginal

publications.

eBased

onthemeasurementofopticaldensity

at535and520nm

ofthiobarbituricacid

andhomogenatereactionproducts.

f Thestudywas

conducted

asainteractionbetweenhypoxia

andexercise.Only

thedataonhypoxia

areoutlined.

gThemethodisbased

ontheprinciple

that

LH

inplasm

areactwithleucomethylenebluein

thepresence

ofhem

oglobin

andtheproductionofmethyleneblueismeasuredbyitsabsorbance

at666nm.

hTherewas

nobeneficial

effect

interm

softheoutlined

biomarkersoftheantioxidantintervention.Thedatarepresentthepooleddataofboth

theplaceboandinterventiongroup.

i Themethodisbased

ontheprinciple

that

LH

inserum

orurinereactwithferrousions(Fe2

1)to

produce

ferric

ions(Fe3

1)that

aredetectedcolometricallybyreactionwiththiocyanate.

j MeasuredbyHPLC

astheadduct

form

edbyreactionofurinewith2,4-dinitrophenylhydrazine.

kThelevel

ofTBARSwas

also

measuredin

whole

bloodandthedata(statistical

results)

weresimilar

tothedataobtained

inplasm

a.

Table

3

Lipid

peroxidationproductsandoxidized

DNA

intissues,exhaled

air,andurineofanim

alsexposedto

hypoxia

Exposure

Anim

als

Biomarker

Effect(N

otes)

Ref

Sim

ulatedaltitudeat

4,000m

for4wkwithatraining

protocolincluded

(rats

killedoneday

afterthelast

trainingsession)

MaleWistarrats(6

weeks;

6rats/group)

TBARS(spectrophotometry)

andLH

(whiteandread

portionsofthequadriceps

muscle)

UnalteredlevelsofTBARS(1.14-fold)and

LH

(1.03-fold)a

(70)

Sim

ulatedaltitudeat

5,500m

forperiodsof0h

(controls),12hand1,3,5,

7,14,and21days.

MaleWistarrats(8

weeks;

5rats/group)

TBARS(serum,lung,liver,

heart,kidney,and

gastrocnem

iusmuscle;

spectrophotometry)

TBARSincreasedin

serum

atday

14(1.9-

fold)and21(2.5-fold).IncreasedTBARS

inthelung(day

21),liver

(12hand21

days),heart(1,5,and21days),andkidney

(7,14,and21days).Decreased

TBARSin

thegastrocnem

iusmuscle

(0.84-fold;12h).

(71)

Sim

ulatedaltitudeat

7,576m

6h/d

for5days

MaleSpragueDaw

ley

albinorats(12rats/

group)

TBARS(plasm

a,liver,lung

andheart;

spectrophotometry,

absorbance

at531nm)

TBARSincreasedbyhypoxia

(2.45-fold)

(72)

Sim

ulatedaltitudeat

7,576m

6h/d

for15daysin

a2x2

designwithvitam

inE

supplementation(40mg/rat

per

day)

MaleSpragueDaw

ley

albinorats(8

rats/

group)

TBARS(plasm

a,liver,lung

andheart;

spectrophotometry,

absorbance

at531nm)

TBARSincreasedbyhypoxia

inplasm

a(2.1-

fold),liver

(2.0-fold),lung(2.22-fold),and

heart(2.19-fold)ofnon-supplementedrats.

Complete

protectionoflipid

peroxidation

byantioxidantsupplementation.

(69)

Sim

ulatedaltitudeat

7,620m

6h/d

for1,7,14,and21

days

MaleSprague-Daw

ley

rats(12rats/group)

TBARS(liver

andthigh

muscle;spectrophotometry)

TBARSincreasedin

muscle

atday

1(1.13-

fold),7(1.36-fold)14(1.13-fold),and21

(1.03-fold).In

theliver

TBARSwas

increasedat

day

1(1.41-fold),7(1.92-

fold),14(1.61-fold),and21(1.35-fold)

(73)

Sim

ulatedaltitudeat

7,620m

(6h/d

for1wk)

MaleSprague-Daw

ley

rats(6

rats/group)

TBARS(m

uscle,liver,blood,

plasm

a;spectrophotometry,

absorbance

at532nm)

TBARSincreasedin

plasm

a(1.6-fold),blood

(1.5-fold),muscle

(1.3-fold),andliver

(1.5-fold)

(74)

Sim

ulatedaltitudeat

7,620m

(6h/d

for1wk)

MaleSprague-Daw

ley

rats(6

rats/group)

TBARS(m

uscle,liver,blood,

plasm

a;spectrophotometry,

absorbance

at532nm)

TBARSincreasedplasm

a(1.7-fold),blood

(1.5-fold),muscle

(1.4-fold),andliver

(1.6-fold)

(75)

Sim

ulatedhypobaric

hypoxia

at7,000m

for24h

CD1CharlesRiver

mice(10mice/group)

TBARS(soleusmuscle,

spectrophotometry)

TBARSincreasedin

muscle

(1.92-fold)

(76)

Inhalationof15%

O2in

nitrogen

for2–4wk

Fem

aleWistarrats

(20

weeks)

onnorm

aland

vitam

inEdeficient

diet

TBARS(serum,liver;

spectrophotometry)

IncreasedTBARSin

serum

b(1.32-fold)after

2wkandunalteredafter4wk(1.05-fold).

Nodifference

ingroupsofrats

eating

norm

alandvitam

inEdeficientdiet.

UnalteredTBARSin

theliver

(77)

Table

3

(Continued)

Exposure

Anim

als

Biomarker

Effect(N

otes)

Ref

Interm

ittentsimulatedhypoxia

at5,700m

(90min/d

for9

days)

or6,300m

(30min/d

for15days)

MaleWistarrats

(5months,5rats/group)

TBARS(erythrocytes;

spectrophotometry)and

lipofuscin

(erythrocytes,

fluorescence

microscopy)

The5,700m

protocolincreasedTBARS

(1.70-fold)andlipofuscin

(1.81-fold).

The6,300m

protocolincreasedTBARS

(1.29-fold)andlipofuscin

(2.52-fold)

(78)

Interm

ittentsimulatedhypoxia

at5,700m

(90min/d

for9

days)

or6,300m

(30min/d

for15days)

MaleWistarrats


TBARS(erythrocytes;

spectrophotometry)and

lipofuscin

(erythrocytes,

fluorescence

microscopy)

The5,700m

protocolwas

associated

with

increasedTBARS(1.55-fold)andunaltered

lipofuscin

(1.76-fold),whereasthe6,300m

protocolyielded

unalteredTBARS(1.27-

fold)andincreasedlipofuscin

(2.52-fold).

(79)

Inhalationof12%

oxygen

for

105min

(last15min

during

collectionofbreath)

Fem

aleFisher

rats

(22months)

feda

norm

alor30%

restricted

diet(10–12rats/group)

Breathpentane

Unalteredbreathpentane(1.03-fold)c

(80)

Inhalationof16%

oxygen

for

8weekswithorwith

unlimited

access

toa

runningwheel

MaleWistarRats

(4wk)

8-oxodG

(liver,antibody-

based

detection)

Decreased

hepatic

8-oxodG

levelsin

the

groupsexposedto

hypoxia

with(0.24-fold)

andwithout(0.36-fold)access

toarunning

wheel,relativeto

asedentary

groupat

norm

oxia.

(81)

Inhalationof10%

oxygen

for

3h,3days,or21days

MaleWistarrats


Lipofuscin

(erythrocytesand

spleen,fluorescence)

Decreased

inerythrocytesby3h(0.54-fold),

increasedafter3days(1.94-fold),and

decreased

after21days(0.54-fold)d.

Increasedin

thespleen

after3h(1.64-fold)

and21days(2.40-fold),whereasthe3

dayssample

was

notelevated

(1.48-fold).

(82)

Inhalationof10%

oxygen

for

3hor5days

MaleWistarrats


Lipid

peroxidationmarkers

measuredas

absorbance

at

586nm

followingreaction

withN-M

ethyl-2-

phenylindole

(lung)

Unalteredeffect

after3h(1.39-fold)and

increasedlevel

oflipid

peroxidationafter5

days(2.35-fold)

(83)

aThedifference

representsthemeanoftheread

andwhiteportionofthemuscle

tissue.

bThefold-increase

inserum

represents

themeanofratseatingnorm

alandvitam

inEdepleteddiet.

cThefold-inductionshownrepresentthemeanoftwodifferentnorm

alizations.Datanorm

alized

bycorrectedforCO2-productionshowed

a1.18-fold

increased,whereasdatanorm

alized

forflow

rate

and

bodymassrevealedless

breath-pentaneproductionbyhypoxia

(0.87-fold).Thedatarepresentthemeanofratsonnorm

alandrestricted

diet.

dThesedatarepresentem

issionat

310nm

(sam

eas

usedforthespleen

samples).Analysisofem

issionsat

360nm,415nm,and440nm

yielded

differentresults.A

calculationoftheaverageofthefour

measurementsindicates

noeffect

ofthehypoxia;0.94-fold

(3h),1.05-fold

(3days),1.00-fold

(21days).

Table

4

Descriptionofassociationcriteria

andremarksabouttheassociationbetweenhypoxia

andoxidativestress

Criteria

Description

Rem

arks

Specificity

ofthe

biomarker

Describes

theassociationbetweentheexposure

and

effect

ofthebiomarker.Specificbiomarkersareonly

influencedbyconfoundingfactors

ortechnical

artifactsto

minim

aldegree.

Alargeproportionofthestudiesonhypoxia

andoxidativestress

haveusedassaysforlipid

peroxidationproductsthat

areunspecificandshould

notbeusedforin

vivo

studies(e.g.

TBARS,LH).Several

studieshaveusedantibody-based

assaysforthedetectionoflipid


DNA

bases,whichareless

specificthan

chem

ical

detection.

Validation

statusofthe

biomarker

Describes

thestatusofthebiomarker

interm

sof

acceptedcommon(true)

values

andthepredictive

valueofthebiomarker

inrelationto

risk

ofdisease.

This

should

beinvestigated

incohortstudiesin

order

toavoid

risk

ofreverse

causality.

Althoughregarded

asan

unspecificmeasurementoflipid

peroxidation,elevated

levelsof

TBARSmeasuredbyspectrophotometrictechniques

areassociated

withhigher

risk

of

cardiovascularevents.Highurinaryexcretionof8-oxodG

isassociated

withincreased

risk

oflungcancerin

non-smokinghumans.

Studydesign

Crossover

andparallelstudydesignsthat

controlfor

theeffect

ofconfoundingfactors

(e.g.ultraviolet

radiationin

highmountains),orrandom

or

system

atic

variation(diurnal

orseasonal

variation)

over

timearemore

reliable

typeofstudiesthan

investigationsthat

arebased

onsequential

designs.

Allstudiesonhighaltitudehypoxia

arebased

ontheweaksequential

designs,whereas

studiesonacute

hypoxia

arebettercontrolled.

Plausibility

Theplausibilityrelatesto

themechanism(s)of

toxicity.Theremightbedifferentmechanismsof

actiondescribingtheeffect

ofvariousbiomarkersof

oxidized

biomoleculesanditis

possible

that

there

aredifferentmechanismsofactionin

different

tissues,even

thoughtheeffect

onthebiomarker

is

thesame.

Several

mechanismshavebeenproposedfortheassociationbetweenhypoxia

andoxidative

stress,includingreductivestress,inflam

mation,highcatecholamineproduction,episodes

oftissueanoxia/reoxygenationbecause

ofdem

andingphysicalperform

ance,andxanthine

oxidasemetabolism

withaccumulationofhypoxanthine.

Allofthesestressors

havebeen

associated

withelevated

generationofROS.Itis

possible

that

themechanismscausing

oxidativestress

differbetweenacute

andlong-term

hypoxia

andtheeffect

of

compensatory

mechanismsis

expectedto

beofincreasingim

portance

inlong-term

hypoxia.

Strength

ofthe

association

Largedifferencesbetweenexposedandunexposed

subjectsareless

likelyto

becausedbyweak

unmeasuredconfoundingorother

sources

ofmodest

bias.

Thereis

approxim

ately45%

difference

inlipid


DNA

betweenexposedandnon-exposedsubjects.Those

studiesshowingthelargesteffect

reportless

than

4-fold

elevated

levelsofoxidized

biomolecules.

Dose-response

relationship

Thedose-response

relationship

relatesto

theeffect

beingincreasedlinearlyornon-linearlyandthus

requires

differentlevelsofexposure.

Mostofthestudieshavenotassessed

theeffect

ofhypoxia

interm

sofintensity

or

duration.However,acrudeassessmentindicates

that

short-term

andmildexposuresare

less

proneto

beassociated

withelevated

levelsofoxidized

DNA

andlipids.

Analogy

Differentexposuresmay

elicitthesameeffect,which

could

becausedbyidenticalmechanismsofaction

orthesameendpointcanbealteredbydifferent

mechanisms.

Exposure

tohyperbaric

oxygen

pressure

isassociated

withoxidativestress

andpreexposure

tohyperbaric

oxygen

pressure

canreduce

themortalityandmorbidityofhypoxia.The

oxidativestress

effect

ofstrenuousexercise

andhypoxia

appears

tobesimilar

anditis

hypothesized

that

exercise

aggravates

theeffect

ofacute

hypoxia.

Intervention

Theeffect

should

bereducedbyinterventionwith

exposuresorcompoundsthat

affect

themechanism

ofaction.

Theevidence

that

antioxidantsreduce

thelevelsofoxidized

DNA

andlipidsfollowing

hypoxia

isequivocal.However,itshould

beem

phasized

that

studiesin

thisarea

areof

limited

valuebecause

ofpoordesignorunspecificbiomarkers.

HPLC-based methods have generated the opposite or no effect

(15, 16). These data can be interpreted as bias toward false pos-

itive associations between hypoxia and oxidative stress because

the antibodies detect the alterations of other cross-reacting prod-

ucts, but it might also imply that 8-oxodG is a poor marker of

hypoxia-induced oxidative stress.

There is a range of biomarkers of lipid peroxidation prod-

ucts. Some of the biomarkers have been severely criticized and

should be avoided for in vivo detection of lipid peroxidation

(17). These include the simple thiobarbituric acid reactive sub-

stances (TBARS) assay that should be dismissed because most

TBARS are not related to lipid peroxidation, whereas improved

methods using HPLC purification steps are more reliable assays

(18). The specificity of in vivo measurements has also been

questioned for the conjugated diene assay and the simple assays

for determination of lipid hydroperoxides (LH), including the

ferrous oxidation-xylenol orange (FOX) assay and similar

assays that are based on measurements of absorbance of oxida-

tion products, because they are unspecific measurements of lipid

peroxidation (19). Unfortunately, there appears to be a wide-

spread use of these assays in studies of hypoxia in humans

(Table 2) and animal experimental models (Table 3). An exam-

ple of bias toward significant positive association of an assay

with low reliability comes from a study of high altitude hypoxia

where lipid peroxidation was measured by the FOX assay,

whereas there was no effect in terms of the more demanding

technique measuring TBARS-adducts by HPLC with fluorometric

detection (20). The isoprostanes are probably the best available

biomarker of lipid peroxidation when measured by mass spec-

trometry with stable isotope dilution. However, detection of iso-

prostanes by immunoassays is the most widely used methods

because they are technically simple and cheap (18, 21). An alter-

native to the measurement of lipid peroxidation products in bod-

ily fluids is the assessment of hydrocarbons (pentane and ethane)

in exhaled air, although the validity and use of this biomarker is

hampered because the hydrocarbon gases are minor end products

of lipid peroxidation, there is not consensus on the background

level, and they are difficult to measure (18, 19, 22). Results from

this assay have only been reported in one of the pioneering

investigations of hypoxia-induced oxidative stress that showed

increased concentration of breath pentane in humans after a stay

at 5,000 m for 4 weeks (23). Lastly, lipid peroxidation has also

been measured as the accumulation of lipofuscin in muscle tissue

following an expedition to the Himalayas (24).

Validation Status of Biomarkers

The validation status of biomarkers implies a broadly

accepted notion among researchers that the method provides

reliable measurements that are reproducible and have a predic-

tive value in terms of health effect. Although a biomarker may

be specific, there may not be consensus about normal levels in

healthy humans. Presently, it is commonly accepted that the

true level of oxidized guanines in DNA is in the range of 0.3–

4.2 lesions/106 dG and there exist reference values for comet

assay endpoints (25, 26), whereas it remains challenging to

compare results of urinary excretion of 8-oxodG because the

data are reported in different units such as the concentration

and total excretion over a period of time. Typical levels of

plasma or serum LH and isoprostanes are known (21, 27).

Biomarkers are used as intermediate endpoints that measure

events in the mechanism of the disease. Therefore, the predic-

tive value is an important feature of the validity of biomarkers.

Oxidative stress is commonly regarded to be associated with

various diseases such as cancer, coronary artery disease, and di-

abetes, but the biomarkers of oxidative stress may be elevated

in patients as a consequence of the disease (28). The predictive

value of biomarkers should be evaluated in prospective studies

that may be a biobank-based type of cohort design (9). Using

this type of approach, it has been shown that urinary excretion

of 8-oxodG is a risk marker of lung cancer in non-smokers

(29). Elevated comet assay endpoints in MNBC appear to be

associated with increased risk of various cancers in case-control

studies, but this could be due to reverse causality (30). The pre-

dictive value of lipid peroxidation markers has been investi-

gated in a few longitudinal studies. Patients with stable coro-

nary artery disease and hemodialysis patients with high serum

levels of TBARS had increased risk of developing cardiovascu-

lar events (31, 32). In addition, high concentrations of plasma

lipid peroxidation products (denoted as malondialdehyde meas-

ured by a commercially available kit) was associated with

increased mortality in elderly institutionalized persons (33). To

the best of our knowledge, the predictive value of isoprostanes

has not been investigated in prospective studies, although there

is growing belief that it is the most valuable measurement of

the currently available biomarkers of lipid peroxidation (27).

Study Design

All the field studies in mountains in this survey have sequen-

tial design with respect to the effect of hypoxia (12–16, 20, 23,

24, 34–41), although some of the studies have been well con-

trolled in regard to effects related to supplementation with anti-

oxidants (12–14). Sequential study designs are also used to a

great extent in investigations of hypoxia by exposure in cham-

bers or masks (42–46), but there are also well-controlled studies

(47–51). Four of the well-controlled studies reported a relation-

ship between exposure to hypoxia and oxidative damage to both

DNA and lipids (47, 48, 50, 51), whereas one investigation

found no effect of hypoxia (49). Data based on sequential stud-

ies indicate hypoxia-induced effects on at least one biomarker

of lipid or DNA oxidation (12–16, 20, 23, 24, 34–42, 45, 46),

except two studies that happens to be short-term (less than 10

min) exposure to hypoxia (43, 44).

Biological Plausibility

Several mechanisms of hypoxia-induced oxidative stress at

high altitude have been put forward, including exercise, ultra-

violet light, lack of dietary antioxidants, increased catechol-

amine production, anoxia/reoxygenation, xanthine oxidase me-

718 MØLLER ET AL.

tabolism of accumulated hypoxanthine, and reductive stress (3).

Exposure to ultraviolet light and insufficient supply of dietary

antioxidants should be considered as independent exposure vari-

ables that might contribute to the increased level of oxidative

stress at high altitude. In general, hypoxia appears to generate

oxidative stress by a variety of mechanisms; some of the mech-

anisms can explain the effect observed by acute (hours) hy-

poxia, whereas others are likely to be more important during

prolonged (days and weeks) exposure to hypoxia.

Acute hypoxia per se appears to induce a state of reductive

stress that is characterized by accumulation of reducing equiva-

lents because of the inability to transfer electrons to oxygen in

the electron transport chain (52). The accumulation of electrons

in the electron transport chain may generate a variety of ROS

via, for example, the ubiquinone-ubiquinol redox couple (3).

Indeed, hypoxia generates a burst of ROS in skeletal muscles

that have high content of mitochondria (53). Moreover, mito-

chondria are considered to be involved in the oxygen sensing

where complex III of the electron transport chain generates

ROS in hypoxia, which act as signaling agents that trigger gene

expression via stabilization of the hypoxia-inducible factor (54).

It should be noted that if the electron transport chain is the

major source of ROS in hypoxia, nuclear DNA damage arises

only if the ROS are lipid soluble and stable molecules such as

H2O2 molecules that are able to cross the mitochondrial and nu-

clear membranes. Although the mitochondrial production of

ROS may explain the acute effect of hypoxia in mitochondria-

rich tissues or MNBC, this mechanism cannot readily explain

the increased levels of lipid peroxidation products in erythro-

cytes. It has been proposed that elevated levels of lipid peroxi-

dation products in that compartment, or plasma or serum, may

originate from higher rate of polyunsaturated fatty acid oxida-

tion in red cell membranes (45).

Prolonged hypoxia appears to activate the inflammation sys-

tem, determined from elevated concentrations of inflammatory

mediators (55, 56). The association between hypoxia, inflamma-

tion and oxidative stress is in accordance with the hypothesis

that inflammatory diseases are associated with oxidative stress

and elevated levels of oxidative damage to DNA and lipids.

Strength of the Association

In the assessment of epidemiological studies, the strength of

the association mainly serves to rule out that associations are

due to weak unmeasured confounding or other sources of mod-

est bias (6). The same argument can be applied in molecular ep-

idemiology, where we will have higher confidence in a true

effect of the biomarkers if the magnitude of the exposure-effect

relationships is large. The magnitude of the effect can be visual-

ized as the ratio and 95% confidence interval (CI) relative to

baseline samples or unexposed control subjects as done in Tables

1 and 2 for oxidation products of lipids and DNA in tissues of

humans, which shows that the effect is less than 2-fold in the

majority of the studies. An overall assessment of the strength of

the association, based on the effects reported in Tables 1 and 2,

indicates that exposure to hypoxia on average is associated with

45% (95% CI: 28–65%) higher levels of oxidized DNA lesions

and lipid peroxidation products. A further assessment, where the

analysis only include the best validated endpoints (ENDOIII and

FPG sites, urinary excretion of 8-oxodG by HPLC, and 8-iso-

PGF2a), shows that exposure to hypoxia is associated with 32%

(95% CI: 5–65%) higher levels of these biomarkers (15, 16, 40–

42, 47, 49, 57). Consequently, it appears that the strength of the

association might be overestimated when assessed by unspecific

endpoints, but even the best available endpoints point toward an

association between exposure to hypoxia and elevated levels of

oxidation products of DNA and lipids.

The strength of the association can also be compared with

other well-known exposures. It has been shown that a 3-day

stay at 4,600 m above SL or 2 h of inhalation of 10% oxygen

resulted in a net increase of 700 and 1,680 FPG sites/diploid

MNBC (16, 47). In addition, a shorter (60 min) and less severe

exposure (12.6% oxygen, corresponding to �4,100 m altitude),

only yielded a net increase of 55 FPG sites/diploid MNBC (cor-

responding to 0.01 sites/106 bp). In comparison, we have previ-

ously observed net increases in FPG sites of 360 and 900

lesions/diploid cell following exposure to air pollution in Co-

penhagen in two different studies (58, 59). In a study of air pol-

lution exposed populations in Benin we found a net difference

of �1,000 FPG sites per diploid cell (60). These comparisons

suggest that the effect elicited by short-term hypoxia is compa-

rable to that generated by urban air pollution.

The determination of the strength of the association of

urinary 8-oxodG excretion suggests a similar pattern of effect

although it is difficult to compare the data because changes in

8-oxodG urinary excretion rate depend on the way it has been

sampled. Results of 8-oxodG in urine that has been collected

over time (e.g., 24 h) and spot samples are difficult to compare.

In our study of high altitude hypoxia, the excretion of 8-oxodG

increased from 214 to 283 pmol/kg bodyweight/24 h (16). This

can be compared with the range observed among putatively

healthy non-smoking humans having a mean excretion of 213

pmol/kg bodyweight/24 h (95% CI: 190–236 pmol/kg body-

weight/24 h), which depends on the age and sex (61). Exposure

to hypoxia, thus, elicits an effect associated with urinary excre-

tion of 8-oxodG that deviates from the normal range. These val-

ues cannot directly be compared with the urinary excretion

reported as the concentration in spot urine (12) or the amount

excreted per unit creatinine (13), although the increases relative

to baseline samples in these studies were of similar size as our

estimate.

Dose-Response Relationship

Quite remarkably, it appears that there are no studies that

only have investigated the effect of different concentrations of

oxygen in inspired air for a fixed period of time. Most of the

studies have used a single exposure level, or designs that

include alterations in the exposure size (level of hypoxia) and

719HYPOXIA-INDUCED OXIDATIVELY DAMAGED DNA AND LIPIDS

duration (length of exposure). Probably the most convincing ex-

posure assessment would be an integrated dose over time such

as pack-years used in the exposure assessment of smoking.

However, the design of the studies on hypoxia precludes that

way of exposure assessment because the scenarios are difficult

to compare. For instance, oxygen level decreased over time in

studies of the ascend to the summit of San Pedro y San Pablo

volcano (46) and the simulated ascend to Mt Everest (37), but

it is impossible to split the time component from the effect of

hypoxia.

Figure 1 outlines the results of an overall analysis where

studies have been stratified into those with exposures lasting 1

h or less (43–45, 48), between 1 and 24 h (16, 42, 46, 47, 50,

51), and between 24 h and 8 weeks (12–16, 20, 23, 34–41).

This analysis indicates that the effect observed after days or

weeks of hypoxia is larger than at earlier time point, but it

should also be recognized that most of these studies are poorly

controlled and it may just be an effect of confounding factors.

Longer period of times (more than 8 weeks of high altitude hy-

poxia) have been investigated in less detail with unequivocal

results. One study showed elevated levels of lipofuscin in mus-

cle tissue (24), whereas another study indicated unaltered levels

of oxidized DNA in muscle tissue (40). The third of the studies

of long stay in high mountains showed unaltered whole-blood

TBARS levels after 13 months stay at 4,000–4,500 m (39).

An assessment of the effect of the level of hypoxia is

depicted in Fig. 2. This analysis does not indicate a strong rela-

tionship between the level of exposure and effect in terms of

oxidized lipids and DNA in various tissues and urine of

humans.

Analogy

Experiencing reduced oxygen supply is unique and analogues

exposures are difficult to find. However, the physical limitation

of strenuous exercise is the oxygen supply and it can therefore

be considered as an analogy. It is also well known that fast

ascends in mountains is a risk factor for the development alti-

tude illnesses (1, 2). Interestingly, there are studies showing an

aggravation of some biomarkers of oxidative stress by exercise

in mountain settings such as SB in MNBC (16) and serum con-

jugated dienes (36), whereas another study did not find such an

interaction in a controlled study with a relative modest exposure

of 16% oxygen for 2 h (51).

Hyperbaric oxygen treatment is another type of analogous

exposure that is associated with oxidative stress, increased lev-

els of lipid peroxidation products in plasma, urinary excretion

of 8-oxodG, and SB and FPG sites in leukocytes of humans

(62–65). In addition, it has been shown that the DNA damaging

effect in leukocytes can be inhibited if the subjects are preex-

posed to hyperbaric oxygen pressure (66). Prolonged exposure

to hyperbaric oxygen treatment is detrimental to rats, but rats

become less susceptible to hyperoxia-induced lung damage, and

have increased survival rate of lethal exposure of hyperoxia if

they have been preexposed to hypoxia (67, 68). These observa-

tions indicate that hypoxia and hyperoxia are associated with

oxidative stress, and although they may not cause oxidative

stress by identical mechanisms, they appear to elicit the same

type of adaptation of the antioxidant defense system.

Figure 1. Relationship between exposure to hypoxia and effect

of oxidative stress in terms of oxidized DNA or lipids in blood,

urine or exhaled air of humans exposed to hypoxia. For each

study (represented by one diamond), the fold-increase relative

to baseline values represents the geometric mean of DNA dam-

age or lipid peroxidation products in that study. The horizontal

lines indicate the mean of the groups (period � 1 h, 1 h \period � 24 h, 24 h \ period \ 8 weeks). Triangles and

whiskers represent the mean and 95% confidence intervals of

the transformed data. The data shows a trend in the exposure-

effect relationship (Ptrend 5 0.002).

Figure 2. Relationship between severity of hypoxia and effect

of oxidative stress in terms of oxidized DNA or lipids in blood,

urine or exhaled air of humans exposed to hypoxia. For each

study (represented by one diamond), the fold-increase relative

to baseline values represents the geometric mean of DNA dam-

age or lipid peroxidation products in that study. The horizontal

lines indicate the mean of the groups. Triangles and whiskers

represent the mean and 95% confidence intervals of the trans-

formed data. The 95% confidence intervals for the studies in

the lowest group (�2,500 m) are relative large (0.09- to 27.8-

fold) and are not shown in the figure. There is no trend in the

exposure-effect relationship (Ptrend 5 0.10).

720 MØLLER ET AL.

Intervention

The acceptance that hypoxia causes oxidative stress with oxi-

dations of biomolecules must relay heavily on the ability to

alleviate or prevent this condition by intervention with protec-

tive actions. There are at least two different revenues of inter-

vention as follows: (1) increasing the activity of the antioxidant

defense and DNA repair systems; (2) supplementation of ROS

scavenging agents.

The results of antioxidant supplementation to hypoxia-

exposed subjects have not been too promising so far. No effect

of antioxidant supplementation in terms of lower levels of oxi-

dized DNA or lipid peroxidation products were observed by

high altitude exposure or cold weather training in moderate alti-

tude (13–15), although a pioneering study revealed an effect of

vitamin E supplementation on the level of lipid peroxidation

(23). Supporting evidence from an animal experiment study on

vitamin E supplementation is unconvincing considering that the

dose of vitamin E was highly unrealistic for humans (the vita-

min E dose/kg bodyweight was 3,200-fold higher than the rec-

ommended daily allowance in humans) and the assay was the

spectrophotometric version of the TBARS method (69).

Although the effect of antioxidant supplementation may seem

discouraging at first sight we regard this field of phytochemical

supplementation to be preliminary. More studies are warranted,

but it should be emphasized that exposure to hypoxia may only

be associated with a 50% increase in the levels of oxidized lip-

ids and DNA damage products. This means that the statistical

power of studies is crucial.

SUMMARY

Table 4 provides a summary of the considerations on the cri-

teria of association between hypoxia and oxidative damage to

lipids and DNA bases. The specificity and validity of the DNA

damage products are more convincing than the lipid peroxidation

products, although some of the latter are biomarkers high qual-

ity. Importantly, an analysis restricted only to those of studies

that have measured the most reliable endpoints shows that sam-

ples from humans have higher levels of these biomarkers follow-

ing exposure to hypoxia. Taken into consideration the dose-

response, which is depicted in Fig. 1, we believe that there is

persuasive evidence from studies in humans for an association

between hypoxia and oxidative stress in terms of oxidized DNA

and lipids. In addition, the biological plausibility and analogy

with similar types of exposures strengthen this notion. Still, the

major limitation remains to be the few properly controlled stud-

ies in the investigations of high altitude hypoxia because the

exertion, ultraviolet radiation and reduced antioxidant intake at

high altitude might be more important than reductive stress.

Unfortunately, most of the animal experimental models should

be interpreted very cautiously because the biomarker assays are

poor, especially the assays for the lipid peroxidation products.

Future studies on the association between hypoxia and oxida-

tive damage to DNA and lipids should focus on use of highly

validated biomarkers and well-controlled experimental designs.

This may show that the effect of hypoxia is weaker than the

45% increase in biomarkers that we find in this survey. The

strongest experimental evidence for the association between hy-

poxia and oxidative stress will come from intervention studies.

Assuming only a 32% effect in biomarkers of oxidative stress,

intervention studies will be challenging because it requires

many subjects and biomarkers with low assay variation. The

effect of defense mechanisms such as the DNA repair system

needs to be addressed thoroughly in order to clarify possible

underestimations of the effect of hypoxia in terms of oxidized

DNA in steady-state situation of prolonged hypoxia.

ACKNOWLEDGEMENTS

The authors (LR, SL, and PM) of this article are partners of

ECNIS (Environmental Cancer Risk, Nutrition and Individual

Susceptibility), a network of excellence operating within the

European Union 6th Framework Program, Priority 5: ‘‘Food

Quality and Safety’’ (Contract No 513943).

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Hypoxia and oxidation levels of DNA and lipids in humans and animal experimental models