Degradation

of

by

Arthrobacter

sp.

strain

A

Gram-positive

bacterial

strain

capable

of

aerobic

biodegradation

of

-LRB-

-RRB-

as

the

sole

source

of

carbon

and

energy

was

isolated

by

selective

enrichment

from

soil

samples

collected

near

an

industrial

site

The

organism

designated

strain

was

identified

as

a

member

of

the

genus

Arthrobacter

on

the

basis

of

ribosomal

RNA

gene

sequence

analysis

Arthrobacter

strain

was

able

to

mineralize

up

to

concentrations

of

mM

in

batch

culture

Stoichiometric

release

of

fluoride

ions

was

observed

suggesting

that

there

is

no

formation

of

halogenated

dead-end

products

during

metabolism

The

degradative

pathway

of

was

investigated

using

enzyme

assays

and

identification

of

intermediates

by

gas

chromatography

-LRB-

GC

-RRB-

GC

mass

spectrometry

-LRB-

MS

-RRB-

high-performance

liquid

chromatography

and

liquid

chromatography

MS.

Cell-free

extracts

of

cells

contained

no

activity

for

catechol

or

catechol

which

indicates

that

the

pathway

does

not

proceed

through

a

catechol

intermediate

Cells

grown

on

oxidized

hydroquinone

and

hydroxyquinol

but

not

During

metabolism

hydroquinone

accumulated

as

a

product

Hydroquinone

could

be

converted

to

hydroxyquinol

which

was

further

transformed

into

maleylacetic

acid

and

β-ketoadipic

acid

These

results

indicate

that

the

biodegradation

of

starts

with

a

monooxygenase

reaction

that

yields

benzoquinone

which

is

reduced

to

hydroquinone

and

further

metabolized

via

the

β-ketoadipic

acid

pathway

Introduction

During

the

past

decades

widespread

application

of

fluoroaromatic

compounds

as

agrochemicals

and

pharmaceuticals

has

lead

to

an

increased

occurrence

of

environmental

contaminants

containing

fluorine

-LRB-

Key

et

al.

-RRB-

Fluorinated

compounds

are

rare

in

nature

-LRB-

Harper

and

O'Hagan

-RRB-

The

stability

of

the

carbon

fluorine

bond

-LRB-

kcal/mol

in

compared

to

kcal/mol

for

the

carbon-chlorine

bond

in

-RRB-

makes

most

fluorine-containing

compounds

much

more

resistant

to

biodegradation

than

their

unsubstituted

analogs

-LRB-

Key

et

al.

-RRB-

Furthermore

the

van

der

Waals

radius

of

fluorine

is

small

-LRB-

Å

in

between

that

of

a

hydrogen

and

an

oxygen

-RRB-

Yet

the

electronegativity

of

fluorine

causes

strong

polarization

of

C

F

bonds

and

fluorine

substituents

may

be

involved

in

biological

interactions

-LRB-

Howard

et

al.

-RRB-

Regardless

of

the

recalcitrance

of

most

fluoroaromatics

to

biodegradation

several

bacterial

cultures

have

been

described

that

aerobically

degrade

fluorobenzoic

acids

-LRB-

Oltmanns

et

al.

Engesser

et

al.

Harper

and

Blakley

Schlomann

et

al.

-RRB-

Research

on

bacterial

fluorophenol

degradation

has

been

limited

to

studies

with

whole

cells

cell

extracts

or

purified

enzymes

from

Rhodococcus

species

that

were

obtained

by

enrichment

with

other

aromatic

compounds

as

a

growth

substrate

-LRB-

Boersma

et

al.

Bondar

et

al.

Finkelstein

et

al.

-RRB-

The

fluorobenzene-degrading

organism

Rhizobiales

could

grow

on

-LRB-

-RRB-

but

information

on

the

pathway

of

metabolism

is

lacking

Cometabolic

degradation

of

difluorophenols

and

trifluorophenols

by

several

Rhodococcus

species

is

initiated

by

a

phenol

hydroxylase

that

catalyzes

ortho-hydroxylation

resulting

in

the

formation

of

the

respective

fluorocatechol

which

is

then

cleaved

by

an

intradiol

dioxygenase

to

produce

fluoromuconate

-LRB-

Bondar

et

al.

-RRB-

Conversion

of

by

whole

cells

of

the

phenol-degrading

organism

Rhodococcus

opacus

resulted

in

the

formation

of

and

fluoromuconates

-LRB-

Finkelstein

et

al.

-RRB-

Yeasts

and

fungi

that

are

able

to

cometabolically

transform

fluorinated

phenols

have

also

been

described

Whole

cells

of

Exophiala

jeanselmei

transformed

into

and

-LRB-

Boersma

et

al.

-RRB-

Penicillium

frequentans

metabolized

monofluorophenols

in

the

presence

of

glucose

or

phenol

The

metabolism

of

meta-or

para-fluorophenols

yielded

the

corresponding

catechol

and

-LRB-

Hofrichter

and

Schreibner

Hofrichter

et

al.

-RRB-

of

these

organisms

could

utilize

fluorophenols

as

a

growth

substrate

To

our

knowledge

studies

on

the

metabolism

of

fluorophenols

by

a

bacterial

culture

that

is

capable

of

using

such

as

compound

as

a

sole

source

of

carbon

and

energy

have

not

been

reported

In

the

present

paper

we

describe

the

isolation

and

characterization

of

a

bacterial

strain

growing

on

as

the

sole

source

of

carbon

and

energy

Based

on

the

identification

of

several

intermediates

a

metabolic

route

for

the

degradation

of

by

this

strain

is

proposed

Materials

and

methods

Media

and

growth

conditions

NB

medium

contained

g

of

Nutrient

Broth

-LRB-

Difco

-RRB-

per

liter

Mineral

salts

medium

-LRB-

MM

-RRB-

contained

per

liter

g

g

g

g

-LRB-

-RRB-

ml

trace

metals

solution

-LRB-

Janssen

et

al.

-RRB-

and

mg

of

yeast

extract

-LRB-

Difco

Laboratories

-RRB-

When

required

the

solid

medium

was

obtained

by

adding

g/l

of

Difco

agar

Strain

was

grown

at

°C

on

a

rotary

shaker

-LRB-

rpm

-RRB-

Cultures

were

grown

in

flasks

filled

to

of

their

volume

and

were

closed

with

Teflon-lined

screw

caps

E.

coli

cells

were

grown

in

Luria

Bertani

medium

-LRB-

LB

-RRB-

at

°C

on

a

rotary

shaker

Enrichment

and

isolation

of

cultures

A

variety

of

soil

samples

collected

from

different

sites

in

The

Netherlands

that

are

contaminated

with

halogenated

aliphatic

compounds

-LRB-

such

as

monochlorobenzene

hexachlorobenzene

and

trichloropropane

-RRB-

were

used

as

the

initial

inoculum

for

the

enrichments

The

soil

samples

were

used

to

inoculate

flasks

containing

ml

of

sterile

minimal

salts

medium

and

mM

of

supplied

in

the

liquid

phase

as

the

sole

carbon

and

energy

source

The

cultures

were

incubated

at

room

temperature

on

a

rotary

shaker

-LRB-

rpm

-RRB-

and

of

the

suspension

was

transferred

to

a

new

flask

containing

fresh

medium

every

days

During

this

time

growth

-LRB-

optical

density

at

nm

-RRB-

and

liberation

of

fluoride

were

monitored

Samples

of

the

enrichment

culture

were

periodically

spread

onto

minimal

salts

agar

plates

containing

mM

and

onto

NB

plates

as

soon

as

growth

on

was

established

Pure

cultures

were

obtained

by

repetitive

streaking

onto

solid

MM

containing

and

tested

separately

for

growth

on

mM

liquid

medium

Growth

and

fluoride

release

were

again

monitored

to

verify

degradation

Strains

capable

of

degradation

as

a

sole

source

of

carbon

and

energy

were

used

for

further

experiments

Strain

was

deposited

at

Centraalbureau

voor

Schimmelcultures

Utrecht

The

Netherlands

under

accession

number

NCCB

Sequencing

of

the

rRNA

gene

For

cloning

of

the

ribosomal

ribonucleic

acid

-LRB-

rRNA

-RRB-

gene

a

single

colony

of

strain

was

directly

used

for

polymerase

chain

reaction

-LRB-

PCR

-RRB-

amplification

The

primers

-LRB-

-RRB-

and

-LRB-

Marchesi

et

al.

-RRB-

were

used

for

PCR

amplification

The

PCR

reaction

mixture

-LRB-

μl

-RRB-

contained

Taq

PCR

buffer

mM

pmol

of

each

appropriate

primer

mM

of

each

deoxyribonucleotide

triphosphate

U

Taq

DNA

polymerase

and

biomass

of

strain

The

PCR

conditions

were

°C

for

min

followed

by

min

at

°C

min

at

°C

min

at

°C

and

min

at

°C

The

resulting

fragments

were

cloned

into

the

vector

-LRB-

Invitrogen

Carlsbad

CA

-RRB-

and

transformed

into

E.

coli

cells

The

transformed

cells

were

plated

on

LB

plates

containing

mg/ml

of

ampicillin

and

the

positive

colonies

were

used

for

plasmid

isolation

and

sequencing

Phylogenetic

analysis

Alignments

of

the

rRNA

gene

were

made

using

sequences

downloaded

from

the

Ribosomal

database

project

II

-LRB-

RDP

II

Cole

et

al.

-RRB-

after

searching

for

nearest

neighbors

using

the

sequence

match

tool

Further

searches

were

conducted

using

BLAST

and

FASTA

of

the

European

Molecular

Biology

Laboratory

-LRB-

EMBL

-RRB-

database

for

rRNA

gene

sequences

that

are

closely

related

to

the

rRNA

gene

of

strain

but

not

available

from

the

RDP

II

Alignments

were

subsequently

made

using

the

profile

alignment

option

in

ClustalX

-LRB-

Thompson

et

al.

-RRB-

refined

using

BioEdit

ver

-LRB-

Hall

-RRB-

and

subsequently

parsed

through

Gblock

-LRB-

Castresana

-RRB-

to

remove

ambiguously

aligned

sections

and

increase

the

robustness

of

the

data

Phylogenetic

trees

were

determined

using

the

neighbor-joining

-LRB-

NJ

-RRB-

method

Evolutionary

distances

for

the

global

tree

were

calculated

using

the

model

with

a

transition/transversion

ratio

of

-LRB-

Fig.

-RRB-

Further

trees

were

constructed

in

Phylip

version

-LRB-

J.

Felsenstein

-RRB-

using

maximum

parsimony

and

maximum

likelihood

methods

All

NJ

trees

were

tested

statistically

by

means

of

bootstrap

analysis

Fig.

tree

of

the

rRNA

gene

sequence

of

strain

The

scale

bar

represents

fixed

mutation

per

site

Bootstrap

values

were

derived

from

analyses

The

DNA

sequences

were

aligned

using

ClustalX

and

the

tree

was

constructed

by

the

neighbor-joining

program

from

a

similarity

matrix

of

pairwise

comparisons

made

by

using

the

algorithm

Tree

a

shows

the

global

position

of

the

strain

while

tree

b

shows

the

Bayesian

tree

and

the

more

precise

relationship

of

strain

to

other

members

of

the

genus

Arthrobacter

The

values

in

parenthesis

in

this

tree

are

the

posterior

probabilities

for

the

nodes

that

show

where

resides

Preparation

of

cell

extracts

Cells

were

grown

in

MM

with

mM

harvested

by

centrifugation

at

g

for

min

washed

with

ice-cold

TD

buffer

-LRB-

M

Tris

HCl

pH

and

M

-RRB-

and

stored

at

°C

until

further

use

The

frozen

cells

were

thawed

resuspended

in

TD

buffer

and

incubated

with

lysozyme

-LRB-

mg/ml

-RRB-

for

h

at

°C

A

French

press

was

used

to

disrupt

the

cells

and

the

crude

extracts

were

centrifuged

at

g

for

min

to

separate

the

soluble

from

the

particulate

fraction

The

supernatant

was

used

for

further

experiments

Protein

concentrations

were

determined

with

the

Biorad

protein

assay

kit

Enzyme

assays

Catechol

and

catechol

activities

were

measured

spectrophotometrically

at

and

nm

respectively

The

reaction

mixtures

contained

mM

catechol

TD

buffer

and

cell-free

extract

-LRB-

mg

of

protein

-RRB-

in

a

final

volume

of

ml

was

measured

as

catechol

but

with

instead

of

catechol

as

the

substrate

The

monooxygenase

activity

was

measured

spectrophotometrically

by

following

the

consumption

of

NADH

at

nm

in

a

reaction

mixture

containing

cell-free

extract

-LRB-

mg

protein

-RRB-

mM

mM

NADH

and

buffer

in

a

total

volume

of

ml

The

observed

rates

were

corrected

for

substrate-independent

NADH

oxidation

Hydroxymuconic

semialdehyde

dehydrogenase

was

measured

at

nm

Reaction

mixtures

contained

-LRB-

in

a

final

volume

of

ml

-RRB-

about

mM

freshly

prepared

hydroxymuconic

semialdehyde

TD

buffer

mM

NAD

and

cell-free

extract

-LRB-

mg

of

protein

-RRB-

Hydroxymuconic

semialdehyde

was

obtained

by

incubation

of

catechol

with

the

cell-free

extract

of

Pseudomonas

putida

as

described

previously

-LRB-

Mars

et

al.

-RRB-

Hydroquinone

dioxygenase

was

assayed

spectrophotometrically

by

monitoring

the

change

in

absorbance

between

and

nm

in

a

reaction

mixture

of

ml

final

volume

containing

mM

hydroquinone

TD

buffer

and

cell-free

extract

-LRB-

mg

of

protein

-RRB-

Hydroquinone

hydroxylase

and

hydroxyquinol

dioxygenase

were

assayed

by

using

a

fiber

optic

oxygen

sensor

Reaction

mixtures

contained

mM

of

substrate

MM

and

cell

suspension

-LRB-

mg/ml

protein

-RRB-

in

a

final

volume

of

ml

Oxygen

uptake

measurements

Strain

was

grown

with

glucose

or

as

the

sole

carbon

source

and

harvested

by

centrifugation

at

g

for

min

at

°C

Cells

were

resuspended

in

MM

and

consumption

was

measured

with

a

fiber

optic

oxygen

sensor

-LRB-

ProSense

BV

Hannover

Germany

-RRB-

All

reactions

were

performed

in

a

stirred

vessel

at

room

temperature

The

reaction

mixtures

contained

mM

of

substrate

-LRB-

hydroquinone

hydroxyquinol

or

catechol

-RRB-

MM

and

cell

suspension

-LRB-

mg/ml

protein

-RRB-

in

a

final

volume

of

ml

Analytical

methods

For

capillary

gas

chromatography

-LRB-

GC

-RRB-

ml

samples

were

extracted

with

ml

of

diethyl

ether

A

model

gas

chromatograph

-LRB-

Hewlett-Packard

-RRB-

equipped

with

a

flame

ionization

detector

and

a

column

-LRB-

Agilent

m

mm

μm

-RRB-

were

used

for

the

analysis

GC-mass

spectrometry

-LRB-

MS

-RRB-

analysis

was

carried

out

with

a

model

mass

selective

detector

-LRB-

Hewlett-Packard

-RRB-

coupled

to

a

HP

series

injector

and

a

column

-LRB-

Agilent

m

mm

μm

-RRB-

High-performance

liquid

chromatography

-LRB-

HPLC

-RRB-

was

carried

out

using

a

Chrompack

column

-LRB-

cm

mm

-RRB-

connected

to

a

Jasco

detector

which

monitored

absorbance

at

and

nm

and

operated

with

Jasco

pumps

and

a

Jasco

sampler

The

mobile

phase

was

water/acetonitrile

-LRB-

-RRB-

mM

potassium

phosphate

-LRB-

pH

-RRB-

and

mg/l

sodium

dodecyl

sulfate

and

the

flow

rate

was

ml/min

Liquid

chromatography

-LRB-

LC

-RRB-

MS

was

carried

out

with

a

ZMD

Micromass

spectrometer

equipped

with

a

XTerra

MS

Symmetry

Shield

column

-LRB-

mm

-RRB-

a

Waters

photodiode

array

detector

and

a

Waters

separations

module

Samples

of

μl

were

analyzed

and

compounds

were

isocratically

eluted

at

a

flow

rate

of

ml/min

with

a

solution

of

water/acetonitrile

-LRB-

-RRB-

and

mM

formic

acid

Concentrations

of

free

fluoride

in

the

culture

supernatants

were

measured

with

a

fluoride

electrode

-LRB-

model

Thermo

Russell

Scotland

-RRB-

Fresh

sodium

fluoride

standards

were

prepared

for

calibration

curves

Chemicals

-LRB-

-RRB-

was

obtained

from

Sigma-Aldrich

-LRB-

Steinheim

Germany

-RRB-

All

chemicals

were

of

the

highest

purity

grade

available

-LRB-

Sigma-Aldrich

Acros

Organics

Geel

Belgium

-RRB-

The

compound

was

kindly

provided

by

Dr.

Erik

de

Vries

Purity

of

and

hydroquinone

was

checked

by

HPLC

Nucleotide

sequence

accession

numbers

The

rRNA

sequence

of

strain

was

deposited

at

GenBank

with

the

accession

no

Results

Isolation

of

a

bacterium

To

obtain

a

bacterial

culture

that

is

able

to

use

as

carbon

and

energy

source

for

growth

enrichments

were

performed

and

followed

over

time

Two

months

of

selective

enrichment

by

repeated

transfer

of

samples

from

a

culture

that

displayed

growth

on

to

fresh

containing

media

resulted

in

a

microbial

consortium

that

was

capable

of

growth

on

as

a

sole

source

of

carbon

and

energy

Samples

of

the

consortium

were

repeatedly

plated

onto

NB

agar

plates

and

MM

agar

plates

containing

This

procedure

resulted

in

the

isolation

of

three

pure

strains

named

and

The

strains

were

restreaked

on

MM

supplemented

with

and

inoculated

in

liquid

cultures

with

mM

Fluoride

liberation

was

observed

for

all

three

strains

but

not

in

control

incubations

to

which

no

bacterial

inoculum

was

added

All

the

three

strains

showed

growth

as

monitored

at

nm

and

thus

were

able

to

use

as

a

sole

carbon

and

energy

source

After

days

of

incubation

in

liquid

culture

supplied

with

mM

strain

reached

fluoride

release

and

an

optical

density

-LRB-

OD

-RRB-

of

at

nm

Strain

released

of

the

fluorine

and

reached

an

of

while

strain

reached

an

OD

of

and

released

of

the

theoretical

amount

of

fluoride

ions

When

a

mixed

culture

of

the

three

strains

in

liquid

media

was

used

an

of

and

fluorine

release

were

measured

after

days

Apparently

the

mixed

culture

contained

even

after

prolonged

adaptation

organisms

with

varying

efficiencies

of

utilization

Because

strain

showed

the

highest

degradation

rates

combined

with

stoichiometric

release

of

fluoride

this

organism

was

chosen

for

further

studies

Microbiological

characterization

Strain

is

a

Gram-positive

motile

bacterium

with

a

rhodococcus

lifecycle

The

optimal

temperature

for

growth

is

°C

The

rRNA

gene

sequence

was

determined

Initial

searches

against

the

RDP

II

and

EMBL

rRNA

databases

resulted

in

very

close

associations

with

members

of

the

genus

Arthrobacter

the

closest

of

which

were

the

quinaldine-degrading

strain

Arthrobacter

sp.

-LRB-

Overhage

et

al.

-RRB-

and

the

actinobacterium

Arthrobacter

nitroguajacolicus

sp.

nov

-LRB-

Kotouckova

et

al.

-RRB-

The

rRNA

gene

sequences

of

these

organisms

were

greater

than

identical

to

's

The

phylogenetic

analysis

places

the

isolates

in

the

phylum

Actinobacteria

and

the

genus

Arthrobacter

-LRB-

Fig.

-RRB-

The

subtree

that

were

obtained

shows

the

detailed

relationship

of

isolate

to

other

members

of

the

genus

Arthrobacter

The

topologies

of

all

the

trees

that

were

obtained

during

statistical

analysis

were

in

agreement

and

clearly

placed

this

isolate

in

the

genus

Arthrobacter

-LRB-

data

not

shown

-RRB-

Furthermore

the

change

in

form

during

the

growth

cycle

between

rod

and

coccus

is

typical

of

the

lifecycle

of

the

Arthrobacter

genus

Catabolic

activities

of

Arthrobacter

sp.

strain

To

study

the

degradation

potential

of

strain

a

range

of

organic

compounds

were

tested

as

growth

substrates

Cells

were

inoculated

into

MM

and

different

organic

substrates

were

added

at

a

concentration

of

mM

After

incubation

growth

substrate

disappearance

and

halide

release

were

measured

Growth

and

substrate

removal

were

found

when

catechol

hydroquinone

hydroxyquinol

benzoate

phenol

acid

and

were

used

as

substrates

The

organism

did

not

grow

on

fluoroacetate

trifluoroacetate

fluoroacetamide

trifluoroethanol

or

on

The

fact

that

strain

is

capable

of

growth

on

catechol

hydroquinone

and

hydroxyquinol

but

not

on

indicates

that

is

not

the

most

likely

intermediate

in

the

pathway

although

toxic

effects

could

also

play

a

role

To

test

the

range

of

concentrations

tolerated

by

strain

experiments

were

conducted

in

sealed

flasks

with

at

concentrations

of

to

mM

as

a

sole

carbon

and

energy

source

Control

assays

without

showed

no

growth

or

release

of

fluoride

and

sterile

controls

showed

no

abiotic

loss

of

Between

and

mM

the

substrate

was

completely

consumed

stoichiometric

release

of

fluoride

was

observed

and

biomass

increased

linearly

with

the

amount

of

added

-LRB-

Fig.

-RRB-

This

indicates

that

the

degradation

of

by

strain

does

not

give

large

amounts

of

fluorinated

dead-end

products

Concentrations

of

above

mM

caused

a

toxic

effect

on

the

growth

of

-LRB-

Fig.

-RRB-

The

use

of

mM

promoted

growth

but

a

longer

lag

time

was

observed

At

mM

no

growth

occurred

even

after

days

of

incubation

Fig.

release

and

biomass

formation

by

strain

with

different

concentrations

of

a

Stoichiometric

fluoride

release

Symbols

squares

optical

density

at

nm

circles

fluoride

concentration

b

Growth

in

the

presence

of

different

levels

of

The

initial

concentrations

used

were

triangles

mM

circles

mM

diamonds

mM

squares

mM

Growth

on

and

formation

of

metabolites

A

batch

culture

of

strain

supplied

with

mM

as

the

only

source

of

carbon

and

energy

was

monitored

in

time

Growth

was

accompanied

by

an

increase

in

biomass

decrease

in

and

formation

of

fluoride

-LRB-

Fig.

-RRB-

After

h

there

was

complete

conversion

of

mM

and

mM

fluoride

was

formed

indicating

that

there

was

no

transient

accumulation

of

fluorinated

intermediates

over

the

whole

growth

period

When

cells

grown

on

were

incubated

in

the

presence

of

the

iron

chelator

dipyridyl

no

degradation

occurred

and

no

fluoride

was

released

in

the

medium

This

indicates

that

initial

or

further

enzymes

involved

in

metabolism

require

ferrous

ions

for

activity

Fig.

of

and

fluoride

liberation

in

a

batch

culture

of

strain

The

carbon

source

used

is

mM

Symbols

triangles

concentration

diamonds

optical

density

squares

fluoride

concentration

To

isolate

intermediates

of

the

degradation

of

samples

from

a

batch

culture

containing

mM

were

taken

at

appropriate

intervals

and

analyzed

by

GC

HPLC

and

LC-MS

Metabolites

that

were

detected

were

numbered

in

order

of

time

of

appearance

in

the

culture

GC

analysis

indicated

that

at

least

four

metabolites

were

formed

and

degraded

over

time

-LRB-

Table

-RRB-

Metabolite

I

the

earliest

product

that

was

observed

had

the

same

retention

time

as

an

authentic

hydroquinone

standard

and

metabolite

II

had

the

same

retention

time

as

a

standard

of

hydroxyquinol

GC-MS

analysis

showed

that

the

mass

spectrum

of

metabolite

I

was

indeed

similar

to

that

of

the

hydroquinone

standard

with

a

molecular

ion

peak

at

and

at

m/z

Metabolite

II

gave

a

molecular

ion

peak

at

and

m/z

which

is

typical

for

hydroxyquinol

and

the

spectrum

coincided

with

that

of

a

standard

Metabolites

V

and

VI

were

detected

by

GC

but

did

not

ionize

in

GC-MS

under

the

conditions

tested

and

were

not

identified

Table

times

in

GC

and

HPLC

analyses

of

metabolic

intermediates

formed

by

cells

of

strain

growing

on

and

of

some

authentic

standardsCompoundGC

retention

time

-LRB-

min

-RRB-

HPLC

retention

time

-LRB-

min

-RRB-

am/z

of

observed

fragment

ions

detected

by

IV

VII

aElution

followed

at

nmbAnalyzed

after

separation

on

a

Symmetry

Shield

column

-LRB-

mm

mm

-RRB-

as

described

in

Materials

and

methods

for

LC-MS

analysisc

Not

determineddLC-MS

analysis

When

the

supernatant

of

cells

growing

on

mM

were

subjected

to

HPLC

analysis

four

peaks

were

detected

-LRB-

Table

-RRB-

Metabolite

I

had

the

same

retention

time

as

the

hydroquinone

standard

and

metabolite

II

coeluted

with

an

authentic

standard

of

hydroxyquinol

Hydroquinone

was

detected

in

culture

supernatants

by

HPLC

between

and

h

-LRB-

Fig.

-RRB-

LC-MS

analysis

of

the

extracted

samples

of

the

supernatant

revealed

the

presence

of

a

compound

-LRB-

VII

-RRB-

of

which

the

negative

ion

spectrum

shows

a

molecular

ion

peak

at

m/z

and

a

peak

at

m/z

which

are

expected

for

the

negative

ionization

of

Fig.

of

hydroquinone

during

growth

of

strain

on

mM

The

optical

density

-LRB-

diamonds

-RRB-

concentration

-LRB-

squares

-RRB-

and

hydroquinone

concentration

-LRB-

x

marks

-RRB-

are

indicated

Hydroquinone

was

identified

by

GC-MS

and

quantified

by

GC-FID

The

above

results

suggest

that

is

initially

converted

to

hydroquinone

The

most

likely

enzyme

involved

in

such

a

conversion

is

a

monooxygenase

Enzyme

activities

To

test

inducibility

of

enzymes

involved

in

degradation

cells

of

Arthrobacter

sp.

strain

were

grown

on

glucose

or

washed

and

tested

for

oxygen

consumption

in

the

presence

of

different

substrates

Washed

suspensions

of

cells

grown

on

rapidly

oxidized

hydroquinone

and

-LRB-

hydroxyquinol

-RRB-

without

a

lag

Catechol

and

did

not

stimulate

oxygen

uptake

-LRB-

Table

-RRB-

This

result

indicates

that

and

catechol

are

not

likely

intermediates

and

that

hydroquinone

and

hydroxyquinol

are

Glucose-grown

cells

did

not

oxidize

any

of

the

aromatic

substrates

tested

with

the

exception

of

hydroxyquinol

This

observation

implies

that

hydroxyquinol

may

be

converted

by

a

constitutive

oxygenase

whereas

most

other

enzymes

seem

inducible

Cells

grown

with

hydroxyquinol

showed

complete

conversion

of

benzenetriol

in

the

presence

or

absence

of

dipyridyl

indicating

that

the

putative

hydroxyquinol

oxygenase

does

not

require

ferrous

ions

to

be

active

and

that

oxidation

beyond

this

compound

is

not

inhibited

by

the

chelator

The

formation

of

hydroquinone

which

can

be

readily

oxidized

by

cells

is

in

agreement

with

the

involvement

in

degradation

of

a

monooxygenase

that

is

induced

by

Table

oxidized

by

and

glucose

grown

cells

of

Arthrobacter

sp.

strain

uptake

-LRB-

μmol/min/mg

of

protein

-RRB-

by

whole

cells

after

growth

0.001Hydroxyquinol0.1950.1564-Fluorocatechol

Catechol

a

Oxygen

consumption

was

measured

with

an

oxygen

sensor

as

described

in

Materials

and

Methods

All

substrate

were

used

at

a

concentration

of

mM

To

study

in

more

detail

the

degradation

pathway

of

by

strain

cell-free

extracts

of

grown

on

were

investigated

for

the

presence

of

several

enzyme

activities

-LRB-

Table

-RRB-

When

catechol

and

were

tested

for

no

activity

was

found

Furthermore

no

activity

was

detectable

for

dioxygenase

Consequently

the

pathway

does

not

proceed

through

catechol

or

a

substituted

catechol

When

cell

extracts

were

assayed

for

monooxygenase

activity

could

be

found

in

the

presence

of

NADH

but

not

in

the

presence

of

NADPH

as

the

reducing

cosubstrate

This

observation

indicates

that

the

cells

contain

an

NADH-dependent

monooxygenase

activity

HPLC

assays

of

incubations

of

cell

extracts

with

showed

that

substrate

conversion

was

complete

The

formation

of

the

putative

monooxygenase

was

induced

by

because

no

activity

was

found

in

extracts

of

cells

grown

in

the

presence

of

glucose

Table

activities

of

enzymes

in

crude

extracts

of

strain

pregrown

in

or

glucoseAssay

substrateEnzyme

testedSpecific

activity

-LRB-

U/mg

of

protein

-RRB-

after

growth

HydroquinoneDioxygenase

Hydroxymuconic

semialdehydeDehydrogenase

When

hydroquinone

was

used

as

an

assay

substrate

its

typical

spectrophotometric

peak

at

nm

was

not

replaced

by

a

peak

absorbing

at

to

nm

which

would

have

pointed

to

formation

of

hydroxymuconic

semialdehyde

Thus

no

hydroquinone

dioxygenase

was

induced

or

its

product

was

rapidly

further

converted

The

latter

was

ruled

out

by

the

observation

that

added

hydroxymuconic

semialdehyde

was

not

converted

by

cell

extracts

of

strain

indicating

that

no

hydroxymuconic

semialdehyde

dehydrogenase

was

induced

during

growth

in

the

presence

of

These

results

make

it

unlikely

that

hydroxymuconic

semialdehyde

is

an

intermediate

in

the

pathway

by

strain

When

cell

extracts

were

assayed

for

hydroxyquinol

degradation

complete

conversion

of

the

substrate

was

observed

with

HPLC

When

the

reaction

was

monitored

spectrophotometrically

the

peak

at

nm

typical

of

hydroxyquinol

was

substituted

by

a

peak

at

nm

typical

of

maleylacetate

This

indicates

the

involvement

of

a

hydroxyquinol

oxygenase

that

converts

hydroxyquinol

into

maleylacetate

which

is

further

converted

into

Discussion

This

study

reports

the

isolation

and

characterization

of

Arthrobacter

strain

an

organism

capable

of

growth

with

as

a

sole

source

of

carbon

and

energy

The

biodegradation

of

by

strain

was

analyzed

by

GC

GC-MS

HPLC

LC-MS

oxygen

uptake

experiments

and

measurements

of

enzymatic

activities

A

metabolic

pathway

is

proposed

on

the

basis

of

the

results

of

this

study

-LRB-

steps

and

ortho-cleavage

-RRB-

and

by

analogy

with

other

systems

-LRB-

Fig.

-RRB-

We

suggest

that

the

degradation

of

in

strain

starts

with

the

conversion

by

a

monooxygenase

to

benzoquinone

which

is

immediately

reduced

to

hydroquinone

Hydroquinone

then

undergoes

a

further

hydroxylation

to

form

hydroxyquinol

This

benzenetriol

is

the

ring

fission

substrate

which

is

transformed

by

ortho-cleavage

and

yields

maleylacetate

which

is

further

converted

to

Fig.

pathway

for

degradation

by

Arthrobacter

sp.

strain

Hydroquinone

and

hydroquinole

were

identified

as

intermediates

by

GC-MS

Up

to

now

two

main

metabolic

routes

for

the

aerobic

degradation

of

halogenated

phenols

have

been

described

in

the

literature

In

bacteria

that

degrade

mono

and

dichlorophenols

a

degradation

pathway

is

usually

observed

in

which

the

substituted

phenol

is

hydroxylated

to

the

corresponding

catechol

which

is

followed

by

ortho-cleavage

of

the

aromatic

ring

-LRB-

Haggblom

Hollender

et

al.

Wieser

et

al.

-RRB-

On

the

other

hand

biodegradation

pathways

in

which

the

substituted

phenol

is

converted

via

hydroquinone

to

maleylacetate

have

been

found

mainly

in

organisms

that

grow

on

polyhalophenols

or

-LRB-

Kiyohara

et

al.

Xun

et

al.

Kadiyala

and

Spain

Nordin

et

al.

Perry

and

Zylstra

-RRB-

Most

studies

have

focused

on

the

organisms

and

pathways

that

involve

degradation

via

catechols

A

clear

indication

for

the

first

step

in

the

catabolic

pathway

of

strain

was

obtained

by

mass

spectroscopic

analysis

of

culture

supernatants

which

indicated

the

formation

of

hydroquinone

as

an

early

nonfluorinated

intermediate

This

result

in

combination

with

the

enzyme

assays

and

oxygen

uptake

experiments

suggests

the

involvement

of

an

inducible

monooxygenase

that

is

NADH

dependent

although

the

first

expected

product

of

this

reaction

i.e.

benzoquinone

was

not

detected

Monooxygenation

of

an

aromatic

substrate

carrying

an

electron-withdrawing

halogen

group

or

a

nitro-substituent

can

result

in

simultaneous

hydroxylation

and

dehalogenation

or

nitrite

removal

This

has

for

example

been

described

for

the

conversion

of

tetrafluoro-p-hydroxybenzoate

by

para-hydroxybenzoate

hydroxylase

-LRB-

Husain

et

al.

-RRB-

and

the

oxidation

of

by

a

monooxygenase

from

Ralstonia

eutropha

-LRB-

Xun

and

Webster

-RRB-

The

quinone

that

is

produced

is

chemically

reduced

by

NADH

to

a

hydroxyquinol

leading

to

a

net

stoichiometry

of

two

NADH

oxidized

per

halide

or

nitrite

that

is

released

-LRB-

Fig.

-RRB-

As

in

our

case

no

transient

accumulation

of

a

benzoquinone

is

usually

observed

indicating

that

benzoquinone

reduction

is

rapid

Other

examples

of

halophenol

degradation

with

formation

of

hydroquinone

derivatives

are

the

degradation

of

trichlorophenol

to

by

a

strain

of

Pseudomonas

pickettii

-LRB-

Kiyohara

et

al.

-RRB-

the

degradation

of

pentachlorophenol

by

a

Sphingobium

sp.

through

hydroxylation

by

a

flavoprotein

monooxygenase

-LRB-

Xun

and

Orser

-RRB-

and

the

degradation

of

by

an

Arthrobacter

sp.

-LRB-

Bae

et

al.

-RRB-

Our

results

further

indicate

that

hydroquinone

is

not

the

ring

fission

substrate

because

no

enzymatic

activities

for

hydroquinone

dioxygenase

or

hydroxymuconic

semialdehyde

dehydrogenase

were

found

Instead

upon

conversion

of

hydroquinone

by

strain

we

observed

the

formation

of

a

transient

metabolite

that

was

identified

as

hydroxyquinol

indicating

that

a

second

hydroxylation

takes

place

before

ring

fission

The

production

of

hydroxyquinol

could

be

due

to

the

action

of

a

separate

hydroquinone

monooxygenase

or

it

could

be

caused

by

a

second

hydroxylation

step

by

the

monooxygenase

Conversion

of

hydroquinone

to

hydroxyquinol

before

ring

fission

was

also

suggested

for

strains

Arthrobacter

that

degrade

-LRB-

Perry

and

Zylstra

-RRB-

and

-LRB-

Nordin

et

al.

-RRB-

Ring

fission

of

hydroxyquinol

would

produce

maleylacetate

which

we

did

not

detect

as

an

intermediate

in

degradation

but

a

transient

metabolite

was

found

with

mass

properties

identical

to

those

of

which

is

expected

to

be

the

next

intermediate

in

hydroxyquinol

degradation

Most

cometabolic

transformations

of

fluorophenols

described

in

the

literature

involve

the

initial

action

of

a

phenol

hydroxylase

that

results

in

the

formation

of

fluorocatechols

which

are

subsequently

transformed

into

fluoromuconates

through

a

catechol

The

conversion

usually

proceeds

with

lactonization

and

elimination

of

fluoride

-LRB-

Boersma

et

al.

Bondar

et

al.

Finkelstein

et

al.

Boersma

et

al.

Bondar

et

al.

-RRB-

Our

results

suggest

that

Arthrobacter

strain

which

grows

on

possesses

a

new

pathway

for

the

degradation

of

that

involves

immediate

defluorination

by

a

monooxygenase

Strain

avoids

the

accumulation

of

possible

toxic

metabolites

such

as

or

by

this

initial

dehalogenation

O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O

biodegradation hydroquinone monooxygenase fluoride


Association

between

long

travel

and

venous

thromboembolic

disease

a

systematic

review

and

meta-analysis

of

case-control

studies

The

term

economy-class

syndrome

defines

an

infrequent

episode

of

venous

thromboembolism

-LRB-

VTED

-RRB-

related

to

a

long

travel

namely

by

plane

However

this

relation

has

not

clearly

been

demonstrated

by

investigators

We

carried

out

a

systematic

review

and

a

meta-analysis

of

cases-control

studies

that

had

studied

this

topic

We

realised

a

systematic

review

of

the

literature

and

selected

all

the

case-control

studies

published

Two

authors

carried

out

a

methodological

evaluation

according

to

the

Scottish

Intercollegiate

Guidelines

Network

items

-LRB-

concordance

was

analysed

by

weighted

kappa

index

-RRB-

and

a

systematic

analysis

of

the

potential

biases

of

each

study

was

assessed

We

carried

out

the

meta-analysis

with

the

data

extracted

from

the

studies

We

recovered

eight

cases-control

studies

The

relation

between

the

antecedent

of

a

long

travel

and

subsequent

VTED

varied

from

OR

to

OR

and

was

found

to

be

significant

in

four

studies

The

studies

were

highly

heterogeneous

in

methodology

and

so

the

results

obtained

about

the

relation

between

the

long

travel

and

the

VTED

and

the

score

at

Two

meta-analysis

were

carried

out

only

with

travels

by

plane

in

which

the

relation

was

not

significant

-LRB-

OR

CI

-RRB-

and

with

all

types

of

transport

with

a

slightly

significant

relation

-LRB-

OR

-RRB-

We

may

deduce

from

this

systematic

review

that

there

does

exist

a

weak

association

between

episodes

of

VTED

and

a

long

travel

but

not

by

plane

specifically

The

heterogeneity

and

the

methodological

quality

of

the

studies

published

preclude

of

more

robust

conclusions

Introduction

Venous

thromboembolic

disease

-LRB-

VTED

-RRB-

has

been

associated

with

many

risk

factors

one

of

which

is

the

antecedent

of

a

long

travel

most

often

by

plane

This

association

was

previously

suggested

by

Homans

-LSB-

-RSB-

and

was

believed

so

self-evident

that

it

gave

rise

to

the

term

economy-class

syndrome

-LSB-

-RSB-

Subsequently

the

description

of

clinical

cases

related

to

other

means

of

transport

led

to

the

coining

of

another

more

appropriate

generic

term

traveller

's

thrombosis

-LSB-

-RSB-

The

scenario

for

this

syndrome

has

varied

from

the

alarm

caused

by

the

apparently

high

frequency

of

fatal

pulmonary

embolism

-LRB-

PE

-RRB-

occurring

after

a

prolonged

flight

as

reported

in

the

media

to

the

scepticism

of

a

weak

although

likely

association

reported

in

various

scientific

studies

The

published

reports

have

ranged

from

descriptions

of

individual

cases

to

cohort

studies

and

even

clinical

trials

However

the

low

incidence

of

episodes

of

VTED

after

a

long

travel

makes

the

design

of

a

case-control

study

particularly

useful

for

evaluating

the

suggested

association

-LSB-

-RSB-

Our

objective

was

to

carry

out

a

systematic

review

and

meta-analysis

of

case-control

studies

to

analyse

the

association

between

long

travels

and

the

development

of

VTED

Materials

and

methods

We

carried

out

a

bibliographic

search

using

a

combination

of

keywords

and

MeSH

headings

in

the

Medline

Embase

and

Cochrane

Library

bibliographic

databases

We

selected

only

case-control

studies

in

any

language

with

no

limit

on

the

date

of

publication

and

performed

a

cross

search

of

the

references

cited

in

these

studies

The

corresponding

author

named

in

each

study

was

requested

to

inform

us

of

any

relevant

data

that

had

not

been

described

in

the

original

manuscript

Two

authors

-LRB-

JTS

and

AJP

-RRB-

carried

out

a

descriptive

analysis

of

the

studies

identified

in

the

search

and

evaluated

their

quality

according

to

the

checklist

proposed

by

the

Scottish

Intercollegiate

Guidelines

Network

-LSB-

-RSB-

Concordance

in

the

evaluation

of

the

items

on

this

list

was

analysed

using

the

respective

weighted

kappa

indexes

We

also

analysed

the

limitations

and

potential

biases

that

might

reduce

the

validity

of

the

studies

examined

The

sources

of

bias

analysed

were

grouped

into

three

categories

exposure

misclassification

-LRB-

recall

bias

-RRB-

selection

bias

-LRB-

including

detection

bias

effect

misclassification

survival

bias

self-selection

bias

and

Berkson

's

bias

-RRB-

and

confounding

factors

Memory

bias

was

defined

as

the

fact

that

when

subjects

know

they

have

suffered

VTED

it

makes

them

more

likely

to

remember

prior

exposure

to

the

antecedent

of

a

long

travel

Effect

misclassification

was

minimised

by

considering

only

those

studies

in

which

VTED

was

diagnosed

by

objective

complementary

examination

Detection

bias

was

suspected

when

the

odds

ratio

-LRB-

OR

-RRB-

in

the

days

immediately

after

the

travel

was

less

than

that

corresponding

to

subsequent

days

We

only

selected

data

published

for

VTED

incidence

at

days

after

the

travel

because

was

the

time

lapse

most

frequently

analysed

in

the

studies

Survival

bias

was

considered

to

be

that

occurring

when

the

study

included

prevalent

cases

which

could

give

rise

to

confounding

of

the

variables

related

to

the

origin

and

those

concerning

the

prognosis

of

the

disease

Self-selection

bias

was

considered

to

be

present

if

the

controls

participated

on

their

own

initiative

in

the

study

and

were

not

selected

in

a

consecutive

or

random

way

thus

they

might

be

related

to

the

results

being

sought

Berkson

's

bias

would

be

derived

from

the

use

of

hospital

patients

as

controls

The

confounding

factors

were

judged

to

be

influential

if

no

account

were

taken

of

the

possibility

that

the

exposure

factor

and

the

disease

might

be

related

through

a

third

variable

which

was

related

to

each

of

the

other

two

From

the

results

included

in

the

published

studies

and

the

data

supplied

by

the

different

authors

we

were

able

to

perform

a

meta-analysis

of

the

studies

that

had

estimated

the

risk

ratio

of

deep-vein

thrombosis

-LRB-

DVT

-RRB-

and

of

DVT

and/or

PE

either

after

a

travel

-LRB-

by

any

means

of

transport

-RRB-

or

only

after

a

travel

by

plane

The

OR

was

used

as

a

measure

of

association

with

a

confidence

interval

of

The

hypothesis

of

homogeneity

was

evaluated

using

X

-LSB-

-RSB-

and

Der

Simonian

Laird

tests

and

fixed

and

random

effects

methods

were

used

respectively

for

the

cases

of

homogeneity

and

heterogeneity

between

the

studies

We

assessed

possible

publication

bias

using

Begg

and

Egger

tests

and

carried

out

a

sensitivity

analysis

by

evaluating

the

influence

of

the

absence

of

each

individual

study

on

the

global

measure

of

association

of

all

the

other

studies

The

computer

softwares

used

for

this

study

were

SPSS

-LRB-

SPSS

Chicago

IL

USA

-RRB-

and

EpiDat

-LSB-

-RSB-

Results

Nine

case-control

studies

were

recovered

of

these

we

analysed

the

data

from

seven

-LSB-

-RSB-

as

the

other

one

-LSB-

-RSB-

corresponded

to

a

preliminary

analysis

for

one

of

the

seven

One

of

these

-LSB-

-RSB-

was

made

up

of

three

sub-studies

but

for

reasons

of

consistency

we

only

included

the

data

for

two

of

these

in

the

meta-analysis

as

in

the

third

one

the

authors

assessed

patients

outpatients

and

inpatients

with

PE

Table

shows

the

main

characteristics

of

all

these

studies

The

relation

between

the

antecedent

of

a

long

travel

and

subsequent

VTED

varied

from

OR

to

OR

and

was

found

to

be

significant

in

four

studies

-LSB-

-RSB-

In

them

the

OR

ranged

from

-LRB-

-RRB-

to

-LRB-

-RRB-

Table

characteristics

of

the

case-control

studies

evaluating

the

association

between

a

long

travel

and

venous

thromboembolic

diseaseStudy

yearFerrari

Wolde

of

of

casesPatients

admitted

with

VTEDDVT

symptomatic

outpatientsEmployees

of

the

World

Bank

with

confirmed

DVTDVT

symptomatic

outpatientsDVT

symptomatic

outpatientsDVT

outpatients

PE

outpatients

and

hospital

admissionsPatients

with

VTED

during

previous

months

and

examined

for

possible

thrombophiliaVTED

outpatients

and

admissionsOrigin

of

controlsPatients

admitted

in

Cardiology

Department

age-matchedViral

syndrome

matched

by

age

and

genderEmployees

of

the

World

Bank

without

DVT

matched

by

month

and

year

of

diagnosisHospital

attention

for

compatible

symptoms

DVT

excludedHospital

attention

for

compatible

symptoms

DVT

excludedHospital

attention

for

compatible

symptoms

DVT

or

PE

excludedFriends

or

partners

volunteers

of

hospital

patientsPartners

of

casesNo

of

travel/cases

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

No

of

travel/controls

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

OR

-LRB-

CI

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Type

of

transportVariousVariousOnly

planeVariousVariousVariousOnly

planeVariousDuration

of

travelAt

least

h

-LRB-

not

stratified

-RRB-

Not

stated

-LRB-

long

travel

-RRB-

Not

statedStratified

into

and

hours

hoursStratified

from

to

hStratified

into

hAt

least

h

stratified

into

hLapse

between

travel

and

VTED

-LRB-

weeks

-RRB-

limitationsBerkson

's

bias

Controls

age

matchedNot

specific

to

assess

risk

of

travel

Confounding

Controls

matched

by

age

and

genderOnly

international

travels

were

evaluated

-LRB-

not

duration

of

travel

-RRB-

Selection

of

controlsConfounding

Not

differentiated

by

duration

of

travelBerkson

's

biasSurvival

bias

Matched

by

age

gender

and

academic

level

Self

selection

bias

Recall

biasMemory

bias

NO

objective

clinical

information

about

controls

With

respect

to

means

of

transport

two

studies

-LSB-

-RSB-

only

assessed

travel

by

plane

while

two

-LSB-

-RSB-

MEGA

examined

separately

each

means

of

transport

Another

study

-LSB-

-RSB-

distinguished

travel

by

plane

from

all

other

means

of

transport

while

another

two

-LSB-

-RSB-

considered

various

means

of

transport

jointly

but

did

not

distinguish

those

corresponding

to

cases

from

controls

and

in

one

study

-LSB-

-RSB-

any

means

of

transport

was

eligible

for

consideration

without

further

specification

The

duration

of

the

travel

was

addressed

in

different

ways

two

studies

-LSB-

-RSB-

made

no

reference

to

this

question

two

studies

-LSB-

-RSB-

only

considered

travels

with

a

duration

exceeding

h

and

another

those

exceeding

h

-LSB-

-RSB-

The

other

three

studies

-LSB-

-RSB-

considered

different

durations

The

lapse

of

time

between

the

travel

and

the

diagnosis

of

VTED

was

weeks

in

one

study

-LSB-

-RSB-

and

weeks

for

the

others

except

that

of

Samama

et

al.

-LSB-

-RSB-

which

did

not

specify

the

period

exactly

and

MEGA

study

-LSB-

-RSB-

that

included

weeks

before

In

the

study

by

Dimberg

et

al.

-LSB-

-RSB-

two

periods

were

analysed

-LRB-

and

days

-RRB-

but

in

our

own

analysis

only

the

data

corresponding

to

days

were

taken

into

consideration

this

period

being

equivalent

to

the

weeks

used

in

the

other

studies

Four

studies

-LSB-

-RSB-

analysed

cases

of

both

DVT

and

of

PE

while

the

others

only

considered

patients

with

clinical

presentation

of

VTED

in

the

form

of

DVT

The

selection

of

cases

was

generally

that

of

patients

with

symptoms

of

DVT

and/or

PE

One

study

-LSB-

-RSB-

used

a

register

of

days

off

work

kept

by

medical

insurance

companies

for

the

staff

of

the

company

in

which

the

study

was

carried

out

The

rate

of

prior

history

of

a

long

travel

among

the

cases

ranged

from

-LSB-

-RSB-

to

-LSB-

-RSB-

Diverse

methods

were

used

for

selection

of

controls

Three

studies

-LSB-

-RSB-

selected

outpatients

who

after

attending

hospital

with

symptoms

compatible

with

DVT

and/or

PE

were

found

not

to

have

these

diseases

in

another

study

-LSB-

-RSB-

the

controls

were

hospital

patients

matched

by

age

who

had

been

admitted

for

other

reasons

during

the

same

period

of

time

as

the

cases

Two

studies

chose

outpatient

controls

matched

by

age

and

sex

with

the

cases

one

selecting

patients

with

symptoms

of

respiratory

viriasis

-LSB-

-RSB-

and

the

other

using

relatives

and

friends

of

patients

who

had

suffered

VTED

-LSB-

-RSB-

Another

study

-LSB-

-RSB-

chose

the

partners

of

the

cases

as

controls

Finally

Dimberg

et

al.

-LSB-

-RSB-

chose

ten

control

cases

matched

by

month

and

year

of

diagnosis

of

DVT

from

among

the

staff

of

the

same

company

that

employed

the

cases

The

study

by

Ferrari

et

al.

-LSB-

-RSB-

even

used

different

exclusion

criteria

for

cases

and

for

controls

such

that

anticoagulant

treatment

was

an

exclusion

criterion

for

the

former

but

not

for

the

latter

The

rate

of

prior

occurrence

of

a

long

travel

among

the

controls

ranged

from

-LSB-

-RSB-

to

-LSB-

-RSB-

With

respect

to

the

possible

biases

present

in

each

of

the

studies

we

highlight

the

following

Recall

or

classification

bias

the

study

by

Martinelli

et

al.

-LSB-

-RSB-

could

suffer

from

bias

in

this

sense

as

one

of

the

exclusion

criteria

was

the

absence

of

a

prior

occurrence

of

VTED

during

the

previous

months

In

general

this

bias

is

consubstantial

with

the

studies

of

cases

and

controls

such

that

the

cases

tend

to

relate

the

disease

with

the

antecedent

of

interest.Detection

bias

only

one

study

-LSB-

-RSB-

analysed

two

periods

of

time

in

which

DVT

had

occurred

after

a

long

travel

namely

at

and

days

The

OR

for

the

former

period

was

greater

than

that

for

the

latter

and

thus

this

form

of

bias

was

not

present

here.Misclassification

bias

in

all

the

studies

only

cases

of

VTED

that

had

been

objectively

diagnosed

and

for

which

the

appropriate

complementary

examinations

were

taken

into

consideration

and

thus

their

correct

classification

was

ensured

In

three

-LSB-

-RSB-

the

controls

were

derived

from

persons

who

having

sought

medical

treatment

for

symptoms

compatible

with

DVT

and/or

PE

were

found

not

to

suffer

either

of

these

after

the

same

objective

examination

used

to

confirm

the

cases

In

others

the

controls

presented

no

symptoms

of

DVT

and/or

PE

and

so

were

also

assumed

to

be

true

controls.Survival

bias

this

bias

type

could

have

occurred

in

the

study

by

Martinelli

et

al.

-LSB-

-RSB-

as

the

cases

chosen

were

those

patients

who

had

suffered

an

episode

of

venous

thromboembolism

during

the

previous

years

and

for

whom

a

study

of

thrombophilia

was

performed

thus

there

was

in

fact

a

selection

of

all

the

potential

incidental

cases

of

DVT

during

this

period.Self-selection

bias

in

the

study

by

Martinelli

et

al.

-LSB-

-RSB-

the

controls

were

recruited

from

the

relatives

and

friends

of

the

cases

a

circumstance

that

could

have

negatively

influenced

the

relation

being

studied

as

the

sample

was

obtained

from

a

population

that

was

socially

related

to

the

cases

and

thus

exposure

factors

could

have

been

shared

In

MEGA

study

-LSB-

-RSB-

the

controls

were

partners

of

the

cases

and

the

evaluated

relation

might

have

been

diminished.Berkson

's

bias

this

bias

might

have

been

present

in

the

study

by

Ferrari

et

al.

-LSB-

-RSB-

as

the

controls

chosen

were

patients

admitted

to

hospital

with

chest

pain

which

could

have

meant

they

had

a

lower

probability

of

prior

travel

this

pathology

was

therefore

a

potential

limiting

factor

Other

studies

-LSB-

-RSB-

could

also

have

been

affected

by

this

form

of

bias

in

that

the

controls

they

chose

were

patients

who

had

attended

hospital

with

symptoms

compatible

with

those

of

DVT

and/or

PE

objective

examination

subsequently

determined

that

neither

pathology

was

present

The

study

by

ten

Wolde

et

al.

-LSB-

-RSB-

included

cases

of

VTED

present

in

patients

who

had

been

admitted

to

hospital

for

other

reasons

which

reduced

the

number

of

days

before

the

episode

of

VTED

in

which

the

patient

could

have

travelled

The

methodological

evaluation

of

the

studies

is

shown

in

Table

Overall

the

evaluation

was

appropriate

in

five

studies

-LSB-

-RSB-

while

results

were

less

satisfactory

in

three

-LSB-

-RSB-

mainly

because

of

the

inadequate

choice

of

controls

and

because

the

study

design

did

not

enable

the

authors

to

achieve

a

good

estimate

of

the

risk

being

studied

There

was

a

high

degree

of

agreement

between

the

reviewers

concerning

the

quality

criteria

of

the

cases

and

controls

-LRB-

weighted

kappa

index

-RRB-

Table

evaluation

of

the

studies

included

in

the

systematic

review

in

accordance

with

SIGN

criteriaCharacteristicFerrari

Wolde

validityClear

appropriate

questionsGAGGGGAGSelection

of

subjectsCases

and

controls

from

comparable

populationsAAA-GA-GGAPGIdentical

exclusion

criteria

for

cases

and

controlsP-AAAGGGAGParticipation

rate

by

cases

and

between

participants

and

non-participantsNSNSNSNSNSNSNSNSCases

are

defined

and

clearly

differentiated

from

controlsA-GGPAGG-APG-AIt

is

clearly

stated

that

the

controls

are

non-casesAGPPG-AG-APPEvaluationKnowledge

of

exposure

did

not

influence

designation

of

casesAAAPG-APAAThe

exposure

is

measured

in

a

standard

valid

wayAPA-PG-AG-AG-APAConfoundingIdentification

of

main

confounding

factorsP-APAP-AAA-PP-AAStatistical

analysisIdentification

of

confidence

intervalsYesYesYesYesYesYesYesYesOverall

assessmentControl

of

bias

and

confounding

factors

Confidence

that

the

overall

effect

is

due

to

the

exposure

being

investigated

The

results

are

applicable

to

the

target

group

of

patients

being

studied

A

single

evaluation

is

shown

when

the

two

reviewers

agree

otherwise

both

evaluations

are

given.G

Good

A

adequate

P

poor

NS

not

stated

Results

of

the

meta-analysis

The

studies

were

varied

as

regards

the

episode

of

VTED

included

-LRB-

DVT

and/or

PE

-RRB-

the

controls

chosen

the

means

of

transport

evaluated

and

the

estimated

duration

of

the

travels

These

variations

influenced

the

performance

of

the

meta-analysis

Thus

we

decided

to

carry

out

two

meta-analyses

on

the

basis

of

the

similarity

between

study

designs

i.e.

on

the

one

hand

studies

of

patients

with

DVT

-LRB-

with

or

without

PE

-RRB-

considering

any

type

of

transport

and

on

the

other

hand

those

studies

examining

only

cases

in

which

the

subjects

travelled

by

plane

-LRB-

Table

-RRB-

Table

obtained

in

the

studies

by

type

of

transport

All

types

of

transportOnly

planeOther

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Ten

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

NANADVT

and/or

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Not

applicable

NS

not

statedResults

are

expressed

as

number

of

patients

travelling

total

-LRB-

-RRB-

aArya

Travels

of

more

than

hbTen

Wolde

Restricted

to

patients

capable

of

travellingcDimberg

More

specific

case

definition

of

the

two

analyseddCannegieter

Venous

thromboembolism

episodes

within

the

weeks

after

travel

Meta-analysis

of

studies

including

patients

with

DVT

or

DVT+PE

all

types

of

transport

Six

studies

-LSB-

-RSB-

were

examined

The

Der

Simonian

Laird

test

of

heterogeneity

gave

a

result

of

p

with

an

estimated

total

variance

due

to

variance

between

the

studies

The

global

estimator

with

a

model

of

random

effects

produced

an

OR

of

-LRB-

CI

-RRB-

No

publication

bias

was

recorded

-LRB-

the

Begg

and

Egger

tests

results

were

not

significant

-RRB-

The

sensitivity

analysis

revealed

that

no

study

influenced

significantly

on

the

final

OR

Figure

shows

the

forest

plot

graph

corresponding

to

this

meta-analysis

Fig.

plot

for

studies

that

evaluated

the

antecedent

of

any

type

of

transport

and

only

travels

by

plane

a

Travels

by

any

type

of

transport

b

Travels

by

plane

Meta-analysis

of

studies

including

patients

with

DVT

or

DVT+PE

only

travels

by

plane

This

meta-analysis

included

five

studies

-LSB-

-RSB-

The

results

of

the

Der

Simonian

Laird

heterogeneity

test

were

not

significant

which

suggests

there

was

little

heterogeneity

between

the

variances

of

the

studies

The

OR

estimator

produced

a

non-statistically

significant

value

of

-LRB-

CI

-RRB-

Again

no

publication

bias

was

detected

and

the

sensitivity

analysis

showed

that

study

was

influential

on

the

OR

Figure

shows

the

forest

plot

graph

corresponding

to

this

meta-analysis

Discussion

The

results

from

our

analysis

reveal

a

slight

association

between

long

travels

and

the

development

of

DVT

or

PE

although

this

association

disappears

when

only

travels

by

plane

are

considered

The

published

case-control

studies

were

characterised

by

considerable

heterogeneity

especially

in

the

selection

of

controls

and

there

were

some

errors

in

the

study

design

that

could

have

led

to

bias

in

estimating

the

effect

of

the

association

being

studied

A

meta-analysis

of

case-controls

studies

has

recently

been

published

In

that

study

we

did

not

find

any

relationship

between

a

long-distance

travel

and

venous

thromboembolic

disease

when

all

the

means

of

transportation

were

valued

or

only

by

plane

but

not

all

published

studies

were

included

-LSB-

-RSB-

The

pathogenic

basis

of

the

economy

class

syndrome

lies

in

a

series

of

predisposing

environmental

factors

within

aircraft

-LRB-

low

humidity

relative

hypoxia

and

low

barometric

pressure

-RRB-

that

would

facilitate

the

relative

dehydration

of

passengers

these

factors

would

be

accompanied

by

mechanical

ones

such

as

immobility

the

narrowness

of

the

seats

and

the

prolonged

maintenance

of

a

seated

position

with

flexion

and

compression

of

the

poplitea

region

-LSB-

-RSB-

The

description

of

episodes

of

DVT

related

to

other

means

of

transport

produces

a

loss

of

plausibility

of

the

environmental

factors

associated

solely

with

travels

by

plane

and

puts

the

prime

focus

on

purely

mechanical

factors

and

on

the

characteristics

of

the

patients

themselves

Moreover

a

recently

published

study

-LSB-

-RSB-

found

no

variation

at

all

between

different

coagulation

factors

after

exposure

to

the

environmental

conditions

resembling

in

a

prolonged

travel

by

plane

Nevertheless

in

one

recent

systematic

review

Philbrick

et

al.

-LSB-

-RSB-

emphasised

the

presence

of

prothrombotic

risk

factors

and

a

flight

duration

longer

than

h

as

predisposing

to

suffer

a

VTE

episode

The

patient-related

variables

refer

to

a

greater

frequency

of

VTED

risk

factors

Thus

various

studies

have

reported

that

a

high

percentage

of

the

patients

involved

were

obese

higher

or

smaller

aged

more

than

years

had

antecedents

of

VTED

were

taking

oral

contraceptives

or

presented

a

state

of

hypercoagulability

or

thrombophilia

-LSB-

-RSB-

This

result

implies

that

there

is

a

relation

between

transport-related

factors

and

those

concerning

to

the

patients

themselves

and

that

this

is

relevant

to

the

development

of

an

episode

of

VTED

Studies

that

have

sought

to

analyse

this

association

have

obtained

divergent

results

mainly

because

of

the

different

study

designs

this

would

explain

the

high

variability

in

the

OR

obtained

which

ranged

from

to

Moreover

these

studies

were

heterogeneous

in

various

methodological

respects

and

this

could

have

influenced

the

final

results

some

-LSB-

-RSB-

matched

the

cases

and

controls

by

sex

and

age

which

limits

the

influence

of

these

variables

in

determining

the

association

under

study

in

the

study

by

Martinelli

et

al.

-LSB-

-RSB-

the

cases

were

a

selection

of

all

the

possible

incident

cases

within

a

given

period

in

another

-LSB-

-RSB-

the

controls

selected

were

not

derived

from

the

same

population

as

that

of

the

cases

the

means

of

transport

and

the

duration

of

the

travel

as

well

as

the

type

of

VTED

episode

evaluated

were

different

in

all

the

studies

and

in

general

no

multivariate

or

stratified

analysis

was

carried

out

to

attribute

the

increased

risk

of

developing

an

episode

of

VTED

to

the

long

travel

An

interesting

aspect

from

the

methodological

point

of

view

is

the

possible

selection

bias

that

might

have

entered

these

studies

those

patients

-LRB-

the

cases

-RRB-

who

travelled

presented

fewer

VTED

risk

factors

than

did

the

controls

i.e.

they

were

basically

more

healthy

Not

only

could

this

have

led

to

the

relation

between

the

development

of

an

episode

of

VTED

and

the

antecedent

of

having

made

a

long

travel

being

underestimated

but

it

might

even

appear

that

the

prior

travel

had

a

protective

effect

as

was

the

case

in

the

study

by

Dimberg

et

al.

-LSB-

-RSB-

One

way

of

minimising

this

type

of

bias

could

have

been

to

include

only

those

patients

who

would

physically

have

been

capable

of

making

a

long

travel

this

was

only

actually

done

in

the

case

of

the

study

by

ten

Wolde

et

al.

-LSB-

-RSB-

Another

possibility

could

be

to

adjust

for

this

variable

-LRB-

which

might

be

considered

that

of

comorbidity

-RRB-

by

means

of

multivariate

analysis

Given

the

methodological

heterogeneity

of

the

published

studies

of

cases

and

controls

it

was

not

possible

to

carry

out

a

detailed

meta-analysis

of

different

aspects

of

the

relation

under

study

and

so

its

scope

was

limited

to

those

studies

that

assessed

the

association

between

DVT

or

between

DVT

and

PE

and

prolonged

travels

by

plane

or

by

any

other

form

of

transport

In

any

case

the

association

that

was

found

in

the

latter

case

was

only

weak

and

there

was

a

large

degree

of

variation

in

the

methodological

quality

applied

in

the

various

studies

A

noteworthy

question

is

the

antecedent

of

a

prolonged

travel

made

by

the

controls

with

variations

in

all

the

studies

of

except

in

the

case

of

the

study

by

Dimberg

et

al.

-LSB-

-RSB-

in

the

latter

case

the

scenario

chosen

for

analysis

led

to

the

choice

of

a

population

in

which

a

high

proportion

-LRB-

-RRB-

made

international

travels

an

aspect

that

could

have

weakened

any

potential

association

Similarly

the

rate

of

incidence

of

a

lengthy

travel

among

the

cases

ranged

from

to

although

in

two

studies

this

figure

was

surprisingly

high

in

that

by

Dimberg

et

al.

-LSB-

-RSB-

and

in

the

study

by

Ferrari

et

al.

-LSB-

-RSB-

The

methodological

quality

of

the

studies

examined

in

this

systematic

review

means

that

we

must

be

cautious

concerning

the

results

reported

Although

there

does

seem

to

be

a

likely

relation

between

a

long

travel

and

the

development

of

an

episode

of

VTED

such

an

association

must

be

of

such

a

magnitude

that

a

small

bias

or

modification

to

the

study

could

increase

or

decrease

the

strength

of

the

association

recorded

In

conclusion

we

may

deduce

from

this

systematic

review

that

there

does

exist

a

real

but

weak

association

between

episodes

of

VTED

and

the

antecedent

of

a

lengthy

travel

and

this

relation

with

the

travels

by

plane

is

only

nearly

significant

The

heterogeneity

and

the

methodological

quality

of

the

studies

published

on

the

question

limit

the

robustness

of

the

conclusions

obtained

O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O