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Dioxinlike
Properties
of
a
Trichloroethylene
Combustion-Generated
Aerosol
S.
Alex
Villalobos,I
Michael
J.
Anderson,1
Michael
S.
Denison,2
David
E.
Hinton,1
Katherine
Tullis,2
Ian
M.
Kennedy,3
A.
Daniel
Jones,6
Daniel
P.
Y.
Chang,5
GoSu
Yang,3
and
Peter
Kelly4
'Department
of
Anatomy,
Physiology
and
Cell
Biology,
School
of
Veterinary
Medicine,
2Department
of
Environmental
Toxicology,
3Department
of
Mechanical
and
Aeronautical
Engineering,
4Department
of
Chemistry,
5Department
of
Civil
and
Environmental
Engineering,
and
6Facility
for
Advanced
Instrumentation,
University
of
California,
Davis,
CA
95616
USA.
Con,ventional
chemic.l
anal
of
incineration
by6-products
identify
comPp
of
ity
but
often
fail
to
indicate
theJ
t
of
other
ch
tat.ma
p
l
is
a
pe
vious
repo
-c
s
fiom
soot
aOsolsforned
d
i
l
ti
d
ene
(TCE)
and
:pyrolysis
of
phastics
exhibited
a
di
T
resone
t
a
hat
adnocyte
y.
To
verify
this
dioxinlkre
effect,
the
ct,
i
polr
nd
nonpolar
fc
ions,
some
containing
pim
hlo
ated
aromatic
hydarbons,were
evaluated
for
toxicity
using
an
embr.o
ay,
for
atie
using
primr.y
liver.
cd
culoM..
,.-.a
for
the
a.iifty
to
tansform
he
aryl
hydroarbon..
iec
tor
its-oo
:sDNA..:bnin
Jr
sIn
.liver
cytosolin
a
ge
ret.
a
t
in
ay.
Each
of
th`Aies:e
mays
dete.:..)
embrs
and
primary
iver
cl
c
of
ranbo
twut(O
y
m
concentrations
of
extmact
rnng
fm
0.05
to
45
I
&CaiotOc
ipenad
,
and
adjacent
p
eritonealdema
occurred
after
exp
of
embryos
to
of
7
pg/I
or
greater.
These
same
cKposure
levds
were
ated
with
bn
ni
dlopment
ad
at
the
higher
concentrtions,
death.
Some
of
the
fatosere
toxic
b.t
n
a
toxic
s
the
whole
extact.
Inlivr
cells,
total
cellular
protein
an
ca
tte
o
acivite
not
altered
in
vt
o
expos
u
whole
e
c(0
25
p
w
ctochrome
P4501AI
protein
ande
In
the
pres-
ence
of
whole
extact,
estadio
d
et
vitel
n
s
y
es
was
c
O
tie
f
only
fraton
I
(nonpolar)
showed
a
similar
trend,
u
viteioe
in
synthesis
i
was
not
significat
The
soot
ect
and
fractions
bound
to
the
Ah
receptor
ad
.showed.
a
sirf
candy
positve
result
in
the
gel
r
n/DNA
bindnt.g
test.
C.hei
..c...y.
..sg
with
detection
l-Wiiwmo
Z37
e
h-
rAnge
did
not
show
presence
of
e
c
pn.
Ou
s
idc
f
...-
ge
~
~~~~~~~~~~.
...
..
dated
with
TCE
-cmb
a
iginally
trgt
f
anlyi
p
iae
sknh
through
diozxnllke
mechanis.
:4
wonkb:
Ah
receptor,
a
t
,
complix
dna
like
toxiity,
d?xi
-response
eleent
binig
emro
lli
incom
c
bustion
by-prod
s,
liver,
trico
hylene,
vviro
b
1047
743
(1996)
Incineration
has
been
widely
used
as
a
means
for
disposal
of
municipal,
hospital,
and
industrial
hazardous
wastes.
Its
use
has
been
curtailed
in
recent
years
because
of
concern
about
the
emission
of
toxic
by-
products
associated
with
the
soot
particles,
especially
chlorinated
phenols,
aromatic
hydrocarbons,
polychlorinated
dibenzodi-
oxins,
and
dibenzofurans
(1-3).
These
emissions
arise
from
improper
operation
of
incinerators
or
from
transients
(4-6)
in
operation
during
which
inadequate
temper-
ature
and
mixing
conditions
in
the
combus-
tion
zone
may
lead
to
incomplete
combus-
tion.
These
transient
discharges,
also
known
as
puffs,
are
characterized
by
large
transient
emissions
of
soot
and
toxic
volatile
organic
hydrocarbons
(47).
Although
they
are
rel-
atively
rare
during
incinerator
operation,
puffs
contribute
a
major
fraction
of
the
toxic
compounds
in
incinerator
effluent.
For
example,
Wendt
(3)
demonstrated
in
a
toluene-fed
kiln
that
puffs
can
emit
approx-
imately
10,000
ppm
of
hydrocarbons
for
a
period
of
about
20
seconds.
Depending
on
the
precursor
chemistry,
additional
reac-
tions
downstream
of
the
high
temperature
regions
may
lead
to
the
formation
of
diox-
ins
(8).
Atmospheric
transport
of
incinera-
tor
emissions
may
result
in
wide-spread
dispersal
and
subsequent
deposition
of
these
particles
in
various
environmental
matrices
(9)
including
soil,
water,
and
veg-
etation
(10).
Dioxin
and
dioxinlike
compounds
con-
stitute
a
diverse
and
important
group
of
con-
taminants
widely
spread
in
the
environment,
where
they
persist
as
complex
mixtures
(7,11,12).
One
particular
compound,
2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD),
has
been
the
subject
of
consider-
able
concern
with
regard
to
incinerator
emis-
sions.
TCDD
and
related
halogenated
aro-
matic
hydrocarbons,
including
2,3,7,8-tetra-
chlorodibenzofuran
(TCDF),
produce
a
wide
variety
of
species-
and
tissue-specific
toxic
and
biological
effects,
such
as
teratoge-
nesis,
immunotoxicity,
hepatotoxicity,
tumor
promotion,
and
induction
of
numerous
enzymes,
including
microsomal
cytochrome
P4501A1
(CYPlAI)
(7,13).
Many
hazardous
waste
sites
contain
chlo-
rinated
solvents,
induding
trichloroethylene
(TCE).
For
example,
the
McClellan
Air
Force
Base
in
Sacramento
(California)
con-
tains
soil
that
is
heavily
contaminated
by
TCE;
it
was
used
as
a
cleaning
agent
on
air-
craft.
Earlier
experiments
by
Blankenship
et
al.
(14)
found
that
extracts
from
soot
aerosols
formed
during
the
combustion
of
TCE
exhibited
a
dioxinlike
response
when
subjected
to
a
keratinocyte
bioassay.
These
experiments
showed
that
all
of
the
hazardous
material
was
associated
with
the
aerosol
and
that
little
was
found
in
the
gas
phase
of
the
flames.
Chemical
analyses
of
the
soot
extracts
indicated
that,
at
picomole
levels,
TCDD/
TCDF
were
not
detected,
suggesting
that
chlorinated
fulvalenes,
among
other
chlori-
nated
hydrocarbons,
were
major
components
of
the
mixture
and
that
these
may
have
been
responsible
for
the
toxic
response.
Because
of
its
environmental
importance
and
in
view
of
the
previous experience
with
toxic
TCE
aerosols,
TCE
was
chosen
as
the
model
waste
for
this
study.
Although
conventional
chemical
analy-
ses
of
incineration
by-products
identify
compounds
of
known
toxicity,
they
often
fail
to
indicate
the
presence
of
other
chemi-
cals
which
may
also
pose
health
risks.
The
purpose
of
the
present
investigation
was
to
verify
whether
materials
with
dioxinlike
properties
were
present
in
the
chemically
Address
correspondence
to
I.M.
Kennedy,
Department
of
Mechanical
and
Aeronautical
Engineering,
University
of
California,
Davis,
Davis,
CA
95616
USA.
Trout-specific
antibodies
and
standards
were
gifts
from
Michael
Miller,
West
Virginia
University,
and
Ray
Simon,
formerly
of
the
U.S.
Fish
Health
Center,
Kearneysville,
West
Virginia.
Anti-scup
CYPlAI
(Mab
1-12-3)
was
a
gift
of
John
Stegeman,
Woods
Hole
Oceanographic
Institution.
2,3,7,8-Tetrachlorodibenzo-p-dioxin
was
obtained
from
S.
Safe,
Texas
A&M
University.
A.V.
thanks
Miguel
Gonzilez-Doncel
and
Swee
Teh
for
their
assistance
in
the
histological
preparations
and
evalu-
ations.
This
research
was
supported
by
the
NIEHS
Superfund
Basic
Research
Program
(P42ES04699),
by
the
Ecotoxicology
Program
of
the
University
of
California
Toxic
Substances
Research
and
Teaching
Program,
and
by
the
US
EPA*UC
Davis
Center
for
Ecological
Health
Research
(R819658).
Received
29
March
1996;
accepted
10
April
1996.
Volume
104,
Number
7,
July
1996
.
Environmental
Health
Perspectives
734
Articles
-
Toxicity
of
trichloroethvlene
soot
complex
TCE
soot
mixture
and
its
frac-
tions.
Dioxinlike
effects
(e.g.,
cardiotoxicity
and
yolk
sac
edema)
have
been
investigated
in
medaka
(Oryzias
latipes)
embryos.
Biological
potency
has
been
demonstrated
in
vitro
both
by
measuring
interference
of
compounds
from
the
mixture
with
estrogen
receptor
using
rainbow
trout
(Oncorhynchus
mykiss)
liver
cells
and
by
monitoring
the
mixture's
binding
affinity
to
the
Ah
recep-
tor
and
further
ability
to
convert
it
into
its
DNA
binding
form.
Methods
Flame
Conditions
and
Chemical
Analysis
Unmixed
or
poorly
mixed
combustion can
be
modeled
in
a
well-defined
laboratory
experiment
with
a
laminar
diffusion
flame.
Poor
mixing
with
relatively
long
residence
times
in
an
incinerator
is
then
modeled
by
increasing
the
flame
length
beyond
the
point
at
which
soot
breaks
through
the
flame
tip.
The
nature
of
the
compounds
that
are
emitted
from
these
flames
is
typical
of
the
material
that
could
be
found
in
puffs
from
incinerators.
A
mixture
of
TCE
and
methane
(CH4)
was
burned
in
a
laminar
diffusion
flame.
TCE
vapor
was
generated
by
passing
CH4
through
an
impinger
containing
liquid
TCE
that
was
maintained
at
a
constant
temperature.
The
mole
fraction
of
TCE
in
the
methane
was
0.51;
the
flow
rates
were
696
ml/min
of
CH4
and
734
ml/min
of
TCE.
This
mixture
was
supplied
to
an
axisymmetric
laminar
diffusion
flame
burn-
er.
The
co-flow
burner
assembly
consisted
of
a
circular
Plexiglas
chamber
with
a
67-
mm
inside
diameter.
The
round
nozzle
was
made
of
thin-walled
stainless
steel
tubing
with
a
6-mm
outside
diameter.
Soot
was
collected
from
the
post-flame
gases
with
a
47-mm
PTFE-coated
glass
fiber
filter
in
line
with
a
sorbent
tube.
The
sorbent
tube
was
prepared
by
pack-
ing
100
mm
lengths
of
Pyrex
glass
tubing
(12
mm
O.D.)
with
3.5
g
of
Carbotrap
C.
Glass
wool
plugs
were
inserted
into
both
ends.
The
filters
were
Soxhlet
extracted
for
16
hr
with
250
ml
of
dichloromethane
(CH2Cl2)
using
anhydrous
sodium
carbon-
ate
(Na2CO3)
to
neutralize
adsorbed
acids.
CH2Cl2
extracts
were
roto-evaporated
to
a
volume
of
10
ml,
divided
into
10
aliquots,
and
stored
at
-
200C.
Each
aliquot
was
dry-
evaporated
under
a
stream
of
nitrogen
at
25°C
and
reconstituted
in
1
ml
of
analytical
grade
dimethylsulfoxide
(DMSO)
for
bio-
assays.
An
individual
aliquot
was
applied
to
a
silica
gel
column
and
four
fractions
were
eluted
with
different
solvents
including
fraction
1
(nonpolar
compounds)
with
n-
hexane,
fraction
2
(primarily
PAHs
and
chlorinated
PAHs)
with
n-hexane/CH2Cl2
(3:2
v:v),
fraction
3
(intermediate
polarity)
with
CH2Cl2,
and
fraction
4
(polar
com-
pounds)
with
methanol.
Control
fractions,
prepared
by
Soxhlet
extractions
of
blank
cellulose
extraction
thimbles,
were
obtained
using
identical
laboratory
procedures.
Analyses
were
performed
on
extracts
and
fractions
using
a
VG
Trio-2
mass
spectrom-
eter
coupled
to
a
Hewlett
Packard
5890
gas
chromatograph.
Separations
were
performed
using
a
30-m
DB-17
capillary
column
with
helium
as
carrier
gas.
Electron
ionization
(70
eV)
mass
spectra
were
obtained;
compounds
were
quantified
based
upon
average
molar
response
factors
obtained
for
a
series
of
PAHs
and
chlorinated
aromatic
standards.
Embryo
Toxicity
Assay
Egg
collection
and
broodstock
mainte-
nance
followed
the
procedure
described
by
Marty
et
al.
(15).
Medaka
female
brood-
stock,
maintained
at
25°C
under
a
16
hr
light:8
hr
dark
photoperiod
stimulating
continuous
egg
production,
were
individu-
ally
netted
and
eggs
<5
hr
old
were
careful-
ly
removed
from
extruded
clusters.
Filaments
that
attached
adjacent
eggs
were
broken
by
gently
rolling
clusters
between
moistened
finger
tips.
Individual
(blastula
stage)
eggs
were
kept
in
continuously
aerat-
ed
embryo
rearing
medium
(ERM)
(16).
Embryo
exposures
were
repeated
until
the
whole
TCE
soot
extract
and
individual
fractions
were
tested.
Each
exposure
was
conducted
as
a
completely
randomized
design
(17)
which
consisted
in
pooling
eggs
and
distributing
them
(n
=
8)
by
stratified
random
assortment
to
individual
20
ml
borosilicate
vials
(Fisher
Scientific,
Pittsburgh,
Pennsylvania)
in
each
of
four
replicates.
Each
vial
contained
2
ml
of
solu-
tion
and
18
ml
of
air
space.
A
double
layer
of
teflon
tape
(Scientific
Instruments,
Randallstown,
Maryland)
and
screw-type
lid
were
used
to
hermetically
seal
each
vial.
For
each
experiment,
vials
were
coded
for
blind
study
except
for
one
additional
ERM
replicate
(known
control,
not
included
in
statistical
analysis),
which
served
as
a
refer-
ence
for
time
of
normal
development.
Due
to
the
hazardous
nature
of
this
complex
mixture
and
to
the
blind
randomized
exper-
imental
design,
embryos
were
maintained
in
vials
under
static
(non-renewal)
condi-
tions
for
duration
of
embryonic
develop-
ment
(8
days).
After
exposure
and
rinsing
in
clean
ERM,
embryos
were
transferred
to
clean
vials
and
allowed
to
complete
their
development.
Static
non-renewal
conditions
have
been
used
when
testing
dioxin,
dioxin-
like
compounds,
and
other
complex
mix-
tures
(17-20).
Oxygen
requirements
dur-
ing
medaka
development
in
a
closed
system
(no
access
to
free
air),
are
approximately
23
ml
of
ERM/egg
(21).
Since
dissolved
oxy-
gen
in
air
is
25-30
times
greater
than
in
ERM,
sufficient
aeration
was
provided
given
the
eggs:ERM/eggs:air
ratio.
For
exposure,
soot
whole
extract
(WE)
and
fraction
stock
solutions
were
dissolved
in
ERM
(pH
7
±
0.2)
using
DMSO
(WE)
or
DMSO/fraction
as
vehide
solvents.
All
vehicle
concentrations
were
restricted
to
500
pIll
(0.05%
v/v).
This
concentration
has
shown
in
pilot
tests
to
produce
no
embryon-
ic
toxicity.
Estimated
maximum
concentra-
tion
of
incomplete
combustion
by-products
was
0.09
pg/pl
of
vehicle
(i.e,
500
p,l/
x
0.09
pg/pl
=
45
pg/I).
The
range
of
interest
in
these
pilot
studies
was
determined
between
a
stock
solution
of
500
pl
carrier
(containing
WE
soot)
in
1
liter
ERM,
and
a
respective
dilution
of
1:100
(1
ml
of
stock
in
100
ml
ERM).
The
intermediate
concentra-
tions
were
chosen
so
that
there
could
be
5
equidistant
intervals
in
a
log
scale,
based
on
the
absolute
difference
(2.0)
between
log
45
pg/l,
and
log
0.45
pg/l.
This
conversion
resulted
in
intervals
of
0.4
log
units,
which
when
reconverted
(antilog)
to
a
linear
scale
gave
concentrations
of
45,
18,
7.2,
2.7,
0.9,
and
0.45
pg/l.
Controls
consisted
of
embryos
exposed
to
vehide
or
ERM
alone.
Embryos
were
observed
daily
under
a
dissecting
microscope
for
normal
and
abnormal
development.
Mortality
and
sub-
lethal
endpoints
including
pericardial
and
peritoneal
edema,
eye
and/or
subdermal
edema,
hemostasis,
yolk
resorption,
cephal-
ic
and
spinal
deformities,
and
hatching
suc-
cess
were
observed.
The
transparent
chorion
of
medaka
embryonated
eggs
permits
direct
visualization
of
heart
beat.
Cardiac
activity
was
monitored
by
averaging
heart
rate
(in
beats
per
minute
±
SD)
of
at
least
three
embryos
per
vial.
This
monitoring
was
done
daily
until
hatching.
Evaluation
was
continued
through
the
first
4-5
days
after
hatching.
Development,
induding
swim
(or
air)
bladder
inflation
and
swimming
activi-
ty,
was
monitored.
A
hatchling
was
consid-
ered
normal
if
it
swam
vigorously,
and
had
normal
gross
morphology
and
an
inflated
swim
bladder.
Medaka
hatchlings
inflate
swim
bladders
within
24
hr
(15).
To
con-
firm
and
extend
observations
with
the
dis-
secting
microscope,
a
limited
number
of
normal
and
abnormal
embryos/larvae
were
fixed
in
10%
buffered
formalin,
dehydrated
in
a
graded
ethanol
series
and
embedded
in
complete
glycolmethacrylate
monomer
(22).
Sections
(4
pm
thickness)
were
cut
on
an
LKB
Historange
microtome,
mount-
ed
to
glass
slides,
and
stained
with
hema-
toxylin
and
eosin
(H&E)
or
toluidine
blue.
Environmental
Health
Perspectives
*
Volume
104,
Number
7,
July
1996
735
Articles
-
Villalobos
et
al.
Serial
sectioning
was
performed
to
validate
locations
within
a
given
embryo/larva.
For
statistical
purposes,
all
embryos
that
failed
to
hatch
were
considered
abnor-
mal.
Differences
from
the
controls
were
identified
with
Wilcoxon's
sign-rank
test
(p<0.05),
using
the
JMP
statistical
soft-
ware
package
(SAS
Institute,
Cary,
North
Carolina).
The
additional
ERM
replicate
was
excluded
from
statistical
calculations.
Liver
Cell
Assays
Sexually
immature
male
and
female
rain-
bow
trout
(400-600
g
mean
weight)
from
Mt.
Lassen
trout
farm
(Red
Bluff,
California)
were
housed
in
a
large
(4
x
1.7
x
1
m)
concrete
tank
at
the
Institute
of
Ecology
aquaculture
facility
at
UC-Davis.
Gonadosomatic
indices
(gonad
weight/
body
weight
x
100)
ranged
between
0.25
and
0.75%.
Fish
were
held
under
natural
photoperiod
in
constant
flow
(Lake
Berryessa,
California)
water
at
temperatures
between
14
and
15°C
and
fed
Silver
Cup
trout
pellets
at
approximately
1%
body
weight/day.
Fish
were
acclimated
to
the
above
holding
conditions
at
least
2
weeks
before
experimentation.
Medium
199,
L-glutamine,
anti-
biotic-antimycotic
solution,
buffer
salts,
anti-rabbit
IgG
alkaline
phosphatase
conju-
gated
antibodies,
p-nitrophenyl
phosphate
(PNPP),
pyruvate,
NADH,
and
NADPH
were
purchased
from
Sigma
(St.
Louis,
Missouri).
17,B-Estradiol
was
purchased
from
Steraloids
(Wilton,
New
Hampshire).
Anti-
mouse
IgG
horseradish
peroxidase-conjugat-
ed
antibody
was
purchased
from
Amersham
(Arlington
Heights,
Illinois).
Tween
20,
enzyme
immunoassay
grade
nonfat
dry
milk,
and
3,3',5,5'-tetramethylbenzidine
(TMB)
solution
were
purchased
from
Bio-Rad
(Burlingame,
California).
Diethanolamine
was
purchased
from
Aldrich
(Milwaukee,
Wisconsin),
collagenase
(269
U/mg)
from
Worthington
Biochemicals
(Newark,
New
Jersey),
and
7-ethoxyresorufin
and
resorufin
from
Molecular
Probes
(Eugene,
Oregon).
All
other
chemicals
were
of
analytical
grade.
Cells
were
isolated
following
a
two-step
perfusion
technique
(23)
with
the
follow-
ing
modifications:
no
heparin
was
injected
into
the
animals
and
the
perfusion
medium
was
a
calcium-free
HEPES
buffered
Hank's
salt
solution,
pH
7.6
(24).
Following
liver
digestion
and
tissue
disassociation,
cells
were
washed
two
times
and
resuspended
in
medium
199
(see
below).
Viability
was
assessed
by
phase
microscopy
and
trypan
blue
dye
exclusion.
Typically
90%
or
more
of
the
cells
were
viable.
Cell
cultures
followed
procedures
of
Pesonen
and
Andersson
(25)
with
one
exception:
HEPES
buffered
medium
199
at
pH
7.6
contained
no
additional
Na2HPO4
because
high
concentrations
caused
precipi-
tation
and
interfered
with
the
ELISA
assays.
Cells
were
plated
on
60-
or
100-mm
diame-
ter
Falcon
polystyrene
tissue
culture
dishes
(Beckton
Dickinson,
Oxnard,
California)
at
a
concentration
of
approximately
1.65
x
105
cells/cm2
and
placed
in
a
humidified
Ambi-
Hi-Low
incubator
(Baxter,
McGaw
Park,
Illinois)
at
1
5°C
in
air
atmosphere.
Cells
were
allowed
to
attach
to
tissue
culture
dishes
and
acclimate
to
culture
con-
ditions
for
24
hr
before
the
first
media
change
and
dosing.
Cells
were
then
treated
with
fresh
medium
199
containing
either
DMSO
alone
(control),
WE
(0.6-25
pg/I),
or
each
of
the
fractions
(in
DMSO).
Due
to
the
use
of
4
fractions
and
testing
of
each
with
cells
from
a
single
trout,
a
single
con-
centration
(1
1.25
pg/l)
was
used.
The
total
concentration
of
DMSO
in
the
media
was
maintained
at
0.05%
(v/v)
as
described
above.
Simultaneously,
1
pM
17,-estradiol
or
an
equivalent
volume
of
ethanol
(carrier
control)
was
added
to
the
medium.
Cells
from
control
and
treatment
groups
were
always
obtained
from
the
same
fish.
After
48
hr
of
exposure,
cells
were
gen-
tly
scraped
off
the
dishes
with
a
teflon
rod
and
placed
in
individual
centrifuge
tubes.
Tubes
were
centrifuged
at
150g
for
2
min
at
40C
to
separate
media
from
cells.
Resultant
cell
pellet
was
resuspended
in
1
ml
of
0.1
M
phosphate
buffer,
pH
7.5
(80mM
Na2HPO4,
20
mM
NaH2PO4)
with
20%
glycerol
and
sonicated
for
5
sec
on
ice.
Cell
homogenates
and
media
were
immediately
frozen
on
dry
ice
and
stored
at
-80°C
until
assays
were
performed.
Determinations
of
vitellogenin
(Vg)
and
albumin
(Alb)
released
into
the
cell
culture
media
and
cellular
CYPlAl
content
were
estimated
by
indirect
ELISA
as
described
(26,27)
using
monoclonal
(MAb)
anti-trout
Vg
(MAb
SD6C)
(28),
polyclonal
rabbit
anti-trout
Alb,
and
anti-scup
CYPlAl
(MAb
Table
1.
List
of
the
major
incomplete
combustion
by-products
present
in
trichloroethylene
whole
extract
Retention
time
(min)
Monoisotopic
m/z
Tentative
identification
%
of
TIC
areaa
17.17
248
C6HCI5,
pentachlorobenzene
5.1
20.37
282
C6C16,
hexachlorofulvene
1.0
20.75
282
C6C16,
hexachlorobenzene
12.3
21.32
272
C8HCI5
0.5
21.62
296
C7H2C16,
heptachlorobicyclo-
0.5
[2.2.1
]hepta-2,5-diene
21.85
310
C8H4C16
1.6
22.15
296
C7H2C16,
heptachlorobicyclo-
0.7
[2.2.1]hepta-2,5-diene
23.08
342
C8HCI7
0.9
24.13
306
C8C16
1.0
24.23
342
C8HCI7
1.0
24.55
330/264
C7HCI7/CloH4cl4
0.9
25.62
330
C7HCI7
1.4
25.85
322
C9H4CI6
0.6
26.20
376
C8C18
3.6
27.08
300/376
C10H5CIYC8CI8
1.2
27.23
298
C10H3C15
2.1
27.55
300
C10H5C15
0.8
27.67
300
C10H5C15
1.0
27.90
298
CiOH3CI5
0.9
28.17
298
C10H3C15
0.6
29.58
298
C10H3C15
0.9
30.92
334
C1OH4C16
3.2
31.05
334
C1OH4C16
2.3
32.07
332
C1oH2CI6
1.4
33.02
332
C10H2C16
1.0
33.40
332
C1OH2C16
1.6
36.23
366
C1OHC17
5.5
37.02
366
CIOHCI7
3.7
43.30
400
C1oC18
5.2
45.15
390
C12HC17
0.8
aListed
peaks
account
for
63%
of
area
of
total
ion
chromatogram
(TIC),
with
ClOHXCI8
x
compounds
repre-
senting
-30%.
The
remaining
area
is
distributed
among
at
least
200
smaller
peaks.
Volume
104,
Number
7,
July
1996
*
Environmental
Health
Perspectives
736
Articles
.
Toxicity
of
trichloroethylene
soot
1-12-3).
Dilutions
of
media
or
cell
extracts
(10-100-fold)
in
phosphate
buffered
saline,
pH
7.5
(PBS:
80mM
Na2HPO4,
20
mM
NaH2PO4,
100
mM
NaCI)
were
used.
Ethoxyresorufin
O-deethylase
(EROD)
activity
of
whole
cell
homogenates
followed
method
of
Burke
et
al.
(29)
adapted
for
microplate
format
(Cambridge
microtiter
plate
fluorometer,
model
7620).
Briefly,
fluorescence
(excitation
530
nm
and
emis-
sion
585
nm)
in
80-100
pg
of
whole
cell
homogenates,
incubated
in
100
mM
potas-
sium
phosphate
buffer,
pH
8.0
(90
mM
K2HPO4,
10
mM
KH2PO4,
0.25
pM
ethoxyresorufin,
and
0.5
mM
NADPH)
to
a
final
reaction
volume
of
0.2
ml,
were
recorded
at
30-40
sec
intervals
over
5
min
at
24°C.
Determinations
of
cellular
lactic
dehydrogenase
(LDH)
activity
were
made
following
the
method
of
Bergmeyer
and
Berndt
(30).
Protein
concentrations
of
cell
homogenates
were
determined
using
the
Bio-Rad
DC
protein
assay
kit,
with
bovine
serum
albumin
(BSA)
as
the
standard.
Each
exposure
group
of
liver
cells
con-
sisted
of
three
to
four
dishes
per
treatment,
with
duplicate
determinations
per
dish.
Significant
differences
between
means
of
various
treatment
groups
were
determined
by
ANOVA
(p<0.05)
and
means
were
con-
trasted
using
Dunnett
with
control
group
and
Tukey-Kramer
methods.
All
statistical
analyses
were
performed
using
the
JMP
procedure
of
SAS
software
(SAS
Institute).
Gel
Retardation/DNA
Binding
Assay
Based
on
the
ability
of
Ah
receptor
(AhR)
ligands
to
convert
this
receptor
to
its
DNA
binding
form,
a
gel
retardation
assay
was
used
to
measure
the
amount
of
inducible
protein
[32P]DNA-complex.
This
provided
an
indirect
way
to
detect
dioxinlike
chemi-
cal(s).
Guinea
pig
hepatic
cytosol
was
used
as
the
source
for
the
receptor,
based
on
pre-
vious
determinations
which
indicated
that
this
species
is
the
most
optimal
for
the
trans-
formation
and
DNA
binding
analyses
of
lig-
and:AhR
complexes
(31).
In
the
assay,
hepatic
cytosol
prepared
from
male
Hartley
guinea
pigs
(250-300
g;
Michigan
Department
of
Public
Health,
Lansing,
MI),
was
suspended
in
ice-cold
HEDG
buffer
(25
mM
HEPES,
pH
7.5,
1
mM
EDTA,
1
mM
dithiothreitol,
10%
(v/v)
glycerol)
and
aliquots
were
stored
at
-80°C
as
previously
described
(32,33).
Protein
concentrations
were
measured
by
the
method
of
Bradford
(34)
using
BSA
as
(D);
. : : :
(.
E)
/
K z --
~~~~~~~~~~~~~~~~~~~~.
..
......
.........
Ca
CJ1H
I
ci
/acl
t1
/
/
..7
c
PC!
* :
./
I
.3
I
_
_n.
-.----.-.--.
Figure
1.
Total
ion
chromatogram
of
trichloroethylene
soot
whole
extract.
More
than
250
incomplete
com-
bustion
by-products
were
formed
during
pyrolysis.
(A)
Hexachlorofulvene,
(B)
pentachlorobenzene,
(C)
hexachlorobenzene,
(D)
octachlorostyrene,
(E)
octachlorofulvalene.
the
standard.
For
gel
retardation
analysis,
125
pl
cytosol
(16
mg
of
protein/ml)
was
incubated
with
DMSO
(20
pl/mI),
15
nM
TCDD
in
DMSO
or
an
aliquot
(2.5
pI)
of
the
soot
WE
or
fractions
(in
DMSO)
for
2
hr
at
20°C.
Gel
retardation
analysis
of
the
samples
was
carried
out
using
[32P]-labeled
dioxin-responsive
element
(DRE)-contain-
ing
DNA
oligonucleotide
as
described
by
Helferich
and
Denison
(33)
and
the
result-
ing
protein-DNA
complexes
were
detected
following
autoradiography
of
dried
gels.
Quantitation
of
the
inducible
protein-DNA
complex
was
carried
out
as
described
by
Denison
and
Yao
(32).
The
2,3,7,8-tetra-
chlorodibenzo-p-dioxin
(TCDD)
was
obtained
from
S.
Safe
(Texas
A&M
University)
and
[y-32P]
ATP
(6,000
Ci/mmol)
from
New
England
Nuclear.
Molecular
biological
reagents
were
obtained
from
New
England
Biolabs.
Results
Analytical
Chemistry
Combustion
of
the
TCE/CH4
mixture
pro-
duced
a
flame
characterized
by
heavy
soot
production,
approximately
100
mg/g
of
fuiel
burned.
CH2CI2
extracts
of
the
soot
had
a
dark
blue
color,
which
may
be
attributed
to
the
presence
of
significant
amounts
of
chlo-
rinated
fulvalenes
(C1OHXCl8-x,
depending
on
number
of
H
and
Cl
substitutions,
or
C10C18=
octachlorofulvalene),
structural
isomers
of
naphthalenes
(14).
CIOHXCI
8x
compounds
represented
-30%
of
the
total
ion
chromatogram
(Table
1).
GC/MS
analysis
of
the
WE
indicated
that
over
250
organics
(Fig.
1)
were
formed
during
TCE
pyrolysis.
Nearly
all
were
chlorinated
mono-
and
polyunsaturated
aliphatics,
cyclic
poly-
enes
1-,
2-,
and
3-ring
aromatics,
phenols,
fulvenes
(structural
isomers
of
benzene),
and
the
above
mentioned
fulvalenes.
With
a
limit
of
detection
of
about
1
pM
in
the
extract,
no
polychlorinated
biphenyls
(2,3,7,8-TCDD
or
TCDF)
were
detected.
Embryo
Toxicity
Assay
The
combined
percentage
of
normal
devel-
opment
for
all
controls
was
above
90
(Table
2).
Greatest
toxicity
was
seen
after
exposure
to
WE
(Table
2
and
Fig.
2).
Based
on
the
nominal
concentrations
previously
estimat-
ed,
the
observed
WE
concentration
in
which
50%
of
larvae
(EC50)
showed
signs
of
abnor-
mality
was
7.2
pg/l,
while
at
2.7
pg/l,
no
effect
was
observed.
The
calculated
EC50
was
-4.3
pg/l
(y
=
-51.64
log
x
+
136.75,
r2
=
0.92).
The
two
higher
WE
concentrations
proved
lethal
to
most
embryos.
The
few
embryos
that
hatched
were
extremely
weak
and
did
not
inflate
swim
bladders.
Statistical
analyses
(Wilcoxon's
sign-rank
test)
showed
Environmental
Health
Perspectives
*
Volume
104,
Number
7,
July
1996
737
Articles
*
Villalobos
et
al.
significant
differences
at
and
above
7.2
pg/l.
The
toxicity
trend
observed
after
exposure
to
the
individual
fractions
indicated
that
frac-
tions
3
and
1
were
the
most
toxic,
while
frac-
tions
2
and
4
had
no
significant
effects.
However,
none
of
the
toxic
fractions
were
as
toxic
as
WE.
For
fraction
1,
approximately
40%
of
embryos
exposed
to
45
pg/l
devel-
oped
abnormally,
while
lower
concentrations
showed
variable
effects
(Tables
2
and
3;
Fig.
2).
Results
with
fractions
2
and
4
were
simi-
lar:
no
more
than
29%
of
the
exposed
embryos
developed
abnormally
regardless
of
concentration
(Table
2).
Fraction
3
was
slightly
more
toxic
than
WE
over
the
range
of
0.45-2.7
pg/l,
and
became
less
toxic
at
higher
concentrations
(approximately
65%
and
50%
of
embryos
exposed
to
18
and
45
pg/l
were
abnormal)
(Tables
2
and
3;
Fig.
2).
The
predominant
embryonic
defect
was
edema,
pronounced
in
pericardial
cavity
but
also
present
in
the
peritoneal
cavity
and
yolk
sac
(Table
3
and
Fig.
3).
Embryonic
mortal-
ity
was
rarely
seen.
Within
the
first
6
days
of
exposure,
13
(1%)
out
of
a
combined
total
of
1280
embryos
died.
Of
these,
only
6
(4
deaths
in
48
hr
or
less
and
2
delayed
hatch-
ings)
were
observed
in
controls.
During
these
first
6
days,
no
symptoms
of
cardiovas-
cular
toxicity
(i.e.,
bradycardia
or
tachycar-
dia)
that
would
indicate
formation
of
edema
were
apparent
(data
not
shown).
Two
to
four
days
later,
depending
on
concentration,
mild
pericardial
edema
appeared
and
pro-
gressed
rapidly,
often
leading
to
death
before
hatching
(Fig.
3).
In
these
severely
affected
embryos,
the
process
of
heart
chamber
for-
mation
observed
as
a
shunt
of
blood
from
left
to
right
was
apparently
terminated,
and
a
pulsatile
single
tube
had
appeared
in
indi-
vidual
fish
who
had
earlier
shown
evidence
of
more
developed
heart
formation.
Other
lesions
included
hemostasis,
a
severe
darken-
ing
over
brain,
and
larger
than
normal
yolk
sac.
Cephalic/spinal
abnormalities
were
rare
(<0.5%).
The
highest
concentration
of
WE
compatible
with
control
hatch
frequency
was
7.2
pg/l.
Fifty
percent
of
hatchlings
exposed
to
this
concentration
showed
nor-
mal
structure
and
were
able
to
inflate
swim
bladders
and
move
about.
The
remainder
could
not
inflate
swim
bladders;
edemas
became
more
severe,
often
extending
from
pericardial
and
peritoneal/yolk
sac
areas
to
the
eyes
(Fig.
4).
Finally,
these
hatchlings
could
not
swim
or
maintain
equilibrium.
In
embryos
showing
no
evidence
of
gross
alterations,
light
microscopy
revealed
additional
lesions.
The
lower
concentrations
of
WE
(0.45
and
0.90
pg/I)
caused
no
apparent
lesions,
but
2.7
pg/l
was
associated
with
mild
hepatocyte
glycogen
depletion
in
liver
hepatocytes.
At
concentrations
of
7.2
pg/l
and
above,
changes
of
greater
magni-
E
0
Co
-
co
CO
=
-0
0C
.1;
0
0
.2
125
100
75
50
25
ERM
control
Solvent
control
0.45
09
.
72
1
-8
5
0.9
2.7
7.2
18
45
Soot
by-products
concentration
(lag/l)
Figure
2.
Effect
of
incomplete
combustion
by-products
from
trichloroethylene
soot
whole
extract
(bars)
and
fractions
1
and
3
on
the
development
of
medaka
after
static
non-renewal
exposures
at
embryonic
stages.
Each
point
represents
the
mean
of
four
replicates
±
SE,
eight
embryos
per
replica.
In
fraction
1
(0.45
pg/I),
one
replicate
was
lost
due
to
bacterial
infection.
Significant
(p<0.05,
Wilcoxon's
sign-rank
test)
abnormalities
compared
to
controls
were
seen
at
concentrations
.7.2
pg/l
for
whole
extract
(*),
45
pg/I
for
Fl
(+),
and
>18
pg/I
for
F3
(#).
Table
2.
Percentage
of
normal
larval
development
(4-5
days
after
hatch)
of
medaka
exposed
to
trichloroethylene
soot
whole
extract
(WE)
and
its
fractions
(l14)a
Concentration
(Ug/I)
WE
F1
F2
F3
F4
0
Controlb
90.6
±
3.1
96.9
±
3.1
87.5
±
5.1
92.5
±
5.6
87.5
±
7.2
0
Solventc
93.8
±
3.5
93.8
±
3.6
87.5
±
5.1
84.4
±
6.0
93.8
±
6.3
0.45
90.6
±
6.0
95.8
±
3.6
78.1
±
6.0
75.0
±
7.2
71.9
±
11.8
0.90
87.5
±
5.1
87.5
±
7.2
81.3
±
8.1
65.6
±
7.9
87.5
±
7.2
2.7
71.9
±
6.0
75.0
±
1.0
71.9
±
6.0
68.8
±
10.8
90.6
±
6.0
7.2
50.0
±
9.0*
93.8
±
3.6
78.1
±
6.0
81.3
±
8.1
87.5
±
5.1
18.0
0*
84.4
±
7.9
84.4
±
7.9
34.4
±
7.9*
84.4
±
7.9
45.0
0*
59.4
±
9.4*
71.9
±
6.0
50.0
±
5.1*
71.9
±
12.9
aValues
represent
the
mean
of
4
replicates
±
SE;
number
of
embryos
per
replica
=
8.
One
replicate
from
Fl
(0.45
pg/I)
was
lost
due
to
possible
bacterial
infection.
Static
non-renewal
exposures
on
embryos
-10
hr
old
(blastula
stage),
until
8
days
(after
completion
of
organogenesis).
bControl
was
embryo
rearing
medium
(ERM).
CERM-DMSO
or
ERM-eluting
solvent
in
DMSO
(0.05%
v/v).
*Statistically
significant
(p
<0.05),
Wilcoxon's
sign-rank,
compared
to
respective
controls.
tude
were
seen
in
both
liver
and
heart.
Since
7.2
pg/l
was
the
experimental
EC50,
analysis
was
divided
into
two
groups,
depending
on
the
presence
or
absence
of
pericardial
edema.
Moderate
glycogen
depletion
char-
acterized
livers
of
embryos
which
showed
no
edema,
suggesting
that
the
former
was
the
more
sensitive
morphologic
indicator
of
exposure.
Although
heart,
kidney,
and
gut
were
examined,
no
other
significant
alter-
ations
were
seen.
More
advanced
structural
alterations
of
the
liver
accompanied
pericar-
dial
edema.
These
included
severe
glycogen
depletion,
mild
lipidosis,
and
occasional
enlarged
hepatocytes.
In
embryos
which
developed
pericardial
edema
but
showed
no
regression
to
tubular
heart,
walls
of
sinus
venosus
and
atrium
were
edematous.
This
localized
cardiac
edema
was
characterized
by
a
subendothelial
accumulation
of
fluid
in
the
sinus
venosus,
dilated
sinoatrial
com-
partment,
and
apparent
enlargement
of
sev-
eral
endothelial
cell
nuclei.
Ventricle
and
bulbus
arteriosus
were
apparently
not
affect-
ed.
Since
death
followed
when
concentra-
tions
>7.2
pg/l
were
used,
histological
alter-
ations
are
not
presented
for
those
fish.
Liver
Cells
Assays
At
concentrations
between
0.05
and
1.2
pg/l
and
in
the
absence
of
17f-estradiol
in
the
culture
media,
WE
induced
EROD
Volume
104,
Number
7,
July
1996
*
Environmental
Health
Perspectives
738
Articles
c
Toxicity
of
trichloroethylene
soot
Table
3.
Percent
(%)
of
medaka
embryos/larvae
with
selected
abnormalities
after
continuous
exposure
to
trichloroethylene
soot
whole
extract
(WE)
and
fractions
1
and
3
(Fl,
F3)
Pericardial/other
Abnormal
Death
resulting
Delayed/
Concentration
(pg/I)
edema
larval
activity
from
edema
incomplete
hatch
WE
Controla
0
0
0
3
Solventb
3
3
3
3
0.45
6
0
6
3
0.90
0
9
0
3
2.70
0
18
0
0
7.20
50
44
50
0
18.0
100
6c
88
6
45.0
100
3c
88
9
F1
Controla
0
3
0
0
Solventb
0
3
0
0
0.45
3
0
3
0
0.90
0
9
0
3
2.70
0
12
0
0
7.20
0
3
0
0
18.0
3
9
0
3
45.0
12
34
9
0
F3
Controla
0
6
0
0
Solventb
0
9
0
3
0.45
0
25
0
0
0.90
3
28
3
0
2.70
9
22
0
0
7.20
0
19
0
0
18.0
9
38
6
0
45.0
6
22
6
3
aControl
was
embryo
rearing
medium
(ERM).
bERM-DMSO
or
ERM-eluting
solvent
in
DMSO
(0.05%
v/v).
Values
represent
mean
of
nearest
whole
num-
ber
from
four
replicates,
except
for
0.45
pg/l
of
Fl
(loss
of
1
replicate).
cEdemas
produced
the
bulk
of
late
embryonic
mortality.
a
0
YS
-
Figure
3.
Normal
(A)
and
abnormal
(B)
late-stage
(216
hr)
medaka
embryos
after
control
and
trichloroethyl-
ene
whole
extract
treatments.
Note
how
pericardial
edema
(small
arrows)
results
in
separation
of
embryo
proper
from
yolk
sac.
E,
Eye;
H,
heart;
0,
oil
droplet;
YS,
yolk
sac.
Bar
=
100
pm.
activity.
This
induction
was
maximal
at
0.6
pg/l.
CYPIA1
protein
synthesis
was
signifi-
cantly
increased
at
the
three
higher
concen-
trations
(Fig.
5).
More
WE
was
required
to
cause
a
detectable
rise
in
CYPIA1
protein
than
for
a
rise
in
EROD
activity.
Only
fraction
1
showed
a
significant
increase
in
CYPlAl
protein
level
and
EROD
activity
(29
and
45%,
respectively).
All
concentrations
from
0.6
to
25
pg/l
of
WE
depressed
trout
liver
cell
response
to
17p-estradiol
relative
to
the
17j-estradiol-
only
positive
control.
Inversely,
CYPlAl
protein
was
induced
in
a
concentration
dependent
manner
with
increasing
concen-
trations
of
extract,
with
CYPlAI
protein
synthesis
maximal
at
25
pg/l
(Fig.
6).
However,
EROD
activity
at
all
concentra-
tions
tested
was
not
significantly
different
from
carrier
or
positive
(17,B-estradiol-only)
controls
(data
not
shown).
Mean
CYPlAl
protein
level
was
higher,
but
not
significant-
Figure
4.
Normal
(A),
and
abnormal
(B-D)
medaka
larvae
after
control
and
trichloroethylene
whole
extract
treatments.
Note
in
B
and
C
pericardial
(PE),
peritoneal/visceral
(VE),
and
eye
(EE)
edema
in
larvae
that
managed
to
inflate
swim
bladder
(SB)
and
all
of
the
above
plus
no
swim
bladder
inflation
in
D.
H,
Heart;
0,
oil
droplet;
YS,
yolk
sac.
Severe
edema
preceded
death.
Bar
=
1
mm.
ly,
at
the
3.95
pg/l
WE
in
the
absence
of
17p-estradiol
(Fig.
6).
Significant
depression
of
albumin
synthesis
(20-30%)
was
seen
only
at
the
higher
concentrations
(3.95-25
pg/i)
of
WE.
However,
the
viability
of
cells
exposed
to
all
concentrations
of
WE
was
confirmed
by
phase
contrast
microscopy,
cellular
protein,
and
cellular
LDH
activity
Environmental
Health
Perspectives
*
Volume
104,
Number
7,
July
1996
F
N
eooo'
t1-I
r.:"
739
Articles
*
Villalobos
et
al.
per
dish.
Typically,
of
the
322
x
106
±
73
x
106
(mean
±
SD)
liver
cells
harvested
per
fish,
90%
or
more
were
viable.
Soot
frac-
tions
1-4
were
tested
for
effects
on
vitel-
logenin
synthesis
as
above;
only
fraction
1
depressed
mean
vitellogenesis
(30%).
DNA
Binding
Gel
retardation
analysis
of
guinea
pig
hepatic
cytosol
which
had
been
incubated
with
WE
or
soot
fractions
resulted
in
the
formation
of
a
soot-inducible
protein-32P-
DNA
complex
(compared
to
the
control
solvent
fractions)
that
migrated
to
the
same
position
as
that
of
the
TCDD-inducible
complex
(Fig.
7).
We
have
previously
shown
(32)
that
the
TCDD-inducible
pro-
tein-DNA
complex
in
this
position
repre-
sents
the
high
affinity
binding
of
trans-
formed
TCDD-(AhR)
complex
to
double-
stranded
32P-labeled
DRE.
Results
indicate
that
not
only
does
WE
gontain
a
chemi-
cal(s)
which
exhibits
dioxinlike
activity
(i.e.,
it
binds
to
AhR
activating
its
transfor-
mation
and
DNA
binding),
but
that
each
of
the
fractions
tested
positive
in
this
assay.
Discussion
Transient
emissions
of
soot
and
toxic
volatile
organic
hydrocarbons
or
"puffs"
(4-6)
were
modeled
in
a
well-defined
labo-
ratory
experiment
with
a
laminar
diffusion
flame.
A
very
complex
mixture
of
halo-
genated
and
nonhalogenated
aromatic
hydrocarbons
was
found
in
association
with
the
aerosol
that
escaped
the
flame
in
the
same
manner
that
transient
puffs
escape
the
oxidation
zone
of
an
incinerator.
Although
the
total
amounts
of
these
emis-
sions
may
be
small
in
practice,
the
present
analysis
has
revealed
that
their
potential
toxicity
may
be
significant.
While
dioxins
and
furans
are
among
the
compounds
of
greatest
concern
that
can
be
found
in
the
effluent
of
hazardous
waste
incinerators,
and
while
significant
amounts
may
be
released
to
the
environment
in
this
way
(35),
attention
should
not
be
exclusive-
ly
directed
toward
these
compounds.
Harris
et
al.
(19,20)
found
that
certain
PCB
con-
geners
and
dioxin,
extracted
from
Lake
Ontario
rainbow
trout
skeletal
muscle,
were
toxic
to
medaka
embryos.
These
com-
pounds
are
present
in
Great
Lakes
biota
at
concentrations
ranging
from
parts
per
tril-
lion
to
parts
per
billion.
It
has
been
pro-
posed
that
these
non-ortho-substituted
PCBs
may
contribute
more
to
the
overall
toxicity
than
dioxins,
which
are
present
at
lower
orders
of
magnitude.
Nearly
all
chemical
species
in
the
mix-
ture
studied
herein
were
heavily
chlorinat-
ed
(4-,
5-,
6-Cl)
and
sometimes
perchlori-
nated.
They
included
benzenes,
styrenes,
._
CL
o.
.5
a,
KL
6
20
=
@
15
S
Is
IL
K
10
=5
0.5
0
E
I
C,
ja0
CD
:
a
.02
0
.0
-a
'4
(a
5
4
3
2
o
1.5
E
C)
LO
0
CD
.0
0
.0
-S0.5
CO)
wj
1
I
A
n
Carrier
0.05
0.15
0.3
control
Soot
WE
(igg/l)
Figure
5.
Soot
whole
extract
(WE)
induces
ethoxyresorufin
0-deethylase
(EROD)
activity
and
CYPlAl
protein
in
rainbow
trout
liver
cells.
Error
bars
=
standard
deviation.
Means
with
the
same
letter
are
not
significantly
different
(p<0.05,
ANOVA).
Number
of
dishes
per
treatment
=
3-4,
with
duplicate
determinations
per
dish.
fulvenes,
butadienes,
fulvalenes,
cyclopen-
tadienes,
naphthalenes,
acenaphthylenes,
and
phenols.
Although
many
compounds
still
remain
unidentified,
it
is
very
likely
that
these
as
yet
unidentified
organics
were
configurational
isomers
of
the
main
com-
pounds
just
mentioned,
given
the
possible
mathematical
combinations
of
chlorine
substitutions
across
the
many
double
bonds.
Despite
the
absence
of
2,3,7,8-
TCDD
and
-TCDF,
it is
conceivable
that
other
chlorinated
dioxins,
dibenzofurans,
and
related
chemicals
were
present.
Results
of
this
study
confirm
and
extend
previous
work
showing
the
presence
of
dioxinlike
compounds
(14)
in
this
com-
plex
soot
mixture
and
demonstrate
that
the
WE
and
fractions
3
and
1
(products
of
mixed
polarity
and
no
polarity,
respective-
ly)
caused
toxicity
and
exhibited
biological
activity.
The
major
developmental
toxicity
endpoint
of
this
study
was
edema
of
peri-
cardial
cavity
with
extension
to
peritoneal
cavity
and
yolk
sac.
Severe
pericardial
edema
was
accompanied
by
an
uncoiling
of
the
fused
endocardial
tube.
This
defect
resulted
in
a
reversal
of
initial
chamber
for-
mation
to
that
of
a
single,
pulsatile
tube.
The
latter,
normally
seen
at
an
earlier
stage
a0
.a
~~~~~
ia
in
Soot
WE
(gg/l)
+
17P-Estradiol
(1
gM)
Figure
6.
Effect
of
various
concentrations
of
whole
extract
(WE)
on
vitellogenin
and
CYPlAl
protein
levels
in
rainbow
trout
liver
cells
simulta-
neously
exposed
to
1
pM
17p-estradiol
or
carrier
control.
Error
bars
=
standard
deviation.
Significant
(p<0.05,
ANOVA)
depression
of
vitel-
logenin
(all
concentrations)
and
increase
in
CYPlAl
protein
(all
concentrations),
indicated
by
asterisks,
is
relative
to
17f-estradiol-only
control.
Number
of
dishes
per
treatment
=
3-4,
with
dupli-
cate
determinations
per
dish.
of
development,
was
also
accompanied
by
apparent
rupture
of
the
posterior
pericar-
dial
membrane
with
release
of
fluid
into
peritoneal
cavity.
These
changes
resembled
those
reported
after
exposure
to
dioxin
or
dioxinlike
compounds
(12,38-41)
by
late
embryo
and
larval
stages
of
rainbow
(36)
and
lake
trout
(37),
medaka
(18),
chick,
fish-eating
birds
(terns,
herons,
double
crested
cormorants,
and
herring
gulls),
and
rodents.
Furthermore,
the
generation
of
toxicity
in
medaka
embryos
exposed
to
TCE
soot
resembled
that
of
TCDD,
where
early
development
proceeded
normally
and
was
followed
by
a
gradual
progression
of
cardiotoxicity.
Histopathological
studies
have
suggested
that
edema
of
endothelial
cells
and
myocar-
dial
interstitium
was
an
important
early
stage
in
cardiotoxicity
(37).
Interestingly,
juvenile
yellow
perch
(Perca
flavescens),
respond
more
aggressively
with
myocyte
necrosis,
hypertrophy,-
and
hyperplasia
of
pericardial
mesothelium
as
well
as
fibrinous
Volume
104,
Number
7,
July
1996
*
Environmental
Health
Perspectives
740
Articles
-
Toxicity
of
trichloroethylene
soot
a a
c c
4_
0
0
0
0
co CO
coJ
-
L
al
_7
X
Lu
co
c
co
c
0
..
....
.X.
.....=
.
. x
Figure
7.
Soot
and
soot
fractions
stimulate
arylhy-
drocarbon
receptor
(AhR)
transformation
and
DNA
binding.
Guinea
pig
cytosol,
incubated
in
the
presence
of
15
nM
TCDD,
whole
extract,
various
control
solvents
or
soot
solvent
fractions,
was
mixed
with
32P-labeled
DRE
oligonucleotide
and
specific
protein-DNA
complexes
resolved
by
gel
retardation
as
described
in
Methods.
The
arrow
indicates
the
position
of
the
inducible
AhR:DNA
complex.
The
following
are
densitometry
readings
(%)
of
the
chemically
induced
bound
complexes
relative
to
that
obtained
with
TCDD.
Control
read-
ings
were
subtracted
as
background.
TCDD
(100%);
whole
extract
(55%),
Fl
(69%),
F2
(52%),
F3
(62%),
F4
(34%).
pericarditis
(42).
Although
our
initial
histo-
logic
analyses
have
not
revealed
altered
endothelial
morphology,
it
is
possible
that
fluid
loss
occurred
through
this
tissue
into
pericardial
and
peritoneal
cavities.
The
occurrence
of
edema
in
mammals,
birds,
and
fish
by
TCDD
and
related
compounds
(37)
suggests
a
common
mechanism
related
to
endothelial
dysfunction
(43).
Immuno-
histochemical
studies
have
localized
CYPlAl
to
endothelium
of
heart
in
scup
(Stenotomus
chrysops)
(44)
and
salmonids,
and
embryonic
induction
occurs
commonly
in
endothelial
cells
(45).
It
is
possible
that
CYPlA
induction
(mediated
through
AhR
activation)
in
our
study
could
have
led
to
oxidative
injury
and
loss
of
endothelial
integrity.
Octachlorofulvalene
appears
to
be
a
potent
inhibitor
and
substrate
of
certain
glutathione
S-transferases
(GSTs)
(46),
as
are
many
extensively
chlorinated
com-
pounds.
Perhaps
some
embryo
toxicity
may
be
related
to
changes
in
cellular
redox
status
resulting
from
depletion
of
reduced
glu-
tathione
or
GST
inactivation.
Embryonic
chick
edema
after
TCDD
or
toxic
PCB
congener
exposure
suggested
that
increased
prostaglandin
synthesis,
as
a
sequel
to
AhR
activation,
could
mediate
CYPlA
induction
and
cardiotoxicity.
Such
a
relationship
was
suggested
by
the
ability
of
benoxaprofen,
an
anti-inflammatory
drug,
to
reduce
toxicity
in
3,4,3',4'-tetra-
chlorobyphenyl-treated
embryos
without
affecting
CYPIA
induction,
supporting
a
role
for
arachidonic
acid
metabolites
(prostaglandins,
leukotrienes,
etc.)
as
medi-
ators
in
toxicity,
rather
than
induction
itself
(38).
Wisk
and
Cooper
(47)
exposed
medaka
embryos
to
dioxin
(.10
ng/l)
or
beta-naphthoflavone
(BNF;
50
pg/1)
and
found
increased
activity
of
benzo(a)pyrene
hydroxylase.
Induction
of
these
CYP1A-
associated
enzymes
over
a
period
of
days
suggests
that
embryos
have
an
intact
AhR-
mediated
activation
pathway.
However,
while
benzo(a)pyrene
hydroxylase
induc-
tion,
hemorrhage,
and
edema
were
seen
in
some
medaka
after
dioxin
treatment,
others
showed
similar
induction
but
no
vascular
changes
at
nontoxic
levels
of
BNF.
This
suggests
that
CYPlA
induction
is
not
a
prerequisite
of
cardiotoxicity.
Nevertheless,
the
importance
of
AhR
mediated
events
in
embryonic
cardiovascular
toxicity
needs
further
study.
While
we
are
not
aware
of
these
types
of
studies
in
fish,
investigations
in
other
animal
models
have
shown
interaction
between
the
CYPIA-AhR
system
and
other
CYP
iso-
forms.
These
linkages
involve
metabolic
alterations
of
endogenous
substrates
through
biochemical
pathways,
which
include
antioxidant
enzymes,
metallothioneins,
heat
shock
proteins,
steroid
receptors,
oncogenes,
tumor
suppresor
genes,
glutathione,
and
GSTs
(45).
Possible
involvement
of
rodent
CYP
1
B
1
in
edematous
lesions
and
overall
dioxinlike
toxicity,
depending
on
tissue-
specificities,
is
being
investigated.
Although
highly
inducible
by
TCDD/PAHs
(via
AhR)
and
involved
in
PAH
metabolism
(48),
the
presence
of
CYPIBI
in
fish
remains
to
be
demonstrated.
Edematous
spaces,
devoid
of
cells,
were
observed
in
heart,
peritoneum,
and
skin
of
medaka
embryos,
a
condition
similar
to
that
of
chickens
exposed
to
TCDD
and
toxic
PCBs
(38,39).
We
cannot
state
whether
the
developing
medaka
has
white
blood
cells
capable
of
emigration
into
extravascular
spaces,
and
we
cannot
rule
out
a
compound-induced
cytopenia.
Other
possible
mechanisms
underlying
edema
continue
to
be
investigated.
In
situ
nuclear
magnetic
resonance
analyses
from
our
labo-
ratory
suggest
that
transient
depression
of
certain
energy
phosphate
metabolite
levels
(mainly
ATP)
may
lead
to
deficient
ion
translocation
and
consequent
edema
(Villalobos,
in
preparation).
We
are
also
focusing
attention
on
the
relative
abun-
dance
of
basement
membrane
components
in
control
versus
treated
medaka
embryos.
While
WE
adversely
affected
normal
development
in
a
concentration
dependent
manner,
various
concentrations
of
fractions
3
or
1
did
not
exhibit
such
a
relationship.
This
finding
may
be
related
to
solubility
but
has
persisted
over
repeated
assays.
Perhaps
combustion
by-products
of
non-
polar
and/or
intermediate
polarity
act
syn-
ergistically
in
the
WE
to
produce
effects
whose
impact
was
not
apparent
when
a
sin-
gle
fraction
was
assayed.
However,
syner-
gism
has
not
been
specifically
tested.
Moreover,
direct
comparisons
of
the
toxici-
ty
of
combined
fractions
with
WE
are
complicated
by
losses
of
volatile
com-
pounds
or
the
reactivity
of
constituents
like
the
chlorinated
fulvenes
and
fulvalenes.
Thus,
evaluation
of
the
toxicity
of
individ-
ual
fractions
should
be
viewed
as
a
qualita-
tive
guide
indicative
of
the
polarity
of
the
most
toxic
components
of
WE.
In
vitro
observations
revealed
no
direct
cellular
toxicity
but
vitellogenin
in
medium
was
reduced.
Fish
liver
cells
are
sensitive
indicators
of
exposure
to
aquatic
pollutants
that
have
dioxinlike
activity
(49-52).
Hepatocytes
and
biliary
epithelial,
and
endothelial
cells
contain
the
readily
inducible
enzyme
CYP1A1
(45).
The
liver
plays
a
key
role
in
reproduction
in
fish,
being
a
component
of
the
hypothalamic,
pituitary,
gonadal,
and
liver
reproductive
axis
(53).
In
these
oviparous
vertebrates,
the
egg
yolk
precursor
protein
vitellogenin
is
synthesized
in
the
liver
and
transported
by
the
circulatory
system
to
the
developing
oocytes.
Vitellogenesis
is
under
direct
con-
trol
of
estrogens
(54),
and
since
CYPlAI-
inducing
compounds
such
as
dioxin
are
known
antiestrogens
in
mammals
(55),
the
possibility
exists
that
vitellogenesis
and
gonadal
maturation
could be
disrupted
in
exposed
fish.
At
the
concentrations
tested,
WE
was
not
overtly
toxic
to
liver
cells
but
induced
dioxinlike
effects.
EROD
activity
and
the
amount
of
CYPlAI
protein
increased
in
a
concentration-dependent
manner,
con-
firming
the
dioxinlike
activity
of
compo-
nent(s)
of
the
extract.
The
EROD
activity
assay
proved
more
sensitive
in
detecting
significant
changes
in
CYPlAI
expression
at
low
WE
concentrations
than
the
CYP
lAl
ELISA
assay.
17P-Estradiol
may
have
had
an
inhibitory
or
antagonistic
effect
upon
EROD
and
CYPlAl
protein
induction
in
cultured
liver
cells,
as
has
been
previously
demonstrated
in
vivo
with
femi-
nized
brook
trout
(56)
and
in
mouse
fetal
cell
cultures
(57).
Higher
concentrations
of
WE
significantly
increased
CYP
IAI
protein,
but
EROD
activity
remained
unchanged.
At
concentrations
above
0.6
pg/l,
components
of
the
WE
may
have
competitively
inhibited
binding
of
ethoxyresorufin
to
CYPlAI.
Substrate
inhibition
by
PCBs
in
fish
liver
cell
EROD
assays
has
been
demonstrated
in
vivo
and
in
Environmental
Health
Perspectives
*
Volume
104,
Number
7,
July
1996
741
Articles
-
Villalobos
et
al.
vitro
(58,59).
These
effects
underscore
the
importance
of
conducting
direct
measure-
ments
of
enzyme
concentration
in
addition
to
enzyme
activity.
Trout
liver
cells
exposed
simultaneously
to
noncytotoxic
concentrations
of
17,1-
estradiol
and
WE
showed
much
less
vitel-
logenin
in
medium
than
did
similar
cells
exposed
to
173-estradiol
alone.
Vitellogenin
levels
and
CYPlAl
protein
appeared
to
be
negatively
correlated.
Higher
concentra-
tions
(3.95-25
pg/l)
of
the
extract
may
affect
the
secretory
capacity
of
liver
cells;
however,
even
at
the
0.6
pg/l
concentration
(where
albumin
synthesis
was
not
depressed)
vitellogenin
production
was
still
compromised.
From
the
fractions,
only
fraction
1
showed
an
effect
on
CYPlAI
protein
or
EROD
activity
(both
increased),
or
vitellogenin
(reduced).
CYPlAI
induc-
ing
compounds
may
suppress
vitellogenin
production
in
fish
liver
cells
by
an
antiestro-
genic
mechanism
mediated
through
the
AhR,
similar
to
that
described
in
mammals
(60).
We
investigated
whether
this
mecha-
nism
might
apply
to
teleost
liver,
since
AhR
has
been
identified
in
this
organ
(45).
Numerous
studies
have
revealed
that
most
of
the
critical
and
sensitive
toxic
and
biological
responses
to
TCDD
and
related
compounds
are
mediated
by
its
soluble
AhR,
to
which
these
chemicals
bind
with
high
affinity
(7,13,61).
After
ligand
binding,
the
halogenated
aromatic
hydrocarbon:AhR
complex
undergoes
transformation
into
its
DNA
binding
form
and
translocates
into
the
nucleus
(62,63).
The
transformed
com-
plex
associates
with
a
specific
DNA
sequence,
the
dioxin
responsive
element
(DRE),
resulting
in
transcriptional
activa-
tion
of
adjacent
responsive
genes
(63-66).
Since
previous
studies
have
demonstrated
a
high
correlation
between
binding
of
a
chem-
ical
to
the
AhR
and
its
degree
of
toxicity,
the
relative
biological/toxicological
potency
of
complex
mixtures
of
chemicals
can
be
esti-
mated
by
measuring
the
ability
of
an
unknown
chemical/mixture
to
activate
the
AhR
or
an
AhR-dependent
response
(61,67).
Previously,
we
have
utilized
a
gel
retardation
DNA
binding
assay
to
demon-
strate
that
transformed
TCDD:AhR
com-
plexes,
formed
in
vitro,
can
bind
to
a
DRE
oligonucleotide
specifically
and
with
high
affinity,
mimicking
that
which
occurs
in
vivo
(32,65,66).
Since
there
appears
to
be
an
excellent
correlation
between
the
ability
of
a
given
chemical
to
stimulate
AhR
trans-
formation/DNA
binding
and
its
ability
to
activate
gene
expression,
this
technique
has
been
utilized
as
a
sensitive
bioassay
for
the
detection
of
dioxinlike
chemicals
(33).
The
gel
retardation
assay
results
indi-
cated
that
WE
contains
dioxinlike
chemi-
cals
which
not
only
bind
to
the
AhR
but
also
induce
its
transformation
and
DNA
binding.
The
formation
of
inducible
pro-
tein-DNA
complexes
by
each
soot
fraction
implies
that
the
soot
must
contain
numer-
ous
AhR
ligands.
Given
the
correlation
between
the
ability
of
a
given
chemical
to
stimulate
AhR
transformation/DNA
bind-
ing
and
its
ability
to
activate
gene
expres-
sion,
our
results
suggest
that
WE
and
frac-
tions
might
also
alter
gene
expression
in
mammals.
In
addition,
given
the
role
of
the
AhR
in
mediating
toxicity
of
these
chemi-
cals
(7,13,61),
it
is
very
likely
that
some
of
the
toxicity
produced
by
these
compounds
was
AhR-mediated.
Fractions
2
and
4
were
not
associated
with
developmental
car-
diotoxicity
but
did
bind
to
the
AhR
induc-
ing
its
transformation
and
DNA
binding.
While
these
processes
may
lead
to
car-
diotoxicity,
mediating
factors
are
not
known
and
need
investigation.
In
summary,
CH2C12
extracts
of
TCE
combustion
aerosol
proved
toxic/bioactive
using
a
battery
of
bioassays.
The
pattern
of
toxicity
was
identical
to
that
previously
reported
for
dioxin.
Chemical
analyses
per-
formed
herein
documented
the
presence
of
at
least
250
chlorinated
incomplete
com-
bustion
by-products
in
the
whole
soot
extract,
but
the
obvious
target
compounds,
TCDD
and
TCDF,
were
not
present
at
detectable
(picomole)
levels.
These
results
indicate
that
an
array
of
toxic
effects
may
arise
from
substances
other
than
those
tar-
geted
by
conventional
chemical
analyses.
They
also
suggest
a
need
for
bioassay-
directed
assessments
of
toxicity/biological
potency
in
complex
mixtures.
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Number
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July
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743
