Not to be citcd without prior referencc to thc authors
ICES STATUTORY
f.,IEETING
1993
C.M. 1993/E:15
between EROD and GSH -T activities and the presence of organochlori-
Correlation
nes in the Iiver of dab from the Belgian continental shelf.
by
P. Roosc, K. Coorcman
and W. Vynckc
Rijksstation voor Zeevisserij, Ankerstraat
1, 8400 Oostende
Abstract
•
•
The cthoxyrcsorufin - 0- dcethylase
(EROO)
and glutathione -S- transferase
(GSH - T)
activities in the liver of dab (Limallda lil1l all da) were corrclated with the concentration of 10 chlorobiphenyl (CB) congeners (IUPAC nrs 31, 28, 52, 101, 105, 118, 138,
153, 156 and 180), hexachlorobenzene
(HCB) and p,p'-dichlorodiphenyldichloroethylene (p,p'-OOE). The sampies of dab were obtainid from the Belgian Continental
Shelf in March, May, September and Occember 19~h. The fat content of dab liver
showcd a ,spe.ci fi (pattern, with the lowest conccntrations in carly spring and the
highcst conccntrations
in carly autumn. Similar organochlorinc
patterns were
observed in all sam pies and no seasonal variation was observed in thc CB concentration as dctermincd' on thc bassis of total extractablc lipid. EROO and GSH - T
activities exhibited no apparent seasonal variation: No significant correlations could
be found betwcen the concentration of the organochlorincs and thc EROO activitics.
However, in several cases a sig~ificant correlation could bc found betwcen the GSHT activities and the organochlorine concentrations.
1. Introduction
The use of enzymatic activity as a rapid screening
environmental
decade.
pollution,
Especially
has been
enzymes
method
the subject of extensive
associated
possible
study during the last
with the cytochrome ,P - 450 monooxygenase
system such as ethoxyresoruCin-O-deethylase
(EROO)
have proven to be weil suited
for this purpose (Payne et aI., 1987; Jiminez and Stegeman,
1992). The monooxygenase
in determining
1990; Goksoyr and Förlin,
system and conjugating enzymes
are important
detoxification processes in animals. Two types of enzymes can be distinguished:
I enzymes
e.g. EROO
and phase 11 enzymes
The phase I enzymes are responsible
to exogenous
products,
e.g. glutha.tione
transferase
for the
phase
(GSH-T).
for the addition of a more polar reactive group
such as apolar
organic
contaminants,
rendering
it a more
1
"
\
t
,
'i ....
suitable substrate for phase II enzymes. The phase II enzymes create conjugates with
endogenous
elevated
molecules
EROD
which can be more readily excreted. Several authors reported
activities
rinated biphenyls (PCB's)
in the presence
of organic
(Payne et aL, 1987; Jimine'z and Stegeman,
aI., 1991; Goksoyr and Förlin, 1992). Moreover,
the Joint
Monitoring
pollutantssuch as polychlo-
Group
indicator for environmental
(JMG)
of the
ERGO
Oslo
has been recommended
and Paris
and Stegeman,
•
by
as an
has been payed
as indicators of environ-
1990). Although it has been demonstrated
that GSH-T adivity in fish liver increases
pounds (George
Commissions
pollution. In contrast very' little attention
to phase 11 enzymes (e.g. glutathione S-transferase (GSH-T»
mental pollution (Jimenez
1990; Galgani et
after administration
of xenobiotic
com-
and Young, 1986; Fair, 1986, Davies, 1985, Boon et aL, 1992). Van
Veld and Lee (1989) reported that GSH-T activity in the European
flounder (Platiclz-
tys fleslls) did not follow a pollution gradient and that the enzyme was therefore not
suited as an indicator. In the present study the seasonal
hexachlorobenzene
(HCB),
nyldichloroethylene
were examined
correlations
polychlorinated
variations of EROD,
biphenyls (CB's)
GSH-T,
and p,p'-dichlorodiphe-
in the liver of dab (Limallda
between enzymatic activities and concentrations
limallda)
and the
of organochlorines
were
analysed.
2. Materials
and methods
2.1. Chemicals
•
The chlorobiphenyl
(CB)
congeners
materials
certified reference
IUPAC nrs 28,52,101,118,138,153
purchased
from the Community
Bureau
and 180 were
of
Reference
(BCR).
The
chlorobiphenyl
dichloroethylene
pure isomers
congeners
(p,p' -DDE)
(Promochem,
Ethoxyresorufin
IUPAC
nrs 31,105
and
and hexachlorobenzene
156, p,p'-dichlorodiphenyl(HCB)
were purchased
as
Wesei).
was synthesized
according
to the method
of Prough
et aL (1977),
modified by Klotz et a1. (1984). Purity was controlled bY,thin-Iayer and reversed phase
liquid chromatography
with ethoxyresorufin
from the Sigma chemical company as a
reference.
All others chemicals were of research grade quality.
2
.\,
t' •
2.2. Sampling
The sam pIes
of dab were obtained
May, September
and Oecember
from the Belgian
Contincntal
Shelf in March,
1992 during scientific cruises of the oceanographic
vessel Belgica. At least 12 sampIes of dab liver per period were collected for chemical
and biochemical
analysis. Although sampling occured on several locations,
site (site n· 120 (Westdiep)
as is described
in Cooreman
correlation analysis between chemical and biochemical
only one
et aL, 1993) was used for
data.
2.3. Biochemical analysis
EROO
•
and GSH-T activities were measured
onboard
activity was f1uorimetrically
the method
of Burke and Mayer (1974). The GSH- T activity was spectrophotome-
detailed
(1993),
according
fish livcrs.
The EROO
trically determined
determined,
in freshly collccted
according to a modified method of Warholm
information
on the biochemical
The livers were subsequently
to a modification
et aL (1985). More
analysis can be found in Cooreman
stored
of
et al.
by freezing (-20°c) prior to chemical
analysis.
2.4. Chemical analysis
2.4.1. Extraction and clean-up
Extraction was based on the total lipid extraction of Bligh and Oyer (1959). Thc lipid
material
•
that was used for the determination
hexane (see also Vandamme
(1982))
of the fat content was redissolvcd
and c1eaned
on a f10risil column (deactivated
during 16 hours at 420 ·C and activated with 1.5 % water). The organochlorines
subsequently
naphtalene
in
were
eluted with hexane. After addition of the internal standard tetrachloro(TCN) and isooctane as a keeper, the hexane fraction was concentrated
a rotary evaporator
in
and further under a stream of nitrogen to 1 mI. This solution was
analysed by gaschromatography.
Quality control was assured using certified reference
materials
and
(CRM
349,
BCR)
exercises (ICES/OSPARCOM
2.4.2. Chromatographie
participation
intercalibration
in
international
intercalibration
for CB's in marine media).
eonditions
'.
3
--- -- - - - - - - - - I
.
~
I' •
Instrument:
CE 8160
Column:
J&W DB 17, lenght: 60 m, internal diameter:
0.25 mm, film thickness:
0.25 11m.
Carrier gas:
Helium, 150 kPa
Injection:
1 111 splitless, splitter open 1 mm 10 after injection.
Temperatures:
Injector: 235 ·C, Detector:
310
re: 90 ·C for 2 min, 1st rate: 15
265 ·C, Isothermal:
C, Oven program:
C/min to 150, 2
10 min, 3th rate: 15
nd
initial temperaturate: 3 C/min to
C/min to 275
C, Isother-
mal: 10 min.
Detection:
Ni 63 electron capture detector.
3. ResuIts
3.1. Seasonal variation of fat.
The results of the fat analysis can be found in table 1. The fat content of the liver
showed a specific pattern, reaching its lowest concentration
rising towards a peak concentration
in early spring, stcadily
in late summer and early autumn and decreasing
again during winter (figure 1).
Seasonal
va~lat10n
1n the rat content or the
11ve~
50
<40
•
30
20
10
A
B
c
o
Figure 1. Seasonal variation of the fat content in dab liver. The X axis represents the sampling
periods (A=March, B=May, C=Scptember and D= December). The Y axis represents the fat
content of the liver expressed as % fat (mean and standard deviation ).
4
.,
,t
3.2. Seasonal variation
Thc rcsults
01' CB's, HCB and p,p'. DDE.
of thc chemical
analyses
varied between 209 ng/g extractable
(expressed
can be found in table
1. The
lipid and 899 ng/g extractable
conccntration
lipid for the CB's
as the sum of the CB's nrs 28, 31, 56, 101, 105, 118, 138, 153, 156 and
180), 3 ng/g extractable
lipid and 35 ng/g extractable
lipid for HCB and between 15
ng/g extractable lipid and 61 ng/g extractable lipid for p,p'-DDE. Among the CB congeners nrs 153 and 138 were always present in the largest concentrations.
Moreover,
the organochlorine
to be con-
patterns (figure 2) of the different sam pies
appeared
stant.
.
..
•
IifIO
. ..
...
.~ =..
u
'"
=~
... ...
j
I
,.'
I
I
I
.'
I
""lol\)
'M
•
(
LI
l__
~
'_L-.,-,,_.L.'.--I._.L.'_...L_.L.'. - - 1 . _ 1
1 . -- - L - L - . _ L - - - 1 . - - L . - - . L .__ - J _
~
~
~
"
:
~
_
=
~
:
"
=
"
G
~
M
.. _ L - . _ l - _.... -1_.-i-_.L-_.1.- _ _ l _ ' - . - L . ~
N
N
~
~
;
;
n
~
=
Figure 2. Chromatogram illustrating the typical CB pattern that was f~und in the liver of clab
(Limanda limanda).
Further , no seasonal
variation
appeared
in the CB concentration
as determined
on
the basis of total extractable lipids (figure 3). This was statistically confirmed using an
ANOVA
test ( P
=
0.8712).
Plotting the total CB content of the liver (calculated
the liver and the CB concentration
on the basis of the fat content of
of the fat, see table 1) against the period of sam'.
5
."
.'
pling (figure 4) resulted in an entirely different picture. As with the seasonal
of the fat content (figure 1), the total CB concentration
summer, reached a peak concentration
increased
variation
during spring and
in late summer and early autumn, decreased
again during wintern and reached the lowest concentration
in carly spring.
COllparison of th. CB canc. in 'at of the l1ver p.r s •• saß
•
A
c
B
D
Figure 3. Seasonal variation of the CB concentration in dab livcr. The X axis represents the
sampling periods (A=March, B=May, C=September and D= December). The Y axis reprcsents
the CB conccntration, expressed as ng/g extractable lipid (mean and standard deviation).
Sel.anal ",ariatiD" 0' the
ca
cancentratlon
•
A
B
c
D
Figure 5. Seasonal variation of the CB concentration in dab liver. The X axis represents the
sampling periods (A=March, B=May, C=September and D= December). The Y axis represents
the CB concentration, expressed as ng/g totalliver weight (mean and standard deviation).
'.
6
. '.
\
"
TABLE 1
Results of the chemical and biochemical analyses for the different periods.
Period
Sampie
nr.
EROD activity
••• pm/mg.min
March '92
May'92
September '92
2
3
4
5
6
7
8
9
10
11
12
1
2
3
4
5
6
7
8
9
10
11
12
1
2
3
4
5
6
7
8
9
GSH-T avtivity
•••• nm/mg.min
SUMCB
IICB
•• ng/g
•• ng/g
3639
1933
3757
382
850
737
156
626
120
2166
967
57
146
98
91
467
214
114
301
153
231
256
712
628
544
273
352
209
423
673
565
210
107
11
298
127
89
342
296
226
570
367
357
630
647
379
229
208
167
88
178
171
247
213
207
443
195
250
260
266
399
228
899
763
471
350
403
423
549
640
• n.d.
• n.d.
• n.d.
12
• n.d.
• n.d.
• n.d.
• n.d.
• n.d.
• n.d.
• n.d.
• n.d.
59
• n.d.
o n.d.
o n.d.
38
• n.d.
• n.d.
59
o n.d.
430
280
579
528
379
374
458
649
430
419
13
14
14
5
9
8
8
35
21
13
8
12
5
8
13
18
10
13
11
10
6
10
15
9
5
6
8
6
3
4
10
5
4
p,p'-DDE
•• ng/g
48
50
35
28
31
16
27
53
47
30
30
38
21
15
46
50
42
61
48
27
15
35
24
28
45
30
37
20
42
21
46
21
18
17
20
20
30
% Fat in
Livcr
16.3
14.7
14.9
20.1
7,1
33.6
18.5
8.5
12.6
8.0
7.8
8.5
26.8
12.8
27.5
22.6
28.3
21.8
14.7
32.6
37.8
23.9
27.6
22.9
24.5
37.8
29.6
33.0
44.1
41.3
33.3
27.9
44.4
39.6
323
3
278
10
31.1
440
4
328
11
30.6
5·
203
382
12
• n.d.
36.6
5
417
584
26
13
29.6
42
521
9
°n.a.
1
December '92
• n.d.
46
33.4
*n.8.
503
8
2
• n.d.
15.5
62
·n.a.
615
9
30
3
25.5
*n.3.
32
527
6
4
• n.d.
15.2
56
*n.8.
7
502
5
• n.d.
33.6
56
·n.a.
7
494
38
6
19.0
88
·n.8.
5
506
7
• n.d.
o n.d.- not detectable; n.a.- not analysed
•• ng/g is expressed as ng per g extractable lipid
••• pm/mg.min is expressed as picoJT!oles resorufin production per minute and per mg protein
•••• nm/mg.min is cxpressed ~s nanomoles S.2,4-dinitrophenylglutathione production per minute and per mg protein
7
.'. ,
".
3.3. Seasonal variation of EROD and GSH - T activities
The results of the biochemical
have
been
extensively
data can be found
discussed
by Cooreman
table 1. The results for EROD
In
et
al.
(1993).
Enhanced
EROD
activitics were observed
in March, with considerable
differences between
dual
the
no or very little activity was
activities.
observed.
During
other
sampling
periods
The GSH-T activities showed little difference
the indivi-
for the three periods,
with
the exception of slightly elevated activities in September.
3.4. Correlation
analysis between chemical and biochemical data.
3.4.1. EROD activity vs organochlorine
•
EROD
activities were plotted against the concentration
as ng/g liver ) and the results
correlation
test). The correlation
case and which would suggest
EROD
concentrations.
were statistically
activity and the concentration
activities were plotted
für correlation . (Pearson
r) did not exceed 0.47 in any
is no significant correlation
against
organochlorine
concentrations
•
total CB (r=0.61)
and GSH-T and p,p'-DDE (r=-O.77)
corrclation
between
GSH- T
and p,p'-DDE
and GSH-T and HCB (r=-0.58)
the da ta for September,
available for December
no correlation
In
was obtained.
(expressed
(Pearson
tion test). The results (table 2) ~uggested a negative linear correlation
a positive linear
between
the
concentrations.
ngl g li ver ) and the results were statistically analysed for correlation
T and total CB's (r=-0.75)
(expressed
of the organochlorines.
3.4.2. GSH - T activity vs organochlorine
GSH- T
analysed
coefficient (Pearson
that there
of organochlorines
as
correla-
bet ween GSH-
in March (fig.5) and
(r=0.78),
GSH- T
May (fig.6). When
Unfortunately,
and
comparing
no da ta were
as was the case with EROD.
8
~
.
."
.'
TABLE 2
Rcsults of the correlation analysis for GSH-T and organochlorincs
Date
March '92
May'92
Comparison
• r
GSH-T vs Sum CB
-0.75
GSH-T vs HCB
-0.32
GSH- T vs p,p' DDE
-0.77
GSH-T vs Sum CH
0.61
GSH-T vs HCB
0.58
GSH- T
•
September '92
VS
p,p' DDE
0.78
GSH-T vs Sum CB
0.00
GSH-T vs HCB
0.10
GSH-T
vS
p,p' DDE
-0.03
• Pearson correlation coeffficient
...
.
"
•
.. ...
,
ZlO
...
Li_
11..'"
Figure 5. GSH-T activity plotted against the concentration of total CB in March (Ieft) and
May (right). The X axis represents the GSH-T activity, expressed as nanomoles S-2,4-dinitrophenolglutathione production per minute and per mg protein. The Y axis represents thc organochlorine concentration, expressed as ng per g liver.
9
."
-'.
...
.....,
"
.
Figure 6. GSH-T activity plotted against the concentration of p,p'-DDE in March (Ieft) and
May (right). The X axis represents the GSH-T activity, expressed as nanomoles S-2,4-dinitrophenolglutathione production per minute and per mg protein. The Y axis represents tbe organochlorine concentration, expressed as ng per g liver.
4. Discussion
The concentration
levels of the organochlorines
were similar to those
Knickmeyer and Steinhart (1990). Fig.3 shows that the CB concentration
dab liver remained
decreased
In
constant during the year. The hepatic fat concentration
two and a half fold between September
that the hepatic CB concentration
•
reported
normalised
by
the fat of
however
and March (fig.1). This implies
to the wetweight of the liver varies in a
constant ratio to the fat content (figA). Maximum values for the total CB concentration per mg wet weight were found in September.
tion decreased
seasonal
Towards March the CB concentra-
to approx. 50 ng/g wetweight. These resuIts c1early indicate tbat the
variation of tbe CB content in the Iiver is related to the variation of the fat
conte nt in the liver. The decrease' of fat in the coldest perio'd of the year is most
probably caused by the changing metabolie
food shortages
fat is metabolised.
status of the animal. To compensate
for
The joint variation of fat and CB content points to
astate of equilibrium between the CB concentration
in the fat of the liver and other
parts of the animal and maybe particulate organic matter outside the anima!. Equilibrium partitioning
reported
of CBs between
body lipids, blood
and the ambient
by Duinker and Boon (1985) as the dominating
water was
process regulating the CB
".
10
"
."
content
in gill- breathing
animals.
The
elimination
chemicals out of the liver during fat metabolism
of CBs
presumably
could be responsible
induction during winter. A significant increase in EROO
coldest period of the year (Coorcman
and
other
for the EROO
activity was observed in the
ct a1., 1993). The scasonal
variation of EROO
seems to bc inverscly related to the variation of CB and fat. On the other hand an
in EROO
increase
conte nt
began
EROO
and simultaneous
gives room
to decrease.
However
increase
to the suggestion
in Oecember
no induction
when the fat and CB
was observed.
that the CBs
in the fat are
systems. Although
correlation
to previous
results reported
could be demonstrated
in literature
of
in summer
weil isolated
and
similar results
expected for HCB and p,p'-OOE, this could not be demonstated
hand. Contrary
The decrease
of hepatic CB and fat concentrations
to cause induction of the defence
available
•
activity could be expected
not
were
with the results at
(Galgani
et a1., 1991), no
between EROO activities and the conccntration
of
organochlorines.
The seasonal
variations of the correlation
and between GSH-T and p,p'-OOE may refer to complex biochemical
processes. The
respective
organochlorinc
decrease
concentration
contaminants
(Andersson
and increase
in enzyme
the inhibition
is caused
found bctween
et a1. (1982)
peroxidation
and
CBs
Thomas
in liver homogenates
Atlantic croacker (Micropogonias
our results, CB's are liberated
production.
clear seasonal
decrease
examined
or by other
et a1., 1985 ; Boon et a1., 1992). Based on these results an inhibitory role
correlation
Thomas
by thc compounds
is not clear. GSH-.T induction by PCBs in fish was previously reported
for CBs on GSH-T should bc considcred
the
activity with increasing
in March and May points to enzyme' inhibition and induction (fig.5 &
fig.6). Whether
•
coefficients between GSH- T and total CBs
Experiments
as unlikely to occur. This could imply that
and
GSH-T
and Wofford
and microsomes
was establishcd
(1984)
elevated
lipid
from mullet (Mugil cephalus) and
undulatus) exposed to a PCB mixture. According to
from liver fat in March, which may enhance
with musseis performed
by Viarengo
variations with enhanced oxidative stress
in the concentration
observed
by coincidence.
of peroxide
scavengers
~n
peroxide
et a1. (1991) showed
musseis accompanied
(e.g.
glutathion,
by a
vitamin
E,
others) and antioxidant enzyme activities in winter. In May an inversed pattern was
observed. Based on these results two mechanisms
could influence the level of GSH-T
11
activitics in dab livcr
variation of the glutathion content and inhibition by peroxides.
The above considerations
organpchlorines
mechanisms
and the presencc of a correlation
between GSH -T and the
examined, stress the need for further research
on the toxicokinetical
that influence the cellular metabolism.
References
Andersson,
T., Personen, M. and Johansson,
P-450-dependent
transferase
monooxygenase,
C. Differential induction of cytochromc
glutathione
S-transfcrase
and UDP
glucuronosyl-
activities in the liver of the rainbow trout by ß-naphtoflavone or Clophen
ASO. BIOCHEM.
PHARMACOL.
34:3309-3314, (1985).
Bligh, E.G. and Dyer, W.J. A rapid method
for organic compounds.
CAN. J. BlOCHEM.
Boon,J.P., E\'erearts,
J.M., Hillebrand,
of total lipid extraction and purification
PHYSIOL.
37:911-917, (1959).
M.T.J., Eggens,
M.L., Pijnenburg,
Goks 0yr, A. Changes in the levels of hepatic biotransformation
globin levels in fern ale
plaice (Plcurollcctes
J. and
enzymes and haemo-
platessa) after oral administration
technical polychlorinated . biphenyl mixture (Clophen
of a
A40). SCI. TOTAL ENVIRON.
114:113-133, (1992).
Burke, M.D. and Mayer, R.T. Ethoxyresorufin:
ma O-dealkylation
Direct fluorometric
assay of mlcroso-
which is preferential1y inducible by 3-methylcholanthrcnc.
DRUG.
METAB. DISP. 2:583-588, (1974).
Cooreman,
C., Roose, P. and Vyncke, W. EROD
monitoring
In
dab from thc Bclgian
continental shelf. ICES E:15:(1993).
Davies, P.E. Thc toxicologie and mctabolism
enzymatics
and detoxication
in Salmo
of chlorothalonil
spp and Galaxias
In
fish. 111. Metabolism
spp. AQUAT.
TOXICOL.
7:277 -300, (1985).
'.
12 .
1
.. ---. "
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\.,
Duinker, J.C. and Boon, J.P. PCB congeners in the marine cnvironment-A review. In:
Organic
Micropol/utants
Environment.
in Aql/atic
Symposium, edited by Commission
of the European
sion of the European
1985, p. 187-205.
Communities,
Fair, P.H. Interaction
and benzo(a)pyrene
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hydroxylase
ENV. CONT. TOXICOL.
Galgani,
EROD
F., Bocquene,
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