induction of hepatic mixed function oxidase activity in trout (salvelinus fontinalis) by aroclor...
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TOXICOLOGY AND APPLIED PHARMACOLOGY 63, 166-172 (1982)
Induction of Hepatic Mixed Function Oxidase Activity in Trout (Salvelinus fontinalis) by Aroclor 1254 and Some Aromatic
Hydrocarbon PCB Replacements
R. F. ADDISON, M. E. ZINCK, D. E. WILLIS, AND J. J. WRENCH
Marine Ecology Laboratory, Bedford Institute of Oceonogrophy, Dartmouth, Nova Scotia B2Y 4A2, Canada
Received May 8, 1981; accepted December 16, 1981
Induction of Hepatic Mixed Function Oxidase Activity in Trout (Solvelinus fontinolis) by Aroclor 1254 and Some Aromatic Hydrocarbon PCB Replacements. ADDISON, R. F., ZINCK,
M. E., WILLIS, D. E., AND WRENCH, J. J. (1982). Toxicol. Appl. Phormacol. 63, 166-172. Groups of brook trout (Salvelinus fontinafis) were fed a polychlorinated biphenyl (PCB) mixture (Aroclor 1254) or three PCB replacements based on phenylxylylethane or diisopro- pylnaphthalene mixtures at dose levels of 30 and 100 pg *g-l. Both dose levels of all the materials tested induced hepatic ethoxyresorufin 0-deethylase activity; ethoxycoumarin O- deethylase activity was induced by Aroclor 1254 and one of the phenylxylylethane mixtures. Benzphetamine N-demethylase activity was not induced by any treatment. Effects on other indices of MFO induction, such as protein content and cytochrome P-450 content, were vari- able. The fish accumulated from 13 to 46% of the Aroclor 1254 dose but only 2 to 7% of the PCB replacements. All components of the phenylxylylethane mixtures were accumulated but some major components of the diisopropylnaphthalenes were not,
Phenylxylylethanes and diisopropylnaph- thalenes were introduced in Japan in the early 1970s as replacements for polychlori- nated biphenyls (PCBs) in pressure-sensitive copying paper and in some electrical appli- cations. Several toxicological studies have shown these substitutes to be generally less persistent and more degradable than the PCBs (Iwahara, 1974; Kawai et al., 1977; Yoshida and Kojima, 1978a,b). Neverthe- less, within about 5 years of their introduc- tion, the diisopropylnaphthalenes were re- ported as contaminants of estuarine mud and biota (Sumino, 1977). Since the induction of mixed function oxidases (MFO) indicates a sublethal effect, we describe here the ef- fects of feeding three commercial prepara- tions containing these PCB replacements on trout (Salvelinus fontinafis) hepatic MFOs, and we compare these effects with those of Aroclor 1254.
METHODS
The polychlorinated biphenyl mixture, Aroclor 1254, was supplied by Monsanto Company. The three Japa- nese PCB replacements (obtained from Dr. K. Sumino) were SAS-295 and SAS-296, both manufactured by the Nisseki Company, and KMC-A manufactured by the Kreha Company. The approximate composition of these materials is shown in Table 1.
Two feeding experiments were carried out 3 months apart, based on methods described previously (Addison et al., 1977) using doses of these materials of 30 and 100 pg. g-’ body weight (“low dose” and “high dose,” respectively). In each experiment, gonadally immature trout of similar weights were randomized among five groups of eight and were kept separately in flowing fresh water at 15’C and offered ad libitum Ewoz trout grower (Rundle Feed Mill, Palmerston, Ontario) throughout the treatment. Mean fish weights differed between the two experiments. Each fish was force fed a single gelatin capsule containing 0.1 ml of an organochlorine-free fish oil containing appropriate amounts of the dose; control fish were fed the encapsulated oil alone. Five days later the fish were killed and analyzed for indices of hepatic MFO activity including liver weight, protein content,
0041-008X/82/050166-07$02.00/0 Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form rcssrwd.
166
MFO INDUCTION BY PCB REPLACEMENTS 167
TABLE 1
COMPOSITION OF THE PCB REPLACEMENTS SAS-295” AND SAS-296” AND KMC-Ab
Material Composition (90, wt or vol not specified)
SAS-295 1 -Phenyl- 1 -( 3,4-xylyl)-ethane 1 -Phenyl- 1 -( 2,4-xylyl)-ethane Other phenylxylylethanes and
phenyl (ethylphenyl) ethanes ,y
SAS-296
KMC-A
1 -Phenyl- 1 -( 3,4-xylyl)-ethane 1 -Phenyl- 1 -( 2,4-xylyl)-ethane Other phenylxylylethanes and ]
33 60
phenyl (ethylphenyl) ethanes [ Trace
Monoisopropylnaphthalenes 2.5 Diisopropylnaphthalenes 86.9 Triisopropylnaphthalenes 6.5 Isopropylmethylnaphthalenes 4.1
93 Present
Trace
’ Nisseki Co. b Kreha, Co. ’ Data from Hesegawa et al. (1973).
and microsomal cytochrome P-450 (Addison er al., 1977). Benzphetamine N-demethylase was determined in the postmitochondrial (10,OOOg) supernatant fraction (PMS), essentially as described for aminopyrine N-de- methylase (Mazel, 1972); temperature and pH optima were 22°C and 8.0, and the reaction was linear with time up to 30 min and with protein concentration up to 2 mgeml-‘. Ethoxycoumarin O-deethylase was an- alyzed in the PMS (in the high-dose experiment only) as described previously (Addison et al., 1977). Micro- somal ethoxyresorufin Odeethylase was determined (Burke and Mayer, 1974) at 25°C; the pH optimum was 8.0, and the reaction was linear over time up to 5 min and with protein concentrations up to 1 mg -ml-‘.
Residues of the materials fed were analyzed in fillets from the experimental fish, since this tissue contains the bulk of residues of PCBs and similar material fed to trout (Addison et al., 1977; Guiney and Peterson, 1980). Samples of control fillet were fortified with appropriate amounts of the materials studied to determine recoveries through the analytical methods. PCBs were determined by standard methods (Addison et al., 1972). Phenylxy- lylethanes and diisopropylnaphthalenes were extracted and cleaned up by Florisil column chromatography in the same way as the PCB analysis. Eluates of the PCB replacements from Florisil were analyzed by gas-liquid chromatography (GLC) with flame ionization detection (FID) in a Hewlett-Packard instrument fitted with a 10-m X 0.25-mm (i.d.) wall coated open tubular column coated with SP-2100 (Supelco, Inc.). Because the com- ponents of SAS-295 and SAS-296 were not selectively deposited in fish tissues, these materials were estimated by comparing mean peak heights in the sample to those
in the standard. The components of KMC-A were ac- cumulated selectively, and this material was estimated by comparing the two major peaks deposited in the fish with those in standard material. A rough estimate of the total residue burden-and hence of the efficiency of accumulation from the diet-was calculated by multi- plying fillet concentrations by fish weight, and assuming that fillet concentrations represent “whole fish” concen- trations. Selected samples were analyzed by coupled gas chromatography-mass spectrometry (GC-MS) with a Finnigan 4000 system fitted with a 25 m X 0.25 mm (i.d.) wall coated open tubular column coated with SP- 2100.
RESULTS
The results are summarized in Table 2. None of the fish showed any external or
behavioral signs of stress caused by experi- mental feeding. Fish weights did not differ significantly between treatments within ei- ther experiment. Liver weight, expressed as a fraction of total body weight, was not af- fected by any of the treatments at the low- dose level, but at the higher level liver weight was increased by feeding SAS-295, SAS- 296, and KMC-A. This increase in relative liver size could not be attributed to an in- crease in protein content; at the low dose,
TABL
E 2
c 2 IN
DICE
S OF
HE
PATI
C M
IXED
FU
NCTI
ON
OXI
DASE
AC
TIVI
TY
IN
BRO
OK
TRO
UT (
Salve
linus
fo
ntin
alis)
FE
D PC
Bs
OR
PCB
REPL
ACEM
ENT
Varia
ble
Dos
e (p
g e g
-’ bo
dy w
) C
ontro
l Ar
oclo
r 12
54
Trea
tmen
t an
d m
ater
ial
fed
SAS-
295
SAS-
296
KMC-
A
Note
. R
esul
ts a
re m
eans
+
SD (
n =
8).
’ Diff
eren
ces
betw
een
grou
p no
t an
alyz
ed
stat
istic
ally
. * M
axim
um
valu
e.
* Si
gnifi
cant
ly
diffe
rent
fro
m c
ontro
l va
lue
(p
< 0.
05).
** S
igni
fican
tly
diffe
rent
fro
m c
ontro
l va
lue
(p <
0.
01).
Fish
wei
ght
Live
r %
bod
y we
ight
PMS
prot
ein
(mg
e g li
ver-‘
)
Micr
osom
al
prot
ein
(mg
. g li
ver-‘
)
Cyt
ochr
ome
P-45
0 (n
mol
* m
g m
ic.
prot
ein-
‘)
Benz
phet
amin
e N
- de
met
hyla
se (
nmol
pr
oduc
t fo
rmed
* m
g PM
S pr
otei
n-’
. min
-‘)
Etho
xyre
soru
fin
O-
deet
hyla
se (
pmol
pr
oduc
t fo
rmed
. m
g m
icro
som
al
prot
ein-
’ m
itt-‘)
Etho
xyco
umar
in
O-
deet
hyla
se (
pmol
pr
oduc
t fo
rmed
- m
g PM
S pr
otei
n-’
* min
-‘)
Res
idue
con
cent
ratio
ns
in
fille
t (fi
g * g
-’ we
t wt
)
30
100 30
100 30
100 30
100 30
100 30
100 30
1.55
f
0.55
10
0 2.
87 f
0.
72
100
16.6
3 +
4.05
30
100
161
* 29
16
8 f
26
174
+ 20
18
5 +
34
284
f 60
26
7 +
39
280
+ 47
28
5 +
44
1.9
f 0.
2 1.
9 +
0.2
1.9
+ 0.
3 2.
0 +
0.3
1.9
+ 0.
2 1.
8 +
0.2
2.1
+ 0.
2*
2.1
+ 0.
2*
96.8
-+
5.6
81.1
+
5.1*
* 79
.4
+ 5.
0**
83.7
+ 8
.9**
12
.0 k
6.
7 80
.4 +
6.
1**
80.0
+
4.7:
: 68
.6
+ 4.
5
12.7
+
2.8
10.9
+
2.8
10.9
+
1.1
10.3
+
1.9*
6.
7 +
1.3
11.6
+
1.9*
* 13
.1 +
1.
9**
7.7
f 2.
1 0.
53 +
0.1
3 0.
69 +
0.3
6 0.
11 f
0.
26*
0.67
+
0.18
0.
81 +
0.2
2 0.
39 +
0.0
9**
0.49
f
0.13
**
0.56
+
O.ll
*
0.14
5 +
0.01
9 0.
152
f 0.
046
0.16
3 ?
0.04
4 0.
156
+ 0.
037
0.21
8 f
0.02
8 0.
220
f 0.
063
0.22
6 f
0.06
2 0.
206
+ 0.
050
0.32
f
0.16
13
.6 +
3.
3 0.
09
+ 0.
03
13.5
+
3.0
12.2
k
13.7
* 17
.8 +
10
.2**
35.8
+
13.6
**
3.05
+
1.77
* 5.
19 I
k 2.
21**
19.5
+
4.20
0.82
+
0.21
2.
19 +
0.
83
6.63
2
3.93
**
3.02
+
1.93
* 7.
76 +
3.
85**
5.
71 f
3.
15*
25.3
f
5.43
**
16.4
f
2.49
1.60
+ 0
.92
<14b
3.
17 +
2.
09
6.86
f
2.41
174
+ 22
29
7 f
55
2.0
f 0.
1 2.
2 +
0.1*
*
61.1
+ 4
.4**
70
.2 +
15
.0
9.0
+ 2.
0**
9.8
+ 2.
2**
Lo
0.61
+ 0
.45
0.44
+ 0
.06*
* g E
0.14
2 zk
0.0
41
2 0.
258
+ 0.
066
m
MFO INDUCTION BY PCB REPLACEMENTS 169
’ I
SAS-295 -3.4 a
LL 2.4 \ b J.- -PRISTANE
-
SAS - 296
I.4 -2.4
C
A.4
TIME -
d
FIG. 1. Gas chromatograms of standard SAS-295 and SAS-296 (Figs. la and c, respectively) identifying the positions of Me substitution on the xylyl ring and of extracts of fillets from trout fed SAS-295 or SAS-296 as described in text (Figs. 1 b and d, respectively), show- ing pristane (a normal component of trout fed marine oils).
the PMS and microsomal protein contents of the livers from treated fish were usually lower than those of controls, and at the higher dose some experimental treatments caused higher protein contents. Microsomal protein expressed as a fraction of PMS frac- tion remained fairly constant at 12 to 14% and did not vary significantly between treat- ments. The cytochrome P-450 content of microsomal protein was slightly increased by the low dose of SAS-295 and was consis- tently lowered by the high dose of all the materials fed. This latter observation may have reflected the unusually low microsomal protein level in livers of control group fish in the high-dose experiment. Cytochrome P- 450 levels vary with sex in gonadally mature trout, but not in immature fish (Stegeman and Chevion, 1980). No significant differ- ences in cytochrome P-450 levels between the sexes were found in either experiment,
presumably because these fish were not ma- ture.
Activities of the 0-deethylase enzymes (expressed on either fresh weight or protein basis) showed the most consistent response. The low and high doses of Aroclor 1254 in- creased ethoxyresorufin 0-deethylase activ- ity (per unit protein) approximately eight- and six-fold, respectively. All the PCB re- placements at both dose levels significantly increased the activity of this enzyme, when expressed per unit protein. When expressed per unit liver fresh weight, only the dose of KMC-A failed to induce activity. Ethoxy- coumarin 0-deethylase activity per unit pro- tein was increased by feeding Aroclor 1254 and SAS-296; activity per unit liver fresh weight was increased by all materials except KMC-A. Benzphetamine IV-demethylase per unit protein remained fairly constant and was not affected by any treatment; when activity was expressed per unit liver fresh weight, the low dose of KMC-A significantly reduced it.
Residues of the materials fed were found in fish from all of the experimental treat- ments. Control fish contained small amounts of PCB (probably accumulated from the traces present in the fish chow; unpublished data); these residues were calculated as Ar- oclor 1254. The experimental fish which were fed Aroclor 1254 deposited it in con- centrations of about 13.5 pg. g-’ (in both experiments), which corresponded to ap- proximately 46 and 13% absorption from diet in the low and high-dose experiments. respectively. Chromatograms of the fish ex- tracts closely resembled those of standards, suggesting that there had been little selec- tivity in the deposition of PCB components.
Residues of SAS-295 and SAS-296 could be recovered efficiently (97.5 and 96.9%, re- spectively; each estimate the mean of four analyses) from fortified control samples, and chromatograms of residues of these mate- rials in the treated fish were similar to those of standards (Fig. 1). They were accumu- lated much less efficiently than Aroclor
170 ADDISON ET AL.
1254: roughly 2 to 3% of dose fed for SAS- 295, and 2 to 4% for SAS-296.
KMC-A could be recovered efficiently from fortified control samples (90.3% mean of four estimates) with no differences in peak heights between standard and fortified ma- terial. Residues in KMC-A-fed fish differed considerably from standards, in that one group of peaks was appreciably reduced (cf. Figs. 2a and b). GC-MS analysis showed these and some early eluting components to have m/e of 2 12 corresponding to diisopro- pylnaphthalenes; substitution positions could not be identified. KMC-A concentrations in the fish were based on comparisons of peaks A and B (Fig. 2a) in sample and in standard. Peak B had m/e of 226, consistent with an “extra” CHr function, possibly attributable to impurities in the naphthalene or the al- kylating agent from which this material was prepared. Since some major components of KMC-A were not deposited in the fish, the residue concentrations shown in Table 2 are approximations for comparative purposes only. Furthermore, the small amount of res- idues resulting from the low dose prevented reliable estimates, and a maximum value is shown.
The major peak found in control fish elut- ing with KMC-A, SAS-295, and SAS-296 was identified by its GLC and mass spec- troscopic properties as the isoprenoid hydro- carbon pristane.
DISCUSSION
The values of various indices of MFO ac- tivity (Table 2) are similar to those we have found previously in brook trout (Addison et al. 1977, 1981). Benzphetamine N-demeth- ylase and ethoxyresorufin O-deethylase ac- tivities were lower than those reported for rainbow trout (Salmo gairdnerii) held under similar conditions (Elcombe and Lech, 1978), which suggests that considerable differences in basal MFO activity exist between species. Some differences in indices of MFO activity
-
t z i? 3 a 0 L
-
II
!L.Ali m/e 2%
.m/e 212
a
,m/e 226
b
,PRISTANE
C
TIME -
FIG. 2. Gas chromatograms of (a) standard KMC-A, identifying m/e values of selected components deter- mined by GC-MS, (b) residues extracted from fillets of trout fed 100 pg’g-’ KMC-A showing peaks (A) and (B) used for quantitation; (c) extracts of a control group fillet showing pristane (a normal component of trout fed marine oils).
also existed between the control groups in the two experiments; although both groups of fish were from the same stock, those in the high-dose experiment were larger and
MFO INDUCTION BY PCB REPLACEMENTS 171
older than those in the lower-dose experi- ment. Size- or age-related effects on MFO activity have been reported elsewhere (Stegeman and Chevion, 1980; Forlin, 1980).
Aroclor 1254 was the most effective in- ducer of the O-deethylase system, and the extent of induction we found (six- to eight- fold for ethoxyresorufln O-deethylase and a doubling of ethoxycoumarin O-deethylase) was similar to that reported previously (El- combe and Lech, 1978; Addison et al., 198 1). All the PCB replacements were about equally effective in inducing ethoxyresorufin O-deethylase, but less so than Aroclor 1254. Since ethoxyresorufin O-deethylase activity appears to be associated with a cytochrome P-448-based MFO system, it seems that all the materials tested belong to the series of compounds which induce this, and not a cy- tochrome P-450 system. This hypothesis is supported by the benzphetamine demethyl- ase analyses: this enzyme is associated with a cytochrome P-450 system, and it was not induced by any of the materials fed. These results are also consistent with the conclu- sion that trout hepatic MFOs do not respond to compounds which induce cytochrome P- 450-based systems in mammals (Addison et al., 1978; Lech and Bend, 1980).
Aroclor 1254 was accumulated from the diet and deposited in the fillet at about 46 and 13% efficiency in the low-dose and high- dose experiments, respectively; this nonlin- earity in accumulation (and in the resulting MFO induction) is consistent with other ob- servations (Forlin and Lidman, 1978; Ad- dison et al., 1981). PCB replacements were accumulated and deposited in the fillet only about one-fifth as efficiently. These results are consistent with the few reports describ- ing the behavior of these materials. Thus, carp rapidly eliminated an accumulated bur- den of diisopropylnaphthalenes analogous to KMC-A with a half-life (whole organism) of 56 hr at 25’C (Yoshida and Kojima, 1978a). For comparison, goldfish at 21- 23°C eliminated a PCB mixture (Clophen A50) with a half-life of 500 hr or more
(Hattula and Karlog, 1973). Diisopropyl- naphthalene mixtures have a short half-life in mice (Iwahara, 1974) as do individual mono- and di-isopropylnaphthalenes in the rat (Kojima et al., 1978, 1979; Kojima and Maruyama, 1979). These compounds may be metabolized in both fish and mammals, generally to a mixture of polar products (Iwahara, 1974; Yoshida and Kojima, 1978a). The selectivity in the deposition of some KMC-A components observed in the present experiments, and by Sumino (1977), is consistent with possible metabolic degra- dation. However, in absence of any infor- mation about the pharmacokinetics of me- tabolites, we cannot ascribe MFO induction to the parent compounds or their metabo- lites.
Other toxicological studies on these PCB replacements have focused mainly on the diisopropylnaphthalenes and have shown that these compounds have relatively low acute toxicity. They are not obviously tera- togenic to mice, even at relatively high dose levels (Kawai et al., 1977), and only at very high levels (several g. kg body wt-‘) do they cause changes in serum enzyme levels which might indicate some liver damage in fish (Ozaki and Ikeda, 1976). The present results extend these toxicological observations and show that ingestion-which is a significant route of exposure of fish to lipophilic pol- lutants (Addison, 1976)---of these materials may exert some sublethal effects.
ACKNOWLEDGMENTS
We thank Dr. K. Sumino for the PCB replacement samples, and Mr. J. Leonard for GC-MS analyses.
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