Document 99zEBKbDB8oL6Eo39zBmn6pbD
Metabolism of a Polychlorinated Biphenyl (Aroclor 1254) Mixture in the Rat
by D. L. Grant, W. E. J. Phillips, and D. C. Villeneuve
Research Laboratories, Food and Drop Directorate Department of National Health and Welfare Tunne.y's Pasture, Ollau>a .1, Ontario
Polychlorinated biphenyls (PCB's) have been used in industry
since at leant 1930. In 1955, von Octtingrn (1) reviewed the
literature on the toxicity of these compounds. Industrial
workers have developed lesions of chloracne as a result of
leaking vapours of a chlorinated biphenyl (Aroclor *) used as
a heat-exchange material (2).
'
Today the problem is not restricted to exposure of industrial
workers as polychlorinated biphenyls are widely dispersed in fish
and wild life (3-11). Westoo et al. (11) found low levels of PCB
(90%, <0.1 mg per kg fat) in a large number of foodstuffs. They
also reported that all 22 samples of human milk analyzed
contained PCB residues with 11 samples between 0-0.5 and the
other 11 between 0.6-1.0 mg per kg fat. Therefore, there is a
need to 6tudy the metabolism, distribution, storage, effect on
reproduction, etc., of PCB's in mammals. This paper will report
on the metabolism and distribution of Aroclor 1254 in normal and
carbon tetrachloride-treated rats.
Experimental
.
Experiment 1
The distribution of PCB's in various tissues of the rat and
the effect of liver damage on their metabolism, was studied in
twenty male wistar rats randomized into four groups and treated
as described in TABLE I.
Experiment II
The effect of Aroclor 1254 on the organ weights, fat content of the liver and on the potentiation of the toxicity of carbon tetrachloride was investigated in thirty eight Wistar rats divided into four groups and treated as described in TABLE II.
In Experiments I and II, the rats were housed in air conditioned rooms and were supplied with food and water ad
libitum. The rats were killed by decapitation,- blood collected, and brain, liver, heart, spleen, kidneys, testes and omental fat removed, weighed and frozen pending analyses,
Aroclor^ , Monsanto Company (U.S.A.), registered trade name
for polychlorinated biphenyls.
..
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It Toxfro1o|ry.
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Group
Day 1
Day 2
Day 3
Day 4
Day 0
Day 24
Each group con "Corn oil, 2 ml
1+1 CCI4:cor qA 500 mg per m
orally at 500 Corn oil, 1 ml rA 1*1 CCl4:cor
Group* 1 Group15 2 Group*3 3
Group 4
t
Eive male rats "Fifteen male r
on days 8, 12, CA 500 mg per m .orally at 500 . aNine male rats gA U1 CCl4:cor
day 1 and 1 ml
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TABLE I
Doeing regimen for experiment I
Group
1 234
Day 1 Day 2 Day 3 Day 4 Day 8 Day 24
Oilb PCBd Oil Killed
--
Oilb PCBd Oil
e Oil Killed
CC4lf PCBd
f CCI4
Killed
* .
--
CCl4 PCBd
f CC14
f CCI4
Killed
Each group contained five male rate, Wietar strain, av. b.w. 375 gCorn oil, 2 ml per kg, administered orally. A 1+1 CCl4:com oil solution, 2 ml per kg, administered orally.
A 500 mg per ml com oil solution of Aroclor 1254, administered orally at 500 mg per kg. Corn oil, 1 ml per: kg, administered orally. fA Itl CCl4:com oil solution, 1 ml per kg, administered orally.
1`
TABLE II
.
t
l ' ' Dosing regimdn"for experiment II '
1
'i
Group 1 Groupb 2 Groupd 3
Group d 4
Nil ;
PCBc on day 2
CC1 on days 1, 3, 8, 12, 16, 20, 24, 28, 32, 36, 4
40, 44, and 47
'
ccf4 on days 1, 3, and 8 and PCBc on day 2
Five male rats, Wietar strain, av. b.w. 410 g,' killed on day 1. bFifteen male rats, Wistar strain, av. b.w, 440 g, 5 killed
on days 8, 12, and 47. CA 500 mg' per ml corn oil solution of Aroclor 1254 administered -orally at 500 mg per kg.
Nine male rats, Wistar strain, av. d.w. ^ g. eA 1 + 1 CCl4*.com oil solution, 2 ml per kg administered orally on
day 1 and 1 ml per kg on other dayo.
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Polychlorinated jbiDbenyl analyses. Tissues were blended with 100 ml hexanc'and 50 9 anhydrous" sodium sulfate for 10 minutes. The extracts were filtered, concentrated to 10 ml, 3 ml of cone. H2S04 " fuming H2 S04 (1:1) added and shaken. The hexane solutions were dried and neutralized with 4 g of anhydrous sodium sulfate-sodium carbonate (10:1). The solutions were subjected to GLC-EC analyses on a Varian Aerograph Model 600D gas chromatograph, fitted with a coiled 4 x 1/4" O.D. glass column containing Chromosorb W, 80-100 mesh, coated with 4% SE-30 and 6X OF-1. The nitrogen flow rate was 120 ml per minute with column and injection temperatures of 193 and 225<>c, respectively. Aroclor 1254 standards and test solutions were chromatographed and the concentration of PCB*s in the test solutions determined by comparing the heights of the six major peaks of the standards with corresponding peak heights of the test solutions.
Problems were not encountered with sensitivity during analyses and the lower limit of detection would depend on the tissue being analyzed. Hexane and acetone gave equal recoveries of Aroclor residues. Quadruplicate analyses of a rabbit liver gave values of 431, 438, 464, and 474 ppm. Recoveries of spiked samples were greater than 95X.
Lipid was determined by the method of Bligh and Dyer (12).
Results and Discussion
Experiment I
From day 1 to day 4 the rats dosed with corn oil and Aroclor 1254 (groups 1 and 2) or carbon tetrachloride and Aroclor 1254 (groups 3 and 4) lost an average of 19 and 55 g (b. w.), respectively. On day 8, group 2 rats had regained their lost weight, while group 4 rats continued to lose weight. Four of the five rats in group 4 which received the oral dose of carbon tetrachloride on day 8 died within the next 24 hours. However, by day 24 the 1 remaining rat in group 4 and the five rats in group 2 had gained 20 and an average of 30 g, respectively. The livers of the group 2 rats were enlarged and averaged 5.32*0.3038 of their body weight.
Residues of Aroclor 1254 (expressed as ppm wet tissue, TABLE III) . were found in all tissues analyzed, with fat and blood having the greatest and least concentration, respectively.
The residues in the blood, testes, liver, kidney and heart were significantly greater in the group 3 rats than in the group 1 rats. This shows that the liver is the main site of Aroclor 1254 metabolism and rats with carbon tetrachloride damaged livers are not able to metabolize this mixture as rapidly as rats with normal livers. The residues in the spleen, brain and fat were similar for both groups. The residues in the brain, liver,
1M
spleen, blood, (killed on d respectively, < values show Various tissue
Resi<
Group
Blood Testes Heart Spleen Kidney Brain Liver Liverd Fat
1. 19. 24. 29. 31. 39. 115. 1860. 996.
Mean of five va ^Standard error
^Single value. "Ppm on a fat be
GLC-EC trac residue found
1. The amount the total resic
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spleen, blood, testes, heart, kidney and fat of group 2 rats
{killed on day 24) were 10, 16, 20, 21, 22, 2*4, 36 and 67%,
respectively, of those of group 1 (killed on day 14) . These
values show that the PCB residues were being cleared from the
various tissues at different rates.
.
TABLE IH
Residues in tissues of rats orally dosed with Aroclor 1254 (500 mg/kg)
Residue found (ppm, wet tissue)
Group
1
2
34
Blood
1.96ai: 0.23b 0.42 i 0.07
3.85 1 0.46
0.25
Testes
19.22 0.59
4.30 0.44
33.181 1.35 ^5.62
Heart s*-.
Spleen '
Kidney
24.16 29.17 31. m*
2 84 3,44 1
5.831 0.53
62.401
1* ** .. ;irfl .
5.821 1.17 1 *36.601
2.09 11.201 1.76
57.301
4.37 , 4.39 3.91 '
6.17 11.08
Brain
39.981 5.91
4.011 0. 31
41.911 3.30 1 5.96
Liver
115.661 10.55 18.851 1.65 796.471 64.96
18.79
Liver15 1868.141166.63
.--
6137.641556.06
Fat 996.161 98.58 672.661155. 12 900.461106.16 1149.05
Mean of five values. Standard error of the mean. Single value. Ppm on a fat basis.
GLC-EC tracings from 24 ng of Aroclor 1254 and from the residue found in the liver of a group 2 rat, are 9hown In Figure 1. The amount (%) that each of the six major peaks contribute to the total residue in the tissues is presented in TABLE IV.
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The amount {* 1254 standai
Peak number Aroclor 1254
Spleen Testes Liver Kidney Fat Brain Blood Heart
Spleen Testes Liver Kidney Fat Brain Blood Heart
* ' `T.^ W tm'jm ipmh
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TABLE IV
The amount <*) that each of the six major peaks of the Aroclor 1254 standard contribute to the total residue in the tissues
Peak number
Percent 123456
Percent 123456
Aroclor 1254 12 25 18 17 15 13
12 25 18 17 15 13
l Group 1
Group 2
Spleen
6 21 9 22 21 21
3 17 2 11 34 32
Testes
3 15 6 21 27 28
1 19 2 11 35 32
Liver .. i - . t
2 11
3 ^ 30 28 27 . i .
0 25 2 18 26 28
Kidney . y-4 >1 .y* Mr
Fat
8 21 11
i. -v 7 25 11
21 19 19
<4* . * 25 17 16
0 18 2 14 34 32
.if'-
*
1 19 4 20 28 28
Brain
1 13 5 23 30 29
2 19 4 12 34 29
Blood
4 13 6 ; 24 26 26
1 20 3 12 33 31
Heart
4 17 6 21 26 26
.1 17 2 8 31 29
Spleen Testes
Group 3
i
7 25 14 19 18 17
. r 6 23 14 !19 19 19
Group 4 1
1 20 4 12 31 32
Liver
4 21 11 21 22 21
0 19 4 20 26 31
Kidney
8 22 15 18 18 17
1 21 4 8 32 34
Fat ` 10 29 16 21 13 12
1 20 5 20 26 28
Brain Blood
6 25 13 ' 20 18 18 5 22 14 22 18 18
1 22 4 11 30 31 2 22 3 11 29 33
Heart
5 27 15 18 18 17 i ii
1 20 - I, ..
3 10 32 34
I IT*
107
.
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limirc 1 and TABLE IV show that the components of the Aroclor 1254 mixture with the shorter retention times, peaks 1, 2 and 3, and presumably with the lowest chlorine contents (10) were metabolized to a greater degree than those with the longer retention times. This observation agrees with that reported in studies with Phenochlor DP6* fed to Japanese Quail (7). in group 2, peak 2 accounted for 25, 20, 19 and 19% of the residue in the liver, blood, brain and testes, respectively, whereas in group 1 the percents were 11, 13, 13 and 15%, respectively. Thus, the metabolism of this component (peak 2) was very slow between day 4 and 24 or one or more of the other components of the mixture were metabolized to the peak 2 component. The residue pattern in the fat of animals treated with carbon tetrachloride (Group 3) was similar to that of the standard Aroclor 1254 indicating little metabolism.
Expression of the Aroclor 1254 residues in the tissues relative to those in blood (TABLE V) shows that the ratios depend on the length of time following the oral dose (kidney, brain, live'' and fat, group 1 vs 2) and the metabolic activity . of the liver (liver and fat, group 1 ve 3) .
TABLE V
Concentrations of the Aroclor 1254 residues in tissues relative to those in blood
Group .
1 j 2;
3
4
Blood Testes Heart Spleen Kidney Brain Liver Fat
:
1.00 9.81,
j 1.00 I 10.24
12.33
13.88
14.88
: 13.86
; 15.89/ ,
26.67
20.40
9.55
59.17
44.88
508.24
1601.57
1.00 '
1.00
> 8.62
22.48
16.21
24.68
9.51
--
14.90
44.32
10.89
23.84
206.88
75"Tl6
233.89
4596.20
;
. .
Phenochlor DP6, Prodelec's (Prance) registered trade name for polychlorinated biphenyls.
: toe
'
However, since jcould be used Iproviding the 'estimates. i Experiment II
In Experime therefore expe Aroclor 1254 (a and (b) alters
Eight of th receiving the rats in group 3 it is conclud carbon tetrachl phenobarbital (
Treatment changes in orga increase in liv
Signiflean in organs
Day killed
Organ
Spleen
Liver
Heart
Kidney
Testes
^Expressed as % "Expressed as ac
,^,1, n11,jT';<vn"it '* -111 W"'
1, ;
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,
However, since blood samples are relatively eaey to obtain they could be used for estimating the residue in other tissues, providing the investigator is aware of the limitations of the
estimates.
,
$ii i
*
Experiment II
In Experiment I no rats received carbon tetrachloride alone, therefore experiment II was conducted to determine whether Aroclor 1254 (a) potentiates the toxicity of carbon tetrachloride
and (b) alters the weights of other organs besides the liver.
Eight of the 9 rats in group 4 died within 24 hours of receiving the carbon tetrachloride on day 6, while none of the rats in group 3 died up to day 47, when they were killed. Thus, it is concluded that Aroclor 1254 potentiated the toxicity of carbon tetrachloride in a manner similar to that reported for phenobarbital (13-15) and for DDT (13, 15).
Treatment with Aroclor 1254 evoked a number of significant changes in organ weights (TABLE VI), the most consistent being an increase in liver weight.!
TABLE VI
Significant differences (Student Fisher's t te6t) found in organs weights from rats treated with Aroclor 1254^ and untreated rate
Day killed
' 8 12 47
Organ
Probability
Spleen Liver Heart Kidney Testes
''
'. ' nil
< 0.005a,b nil
nil <0.005a,b <0.05b
<0.01a <0.005a,b <0.05b
` - ' ; <o.osa
-
1 -<0.05^,3---' :
! j Ii
! i
; ;
;; 1I
1
, ; ;
f
;
^Expressed as % body weight. ^Expressed as actual organ weight.
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This agrees with results from experiments in this laboratory on rabbits treated with PCB's, which will be reported elsewhere. More differences were observed on day 12 than on days 8 and 47. The only organ to significantly decrease in weight was the spleen on day 47.
The results of the lipid analyses of some livers from experiments I and II are presented in TABLE VII.
TABLE VII
Percent lipid of rat livers from experiments I and II
Treatment
i i Lipid (X)
Nil, killed on day 1
Aroclor 1254 on day,2, killed on day 4d
|1 ' \1 i; .
'!
Carbon tetrachloride on day 1,
killed on day 4e
1
Aroclor 1254 on day^2,
,
killed on day 8r
. /i
Aroclor 1254 on day 2,
j
killed on day 121
*
4.45b0.008
6.19 0.23
r 13.22 1.17
1 1 . ' r'i .1. <
8.35 0.99 7.78 0.38
i
l 1
^Experiment II, group 1. Mean of five values. cStandard error of the mean.
Experiment I, group 1. Experiment I, group 3. Experiment II, group 2.
In experiment II, the lipid content of livers from rats killed 6 and 10 days after receiving the Aroclor were significantly higher than the controls (P<0.005, student Fisher's t test). Tanaka et al. (16) reported that chlorobiphenyls given orally (0.1 g per kg per day) to rats for four weeks caused loss of body weight, hepatomegaly ,and marked increase in serum lipid components. As expected, livers from rats which received the carbon tetrachloride and Aroclor 1254 had a very high content of lipid.
I ,
A number of the tissues from the rats killed on day 47 were analyzed for PCB residues (TABLE VIII).
HO
Residue: adminir.t
Residue Groups
pprr
Blood Heart Kidney Brain Liver Fat
0 2 3 4 16 397
?Group 2 of E Eean of five cStandard err
The residues were 9, 11, 1 group 1, e>: ratios again following di individual Ar amount <<) ` total residue 12, 25, 18, 1
Male rats v were found concentration i different from all components
residues were Aroclor 1254 re kidney and fat
respectively ir the size of
Aroclor 1254 we
tetrachloride i
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TABLE VIII
Residues in tissues of rats 45 days after being orally administered a single dose of Aroclor 1254 (500 mg/kg)
Residue Group0
Blood Heart Kidney Brain Liver Fat
ppm, wet tissue
Relative to Blood
2 bc 0.18 *0.05
. i1 .
2 . 1.00
`
2.71 0.61
15.06
3.39 0.29
18.83
4.19 0.34
23.28
.
16.04 3.79
89.11
397.30 41.86
2207.22
fGroup 2 of Experiment II killed on day 47.
tViean of five values.
.
cStandard error of the mean.
The residues in the blood# heart, kidney, brain, liver and fat
were 9, 11, 11, 10, 14 and 40X respectively, of those found in
group 1, Experiment I rats (TABLE I). The tissue-blood residue
ratios again show these values depend on the length of time
following dosing.
The uneven rate of metabolism of the
individual Aroclor components was again noted. For example, the
amount (%) that each of the six major peaks contributed to the
total residue in the fat was 0, 15, 1, 12, 40 and 34, compared to
12, 25, 18, 17, 15, and 13 for Aroclor 1254 standard.
Summary
Male rats were orally dosed with Aroclor 1254 and residues were found in all tissues analyzed, with the greatest concentration in the fat. The GLC-EC pattern of the residues was different from the standard mixture administered, indicating that
all components were not metabolized at the . same rate. Higher residues were found in the carbon tetrachloride-treated rats. Aroclor 1254 residues in the brain, spleen, blood, testes, heart, kidney and fat were reduced by 90, 84, 80, 79, 78, 76, 64 and 33JG
respectively in 20 days, Aroclor 1254 significantly increased the sizo of the liver and also the percent lipid in the liver.
Aroclor 1254 was found to potentiate the toxicity of carbon tetrachloride in the rat.
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Acknowledgment
We acknowledge C.R. Westerby for technical assistance and B.B. Coldwell and K.A. McCully for review of this manuscript.
References
1. von Oettingen, W.F., Public Health Service Publication No,
414, 306 (1955).
2. Meigs, J.W., Albom, J.J., and Kartin, B.L., J. Am. Med.
t Assoc. J1J>4, 1417 (1954) .
.
3. Risebrough, R.W., Reiche, P., Peakall, D.B., Herman, S.G., and Kirven, M.N., Nature 220, 1098 (1968).
4. Jensen, New Scientist 3^,* 612 (1966).
5. Holmes, D.C., Simmons, J.H., Tatton, J.o'G., Nature 216, 227
(1967).
-----
6. Holden A.V., and Marsden, K,f Nature 216, 1274 (1967).
7. Koeman, J.H., Ten Noever De Brauw, M.C., and DeVos, R.II., Nature 221_, 1 126 (1969).
8. Jensen, S., Johnels, A.G., Olsson, M., and Otterlind, '''G., Nature 224, 247 (1969).
9. Duke, T.W., Lowe, J.I., and Wilson, A.J., Jr., Dulletin Environmental Contamination and Toxicology 5, 171 (1970).
i
10. Bagley, G. E., Reichel, W. L., and Cromartie, E., J. Assoc.
Offic. Anal. Chem. 5J3, 251 (1970).
;
11. Westoo, G., Noren, K., and Andersson, M., Varfoda 22, 9 (1970) .
12. Bligh, E.G., and Dyer, W.J., Can. J. Biochem. and Physiol.
37, 91 1 (1959) .
,
13. McLean A.E.M. and McLean, E.K., Biochem. J. 100, 564 (1966).
14. Farber, T.M., Heider, A., Peters, E.L. , Ritter, D.L., Disraely, M,, and Van Loon, E.J., Ninth Annual Meeting, Society of Toxicology, Atlanta, Georgia, March 15-19 (1970).
15. Cawthorne, M.A., Dunyan, J., Sennitt, M.V., and Green, J., Br. J. Nutr, 24, 357 (1970).
16. Tanaka, K., Fujita, S., Komatsu, F,, and Tamura, N., FukuokaIgaku-Zasshi 60, 544 (1969),
112
Cl)
i L
Evidence (PCB's) these po . have bcc (2), In birds, t fish, an at this sediment toxicity of this was not laborato effects (Lelostc we mease spot.
Regie
1 Gulf
rBullflin of
VoL 6, No. 2
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